Effects Of JNK Inhibitor On Hyperthermia-induced Cell Injury In Cultured Bovine Mammary Epithelial Cells

The JNK pathway was important in controlling programmed cell death or apoptosis,and it was a heated discussion about the regulation of JNK pathway.In this study,we investigated the effects of JNK specific inhibitor SP600125 on hyperthermia-induced the bMECs by analyzing the cell viability,apoptosis,oxidative damage and the expressions of related genes and proteins in response to the mutual role of the JNK pathway and apoptosis of bMECs under heat stress from the cellular level.This thesis includes three sections.1.Effects of JNK inhibitor on bovine mammary epithelial cells viability and apoptosis under hyperthermia.Cells were cultured in DMEM medium with different inhibitor SP600125 concentrations(0,20,40,60pmol/L)for 30min.The experiment group,cell was put into incubator at 41 ?,0.5h then recoveried 6h at a temperature of 37?.Cell viability was detected by MTT assay.Cell apoptosis was investigated by an Annexin V/PI double staining kit and flow cytometry.The result showed that:compared with the 37? groups,the cell viability was decreased and the ratio of cell apoptosis was inecreased in 41? groups.When added to 20?mol/L SP600125,the cell viability was higher significantly than without inhibitor in the 41 ? groups(P<0.05).After when added to 40?mol/L,the cell viability was very close to the group of 20?mol/L group.Until added to 60 ?mol/L,the cell viability was the lowest.Compared to 0?mol/L at 41 ?,adding SP600125 at 20,40 and 60pmol/L reduced the apoptosis rate by 37,30 and 27%,respectively(P<0.01)under hyperthermia.And the group of 20?mol/L was lower than the groups of 40 and 60?mol/L significantly at the 41?groups(P<0.05).It suggested that there was a heat stress under hyperthermia-treated of 41 ?and 0.5h,and cells subjected to the apoptosis.In order to make sure the accuracy of this experiment,we used two level of inhibitor(20 and 40 ?mol/L)to further study.2.Effects of JNK inhibitor on bovine mammary epithelial cells oxidative damage under hyperthermia.Cells were cultured in DMEM medium with different inhibitor SP600125 concentrations(0,20,40pmol/L)for 30min.The experiment group,put the cell into incubator at 41 ?,0.5h then recovery 6h at a temperature of 37?,determination of the activities of SOD and GSH,the content of MDA,the level of ROS,and the mRNA and protein expressions of theHSP72.The result showed that:compared with the37? groups,the activities of SOD and GSH were improved and the ROS level and MDA content were declined in 41? groups.And the expressions of HSP72 was increased in experimental groups.Under hyperthermia-treated,the ROS level(P<0.01)and MDA content(P<0.05)were significantly decreased in 20?mol/L group compared to without inhibitor.However,the activities of SOD(P<0.01)and GSH(P<0.05)were just reverse.After hyperthermia and adding to 20?mol/L SP600125,the mRNA expressions of HSP72 were improved by 21.9%(P<0.01),and the protein expression of HSP72 was improved by 54.4%(P<0.05)than 0?mol/L group.Thus,the hyperthermia induced the cells oxidative damage,the up-regulation of HSP72 had a protective effect oncells,and suppressed JNK could remission the bMECs oxidative damage.3.Effects of JNK pathway on related genes and proteins expression under hyperthermia-induced bovine mammary epithelial cells apoptosis.The test processwas the same as above.The related genes and proteins of Bax,Bcl-2,JNK,p-JNK,p53 and PUMA were detected by RT-qPCR and Western blot.The results showed that:compared to the 37? groups,the expressions of p-JNK,JNK,Bax/Bcl-2,P53and PUMA were increased in 41? groups,respectively.After hyperthermia and adding to 20?mol/L SP600125,the mRNA expression of JNK was down-regulation significantly than without inhibitor(P<0.01).And the mRNA expression of Bax/Bcl-2 was significantly decreased by 14.7%than the 0?mol/L at 41? groups(P<0.01).And the mRNA expression of P53 and PUMA were significantly.declined by 15.6%(P<0.01)and 13.4%(P<0.01),respectively.Compared to 0?mol/L group,the protein concentrations of p-JNK was decreased by 36.0%(P<0.01),and the protein concentrations of P53 and PUMA were lower and the protein concentretion of Bax was declined by 31.4%(P<0.01)in 20?mol/L group at 41? groups,respectively.It proved that hyperthermia aroused the cells heat stress,activated the JNK pathway,and induced the cells apoptosis.After inhibited the JNK,the related genes were down-regulation inordinately.Thus,hyperthermia induced the bMECs apoptosis via JNK pathway.