Carbon monoxide (CO) is an important signaling molecule participating in multiple biological functions. Previous studies have confirmed the valuable roles of CO in cancer therapies. If the CO concentration and distribution can be controlled in tumors, new cancer therapeutic strategy may be developed to benefit the patient survival.2. Co/Ru-UiO-67 can transform tumor CO2 into CO at both cellular level and living tissues, which consequently interacts with relevant signaling pathways  to adjust tumor microenvironment. With proper PEGylation  and attachment of a tumor-homing peptide (F3), functionalized Co/Ru-UiO-67 could accumulate strongly in triple-negative MDA-MB-231 breast tumors, witnessed by positron emission tomography (PET) imaging after the addition of radioactive zirconium-89 (89Zr) into Co-UiO-67. When applied in vivo, Co/Ru-UiO-67 could alter the local hypoxic condition of MDA-MB-231 tumors, and work synergistically with tirapazamine (TPZ).In this study, a UiO-67 type metal–organic framework (MOF) nanoplatform was produced with cobalt and ruthenium ions incorporated into its structure (Co/Ru-UiO-67). Co/Ru-UiO-67 had a size range of 70–90 nm and maintained the porous structure, with cobalt and ruthenium distributed uniformly inside. Co/Ru-UiO-67 was able to catalyze carbon dioxide into CO upon light irradiation in an efficient manner with a catalysis speed of 5.6 nmol/min per 1 mg Co/Ru-UiO-67. Due to abnormal metabolic properties of tumor cells, tumor microenvironment usually contains abundant amount of COThis nanoscale UiO-67 MOF platform can further our understanding of CO functions while produce CO in a controllable manner during cancer therapeutic administration.The online version contains supplementary material available at 10.1186/s12951-022-01704-2. As a gaseous signaling molecule, carbon monoxide (CO) is actively involved in various physiological or pathological processes –3. With 2 to CO upon light irradiation [Metal organic framework (MOF) materials, which are composed from metal ion clusters and organic bridging ligands, can be generated from different methods. Nanoscale MOF (nMOF) possessed porous structure, and combined the advantages of metal ions and the organic ligands, which were frequently used for catalysis , sensingadiation . From thTirapazamine (TPZ), which belongs to the category of hypoxia-activated prodrugs (HAPs) , was sel2, since its catalytic energy barrier was relatively low (0.86 eV) [2 in the tumor microenvironment (TME) and convert it to CO, which can partially alter the TME. To improve the whole light catalysis sensitivity, ruthenium (II) was also loaded inside the UiO-67 structure to serve as a photosensitizer [Our goal in this study is to develop a nMOF platform to catalyze carbon dioxide to CO in vivo. Inspired by the previous findings, we hope to build an in situ CO “generator”, which can not only impose selective cancer cell killing, but also be traceable by different imaging techniques. UiO-67 type nMOF, which is here composed of zirconium clusters and 2,2-bipyridine-4,4′-dicarboxylic acid, was used as the core material here. Based on multiple previous reports –22, UiO-0.86 eV) . Moreove0.86 eV) , 24. Thinsitizer . The cob111In or 225Ac for theranostic utilization [89Zr) [Nucleolin in nucleus regulates the synthesis of rRNA and biogenesis of ribosome. However, nucleolin translocation from nucleus to cell membrane is considered as a tumorigenic event. It was reported that multiple types of cancer cells and activated (angiogenic) endothelial cells demonstrated the overexpression of nucleolin on their membrane surface , 26. Thulization , 28. Conlization . A positn [89Zr) , was embn [89Zr) . After t2/g, which revealed its suitability for cargo accommodation. After the loading of different cargos , the specific surface area changed to 162.3 m2/g, again validating that all the cargos were actually on board. Also, based on the thermogravimetric analysis (TGA)  was developed with satisfactory catalytic efficacy.The CO transformation properties of Co/Ru-UiO-67 was subsequently investigated. When irradiated by an 8 W LED light at a distance of 5 cm, the CO production could be clearly observed (witnessed as the continuous air bubble formation) in the DMF Co/Ru-UiO-67 suspension with saturated COAfter the PEGylation with Py-PAA-PEG and tumor-targeting F3 peptide, the material interactions with cancer cells (i.e. triple-negative breast cancer cells MDA-MB-231) and control cells (murine fibroblasts L929) were investigated. Learned from the flow cytometry results  was incorporated into Co/Ru-UiO-67, the quantitative kinetic evaluation of material interactions with MDA-MB-231 cells was given , and the specificity activity was ~ 185 MBq/mg. The stability of 89Zr-Co-UiO-67 was quite good, since  > 99% 89Zr stayed on 89Zr-Co-UiO-67 post a 120 h incubation of mouse serum  of Co-UiO-67). After the radiolabeling and PEGylation, 89Zr-Co-UiO-67 was used in MDA-MB-231 tumor-bearing mice. In vivo investigation by PET imaging indicated a rapid accumulation of 89Zr-Co-UiO-67@Py-PAA-PEG-F3 in MDA-MB-231 tumors  values were obtained from PET and biodistribution studies . However, the relationship between CO and CD8+ T cells in cancer therapeutics should be further explored, and we hope to use the Co/Ru-UiO-67 platform as a good CO “director” to simplify the CO-immune system investigations in cancer. Meanwhile, no significant tissue morphological change or blood biochemical factor alteration was observed in all groups, indicating the safety and biocompatibility of the nanoparticles used here, which was further confirmed by H&E staining of major organs  was developed for “targeted” synergistic tumor treatment based on light-initiated CO catalysis. Our findings indicated that Co/Ru-UiO-67 nanoplatform could produce CO in a controllable manner both in vitro and in vivo, and the formed CO could affect the downstream signaling to enhance the treatment power of TPZ. With the proper surface modification and attachment of a tumor-targeting peptide (F3), Co/Ru-UiO-67 could selectively enter triple-negative breast cancer cells and have a long cellular retention. In vivo PET imaging investigations also demonstrated the good pharmacokinetic performance of these Co/Ru-UiO-67 conjugates. The primary benefit of this Co/Ru-UiO-67 nanoplatform lies in its ability to fine tune the tumor microenvironment. Therefore, when applied in vivo, it can work with various drug cargos (e.g. TPZ in this report) to efficiently activate the killing functions of the drugs. The combination of Co/Ru-UiO-67@Py-PAA-PEG-F3 (10 mg/kg) with TPZ showed a more than 70% tumor suppression rate in the subcutaneous MDA-MB-231 tumor model with low systemic toxicity. These findings indicated that the synergistic work between CO-release platform and activatable drugs, was really a potential therapeutic approach for TNBC treatment.2bpydc) and 4,4′-Biphenyldicarboxylic acid (H2bpdc) were purchased from Chemsoon . Zirconium oxychloride octahydrate was acquired from Honeywell . Polyacrylic acid (PAA) was obtained from Ruixibio . F3 peptide with cysteine at C-terminal (sequence: KDEPQRRSARLSAKPAPPKPEPKPKKAPAKKC) was synthesized by QYaoBio . Fluorescein isothiocyanate (FITC), cobalt chloride (CoCl2), and pyrene (Py)-amine were got from Thermo-Fisher. Antibodies against CD31, GAPDH, Jagged 1 etc. were all got from Abcam. All the other chemicals and solvents were acquired from Sigma-Aldrich and used without further purifications.2,2′-Bipyridine-5,5′-dicarboxylic acid  was dissolved in a mixed solution of DMF (10 mL) and acetic acid (0.5 mL). Sonication of 30 min was carried out to form a transparent solution. Subsequently, H2bpydc (0.012 mmol) and H2bpdc (0.028 mmol) were added into the solution and the whole mixture was transferred into a 25 mL Teflon autoclave and reacted under 90 ℃ for 12 h. The resulting UiO-67 materials were washed repeatedly by DMF and ethanol (three times at least for each solvent). To acquire Co/Ru-UiO-67, 10 mg of UiO-67 was suspended in 10 mL of THF, and CoCl2 (0.15 mmol) along with trisruthenium(II) hexafluorophosphate (0.1 mmol) was added into the suspension. With overnight stirring at room temperature under nitrogen gas protection, Co-UiO-67 (without Ru(II) added) and Co/Ru-UiO-67 were obtained after repeated THF, ethanol, and Millipore water washing and resuspended in water for further treatment.UiO-67 was synthesized with a previously established method with slight modifications . BrieflyThe size of UiO-67 conjugates was measured by JEM-2100 transmission electronic microscopy  and the dynamic laser scattering (DLS) measurement was carried out on Zetasizer Nano ZS . UV–Vis spectra were recorded in a Perkin-Elmer Lambda 390 spectrometer. X-ray photoelectron spectroscopy (XPS) for UiO-67 was conducted on a PHI5000 VersaProbe . Thermogravimetric analysis (TGA) was carried out on a Pyris 1 differential scanning calorimeter (Perkin-Elmer).To load TPZ into Co-UiO-67 or Co/Ru-UiO-67, Co-UiO-67 or Co/Ru-UiO-67 (10 mg) and TPZ (5 mg) were stirred in a mixed solution of DMSO and ethanol (0.5 mL + 0.5 mL) for 12 h. Since the physical properties and physiological performance of Co-UiO-67 and Co/Ru-UiO-67 were quite similar, we used Co/Ru-UiO-67 under light catalysis condition. Other than that, Co-UiO-67 was selected as the “target” material in the relevant evaluations for clarity reasons. Centrifugation (5000 rpm* 20 min) was used to separate TPZ-loaded Co/Ru-UiO-67 (or Co-UiO-67) from free TPZ. The precipitates were washed with ethanol for three times and resuspended in Millipore water. After all the cargo