EPOR activation attenuates colitis via enhancing LC3B-dependent apoptotic cell clearance.

Systemic immune activation and excessive inflammatory response, induced by intestinal barrier damage, are the major characteristics of inflammatory bowel disease (IBD). Excessive apoptotic cell accumulation leads to the production of a large number of inflammatory factors, further aggravating IBD development. Gene set enrichment analysis data showed that the homodimeric erythropoietin receptor (EPOR) was highly expressed in the whole blood of patients with IBD. EPOR is specifically expressed in intestinal macrophages. However, the role of EPOR in IBD development is unclear. In this study, we found that EPOR activation significantly alleviated colitis in mice. Furthermore, in vitro, EPOR activation in bone marrow-derived macrophage (BMDMs) promoted microtubule-associated protein 1 light chain 3B (LC3B) activation and mediated the clearance of apoptotic cells. Moreover, our data showed that EPOR activation facilitated the expression of phagocytosis- and tissue-repair-related factors. Our findings suggest that EPOR activation in macrophages promotes apoptotic cell clearance, probably via LC3B-associated phagocytosis (LAP), providing a new mechanism for understanding pathological progression and a novel potential therapeutic target for colitis.

Oridonin ameliorates caspase-9-mediated brain neuronal apoptosis in mouse with ischemic stroke by inhibiting RIPK3-mediated mitophagy.

Neuronal loss is a primary factor in determining the outcome of ischemic stroke. Oridonin (Ori), a natural diterpenoid compound extracted from the Chinese herb Rabdosia rubescens, has been shown to exert anti-inflammatory and neuroregulatory effects in various models of neurological diseases. In this study we investigated whether Ori exerted a protective effect against reperfusion injury-induced neuronal loss and the underlying mechanisms. Mice were subjected to transient middle cerebral artery occlusion (tMCAO), and were injected with Ori (5, 10, 20 mg/kg, i.p.) at the beginning of reperfusion. We showed that Ori treatment rescued neuronal loss in a dose-dependent manner by specifically inhibiting caspase-9-mediated neuronal apoptosis and exerted neuroprotective effects against reperfusion injury. Furthermore, we found that Ori treatment reversed neuronal mitochondrial damage and loss after reperfusion injury. In N2a cells and primary neurons, Ori (1, 3, 6 µM) exerted similar protective effects against oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury. We then conducted an RNA-sequencing assay of the ipsilateral brain tissue of tMCAO mice, and identified receptor-interacting protein kinase-3 (RIPK3) as the most significantly changed apoptosis-associated gene. In N2a cells after OGD/R and in the ipsilateral brain region, we found that RIPK3 mediated excessive neuronal mitophagy by activating AMPK mitophagy signaling, which was inhibited by Ori or 3-MA. Using in vitro and in vivo RIPK3 knockdown models, we demonstrated that the anti-apoptotic and neuroprotective effects of Ori were RIPK3-dependent. Collectively, our results show that Ori effectively inhibits RIPK3-induced excessive mitophagy and thereby rescues the neuronal loss in the early stage of ischemic stroke.

Erythropoietin signaling in peripheral macrophages is required for systemic ß-amyloid clearance.

Impaired clearance of beta-amyloid (Aß) is a primary cause of sporadic Alzheimer's disease (AD). Aß clearance in the periphery contributes to reducing brain Aß levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aß-degrading enzymes, and Aß clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aß-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aß levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aß clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Salidroside ameliorates orthopedic surgery-induced cognitive dysfunction by activating adenosine 5'-monophosphate-activated protein kinase signaling in mice.

