Protection Mechanism Of Niacin On Low PH Induced Apoptosis In Rumen Epithelial Cells

With the rapid development of animal husbandry,rumen acidosis in ruminants is a common nutritional metabolic disease,which causes serious economic losses in animal husbandry.The main causes of rumen acidosis are the decrease of rumen fluid pH,the rumen epithelium is eroded by acid,and the rumen barrier function is destroyed.Bacteria,viruses and toxic metabolisms in the rumen enter the blood circulation through the rumen barrier,causing systemic inflammation and seriously leading to the death of animals.It was found that the excessive apoptosis of rumen epithelial cells may be the main factor causing the loss of rumen epithelial barrier function.Low pH is the main factor of rumen acidosis.However,whether low pH leads to apoptosis of epithelial cells and through which apoptotic pathway has not been reported.As an important B vitamin,niacin plays a certain role in stabilizing the stability of rumen microorganisms,regulating rumen fermentation and inhibiting the occurrence of rumen acidosis.However,it is still unclear whether nicotinic acid can regulate the apoptosis of rumen epithelial cells treated with low pH.Therefore,this study was conducted for this purpose.In this study,rumen epithelial cells of adult Boer goats were used as the research subjects,and the effects of low pH and niacin on rumen epithelial cell viability,tight juncture and apoptotic genes were studied by in vitro culture technique.Cell apoptosis was detected by MTT cell viability assay and Annexin V FITC-flow cytometry.m RNA expression levels of apoptosis-related genes were analyzed by real-time quantitative PCR assay.The test results are as follows:（1）Effect of low pH and niacin culture for different time on rumen epithelial cell viability.The medium containing 0,20,40,80,160 and 320 mmol/L of different niacin was prepared with pH of 7.4,5.8,5.5,5.2 and 5.0,respectively.Rumen epithelial cells were treated for 3,6,12 and 24 h,respectively,and the activity of rumen epithelial cells was determined by MTT.The results showed that the rumen epithelial cells treated with low pH medium decreased the cell viability,Inhibiting the growth of cells,the longer the treatment time,the greater the damage to cells,5.8 The cell viability at 3 h was 75.6%（P<0.05）,the cell viability was lower than 50%after 12 h treatment（P<0.05）.The rumen epithelial cells were treated with a low concentration（20～40 mmol/L）of niacin in a short period of time,and the activity of cells was improved and the growth of cells was promoted,especially at 12 h.However,high concentration（80～320 mmol/L）of niacin reduced the activity of cells and had a toxic effect on cells.Therefore,20 mmol/L niacin treatment for 12 h and 5.8 treatment for 3 h were selected as subsequent experiments.（2）Effect of low pH and niacin supplementation on rumen epithelial cell viability.Rumen epithelial cells were treated with 20 mmol/L nicotinic acid as pre-protection,niacin as treatment,and nicotinic acid and low pH at the same time to explore the effect of niacin on rumen epithelial cell viability under low pH treatment.The results showed that the results of the niacin preprotection test were the same when the pH was 5.8 and 5.5.The rumen cells treated with 20 and 40 mmol/L nicotinic acid for 12 h significantly increased the cell viability（P<0.05）,80～320 mmol/L nicotinic acid significantly decreased the activity of rumen cells（p<0.05）.When pH was 5.2 and 5.0,20～320 mmol/L niacin significantly decreased cell viability（P<0.05）.The niacin treatment trial had the same results at pH 5.8 and 5.5,The rumen cells treated with 20 and 40 mmol/L niacin for 12 h significantly increased the cell viability（P<0.05）,80～320 mmol/L niacin significantly decreased the activity of rumen cells（P<0.05）,When pH was 5.2 and 5.0,20～320 mmol/L niacin significantly decreased cell viability（P<0.05）..At pH 5.8,5.5,5.2 and 5.0,20～320mmol/L of niacin significantly reduced cell viability（P<0.05）.Analysis of the reasons for the structural changes of niacin under low pH conditions,and then activate the release of endoplasmic reticulum Ca²⁺into the cytoplasm,activate the apoptosis of the endoplasmic reticulum,leading to cell apoptosis.（3）Effects of niacin on tight junction proteins,apoptosis and related genes in rumen epithelial cells treated with low pH.It was found that pH 5.8 treatment for 3 h increased the number of early,late and total apoptosis of rumen epithelial cells（P<0.05）,The expression of tight junction protein Occludin-1,ZO-1,Claudin-1/7 and m RNAs of apoptotic genes MCT1,p53,PARP-1,Fas,AIF,Caspase-3/8/9 and Bax increased（P<0.05）,The ratio of Bcl-2/Bax decreased（P<0.05）.Rumen epithelial cells were treated with 20mmol/L nicotinic acid for 12 h for pre-protection,5.8 After another 3 hours of treatment,Early,late and total apoptosis of rumen epithelial cells decreased（P<0.05）,The expression of Occludin-1,ZO-1,Claudin-1/4/7 tight junction protein,PARP-1,Bax,Caspase-3/8/9apoptotic genes decreased（P<0.05）,The ratio of Bcl-2/Bax increased（P<0.05）.These results indicated that niacin alleviated the damage of low pH to tight junction protein in rumen epithelial cells and inhibited the apoptosis of rumen epithelial cells induced by low pH through endogenous apoptosis and exogenous apoptosis pathway.