Melatonin Modulates Microglia Activation in Neuroinammation by Regulating the ER Stress/PPARd/SIRT1 Signaling Pathway

Background: Activated microglia-mediated neuro-inammation plays a vital aspect in regulating the micromilieu of the central nervous system. Neuro-inammation involves distinct alterations of microglial phenotypes, containing nocuous pro-inammatory (M1) phenotype and neuroprotective anti-inammatory (M2) phenotype. Currently, there is no effective treatment for modulating such alterations. Little evidence shows that melatonin prevents the detrimental cascade of activated microglia-mediated neuro-inammation. Methods: The expression levels of M1/M2 marker of primary microglia inuenced by Melatonin were detected via qPCR. Functional activities were explored by western blotting, luciferase activity, EMSA, and ChIP assay. Structure interaction was assessed by molecular docking and LIGPLOT analysis. ER stress detection was examined by ultrastructure TEM, calapin activity, and ERSE assay. The neurobehavioral evaluations and immunouorescence staining in animals were used for investigation of Melatonin on the neuroinammation in vivo. Results: Melatonin had targeted on Peroxisome Proliferator Activated Receptor Delta (PPARd) activity, boosted LPS-stimulated alterations in polarization from the M1 to the M2 phenotype, and thereby inhibited NFkB–IKKb activation in primary microglia. The PPARd agonist L-165041 or over-expression of PPARd plasmid (ov-PPARd) showed similar results. Molecular docking screening, dynamic simulation approaches, and biological studies of melatonin showed that the activated site was located at PPARd (phospho-Thr256-PPARd). Furthermore, we found that activated microglia had lowered PPARd activity as well as the downstream SIRT1 formation via enhancing ER stress. Melatonin, PPARd agonist and ovPPARd all effectively reversed the above-mentioned effects. Melatonin blocked ER stress by regulating calapin activity and expression in LPS-activated microglia. Additionally, melatonin or L-165041 ameliorated the neurobehavioral decits in LPS-aggravated neuroinammatory mice through blocking microglia activities, and also promoted phenotype changes to M2-predominant microglia. Conclusions Melatonin suppressed neuro-inammation in vitro and in vivo by tuning microglial activation through the ER stress-dependent PPARd/SIRT1 signaling cascade. We proposed that this treatment strategy is an encouraging pharmacological approach for the remedy of neuro-inammation associated disorders. Mobility Shift Assay; HFD High-Fat Diet: IR: Ischemia Or Reperfusion; Knock-Out of Proliferator result is a two-signal assay system that enables normalization of experimental signal (rey luciferase) to a constitutively expressed control (Renilla luciferase). All results are presented as mean±SD, n ≥ 5, *p <0.05. compared to the Control group. After formaldehyde cross-linking, the PPARd-binding DNA fragments were recovered by immunoprecipitation using PPARd antibodies. Puried precipitates and input DNA were analyzed by PCR using primers specic for SIRT1 promoters. The cross-links were reversed, and the recovered DNA population was analyzed by PCR with primers designed for detecting SIRT1. An amplied PCR fragment is visible in the DNA immunoprecipitated with PPARd antibody in SIRT1 promoters. There is a subtle detection of PPARd band in the LPS-treated groups which are reversed by Melatonin. PCR products were subjected to gel electrophoresis and visualized by ethidium bromide staining; 10% aliquot of the pre-cleared chromatin was regarded as an input. This experiment has been replicated at least four times with independent assays. All experiments were repeated at least four times. Cells were exposed to Melatonin, L165041 or transfected with pcDNA3-PPARd, pcDNA3 for overexpression PPARd, followed by stimulation with LPS for 24 h. Cells were evaluated by Western blotting. All experiments were repeated at least ve times.


Introduction
Microglia are known as resident macrophages in the central nervous system (CNS) and maintain its homeostasis by modulating phenotypic expressions. Microglial cells monitor the CNS microenvironment and respond to danger signals in physiological conditions. [1] Activated microglial cells undergo morphological transformation to ameboid, rod-shaped and rami ed forms. They have expanded cell size, thickened proximal processes, and reduced rami cation of distal branches. Activated microglia, when under in uence of released in ammatory mediators, result in self-perpetuating damage to neurons. [25] Serotonin is acetylated and then methylated to form melatonin within the pineal gland.
