Jasminum grandiflorum flower extract (JGF) using LC-MS/MS and explored the possible molecular mechanisms against acute renal failure through pharmacological network analysis. Furthermore, the possible molecular mechanisms of JGF against acute renal failure were verified in an in vivo nephrotoxicity model caused by cisplatin. LC-MS analysis furnished 26 secondary metabolites. Altogether, there were 112 total hit targets for the identified metabolites, among which 55 were potential consensus targets related to nephrotoxicity based on the network pharmacology approach. Upon narrowing the scope to acute renal failure, using the DisGeNET database, only 30 potential targets were determined. The computational pathway analysis illustrated that JGF might inhibit renal failure through PI3K-Akt, MAPK signaling pathway, and EGFR tyrosine kinase inhibitor resistance. This study was confirmed by in vivo experiment in which kidneys were collected for histopathology and gene expression of mitogen-activated protein kinase 4 (MKK4), MKK7, I-CAM 1, IL-6, and TNF receptor-associated factor 2 (TRAF2). The animal-administered cisplatin exhibited a substantial rise in the expression levels of the MMK4, MKK7, I CAM 1, and TRFA2 genes compared to the control group. To summarize, J. grandiflorum could be a potential source for new reno-protective agents. Further experiments are needed to confirm the obtained activities and determine the therapeutic dose and time.Cisplatin (CP) is a powerful chemotherapeutic agent; however, its therapeutic use is restricted due to its nephrotoxicity. In this work, we profiled the phytoconstituents of  Cisplatin is a powerful chemotherapeutic drug that can be used to cure tumors. However, CP has a number of side effects, including nephrotoxicity, which is linked to a high morbidity and mortality rate ,2. NephrTherefore, antioxidants and free radical scavengers can aid in the prevention of cisplatin-induced nephrotoxicity. Cisplatin causes apoptosis by disrupting DNA . Many siCisplatin is also seen in numerous cell lines as renal epithelial cells, which stimulate these three pathways ,15. InflJasminum grandiflorum Linn. (Family: Oleaceae) is widely utilized for both decorating and therapeutic value. J. grandiflorum flower (JGF) is traditionally used to treat mental illness, wounds, infections, skin injuries such as conjunctivitis and dermatitis, dysmenorrhea, spasms, ulcers, and cancer. In China, JGF is also extensively drunk as a heat-clearing and purifying beverage [J. grandiflorum [Plants and their components are a better choice for treating ailments than any manufactured chemical . The medbeverage ,23. Sevediflorum ,25.Jasminum species and different organs have been investigated, the active metabolites of J. grandiflorum flowers (JGF) growing in Egypt have never been studied [Although other  studied ,27. HereJasminum grandiflorum Linn. flower powder was made from dried fine crushed flowers collected from Keram fields in El-Behaira Governorate, Egypt. Dr. Mohammed El-Gebaly (Consultant in Orman Garden) verified the plant, and a voucher sample (# 3.10.16.6) was preserved at the Department of Pharmacognosy, Cairo University.J. grandiflorum flower extract (JGF).The powdered flower material (1 kg) was extracted with 80% aqueous methanol by percolation (3 × 3 L). The extract was concentrated under reduced pressure to afford a residue (108.8 g) of dry A Thermo Finnigan Surveyor Plus HPLC apparatus connected to a quaternary pump, a Surveyor UV–Vis photodiode array detector, and an LCQ-Duo ion trap mass spectrometer, via an electrospray ionization source, was utilized. The experiment was performed on a Gemini C18 110 Å . A gradient of water and acetonitrile (ACN) (0.1% formic acid each) was applied from 5% to 30% ACN over 90 min, with a flow rate of 1 mL/min and a 1:1 split before the ESI sourceTM 2.0.7 software. The negative ion mode operating parameters of the MS were employed [The injection volume was 5 μL with 1 mL/min flow rate. For the negative ionization mode, the following were the ESI conditions: 350 °C, 35 psi nebulizer pressure, 10 L/min N2 drying gas flow rate, mass range 50–2000 m/z. The system was controlled with Xcaliburemployed .http://pubchem.ncbi.nlm.nih.gov/ accessed on 13 March 2022) was utilized to obtain the chemical data of identified compounds . The 3D molecular structure files were uploaded to the Binding DB database (https://www.bindingdb.org/bind/index.jsp accessed on 20 March 2022), an online tool for identifying drug targets. We obtained anticipated drug targets for each metabolite using this web tool. We identified the target genes with a normalized fit score of >0.7 as prospective JGF targets after combining the duplicate data [All the compounds of JGF by LC/MS/MS analysis were considered as candidate compounds. As an online chemical database, PubChem Database (ate data .https://www.ncbi.nlm.nih.gov/gene/ accessed on 22 March 2022) [https://www.uniprot.org/help/uniprotkb accessed on 25 March 2022) [Using the NCBI Gene database (ch 2022) , and thech 2022) , the gench 2022) . The keyhttps://string-db.org/cgi/network?taskId=bIDN4htc9NBY&sessionId=bZWvNlZHMn9h accessed on 27 March 2022) [The PPI data were acquired from the STRING database (ch 2022) , as a rech 2022) .p < 0.05. Nodes represent targets, compounds, and renal diseases in the graphical network, while edges represent corresponding interactions.The Cytoscape network analysis program version 3.9.0  was used to construct compound–target, protein–protein interaction (PPI), and compound–target–disease networks . The difhttps://www.genome.jp/kegg accessed on 4 April 2022) [http://bioinformatics.sdstate.edu/go/ accessed on 7 April 2022) [The represented pathways related to JGF and acute renal failure were retrieved by the KEGG (Kyoto Encyclopedia of Genes) enrichment analyses (il 2022)  and Geneil 2022)  to invesThe experiment was performed under the guidelines of the Research Ethical Committee . Male Sprague Dawley (230–260 g) rats were housed under standard conditions of temperature (22 ± 1 °C) and humidity (50–55%), and a 12-h light/dark cycle. There was plenty of food and drink.The rats were divided into four groups (15 each) at random: the first group was given saline (2.5 mL/kg) intraperitoneally (IP) ; for the second group, a single dosage of 5 mg/kg CP (Pfizer Company) (1 mg/mL sterile concentrate) was given IP (CP group). For 14 days, the third group was given 40 mg/kg JGF dissolved in water orally (JGF group). The fourth group was given 40 mg/kg JGF dissolved in water orally for two weeks, with a single CP  dosage on the tenth day. All rats were weighed and anesthetized before decapitation 24 h after the last treatment. Sera were separated after blood samples were collected. The kidneys were removed right away in an ice-cold saline solution. For histopathological and gene expression research, portions of kidneys were sliced into small pieces.Blood urea nitrogen (BUN) was monitored spectrophotometrically following Tabacco et al. . In summThe concentration of MDA was determined in tissues using the method previously established by Satoh  and OhkaGSH concentration was measured using Beutler’s method . The prohttps://frodo.wi.mit.edu/; accessed on 29 December 2021 −ΔΔCt) was used for the calculation of fold induction values [TRIzol reagent  was used to purify the RNA. Thermo Scientific Maxima First Strand DNA Synthesis Kit with dsDNase was then used to make complementary DNA . The control gene was the B actin gene. Primer 3 PLUS software was used to create gene primer sets , were used to stain sections cut using a rotary microtome at 5–7 μm thickness.p < 0.05.All the tests were done in triplicate, and the data were reported using SPSS software version 26 as means and standard deviation (SD) . ANOVA was used to compare the parameters obtained from the various tested groups. The statistical significance of the received data was assessed using A total of 26 secondary metabolites were tentatively identified in JGF using LC-MS/MS. The main compounds are divided into several subclasses, including secoiridoids, flavonoids, and phenolic and organic acids. Comprehensive profiling is presented in A total of 112 targets were found among the 26 identified chemicals in the NCBI Gene and UniProtKB/Swiss-Prot databases. A total of 55 potential renal disease targets were identified from the DisGeNet database, among which only 30 targets are related to acute renal failure. Taking the intersection of the 112 potential targets and the compounds in JGF into account, we constructed a compound–target network using Cytoscape , which iThe 112 possible therapeutic target genes were loaded into the STRING database, which supplied predicted interaction data, and then imported into Cytoscape 3.9.0 for analysis and network construction . In the The DisGeNET online database was used to interpret the associated diseases with the identified 112 target hits of the JGF-identified compounds. Upon filtration of the results to narrow the scope to urogenital disorders, a total of 47 genes were obtained . To direThe combination and merging of the JGF–compounds, compounds–targets, and targets–non-cancer renal diseases networks formed the complete pharmacology network that correlates the identified compounds from JGF, as described in To investigate the probable pathways of JGF on kidney disorders, the pathway enrichment of 112 probable targets was targeted by 26 identified compounds interacting with renal diseases using the KEGG and ShinyGO databases. The pathways analysis conducted with the KEGG diagram illustrated the top 24 pathways annotated by the target genes. Upon focusing on the pathways involved in acute renal failure disorders, three signaling pathways were identified: PI3K-Akt, MAPK, and EGFR tyrosine kinase inhibitor resistance. Each pathway diagram is illustrated to elaborate the specific genes among our targets involved directly in the nominated pathways as described by the KEGG labeled diagrams .Using GO terms, the gene ontology results are illustrated as networks , represep < 0.05) at the end of the experiment. Interestingly, JGF treatment alone increased the body weight