Metformin attenuates hypothalamic inflammation via downregulation of RIPK1-independent microglial necroptosis in diet-induced obese mice

Necroptosis, a form of programmed cell death, accounts for many inflammations in a wide range of diseases. Diet-induced obesity is manifested by low-grade inflammation in the mediobasal hypothalamus (MBH), and microglia are implicated as critical responsive components for this process. Here, we demonstrate that microglial necroptosis plays a pivotal role in obesity-related hypothalamic inflammation, facilitating proinflammatory cytokine production, such as TNF-α and IL-1β. Treatment with the anti-diabetic drug metformin effectively reduces the obese phenotypes in the high-fat diet (HFD)-fed mice, attributing to remission of hypothalamic inflammation partly through repressing microglial necroptosis. Importantly, using the receptor-interacting protein kinase 1 inhibitor, necrostatin-1s, could not suppress the microglial inflammation nor prevent body weight gain in the obese mice, indicating that the microglial necroptosis is RIPK1-independent. Altogether, these findings offer new insights into hypothalamic inflammation in diet-induced obesity and provide a novel mechanism of action for metformin in obesity treatment.

Shuangxinfang prevents S100A9-induced macrophage/microglial inflammation to improve cardiac function and depression-like behaviour in rats after acute myocardial infarction

Background Depression is a common complication of cardiovascular disease, which deteriorated the cardiac function. Shuangxinfang (Psycho-cardiology Formula, PCF) was reported to alleviate myocardial ischemia injury and improve depression-like behavior. Interestingly, our previous proteomics study predicted that the protein S100A9 appeared as an important target, and macrophage/microglial inflammation might be involved in the process of PCF treating depressive disorder induced by acute myocardial infarction (AMI). The aim of this study is to validate the proteomics results. Methods AMI rat models were established in vivo, followed by the administration of PCF or ABR-215757 (also named paquinimod, inhibiting S100A9 binding to TLR4) for 5 days. Forced swimming test (FST) and open field test (OFT) were applied to record depression-like behavior, and echocardiography was employed to evaluate cardiac function. Morphological changes of cardiomyocytes were assessed by HE staining and TUNEL staining on day 7 after cardiac surgery, as well as masson trichrome staining on day 21. Hippocampal neurogenesis was determined by Nissl staining, while 5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) in the hippocampus were detected as biochemical indicators of depression. Myocardial and hippocampal expression of inflammatory factors were analyzed by western blotting, immunofluorescence, and ELISA. The activation state of macrophage and microglia was assessed via immunoreaction respectively using CD68 and Iba1. For in vitro confirmation, BV2 cells were primed with recombinant protein S100A9, and then pretreated with PCF serum, to determine alterations in microglial activation and inflammation. Results Rats in the AMI group showed heart function deterioration, as well as depression-like behavior. Coronary ligation not only brought about myocardial inflammation, cell apoptosis and fibrosis, but also reduced the neurogenesis and decreased the content of 5-HT. PCF could ameliorate the pathological and phenotypic changes of the heart and brain, and inhibited the expression of S100A9/TLR4/NF-κB pathway, the activation of microglial cell and the secretion of IL-1β and TNF-α raised by AMI. ABR-215757 showed therapeutic effect and molecular biological mechanisms similar to PCF. Pretreatment with PCF serum in vitro was proved to efficiently block the hyperactivation of BV2 cells and increasement of cytokine contents induced by recombinant protein S100A9. Conclusion We identify S100A9 as a novel and potent regulator of inflammation in both heart and brain. Macrophage/microglia inflammation mediated by S100A9 is considered as a pivotal pathogenic in depression post-AMI, as well as a major pathway for the treatment of PCF, suggesting that PCF is a promising therapeutic candidate for psycho-cardiology disease.

Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K2 sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.

Selective targeting of the TLR2/MyD88/NF-κB pathway reduces α-synuclein spreading in vitro and in vivo

Pathways to control the spreading of α-synuclein (α-syn) and associated neuropathology in Parkinson’s disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are unclear. Here, we show that preformed α-syn fibrils (PFF) increase the association between TLR2 and MyD88, resulting in microglial activation. The TLR2-interaction domain of MyD88 (wtTIDM) peptide-mediated selective inhibition of TLR2 reduces PFF-induced microglial inflammation in vitro. In PFF-seeded A53T mice, the nasal administration of the wtTIDM peptide, NEMO-binding domain (wtNBD) peptide, or genetic deletion of TLR2 reduces glial inflammation, decreases α-syn spreading, and protects dopaminergic neurons by inhibiting NF-κB. In summary, α-syn spreading depends on the TLR2/MyD88/NF-κB pathway and it can be reduced by nasal delivery of wtTIDM and wtNBD peptides.

