The Mechanism Of Estrogen Affecting Cell Autophagy In Glucose Metabolism Of Bovine Neutrophil

Peripheral estrogen(E2)and neutrophils(PMNs)in the perinatal period of cattle play a key role in the physiological adaptation process of maternal initiation of delivery and postpartum recovery.The PMN mitochondria content is low,and the cell's self-energy level is limited.It requires continuous intake and utilization of exogenous glucose or lipids to maintain its mature differentiation and immune activation.However,under the perinatal energy balance(NEB)state,whether the characteristic dynamic changes of E2 are highly correlated with the immune activity of PMNs,its internal mechanism remains to be revealed.This study used in vitro induction tests,using Western Blot,qRT-PCR,cellular immunofluorescence,flow cytometry and other technologies to explore the autophagy mechanism and immune activity effects of E2 regulating PMNs glucose metabolism,thus explaining the perinatal NEB status of cattle Lower E2 regulates the mechanism of PMNs glucose metabolism.In this experiment,the effect of E2 on the immune activity of PMNs was first studied.After the cells were separated,low concentration E2(L-E2;20 pg/mL),medium concentration E2(M-E2;450 pg/mL),and high concentration E2(H-E2;880 pg/mL)induced cells for 2h,in which the estrogen receptor antagonist ICI182780(10-6M)was added in the H-E2 group and pretreated for 1h,and the cells and culture medium were collected for subsequent experiments.Secondly,the effect of E2 on the autophagy activity of PMNs in a low-sugar state was studied.L-E2,M-E2,and L-E2 induced cells were added to the low-concentration glucose(L-G;2.5 mM)culture system for 2 hours and the cells were collected for subsequent experiments.Finally,the effect of E2 on the autophagy activity of PMNs in high glucose state was studied.In the high concentration glucose(H-G;25 mM)culture system,L-E2,M-E2,and L-E2 were added to induce cells for 2h and the cells were collected for subsequent experiments.The results show that E2 promotes the expression of ER? and GPR30(p<0.01)but inhibits ER?(p<0.01);L-E2 activates p65 and promotes the release of downstream cytokines(p<0.01);H-E2 inhibits p65 activation(p<0.05)and inhibit the release of cytokines(p<0.01).Compared with H-E2 group,the addition of ICI promoted p-p65 protein expression(p<0.01)and cytokine release(p<0.01).L-E2 activated AMPK(p<0.01)while H-E2 inhibited AMPK(p<0.01).Compared with H-E2 group,ICI inhibited AMPK protein expression after adding ICI(p<0.01).Compared with the LG group,the ATP and MDA content of the L-G+E2 group was significantly increased(p<0.01),SOD content,ROS content,GLUT1,GLUT4 protein expression and apoptosis were significantly reduced(p<0.01),with the E2 concentration Increasing autophagy-related proteins Beclinl,Atg5,LC3B-? showed a concentration-dependent increase(p<0.01),p62 showed a concentration-dependent decrease(p<0.01).Compared with the HG group,there was no change in apoptosis in the H-G+E2 group(p>0.05),GLUT1,GLUT4 protein expression,ATP content,ROS content,and MDA content were significantly reduced(p<0.01),and SOD content was significantly increased(p<0.01),with the increase of E2 concentration,the autophagy related proteins Beclinl,Atg5,LC3B-? increased in a concentration-dependent manner(p<0.01),and p62 showed a concentration-dependent decrease(p<0.01).In conclusion,E2 relies on ERa and GPR30 receptors,and plays an important role in protecting PMNs from impairment of glucose metabolism by promoting autophagy.