The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner. App mRNA is expressed continuously starting at embryonic day 9.5 (E9.5) coinciding with the initiation of neurogenesis and neuronal differentiation (The Amyloid Precursor Protein (APP) is the precursor that generates the Aβ peptide, whose accumulation is associated with Alzheimer’s disease AD; . As an antiation .Structurally, APP is a type I transmembrane protein, which possesses a large extracellular amino acids sequence, an α−helix transmembrane sequence and a relatively short intracellular C-terminal sequence . Based oDrosophila brain development the fly homolog of APP, called APPL, functions as key component of the neuronal Wnt-PCP signaling pathway and regulates the axonal outgrowth in fly mushroom body  signaling pathway and the calcium pathway. Wnt signaling regulates various features during development such as cell proliferation, migration, and differentiation . RecentlDrosophila and mouse embryonic primary cortical neurons as models to explore the APP-Wnt interactions during development. We provide evidence that the CRD of APP is a conserved binding domain for both canonical and PCP Wnt ligands. Furthermore, APP trafficking and expression is regulated by Wnts through the CRD, which in turn is required for APP to regulate axonal and dendritic growth and branching.The presence of CRD in APP, the reported involvement of Wnt signaling in APP processing and the importance of Wnt signaling during development suggested to us that APP may be a novel class of Wnt receptor regulating neuronal development. We used Drosophila APPL has been implicated in neural development . In contrast to control MBs, 17% of male appl null mutant flies  displayed a loss of the MBb-lobe  to the complete loss of vang , while Wnt5 nulls showed β-lobe loss only in 5% of the brains examined, suggesting that Wnt5 is largely dispensable for β-lobe growth. Surprisingly, Wnt5-/-; vang-/- double mutants showed an almost complete rescue of vang loss of function  exogenous APP/APPΔCRD was detectable using either anti-APP or anti-flag antibodies  and pLv-pSyn1-mAPPΔCRD-Flag-IRESeGFP (flag-tagged mAPPΔCRD). Primary cortical neurons from tibodies . In neurtibodies . In conttibodies . Finallytibodies  and 4 hrtibodies . Similartibodies . All thetibodies .In summary, these data show that the CRD of mAPP is critical for Wnt3a/5a binding and mediates the effects of Wnts on mAPP trafficking and expression.APP and its proteolytic products have been reported to affect neurite outgrowth during development  in diffeWhile we found no effect on initial outgrowth at DIV2 , at DIV3To further analyze neurite outgrowth, we examined axonal and dendritic growth at DIV7. By this stage, mAPP knockout neurons showed an increased ACI . In contFinally, we examined dendritic growth at DIV7. We observed no difference in total dendrite length or the size of the longest dendrite , but obsThe Wnt pathway plays an important role in regulating neurite development, therefore we tested if the APP CRD is important for the interaction between APP and Wnt in regulating neurite outgrowth. To address this question, we analyzed neurite outgrowth in APP-KO primary cortical neurons rescued with APP-WT or APP-ΔCRD plasmids after Wnt3a/5a treatment. On DIV3 Wnt3a treatment significantly increased total axon length, the length of longest axon and axon branch tips in the presence of fl-APP but not APP-Δ-CRD . These dHere, we identify a previously unknown conserved Wnt receptor function for APP proteins. We show that APP binds both canonical and non-canonical Wnt ligands via a conserved CRD and that this binding regulates the levels of full-length APP by regulating its intracellular trafficking from early endosomes to the trans Golgi network versus the lysosome. Finally, we show that APP through the CRD regulates neurite growth and axon branching complexity in primary mouse cortical neurons.A function for APP as a cell surface receptor has be proposed for quite a long time . The firDrosophila APPL loss reduced axonal growth, the comparison of axonal outgrowth and branching in primary cortical neuron derived from mAPP wild type or mAPP knock out mice at DIV2, DIV3 and DIV7 showed that loss of mAPP significantly accelerated axonal maturation. Specifically, we found that the initial phase of axonal growth at DIV2 is unaffected, but that APP mutant axons grow longer at DIV3 and then show increased axon complexity at DIV7. We therefore suggest that the conflicting data in the literature may arise from examining different types of neurons at different time points, where the requirement of APP may differ in a context-specific manner. We speculate that this context specificity may in part be due to the levels and types of Wnt ligands present in the environment.APP has been extensively reported to be involved in regulating neurite outgrowth , with coApp mRNA levels upon Wnt treatment. With regard to the role of APP processing in Alzheimer’s disease, recently published work suggests that an imbalance between Wnt3a/canonical signaling pathway and the Wnt5a/PCP signaling pathway at the initial step of amyloid beta production could trigger a vicious cycle favoring the amyloidogenic processing of APP  via PhiC31-mediated transgenesis.Adult fly brains were dissected in phosphate buffered saline (PBS) and fixed in 3.7% formaldehyde in PBT (PBS+ 0.1% Triton100-X) for 15 min. Then, the brains were washed three times in PBT and blocked in PAX-DG for 1 hr at RT. The samples were later incubated with the primary antibody overnight at 4°C. After incubation, the brains were washed three times with PBT and incubated with an ALEXA Fluor secondary antibodies (Life technologies) for 2 hr at RT. After three times washes in PBT, the brains were mounted in Vectashield  mounting medium. The following antibodies were used: mouse anti-FasII , 1/50), rabbit anti-GFP . The mounted brains were imaged on a LEICA DM 6000 CS microscope coupled to a LEICA CTR 6500 confocal system and a Nikon A1-R confocal (Nikon) mounted on a Nikon inverted microscope (Nikon). The pictures were then processed using ImageJ.All experiments were done according to policies on the care and use of laboratory animals of European Communities Council Directive (2010/63). The protocols were approved by the French Research Ministry following evaluation by a specialized ethics committee (dossier number 4437). APP knock out mice were a gift from the De Strooper lab. Cortical primary neuron cultures were prepared from embryonic day 16.5 mice (APP wild or APP mutant), as described previously .5cells/mL). Fifty µL (50 µL par well) of the adequate lentiviral dilution in the medium of interest must be ready in tubes. Seed 150 µL of the cell preparation to each well. Immediately add 50 µl of the diluted lentiviral preparation to each well . Mix slowly the cells-lentivirus suspension by pipetting. Incubate 1 hr at 37°C. Finally add 800 µL of culture medium to each well and incubate for 3 additional days before any analysis.Virus  transduction was performed during seeding in 24-well plates (4x105cells/mL) in 24 wells plates with coverslip coated with PDL 24 hr before. All procedure follow the protocol from Lipofectamine 3000 transfection reagent  with little modified, each well transfection with 500 ng corresponding plasmid, medium was refreshed 5–6 hr after transfection.Plasmids  transfection was performed at the onset of cell seeding (4x10Wnt5a (400 ng/ml) , Wnt3a (150 ng/ml) , and PBS/BSA (control) addition performed at DIV7. In all experiments related to inhibitor, cells will be treated with inhibitor 1 hr after Wnt addition , LY294002  and a DMSO (0.05%DMSO in culture medium) group will be set as control. Protein or RNA samples collected after 4 hr of Wnt treatment.For the Aβ 40/42 detection, first, culture medium refreshed with Wnt3a (50 ng/ml or 150 ng/ml) or Wnt5a (100 ng/ml or 400 ng/ml) on DIV7 primary cortical neurons. Then, supernatant was collected after 4 hr Wnt3a/5a treatment for Aβ detection, and refreshed with medium without Wnt3a/5a. At last, the second round supernatant collection performed after additional 24 hr culture. The procedure of Aβ detection is based on the protocol from V-PLEX Plus Aβ Peptide Panel 1 (6E10) Kit .Amino acids with underscore are deleted in mouse, human APPΔCRD or APPLΔCRD.CGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVRVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN.MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGKLNMHMNVQNGKWESDPSGTKTCIGTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHTHIVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDSVDSADAEEDDSDVWWGGADTDYADGGEDKVVEVAEEEEVADVEEEEADDDEDVEDGDEVEEEAEEPYEEATERTTSTATTTTTTTESVEEVVRVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPHHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHPFGVDSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN.MLPSLALLLLAAWTVRALEVPTDGNAGLLAEPQIAMFceagqiyqpqylseegrwvtdlskkttgptclrdkmdlldyckkaypnrditnivesshyqkiggwcrqgalnaakckgshrwikpfrclgpfqsdallvpegclfdhihnasrcwpfvrwnqtgaaacqergmqmrsfamllpcgisvfsgvefvccpkhfktdeihvkktdlpvmpaaqinsandelvmndeddsndsnyskdaneddlddeddlmgddeeddmvadeaataggspntgssgdsnsgslddinaeydsgeegdnyeedgagseseaeveaswdqsggakvvslksdssspssapvapapekapvksesvtstpqlsasaaafvaansgnsgtgagappstaqptsdpyfthfdphyehqsykrleeshrekvtrvmkdwsdleekyqdmrladpkaaqsfkqrmtarfqtsvqaleeegnaekhqlaamhqqrvlahinqrkreamtcytqalteqppnahhvekclqkllralhkdrahalahyrhllnsggpggleaaaserprtlerlididravnqsmtmlkrypelsakiaqlmndyilalrskddipgsslgmseeaeagildkyrveierkvaekerlrlaekqrkeqraaereklreeklrleakkvddmlksqvaeqqsqptqsstqsqaqqqqqekslpgkelgpdaalvtaanpnlettksekdlsdteygeatvsstkvqtvlptvdddavqravedvaaavahqeaepqvqhfmthdlghressfslrrefaqhahaakegrnvyftlsfagialmaavfvgvavakwrtsrsphaqgfievdqnvtthhpivreekivpnmqingyenptykyfevke.mcaalrrnlllrslwvvlaigtaqvqaasprwepqiavlTCCATCATGAAGTGTGACGT-3' and anti-sense 5'- GAGCAATGATCTTGATCTTCAT −3', mAPP, sense 5'- CATCCAGAACTGGTGCAAGCG-3' and anti-sense 5'- GACGGTGTGCCAGTGAAGATG −3' GAPDH, sense 5'- GCTGCCAAGGCTGTGGGCAAG-3' and anti-sense 5'- GCCTGCTTCACCACCTTC −3'.Cells were lysed for RNA or protein extraction and then subjected to qRT-PCR or western blots as previously described . The detWestern blots were performed follow the user guide of Mini Gel Tank  with little modified. Briefly, Protein samples collected from total cell lysates with RIPA buffer, supernatant were collected after centrifugation, denatured samples were loaded separated on the 4–12% polyacrylamide gels (SDS-PAGE)  and then transferred to the 0.42 µm nitrocellulose membranes, blots visualization performed after primary and secondary antibody incubation.Human embryonic kidney (HEK293) cells (provided from Dr. Marie-Claude Potier) were purchased from ATCC, regularly mycoplasma test performed by the CELIS core facility of ICM. For the immunoprecipitation experiments, HEK293in 10 cm dish (70% confluent) were transfected with pCDNA3-MmApp-FLAG-IRES-eGFP, pCDNA3-mAPPΔCRD-FLAG-IRES-eGFP, pCDNA3-Wnt5a-myc, pCDNA-Wnt3A-V5 or co-transfected APP or APPΔCRD with Wnt3a or Wnt5a. Three days after transfection, cells were collected with NP-40 lysis