The Effect Of Lipopolysaccharide On Oxidative Stress And Autophagy In Bovine Hepatocytes During SARA And The Regulation Of Taurine

SARA(Subacute ruminal acidosis)is a common disease in dairy industry.It is usually caused by low p H value in remen which is a consequence of fermentation of lots of carbohydrates.Rumen derived LPS enter liver from portal vein and induced damaged liver and decreased production performance in cows,which bring huge economic loss to dairy industry.Tissue and cell inflammation,apoptosis,oxidative stress and autophagy are often accompanied by occurrence and development of organ damage,regulating damage process.As a drug widely used for cytoprotection,taurine has several applications such as anti-inflammatory and anti-oxidative stress,and can regulate Nrf2/HO-1 signaling pathway as well as autophagy process.In this study,we aim to investigate oxidative stress and autophagy related data in blood and liver tissue of cows given high grain diet.In cell experiments,LPS was used to stimulate bovine hepatocytes to detect the level of oxidative stress and autophagy,NAC was added to inhibit the level of ROS to detect the effect of inhibiting ROS on the level of autophagy caused by LPS.Finally,taurine was used to prevent cells to detect its regulatory effects on oxidative stress and autophagy in bovine hepatocytes caused by LPS.1 High grain diet induced oxidative stress and autophagy in liver tissue of cows in SARALong-term feeding of high-concentration diets will make a large amount of carbohydrates ferment in the rumen,producing a large amount of volatile fatty acids(VFA)and lactic acid(LA)and cause a drop in rumen p H value,which will lead to subacute ruminal acidosis.The purpose of this experiment is to study the effects of high-concentration diets on the oxidative stress and autophagy levels in the liver of dairy cows.In this experiment,12 mid-lactating Holstein cows were used as the research object,and randomly divided into two groups: a high-concentration diet group(HC)fed with a concentration to thickness ratio of 6:4 and a low-concentration diet group fed with a concentration to thickness ratio of 4:6.The expression of antioxidant enzymes in blood were detected by western blot,q PCR and antioxidant enzyme detection kits.The results showed that feeding high-concentration diet reduced p H value in rumen fluid and increased level of LPS in blood.At the same time,compared with the low-concentration diet group,the expression of autophagy related proteins and genes increased.The content of antioxidant enzymes in blood was detected by detection kit and results showed that the content of antioxidant enzymes in blood was reduced.This experiment showed that rumen-derived LPS caused oxidative stress and autophagy in liver of dairy cows.2 LPS induced oxidative stress and autophagy in bovine hepatocytes in dose dependent mannerThere was no report about LPS induced oxidative stress and autophagy in bovine hepatocytes and many study reported that under some stress condition,ROS has an important regulatory effect on autophagy.Our study treated bovine hepatocytes with different concentrations of LPS(2,4,8,10,12 μg/m L)for 6 h,western blot assay,acridine orange staining,ROS,MDA and T-AOC detective kit were used to detect change of oxidative stress and autophagy related data.The results showed that the level of ROS and the activity of MDA were elevated while the T-AOC was reduced in bovine hepatocytes induced by LPS.The protein expression of LC3Ⅱ,Beclin1,Atg5 were increased while the protein expression of p-Nrf2,HO-1 and p62 were decreased.The results of acridine orange staining showed that LPS induced increased number of autolysosomes in cytoplasm.Both oxidative stress and autophagy were changed in LPS concentration dependent manner and we get the best result at the concentration of 12 μg/m L LPS,which was selected for further experiment.Then we used NAC,an inhibitor of ROS production,to suppress ROS level in bovine hepatocytes with LPS treatment.There were four groups in our experiments: control group,LPS group,10 m M NAC treatment for 6 h followed by treatment with 12 μg/m L LPS for 6 h,15 m M NAC treatment for 6 h followed by treatment with 12 μg/m L LPS for 6h.Western blot,ROS,MDA and T-AOC detective kit were used to detect change of oxidative stress and autophagy related data.The results showed that NAC significantly reduced the levels of intracellular ROS and MDA caused by LPS,and restored the T-AOC of bovine hepatocytes.Compared with LPS group,pretreatment of NAC did not significantly change the expression of autophagy related proteins LC3Ⅱ,Atg5,Beclin1 and p62.The above results indicate that LPS treatment could induce autophagy and oxidative stress in bovine hepatocytes in concentration-dependent manner,and LPS-induced autophagy in bovine hepatocytes was not ROS-dependent.3 Effect of taurine on LPS induced oxidative stress and autophagy in bovine hepatocytesTaurine is a kind of sulfur amino acid which is rich in mammal body and used for anti-inflammatory,anti-oxidative stress and cytoprotection.In this study,we aim to investigate the regulation of taurine on LPS induced oxidative stress and autophagy in bovine hepatocytes.We divided cells into five groups: control group,LPS treatment group,5 m M taurine treatment for 6 h followed by LPS treatment for 6 h,10 m M taurine treatment for 6 h followed by LPS treatment for 6 h,20 m M taurine treatment for 6 h followed by LPS treatment for 6 h.Western blot,immunofluorescence assay,acridine orange staining,ROS,MDA and T-AOC detective kit were applied.The results showed that compared with LPS group,taurine treatment reduced protein expression of LC3Ⅱ,Atg5,Beclin1 and increased protein expression of p62,p-Nrf2 and HO-1.The level of ROS and MDA were also reduced while the T-AOC level increased.Immunofluorescence assay showed LPS decreased p-Nrf2 expression in nucleus and cytoplasm and make Nrf2 translocate from nucleus to cytoplasm,but pretreatment with taurine increased p-Nrf2 level in both nucleus and cytoplasm and promoted Nrf2 translocate to nucleus.Our study indicate that taurine can protect bovine hepatocytes from LPS induced oxidative stress and autophagy.