compared to the control and cisplatin groups. In addition, treatment of JGF in combination with CP attenuated the deleterious effects of CP and increased the declined body weight . However, JGF combined with CP completely reversed the elevated levels, which returned to normal levels in the control group  compared to the control group. Pretreatments with JGF inverted the elevated levels, leading to a complete setback to normal values  were also represented by aglycones and glycosides.Flavonoids are essential in a wide range of nutraceutical, pharmacological, medical, and cosmetic uses because they are linked to a wide range of health-promoting effects . This isCisplatin-induced inflammatory cytokines primarily rely on forming reactive oxygen species (ROS), NFκB activation, and p38 MAPK activation. TNF-α and IL-1 substantially activate the stress-activated group of MAPKs (JNK and p38) . This waThe present study suggests that cisplatin induced TRAF2 overexpression is the cause of nephrotoxicity and apoptosis. The reduction in TRAF2 expression in kidney tissues after JGF administration in CP-treated rats implies that JGF may safeguard against nephrotoxicity caused by CP through regulating apoptotic pathways. The TRAF2 adapter protein is recruited when TNF receptors are activated ,63. TRAFAccording to a previous study, JNK genes have an essential role in modifying the pro- and anti-apoptotic proteins in the mitochondria in the nephrotoxicity caused by chemotherapy . By blocThe MKK7 is mainly triggered by cytokines, whereas MKK4 is activated primarily by environmental stress . In the JGF showed a significant nephroprotective effect in rats, and its nephroprotection mechanism could be attributable to the presence of a variety of phytochemicals, including secoiridoids, flavonoids, and phenolic and organic acids with antioxidant properties and free-radical-scavenging properties. Therefore, this plant could be useful in treating renal failure caused by nephrotoxic drugs.J. grandiflorum were detected using HPLC-PDA-MS/MS, including quercetin, myricetin, kaempferol, isorhamnetin flavonoids, and other secoiridoid derivatives. Our findings could contribute to the understanding of the molecular mechanisms of JGF in CP nephrotoxicity, which were confirmed by in vivo research that supported the molecular processes predicted by the network pharmacology method. Compared to the control group, a single dose of CP to rats caused nephrotoxicity, which was related to a considerable rise in BUN and serum creatinine and a significant increase in MDA in renal tissues. The expression levels of the MMK4, MKK7, I CAM 1, and TRFA2 genes were considerably higher in the cisplatin-treated animals than in the control. Furthermore, the histological investigation revealed that cisplatin induced significant tubular cell lining vacuolation and intratubular cast deposition in the renal tubules. JFG treatment reduced cisplatin-induced gene expression changes and kidney structural and functional abnormalities. Gene expression data were also corroborated by histological investigation of kidney tissues. Our study suggests that the anti-inflammatory effects of JGF can protect from CP-induced nephrotoxicity through lowering oxidative stress and repairing the histopathological changes against cisplatin use. However, further studies should be carried out in the future to reveal the clinical effectiveness of J. grandiflorum as a renal protective drug in preclinical and clinical trials.The metabolites in


Diabetic retinopathy (DR), one of the most common and severe microvascular complication of diabetes mellitus (DM), is mainly caused by diabetic metabolic disorder. So far, there is no effective treatment for DR. Eriocauli Flos, a traditional Chinese herb, has been used in treating the ophthalmic diseases including DR. However, the active ingredients and molecular mechanisms of Eriocauli Flos to treat diabetic retinopathy remain elusive.Here, the systems pharmacology model was developed via constructing network approach. 8 active components which were screened by oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18) and 154 targets were selected from Eriocauli Flos through TCMSP database. Another 3593 targets related to DR were obtained from Genecards, OMIM, TTD, and Drugbank databases. The 103 intersecting targets of DR and Eriocauli Flos were obtained by Draw Venn Diagram. In addition, protein-protein interaction network was established from STRING database and the compound-target network was constructed by Cytoscape which screened top 12 core targets with cytoNCA module. Then the overlapping targets were analyzed by GO and KEGG enrichment. Moreover, two core targets were selected to perform molecular docking simulation. Subsequently, CCK8 assay, RT-PCR and Western blotting were applied to further reveal the mechanism of new candidate active component from Eriocauli Flos in high glucose-induced HRECs.The results showed that the overlapping targets by GO analysis were enriched in cellular response to chemical stress, response to oxidative stress, response to reactive oxygen species, reactive oxygen species metabolic process and so on. Besides, the overlapping targets principally regulated pathways such as AGE-RAGE signaling pathway in diabetic complications, lipid atherosclerosis, fluid shear stress and atherosclerosis, and PI3K-Akt signaling pathway. Molecular docking exhibited that VEGFA and TNF-α, had good bindings to the great majority of compounds, especially the compound hispidulin. In vitro, hispidulin ameliorated high-glucose induced proliferation by down-regulating the expression of p-ERK, p-Akt, and VEGFA; meanwhile inhibited the mRNA levels of TNF-α.In this study, through network pharmacology analysis and experimental validation, we found that hispidulin maybe has a potential targeted therapy effect for DR by decreasing the expression of p-Akt, p-ERK, and VEGFA, which resulted in ameliorating the proliferation in HRECs.The online version contains supplementary material available at 10.1186/s12906-022-03593-2. Eriocauli Flos, a Chinese herbal medicine, is traditionally used in ophthalmic diseases with the function of coursing wind, dissipating heat and improving eyesight in the theory of traditional Chinese medicine [Eriocauli Flos mainly belong to flavono, lavones, isoflavones, xanthones, naphthopyranones and phenolic acids, with pharmacology effects including anti-inflammatory and anti-bacterial [Eriocauli Flos, has been reported had the anti-inflammatory, anti-cancer, anti-oxidative stress effects [Diabetic retinopathy (DR) is a common complication of diabetes with microvascular lesions that is featured by vascular endothelial injury including pericyte loss, endothelial cell proliferation, basement membrane thickening, retinal ischemia and hypoxia, and angiogenesis , 2. The medicine . The ingacterial , 11. His effects . For exa effects  and anot effects . HoweverEriocauli Flos against DR. Then, hispidulin was screened as the most effective compound from Eriocauli Flos by molecular docking. Furthermore, the effects of hispidulin against DR was further investigated by quantitative RT-PCR and Western blotting to test the mRNA and protein expression levels of p-Akt, p-ERK, and VEGFA. This study provided the theoretical foundation and laid the theoretical foundation for the subsequent experiment validation.Network pharmacology, combining system biology with computer technology, systematically observes the influence of drugs on diseases through network data analysis on the basis of “drug target of disease gene” interaction, reveals the molecular mechanism of drug action on diseases, as well as provides new ideas and directions for new drug research and development , 16. MolHispidulin (1447-88-7) was purchased from Victory Biological Technology, China and D-glucose (G8270) was from Sigma, USA.Eriocauli Flos were gathered by using Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) (http://sm.nwsuaf.edu.cn/lsp/tcmsp.phpphp), [https://www.uniprot.org/ ) [All the compounds of phpphp),  and the phpphp), . The tart.org/ )  to be fuhttps://www.genecards.org/), [https://omim.org/), [http://bidd.nus.edu.sg/group/ttd/ttd.asp) [Eriocauli Flos and DR were displayed by Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) [The targets associated with DR were predicted via GeneCards (s.org/),  Online Mm.org/),  Therapeuttd.asp)  and Drugttd.asp) . All tars/Venn/) .Eriocauli Flos and DR were constructed into Cytoscape 3.8.0 to construct and analyze the network [To better acknowledge the interaction relationship between compounds and targets, the 8 active compounds and 103 intersection targets of  network . Each nohttps://string-db.org/) [Eriocauli Flos and DR. To build the interaction network, minimum required interaction score > 0.9 was set and disconnected nodes in the network were hidden and 89 targets were obtained. Each node represents a protein and its structure, and each edge represents the associations between the different protein. And the PPI results were output as a TSV file which was then imported to Cytoscape 3.8.0 for further network analysis by using the cytoNCA plug-in, [Using STRING11.0 database (db.org/)  to prediplug-in,  and eachplug-in, . Hence, http://geneontology.org/), [www.kegg.jp/kegg/kegg1.html), [GO database (y.org/),  includin1.html),  a knowle1.html), .https://pubchem.ncbi.nlm.nih.gov/) [. The 3D structures of proteins (targets) were obtained from RCSB PDB database (http://www.rcsb.org/pdb/) [The 2D structures of ligands (compounds) were downloaded from Pubchem database (ih.gov/)  and TCMSrg/pdb/)  and wererg/pdb/) . The 3D rg/pdb/) .Human retinal endothelial cells (HRECs) were obtained from BNCC and the cells previously were grown in Dulbecco’ Modified Eagle’s Medium  supplemented with 10% fetal calf serum  and 1% (v/v) penicillin–streptomycin  at 37 °C in 5% CO2 incubator. For the hispidulin treatment study, HRECs were exposed to normal glucose  and high glucose  with or without hispidulin for 48 h according to previous research about high glucose model establishment in HRECs. All the cells in the experiment were used from 3 to 6 passages.3 cells per well