Repairment Of Glutamic Acid On The Damage By Heat Stress In Dairy Cattle Intestinal Epithelial Cells

Heat stress is an important factor that threatens dairy production.Relevant research believe that the damage of intestinal epithelial is core problem in dairy production by heat stress.It is an effective way to cope with heat stress by protecting intestinal epithelium.Glu is the main source of energy for intestinal tract of dairy cows,playing an important role in maintaining the health of intestinal tract.Therefore,in this study,we attempted to explore the mechanism for Glu in repairing heat-damaged IEC,to find an effective solution the problem of heat stress in the dairy industry.The intestinal epithelial were isolated by enema digestion,mechanical scraping and enzymatic hydrolysis.The results showed that the number of IEC obtained by mechanical scraping and enzymatic hydrolysis was high and the cell activity was high.The cells were purified with cell scraping,differential adhesion and digestion method in difference,the cell reproduction curve was determined by CCK-8,and the cells were identified by HE staining.The results show that the purity of IEC is high after using the differential adherence method,which is typical of “paving stone”,the growth curve is “S” type,can be used in subsequent tests.Cows with good growth status were treated at control group(37?)and heat treatment group(40?,42?)for 0.5 h,1 h,1.5 h,2 h,3 h,4 h,6 h,8 h.Then,the levels of GSH-PX,LDH,SOD and MDA in the supernatant were measured.And the mRNA relative expression levels of heat shock proteins,intercellular junction proteins,inflammatory factors,and apoptotic protein.The results showed that the IEC was in a hot environment,the levels of GSH-PX and SOD decreased,the levels of LDH and MDA increased.The mRNA relative expression of HSP27,HSP70 and HSP90 was significantly higher at 42? for 6 h than other treatment groups(P<0.05).The mRNA relative expression of occludin in the heat treatment group was significantly lower than that in the control group(P<0.05).The mRNA relative expression of IL-10 was low at 42? for 6 h.The ratio of Bcl-2 and Bax was highest at 40?,42? for 1 h,4 h,and decreased after 4 h,and decreased to 8 h.So when the cells were treated with 42?for 6 h,the damage was obvious,the thermal damage model was successfully established.The epithelial cells were cultured at 42?for 6 h,and then cultured with different concentrations of Glu(Glu concentrations of 4,8,12,16,20,28,36 mmol/L).After 12 hours of culture,cell supernatants and cells were collected.The levels of antioxidant indexes in supernatant and the levels of heat shock protein,connexin,inflammatory factor and apoptotic protein were detected,to study the effects of different concentrations of Glu on the repair of heat-damaged cells.The results showed that the value of OD was the highest when the Glu concentration was 8 mmol/L.When the Glu concentration was 8,12,16,20,28,36 mmol/L,the content of GSH-PX and SOD decreased,the content of LDH was the lowest when the Glu concentration was 28 mmol/L,when the Glu concentration was 20 mmol/L,the MDA content was the lowest.The mRNA level of relative expression of HSP27,HSP70 and HSP90 was highest when the concentration of Glu was 36 mmol/L.When the Glu concentration was 4 mmol/L,the relative expression of claudin-1 was the lowest.When the Glu concentration was 8 mmol/L,the relative expression of occludin was the highest.The relative expression of IL-1? mRNA was lower in the lower concentration group than in the heat injury group.The relative expression of anti-inflammatory factor IL-10 gene was higher than that in the heat injury group when the Glu concentration was 28 mmol/L.The relative expression levels of Bcl-2 and Bax were lower than those in the heat injury group at low Glu concentration,however,the value of Bcl-2/Bax is higher than that of the thermal injury group at Glu concentrations of 4,16,and 36 mmol/L.Thus,Glu can repair damaged IEC,the mechanism of repairing could be enhanced the antioxidant capacity of cells by promoting the production of antioxidant enzymes,regulated the expression of heat shock proteins,promoted the expression of connexins in epithelial cells,inhibit the expression of inflammatory factors,attenuated apoptosis,and enhanced cell viability.In conclusion,IEC of dairy cows with high purity,good viability and strong proliferative ability is successfully obtained by mechanical scraping and enzymatic separation of cells and differential attachment method.When IEC of dairy cows is treated at 42? for 6 hours,heat stress model is successfully established.The adding of different concentrations of Glu can repair IEC of heat-damaged dairy cows.