Tangshen Formula Alleviates Diabetic Kidney Injury By Regulating Canonical NLRP3 Inflammasome Pathway

Diabetic kidney disease(DKD)is a sever microvascular complication of diabetes,and also the main cause of end-stage renal disease(ESDR).Systemic and local persistent low-grade inflammation caused by imbalanced innate immunity plays an important role in the development of DKD,The inappropriate activation of inflammasome is involved in the imbalanced status of innate immunity.Inflammasome is a multi-protein complex composed of cytoplasmic pattern recognition receptor,ASC protein and caspase-1,which can be activated by the stimulation of a variety of intracellular dangerous signals.Activated caspase-1 is the functional unit of inflammasome,which promotes the production of pro-inflammatory factors(IL-1?and IL-18)and leads to a special programmed cell death-pyroptosis.Among the known types of inflammasome,NLRP3 inflammasome is most widely and deeply studied.The canonical NLRP3 inflammasome pathway depends on the activation of caspase-1.Many studies proved that the mature and release of IL-1?/IL-18 induced by caspase 1(NLRP3/caspase 1/IL-1?/IL-18)are closely related to renal endothelial dysfunction,tubulointerstitial fibrosis and podocyte damage of DKD,but the role of caspase 1-induced pyroptosis in DKD remains to be further explored.At present,the treatment of DKD is limited.As the inhibition of NLRP3 inflammasome shows protective effects in renal,targeting NLRP3 inflammasome is expected to be a new strategy for DKD therapy.The traditional Chinese medicine Tangshen formula(TSF)is developed by Professor Li Ping's group,which based on the previous experiences and clinical practices.TSF was observed to reduce the urinary protein and improve the glomerular filtration rate(GFR)of T2DKD patients in our previous RCT trial.The basic studies proved the multi-effects of TSF on renal protection,such as anti-inflammation,anti-fibrosis,anti-oxidation and regulation of dyslipidemia.The aim of this study is to further explore the mechanism of TSF for DKD therapy,focusing on its effects on the canonical NLRP3 inflammasome pathway and pyroptosis.Purpose:1.To clarify that inflammation and pyroptosis caused by abnormal activation of canonical NLRP3 inflammasome pathway lead to diabetic kidney injuries.2.To confirm that TSF alleviates inflammation and pyroptosis by regulating the canonical NLRP3 inflammasome pathway.Method:1.Animal experiment groups,establishment of DKD model and TSF administration:thirty-nine SD rats of 6-week-age were randomly divided into control(number=13),DKD(number=13)and DKD+TSF group(number=13).DKD models were established by right nephrectomy and single intraperitoneal injection of streptozotocin(STZ)one week after operation.About 72h after STZ injection,rats with glucose over 16.7mmol/L were confirmed to be in diabetic state.Rats in control group were performed sham operation and intraperitoneal injection of sodium citrate buffer.Administration was performed 72 hours after STZ/sodium citrate buffer injection.Rats of TSF+DKD group were treated with TSF(2.4g/kg body weight/day)for 20 weeks,the other two groups were given distilled water.Blood glucose,body weight and urine volume were measured during the experiment.Serum creatinine,urea nitrogen,serum IL-1? level and 24-hour urine protein(24-UP)level were measured at the end of the experiment.Kidney tissues were collected for follow-up experiment.2.Histological examination:the H&E,Masson and PAS stains were performed on kidney sections to evaluate the pathological damages;the expression of NLRP3 and IL-1? in renal tissue was detected by immunohistochemistry(IHC);the mRNA and protein expression of NLRP3,caspase 1,ASC and IL-1? in renal were detected by RT-PCR and western blot.3.Screening stimulating conditions and CCK8 experiment:HK-2 cells were stimulated by AGEs of different concentrations.The IL-1? level in supernatant and caspase-1 activity in cell lysate were detected at three time points(12h,24h,48h),so as to screen the best stimulating conditions.The intervened concentration of TSF was determined according to CCK8 experiment and our previous studies.4.Vitro experiment groups,intervention and indicators to be tested:HK-2 cells were divided into control group,AGEs group,AGEs+TSF group,AGEs+TSF+nigericin(nig)group.The AGEs group includes three subgroups:12h,24h and 48h.Two subgroups were set in AGEs+ TSF group and AGEs+TSF+nig group,according to different concentrations of TSF intervention(250ug/ml and 500ug/ml).In AGEs+TSF group and AGEs + TSF+nigericin group,cells were treated by AGEs plus TSF for 48 hours.Nigericin was added one hour before cell collection.At the end of the experiment,the level of IL-1? in the supernatant and the activity of caspase-1 in the lysate were measured.The protein was extracted and the expressions of NLRP3,ASC,caspase-1 and IL-1? were tested by western blot.TUNNEL and PI stain were performed to detect the DNA injury and membrane integrity preliminarily.5.Vitro experiment groups,intervention and detection of pyroptosis:HK-2 cells were divided into control group,AGEs group,AGEs+Ac YVAD cmk group and AGEs+TSF group.The intervention of AGEs was 400ug/ml for 48h,and the TSF was 500ug/ml for 48h.In AGEs+Ac YVAD cmk group,cells were pre-treated by Ac YVAD cmk 30 minutes before AGEs stimulation.At the end of the experiment,the LDH and ATP levels were detected by the commercial kits.Stains with calcein AM/PI were performed to observe cell death.The caspase 1 was detected by immunofluorescence assay on the fixed cells.The cleaved caspase 1,GSDMD/GSDMD-N and NLRP3 were detected by WB.Results:1.TSF reduced the 24h-UP level,alleviated the systemic inflammatory status(serum IL-? level)and pathological damages(fibrosis,expanded mesangial area,tubular damage).It also inhibited the mRNA and protein expression of the components of NLRP3 inflammasome in kidney,as well as its downstream IL-1?.2.TSF inhibited the activation of NLRP3 inflammasome in AGEs stimulated HK-2 cells,illustrated by the decreased IL-1? in supernatant,the decreased caspase-1 activity and the down-regulated NLRP3 inflammasome protein expression.These inhibition effects of TSF can be restored by NLRP3 inflammasome agonist(nigericin).TSF reduced membrane and DNA damage induced by AGEs.3.TSF inhibited the pyroptosis of HK-2 cells induced by AGEs.Compared with the control group,HK-2 cells with AGEs stimulating showed increased LDH release,decreased ATP concentration,increased PI positive cells,decreased Calcein-AM positive cells,increased caspase-1 positive cells,as well as upregulated expression of GSDMD/GSDMD-N,cleaved-caspase 1(p20)and NLRP3.Pre-treatment with Ac YVAD cmk(caspase-1 inhibitor)could significantly reduce the dead cells,as well as the protein expression of pyroptosis pathway(NLRP3/caspase 1/GSDMD).All above proved that AGEs induced caspase-1 dependent cell death-pyroptosis.With TSF intervention,the number of pyroptotic cell decreased significantly,and the expression of pyroptosis pathway molecules were inhibited.Conclusion:1.TSF reduces the generation of inflammatory factor and alleviates diabetic kidney injury by inhibiting the activation of canonical NLRP3 inflammasome pathway.2.AGEs induces caspase 1-dependent pyroptosis in HK-2 cells.TSF inhibits pyroptosis by regulating the canonical NLRP3 inflammasome pathway.