Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.     

Docosahexaenoic acid protects human retinal pigment epithelial cells against oxidative stress-induced apoptosis

AIM: To observe the effect of docosahexaenoic acid(DHA) on H₂O₂-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism. METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H₂O₂ was used to mimic the oxidative stress condition. The cells were treated with 30~100μmol/L DHA for 4~24 h. The expression of heme oxygenase-1(HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The enzymic activity of HO-1 was measured by colorimetry. Production of reactive oxygen species(ROS) was determined by fluorescent probe. Activation of NF-E2-related factor 2(Nrf2) was examined by immunofluorescence method. Apoptosis of ARPE-19 cells was analyzed by flow cytometry. RESULTS: The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment. Meanwhile, nuclear translocation of Nrf2 was also observed. Apoptosis appeared and ROS was produced upon H₂O₂ incubation. In contrast, DHA at 100μmol/L significantly abrogated H₂O₂-induced apoptosis and ROS production. Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H₂O₂-induced apoptosis and ROS production. CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase-1 expression after Nrf2 activation.     

Acetyl-L-carnitine attenuates H2O2-induced oxidative damage of PC12 cells

AIM: To investigate the effect of acetyl-L-carnitine (ALC) on H₂O₂-induced oxidative damage in PC12 cells and its possible mechanism. METHODS: A moderate oxidative damage PC12 cell model was induced by exposure of the PC12 cells to H₂O₂. ALC at different concentrations (100, 200 and 400 μmol/L) was applied to the PC12 cells cultured in vitro, and CCK8 assay was used to detect the cell viability. The cells were divided into control group, H₂O₂ group, and low-ALC, medium-ALC and high-ALC groups. The apoptosis of the cells was analyzed by flow cytometry. The protein levels of Nrf2 and cleaved caspase-3 were determined by Western blot. The nuclear translocation of Nrf2 was observed by immunofluorescence staining. RESULTS: ALC at different concentrations (100, 200 and 400 μmol/L) significantly inhibited H₂O₂-induced PC12 cell apoptosis, and the medium concentration group had the best effect. Compared with H₂O₂ group, low, medium and high concentrations of ALC significantly increased the viability of the PC12 cells induced by H₂O₂, inhibit cell apoptosis (P<0.05), significantly down-regulated the protein level of cleaved caspase-3 (P<0.05), up-regulated the protein level of Nrf2 (P<0.05), and promoted the translocation of Nrf2 from the cytoplasm to the nucleus. CONCLUSION: Acetyl-L-carnitine attenuates H₂O₂-induced oxidative damage of PC12 cells, inhibits the apoptosis and increases the viability, which is related to the activation of Nrf2 signaling pathway.     

Effects of 6-gingerol on apoptosis of rat nucleus pulposus cells

AIM: To study the effect of 6-gingerol on the apoptosis of rat nucleus pulposus cells and its possible mechanism. METHODS: Rat nucleus pulposus cells were isolated and cultured. The effects of 6-gingerol and hydrogen peroxide (H₂O₂) at different concentrations on the viability of nucleus pulposus cells were measured by CCK-8 assay. After 6-gingerol treatment, the protein level of p-Akt was determined by Western blot. The cells were divided into 4 groups:control group, H₂O₂ group, 6-gingerol group (6-gingerol + H₂O₂) and LY294002 group (6-gingerol + H₂O₂ + LY294002). The apoptotic rate and the levels of reactive oxygen species (ROS) were analyzed by flow cytometry. TUNEL fluorescence staining was used to observe the number of apoptotic cells. The morphological changes of mitochondria were observed under transmission electron microscope, and Western blot was used to determine the protein levels of caspase-3, Bcl-2, Bax, p-Akt, Akt and p53. The mRNA expression of aggrecan and type II collagen was measured by RT-qPCR. RESULTS: The results of CCK-8 assay showed that the optimal concentration of 6-gingerol for promoting the viability of rat nucleus pulposus cells was 24 mg/L, and the exposure condition of H₂O₂ at 80 μmol/L for 6 h was appropriate for establi-shing the cell damage model. 6-Gingerol increased the protein level of p-Akt in a time-dependent manner. The apoptotic rate, ROS level and TUNEL positive cells in H₂O₂ group were significantly increased compared with control group. The mitochondrial edema was obvious in H₂O₂ group compared with control group. The protein levels of pro-apoptotic molecules caspase-3, Bax and p53 were significantly increased, while anti-apoptotic protein Bcl-2, and mRNA expression of aggrecan and type II collagen were significantly decreased compared with control group (P<0.05). 6-Gingerol exerted a protective effect against H₂O₂-induced apoptosis and promoted the expression of anti-apoptotic proteins. However, this effect was weakened after treatment with PI3K/Akt signaling pathway inhibitor LY294002. CONCLUSION: H₂O₂ induces damage and dysfunction of rat nucleus pulposus cells, and 6-gingerol may inhibit H₂O₂-induced apoptosis of nucleus pulposus cells by activation of PI3K/Akt signaling pathway.     