buffer, then sample supernatant was collected after >12000 rpm centrifugation for 20 min at 4°C, 450 µl supernatant was incubated with primary antibody overnight at 4°C, then Protein G sepharose beads (Thermo Fisher Scientific) were added to the sample to capture protein-antibody complex by rotating 2 hr at room temperature, then washed four times with the lysis buffer, and resuspended with loading buffer then denatured at 95 degree for 10 min, blots visualized after western blot procedure as described before.At DIV 7, cultured primary neurons in 24 wells were washed once with 1X PBS, then fixed in 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 10 min. After three times washing with 1X PBS, cells were blocked with 10% normal donkey or goat serum in 1 X PBS for 30 min at RT followed by three times washing in 1 X PBS. Thereafter, cells were incubated with primary antibodies diluted in 1 X PBS containing 1% normal donkey or goat serum for 2–3 hr at RT. three times washing with 1 X PBS, incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647  in 1 X PBS containing 1% normal donkey or goat serum for 1 hr at RT. Washed with 1 X PBS for three times, then counterstained the slides with DAPI  and mounted by using Vectashield (Vector) after rinsing. Primary antibodies used in this study were rabbit anti-APP , mouse anti-rab5 , mouse anti-rab11a , mouse anti- Golgin-97 , rat anti-Lamp1 . After staining, images were obtained by using confocal microscope (Olympus FV-1200 or Leica SP8). The percentage of APP or APP-ΔCRD co-localizing with rab5, rab11, Golgin-97 and Lamp1 was calculated using JACOP .Statistical analyses were performed using GraphPad Prism software . Differences between groups were compared using the Student’s t test, One-way ANOVA and Mann Whitney two-sample test (two-tail) as appropriate. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.] and these findings were the reason why we tested if APP might directly interact with Wnt ligands. We have now performed IF and WB with β-catenin, Dvll and Dvl2 antibodies. Unfortunately, only the Β-catenin antibody worked in WB experiments on primary neurons. We find that Wnt3a treatment increases levels of β-Catenin in control but not APP-KO primary neurons, consistent with previous findings in cell lines that APP enhances canonical Wnt signaling. These data are now presented in Figure 4—figure supplement 1 and on page 8 of the revised manuscript.3. It has been demonstrated that canonical Wnt signaling inhibits neurite outgrowth. In Figure 6, it was shown that mAPP knockout neurons exhibited increased axonal outgrowth, which was rescued by the fl-mAPP but not by the form lacking the CRD at DIV3. Therefore, the authors should determine whether the APP effect on neurite outgrowth is associated with an activation of canonical Wnt signaling. Specifically, does the fl-mAPP facilitate activation of canonical Wnt signaling in neurons? If yes, does the APP lacking the CRD lost its ability on canonical Wnt signaling?This is an excellent suggestion to disentangle the effects of each of the two pathways on APP-mediated neurite outgrowth. To address this issue we treated neurons expressing either fl-APP or APP-Δ-CRD with Wnt3a or Wnt5a and measured the effect on APP-dependent axon and dendrite growth on DIV3 and DIV7.On DIV 3 we found that at DIV3 Wnt3a treatment significantly significant increased total axon length, the length of longest axon and axon branch tips in the presence of fl-APP but not APP-Δ-CRD . These data suggest that APP mediates Wnt3a effects on axonal outgrowth. In contrast to Wnt3a, Wnt5a treatment had no effect in the presence of fl-APP but resulted in increased axonal length when the CRD domain was removed. These data show that APP normally protects axons from the effects of Wnt5a at this developmental stage and that the CRD is also required for this. Thus, APP promotes Wnt3a and antagonizes Wnt5a effects on neurons at DIV 3 via the CRD domain.Similarly, on DIV7 we found a positive Wnt3a effect on axon complexity which was observed in the presence of fl-APP but not APP-Δ-CRD. In contrast Wnt5a had no effect. Finally, we found that treatment with either Wnt3a or Wnt5a decreases the number of the main dendrites in the presence of fl-APP and the effect is absent or even reversed in the presence of APP-Δ-CRD.Together, these data show that the CRD of APP is required for Wnt3a/5a to regulate neurite outgrowth in cortical primary neurons. We will add these data to the revised manuscript.These data are now presented in Figure 8 and on pages 11-12 of the revised manuscript.4. It is unclear whether the interactions between Wnt ligands and APP modulate Aβ production, and whether Wnt3a and Wnt5a have different effects on Aβ production after their binding to the CRD of APP.We have tested Aβ40 and Aβ 42 production in primary cortical neurons after 4 hours treatment of Wnt3a or Wnt5a at DIV7. Briefly, after a 4-hour treatment with Wnt3a/5a, supernatant was collected at 4 hours and at 24hours for Aβ detection using MSD kit. Our data shows that a 4-hour Wnt3a/5a treatment has a long-lasting effect on Aβ40/42 production and that those effects are antagonistic. Consistent with Wnt3a favoring recycling of APP we found that Wnt3a treatment significantly decrease Aβ40/42 production. In contrast, Wnt5a treatment, which indices APP internalization into acidic compartments, resulted in an increase in Aβ40/42 producing. Importantly, these data are in accordance with other reports that Wnt/catenin pathway favors non-amyloidogenic APP processing while Wnt/PCP signaling does the opposite.These data are now presented in Figure 4—figure supplement 2 and on pages 8-9 of the revised manuscript.Minor comments:1. The authors should provide detail information on the APP plasmid construct hAPPΔCRD. Specifically, which amino acid residues are removed from the extracellular portion of APP?The exact amino acid sequence of APP/APP-ΔCRD within plasmids or virus we used in our experiments have been added to the revised manuscript in the Materials and methods section pages 18-20.2. It has been reported that APP co-activates both the canonical and noncanonical Wnt pathways through physical interactions with Wnt co-receptors LRP6 and Vangl2, respectively (ref. 24). Interestingly, some LRP6-binding sites are also located in the CRD of APP. Therefore, more discussions are needed to address this relevance.A discussion of this interesting point was added to the revised manuscript on pages 13-14.Significance:APP is central to the study of AD. It is also established that dysregulation of Wnt signaling contributes to AD pathogenesis. Therefore, the study on the interactions between APP and Wnt ligands is significant, and the finding of Wnt ligand-binding to APP described in this manuscript is novel and interesting. However, in Figure 3F and Figure 4F, the concentrations of Wnt5a and Wnt3a were 400 ng/ml and 150 ng/ml, respectively, for modulating APP level/stability in DIV7 primary cortical neurons. These are quite high concentrations and difficult to be reached at pathophysiological conditions (particularly for Wnt5a). Therefore, the pathophysiological significance of the interactions between Wnt ligands and APP is unclear.We thank the reviewer for the positive comments. We note that the concentrations we used mirror (and are even less than in some cases) what is used in the literature which is why we used them. However, we have now tested different doses in the Aβ40/42 experiments and find similar effects for Wnt3a and the same trend for lower doses of Wnt5a.These data are now presented in Figure 4—figure supplement 2 and on pages 8-9 of the revised manuscript.Reviewer #2Evidence, reproducibility and clarity:Drosophila genetic model, APP KO, and a neuronal lentivirus-mediated transduction model. They found that Wnt-mediated function requires the region of extracellular Cysteine Rich Domain. Experiments in this study were well designed and the data were compelling. The conclusion is supported by the experimental results.The manuscript submitted by Liu and colleagues described an interesting observation that the amyloid precursor protein (APP) binds to Wnt ligands Wnt3a and Wnt5a; this binding regulates APP protein level and neuronal development in vivo and in vitro. Authors used multiple approaches to show that APP is a Wnt receptor for Wnt5a and Wnt3a, including the use of a We thank the reviewer for the positive comments on the quality of the work.Major Concerns:The authors should perform dot blot, solid phase, and/or biolayer interferometry analysis experiments to measure the binding affinity of Wnts binds to APP.Wnts are notoriously “sticky” proteins in vitro and we were not convinced that such experiments will yield easy to interpret results. Nonetheless, we did attempt solid-phase measurements using commercial ELISA kits. We performed the solid-phase experiment with pre-coated anti-flag ELISA plate . First, we tested if the anti-flag plate work by detecting the APP-flag from cell lysate of HEK 293 transfected with APP-flag or GFP control plasmid and follow the official manual. Briefly, 100ul cell lysate (APP-flag or GFP) of gradient concentration (100%-0.78125%) was added to the wells and incubated for 2 hours at room temperature. After washing, anti-APP antibody was added for another 1hour, then incubated with secondary HRP antibody for 1 hour before adding the TMB substrate. Absorbance was read on a microplate reader at 370 nm. Results, shown in panel A of Then we tried to test the binding affinity between APP-flag and Wnt3a. Different concentrations of Wnt3a (100nM-0.046nM) were added to the wells for 2-3 hours after the incubation of APP-flag, GFP or APP-deltaCRD cell lysate, or PBT to control for binding of Wnt3a alone to the plate. Similarly, we used an “antibody only” test where no Wnt3a was added. This was followed by adding Wnt3a antibody and HRP-conjugated secondary antibody, absorbance was read at 370nm after TMB substrate incubation. As shown in panel B of Significance:This study reveals a new function of APP as a receptor for Wnt and may provide some pieces of evidence to explain why abnormal Wnt pathway regulates Aβ generation and accumulation in AD.We agree with the reviewer that the physiological function of APP as a Wnt co-receptor is likely to be key to its role in AD and have now new data showing the effects of Wnts on Aβ production in neurons.Reviewer #3Evidence, reproducibility and clarity:While amyloid-β (Aβ) cleaved from amyloid precursor protein (APP) is a key molecule in the pathogenesis of Alzheimer’s disease (AD), APP and its homologues have been shown to mediate various aspects of neuronal development and activity. In this manuscript, Authors have found that Wnt3a and Wnt5a bind to cysteine rich domain (CRD) of APP and differently regulates APP trafficking and levels, which influence axonal and dendritic growth of neurons. Although their finding is potentially important, they lack the impact because the links between APP and Wnt pathways have been reported. Also, there are several concerns as follow.We thank the reviewer for