loading, Co/Ru-UiO-67 or Co-UiO-67 was conjugated with pyrene-derived PEG molecules. The surface-modifying PEG (Py-PAA-PEG) was synthesized using a previously established method . In the 89Y89Zr reaction with a specific activity of 352.2 ± 26.4 MBq/μg of Zr (n = 5). Zirconium-89 chloride was obtained from 89Zr-oxalate after the heat vaporization  of oxalate by the addition of concentrated HCl. During the UiO-67 nMOF synthesis, 148 MBq of 89ZrCl4 was added into a 2-mL reaction mixture while the same reaction condition was maintained for UiO-67. After obtaining the 89Zr-incorporated UiO-67, cobalt embedding, Py-PAA-PEG-Mal functionalization and F3 conjugation was also carried out for in vitro and in vivo evaluation of 89Zr-Co-UiO-67 conjugates.Zirconium-89 oxalate was obtained from DC AMS Pharma , which was produced via +) and L929 (nucleolin−) cells) were used for the measurement of cell interactions. The material concentration of 100 μg/mL was chosen to evaluate the material-cell dynamics. Flow cytometry data collection post 1 h material incubation was conducted on a BD FACScalibur and the data were processed by the FlowJo software (vX.0.7). Confocal fluorescence microscopy was done on a Zeiss LSM 980 with Airyscan 2, taken at 200 × magnification.The dynamic interactions between Co/Ru-UiO-67 or Co-UiO-67 nMOFs and different types of cells were assessed in three ways. Flow cytometry and confocal fluorescence microscopy were used for non-radioactive Co/Ru-UiO-67 or Co-UiO-67 conjugates. FITC was covalently attached on the PEGylated Co/Ru-UiO-67@Py-PAA-PEG or Co-UiO-67@Py-PAA-PEG via the intermediate linker of 2-mercaptoethylamine. Since the attachment of F3 peptide was to introduce the nucleolin targeting capacity, two cell types with distinct nucleolin expression profiles , a portion of cell suspension was taken and treated with 0.25 M sodium citrate (pH = 2) to remove the material binding to the cell surface. Cells were centrifuged and collected to count the radioactivity inside the cells, which was transferred to the material concentration per cell numbers. In addition, when the cellular internalization level of 89Zr-Co-UiO-67 conjugates reached the plateau, material-containing MDA-MB-231 cells were resuspended in PBS (still at the cell density of 2 × 105 cells/mL), while a portion of the cell suspensions were taken and centrifuged, since the radioactivity in the supernatant represented the efflux percentage of the materials once they got internalized into the cells.In addition, the interaction of he cells . The dyn2 saturated solution (the distance between the LED and the solution was 5 cm). At different time post incubation, a portion of the solution was taken from the mixture, and the CO concentration was quantified by either a UV measurement from myoglobin incubation or a fluorescence analysis from interactions with COP-1 (carbon monoxide probe 1) [2 and an irradiation time of 10 min.To test the CO catalytic capacity of Co/Ru-UiO-67, an 8-W LED was used to irradiate Co-UiO-67 suspended in a COprobe 1) . When Co2 for 10 min. Post laser irradiation, CO relevant signaling changes  were measured by Western blot. In addition, mitochondria membrane hyperpolarization condition for treated cells was evaluated by the JC-1 probe. The cellular ROS level was measured by a dichloro-dihydro-fluorescein diacetate (DCFH-DA) kit, and the cellular ATP level was tested by a luminescent ATP detection kit . These all reflected the cellular changes post the formation of CO from light catalysis.MDA-MB-231 cells were incubated with 50 μg/mL Co/Ru-UiO-67@Py-PAA-PEG and Co-UiO-67@Py-PAA-PEG-F3. At 1 h post-incubation, the cells were irradiated by the 473 nm laser at 0.25 W/cmAll animal experiments were conducted following the approved protocols of Nanjing University Institutional Animal Care and Use Committee. These protocols also conform to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. Female nude mice (6–10 weeks) were purchased from Gem Pharmatech  for in vivo experiments. The mice were maintained at five per cage and kept at a specific pathogen-free (SPF) animal facility with 12 h light/dark cycles. Strict temperature and humidity control were available, along with the proper food and water supply.89Zr-Co-UiO-67 conjugates (i.e. 89Zr-Co-UiO-67@Py-PAA-PEG and 89Zr-Co-UiO-67@Py-PAA-PEG-F3) were administrated into MDA-MB-231 tumor-bearing mice at the dosage of 3–5 MBq per mouse (n = 4) for PET imaging. The acquisition of 40 million events per time point were adopted in a Siemens Inveon rodent PET/CT scanner. The scanning was carried out at 1 h, 4 h and 24 h post-injection to evaluate the pharmacokinetic profile of 89Zr-Co-UiO-67 conjugates when the animals were anaesthetized by isoflurane + oxygen. The body temperature and heart-beat condition of the tested animals were also monitored in a real-time manner to avoid possible anesthesia-induced deaths. The quantification of organ radioactivity was conducted in the Inveon Research Workplace (IRW) software . Immediately after the last PET scanning, the animals were euthanized to collect the major organs for radioactivity counting using a Perkin-Elmer Wizard2 gamma counter. Both the PET quantification and gamma counting results were shown by the unit of % injection dose per gram (%ID/g).PEGylated To test the possible side effects or toxicity from Co-UiO-67 conjugates, PEGylated Co-UiO-67 was administered into female Balb/c mice (n = 5) at a dose of 10 mg/kg body weight. The body weights of the tested mice were monitored for 7 days. At 7 days post injection, the mice were euthanized and the major organs were taken for hematoxylin and eosin (H&E) staining. In addition, mini chemistry panel test was conducted to investigate the overall health impact of Co-UiO-67 conjugates to the test animals at day 1 and day 7.6 MDA-MB-231 cells were injected into female nude mice (6 – 10 weeks in age). And the treatment started when the tumor volume reached ~ 150 mm3. MDA-MB-231 tumor bearing mice were randomly divided into different groups (n = 5 per group). Since the PET imaging confirmed the good tumor-targeting efficacy of F3 peptide, PEGylated and F3 conjugated UiO-67 nMOFs were used here for combinational therapeutic purposes. All the therapeutics were administered intravenously, and the treatment groups included: PBS, Co/Ru-UiO-67@Py-PAA-PEG-F3 (10 mg/kg), TPZ-Co/Ru-UiO-67@Py-PAA-PEG-F3 (10 mg/kg), Co/Ru-UiO-67@Py-PAA-PEG-F3 (10 mg/kg) + laser irradiation, and TPZ-Co/Ru-UiO-67@Py-PAA-PEG-F3 (10 mg/kg) + laser irradiation. Therapeutic administration was performed on day 0 and day 7, and in irradiation groups, mice were irradiated with an 8-W LED light for 1 h each time (distance 5 cm) at 4 h post-injection of therapeutics (mice were anesthetized by a 1.5% isoflurane/oxygen flow during the irradiation process). Moreover, since 89Zr could be effectively embedded into UiO-67 structure, and 89Zr was known to have relatively strong Cerenkov luminescence [89Zr-TPZ-Co/Ru-UiO-67@Py-PAA-PEG-F3  to see whether Cerenkov luminescence from 89Zr could serve as a light trigger for CO catalysis using Co/Ru-UiO-67.For cancer therapy studies, 2 × 10nescence , an addiAll data were presented in the form of mean ± SD (standard deviation). GraphPad Prism 7.0 was used to analyze the data. One-way ANOVA or student’s t-test was used for the difference analysis of the experimental data. A value of P < 0.05 (*) was considered as statistically significant.Additional file 1: Figure S1. The scanning electronic microscopy (SEM) images of UiO-67 and PEGylated TPZ-loaded Co/Ru-UiO-67. Figure S2. The dynamic laser scattering (DLS) measurements of Co/Ru-UiO-67 and PEGylated Co/Ru-UiO-67. Figure S3. The UV-Vis spectra of cobalt chloride and cobalt-embedded UiO-67. Figure S4. The cumulative release of ruthenium and cobalt from Co/Ru-UiO-67 at different pH values. Figure S5. The FT-IR spectra of UiO-67 conjugates and PEGylated Co/Ru-UiO-67. Figure S6. The thermogravimetric analysis (TGA) of TPZ-Co/Ru-UiO-67 and PEGylated TPZ-Co/Ru-UiO-67. Figure S7. The absorbance curve of dexoy-myoglobin (Mb) before and after the incubation with CO. Figure S8. The cell toxicity of Co-UiO-67@Py-PAA-PEG-F3 and Co/Ru-UiO-67@Py-PAA-PEG-F3 in MDA-MB-231 cells (measured by an MTT assay in the dark). Figure S9. The measurement of ATP level in MDA-MB-231 cells post Co/Ru-UiO-67@Py-PAA-PEG-F3 treatment and laser irradiation. Figure S10. The measurement of mitochondrial membrane potential by JC-1 in MDA-MB-231 cells treated with TPZ-Co/Ru-UiO-67@Py-PAA-PEG-F3 with or without laser irradiation. Figure S11. The blood circulation profiles of 89Zr-Co-UiO-67@Py-PAA-PEG-F3 and 89Zr-Co-UiO-67@Py-PAA-PEG in mice. Figure S12. The impact of Co/Ru-UiO-67@Py-PAA-PEG-F3 to the infiltration of CD8+ T cells to the tumor site. Blue: DAPI. Green: CD8+ T cells. Length bar: 50 μm. Figure S13. The H&E staining of representative tissue slices before and after the 10 mg/kg treatment of Co/Ru-UiO-67@Py-PAA-PEG-F3. Figure S14. The blood chemical measurement of normal mice and mice at 1 day and 1 week post the treatment with Co/Ru-UiO-67@Py-PAA-PEG-F3 (10 mg/kg). Figure S15. Body weight curve of normal mice treated with Co/Ru-UiO-67@Py-PAA-PEG and PBS intravenously. Table S1. The region-of-interest (ROI) analysis of major organs injected with 89Zr-Co-UiO-67@Py-PAA-PEG-F3 and 89Zr-Co-UiO-67@Py-PAA-PEG in PET images (n = 4).Additional file 2. Video S1. CO catalysis condition post light irradiation of Co/Ru-UiO-67 with saturated CO2 in DMF.Additional file 3. Video S2. CO catalysis condition post light irradiation of UiO-67 with saturated CO2 in DMF.


