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2.
Heliyon ; 10(10): e30195, 2024 May 30.
Article in English | MEDLINE | ID: mdl-38784565

ABSTRACT

Hypoxia-induced apoptosis of bone marrow mesenchymal stem cells (BMSCs) limits the efficacy of their transplantation for steroid-induced osteonecrosis of the femoral head (SONFH). As apoptosis and RNA methylation are closely related, exploring the role and mechanism of RNA methylation in hypoxic apoptosis of BMSCs is expected to identify new targets for transplantation of BMSCs for SONFH and enhance transplantation efficacy. We performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA-seq on a hypoxia-induced apoptosis BMSC model and found that the RNA methyltransferase-like 3 (METTL3) is involved in hypoxia-induced BMSC apoptosis. The expression of METTL3 was downregulated in BMSCs after hypoxia and in BMSCs implanted in osteonecrosis areas. Knockdown of METLL3 under normoxic conditions promoted apoptosis of BMSCs. In contrast, overexpression of METTL3 promoted the survival of BMSCs under hypoxic conditions, and overexpression of METTL3 promoted the survival of BMSCs in the osteonecrosis area and the repair of the osteonecrosis area. Regarding the mechanism, the m6A levels of the mRNAs of anti-apoptotic genes Bcl-2, Mcl-1, and BIRC5 were significantly increased upon the overexpression of METTL3 under hypoxic conditions, which promoted the binding of Bcl-2, Mcl-1, and BIRC5 mRNAs to IGF2BP2, enhanced the mRNA stability, and increased the protein expression of the three anti-apoptotic genes. In conclusion, overexpression of METTL3 promoted m6A modification of mRNAs of Bcl-2, Mcl-1, and BIRC5, promoted the binding of IGF2BP2 to the above-mentioned mRNAs, enhanced mRNA stability, inhibited hypoxia-induced BMSC apoptosis, and promoted repair of SONFH, thereby providing novel targets for transplantation of BMSCs for SONFH.

3.
Apoptosis ; 29(5-6): 882-897, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38491252

ABSTRACT

Bone marrow mesenchymal stem cell (BMSC) transplantation is a promising regenerative therapy; however, the survival rate of BMSCs after transplantation is low. Oxidative stress is one of the main reasons for the high apoptosis rate of BMSCs after transplantation, so there is an urgent need to explore the mechanism of oxidative stress-induced apoptosis of BMSCs. Our previous transcriptome sequencing results suggested that the expression of P53-induced nuclear protein 1 (TP53INP1) and the tumor suppressor P53 (P53) was significantly upregulated during the process of oxidative stress-induced apoptosis of BMSCs. The present study further revealed the role and mechanism of TP53INP1 and P53 in oxidative stress-induced apoptosis in BMSCs. Overexpression of TP53INP1 induced apoptosis of BMSCs, knockdown of TP53INP1 alleviated oxidative stress apoptosis of BMSCs. Under oxidative stress conditions, P53 is regulated by TP53INP1, while P53 can positively regulate the expression of TP53INP1, so the two form a positive feedback loop. To clarify the mechanism of feedback loop formation. We found that TP53INP1 inhibited the ubiquitination and degradation of P53 by increasing the phosphorylation level of P53, leading to the accumulation of P53 protein. P53 can act on the promoter of the TP53INP1 gene and increase the expression of TP53INP1 through transcriptional activation. This is the first report on a positive feedback loop formed by TP53INP1 and P53 under oxidative stress. The present study clarified the formation mechanism of the positive feedback loop. The TP53INP1-P53 positive feedback loop may serve as a potential target for inhibiting oxidative stress-induced apoptosis in BMSCs.


Subject(s)
Apoptosis , Mesenchymal Stem Cells , Oxidative Stress , Tumor Suppressor Protein p53 , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Apoptosis/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Animals , Heat-Shock Proteins/metabolism , Heat-Shock Proteins/genetics , Humans , Bone Marrow Cells/metabolism , Bone Marrow Cells/cytology , Ubiquitination , Carrier Proteins/genetics , Carrier Proteins/metabolism , Phosphorylation , Cells, Cultured , Feedback, Physiological , Mice
4.
Bone Res ; 11(1): 27, 2023 May 22.
Article in English | MEDLINE | ID: mdl-37217464

