| Rotavirus(RV)is one of the main pathogens causing gastroenteritis in young animals.It can infect intestinal mucosal epithelial cells,leading to dysfunction of the intestinal mucosal mechanical barrier,causing diarrhea and even death in the body.Previous studies have shown that gut microbiota metabolites can regulate the expression and distribution of tight junction(TJ)related proteins in intestinal mucosal epithelial cells,thereby regulating the mechanical barrier function of the intestinal mucosa.Butyrate is short chain fatty acids(SCFAs,also known as volatile fatty acids)that are metabolites produced by the gut microbiota through glycolysis and pentose phosphate pathways by degrading indigestible dietary fiber.Sodium butyrate(SB)is a form of sodium butyrate.As the main energy source of intestinal mucosal epithelial cells,SB plays an important role in regulating innate immune responses such as oxidative stress and promoting intestinal mucosal mechanical barrier function.However,the effect and mechanism of SB on intestinal mucosal mechanical barrier dysfunction induced by RV infection are still unclear.Therefore,this study used newborn piglet jejunal epithelial cells(IPEC-J2)as an experimental model to investigate the types of intestinal mucosal epithelial cell damage induced by RV infection;Using RV infected IPEC-J2 cells as an experimental model,we investigated the alleviating effect and mechanism of SB on RV induced IPEC-J2 cell damage,and explored whether SB alleviates RV induced cell damage through the AMPK/Nrf2/HO-1 signaling pathway,and whether SCFAs receptor GPR109 A is involved in this process.The main research findings are as follows:1.The effect of SB on RV-induced IPEC-J2 cell injuryThis experiment used different concentrations of SB(0,2,and 4 m M)to treat IPECJ2 cells before and after inoculation with 10-fold multiple infection(MOI)RV for 24 hours.The cells were divided into NC group(0 m M SB+0 MOI RV),RV group(0 m M SB+10MOI RV),2 m M SB group(2 m M SB+10 MOI RV),and 4 m M SB group(4 m M SB+10MOI RV),with 6 replicates per treatment group.The aim was to investigate the types of RV induced cell damage and the relief effect of SB on RV induced cell damage.The results showed that RV infection induced severe damage to the TJ structure of IPEC-J2 cells,with a significant increase in intracellular ROS levels and a decrease in cellular antioxidant capacity;The appropriate concentration of SB can effectively alleviate RV induced TJ structural damage in cells,reverse the distribution defects of TJ related proteins and the downregulation of protein expression levels;And significantly reduce intracellular ROS levels,effectively increase the activity of antioxidant enzymes(T-SOD,CAT,GSH-px),and reduce MDA content.The results indicate that the appropriate concentration of SB can effectively alleviate oxidative stress and TJ damage in IPEC-J2 cells induced by RV infection.2.SB alleviated RV-induced IPEC-J2 cell injury by activating AMPK/Nrf2/HO-1signaling pathwayThis experiment further detected the protein expression levels of related antioxidant signaling pathways on the basis of Experiment 1,aiming to explore the specific mechanism of SB alleviating RV induced IPEC-J2 cell damage.The results showed that SB may improve the antioxidant capacity of IPEC-J2 cells by activating the AMPK/Nrf2/HO-1signaling pathway to alleviate cell TJ damage induced by RV infection.To confirm the above results,further experiments were conducted using AMPK activator metformin and inhibitor compound C.In the metformin experiment,IPEC-J2 cells were first pretreated with metformin for 6 hours,and then inoculated with 10 MOI RV for 1 hour before continuing infection for 24 hours.They were divided into NC group(0m M SB+0 MOI RV),RV group(0 m M SB+10 MOI RV),and AMPK activation group(Metformin+10 MOI RV).Each treatment group had 6 replicates,with the aim of exploring whether AMPK activation effectively alleviates RV induced oxidative stress and TJ injury in IPEC-J2 cells.In the compound C experiment,IPEC-J2 cells were first pretreated with compound C for 6 hours,and then treated with 2 m M SB for 24 hours before and after 10 MOI RV inoculation(1 hour).The cells were divided into NC group(0 m M SB+0 MOI RV),RV group(0 m M SB+10 MOI RV),2 m M SB group(2 m M SB+10 MOI RV),and AMPK inhibition group(2 m M SB+10 MOI RV+compound C),with 6 replicates per treatment group,The aim is to investigate whether SB alleviates RV induced oxidative stress and TJ damage in IPEC-J2 cells and is associated with the AMPK pathway.The results showed that AMPK activators significantly increased TJ protein expression,promoted Nrf2 nuclear translocation levels,and increased antioxidant enzyme activity.In addition,AMPK inhibitors significantly blocked the beneficial effect of SB on alleviating RV induced cell damage.The results indicate that SB mediates Nrf2 nuclear translocation by activating AMPK,promotes downstream antioxidant related protein expression,enhances the antioxidant capacity of IPEC-J2 cells,and alleviates cellular oxidative stress and TJ damage induced by RV infection.3.SB regulates AMPK/Nrf2/HO-1 signaling pathway in IPEC-J2 cells in a GPR109 Adependent mannerThis experiment used small interfering RNA(siRNA)of GPR109 A to knock down the expression of GPR109 A.In the siRNA assay,IPEC-J2 cells were first treated with siRNA negative control(referred to as Ctrl siRNA)or GPR109 A siRNA for 12 hours,and then treated with 2 m M SB for 24 hours before and after 10 MOI RV inoculation(1 hour).The cells were divided into Ctrl siRNA groups(0 m M SB+0 MOI RV,0 m M SB+10 MOI RV,2m M SB+10 MOI RV)and GPR109 A siRNA groups(0 m M SB+0 MOI RV,0 m M SB+10MOI RV,2 m M SB+10 MOI RV).Each processing group has 6 replicates,The aim is to investigate whether SB alleviates RV induced IPEC-J2 cell damage by regulating the AMPK/Nrf2/HO-1 signaling pathway through its receptor GPR109 A.The results showed that GPR109 A siRNA treatment significantly reduced the promoting effect of SB on cell TJ and the regulatory effect of antioxidant enzyme activity,significantly blocking the beneficial effect of SB on alleviating RV induced cell damage.The results indicate that SB exerts a protective effect on cells through GPR109 A,enhancing the antioxidant capacity of IPEC-J2 cells to resist RV infection,thereby alleviating cell TJ damage.In summary,this study confirms that RV infection induces an increase in ROS content in IPEC-J2 cells,leading to oxidative stress and cell TJ damage;In addition,SB can alleviate RV induced cellular oxidative stress and TJ injury by activating the AMPK/Nrf2/HO-1 signaling pathway,which depends on the effective activation of GPR109 A by SB.This also indicates that GPR109 A is one of the effective receptors of SB in IPEC-J2 cells induced by RV infection. |
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