Dissecting Molecular Mechanisms Underlying H2O2-induced Apoptosis of Mouse Bone Marrow Mesenchymal Stem Cell: Role of Mst1 Inhibition.

Abstract BackgroundBone marrow mesenchymal stem cell (BM-MSC) has been shown to treat pulmonary arterial hypertension (PAH). However, excessive reactive oxygen species (ROS) increases the apoptosis of BM-MSCs, leading to poor survival and engraft efficiency. Thus, improving the ability of BM-MSCs to scavenge ROS may considerably enhance the effectiveness of transplantation therapy. Mammalian Ste20-like kinase 1 (Mst1) is a pro-apoptotic molecule which increases ROS production. The aim of this study is to uncover the underlying mechanisms the effect of Mst1 inhibition on the tolerance of BM-MSCs under H2O2 condition.MethodsMst1 expression in BM-MSCs was inhibited via transfection with adenoviruses expressing a short hairpin (sh) RNA directed against Mst1 (Ad-sh-Mst1) and exposure to H2O2. Cell viability was detected by Cell counting Kit 8 (CCK‑8) assay, and cell apoptosis was analyzed by Annexin V-FITC/PI, Caspase 3 Activity Assay kits, and pro caspase 3 expression. ROS level was evaluated by the ROS probe DCFH-DA, mitochondrial membrane potential (ΔΨm) assay, SOD1/2, CAT, and GPx expression. Autophagy was assessed using transmission electron microscopy, stubRFP-sensGFP-LC3 lentivirus and autophagy-related protein expression. The autophagy/Keap1/Nrf2 signal in H2O2-treated BM-MSC/sh-Mst1 was also measured.ResultsMst1 inhibition reduced ROS production, increased antioxidant enzyme SOD1/2, CAT, GPx expression, maintained ΔΨm, and alleviated cell apoptosis in H2O2-treated BM-MSCs. In addition, this phenomenon was closely correlated with the autophagy/Keap1/Nrf2 signal pathway. Moreover, the antioxidant pathway Keap1/Nrf2, was also blocked when autophagy was inhibited by the autophagy inhibitor 3-MA. However, Keap1 or Nrf2 knockout via siRNA had no effect on autophagy activation or suppression.ConclusionMst1 inhibition mediated the cytoprotective action of mBM-MSCs against H2O2-induced oxidative stress injury. The underlying mechanisms involve autophagy activation and the Keap1/Nrf2 signal pathway.

Download Full-text

Dissecting Molecular Mechanisms Underlying H2O2-induced Apoptosis of Mouse Bone Marrow Mesenchymal Stem Cell: Role of Mst1 Inhibition.

10.21203/rs.3.rs-45400/v2 ◽

2020 ◽

Author(s):

Qian Zhang ◽

Xianfeng Cheng ◽

Haizhou Zhang ◽

Tao Zhang ◽

Zhengjun Wang ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Cell Apoptosis ◽

