| In the process of livestock and poultry breeding,animals are often stimulated by environmental factors such as bacteria,viruses and toxins,resulting in liver damage,hepatocyte necrosis and hepatic dysfunction,and ultimately inhibiting the growth of livestock and poultry,which results in great economic losses to animal husbandry.Liver is not only an important metabolic organ,but also participates in immune response.Kupffer cells(KCs)are the main macrophages in the liver.As the first line of defense in the liver,KCs can remove foreign substances such as bacteria and toxins,and play an important role in liver injury by releasing various inflammatory mediators.Studies have shown that activated macrophages leads to changes in lipid metabolism and energy homeostasis.In addition,some lipid metabolic factors may modulate the inflammatory response of macrophages.The imbalance of lipid metabolism in macrophages and its dysfunction are mutually causal.However,until now,the relationship between the inflammatory function of KCs and lipid metabolism,as well as how to regulate KCs inflammation through lipid mediation is not clear.Therefore,this study first explored the effects of LPS on liver and lipid content of KCs through in vivo and in vitro inflammation model,and revealed that expression of GPAT3 in inflammatory KCs was significantly changed.Secondly,some experimental methods,such as interference,knockout and inhibitor were used to explore the effects of GPAT3 on liver and KCs inflammation in mice and its mechanism.Finally,the effects and mechanism of DHA and AA on LPS-induced KCs inflammation and pyroptosis in mice were investigated.1 LPS induced lipid deposition in mouse liver and lipid metabolism remodeling in Kupffer cellsSPF male mice aged 6-8 weeks were randomly divided into two groups:control group and LPS group.LPS group was injected intraperitoneally with 5 mg/kg LPS,and control group was injected with equal volume of normal saline.After 12 h,plasma and liver were collected for subsequent analysis.The levels of plasma alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)were significantly increased after LPS treatment(P<0.05);In addition,the expression of genes associated with liver inflammation was significantly increased in LPS-treated mice(P<0.05).These results indicated that LPS successfully induced liver injury and established inflammation model in mice.After that,plasma triglyceride(TG)levels in mice were detected and LPS significantly increased plasma TG content(P<0.05).Oil red staining and TG content detection also showed that LPS significantly increased TG content in liver(P<0.05).Kupffer cells(KCs)are the first barrier of immune defense in liver,so we used Kupffer cell line to further study.We found that LPS caused the accumulation of TG and lipid droplets in KCs(P<0.05).Lipidomics analysis showed that lipid components were significantly changed between LPS and control group(P<0.05).Lipidomics analysis showed that the content of TG,fatty acids and phospholipids in LPS-treated Kupffer cells were significantly changed compared with the control group(P<0.05),meanwhile,the content of polyunsaturated fatty acids decreased significantly after LPS treatment(P<0.05).In addition,the results showed that metabolism was the main category in KEGG analysis,and lipid metabolism was the most influential metabolic pathway in LPS-treated Kupffer cells(KCs)by Transcriptomic analysis.Which indicates the importance of lipid metabolism in LPS-stimulated KCs.Many genes involved in the synthesis and utilization of triglyceride(TG)were significantly changed in LPSactivated Kupffer cells.And GPAT3 was the most significantly up-regulated gene after LPS treatment(P<0.05).These results indicated that LPS induced liver injury and inflammation,and increased TG content in plasma and liver.In addition,LPS stimulated KCs,resulting in accumulation of TG and lipid droplets,inducing lipid metabolism remodeling and GPAT3 was significantly upregulated.2 Interference with GPAT3 improves LPS-induced inflammation and mitochondrial damage in Kupffer cellsIn the previous chapter,we found that GPAT3 was the most significantly changed gene among the up-regulated lipid metabolism genes after LPS treatment(P<0.05).In this chapter,we further verified by qPCR and Western blot,and the results showed that LPS treatment significantly increased(P<0.05)GPAT3 expression in KCs.In addition,GPAT3 mRNA expression was significantly increased after KCs inflammation induced by TNF-α(P<0.05).GPAT3 catalyzed the production of glycerol-3-phosphate into lysophosphatidic acid(LPA).ELISA results showed that LPA content and lysophosphatidylcholine(LPC)level of KCs were significantly increased after LPS treatment(P<0.05).These results suggested that GPAT3 may play an important role in LPS-induced Kupffer cell inflammation.Transfection of GPAT3 siRNA showed that GPAT3 deletion significantly improved LPS-induced KCs inflammation(P<0.05).In addition,GPAT3 deletion significantly inhibited the accumulation of TG and lipid droplets in LPS-treated KCs(P<0.05).Transcriptome analysis showed that LPS significantly reduced the expression of Cpt1a in Kupffer cells(P<0.05),qPCR analysis further verified suggesting that LPS may affect mitochondrial function.Flow cytometry and fluorescence staining showed that Interference with GPAT3 significantly improved LPS-induced KCs mitochondrial dysfunction(P<0.05).Transmission electron microscopy(TEM)further showed that LPS induced mitochondrial swelling and cristbreaking in Kupffer cells,which was improved by GPAT3 deletion.Finally,FSG67,an inhibitor of GPAT,was used to treat KCs,and FSG67 significantly reduced LPS-induced KCs inflammation(P<0.05).These results