ABSTRACT

The specific pathogenesis of steroid-induced osteonecrosis of the femoral head (SONFH) is still not fully understood, and there is currently no effective early cure. Understanding the role and mechanism of long noncoding RNAs (lncRNAs) in the pathogenesis of SONFH will help reveal the pathogenesis of SONFH and provide new targets for its early prevention and treatment. In this study, we first confirmed that glucocorticoid (GC)-induced apoptosis of bone microvascular endothelial cells (BMECs) is a pre-event in the pathogenesis and progression of SONFH. Then, we identified a new lncRNA in BMECs via lncRNA/mRNA microarray, termed Fos-associated lincRNA ENSRNOT00000088059.1 (FAR591). FAR591 is highly expressed during GC-induced BMEC apoptosis and femoral head necrosis. Knockout of FAR591 effectively blocked the GC-induced apoptosis of BMECs, which then alleviated the damage of GCs to the femoral head microcirculation and inhibited the pathogenesis and progression of SONFH. In contrast, overexpression of FAR591 significantly promoted the GC-induced apoptosis of BMECs, which then aggravated the damage of GCs to the femoral head microcirculation and promoted the pathogenesis and progression of SONFH. Mechanistically, GCs activate the glucocorticoid receptor, which translocates to the nucleus and directly acts on the FAR591 gene promoter to induce FAR591 gene overexpression. Subsequently, FAR591 binds to the Fos gene promoter (-245∼-51) to form a stable RNA:DNA triplet structure and then recruits TATA-box binding protein associated factor 15 and RNA polymerase II to promote Fos expression through transcriptional activation. Fos activates the mitochondrial apoptotic pathway by regulating the expression of Bcl-2 interacting mediator of cell death (Bim) and P53 upregulated modulator of apoptosis (Puma) to mediate GC-induced apoptosis of BMECs, which leads to femoral head microcirculation dysfunction and femoral head necrosis. In conclusion, these results confirm the mechanistic link between lncRNAs and the pathogenesis of SONFH, which helps reveal the pathogenesis of SONFH and provides a new target for the early prevention and treatment of SONFH.

5.
Commun Biol ; 6(1): 365, 2023 04 03.
Article in English | MEDLINE | ID: mdl-37012358

ABSTRACT

The osteonecrotic area of steroid-induced avascular necrosis of the femoral head (SANFH) is a hypoxic microenvironment that leads to apoptosis of transplanted bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism remains unclear. Here, we explore the mechanism of hypoxic-induced apoptosis of BMSCs, and use the mechanism to improve the transplantation efficacy of BMSCs. Our results show that the long non-coding RNA AABR07053481 (LncAABR07053481) is downregulated in BMSCs and closely related to the degree of hypoxia. Overexpression of LncAABR07053481 could increase the survival rate of BMSCs. Further exploration of the downstream target gene indicates that LncAABR07053481 acts as a molecular "sponge" of miR-664-2-5p to relieve the silencing effect of miR-664-2-5p on the target gene Notch1. Importantly, the survival rate of BMSCs overexpressing LncAABR07053481 is significantly improved after transplantation, and the repair effect of BMSCs in the osteonecrotic area is also improved. This study reveal the mechanism by which LncAABR07053481 inhibits hypoxia-induced apoptosis of BMSCs by regulating the miR-664-2-5p/Notch1 pathway and its therapeutic effect on SANFH.


Subject(s)
Femur Head Necrosis , Mesenchymal Stem Cells , MicroRNAs , Humans , Femur Head Necrosis/chemically induced , Femur Head Necrosis/genetics , Femur Head Necrosis/therapy , Mesenchymal Stem Cells/metabolism , Apoptosis/genetics , Hypoxia/metabolism , Steroids/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism
6.
Exp Mol Med ; 54(11): 1991-2006, 2022 11.
Article in English | MEDLINE | ID: mdl-36380019

ABSTRACT

Bone marrow mesenchymal stem cells (BMSCs) have been used in the treatment of early steroid-induced osteonecrosis of the femoral head (SONFH). However, the hypoxic microenvironment in the osteonecrotic area leads to hypoxia-induced apoptosis of transplanted BMSCs, which limits their efficacy. Therefore, approaches that inhibit hypoxia-induced apoptosis of BMSCs are promising for augmenting the efficacy of BMSC transplantation. Our present study found that under hypoxia, the expression of the long noncoding RNA (Lnc) transmembrane protein 235 (Tmem235) was downregulated, the expression of Bcl-2-associated X protein was upregulated, the expression of B-cell lymphoma-2 protein was downregulated, and the apoptotic rate of BMSCs was over 70%. However, overexpression of Lnc Tmem235 reversed hypoxia-induced apoptosis of BMSCs and promoted their survival. These results demonstrated that Lnc Tmem235 effectively inhibited hypoxia-induced apoptosis of BMSCs. Mechanistically, we found that Lnc Tmem235 exhibited competitive binding to miR-34a-3p compared with BIRC5 mRNA, which is an inhibitor of apoptosis; this competitive binding relieved the silencing effect of miR-34a-3p on BIRC5 mRNA to ultimately inhibit hypoxia-induced apoptosis of BMSCs by promoting the expression of BIRC5. Furthermore, we cocultured BMSCs overexpressing Lnc Tmem235 with xenogeneic antigen-extracted cancellous bone to construct tissue-engineered bone to repair a model of early SONFH in vivo. The results showed that overexpression of Lnc Tmem235 effectively reduced apoptosis of BMSCs in the hypoxic microenvironment of osteonecrosis and improved the effect of BMSC transplantation. Taken together, our findings show that Lnc Tmem235 inhibited hypoxia-induced apoptosis of BMSCs by regulating the miR-34a-3p/BIRC5 axis, thus improving the transplantation efficacy of BMSCs for treating early SONFH.