[36] However, the molecular mechanisms of melatonin in neuro-in ammation-induced microglia inactivation and relief of ER stress are yet to be well studied.
Here, we aimed to evaluate the deleterious response of microglial activation by LPS administration. Such a response was rescued by neuroprotective effects of melatonin. We intended to investigate how melatonin attenuates LPS-induced neuro-in ammation and alleviates the impairments due to ER stress.
Our results showed that melatonin treatment markedly abolished the LPS-evoked switch of microglial polarization from the M1 to M2 phenotype. Melatonin also reduced the ER stress and its associated proin ammatory mediators via the PPARd/SIRT1 related signaling pathway. Melatonin may therefore be a potent therapeutic strategy to treat neuro-in ammation-related diseases.

Materials And Methods
The methods listed below have been published before but are repeated here for clarity and shown in Supplementary material methods. [22,23,36] Chemicals and materials LPS (Escherichia coli 0111: B4) was purchased from Sigma-Aldrich. Melatonin was also purchased from Sigma-Aldrich. L-165041 is a phenyloxyacetate PPARδ receptor agonist from R&D. The calpain inhibitors were obtained from BIOMOL and from Merck Biosciences.

Cell culture
Microglia were extracted from the Sprague-Dawley rats aged 1-3 days and cultured according to procedures previously described. [22,23,36] In brief, culture media were rst replaced 2 days after preparation, and media were replaced every 2 to 3 days thereafter. Before the experiment, the percentages of the primary rat microglial cells were estimated with Iba1 staining, and con rmed >97% in purity.
To study gene expression, total RNA was extracted from BV2 cell lines, primary microglia culture, or brain cortical tissue using the Trizol reagent. The isolated and puri ed RNA was reverse-transcribed into cDNA using cDNA synthesis kit according to standard protocols. Quantitative PCR (qPCR) was done using synthetic primers and SYBR Green on a Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Incubated at 50 °C for 2 min and 95 °C for 10 min, target samples were allowed to 35 cycles at 95 °C for 15 secs and 60 °C for 1 min. Table I showed the list of qPCR primers. The data were presented as the fold-change of the target gene relative to GAPDH.

Immuno-blotting
Whole-cell lysates of microglia were prepared as previously described for immuno-blotting. [22,23,36] Antibodies used are shown in Supplementary Tables 1 and 2. Assessment was conducted using ECL (Amersham) and by chemiluminescence on Kodak X-Omat lm. The data was repeated 5 times independently.

Transmission electron microscopy (TEM)
The procedure was executed previously described. [22,23,36] The targeted samples under different treatment were xed with 4% glutaraldehyde and then 2.5% paraformaldehyde dissolved in 0.1M sodium cacodylate, and then further post-xed in 1% osmium tetraoxide, dehydrated in ethanol, and embedded in araldite. Sections on grids were counter-stained with uranyl acetate and lead citrate, and images validated with a JEM 1200 EX TEM (JEOL, Peabody, MA, USA) at an accelerating voltage of 80 kV.

Statistical analyses
The calculated data were presented as mean ±standard deviation. All the data were analyzed by analysis of variance (ANOVA) and post hocked by Fisher's least signi cant difference test. Statistical signi cance was set at p <0.05.

Results
Melatonin reciprocally suppresses proin ammatory mediators and augments anti-in ammatory response in LPS-treated mouse primary microglia First, we handle LPS-induced primary microglia cells to check the power of melantonin in the antiin ammatory action. Melatonin showed no signs of toxicity on microglia at concentrations up to 1 mM (data not shown). Treatment with melatonin signi cantly inhibited LPS-induced microglial activation ( Figure.1A). Moreover, it dramatically diminished mRNA levels of M1 markers, such as CD86, COX-2, iNOS, IL-1b, IL-6, TNF-a, and CCL2, compared with the LPS groups ( Figure.1Aa-h). IFNg showed opposite effects. Furthermore, expressions of M2 markers, like CD206, IL-4, GM-CSF, Arginase-1, IGF-1, TGF-b1, YM1/2, and IL-10 increased after melatonin application (Figure1Ai-p). Results showed that melatonin enhanced microglial M2 polarization. NF-κB is a main transcription factor of M1 macrophages, and it is essential for the ignition of a series of in ammatory associated genes or mediators. It is also crucial l for both M1-and M2-like microglial differentiations. After LPS-treated, we found remarkable increases in the phosphorylation of IKKb and NFκB, but downregulation of IκB. In contrast, melatonin forcefully abolished LPS-induced phosphorylations of IKKb and NFκB ( Figure.1Ba). Quantitative results are shown in Figure   1Bb-d. Moreover, the luciferase-based approach for in vivo measurement of NF-κB promoter activity also showed that melatonin markedly blocked NF-κB activity ( Figure.1Be). These ndings suggested that the IKKb and NFκB participated in the phenotypic shift of microglial polarization through inhibition of IKKb/NFκB pathway.
