Effects Of Heat Stress On Function Of Bovine Mammary Epithelial Cells And The Alleviative Effects Of Taurine

As the global warming effect intensifies,dairy industry has a significant impact.As we know,sweat glands of cows are not developed,skin evaporation heat loss less.In high temperature environment,they are susceptible to heat stress.This will not only reduce the productivity of cows,but also increase the incidence of metabolic disorders,and even cause the death of cows,which bringing huge economic losses to farmers and causing a series of animal welfare problems.In addition,many studies have shown that heat stress can induce oxidative stress,apoptosis and other events in bovine mammary epithelial cells in vitro.Taurine was first isolated from bovine bile and has a variety of biological and pharmacological activities.Previous studies have shown that taurine plays an important role in metabolism,nutrition,and antioxidant and anti-inflammatory events.It is a powerful protective agent in the event of disease caused by oxidative damage and multiple stresses.However,the mechanism of taurine on mammary epithelial cells of dairy cows under heat shock is not clear.In this study,on the basis of comparing mammary tissue morphology and m RNA abundance and protein expression level of heat shock protein of dairy cows in spring and summer,heat treatment model of mammary epithelial cells was established in vitro.Through the study of cell morphology,structure and function,antioxidant capacity,autophagy and apoptosis were focused,the functional gene and protein expression level were detected,furthermore,the mechanism of related signaling pathways was studied.The effects of heat treatment on functional metabolism of mammary epithelial cells and the alleviating effect and molecular regulation mechanism of taurine were investigated.We obtained the following results:1.Effects of high temperature on production,mammary tissue morphology and heat shock protein of dairy cowsAccording to the analysis of the average daily milk yield and milk quality of a dairy farm in Fujian province in 2018,the average daily milk yield in July,August and September was 28.4kg,29.3 kg and 28.8 kg,respectively.Experiment 1: Four cows in spring and four cows in summer were selected,whose mammary glands were healthy.The spring group was Control group,and the summer group was heat stress(HS)group.In the Control group,cows were exposed to an ambient temperature of 19.6℃.In HS group,cows were exposed to 29.9℃.Respiration rate and mammary gland surface temperature were measured before slaughter,and mammary gland tissue was collected after slaughter.The results showed that when the temperature was 19.6℃,the respiration rate of cows was 24 times /min.When the temperature was 29.9℃,the respiration rate of cows reached 114.5 times /min.It shows that cows in HS group are in heat stress state in summer.Compared with the control group,the mammary gland surface temperature in HS group increased significantly(P < 0.01).Quantitative Real-Time PCR of heat shock protein 70(HSP70)and heat shock protein90(HSP90)m RNA expression showed that the HSP70 and HSP90 m RNA expression levels in HS group were significantly higher than those in Control group.Furthermore,HSP90 was expressed in the mammary gland tissue both in Control group and in HS group,and the protein level of HSP90 in the HS group was significantly elevated in comparison with the Control group(P < 0.05).2.Establishment of heat stress model of mammary epithelial cells in vitro and effects of heat treatment on cellular functionIn this study,MAC-T(a bovine mammary epithelial cell line)cells were used as target cells to construct the heat stress model of bovine mammary epithelial cells in vitro.Experiment 2: MAC-T cells was digested by trypsinase and inoculated into 6-well or 96-well plates when the cell growth density was about 80%.When the cell growth reached 70%,the cells were replaced with serum-free medium for 24 h,and then the heat stress(HS)group were placed in a cell incubator at 42.5℃ for heat treatment for 1h,then moved to 37℃incubator for further culture.Cells in the Control group were cultured at 37℃.First,bovine mammary epithelial cells were identified by immunofluorescence staining for keratin-18.Then,the effect of heat treatment on the activity of mammary epithelial cells of dairy cows was detected.It showed that mammary epithelial cells were treated with heat shock at 42.5℃for 1 h,and then cultured at 37℃ for 12 h,the cell activity decreased most obviously.Furthermore,the results showed that after 1h heat exposure at 42.5℃,the m RNA expression levels of HSF1 and HSP90 were different at different time when cells were recovered at 37℃.After 12 h recovering,the m RNA expression levels of the two genes were observably elevated in comparison with the Control group(P < 0.01).Heat treatment also had an