Subject(s)
Mesenchymal Stem Cells , MicroRNAs , Osteonecrosis , RNA, Long Noncoding , Apoptosis , Femur Head , Hypoxia/metabolism , Mesenchymal Stem Cells/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Osteonecrosis/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/metabolism , Steroids/adverse effects , Animals
7.
Apoptosis ; 27(9-10): 762-777, 2022 10.
Article in English | MEDLINE | ID: mdl-35779185

ABSTRACT

Bone marrow mesenchymal stem cells (BMSCs) have strong regenerative potential and show good application prospects for treating clinical diseases. However, in the process of BMSC transplantation for treating ischemic and hypoxic diseases, BMSCs have high rates of apoptosis in the hypoxic microenvironment of transplantation, which significantly affects the transplantation efficacy. Our previous studies have confirmed the key role of long non-coding RNA Tmem235 (LncRNA Tmem235) in the process of hypoxia-induced BMSC apoptosis and its downstream regulatory mechanism, but the upstream mechanism by which hypoxia regulates LncRNA Tmem235 expression to induce BMSC apoptosis is still unclear. Under hypoxic conditions, we found that the level of LncRNA Tmem235 promoter histone H3 lysine 27 trimethylation modification (H3K27me3) was significantly increased by CHIP-qPCR. Moreover, H3K27me3 cooperated with LncRNA Tmem235 promoter DNA methylation to inhibit the expression of LncRNA Tmem235 and promote apoptosis of BMSCs. To study the mechanism of hypoxia-induced modification of LncRNA Tmem235 promoter H3K27me3 in the hypoxia model of BMSCs, we detected the expression of H3K27 methylase and histone demethylase and found that only histone methylase enhancer of zeste homolog 2 (EZH2) expression was significantly upregulated. Knockdown of EZH2 significantly decreased the level of H3K27me3 modification in the LncRNA Tmem235 promoter. The EZH2 promoter region contains a hypoxia-responsive element (HRE) that interacts with hypoxia-inducible factor-1alpha (HIF-1α), which is overexpressed under hypoxic conditions, thereby promoting its overexpression. In summary, hypoxia promotes the modification of the LncRNA Tmem235 promoter H3K27me3 through the HIF-1α/EZH2 signaling axis, inhibits the expression of LncRNA Tmem235, and leads to hypoxic apoptosis of BMSCs. Our findings improve the regulatory mechanism of LncRNA Tmem235 during hypoxic apoptosis of BMSCs and provide a more complete theoretical pathway for targeting LncRNA to inhibit hypoxic apoptosis of BMSCs.


Subject(s)
Mesenchymal Stem Cells , RNA, Long Noncoding , Apoptosis/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Histone Demethylases/genetics , Histone Demethylases/metabolism , Histones/genetics , Histones/metabolism , Humans , Hypoxia/genetics , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lysine/genetics , Lysine/metabolism , Mesenchymal Stem Cells/metabolism , Promoter Regions, Genetic , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism
8.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 36(3): 362-369, 2022 Mar 15.
Article in Chinese | MEDLINE | ID: mdl-35293179

