Study On The Molecular Mechanism Of Anti-Atherosclerosis Effect Of Compound Xinta Flower Based On Network Pharmacology

Objective:Compound Xinta flower is a traditional Chinese medicine prescription composed of three traditional Chinese medicines:Ziziphorae Bungeanae Herba,Hyssopi Herba and Origani Herba,which has been used in the prevention and treatment of cardiovascular disease for more than 20 years and has good clinical effect.In this study,the effective components,key targets and main signaling pathways of compound Xinta flower were screened out by network pharmacology technology and topology analysis.The pharmacological effect of anti-atherosclerosis was determined by experimental animal model.according to the network of"component-target-pathway"and molecular biological methods,the mechanism of action was elucidated from the aspects of anti-inflammation,anti-oxidation and lipid regulation.It provides a scientific basis for the multi-component,multi-target and multi-channel pharmacodynamic mechanism of compound Xinta flower.Methods:1.Through the early literature mining,using network pharmacology technology,using TCMSP and other traditional Chinese medicine pharmacological database to screen the effective components of compound Xinta flower,through SwissTargetPrediction and other databases for target prediction and analysis.The target was introduced into KOBAS and other servers for target annotation and analysis,combined with FunRich,KEGG and other databases for path analysis,so as to construct the"component-target-pathway"network.2.The rat model of atherosclerosis induced by high fat emulsion combined with vitamin D₃ was established.The animals were randomly divided into normal group,model group,positive group?0.45mg/kg?,compound Xinta flower low dose group?6.25g/kg?,middle dose group?12.5g/kg?and high dose group?25g/kg?.After the last administration,the corresponding target organs were examined by ultrasonography,and blood lipids?TC,TG,LDL,HDL?were measured to analyze the aorta pathologically according to the results of ultrasound.3.According to the results of network pharmacology component-target-pathway analysis,combined with pharmacodynamic indicators,the main pharmacological mechanism of compound Xinta flower may be related to anti-inflammatory and anti-oxidation.?1?Proinflammatory factors?IL-1?,TNF-?,IL-6,hs-CRP,LP-PLA2?,anti-inflammatory factors?IL-10?,plaque stabilization factors?MPO,MMP9?and oxidation factors?SOD,MDA,LDH?were detected from inflammatory factors.To observe the anti-inflammatory and antioxidant effects of compound Xinta flower.The pro-inflammatory genes?IL-1?,TNF-?,TGF-??,anti-inflammatory genes?IL-10?,plaque stability genes?MPO,MMP-2,MMP-9,MMP-12?,adhesion genes?RHO,ICAM-1,VCAM-1?,The expression of vascular growth genes?VEGF,VEGFA,VEGFR2?and apoptosis genes?Caspase-3?were detected.And combined with Western blot method to detect the protein expression of the above inflammatory factors,so as to determine the signaling pathway of its role.?2?The pharmacodynamic antioxidant index showed that the antioxidant effect of compound Xinta flower was unique,so the experimental study in vitro was carried out.The oxidative damage model of HUVEC cells induced by H₂O₂ was established to study the antioxidant effect of compound Xinta flower in the mechanism of anti-atherosclerosis.Starting with oxidation factors,the dosage of compound Xinta flower and the dose of H₂O₂ oxidative damage were determined.The cell survival rate,oxidative factors?SOD,MDA,LDH,CAT,GSH-Px?and apoptosis?Hoechst 33258 staining,AO staining?were detected to observe the antioxidant and anti-apoptotic effects of compound Xinta flower.The expression of pro-apoptosis genes?Bax,Caspase-3?and anti-apoptosis genes?Bcl-2?were detected by RT-PCR.The protein expression of the above apoptosis factors was detected by Western blot method,so as to determine the signaling pathway of the above apoptosis factors.Results:The results of network pharmacology experiment:the network diagram of anti-atherosclerotic effect and mechanism of compound Xinta flower was constructed by using network pharmacology technology,and 316 compounds,117 targets and 290signaling pathways were screened out.Among them,16 active components,39 main targets and 43 main pathways are closely related to atherosclerosis.Pharmacodynamic results:?1?according to the results of ultrasound and four items of blood lipid,it is suggested that the atherosclerotic model is successful.Compared with the normal group,the arterial wall of the model group was thickened and there were ridged plaques.Compared with the model group,the therapeutic effect of compound Xinta flower was as follows:positive group?high dose group>middle dose group>low dose group,arterial wall thickening and spinal plaques were less,visual acuity was significantly improved,There was no significant difference between the positive group and the high dose group,but the effect of the positive group was better than that of the middle and low dose group.?2?The results of blood lipid showed that TC,TG,LDL and HDL in the model group were significantly higher than those in the normal group,TC,TG,LDL were significantly lower and HDL were significantly higher in the compound Xinta flower group than those in the model group.Among them,it is the most significant to reduce total cholesterol.?3?The histopathological results showed that the intimal thickness did not increase,the intima was intact,and the middle smooth muscle cells arranged neatly and did not proliferate in the normal group.Compared with the normal group,the intimal thickness of the model group was significantly increased,the endothelial cells were incomplete and exfoliated,the smooth muscle cells proliferated and the structure was disordered,and the lipid accumulation was serious.Compared with the model group,the intimal thickness was reduced in the high dose group,and the smooth muscle cells proliferated in a small amount without disorder in the high dose group.the smooth muscle cells in the middle dose group were slightly proliferated and disordered,and the intimal thickness was increased and the endothelial cells were exfoliated in the low dose group.Smooth muscle cells were partially proliferated,calcified and disordered,but the degree of atherosclerosis in each group was significantly lower than that in the model group.Compared with the model group,the intima of the positive group did not increase significantly,the endothelial cells did