Anti-inflammatory and neuroprotective effects of natural cordycepin in rotenone-induced PD models through inhibiting Drp1-mediated mitochondrial fission.

Accumulating evidences suggest that inflammation-mediated neurons dysfunction participates in the initial and development of Parkinson's disease (PD), whereas mitochondria have been recently recognized as crucial regulators in NLRP3 inflammasome activation. Cordycepin, a major component of cordyceps militaris, has been shown to possess neuroprotective and anti-inflammatory activity. However, the effects of cordycepin in rotenone-induced PD models and the possible mechanisms are still not fully understood. Here, we observed that motor dysfunction and dopaminergic neurons loss induced by rotenone exposure were ameliorated by cordycepin. Cordycepin also reversed Drp1-mediated aberrant mitochondrial fragmentation through increasing AMPK phosphorylation and maintained normal mitochondrial morphology. Additionally, cordycepin effectively increased adenosine 5'-triphosphate (ATP) content, mitochondrial membrane potential (MMP), and reduced mitochondrial ROS levels, as well as inhibited complex 1 activity. More importantly, cordycepin administration inhibited the expression of NLRP3 inflammasome components and the release of pro-inflammatory cytokine in rotenone-induced rats and cultured neuronal PC12 cells. Moreover, we demonstrated that the activation of NLRP3 inflammasome within neurons could be suppressed by the mitochondrial division inhibitor (Mdivi-1). Collectively, the present study provides evidence that cordycepin exerts neuroprotective effects partially through preventing neural NLRP3 inflammasome activation induced by Drp1-dependent mitochondrial fragmentation in rotenone-injected PD models.

Preservation of mitochondrial homeostasis is responsible for the ameliorative effects of Suhuang antitussive capsule on non-resolving inflammation via inhibition of NF-κB signaling and NLRP3 inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Suhuang antitussive capsule (Suhuang), one of traditional antitussive Chinese patent medicines, has been used for the treatment of post-infectious cough and cough variant asthma in clinical practice. It has been demonstrated to show numerous biological actions including antitussive and anti-inflammatory effects. AIM OF THE STUDY: This study aims to investigate the effects of Suhuang on non-resolving inflammation and its underlying molecular mechanism. MATERIAL AND METHODS: In vitro, mitochondrial membrane potential and ROS were detected by flow cytometry analysis. mtDNA release and mPTP fluorescence were determined by Q-PCR and fluorescence microplate reader analysis. Cytochrome C release and 8-OHdG levels were evaluated by ELISA. Additionally, the effects of Suhuang on Drp1, MMP9, IκBα/NF-κB and NLRP3/ASC/Caspase-1 expression were determined by Q-PCR, gelatin zymography or immunoblot analysis. In vivo, C57/BL6 mice were orally administrated for 2 weeks with Suhuang, then lung injury was induced by LPS. Inflammatory mediators mRNA, histological assessment and NF-κB/Caspase-1/IL-1ß levels were evaluated by Q-PCR, H&E staining and immunoblot analysis. Two sepsis models of mice were further used to evaluate its anti-inflammatory effects. RESULTS: Suhuang restored mitochondrial homeostasis by inhibiting Drp1 activation and mitochondrial fission. Besides, Suhuang reduced mPTP opening, mitochondrial membrane potential collapse, ROS overproduction and mtDNA release. Moreover, Suhuang down-regulated MMP9 expression. As a consequence of preserved mitochondrial homeostasis, Suhuang inhibited NF-κB pathway activation by prevention of NF-κB-p65 phosphorylation and IκBα degradation. Suhuang also limited NLRP3 inflammasome activation by blocking NLRP3-ASC interaction and promoting NLRP3 ubiquitination degradation. Drp1 knockdown in vitro diminished the inhibitory effects of Suhuang on inflammatory responses, indicating the essential role of Drp1 in the Suhuang's activity. Consistently, the therapeutic effects of Suhuang were confirmed in LPS-inhaled mice, which recapitulated the protective actions of Suhuang in mitochondrial homeostasis in vitro. Additionally, two sepsis models of mice confirmed the inhibitory effects of Suhuang on uncontrolled inflammation. CONCLUSIONS: Altogether, our work reveals that Suhuang inhibits non-resolving inflammation through inhibition of NF-κB signaling and NLRP3 inflammasome activation by preserving mitochondrial homeostasis, providing new pharmacological data for the clinical use of Suhuang. Our study also suggests mitochondrial homeostasis as a potential intrinsic regulatory strategy for treating inflammatory diseases.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. AIM OF THE STUDY: In this study, we aimed to observe the effects of OP-D on the diabetic myocardium. MATERIALS AND METHODS: Leptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. RESULTS: Blood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. CONCLUSIONS: Our findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.

Inhibition of Drp1-mediated mitochondrial fission improves mitochondrial dynamics and bioenergetics stimulating neurogenesis in hippocampal progenitor cells from a Down syndrome mouse model.

Functional and structural damages to mitochondria have been critically associated with the pathogenesis of Down syndrome (DS), a human multifactorial disease caused by trisomy of chromosome 21 and associated with neurodevelopmental delay, intellectual disability and early neurodegeneration. Recently, we demonstrated in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice -a widely used model of DS - a severe impairment of mitochondrial bioenergetics and biogenesis and reduced NPC proliferation. Here we further investigated the origin of mitochondrial dysfunction in DS and explored a possible mechanistic link among alteration of mitochondrial dynamics, mitochondrial dysfunctions and defective neurogenesis in DS. We first analyzed mitochondrial network and structure by both confocal and transmission electron microscopy as well as by evaluating the levels of key proteins involved in the fission and fusion machinery. We found a fragmentation of mitochondria due to an increase in mitochondrial fission associated with an up-regulation of dynamin-related protein 1 (Drp1), and a decrease in mitochondrial fusion associated with a down-regulation of mitofusin 2 (Mnf2) and increased proteolysis of optic atrophy 1 (Opa1). Next, using the well-known neuroprotective agent mitochondrial division inhibitor 1 (Mdivi-1), we assessed whether the inhibition of mitochondrial fission might reverse alteration of mitochondrial dynamics and mitochondrial dysfunctions in DS neural progenitors cells. We demonstrate here for the first time, that Mdivi-1 restores mitochondrial network organization, mitochondrial energy production and ultimately improves proliferation and neuronal differentiation of NPCs. This research paves the way for the discovery of new therapeutic tools in managing some DS-associated clinical manifestations.

Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression.

BACKGROUND: The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. RESULTS: We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. CONCLUSION: Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.