Caspase 3 ◽

Signal Pathway ◽

Cell Counting ◽

Ros Production ◽

Stress Injury ◽

Underlying Mechanisms

Abstract Background: Bone marrow mesenchymal stem cell (BM-MSC) has been shown to treat pulmonary arterial hypertension (PAH). However, excessive reactive oxygen species (ROS) increases the apoptosis of BM-MSCs, leading to poor survival and engraft efficiency. Thus, improving the ability of BM-MSCs to scavenge ROS may considerably enhance the effectiveness of transplantation therapy. Mammalian Ste20-like kinase 1 (Mst1) is a pro-apoptotic molecule which increases ROS production. The aim of this study is to uncover whether Mst1 inhibition enhanced the tolerance of BM-MSCs under H2O2 condition and the underlying mechanisms. Methods: Mst1 expression in BM-MSCs was inhibited via transfection with adenoviruses expressing a short hairpin (sh) RNA directed against Mst1 (Ad-sh-Mst1) and exposure to H 2 O 2 . Cell viability was detected by Cell counting Kit 8 (CCK-8) assay, and cell apoptosis was analyzed by Annexin V-FITC/PI, Caspase 3 Activity Assay kits, and pro caspase 3 expression. ROS level was evaluated by the ROS probe DCFH-DA, mitochondrial membrane potential (ΔΨm) assay, SOD1/2, CAT, and GPx expression. Autophagy was assessed using transmission electron microscopy, stubRFP-sensGFP-LC3 lentivirus and autophagy-related protein expression. The autophagy/Keap1/Nrf2 signal in H 2 O 2 -treated BM-MSC/sh-Mst1 was also measured. Results: Mst1 inhibition reduced ROS production, increased antioxidant enzyme SOD1/2, CAT, GPx expression, maintained ΔΨm, and alleviated cell apoptosis in H 2 O 2 -treated BM-MSCs. In addition, this phenomenon was closely correlated with the autophagy/Keap1/Nrf2 signal pathway. The autophagy inhibitor, the antioxidant pathway Keap1/Nrf2, was also blocked when autophagy was inhibited by 3-MA. However, Keap1 or Nrf2 knockout via siRNA had no effect on autophagy activation or suppression. Conclusion: Mst1 inhibition mediates the cytoprotective benefit of mBM-MSCs against H 2 O 2 oxidative stress injury. The underlying mechanisms involve autophagy activation and the Keap1/Nrf2 signal pathway.

Download Full-text

Dissecting molecular mechanisms underlying H2O2-induced apoptosis of mouse bone marrow mesenchymal stem cell: role of Mst1 inhibition

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02041-7 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Qian Zhang ◽

Xianfeng Cheng ◽

Haizhou Zhang ◽

Tao Zhang ◽

Zhengjun Wang ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Cell Apoptosis ◽

Caspase 3 ◽

Signal Pathway ◽

Cell Counting ◽

Ros Production ◽

Stress Injury ◽

Underlying Mechanisms

Abstract Background Bone marrow mesenchymal stem cell (BM-MSC) has been shown to treat pulmonary arterial hypertension (PAH). However, excessive reactive oxygen species (ROS) increases the apoptosis of BM-MSCs, leading to poor survival and engraft efficiency. Thus, improving the ability of BM-MSCs to scavenge ROS may considerably enhance the effectiveness of transplantation therapy. Mammalian Ste20-like kinase 1 (Mst1) is a pro-apoptotic molecule which increases ROS production. The aim of this study is to uncover the underlying mechanisms the effect of Mst1 inhibition on the tolerance of BM-MSCs under H2O2 condition. Methods Mst1 expression in BM-MSCs was inhibited via transfection with adenoviruses expressing a short hairpin (sh) RNA directed against Mst1 (Ad-sh-Mst1) and exposure to H2O2. Cell viability was detected by Cell Counting Kit 8 (CCK-8) assay, and cell apoptosis was analyzed by Annexin V-FITC/PI, Caspase 3 Activity Assay kits, and pro caspase 3 expression. ROS level was evaluated by the ROS probe DCFH-DA, mitochondrial membrane potential (ΔΨm) assay, SOD1/2, CAT, and GPx expression. Autophagy was assessed using transmission electron microscopy, stubRFP-sensGFP-LC3 lentivirus, and autophagy-related protein expression. The autophagy/Keap1/Nrf2 signal in H2O2-treated BM-MSC/sh-Mst1 was also measured. Results Mst1 inhibition reduced ROS production; increased antioxidant enzyme SOD1/2, CAT, and GPx expression; maintained ΔΨm; and alleviated cell apoptosis in H2O2-treated BM-MSCs. In addition, this phenomenon was closely correlated with the autophagy/Keap1/Nrf2 signal pathway. Moreover, the antioxidant pathway Keap1/Nrf2 was also blocked when autophagy was inhibited by the autophagy inhibitor 3-MA. However, Keap1 or Nrf2 knockout via siRNA had no effect on autophagy activation or suppression. Conclusion Mst1 inhibition mediated the cytoprotective action of mBM-MSCs against H2O2-induced oxidative stress injury. The underlying mechanisms involve autophagy activation and the Keap1/Nrf2 signal pathway. Graphical abstract

Download Full-text

Dissecting Molecular Mechanisms Underlying H2O2-induced Apoptosis of Mouse Bone Marrow Mesenchymal Stem Cell: Role of Mst1 Inhibition.

