Study On The Regulatory Mechanism Of Autophagy On Podocyte Injury Induced By Activation Of NLRP3 Inflammasome

Objectives:Renal podocyte injury is one of the main causes of proteinuria in patients with lupus nephritis(LN).Inflammatory bodies of the NLR family Pyrin domain protein 3(NLRP3)are excessively activated in renal podocytes of LN patients,and the degree of activation is positively correlated with the degree of renal podocyte injury.Autophagy also plays a key regulatory role in the process of this injury,but how it plays a regulatory role in renal podocyte damage caused by excessive activation of NLRP3 Inflammasome remains to be studied.Therefore,this experiment is based on the fact that mouse podocytes are widely used to establish a cellular model for the activation of inflammatory corpuscles,so mouse podocytes can be used as the research object.The aim is to provide potential research and theoretical basis for improving renal function and improving quality of life in patients with LN.Methods:1.Western blot(WB)was used to evaluate the expression of Ig G in peripheral blood of LN patients on NLRP3 Inflammasome related protein in mouse renal podocytes;And changes in the expression of podocyte damage marker protein(podocin)at the protein level;2.Using different concentrations of LPS and 100 m M ATP to jointly induce mouse renal podocytes to construct an NLRP3 Inflammasome activation model,real-time quantitative PCR(q PCR)and WB experiments were used to detect NLRP3 Inflammasome related proteins at the m RNA and protein levels,respectively;At the same time,pay attention to the expression of podocin at the protein level.To clarify that this injury originates from the overactivation of NLRP3 Inflammasome,this experiment introduced a NLRP3 Inflammasome specific blocker(MCC950)under the co-induction of LPS and ATP,and detected the expression of NLRP3 Inflammasome related proteins and podocin at the protein level through WB;3.Set NC 、 LPS+ATP 、 autophagy inhibitor(3-MA)+LPS+ATP 、 autophagy activator(RAPA)+LPS+ATP as experimental groups,and detect the changes in cell viability in different experimental groups through CCK-8 experiment;WB and q PCR were used to detect the expression of autophagy and NLRP3 Inflammasome related proteins at the m RNA and protein levels,as well as the expression of the cytokine interleukin-18 downstream of NLRP3 Inflammasome at the m RNA level;Results:1.Compared with NC group,the expression levels of NLRP3 protein and ASC protein in mouse renal podocytes induced by different concentrations of LN patient Ig G were significantly increased in each treatment group(P<0.001).The expression of NLRP3 and ASC proteins was significantly increased(P<0.001)and the expression of podocin protein was significantly decreased(P<0.001)after Ig G treatment with 200μg/m L in mouse renal podocytes.These results suggest that Ig G from LN patients can induce NLRP3 Inflammasome activation and cell damage in mouse renal podocytes;2.After inducing mouse renal podocytes with different concentrations of LPS and100 m M ATP,the results of WB and q PCR experiments showed that compared with NC group,the protein and m RNA expression levels of NLRP3 and ASC were significantly increased in mouse renal podocytes induced by 2 μg/m L LPS and 100 m M ATP(P<0.05),indicating that NLRP3 Inflammasome were activated;The expression level of podocin protein was significantly decreased(P<0.01),indicating that mouse renal podocytes were damaged.Compared with the LPS and ATP induced groups,the expression of NLRP3 Inflammasome related protein was significantly decreased(P<0.001)and the expression of podocin protein was significantly increased(P<0.001)in the renal podocytes of mice introduced into the MCC950 group under the co-induction of LPS and ATP,indicating that the establishment of a successful model of LPS and ATP induced renal podocyte injury and activation of NLRP3 Inflammasome can be used for further experimental study;3.Compared with NC group,LPS+ATP group significantly decreased cell viability(P<0.05);Compared with the LPS+ATP group,the activity of renal podocytes in the 3-MA+LPS+ATP group significantly decreased(P<0.001).Compared with LPS+ATP group,RAPA+LPS+ATP group significantly increased the cell viability of mouse podocytes(P<0.05);Treatment of renal podocytes with RAPA or 3-MA alone can change the autophagic state,but does not cause changes in cell viability.It is suggested that inducing autophagy can alleviate podocyte damage caused by NLRP3activation;4.Compared with the LPS+ATP group,the expression of NLRP3、ASC、caspase1、p62 protein and m RNA was significantly increased(P<0.001),the expression of IL-18 m RNA was significantly increased(P<0.05),the expression of LC3 m RNA was significantly decreased(P<0.001),and the expression of podocin,LC3 II protein was significantly decreased(P<0.05)in the 3-MA+LPS+ATP group.Compared with the LPS+ATP group,the results in the RAPA+LPS+ATP group were contrary to the above.The expressions of NLRP3、ASC、caspase1、and p62 were significantly lower at the m RNA and protein levels(P<0.01),LC3 was significantly higher at the m RNA levels(P<0.001),LC3Ⅱ 、 podocin protein expression was significantly higher(P<0.001),and IL-18 expression was significantly lower at the m RNA level(P<0.001).Conclusions:1.The peripheral blood serum Ig G of LN patients can induce the activation of NLRP3 inflammasome in mouse renal podocytes and cause damage to mouse renal podocytes;2.The use of LPS and ATP can induce the activation of NLRP3 inflammasome in mouse renal podocytes,leading to podocyte damage;3.Inhibiting autophagy can enhance the activation of NLRP3 inflammasome in mouse renal podocytes induced by LPS and ATP,reduce the expression of podocin protein and m RNA,exacerbate damage to renal podocytes,and reduce cell viability.Activating autophagy can alleviate the activation of NLRP3 inflammasome in mouse renal podocytes induced by LPS and ATP,increase the expression of podocin protein and m RNA,alleviate renal podocyte damage,and improve cell viability.It may be effective in treating renal podocyte damage in LN patients.