This study provides scientific support for the clinical use of YZD and justifies further investigations into its effects in AD. Furthermore, it demonstrates the utility of network pharmacology in elucidating the biochemical mechanisms underlying the beneficial effects of traditional Chinese medicines (TCM).Yuan-Zhi Decoction (YZD) is a traditional Chinese medical formulation with demonstrated clinical benefits in Alzheimer’s disease (AD). We used liquid chromatography coupled with mass spectrometry to identify 27 unique chemical components of YZD. Analyzing these using network pharmacology and molecular docking models identified 34 potential interacting molecular targets involved in 26 biochemical pathways. When tested in an animal model of AD, the APP/PS1 transgenic mice showed measurable improvements in spatial orientation and memory after the administration of YZD. These improvements coincided with significantly reduced deposition of Aβ plaques and tau protein in the hippocampi in the treated animals. In addition, a decreased BACE1 and beta-amyloid levels, a downregulation of the p-GSK-3β/GSK-3β, and an upregulation of the PI3K and p-AKT/AKT pathway was seen in YZD treated animals. These  Alzheimer’s disease (AD) is an irreversible, chronic, and relentless neurodegenerative disease, characterized by memory loss, psychoses, affective disorders, and behavioral alterations . NumerouConsidering the multifactorial causation of AD, pharmacotherapy approaches targeting a single molecule may not optimal. And As an example, although the anti-amyloid antibody, aducanumab, developed by Biogen, has been shown to lower the number of amyloid plaques in the brain, there is no evidence that it improves cognitive outcomes. Thus, the accelerated approval of aducanumab by the FDA remains controversial . Given tPolygala tenuifolia Willd. (yuan-zhi in Chinese), Panax ginseng C. A. Mey. (ren-shen in Chinese), Acorus tatarinowii Schott (shi-chang-pu in Chinese), Notopterygium incisum Ting ex H. T. Chang (qiang-huo in Chinese), Asrum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. (xi-xin in Chinese), Ephedra sinica Stapf (ma-huang in Chinese), Paeonia lactiflora Pall. (chi-shao in Chinese), Atractylodes macrocephala Koidz. (bai-zhu in Chinese). This formulation has been in use to treat a variety of medical issues. Recent pharmacological studies have demonstrated that many ingredients of YZD have beneficial effects in AD. Yuanzhi  is an ingredient of multiple formulas use to managed age related memory impairment (Yuan-Zhi Decoction (YZD) is a natural plant-derived TCM formulation. It can be traced back to the book “Qi xiao liang fang” written by Fang Xian during the Ming Dynasty. YZD is a mixture of eight Chinese herbs, namely: pairment . Shichanpairment . Xixin dpairment . HoweverNetwork pharmacology, first proposed by Andrew Hopkins in 2007 is a relatively novel approach in elucidating the mechanism of action of multicomponent drugs . Its corMolecular docking studies use computer simulations to study the interactions between receptors and small molecules potentially binding to them. By predicting certain features, such as the location and affinity of these interaction it is an invaluable tool in drug design and screening of chemical compounds . RecentlIn the study presented here we systematically identified chemical constituents of YZD using liquid chromatography/mass spectrometry and explored possible targets and related pathways with potential relevance to the treatment of AD. The validity of this approach was confirmed by behavioral evaluation, microscopic analysis of the brains, and by Western blotting.via a Milli-Q water purification system . 3,6′-disinapoyl sucrose, Ginsenoside Re, β-Asarone, Asarinin, Notopterol, Ephedrine, Paeoniflorin, and Atractylenolide II were purchased from National Institutes for Food and Drug Control . The purity of each reference standard was above 98%. All eight herb constituents of YZD, including Polygala tenuifolia Willd., Panax ginseng C. A. Mey., Acorus tatarinowii Schott, Notopterygium incisum Ting ex H. T. Chang, Asrum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., Ephedra sinica Stapf, Paeonia lactiflora Pall., and Atractylodes macrocephala Koidz. were purchased from the Hebei Renxin Pharmaceutical Co., LTD., and authenticated by Deputy Chief Pharmacist Jing He . Their voucher specimens were deposited at the author’s laboratory.HPLC-grade acetonitrile was obtained from Merck KgaA . HPLC-grade formic acid was supplied by Macklin Biochemical Co., Ltd. . Ultra-pure water (18 MΩ cm) was prepared g for 10 min at 4°C, the supernatant was filtered through a 0.22 μm membrane filter, and used for further analysis.Raw herbs contained in the formula  were soaked for 2 h and extracted by reflux extraction using a 10-fold mass of 70% ethanol aqueous solution. This process was repeated twice. Raw extracts were filtered with a 200-mesh sieve, combined, and concentrated under vacuum, resulting in 60 g thick paste. The extract was obtained with a yield of 33.7% and kept frozen at −20°C until use. A 1.0 g of this paste was sonicated in 50 ml of methanol/water mixture  for 5 min, centrifuged at 1,500×− and [M + H]+ were selected as precursor ions and subjected to target-MS/MS analyses with different collision energies ranging from 10 to 60 V to acquire sufficient product ions. MS spectra were recorded over the m/z range of 50–1,000. The Compound Discoverer 3.1 platform was used to perform retention time correction, peak identification, peak extraction, and other tasks. Data was processed using the Thermo mzCloud online database and Thermo mzVault local database to identify substances based on the secondary mass spectrum information.Chromatographic separation was carried out on a Thermo Ultimate 3000LC, Q Exactive HF LC-MS system  utilizing an Agilent Zorbax Eclipse C18 column  at 30°C with a flow rate of 0.3 ml/min. The mobile phase was 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The gradient elution program was performed as follows: 0–2 min, 5% B; 2–6 min, 5–30% B; 6–7 min, 30% B; 7–12 min, 30–78% B; 12–14 min, 78% B; 14–17 min, 78–95% B; 17–20 min, 95% B; 20–21 min, 95–5% B; 21–25 min, 5% B. 2 μL of sample solution was injected for analysis. Mass detection was performed using an Thermo Ultimate 3000LC Q Exactive HF mass spectrometer  equipped with a ESI source operating in both positive and negative mode with the following operating parameters: drying gas (N2) temperature: 325°C, sheath gas flow rate: 45 arb; auxiliary gas flow rate: 15 arb, purge gas flow rate: 1 arb, electrospray voltage: 3.5 KV, capillary temperature: 330°C, S-Lens RF Level: 55% under positive or negative mode. The nozzle voltage was set at +500 V or −1000 V, respectively. The quasimolecular ions [M-H]https://www.ncbi.nlm.nih.gov/pubmed/), SEA (https://sea.bkslab.org), STITCH (https://stitch.embl.de/), PharmMapper (https://lilab-ecust.cn/pharmmapper/), and SwissTarget Prediction databases (http://www.swisstargetprediction.ch/) to create a comprehensive list of molecules present in YZD that could mediate beneficial effects in AD.Briefly, all available Chinese medicine network pharmacology research databases  were searched for previously identified chemical components of YZD. We also prepared a list a list of all chemical components identified by combined LC-MS analysis. This comprehensive list of YZD components was used to search the PubChem (Homo sapiens”. The following databases were queried: Drugbank database (https://www.drugbank.ca/); GeneCards database (https://www.genecards.org/); OMIM database (http://omim.org/); DisGeNET database (http://www.disgenet.org/), and GEO database (https://www.ncbi.nlm.nih.gov/geo/). Potential AD-related molecular targets identified from these searches were merged into a single list and duplicates were removed, resulting in a list of potential drug targets in AD.To identify potential AD-related therapeutic targets we conducted multiple database searches with the search term “Alzheimer’s Disease” and limiting searches to “The list of potential drug targets in AD (described in 2.4.2) was intersected with a list of known or presumed molecular constituents of YZD (described in 2.4.1) using the Omic Share Tools. Molecules present in both lists represented molecules in YZD that were likely to be responsible for its beneficial effects in AD. The Omic Share Tools were used to draw Venn diagrams.https://string-db.org/), with the species limited to Homo sapiens.To clarify relationships between the proteins identified in this process their protein-protein interaction (PPI) network was constructed and analyzed using the STRING database (http://www.genome.jp/kegg/)   term performance analysis were conducted on proteins involved in the network using the Kyoto Encyclopedia of Genes and Genomes   database/, v6.7)  RelevantTo facilitate the interpretation of the complex relationships between putative beneficial components of YZD and known or presumed therapeutic targets in AD, an interaction network was created using the Cytoscape software . This provided an approach for data integration, analysis, and visualization for complex network analysis. http://www.rcsb.org) database and saved in pdb format. The Maestro software package was used to create a representation of these key proteins, including the removal of repeat units, hydrogenation and charge treatment. Then the docking site of various YZD-derived chemicals on the target proteins was determined, and a grid file was prepared. Models containing the selected protein and the interacting YZD chemical were crated by molecular docking, using the docking score  as the scoring standard.To further verify the reliability of the target prediction results, a group of key molecules was selected based on their importance in the“component-target-pathway” network. Three-dimensional structures of the selected target proteins were downloaded from the RCSB  85Dbo mice (APP/PS1) on a C57BL/6 background were obtained from Beijing HFK Bioscience Co. Ltd. These double transgenic animals express both a human-mouse chimeric APP gene (Mo/Hu APP695) carrying the Swedish mutation (Swedish KM594/595NL) and a human PS1 gene with a mutation in its ninth exon (dE9). APP/PS1 mice and wild-type littermates were genotyped by PCR analysis of genomic DNA from tail biopsies. Animals were maintained at 22 ± 2°C with a 12-h light-dark cycle  with free access to food and water. All experiments were approved by the Animal Care and Use Institutional Committee of the General Hospital of the Northern Theater Command. APP/PS1 transgenic AD mice were randomly divided into five groups. Animals in the three YZD treatment groups received 0.11 g/kg, 0.32 g/kg, or 0.96 g/kg YZD. Two control groups were given either 4 mg/kg Donazepil or water. A group of wild type mice (WT), sham treated with the same volume of water was used as an additional control group. Treatment or sham treatment was administered After the 3 months treatment period the Morris water maze (MWM) test was used to assess cognitive behavior. The test consisted of a one-day conditioning test, a five-day spatial acquisition test, and a one-day probe trial test. Tests were performed in a circular pool (120 cm in diameter and 50 cm in height) filled with water at 20–22°C. The pool was filled to a depth of 26 cm with water containing an opaque nontoxic Titanium dioxide die, and placed in a dimly lit, soundproof test room surrounded by distinct extra maze cues. The pool was equally divided into four quadrants. A white hidden circular platform (12 cm in diameter and 25 cm high) was placed randomly under a quadrant of the pool, submerged 1 cm below the surface of the water. In the conditioning test, mice swam freely in the pool for 60 s. Each mouse was placed in the pool in the four different quadrants every day. The timer was set to 60 s and automatically stopped once the mouse found the platform and remained on it for 5 s. Animals unable to locate the platform themselves withing 60 s were manually moved to the platform and allowed to stay on it for 20 s. The probe trial test was performed on the 7th day when the platform was removed. Mice were allowed to swim for 60 s, searching for the missing platform at its last know location. The escape latency, distance, speed, and the number of times animals crossed the platform were recorded using a video camera-based Supermaze System .Mice were anesthetized with sodium pentobarbital (50 mg/kg), killed, and brains were harvested for Western blot analysis. Hippocampi, isolated from the brain, were homogenized in RIPA lysis buffer  containing a protease and phosphatase inhibitor