Agonizing PPARd using L16504, or overexpression of PPARd promotes M1-to-M2 polarization of primary cultured microglia A number of reports have displayed the important roles of PPARa and PPARg in microglial-induced in ammation and phenotype shift. Yet, how PPARd alters LPS-induced microglial polarization in the activated microglial remains unclear. In addition, simultaneous high-throughput screening with mass spectrometry showed that PPARd had played a crucial role for microglia activation. Here, to elucidate how to regulate activated microglia, we used the pharmacological induction method based on PPARd speci c agonist L165041 and gene overexpression PPARd plasmid (ovPPARd). L165041 and ovPPARd were found to inhibit LPS-induced M1 microglial polarization in mouse primary microglia. The LPS treatment accelerated mRNA expression of in ammatory cytokines (M1 markers, CD86, iNOS, COX-2, IL-1b, IL-6, TNF-a, and CCL2), and pretreatment with melatonin attenuated mRNA expression of these in ammatory cytokines ( Figure.2Ba-h). IFNg on the other hand, showed the opposite effects. In mouse primary microglia, LPS-stimulated microglial polarization was associated with a drop in mRNA expressions of M2 markers (CD206, IL-4, GM-CSF, Arginase-1, IGF-1, TGF-b1, YM1/2, IL-10). However, melatonin pretreatment elevated mRNA expressions of all the aforementioned markers ( Figure.2Bi-p). In addition, ELISA showed that melatonin treatment enhanced the release of IL-10 in LPS-treated mouse primary microglia (data not shown). Melatonin suppressed M1 microglia polarization, promoted M2 microglia polarization, and attenuated in ammatory responses in activated microglia. According to the above-mentioned ndings, NF-κB pathways are involved in M1 polarization roles and involved in the regulation of microglial activation. After LPS induction, marked enhancement in phosphorylation was found in IKKb and NFκB, but marked decrement was found in IκB. In contrast, L165041 or transfection of ovPPARd plasmid signi cantly inhibited LPS-induced phosphorylation of IKKb and NFκB ( Figure.2Ba-b). The quantitative results are shown in Figure.2Bc-e. Moreover, NF-κB luciferase activity was markedly blocked by L165041 and the transfection of ovPPARd. These ndings suggested that PPARd activation participated in the phenotypic shift of microglial polarization by suppressing IKKb/NFκB pathway. Molecular docking, dynamic simulation studies and biochemical evidence show activation properties of Melatonin targeting PPARd.