ABSTRACT

Objective: To investigate the effect of LOC103693069 on hypoxic apoptosis of bone marrow mesenchymal stem cells (BMSCs). Methods: BMSCs from 1-week-old Sprague Dawley rat bone marrow were isolated, cultured, and passaged by the whole bone marrow adherent culture method. After identification of adipogenic, chondrogenic, and osteogenic differentiation, the 3rd generation cells were treated with hypoxia under 5%O 2, 1%O 2, and anaerobic conditions. After 48 hours, the cell viability, apoptosis, and apoptosis-related proteins [hypoxia inducible factor 1α (HIF-1α), Caspase-3, B cell lymphoma/leukemia 2 (Bcl-2)] expressions were detected, and normal BMSCs were used as controls. Based on the research results, the concentration group with the most obvious apoptosis was selected and used for subsequent experiments. After 48 hours of hypoxia treatment, BMSCs were taken and analyzed by gene chip and real-time fluorescence quantitative PCR (qRT-PCR) to screen the most significantly down-regulated gene and construct their high-expression, low-expression, and negative control lentiviruses; BMSCs were transfected with the different lentiviruses, respectively. After qRT-PCR detection confirmed that the transfection was successful, the BMSCs were treated with hypoxia for 48 hours to observe the cell viability and the expressions of apoptosis-related proteins. Results: After cell viability, apoptosis, and apoptosis-related proteins were detected, cell apoptosis was the most significant under anaerobic conditions after 48 hours. The above indicators were significantly different from other groups ( P<0.05), and this group was used for treatment conditions for subsequent experiments. Gene chip analysis showed that after 48 hours of hypoxia treatment, AC125847.1, LOC102547753, AABR07017208.2, and LOC103693069 were significantly down-regulated in BMSCs, and the expressions of LOC103693069 was the most significant down-regulation detected by qRT-PCR ( P<0.05). It was selected to construct lentivirus and transfect BMSCs. Afterwards, qRT-PCR detection showed the successful transfection into the cells. After hypoxia treatment, the apoptosis rate and the expressions of apoptosis-related proteins of BMSCs overexpressed by the gene were significantly reduced ( P<0.05). Conclusion: LOC103693069 can relieve the hypoxic apoptosis of BMSCs.


Subject(s)
Mesenchymal Stem Cells , Osteogenesis , Animals , Apoptosis , Bone Marrow Cells , Hypoxia , Rats , Rats, Sprague-Dawley
9.
Biosci Rep ; 42(1)2022 01 28.
Article in English | MEDLINE | ID: mdl-34981121

ABSTRACT

Oxidative stress damage is a common problem in bone marrow mesenchymal stem cell (BMSC) transplantation. Under stress conditions, the mitochondrial function of BMSCs is disrupted, which accelerates senescence and apoptosis of BMSCs, ultimately leading to poor efficacy. Therefore, improving mitochondrial function and enhancing the antioxidative stress capacity of BMSCs may be an effective way of improving the survival rate and curative effect of BMSCs. In the present study, we have confirmed that overexpression of nicotinamide mononucleotide adenylyl transferase 3 (NMNAT3) improves mitochondrial function and resistance to stress-induced apoptosis in BMSCs. We further revealed the mechanism of NMNAT3-mediated resistance to stress-induced apoptosis in BMSCs. We increased the level of nicotinamide adenine dinucleotide (NAD+) by overexpressing NMNAT3 in BMSCs and found that it could significantly increase the activity of silent mating type information regulation 2 homolog 3 (Sirt3) and significantly decrease the acetylation levels of Sirt3-dependent deacetylation-related proteins isocitrate dehydrogenase 2 (Idh2) and Forkhead-box protein O3a (FOXO3a). These findings show that NMNAT3 may increase the activity of Sirt3 by increasing NAD+ levels. Our results confirm that the NMNAT3-NAD+-Sirt3 axis is a potential mechanism for improving mitochondrial function and enhancing antioxidative stress capacity of BMSCs. In the present study, we take advantage of the role of NMNAT3 in inhibiting stress-induced apoptosis of BMSCs and provide new methods and ideas for breaking through the bottleneck of transplantation efficacy of BMSCs in the clinic.


Subject(s)
Mesenchymal Stem Cells , Nicotinamide-Nucleotide Adenylyltransferase , Sirtuin 3 , Apoptosis/genetics , Bone Marrow Cells/metabolism , Mesenchymal Stem Cells/metabolism , Mitochondria/genetics , Mitochondria/metabolism , NAD/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Oxidative Stress/genetics , Sirtuin 3/genetics , Sirtuin 3/metabolism
10.
Cell Death Dis ; 12(10): 940, 2021 10 13.
Article in English | MEDLINE | ID: mdl-34645791

