Study On The Inhibition Of Vascular Smooth Muscle Cell Proliferation By Chuanxudan Saponin B Through TOP2a/PTEN Pathway

Objective:Abnormal proliferation and migration of vascular smooth muscle cells(VSMCs)is the pathological basis of vascular lesions.Inhibition of abnormal proliferation of VSMCs can effectively reverse vascular remodeling,improve vascular function,and slow down atherosclerosis(AS)process.Therefore,screening of traditional Chinese medicine compounds to inhibit the abnormal proliferation and migration of VSMCs is of great significance in the prevention and treatment of vascular diseases.The application of Lonicerae japonica in cardiovascular disease has been recorded for a long time,and it has the pharmacological function basis in the treatment of cardiovascular disease.There is no obvious difference in the function,usage and dosage of Lonicerae japonica and Lonicerae flos,so they are not standardized in clinical use and mixed use,which leads to the controversy between the north and the south for Lonicerae japonica and Lonicerae flos.Therefore,it is necessary to make clear the difference between them and their respective attribution,so as to make their respective positioning and play their respective medical value.We screened the differential components of Lonicerae japonica and Lonicerae flos through network pharmacology,and verified the pharmacological effects of the key differential components of Lonicerae japonica and.Lonicerae flos.This study is beneficial to further compare the composition difference between Lonicerae japonica and Lonicerae flos,and it is of great significance to develop new medicinal value of Lonicerae flos.Methods:1.TCMSP,TCMID and Swiss Target Prediction database were used to screen the active components of Lonicerae Japonicae flos and Flos Lonicerae.Cytoscape 3.5.1software was used to construct the component-target network diagram,and key nodes of compounds were screened based on the network diagram,and compounds with key nodes of Flos Lonicerae were screened based on literature for follow-up research.2.Network pharmacology analysis: to predict the DB target on Chem Mapper database,compound docking with the target protein by i GEMDOCK software,according to the score to select the key target protein,the key target protein by the STRING database building,interactions between the key targets for enrichment and KEGG pathway analysis GO protein regulation network of biological processes and pathways.3.Anangiotensin II(Ang-II)was used to induce VSMCs proliferation model.The effects of DB on the cell activity,proliferation and migration of VSMCs were detected by MTT assay and EDU assay.The effect of DB on the cell cycle of VSMCs was measured by flow cytometry.The effect of DB on the phenotypic transformation marker protein of VSMCs was measured by Western blot and immunofluorescence.4.He staining was used to observe the intima and media of the aorta.The expression of PTEN and contractile protein ?-SMA were detected by immunohistochemistry and Western blot.5.According to the predicted targets,the expression of the key target protein was detected by Western blot,and the key target protein was incubated with PTEN inhibitor(OV-ohpic)and silenced with small interfering RNA to detect the expression of PTEN and its corresponding proteins.Result:1.In the TCMSP database,there were 18 active compounds of Lonicerae Japonicae flos and 15 active compounds of Lonicerae japonicae.In the network diagram of Lonicerae Japonicae flos,8 compounds,such as luteolin and quercetin,were the key nodes,and flavonoids were the main ones.In the network diagram of active compounds--targets of Lonicera japonicae,14 compounds,including macranthoidin b and DB,were the key nodes,phenolic acids and saponins were the main components,and DB was selected as the follow-up study object.2.DB has four target proteins,and DNA topoisomerase 2a(TOP2a)was selected as the follow-up study object according to the lowest molecular docking energy value.TOP2a-related proteins were predicted,and 10 related proteins were obtained.After GO enrichment analysis and KEGG pathway analysis,TOP2 a may be involved in the regulation of VSMCs cell cycle and proliferation.3.MTT assay showed that DB inhibited VSMCs activity in a dose-dependent manner.EDU method was used to detect the obvious inhibitory effect of DB(3,10,30?mol/L)on VSMCs proliferation.DB can inhibit the migration of VSMCs to some extent.Flow cytometry showed that DB might block the synthesis of VSMCs from G1 phase to S phase,thus affecting cell proliferation.Western blot results showed that DB down-regulated the expression of protein PCNA.DB significantly increased the expression of contractile proteins ?-SMA and SM22?,and significantly inhibited the expression of synthetic protein OPN.The results showed that DB could significantly inhibit the transformation of VSMCs to synthetic phenotype.4.The results of animal experiments showed that :(10,60mg/kg)DB could inhibit the new intima and up-regulate the expression of PTEN and contractile protein ?-SMA.5.DB down-regulated the expression of TOP2 a and p-Akt protein,and up-regulated the expression of PTEN protein.After OV-ohpic inhibited the expression of PTEN protein,the inhibitory effect of DB on the cell activity of VSMCs was decreased,the up-regulation effect of PTEN protein was significantly decreased,and TOP2 a protein was up-regulated to a certain extent.The expression of TOP2 a protein was significantly decreased after si TOP2 a was transfected into VSMCs.DB still had a certain inhibitory effect on TOP2 a in VSMCs transfected with si TOP2 a,but the up-regulation effect on PTEN decreased.Conclusion:(1)DB inhibited the transformation,proliferation and migration of VSMCs to synthetic phenotype,and inhibited the formation of new intima.(2)DB may regulate the proliferation of VSMCs by up-regulating the expression of PTEN and down-regulating the expression of TOP2 a,regulating the cell cycle.