the positive comments on the potential importance of the work. We note that both reviewers 1 and 2 described the work as significant and novel. While we agree that a link between APP and Wnt signaling has been described, and our group was one of the first to demonstrate this in vivo, we stress that the exact nature of this link has remained entirely unclear. Showing that APP directly interacts with Wnts ligands and examining why those ligands might have antagonistic effects on APP-dependent processes is a very significant step forward in providing a clear link between the physiological function of APP at the molecular and cellular levels and its potential role in AD. The lack of this link has hitherto been a source of major controversy in the field as to whether APP’s physiological function had any relevance to AD at all. Our data, as well as new experiments we performed provide very strong evidence that this is the case.Major Concerns:1. Although authors described that APPL mediates Wnt5a function in axonal growth through Figure 1, it is too conclusive because the effect of Wnt5 is not so evident in the figure. The results should be confirmed using Wnt5 overexpression approach in addition to knockout.Drosophila is a helpful experiment. The loss of Wnt5 causes little effects on mushroom body β lobe outgrowth, but completely rescues loss Of Vang, but not loss of Appl. These are very strong genetic epistasis data that allow clear conclusions that the effect of Wnt5 on the pathway absolutely requires APP.We do not agree that Wnt5 overexpression in 2. It is insufficiently addressed how deletion of APP-CRD influence the β-catenin canonical pathway, the PCP signaling pathway and/or the calcium pathway in Wnt signaling.We and others have previously shown that APP is a key component in both Wnt canonical and non-canonical signaling groups . We have now performed IF and WB with β-catenin, Dvll and Dvl2 antibodies. Unfortunately, only the Β-catenin antibody worked in WB experiments on primary neurons. We find that Wnt3a treatment increases levels of β-Catenin in control but not APP-KO primary neurons, consistent with previous findings in cell lines that APP enhances canonical Wnt signaling. These data are now presented in Figure 4—figure supplement 1 and on page 8 of the revised manuscript.3. Does Wnt induce the endocytosis of Frizzled and LRP5/6 or specific APP? Will be those Wnt receptors co-localized with APP after exposure to Wnt?Drosophila Vang and APPL in a CRD-dependent manner  at 2 hours after Wnt addition. We found that Wnt treatment induces re-localization of APP at already at 2 hours with the same pattern observed at 4 hours: Wnt3a caused re-loclaization of APP to the TGN in a CRD-depedent manner while Wnt5a treatment caused re-localization to the lysosome in a CDR-depdent manner. In terms of recycling endosomes, we used Rab11 as a marker (the Rab4 antibody did not work in our hands) We find that neither Wnt3a nor Wnt5a has an effect on the fraction of APP localized to Rab11+ recuclcing endosomes, either at 2 hours or at 4 hours.These data are now presented in Figure 3—figure supplement 3 and on pages 7, 8, 9, 10 of the revised manuscript.5. While Wnt may influence APP processing, there is no attempt to assess how deletion of APP-CRD influence APP processingWe are not quite sure what precise modification the reviewer is exactly referring to with the term APP “processing”. We assumed the reviewer is referring to Aβ production. We therefroe tested Aβ40 and Aβ 42 production in primary cortical neurons after 4 hours treatment of Wnt3a or Wnt5a at DIV7. Briefly, after a 4-hour treatment with Wnt3a/5a, supernatant was collected at 4 hours and at 24hours for Aβ detection using MSD kit. Our data shows that a 4-hour Wnt3a/5a treatment has a long-lasting effect on Aβ40/42 production and that those effects are antagonistic. Consistent with Wnt3a favoring recycling of APP we found that Wnt3a treatment significantly decrease Aβ40/42 production. In contrast, Wnt5a treatment, which indices APP internalization into acidic compartments, resulted in an increase in Aβ40/42 producing. Importantly, these data are in accordance with other reports that Wnt/catenin pathway favors non-amyloidogenic APP processing while Wnt/PCP signaling does the opposite.These data are now presented in Figure 4—figure supplement 2 and on pages 8-9 of the revised manuscript.To address a role for the CRD in this process, we attempted to restore Fl-APP or APP-ΔCRD expression to APP-KO neurons using viral transduction. This proved to be very difficult as levels of both proteins were significantly lower than what is normally present, especially that of APP-ΔCRD, which was expressed at much lower levels  precludi6. Because different concentrations of Wnt3a and Wnt5a are used at one dosage, it might be difficult to compare their effects properly.Different concentrations (and even higher than those we use) of Wnt3a and Wnt5a are used in published literature on the topic . However, we have now tested different doses in the Aβ40/42 experiments and find similar effects for Wnt3a and the same trend for lower doses of Wnt5a.These data are now presented in Figure 4—figure supplement 2 and on pages 8-9 of the revised manuscript.7. There is no evidence showing that endogenous levels of Wnt can directly bind to APP-CRD. Binding affinities of Wnt3a and Wnt5a to APP-CRD should be examined.Wnts are notoriously “sticky” proteins in vitro and we were not convinced that such experiments will yield easy to interpret results. Nonetheless, we did attempt solid-phase measurements using commercial ELISA kits. We performed the solid-phase experiment with pre-coated anti-flag ELISA plate . First, we tested if the anti-flag plate work by detecting the APP-flag from cell lysate of HEK 293 transfected with APP-flag or GFP control plasmid and follow the official manual. Briefly, 100ul cell lysate (APP-flag or GFP) of gradient concentration (100%-0.78125%) was added to the wells and incubated for 2 hours at room temperature. After washing, anti-APP antibody was added for another 1hour, then incubated with secondary HRP antibody for 1 hour before adding the TMB substrate. Absorbance was read on a microplate reader at 370 nm. Results, shown in panel A of Then we tried to test the binding affinity between APP-flag and Wnt3a. Different concentrations of Wnt3a (100nM-0.046nM) were added to the wells for 2-3 hours after the incubation of APP-flag, GFP or APP-deltaCRD cell lysate, or PBT to control for binding of Wnt3a alone to the plate. Similarly, we used an “antibody only” test where no Wnt3a was added. This was followed by adding Wnt3a antibody and HRP-conjugated secondary antibody, absorbance was read at 370nm after TMB substrate incubation. As shown in panel B of 8. Other molecules might interact with APP-CRD in addition to Wnt. Additional experiments are necessary to investigate whether Wnt-APP interaction mediates the phenotypes for neurite outgrowth in Figure 6 and 7.This is an excellent suggestion to disentangle the effects of each of the two pathways on APP-mediated neurite outgrowth. To address this issue we treated neurons expressing either fl-APP or APP-Δ-CRD with Wnt3a or Wnt5a and measured the effect on APP-dependent axon and dendrite growth on DIV3 and DIV7.On DIV 3 we found that at DIV3 Wnt3a treatment significantly significant increased total axon length, the length of longest axon and axon branch tips in the presence of fl-APP but not APP-Δ-CRD . These data suggest that APP mediates Wnt3a effects on axonal outgrowth. In contrast to Wnt3a, Wnt5a treatment had no effect in the presence of fl-APP but resulted in increased axonal length when the CRD domain was removed. These data show that APP normally protects axons from the effects of Wnt5a at this developmental stage and that the CRD is also required for this. Thus, APP promotes Wnt3a and antagonizes Wnt5a effects on neurons at DIV 3 via the CRD domain.Similarly, on DIV7 we found a positive Wnt3a effect on axon complexity which was observed in the presence of fl-APP but not APP-Δ-CRD. In contrast Wnt5a had no effect. Finally, we found that treatment with either Wnt3a or Wnt5a decreases the number of the main dendrites in the presence of fl-APP and the effect is absent or even reversed in the presence of APP-Δ-CRD.Together, these data show that the CRD of APP is required for Wnt3a/5a to regulate neurite outgrowth in cortical primary neurons. We will add these data to the revised manuscript.These data are now presented in Figure 8 and on pages 11-12 of the revised manuscript.9. It is more informative to assess how APP overexpression influences Wnt signaling.We are not in full agreement with this comment. Overexpression is rarely, if ever, more informative than loss of function when trying to determine the physiological function of a protein. There is a very good reason why overexpression models of APP have so far failed to yield profound insight into its physiological activity or to properly model AD.Significance:It is significant to define physiological roles of APP. It is interesting if Wnt3a and Wnt5a differently mediate Wnt pathway through APP.We thank the reviewer for this positive comment on the significance of our work.Referenceset al. (2013). The Drosophila Homologue of the Amyloid Precursor Protein Is a Conserved Modulator of Wnt PCP Signaling. PLoS Biol. 11, e1001562. Available at: https://dx.plos.org/10.1371/journal.pbio.1001562 .1. Soldano, A., Okray, Z., Janovska, P., Tmejová, K., Reynaud, E., Claeys, A., Yan, J., Atak, Z.K., De Strooper, B., Dura, J.-M., et al. (2011). Wnt5a is a transcriptional target of Dlx homeogenes and promotes differentiation of interneuron progenitors in vitro and in vivo. J. Neurosci. 31, 2675–2687. Available at: www.jneurosci.org .2. Paina, S., Garzotto, D., DeMarchis, S., Marino, M., Moiana, A., Conti, L., Cattaneo, E., Perera, M., Corte, G., Calautti, E., et al. (2018). A role for APP in Wnt signalling links synapse loss with β-amyloid production. Transl. Psychiatry 8, 179. Available at: http://www.nature.com/articles/s41398-018-0231-6 .3. Elliott, C., Rojo, A.I., Ribe, E., Broadstock, M., Xia, W., Morin, P., Semenov, M., Baillie, G., Cuadrado, A., Al-Shawi, R., 9, 8. Available at: https://molecularbrain.biomedcentral.com/articles/10.1186/s13041-016-0189-3 .4. Horigane, S.I., Ageta-Ishihara, N., Kamijo, S., Fujii, H., Okamura, M., Kinoshita, M., Takemoto-Kimura, S., and Bito, H. (2016). Facilitation of axon outgrowth via a Wnt5a-CaMKK-CaMKIα pathway during neuronal polarization. Mol. Brain 32, 11–17. Available at: http://www.tandfonline.com/doi/full/10.3109/08977194.2013.875544 .5. Clark, C.E.J., Richards, L.J., Stacker, S.A., and Cooper, H.M. (2014). Wnt5a induces Ryk-dependent and -independent effects on callosal axon and dendrite growth. Growth Factors 74, 797–817. Available at: https://onlinelibrary.wiley.com/doi/10.1002/dneu.22102 .6. Li, L., Fothergill, T., Hutchins, B.I., Dent, E.W., and Kalil, K. (2014). Wnt5a evokes cortical axon outgrowth and repulsive guidance by tau mediated reorganization of dynamic microtubules. Dev. Neurobiol.


