Effects Of Melatonin On Adipocyte Endoplasmic Reticulum Stress Induced Inflammation And Molecular Mechanisms

Melatonin,as a multi-functional biological factor,has various regulatory effects including anti-oxidation,anti-inflammatory,and participation in immune regulation,but it is unknown whether melatonin affects inflammation induced by endoplasmic reticulum stress.In this study,3T3-L1 adipocytes were treated with tunicamycin to establish an endoplasmic reticulum stress model to induce inflammation,and after treatment of melatonin,their effects on different aspects of endoplasmic reticulum stress and inflammation and their mechanisms of action were examined.The main results are listed below:1.Melatonin relieves endoplasmic reticulum stress by upregulating circadian rhythms.After treatment of mature adipocytes with 1 ?mol/L melatonin,the mRNA expression of the circadian clock genes CLOCK and BMAL1 was significantly increased(P<0.05),PER1,PER2,CRY1,CRY2,and REV-ERB.mRNA expression was significantly decreased(P<0.05),and endoplasmic reticulum stress and inflammation-related gene expression were also significantly reduced;melatonin treatment was found to significantly reduce the calcium ion fluorescence intensity and alleviate the endoplasmic reticulum after staining with a calcium ion probe.Mesh stress: It was found that melatonin promotes the biological rhythm by down-regulating the DNA methylation of the circadian clock gene PER1 by measuring the DNA methylation level of PER1 before and after MT treatment,in which the Methyl-CpG-binding domain 3 expression was significantly changed;then interfered with PER1 in cells found after the endoplasmic reticulum stress critical pathway molecules ATF6 mRNA levels increased significantly(P<0.05),the use of bioinformatics prediction found two ATF6 promoter region can be combined with the E-box site of PER1 and the relative fluorescence activity of the two E-box sites can be significantly increased by perturbing the PER1 gene by luciferase activity assay(P<0.05).PER1 regulates ATF6 to relieve endoplasmic reticulum stress through negative transcription.2.Melatonin relieves the inflammatory response mediated by endoplasmic reticulum stress.In order to detect the effect of melatonin on endoplasmic reticulum stress-mediated inflammation,we used 2 ug/mL tunicamycin(TM)to treat cells for 12 hours to construct a model of endoplasmic reticulum stress while using faded black After treatment for 24 h,the mRNA and protein expression levels of the endoplasmic reticulum stress markers ATF6,PERK,IRE1,GRP78,and CHOP were significantly up-regulated(P<0.05).The inflammation-related genes IL-6,TNF-?,and NF-?B mRNA and protein expression of NLRP3 and IL-1? were also significantly up-regulated(P<0.05),indicating that endoplasmic reticulum stress induced inflammation successfully,and MT treatment could alleviate this effect;in addition,endoplasmic reticulum stress was used.Inhibitor 4-phenylbutyric acid(4-PBA)treatment,found that when endoplasmic reticulum stress was inhibited,inflammation-related gene expression was significantly downregulated.By injecting 20 mg/kg melatonin and 1 ?g/g tunicamycin in vivo,the expression of endoplasmic reticulum stress and inflammation-related genes in adipose tissue was detected to be consistent with the cell level,and H&E was performed on the tissue section.Staining revealed that inflammatory infiltrates were evident under endoplasmic reticulum stress conditions,and the level of immunohistochemistry of ATF6 was significantly increased,and the immunofluorescence intensity of IL-6,TNF-?,GRP78,and ATF6 is also significantly increased,while MT could inhibit these effects.The above shows that MT can relieve endoplasmic reticulum stress-mediated inflammation.3.Melatonin alleviates endoplasmic reticulum stress-induced M1 macrophage polarization-mediated inflammation.Adipocyte inflammation is closely related to the role of macrophages.In this experiment,adipocytes and macrophages were co-cultured.Lipopolysaccharide(LPS)and interleukin-4(IL-4)were used to induce M1 and M2 macrophages,respectively.Maturation,overexpression of ATF6,a key pathway factor for endoplasmic reticulum stress,through the detection of M1 macrophage marker genes IL-6,TNF-a,IFN-? and IL-12,and M2 macrophage marker genes CD163,CD206 mRNA and protein expression of IL-10 and IL-4 were found to significantly upregulate the expression of surface marker genes on M1 macrophages under the condition of endoplasmic reticulum stress in adipocytes,and the expression of surface marker genes on M2 macrophages was significantly downregulated;Immunofluorescence staining of IFN-? and CD163 showed that adipocyte endoplasmic reticulum stress can promote the fluorescence intensity of IFN-? and weaken the fluorescence expression of CD163,and these phenomena were reversed after the addition of melatonin.In addition,after overexpressing ATF6,the mRNA and protein levels of the inflammatory body-related genes NLRP3,Pro-caspase1,Caspase1,Pro-IL-1?,and IL-1? were significantly upregulated(P<0.05).After MT treatment,the effects were reversed,suggesting that adipocyte endoplasmic reticulum stress promotes inflammation by activating the expression of inflammasome,whereas MT can alleviate this effect.The above results demonstrate that melatonin relieves ER-mediated inflammatory responses.In summary,this experiment clarifies the regulation of melatonin in adipocyte endoplasmic reticulum stress and inflammation,that is,melatonin inhibits endoplasmic reticulum stress-induced inflammatory response by increasing the gene rhythm of the biological clock and discovers that DNA methylation,transcriptional regulation,and the mechanism of macrophage polarization are involved.This study provides a new target for the treatment of adipocyte inflammation and provides a theoretical basis for studying the genetic regulation of animal fat deposition.