ABSTRACT

Novel therapies for the treatment of early steroid-induced osteonecrosis of the femoral head (SONFH) are urgently needed in orthopedics. Transplantation of bone marrow mesenchymal stem cells (BMSCs) provides new strategies for treating this condition at the early stage. However, stress-induced apoptosis of BMSCs transplanted into the femoral head necrotic area limits the efficacy of BMSC transplantation. Inhibiting BMSC apoptosis is key to improving the efficacy of this procedure. In our previous studies, we confirmed that Parkinson disease protein 7 (PARK7) is active in antioxidant defense and can clear reactive oxygen species (ROS), protect the mitochondria, and impart resistance to stress-induced apoptosis in BMSCs. In this study, we investigated the mechanism driving this PARK7-mediated resistance to apoptosis in BMSCs. Our results indicate that PARK7 promoted the disintegration of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like echinacoside-associated protein 1 (Keap1) complex. The free Nrf2 then entered the nucleus and activated the genetic expression of manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), and other antioxidant enzymes that clear excessive ROS, thereby protecting BMSCs from stress-induced apoptosis. To further explore whether PARK7-mediated resistance to stress-induced apoptosis could improve the efficacy of BMSC transplantation in early-stage SONFH, we transplanted BMSCs-overexpressing PARK7 into rats with early-stage SONFH. We then evaluated the survival of transplanted BMSCs and bone regeneration in the femoral head necrotic area of these rats. The results indicated that PARK7 promoted the survival of BMSCs in the osteonecrotic area and improved the transplantation efficacy of BMSCs on early-stage SONFH. This study provides new ideas and methods for resisting the stress-induced apoptosis of BMSCs and improving the transplantation effect of BMSCs on early-stage SONFH.


Subject(s)
Apoptosis , Femur Head Necrosis/chemically induced , Femur Head Necrosis/metabolism , Mesenchymal Stem Cells/pathology , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Protein Deglycase DJ-1/metabolism , Steroids/adverse effects , Animals , Cell Survival , Cellular Microenvironment , Femur Head Necrosis/diagnostic imaging , Kelch-Like ECH-Associated Protein 1/metabolism , Lentivirus/metabolism , Male , Mesenchymal Stem Cells/metabolism , Methylprednisolone/adverse effects , Protein Binding , Rats, Sprague-Dawley , Signal Transduction , X-Ray Microtomography
11.
Open Life Sci ; 16(1): 969-980, 2021.
Article in English | MEDLINE | ID: mdl-34595348

ABSTRACT

The main cause of steroid-induced necrosis of femoral head (SNFH) is excessive glucocorticoid (GC) intake. The aim of this article was to investigate the role of lncRNA NEAT1 as a molecular sponge to adsorb miR-23b-3p and regulate CYP1A2 in SNFH. Fluorescence in situ hybridization was used to localize lncRNA NEAT1. Human bone marrow mesenchymal stem cells (hBMSCs) were collected from patients with SNFH. The expression of lncRNA NEAT1, miR-23b-3p and CYP1A2 in hBMSCs were intervened. Compared to the control group, the lncRNA NEAT1 and CYP1A2 expression in the SNFH group was increased, while miR-23b-3p expression was decreased. GCs could inhibit the osteogenic differentiation of hBMSCs and upregulate the expression of lncRNA NEAT1. Knockdown of lncRNA NEAT1 could promote the proliferation and osteogenic differentiation of hBMSCs in the SNFH group. Overexpression of miR-23b-3p could partially counteract the effect of lncRNA NEAT1 on hBMSCs. CYP1A2 was confirmed to be a target of miR-23b-3p. Overexpression of CYP1A2 could partially rescue the effect of miR-23b-3p overexpression on hBMSCs. In conclusion, lncRNA NEAT1 as a ceRNA can adsorb miR-23b-3p and promote the expression of CYP1A2, which then inhibits the osteogenic differentiation of hBMSCs and promotes the progress of SNFH.

12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(2): 105-112, 2021 Feb.
Article in Chinese | MEDLINE | ID: mdl-33504415

ABSTRACT

Objective To investigate the effect of oxidative stress preconditioning on the oxidative stress-induced damage of bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were isolated and cultured by density gradient centrifugation combined with adherence method. The cells were divided into three groups: control group (control medium), oxidative damage group (treated with 1000 µmol/L H2O2 for 24 hours), and preconditioning group (preincubated with 50 µmol/L H2O2 for 8 hours before treatment with 1000 µmol/L H2O2 for 24 hours). DCFH-DA staining was used to analyze the level of reactive oxygen species (ROS). Mitochondrial membrane potential was measured by JC-1 staining. DNA damage was detected by TUNEL. Malondialdehyde (MDA) content was detected by thiobarbituric acid (TBA) method, and superoxide dismutase (SOD) activity was detected by water soluble tetrazolium-1 (WST-1) assay. CCK-8 assay was used to detect cell viability and flow cytometry to detect cell apoptosis. Results Compared with the oxidative damage group, the preconditioning group had reduced ROS level, reduced DNA damage, higher mitochondrial membrane potential, significantly decreased MDA content, increased SOD activity, increased cell viability, and significantly decreased apoptosis. Conclusion Oxidative stress preconditioning can enhance the anti-oxidative stress ability of BMSCs and promote its survival under oxidative stress.