were seeded into 96- well plate and cells were exposed to normal glucose (5 mM) with or without hispidulin (5-80 μM) for 24 h to assess cytotoxicity. Then 5 × 103 cells per well were seeded into 96- well plate and cells were treated with glucose (5 and 30 mM) with or without different concentrations of hispidulin (5-40 μM) for 48 h after the cells adhered for 24 h, Cell viability was measured following the manufacturer’s instructions.The viability of HRECs was detected by CCK-8 kit . Briefly, 8 × 105 HRECs were seeded into six-well plates. After treatment with Hispidulin for 48 h, total RNA was extracted using Trizol reagent  and was reverse-transcribed into cDNA with ReverTra Ace qPCR RT Master Mix . Finally, 2 μl cDNA was used for fluorescence quantitative PCR analysis by using SYBR Green Realtime PCR Master Mix . The relative CT method (△△CT) was used to calculate the relative transcriptome level, and the normalized target protein number was compared with the endogenous reference gene GAPDH. The specific primers used were as follows: human TNF-α , human VEGFA , humanGAPDH .For the quantitative real-time PCR of VEGFA and TNF-a, 2.0× 10The cells were lysed with RIPA lysate  containing a protease inhibitor, phosphatase inhibitor and PMSF. Then, lysates were centrifuged at 14,000 rpm, for 10 min at 4 C, and the supernatants were extracted, and resuspended in the 5× loading buffer  to prepare the protein samples. The electrophoresis was performed on SDS-PAGE and then the proteins were transferred to PDVF membranes . Following blocked of the membranes with 5% skim milk for 1 h and the membranes were then incubated overnight at 4 °C with primary antibodies as follows: VEGFA , ERK , p-ERK , Akt  and p-Akt . Then membranes were incubated by the addition of a secondary antibody for 1 h at room temperature after another three times wash with TBST. Finally, the protein expression was detected by using ECL chemiluminescence substrate .P < 0.05 was considered as statistically significant.All data were shown as mean ± SD and analyzed using one-way ANOVA and followed by Dunnett’ s multiple comparison test. Eriocauli Flos from TCMSP database, containing 154 targets, reached the criteria of OB ≥ 30% and DL ≥ 0.18. The goals  were narrowed down by betweenness, closeness and degree parameter of cytoNCA module , 49 molecular functions (MFs) and 153 cellular components (CCs). The top 10 enrichment results of BP, MFs and CCs were listed in the Fig. We selected VEGFA and TNF-α, from the core targets, which was closely related to DR, to dock with the 8 compounds. Generally, the value of vina scores exhibits the binding activity between a protein and a compound and the lower value represents greater affinity. The results indicated that hispidulin, among the 8 compounds, had the highest affinity with VEGFA and the second highest affinity with TNF-α, and the affinity of VEGFA to each component was higher than that of the original ligand (HT7) while the affinity of TNF-α to the original ligand (VGY) was opposite Table . In addiD-glucose) for 24 h. As shown in the Fig. To evaluate the effect of hispidulin cytotoxicity on HRECs, we measured the cell viability of HRECs under normal glucose condition (5 mmol/L To further examine the anti-inflammatory and anti-angiogenic effect of hispidulin on the regulation of VEGFA and TNF-α expression under high glucose conditions, we compared the transcription levels of VEGFA and TNF-α genes. The results demonstrated that the mRNA expression levels of VEGFA and TNF-α were increased after high glucose-stimulated HRECs culture for 48 h. Oppositely, the treatment of hispidulin significantly reduced the expression levels of VEGFA and TNF-α in a dose-dependent manner compared with high glucose group (Fig. Eriocauli Flos and DR. Furthermore, phosphorylation of Akt and ERK1/2 which play a vital role in the cell proliferation. Consequently, we found that treated with hispidulin showed down-regulating the expression of VEGFA only in the concentration of 20 μM, but decreasing phosphorylation of Akt and ERK1/2 in a dose-dependent manner (Fig. Through the network pharmacology analysis, the genes of VEGFA was predicted as the most crucial targets of ner Fig. .Fig. 9EfEriocauli Flos in diabetic retinopathy. Through the PPI network and cytoNCA module analysis, two core genes VEGFA and TNF-α, were identified as major hub genes targets for DR, which has been paid to the key regulators in DR, [Eriocauli Flos may modulate DR by regulating multiple pathways such as the AGE-RAGE and the PI3K-Akt signaling pathway in diabetic complications. Previous reports have indicated that the interaction between AGE and its receptor RAGE leaded to loss of retinal pericytes, and triggered inflammatory response and blood vessel formation [In this study, we employed a systematic network pharmacology method to explore the molecular mechanism of s in DR,  and alsos in DR, . In addis in DR, , 41. Accormation . In addiormation . Moreoveormation  but in tormation  which prEriocauli Flos played a significant role in DR through multi-targets, multi-pathways approach. And hispidulin, a vital compound from Eriocauli Flos, exerts an anti-angiogenesis effect by down-regulating the expression of `p-AKT, p-ERK, and VEGF in vitro, therefore suggesting that hispidulin have potential as anti-VEGF agent in the therapy of DR. However, in our current study, it’s just simple verification of relevant targets and pathways in network pharmacology from the perspective of cell proliferation and the expression of VEGFA, and further in vivo studies are necessary to confirm the anti-angiogenic effect of hispidulin and to explore the underlying mechanisms of DR such as the effects on oxidative stress in terms of GO enrichment analysis.Through network pharmacology analysis and by mining the data step by step, it was much clearer that Additional file 1.Additional file 2.


Hedyotis diffusa Willd (HDW) is one of the most well-known herbs used in the therapy of cancer. However, the potential mechanisms of its antiangiogenic effects have not been fully explored. Here, we applied a network pharmacology approach to explore the potential mechanisms of HDW against liver cancer angiogenesis (LCA) and used a mouse orthotopic liver cancer model for experimental verification accordingly.  The effective components, primary active compounds, and possible targets in the therapy of LCA were predicted using network pharmacology and bioinformatics. In vivo testing of the pharmacodynamic foundation of HDW in the treatment of LCA was performed. Hepa1-6 cells were implanted in C57BL/6 mice to establish an orthotopic liver cancer model to evaluate the antitumor and antiangiogenesis effects of the drug. Furthermore, protein levels were evaluated by western blotting, immunofluorescence, and immunohistochemistry. β, IL-17, hypoxia inducible factor-1α (HIF-1α), and tumor necrosis factor-α (TNF-α). Importantly, we preliminarily verified the results of the network pharmacology analysis in vivo.  We firstly confirmed the therapeutic effect of HDW on LCA and subsequently screened 7 active compounds from HDW according to their pharmacokinetic properties. Network analysis and enrichment analysis indicated that these compounds exhibit antiangiogenic effect by acting on multiple targets and thereby regulating multiple pathways mainly involved in Akt1, IL-6, IL-1 Collectively, our work initially explored the therapeutic mechanism of HDW on tumor angiogenesis, which lays an experimental reference for further exploring its pharmacological action and its clinical application. Angiogenesis is an essential factor in tumor progression. Tumor cells can get the nutrients and oxygen they need for sustained angiogenesis. Angiogenesis plays a vital role in promoting tumour growth and metastasis . As one Hedyotis diffusa Willd (HDW) has been recorded for thousands of years in clinical applications. It is widely used for heat clearing, detoxification, and removal of blood stasis [Increasing evidence has shown that Chinese medicinal active compounds offer unique advantages due to the synergy effect of multicompounds, multitargets, and minor adverse reactions in the antitumor aspect . As a trd stasis . Moreoved stasis –12. Exisd stasis . HoweverAs an emerging subject, network pharmacology can comprehensively and systematically clarify the mechanism and targets of Chinese medicinal active compounds, which are fully compatible with the characteristics of traditional Chinese medicine (TCM) in terms of multicomponent, multitarget, and multipathway properties , 14. In Hedyotis diffusa Willd injection was purchased from Keyuan Pharmaceutical Co., Ltd. . The following primary antibodies were used in this study: Akt1, p-Akt1, mTOR, p-mTOR, STAT3, and p-STAT3 ; CD31, HIF-1α, and GAPDH ; and α-SMA . The fluorescein isothiocyanate (FITC)-conjugated secondary antibodies and all horseradish peroxidase-conjugated secondary antibodies were obtained from Proteintech . The mouse ELISA kit was purchased from PeproTech Inc. . Hepa1-6 tumor cells were obtained from iCell Bioscience Inc.  and cultured according to the manufacturer's instructions.https://www.uniprot.org/) to calibrate the gene names of all targets, using humans as the chosen species [All chemical compounds of HDW were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database . After t species .https://www.genecards.org/) and (2) the NCBI gene database (https://www.ncbi.nlm.nih.gov/). Our investigation searched these human illness target databases for targets associated with LCA using the keywords “liver cancer angiogenesis,” “hepatocellular carcinoma angiogenesis,” and the species “homo sapiens.” After merging and deleting the genes in the two databases, the target genes associated with LCA were obtained; common targets associated with LCA and potential targets of bioactive substances were chosen as HDW's targets against LCA. A Venn diagram of the intersection between the target of the drug and the disease was established using Venny 2.1.The LCA targets were obtained from two different sources , 18: (1)https://string-db.org/cgi/input.pl) was used to collect possible protein-protein interactions (PPI) by uploading 116 common targets related to LCA and putative targets of active compounds. Species were limited to “homo sapiens” with a confidence score >0.4. Then, we imported the database from STRING into Cytoscape (version 3.7.1) to construct a PPI network for further analysis [The Search Tool for the Retrieval of Interacting Genes  Enrichment Analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analysis. (1) GO enrichment analysis was performed by using the ClusterProfiler package of R4.0.3 and mainly included biological process (BP), molecular function (MF), and cellular component (CC). GO terms with the STRING database-corrected  network . (2) KEG network .6 Hepa1-6 tumor cells were suspended in 50 μL Dulbecco's Modified Eagle Medium (DMEM) (containing 20% Matrigel) and orthotopically injected into the liver of C57BL/6 mice under anesthesia with tribromoethanol  [C57BL/6 mice  were purchased from Beijing SiPeiFu Biotechnology Co., Ltd. and fed in a pathogen-free vivarium under standard conditions. All animal experimental protocols and principles were approved by the Animal Care and Use Committee of Jiangsu Vocational College of Medicine. To establish an orthotopic liver cancer model, 1 × 10ts, USA) . After 7α-SMA (1 : 500). Following washing several times, the samples were incubated for 1 h at room temperature with secondary antibodies (diluted 1 : 200) conjugated with FITC and 4,6-diamidino-2-phenylindole (DAPI) for counterstaining nuclei. The coverslips were mounted on glass slides, and the immunofluorescence staining was visualized and photographed using a Zeiss inverted fluorescence microscope.Immunofluorescence (IF) staining has been previously described . Mouse lα (1 : 200) at 4°C, biotin-conjugated secondary antibodies, and horseradish peroxidase-conjugated streptavidin successively. Then, the sections were stained with diaminobenzidine and counterstained with hematoxylin. The staining density was assessed as in our previous study [An immunohistochemical assay was conducted according to our previous study with slight modifications . In brieus study . The detThe inflammatory factor levels in serum were detected with enzyme-linked immunosorbent assay kits  according to the manufacturer's instructions.α (1 : 1000)) and secondary antibodies (1 : 5000) were added for electrochemiluminescence (ECL) coloration, and the image was semiquantitatively analyzed with alpha SP image analysis software. GAPDH was used as an invariant control for equal loading of total proteins. The proposed blots are representative of three independent experiments.The western blotting assay was done as previously described in our study . In briet-test or one-way ANOVA, as appropriate. Values of p  <  0.05 were considered statistically significant.Data were presented as means ± standard deviation (SD). The statistical significance of the difference was evaluated by the unpaired Student's To investigate the treatment effect of HDW on LCA, we established a mouse orthotopic liver cancer model and subsequently evaluated the attenuation effect of HDW on tumor growth and angiogenesis. The in vivo experimental schema is depicted in As shown in TCMSP yielded a total of 7 active compounds of HDW after ADME screening with OB ≥ 30% and DL ≥ 0.18. All 7 compounds were validated as promising bioactive molecules for future research. Detailed information is presented in 174 putative targets linked to 7 compounds of HDW were collected. A total of 2116 genes related to LCA were obtained. Through Venny analysis, 116 common targets of HDW in the treatment of LCA were obtained .β, HIF-1α, EGFR, JUN, and CASP3  and species limited to “homo sapiens.” After that, the PPI network was established, as shown in nd CASP3 .https://david.ncifcrf.gov/home.jsp). GO analysis includes biological processes (BP), cellular components (CC), and molecular functions (MF), which together describe the functions of gene products. The top 10 significantly enriched GO targets are presented  in α signalling pathway, IL-17 signalling pathway, and so on.To further explore the underlying mechanisms of HDW against LCA, we performed GO enrichment and KEGG pathway analysis, executed using the DAVID database  expression at the gene level [Therefore, Akt1 is a serine/threonine-specific protein kinase, a member of the protein kinase B family, and a crucial protein in the PI3K/Akt signalling pathway . As an ene level  or indirne level .α and IL-17. TNF-α is a cytokine produced by macrophages or activated monocytes and plays a significant role in inflammatory response and tumor angiogenesis [According to the results of GO and KEGG enrichment analysis, the targets of HDW's antiangiogenic effect in liver cancer are primarily involved in signalling pathways such as TNF-ogenesis . IL-17 iogenesis , 34. It ogenesis .α, and IL-1β in the orthotopic liver cancer mouse model, and these results were dose-dependent to some extent. It suggests that HDW may attenuate tumor angiogenesis by lowering the levels of inflammatory factors. However, the exact mechanism needs further study.This research adopts the in vivo experiment to further validate the pharmacodynamic effects of HDW on anti-liver cancer and angiogenesis. The result shows that the injection of HDW could significantly reduce the liver lesion area and the serum levels of IL-6, IL-17, TNF-α, a core molecule that induces tumor angiogenesis [α transcription. Similarly, phosphorylated STAT3 in the nucleus enhances HIF-1α transcription, which in turn promotes the transcription of several proangiogenic genes such as VEGF, ultimately contributing to tumor angiogenesis [α via modulating the level of phosphorylated Akt1 and STAT3 in several key signalling pathways, which ultimately affect tumor angiogenesis.In addition, it was known that the phosphorylation of Akt1 and STAT3 regulates the transcription of HIF-1ogenesis –38. Phosogenesis , 38. By However, the shortcomings of this study inevitably exist. Specifically, the limitations of this study are as follows: the selection of the database is not comprehensive enough, the positive control group is lacking, and the validation of in vivo targets is not deep enough. In the next study, these shortcomings need to be further improved.β, as well as signalling pathways such as IL-17 and TNF-α via key active components such as DMQ, MMA, HHTCA, poriferasterol, stigmasterol, β-sitosterol and quercetin. It reflects the antitumor and antiangiogenesis effects of HDW that are multicomponent, multitarget, and multipathway. The result of the in vivo experiment further confirms the antiangiogenic effect of HDW, and it is supposed that its mechanism may be related to the inhibition of inflammatory factor production, which affects Akt1 and STAT3 phosphorylation, providing a reference for future studies on HDW's antiangiogenic mechanism.In conclusion, HDW may affect key targets such as Akt1, HIF-1, IL6, and IL-1


Renal osteodystrophy (ROD) secondary to chronic kidney disease is closely associated with osteoporosis and fractures. Based on the theory of “kidney governing bones” in traditional Chinese medicine (TCM), treating bone diseases from the perspective of the kidney has become a basic principle of treating ROD. However, there are many kidney-tonifying herbs and their mechanisms of treating ROD are not clear. Therefore, our study intends to use data mining and network pharmacology to study the commonly used kidney-tonifying herbs, as well as their active ingredients and mechanisms of treating ROD.  We established a clinical ROD database by searching PubMed, CNKI, and other databases and screened out a core herbal combination of treating ROD. Furthermore, by using databases such as Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and GeneCards, we obtained active ingredients and targets of the core herbal combination and ROD targets. The STRING website and Cytoscape software were then used to obtain information on key active ingredients and key targets. Finally, we conducted GO and KEGG analyses using the Metascape website and molecular docking using the AutoDock Vina software.  Our study eventually included 58 prescriptions and 116 herbs of treating ROD. Through data mining, we found that yin-yang-huo, du-zhong, and bu-gu-zhi (YDB) constituted a core herbal combination to treat ROD. Network pharmacology showed that YDB mainly acted on targets such as estrogen receptor alpha through active ingredients such as quercetin by mitogen-activated protein kinase and other signaling pathways.  Many ingredients, targets, and pathways are involved in the treatment of YDB for ROD. Specifically, the flavonoids contained in YDB have great potential for ROD treatment. In 2005, Kidney Disease: Improving Global Outcomes (KDIGO) proposed the concept of chronic kidney disease-mineral and bone disorder (CKD-MBD), which was intended to emphasize the systemic mineral and bone metabolic disorders caused by CKD . CKD-MBDROD, which is very common in CKD patients, has already occurred in the early stage of CKD . As the The theory of “kidney governing bones” originated in the Yellow Emperor's Classic of Internal Medicine (“hangdi neijing” in Chinese), which suggests that “treatment from the kidney” is an important idea for treating bone diseases . The ancBy using modern computing technology, data mining can analyze medication patterns from clinical prescription data, so as to find potential relationships between herbs and diseases and between different herbs, which is an important embodiment of TCM informatization .Based on the “multi-ingredient and multitarget” nature of herbs and the multigene pathogenesis of disease, network pharmacology reveals the complex bioinformatics network of herb-ingredient-target-disease through high-throughput screening and can predict the potential mechanisms of herbs –17. Therhttps://pubmed.ncbi.nlm.nih.gov/), Web of Science (https://webofscience.com), CNKI (https://www.cnki.net/), Wanfang data (https://www.wanfangdata.com.cn/index.html), and VIP database (https://www.cqvip.com/). Based on these databases, we conducted a systematic search using two sets of keywords. The first set of keywords included “ROD” OR “renal bone