Study On The JNK-mediated Apoptosis Mechanism Of Rumen Epithelial Cells Induced By Low PH

Background:Ruminal acidosis is the most common nutritional metabolic disease which was caused by the rapidly dropped ruminal pH due to long-term feeding of high-concentrate diets in the production of ruminants.Rumen acidosis further caused the disturbance of rumen microbiota,the decline of rumen degradability,and the serious economic losses.Under the circumstance of long-termed exposed to a low pH environment,the structure of the rumen epithelium is destroyed,which is manifested as necrosis of rumen wall mucosa epithelial cells,widening of intercellular spaces,relaxation of tight links,and thinning of the rumen epithelium.Furthermore,acidosis will also cause increased osmotic pressure,which thereby weakened the barrier function of the rumen epithelium,caused the penetration of harmful substrates into blood circulation,and seriously induced physiological death.However,the mechanism of apoptosis of rumen epithelial cells under acidotic conditions has rarely been reported.JNK belongs to the serine/threonine protein kinase,is one of the important members of the MAPK family,which ubiquitously existed in mammals.The JNK signaling pathway can be activated by cytokines,stress,changes in osmotic pressure,and other factors that participate in biological responses such as cell proliferation and differentiation,cytoskeleton construction and apoptosis.In recent years,studies have found that the activation of the JNK signaling pathway can effectively regulate cell apoptosis,and is also an important pathway for inducing cell apoptosis under acidic conditions,SP600125 as a JNK specific inhibitor can inhibit JNK signaling pathway activation.Previous studies of our team researched the stimulation of rumen epithelial cells by organic acids(acetic acid:propionic acid:butyric acid=6:3:1),and found that the low pH treat significantly promoted the apoptosis of rumen epithelial cells compared with the control group pH 7.4.However,the underlying mechanism of acidity and apoptosis is still unknown.In order to deeply understand the effect of H~+on the apoptosis of rumen epithelial cells,the inorganic acid HCl was used to adjust the pH of the cell culture medium through in vitro experiments,and to explore the mechanism of low pH regulating the apoptosis of rumen epithelial cells.(1)Impacts of low pH treatment on rumen epithelial cell viability.In this experiment,pH of culture medium was regulated by HCl and the pH was gradiently set as 7.4,6.8,5.8,5.5,and 5.0.Rumen epithelial cell viability was further measured after 3h treating by CCK-8method and results indicated that,when the pH is lower than 5.8,the cell viability is significantly lower than that of the pH7.4 and pH=6.8 treatment groups,so pH 5.8 is selected as acidosis model for further comparison experiments.(2)Toxicity analysis of SP600125 on rumen epithelial cells.The pH 5.8 of the medium was selected as the control treatment,and test treats were gradiently supplement of SP600125(0,2.5,5,10,20,40μmol/L)to examine the effect of inhibitor SP600125 on the relative viability of rumen epithelial cells.Results showed that,SP600125 supplement significantly increased cell viability in acid environment excel 2.5μmol/L,and adding 20μmol/Linhibitor SP600125 to the acid treatment for 3 h,the cell viability showed the highest and significantly higher than that in the acid treatment.Therefore,20μmol/L SP600125supplement was selected as the optimum treatment for further analysis.(3)Effects of low pH treatment and SP600125 supplement on the anti-oxidant capacity of rumen epithelial cells.The experiment was then divided into 3 treats,the cells treated with basal medium(pH7.4)were the control group,the basal medium(pH 5.8)adjusted with HCl was considered as the low pH treatment,and the low SP group(Hereinafter referred to as SP group.(First add 20μmol/L SP600125 to pH 7.4 medium culture for 3 h,then add pH 5.8medium to culture cells).The effects of three different treatments on the redox state of cells were detected,and ROS and activities of T-AOC,T-SOD and GSH-PX in rumen epithelial cells were measured respectively.Results showed,compared with the control group,the intracellular ROS content in the low pH group increased(P<0.05).T-AOC,T-SOD and GSH-PX decreased(P<0.05);compared with the low pH group,the intracellular ROS,T-AOC,T-SOD and GSH-PX in the SP group had no significant effect.The results showed that:Low pH stimulation significantly increased ROS production in rumen epithelial cells,causing oxidative damage to rumen epithelial cells.(4)Effects of low pH and adding SP600125 on apoptosis of rumen epithelial cells and related genes and proteins.Results indicated that,the number of early apoptotic cells,late apoptotic cells,and cell apoptosis index were increased in the low pH group after 3 h treatment(P<0.05)compared with the control group.SP group could effectively reduce the number of early and late apoptotic cells,decrease the apoptotic index(P<0.05),alleviate cell apoptosis,and have a protective effect on cell damage caused by low pH.The low pH group significantly up-regulated the gene expression levels of Caspase-3,Caspase-9,Caspase-8,Cyt-C,Bax,p53,PARP,CHOP,JNK(P<0.05),significantly down-regulated the gene expression levels of Bcl-2,Bcl-2/Bax,and IRE1(P<0.05);significantly up-regulated the protein expression levels of Caspase-3,Cyt-C,IRE1,and JNK(P<0.05).),significantly down-regulated the protein expression levels of Bcl-2 and Bcl-2/Bax(P<0.05),but had no significant effect on the protein expressions of Caspase-9,Caspase-8,Bax,p53,PARP and CHOP(P>0.05).Compared with the low pH group,the SP group significantly down-regulated the gene expression levels of Caspase-9,Cyt-C,IRE1,p53,CHOP and JNK(P<0.05).No significant effect(P>0.05),significantly down-regulated the protein expressions of Caspase-3,Caspase-9,Cyt-C,IRE1,JNK,p-JNK(P<0.05),significantly up-regulated Bcl-2,Bcl-2/Baxprotein expression level(P<0.05).No significant effect on the protein expression of Caspase-8,Bax,p53,PARP,CHOP(P>0.05).Conclusion:low pH treatment on rumen epithelial cells caused the cellular apoptosis process through the decrease of relative cell viability and the antioxidant capacity,while the increase of intracellular ROS content,the upregulation of JNK、p-JNK expression,and further up-regulated the relative expression of genes and proteins that related to cell apoptosis.The addition of SP600125 can significantly inhibit the above events.Therefore,low pH induces apoptosis of rumen epithelial cells,possibly by increasing intracellular oxidative stress and activating the JNK signaling pathway to regulate apoptosis-related factors and cause apoptosis.