Perioperative neurocognitive disorders (PND) are the most common postoperative complications with few therapeutic options. Salidroside, a plant-derived compound, has gained increased attention as a treatment for various neurological diseases and particularly as a modifier of microglia-mediated neuroinflammation. However, the effect of salidroside on orthopedic surgery-induced cognitive dysfunction and the underlying mechanisms are largely unknown. Here, we found that salidroside greatly attenuated cognitive impairment in mice after orthopedic surgery. Neuroinflammation in the mouse hippocampus was also attenuated by salidroside. Meanwhile, salidroside treatment induced a switch in microglial polarization to the anti-inflammatory phenotype. In vitro, salidroside suppressed the expression of proinflammatory cytokines and induced a switch in microglial phenotype to the anti-inflammatory phenotype. Mechanistically, molecular docking studies revealed the potential AMPK activation activity of salidroside. And salidroside did up-regulated the AMPK pathway proteins. Moreover, AMPK antagonist abolished the effects of salidroside in vivo and in vitro. Taken together, our results demonstrated that salidroside effectively suppressed PND by suppressing microglia-mediated neuroinflammation through activating AMPK pathway, and it might be a novel therapeutic approach for PND.

Oridonin Ameliorates Traumatic Brain Injury-Induced Neurological Damage by Improving Mitochondrial Function and Antioxidant Capacity and Suppressing Neuroinflammation through the Nrf2 Pathway.

Traumatic brain injury (TBI) is a global public health concern, and few effective treatments for its delayed damages are available. Oridonin (Ori) recently has been reported to show a promising neuroprotective efficacy, but its potential therapeutic effect on TBI has not been thoroughly elucidated. The TBI mouse models were established and treated with Ori or vehicle 30 min post-operation and every 24 h since then. Impairments in cognitive and motor function and neuropathological changes were evaluated and compared. The therapeutic efficacy and mechanisms of action of Ori were further investigated using animal tissues and cell cultures. Ori restored motor function and cognition after TBI-induced impairment and exerted neuroprotective effects by reducing cerebral edema and cortical lesion volume. Ori increased neuronal survival, ameliorating gliosis and the accumulation of macrophages after injury. It suppressed the increased production of reactive oxygen species, lipid peroxide, and malondialdehyde and reversed the decrease of mitochondrial membrane potential and adenosine triphosphate content, which was also identified in oxidatively stressed neuronal cultures. Further, Ori inhibited the expression of nucleotide-binding domain leucine-rich repeats family protein 3 (NLRP3) inflammasome proteins and NLRP3-dependent cytokine interleukin-1ß that can be induced by oxidative stress after TBI. Regarding underlying mechanisms, Ori significantly enhanced expression of key proteins of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway. Our results demonstrated that Ori effectively improved functional impairments and neuropathological changes in animals with TBI. By activating the Nrf2 pathway, it improved mitochondrial function and antioxidant capacity and suppressed the neuroinflammation induced by oxidative stress. The results therefore suggest Ori as a potent candidate for managing neurological damage after TBI.

Inhibition of Smad3 in macrophages promotes Aß efflux from the brain and thereby ameliorates Alzheimer's pathology.

Impaired amyloid-ß (Aß) clearance is believed to be a primary cause of Alzheimer's disease (AD), and peripheral abnormalities in Aß clearance have recently been linked to AD pathogenesis and progression. Data from recent genome-wide association studies have linked genetic risk factors associated with altered functions of more immune cells to AD pathology. Here, we first identified correlations of Smad3 signaling activation in peripheral macrophages with AD progression and phagocytosis of Aß. Then, manipulating the Smad3 signaling regulated macrophage phagocytosis of Aß and induced switch of macrophage inflammatory phenotypes in our cell cultures. In our mouse models, flag-tagged or fluorescent-dye conjugated Aß was injected into the lateral ventricles or tail veins, and traced. Interestingly, blocking Smad3 signaling efficiently increased Aß clearance by macrophages, reduced Aß in the periphery and thereby enhanced Aß efflux from the brain. Moreover, in our APP/PS1 transgenic AD model mice, Smad3 inhibition significantly attenuated Aß deposition and neuroinflammation, and ameliorated cognitive deficits, probably by enhancing the peripheral clearance of Aß. In conclusion, enhancing Aß clearance by peripheral macrophages through Smad3 inhibition attenuated AD-related pathology and cognitive deficits, which may provide a new perspective for understanding AD and finding novel therapeutic approaches.