suggest that GPAT3 expression is significantly increased in inflammatory Kupffer cells,while GPAT3 deletion and the inhibitor FSG67 can ameliorate LPS-induced KCs inflammation and mitochondrial dysfunction.3 GPAT3 deletion improves LPS-induced hepatic injury and inflammation in miceThis chapter further explored the effects of GPAT3 on inflammation through in vivo experiment.First,we used GPAT3 knockout mice and intraperitoneal injection of LPS to analyze its effects on liver inflammation.The results showed that plasma levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)in GPAT3 knockout mice were significantly decreased after LPS treatment(P<0.05),and the contents of IL-1α and TNF-α in plasma were significantly decreased(P<0.05).HE staining showed that GPAT3 knockout improved LPS-induced liver inflammatory cell infiltration and vacuolation.Primary Kupffer cells(KCs)and hepatocytes were isolated for further analysis,the results showed that GPAT3 deletion significantly reduced the expression of inflammatory factor IL-1α,IL-6 and MCP-1(P<0.05).Meanwhile,the mitochondrial membrane potential was significantly increased after GPAT3 knockout(P<0.05).In addition,inflammatory markers in primary hepatocytes were also significantly inhibited after GPAT3 knockout under LPS treatment(P<0.05).Finally,mice were intraperitoneally injected with GPAT3 inhibitor FSG67,and FSG67 treatment significantly inhibited LPS-induced liver injury and inflammatory response(P<0.05).CD68 is one of the macrophages marker,and the expression of CD68 is closely related to activated KCs.Immunohistochemical results showed that inhibition of GPAT3 significantly reduced LPS-induced CD68 expression,indicating that FSG67 inhibited LPS-induced activation of KCs M1 type.These results suggested that GPAT3 deficiency alleviates LPS-induced liver injury,inflammation of primary KCs and hepatocyte,and significantly increases mitochondrial membrane potential of primary KCs.4 GPAT3-mediated LPA promotes Kupffer cells inflammation and mitochondrial damage through the P-ERK/P-C-Jun pathwayThis chapter explored the potential mechanism of GPAT3 in LPS-induced inflammation of Kupffer cells(KCs).The results showed that the loss of GPAT3 significantly reduced LPS-induced accumulation of lysophosphatidic acid(LPA)(P<0.05),which suggested that LPS up-regulation of GPAT3 leads to accumulation of LPA content and may promote KCs inflammatory response.Therefore,we tested our hypothesis by treating KCs with LPA.It was found that LPA treatment resulted in inflammation of KCs and decreased mitochondrial membrane potential(P<0.05).We further demonstrated in vivo that plasma levels of IL-6 and IL-1β were significantly increased in mice of LPA treatment(P<0.05),and damage and inflammation of liver also develops(P<0.05).Further,we explored the mechanism of LPA promoting KCs inflammation.The results showed that LPA significantly increased the protein expression of PKCθ/P-ERK1/2 and P-c-Jun in KCs(P<0.05).Consistent with the above,GPAT3 deletion significantly inhibited the protein expression of PKCθ/P-ERK1/2 and P-c-Jun in LPS-stimulated KCs(P<0.05).Treatment with ERK inhibitor U0126 further confirmed that LPA functions a role through P-ERK1/2 and P-C-Jun signaling pathways(P<0.05).These results indicated that GPAT3-mediated LPA promotes KCs inflammation and mitochondrial damage through PKCθ/P-ERK/P-c-Jun signaling pathway.5 DHA/AA promotes the interaction between GPR120 and NLRP3 to alleviate LPSinduced pyroptosis in Kupffer cellsIn chapter 4,we found that the contents of fatty acids in LPS-treated Kupffer cells(KCs)were significantly changed,and the content of polyunsaturated fatty acids decreased significantly after LPS treatment.Docosahexaenoic acid(DHA)and arachidonic acid(AA)are typical representatives of omega-3 and omega-6 polyunsaturated fatty acids,respectively.DHA has been reported to have anti-inflammatory effects,while the effect of AA on inflammation is not clear at present.Pyroptosis is a kind of inflammatory necrosis of cells,which is related to many inflammatory diseases.The effect and mechanism of DHA and AA on pyroptosis of KCs has not been reported.The purpose of this study was to investigate the effects of DHA and AA on LPS-induced inflammation and pyroptosis of KCs and its mechanism.The results showed that DHA/AA improved LPS-induced KCs pyroptosis by inhibiting the expression of NLRP3 inflammasome complex,IL-1β,IL-18 and GSDMD.In addition,the results of PI fluorescence staining also confirmed this conclusion.We also found that silencing GPR120 eliminated the anti-pyroptosis effects of DHA/AA in LPS-induced KCs(P<0.05),suggesting that DHA/AA functions through GPR120 signaling pathway.LPS treatment significantly reduced the protein expression of GPR120 on cell membrane(P<0.05),and the protein expression of GPR120 was significantly increased in cytoplasm(P<0.05).,which suggested that GPR120 was endocytosis from cell membrane into cytoplasm under LPS stimulation.In addition,LC-MS/MS analysis and immunofluorescence showed that GPR120 was bound to NLRP3 in LPS-stimulated KCs.The immunoprecipitation results showed that DHA/AA promoted the interaction between GPR120 and NLRP3 in LPSstimulated KCs,thereby inhibiting the assembly of NLRP3 inflammasome complex.In addition,DHA/AA significantly alleviated LPS-induced liver injury of mice in animal experiments(P<0.05).Also,we further demonstrated the anti-pyroptosis effects of DHA and AA on primary KCs.These results suggested that DHA/AA inhibits the assembly of inflammasome complex by promoting endocytosis of GPR120 and interacting with NLRP3,thereby alleviates LPSinduced KCs coke death. |
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