| Neutrophils(PMN)dysfunction caused by negative perinatal energy balance(NEB)status and abnormal fluctuation of estrogen(17β-estradiol,E2)in cattle is closely related to perinatal disease susceptibility,while cellular energy status.It is the basis for maintaining the normal function of PMN.However,in the low-glucose microenvironment formed by NEB in the perinatal period,the effect of E2 on the energy status of PMN and the specific regulatory mechanism are still unclear.In this study,three simmental ovariectomized cattles were selected,and PMN were obtained by jugular vein puncture and percoll density gradient separation for the following experiments.The purpose of this study was to explore the potential mechanism of E2 regulating PMN energy metabolism in cattle perinatal NEB state by evaluating the regulatory effect of E2 on the energy state and cellular metabolic pathways of cattle circulating PMN in vitro.(1)PMN was cultured with low glucose(2.5 m M)and normal glucose(5.5m M)and induced with E2(0,20,200 and 450 pg/m L)for 0 h,2 h,4 h,6 h,8 h and12 h,respectively.Cell viability assay showed that E2 had concentration-dependent(0-450 pg/m L)protective effect(P < 0.01),and the protective effect was most significant at 6 h(P < 0.01).In the downstream experiment,450 pg/m L E2 was used to induce PMN for 6 h.(2)PMN was induced with 450 pg/m L E2 under low glucose conditions for 6h,and the detection of autophagy and apoptosis protein(Western Blot)showed that,E2 enhanced the expression of PMN autophagy proteins LC3,p62 and Beclin1(P <0.01),increased the ratio of apoptotic protein Bcl-2/Bax(P < 0.01).These results suggest that E2 can resist the decline of cell viability induced by low glucose stress by promoting autophagy and inhibiting apoptosis.(3)PMN was induced by LPS for 6 h under low glucose conditions,and the intracellular glycogen content(biochemical method),ATP content(ELISA)and apoptosis rate(flow cytometry)were detected,the results showed that E2 inhibited GSK-3β activity and PMN apoptosis(P < 0.05)and increased the contents of glycogen and ATP(P < 0.05).These results suggest that E2 can inhibit GSK-3βactivity to maintain glycogen and ATP levels,and mitigate the negative effects of low glucose stress in cells.(4)To evaluate the effects of E2 on metabolic status and metabolic pathway characteristics under low glucose and LPS conditions,energy metabolic pathway inhibitors were used.Energy metabolism pathway related enzymes(ELISA and biochemical assays)showed that 2-DG(glycolysis inhibitor)inhibited PFK1 activity and decreased glycogen and ATP content(P < 0.01);6-AN(pentose phosphate pathway inhibitor)inhibited G6 PDH activity and increased glycogen content(P <0.01),but ATP content remained unchanged;Oligomycin A(oxidative phosphorylation inhibitor),TFA(tricarboxylic acid cycle inhibitor),etomoxir(fatty acid oxidation inhibitor)and BPTES(glutamine inhibitor)decreased ATPS,CS,CPT-1 activity and glutamine content,respectively(P < 0.05),increased glycogen content and decreased ATP production(P < 0.05),and E2 could reverse this trend and increase ATP and glycogen contents(P < 0.05).These results suggest that pentose phosphate pathway,mitochondrial metabolism and glutamine metabolism may determine the glycogen content of PMN under low glucose stress.PMN provides ATP to cells through glycolysis,mitochondrial metabolism and glutamine metabolism,while pentose phosphate pathway contributes less to ATP.E2 can provide ATP and glycogen to cells through PMN energy metabolism conversion.Low glucose significantly decreased PFK1 activity and glutamine content in resting PMN(P < 0.01),significantly increased the activities of G6 PDH,ATPS,CS and CPT-1(P < 0.05),E2 also decreased the activity of PFK1 and G6PDH(P < 0.01),enhanced ATPS,CS and CPT-1 activities,but increased glutamine content(P < 0.05).E2 can enhance glutamine and mitochondrial metabolism(oxidative phosphorylation,fatty acid oxidation and tricarboxylic acid cycle),inhibit glycolysis and pentose phosphate pathways,and promote glycogen storage and ATP production of resting PMN in low glucose environment.After PMN activation,PFK1 and G6 PDH activities and glutamine content increased(P < 0.01),ATPS,CS and CPT-1 activity decreased(P <0.05);E2 decreased PFK1 and G6 PDH activities and glutamine content(P < 0.01),enhanced ATPS,CS and CPT-1 activity(P < 0.05).These results indicated that E2 inhibited glycogen decomposition and promoted ATP production by changing the transformation of mitochondrial metabolism to glucose metabolism and glutamine metabolism pathways in activated PMN.In conclusion,E2 can enhance the glutamine metabolism and mitochondrial metabolism of PMN in low-glucose environment,inhibit glucose catabolism(glycolysis and pentose phosphate pathways),and reverse the conversion of activated PMN from mitochondrial metabolism to glucose metabolism and glutamine metabolism.E2 plays a role in maintaining the survival and immune potential of PMN cells under low glucose stress by inhibiting GSK-3β activity and promoting autophagy and increasing glycogen content. |
