The Mechanism Of Wenjing Tongluo Decoction Inhibiting Angiogenesis In Intervention Of Knee Osteoarthritis Was Studied Based On Genome-wide Library

Objective:The mouse model of knee osteoarthritis was established by ACLT method,and then Wenjingtongluo Decoction was administered to determine the regulation effect of Wenjingtongluo Decoction on related inflammatory factors in the course of osteoarthritis.The whole genome library of mouse vascular endothelial cells was constructed to analyze the possible target of Wenjingtongluo Decoction to delay the progression of knee osteoarthritis by inhibiting vascular dysplasia and invasion.Under the guidance of collaterology theory,the efficacy of Wenjing Tongluo Decoction compound was disintegrated.After comparative analysis,the best dissolving group of the compound to inhibit vascular proliferation was determined,which provided data support for the formulation optimization of Wenjing Tongluo Decoction.Method:(1)Anterior cruciate ligament dissection(ACLT)was used to construct a mouse model of knee osteoarthritis.HE staining was used to observe the morphological changes of knee surface cartilage in mice model of knee osteoarthritis.Mankin’s score was used to evaluate the morphological differences of the surface cartilage of the knee joint in each group.The expressions of TNF-α,IL-6,IL-1β and MMP13 in the knee surface cartilage of mice model of knee osteoarthritis were detected by ELISA.The expressions of TNF-α,IL-6,IL-1β and MMP13 in serum of mice with knee osteoarthritis were determined by ELISA.The above methods were used to determine the regulatory effect of Wenjing Tongluo Decoction on the related factors in the course of knee osteoarthritis in mice.(2)Immortalized mouse umbilical vein endothelial cells were constructed by SV40 lentivirus transfection.Cas9 blast lentivirus was transferred and mouse umbilical vein endothelial cells with stable Cas9 expression were screened by blast insecticide.After lentiviral transmutation of the whole genome library,the successful whole genome library cell lines were screened with purinomycin.This division of labor provides the basis for subsequent reference transcriptomic sequencing research.(3)After the intervention of Wenjingtongluo Decoction in whole genome library cells,the main differential genes before and after the intervention were screened out through reference transcriptomic sequencing.KEGG enrichment analysis and GO enrichment analysis were used to identify the target and signal pathway of Wenjingtongluo Decoction in inhibiting the proliferation of vascular endothelial cells.(4)Vascular endothelial cells were extracted from the knee joint of mice with osteoarthritis by gradient digestion centrifugation,and Wenjing Tongluo Tang compound was disassembled according to the prescription principles and medication rules.Cell scratch test,CCK8 test,lumen formation test,WesternBlot and other experimental methods were used.After parallel comparison,the combination of disassembling prescription with the strongest inhibitory effect on the proliferation of vascular endothelial cells was determined.To provide data support for the formulation optimization of TCM compounds.Result:(1)Wenjing Tongluo Decoction can effectively alleviate the destruction process of knee joint surface cartilage in mice with knee osteoarthritis.At the same time,the expression of inflammatory factors(TNF α,IL-6,IL-1β,MMP13)in the surface cartilage of knee joint and serum of osteoarthritis mice was inhibited effectively.(2)Reference transcriptome sequencing results showed that the signaling pathways involved in differential genes before and after the intervention of Wenjing Tongluo Tang drug-containing serum in mouse vascular endothelial cells mainly included cell process,intracellular environment,genetic information,human disease,cell metabolism,organic system 6.Among them,hypoxia inducible factor 1(HIF1)signaling pathway is related to the intracellular environment.The signaling pathways related to cell process include:tight junction,adhesion patch,cell cycle,cell apoptosis,etc.Signaling pathways associated with human disease include:small cell lung cancer,proteoglycan in cancer,cancer-related signaling pathways,pancreatic cancer,non-small cell lung cancer,hypertrophic cardiomyopathy,dilated cardiomyopathy,bacterial invasion of epithelial cells,and arrhythmogenic right ventricular cardiomyopathy.Signaling pathways related to cell metabolism include glycolysis/glycogen generation,carbon metabolism,amino acid biosynthesis,etc.The signaling pathways associated with organic systems are axon-oriented.(4)The inhibition effect of Wenjing Tongluo Decoction compound on the proliferation of vascular endothelial cells was the most obvious.The inhibition effect of regulating qi and removing phlegm and regulating collaterals and searching wind and regulating collaterals were obviously better than that of warming Jing and regulating collaterals and promoting blood circulation and regulating collaterals.The drugs of Soufeng and collaterals had the most obvious inhibition on the lumen-forming ability of vascular endothelial cells,and the drugs of dampness and collaterals and qi and phlegm and collaterals had a certain promotion effect on the lumen-forming ability of vascular endothelial cells.The inhibition effect of Wenjing Tongluo Decoction compound on VEGF is obvious due to the efficacy of different prescription groups.At the same time,regulating qi and removing phlegm and regulating collaterals and seeking wind and regulating collaterals also have obvious inhibitory effects on VEGF.Soufeng and Tongluo herbs had the most obvious inhibition effect on HIF1α,and Wenjing and Tongluo herbs and Wenjing and Tongluo Decoction compound also effectively inhibited the expression of HIF1α in vascular endothelial cells.Conclusion:In vivo experiments confirmed that Wenjing Tongluo decoction can effectively inhibit the expression of osteoarthritis-related factors,so as to protect articular cartilage and delay the process of osteoarthritis.Whole genome sequencing and in vitro experiments showed that the signal pathway that had the highest correlation with the inhibition of vascular dysplasia by Wenjing Tongluo Decoction was the hypoxia-inducing factor signal pathway.In the study of efficacy-dissolving prescription,it was found that the inhibition effect of regulating qi and dispelling phlegm and searching wind and regulating collaterals-on vascular endothelial cells was obviously better than other dissolving prescription groups,but there was still a certain gap between them and Wenjing Tongluo Decoction compound.