disease” OR “high turnover bone disease” OR “low turnover bone disease” OR “adynamic bone disease” OR “osteomalacia”. The second set of keywords included  AND ” OR “bone biopsy” OR “bone metabolism” OR “bone remodeling” OR “bone pain”). Then, we screened and identified all relevant studies with TCM prescriptions.Our prescriptions were obtained from databases including PubMed , and case report studies were included. Studies with same prescriptions were included only once.Animal or cell experimental studies, purely theoretical studies, studies with unknown prescription or single herb, and studies on the treatment of acupuncture and external use of TCM were excluded.Kidney-tonifying herbs are mostly kidney meridian herbs, and its definition is vague in clinical practice. Therefore, we expanded the search scope and extracted kidney meridian herbs by referring to “Chinese Pharmacopoeia” (2020 Edition) and “Chinese Materia Medica” in the prescriptions of treating ROD; additionally, standardizing their names based on these two books, for example, standardizing “salt du-zhong” as “du-zhong”.The apriori algorithm was used in SPSS Modeler 18.0 software to conduct an association analysis on kidney meridian herbs [https://old.tcmsp-e.com/tcmsp.php) database was used to search active ingredients of the core herbal combination [https://www.uniprot.org/) [First, the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform   to matchhttps://www.megabionet.org/tcmid/) [https://www.swissadme.ch/) [https://www.swisstargetprediction.ch/) [For herbs that were not included in the TCMSP database, their ingredients can be obtained from the Traditional Chinese Medicine Integrated Database  , CNKI, adme.ch/) , and we dme.ch/) . Next, tion.ch/) . At lasthttps://www.genecards.org/) [https://omim.org/) [We searched the GeneCards database (ds.org/)  and the im.org/)  for ROD We obtained the common targets that were included in both herb targets and disease targets by taking the intersection and found the ingredients containing these common targets. Cytoscape 3.7.1 software was then used to construct the network of these active ingredients and targets . Furtherhttps://cn.string-db.org/) [Homo sapiens” and the item of “the max number of interactors to show” to “1st shell no more than 20 interactors and 2nd shell no more than 20 interactors”, and hid disconnected nodes in this network. Then, we saved the results in a TSV file and imported it into the Cytoscape 3.7.1 software to construct a PPI network diagram of the targets of YDB in treating ROD. Information about key targets can be obtained by screening topological parameters.First, we expanded targets by importing common targets into the SRTING 11.5 website (db.org/) , set thehttps://metascape.org/) [https://www.bioinformatics.com.cn/, an online platform for data analysis and visualization.GO and KEGG enrichment analyses were performed on targets obtained in pe.org/) . GO analhttps://pubchem.ncbi.nlm.nih.gov/) to obtain 2D structures of key active ingredients. We then converted them to 3D structures, minimized their energy, and saved them in mol2 formats by using ChemBio 3D Ultra software [https://www.rcsb.org/) was used to download protein structures of the key targets [Molecular docking verification was based on key active ingredients and key targets obtained under sections software . Next, tsoftware  and conv targets . Next, t targets  to remov targets , and docIn 56 studies, 58 prescriptions containing 116 Chinese herbs were included in our study. These herbs have been used for a total 613 times in different prescriptions. Among these 116 herbs, there were 60 kidney meridian herbs , which have been used for a total of 317 times . In addition, among the 60 kidney meridian herbs, there were 12 herbs with frequencies more than 10 times, which were shu-di-huang, yin-yang-huo, bu-gu-zhi, du-zhong, niu-xi, shan-yao, etc. Their frequencies are shown in Two groups of herbal combinations were obtained under By using the TCMSP database, 28 active ingredients and 529 protein targets of du-zhong and 23 active ingredients and 508 protein targets of yin-yang-huo were obtained. After deduplication, 47 active ingredients and 242 protein targets were remained, of which 229 protein targets were matched as gene names using the UniProt website.By using TCMID and referring to CNKI and PubMed databases, a total of 40 active ingredients of bu-gu-zhi were obtained. According to the steps in 2.5.1. and screening out targets with “probability ≥ 0.100”, 24 active ingredients and 266 gene names of protein targets were obtained. Then, ingredients and targets obtained in the previous steps were combined and deduplicated, and 71 active ingredients and 424 protein targets with gene names were remained for the treatment of ROD with YDB.By searching the TCMSP database, we only found information about du-zhong and yin-yang-huo. According to We obtained 201 targets from the OMIM database and 157 targets from the GeneCards database, and a total of 354 targets were remained after deduplication.We obtained 31 common targets that were contained in both 424 herbal targets and 354 disease targets, which were considered to be the potential targets of YDB in treating ROD. According to Degree, betweenness centrality, and closeness centrality are all network topology parameters used to measure the importance and centrality of nodes , 37. Theβ-catenin binding, among which protein domain-specific binding enriched the most genes. BP mainly focused on response to hormone, regulation of kinase activity, regulation of epithelial cell proliferation, cellular response to organic cyclic compounds, and regulation of mitogen-activated protein kinase (MAPK) cascade. There were 13 clustering results of CC, which mainly concentrated in transcription regulator complex, membrane raft, apical part of cell, clathrin-coated endocytic vesicle membrane, cell projection membrane, and nuclear membrane.The 71 targets were imported into the Metascape website for GO and KEGG pathway enrichment analyses, respectively. GO enrichment analysis of YDB in the treatment of ROD showed that there were 74 items in MF, 854 items in BP, and 51 items in CC. β (TGF-β) signaling pathway, ovarian steroidogenesis, and Wnt signaling pathway.The pathways for YDB to treat ROD mainly enriched in 157 pathways, and there were 19 clusters in total after the hierarchical cluster method, ranked by the −lgP value from the largest tothe smallest, as shown in Figure 7(d). It can be seen that the molecular pathways mainly concentrated in the MAPK signaling pathway, 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway, PTH synthesis, secretion, and action, calcium signaling pathway, transforming growth factor-−1), as shown in −1 can be considered to have the binding activity. Binding energy < −5.0 kcal mol−1 indicates a good binding activity, and binding energy < −7.0 kcal mol−1 indicates a strong binding activity [−1, indicating that these ingredients and targets generally had strong binding activities. Among them, the targets with higher binding affinities were concentrated in ESR1, TNF, and ALB and the highest binding affinities were genistein and ALB, with the binding energy of −9.3 kcal mol−1, followed by quercetin and TNF , genistein and ESR1 , and luteolin and ALB . The binding modes are shown in We conducted molecular docking verification of 6 key active ingredients and 6 key targets, and a total of 36 results were obtained, which were expressed by the lowest binding energies  is a key molecule that promotes bone resorption [α, and IL1, which play a crucial role in enhancing RANKL expression and stimulating osteoclast activation [−1, and the docking abilities with TNF were the strongest. Isoneobavachalcone and isobavachin are components of bu-gu-zhi, which can also affect bone remodeling. The study of Lee et al. showed that isobavachin can inhibit RANKL signaling, and isoneobavachalcone can affect the activity of cathepsin K (CTSK), a specific protease secreted by osteoclasts and responsible for the degradation of collagen type I in the bone matrix [Quercetin, genistein, luteolin, and kaempferol all belong to flavonoids, which have been developed into nutraceuticals due to the abilities of anti-inflammatory, improving oxidative stress, and regulating the activities of key cellular enzymes . Flavonosorption . Osteoprsorption . Genistesorption . The stusorption . Luteolitivation , 56–58. e matrix .Besides TNF, IL6, and IL1B, the rest three targets including EGF, ESR1, and ALB also play an important role in regulating bone remodeling. Specifically, EGF can promote the proliferation of osteoblasts and osteoclasts . Howeverβ signaling pathway. These pathways are ways for the therapeutic effects of kidney-tonifying herbs and also suggest the pathogenesis of ROD.As mentioned earlier, ROD is actually the abnormal bone remodeling caused by CKD . The proα, IL6, and IL1B can regulate bone remodeling by activating the MAPK signaling pathway [β, BMP, wingless related MMTV integration site (Wnt), FGF23, and IGF-1 [β/BMP signaling pathway: TGF-β/BMP belongs to the TGF-β superfamily and also plays crucial roles in bone remodeling [β on bone remodeling are complicated. In vivo, the interactions with other growth factors in the bone environment and the environment as such determine the final outcome of TGF-β on bone remodeling [β-catenin dependent [β-catenin signaling pathway [β-catenin is also involved in kidney damage and repair [β-catenin is found [β-catenin pathway is closely related to the pathogenesis of ROD.(1) MAPK signaling pathway: it involves in the biological process of osteoblasts and osteoclasts from development to maturity, and the transmission of multiple molecule signals , 67. Bot pathway , 62, 68. pathway . Moreovend IGF-1 . Therefond IGF-1 . (3) TGFmodeling , 71. Themodeling . The majmodeling . After tmodeling . (4) Wntmodeling . The rolependent . Many he pathway , 76. Besd repair . In manyis found . Therefoβ, Wnt, and other pathways were obtained in our study. In conclusion, our study reveals the “multicomponent, multitarget, and multipathway” effects of YDB in the treatment of ROD through data mining and network pharmacology based on the theory of “kidney governing bones” in TCM. Moreover, the potential roles of flavonoids in the treatment of ROD are worth further study.By data mining, we concluded that YDB is the core herbal combination for ROD treatment. Furthermore, network pharmacology showed that the key targets of therapeutic effects were concentrated on ESR1, IL6, TNF, ALB, EGF, and IL1B, and the key components were concentrated on quercetin, genistein, isoneobavachalcone, isobavachin, luteolin, and kaempferol, which all belong to flavonoids, suggesting their great potential in the treatment of ROD. Therefore, we suggest that more research should be devoted to the effects and mechanisms of flavonoids on bones and kidneys under the circumstance of ROD or CKD-MBD in the future. The essence of ROD is the abnormal bone remodeling caused by CKD, which is regulated by a complicated network involving multiple molecules and pathways. Through KEGG pathway enrichment analysis, MAPK, PTH, TGF-


