| The perinatal cows often stay in a state of negative energy balance, some evenhave the metabolism disorder, like ketosis and fatty liver. Meanwhile, the NEB willcause the fat mobilization, resulting in high plasma NEFAs. This is the result ofcows’ compensatory adjustment, but high concentration of NEFAs is the importantrisk factors for the ketosis, fatty liver and other common metabolic disorders. NEFAscan supply energy through oxidation in the liver to anesis the energy lack. Studieshave shown that NEFAs can cause oxidative stress and liver damage, but thepathogenesis is unclear. In this study, we detected the level of oxidative stress andapoptosis in hepatocytes treated with different concentration of NEFAs in vitro, andanalyzed the mechanisms how NEFAs produce these effects on hepatocytes. Theresults will provide a theoretical basis in developing new drugs and the prevention ofmetabolic disorders in cows.We detected some oxidative and antioxidant indexes of hepatocyte treated withdifferent concentration of NEFAs in vitro, aiming at studying the relationshipbetween NEFAs and oxidative stress in hepatocytes. The results showed that treatingthe hepatocytes with high concentration of NEFAs could significantly improve theROS levels and contents of SOD and GSSG in hepatocytes in vitro, whilesignificantly reducing CAT, GSH-Px and SOD activities, as well as TAC and GSHcontents. The results showed that high concentrations of NEFAs could causeoxidative stress in hepatocytes and then promote the hepatocytes apoptosis, while theglucose and NAC protected hepatocytes against the oxidative stress caused byNEFAs, suggesting that high concentration of NEFAs could cause the oxidativestress then apoptosis in hepatovytes during energy lack.We detected the activity of p38α and the mRNA level of p38in hepatocytes treatd with NEFAs for24h to seek the correlation between NEFAs and p38. Theresults indicated that NEFAs improved the activity and mRNA expression levels ofp38, suggesting that NEFAs could activate p38with the measurement correlation.We detected the best time for p38activation through treating the hepatocytes withNEFAs for0h,0.5h,1h,3h,6h,9h,12h and24h respectively. The resultsindicated that the p38was activated highest in9h by phosphorylation, whichsuggested we should choose9h as the best time. Then, we detected the expressionlevels and the transcriptional activity of transcription factors p53and Nrf2which arethe p38downstream genes. The results showed that NEFAs increased the nuclearlocalization and transcriptional activity of p53, while decreased Nrf2, indicating thatthe transcription factors p53and p38were involved in the p38signal pathwaymediated cow hepatocytes apoptosis caused by NEFAs in vitro. Even more, wedetected the expression levels of some apoptosis related genes p38, p-p38, Caspase3, Cleaved Caspase3, Caspase9, Bax, Bcl-2, cleaved PARP. And the results showedthat NEFAs promoted the expression of pro-apoptosis genes, while decreased theexpression of anti-apoptosis genes. The results above showed that NEFAs couldcause the apoptosis of hepatocytes through activating the p38signal pathway, thenpromoting the expression of p53and some pro-apoptosis genes and inhibit theexpression of Nrf2and some anti-apoptosis genes.Summing up the above results, we can conclude that high concentration ofNEFAs cows can cause oxidative stress in hepatocytes in vitro. The oxidative stresscaused by NEFAs further activates p38MAPK signaling pathway to promote theexpression, nuclear localization and transcriptional activity of p53, inhibit theexpression, nuclear localization and transcriptional activity of Nrf2. The regulationof P53and Nrf2will promote the expression of pro-apoptotic genes, while reducethe expression of antiapoptotic genes, resulting in hepatocytes apoptosis. Theantioxidant NAC, GLU and p38MAPK inhibitor SB203580alleviated hepatocyteapoptosis induced by NEFAs, indicating that NEFAs can activate the ROS-p38-p53/Nrf2signaling pathway to induce the apoptosis of dairy hepatocytesin vitro. |
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