hUMSCs restore ovarian function in POI mice by regulating GSK3ß-mediated mitochondrial dynamic imbalances in theca cells.

Premature ovarian insufficiency (POI), a major cause of female infertility, is defined as follicular atresia and a rapid loss of germ cells in women of reproductive age due to ovarian failure. Recently, findings from several studies have indicated that human umbilical cord mesenchymal stem cells (hUMSCs) can alleviate ovarian dysfunction resulting from POI. However, the mechanisms underlying this effect require further clarification. In this study, a mouse model of POI was established as achieved with an intraperitoneal injection of cyclophosphamide (CTX) into female C57BL/6J mice in vivo. These POI mice received a 1-week intervention of hUMACs. In addition, an in vitro POI model was also included. The cultured supernatants of hUMSCs and glycogen synthase kinase 3 beta (GSK3ß) inhibitor (SB216763) were used to treat theca cells (TCs) exposed to CTX. Hematoxylin and Eosin (H&E) staining and Enzyme-linked immunosorbent assay (ELISA) were used to assess ovarian structure and morphology, as well as endocrine function in these POI mice. Based on results from the ELISA and JC-1 labeling, CTX exerted significant detrimental effects on testosterone levels and the mitochondrial membrane potential in TCs. Subsequently, Western Blot, Immunofluorescence staining (IF), and Quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate various indicators of testosterone synthesis function and mitochondrial dynamics in ovaries and TCs of POI mice. In vivo, dysfunctions in ovarian structure and function in the POI mouse model were effectively restored following hUMSCs treatment, and abnormalities in hormone synthesis were significantly reduced. Furthermore, when the stem cell supernatants of hUMSCs were applied to TCs in vitro we found that GSK3ß expression was reduced, the imbalance of mitochondrial dynamics was alleviated, and the ability of mitochondrial testosterone synthesis was increased. Taken together, our results indicate that hUMSCs treatment can restore the imbalance of mitochondrial dynamics and restart testosterone synthesis of TCs by suppressing GSK3ß expression, ultimately alleviating POI damage.

Modified Hu-lu-ba-wan protects diabetic glomerular podocytes via promoting PKM2-mediated mitochondrial dynamic homeostasis.

BACKGROUND: Mitochondrial dysfunction is implicated in the progression of diabetic kidney disease (DKD). Damaged mitochondria produce excessive reactive oxygen species (ROS) that can cause apoptosis. Mitochondrial dynamics control the quality and function of mitochondria. Targeting mitochondrial dynamics may reduce ROS-induced apoptosis and improve renal injury in DKD. Modified Hu-lu-ba-wan (MHLBW) shows distinct clinical effects on DKD patients, which are related to its role in antioxidant stress modulation. However, the relevant mechanisms of MHLBW have not been clearly explored. PURPOSE: This study was aimed to evaluate the therapeutic effects of MHLBW on spontaneous DKD mice and clarify the potential mechanisms. METHODS: The main components of MHLBW were identified by HPLC. Using db/db mice as DKD models, we evaluated the therapeutic effects of MHLBW on mice after an 8-week administration. We investigated the molecular mechanism of MHLBW in regulating mitochondrial dynamic homeostasis, podocyte apoptosis, and glomerular damage. After that, computational docking analysis and in vitro experiments were conducted for further mechanism verification. RESULTS: Intragastric administration of MHLBW for 8 weeks in db/db mice significantly improved glucose metabolism, basement membrane thickening, mesangial expansion, glomerular fibrosis, and podocyte injury. MHLBW can reverse podocyte apoptosis via promoting mitochondrial dynamic homeostasis, which was related to regulating the PKM2/ PGC-1α/Opa1 pathway. Berberine (BBR), one of the components of MHLBW, exhibited preeminent affinity with PKM2 as reflected by computational docking analysis. In cultured podocytes, BBR can also prevent apoptosis by promoting PKM2-mediated mitochondrial dynamic homeostasis. CONCLUSION: Our study demonstrates that MHLBW can treat DKD by inhibiting glomerular damage and podocyte apoptosis through positive regulation of PKM2-mediated mitochondrial dynamic homeostasis. These results may provide a potential strategy against DKD.

SIRT4 Protects Müller Glial Cells Against Apoptosis by Mediating Mitochondrial Dynamics and Oxidative Stress.

SIRT4 is a member of the sirtuin family, which is related to mitochondrial function and possesses antioxidant and regulatory redox effects. Currently, the roles of SIRT4 in retinal Müller glial cells, oxidative stress, and mitochondrial function are still unclear. We confirmed, by immunofluorescence staining, that SIRT4 is located mainly in the mitochondria of retinal Müller glial cells. Using flow cytometry and Western blotting, we analyzed cell apoptosis, intracellular reactive oxygen species (ROS) levels, apoptotic and proapoptotic proteins, mitochondrial dynamics-related proteins, and mitochondrial morphology and number after the overexpression and downregulation of SIRT4 in rMC-1 cells. Neither the upregulation nor the downregulation of SIRT4 alone affected apoptosis. SIRT4 overexpression reduced intracellular ROS, reduced the BAX/BCL2 protein ratio, and increased the L-OPA/S-OPA1 ratio and the levels of the mitochondrial fusion protein MFN2 and the mitochondrial cleavage protein FIS1, increasing mitochondrial fusion. SIRT4 downregulation had the opposite effect. Mitochondria tend to divide after serum starvation for 24 h, and SIRT4 downregulation increases mitochondrial fragmentation and oxidative stress, leading to aggravated cell damage. The mitochondrial division inhibitor Mdivi-1 reduced oxidative stress levels and thus reduced cell damage caused by serum starvation. The overexpression of SIRT4 in rMC-1 cells reduced mitochondrial fragmentation caused by serum starvation, leading to mitochondrial fusion and reduced expression of cleaved caspase-3, thus alleviating the cellular damage caused by oxidative stress. Thus, we speculate that SIRT4 may protect retinal Müller glial cells against apoptosis by mediating mitochondrial dynamics and oxidative stress.

