Molecular Mechanism Of LncRNA NEAT1 Regulating Palmitic Acid-induced Pyroptosis Of Hepatocyte

Objective:The prevalence of obesity and metabolic syndrome has rapidly risen in China.Non-alcoholic fatty liver disease(NAFLD)has emerged as the first significant chronic liver disease and the factors that contributed to impaired liver biochemical markers in healthy physical examinations because of the increased incidence of obesity and metabolic syndrome.In the absence of high alcohol consumption or other induces,NAFLD is characterized by excessive lipid deposition and steatohepatitis in the liver.The accumulation of toxic lipids is triggered by an imbalance in lipid homeostasis in liver cells,which either ultimately resulted in organelle dysfunction and cellular damage through apoptosis,necrosis,or pyroptosis.The cause and mechanism of NAFLD disease development is hepatocyte lipotoxicity.It is now acknowledged to be lipotoxicity resulting in organ dysfunction,abnormal intracellular signaling pathway activation,persistent inflammatory response,and cell death.Pyroptosis,a programmed form of inflammatory cell death,is crucial to the development of lipotoxicity.Pyroptosis inhibition might be used as a NAFLD therapy.Long non-coding RNAs(lnc RNAs)serve a critical regulatory function in a variety of biological signaling pathways,as well as an important physiological role in the process of substance metabolism and immune response.The regulation of endocrine and metabolic disorders,obesity,heart disease,and other diseases have been linked to the lnc RNA para-nuclear enriched transcript 1(NEAT1),which is also connected to the regulation of chemokines,cytokines production,and activation of the inflammasome.Further research is necessary to determine whether NEAT1 contributes to pyroptosis brought on by lipotoxicity in hepatocytes and the precise molecular process by which NEAT1 controls pyroptosisrelated inflammation.Our research seeks to identify the molecular mechanisms underlying the regulation of NEAT1 in hepatocyte pyroptosis induced by lipotoxicity.Methods:1.Hep G2 cells were treated with palmitic acid(PA),oleic acid(OA),and their mixture ratio of 1:2,respectively.The cell counting kit 8(CCK-8)was employed to evaluate the survival rate of Hep G2 that had been exposed to various free fatty acids;the contents of triglyceride(TG)in the supernatant were detected by the TG enzyme analysis kit;Quantitative real-time fluorescence polymerase chain reaction(q RT-PCR)was performed to detect the relative m RNA expression levels of apoptosis,inflammation,and fibrosis-related genes,and other related genes to determine the appropriate reagents to induce the cell model of lipid toxic damage in vitro.2.Hep G2 cells were treated with a gradient concentration of PA,and lipid deposition was observed using oil red O staining,and isopropyl alcohol was used to melt intracellularly stained lipid drops.The content of TG released into the supernatant was detected by TG enzyme assay.Lactate dehydrogenase(LDH)release assay kit was used to detect the amount of LDH released by cells to determine the appropriate concentration of PA-induced hepatocyte model of lipid toxic injury in vitro.3.Hep G2 cells were treated with 0.25mmol/L PA,and the protein expressions of pyroptosis-related inflammatory cytokines IL-1β and IL-18 were detected by enzymelinked immunosorbent assay(ELISA).The expression levels of NLRP3 and Cleaved caspase-1 p20 were detected by Western blot.The relative gene expression of NEAT1 was detected by q RT-PCR.4.The liposome was used to transfect overexpressed plasmid(pc DNA3.1-NEAT1)or small interfering RNA(si-NEAT1)to detect the effect of knockdown or overexpression of NEAT1 on PA-induced pyroptosis of Hep G2 cells,and q RT-PCR was used to detect the transfection efficiency.Using CCK-8 to detect the effect of NEAT1 knockdown or overexpression on Hep G2 cell viability after PA stimulation.The effects of knockdown or overexpression of NEAT1 on pyroptosis of Hep G2 cells stimulated by PA were determined by LDH release assay,ELISA,and Western blot.5.The effect of NEAT1 overexpression and NLRP3 knockdown on PA-induced pyroptosis of Hep G2 cells was detected by transfecting the overexpressed plasmid q RTPCR(pc DNA3.1-NEAT1)and/or small interfering RNA(si-NLRP3)with liposome,and the transfection efficiency was detected by q RT-PCR.The effect of overexpression of NEAT1 and/or NLRP3 knockdown on Hep G2 cell viability after PA stimulation was detected by CCK-8.The effects of overexpression of NEAT1 and/or NLRP3 knockdown on pyroptosis of Hep G2 cells after PA stimulation were determined by LDH release assay,ELISA,and Western blot.Results:1.Compared with the blank control group,the cell survival rate in the PA group was significantly decreased,and the genes associated with apoptosis(Bax,Bcl-2,and Cleaved caspase-3),fibrosis(α-SMA and Col.I),and inflammation(NLRP3 and TNF-α)had significantly increased in m RNA levels(P<0.05).Compared with the PA group,the survival rate of cells in the PA group supplemented with twice the concentration of OA was increased(P > 0.05),and the m RNA levels of apoptosis,fibrosis,and inflammation genes were significantly down-regulated(P<0.05).2.With the increase in PA concentration,the survival rate of Hep G2 cells decreased,and the contents of TG and LDH in the cell supernatant increased.The results of oil red O staining showed that 0.25mmol/L PA significantly induced lipid deposition(P<0.05),which suggest that 0.25mmol/L PA treatment of Hep G2 cells for24 h can successfully established an in vitro model of lipotoxicity of hepatocyte.3.Compared with blank control group,0.25mmol/L PA induced significantly upregulated expression of IL-1β,IL-18,NLRP3,Cleaved caspase-1 p20,and long noncoding RNA NEAT1 gene in Hep G2 cells(P<0.01).4.Compared with the vector+PA group,NEAT1 and NLRP3 gene expressions in the NEAT1 overexpression group were significantly up-regulated,the cell survival rate was decreased,LDH,IL-1β,and IL-18 were significantly increased,and NLRP3 protein expression was significantly increased(P<0.05).There were no significant changes in Cleaved caspase-1 p20 protein expression(P > 0.05).Compared with the si-NC+PA group,gene expressions of NEAT1 and NLRP3 were significantly down-regulated,the cell survival rate was increased,LDH,IL-1β,and IL-18 were significantly decreased,and NLRP3 and Cleaved caspase-1 p20 protein expressions were decreased in NEAT1down-regulated group(P<0.05).5.Compared with Vector+si-NC+PA group,NLRP3 gene expression was significantly down-regulated,LDH,IL-1β,and IL-18 were significantly decreased,Cleaved caspase-1 p20 protein expression was decreased(P<0.05),and NLRP3 protein expression was decreased.But there was no statistical significance(P > 0.05);Compared with the pc DNA3.1-NEAT1+si-NC+PA group,Knockdown of NLRP3 based on overexpression of NEAT1 significantly reversed the promotion of overexpression of NEAT1 on NLRP3 gene,cell LDH,IL-1β and IL-18,NLRP3 protein and Cleaved caspase-1 p20 protein expression.The reduction of cell survival rate was reversed(P<0.05).Conclusions:In the cell model of lipotoxicity induced by PA,NEAT1 could regulate the activation of the inflammasome by targeting the expression of NLRP3 and enhancing PA-induced pyroptosis of hepatocyte.