Zerumbone ameliorates behavioral impairments and neuropathology in transgenic APP/PS1 mice by suppressing MAPK signaling.

BACKGROUND: Alzheimer's disease (AD) is a major clinical problem, but there is a distinct lack of effective therapeutic drugs for this disease. We investigated the potential therapeutic effects of zerumbone, a subtropical ginger sesquiterpene, in transgenic APP/PS1 mice, rodent models of AD which exhibit cerebral amyloidosis and neuroinflammation. METHODS: The N9 microglial cell line and primary microglial cells were cultured to investigate the effects of zerumbone on microglia. APP/PS1 mice were treated with zerumbone, and non-cognitive and cognitive behavioral impairments were assessed and compared between the treatment and control groups. The animals were then sacrificed, and tissues were collected for further analysis. The potential therapeutic mechanism of zerumbone and the signaling pathways involved were also investigated by RT-PCR, western blot, nitric oxide detection, enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, and flow cytometry analysis. RESULTS: Zerumbone suppressed the expression of pro-inflammatory cytokines and induced a switch in microglial phenotype from the classic inflammatory phenotype to the alternative anti-inflammatory phenotype by inhibiting the mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B signaling pathway in vitro. After a treatment period of 20 days, zerumbone significantly ameliorated deficits in both non-cognitive and cognitive behaviors in transgenic APP/PS1 mice. Zerumbone significantly reduced ß-amyloid deposition and attenuated pro-inflammatory microglial activation in the cortex and hippocampus. Interestingly, zerumbone significantly increased the proportion of anti-inflammatory microglia among all activated microglia, potentially contributing to reduced ß-amyloid deposition by enhancing phagocytosis. Meanwhile, zerumbone also reduced the expression of key molecules of the MAPK pathway, such as p38 and extracellular signal-regulated kinase. CONCLUSIONS: Overall, zerumbone effectively ameliorated behavioral impairments, attenuated neuroinflammation, and reduced ß-amyloid deposition in transgenic APP/PS1 mice. Zerumbone exhibited substantial anti-inflammatory activity in microglial cells and induced a phenotypic switch in microglia from the pro-inflammatory phenotype to the anti-inflammatory phenotype by inhibiting the MAPK signaling pathway, which may play an important role in its neuroprotective effects. Our results suggest that zerumbone is a potential therapeutic agent for human neuroinflammatory and neurodegenerative diseases, in particular AD.

Selective suppression of the JNK-MMP2/9 signal pathway by tetramethylpyrazine attenuates neuropathic pain in rats.

BACKGROUND: Activated astrocytes release matrix metalloproteinase-2/9 (MMP-2/9) to induce central sensitization and maintain neuropathic pain. However, the mechanisms involved in the activation of MMP-2/9 on astrocytes during pain remain poorly understood. Meanwhile, there is a lack of effective treatment to inhibit the activation of MMP-2/9 on astrocytes. In this study, we aim to investigate the effect of tetramethylpyrazine (TMP), a natural compound with analgesic effects but unknown mechanisms, on MMP-2/9 in neuropathic pain. METHODS: The nociception was assessed by measuring the incidence of foot withdrawal in response to mechanical indentation in rats (n = 6). Cell signaling was assayed using western blotting (n = 6) and immunohistochemistry (n = 5). The astrocyte cell line C8-D1A was cultured to investigate the in vitro effects. RESULTS: TMP significantly attenuated the maintenance of chronic constrictive injury (CCI)-induced neuropathic pain, inhibited the activation of astrocytes, and decreased the expression of MMP-2/9. Furthermore, our results indicated that TMP could selectively suppress JNK activity but had no notable effects on ERK and p38. Our study also revealed that the effect of TMP may be dependent on the inhibition of TAK1. CONCLUSIONS: Inhibition of astrocyte activation in the spinal cord by tetramethylpyrazine may have utility in the treatment of CCI-induced neuroinflammation, and our results further implicate JNK-MMP-2/9 as a novel target for the attenuation of neuropathic pain.