The effect of adipose-derived mesenchymal stem cell transplantation on ovarian mitochondrial dysfunction in letrozole-induced polycystic ovary syndrome in rats: the role of PI3K-AKT signaling pathway.

OBJECTIVE: The present study aimed to elucidate how mesenchymal stem cells (MSCs) application could efficiently attenuate pathological changes of letrozole-induced poly cystic ovary syndrome (PCOS) by modulating mitochondrial dynamic via PI3K-AKT pathway. METHODS: Thirty-two female rats were randomly divided into four experimental groups: Sham, PCOS, PCOS + MSCs, and PCOS + MSCs + LY294002. The Sham group received 0.5% w/v carboxymethyl cellulose (CMC); the PCOS group received letrozole (1 mg/kg, daily) in 0.5% CMC for 21 days. Animals in the PCOS + MSCs group received 1 × 106 MSCs/rat (i.p,) on the 22th day of the study. In the PCOS + MSCs + LY294002 group, rats received LY294002 (PI3K-AKT inhibitor) 40 min before MSC transplantation. Mitochondrial dynamic gene expression, mitochondrial membrane potential (MMP), citrate synthase (CS) activity, oxidative stress, inflammation, ovarian histological parameters, serum hormone levels, homeostatic model assessment for insulin resistance (HOMA-IR), insulin and glucose concentrations, p-PI3K and p-AKT protein levels were evaluated at the end of the experiment. RESULTS: PCOS rats showed a significant disruption of mitochondrial dynamics and histological changes, lower MMP, CS, ovary super oxide dismutase (SOD) and estrogen level. They also had a notable rise in insulin and glucose concentrations, HOMA-IR, testosterone level, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, ovarian malondialdehyde (MDA) content as well as a notable decrease in p-PI3K and p-AKT protein levels compared to the Sham group. In the PCOS + MSCs group, the transplantation of MSCs could improve the above parameters. Administration of LY294002 (PI3K-AKT pathway inhibitor) deteriorated mitochondrial dynamic markers, oxidative stress status, inflammation markers, hormonal levels, glucose, and insulin levels and follicular development compared to the PCOS + MSCs group. CONCLUSIONS: This study demonstrated that the protective effects of MSC transplantation in regulating mitochondrial dynamics, promoting mitochondrial biogenesis, competing with redox status and inflammation response were mainly mediated through the PI3K-AKT pathway in the PCOS model.

Role of mitochondrial dynamic-related protein 1 on inflammation after intracerebral hemorrhage in mice / 中国免疫学杂志

Objective:To investigate the effect and mechanisms of mitochondrial dynamic-related protein 1(Drp1)on the in-flammatory response after intracerebral hemorrhage(ICH)in mice.Methods:Western blot was used to detect the expressions of Drp1 and phosphorylated Drp1(p-Drp1)in brain tissues after ICH,and the optimal concentration of selective Drp1 inhibitor(Mdivi-1)were screened.The mice were randomly divided into sham group,ICH group,ICH+solvent control group(ICH+Vehicle group)and ICH+Mdivi-1 group.The neurological function was evaluated by mNSS,the content of brain water was measured by wet-dry weight method,HE and Nissl staining were used to observe the morphological changes of brain tissues,neutrophil infiltration were observed by MPO staining,and the expression of inflammatory cytokines(IL-6,TNF-α)and mitochondrial membrane marker proteins TOM20,COX4Ⅰ1 were detected by Western blot.Results:Compared with sham group,the expression of p-Drp1 was significantly increased at 12 h after ICH(P<0.01).Compared with ICH+Vehicle group,the neurological function score(P<0.01)and brain water content(P<0.05)of ICH+Mdivi-1 group were increased,the protein expressions of IL-6 and TNF-α and the number of MPO positive cells around hematoma in ICH+Mdivi-1 group were significantly increased(P<0.05),and the expressions of mitochondrial membrane proteins TOM20 and COX4Ⅰ1 in ICH+Mdivi-1 group were significantly elevated(P<0.01).Conclusion:Inhibition of Drp1 can inhibit mito-chondrial division and aggravate inflammatory injury after ICH in mice.Therefore,Drp1 may reduce the inflammation after ICH.

Recent advance in dysfunction of mitochondrial dynamics and pathogenesis of Alzheimer's disease / 中华神经医学杂志

The pathogenesis of Alzheimer's disease (AD) has not been fully elucidated.Mitochondria play critical roles in neuronal function;and damage of hippocampal neurons by dysfunction of mitochondrial dynamics is the major pathological mechanism of AD.Improvement of mitochondrial dynamics imbalance in time should alleviate or reverse damage of hippocampal neurons.After a brief overview of basic mechanisms involved in regulation ofmitochondrial fission,fusion,and transportion,and how mitochondrial dynamics affects AD,this review article focuses on discussing the role of key sites such as Drpl,Mfnl/2,Opal and Miro/Milton in regulating mitochondria dynamics and their effects on