In this study, 55 sesquiterpene pyridine alkaloids from Tripterygium were subjected to identification of pharmacophore characteristics and potential targets analysis. Our results revealed that the greatest structural difference of these compounds was in the pyridine ring and the pharmacophore model-5 (Pm-05) was the best model that consisted of three features including hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), and hydrophobic (HY), especially hydrophobic group located in the pyridine ring. It was proposed that 2- nicotinic acid part possessing a pyridine ring system was not only a pharmacologically active center but also a core of structural diversity of alkaloids from Tripterygium wilfordii. Furthermore, sesquiterpene pyridine alkaloids from Tripterygium were predicted to target multiple proteins and pathways and possibly played essential roles in the cure of Alzheimer's disease, breast cancer, Chagas disease, and nonalcoholic fatty liver disease (NAFLD). They also had other pharmacological effects, depending on the binding interactions between pyridine rings of these compounds and active cavities of the target genes platelet-activating factor receptor (PTAFR), cannabinoid receptor 1 (CNR1), cannabinoid receptor 1 (CNR2), squalene synthase (FDFT1), and heat shock protein HSP 90-alpha (HSP90AA1). Taken together, the results of this present study indicated that sesquiterpene pyridine alkaloids from Tripterygium are promising candidates that exhibit potential for development as medicine sources and need to be promoted.Sesquiterpene pyridine alkaloids are a large group of highly oxygenated sesquiterpenoids, which are characterized by a macrocyclic dilactone skeleton containing 2- nicotinic acid and dihydro- Tripterygium, such as Tripterygium wilfordii Hook. f. (TWHF), a typical traditional Chinese medicine plant, have been widely used to treat autoimmune diseases and neurodegenerative diseases including rheumatoid arthritis, systemic lupus erythematosus, and Parkinson's disease in China [T. wilfordii responsible for the overall curative effects [Traditional Chinese medicine (TCM) and natural products represent a huge source of diverse new drugs due to their potent and highly varied physiological activities. Plants in the genus in China –3. Chemi effects –6. Tript effects , 8. As t effects . However effects .Alkaloids with diverse structures are an important class of chemical components in TWHF . A totalTripterygium have been extensively studied due to their potential role in a wide spectrum of pharmacological activities, such as anti-inflammatory, antimicrobial and antitumor activities. TWHF alkaloids are less toxic than diterpenoids and have significant medicinal effects [Tripterygium could be utilized as a valuable chemical probe or a chemical moiety for the dissection of complex biological processes, discovery of unknown molecular relationships, and identification of therapeutic target molecules and pathways, which were worthy of in-depth study. The molecular mechanisms induced by sesquiterpene pyridine alkaloids from Tripterygium and the resulting changes in cellular phenotypes are rarely studied.Sesquiterpene pyridine alkaloids from  effects , 16. TheNetwork pharmacology is emerging as a promising strategy which is closely related to the application of multiple omics- and systems biology-based technologies . It is aTripterygium. As a result, the greatest structural difference of 55 sesquiterpene pyridine alkaloids from Tripterygium lied in 2- nicotinic acid part possessing pyridine ring and the pharmacophore model-5 (Pm-05) was the best model that consisted of three features including being hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), and hydrophobic (HY), especially hydrophobic group located in the pyridine ring. 2- nicotinic acid part possessing a pyridine ring system as the pharmacologically active center and the core of structural diversity of alkaloids from Tripterygium wilfordii was proposed. Moreover, sesquiterpene pyridine alkaloids from Tripterygium were predicted to target multiple proteins and pathways and possibly participated in the treatment of Alzheimer's disease, breast cancer, Chagas disease, and NAFLD. They also had other pharmacological effects, depending on the binding interactions between pyridine rings of these compounds and active cavities of the target genes encoding PTAFR, CNR1, CNR2, FDFT1, and HSP90AA1. The elaboration of 2- nicotinic acid part from macrocyclic dilactone skeleton as an active group and prediction of potential targets provide a promising resource for further being utilized as a chemical moiety, lead compound, or active ingredient for future drug discovery.These findings prompted us to systematically investigate the sesquiterpene pyridine alkaloids from Tripterygium established by our research group, data on sesquiterpene pyridine alkaloids from Tripterygium were extracted, and their molecular structures were converted into the standard Canonical SMILES format using PubChem database (https://pubchem.ncbi.nlm.nih.gov) and ChemSpider database (http://www.chemspider.com/), for target prediction of compounds [Based on related literature and the HR-MS-Database of macrocyclic dilactone skeleton alkaloids from ompounds , 20.Tripterygium were taken as starting points for further ligand-based pharmacophore design and were imported in the same window of Discovery Studio software 2016 software. Then, a principal value was set at 2 to make sure that the chemical features of all the ligands were considered when hypothesis space was generated, while a MaxOmitFeat value at zero could force to map all ligand features. Before starting the pharmacophore generation process, the small molecular structures were optimized by Minimize Ligands program. The number of conformers generated for each ligand was limited to a maximum number of 200.The ligand-based pharmacophore approach is a standard and very efficient way to conduct large virtual screening. There are multiple public sources, including OpenPHACTS, ChEMBL, and PubChem, for collecting compounds and activity data . Six traThe Pharmacophores module in the Discovery Studio 2016 software was employed to build the pharmacophore models with qualitative common features . Maximumhttp://targetnet.scbdd.com) is an open web server that could be used for netting or predicting the binding of multiple targets for any given molecule [Tripterygium were predicted using TargetNet with default parameters. To improve the reliability of the target prediction results, only targets with a high probability score (≥0.40) were selected.The TargetNet web server (molecule . TargetNhttp://www.genemania.org) is a user-friendly and flexible web server, for generating gene function hypotheses, analyzing gene lists, and prioritizing genes for functional assays [Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Homo sapiens, and Saccharomyces cerevisiae) in GeneMANIA. The putative target genes were entered into the search bar after selecting “Homo sapiens” from the organism option, and the results were further collated.GeneMANIA  can be utilized to thoroughly clarify and understand the functional and pathway enrichment information of a gene of interest [The DAVID database (interest . Potentiinterest . The R vhttps://omim.org/) is a knowledge source and database for human genetic diseases and related genes [The Online Mendelian Inheritance in Man  to construct and analyze the two-layer networks (compound-target network) and three-layer networks (compound-target -disease network) .Tripterygium, a molecular docking simulation was performed using the program LibDock implemented in Discovery Studio 2016. The crystal structures of the candidate targets were directly downloaded from the Protein Data Bank  and their resolutions varying from 1.0 to 3.5 Å were carefully examined. The higher the resolution is, the better the actual locations of separate atoms can be assigned. To evaluate the binding affinity of each candidate target to the corresponding compound, a docking score was calculated by the customizable scoring function of LibDock.To verify the binding affinity of candidate targets to constituent compounds of sesquiterpene pyridine alkaloids from Tripterygium were collected and sorted. Detailed information consisting of compound name, molecular formula, class, and data sources is shown in Supplementary β-agarofuran sesquiterpenoid  was developed and screened using Common Feature Pharmacophore Generation  of Discovery Studio 2016 and Fit values  was selected on the basis of heat mapping results depicted by Fit values, in which the “amide” feature was replaced by a hydrogen bond acceptor (HBA) feature. The blue circle, where the pyridine ring was formed, might be an important functional feature of the sesquiterpene pyridine alkaloids  nicotinic acid derivatives with functional groups possessing pharmacophore features were analyzed and proposed.As a continuation of the chemical studies of  Figures . All the Figures , and the Figures . Though Tripterygium, the reverse targeting strategy was firstly applied to acquire valuable targets. In the compound-target network of sesquiterpene pyridine alkaloids from Tripterygium, 55 compounds yielded 86 candidate target genes after removing the nonhuman genes, filtering the genes (probability < 0.4), and eliminating all duplicates. The detailed information on these targets is provided in Supplementary For the purpose of target identification of alkaloids characterized by macrocyclic dilactone skeleton from https://string-db.org/). The relationship between the targets is shown in After the construction of a compound-target (C-T) interaction network and calculation of the four topological features, “degree,” “betweenness centrality,” “closeness centrality,” and “clustering coefficient,” 20 known targets were screened, and their detailed information is provided in Supplementary Among the 86 key targets and their interacting proteins, 21.12% had physical interactions, 6.75% showed colocalization, and 29.77% displayed similar coexpression characteristics. Other results, including pathways and predicted and shared protein domains and genetic interactions, are shown in p ≤ 0.05, Supplementary p-value including neuroactive ligand-receptor interaction and pathways in cancer, which are shown in In order to further study the 86 identified target genes, GO and KEGG enrichment analyses were conducted using DAVID database. GO enrichment analysis identified 1230 entries, which were categorized into biological processes, cellular components, and molecular function (excluding pathways that belong to the section of human diseases) to be significantly enriched with putative targets  structures of targets were downloaded in the Protein Data Bank (https://www.rcsb.org/). Detailed information on these targets is provided in Supplementary In order to test and verify interaction between sesquiterpene pyridine alkaloids from Tripterygium plants, sesquiterpene pyridine alkaloids possess various structures and many pharmacological activities, which are a research hotspot [As the representative ingredients in  hotspot , 31. HowTripterygium using computational methodologies. A schematic diagram of the analysis procedures for target gene prediction and a clear explanation of active mechanism is shown in Tripterygium wilfordii alkaloids. And the more significant one was the pyridine ring. As previously shown, pyridine and its derivatives , among the most abundant natural N-heterocyclic compounds, were widely used in agriculture and pharmaceutical as pharmaceuticals, herbicides, and pesticides [Tripterygium wilfordii alkaloids may also become an important source of new drug development.Therefore, we systematically elucidated the pharmacological actions and structural characteristics of sesquiterpene pyridine alkaloids from sticides –34. ThusFor further verification of functional groups of sesquiterpene pyridine alkaloids, biological effects of small molecules result from favorable interactions between the molecules and their target proteins, and chemical functionalities needed for a small molecule to block or activate its target protein could be represented as pharmacophore models . Based oTarget gene identification is the first step in drug discovery and elucidation of structure-activity relationship. Increasing number of active compounds or drugs have been shown to interact with multiple genes or proteins using various in silico target identification approaches , 37. As In addition, the topological analysis of compound-target network yielded the key targets as follows: CYP3A4, PTPN1, PTAFR, CNR2, PTPN2, FDFT1, NR3C1, TBXA2R, CNR1, PLA2G1B, HSP90AA1, PTGDR2, DNMT1, CYP2C9, CASP9, ABCB1, ACHE, NR3C2, PTPN7, and HMGCR. Among them, PTAFR located on plasma membrane was reported to be a chemotactic phospholipid mediator that possessed potent inflammatory, smooth-muscle contractile and hypotensive activities , 39. ABCUnder the premise of obtaining significantly functional groups and potential targets, 55 compounds mapped on all of the pharmacophoric features present in pharmacophore model-5 were finally used in a molecular docking study. Importantly, molecular docking results showed the molecular docking relationship with LibDock Score value between 90 and 150 points accounted for 80 pairs (75.00%) and also confirmed that the target genes PTAFR, CNR1, CNR2, FDFT1, and HSP90AA1 could better combine with most sesquiterpene pyridine alkaloids through the binding interactions between amino acid residues and pyridine rings and were mainly focused on NLRP3 inflammasome activation, IL1B and IL18 secretion, progression of type-2 diabetes, enzymes of the sterol biosynthesis pathway, and drug resistance –44. We sTripterygium are promising compounds for the development of safe and effective multitargeted anticancer or anti-inflammatory medicines. This study also provided novel insights into the experimental directions and challenges for the follow-up study on pharmacodynamic material basis of sesquiterpene pyridine alkaloids from Tripterygium. However, these findings needed to be further substantiated under the experimental system.In summary, we analyzed and screened the relevant pharmacological laws, important targets, and the binding sites of niacin derivatives that exert their pharmacodynamics. Sesquiterpene pyridine alkaloids from


