益气活血通络方调控NR4A1/Hippo/YAP途径抑制肾小管上皮细胞G2/M期阻滞拮抗肾纤维化的机制研究OA

Objective:To investigate the mechanism of Yiqi Huoxue Tongluo Formula regulating the solitary nucleus receptor 4A(NR4A1)/Hippo/Yes kinase-associated protein(YAP)pathway to inhibit the G2/M phase arrest of renal tubular epithelial cells(TEC)in unilateral ureteral obstruction(UUO)mice against renal fibrosis(RF).Methods:A total of 40 male C57/BL6 mice were randomly divided into Sham group(Sham group),model group(UUO group),Yiqi Huoxue Tongluo Formula group(YQHX group),NR4A1 agonist Csn-B group(Csn-B group),with 10 mice in each group.UUO modeling was performed in all groups except sham operation group.HE,Masson,and Sirius red staining were used to observe the pathological changes in the kidney tissues.Immunohistochemistry was employed to detect the expression of α-SMA and Vimentin.Western Blot was used to detect the expression of α-SMA,Vimentin,NR4A1,and proteins related to the Hippo/YAP pathway.Immunofluorescence staining was utilized to detect the co-localization of YAP and TEAD1,as well as Ki67 and p-H3.In vitro,HK-2 cells were randomly divided into blank group(Con group),TGF-β1 group(TGF group),TGF-β1+YQHX group(TGF+YQHX group)and TGF-β1+Csn-B group.Except for the blank group,cells in the other groups were stimulated with TGF-β1 to establish a cell fibrosis model,which was treated for 48 hours,cells were collected.Western Blot was used to detect the expression of α-SMA,Vimentin,NR4A1 and proteins related to the Hippo/YAP pathway.Immunofluorescence staining was used to detect the co-localization of YAP and TEAD1,as well as Ki67 and p-H3.Results:In vivo experiments:compared with the UUO group,the degree of renal fibrosis was alleviated in the YQHX group and the Csn-B group,the expression of α-SMA and Vimentin was significantly decreased(P＜0.05),while the expression of NR4A1 and the protein ratios of p-YAP/YAP,p-MST/MST and p-LATS/LATS was significantly increased(P＜0.05),the co-localization of YAP and TEAD1,as well as p-H3 and Ki67,was weakened.In vitro experiments:compared with the TGF group,the expression of α-SMA and Vimentin was significantly decreased in the TGF+YQHX group and the TGF+Csn-B group(P＜0.05),and the expression of NR4A1 and the protein ratios of p-YAP/YAP,p-MST/MST,and p-LATS/LATS were significantly increased(P＜0.05),the co-localization of YAP and TEAD1,as well as p-H3 and Ki67,was weakened.Conclusion:Yiqi Huoxue Tongluo Formula can antagonize RF by regulating NR4A1/Hippo/YAP pathway to inhibit the G2/M phase arrest of TEC.