Callicarpa americana that catalyzes the oxidation of bartsioside to aucubin, predicted by our model despite neither molecule having been observed in the genus. We also demonstrate aucubin synthase activity in orthologues of Vitex agnus-castus, and the outgroup Paulownia tomentosa, further strengthening the hypothesis, enabled by our model, that the reaction was present in the ancestral biosynthetic pathway. This is the first systematic hypothesis on the epi-iridoid glucosides biosynthesis in 25 years and sets the stage for streamlined work on the iridoid pathway. This work highlights how curation and computational analysis of widely available structural data can facilitate hypothesis-based gene discovery.Countless reports describe the isolation and structural characterization of natural products, yet this information remains disconnected and underutilized. Using a cheminformatics approach, we leverage the reported observations of iridoid glucosides with the known phylogeny of a large iridoid producing plant family (Lamiaceae) to generate a set of biosynthetic pathways that best explain the extant iridoid chemical diversity. We developed a pathway reconstruction algorithm that connects iridoid reports via reactions and prunes this solution space by considering phylogenetic relationships between genera. We formulate a model that emulates the evolution of iridoid glucosides to create a synthetic data set, used to select the parameters that would best reconstruct the pathways, and apply them to the iridoid data set to generate pathway hypotheses. These computationally generated pathways were then used as the basis by which to select and screen biosynthetic enzyme candidates. Our model was successfully applied to discover a cytochrome P450 enzyme from  In the last 100 years, there has been a myriad of reports describing the isolation and structural characterization of natural products from plants. This has yielded a plethora of information on chemical diversity in the literature that is dispersed and underutilized. This is exemplified by iridoid glucosides, plant-derived natural products classified as noncanonical monoterpenes. Iridoid glucosides are found throughout the Asterids  and are Iridoids’ vast chemical diversity is of chemotaxonomic interest, and their numerous reports have been periodically aggregated and reviewed for decades . HoweverCallicarpa americana CYP72 and orthologues in Vitex agnus-castus and Paulownia tomentosa in which we demonstrate biosynthesis of the iridoid glucoside aucubin using bartsioside as a substrate. Notably, neither bartsioside nor aucubin had been previously reported in Callicarpa spp., yet both molecules, as well as the reaction connecting them, are predicted by our model for the genus iridoid pathway.Here, we use a large set of chemical data accumulated for the iridoid glucosides, along with the known phylogeny of a large iridoid producing plant family (Lamiaceae), to computationally generate an accurate set of biosynthetic pathways that best explain the currently observed chemical diversity of iridoids in Lamiaceae. These pathways were used to then select and screen biosynthetic enzyme candidates. We demonstrate the application of our computational model through the discovery and functional characterization of a Paulownia and Aureolaria; fig. 1a). Iridoid glucosides consist of a core nepetalactol scaffold, modified by oxidation and decorated with methyl groups, sugars, phenolics, or terpenes, among other decorations (fig. 1b). The undecorated oxidized scaffold is the basis of iridoid glucosides diversity in Lamiaceae; thus, we removed the decorations from the structures prior to analysis. In total, 64 unique oxidized iridoid scaffolds are identified in Lamiaceae, distributed in 131 unique scaffold-species pairs, as shown in fig. 1a, which also shows representative iridoid glucosides from selected species (fig. 1b).As a first step toward reconstructing the network of iridoid glucoside biosynthetic pathways in Lamiaceae, we compiled literature reports, consisting of 274 structurally distinct iridoids from 19 Lamiaceae genera and 2 outgroups from the Lamiales , followed by alcohol (1), then aldehyde (2), carboxylic acid (3), and finally decarboxylation (4), with the exception of C11, which is known to bypass the alcohol intermediate , and the Manhattan distance between pairs of molecules is directly related to the minimal number of reactions needed to convert one into the other. Double bonds and epoxide rings, however, affect more than one carbon and can coexist with further modifications as long as valence is respected (fig. 2a). Thus, we developed an expanded notation to represent these possibilities: a carbon having a value of i/4 if it is involved in a double bond (see C3 and C4 in fig. 2c) and 3i/4 if it is involved in an epoxide ring (see C7 and C8 in fig. 2c). With this complete representation, a generalized calculation of Manhattan differences, which becomes vectorial in the complex plane, yields an approximate value of 1 in reaction–adjacent molecules  are then chosen, and the rest are discarded , edges (reactions) or both; and which tolerance to use as a cutoff.a). This diversity has been proved by natural product isolation and characterization, creating an incomplete subset of reports of molecules in different species (b). For the pathway reconstruction algorithm to accurately predict biosynthetic pathways from these reports, values for five parameters must be determined (c). To model natural phenomena when only sparse measurements are available, synthetic data sets are typically used to optimize reconstruction parameters. This practice has been used, for example, to train plant phenotyping algorithms (d–f). For that, we first set the basis of an actionable pathway evolution hypothesis (Section 2) and select the outputs that are closer to the in natura pathways (Section 3). We can then optimize the parameters of the pathway reconstruction algorithm using that data set (Section 4). We predict the iridoid pathways using these optimized parameters in Section 5 and use these predictions to facilitate gene discovery in Section 6.In nature, metabolic pathways expand and diverge in different genera, creating chemical diversity a. This d species b. For thtermined c. To modgorithms , model tgorithms , and altgorithms ; the latgorithms . We thusgorithms d–f. For First, we need to develop a model for iridoid pathway evolution. Iridoids act on generalist herbivores , suggestWe developed an actionable pathway evolution hypothesis to recreate biosynthetic pathway evolution of iridoid biosynthesis. The pathway evolution hypothesis follows three practical constraints: random uniform exploration of the chemical space by novel enzyme activity followed by fixation on the substrate present, analogous to Granick’s hypothesis ; an enzyFor the first constraint, we assume that there is a “biochemical reservoir” of enzymes that generates different catalytic activities that appear and disappear in evolutionary time and is only fixed in a species when they have a substrate to act upon . We assuThe implementation of these assumptions is shown in a) directly with the in silico pathways generated by fig. 3d). However, by applying natural data (fig. 3b), we can get networks within which we expect the real biosynthetic pathways to be (fig. 3c). We can then comparethese to the results of fig. 3f; hollow arrow). If the outputs using fig. 3a and d) are similar.If the hypothesis on pathway evolution reasonably mimics the evolution of iridoid diversity in the Lamiaceae, fig. 310-transformed, and z-scaled the matrix and applied a Uniform Manifold Approximation and Projection (UMAP) to reduce the dimensionality of this data set  In contrast, Ajugoideae has highly oxidized decarboxylated iridoids (Ajuga spp.) and a unique set of aldehyde iridoids  and only applied the expansion by one reaction, along with the corresponding best parameters mentioned in Section 4, to include molecules with no immediate connection to another reported molecule. The resulting model contains an individual pathway hypothesis for each of the 19 Lamiaceae genera considered , which implies that once they enter these pathways, iridoid oxidase cannot act on them.Analyzing the pathway hypotheses for each species in these two clades, the diversity of carboxylate iridoids in the Lamioideae is predicted to be mostly due to emergent oxidation of the mussaenosidic and 8-s genera . In contscaffold . Interesa).As shown in fig. 9b). The results of these labeling experiments were not included in our algorithms, but the biochemical steps of the Damtoft–Jensen hypothesis are predicted by our model in all the Lamiaceae species that are predicted to have either aucubin or catalpol.Rigorous isotope labeling experiments led to the hypothesis of a proposed biosynthesis of aucubin and catalpol (Damtoft–Jensen hypothesis or “route II”) in Lamiaceae as well as Scrophulariaceae and families that has not been tested in 25 years ; fig. 9bfig. 9C. americana, which has the highest impact on the prediction of ancestral conditions  and L. album . Within these candidate genes, only 520 orthologous groups were shared  contained four enzymes (three in C. americana) that had Uniprot annotations belonging to the CYP72 family. Since previously reported enzymes that act on iridoid substrates, albeit with different C8 stereochemistry, have been reported to have high homology or belong to the CYP72 family . Neither bartsioside nor aucubin is reported in Callicarpa spp., yet both molecules, as well as the reaction connecting them, are predicted by our model to be in the genus and the ancestral pathway.We further assumed that hydroxylation is catalyzed by cytochrome p450s, so we further cleaned these candidates and kept only ten orthogroups, which had at least one member assigned as a p450 by notation  online. 