Role of miR-486-5p in apoptosis of human bone marrow mesenchymal stem cells induced by hydrogen peroxide

AIM: To investigate the role of microRNA-486-5p (miR-486-5p) in the apoptosis of human bone marrow mesenchymal stem cells (hMSCs) induced by hydrogen peroxide (H₂O₂). METHODS: The hMSCs were cultured in vitro and exposed to serum-free medium and H₂O₂ (10 mmol/L). The changes of miR-486-5p expression in oxidative stress-related apoptosis of hMSCs were measured by real-time PCR. The hMSCs were transfected with miR-486-5p mimic or inhibitor at concentration of 30 nmol/L by Lipofectamine RNAiMAX. The effect of miR-486-5p on H₂O₂-induced decrease in cell viability was evaluated by MTT assay. Hoechst 33342 staining and flow cytometry were applied to determine the role of miR-486-5p in the apoptosis of hMSCs. The protein expression was evaluated by Western blotting. Caspase-3 activity was determined using a caspase-3 activity kit. RESULTS: Compared with control group, the expression of miR-486-5p significantly decreased after treated with H₂O₂ (P<0.05). In addition, over-expression of miR-486-5p in the hMSCs reduced the cell viability, accelerated apoptosis, down-regulated Bcl-2/Bax ratio, caspase-3 enzyme precursor content and phosphorylation of Akt, and activated caspase-3 activity. Conversely, down-regulation of miR-486-5p significantly inhibited H₂O₂-induced cell apoptosis and the caspase-3 activity, increased cell viability and up-regulated Bcl-2/Bax ratio and phosphorylation level of Akt. CONCLUSION: Over-expression of miR-486-5p promotes H₂O₂-induced hMSCs apoptosis, and repression of miR-486-5p protects hMSCs from H₂O₂-induced cellular apoptosis, which may be mediated by regulating Akt signaling pathway.     

Effects of PDGFRα on melanocyte apoptosis induced by hydrogen peroxide

AIM: To investigate the effects of platelet-derived growth factor receptor α (PDGFRα) on melanocyte apoptosis induced by hydrogen peroxide (H₂O₂). METHODS: Melanocyte PIGI was used as the research object. After exposed to H₂O₂ at different concentrations, the cell viability was detected by MTT assay. The PIGI cells were transfec-ted with empty vector pCMV6 or PDGFRα over-expression vector pCMV6-PDGFRα. The transfection efficiency was determined by RT-qPCR and Western blot. The effect of H₂O₂ on the viability of the PIGI cells after over-expression of PDGFRα was measured by MTT assay. The cell apoptosis was analyzed by flow cytometry. The protein levels of p38, p-p38 and cleaved caspase-3 in the cells were detected by Western blot. DCDHF-DA was used to estemate the generation of reactive oxygen species (ROS) in the cells. RESULTS: The viability of PIGI cells decreased after exposed to H₂O₂ (P<0.05), and the half maximal inhibitory concentration of H₂O₂ was 0.7 mmol/L. Transfection with PDGFRα over-expression vector successfully induced high expression of PDGFRα at mRNA and protein levels in the PIGI cells, and increased the viability of the cells with H₂O₂ treatment (P<0.05). Over-expression of PDGFRα decreased the apoptotic rate of PIGI cells treated with H₂O₂ (P<0.05), and the level of ROS in the cells (P<0.05). The protein levels of cleaved caspase-3 and p-p38 were also decreased (P<0.05). CONCLUSION: PDGFRα inhibits the apoptosis of melanocytes induced by H₂O₂, partially reverses the growth inhibition of melanocytes by H₂O₂, and decreases the ROS level. The mechanism may be related to regulating the protein levels of p-p38 and cleaved caspase-3 in the cells.     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.     

