Protection Mechanism Of Niacin On Lactate-induced Apoptosis In Rumen Epithelial Cells

Ruminal acidosis is a common nutritional metabolic disease in ruminant production.Rumen acidosis can damage the structure of the rumen barrier,loss of function,increase the way of ruminal toxic substances entering the blood,and then aggravate the symptom and cause systemic metabolic acidosis.However,its mechanism is still unclear and needs further study.Some studies have found that the damage of rumen barrier structure maybe related to excessive apoptosis of rumen epithelial cells.High concentration of lactate is the main cause of acute rumen acidosis.However,whether lactate can induce apoptosis of rumen epithelial cells has not been reported.Niacin is a B vitamin,which has certain effect on alleviating the symptoms of rumen acidosis.However,it is not clear whether niacin has protective effect on rumen epithelial cell apoptosis.In present study,rumen epithelial cells were used as experimental objects,to investigate the effects of lactate and niacin on cell viability,apoptosis index,redox state,mitochondrial membrane potential,apoptosis-related genes and proteins in vitro to explore the regulation mechanism of lactate and niacin on rumen epithelial cell apoptosis.Using MTT assay to detecte cell viability,Annexin V FITC/PI staining detected cell apoptosis,fluorescence microscopy observated the changes of mitochondrial membrane potential(MMP),flow cytometry determined the changes of the intracellular reactive oxygen species(ROS)and MMP and RT-PCR and western blot analyzed the levels of the apoptosis-related genes and proteins.The results are as follows:(1)Effects of different doses of antibiotics on primary culture of the dult Hu-sheep rumen epithelial cells.Rumen epitheliums were washed with PBS containing different doses of antibiotics to remove microorganisms before primary culture,and screen out the optimum conditions.The results showed that the II group(PBS containing 5 times penicillin streptomycin and 5 times gentamicin and amphotericin B)could obtain the purer tissue,and the growth and adherence of the isolated rumen epithelial cells were better.According to the detection of 7-day growth curve of rumen epithelial passage cells,found that the cells grew in a typical “S” growth.(2)Effects of lactate and niacin on rumen epithelial cells viability.Adding different concentrations(0,100,200,300,400,500 mmol/L)lactate treated rumen epithelial cells at different times(3,6,12,24 h)to observe the effects of lactate on rumen epithelial cells relative viability.The results showed that low concentration(100 mmol/L)lactate can increase the cells relative viability,but high concentrations(200~500 mmol/L)lactate decreased cells relative viability and had inhibited cell growth.According to above results,300 mmol/L lactate treated for 24 h to do next test.Adding different concentrations(0,20,40,80,160,320 mmol/L)niacin in 300 mmol/L lactate treatment for 24 h,to determine the protective effects of niacin on cells damage caused by lactate.Result showed that 20~80 mmol/L niacin can alleviate cell injury caused by lactate,increase cell viability,promote cell growth,of which 40 mmol/L niacin has the most significant effect.160 mmol/L and 320 mmol/L niacin had no obvious protective effect on cell injury,but reduced cell viability and had cytotoxicity.And then,the experiment was divided into 3 groups.The cells treated with basic medium as the control group,cell treated with 300 mmol/L lactate for 24 h as lactate group,cell treated with 300 mmol/L lactate + 40 mmol/L niacin for 24 h as lactate + niacin group,3 repititions per group and following tests.(3)Effects of lactate and adding niacin on redox state and mitochondrial membrane potential.The results of ROS and T-AOC showed that compared with the control group,lactate group intracellular ROS content increased(P<0.05),T-AOC decreased(P<0.05);compared with lactate group,lactate + niacin group intracellular ROS production were decreased(P<0.05),increased antioxidant capacity of cells(P<0.05).The results of MMP showed that compared with the control group,lactate treatment resulted in loss of MMP(P<0.05)and increase of mitochondrial membrane structure damage,and compared with lactate group,lactate + niacin group decreased the loss of MMP(P<0.05)and alleviated the mitochondrial injury of rumen epithelial cells.(4)Effects of lactate and adding niacin on the apoptosis and apoptosis related genes and proteins.The flow cytometry result showed that compared with the control group,300 mmol/L lactate treatment for 24 h can reduce the number of late apoptotic cell,increase the number of early apoptotic cells,and the apoptosis index(P<0.05),induce cell apoptosis,and compared with lactate group,adding 40 mmol/L niacin can decrease the number of late apoptotic cell and early apoptotic cells,and decrease apoptosis index(P<0.05),relieve cell apoptosis,and had protection function on cell injury induced by lactate.Genes and proteins tests results showed that compared with the control group,lactate treatment up-regulated Mct1,p53,PARP-1,Cyt-C,AIF,caspase-9,Fas,caspase-8,caspase-3,mRNAs and proteins expressions(P<0.05),up-regulated Bax mRNA level(P<0.05)and had no significant effect on Bcl-2 mRNA expression and the ratio of Bcl-2/Bax mRNA(P>0.05),down-regulated the expression of Bcl-2 protein(P<0.05)and had no significant effect on Bax protein and Bcl-2/Bax protein ratio(P>0.05),and compared with lactate group,adding niacin significantly decreased the expression of Mct1,p53,PARP-1,Bax,Cyt-C,AIF,caspase-9,Fas,caspase-8,caspase-3,mRNAs and proteins(P<0.05),down-regulated Bcl-2 mRNA expression(P<0.05)and had no significant effect on Bcl-2/Bax mRNA ratio(P>0.05),and up-regulated expression of Bcl-2 protein and Bcl-2/Bax protein ratio(P<0.05).In combination with these results ahowed that 300 mmol/L lactate caused the increase of intracellular ROS content,the decrease of antioxidant capacity,the loss of MMP,the increase of Mct1,p53,PARP-1,Cyt-C,AIF,caspase-9,Fas,caspase-8,caspase-3,mRNAs and proteins expression,and had no difference in ratio of Bcl-2/Bax,indued cell apoptosis.But adding niacin could significantly inhibit the occurrence of these events,and up-regulated the ratio of Bcl-2/Bax protein.Therefore,high concentration lactate induced apoptosis of rumen epithelial cells may through positive regeneration of Mct1 concentration increase,promoting the transport of lactate into cell,increasing intracellular oxidative stress,damaging mitochondria membrane structure and DNA,activating Bcl-independent intrinsic pathway and extrinsis pathway.Adding niacin can relieve lactate-induced rumen epithelial cells apoptosis caused by lactate,its mechanism may through increasing intracellular antioxidant level,relieving injury of mitochondrial and DNA,and preventing the activation of downstream apoptotic events to protect cells from apoptosis.