not fall off completely,and some smooth muscle cells showed a small amount of proliferation,but the structure was stable and not disordered.Compared with the positive group,the effect of the high dose group was not significantly different from that of the positive group,but the positive group was better than the middle and low dose group.?4?The results of body mass index and organ coefficient showed that the body weight of each group was significantly lower than that of the normal group,but there was no significant change in the body weight of the positive group and the administration group compared with the model group.Compared with the normal group,the heart mass of each group did not change significantly,the weight of liver and kidney increased significantly,the weight of aorta in the model group increased significantly,there was significant difference,and there was no difference in the other groups.Pharmacological mechanism results:?1?Anti-inflammatory effect of compound Xinta flower:?1?Compared with the normal group,the levels of pro-inflammatory factors?IL-1?,TNF-?,IL-6,hs-CRP,LP-PLA2?,anti-inflammatory factors?IL-10?and plaque stability factors?MPO,MMP9?in the model group were higher than those in the normal group.Compared with the model group,Compound Xinta flower can reduce pro-inflammatory factors,increase anti-inflammatory factors and stabilize plaques.?2?The results of oxidation index showed that compared with the normal group,SOD decreased,MDA and LDH increased in the model group,and compared with the model group,compound Xinta flower could increase SOD,decrease MDA and LDH.?3?RT-PCR results showed that,compared with the normal group,In the model group,the expression of pro-inflammatory genes?IL-1?,TNF-?,TGF-??was increased,the expression of anti-inflammatory genes?IL-10?was decreased,the expression of plaque stability genes?MPO,MMP-2,MMP-9,MMP-12?was increased,and the adhesion genes(RHO,ICAM-1,The expression of VCAM-1 was increased and vascular growth genes(VEGF,VEGFA,The expression of VEGFR2 and apoptosis gene?Caspase-3?was increased.Compared with the model group,the compound Xinta flower administration group showed a reversal effect.?4?The results of Western Blot showed that the levels of VEGF/PI3K/AKT/NF-?B signaling pathway in the model group were significantly higher than those in the normal group,and the proteins in the signaling pathway were down-regulated by compound Xinta flower compared with the model group.?2?Antioxidant effect of compound Xinta flower:?1?The results of compound new tower flower concentration and H₂O₂concentration showed that:the concentration of compound Xinta flower was?0.25,0.5,1mg/mL?andtheconcentrationofH₂O₂oxidativedamagewas100umol/L.?2?The results of MTT showed that compared with the normal group,the cell survival rate of the model group was decreased,the cells were short fusiform or crumpled into stars,and a large number of suspended cells and cell fragments could be observed.compared with the model group,the cell survival rate of the compound Xinta flower administration group was higher than that of the model group.Cells can be seen in complete form of cells,rare suspended cells and cell fragments.?3?The results of oxidation index showed that compared with the normal group,the model group?SOD,CAT,GSH-Px?decreased,?MDA,LDH?,and compared with the model group,the compound Xinta flower administration group increased?SOD,CAT,GSH-Px?and decreased?MDA,LDH?.?4?The results of apoptosis showed that compared with the normal group,the nuclei of the model group were irregular or chromatin polymerized and divided,dense granular fluorescence could be seen,and a large number of apoptotic and necrotic cells were observed,compared with the model group,the nuclei of the model group were irregular or chromatin polymerized and divided,and a large number of apoptotic and necrotic cells were observed.The compound Xinta flower administration group could reduce the dense staining,shrinkage,dense nuclear and chromosome status,reduce apoptosis,reduce cell death and improve the morphological changes of cells.?5?The results of RT-PCR showed that compared with the normal group,the expression of apoptosis-promoting genes?Bax,Caspase-3?in the model group increased and the expression of anti-apoptosis gene?Bcl-2?decreased,and compared with the model group,the compound Xinta flower administration group showed a reverse effect.?6?The results of Western Blot showed that compared with the normal group,the expression of VEGFR2/AKT/NF-?B/Bax/Caspase-3protein in the model group was up-regulated and the expression of Bcl-2 protein was down-regulated,and compared with the model group,the compound Xinta flower administration group showed a reverse effect.Conclusion:1.The effective components of compound Xinta flower against atherosclerosis were flavonoids and phenolic acids,and the main targets of anti-atherosclerosis were VEGF,MPO,MMP family,PPARD family and so on.The main signal pathways against atherosclerosis are VEGF pathway,PI3K pathway and other signal axes.2.Compound Xinta flower has anti-atherosclerotic effect,can reduce TC,TG,LDL,increase HDL;reduce arterial wall thickness,reduce plaques,reduce aortic intimal thickness,reduce smooth muscle cell proliferation,calcification,disorder and other pathological processes.3.The anti-atherosclerotic mechanism of compound Xinta flower may be related to its anti-inflammatory and antioxidant effects:Compound Xinta flower could decrease the content of pro-inflammatory factor and gene protein,increase the content and gene expression of anti-inflammatory factor,decrease the content and gene expression of plaque factor,and decrease the gene and protein expression of adhesion factor and angiogenesis factor.It can reduce the aggregation and adhesion of inflammatory factors,reduce the proliferation and migration of smooth muscle cells and improve the stability of plaques,and play an anti-inflammatory role by down-regulating the VEGF/PI3K/Akt/NF-?B signal axis.Compound Xinta flower can regulate the content of oxidation factor,increase the activity of scavenging oxygen free radical,reduce the product of lipid peroxidation,decrease the expression of pro-apoptosis factor gene and protein,and increase the expression of anti-apoptosis factor gene and protein.Maintain the integrity of cell membrane and mitochondria,inhibit apoptosis,protect vascular endothelial cells.And by down-regulating the VEGFR2/Akt/Bax signal axis,play an antioxidant role.