Background: Coronavirus Disease 2019 (COVID-19) is still a relevant global problem. Although some patients have recovered from COVID-19, the sequalae to the SARS-CoV-2 infection may include pulmonary fibrosis, which may contribute to considerable economic burden and health-care challenges. Convalescent Chinese Prescription (CCP) has been widely used during the COVID-19 recovery period for patients who were at high risk of pulmonary fibrosis and is recommended by the Diagnosis and Treatment Protocol for COVID-19 . However, its underlying mechanism is still unclear.Methods: In this study, an integrated pharmacology approach was implemented, which involved evaluation of absorption, distribution, metabolism and excretion of CCP, data mining of the disease targets, protein-protein interaction (PPI) network construction, and analysis, enrichment analysis, and molecular docking simulation, to predict the bioactive components, potential targets, and molecular mechanism of CCP for pulmonary fibrosis associated with SARS-CoV-2 infection.Results: The active compound of CCP and the candidate targets, including pulmonary fibrosis targets, were obtained through database mining. The Drug-Disease network was constructed. Sixty-five key targets were identified by topological analysis. The findings of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation suggested that the VEGF, Toll-like 4 receptor, MAPK signaling pathway, and TGF-β1 signaling pathways may be involved in pulmonary fibrosis. In the molecular docking analyses, VEGF, TNF-α, IL-6, MMP9 exhibited good binding activity. Findings from our study indicated that CCP could inhibit the expression of VEGF, TNF-α, IL-6, MMP9, TGF-β1 via the VEGF, Toll-like 4 receptor, MAPK, and TGF-β1 signaling pathways.Conclusion: Potential mechanisms involved in CCP treatment for COVID-19 pulmonary fibrosis associated with SARS-CoV-2 infection involves multiple components and multiple target points as well as multiple pathways. These findings may offer a profile for further investigations of the anti-fibrotic mechanism of CCP. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly discovered coronavirus responsible for COVID-19, which causes atypical pneumonia progressing to acute respiratory distress syndrome (ARDS) and acute lung injury . After SCurrently, there are few anti-fibrotic drugs available with clinically positive results, and a limited number of such agents are under investigation . AlthougTraditional Chinese medicines (TCM) have been widely used to treat lung diseases. Recently, several meta-analyses have shown that TCM exerted positive effects on PF, such as delaying the decline of pulmonary function and improving the QoL, with a good safety profile . In addiAlthough clear clinical benefits exist, very little has been elucidated about the potential molecular mechanism involved. Network pharmacology is a branch of pharmacology that uses network methods to analyze the synergistic relationship among drugs and diseases and targets via “multi-component, multi-target, multi-pathway” analyses, and can build a multi-dimensional network model of “drug–component–target–disease” to explore the relationship between drugs and diseases . This sthttp://pubchem.ncbi.nlm.nih.gov), China Knowledge Network, PubMed and BATMAN-TCM) and Shanghai Institute of Organic Chemistry of CAS. Chemistry Database [DB/OL].CCP contains 18 types of Chinese herbal medicine . All ingThe critical parameters of oral bioavailability (OB), drug-likeness (DL), and drug half-life (HL) were used to screen the active components of CCP. OB is an essential indicator for objective evaluation of the internal quality of drugs. OB defines the percentage of an orally administered dose of unchanged drug that reaches the systemic circulation and represents the convergence of the absorption, distribution, metabolism, and excretion (ADME) process. High OB is often a key indicator to determine the “drug-like” properties of bioactive molecules as therapeutic agents. Molecules with OB >30% were considered to have good OB in the present study . DL is aWe screened for potential targets of the herbs constituting CCP in the TCMSP database. If there was no corresponding drug target in the database, we determined the molecular structural formula based on available chemical formulas from the literature and predicted their potential targets based on the spatial structure of the molecular structure formula in the Swiss-target prediction. The final potential targets of these herbs were obtained by screening the corresponding drug targets for constituents and removing duplicates. The qualitative targets were matched to the UniProt database for normalization . In thisTo further explore the mechanisms of CCP's treatment effects on PF of patients with COVID-19 during the rehabilitation stage, we established a network drug-disease map to show the association between the active components in the CCP and its potential targets using Cytoscape v3.7.1. The components and targets were represented by triangles and circles, respectively, and the interaction between the two was shown by a connecting line. Overlapping portions between drug targets and disease targets are represented by Wayne's diagram. To explain the interaction between target proteins, overlapping target proteins between CCP and PF were uploaded to STRING to obtain information on protein-protein interaction (PPI) . We selehttps://dockthor.lncc.br/v2/). A similar result occurs if molecular docking is relatively precise.The above-mentioned PPI analysis generated potential hub genes active in PF treatment. Binding activity between the active drug components and the hub genes were evaluated by flexible molecular docking using Surflex-Dock software . SurflexThe active components of CCP were retrieved from the TCMSP database based on three parameters . Ultimately, 308 related components were identified as active ingredients in the CCP. Milkvetch root , Tangshen , Largehead atractylodes rhizome , Fourleaf ladybell root , Coastal glehnia root , Dried tangerine peel , Indian bread , Dwarf lilyturf tuber , Radix Salviae Miltiorrhizae , Thunberbg fritillary bulb , Leech , Hawthorn fruit , Medicated leaven , Germinated barley , Common yam rhizome , Ground beetle , Radix glycyrrhiza , and Pinellia tuber . Leech and Ground beetle were obtained by literature mining via China National Knowledge Infrastructure (CNKI) . The pot.Targets from 14 Chinese herbs were available in the TCMSP database. Targets from Leech (SZ) were available from a previous report . TargetsAKT1, TNF, IL6, TP53, VEGFA, IL1B, MMP9, EGFR, CCL2, PTGS2, STAT3, EGF, SRC, MMP2, FOS, CAT, HMOX1, ICAM1, MMP1, TGFB1) represented potential key targets for the therapeutic effects of CCP (The intersection (65 common targets) between drug targets and disease targets is shown in s of CCP .p < 0.05) comprising 336 biological processes (BP), 30 cellular components (CC), and 27 molecular functions (MF). The top 20 entries were selected from BP, CC, and MF, respectively, in order of -lgP value  were obtained from the KEGG pathway enrichment analysis using DAVID data. The first 20 entries were selected according to the–lgP value to draw a bubble diagram ; Toll-like 4 receptor pathway ; MAPK pathway ; and in the NOD-like receptor pathway .In total, 93 terms , α-smooth muscle actin (α-SMA), and tumor necrosis factor-α (TNF-α), inhibit alveolar cell apoptosis and reduce lung tissue inflammation and fibrosis injury in rats, which can effectively improve lung fibrosis  are not included in the TCMSP platform. The targets of these components need to be further studied. Quercetin is known to possess marked antioxidative, anti-inflammatory, and antifibrotic capacities. Dietary quercetin supplementation also decreases chronic systemic inflammation . Quercetfibrosis . Kaempfefibrosis . Luteolifibrosis .IL-6 gene. IL-6 is a complex ∼25-kDa cytokine, acting in both pro- and anti-inflammatory capacities (According to the results of the PPI network analysis and the topological property analysis of the "Drug–disease" network, the targets of action of CCP were the cytokine IL-6, members of the MAPK family, and PTGSE or prostaglandin G/H synthase. The results of GO functional enrichment analysis revealed that CCP components were mainly involved in the cell communication, endogenous stimulus regulation, apoptosis, programmed cell death, steroid hormone stimulus, signal transduction, regulation of catalytic activity, wounding regulation of cell proliferation, regulation of chemokine production and multicellular organismal macromolecule metabolic process. The KEGG pathway enrichment mainly involved Toll-like 4 receptor signaling and immune response pathways. Additionally, the NOD-like receptor signaling pathway and the Graft-versus-host disease pathway involved the pacities . Importapacities .The results of this study suggested that CCP may have an interventional effect on viral infections and lung injury. The pleotropic cytokine, TNF-α, plays a significant role in the pathogens of chronic inflammatory diseases. The MAPK signaling pathway controls diverse cellular processes in response to a variety of extracellular stimuli . Taken tWe then performed docking studies for TOP, MMP2, MMP9, IFNG, SELE, PLAU, VEGFA, HMOX1, F2, TNF, TP53, PPARG, PIK3CG, IL6, PTGS2, HSP90AB1, EGFR, using the critical ingredients quercetin, kaempferol, and luteolin as ligands. Molecular docking is used to evaluate whether ligands and proteins may bind thermodynamically. Overall, the scores of the active compounds with the key targets were for the majority positive with scores greater than 3, demonstrating that quercetin, kaempferol, and luteolin exhibited good binding properties with VEGFA, TNF, MMP9, and IL-6, respectively, all of which play an essential role in PF.The pathological process of PF could be roughly divided into three stages. The first stage involves the diffuse damage of vascular endothelial cells and alveolar epithelial cells by pathogenic factors, which initiates the inflammatory immune response. Second, inflammatory cells release a variety of cytokines and inflammatory mediators, expanding tissue damage and causing interstitial hyperplasia. The third step involves the migration and proliferation of fibroblasts and endothelial cells and the metabolic disorders of collagen and other ECM components, which further aggravate inflammatory damage and proliferation in a positive feedback manner. Eventually, the process could lead to the replacement and reconstruction of normal lung tissue. These three processes exist simultaneously, which are interrelated and interact .VEGF, TNF-α, IL-6, MMP9, and TGF-β1 via the VEGF, Toll-like 4 receptor, MAPK, and TGF-β1 signaling pathways. The potential mechanisms involved in CCP activity are summarized in VEGF-A is considered a critical factor in the pathogenesis of PF . VEGFR iThe findings suggest that CCP treatment of COVID-19 PF associated with SARS-CoV-2 infection involves multiple components and multiple target points as well as multiple pathways. Such proteins should be interesting to future studies that provide a novel direction for the mechanisms of PF associated with SARS-CoV-2 infection development and a new intervention target for clinical investigations, covering these gaps in research to be able to draw more meaningful conclusions about the benefits of CCP. These findings may offer a rationale for further investigations of the anti-fibrotic mechanisms of CCP therapy.