Inhibitory Effect of Gualou Guizhi Decoction on Microglial Inflammation and Neuron Injury by Promoting Anti-Inflammation via Targeting mmu-miR-155

Gualou Guizhi decoction (GLGZD) treatment exerts neuroprotective effects and promotes spasticity following ischemic stroke. However, the molecular mechanism of GLGZD treatment on ischemic stroke remains unclear. Our previous study indicated that GLGZD ameliorates neuronal damage caused by secondary inflammatory injury induced by microglia. In the present study, we investigate the potential mechanism of GLGZD treatment on neuron damage induced by neuroinflammation via mmu-miR-155 in vitro. The HT22 cell line and the BV2 cell line were exposed to oxygen/glucose-deprive (OGD) conditions; the conditioned medium was prepared using the supernatants from OGD-stimulated BV2 cells after pretreating with GLGZD. Cell viability was determined by MTT assays; levels of released inflammatory cytokines were assessed using the BioPlex system. mmu-miR-155 and its targeting genes were detected using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression of anti-inflammatory proteins was evaluated by Western blotting. DAPI staining was used to test the apoptotic cells. Our results showed that GLGZD pretreatment significantly induced IL10 release and decreased the production of TNF-α, IL6, and IFN-γ. In addition, GLGZD markedly attenuated mmu-miR-155 expression and its downstream SOCS1, SMAD2, SHIP1, and TAB2 expression levels. The DAPI-stained apoptotic cell death and caspase-3 activation in HT22 cells exposed to the conditioned medium were reversed by GLGZD treatment. Our findings suggested that GLGZD pretreatment downregulates the mmu-miR-155 signaling, which inhibits microglial inflammation, thereby resulting in the suppression of neuron apoptosis after OGD stress. The underlying mechanisms may provide the support for GLGZD treatment of cerebral ischemic injury.

Pharmacological, Biochemical and Molecular Investigations in 6-OHDA Induced Parkinson’s Disease Rats on the Protection Benefits of Mangiferin in Behavioral, Inflammatory and Oxidative Biomarkers

Background: Persistent up regulation of NF-κB leads to chronic inflammation and subsequent microglial activation and takes neurons towards death by activating death receptor domains and the p53 pathway. Thus, inhibition of NF-κB may lead to more effective treatment for Parkinson’s disease. Therefore, we have used mangiferin, specific inhibitor of NF-κB in this study. Method: The study utilized male Wistar rats weighing 200-250 gm (n=8 in each group). Stereotactic surgery of rats was done to induce 6-OHDA lesioning in rats. On day 42, rats were subjected to behavioural studies to evaluate effect of mangiferin and their brains were taken out after euthanasia to perform biochemical and molecular studies. Results: Mangiferin significantly increases locomotor parameters in 6-OHDA lesioned rats. It also decreases activity of Cyclooxygenase enzyme which then leads to decrease concentration of inflammatory cytokines. Microglial inflammation was also substantially reduced by reducing MPO concentration. Oxidative stress burden was also reduced after treatment with mangiferin as indicated by increase in Total Antioxidant Capacity, SOD and Catalase and reduction in concentration of MDA. Treatment with mangiferin also reduces burden of oxidative stress by increasing the activity of NRF2/ARE pathway. Activity of Caspase 3 and 9 was also significantly reduced after treatment with mangiferin. Significant decrease in activity of both Cox1 and Cox 2 was also observed. Maximum improvement in all parameters was observed in rats treated with grouping of mangiferin 45mg.kg-1 and levodopa 10mg.kg-1. Treatment with levodopa alone has no significant effect on biochemical and molecular parameters though it significantly improves behavioural parameters. Conclusion and Implications: Results of this study suggest that mangiferin has protective effect in hemi-parkinsonian rats by inhibiting NF-κB. Current treatment of Parkinson’s disease does not target the underlying problem of the disease. Therefore, combination therapy of mangiferin and levodopa can be helpful in better management of Parkison’s.