Molecular docking emerges as a paramount tool for drug discovery, and it can miniature interactions between small molecules and ligand-proteins at the atomic level. That yields the characterization of their interacting behavior as well as their biochemical processes of known three-dimensional structure. Dynamic simulation based on a computer program allows the evaluation and understanding of transient physical and chemical processes of the time-varying behavior. In the present study, we used the approaches of both docking and dynamic simulation to investigate the potential interactions between melatonin and PPARd. Results appeared that the key residues within the PPARd catalytic site had interacted with melatonin, and the binding took place in the tunnel of the active site ( Figure.3A-B). Crystal structures of PPARd showed that the highly conserved Thr256 residue in PPARd was located within the active site, which was catalytically required in stabilizing the functional activity of the molecule. Changes in both conformation and dynamics likely occurred when melatonin was bound to PPARd, facilitating melatonin for targeting the active site. A computer program, bioinformatics LIGPLOT, generates schematic 2-D representations of protein-ligand complexes. It showed that interplay with the candidate site contained the Thr256 residue ( Figure.3C). Furthermore, speci c antibody targeting phosphorylation of PPARd (Thr 256) in microglia was markedly induced by melatonin after 30 minutes of treatment ( Figure.3D). The response gradually lessened after 60 minutes. Importantly, L165041 or transfection of ovPPARd evoked phospho-Thr256-PPARd as detected by western blotting. Moreover, LPS-induced phospho-PPARd (Thr256) translocated to the nucleus was enhanced markedly by melatonin induction as shown in confocal uorescence images ( Figure.3E). In summary, these results implicated a possible interaction between melatonin and PPARd based on structure molecular docking as well as phospho-PPARd (Thr256) activities. Melatonin is therefore a direct target of phospho-PPARd (Thr256), and our methods worked well in predicting interactions of melatonin with its target ligand protein PPARd.
PPARd agonists have potential neuroprotective effects. Bene cial roles of SIRT1 are known in neuroin ammation-related diseases. We aimed to predict in silico promoter binding sites to elucidate possible mechanisms. The SIRT1 promoter-anking region (−345~−384) containing the cis-acting elements PPARd DNA binding activity in silico was predicted in the laboratory ( Figure. LPS reduced PPARd activity, which could be reversed by melatonin. In addition, our previous study showed that apoptosis episode in LPS-induced microglia is regulated by calpain-II activity under conditions of ER stress. [22] In that study, exposures to Calpain inhibitor (Z-Val-Phe-CHO) also markedly reversed PPARd expressions. These suggested a strong standing point of PPARb in transcriptional activity in response to LPS-induced ER stress in activated microglia. For the purpose to demonstrate that PPARb directly regulates SIRT1 expression, DNA binding activity of the SIRT1 promoter was measured. As shown in Figure.4D, EMSA assay showed that LPS had suppressed PPARd binding activity at the SIRT1 promoter binding site across time frames. Noticeably, pretreatment with melatonin enhanced DNA binding activity ( Figure.4D; left). On the other hand, pretreatment with PPARd agonist L165041 or transfection with PPARd (ovPPARd) intensi ed similar PPARd binding activity ( Figure.4D; right). In ChIP experiments with an anti-PPARd polyclonal antibody, PCR ampli cation at the speci c PPARd locus of the SIRT1 promoter obtained with chromatin puri ed from control cells gave a strong signal. In contrast, controls with immunoprecipitation in normal rabbit serum (IgG) gave only a weak background signal. In LPS-treated microglia, the signal reduced gradually after 1 hr. But melatonin pre-treatment had selectively enriched speci c DNA-binding proteins PPARd along with their DNA targets SIRT1 ( Figure.4E). We further studied the aspect of PPARd in the amplitude of SIRT1 and the regulatory relationship between PPARd and the calpain signals. As shown in Figure.4F, microglia treated with LPS resulted in lowered expression of SIRT1 protein. Melatonin or PPARd agonist L165041 effectively increased the expression of SIRT1. Moreover, the calpain inhibitor Z-Leu-Leu-CHO markedly enhanced the expression. The transfection of ovPPARd in activated microglia also led to a signi cant induction of the SIRT1 protein expression. These results implied that melatonin-enhanced PPARd regulated SIRT1 signaling pathway involved in LPStriggered microglial activation. Our ndings reinforced the hypothesis that melatonin tuned the PPARd binding to the SIRT1 promoter via the calpain signaling cascade.
