Effects Of Glutamine On Repairing Injury Bovine Rumen Epithelial Cells

Because of its unique and complex physiological function,Glutamine(Glutamine,Gln)has become a heated research focus in disciplinary fields such as the nutrition,physiology and nutritional immunology,particularly it performs a significant role in repairing the pathological intestinal structure and maintaining health condition.Furthermore,the problem that low rumen pH can give rise to rumen epithelium some injury is still existing and whether the Gln has some functions in repairing the damaged REC caused by low rumen pH remains unknown,accordingly,we attempted to explore the mechanism for glutamine in rumen epithelial cell growth and repairing the damaged intestinal structure of rumen.The rumen epithelial tissue was firstly washed with 9 different doses of antibiotics and then digested using two various methods: I)continuous digestion using the reagents of 0.25% trypsin and 0.02% EDTA;and II)firstly digested with 0.1%collagenase I,and next with the reagents of 0.25% trypsin and 0.02% EDTA at 37℃.The results demonstrated that the microbes from rumen epithelium can be effectively removed after the washing with 6-time concentration of penicillin and streptomycin and 6-time of concentration of gentamycin respectively.In addition,more and higheractivity rumen epithelial cells can be achieved from the washed rumen epithelium with the second digestion.The cells were purified with three different operations of cell scraping,differential adhesion and digestion method in difference,then the corresponding cell growth curve was drawn,and finally the cells were identified using the HE staining and the technology of cell flow.The result implied that the purified rumen epithelial cells with differential adhesion were alike as the typical paving stone and the "S" type of growing curve,and higher-activity;the cells whose purity reached above 95% can be utilized in the following experiment.For the purpose of establishing the injury model of REC,the four mediums were prepared according to the occurrence of bovine subacute rumen acidosis,rumen pH value and volatile fatty acids in rumen fluid:Ⅰ)normal medium,pH 7.2;Ⅱ)the medium with lactic acid,pH5.5;and Ⅲ)the medium adjusted with VFA of 100-time of liquid culture,pH5.5;Ⅳ)the medium adjusted with 100-time VFA and liquid lactic acid,pH5.5.The CCK-8 cell activity and apoptosis were measured after the cellsculturing for 3hr,5hr,8hr,and 12 hr,24hr,separately.In the following,the level of LDH,MDA,SOD and NO in cell supernatant and mRNA of the cell tumor necrosis factor alpha(TNF-alpha)by fluorescence quantitative PCR,inflammatory factor(beta IL-1,IL-8)intercellular junction protein(Claudin-1,occludin),cell surface receptors(TLR-2,TLR-4)were measured.The results showed that vacuolar degeneration and apoptosis increased,adherent cells gradually float after the treatment of acid for 12 hr.The level of LDH in cell supernatant was significantly increased(P<0.05),however,the SOD decreased significantly(P<0.05);Meanwhile,the relative expression quality of m RNA for TNF-α,L-1β 、 IL-8 was significantly increased(P>0.05),while the mRNA of occluding decreased(P<0.05)especially for the increased mRNA of cell surface receptors(TLR-2,TLR-4)at 3hr time site post cells culturing.With volatile fatty acid and lactic acid treatment of REC for 3hr induce a obvious injury and successfully established the injury model.According to the rumen acidosis tumor gastric juice pH value,use 100 times volatile fatty acid and lactic acid adjust the liquid culture medium pH equal to 5.5,and then in mediums containing different concentrations of Gln(Gln 4,8,12,32mM/L)for3hr.Injury Cells and supernatant were collected after incubation for12 hr so as to detect the levels of both LDH,SOD,MDA and mRNA of tumor necrosis factor alpha cells by fluorescence quantitative PCR technique to extract RNA(TNF-alpha),inflammatory factor(IL-1,IL-8),intercellular junction protein(Claudin-1,occludin),cell surface receptors(TLR-2,TLR-4).The results demonstrated that the Gln concentration is 8m M/L,the content of LDH in the supernatant was the lowest,and mRNA expression of TLR-2,TNF-alpha,TLR-4 decreased significantly(P<0.05),the mRNA expression of Claudin-1 rising significantly(P<0.05).Conclusions: Gln can promote the cell’s antioxidant capacity by promoting the production of antioxidant enzymes,promote the expression of connexin in epithelial cells,inhibit the expression of proinflammatory cytokines in the inflammatory pathway.Gln had the function of repairing the acid injury REC for 3 hr.