Subject(s)
Mesenchymal Stem Cells , Apoptosis , Bone Marrow Cells , Hydrogen Peroxide/toxicity , Malondialdehyde , Oxidative Stress , Reactive Oxygen Species
13.
J Cell Biochem ; 122(2): 222-234, 2021 02.
Article in English | MEDLINE | ID: mdl-32918333

ABSTRACT

Oxidative stresss in the microenvironment surrounding lesions induces apoptosis of transplanted bone-marrow-derived mesenchymal stem cells (BMSCs). Hence, there is an urgent need for improving antioxidative-stress processes of transplanted BMSCs to further promote their survival. The present study reports the role and mechanism of Parkinson's disease protein 7 (PARK7) in enhancing antioxidative activity in BMSCs. We used a PARK7 lentivirus to transfect BMSCs to up- or downregulate PARK7, and then used H2 O2 to simulate oxidative stress in BMSCs in vitro. Overexpression of PARK7 effectively reduced reactive oxygen species and malondialdehyde, protected mitochondrial membrane potential, and resisted oxidative-stress-induced apoptosis of BMSCs, but the expression of PARK7 was downregulated, these results were reversed. At the same time, we also found that overexpression of PARK7 increased extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation and nuclear translocation, as well as upregulated Elk1 phosphorylation and superoxide dismutase (SOD) expression. In contrast, when U0126 was used to block the ERK1/2 pathway, ERK1/2 and Elk1 phosphorylation levels were downregulated, ERK1/2 nuclear translocation and SOD content were significantly reduced, and PARK7-overexperssion-induced antioxidative activity was completely blocked. Collectively, our results suggest that PARK7 overexpression increased antioxidative-stress processes and survival of BMSCs subjected to H2 O2 via activating the ERK1/2 signaling pathway. Our findings may guide the development of a PARK7-specific strategy for improving the transplantation efficacy of BMSCs.


Subject(s)
Mesenchymal Stem Cells/metabolism , Parkinson Disease/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Butadienes , Humans , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/physiology , Malondialdehyde/metabolism , Membrane Potential, Mitochondrial/genetics , Membrane Potential, Mitochondrial/physiology , Nitriles , Oxidative Stress/genetics , Oxidative Stress/physiology , Parkinson Disease/genetics , Protein Deglycase DJ-1/genetics , Protein Deglycase DJ-1/metabolism , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism
14.
Acta Orthop Belg ; 87(4): 745-750, 2021 Dec.
Article in English | MEDLINE | ID: mdl-35172442

ABSTRACT

Open and comminuted mid-distal fractures often result from high-energy trauma, and a concomitant poor blood supply often leads to skin necrosis, infection, and bone union. To circumvent such complications, we used limited-reduction and bilateral-external fixators to treat open and comminuted mid-distal tibial fractures with compromised soft tissue. A retrospective series of 34 patients who had open and comminuted mid-distal tibial fractures and treated by bilateral-external fixators with limited-internal fixation were analyzed. Patients were followed for 10-25 months (mean: 12 months) post-treatment and osseous union was achieved in each case. The average union time was 16.3 weeks. Based on the Johner- Wruhs criteria, the retrospective series consisted of 21 'excellent' cases, 8 'good' cases, 4 'fair' cases, and a 'poor' case. The total percentage of 'excellent' and 'good' cases of fracture recovery was 85.29%. Bilateral-external and limited-internal fixators pro- vided high bone union rate and excellent ankle-joint motion. Hence, it is an appropriate surgical approach for treating open and comminuted mid-distal tibial fractures with compromised soft tissue.


Subject(s)
Fractures, Comminuted , Fractures, Open , Tibial Fractures , Bone Plates , External Fixators , Fracture Fixation , Fracture Fixation, Internal , Fractures, Comminuted/diagnostic imaging , Fractures, Comminuted/surgery , Fractures, Open/diagnostic imaging , Fractures, Open/surgery , Humans , Retrospective Studies , Tibial Fractures/diagnostic imaging , Tibial Fractures/surgery , Treatment Outcome
15.
J Orthop Surg Res ; 15(1): 392, 2020 Sep 09.
Article in English | MEDLINE | ID: mdl-32907609

