The Role Of Microglial Melatonin Type 1(MT1) Receptor On Neuroinflammation And The Mechanisms Underlying It

Aim:To explore the function of microglial melatonin type 1(MT1)receptor on microglial activation and the potential mechanisms underlying it,and to further investigate the roles and mechanisms of MT1 receptor in the development of neuroinflammation and Parkinson’s disease(PD).Methods: In vitro,lipopolysaccharide(LPS)(100 ng/m L)was used to stimulate the microglial cell line BV2 for 24 h to obtain a cell model of neuroinflammation,and then the conditioned medium of LPS-stimulated BV2 cells were collected to culture dopaminergic(DA)neuron cell line MES23.5 to acquire the inflammation-induced DA neuron death cell model.In vivo,wild-type C57BL/6 mice were injected with stereotactic brain injection,and LPS(1 mg/m L)was injected into the mice substantia nigra(SN)to obtain neruoiflammationinduced PD mouse model.In vitro,we knocked down Mtnr1 a which encoding the MT1 receptor in BV2 with a small interfering RNA si-Mntr1 a,and then the cells were stimulated with LPS for 12 h.After that,Western blot was performed to detect the protein expressions of pro-inflammatory factors like nitric oxide synthase(i NOS)and cyclooxygenase-2(COX-2).Real-time quantitative PCR(q RT-PCR)was performed to assess the m RNA levels of i NOS,COX-2,prostaglandin-6(IL-6)and tumor necrosis factor alpha(TNFα)to evaluate whether the absence of the MT1 receptor could aggravate neuroinflammation.To further explore the function of MT1 receptor in neuroinflammation,we pretreated BV2 cells with different concentrations of MT1 agonist Ramelteon(10 μM,50 μM,100 μM)for 12 h,and then stimulated with LPS for 12 h.Then,western blot was carried out to detect the protein levels of pro-inflammatory factors i NOS and COX-2 to determine whether MT1 receptor activation can inhibit LPS-induced microglial activation.We used small interfering RNA siMtnr1 a to slience the gene Mtnr1 a encoding MT1 receptor on BV2 cells or pretreating BV2 cells with Luzindole,an antagonist of MT1 receptor for 12 h,and then the cells were conducted with MT1 receptor agonist Ramelteon(100 μM)followed by the LPS stimulation for 12 h.Thereafter,Western blot was used to detect the expressions of the inflammatory proteins i NOS and COX-2 to investigate whether the anti-inflammatory effects of MT1 activation was receptor-dependent.Glucose detection kits and lactate detection kits were used to assess the effects of Mtnr1 a knockdown or MT1 receptor activation on glucose consumption and extracellular acid production in BV2 cells with LPS stimulation.Transcriptome sequencing was used to find the changes of metabolic pathways and key metabolic enzymes between Mtnr1 a knockdown microglia and normal microglia.QRT-PCR and Western blot were used to estimate the effect of MT1 receptor on the regulation of key enzymes in glycolysis and tricarboxylic acid cycle(TCA).In vivo,before and after LPS treatment,we gave mice MT1 receptor agonist Ralmelteon(20 mg/kg/d)for 7 days by intraperitoneal injection,followed by Western blot and Immunohistochemical to evaluate the expressions of tyrosine hydroxylase(TH),microglia marker(IBA1),and astrocyte marker(GFAP)in the substantia nigra(SN)of mice.Results:In vitro,Western blot and q RT-PCR experiments indicated that the defiency of Mtnr1 a greately aggravated LPS-induced releases of pro-inflammatory factors i NOS,COX-2,IL-6,and TNFα,while MT1 receptor agonist Ramelteon can inhibit them in a dosedependent manner.Western blot results indicated that knocking down Mtnr1 a or pretreatment of MT1 receptor antagonist Luzindole on BV2 cells could significantly reverse the inhibitory effects of MT1 agonist Ramelteon on LPS-induced neuroinflammation.The results of the glucose and the lactic acid detection kits showed that the activation of MT1 receptor could inhibit LPS-induced microglia from oxidative phosphorylation to aerobic glycolysis,conversely,Mtnr1 a knockdown could aggravate LPS-induced microglia from oxidative phosphorylation to aerobic glycolysis.Compared to the normal control microglia,transcriptome sequencing results showed that the pyruvate dehydrogenase complex α1 subunit(PDHA1)was transcriptionally inhibited in the Mtnr1 a knockdown microglia.MT1 can positively regulate the expression of PDHA1 which is the key enzyme of tricarboxylic acid cycle,thereby promoting the metabolic state from glycolysis to tricarboxylic acid cycle(TCA).Western blot and q RT-PCR were used to verify the transcriptome sequencing results.In vivo,immunohistochemistry and western blot showed that in mice SN,MT1 receptor agonist Ramelteon could significantly inhibit LPS-induced increase of microglial marker IBA1 and astrocyte marker GFAP,indicating the inhibitory effects of MT1 activation on neuroinflammation induced by LPS in vivo.Most importantly,Ramelteon significantly remitted the loss of dopaminergic(DA)neurons caused by neuroinflammation.Conclusion: In this study,we assessed the potential protective effects of MT1 activation in inflammatory factor-induced DA neurons damage.Activation of nonselective MT1 agonist Ramelteon remarkably inhibits LPS-induced microglial activation,while silencing of Mtnr1 a aggravates microglia-mediated neuroinflammation both in vivo and in vitro.Moreover,we found that microglial metabolic reprogramming might participate in the inhibitory effects of MT1 receptor on neuroinflammation.