Expression And Significance Of Interleukin 35 In Sepsis Induced Acute Kidney Injury

Acute kidney injury(AKI)caused by sepsis is one of the most common complications of sepsis,the pathogenesis is not clear,the clinical treatment is limited,and the mortality is high.To investigate the pathogenesis of septic AKI is a hot issue in the field of kidney disease and critical illness.In addition to the usual pathogenesis:microcirculatory blood flow abnormalities,inflammation,adaptive response of cells to injury and microparticles,etc.It is believed that immune inflammatory response plays an important role in sepsis AKI.Regulatory T cells(Treg)plays a key role in immune suppression in sepsis in the state,it is mainly through cell contact mechanism and cytokine secretion mode of inhibition mediated apoptosis and immune paralysis of immune cells,the inhibition of Treg can improve the prognosis of sepsis.TGF-and IL-10 are the major inhibitory cytokines of Treg secreted,and the novel cytokine IL-35 has stronger inhibitory effect.It can inhibit the proliferation of T cells and the differentiation of Thl7 and the production of IL-17,significantly reduce the inflammatory response,and participate in the regulation of inflammation in a variety of infectious diseases and autoimmune diseases.It is also a new hot spot in the research of immunity,which is related to cell apoptosis.This study intends to proceed from the immune and cytokine IL-35,to observe sepsis AKI mortality and AKI incidence,pathological changes of renal tissue,to investigate the changes of inflammatory factor IL-35 and its relationship with renal tissue injury,provide a new theoretical basis for elucidate the mechanism of sepsis AKI.Methods1.The relationship between the incidence of AKI and mortality in septic miceAcute renal injury model induced by cecal ligation and puncture(cecal ligation and puncture,CLP).The model was replicated 12 times,and each time the mice were taken for 20 mice.The number and size of cecal perforation were adjusted,and the mortality rate of 24h mice was different.The mice were sacrificed after 24h,and the levels of Cr,BUN,ALT and AST were detected.Renal tissue PAS staining was used to evaluate renal tissue injury,and Tunel staining was used to detect the apoptosis of renal cells.Correlation between mortality and incidence of postoperative AKI was analyzed by Pearson.2.The expression of IL-35 in mouse sepsis AKIC57 male mice were induced by cecal ligation and puncture to induce septic AKI.The septic group of 11 mice were induced by AKI for the first time after operation for 24 hours,and the control group was treated with sham operated mice(AKI)in the control group(n =8).Immunohistochemical method was used to detect the expression of IL-35 two subunit IL12A and EBI3 in the kidney,and the expression of IL12A and EBI3 in the kidney was analyzed by immunofluorescence.RT-PCR method was used to detect the expression level of IL12A and EBI3 in the subunit of IL-35,and the level of IL-35 protein was detected by ELISA in mRNA.The correlation between IL-35 level and serum Cr,BUN,ALT and AST levels was analyzed by Pearson.3.IL-35 changes of renal tubular epithelial cells stimulated by LPSThe cells were cultured in DMEM culture medium containing 10%fetal bovine serum,and cultured in medium of 5%C02 and culture medium at 37 DEG C for a density of up to 90%,and then switched to serum-free DMED for 24 h.LPS cells were stimulated with h 0,4 h,8 h,16 h and 24 h,respectively.The cells were collected and cultured with 20ug/ml.The level of LDH in the supernatant was detected,the survival rate of the cells was detected by MTT method,the apoptosis rate was detected by flow cytometry,and the expression of IL12A and EBI3 in the IL-35 subunit of mRNA was detected by RT-PCR.The Lipofectamine with liposome2000 5ul plus IL-35siRNA8ul,siRNA8ul control HKC transfected HKC cells were co cultured with 24h rIL-35 cells,the transfection efficiency was observed by fluorescence microscope.To detect the expression of RT-,PCR subunit EBI3 mRNA.And then to 20ug/ml stimulation of LPS cells were 24 h cells were divided into control group,HKCLPS group,RIL-35LPS group and control group,SiRNALPS IL-35siRNALPS group.The levels of LDH,cell survival rate and apoptosis rate were determined by the same method.Results1.The pathological changes of AKI and the occurrence of AKI in sepsisPAS staining of septic AKI mice renal pathological changes showed that each flat,vacuolar degeneration in renal tubular epithelial cells,some fall off.Renal interstitial edema,a little inflammatory cell infiltration.TUNEL staining showed that the renal tubular epithelial cells of septic AKI mice were more positive than those of the cells.Biochemical detection of AKI is often accompanied by liver function ALT,AST increased significantly.And the higher the mortality of sepsis mice,the higher the incidence of AKI,Pearson correlation analysis confirmed that the incidence of sepsis AKI and mortality(r= 0.893,p=0.001)was significantly negative correlation.2.IL-35 can be expressed in renal tubular epithelial cells,which is associated with abnormal renal functionImmunohistochemistry showed that septic AKI group and control group in the kidney of IL12A and EBI3 subunits were expressed in renal tubular epithelial cells,but there was no expression of glomerular and renal small vessel in the two subunits,semi quantitative counting confirmed septic group AKI IL12A and EBI3 were significantly lower than those of control group(p<0.05).Immunofluorescence assay showed that IL12A and EBI3 subunits were co expressed in the renal tubular epithelial cells of septic AKI group and control group.At the same time,the expression of IL12A and EBI3 in the kidneys of septic AKI group was significantly lower than that in control group(mRNA)(p<0.05).The level of IL-35 protein in renal tissue of sepsis group was 182.39 + ng/mgTP,which was significantly lower than that of control group(260.87 + 69.03 ng/mgTP)(AKI)(p<0.05).Correlation analysis demonstrated that Pearson,IL-35 in kidney of protein level and serum Cr(r=-0.584,p=0.009),ALT(r=-0.549,p=0.015),AST(r=-0.475,p=0.04)are negatively correlated,but not correlated with the serum level of BUN(r=-0.437,p=0.061).3.Inhibiting the expression of IL-35 can increase the apoptosis rate of renal tubular epithelial cellsThe results of in vitro experiments showed that the LDH level of HKC increased and the apoptosis rate increased with the prolongation of stimulation time after LPS stimulation.IL-35mRNA was not expressed in the cultured human renal tubular epithelial cells,and the expression of LPS was increased firstly and then decreased after stimulation.Intervention experiment found that LPS stimulated HKC cells after 24 hours,compared with the control group rIL-35 group,control group siRNA,IL-35siRNA supernatant LDH levels were significantly higher(p<0.05).The apoptosis rate of HKC + LPS group,rIL-35 group and control siRNA group was not significantly different(p>0.05),but the apoptosis rate of IL-35siRNA group was significantly higher than that of other groups(p<0.05).ConclusionsThe results of in vivo and in vitro experiments showed that renal tubular epithelial cells could express inflammatory factor IL-35.The mRNA and protein levels of IL-35 in sepsis AKI were lower than those in the control group,IL-35 was negatively correlated with renal function and liver function,but also negatively correlated with the apoptosis of renal tubular epithelial cells after LPS stimulation.Inhibition of IL-35 expression can increase the apoptosis of renal tubular epithelial cells.It is suggested that the expression of IL-35 in renal tubular epithelial cells may play a protective role in the apoptosis of renal tubular epithelial cells by AKI.