The Effect Of Traditional Chinese Medicine On Pyroptosis By Regulating SGK-1/TLR4 Signal Pathway

Objective:To observe the expression of NOD-like receptor 3(NLRP3),cysteine-aspartic acid protease 1(Caspase-1)and Interleukine-1 beta(IL-1?),which are marks of pyroptosis.And to test whether the changes of serum and glucocorticoid induced kinase-1(SGK-1),Toll-like receptor 4(TLR4),Nuclear factor ?B(NF-?B)are associated with them.And thus,to investigate the mechanism of pyroptosis in obstructed kidney,and how traditional Chinese medicine works against it to provide an evidence for clinical application.Methods: The 48 male Wistar rats weighting(180±20)g were fed a normal diet for a week.And then,the rat were randomly assigned to one of the groups as follows: the sham-operation group(Sham group),the unilateral ureteral obstruction group(UUO group),the Eplerenone treatment group(EPL group)and the traditional Chinese medicine group(ZY group),12 rats each group.Under anesthesia with 10% chloral hydrate(3ml/kg),the the left side of abdominal skin of the rats in UUO group,EPL group and ZY group was sliced,the left ureter was isolated,ligated at two locations,cut between the ligatures,and then Sutured.A sham operation was performed in the sham group,which only had their ureters isolated but not ligated.After surgery,EPL group was treated with Eplerenone at the dosage of 100 mg/kg.d,ZY group with traditional Chinese medicine at 13.5g /kg·d,And the other groups recevied the same volume of physical saline.After 10 days of treatment,the rats were decapitated,the left kidneys were harvested and weighted.A segment of the kidney were fixed in 4% paraformaldehyde and the others were kept in-70 ? for protein extraction.Pyroptosis was examined by TdT-mediated dUTP Nick-End Labeling(TUNEL).And the protein expression of SGK-1,TLR4,NF-?B,NLRP3,Caspase-1 and IL-1?in renal tissues was examined by immunohistochemistry and Western blot analysis.Results:1.The general condition of the ratsThe rats in the sham-operated group were generally in good condition,with no significant changes in drinking,eating,etc.The renal/body weight was(0.0030±0.0004)g.But the intake and weight of rats in UUO group were decreased.Renal/body weight was(0.0138±0.0030)g,which was significantly higher than that in the sham operation group(P<0.01).The general condition of rats in EPL group and ZY group seems better than UUO group,and the renal/weight were(0.0080±0.0031)g and(0.0095±0.0041)g respectively,both decreased(P<0.05).2.Pyroptosis in kidney tissue sections identified by the d UTP nick-end labeling(TUNEL)assay.The positive rate in the UUO group was(36.79±4.67)%,which was significantly higher than that in the sham group(2.69±1.01)%(P<0.01).The positive rates in the two treatment groups were(22.35±3.26)% and(24.08±5.06)% respectively,and the number of TUNEL-positive cells decreased compared with that in UUO group(P<0.05).3.The expressions of SGK-1,TLR4,NF-?B,NLRP3,Caspase-1 and IL-1? characterized by immunohistochemistryOnly few SGK-1,NF-?B,NLRP3,Caspase-1 and IL-1?positive cells were observed in sham group,which were significantly increased in renal tubular epithelial cell in UUO group.EPL or traditional Chinese medicine treatment resulted in lower positive cells than those in UUO group.We detect few TLR4 staining in glomerulus and tubulointerstitial in sham group,in contrast to a marked expression of that in UUO group.And the expression of TLR4 attenuated under EPL or traditional Chinese medicine treatment.4.The Protien expressions of SGK-1,TLR4,NF-?B,NLRP3,Caspase-1 and IL-1? detected by western blot.Western blot analysis demonstrated that the expressions of SGK-1,TLR4,NF-?B,NLRP3,Caspase-1 and IL-1? significantly higher in the UUO group than that in the sham group(P<0.05)and were lower in the EPL group and ZY group(P<0.05).Protein expression of TLR4 was increased in UUO group(P<0.05)and markedly decreased in EPL group(P<0.05),which was even lower than sham group.And the expression of TLR4 in ZY group was lower than that in UUO group but higher than that in sham group(P<0.05).Conclusions:1.Aldosterone increased the expression of NF-?B through SGK-1/TLR4 pathway,triggered the activation of NLRP3,and led to Caspase-1 induced pyroptosis.2.Eplerenone suppressed the pyroptosis through the inhibition of Aldosterone,which can decrease the express of TLR4 and SGK-1.3.Traditional Chinese medicine could decrease Caspase-1 induced pyroptosis through inhabiting the expression of NLRP3,which may involved the down-regulation of TLR4 and SGK-1.