| The small intestine is the central organ of the stress response and the main place for digestion and absorption of nutrients.High temperature external stress can cause serious damage to the intestinal mucosa.To study how to reduce heat stress-induced small intestinal mucosal damage is beneficial to ensure the integrity of the small intestinal mucosa structure and function.By increasing feed remuneration and improving animal growth performance,it is beneficial to reduce the huge economic losses caused by heat stress to the animal husbandry.In this study,a heat stress model of goat small intestinal epithelial cells was established in vitro,and pretreatment with resveratrol?RES?was performed to explore whether it can protect goat small intestinal epithelial cells from heat stress injury.Combine molecular methods to explore the molecular mechanism of resveratrol's anti-oxidant and anti-inflammatory effects in goat small intestinal epithelial cells,with a view to providing scientific basis for later feed research and development.Exp.1 Effects of resveratrol on the antioxidant capacity and HSP70 expression of GIE cells induced by heat stressThe well-growing fourth-generation GIE cells were used for the experiment and were divided into five groups:blank control group?37??,heat stress group?42??,and 42?resveratrol group(5,15,30?mol·L-1);6 hours heat stress treatment was carried out 6 hours after pretreatment.After the detection of resveratrol cytotoxicity by CCK-8 method.The effects of resveratrol on heat stress-induced antioxidant performance were measured using biochemical methods and flow cytometry.Real-time PCR was used to detect HSP70 gene expression.The results showed that resveratrol pretreated GIE cells can significantly increase the activity of T-AOC,SOD,GSH-PX?P<0.01?,inhibit MDA and ROS production?P<0.01?,and reduce HSP70 gene expression?P<0.01?,Thereby inhibiting heat stress-induced oxidative stress in GIE cells.Exp.2 Effect of resveratrol on transcription of immune factors in GIE cells induced by heat stressThis test uses enzyme-linked immunosorbent assay?ELISA?to detect the effect of resveratrol on the secretion of cytokines induced by heat stress;real-time fluorescence quantitative PCR?RT-PCR?is used to detect the resveratrol on heat stress-induced IL Effects of-1?,TNF-?,IL-8 and NF-?B gene transcription.The results showed that resveratrol could inhibit the secretion of IL-1?,IL-2,IFN-?and TNF-?in goat small intestinal epithelial cells induced by heat stress?P<0.01?,and also reduce IL-1?and TNF-?,IL-8 and NF-?B gene transcription levels?P<0.01?.Exp.2 Resveratrol activates PI3K/AKT-Nrf2-HO-1 to enhance cell antioxidant capacityThis experiment using Western blot test resveratrol treatment cells in different concentrations(0,5,15,30?mol·L-1)and different time?0,1,3,6,9 and 12 h?after HO-1,Nrf2,PAKT,AKT,PCDA,GAPDH protein expression,using the method of CCK 8 and flow cytometry to detect AKT inhibitors and resveratrol after preprocessing govemment cell cell vitality and apoptosis.The results showed that resveratrol could significantly promote ho-1 expression,Nrf2 nucleation and AKT phosphorylation in a dose-and time-dependent manner.After treatment with the addition of AKT inhibitor,Nrf2 nuclear shift,ho-1 expression and AKT phosphorylation were significantly reduced.Meanwhile,after the heat stress of GIE cells,the protective effect of resveratrol was blocked,indicating that resveratrol can resist the oxidative stress damage induced by heat stress in GIE cells through the PI3K/akt-nrf2-ho-1 signaling pathway. |
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