METTL3-dependent N6-methyladenosine modification is involved in berberine-mediated neuroprotection in ischemic stroke by enhancing the stability of NEAT1 in astrocytes.

Ischemic stroke (IS) is one of the principal causes of disability and death worldwide. Berberine (BBR), derived from the traditional Chinese herbal medicine Huang Lian, has been reported to inhibit the progression of stroke, but the specific mechanism whereby BBR modulates the progression of ischemic stroke remains unclear. N6-methyladenosine (m6A) modification is the most typical epigenetic modification of mRNA post-transcriptional modifications, among which METTL3 is the most common methylation transferase. During the study, the middle cerebral artery occlusion/reperfusion (MCAO/R) was established in mice, and the mice primary astrocytes and neurons induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was simulated in vitro. Level of LncNEAT1, miR-377-3p was detected via RT-qPCR. The levels of Nampt and METTL3 were measured by Western blot. CCK8 and LDH assay was performed to detect cell viability. Here, we found that berberine alleviates MCAO/R-induced ischemic injury and up-regulates the expression of Nampt in astrocytes, miR-377-3p inhibits the expression of Nampt in astrocytes after OGD/R, thus promoting neuronal injury. NEAT1 binds to miR-377-3p in OGD/R astrocytes and plays a neuronal protective role as a ceRNA. METTL3 can enhance NEAT1 stability in OGD/R astrocytes by modulating m6A modification of NEAT1. Taken together, our results demonstrate that berberine exerts neuroprotective effects via the m6A methyltransferase METTL3, which regulates the NEAT1/miR-377-3p/Nampt axis in mouse astrocytes to ameliorate cerebral ischemia/reperfusion (I/R) injury.

Advancement in Beneficial Effects of AVE 0991: A Brief Review.

AVE 0991, a non-peptide analogue of Angiotensin-(1-7) [Ang-(1-7)], is orally active and physiologically well tolerated. Several studies have demonstrated that AVE 0991 improves glucose and lipid metabolism, and contains anti-inflammatory, anti-apoptotic, anti-fibrosis, and anti-oxidant effects. Numerous preclinical studies have also reported that AVE 0991 appears to have beneficial effects on a variety of systemic diseases, including cardiovascular, liver, kidney, cancer, diabetes, and nervous system diseases. This study searched multiple literature databases, including PubMed, Web of Science, EMBASE, Google Scholar, Cochrane Library, and the ClinicalTrials.gov website from the establishment to October 2022, using AVE 0991 as a keyword. This literature search revealed that AVE 0991 could play different roles via various signaling pathways. However, the potential mechanisms of these effects need further elucidation. This review summarizes the benefits of AVE 0991 in several medical problems, including the COVID-19 pandemic. The paper also describes the underlying mechanisms of AVE 0991, giving in-depth insights and perspectives on the pharmaceutical value of AVE 0991 in drug discovery and development.

Astrocytes-derived exosomes pre-treated by berberine inhibit neuroinflammation after stroke via miR-182-5p/Rac1 pathway.

BACKGROUND: Our previous studies have shown that berberine can improve the nerve function deficits in ischemic stroke by inhibiting inflammation. The cellular communication between astrocytes and neurons via exosomes might affect neurological function after ischemic stroke, which plays a vital role in the therapy of ischemic stroke. OBJECTIVE: The present study focused on the effects of exosomes released from astrocytes induced by the glucose and oxygen deprivation model with berberine pretreatment (BBR-exos) treatment for ischemic stroke and its regulatory mechanism. METHODS: Oxygen-glucose-deprivation/Reoxygenation (OGD/R)-treated primary cells were used to mimic cerebral ischemia/reperfusion conditions in vitro. With the treatment of BBR-exos and exosomes released from primary astrocytes induced by the glucose and oxygen deprivation model (OGD/R-exos), the cell viability was detected. C57BL/6J mice were used to establish middle cerebral artery occlusion/reperfusion (MCAO/R) model. The anti-neuroinflammation effects of BBR-exos and OGD/R-exos were evaluated. Subsequently, the key miRNA in BBR-exos was identified by exosomal miRNA sequencing and cell validation. miR-182-5p mimic and inhibitors were provided to verify the effects in inflammation. Finally, the binding sites between miR-182-5p and Rac1 were predicted online and verified by using a dual-luciferase reporter assay. RESULTS: BBR-exos and OGD/R-exos both improved the decreased activity of OGD/R-induced neurons, and decreased the expression of IL-1ß, IL-6 and TNF-α (all P ＜ 0.05), which reduced neuronal injury and inhibited neuroinflammation in Vitro. And BBR-exos showed better effects (P ＜ 0.05). The same effect has been verified in vivo experiments: BBR-exos and OGD/R-exos both reduced cerebral ischemic injury and inhibited neuroinflammation in MCAO/R mice (all P ＜ 0.05). Likewise, BBR-exos showed better effects (P ＜ 0.05). The exosomal miRNA sequencing results showed that miR-182-5p was highly expressed in BBR-exos and inhibited neuroinflammation by targeting Rac1 (P ＜ 0.05). CONCLUSION: BBR-exos can carry miR-182-5p to injured neurons and inhibit the expression of Rac1, which could inhibit neuroinflammation and improved brain injury after ischemic stroke.