Melatonin circumvented ER stress-related markers in LPSactivated microglia To directly investigate the effects of melatonin on activated microglia by ER stress induction, we assessed the morphology of ER ultrastructures using the transmission electron microscopy (TEM). As shown in Figure.5A, we found dilated ER in LPS-treated cells. Pretreatment of melatonin rescued the effect, seen as less distention and fragmented organelles. Moreover, on exposure to L165041, we found similar phenomenon. We next examined the response of ER stress-related unfolded proteins in activated microglia with or without melatonin or L165041. As shown in Figure.5B, LPS exaggerated the response in phospho-PERK, phospho-eIF2a and GADD153 in primary and BV-2 microglial cells. Melatonin or L165041 markedly lowered expressions of above said ER stress associated markers in activated microglia. Melatonin or L165041 fundamentally had no appreciable effect on ER stress associated markers (data not shown). We further demonstrated that PPARd antagonist GSK3787 combined with LPS had aggravated the activated microglia ( Figure.5B). Additionally, we examined ER stress activity using the ER stress-response elements (ERSE) reporter system. We found that LPS induced ER stress activity, and melatonin completely abolished the reporter activity. On exposure to L165041 (10 mM), the downregulation effect was observed ( Figure.5C). However, GSK3787 (5 mM) alone or in combination with LPS-induction enhanced ER stress activity. Tunicamycin treatment was taken as a positive control for the ER stress induction. These results indicated that melatonin had blocked ER stress in activated microglia.
Melatonin blocked induction of calpain protein and activity in LPS-activated microglia.
Calpain is renowned for the involvement of ER stress induced neurodegeneration. First, we examined whether melatonin blocked the calpain activity in activated microglia and its associated calpain protein expressions. As shown in Figure.6A, LPS increased calpain activity in primary microglia, starting at 30 minutes and beyond. Combined with calpain inhibitor (Z-Leu-Leu-CHO; 10 mM), LPS markedly reduced calpain activity. LPS also increased microglial calpain activity, and that was signi cantly reduced by melatonin or L165041. However, the antagonist GSK3787 showed no effect ( Figure.6B). In addition, transfection of ovPPARd attenuated calpain activity. Gene silencing of PPARd by shPPARd on the other hand, produced no effect ( Figure.6C). Regarding expressions of calpain associated proteins, Calpain-I and Calpain-II were induced in LPS-activated microglia, and their inductions were reversed by melatonin ( Figure.6D). We further examined whether melatonin suppressed the interaction between Calpain and PPARd. As shown in Figure.6E, the interaction of Calpain with PPARd (as shown by immunoprecipitation assay), which was modest in the control cells after LPS stimulation, became signi cantly lower for 90 minutes in the presence of melatonin. The 10% input displayed for protein visualization was used as a positive control for the comparison. In addition, confocal microscopy revealed greater co-localization of Calpain-I/II with PPARd in microglia after LPS exposures for 2-4 hr (Figure.6F). Such co-localization was markedly suppressed by melatonin. These results indicated that melatonin inhibited LPS-induced Calpain/PPARd interaction, leading to cleavage of PPARd.
Melatonin therapy restrains microglia activation and enhances PPARd expression.
In ammatory conditions and nerve injuries alter gait, suppress general locomotion and voluntary wheel running, reduce social interactions, and/or induce anxiety-like behavior. [37] To assess in-vivo effects of melatonin and L165041 in the activated microglia, we conducted neurobehavioral evaluations using Ethovision, Rotarod, and CatWalk gait analysis. In Ethovision assessments, either in the short term and long-term memory, we found that LPS injections aggravated the neurobehavior. Either melatonin or L165041 had alleviated such aggravations ( Figure.7A;7C). Gross motor function tests (rotarod), related to motor coordination and balance, also showed similar trends ( Figure.7B). In the Catwalk gait analysis, deterioration of gait balance was triggered by LPS injection, and improvement was found after melatonin and L165041 injections (Table4). To further investigate whether administration of melatonin exhibits neuroprotective effects in rats with activated microglia, the extent of in ammation was assessed with immuno uorescence staining. On day 7 after LPS-induction, the sham group showed only minimal sized in ammatory areas ( Figure.7D). Compared with the LPS group, melatonin (5 mg/kg, i.p.) signi cantly reduced the overwhelmingly activated microglia volume (P <0.01) (Figure.7E). Simultaneously, we also found similar effects with the PPARd agonist L165041 (2 mg/kg, i.p.) ( Figure.7F). Quanti cation is shown in Figure.7G. This nding suggested that melatonin uniquely reduced neuroin ammatory conditions in activated microglia, via a PPARd regulatory mechanism.