ABSTRACT

OBJECTIVE: To investigate the effects of low-concentration hydrogen peroxide pretreatment on the anti-oxidative stress of the bone marrow mesenchymal stem cells (BMSCs). METHODS: Rabbit BMSCs were isolated and cultured by density gradient centrifugation combined with the adherence method. Then, the third generation of well-grown BMSCs was continuously treated with 50-µM hydrogen peroxide (H2O2) for 8 h as the optimal pretreatment concentration and the BMSCs were continuously applied for 24 h with 500 µM H2O2, and the optimal damage concentration was determined as the oxidative stress cell model. The experiment was divided into three groups: control group, high-concentration H2O2 injury group (500 µM), and low-concentration H2O2 pretreatment group (50 µM + 500 µM). In each group, the DCFH-DA fluorescence probe was used to detect the reactive oxygen species (ROS). ELISA was used to detect the activity of superoxide dismutase (SOD) and catalase (CAT), and the TBA method was used to detect malondialdehyde (MDA). The mitochondrial membrane potential was detected by JC-1. The cell viability was detected by CCK-8 method, while flow cytometry and TUNEL/DAPI double staining were performed to detect cell apoptosis. Hence, the effect of H2O2 pretreatment on the anti-oxidative stress of BMSCs was investigated. One-way analysis of variance was performed using SPSS 19.0 statistical software, and P < 0.05 was considered statistically significant. RESULTS: A large number of typical BMSCs were obtained by density gradient centrifugation and adherent culture. The oxidative stress cell model was successfully established by 500-µM H2O2. Compared with the high-concentration H2O2 injury group, the low-concentration H2O2 pretreatment reduced the production of ROS [(62.33 ± 5.05), P < 0.05], SOD and CAT activities significantly increased (P < 0.05), and MDA levels significantly decreased (P < 0.05). The mitochondrial membrane potential fluorescence changes, the ratio of red/green fluorescence intensity of the high-concentration H2O2 injury group was less, and the ratio of the low-concentration H2O2 pretreatment group was significantly higher than that. The ratio of red/green increased by about 1.8 times (P < 0.05). The cell viability and survival rate of BMSCs were significantly increased in low-concentration H2O2 pretreatment group (P < 0.05), and the cell apoptosis rate was significantly decreased (P < 0.05). CONCLUSION: Pretreatment with low-concentration H2O2 can enhance the anti-oxidative stress ability and reduce their apoptosis of BMSCs under oxidative stress.


Subject(s)
Hydrogen Peroxide/pharmacology , Mesenchymal Stem Cells/metabolism , Oxidative Stress/drug effects , Animals , Apoptosis/drug effects , Catalase/metabolism , Cell Culture Techniques , Cell Survival/drug effects , Cells, Cultured , Centrifugation, Density Gradient , Dose-Response Relationship, Drug , Hydrogen Peroxide/administration & dosage , Malondialdehyde/metabolism , Mesenchymal Stem Cells/physiology , Rabbits , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism
16.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(5): 621-629, 2020 May 15.
Article in Chinese | MEDLINE | ID: mdl-32410431

ABSTRACT

OBJECTIVE: To investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD +) levels. METHODS: The bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H 2O 2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H 2O 2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H 2O 2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD + and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-ß-galactosidase (SA-ß-gal) staining and TUNEL staining] were detected after 24 hours of treatment. RESULTS: The rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased ( P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H 2O 2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD + and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-ß-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant ( P<0.05). CONCLUSION: NMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD + levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.


Subject(s)
Mesenchymal Stem Cells , Nicotinamide-Nucleotide Adenylyltransferase , Animals , Bone Marrow Cells , Cells, Cultured , Mitochondria , NAD , Nicotinamide Mononucleotide , Nucleotidyltransferases , Oxidative Stress , Rabbits
17.
Cell Death Dis ; 11(1): 42, 2020 01 20.
Article in English | MEDLINE | ID: mdl-31959744

ABSTRACT

Survival and stemness of bone marrow mesenchymal stem cells (BMSCs) in osteonecrotic areas are especially important in the treatment of early steroid-induced osteonecrosis of the femoral head (ONFH). We had previously used BMSCs to repair early steroid-induced ONFH, but the transplanted BMSCs underwent a great deal of stress-induced apoptosis and aging in the oxidative-stress (OS) microenvironment of the femoral-head necrotic area, which limited their efficacy. Our subsequent studies have shown that under OS, massive accumulation of damaged mitochondria in cells is an important factor leading to stress-induced apoptosis and senescence of BMSCs. The main reason for this accumulation is that OS leads to upregulation of protein 53 (P53), which inhibits mitochondrial translocation of Parkin and activation of Parkin's E3 ubiquitin ligase, which decreases the level of mitophagy and leads to failure of cells to effectively remove damaged mitochondria. However, P53 downregulation can effectively reverse this process. Therefore, we upregulated Parkin and downregulated P53 in BMSCs. We found that this significantly enhanced mitophagy in BMSCs, decreased the accumulation of damaged mitochondria in cells, effectively resisted stress-induced BMSCs apoptosis and senescence, and improved the effect of BMSCs transplantation on early steroid-induced ONFH.


