Farrerol Inhibits Balloon Injury-induced Intimal Hyperplasia In Rats Via Regulating Of The NLRP3 Inflammasome Activities By Targeting Grp78 In VSMCs

Objective:Intimal hyperplasia（IH）is an important cause resulting in vascular surgery-induced restenosis,which is still an important clinical problem to be solved urgently.The activation of local inflammation response is one of the mechanisms of action by the damaged vascular cells against injury.In our previous study,farrerol,an active ingredient from“man shan hong”,was demonstrated to inhibit the balloon injury-induced IH in rats model and suppress the release of some inflammatory cytokines,including IL-1β,in H₂O₂-induced vascular smooth muscle cells（VSMCs）.It was basically confirmed that farrerol could directly bind to the glucose-regulated protein 78（Grp78）,but the mechanism of action has not been elucidated.Therefore,in the present study,AngiotensinⅡ（AngⅡ）-induced inflammation model in A7r5 cells and balloon-injured intimal hyperplasia model of common carotid artery in rats were performed to investigate the mechanism underlying the inhibitory effects of farrerol against IH by targeting Grp78 to regulate VSMCs inflammation response,so as to provide data support to clarify the targets and mechanisms of flavonoids including farrerol,and lay a foundation for their further development and utilization.Methods:1.To demonstrate the effect of farrerol on NLRP3 inflammasome activity and NF-κB signaling pathway in AngⅡ-induced A7r5 cells,the expression of nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3（NLRP3）and cysteinyl aspartate specific proteinase 1（Caspase-1）,the secretion of interleukin18 and 1β（IL-18,IL-1β）,and the expression of p65 protein of Nuclear factor kappa-B（NF-κB）in the nucleus were investigated.2.Grp78 protein was silenced using si Grp78 and overexpressed using pc DNA3.1（+）-Grp78 plasmids,and the relationship between Grp78 protein and NLRP3inflammasome activity and NF-κB signaling pathway in A7r5 cells was investigated.3.A7r5 cells were treated with cycloheximide（CHX,a protein synthesis inhibitor）,MG-132（a proteasome inhibitor）or Bafilomycin A1（Baf-A1,a V-ATPase inhibitor）to investigate the mechanism underlying farrerol-induced reduction of Grp78 expression.4.The protein expression of Grp78,NLRP3 and caspase-1 were detected using western blot（WB）;the secretion of IL-1βand IL-18 were detected by ELISA;the nuclear translocation of NF-κB p65 was examined by immunofluorescence assay.5.The balloon-induced carotid artery injury rat model in male Sprague-Dawley rats was established.Farrerol（18 mg/kg,36 mg/kg）and 4-PBA（positive drug）were mixed with 25%pluronic F-127,respectively,which were applied around the injured arteries.The vascular segments at the surgical site were taken at a specific time after operation.6.The intimal hyperplasia was detected using HE staining,the protein expression of Grp78 and NLRP3 in injured arteries was detected by immunohistochemistry.Results:1.Farrerol effectively reduces the secretion of IL-1βand IL-18,the protein expression of Caspase-1 and NLRP3 in A7r5 cells and the expression of p65 in the nucleus induced by AngⅡ,suggesting that farrerol inhibits AngⅡ-induced inflammatory response in A7r5 cells by regulating the NF-κB-NLRP3 inflammasome pathway.2.Farrerol effectively inhibits the Grp78 expression in AngⅡ-induced A7r5 cells.si Grp78 effectively reduces the secretion of IL-1βand IL-18,the protein expression of Caspase-1 and NLRP3 in AngⅡ-induced A7r5 cells,as well as the expression of p65 in the nucleus.3.Farrerol effectively inhibits the secretion of inflammatory factors IL-1βand IL-18,and the protein expression of Caspase-1 and NLRP3 induced by Grp78 overexpression in A7r5 cells,as well as the expression of p65 in the nucleus.These results demonstrated that farrerol inhibited AngⅡ-induced VSMCs inflammation by targeting Grp78 and regulating the NF-κB-NLRP3 inflammasome pathway.4.Farrerol decreased the expression of Grp78 significantly examined by treatment with CHX,suggesting that farrerol treatment promotes the degradation of Grp78;the lysosomal inhibitor Baf-A1 effectively restore farrerol-induced decrease of Grp78.In contrast,the proteasome inhibitor MG-132 failed to rescue the Grp78 level,suggesting that farrerol accelerates the degradation of Grp78 may via a lysosomal pathway.5.The results of HE staining and immunohistochemistry showed that balloon injury induced intimal hyperplasia and high expression of Grp78 and NLRP3 in the neointima of the common carotid artery of rats.Farrerol effectively inhibits the intimal hyperplasia along with the expression of Grp78 and NLRP3 in the common carotid artery of rats induced by balloon injury.Conclusion:Farrerol inhibits balloon injury-induced intimal hyperplasia in rats via regulating of the NF-κB signaling pathway in VSMCs to inhibit the activation of NLRP3 inflammasome and reduce the release of inflammatory factors such as IL-1βby inducing Grp78degradation in VSMCs via the lysosomal pathway.