A Study On The Molecular Mechanisms Of Melatonin On Adipocyte Proliferation And Inflammatory Response

Adipose tissues play an important role in the regulation of energy storage and homeostasis.Melatonin is an amphiphilic neurohormone synthesized and secreted by the pineal gland mainly during the dark period of the circadian cycle.It regulated a number of neuroendocrine and physiological processes in the central nervous system as well as in peripheral tissues in mammals.Studies have shown circadian rhythm involved in proliferation,differentiation,lipid metabolism and apoptosis of adipocytes.The disorder of circadian rhythm closely related with unbalance homeostasis of adipose tissue.However,the effects of melatonin in this progression still be unclear.In this study,melatonin was used to treat adipose tissue and adipocytes of mice.The effects of melatonin on adipocyte proliferation and circadian rhythm were investigated,and this study showed melatonin regulated the cross talking of adipocytes and macrophages via the extracellular vesicles.Further revealed the underlying regulating mechanisms of adipocytes inflammation and pyroptosis by melatonin.The main results are listed as followed:1.Melatonin increased the mRNA level and amplitude of circadian rhythm marker genes Bmal1 and Clock.The positive EDU cells were also elevated with Melatonin treatment.The rhythmic expression of cell proliferation indicators c-Myc and Cyclin E were increased with melatonin.In addition,the gene Clock can combine with the E-box domain of c-Myc,and melatonin treatment enhanced this combination and promoted the translation of c-Myc.Melatonin further accelerate the proliferation of adipocytes.Moreover,three protein CLOCK,c-Myc and HDAC3 formed a complex.The complex played key role in the regulation of adipocyte proliferation.Melatonin alleviated cell proliferation inhibition in HFD-induce obese model and jet-lagged mice model via the regulation of circadian genes,such as Clock.2.Melatonin inhibited obese-induced higher expression of inflammation indicators: IL-6,TNF? and IL-1?.Melatonin increased the concentration of Citrate and ?-KG which are the important core intermediates of TCA cycle.Deep RNA-seq analyzation revealed IDH2,the key regulator of TCA cycle,participated in the inhibition regulation of melatonin on adipose tissue inflammation.And melatonin blocked the expression of TNF? via the regulation of connection between Sirt1 and IDH2 protein.In addition,melatonin enhanced the mRNA expression level and activity of IDH2;Increased the ?-KG content of adipocytes and melatonin also inhibited adipocyte inflammation.This was conducted by activating the demethylation which was mediated by Tet enzyme family.Moreover,melatonin elevated the exosomal ?-KG transferring from adipocytes to macrophages,which promoted the polarization of M2 macrophages and alleviated adipose tissue inflammation.The study also showed melatonin reduced circadian rhythm disorder induced adipose tissue inflammation by using jet lag model.3.RNA-sequencing method was used to investigated the inflammation related genes which were regulated with melatonin.There is an enrichment of NLRP3 inflammasome and NF-?B signal.Melatonin treatment decreased the expression levels of NLRP3,ASC,Caspase1 and IL-1?.The apoptosis induced by LPS was also reduced.The TUNEL positive cells caused by Caspase 1 were reduced with melatonin treatment along with the decrease secretion of LDH,and this process is not depended on the activation of Caspase 3.Additionally,melatonin inhibited the mRNA level of GSDMD,the core gene in pyroptosis,and further alleviated pyroptosis.Melatonin suppressed the activation of NF-?B signals and inhibited the nuclear translocation,which led to the decrease expression of IRF7 that located in the downstream.Meanwhile,melatonin inhibited pyroptosis via regulating the interaction of IRF7 and GSDMD.In summary,this study showed the regulation mechanisms of melatonin promoted adipocytes proliferation via circadian rhythm genes and Clock/c-Myc/HDAC3 signals.And investigated the underlying mechanisms that melatonin activated ?-KG mediated DNA demethylation,and melatonin alleviated adipose tissue inflammation by exosome transportation.The study also revealed the regulation of melatonin in inactivation of NLRP3 inflammasome and GSDMD mediated pyroptosis.Finally,this study illustrated the function of melatonin in maintaining adipose tissue homeostasis.This study will lay a molecular basis for the mouldability of lipid homeostasis,and give important theoretical basis for the improvement of meat quality.