Subject(s)
Femur Head Necrosis/metabolism , Femur Head Necrosis/pathology , Mesenchymal Stem Cells/metabolism , Mitophagy , Steroids/adverse effects , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolism , Wound Healing , Animals , Apoptosis , Cellular Senescence/genetics , Disease Models, Animal , Femur Head Necrosis/chemically induced , Femur Head Necrosis/genetics , Gene Expression Regulation , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/ultrastructure , Mitochondria/pathology , Mitochondria/ultrastructure , Mitophagy/genetics , Models, Biological , Osteogenesis , Oxidative Stress/genetics , Rabbits , Tumor Suppressor Protein p53/genetics , Ubiquitin-Protein Ligases/genetics
18.
J Cell Biochem ; 120(12): 19902-19914, 2019 12.
Article in English | MEDLINE | ID: mdl-31347718

ABSTRACT

Clinically, bone marrow mesenchymal stem cells (BMSCs) have been used in treatment of many diseases, but the local oxidative stress (OS) of lesion severely limits the survival of BMSCs, which reduces the efficacy of BMSCs transplantation. Therefore, enhancing the anti-OS stress ability of BMSCs is a key breakthrough point. Preconditioning is a common protective mechanism for cells or body. Here, the aim of this study was to investigate the effects of OS preconditioning on the anti-OS ability of BMSCs and its mechanism. Fortunately, OS preconditioning can increase the expression of superoxide dismutase, catalase, NQO1, and heme oxygenase 1 through the nuclear factor erythroid 2-related factor 2 pathway, thereby decreased the intracellular reactive oxygen species (ROS) levels, relieved the damage of ROS to mitochondria, DNA and cell membrane, enhanced the anti-OS ability of BMSCs, and promoted the survival of BMSCs under OS.


Subject(s)
Mesenchymal Stem Cells/physiology , NF-E2-Related Factor 2/metabolism , Oxidative Stress/physiology , Animals , Apoptosis/drug effects , Bone Marrow Cells/cytology , Catalase/metabolism , Cells, Cultured , DNA Damage , Dose-Response Relationship, Drug , Hydrogen Peroxide/administration & dosage , Hydrogen Peroxide/pharmacology , Male , Malondialdehyde/metabolism , Membrane Potential, Mitochondrial/drug effects , Mesenchymal Stem Cells/drug effects , Oxidative Stress/drug effects , Rabbits , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism
19.
Exp Ther Med ; 17(4): 3021-3028, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30936973

ABSTRACT

The present study investigated the key genes, which cause switch from adipogenic to osteogenic differentiation of human mesenchymal stem cells (hMSCs). The transcriptomic profile of hMSCs samples were collected from Array Express database. Differential expression network was constructed by calculating the Pearson's correlation coefficient and ranked according to their topological features. The top 5% genes with degree ≥2 were selected as ego genes. Following the KEGG pathway enrichment analysis and the relevant miRNAs prediction, the miRNA-mRNA-pathway networks were constructed by combining the miRNA-mRNA pairs and mRNA-pathway pairs together. In total, we obtained 84, 119, 94 and 97 ego-genes in B, BI, BT and BTI groups, and DLGAP5, DLGAP5, NUSAP1 and NDC80 were the ego-genes with the highest z-score of each group, respectively. Beginning from each ego-gene, we identified 2 significant ego-modules with gene size ≥4 in group BI, and the ego-genes were PBK and NCOA3, respectively. Through KEGG pathway analysis, we found that most of the pathways enriched by ego-genes were associated with gene replication and repair, and cell proliferation. According to the miRNA prediction results, we found that some of the predicted miRNAs have been validated to be the regulatory miRNAs of these corresponding mRNAs. Finally we constructed a miRNA-mRNA-pathway network by integrating the miRNA-mRNA and mRNA-pathway pairs together. The constructed network gives us a more comprehensive understanding of the mechanism of osteogenic differentiation of hMSCs.

20.
J Cell Biochem ; 120(2): 1427-1434, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30335890

ABSTRACT

The aim of this study is to observe the changes in osteoprotegerin (OPG) and receptor activator for the nuclear factor-κB ligand (RANKL) in a rabbit model, and to explore the therapeutic effect of tissue engineering bone on femoral head necrosis. A total of 60 rabbits were randomly divided into 5 groups. The necrosis model of femoral head defects was created by dexamethasone combined with a horse serum injection. The model of femoral head necrosis was reconstructed by tissue engineering bone. The protein expressions of OPG and RANKL were detected by Western blot analysis. The expression of OPG and the RANKL protein in group E was higher than that in the other 4 groups (P < .05); there was no significant difference between groups C and D ( P > .05). The expression of OPG protein in the rabbit femoral head necrosis group was improved by xenogeneic antigens of cancellous bone/lentiviral-basic fibroblast growth factor/mesenchymal stem cells, which were expected to be used as an effective tissue engineering material to repair the necrosis of the femoral head.

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