Ginsenoside Rb1 Alleviates Renal Cell Pyroptosis Induced By 3-chloro-1,2-propanediol Through Mitophagy

Pyroptosis was a form of programmed cell death caused by inflammasomes and was closely associated with a variety of diseases.3-chloro-1,2-propanediol(3-MCPD)was a heat-induced food processing contaminant that the most sensitive target organs are the kidneys and male reproductive organs.Notably,both in vivo and in vitro studies have shown that 3-MCPD triggered the NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome and caused renal cell pyroptosis.Research has demonstrated that ginsenoside Rb1(Gs-Rb1)has a variety of pharmacological effects on people,including antioxidant,anti-inflammatory,immunomodulatory and neuroprotective effects.Mitochondria were important organelles for energy production and metabolism in eukaryotic cells,and damage to mitochondria leads to mitochondrial dysfunction and accumulation of reactive oxygen species(ROS)in mitochondria.Damaged or dysfunctional mitochondria can be degraded by mitophagy to maintain intracellular homeostasis.Lysosomal biosynthesis and autophagy were both transcriptionally regulated in large part by TFEB.Previously,we found that Gs-Rb1 could ameliorate 3-MCPD-induced liver injury by stimulating autophagic flux,suggesting that Gs-Rb1 may be a potential natural protector that counteract the toxicity of 3-MCPD.However,the protective effect of Gs-Rb1 against3-MCPD-induced pyroptosis and its regulatory mechanism remained unclear.Consequently,it is meaningful to further investigate whether Gs-Rb1 can ameliorate 3-MCPD-induced renal cell pyroptosis and its targets of action on the prevention and mitigation of 3-MCPD-induced renal injury,while providing a certain theoretical basis.The objective of this research was to examine the protective effect of Gs-Rb1 against 3-MCPD-induced pyroptosis and to elucidate in depth the specific mechanism by which Gs-Rb1 ameliorates 3-MCPD-induced pyroptosis.The main studies and results are as follows.(1)Gs-Rb1 alleviates 3-MCPD-induced pyroptosis in ICR mouse kidney cells through activation of mitophagy.Gs-Rb1 was determined to alleviate 3-MCPD-induced kidney injury in ICR mice by H&E staining,blood urea nitrogen(BUN),creatinine(CRE)and urine albumin creatine(UA)content assay and mouse kidney weight ratio;by using an enzyme-linked immunosorbent assay,the release of the inflammatory factors IL-1β and IL-18 in mouse serum and cell culture supernatant was discovered,and the results showed that Gs-Rb1 could significantly reduce 3-MCPD-induced inflammatory factors.The results showed that Gs-Rb1 significantly reduced 3-MCPDinduced IL-1β and IL-18 release;the results of serum lactate dehydrogenase(LDH)release assay in mice also showed that Gs-Rb1 significantly reduced 3-MCPD-induced LDH release;the results of Western blot assay for pyroptosis-related proteins in mouse kidney tissues determined that Gs-Rb1 significantly inhibited 3-MCPD-induced NLRP3 activation and markedly down-regulated gasdermin D-N(GSDMD-N)and Cleaved Caspase-1 protein expression levels;and the producition of ROS in ICR mouse kidney cells was examined by using the fluorescent probe DCFH-DA,and the results showed that Gs-Rb1 could significantly inhibit 3-MCPD-induced ROS accumulation;by examining the levels of microtubule-associated protein1 light chain 3(LC3),sequestosome 1(P62),PTEN induced putative kinase 1(PINK1),and Parkinson disease protein 2(Parkin),four mitophagy-related proteins.The results showed that Gs-Rb1 significantly up-regulated LC3-Ⅱ,PINK1,and Parkin protein expression and downregulated P62 protein expression;while the use of autophagy inhibitor Chloroquine(CQ)reversed these trends and up-regulated NLRP3,GSDMD-N,and Cleaved caspase-1 protein expression levels;and PINK1 and LC3 Co-Location detected using tissue immunofluorescence staining.The results showed that Gs-Rb1 significantly promoted the co-localization of PINK1 and LC3,however,the addition of CQ inhibited the effect of Gs-Rb1 in promoting the co-localization of PINK1 and LC3.The above experimental results demonstrated that Gs-Rb1 could alleviate 3-MCPD-induced renal cells pyroptosis in ICR mice by activating mitophagy.(2)Gs-Rb1 alleviated 3-MCPD-induced NRK-52 E cell pyroptosis by promoting mitophagy.The non-toxic dose of Gs-Rb1 was determined by methyl thiazolyl tetrazolium(MTT)and LDH assays and appropriate concentrations were selected for subsequent experiments,and Gs-Rb1 was shown to mitigate 3-MCPD-induced NRK-52 E cell death;and ELISA was used to measure the release of IL-1β and IL-18 in the cell supernatant,indicating that Gs-Rb1 inhibited 3-MCPD in NRK-52 E cells induced IL-1β and IL-18 release in NRK-52 E cells;assaying the effect of Gs-Rb1 on intracellular pyroptosis protein expression,the results likewise showed that Gs-Rb1 significantly inhibited the protein expression of NLRP3,GSDMD-N and Cleaved caspase-1 in vitro;assaying the effect of Gs-Rb1 on intracellular mitophagy protein expression,it was likewise found that Gs-Rb1 also significantly increased the expression of the proteins PINK1,Parkin,LC3-II,and P62,decreased the expression of P62,and suppressed NLRP3.,and inhibited NLRP3,GSDMD-N,and Cleaved caspase-1 protein expression in vitro,while this phenomenon were abolished by the addition of mitophagy inhibitor Cyclosporin A(Cs A)in vitro;cellular immunofluorescence staining identified the co-expression of PINK1 and LC3 in NRK-52 E cells.The co-localization of PINK1 and LC3 in NRK-52 E cells was significantly increased by Gs-Rb1,while the presence of mitophagy inhibitor(Cs A)also abolished this phenomenon in vitro;Using fluorescent sensors DCFH-DA and JC-1,the intracellular ROS levels and mitochondrial membrane potential(MMP)were detected,and the results also showed that Cs A pretreatment reversed Gs-Rb1 inhibition of ROS accumulation and decrease in MMP.These results suggest that Gs-Rb1 can inhibit 3-MCPD-induced NRK-52 E cell pyroptosis by promoting mitophagy.(3)Gs-Rb1 alleviated 3-MCPD-induced NRK-52 E cell pyroptosis by promoting mitophagy through the TFEB signaling pathway.It was found that Gs-Rb1 increased TFEB expression in mouse kidney and NRK-52 E cells,promoted nuclear TFEB protein expression and decreased cytoplasmic TFEB protein expression,indicating that TFEB may be involved in regulating 3-MCPD-induced pyroptosis alleviated by Gs-Rb1.The expression levels of the pyroptosis-related proteins NLRP3,GSDMD-N,and Cleaved Caspase-1 and mitophagy-related proteins LC3,P62,PINK1,and Parkin were found by small interference with TFEB-treated NRK-52 E cells,and the results showed that NRK-52 E cells with pretreatment knockdown of TFEB,the expression of the proteins LC3-II,NLRP3,GSDMD-N,and Cleaved caspase-1 increased,while that of PINK1 and Parkin decreased;similarly,the results of Mito-Trccker and LC3 co-localization showed that Gs-Rb1 promoted Mito-Tracker and LC3 co-localization,while si-TFEB reversed this phenomenon.The results indicated that Gs-Rb1 activated mitophagy through TFEB signaling pathway and thus improved 3-MCPD-induced NRK-52 E cells pyroptosis.