The Mechanism Of Farrerol Directly Targets Grp78 To Modulate The Phenotypic Conversion Of Vascular Smooth Muscle Cells

Objective:Our previous studies found that farrerol,a natural active compound,has been proved to be effective in inhibiting balloon injury-induced intimal hyperplasia（IH）of the common carotid artery in rats,which is due in part to its inhibitory effects on the proliferation of vascular smooth muscle cells（VSMCs）.glucose-regulated protein 78（Grp78）may be the direct target protein of farrerol,but whether this compound inhibits VSMCs proliferation by targeting Grp78 and the potential mechanism of action remain unclear.In our present study,the regulation and underlying mechanism of farrerol on phenotypic transformation of VSMCs（proliferation,migration,and inflammation）via directly targeting Grp78 were demonstrated using molecular biology methods and bioinformatics methods.Methods:1.The direct binding properties of farrerol to Grp78 and the effects on the function of Grp78 were investigated using cell thermal transition assay（CETSA）combined with western blotting,as well as molecular docking technique and adenosine triphosphatase（ATPase）activity detection method.The human recombinant pure Grp78,the total proteins and A7r5 cell were used for the above detection.2.The target proteins of farrerol inhibiting intimal hyperplasia（IH）were predicted,and the related key signal pathways were analyzed by means of bioinformatics tools,such as STRING database,GO bioannotation analysis and KEGG pathway analysis.The interaction of Grp78 with the proteins in this key pathway were predicted.3.The effects of farrerol and Grp78 si RNA on the phenotypic transition of A7r5 cells,including proliferation,migration,inflammation,and other phenotypes,were investigated using CCK8,Edu staining,scratch test,enzyme-linked immunosorbent assay（ELISA）,flow cytometry,and small interfering RNAs（si RNAs）transfection.The expression of Grp78 and several key proteins in the PI3K/Akt/m TOR signaling pathway,in A7r5 cells pre-treatment with farrerol,Grp78 si RNA or farrerol in combination with Grp78 si RNA,was detected using Western Blot.Results:1.The results of thermal stability and CETSA test showed that farrerol(75μmol·L^-1,150μmol·L^-1,300μmol·L^-1)increased the thermal stability of Grp78 in a concentration-dependent manner,at the levels of pure protein,total proteins and living cells,suggests that farrerol binds to Grp78 directly.The further molecular docking and ATPase activity assay showed that farrerol binds to the ATPase domain of Grp78 at the ATPase domain,and inhibit its ATPase activity.2.GO analysis results suggest that farrerol may inhibit IH by regulating diverse biological processes（BP）and molecular functions（MF）.The BP was mainly involved protein kinase activity and protein serine/threonine kinase activity;MF was mainly involved the negative regulation of apoptosis and protein phosphorylation.KEGG signal pathway enrichment analysis results showed that the PI3K/Akt/m TOR pathway was highly enriched among the 43 predicted pathways（p<0.05）.3.In 10%fetal bovine serum（FBS）-induced A7r5 cells,the results of CCK8 test,Ed U staining assay,scratch assay and flow cytometry showed that farrerol and Grp78 si RNA pre-treatment inhibited cell proliferation,induced cell cycle arrest in the G0/G1 phase,inhibited cell migration,and suppressed the expression Grp78 and activation of the PI3K/Akt/m TOR pathway.Treatment of farrerol combined with Grp78 si RNA show more inhibitory effects in A7r5 cells than the treatment of farrerol or Grp78 si RNA alone.4.In H₂O₂(300μmol·L^-1)-induced A7r5 cells,farrerol and Grp78 si RNA pre-treatment reduced the release of IL-1βand TNF-α;and reversed the suppression of m TOR,but not PI3K,in response to H₂O₂stimulation.Treatment of farrerol combined with Grp78si RNA show stronger effects in A7r5 cells than the treatment of farrerol or Grp78 si RNA alone.Farrerol,but not Grp78 si RNA,can reverse H₂O₂-induced down-regulation of p-Akt in A7r5 cells.Conclusion:Farrerol directly targets Grp78 at the ATPase domain to inhibit the cell proliferation and migration of VSMC via PI3K/Akt/m TOR signaling pathways,and inhibit the inflammatory response of VSMC via the m TOR signaling pathway.This study confirmed that Grp78 was a direct target protein of farrerol inhibiting the proliferation and inflammatory response of VSMC.