The Effect Of The Huoxue Jiedu Huayu Recipe On Pyroptosis Of Glomerular Mesangial Cells In The Contralateral Kidney Of UUO Rats

Objectives:To observe the effect of the Huoxue Jiedu Huayu recipe on pyroptosis of glomerular mesangial cells in the contralateral kidney of unilateral ureteral obstruction（UUO）rats.Methods:1.Wistar male rats were randomly divided into 4 groups:sham operation group,UUO group,eplerenone group(100 mg·kg^-1·d^-1)and traditional Chinese medicine group(11.7 g·kg^-1·d^-1).Kidney injury was induced by unilateral ureteral ligation in rats.After 10 days,hematoxylin-eosin staining（HE）and Masson staining were used to observe the pathological changes of kidney,TUNEL staining was used to observe cell DNA damage,and immunohistochemical staining was used to observe mineralocorticoid receptor（MR）,MCP-1,NF-κB expression,immunofluorescence staining to detect the expression of F4/80,NLRP3 and SGK-1 and Western blot method to detect NLRP3,caspase-1p20,pro-IL-1βand mature-IL-1β.2.Human mesangial cells induced with aldosterone 24 hours,then treated with serum containing the Huoxue Jiedu Huayu recipe or eplerenone.The following experiments were performed:TUNEL staining was used to observe cell DNA damage,immunofluorescence was used to detect the activation of MR,NF-κB and NLRP3,and Western blotting was used to detect the activation of NLRP3and the expression of IL-1β.Result:1.Pathological changes in the contralateral kidney in UUO ratsHE and Masson staining showed that there were no changes in glomeruli in the sham group,and the glomerular structures were well preserved,with few inflammatory cells observed in the contralateral glomeruli.In the UUO group,a large number of infiltrated inflammatory cells and collagen deposition were observed.There were few inflammatory cells in the eplerenone group and TCM group,which were less than that in the UUO group.2.TUNEL staining to detect DNA damage in the contralateral glomerular cells TUNEL positive cells were stained brown,indicating cellular DNA damage.There were few positive cells in the Sham group.The number of TUNEL-positive cells in the UUO group were significantly increased compared with that of the sham group（P<0.05）.The number of TUNEL-positive cells in the eplerenone group and TCM group were significantly reduced compared with that of the UUO group（P<0.05）.3.Huoxue Jiedu Huayu recipe and eplerenone inhibited UUO-induced activation of NLRP3 inflammation in the contralateral kidneys of rats and reduced inflammationWestern blot results showed that the expression levels of NLRP3,caspase-1p20,pro-IL-1βand mature-IL-1βin the contralateral kidneys of UUO rats were significantly higher than those in the sham group（P<0.05）.4.Huoxue Jiedu Huayu recipe and eplerenone blocked MR/NF-κB activation and reduced kidney damageImmunohistochemical detection of activation of MR showed that there were fewer nucleated cells in the sham group,and MR was mainly expressed in the cytoplasm with less in the nucleus.The number of MR nucleated cells was significantly reduced in the eplerenone and TCM groups compared with that of the UUO group.Immunohistochemical detection of nuclear translocation of NF-κB showed that there were few positive cells in the sham group.The number of positive cells increased significantly in the UUO group compared with that of the sham group.The number of positive cells decreased significantly in the eplerenone and TCM groups compared with that of the UUO group.5.Huoxue Jiedu Huayu recipe and eplerenone reduced the expression of MCP-1 and F4/80 in the contralateral kidney of UUO rats.Immunohistochemical staining showed that MCP-1 expression in renal tissue of the UUO group was significantly enhanced compared with that of sham group rats.Renal tissue MCP-1 expression was significantly reduced in the eplerenone and TCM groups compared with that of UUO group rats.Immunofluorescence results showed that only a small number of F4/80 positive cells were seen in the glomeruli of the sham group rats;the infiltration of F4/80positive cells in the UUO group was significantly increased.Positive cells were significantly reduced in eplerenone group and TCM group compared with that of UUO group rats.6.Observe aldosterone-induced MR and NF-κB activation in HMC with immunofluorescenceMR was mainly expressed in the cytoplasm of control group.Nuclear-positive cells were significantly increased in aldosterone group.Nucleated cells were decreased in eplerenone group and TCM group compared with aldosterone group.It is shown that mesangial cell is the target for injuries induced by aldosterone via activation of MR.In the control group,NF-κB was mainly expressed in the cytoplasm.In the aldosterone group,the number of nuclear-positive cells was significantly increased.The number of nucleated cells was significantly reduced in the eplerenone group and the TCM group compared with the aldosterone group.7.TUNEL staining to detect DNA damage in human mesangial cellsThe number of TUNEL positive cells in the aldosterone group were significantly increased compared with the control group.The number of positive cells were significantly reduced in the eplerenone group and the TCM group compared with the aldosterone group（P<0.05）.8.Huoxue Jiedu Huayu recipe and eplerenone inhibited aldosterone-induced activation of NLRP3 inflammatoryImmunofluorescence results showed that NLRP3 was weakly expressed in the control group,while NLRP3 expression was significantly increased in the aldosterone group.The NLRP3 expression was decreased in the eplerenone group and the TCM group compared with the aldosterone group.Western blot results showed that NLRP3,caspase-1p20 and pro-IL-1βmolecules were all weakly expressed in the control group.The expression of the aldosterone group was significantly enhanced compared with the control group（P<0.05）.The expressions of NLRP3,caspase-1p20 and pro-IL-1βmolecules in the two treatment groups were significantly reduced compared with the aldosterone group（P<0.05）.Conclusion:1.UUO activate MR to mediate the pyroptosis of contralateral renal mesangial cells.2.NLRP3/caspase-1/IL-1βsignal pathway regulates the pyroptosis of mesangial cells,and aldosterone receptor blocker inhibit the activation of MR and alleviate the injury of contralateral kidney.3.Huoxue Jiedu Huayu recipe inhibit the activation of MR,regulate NLRP3/caspase-1/IL-1βsignal pathway and reduce the injury of contralateral renal mesangial cells.