Influence Of α7nAChR Activation In Adipocytes On Tunicamycin-induced Endoplasmic Reticulum Unfolded Protein Response

Background and ObjectiveObesity is a huge public health problem and closely related to metabolic diseases such as type 2 diabetes,atherosclerosis,hypertension,cancer,asthma,stroke,non-alcoholic fatty liver disease.Expansion of adipose tissue occurs during obesity,including enlargement of adipocytes and the increased number of adipocytes.Now low-grade chronic inflammation in adipose tissue is considered to be involved in the development of these metabolic diseases,and endoplasmic reticulum stress plays a key role in this chronic inflammation.The endoplasmic reticulum is the main assembly site for almost all secreted and integrated membrane proteins.Many physiological and pathological injuries such as abnormal calcium regulation,viral infections,environmental toxins,redox damage,mutant protein expression,acidosis,radiation,aging,the increased synthesis of secreted protein and so on,these disturbances disrupt the protein balance,resulting in impaired endoplasmic reticulum protein folding ability and increased accumulation of unfolded proteins,causing endoplasmic reticulum stress.In order to alleviate endoplasmic reticulum stress and coordinate restoration the function of endoplasmic reticulum,cells initiate a comprehensive pathway called the unfolded protein response that transfers protein folding information from the endoplasmic reticulum to the nucleus by controlling the activity of specific downstream transcription factors.UPR has three sensors that embedded in the endoplasmic reticulum membrane:PERK,IRE 1,ATF6,which play a key role in restoring homeostasis by three pathways:1)focus on translation attenuation for a period of time and prevent new produced proteins from entering the endoplasmic reticulum,resulting in a decrease in overall protein synthesis;2)induce upregulation of endoplasmic reticulum chaperone gene to produce the molecular chaperone required for the correct protein folding in the endoplasmic reticulum;3)in order to adapt to high protein loads,the endoplasmic reticulum compartment proliferates and starts endoplasmic reticulum-associated degradation of unfolded or misfolded proteins(ERAD).The expression level of α7nAChR in the subcutaneous adipose tissue of human obese subjects was decreased,and the activation of α7nAChR showed a significant anti-inflammatory effect.α7nAChR is a member of the non-neuronal cholinergic system and is widely expressed on non-neuronal cells,including adipocytes,T lymphocytes,B lymphocytes,monocytes,and macrophages.Non-neuronal cholinergic system is widely present in peripheral tissues(including liver tissue and muscle tissue)and cells(including adipocytes,aortic endothelial cells and macrophages).The system consists of nAChR and mAChR,acetylcholine,and cholinesterase.The proinflammatory cytokine TNF-α was decreased in a dose-dependent manner with increased nicotine concentration when the α7nAChR agonist.nicotine pretreated rat adipocytes;the α7nAChR-specific agonist PNU-282987 produced significant anti-inflammatory effects when treated human adipocytes in vitro,and reduced the expression of proinflammatory genes such as IL-6,MCP-1 and TNF-α induced by lipopolysaccharide.Therefore,α7nAChR-mediated non-neuronal cholinergic system plays an important role in regulating adipocyte inflammation.Adipocyte endoplasmic reticulum stress is closely related to low-grade chronic inflammation.The purpose of this research is to study the effect of activation ofα7nAChR in non-neuronal cholinergic system on tunicamycin-induced adipocyte endoplasmic reticulum stress,and the specific mechanism by western blot,RT-PCR,immunofluorescence and other techniques.Provide a new therapeutic target and theoretical basis for the treatment of