2 family , we decitsioside . This enVitex grandifolia  defined chemical space, which is a property only found in certain pathways, like xanthine alkaloids  were ground in liquid nitrogen, and total RNA was extracted using the Qiagen Plant RNeasy kit  following manufacturer’s instructions. RNA samples were treated with Ambion TURBO DNase  to remove any residual DNA in the sample. RNA integrity was assessed using an Agilent 2100 Bioanalyzer System  followed by Illumina TruSeq Stranded mRNA Library Preparation  using the Sciclone G3 NGS workstation  by the Research Technology Support Facility at Michigan State University. Libraries were sequenced on an Illumina HiSeq 4000  in 150 nt paired-end mode. Read quality was assessed using FastQC v0.11.5 , 1-deoxy-d-xylulose-5-phosphate reductoisomerase, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, geranylgeranyl pyrophosphate synthase large subunit (GGPPS-LSU), geraniol synthase, 8-hydroxygeraniol oxidoreductase, iridoid synthase, and iridoid oxidase. The best BLAST hits for each of these genes were selected manually and can be found in the EDMOND repository files, in the Biosynthesis.csv file.To identify putative genes involved in the early iridoid pathways of ndidates  for 1-deC. americana and L. album expression data, for each separate species, we took the expression matrix, in fragments per kilobase per million mapped reads, removed zeroes by summing 1 to all the values, scaled by applying logarithm base 2, and generated a z-score subtracting the mean and dividing by the standard deviation in a transcript-wise manner. This yields a center-scaled matrix wherein every transcript has a mean of zero and a standard deviation of 1, allowing a comparison of expression patterns, regardless of the expression level. We decided to follow a “guilt-by-association” approach, in which we assume that enzymes involved in the iridoid pathway will have similar expression patterns to early pathway enzymes. Thus, we grouped transcripts by expression patterns in each species by means of a SOM using the kohonen library v3.0.10 (P < 0.05) in early biosynthetic genes were considered to be coexpressed candidates. Only candidates with a matching orthogroup in both species and a pfam annotation corresponding to the cytochrome p450 family of enzymes were determined to be of interest, and Uniprot annotations were manually explored. Orthogroup OG0001031 contained four transcripts that had Uniprot annotations belonging to the CYP72 family, to which most of the discovered iridoid oxidizing enzymes belong . cDNA was obtained by using SuperScript IV VILO MasterMix , and the target gene was extracted by PCR using primers that contained overhangs with homology to the pESC-Leu2d plasmid Gal10 MCS. The PCR product was gel-purified by the Zymoclean gel DNA recovery kit  and cloned using the ClonExpress II Kit , and purified plasmids were sent for Sanger sequencing . Sequences were found to match perfectly to the sequence extracted from the published transcriptome.Candidate genes were synthesized by Twist Bioscience  and cloned into the Gal10 MCS of a pESC-Leu2d plasmid containing a Cytochrome P450 reductase (CPR) from ia annua . To confSaccharomyces cerevisiae YPL 154C:Pep4KO was used for heterologous expression, and microsomes were prepared as stated previously  at 300 rpm. All reactions were started by adding NADPH and stopped by adding 50 µl of methanol. Samples were centrifuged at 20,000 RCF for 10 min before being transferred to HPLC vials for injection.eviously . Each baSamples were analyzed using an Elute LC system  equipped with an Acquity UPLC C18 column  and coupled via electrospray ionization (ESI) to an Impact II q-TOF . Iridoids were separated at 40°C using a gradient from water (A) to acetonitrile (B), both modified with 0.1% formic acid, at a flow of 0.3 ml/min, redirecting to waste the first 60 s. The program starts with an isocratic flow of 99% A for 1 min, followed by an isocratic step of 95% A for 1.5 min, and then linearly decreases from 95% A in 2.5 min to 80% A in 6 min, then linearly decreases to 60% A in 8 min, and finally decreases to 0% A in 9 min. The column is then washed with 100% B for 2 min and equilibrated in 1% B for another 2 min before the next injection. Ionization was performed via pneumatic-assisted ESI in the negative mode, with a capillary voltage of 3.5 kV and a nebulizer pressure of 2.5 bar. Nitrogen gas was used as drying gas at 350°C and a flow of 11 l/min. The acquisition was set to 6 Hz, from 100 to 1000 m/z, with data-dependent fragmentation and active exclusion after 3 spectra, released after 0.2 min. Fragmentation was triggered on an absolute threshold of 400 and acquired on the most intense peaks with dynamic collision energy from 20 to 50 eV. At the beginning of each run, whereas the LC input was redirected to waste, a sodium formate–isopropanol solution was injected at 0.2 ml/h for the first 60 s of each run, and the m/z values were recalibrated using the expected cluster ion m/z values. Raw MS files were converted to mzXML using Bruker Data Analysis software , and, when needed, extracted ion chromatograms (XICs) were exported to csv using MZMine2 v2.40.1  and plotepi-7-deoxyloganic acid were bought from AnalytiCon Discovery GmbH ; scandoside and geniposidic acid were bought from Aobious Inc. ; bartsioside were bought from Sigma-Aldrich/Merck KGaA ; and aucubin were bought from Extrasynthese . 7-Deoxyloganic acid and a mix of loganin epimers were synthesized as previously reported (Mussaenosidic acid and 8-reported .base (ggplot2 (gplots (VennDiagram library (phangorn v2.6.3 (ape v5.4-1 (dendextend (seqinr v4.2-5 (igraph package v1.2.6 (Matrix library (QuACN library v1.8.0 (uwot package (kohonen library (parallel (snow (Unless otherwise stated, data analysis was performed using R v4.0.3 . Images base , ggplot2(ggplot2 , and gpl (gplots  packages library . Phylogen v2.6.3  and ape e v5.4-1  librariendextend . Nucleotr v4.2-5 , multiplr v4.2-5 , and trer v4.2-5 . Graph pe v1.2.6 , handlin library , and nety v1.8.0 . Dimensi package , and by  library . Paralleparallel  and snowel (snow  librariemsac057_Supplementary_DataClick here for additional data file.


ACADL long-chain acyl-CoA dehydrogenase, not only a key enzyme in the initiation step of fatty acid oxidation, but also plays an important role in adipocyte differentiation. In our study, we found that ACADL promotes intramuscular adipocyte differentiation in goats via the TNF signaling pathway. This study provides a theoretical basis for the study of adipocytes differentiation in goat intramuscular adipocytes.ACADL) is a key enzyme for catalyzing fatty acid oxidation, and studies have shown ACADL is involved in the deposition and differentiation of intramuscular adipocytes. However, the effect of ACADL on intramuscular adipocytes differentiation in goats needs further study. In this study, to explore the mechanism of ACADL on the development of goat intramuscular adipocytes, we constructed an over-expression plasmids and a SI-RNA of ACADL to explore the function of ACADL on the development of goat IMF. It was found that overexpression of ACADL promoted the differentiation of goat intramuscular adipocytes, and promoted the expression of fat cell differentiation marker genes lipoprotein lipase (LPL), peroxisome proliferator activated receptor gamma (PPARγ), APETALA-2-like transcription factor gene (AP2), CCAT enhancer binding protein (CEBPα), preadipocyte Factor 1 (Pref-1) and CCAT enhancer binding protein (CEBPβ), and the opposite trend occurred after interference. In addition, we screened of this related tumor necrosis factor (TNF) signaling pathway by RNA-Seq. So, we validate the signaling pathway with inhibitor of TNF signaling pathway. In summary, these results indicate that ACADL promotes intramuscular adipocytes differentiation through activation TNF signaling pathway. This study provides an important basis for the mechanism of IMF development.Intramuscular fat (IMF) deposits help improve meat quality such as marbling, juicy, flavor and tenderness. Long-chain acyl-CoA dehydrogenase ( CEBPβ; CEBPα; PPARγ, and SREBP-1 [Lamb has low cholesterol and excellent tenderness, so people pay great attention to the traits of mutton, including meat’s color, marbling, tenderness, and flavor. Adipose tissue plays an important role in maintaining energy homeostasis, supporting vital activity of the body . Among t SREBP-1 ,6,7.ACADL is the key enzyme in catalyzing fatty acid oxidation [ACADCL), long chain (ACADL), middle chain (ACADM), and short chain acyl-CoA dehydrogenases (ACADS) [ACADL has been shown to be associated with the deposition and differentiation of yak IMF , and ACADL deficiency can lead to severe hepatic and cardiac lipidosis, hypoglycemia and hepatic insulin resistance [ACADL on intramuscular adipocytes differentiation in goats needs further study.xidation , which b (ACADS) . Styliss (ACADS) . Furthersistance ,12. HoweACADL on intramuscular adipocyte differentiation in goats was studied by overexpression and interference. It was found that overexpression of ACADL promoted the differentiation of goat intramuscular adipocytes, and promoted the expression adipocytes differentiation marker genes LPL, PPARγ, AP2, CEBPα, Pref-1, and CEBPβ, and the opposite trend occurred after interference. To further explore the mechanism, RNA-seq techniques were performed to compare the differences between overexpression ACADL and