β-sitosterol Regulates Autophagy Against The Proliferation Of A7r5 Cells Induced By Angiotensin Ⅱ

Objective:1.To study the effect of β-sitosterol on the proliferation of rat thoracic aortic vascular smooth muscle cell line A7r5 induced by angiotensin Ⅱ(Ang Ⅱ).2.To study the effect of β-sitosterol on autophagy in A7r5 cells induced by Ang Ⅱ.3.To explore the effect and mechanism of β-sitosterol on the proliferation of A7r5 cells induced by Ang Ⅱ by regulating autophagy.Methods:1.The CCK-8 assay was used to detect the toxic effects of Ang Ⅱ and β-sitosterol on A7r5 cells,and the effect of β-sitosterol on the proliferation of A7r5 cells induced by Ang Ⅱ.2.Flow cytometry was used to analysis the effect of β-sitosterol on A7r5 cell cycle and apoptosis induced by Ang Ⅱ.3.The Ad-mcherry-GFP-LC3 B double-labeled adenovirus assay was used to detect the changes of β-sitosterol on the autophagy flux of A7r5 cells induced by Ang Ⅱ.4.Autophagy activator(rapamycin)was used to investigate whether β-sitosterol can affect the proliferation of A7r5 cells by regulating autophagy.5.Pharmmaper,TCMSP,and Gene Cards platforms were used to predict the possible target of β-sitosterol on autophagy,and the DAVID database was used for signaling pathway enrichment analysis.6.Western Blot assay to detect the effect of β-sitosterol on the proliferation-related proteins(PCNA)and autophagy-related proteins(LC3,Beclin-1,ULK1)in A7r5 cells induced by Ang Ⅱ and the changes in the expression of important proteins in Akt/m TOR signaling pathway.Results:1.The proliferation effect of A7r5 cells was the most significant after treatment with 2 μg/m L Ang Ⅱ for 24 h,and the state was stable and non-toxic;1,2,4 μg/m L β-sitosterol were treated with A7r5 cells for6/12/24 h,and the state was stable and non-cytotoxic.2.β-Sitosterol can inhibit the proliferation of A7r5 cells induced by Ang Ⅱ in a time-and concentration-dependent manner,while downregulating the expression of the proliferation-related protein PCNA.3.A7r5 cells induced by Ang Ⅱ were pretreated with β-sitosterol,the number of cells in S phase decreased,the number of cells in G0/G1 phase increased,and the rate of apoptosis increased.4.β-Sitosterol can inhibit Ang Ⅱ-induced autophagy in A7r5 cells in a time-and concentration-dependent manner,while downregulating the expression of autophagy-related proteins LC3,Beclin-1,and ULK1.5.After treatment with rapamycin,the effect of β-sitosterol on the proliferation and autophagy of A7r5 cells induced by Ang Ⅱ was reversed,and the expression of PCNA and LC3 was up-regulated.6.Pharmmaper,TCMSP,Gene Cards platform screened 160β-sitosterol targets and 5786 autophagy-related targets.After the two were crossed,95 β-sitosterol targets that might be related to autophagy were obtained.7.DAVID database analysis results show that the effect ofβ-sitosterol on autophagy may be related to the PI3K/Akt/m TOR signaling pathway.8.β-Sitosterol can increase the levels of p-Akt/Akt and p-m TOR/m TOR in A7r5 cells induced by Ang Ⅱ.9.After treatment with Akt inhibitor(MK2206),the effects ofβ-sitosterol on A7r5 cell proliferation,autophagy and increasing p-Akt/Akt and p-m TOR/m TOR levels induced by Ang Ⅱ were reversed.Conclusion:β-Sitosterol can effectively inhibit the proliferation of A7r5 cells induced by Ang Ⅱ,and the inhibitory effect is dependent on the concentration of 1～4 μg/m L and the treatment time of 6/12/24 h.The mechanism is that β-sitosterol may down-regulate autophagy through Akt/m TOR signaling pathway,thereby inhibiting the proliferation of A7r5 cells induced by Ang Ⅱ.