effect on the morphology of mammary epithelial cells,the cells in the Control group showed normal morphology and typical pebblestone shape.When the cells were heat treated for 1h and recovered for 12 h,the morphology of some cells changed and showed clumps instead of normal adherent growth.Ultrastructural results showed that mitochondria in the Control group were normal,with complete structure and abundant in cristae.In the HS group,mitochondria were enlarged and some cristae had fallen off.The results showed that reactive oxygen specie(ROS)production in the Control group was significantly lower than that in the HS group(P < 0.01).The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in HS group were significantly decreased compared with the Control group(P < 0.01).The activity of catalase(CAT)of mammary epithelial cells in HS group was significantly lower than that in Control group(P< 0.05).In addition,malondialdehyde(MDA)production in HS group were enhanced compared with Control group(P < 0.01).Also,heat treatment can affect the autophagy of mammary epithelial cells of dairy cows.A small number of autophagosomes were found in the Control group,while the number of autophagosomes were significantly increased in the HS group.Further studies showed that heat treatment significantly increased the m RNA expression levels of autophagy-related genes 7(ATG7)and microtubule-associated protein l light chain 3(LC3)(P < 0.05).There was no significant difference in m RNA expression levels of beclin 1(BECN1)and lysosomal-associated membrane protein 2(LAMP2)between the two groups(P > 0.05).Oil red O staining showed that fatty acids were secreted in mammary epithelial cells in both the Control and HS groups,and there was no significant difference between the two groups.The results of triglyceride detection showed that there were no significant difference in triglyceride content of intracellular and cell culture supernatant between Control group and HS group(P > 0.05).However,compared with Control group,the m RNA expression levels of milk protein synthesis gene casein beta(CSN2)and casein kappa(CSN3)in HS group were markedly decreased(P < 0.05).In addition,there was no significant difference in m RNA expression levels of casein alphaS1(CSN1S1)and casein alpha-S2(CSN1S2)between the two groups(P > 0.05).3.Effects of taurine treatment on antioxidant capability and apoptosis of heatexposure mammary epithelial cellsIn order to elucidate the effects and molecular mechanism of taurine supplementation on antioxidant capacity and apoptosis of mammary epithelial cells under heat stress,the experiment 3 was designed as follows: Mammary epithelial cells were randomly divided into5 groups : control(C)group,heat stress(HS)group,low taurine(HS+LTau)group,medium taurine(HS+MTau)group and high taurine(HS+HTau)group.After cell fusion reached about 70%,the cells were cultured under the condition of DMEM/F12 without serum for 24 hours before heat treatment.The HS group was cultured at 42.5℃ for 1 h and recovered at 37℃ for 12 h.The taurine groups were pretreated with 10 m M,50 m M and 100 m M taurine for 2 h,cultured at 42.5℃ for 1 h,and recovered at 37℃ for 12 h.The C Group was cultured continuously at 37℃ without taurine treatment.The results showed that three different doses of taurine(10 m M,50 m M,and 100 m M)were used to study the effect of taurine on heat-induced MAC-T cells.Further studies showed that taurine at different concentrations could significantly inhibit the m RNA expression levels of HSF1 and HSP90.The western blotting assay showed that the protein expression of HSF1 in HS group was significantly increased compared with Control group(P < 0.01).Taurine of 10 m M,50 m M and 100 m M decreased the protein expression of HSF1 at different degrees.At the same time,there was no significant difference in HSP90 protein expression among the five treatment groups(P > 0.05).This study was to investigate low levels of HSF1 were found in both cytoplasm and nucleus in the Control group,while the protein level of HSF1 in cytoplasm and nucleus cells in HS group markedly increased,and translocation of HSF1 was inhibited in HS+LTau,HS+MTau and HS+HTau groups.Ultrastructure were observed that there were complete mitochondrial structure and abundant cristae in the Control group,while the mitochondrial morphology of HS group was significantly changed,the mitochondrial structure was swollen,and some cristae fell off.Taurine of 10 m M and 50 m M can effectively relieved the damage of mitochondrial structure.In addition,the activity of mitochondrial complex I was inhibited by heat exposure in mammary epithelial cells(P < 0.05),and was markedly elevated in the HS + LTau group(P < 0.05).Taurine significantly increased the antioxidant capacity of mammary epithelial cells under heat treatment.Compared with Control group,MDA level in HS group was