cocktail . The homogenized tissue was centrifuged at 12,000 rpm for 10 min at 4°C to remove debris and the protein concentration of each sample was determined using a BCA protein assay (Solarbio Science & Technology Co.). The prepared homogenates were used to estimate the protein expression levels of APP, BACE1, beta-amyloid, pGSK3B/GSK3B, and pAKT/AKT. Samples were electrophoresed using SDS-10% PAGE, transferred to a PVDF membrane, and protein expression was determined using antibodies against APP, BACE1, beta-amyloid, pGSK3B/GSK3B, pAKT/AKT, and actin , respectively. Signal was detected using an ECL method and quantified in a ChemiDoc MP Imaging System .The schematic diagram depicting the steps of all experimental work is shown in Hemispheres prepared for immunohistochemical analyses were immersion fixed in 5% paraformaldehyde, and paraffin embedded brain samples were cut into 3 μm slices using a microtome (Leica RM2235). Slides were deparaffinize with graded ethanol, incubated with the primary antibody overnight at 4°C and with the appropriate fluoroprobe labelled secondary antibody for 1 h at room temperature, protected from light. Antibodies used were: Anti-Amyloid beta 40 Rabbit  (1:500) for the detection of Aβ plaques, and anti-Tau Rabbit  (1:200) for Tau.p value <0.05 was considered statistically significant.The experimental data were analyzed using SPSS 20.0 package . Figures were created by GraphPad Prism 6.0 software . All results are expressed as mean ± standard error of the mean (SEM). Multigroup comparisons in the MWM test were performed using two-way ANOVA. For other experimental data, one-way ANOVA was used, using LSD to test for homogeneity of variance. A non-parametric test was used for unequal variance. A Using a combination of liquid chromatography and mass spectroscopy identified 211 constituents of YZD based on mass, chromatographic behavior, MS/MS data, and fragmentation rules  . Additi3: 370 targets, Ginsenoside-Rg1: 102 targets, Ginsenoside-Re: 103 targets, Ginsenoside-Rb1: 114 targets, Caryophyllene oxide: 285 targets, β-asarone: 339 targets, Aristolactam I: 113 targets, Ephedrine: 109 targets, Pseudoephedrine: 48 targets, Norephedrine: 88 targets, linoleic acid: 546 targets, Paeoniflorin: 77 targets, gallic acid: 434 targets, ellagic acid: 479 targets, albiflorin: 426 targets, chlorogenic acid: 424 targets, Notopterol: 362 targets, Isoimperatorin: 419 targets, Columbianetin: 265 targets, Nodakenin 327: targets, Ostruthin: 474 targets, and Atractylenolide II: 48 targets. After combining and deduplicating the list of potential interaction partners, resulted in 949 predicted targets. Detailed information of these is given in Based on database searches we found that the 27 key components of YZD had the potential to interact with a total of 949 predicted cellular targets. The contribution of the individual herbs is as follows: Polygalaxanthone: 12 targets, Tenuifolin: 43 targets, Ferulaldehyde: 238 targets, Sibiricose A3: 152 targets, citric acid: 376 targets, Ginsenoside-RgDatabase searches identified a total of 231 cellular molecular targets with presumed or proven relevance to AD. Of these 31 were found in the DrugBank database, 87 in the GeneCards database, 43 in the DisGeNET database, and 25 in the OMIM database. After removing duplicates resulted in 169 AD-related targets. The details of these, including additional information, are given in We compared the 949 know molecular targets of the active ingredients of YZD with the 231 AD-related differentially expressed mRNAs identified in p value of less than 1.0e−16. Importing the interaction relationship data into the Cytoscape 3.6.0 software resulted in the PPI network map shown in To investigate protein-protein interaction (PPI) relationships between the identified 34 molecules, a PPI network was built using the String platform and Cytoscape. This network included 34 nodes and 137 edges, with an average degree value of 8.06, and a PPI enrichment p < 0.05) were selected as significant entries based on their p value , molecular function (MF), and cellular components (CC) of these key proteins, GO enrichment analysis was carried out. The top 10 GO terms . The top 10 of these are listed in KEGG pathway enrichment analysis demonstrated that the 34 key targets contributed to 26 distinct pathways . As expected, YZD and Donazepil (DON) treatment significantly decreased BACE1 expression (p < 0.05). These results suggest that the therapeutic effect of YZD in AD may be attributed to the inhibition of BACE1 expression and the reduced production of beta-amyloid.The hippocampi of animals from the MWM test were analyzed for the abundance of several AD-related proteins using Western blot analysis. As shown in pression  and betapression  levels cp < 0.05). Compared to untreated transgenic animals, the levels of PI3K  accumulation is a pathological feature of AD, inducing neuronal amyloidosis and even apoptosis. Therefore, we studied the effect of YZD on Aβ accumulation in the brain of APP/PS1 mice by immunohistochemistry. The resulting images shown numerous brown plaques. Tau protein, the main protein constituent of microtubule structure, maintains the stability of the microtubular system. In the pathogenesis of AD, microtubules depolymerize, resulting in the production of NFTs and the acceleration of the aging of neuronal fibers. Our results showed that the deposition of Aβ plaques was significantly decreased in the hippocampi of YZD-treated APP/PS1 transgenic mice . A similAD is a complex disease affecting multiple biochemical pathways. Given its unclear etiology and pathogenesis, there is an increasing interest in the use of traditional medical formulations in the treatment of the condition . TCM genin vivo efficacy of YZD in improving learning and memory deficits. These experiments demonstrated that YZD administration had a positive impact on spatial learning and memory, evidenced by reducing the time needed to reach a hidden platform during MWM trials. Finally, some of the network pharmacology predictions were validated by Western blot analysis and immunohistochemistry, confirming that several AD-related pathways predicted in silico indeed played a critical role in a live animal model.To the best of our knowledge, this is the first study to systematically investigate the mechanism of action of YZD in combatting AD using network pharmacology analysis. The main chemical constituents of YZD were identified using liquid chromatography coupled to mass spectroscopy. This was followed by network pharmacology studies to assess potential interactions between the detected chemical components and putative AD-related protein targets identified in database searches. With this approach, both the protein targets and candidate active constituents of YZD were predicted in silico. By comparing the list of all potential target proteins affected by the constituents of YZD with a comprehensive list of proteins relevant to the development of AD we identified those proteins YZD is likely to act to deliver its beneficial effects in AD. Next, pathway enrichment analysis was performed to investigate the molecular pathways affected by the 27 components of YZD that were predicted act in AD. Following this computational phase of the work, the APP1/PS1 transgenic mouse model of AD was used to assess the In this study, data from UHPLC-Q Exactive HF mass spectrometry was complemented with TCM database searches to maximize the number of identified chemical constituents of YZD . In totaOligosaccharide esters are a unique class of sugar esters in Polygonum consisting of a sucrose core and connect monosaccharides, mainly glucose and rhamnose, linked with different glycosidic bonds . This ra42-induced neuroinflammation  and is regarded as a promising therapeutic option in the treatment of AD (Some of the identified YZD components were previously shown to be beneficial in AD. Tenuifolin attenuates Amyloid-βammation . This meammation . Ginsenoammation , while gammation . Ginsenoammation  and Notoammation . Other sammation . Albifloammation  and Atra disease . β-Asaro12 cells . β-Asaro animals . Nodakennt of AD .Network pharmacology analysis is able to identify interactions between chemical constituents and their targets/pathways, and establish “compound-target-pathway” maps identifying chemical constituents and the mechanisms associated with their pharmacological activity . Our worAfter network topology analysis 17 of these genes, including IL6, APP, VEGFA, MPO, ITGB1, IL1B, ACE, MME, MAPT, IL4, CXCR2, GRIN2B, VCP, GSK3B, LTB4R, CHRM1, and BACE1 were considered to be the key molecules targeted by the chemical constituents of YZD. These proteins show complex interactions with each other, forming a PPI network. In such networks a change affecting a single molecule can result in a series of functional abnormalities, potentially leading to the emergence of pathologic abnormalities . Our resKEGG pathway enrichment analysis showed that the identified molecules preferentially affected the transcription of RNA polymerase II promoters, the process of apoptosis, cell proliferation, signal transduction, synaptic transmission, neuroactive ligand-receptor interactions, and the PI3K-Akt, HIF-1, and calcium signaling pathways. The PI3K/AKT signaling pathway is involved in regulating cell proliferation, differentiation, metabolism, and apoptosis. It also exerts anti-oxidative, anti-inflammatory, and neuroprotective effects. Altered regulation of this pathway has been found to play a central role in the development of AD . PI3K acWe also conducted behavioral tests to assess the efficacy of YZD in treating learning and memory deficits in aged APP/PS1 transgenic mice. These mice exhibit impairments in spatial learning and memory, as evidenced by their poor performance in MWM tests. Our results showed that YZD treatment restored learning and memory deficits in these animals, demonstrating a protective effect of this formulation in APP/PS1 transgenic AD mice.The PI3K/AKT/GSK-3β signaling pathway plays a crucial role in cell growth, survival, apoptosis, and proliferation, and is also one of the important pathways that lead to the phosphorylation of tau protein and then the development of AD.  Activatiβ-amyloid were also down-regulated significantly, providing experimental confirmation of predictions reached by using network pharmacology and molecular docking analyses. Based on these findings, we propose that YZD may protect against the progression of AD by inhibiting the expression of BACE1, thereby reducing the production of beta-amyloid. The other main action of the formulation is via the regulation of the PI3K/AKT/GSK-3β pathway.Our Western blot experiments indicated that treatment with YZD promoted the expression of PI3K and p-Akt, and reduced the levels of p-GSK3β, proving that YZD can regulate the activated PI3K/AKT/GSK3β pathway. The expression of BACE1 and Although this work shed light on some of the therapeutic mechanisms of YZD in AD, the results need to be interpreted with caution. Additional pharmacologic experiments will be necessary to validate further mechanisms of YZD predicted to occur in our network analysis. Second, network pharmacology fails to identify dose-effect relationships of multi-component formulations. These issues will be addressed in subsequent studies. Finally, further work is needed to study global metabolic changes in other species to fully elucidate the mechanisms by which YZD improves AD. Enhanced metabolomics technologies, such as targeted metabolomics, are required to detect metabolite biomarkers .In summary, multiple compounds in YZD show promise in the potential treatment of AD . Our expHerbal medicines have provided valuable tools in preventing and treating AD for centuries. In this study, we have identified 27 chemical constituents of YZD. Using a network pharmacology approach combined with molecular docking analyses identified some of the mechanisms involved in the beneficial effects of YZD treatment in AD. These involve effects on neuroactive ligand-receptor interaction pathways, the PI3K/AKT signaling pathway, as well as HIF-1 and calcium signaling. The results showed that YZD has potential benefits on learning and memory deficits in APP/PS1 transgenic AD mice, and regulated the apoptotic process and signal transduction. The key proteins of related pathways were quantitatively analyzed using Western blots indicating that the extract of YZD could inhibit or activate key protein pathways. The insights provided by this preliminary work will pave the way for further studies into the potential mechanisms by which YZD acts as a therapeutic agent in AD.


