| Heat stress has become one of the important factors restricting the development of the live pig industry.Heat stress can seriously reduce the performance,body health and reduce the quality of pork.Epigallocatechin gallate(EGCG),as the main polyphenolic substance in green tea,plays an important role in regulating fat deposition,fat metabolism and cell apoptosis.In this study,we constructed the heat stress model of porcine adipocytes in vitro by using subcutaneous and intramuscular preadipocytes as the research materials,to investigate the effects of EGCG on fat deposition and metabolism of porcine subcutaneous and intramuscular preadipocytes by regulating the amp activated protein kinase(AMPK)signaling pathway and its molecular mechanism,as well as its protective effect on cell apoptosis.It provides a scientific theoretical basis for the future development of EGCG as a green and safe feed additive which can alleviate heat stress and improve pork quality by regulating fat deposition and metabolism.The main research results are as follows:1.Effects of heat stress on fat deposition and metabolism of porcine subcutaneous preadipocytes and EGCG regulation mechanism.(1)The heat stress model of porcine subcutaneous preadipocytes was established by high temperature stimulation at 41.5℃ and the induced differentiation test results showed that: heat stress could induce heat shock response after differentiation of porcine subcutaneous preadipocytes,and promote the expression of HSP60,HSP70 and HSP90.Heat stress promoted fat deposition by inhibiting the AMPK-SIRT1-PGC-1α signaling pathway and up-regulated the expressions of PPARγ,CEBPα,AP2 and FAS,and down-regulated the expressions of ATGL,HSL and LPL in subcutaneous preadipocytes after differentiation.(2)Using AMPK’s activator AICAR and inhibitor Compound C to treat heat-stressed porcine subcutaneous preadipocytes respectively and induce differentiation test results showed that: heat stress induced the lipid accumulation of porcine subcutaneous preadipocytes by inhibiting AMPK-SIRT1-PGC-1α signaling pathway.(3)EGCG intervention in heat-stressed porcine subcutaneous preadipocytes and induced differentiation test results showed that: EGCG significantly reduced the expression of heat shock protein and alleviated heat stress.EGCG could down regulate the expression of PPARγ,CEBPα,AP2 and FAS,and up regulate the expression of ATGL,HSL and LPL by activating AMPK-SIRT1-PGC-1α signaling pathway,so as to inhibit the lipid accumulation of porcine subcutaneous preadipocytes under heat stress.2.Effects of heat stress on fat deposition and metabolism of porcine intramuscular preadipocytes and EGCG regulation mechanism.(1)The heat stress model of porcine intramuscular preadipocytes was established by high temperature stimulation at 41.5℃ and the induced differentiation test results showed that: heat stress could induce heat shock reaction of porcine intramuscular preadipocytes after differentiation,and promote the expression of HSP60,HSP70 and HSP90.Heat stress down-regulated the expression of fat synthesis genes PPARγ,CEBPα,AP2 and FAS,and up-regulated the expression of lipolytic genes ATGL,HSL and LPL,after the differentiation of porcine intramuscular preadipocytes by activating the AMPK-SIRT1-PGC-1α signaling pathway,thereby inhibited fat deposits.(2)Using AMPK’s activator AICAR and inhibitor Compound C to treat heat-stressed pig intramuscular preadipocytes respectively and induce differentiation test results showed that: heat stress inhibited adipogenesis of porcine intramuscular preadipocytes by activating AMPK-SIRT1-PGC-1α signaling pathway.(3)EGCG intervention in heat-stressed porcine intramuscular preadipocytes and induced differentiation test results showed that: EGCG significantly reduced the expression of heat shock protein and alleviated heat stress.EGCG inhibited the AMPK-SIRT1-PGC-1α signaling pathway in heat-stressed pig intramuscular preadipocytes,thereby up-regulated the expression of key fat synthesis genes PPARγ,CEBPα,FAS and AP2,and down-regulated the expression of key fat decomposition genes ATGL,HSL and LPL,thereby promoted lipid accumulation in porcine intramuscular preadipocytes under heat stress conditions.3.The effect of heat stress on the apoptosis of porcine subcutaneous and intramuscular preadipocytes and the protective effect of EGCG.(1)Heat stress leaded to a decrease in the viability of porcine subcutaneous and intramuscular preadipocytes,an increase in apoptosis rate,inhibition of BCL-2 gene expression,and promotion of BAX gene expression,which had varying degrees of proapoptotic effects on cell.(2)EGCG increased the viability of heat-stressed porcine subcutaneous and intramuscular preadipocytes,significantly reduced the rate of cell apoptosis,promoted the expression of BCL-2 gene,and inhibited the expression of BAX gene,and had different protective effects on the apoptosis induced by heat stress.In summary,heat stress can promote lipid accumulation of porcine subcutaneous preadipocytes,inhibit adipogenesis of porcine intramuscular preadipocytes,and promote apoptosis of the two kinds of cells.EGCG mediates the AMPK-SIRT1-PGC-1α signaling pathway to improve the ability of cells to resist heat stress,regulates fat deposition and metabolism and has a protective effect on heat stress induced apoptosis.This provides a theoretical basis for the future development of EGCG as a feed additive that can improve pork quality under heat stress. |
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