Berberine Mediates the Production of Butyrate to Ameliorate Cerebral Ischemia via the Gut Microbiota in Mice.

Ischemic stroke (IS) is a vascular disease group concomitant with high morbidity and mortality. Berberine is a bioactive substance and it has been known to improve stroke, but its mechanism is yet to be proven. Mice were fed with BBR for 14 days. Then, the mice were made into MCAO/R models. Neurological score, infarct volume, neuronal damage and markers associated with inflammation were detected. We tested the changes in intestinal flora in model mice after BBR administration using 16SrRNA sequencing. Chromatography-mass spectrometry was used to detect butyrate chemically. Tissue immunofluorescence was used to detect the changes in the microglia and astroglia in the mice brains. Our findings suggest that berberine improves stroke outcomes by modulating the gut microbiota. Specifically, after MCAO/R mice were given berberine, the beneficial bacteria producing butyric acid increased significantly, and the mice also had significantly higher levels of butyric acid. The administration of butyric acid and an inhibitor of butyric acid synthesis, heptanoyl-CoA, showed that butyric acid improved the stroke outcomes in the model mice. In addition, butyric acid could inhibit the activation of the microglia and astrocytes in the brains of model mice, thereby inhibiting the generation of pro-inflammatory factors IL-6, IL-1ß and TNF-α as well as improving stroke outcomes. Our results suggest that berberine may improve stroke outcomes by modulating the gut flora to increase the abundance of butyric acid. These findings elucidate the mechanisms by which berberine improves stroke outcomes and provide some basis for clinical treatment.

Astrocyte-derived exosomal nicotinamide phosphoribosyltransferase (Nampt) ameliorates ischemic stroke injury by targeting AMPK/mTOR signaling to induce autophagy.

Acute ischemic stroke (AIS) is a global cerebrovascular disease with high disability and mortality, which has no effective therapy. Studies have demonstrated that astrocyte-derived exosomes (ADEXs) provided neuroprotection in experimental stroke models. Nevertheless, the role of exosomes derived from oxygen-glucose-deprivation/reoxygenation-stimulated astrocytes (OGD/R-stimulated astrocytes; OGD/R-ADEXs) in AIS remains largely unknown. Here, we found that OGD/R-ADEXs significantly reduced OGD/R-induced neuronal death and promoted neuronal autophagy. These effects were reversed when astrocytes were pretreated with GW4869, an exosome secretion inhibitor, or when hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was knocked down. Neuroprotection was also observed during treatment with OGD/R-ADEXs in vivo. Further studies showed that Nampt, played a vital effect in the regulation of autophagy, was significantly increased in OGD/R-ADEXs. Knockdown of Nampt in astrocytes abolished the above-mentioned effects of OGD/R-ADEXs. Mechanistically, Nampt increased autophagy and decreased cell death by modulating AMPK/mTOR signaling, which recognized as a key signaling pathway of autophagy after AIS. Collectively, these results showed that Nampt released by OGD/R-ADEXs ameliorated acute ischemic stroke during neuronal injury by targeting AMPK/mTOR signaling to induce autophagy. Our study revealed a new key factor in the secretion of exosomes by OGD/R astrocytes, which regulated autophagy and induced neuroprotection in a mouse stroke model.

Protective effects of catalpol on diabetes mellitus-induced male reproductive damage via suppression of the AGEs/RAGE/Nox4 signaling pathway.

AIMS: Diabetes mellitus (DM)-induced reproductive damage is an important cause of infertility for male DM patients, we herein evaluated the effects of catalpol on diabetic reproductive damage through the suppression of the AGEs/RAGE/Nox4 signaling pathway. METHODS: KK-Ay diabetic reproductive damage mice were administered with catalpol for 8 weeks, the testis/body weight ratio, testicular histopathology, the levels of endogenous hormone and the activity of testicular marker enzymes were determined. In vitro, the GC-2 cell injury model was induced by advanced glycation end-products (AGEs) and pretreated with catalpol. Cell viability, apoptosis, and oxidative stress markers were detected and the mechanism based on the AGEs/RAGE/Nox4 pathway was explored. KEY FINDINGS: Catalpol showed remarkable capacity on protecting diabetic reproductive damage by improving the histomorphology of the testes, increasing the testis/body weight ratio and activity of acid phosphatase (ACP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (γ-GT). The reduced testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in DM mice were also reversed with catalpol intervention. Moreover, catalpol showed markedly effects of anti-oxidative in vivo and in vitro, which significantly down-regulated reactive oxygen species (ROS) levels and restored superoxide dismutase (SOD) activity, meanwhile decreased GC-2 cell apoptosis and Bax/Bcl-2 ratio. Moreover, the over-expression of receptors for AGEs (RAGE), NADPH oxidase type 4 (Nox4) and phosphorylation of nuclear transcription factor-κB p65 (NF-κB p65) were suppressed by catalpol. SIGNIFICANCE: Catalpol could alleviate DM-induced male reproductive damage by inhibiting oxidative stress-induced apoptosis and inflammation mediated by AGEs/RAGE/Nox4 signaling pathway.