Using an integrative network pharmacology-based approach and experimental verification, the study showed that JPYSF had therapeutic effects on EMT by regulating multi-pathway, among which one proven pathway was the Wnt3a/β-catenin signaling pathway. These findings provide insights into the renoprotective effects of JPYSF against EMT, which could suggest directions for further research of JPYSF in attenuating renal fibrosis by suppressing EMT.Jian-Pi-Yi-Shen formula (JPYSF), a traditional Chinese medicine, has been recommended to treat renal fibrosis for decades. Previous studies had shown that JPYSF could inhibit epithelial–mesenchymal transition (EMT), an important regulatory role in renal fibrosis. However, the mechanism of JPYSF action is largely unknown. In this study, network pharmacology and experimental verification were combined to elucidate and identify the potential mechanism of JPYSF against renal fibrosis by suppressing EMT at molecular and pathway levels. Network pharmacology was first performed to explore the mechanism of JPYSF against renal fibrosis targeting EMT, and then a 5/6 nephrectomy (5/6 Nx)-induced rat model of renal fibrosis was selected to verify the predictive results by Masson’s trichrome stains and western blot analysis. Two hundred and thirty-two compounds in JPYSF were selected for the network approach analysis, which identified 137 candidate targets of JPYSF and 4,796 known therapeutic targets of EMT. The results of the Gene Ontology (GO) function enrichment analysis included 2098, 88, and 133 GO terms for biological processes (BPs), molecular functions (MFs), and cell component entries, respectively. The top 10 enrichment items of BP annotations included a response to a steroid hormone, a metal ion, oxygen levels, and so on. Cellular composition (CC) is mainly enriched in membrane raft, membrane microdomain, membrane region, etc. The MF of JPYSF analysis on EMT was predominately involved in proximal promoter sequence-specific DNA binding, protein heterodimerization activity, RNA polymerase II proximal promoter sequence-specific DNA binding, and so on. The involvement signaling pathway of JPYSF in the treatment of renal fibrosis targeting EMT was associated with anti-fibrosis, anti-inflammation, podocyte protection, and metabolism regulation. Furthermore, the  Renal fibrosis can lead to the progressive loss of renal function, with the characteristic of the proliferation of renal intrinsic cells, activation of renal interstitial fibroblasts, and deposition of extracellular matrix, which is the most common cause of kidney failure . A largeAstragalus mongholicus Bunge (A. mongholicus), Dioscorea oppositifolia L. (D. oppositifolia.), Salvia miltiorrhiza Bge. (S. miltiorrhiza), Atractylodes macrocephala Koidz. , Cistanche deserticola Ma (C. deserticola), Amomum kravanh Pierre ex Gagnep. (A. kravanh), Rheum palmatum L. , and Glycyrrhiza uralensis Fisch. . Adopting the two-complement five-way method, the two complement mainly consists of “tonifying the kidney” and “tonifying the spleen.” A. mongholicus, A. macrocephala., D. oppositifolia., and C. deserticola were used as spleen and kidney supplements  is derived from the addition and subtraction of “Yupingfeng San” and “Sijing Pills” (recorded in Shengji Zonglu). JPYSF has been prescribed for patients with chronic kidney disease in clinics, which consists of plements . A. mongtipation . A. mongc effect . A. kravd stasis . G. uralfibrosis . HoweverNetwork pharmacology is a theory based on systems biology and has been commonly used in the modern research of TCM in recent years. It emphasizes the multichannel regulation of signaling pathways, which is in accord with the characteristics of multi-components and multi-targets of Chinese medicine. Therefore, network pharmacology has become a new effective approach in TCM research at the molecular level .in vivo, we first explored the main active ingredients, targets, and signal pathways of JPYSF in the treatment of EMT, and then the animal experiment was carried out to verify the effect of JPYSF against renal fibrosis by inhibiting EMT. The research may lay a good theoretical foundation for further study on developing new drugs for renal fibrosis.In this study, the integrated strategy of network pharmacology and verification https://tcmspw.com/tcmsp.php), Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine (TCMIP) v2.0  v2.0 , and preIP) v2.0 . All chehttp://www.swisstargetprediction.ch/) databases, and previous pieces of literature. The candidate’s therapeutic genes related to EMT were acquired from the OMIM (https://www.omim.org/) (https://www.genecards.org/), and Drugbank (https://go.drugbank.com/) using “epithelial–mesenchymal transition” as the keyword. Then, all targets were converted into gene names by the UniProt database (https://www.uniprot.org/).The target screening of components was predicted using TCMIP v2.0, TCMSP, the Swiss Target Prediction (im.org/) , GeneCarhttps://www.string-db.org/). To identify the potential hub nodes of JPYSF in the treatment of EMT, the minimum required interaction score was set as the highest confidence 0.9. “hide disconnected nodes in the network” was ticked in network display options. String_interactions.tsv was then exported. Network visualization and analysis were performed using Cytoscape 3.7.1 software.The interaction networks of the common targets of JPYSF and EMT were constructed by the STRING platform (https://bioinfogp.cnb.csic.es/tools/venny/index.html), and the Venn diagram was then prepared. The coexistent targets were analyzed using R (https://www.r-project.org/) software for the GO and KEGG enrichment analysis. The threshold was set to p < 0.05. The results were visually displayed in the form of bubble charts and histograms. The biological processes (BPs), molecular functions (MFs), and cellular compositions (CCs) were included in the GO enrichment analysis.The coexistent targets of JPYSF and EMT were conducted on VENNY 2.1 website  and were authenticated by Shangbin Zhang. The extraction of JPYSF was conducted as previously described . In brieeviously . The extAll animal experiments were in accord with the ethics committee of Guangzhou University of Chinese Medicine and the National Institutes of Health Guideline for the care and use of laboratory animals. Forty male Sprague–Dawley (SD) rats weighing between 180 and 220 g were supplied by Guangdong Medical Laboratory Animal Center . All the rats were housed under controlled conditions (12-h light/12-h dark cycle) in a specific pathogen-free animal facility with free access to rodent food and drinking water. Either 5/6 nephrectomy (5/6 Nx) or sham operation (sham) was performed on SD rats. The 5/6 Nx operation was conducted in a two-step surgery as described previously . In brieThe kidneys of the rats were fixed with 10% neutral formaldehyde and then paraffin embedding and sectioning were carried out. Pathological changes in the kidney were evaluated by Masson’s trichrome stains. The experimental process and quantitative methods were performed as previously described .The cortex tissues of the kidneys were lysed with RIPA lysis buffer. Equal amounts of lysates were loaded and electrophoresed through 10% SDS-polyacrylamide gels, and then transferred to the PVDF membrane. Following blocking in 5% non-fat milk for 2 h at room temperature, the membranes were incubated with various primary antibodies at 4°C overnight. The primary antibodies included α-SMA , E-cadherin , Wnt3a , β-catenin , and GAPDH . Then, the membranes were incubated in HRP-conjugated secondary antibodies for 2 h at room temperature. HRP activity was visualized using Tanon-6100C .p’ value was less than 0.05.Data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to determine the level of statistical significance followed by Tukey’s multiple comparison tests. There was a significant difference if the ‘A. macrocephala, 16 compounds were in R. palmatum, 67 compounds were in S. miltiorrhiza, 9 compounds were in A. kravanh, 89 compounds were in G. uralensis, 14 compounds were in A. mongholicus, 4 compounds were in C. deserticola, and 16 compounds were in D. oppositifolia. Luteolin was the common compound derived from both S. miltiorrhiza and A. kravanh; Jaranol was the common ingredient of A. kravanh, G. uralensis, and A. mongholicus. Mairin and calycosin co-existed in A. mongholicus and G. uralensis. Quercetin existed in G. uralensis, A. mongholicus, A. kravanh, and C. deserticola. -10,13-Dimethyl-17-[-5-propan-2-yloctan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol was present in A. mongholicus and A. macrocephala. Isorhamnetin, formononetin, and kaempferol were present in A. mongholicus and G. uralensis. β-sitosterol was present in both C. deserticola and R. palmatum. Sucrose was present in A. mongholicus and D. oppositifolia. The aforesaid compounds in JPYSF can be found in There were 232 compounds in JPYSF that were being screened by the conditions of OB value ≥ 30% and DL value ≥ 0.18. Among them, 6 compounds were in Fifty-nine EMT-related targets were identified in the DrugBank database, 4,770 targets in the GeneCards database, and 7 targets in the OMIM database. There were 4796 EMT-related targets after removing the duplicate values . Excludip＜0.05) were obtained . Tubular atrophy and interstitial fibrosis in 5/6 Nx rats were significantly ameliorated in both 2.73 g/kg and 10.89 g/kg JPYSF groups.As shown in p value was in the enriched anti-fibrosis signaling pathway. Therefore, the protein expression of Wnt3a and β-catenin was calculated. The protein levels of both Wnt3a and β-catenin were increased in the model group than those in the sham group, and the changes were significantly reversed by the JPYSF treatment. The western blot results showed that JPYSF administration significantly alleviated EMT by inhibiting the Wnt3a/β-catenin pathway.To investigate whether JPYSF improved kidney fibrosis by restraining EMT, the protein expression of α-SMA and E-cadherin in the kidney tissues of 5/6 Nx rats was detected by western blot. As shown in JPYSF, a classic prescription of hospital preparations, has been used clinically to treat renal fibrosis as an effective and safe TCM. Previous studies confirmed that JPYSF significantly decreased fibrotic area to ameliorate interstitial fibrosis and down-regulated fibrosis-associated protein expression such as fibronectin and type IV collagen in 5/6 Nx rats . These r. Taken together, our results demonstrate that the core components may be the effective ingredients of JPYSF for the EMT treatment.First, network pharmacology was employed to explore the potential active ingredients and targets of JPYSF. Second, the common targets were the intersection targets between the potential targets of JPYSF and EMT-associated targets, and then they were identified to build a protein interaction network. According to the string database, the top 30 targets with action frequency are shown in A. mongholicus, is considered as the monarch drug, the ingredient that provides the principal curative action on the main syndrome in JPYS prescription and has also been identified within JPYSF by HPLC-MS analysis in the current study (Finally, the 5/6 Nx-induced renal fibrosis rat model was selected to evaluate the therapeutic effects of JPYSF on EMT to verify the network pharmacology prediction. As expected, JPYSF treatment significantly ameliorated interstitial fibrosis in 5/6 Nx rats by Masson staining analysis . In our nt study . Since mHowever, there were some limitations of research, such as the core compound with no effective dose, which still needed further experiments to identify. Moreover, the direct regulatory relationship of JPYSF in EMT and the Wnt3a/β-catenin pathway by gene knockout animal experiments and cell experiments would be our future potential work for a better understanding of this signaling pathway.In vivo experiment data showed that JPYSF significantly ameliorated interstitial fibrosis by Masson staining and exerted the regulation of EMT by inhibiting the Wnt3a/β-catenin pathway (In this research, a combining approach of systematic network pharmacology and experimental verification were employed to determine therapeutic targets and pharmacological mechanisms of a complex herbal formulation, JPYSF, against EMT. The results suggested that 17 core compounds such as quercetin, kaempferol, luteolin, naringenin, and 23 core targets such as NCOA2, HSP90AA1, and PTGS1 might play an important role in JPYSF-treated renal fibrosis. The multi-target synergetic mechanisms of JPYSF in EMT mainly consist of four therapeutic aspects including anti-fibrosis, anti-inflammation, podocyte protection, and metabolism regulation.  pathway . In summ pathway .