Danggui Shaoyao San (DSS) was developed to treat the ischemic stroke (IS) in patients and animal models. The purpose of this study was to explore its active compounds and demonstrate its mechanism against IS through network pharmacology, molecular docking, and animal experiment.  All the components of DSS were retrieved from the pharmacology database of TCM system. The genes corresponding to the targets were retrieved using OMIM, CTD database, and TTD database. The herb-compound-target network was constructed by Cytoscape software. The target protein-protein interaction network was built using the STRING database. The core targets of DSS were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Then, we achieved molecular docking between the hub proteins and the key active compounds. Finally, animal experiments were performed to verify the core targets. Triphenyltetrazolium chloride (TTC) staining was used to calculate the infarct size in mice. The protein expression was determined using the Western blot. p < 0.05) and 119 signaling pathways (p < 0.05) in KEGG, mainly including negative regulation of apoptosis, steroid hormone-mediated signaling pathway, neutrophil activation, cellular response to oxidative stress, and VEGF signaling pathway. MAPK1, SRC, and PIK3R1 docked with small molecule compounds. According to the Western blot, the expression of p-MAPK 1, p-AKT, and p-SRC was regulated by DSS.  Compound-target network mainly contained 51 compounds and 315 corresponding targets. Key targets contained MAPK1, SRC, PIK3R1, HRAS, AKT1, RHOA, RAC1, HSP90AA1, and RXRA FN1. There were 417 GO items in GO enrichment analysis ( This study showed that DSS can treat IS through multiple targets and routes and provided new insights to explore the mechanisms of DSS against IS. Stroke has the characteristics of high incidence, high recurrence rate, high fatality rate, high disability rate, and high economic burden, which seriously threatens human health and quality of life. Ischemic stroke (IS) accounts for 70% to 80% of all strokes, which results from sudden interruption of the blood supply to areas of the brain . NeuroloTraditional Chinese medicine (TCM) has played an important part in maintaining health for thousands of years. In recent years, traditional Chinese drugs have been reported to possess protective effects on the nervous system, which has attracted the attention of researchers worldwide . DangguiCompared with the single-target therapeutic effect of chemical drugs, TCM compound ingredients have the overall therapeutic effect, which is usually modulated through various pathways and targets . System According to the description above, DSS is an ideal TCM in the application of treatment to IS. However, the underlying pharmacological mechanisms of DSS on IS treatment remain unclear. In this study, we used the emerging traditional Chinese medicine network pharmacology method to predict the targets of DSS and systematically predict the mechanism of action of DSS in IS. An outline of the method is shown in https://tcmsp-e.com/) was used for the active ingredient screening [pubchem.ncbi.nlm.nih.gov), and the PharmMapper database (http://www.lilabecust.cn/pharmmapper) after identifying the active ingredients [The traditional Chinese medicine systems pharmacology database and analysis platform  database, Comparative Toxicogenomics Database  [http://db.idrblab.net/ttd/) were selected to obtain IS-related target gene [Online Mendelian Inheritance in Man  , and Theget gene . All thehttp://string-db.org/cgi/inpup.pl) with the species set to “Homo sapiens” to construct a protein-protein interaction (PPI) network [The obtained intersection targets were inputted to the STRING database  for GO and KEGG enrichment analyses [The predicted targets were imported into the Database for Annotation, Visualization, and Integrated Discovery . The software AutoDock Vina was used to carry out molecular docking between the key active ingredients and the key targets [The top three key compounds analyzed by “Network Analyzer” were obtained in the TCMSP database in mol2 format, and the top three key target proteins in the PPI network were obtained in the PDB database  and in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male C57/BL6 mice (21–23 g) were used. Transient focal ischemia was induced by right middle cerebral artery occlusion (MCAO) using the intraluminal vascular occlusion method as previously described [-1 and inThe materials of DSS were purchased from Tong Ren Tang Pharmaceutical Company  and were then authenticated by Dr. Weipeng Yang in the China Academy of Chinese Medical Sciences. The DSS dilution was prepared as described previously . In brieRegional cerebral blood flow (CBF) was monitored by the two-dimensional laser speckle imaging before ischemia and after the onset of ischemia (10 min after MCAO). We calculated the blood flow ratio of the two cerebral hemispheres (right/left), and at a ratio less than 20%, the MCAO model was considered successful Figures .n = 7 per group) were anesthetized with 1% pentobarbital sodium, and then, the brains were removed and sectioned coronally at 1-mm intervals to generate 6 slices. The slices were then incubated with 2% solution of 2, 3, 4-triphenyltetrazolium chloride (TTC). The infarct area and the corresponding contralateral area were measured by a blinded observer using the Image-Pro Plus software 5.0 . Infarct size was calculated as a percentage of the size of contralateral hemisphere.Infarct size was measured according to previous methods . Twenty-μg) was electrophoresed on 10% SDS polyacrylamide gels  and then transferred to a polyvinylidene fluoride membrane . The membrane was probed with primary antibody: anti-p-ERK1/2 antibody , anti-p-AKT antibody , and anti-p-SRC antibody . The specific reaction was visualized through the use of a chemiluminescent substrate  [β-Actin was used to verify equal loading. The optical density of protein was measured using ImageJ software  according to the manufacturer's instructions (n = 7 per group).Tissue samples were collected from the ischemic hemisphere at 24 hours and 7 days, respectively, after reperfusion for the Western blot analysis. The samples at 24 hours after reperfusion were used for the detection of phosphorylated MAPK1 (p-ERK1/2) and phosphorylated RAC-alpha serine/threonine-protein kinase (p-AKT). The samples at 7 days after reperfusion were used for the detection of phosphorylated proto-oncogene tyrosine-protein kinase Src (p-SRC). Protein  . β-Actint-test. For three or more groups, the differences were analyzed for statistical significance by the one-way ANOVA followed by Tukey's post hoc test. All the data were expressed as mean ± SD. The statistical analysis was performed with SPSS for Windows, version 21.0 (SPSS Inc.). The p-value <0.05 was considered significant.For two groups, the differences were analyzed for statistical significance by Student's According to the two screening conditions of OB value and DL index, 54 active ingredients of DSS were obtained from TCMSP, including 13 ingredients in PRA, 7 ingredients in CR, 7 ingredients in AMK, 2 ingredients in ASR, 15 ingredients in PCW, and 10 ingredients in AOJ. ASR and PRA share one ingredient. PRA, AOJ, and CR share one ingredient . Then, tThe overlapping targets of DSS-related targets and IS-related targets were considered as potential therapeutic targets for DSS anti-IS. As shown in 315 selected target genes were introduced into the STRING database to construct PPI network . The PPI315 targets screened by the DAVID database were used for GO functional enrichment analysis and KEGG signaling pathway enrichment analysis (FDR < 0.01). As a result, we obtained 417 GO terms, including 288 biological processes (BPs), 86 molecular functions (MFs), and 43 cellular components (CCs). BP mainly includes negative regulation of apoptosis, steroid hormone-mediated signaling pathway, neutrophil activation, and cellular response to oxidative stress. MF mainly includes the activity of steroid hormone receptor and protein tyrosine kinase. CC mainly includes cytoplasm and extracellular bodies. The top 10 screened BP, MF, and CC were selected as bubble charts through R language-related procedures .The enrichment and screening of KEGG pathways resulted in 119 signaling pathways (FDR < 0.01) and 16 pathways related to stroke . The maiThe top 3 key target proteins  in PPI network were selected to dock with the top 3 key active compounds . The PyMOL software was used to visualize the docking results of key active ingredients and core targets, as shown in P=0.0003) , the infarct size was reduced by 39% in the MCAO + DSS group (34.49 ± 2.94) (=0.0003) .p < 0.01) and further significantly increased after LRIC treatment  (P < 0.001), but that in the DSS group was significantly upregulated compared with the MCAO group (p < 0.05) (P < 0.001). DSS treatment for 7 days increased the levels of p-SRC compared with those found in the MCAO group (P < 0.05)  . Moreove < 0.05) . We also < 0.05) .In this study, the anti-IS mechanism underlying the effect of DSS was explored using network pharmacology through data mining and subsequent computational modeling. It was noteworthy that our findings partially elucidate the complex anti-IS mechanism in the DSS, and they provide insight into the integrated understanding of the therapeutic efficacy and pharmaceutical activity of DSS. First, a total of 534 active ingredients were extracted from DSS. The possible targets of DSS were mined using the TCMSP database. PPI data were used to construct the core PPI network. Then, 30 candidate anti-IS targets of DSS were identified as the pivotal hub genes in the core PPI network according to the specific topological importance. In addition, GO and KEGG pathway analyses were carried out to demonstrate the candidate target biological significance after the pivotal hub genes were incorporated into ClueGO. Finally, we evaluate the neuroprotection role of DSS in IS stroke mice and detected the effect of DSS on the major drug targets in IS treatment using the Western blot.Cerebral ischemia-reperfusion injury induces a complex pathophysiological cascade that includes a wide range of aberrant cellular processes . In the β (GSK3β), an apoptosis-related molecule [The increasing number of reports shows that apoptosis may contribute to a significant proportion of neuron death following IS . Mechanimolecule . Caspasemolecule . The repmolecule . In thisInflammation after cerebral ischemia is a complicated pathological process starting from the activation of microglia, circulating leukocyte infiltrate , and releasing of pro-inflammatory mediators mediated by ischemic cells and immune cells , 35. OurAn imbalance between free radical production and antioxidant activity leads to oxidative stress, which is a major pathologic mechanism of secondary brain damage after cerebral ischemia . Our GO Spontaneous neurogenesis and angiogenesis in the post-acute phase are highly coordinated responses and may contribute to the improvement in neurologic function after stroke. Accumulating experimental studies showed that promoting post-ischemic angiogenesis and neurogenesis can improve neurological function . IschemiDSS is a famous herbal formulary from the Synopsis of the Golden Chamber, which was used to treat gynecological disease, especially female abdominal pain. At present, DSS is widely used in neurological diseases , 14. TheDespite abundant new findings in this study, some limitations still exist. First, the corresponding experimental validation covered only a small number of mechanisms. Second, the compounds, targets, and pathways contained in these databases may not be exhaustive. In conclusion, the study, combined with network pharmacology, molecular docking, and animal experiments, offers the systematic mechanism of DSS in the treatment of IS and clearly clarifies the synergy mechanism of DSS multicomponent and multi-target. DSS may contribute to the treatment of IS by diminishing apoptosis, reducing inflammation and oxidative stress, increasing angiogenesis and neurogenesis, and modulating estrogen level. Our results indicate that DSS can not only reduce brain injury in the acute phase of stroke but also promote neurological recovery in the chronic phase of stroke.