obesity and obesity-related diseases.Methods1.3T3-L1 preadipocytes purchased from Cell Bank of Shanghai Academy of Sciences were cultured and induced into adipocytes by the classic hormone cocktail method.The CCK8 method was then used to detect the viability of adipocytes after treatment with different Tm concentrations for 12 h,18 h,and 24 h.RT-PCR and western blot were used to detect the expression levels of endoplasmic reticulum stress markers in adipocytes when different Tm concentrations were treated for 12 h,18 h and 24 h.Then select the appropriate concentration and time to establish a endoplasmic reticulum stress model.2.The used concentration of α7nAChR non-selective agonist Ach、NIC and selective agonist GTS-21 and α7nAChR selective antagonist MLA were 10 μmol/L according to the literature.CCK8 method was used to detect the viability of adipocytes after treatment with 10 μmol/L Ach,NIC,GTS-21 and MLA for 48 h.RT-PCR and western blot were used to detect the levels of endoplasmic reticulum stress markers in adipocytes after treatment with 10 μmol/L Ach,NIC,GTS-21 and MLA for 3 h,6 h,12 h,24 h,36 h,and 48 h.3.3T3-L1 adipocytes were divided into Control group,Tm group,MLA+Tm group,ACh+Tm group,NIC+Tm group,GTS-21+Tm group,MLA+ACh+Tm group,MLA+NIC+Tm group,MLA+GTS-21+Tm group.After treatment with or without MLA for 2 h,then treatment with ACh,NIC,GTS-21 for 36 h,and finally with Tm for 12h,western blot was used to detected the effect of activation ofα7nAChR on UPR signaling pathway.4.3T3-L1 preadipocytes were divided into Control group,Tm group,MLA+Tm group,ACh+Tm group,NIC+ Tm group,GTS-21+Tm group,MLA+ACh+Tm group,MLA+NIC+Tm group,MLA+GTS-21+Tm group.After treatment with or without MLA for 2 h,treatment with ACh,NIC,GTS-21 for 36 h,and finally with Tm for 12 h,western blot was used to detect the effect of activation of α7nAChR on UPR signaling pathway.5.3T3-L1 preadipocytes were divided into the Control group,Tm group,ACh+Tm group,NIC+Tm group,GTS-21+ Tm group.First treated with ACh,NIC,GTS-21 for 36 h,and then treated with Tn for 12 h.Immunofluorescence was used to detect the expression levels of ATF6 and p-eIF2α of the UPR pathway.Result1.The endoplasmic reticulum stress model was established by treatment with 1μg/ml Tm concentration for 12 h.2.Treatment adipocytes with ACh,NIC,GTS-21 and MLA at a concentration of 10μmol/L for 48 h did not affect the viability of adipocytes and did not induce endoplasmic reticulum stress in normal adipocytes.3.Activation of α7nAChR reduced the proteins expression levels of XBP1u,ATF6,p-PERK/PERK,p-eIF2α/eIF2α,p-IRE1α/IRE1α and increased the proteins expression levels of CHOP and XBP1s in 3T3-L1 adipocytes,but had no effect on the expression level of Bip protein.MLA alone used had no effect,but can reverse the effects of ACh,NIC,and GTS-21.4.Activation of α7nAChR reduced the proteins expression levels of Bip,CHOP,XBP1u,ATF6,p-PERK/PERK,p-eIF2α/eIF2α,p-IRE1α/IRE1α and increased the proteins expression levels of XBP1s in 3T3-L1 preadipocyte.MLA alone used had no effect,but can reverse the effects of ACh,NIC,and GTS-21.5.Activation of α7nAChR reduced the nuclear translocation of the ATF6 protein and the cytoplasmic level of p-eIF2α protein of the UPR pathway in 3T3-L1 preadipocytes.Conclusion1.The activation of the non-neuronal cholinergic system does not induce endoplasmic reticulum stress under physiological conditions;2.Activation of α7nAChR inhibits the expression intensity of PERK-eIF2α and ATF6 signaling pathways during acute endoplasmic reticulum stress;3.The activation of α7nAChR inhibits the expression of p-IRE1α/IRE1α protein,but further up-regulated the expression of XBP1s protein during acute endoplasmic reticulum stress,providing a new regulatory target for regulating the intensity of metabolic stress and preventing obesity.