Salvianolic acid B  is a bioactive component of Radix Salviae, which has antiinflammation and antiapoptotic activity in diabetic complications. However, the molecular mechanism of action of Sal B on diabetic peripheral neuropathy (DPN) is unknown. This study was designed to identify a mechanism for Sal B in the treatment of DPN by using a pharmacology network, molecular docking, and in vitro experiments.  Sal B and DPN-related targets from Gene Cards and OMIM platforms were retrieved and screened. Then, an analysis of possible targets with STRING and Cytoscape software was conducted. KEGG signaling pathways were determined using the R software. Subsequently, the binding capacity of Sal B to target proteins was analyzed by molecular docking and in vitro experiments.  A total of 501 targets related to Sal B and 4662 targets related to DPN were identified. Among these targets, 108 intersection targets were shared by Sal B and DPN. After topological and cluster analysis, 11 critical targets were identified, including p38MAPK. KEGG analysis revealed that the AGE-RAGE signaling pathway likely plays an important role in Sal B action on DPN. The p38MAPK protein is a key target in the AGE-RAGE signaling pathway. Molecular docking results suggested that Sal B and p38MAPK have excellent binding affinity . The in vitro experiments revealed that Sal B downregulates the expressions of p-P38MAPK, inflammatory cytokines, and apoptosis targets, which are upregulated by hyperglycemia.  Sal B may alter DPN by inhibiting inflammation and apoptosis activated by p38MAPK. Diabetic peripheral neuropathy (DPN) is the most common microvascular complication in patients with diabetes mellitus (DM), affecting about 50% of cases. It is defined as the emergence of a series of nerve dysfunction in patients with DM. Clinical manifestations include neuropathic pain and sensory disorder or loss. This neuropathic injury greatly affects the patient's quality of life and burdens the family and society. These devastating effects have led to the search for therapeutic solutions –3. CurreIn China, a traditional herb has been widely known and utilized for treating DPN for more than 2000 years because of its prominent efficacy, abundant resources, and low toxicity. Studies showed that traditional herbs lowered patient glucose levels but also relieved the numbness and painful symptoms of DPN –16. ThusWith the rapid progress of bioinformatics and systems biology, a TCM network pharmacology method was created to predict disease-associated genes, detect target profiles of herbal compounds, and elucidate drug-gene-disease comodule associations. This method provides opportunities for discovering bioactive ingredients and biomarkers, potentially revealing scientific evidence of herbal formula-diseases co-action . In thishttps://www.genecards.org) and the Online Mendelian Inheritance in Man database (OMIM: https://www.ncbi.nlm.nih.gov/omim). Gene Cards and OMIM databases are considered comprehensive, authoritative compendiums of human genes and genetic phenotypes, which are widely used in analyzing drug components and disease-related targets [Homo sapiens. Then, the intersecting targets between Sal B and DPN were analyzed using a Venn Diagram. After that, the overlapping targets were imported to the String Database (https://string-db.org). The String dataset is one of the largest PPI (protein-protein interactions) datasets, including text mining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence [Homo sapiens” to ensure the reliability and high confidence of information. All predicted interaction data were uploaded to Cytoscape 3.8.0 software and analyzed by CytoNCA. CytoNCA is a cytoscape plugin for calculation, evaluation, and visualization analysis of multiple centrality measures. Betweenness (BC), Closeness (CC), Degree (DC), Eigenvector (EC), Local Average Connectivity-based method (LAC), and Network (NC) were the major centrality measures. After being evaluated by the above-mentioned standards, the nodes and edges of the data were calculated in the network.To collect the targets of Sal B and DPN, we screened the human gene database  Receptor molecular preparation. The gene of p38MAPK corresponding protein Q16539 was examined using the UniProt platform. Q16539 corresponding protein 4GEO structure was downloaded in ∗PDB format. (3) Molecular docking. The PDB format of the receptor with added polar hydrogen, removed solvent, and organics were saved as the receptor PDBQT format. Then, the mol2 format was transformed to the ligand PDBQT format similarly. Next, the ligand PDBQT-receptor PDBQT format docking box was constructed in Auto Grid, and the docking results were displayed in PYMOL software.The reliability of the predicted interactions between Sal B and p38MAPK was investigated using molecular docking performed with Auto Dock Vina. AutoDock is a molecular docking software that is widely used to predict the binding mode of a ligand in the active site of a protein. The specific procedure is as follows. (1) ligand molecule preparation. The 2D structure of Sal B was explored on the PubChem platform and saved in 2. Cells were cultured with different solutions, and the groups were arranged as follows: control (5.6 mmol/L glucose), high glucose , and Sal B ).RSC96 cells were purchased from Shan-Xi-Fan-Chang-Sheng-Wu and cultivated in DMEM medium  with 10% fetal calf serum  at 37°C in an atmosphere comprising 5% CO5 density in 9.6 cm dishes. After the cells were completely adhered to the wall, the cells were treated with different solutions as the above-mentioned. Then, the cells were incubated with primary antibodies  and secondary antibodies . The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) . After that, the cells were fixed with an embedding medium on a microscope slide and imaged by fluorescent microscopy .Cells were cultured at a 5.0 × 105 density in 9.6 cm dishes and incubated for analysis. The levels of IL-6 and IL-1β in cell supernatants were extracted and quantified with commercial ELISA kits , following the manufacturer's protocol.The cells were cultured at a 5.0 × 105 density in 9.6 cm dishes and then lysed and extracted. The BCA assay was used to quantify proteins. Proteins were loaded on an SDS-PAGE polyacrylamide gel, transferred to an NC membrane , incubated with p-P38MAPK (1 : 1000) antibodies, and subsequently measured by chemiluminescence . Images were analyzed by Image-Lab software .The cells were cultured at a 5.0 × 10P < 0.05 being considered statistically significant . Statistical analyses were performed using the GraphPad 5 software, with In total, we discovered 501 potential targets of Sal B after removing duplicate data. There were 453 targets in OMIM and 51 in Gene Cards. A total of 4662 targets related to DPN were identified after removing duplicate data. There were 409 targets in OMIM and 4352 in Gene Cards. Comparison of the 501 potential targets of Sal B with the 4662 identified with DPN resulted in 108 overlapping targets detected by Venn Diagram .The overlapping targets between Sal B and DPN were uploaded to the STRING platform. The interaction files were imported to Cytoscape software. When the criteria were set at Betweenness (BC): 28.003, Closeness (CC): 0.4049, Degree (DC): 6, Eigenvector (EC): 0.03229, Local Average Connectivity-based method (LAC): 2.125, and Network (NC): 3, the results presented in To further identify a mechanism of action for Sal B on DPN, the ID codes between Sal B and DPN were uploaded to R software, and the top 20 signaling pathways were determined . Among tMolecular docking of Sal B action on p38MAPK was generated with Auto Dock software. The binding energy values of Sal B and p38MAPK  showed that the former interacts with the p38MAPK receptor. The 2D structure of Sal B was downloaded from the PubChem platform and the 3D structure was transformed in ChemOffice . The p38β as targets and verified by in vitro analysis. The level of p-p38MAPK was determined by western blot. Results showed that p-p38MAPK was low in control cells but increased in hyperglycemic conditions. The addition of Sal B lowered the expression of p-P38MAPK, as shown in β, were analyzed by immunofluorescence and ELISA. Results showed that caspase-3, IL-6, and IL-1β were low in controls but increased in hyperglycemic conditions. Again, the addition of Sal B lowered the levels of caspase-3, IL-6, and IL-1β as shown in Figures Combining critical targets and signaling pathway results indicated p38MAPK, Caspase 3, IL-6, and IL-1DPN is the most common complication of DM. Although many studies using in vitro and in vivo models have been done, there is no effective treatment for DPN. Sal B, the main component of Radix Salviae, has been identified as being neuroprotective both in vitro and in vivo . Howeverβ, and TNF-α) released in diabetic complications [β were upregulated after treatment with high glucose. Addition of Sal B results in both p38MAPK and inflammatory cytokines being significantly downregulated compared with untreated SCs. Studies also revealed that the release of apoptosis mediators driven by the phosphorylation of p38MAPK induced neuropathy [Studies have shown that the p38MAPK receptor initiates inflammatory cytokine (IL-6, IL-1ications , 32. Ourications –39. Our uropathy , 41. In The inhibition of inflammation, apoptosis, and phosphorylation of p38MAPK by Sal B has been studied previously , 42–45. In summary, Sal B treatment decreased the upregulation of the p38MAPK receptor in SCs and decreased its mediated inflammation and apoptosis activation. These results provide a theoretical basis for Sal B research and its clinical application in DPN.In conclusion, the pharmacology network, molecular docking, and in vitro experimental analysis identify potential molecular mechanisms of Sal B in the treatment of DPN. Sal B may limit inflammation and apoptosis by adjusting p38MAPK. This provides a theoretical basis for the ameliorative effect of Sal B on DPN.