10.21203/rs.3.rs-45400/v1 ◽

2020 ◽

Author(s):

Qian Zhang ◽

Xianfeng Cheng ◽

Haizhou Zhang ◽

Tao Zhang ◽

Zhengjun Wang ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Cell Apoptosis ◽

Caspase 3 ◽

Signal Pathway ◽

Cell Counting ◽

Ros Production ◽

Stress Injury ◽

Underlying Mechanisms

Abstract Background Bone marrow mesenchymal stem cell (BM-MSC) has been shown to treat pulmonary arterial hypertension (PAH). However, excessive reactive oxygen species (ROS) increases the apoptosis of BM-MSCs, leading to poor survival and engraft efficiency. Thus, improving the ability of BM-MSCs to scavenge ROS may considerably enhance the effectiveness of transplantation therapy. Mammalian Ste20-like kinase 1 (Mst1) is a pro-apoptotic molecule which increases ROS production. The aim of this study is to uncover whether Mst1 inhibition enhanced the tolerance of BM-MSCs under H2O2 condition and the underlying mechanisms. Methods Mst1 expression in BM-MSCs was inhibited via transfection with adenoviruses expressing a short hairpin (sh) RNA directed against Mst1 (Ad-sh-Mst1) and exposure to H2O2. Cell viability was detected by Cell counting Kit 8 (CCK‑8) assay, and cell apoptosis was analyzed by Annexin V-FITC/PI, Caspase 3 Activity Assay kits, and pro caspase 3 expression. ROS level was evaluated by the ROS probe DCFH-DA, mitochondrial membrane potential (ΔΨm) assay, SOD1/2, CAT, and GPx expression. Autophagy was assessed using transmission electron microscopy, stubRFP-sensGFP-LC3 lentivirus and autophagy-related protein expression. The autophagy/Keap1/Nrf2 signal in H2O2-treated BM-MSC/sh-Mst1 was also measured. Results Mst1 inhibition reduced ROS production, increased antioxidant enzyme SOD1/2, CAT, GPx expression, maintained ΔΨm, and alleviated cell apoptosis in H2O2-treated BM-MSCs. In addition, this phenomenon was closely correlated with the autophagy/Keap1/Nrf2 signal pathway. The autophagy inhibitor, the antioxidant pathway Keap1/Nrf2, was also blocked when autophagy was inhibited by 3-MA. However, Keap1 or Nrf2 knockout via siRNA had no effect on autophagy activation or suppression. Conclusion Mst1 inhibition mediates the cytoprotective benefit of mBM-MSCs against H2O2 oxidative stress injury. The underlying mechanisms involve autophagy activation and the Keap1/Nrf2 signal pathway.

Download Full-text

2,5‐hexanedione induces bone marrow mesenchymal stem cell apoptosis via inhibition of Akt/Bad signal pathway

Journal of Cellular Biochemistry ◽

10.1002/jcb.26602 ◽

2018 ◽

Vol 119 (4) ◽

pp. 3732-3743 ◽

Cited By ~ 6

Author(s):

Jingsong Sun ◽

Xiaoxia Shi ◽

Shuangyue Li ◽

Fengyuan Piao

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Cell Apoptosis ◽

Signal Pathway

Download Full-text

Quercetin Stimulates Bone Marrow Mesenchymal Stem Cell Differentiation through an Estrogen Receptor-Mediated Pathway

BioMed Research International ◽

10.1155/2018/4178021 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 11

Author(s):

Xin-Gang Pang ◽

Yu Cong ◽

Ni-Rong Bao ◽

Yong-Gang Li ◽

Jian-Ning Zhao

Keyword(s):

Bone Marrow ◽

Estrogen Receptor ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Osteogenic Differentiation ◽