Radiation pneumonia (RP) is the most common complication of radiotherapy to the thorax and seriously affects the survival rate and quality of life of patients. Radix Salviae Miltiorrhizae (RSM) is an ancient Chinese medicine, whose main pharmacological effect is to promote blood circulation and remove stasis. A growing number of studies have proved that RSM has a good effect on RP. However, the underlying mechanism is still unclear and needs to be fully elucidated.The effective components and predictive targets of RSM were analyzed by Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, and the related targets of RP were predicted by GeneCards database. The common targets of the two targets mentioned above were analyzed by protein-protein interaction on the STRING website, GO and KEGG analysis on the DAVID website, visualization by CytoScape3.7.0, and screening for Hubber gene by cytoHubber plug-in.A search of the TCMSP database revealed that RSM contains 65 chemical constituents and 165 potential protein targets. A total of 2,162 protein targets were found to be associated with RP. The top 10 hub genes were obtained by MCC algorithm for 70 common genes, including TP53, CASP3, MAPK1, JUN, VEGFA, STAT3, PTGS2, IL6, AKT1, and FOS. By analyzing the Gene Ontology, The anti-radiation pneumonia effect of RSM is that it performs molecular functions (protein homodimerization activity) in the nucleus through three biological processes . Through KEGG analysis, the mechanism of RSM treatment of radiation pneumonia may be through PI3K-Akt, HIF-1, TNF signaling pathways.Through network pharmacology analysis, we found the possible target genes of RSM on RP and revealed the most likely signaling pathway, providing theoretical basis for further elucidating the potential mechanism of RSM on RP. Radiation therapy (RT) has become one of the main treatments for thorax malignancies . The lunRadix Salvia Miltiorrhizae  is one of the oldest traditional Chinese medicines in China, with a history of more than 1,000 years of clinical application , 6. RSM Network pharmacology came into being with the development of genomics, proteomics, and systems biology , by miniThe main research process of this research is shown in the flowchart (http://tcmspw.com/tcmsp.php) to retrieve the effective chemical ingredients of RSM and their putative targets. TCMSP is a systematic pharmacological platform for the study of herbal medicines. Its functions include the identification of the chemical constituents of herbal medicines and their corresponding targets. Absorption, distribution, metabolism, and excretion (ADME) are key processes that need to be considered in screening compounds for TCM. Oral bioavailability (OB) and drug similarity (DL) were the most important pharmacokinetic parameters. OB is the dose and rate at which the active ingredient of a drug enters the human bloodstream by mouth, and DL is used to evaluate the structural similarity between a drug and clinical treatment drugs in the drugbank database. The higher the OB and DL, the more likely it is to be an active ingredient in the drug. On the website, we searched for effective compounds by inputting Chinese pinyin “Danshen” and screened out effective compounds according to the criteria of oral bioavailability (OB) >30% and drug likeness (DL) >0.18, and we found their corresponding target genes.We used the Traditional Chinese Medicine System Pharmacology Database    , which ihttp://bioinformatics.psb.ugent.be/webtools/Venn/) to intersect the targets that match the active ingredients of RSM and the targets related to radiation pneumonia. The obtained overlapping genes are the key genes of RSM to act on radiation pneumonia.We used Bioinformatics & Evolutionary Genomics website  to obtain the topological network between them. Cytoscape is an open software platform for visualizing networks of biological pathways and molecular interaction networks, and it has rich plug-in functions.We inputted the abovementioned network data on traditional Chinese medicine, active ingredients of traditional Chinese medicine, target genes, and diseases into the software Cytoscape   , with thhttps://david.ncifcrf.gov/home.jsp, ver. 6.8) (www.kegg.jp/kegg/kegg1.html) enrichment analyzes the potential biological pathways and functions associated with the target. The data were visualized by software R language 3.6.1 and GGplot2 package.We performed Gene Ontology (GO) enrichment analysis and KEGG signal pathway enrichment analysis of intersection genes through the Database for Annotation, Visualization and Integrated Discovery  , 22. GO We have screened 65 active compounds through the website according to the foregoing criteria. The detailed information are shown in We used the key word “radiation pneumonia” and screened more than 2,000 genes related to radiation pneumonia through the website  was used to build a network of 70 common genes, with 70 nodes and 903 edges  is displayed in EGFR, IL4, IL6, MCL1, RELA, MET, TP53, ITGB3, BCL2L1, AKT1, MAPK1, CDKN1A, CCND1, CASP9, GSK3B, BCL2, VEGFA, PIK3CA, MDM2, NOS3, INSR, MYC, IL2), HIF-1 , and TNF  pathway, respectively. The diagram of the above signaling pathways is shown in Additionally, the results of the KEGG Pathway analysis showed that 70 genes were mapped in 50 signal pathways with significant differences (P<0.05), and the top 15 pathways are shown in RP is one of the most challenging clinical complications of radiotherapy for lung malignancies. At present, the molecular mechanism of its pathogenesis mainly has the following points. First, the free radicals produced by radiotherapy can be treated with amphostine. Second, recruit inflammatory cells, which can be treated with the drug celecoxib. Third, cytokines and growth factors, therapeutic drugs TGF-β inhibitors (SM16) are still in basic research. Fourth, angiotensin-converting enzyme inhibitors (ACEI) have a potential role in reducing radiation-induced lung injury. However, the therapeutic effect is not good. The traditional Chinese medicine RSM has been widely used in the treatment of RP in China and many Chinese literature reports have been published, but the mechanism has not been clarified yet.In our study, we obtained 65 effective compounds of RSM from the database and predicted 165 possible target genes, while we screened out 2,162 genes associated with radioactive pneumonia. By intersections of these genes, we obtained 70 common genes, which may regulate the pharmacological effects of these genes on radiation pneumonia. By establishing the network between drugs, effective compounds, target genes, and diseases , which is a key enzyme in prostaglandin biosynthesis and has the dual function of dioxygenase and peroxidase. The expression of PTGS2 is positively correlated with the production of ROS and inflammatory signals in tissues, and inhibiting cox-2 can reduce inflammatory symptoms . The mospathways .PTGS2 and Luteolin are only the tip of the iceberg in the mechanism of resistance of RSM against RP, and its efficacy must be the result of multi-target and multi-pathway action. In order to further reveal the mechanism, we uploaded the obtained 70 common genes to the STRING website to obtain their PPI network, and we obtained the hub genes through two algorithms (MMC and Degree), which were displayed in multiple ways , the Six Talent Peaks Project of Jiangsu Province of China (WSN095).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hemerocallis fulva Flowers (HF) on depressive disorder (DD).  The purpose of our research is to systematically explore the multiple mechanisms of  The components of HF were searched from the literature. The targets of components were obtained from PharmMapper. After that, Cytoscape software was used to build a component-target network. The targets of DD were collected from DisGeNET, PharmGKB, TTD, and OMIM. Protein-protein interactions (PPIs) among the DD targets were executed to screen the key targets. Afterward, the GO and KEGG pathway enrichment analysis were performed by the KOBAS database. A compound-target-KEGG pathway network was built to analyze the key compounds and targets. Finally, the potential active substances and targets were validated by molecular docking.  A total of 55 active compounds in HF, 646 compound-related targets, and 527 DD-related targets were identified from public databases. After treated with PPI, 219 key targets of DD were acquired. The gene enrichment analysis suggested that HF probably benefits DD patients by modulating pathways related to the nervous system, endocrine system, amino acid metabolism, and signal transduction. The network analysis showed the critical components and targets of HF on DD. Results of molecular docking increased the reliability of this study.  It predicted and verified the pharmacological and molecular mechanism of HF against DD from a holistic perspective, which will also lay a foundation for further experimental research and rational clinical application of DD. Depressive disorder (DD) is a severe and occasionally fatal mental disorder that occurs in 4.4% to 20% of the general population. It happens at any time peaking in older adulthood, and it is more prevalent in women than in men. According to the world health organization (WHO) report, depression will be the second most burdensome disease in terms of treatment and care costs by 2020 , 2. TreaTCM is a multicomponent, multitarget, and multipathway therapy, which can achieve its unique therapeutic effect by adjusting the biological network of the human body system. Therefore, it is difficult to detect the exact mechanism of TCM only through traditional experimental methods . With thHemerocallis fulva L. is a perennial herb of the Liliaceae family [Hemerocallis fulva L. are considered to have sweet, cool, and nontoxic properties and to be associated with the spleen, liver, and bladder meridians [Hemerocallis fulva Flowers (HF) have been used to treat various diseases including depression [e family , which ieridians . Previoueridians , flavoneeridians , terpeneeridians , steroideridians , and pheeridians . Accordipression , 15, infpression , insomnipression , hepatospression , and canpression .However, there were few research studies on the network pharmacology of HF, and the mechanism of HF in treating depression was not very clear. In this study, we proposed a network pharmacology method aiming at uncovering the active substances and mechanisms of HF in the treatment of DD. Firstly, the components from HF were searched from the literature. The components were filtered by the metrics of oral bioavailability and drug-likeness. The targets were predicted by PharmMapper. Secondly, protein-protein interactions (PPIs) among the targets associated with DD searched from the DisGeNET, PharmGKB, TTD, and OMIM databases were predicted by the STRING database, and the key targets of DD were acquired. Then, the intersection of the compound-compound target network and PPI network of DD targets was taken to find overlapping targets. After that, the GO and KEGG pathway enrichment analysis were performed by KOBAS. The compound-target-pathway (C-T-P) network was analyzed to obtain the key potential active substances and targets. Lastly, the key targets were further validated by molecular docking. The whole framework is shown in Based on network pharmacology technology, this study aimed to explore the overall regulatory role of multicomponent, multitarget antidepressants in the molecular-level system of HF and to provide a theoretical basis for further experimental study and rational clinical application of HF.http://www.megabionet.org/tcmid/), which records a great deal of information about herbal ingredients, and the Traditional Chinese Medicine Systems Pharmacology Database [http://lsp.nwu.edu.cn/), a unique systemic pharmacology platform for Chinese herbal medicine. Unfortunately, few compounds