Effects Of Paeonol On Toll-like Receptor 4 Protein Expression And Pyroptosis In Renal Ischemia Reperfusion Injury In Rats

Objective:In this study,an animal model of renal ischemic reperfusion injury(RIRI)was established in SD rats.The rats were pretreated with paeonol(Pae)and TAK242,a Toll-like receptor 4 inhibitor,by intraperitoneal injection.To observe the changes of renal function,renal morphology,pyroptosis-related proteins and inflammatory factors in rats,and to explore the role of paeonol in renal I/R injury and its possible mechanism,so as to find an effective target for clinical prevention and treatment of RIRI.Methods:Thirty SPF SD rats,weighing about 170-200 g,were randomly divided into Sham operation group(Sham group),model group(RIRI group),paeonol group(Pae group),TLR4 inhibitor group(TAK242 group)and paeonol +TLR4 inhibitor group(RIRI+TAK242 group),with 6 rats in each group.In Sham group,only bilateral renal pedicles were free without any operation.In the other groups,bilateral renal pedicles were clipped for 45 min and then perfused for 24 h.The levels of serum creatinine(Scr),blood urea nitrogen(BUN)and cystatin C(Cys-C)in each group were detected to understand the degree of renal function damage.Elisa method was used to detect the levels of inflammatory factors(IL-1β,IL-18)in serum,and HE staining was used to observe the morphological changes of kidney tissue.The expression and distribution of TLR4,My D88 and pyroptosis related proteins(NLRP3,Caspase-1,GSDMD,IL-1β,IL-18)in kidney tissue were detected by immunohistochemistry.The expression levels of TLR4,My D88 and pyroptosis related proteins were detected by Western blot.Results:1.Evaluation of modeling effect: The bilateral kidneys of rats in Sham group were always redbrown with smooth and shiny surfaces.In the RIRI group,RIRI+Pae group,RIRI+TAK242 group and Pae+TAK242 group,the bilateral kidneys were red brown before clamping the bilateral renal pedicle,and purple black after clamping the bilateral renal pedicle.After loosening the bilateral renal pedicle,the bilateral kidney color returned to red brown,indicating that the modeling was successful and the modeling effect was good.Renal ischemia-reperfusion was completed in all groups except Sham group.2.Renal function test results of each group: Compared with Sham group,Scr,BUN and Cys-C levels were increased in RIRI group(p < 0.05).Compared with RIRI group,the levels of Scr,BUN and Cys-C in RIRI+Pae group,RIRI+TAK242 group and Pae+TAK242 group were decreased,and the differences were statistically significant(p < 0.05).3.HE staining results: Compared with Sham group,the renal tissue structure of RIRI group was disordered,and the renal tubular epithelial cells were severely degenerated and necrotic.Compared with RIRI group,the degree of renal tissue damage in RIRI+Pae group,RIRI+TAK242 group and Pae+TAK242 group was reduced.4.Immunohistochemical results: Compared with Sham group,RIRI group,RIRI+Pae group,RIRI+TAK242 group and Pae+TAK242group showed that Toll like receptor-4,TLR4),myeloid differentiation factor 88(My D88),Caspase-1,interleukin-18,IL-18)and interleukin-1β(IL-1β)expression increased.Compared with the RIRI group,the expressions of TLR4,My D88,Caspase-1,IL-18 and IL-1β in the RIRI+Pae group,RIRI+TAK242 group and Pae+TAK242 group were decreased.5.ELISA results: Compared with Sham group,the levels of IL-18 and IL-1β in RIRI group,Pae group,TAK242 group and Pae+TAK242 group were increased.Compared with RIRI group,the levels of IL-18 and IL-1β in RIRI+Pae group,RIRI+TAK242 group and Pae+TAK242 group were decreased,and the differences were statistically significant(all P < 0.05)。6.Protein immunoblotting results: Compared with Sham group,the expression levels of TLR4,My D88 and pyroptosis related proteins were increased in RIRI group.Compared with RIRI group,the expression levels of TLR4,My D88 and pyroptosis related proteins in RIRI+Pae group,RIRI+TAK242 group and Pae+TAK242 group were decreased,and the differences were statistically significant(all P<0.05).Conclusions:Renal ischemia-reperfusion injury can cause serious damage of renal tissue structure,decrease of renal function and occurrence of inflammatory reaction.Paeonol pretreatment can improve the renal function injury after RIRI in rats,and the mechanism may be to inhibit TLR4-My D88-NLRP3 signaling pathway,then inhibit pyroptosis,reduce the release of inflammatory factors,alleviate renal ischemia reperfusion injury,and play a protective role in the kidney.