The Role Of Chemerin In Alleviating Placental Oxidative Stress By Regulating The CGAS-STING Pathway In Gestational Diabetes Mellitus

Objective: To explore pathological mechanisms of placental mitochondrial dysfunction and oxidative stress in gestational diabetes mellitus(GDM),and to examine the role of chemerin in regulating cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)signaling pathway,and to investigate the effect and molecular mechanism of chemerin in GDM placental oxidative stress and mitochondrial dysfunction,with the aim of searching for novel therapeutic targets for GDM.Methods: The study was divided into three parts,including clinical sample analysis,cellular molecular mechanism and animal experiments.The first part was performed by collecting placental tissues,peripheral blood and umbilical arterial blood of twenty-five pregnant women with GDM and twenty-five pregnant women with normal glucose tolerence during cesarean section.The mitochondrial structure of placental trophoblastic cells were observed by transmission electron microscopy.The activities of placental antioxidant enzymes,including superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)and the content of lipid peroxidation malondialdehyde(MDA)were measured,respectively.The expressions of disulfide-bond A oxidoreductase-like protein(Dsb A-L)and peroxisome proliferator-activated receptor-gamma coactivator-1α(PGC-1α)in placenta were detected by immunohistofluorescence,immunohistochemistry,western blot and real-time quantitative PCR,respectively.The localization and expressions of chemerin,STING,TANK binding kinase 1(TBK1),interferon regulatory factor 3(IRF3)and their phosphorylated kinases in placenta tissues were assayed by immunohistochemistry and western blot,respectively.The levels of chemerin in plasma of peripheral blood and umbilical arterial blood of the two groups were measured by enzyme-linked immunosorbent assay.In the second part,an insulin-resistance(IR)cell model was constructed on human chorionic trophoblastic HTR-8/SVneo cells and the effect was verified.Recombinant human chemerin(active cytokine)with different concentrations were administrated to the IR cell model for different lengths of time,and the effects of chemerin on the protein expressions of cGAS,STING,TBK1 and IRF3 were detected by western blot.The plasmid RARRES2,a gene encoding chemerin,was transfected into HTR-8/SVneo cells,and the direct protein interaction between chemerin and Dsb A-L was investigated by co-immunoprecipitation.The Dsb A-L gene was silenced in HTR-8/SVneo cells by small interfering RNA technique,and then recombinant human chemerin(active cytokine)was added to the cell culture medium for 72 hours,and the expressions of TBK1 and IRF3 were detected by western blot.In the third part,a GDM animal model was established by feeding C57BL/6J mice with high-fat diet,and the effect of GDM animal model was evaluated.After intraperitoneal injection of the active cytokine chemerin(10 μg/kg)into GDM mice for 4days,the impact of exogenous chemerin on the glucose tolerance in tail vein of GDM mice and on the activities of placental antioxidant enzymes SOD,GSH-Px,CAT and the content of MDA in GDM mice were investigated.The molecular mechanisms of active cytokine chemerin in placental insulin resistance and oxidative stress in GDM mice were examined by western blot.The changes of body weight of mice offsprings from 1 week of age to 8 weeks of age were recorded.Results: 1.Compared with the control group,the mitochondria of placental trophoblastic cells were relatively swollen in GDM,and there were compensatory increases in SOD,GSH-Px and CAT enzyme activities in GDM placenta.There was no statistical difference in MDA content in placenta between the two groups of pregnant women.Compared with the control group,the expressions of Dsb A-L and PGC-1α in placenta were both lower,and the cGAS-STING signaling pathway was activated,and the expression of chemerin was upregulated in the GDM group.In the GDM group,the plasma levels of chemerin in umbilical blood were higher than those in peripheral blood,however there were no statistical differences in the content of chemerin in plasma of umbilical blood or peripheral blood between the two groups of pregnant women.2.The IR cell model was accompanied with oxidative stress,decreased mitochondrial membrane potential,and activated cGAS-STING pathway.The inhibitory effects of active cytokine chemerin on the protein expressions of cGAS,STING and IRF3 were related to the intervention time and concentration gradient.Overexpression of RARRES2 gene upregulated the protein level of Dsb A-L,but there might not be a direct protein interaction between chemerin and Dsb A-L.After silencing the Dsb A-L gene,active cytokine chemerin still down-regulated the protein expressions of TBK1 and IRF3.3.The GDM mice model was constructed successfully,active cytokine chemerin reduced the blood glucose level at 120 minutes after glucose administration in GDM mice,and increased the expressions of protein kinase B(AKT/PKB)and Dsb A-L in placenta of GDM mice,and elevated the activity of antioxidant SOD,and reduced the body weight of GDM mice offsprings from birth to 4 weeks of age,thereby reversing the obese phenotype of the offsprings.Conclusions: The development of GDM was associated with placental oxidative stress,mitochondrial dysfunction,and activation of the cGAS-STING pathway.The active cytokine chemerin regulated the cGAS-STING signaling pathway in a Dsb A-L independent manner,and its regulatory role presented time-dependent and concentration-dependent effect.The elevated expression of chemerin in GDM placenta might be a protective mechanism,and chemerin was beneficial for reducing oxidative stress,improving mitochondrial function,and alleviating insulin resistance.