Background: Yiyi Fuzi Baijiang powder (YFBP) is a traditional Chinese medicine used to treat colorectal cancer, although its bioactivity and mechanisms of action have not been studied in depth yet. The study intended to identify the potential targets and signaling pathways affected by YFBP during the treatment of colorectal cancer through pharmacological network analysis and to further analyze its chemical compositions and molecular mechanisms of action.Methods: The Traditional Chinese Medicine Systems Pharmacology (TCMSP), Traditional Chinese Medicine Integrated Database (TCMID), HitPredict (HIT), and Search Tool for Interactions of Chemicals (STITCH) databases were used to screen the bioactive components and promising targets of YFBP. Targets related to colorectal cancer were retrieved from the GeneCards and Gene Ontology databases. Cytoscape software was used to construct the “herb–active ingredient–target” network. The STRING database was used to construct and analyze protein–protein interactions (PPIs). Afterward, the R packages clusterProfiler and Cytoscape Hub plug-in were used to perform Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of target genes. The results of the network pharmacological analysis were also experimentally validated.Results: In total, 33 active components and 128 target genes were screened. Among them, 46 target genes were considered potential therapeutic targets that crossed the CRC target genes. The network pharmacology analysis showed that the active components of YFBP were correlated positively with CRC inflammatory target genes such as TLR4, TNF, and IL-6. The inflammation-related signaling pathways affected by the active components included the TNF-α, interleukin-17, and toll-like receptor signaling pathways. The active ingredients of YFBP, such as luteolin, β-sitosterol, myristic acid, and vanillin, may exert anti-tumor effects by downregulating SMOX expression via anti-inflammatory signaling and regulation of the TLR4/NF-κB signaling pathway.Conclusion: In the present study, the potential active components, potential targets, and key biological pathways involved in the YFBP treatment of CRC were determined, providing a theoretical foundation for further anti-tumor research. Cancer is a major public health issue and is the main cause of death worldwide . It is aPolyamines such as putrescine, spermidine, and spermine are small alkaline molecules containing two or more primary amino groups; they participate in a variety of cellular functions, including protein synthesis, DNA and RNA structure, protein–RNA interactions, and gene expression . StudiesThe metabolism of natural polyamines is regulated by spermine oxidase (SMOX), spermine/spermidine N1-acetyltransferase (SSAT), and polyamine oxidase (APAO) . SMOX, aFor thousands of years, natural products and their derivatives have played an important role in disease research and treatment . Yiyi FuNetwork pharmacology, which is based on a disease–gene–drug network, is a promising new approach in current drug discovery and development, especially in traditional Chinese medicine research .In the present study, a network pharmacology approach was used to predict the molecular targets and bioactive ingredients of YFBP. The active components of YFBP were screened for the first time, and the CRC-related targets of the active components were analyzed and summarized. Gene Ontology (GO) and biological pathway (KEGG) enrichments were used to study the targets and mechanistic pathways impacted by the active ingredients. The results of network pharmacological analysis were also experimentally validated. Overall, this study aimed to provide a theoretical basis for the molecular mechanism of action of YFBP during CRC .All chemical constituents of YFBP were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database , TraditiThe GeneCards database was used to identify potential targets implicated in CRC. All target compounds were collected from the TCMSP, TCMID, HIT, and Search Tool for Interactions of Chemicals (STITCH) databases. A Venn diagram was used to determine the main targets of YFBP in CRC treatment. The protein–protein interaction (PPI) network analysis was performed using the STRING database.GO and KEGG were used to analyze signaling pathways and biological processes related to the target CRC-related genes. GO functional and KEGG pathway enrichment analyses of target genes were performed using the R package clusterProfiler and Cytoscape Hub plug-in.All network plots were constructed using Cytoscape software. Cytoscape with an in-house plugin was used to evaluate and screen core targets to form a core network that was formed to assess the relationship of KEGG-associated items of YFBP and CRC.2 in DMEM 1640 (Gibco) that contained 10% fetal bovine serum  and 1% penicillin–streptomycin (Gibco). When the cells reached 80% confluence, they were divided and sub-cultured for further passages with 0.25% trypsin and 0.02% ethylene-diamine-tetra-acetic acid . Cell viability was determined using the cell counting kit-8 (CCK-8) assay . Briefly, cells were seeded and treated with different concentrations of luteolin . The cells were cultured at 37°C and 5% COHigh-performance liquid chromatography  was used to measure the content of LUT, SIT, MYA, and VAN in YFBP. A volume of 51.00 g of YFBP was accurately weighed and 100 ml of methanol was added, and the mixture was ultrasonicated for 12 h at 60°C. The extracts were filtered using a filter paper and concentrated to 1.00 g crude drug per ml. The concentrated solution was taken and passed through a 0.22-µm microporous membrane, and the content of LUT, SIT, MYA, or VAN was determined by HPLC. LUT, SIT, MYA, or VAN standards were accurately weighed and dissolved in methanol as the stock solution. Chromatography was performed on a C18 column  at 35°C. LUT was eluted with a mobile phase (methanol/0.1% phosphoric acid water at a ratio of 50:50) at a flow rate of 0.5 ml/min and monitored at 350 nm. SIT was eluted with a mobile phase (methanol/water at a ratio of 55:45) at a flow rate of 0.5 ml/min and monitored at 210 nm. MYA was eluted with a mobile phase (acetonitrile/water at a ratio of 33:67) at a flow rate of 0.5 ml/min and monitored at 270 nm. VAN was eluted with a mobile phase (acetonitrile/0.1% phosphoric acid water at a ratio of 13:87) at a flow rate of 0.5 ml/min and monitored at 281 nm. The chromatograms were recorded and integrated using LabSolutions System software.Cell scratch, invasion, and apoptosis assays were performed as described previously . Briefly7/ml. A total of 2×106 HT-29 cells were subcutaneously injected into the back of the nude mice. Two weeks after tumor formation, qualified nude mice were randomly divided into five groups: control (CTR), LUT (50 mg/kg), SIT (50 mg/kg), MYA (50 mg/kg), and VAN (50 mg/kg). For each drug, the administration groups were given the drug for 21 days (q.d.) after tumor formation. After 21 days of continuous administration, the nude mice were sacrificed, and tumors were isolated for further experimental study.SPF male BALB/c nu/nu mice, weighing 18–22 g , were housed in an animal laboratory at 25°C and 60% humidity. HT-29 cells in the logarithmic growth phase were selected and digested to prepare a single-cell suspension with a density of 1×10All experimental protocols and animal handling procedures were performed in accordance with the Experimental Animal Ethics Committee of Sichuan University.Hematoxylin/eosin (HE) staining of tumor samples followed standard HE staining procedures. The tumor tissue was cut off and fixed in 4% polymethylaldehyde solution for 24 h. After alcohol gradient dehydration, about 6-μm-thick coronal sections were embedded in transparent paraffin, and dewaxing was performed with xylene. Each tissue section was incubated at 62°C for 24 h. After thorough dewaxing, the slide with the tissue sections was passed through the decreasing concentration of alcohol to remove xylene I and II and thoroughly rinsed in water. The slide was stained with a hematoxylin staining solution, and for the differentiation step, 1% hydrochloric acid alcohol was used to remove non-specific background staining and to improve contrast. After differentiation, blueing and thorough rinsing with water were performed. For counterstaining, 5% eosin was used. After counterstaining, the slide was passed through 80%, 95%, and 100% alcohol to remove all water traces and rinsed with xylene, which clears the tissue and renders it completely transparent. The slide was sealed with neutral gum. Optical microscopic random observations of the tumor tissue cortex at ×400 magnification do not overlap the three fields of vision and radiography.The tumor tissue was excised and fixed in 4% polymethylaldehyde solution for 24 h. All the samples were paraffin-embedded and sectioned. Immunostaining for SMOX was performed in tumor slices using rabbit anti-SMOX primary antibodies .The level of SMOX in the serum obtained from BALB/c nu/nu mice 21 days after tumor formation was measured using an ELISA kit , according to the manufacturer’s instructions.−ΔΔCt method for relative quantification. A list of primers designed for qRT-PCR is shown in The qRT-PCR analysis was performed as described in a previous study . BrieflyWestern blotting was performed as described in a previous study . BrieflyThe chromatin immunoprecipitation (ChIP) experiment was performed as previously described . Brieflyp < 0.05 and **p < 0.01.Data are presented as mean ± SEM (standard error of the mean). All data were analyzed by ANOVA using SPSS. The levels of significance were set at *A total of 81 YFBP components were identified from the TCMSP, TCMID, and HIT databases. Based on the quantitative estimate of the drug-likeness (QED) value of drug bank-listed compounds, 0.2 was selected as the threshold for screening . In totaBased on the GeneCards database , a totalThe results of 33 active ingredients and 46 potential target genes were entered into Cytoscape software to obtain a YFBP-CRC potential target gene network . The viop < 0.05, single channel count ≥5). A scatter plot was generated by selecting 15 distinct enriched gene biological function catalogs . A scatter diagram  on HT-29 cell viability was evaluated using a CCK-8-based viability assay after incubating the cells with LUT, SIT, MYA, or VAN for 24 h. As shown in Tumors were isolated at the end of the mouse experiments, representative tumors were photographed, and tumor weights were quantitated. The tumor volume was calculated once a week until the end of the experiment (3 weeks). Tumor size , weight The effect of the YFBP active ingredients on SMOX production was determined by ELISA. LUT, SIT, MYA, and VAN significantly suppressed SMOX secretion . The expTo determine whether the anti-tumor effect of the active ingredients of YFBP is associated with the regulation of the expression of the inflammatory mediator, we assessed the gene expression of TNFα, IL-1β, and IL-6. As shown in Increasing evidence suggests that TLR4 and NF-κB play important roles in mediating inflammatory signaling pathways. To understand the mechanisms underlying the anti-inflammatory effects of the active ingredients of YFBP, the protein expression levels of TLR4 and NF-κB p65 were measured. TLR4 and NF-κB p65 proteins were significantly attenuated by pre-treatment with LUT, SIT, MYA, and VAN . These rAs shown in CRC is the third most common cancer worldwide, and inflammation is an important risk factor for its development and progression . ChronicThe composition of traditional Chinese medicine compounds is complex. It is not enough to study the extraction process only on a single component. At present, there are few studies on the extraction process and active ingredient analysis method of YFBP. The research indicated that Aconiti Lateralis Radix Praeparata has certain toxicity, and in clinical practice, Aconiti Lateralis Radix Praeparata was generally decocted first or for a long time and then mixed with Herba Patriniae for extraction . Coicis Network pharmacology provides a new method to understand the complex pharmacological mechanisms of traditional Chinese medicine . Owing tThe results of bioinformatics analyses indicated that SMOX is highly expressed in CRC and is related to poor prognosis. SMOX is an enzyme containing flavin adenine dinucleotide that catalyzes the oxidative degradation of polyamine spermine to produce spermidine, hydrogen peroxide, and 3-aminopropionaldehyde. The use of polyamines and their metabolites as cancer biomarkers is not new, as this process is necessary for cell proliferation. Many cancers, especially oncogene-driven cancers, may be sensitive to interference from polyamine metabolism . The disSMOX is a highly inducible enzyme in the polyamine catabolism cascade, and its expression is upregulated by inflammatory cytokines (including TNF-α and IL-6) . MoreoveCancer is a chronic disease. When infection and inflammation promote tumor development, signal transduction mechanisms that affect the malignant transformation or factors related to cancer monitoring are involved . NF-κB iCompatibility plays an important role in traditional Chinese medicine. The basic principle of practice is the compatibility of different herbal components with different syndromes. Nevertheless, the different efficacies and potential mechanisms of “prescription compatibility” remain unclear . To studYFBP provides obvious advantages in CRC treatment. Network pharmacology was used to study the biological functions, target genes, and signaling pathways impacted by the YFBP active components during the treatment of CRC. Furthermore, the results of network pharmacological analysis were experimentally validated. The present study revealed that the active ingredients of YFBP, such as LUT, SIT, MYA, and VAN, might exert anti-tumor effects by downregulating SMOX expression through anti-inflammatory effects and the regulation of the TLR4/NF-κB signaling pathway. These results aid in further elucidating the molecular mechanism of YFBP anti-CRC activity and providing a theoretical foundation for further anti-tumor research.