control groups. We selected the TNF-signaling pathway from RNA-Seq results based on KEGG enrichment analysis and ACADL association pathway research reports. We investigated whether ACADL promotes intramuscular fat cell differentiation through the TNF-signaling pathway. Our experiments provide sufficient evidence that overexpression of ACADL promotes intramuscular preadipocytes differentiation in goats by activating the TNF-signaling pathway, and that the promoted trend is restored after the action of TNF-signaling pathway inhibitors (S1029). The above results show that the specific mechanism of ACADL to promote intramuscular adipocyte differentiation is elaborated.In this study, the regulatory effect of 2.Our experiments meet the requirements of ethical treatment of Experimental Animals of China, and meet the requirements of the “List of Ethical Treatment of Laboratory Animals in China”. We purchased 1-year old Jian Zhou Da-er goat from Dageda animal husbandry . Isolation of intramuscular pre-adipocytes and culture of goat pre-adipocytes were both reported previously ,14. BrieACADL gene using the liver tissue of goats as a template . In this study, we successfully built the ACADL overexpression vector (OE: PCDNA3.1-ACADL) with the control group PCDNA3.1. The goat ACADL interference sequence (n = 3) and negative control (NC) sequence were synthesized by Shanghai Gene pharma Bio Company. The information of ACADL shown in In the early stage, we successfully cloned the goat The staining method was performed by Xiong et al. ,16. BrieWhen goat intramuscular preadipocytes growth was confluent to 80%, we transfected ACADL overexpression and siRNA into cells according to the tubrofect transfection reagent  instructions. We aspirated the medium 4 h before transfection, re-added 2 mL of fetal bovine serum medium , incubated for four hours, and transfected 4 μL of transfection reagent , 400 μL of OPTI-MEM , and 1000 ng of no-load PCDNA3.1 or ACADL overexpression plasmid (and corresponding plasmid). Adipocytes were collected 48 h after induction of differentiation.ACADL and silencing ACADL, and other marker genes  for differentiation through qPCR protocol. Ubiquitously expressed transcript gene (UXT) was used as internal housekeeping gene to normalize the mRNA levels. The PCR reaction procedure, including pre-degeneration , degeneration , annealing , and extension , 38 cycles for degeneration, annealing, and extension. The data were analyzed by the 2−ΔΔCt method [Total RNA was extracted by Trizol reagent and reversely transcribed into cDNA. The effect of overexpression t method . The infTotal RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system . Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system .Raw data of fast format were firstly processed through in-house per scripts. In this step, clean data were obtained by removing reads containing adapter, reads 1 containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30, and GC content clean data were calculated. All the downstream analyses were based on the clean data with high quality.p-value less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies http://www.genome.jp/kegg/ (accessed on 8 August 2021). We used cluster Profiler R package to test the statistical enrichment of differential expression genes in KEGG pathways.Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package, in which gene length bias was corrected. GO terms with corrected p < 0.05, “**” p < 0.01. In this study, all experiments were repeated three times.Take advantage graph-pad 5.0 to prepare graph. Variation was considered statistically significant at “*” 3 cells per well. After 0 and 12 h of cell treatment, we added 10 μL MTT reagent  to each well, away from light, and then incubated the cells with 5% CO2 for 4 h at 37 °C. Finally, absorbance was measured at a wavelength of 490 nm.MTT assays were used to detect the effects of TNF pathway inhibitors on cell viability. Goat intramuscular adipocytes were seeded in 96-well plates at a density of 2 × 10ACADL gene was accompanied by a trend of increasing and then decreasing along with the differentiation of intramuscular adipocytes. Suggests that ACADL may be involved in the process of intramuscular fat fraction. To explore the effect of ACADL in regulating the differentiation of goat intramuscular adipocytes, we transfected ACADL over-expression vector (OE) and ACADL-interference (SI) and their corresponding to control vector and negative control (NC) groups into intramuscular adipocytes. The expression level of ACADL was largely upregulated compared to the vector (ACADL significantly upregulated the mRNA levels of LPL (p < 0.01), PPARγ (p < 0.01), CEBPα (p < 0.05), and PREF-1(p < 0.05). However, the mRNA levels of AP2 (p < 0.05), and CEBPβ (p < 0.05) were downregulated , PPARγ (p < 0.05), CEBPα (p < 0.05), and PREF-1 (p < 0.05), while upregulated the expression of SREBP-1 (p < 0.01) and CEBPβ  met the criteria required for transcriptome sequencing , log2FC > 1), a total of 246 genes, of which 136 genes were upregulated and 110 genes were downregulated , cellular component (CC) and biological process (BP), the number of gene-rich entries was metal-ion homeostasis , signaling receptor binding , molecular function regulatory , receptor ligand activity  and ion channel activity  A. So, mopathways B. Genes ACADL overexpression in goat intramuscular adipocytes differentiation. Since the concentration of the inhibitor has an effect on cell activity, as well as adsorption capacity [ACADL expression for the detection of inhibitor efficiency, and the results showed that inhibitor efficiency was optimal at 2.5 μM  staining, but decreased the degree of aggregation of lipid droplets in IMF adipocytes after interference. As is known to all, adipocytes differentiation determined by hyperplasia and hypertrophy of intramuscular adipocytes [PPARγ, CEBPβ, and CEBPα are considered as the important regulators of adipocytes [AP2, SREBP-1, and LPL have been considered positive regulator for adipocyte differentiation, while PREF-1 have been identified as factors negatively regulators for this [ACADL, the expression of LPL, PPARγ, CEBPα, and PREF-1 were enhanced. In general agreement with the expected results, the expression levels of SREBP-1, AP2, and CEBPβ were downregulated after ACADL silencing. These results suggest that ACADL may promote intramuscular adipocyte differentiation through upregulation of LPL, PPARγ, and CEBPα. In IMF adipocytes, whether ACADL directly or indirectly modulates the level of the adipogenesis transcription gene mRNA directly or through molecules requires further exploration.ACADL has an important role in intramuscular adipocytes differentiation. Research shows that glycemia . Long-chglycemia . Researcipocytes . Intramuipocytes . PPARγ, ipocytes . AP2, SRfor this . In thisADADL overexpression treatment, and the upper 136 genes are more than 110 down-regulated genes. ACADL overexpression causes more gene expression and leads to functional changes. We select the TNF-signaling pathway from KEGG enrichment analysis. Interestingly, studies have shown that TNF promotes the differentiation of intramuscular adipocytes by activating the downstream pathways by secreting the TNFα factor [(TNFAIP3), E-selectin (SELE), CC-chemokine 5 (CCL5), and CC-chemokine 20 (CCL20) enriched in this pathway. TNFAIP3 is the main component of the TNF-signaling pathway, TNFAIP3 is the key factor for mediating the secretion of TNFa [TNFAIP3 inhibits the secretion of TNFa in the TNF-signaling pathway, which, in turn, affects the downstream pathway of the TNF signaling pathway to regulate adipocyte differentiation [CCL5 and CCL20 all belong to the chemokines, and play roles in various pathologic conditions [CCL20 [CCL5 selectively activates the downstream JNK pathway of the TNF pathway [ACADL downregulated expression of TNFAIP3, SELE, CCL5, and CCL20.To reveal the potential specific molecular mechanism, RNA-sequencing was performed. Quantitative analysis showed that the number of overexpressed genes was higher than that of the control group (VECTOR). There are 246 differential expression genes (DEGs) intramuscular adipocytes after α factor . Similarα factor ,30. So,  of TNFa . TNFAIP3ntiation ,33. E-sentiation . In addintiation . CCL5 ans [CCL20 , and CCL pathway ,39,40. WACADL in promoting adipocytes differentiation in goats through the TNF-signaling pathway, we added TNF-pathway inhibitors (S1029) to intramuscular adipocytes for further validation. In intramuscular adipocytes, ACADL overexpression activates TNF pathway, which subsequently lead to increased differentiation of intramuscular adipocytes. Interestingly, the TNF inhibitor treatment OE group rescued the phenotype caused by ACADL expression in a specificity dose manner. Preadipocytes are characterized by the number of cells and enlargement. The TNF-signaling pathway plays an important role in both [TNPAIP3, SELE, CCL5, and CCL20 was upregulated, which was contrary to the sequencing data and ACADL overexpression treatment group, and demonstrated that the inhibitor is a potent inhibitor of the TNF pathway. The apparent phenomena showed that the inhibitor treatment group significantly reduced the degree of cell differentiation and lipid droplet aggregation, which had a weakening effect on the ACADL overexpression group. Expression of TNFAIP3, SELE, CCL5, and CCL20 in the inhibitor-treated group showed an upward trend, the fat adipocyte differentiation-related genes also showed a reverse trend, and the expression level of S019 in the OE treatment group showed a weakening trend. Therefore, we speculate that ACADL regulates the TNF-signaling pathway and subsequently affects pre-adipocyte differentiation by altering cellular lipid droplet aggregationTo determine the role of  in both . In the ACADL promotes adipocytes differentiation and silencing ACADL decreased adipocytes differentiation. Further study showed that TNF-signaling pathway is involved in the regulation of ACADL on the differentiation of adipocytes. In summary, ACADL promotes goat adipocytes differentiation by activating the TNF-signaling pathway.The overexpression of