Discussion
Emerging evidence indicates that down-regulation of microglial proin ammatory mediator's expressions and augmentation of the anti-in ammatory factors production both play a pivotal role in neuroprotective recovery after brain injury. Our study was therefore designed to determine whether melatonin, secreted by the pineal gland in the brain, has anti-neuroin ammatory effects in vitro and in vivo based on a LPSinduced neuro-in ammation animal model. The current functionally polarized classi cation of microglia into M1 or M2 remains a subject of debate. The M1/M2 polarization of microglia is largely based on genes associated with M1 markers (CD86, COX-2, iNOS, IFNg, IL-1b, IL-6, TNF-a, CCL2) and M2 markers (CD206, IL-4, GM-CSF, Arginase-1, IGF-1, TGF-b1, YM1/2, IL-10). In response to sensing distinct microenvironmental cues, the polarized M1 and M2 subpopulations can reverse their phenotype and biological functions. Forthcoming evidence revealed that ER stress is essential for the induction and functional alteration in M1 phenotype microglia. Therefore, strategies based on stimulating M1-to-M2 phenotype conversions are potentially useful in treating neuro-in ammation-induced injuries. The main ndings of our study are as follows: 1) Melatonin inhibits microglial activation, promotes M2 polarization, and suppresses in ammatory mediators. 2) Melatonin reduces activated microglia, improves sensorimotor recovery and inhibits the proin ammatory mediators in a mouse model, and ameliorates de ciency of neurobehavior in a LPS-induced neuro-in ammation animal model. 3) Bene cial effects of melatonin-induced PPARd activation in regulating downstream SIRT1 in vivo are associated with the anti-ER stress and anti-in ammatory effects. 4) We further obtained evidence that the application of the PPARd agonist L165041 facilitates microglial phenotypic polarization from M1 to M2 and contributes to the anti-in ammatory effects (Fig. 8).
Microglia represent an important element of the immune system's in ammatory response in the CNS, and they inhibit or promote in ammation dependent on different conditions. Therefore, microglia plays dual roles either in detrimental or bene cial for functional recovery. In our previous study, we found that IL-13 downregulates PPAR-gamma/heme oxygenase-1 via ER stress-stimulated calpain in activated microglia responding to endotoxin injury. [22] Furthermore, the ER stress-related calpain, downregulates the PPARγ/HO-1 pathway via C/EBP-β and that leads to aggravated death of activated microglia via IL-13.
Consequently, the results contributed to the prevention of cerebral in ammation and neuronal injury. [23] Regarding anti-in ammatory effects of melatonin on activated microglia, we studied mouse primary microglia cells or BV2 cells activated by LPS. We found that melatonin had enhanced expression of antiin ammatory mediators like PPARd and SIRT1. Melatonin is known to up-regulate SIRT1 during aging, and it displays pro-oxidant, pro-apoptotic and SIRT1-downregulating properties in cancer cells. [28,[38][39][40] Meanwhile, a connection between melatonin and another mediator component, SIRT1, is becoming clearer; SIRTuin may also mediate other effects.[28, 39,40] Furthermore, in PND7 rat brains, melatonin treatment rescues the hippocampal dentate gyrus (DG) against LPS-induced oxidative stress damage, acute neuro-in ammation, and apoptotic neurodegeneration via the SIRT1/Nrf2 signaling cascade. [29] Furthermore, melatonin through the SIRT1 pathway attenuates LPS-induced acute depressive-like behaviors and microglial NLRP3 in ammatory activation. [41] Consistent with these effects, melatonin also acts as an antidepressant via attenuation of neuro-in ammation by targeting SIRT1 signaling. [42] In this study, we found that SITR1 expression was also markedly increased after melatonin-induction, a nding which is consistent with all previous ndings. However, we provided new information on gene regulation for the transcription factor PPARd. Our results indicated that the neuroprotective effects of melatonin in brain damage is, at least in part, associated with the microglial anti-in ammatory activation via the PPARd/SIRT1 signaling pathway.