Among the proposed targets screened in the c9ALS Drosophila model, we verified 8 unreported genes  whose suppression strongly rescues eye neurodegeneration. Dysregulated pathways identified from CNS and diMN data characterize different stages of disease development. Altogether, our study provides new insights into ALS pathophysiology and demonstrates how AI speeds up the target discovery process, and opens up new opportunities for therapeutic interventions.Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with ill-defined pathogenesis, calling for urgent developments of new therapeutic regimens. Herein, we applied PandaOmics, an AI-driven target discovery platform, to analyze the expression profiles of central nervous system (CNS) samples  from public datasets, and direct iPSC-derived motor neurons (diMNs)  from Answer ALS. Seventeen high-confidence and eleven novel therapeutic targets were identified and will be released onto ALS.AI ( Amyotrophic lateral sclerosis (ALS) is a rare neuromuscular disease resulting from progressive degeneration of motor neurons (MNs) in the brain and spinal cord. With involvement of celebrities, politicians, and athletes worldwide, the Ice Bucket Challenge in 2014 successfully promoted the awareness of this devastating illness. It is the most common MN disease , with thSOD1, TARDBP, C9orf72, and FUS have been extensively characterized. According to a pooled summary of mutation frequency in 111 studies, those four major ALS-associated genes explain 47.7% fALS and 5.2% sporadic ALS (sALS) cases  cases , leavingS) cases .Presently, ALS remains an incurable disease due to an inadequate understanding of disease mechanisms. The United States Food and Drug Administration (FDA) has approved four drugs for the treatment of ALS, including Riluzole, Tiglutik, Edaravone, and Nuedexta. Riluzole - an inhibitor of sodium channel α subunit - is the first FDA-approved neuroprotective agent for ALS and the only drug that prolongs the survival of ALS patients. Beside blocking the glutamatergic transmission, riluzole has a wide range of neural effects, including inhibition of persistent and fast sodium currents, suppression of neurotransmitter release, diminishment of voltage-gated calcium and potassium currents, and potentiation of calcium-dependent potassium current . These aACSL5, KIF5A, ATXN2, and MOBP) , the most common fALS case. The aim of this study is to demonstrate the utilization of the AI-driven target discovery platform – PandaOmics – to identify therapeutic targets for ALS.Thanks to the advancements in genomic profiling techniques, numerous genome-wide association studies have screened for common genetic variants in ALS and have identified novel candidates as either genetic risk factors or biomarkers  . Genomicnd MOBP) . Furthernd MOBP) . Here, wMicroarray and RNA sequencing (RNA-seq) datasets for ALS patients and control samples were retrieved from public repositories and processed by PandaOmics for downstream analysis and target identification. Over 60 ALS-related datasets of various tissue sources were available in PandaOmics , includiThe raw transcriptomic data of CNS comparisons were available in public repositories, which could be retrieved by their series identifiers. In addition, transcriptomic and proteomic profiles of the diMN samples were available to investigators upon request and approval from Answer ALS.via an episomal iPSC reprogramming system. The iPSCs underwent three major stages of differentiation for 32 days to generate the mixture of mature motor neuron populations. Detailed protocol for the diMN generation and standards of quality assurance were described by At present, Answer ALS is the largest collaborative effort in ALS bringing together multiple research organizations and key opinion leaders (KOLs). Over eight hundred ALS patients and one hundred healthy controls from eight neuromuscular clinics distributed across the United States were enrolled in this project. Blood sample was collected at the first visit of each participant. The iPSC lines were generated from peripheral blood mononuclear cells extracted from whole blood SOD1, TARDBP, FUS, and C9orf72) were classified as the fALS group, and those with other or unspecified gene variations as the sALS group, yielding five independent fALS as well as seven independent sALS case-control comparisons  were pooled into a single meta-analysis, yielding a total of six meta-analyses. An illustration of the target identification process is available in de novo molecular design  accelerated the process of target discovery using transcriptomic data and r design . PandaOmL scores . In addiL scores .(ELFC) referred to the log-transformed fold change of enrichment showing how much the top of the list was enriched by known targets, and was calculated by formula (I):The “time machine” approach was applied for the validation of the ability of a model to identify the truly novel targets of the disease of interest. The data before a given year was used as training data and the trained model was then evaluated based on the targets entering the clinical trial after the given year . Two valk was the number of known targets for this disease in top-k (or 0.1 if there were none), and targetsN was the total number of known targets for this disease among the genes that were available for a particular PandaOmics score. And hypergeometric p-value (HGPV) stood for the statistical significance of the effect and showed how likely the same level of enrichment could be achieved from the random distribution and was calculated by formula (II):where targetsELFC and HGPV corresponded to the higher predictive power of the target-disease association  that prioritized the targets based on literature evidence in high-quality journals. To make sure the identified targets were actionable, the Druggable Class filter was switched on. Simultaneously, we customized the Druggability filters to screen targets already associated with small molecules and not considered as essential genes in the Online GEne Essentiality database . A list Similarly, novel ALS targets were identified without prior knowledge by restricting the Druggability filter to a higher novelty level, selecting only the Omics scores, and disabling the Text-based, Financial, and KOL scores . After rThe schematic representation of pathway analyses conducted in the present study is shown in N stood for the total number of pathways defined in Reactome database, K represented the number of dysregulated pathways in the interested biological process, n was the total number of dysregulated pathways in a comparison group, and r represented the number of pathways belonging to the interested biological process. The p-values were adjusted by Bonferroni correction for multiple testing.where 4C2 repeats were generated by injecting pUAST plasmids with 30 G4C2 repeats in a w1118 strain. Details were discussed in 4C2)30/CyO, twi-GAL4, UAS-GFPGMR-GAL4, UAS- ameliorated eye degeneration  worsened motor neuron degeneration in SOD1G93A mice  and sALS (86%) comparisons. NR3C1 encodes a glucocorticoid receptor with a dual role as a modulator of transcription factors and a transcription factor itself. Over 60 drugs are associated with NR3C1, including both agonists and antagonists. NR3C1 agonist and its ligand, glucocorticoids, were widely adopted as the standard treatment for inflammatory diseases (ERR (fly ortholog of NR3C1) strongly abolished eye degeneration  moderately rescued eye degeneration . The suppression of Ire1 in flies impeded eye degeneration , which serves as a critical stress response that copes with endoplasmic reticulum (ER) stress and maintains cell viability. IRE1 signaling was considered to be pathogenic in ALS  family, were upregulated in CNS sALS (83%) comparisons and diMN transcriptomic sALS samples , respectively. In agreement with our results, the inhibition of Shab (fly ortholog of KCNB2 and KCNS3) substantially hampered eye degeneration in the fly model  of CNS sALS comparisons. It encodes a purinergic receptor responding to UDP-glucose and other UDP-sugars coupled to G-proteins, and provides a novel candidate for drug development. The role of P2RY14 in neurodegeneration remains unclear, as both neuroprotective and neurotoxic roles were reported. While it may execute its neuroprotective function by inhibiting the expression of MMP9 in AD  by RNAis reduced eye degeneration in the c9ALS Drosophila model , was selected as a potential target for ALS. Reduction of PPP3CB protein level was detected in diMN fALS samples . Cn stability depended on its interaction with SOD1  exerted suppressive effects  hexanucleotide repeat expansion (HRE) in C9orf72, the most common driver of ALS (4C2 [(G4C2)30] using the UAS/GAL4 system, under the control of GMR-GAL4 30-mediated eye degeneration. To validate our findings, we compared the candidates identified by the PandaOmics analysis and our fly screening results. The severity of eye degeneration was assessed using the scoring scale shown in SLC25A10 and METTL21A. Suppression of 18 of these 26 targets using RNAi strongly or moderately rescued eye degeneration (4C2)30-mediated neurotoxicity. On the other hand, depletion of S6k (fly ortholog of RPS6KB1) moderately enhanced eye degeneration. Representative images of fly eyes whose degenerations were strongly rescued by RNAi are shown in To validate whether the targets we have identified were relevant to the disease, we used the c9ALS l of ALS . This mor of ALS . PreviouGMR-GAL4 , inducedrphology . Using trphology , which ineration , suggestp = 3.26E-7), signal transduction process , and hemostasis . The diMN transcriptomic groups were enriched for dysregulated pathways belonging to the protein metabolism process . The dysregulated pathways in the diMN proteomic groups were overrepresented in the processes of disease , DNA repair , and developmental biology . The details of dysregulated pathways in different biological processes are shown in The Reactome database provides the hierarchical organization of signaling pathways grouped into broader domains of biological functions . TherefoFurthermore, pathways with similar gene contents were connected to form clusters. As shown in UBC, UBB and UBA52), the proteasome genes , and the ER-associated degradation genes .The dysregulated pathways in the diMN ALS groups belonged to different biological processes when compared to the CNS groups. For the diMN transcriptomic comparisons, pathways of the cap-dependent translation, and diseases of the neuronal system were found to be activated in ALS case groups . For theAfter decades of research, the genetic and environmental factors contributing to the etiology of ALS remain inadequately defined. Integrative multi-omics approaches have been employed to dissect the disease pathophysiology . PandaOm2, publications, geroprotectors3 and GenAge database, indicating the association between aging and ALS.With the advance of medical care and improved lifestyles, human life expectancy has been significantly lengthened, which in turn poses significant health-associated challenges, due to the shift in demographic structure toward the aged. Despite multiple risk factors being proposed to contribute to ALS, aging remains one of the most prevalent risk factors and driving forces for developing the disease . IdentifDrosophila model, of which eighteen demonstrated that their depletion has rescued neurodegeneration, while the loss of RPS6KB1 resulted in an opposite effect. This validation confirmed the power of PandaOmics in identifying therapeutic targets with potential roles in ALS neurodegeneration. Although some of the proposed targets were not directly associated with neurodegeneration, all of them have been reported to participate in pathways that may contribute to ALS development. Some well-known ALS-associated genes, such as TARDBP, C9orf72 and FUS, were not included in our target list, as they did not belong to any druggable classes and thus were filtered out. SOD1, ranked among top 10 in the high-confidence genes of diMN proteomic meta-analyses, was also not proposed in the present study, as its role in ALS pathology is well-established. Overall, we demonstrated our AI-enabled target discovery approach in accelerating the novel ALS target discovery process for new therapeutic regimen development.As summarized in We showed that several enriched pathway clusters were closely linked with ALS-driven mechanisms. For example, RNA metabolism was commonly dysregulated in our analysis regardless of tissue type. Altered RNA metabolism was indicated as a key concern in ALS . SeveralC9orf72 mutations that represent the dominant genotype in the fALS comparisons  whose suppression strongly rescued eye neurodegeneration. Future studies are warranted to further define their pathogenic role and potential as therapeutic targets for c9- and other types of ALS using diMNs or mammalian models. To accelerate novel target discovery and drug investigation for ALS, targets identified in this study will be disclosed on ALS.AI. Altogether, the present study offers new insights on how AI speeds up the target discovery process from years to months.In conclusion, this study demonstrates the application of PandaOmics target discovery system to identify and prioritize high-confidence and novel targets for ALS with our latest AI models based on comprehensive omics data analysis. Several well-characterized mechanisms in ALS pathology were found to be dysregulated, including the immune system, RNA metabolism, excitotoxicity, as well as programmed cell death. Seventeen high-confidence and eleven novel therapeutic targets were identified from CNS and diMN samples. CNS data mainly reflects the late-stage signatures of ALS, while results from diMN comparisons are more likely to be attributed to the early-stage signatures. Combining the usage of diMN and post-mortem CNS samples could provide a comprehensive understanding of ALS disease progression. The employment of the The original contributions presented in this study are included in the article/Ethical review and approval were not required as only public available human data and no animal experiments were involved in the study. ALS data were obtained with written consent from every ALS and control patient.FP performed the data analysis, result interpretation, project administration, and writing the original draft. BLi and XL conducted the data analysis, visualization, result interpretation, and writing the original draft. HL performed the result interpretation and validation. GL conducted the result interpretation and website design. QM performed the fly experiments and result interpretation, and wrote the manuscript. JG performed the fly experiments and result interpretation. AS conducted the data curation and processing. IO performed the data curation, methodology and software. JW, FR, and XG performed result interpretation. AA was responsible for methodology and resources. EB and EI performed result interpretation and reviewed the manuscript. KZ provided conceptualization, funding acquisition, supervision for the fly experiments, and wrote the manuscript. BLu performed result interpretation and reviewed the manuscript. JR and MC conducted the data acquisition, result interpretation and reviewed the manuscript. AZ provided the conceptualization, resources, and supervision. All authors have read and agreed to the published version of the manuscript.FP, BLi, XL, HL, GL, AS, IO, JW, FR, AA, and AZ were employed by Insilico Medicine Hong Kong Ltd. XG was employed by 4B Technologies Limited. The remaining 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. The handling editor AS declared a shared parent affiliation with one the authors, MC at the time of the review.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.