Western Blotting ◽

Stem Cell Differentiation ◽

Cell Counting ◽

Mouse Bone Marrow ◽

Alizarin Red

Objectives. The present study aimed to investigate the overall effect of quercetin on mouse bone marrow mesenchymal stem cell (BMSC) proliferation and osteogenic differentiation in vitro. Materials and Methods. BMSCs were treated with different concentrations of quercetin for 6 days. The effects of quercetin on cell proliferation were assessed at predetermined times using Cell Counting Kit-8 (CCK-8) assay. The cells were then treated with quercetin, estrogen, or an estrogen receptor (ER) antagonist (which was also administered in the presence of quercetin or estrogen) for 7 or 21 days. The effects of quercetin on BMSC osteogenic differentiation were analyzed by an alkaline phosphatase (ALP) assay kit, Alizarin Red S staining (ARS), quantitative real-time PCR (qPCR), and western blotting. Results. The CCK-8 and ALP assays and ARS staining showed that quercetin significantly enhanced BMSC proliferation, ALP activity, and extracellular matrix production and mineralization, respectively. The qPCR results indicated that quercetin promoted osterix (OSX), runt-related transcription factor 2 (RUNX2), and osteopontin (OPN) transcription in the presence of osteoinduction medium, and the western blotting results indicated that quercetin enhanced bone morphogenetic protein 2 (BMP2), Smad1, Smad4, RUNX2, OSX, and OPN expression and Smad1 phosphorylation. Treatment with the ER inhibitor ICI182780 blocked the effects of quercetin. Conclusions. Our data demonstrated that quercetin promotes BMSC proliferation and osteogenic differentiation. Quercetin enhances BMP signaling pathway activation and upregulates the expression of downstream genes, such as OSX, RUNX2, and OPN, via the ER.

Download Full-text

MiR-140-3p inhibits the cell viability and promotes apoptosis of synovial fibroblasts in rheumatoid arthritis through targeting sirtuin 3

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02236-5 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Beibei Zu ◽

Lin Liu ◽

Jingya Wang ◽

Meirong Li ◽

Junxia Yang

Keyword(s):

Rheumatoid Arthritis ◽

Cell Viability ◽

Cell Apoptosis ◽

Caspase 3 ◽

Pearson Correlation ◽

Fibrous Tissue ◽

Synovial Fibroblasts ◽

Cell Counting ◽

Sirtuin 3 ◽

Fibrous Tissues

Abstract Background Synovial fibroblasts (SFs) with the abnormal expressions of miRNAs are the key regulator in rheumatoid arthritis (RA). Low-expressed miR-140-3p was found in RA tissues. Therefore, we attempted to investigate the effect of miR-140-3p on SFs of RA. Methods RA and normal synovial fibrous tissue were gathered. The targets of miR-140-3p were found by bioinformatics and luciferase analysis. Correlation between the expressions of miR-140-3p with sirtuin 3 (SIRT3) was analyzed by Pearson correlation analysis. After transfection, cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The expressions of miR-140-3p, SIRT3, Ki67, Bcl-2, Bax, and cleaved Caspase-3 were detected by RT-qPCR or western blot. Results Low expression of miR-140-3p and high expression of SIRT3 were found in RA synovial fibrous tissues. SIRT3 was a target of miR-140-3p. SIRT3 expression was negatively correlated to the expression of miR-140-3p. MiR-140-3p mimic inhibited the MH7A cell viability and the expressions of SIRT3, Ki67, and Bcl-2 and promoted the cell apoptosis and the expressions of Bax and cleaved Caspase-3; miR-140-3p inhibitor showed an opposite effect to miR-140-3p mimic on MH7A cells. SIRT3 overexpression not only promoted the cell viability and inhibited cell apoptosis of MH7A cells but also reversed the effect of miR-140-3p mimic had on MH7A cells. Conclusions The results in this study revealed that miR-140-3p could inhibit cell viability and promote apoptosis of SFs in RA through targeting SIRT3.

Download Full-text