were collected, except for some simple amino acid compounds. So, we consulted a lot of studies [To collect the compounds of HF, we searched the TCM Integrated Database   [Absorption, distribution, metabolism, and excretion (ADME) are used in drug discovery. Appropriate ADME screening can ensure that these candidate compounds have suitable pharmacokinetic properties. In this study, two ADME-related parameters were employed to screen out the active compounds in HF, including gastrointestinal (GI) absorption and drug-likeness evaluated by Lipinski's rule. GI absorption and drug-likeness were obtained from SwissADME (dex.php) . GI absodex.php) . Lipinskdex.php) , 32. Allhttp://lilab.ecust.edu.cn/pharmmapper/), which was an online server that utilized pharmacophore mapping to approach for potential drug target identification. In this study, the targets of each compound obtained from PharmMapper were selected as potential targets. The details about the selected targets are described in The chemical structural formulas of HF active compounds were saved as “mol2.” formatted files and treated with the function of MM2 to optimize the 3D molecular structures by ChemBio3D Ultra 14.0. The 3D molecular structure files of HF were imported into PharmMapper  (http://http://www.disgenet.org/), PharmGKB [https://www.pharmgkb.org/), TTD [http://db.idrblab.net/ttd/), and OMIM (https://omim.org/). We searched these platforms with the keywords “depressive disorder,” “depression,” or “major depressive disorder.” Humans' protein targets were selected in this study. The details about the selected targets are described in The genes associated with targets of DD were collected from DisGeNET  (http://PharmGKB  (https:/g/), TTD  , with the species limited to “Homo sapiens.” PPIs with comprehensive scores >0.7 were reserved in this study.STRING is a database, which can predict protein-protein interactions. The interactions include direct  and indirect  associations. They stem from computational prediction, from knowledge transfer between organisms, and from interactions aggregated from other (primary) databases. The data of protein-protein interaction (PPI) were obtained from the STRING database   was used to show all the above networks. The software is well suited for visualizing molecular interactions in networks. Besides, the tool of NetworkAnalyzer [i. “Node betweenness” represents the number of shortest paths between pairs of nodes passing through node i. “Closeness” is the reciprocal of the sum of distances between node i and the other nodes.Network construction was executed as follows: (1) compound-compound target network was structured by connecting active compounds and corresponding targets; (2) PPI network of DD targets was built by linking DD targets; (3) compound-DD target-KEGG pathway network was established by connecting compounds, overlapping targets between compound targets and core DD targets, and top 15 KEGG pathways. In network interactions, nodes represent compounds, targets, and KEGG pathways, while edges represent the interaction of each other. The network visualization software Cytoscape   is a bioinformatics project aimed to unify the characteristics of genes and genetic products in all species. Semantic lexical criteria that define and describe the function of genes and proteins can be updated for further study. GO has three classifications, including molecular function (MF), biological process (BP), and cell component (CC). MF describes the activity of gene products in molecular biology, such as catalytic or binding activity. BP is a multistep process composed of ordered molecular functions. CC refers to organelles or gene product groups and represents the role of gene products. Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies.p value, enrichment probability, and enrichment score according to the results of various methods. In this study, KOBAS was applied to perform GO and KEGG pathway enrichment analysis. Enriched GO terms and pathways were defined as those p value <0.05. The pvalue was corrected using the method introduced by Benjamini and Hochberg [http://www.bioinformatics.com.cn/), which was a free online data analysis platform.KOBAS is a widely used gene set enrichment (GSE) analysis tool . The curHochberg . It conthttps://www.rcsb.org/). For docking studies with Surflex-Dock, the ligand-binding site protomol was generated using the ligand from the PDB file. The visualization of intermolecular forces between the candidate compound and their potential target was performed on Discovery Studio 2020 program.Molecular docking plays an important role in rational drug design. It is often used to predict the binding sites and binding postures between candidate drugs and targets, as well as to estimate the binding affinity of molecules , 43. SurAfter consulting literature, 81 herbal compounds with structural information were gathered. After the GI absorption and drug-likeness process, some compounds that did not meet the ADME criteria were added back into the database because of their high bioactivity proved in previous studies , 44–52. β-D-glucopyranoside. The average number of targets per component is 56.6, and the mean degree of components per target is 4.8. It clearly showed that HF fit the multicomponent and multitarget characteristics of TCM. Consequently, we not only obtained an approximate observation of the relationship between bioactive compounds and compound targets, but also discovered the potential pharmacological effects of HF from this network.The compound-compound target network is depicted in Based on the gene database DisGeNET, PharmGKB, TTD, and OMIM, there were a total of 527 candidate targets relevant to depressive disorder. After PPI was acquired, there were 446 nodes and 3905 edges in the DD target PPI network . The respvalue <0.05. After GO enrichment analysis of 18 overlapping targets, a total of 42 GO entries were found with the corrected p < 0.05. The 34 pathways belonged to four categories: human diseases (9/34), organismal systems (13/34), environmental information processing (4/34), and metabolism (8/34). Thus, our findings showed that HF integrated multiple signaling pathways to the nervous system, endocrine system, amino acid metabolism, signal transduction, and substance dependence. Based on the results of pathway analysis, it was found that these high-degree pathways were closely related to the DD, such as serotonergic synapse and dopaminergic synapse. The top 15 KEGG pathways are shown in The 18 overlapping targets were further mapped to 34 pathways with β-carboline-3-carboxylic acid, hydroxydihydrobovolide, epigallocatechin gallate (EGCG), prunasin, quercetin-3-O-β-D-galactopyranoside, vitexin, and α-linolenic acid. It indicated that these compounds might play a more important role than others in the treatment of depressive disorder with HF. Five target nodes with values of degree, betweenness, and closeness more than average were selected as key target nodes, namely, MAOA, MAOB, AR, CAMK2A, and GAD2. It indicated that these targets may be crucial to the treatment of depressive disorder with HF.The details of KEGG pathways are described in β-carboline-3-carboxylic acid, and 5-O-p-coumaroylquinic acid butyl ester as examples to show the visualization of intermolecular forces between compounds and MAOA target, which are displayed in To verify the top 5 key targets and their interacting compounds, the molecular docking simulation was carried out by the Surflex-Dock method. As the result in Researchers have conducted a large number of studies to explore the pathogenesis of depression, but its exact pathogenesis is still unclear. The supposed pathogenesis of depression is the epigenetic hypothesis, neurotransmitter hypothesis, neurotrophic regeneration hypothesis, neurokinin hypothesis, neuroendocrine dysfunction hypothesis, and immune system abnormality hypothesis . The resIn the present study, a total of 55 active compounds in HF were screened by pharmacokinetic analysis, 646 compound-related targets were predicted by PharmMapper, and 527 DD-related targets were identified from public databases. After treated with PPI, 219 key targets in the development of DD were acquired. Among these targets, 18 targets were shared between compound-related and DD-related targets. Most entries from GO enrichment play important parts in the central nervous system, which affect different steps of synthesis or transportation of neurotransmitters. The KEGG pathway analysis proved that bioactive compounds from HF exerted a synergistic effect on the treatment of DD through numerous pathways and brain amino acid metabolism, such as amphetamine addiction, serotonergic synapse, dopaminergic synapse, arginine and proline metabolism, HIF-1 signaling pathway, phenylalanine metabolism, alcoholism, calcium signaling pathway, tyrosine metabolism, and tryptophan metabolism.Furthermore, the top 5 key targets and top 12 key compounds were screened from the C-T-P network. These five screening targets have been shown to be highly correlated with depression. Monoamine oxidases (MAOs) play a crucial role during the development of various mental diseases . There aβ), interleukin 6 (IL-6), indoleamine 2,3-dioxygenase (IDO), and upregulation of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in the hippocampus [β-D-galactopyranoside may improve depression-like effects by regulating the hypothalamic-pituitary-adrenal (HPA) axis and reducing the level of oxidative stress in the hippocampus [α-linolenic acid adipose tissue stores were negatively correlated with depression [Hemerocallis extract was mainly related to flavonoids [β-carboline-3-carboxylic acid, (S)-2,4-dibutoxy-3-(hydroxymethyl)cyclopent-2-en-1-one, 5-O-p-coumaroylquinic acid butyl ester, lycoperodine-1, hydroxydihydrobovolide, and prunasin. Subsequent experiments could verify their activity.Meanwhile, some of the screened compounds had been shown to antidepressant activity. It reported that naringin was a neuroactive flavonoid. It possessed functional beneficial neurobehavioral effects including anxiolytic, antidepressant, and memory enhancing . Hesperipocampus . Quercetpocampus . Can et pocampus . In a stpression . The antavonoids , which wβ-carboline-3-carboxylic acid are similar to that of harmine, which was a carboline alkaloid and also was a MAOA inhibitor [Molecular docking experiment is a new method that uses computer simulation of compound structure and related disease targets to execute molecular docking, calculate and analyze the bioactivity of the compound, and screen the material basis of pharmacodynamics. This method could quickly and efficiently discover some new bioactive lead compounds from the database. In this study, molecular docking results showed that targets predicted by PharmMapper were reasonable. Most of the screened compounds and their corresponding targets scored well. The structures of lycoperodine-1 and (-)--1-methyl-1,2,3,4-tetrahydro-nhibitor . The simIn summary, the present study is the first one that combines active components, target prediction, network analysis, and gene enrichment analysis by a network pharmacology method to elucidate the molecular and pharmacological mechanism of HF against DD from a systematic perspective. In this research, we showed multiple targets of HF against DD for the first time. Based on this neural network model, active components, targets, and pathways in depressive disorder were initially explored to provide a preliminary theoretical basis for the design of subsequently targeted drugs. The results of molecular docking confirmed that the present network pharmacology method was reasonable. Nonetheless, more experiments should be implemented to verify the validity of our findings in further pharmacological and molecular research. Moreover, we hope that our study will be useful for antidepression drug discovery.