PPAR activation regulates the in ammatory activity mediated by microglia and astrocyte, protecting neurons from insult, reducing oxidative stress, inhibiting ER stress, and improving mitochondrial functions. In particular, reports from our previous study and from others showed that PPARg is mainly expressed in microglia, and it mediates anti-in ammatory activity by inhibiting oxidative stress, reducing ER stress, and inducing microglial polarization to M2-like phenotype. Melatonin attenuates dysregulation of the circadian clock pathway in mice with ccl4-induced brosis and human hepatic stellate cells through PPARa activation. [43] PPAR-α modulates the anti-in ammatory effect of melatonin in the secondary events of spinal cord injury under PPARα KO mice. This indicates that PPAR-α can contribute to the anti-in ammatory activity of melatonin in SCI. [44] On the other hand, melatonin signi cantly increases the expression of PPAR-γ, a master regulator of adipogenesis, and it promotes cell differentiation into adipocytes. [45] Melatonin attenuates acute pancreatitis-induced liver damages through AKT-dependent PPAR-γ Pathway.
[46] Melatonin suppresses platelet activation and function against cardiac ischemia/reperfusion injury via PPARγ/FUNDC1/mitophagy pathways. This action may provide a novel strategy for cardioprotection in cardiac I/R injury. 50 Currently, there is very little known about the correlation between melatonin and PPARd. To characterize the role of PPARd in melatoninmediated anti-in ammatory activity, we tested the e cacy of melatonin (10 mg/kg) in an experimental model of LPS-induced neuro-in ammation using functional study and histology images to examine activation of microglia response. We found that melatonin markedly reduced the deterioration in neurobehavior and attenuated microglia cells activation. The PPARd agonist, L165041, also possessed similar effects. Based on the structural biology approach, we constituted the melatonin target PPARd (Thr256 site) through molecular docking. In the in vitro study, melatonin activated phospho-PPARd in the early stage, and forward PPARd to regulate SIRT1 protein expressions. Therefore, PPARd is likely a novel therapeutic target for neurodegenerative diseases. In the present study, we reported, for the rst time, that melatonin induces PPARd activation, and that PPARd played a critical role as a master regulator of antiin ammatory action. Our ndings supported the therapeutic potential of melatonin in treating neurodegenerative diseases or neuro-in ammation disorders.

Conclusion
Melatonin, PPARd and its agonist L165041 reduced neurobehavioral de cits induced by LPS injections, exerting anti-in ammatory effects and thwarting ER stress, through the PPARd/SIRT1 signaling pathway. Melatonin and PPARd and its agonist L165041 hopefully are encouraging pharmacological candidates for treating brain disorders associated with neuro-in ammation.

Figure 1
The prevention of LPS-evoked M1 microglial activation and predisposition to M2 polarization by melatonin. Microglia were cultured with Melatonin (0.1 mM) followed by the incubation with LPS (0.1 mg/ml) for 24 hr. Melatonin suppressed the amoeboid "activated" morphology of microglia triggered by LPS.
(1Ba-b) Protein expressions in activated microglial cells as determined by Western blotting. Speci c antibody probe as indicated for p-IKKb, p-NFkB, IκB.
(1Bc-e) The quanti cation analysis for Western blotting images, and normalized with C23 or a-tubulin.
(1Bf) NFkB promoter luciferase activity. The functional activity was examined using the dual luciferase reporter assay kit. The result is a two-signal assay system that enables normalization of experimental signal ( re y luciferase) to a constitutively expressed control (Renilla luciferase). All results are presented as mean±SD, n ≥ 5, *p <0.05. compared to the Control group. (2Bc-e) The quanti cation analysis for Western blotting images, and normalized with C23 or a-tubulin.
(2Bf) NFkB promoter luciferase activity. The functional activity was examined using the dual luciferase reporter assay kit and presented as ratio of normalization of experimental signal ( re y luciferase) to a constitutively expressed control (Renilla luciferase). All data are presented as mean±SD, n ≥ 5, *p <0.05. compared to the Control group.  Melatonin-evoked PPARd activity regulated SIRT1 production.