significantly increased(P < 0.01),while the activities of SOD and GSH-Px were significantly decreased(P < 0.01).The MDA level in taurine group was significantly lower than that in HS group(P < 0.01).There was no significant difference in SOD activity between HS group and HS + LTau group.SOD activity in HS + MTau and HS + HTau groups was significantly higher than that in HS group(P < 0.001).Meanwhile,the activity of GSHPx in HS + LTau group was significantly higher than that in HS group.Moreover,taurine effectively alleviated the apoptosis of mammary epithelial cells under heat stress.The ratio of cell apoptosis increased markedly when the MAC-T cells were treated with heat shock(P< 0.001).The 10 m M,50 m M,and 100 m M of taurine treatments significantly alleviated cell apoptosis in the heat shock-induced MAC-T cells.Compared with Control group,the m RNA abundance of Bcl-2 associated X(BAX)and Caspase3,the ratio of BAX/Bcl2 in HS group were significantly increased(P < 0.05).Different concentrations of taurine alleviated the increase of the m RNA expression of BAX and Caspase3,the ratio of BAX/Bcl2.In addition,compared with Control group,protein expression of Caspase3 and Cleaved Caspase3 were both increased in HS group.After adding taurine,the protein level of Caspase3 and Cleaved caspase3 decreased.In conclusion,taurine can effectively reduce oxidative damage and apoptosis associated with heat stress,so that it can protect MAC-T cells from heat exposure.4.Effects of taurine supplementation on autophagy and milk protein synthesis of mammary epithelial cells under heat shockTo study the effects of taurine supplementation on the autophagy and milk protein synthesis in heat-induced mammary epithelial cells,the experiment design is the same as experiment 3.The results showed that the protein expression level of LC3-Ⅱ in HS group was significantly higher than that in Control group(P < 0.01).The protein expression level of LC3-Ⅱ in HS+MTau group was significantly lower than that in HS group(P < 0.05).Immunofluorescence staining showed that there was no significant difference in CSN3 protein expression between Control group and HS group(P > 0.05).However,the CSN3 protein expression level in HS+LTau and HS+MTau groups was significantly higher than that in Control group and HS group(P < 0.05).Furthermore,we investigated the mammalian target of rapamycin(m TOR)pathways which was related to milk protein synthesis and autophagy.The results of WB showed that p-m TOR protein expression in heat-induced MAC-T cells were not significantly different from that in the Control group.However,the expression of p-m TOR protein in HS+MTau group was significantly higher than that in Control group and HS group(P < 0.01).To sum up,the conclusions of this study are as follows:(1)High temperature significantly increased the respiratory rate and surface temperature of mammary gland,and the m RNA levels of HSP70 and HSP90,and the protein expression level of HSP90 in mammary gland tissue.(2)Heat treatment inhibited cell viability and cell morphology of mammary epithelial cells.Heat treatment caused swelling of mitochondria,loss or disappearance of crists,also significantly increased m RNA expression of HSF1 and HSP90 in mammary epithelial cells.Moreover,heat treatment significantly increased ROS level,significantly decreased SOD,GSH-Px and CAT activities,and increased the accumulation of MDA production,resulting in oxidative damage.Heat treatment also significantly increased the number of autophagosomes in mammary epithelial cells,and the m RNA expression levels of autophagyrelated genes ATG7 and LC3.In addition,heat treatment reduced the m RNA expression of CSN2 and CSN3 genes in bovine mammary epithelial cells,resulting in reduced protein synthesis ability.(3)Taurine supplementation effectively inhibited the migration of HSF1 induced by heat treatment and the increase of HSF1 m RNA and protein levels,which improved the heat tolerance of mammary epithelial cells of dairy cows.Moreover,taurine alleviated oxidative stress,and the damage of mitochondrial structure and the decrease of complex I activity.Moreover,taurine inhibited the apoptosis of mammary epithelial cells induced by heat treatment.(4)Taurine supplementation significantly reduced the number of autophagosomes induced by heat treatment and the protein level of autophagy marker LC3-Ⅱ,which can alleviate the increase of autophagy induced by heat treatment in mammary epithelial cells.Taurine supplementation also increased the m RNA expressions of milk protein synthesis genes CSN1S1,CSN1S2,CSN2 and CSN3,and increased the protein level of CSN3,thus alleviating the decreased milk protein synthesis ability caused by heat treatment in mammary epithelial cells.Taurine can significantly enhance p-m TOR protein expression,thereby inhibiting autophagy and enhancing milk protein synthesis ability.