| Objective:Proteomics and network pharmacology were used to analyze the multi-target signal network of Wenjingtongluo Decoction in regulating angiogenesis,in order to clarify the mechanism of Wenjingtongluo Decoction inhibiting angiogenesis;Based on the ACLT induced KOA mouse model,characteristics of angiogenesis and the related signaling pathways(VEGF-A/VEGFR2,PI3K/AKT)in cartilage of different pathological stages of OA were analyzed,to clarify the correlation between angiogenesis and the pathological process of KOA;Elucidate the molecular mechanism of Wenjingtongluo Decoction in regulating angiogenesis and then intervening KOA,so as to provide experimental basis for Wenjingtongluo Decoction in treating KOA.Method:(1)Effect of Wenjingtongluo decoction containing serum on IHUVEC through Proteomics analysis:IHUVEC were cultured,then different concentrations of Wenjingtongluo Decoction drug contained serum and blank serum were used for intervention,CCK8,scratch assay,Transwell and tube formation assay were performed to verify the effects of Wentongluo decoction on regulating cell proliferation,migration and tube formation;Proteomics analysis was used to reveal the differential proteins after drug contained serum intervened in endothelial cells,and the signaling pathway related proteins with higher enrichment were selected for the next study by GO,KEGG enrichment analysis.(2)Network pharmacology validation:The active ingredients and targets of Wenjingtongluo Decoction were screened through network pharmacology.To explore the key targets and signaling pathways for the treatment of knee OA,and verify whether the related endothelial cell secreted factors are targets for regulating koa by Wenjingtongluo decoction.(3)Pathological characteristics of KOA mouse model induced by ACLT:After the model was established,staining,Immunofluorescence,Immunohistochemistry,Western blot and other experimental methods were used to clarify the angiogenesis in different stages and the relationship between VEGF-A/VEGFR2 and PI3K/AKT pathway and inflammation,cartilage degeneration(Mankin score).(4)Therapeutic effect and mechanism of Wenjingtongluo Decoction on KOA mouse:After the model was established,mice were treated with different concentrations of Wenjingtongluo Decoction by gavage four weeks after ACLT.In order to detect the underlying molecular mechanism of Wenjingtongluo Decoction in regulating angiogenesis,the expression of cartilage matrix components col-II and col-x was detected by immunohistochemically and WB;Changes of VEGF-A/VEGFR2/ERK1/2 and PI3K/AKT pathways were detected by RT-PCR and WB.Result:(1)CCK8,scratch assay,Transwell and angiogenesis assay results indicated that 4%Wenjingtongluo Decoction drug contained serum intervention for 48h can significantly delay the proliferation,migration and angiogenesis of Immortalized Human Umbilical Vein Endothelial Cells.The results of proteomic analysis showed a total of 148 differential proteins after intervention with Wenjingtongluo decoction drug containing serum compared with the control group,of which 87 proteins were upregulated and 61 were downregulated.GO and KEGG enrichment analysis were used to screen the signal pathway and functional proteins,the differential proteins are mainly associated with signaling pathways such as positive regulation of blood vessel endothelial cell migration,negative regulation of blood vessel endothelial cell migration,positive regulation of angiogenesis,negative regulation of apoptotic process,extracellular matrix binding,and the main differential proteins VEGF-A,VEGFR2 were screened for validation.(2)Mapping the drug predicted targets and osteoarthritis disease targets of Wenjingtongluo Decoction by the Venn diagram yielded,a total of 131 common effect targets of Wenjingtongluo Decoction for the treatment of knee OA,and 38 key targets were screened for go and KEGG enrichment analysis,which revealed the related signaling pathways were involved in inflammation,immunity and metabolism.Among these pathways,HIF1 signaling,VEGF,and PI3K-Akt signaling are associated with angiogenesis.(3)The degree of cartilage degeneration in the ACLT induced OA model was time-dependent;The cartilage matrix degradation and inflammation were largely consistent with the degree of osteoarthritis;VEGF-A/VEGFR2 signaling pathway played an irreplaceable role in the development of KOA.Meanwhile,PI3K/AKT signaling was also involved in the development of KOA,but showed an opposite trend with KOA pathological process,which indicated that in this model PI3K/AKT pathway was involved in regulating KOA through other pathways.(4)Wenjingtongluo Decoction decreased the expression of col-x and increased the expression of col-? in the cartilage of KOA mouse(P<0.01),delaying cartilage matrix degradation,it can also reduce the expression of inflammatory mediators COX2 and iNOS(P<0.01),thus delaying OA progression.Initially,it was clarified that Wenjingtongluo Decoction might participate in KOA by regulating vascular invasion through VEGF-A/VEGFR2/ERK1/2 signaling pathway;The specific molecular mechanism underlying of regulation of the PI3K/Akt signaling pathway requires further investigation.Conclusion:In vitro experiments confirmed that Wenjingluo Decoction drug contained serum could inhibit the proliferation,migration,and tube formation of IHUVEC,thus inhibiting angiogenesis;The results of proteomic technique and network pharmacology analysis indicated that Wenjingtongluo Decoction drug contained serum could inhibit angiogenesis from multi pathway and multi targets such as VEGF-A/VEGFR2 and PI3K/AKT;CD31 immunofluorescence staining could clearly showed angiogenesis in cartilage of KOA mice,and VEGF-A/VEGFR2 and PI3K/AKT pathways were involved in regulating angiogenesis in cartilage of KOA through different pathways;Wenjingtongluo Decoction could inhibit angiogenesis and improve cartilage matrix degradation and inflammatory responses in KOA mice,thus delay the progression of KOA by regulating the expression of VEGFA/VEGFR2 and PI3K/Akt signaling pathways. |
PDF Full Text Request