(A) The sequences of PPARd on theSIRT1 promoter region were shown, pointing out the sub fragments which have been used in this study (underlined). Promoter regulation in the SIRT1 promoter-anking region contains the cis-acting elements PPARd (-345 to-384) DNA binding site. (B) The culture of primary microglia was conditioned in serum-free medium overnight either presence or absence of LPS-treated (100 ng/ml) status. Whole cell lysates were blotted and immunostained with antibodies against PPARd and SIRT1. (C) The cells were transiently transfected with the PPRE luciferase reporter plasmid followed by thymidine kinase promoter-driven Renilla-luciferase vector, which were pre-treated with Melatonin or calpain inhibitors (Z-Leu-Leu-CHO; 10 mM). The relative activity was measured in luciferase assay as described in the text. The luciferase activities in means ± SD were calculated by triplicate determinations. (D) The synthetic dsDNA representing PPARd-binding sites in the promoter of SIRT1 gene was analyzed by electrophoretic mobility shift assay (EMSA) using nuclear extracts in control or LPS groups following stimulation of cells with or without Melatonin. The unbound excess radiolabeled probe migrated out of the gel and shown in the bottom. Cells were treated with Melatonin at various time frame, and nuclear PPARd DNA binding activity was analyzed by EMSA. Cells were pretreated with L165041, transfected with ovPPARd, pcDNA3 or calpain inhibitor followed by stimulation with LPS for 1-4 h. At least three independent measurements were done for each data. Arrow points speci c PPARd sequences located on SIRT1 promoter DNA binding site. The PPARd DNA binding labeled probe as indicated. All experiments were repeated at least three times.
(E) PPARd binds to the SIRT1 promoter in vivo. ChIP assay was carried out in control, LPS or Melatonin. After formaldehyde cross-linking, the PPARd-binding DNA fragments were recovered by immunoprecipitation using PPARd antibodies. Puri ed precipitates and input DNA were analyzed by PCR using primers speci c for SIRT1 promoters. The cross-links were reversed, and the recovered DNA population was analyzed by PCR with primers designed for detecting SIRT1. An ampli ed PCR fragment is visible in the DNA immunoprecipitated with PPARd antibody in SIRT1 promoters. There is a subtle detection of PPARd band in the LPS-treated groups which are reversed by Melatonin. PCR products were subjected to gel electrophoresis and visualized by ethidium bromide staining; 10% aliquot of the precleared chromatin was regarded as an input. This experiment has been replicated at least four times with independent assays. All experiments were repeated at least four times.
(F) Cells were exposed to Melatonin, L165041 or transfected with pcDNA3-PPARd, pcDNA3 for overexpression PPARd, followed by stimulation with LPS for 24 h. Cells were evaluated by Western blotting. All experiments were repeated at least ve times.   Neurobehavior assessment after intracerebral injection of melatonin and L 165041. In experimental design of neuro-in ammation model, animals received the neurobehavior assessment 3 days before intracerebral injection and then 3, 7, 14, 21, and 28 days after operation. Mice were intravenously injected with or without Melatonin (5 mg/kg), or L165041 (2 mg/kg) by i.p. injection or local pretreated at 24 hrs after LPS-injection. Animal were sacri ced further to perform histological and immuno uorescence analysis at day 7 after induction. Melatonin (5 mg/kg), and L165041 (2 mg/kg) reduced activated microglia-induced aggravated volume as assessed by immuno uorescence staining. (E-F) Primary antibodies for microglia marker Iba1 and PPARd were conjugated to microglia followed by secondary antibodies coupled with FITC conjugated (green) or TRITC-conjugated (red), respectively as indicated. Co-localization of two labeled antigens was detected as a single image when the images from both channels were overlaid. (red: PPARd; green: Iba1).
(G) Quanti cation analysis for confocal microscope image localization between microglia markers Iba1green; PPARd-red. The image in each section was measured in ve images from each treatment under HFP (n > 5). All data are shown as means ± SD of ve independent experiments. *P < .05 compared to controls; #P < .05 compared to LPS.

Figure 8
Schematic of proposed mechanism for the role of Melatonin in activated microglia and neuroin ammation effects. LPS-induced ER stress downregulated PPARd and NFkB activation, subsequently reduces SIRT1 expression and then triggers the microglia activation and brain damage. In the present study, we provide the evidence to demonstrate that melatonin plays a potential protective role in neuroprotective effects through PPARd/SIRT1 pathway. In addition, PPARd pharmacological agonists L165041 also possessed similar effects. These results suggest that the activation of PPARd/SIRT1 by melatonin could counteract the detrimental effect of LPS. Also, the results suggest melatonin may exert a therapeutic effect for neuroin ammatory disorders.

Supplementary Files
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