Effect of senegenin on H2O2-induced damage in hippocampal neurons of SD rats

AIM: To observe the effect of senegenin (Sen) on hippocampal neuron injuries induced by H₂O₂.METHODS: Hippocampal neurons were isolated from neonatal SD rats. The primarily cultured neurons were divided into control group, H₂O₂ group, Sen group and Sen+H₂O₂ group. The cell viability, the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in the neurons were detected after treated with Sen. The morphological changes of nucleus of the neurons were observed by Hoechst 33258 staining. The mRNA expression of bcl-2 and bax was quantified by real-time PCR. The protein levels of Bcl-2 and bax were measured by Western blotting. The activity of caspase-3 was also assayed.RESULTS: Compared with H₂O₂ group, the levels of antioxidative enzyme were increased in Sen+H₂O₂ group (P<0.05). In addition, mRNA expression of bcl-2 increased and that of bax decreased (P<0.05) in Sen+H₂O₂ group. Moreover, Sen increased the protein level of Bcl-2, and reduced the protein level of Bax and the activity of caspase-3 in the neurons exposed to H₂O₂ (P<0.05).CONCLUSION: The protective effect of Sen on hippocampal neurons with H₂O₂ -induced injury may be involved in the mechanisms of     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

Limb ischemic preconditioning protects human umbilical vein endothelial cells from oxidative stress induced by H2O2

AIM: To investigate the effects of the sera from the rats after limb ischemic preconditioning (LIPC) on human umbilical vein endothelial cells (HUVECs) injured by hydrogen peroxide (H₂O₂). METHODS: The HUVECs were divided into 5 groups: the cells in control group were cultured without any intervention; the cells in model group (M) were damaged by 1 mmol/L H₂O₂ for 2 h; the cells in early preconditioning serum (EPS) group, delayed preconditioning serum (DPS) group or sham limb ischemic preconditioning serum (SPS) group were treated with the corresponding serum at 5% for 12 h, respectively, and then treaed with H₂O₂ for 2 h. The viability of the HUVECs was analyzed by flow cytometry. The lactate dehydrogenase (LDH) in the culture media was detected. The cell adhesion molecules in the HUVECs were detected by real-time PCR. The mRNA and protein expression of heme oxygenase-1 (HO-1) was also determined. RESULTS: The viability of HUVECs incubated with 1 mmol/L H₂O₂ for 2 h significantly decreased compared with the control cells, which was accompanied with the augmentations of LDH in the medium and the cell adhesion molecules in cells, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Preincubation with EPS and DPS derived from the rats subjected LIPC attenuated these injuries. Furthermore, pretreatment with EPS and DPS increased the expression of HO-1 at mRNA and protein levels. CONCLUSION: LIPC protects the HUVECs from H₂O₂-induced injury.     

Protective effect of senegenin on retinal ganglion cells in oxidative stress induced injury

AIM: To evaluate the effect of senegenin (Sen) on H₂O₂-treated retinal ganglion cells (RGCs) and to explore its underlying mechanisms. METHODS: RGCs were retrograde labeled by injection of fluorogold into the superior colliculi of SD rats on the postnatal day 3. On the postnatal days 6 to 8, the retinas were dissociated with papain and cultured. Primary RGCs cultured in vitro were treated with H₂O₂ and/or various doses of Sen. The viability of RGCs was evaluated by counting the fluorescence-labeled neurons under microscope. The morphological changes of the nuclei in the retinal neurons were observed by Hoechst 33258 staining. Western blotting was applied to determine the expression of cleaved caspase-3, cytochrome C and Bcl-2 in cultured retinal neurons. RESULTS: Compared with the control cells, Sen at doses of 10, 20 or 40 μmol/L had no toxicity to RGCs (P>0.05). However, Sen at doses of 80 and 160 μmol/L had significant toxicity to RGCs (P<0.01). Compared with H₂O₂-injured group, Sen at doses of 10, 20 and 40 μmol/L effectively protected against H₂O₂-induced injury in RGCs (P<0.05) with the best efficiency at 40 μmol/L. Hoechst 33258 staining showed that the neuronal apoptosis caused by H₂O₂ was reduced by Sen. The results of Western blotting showed an up-regulation of Bcl-2, and decreased cytochrome C and cleaved caspase-3 levels by Sen in H₂O₂-treated retinal neurons. CONCLUSION: Sen is able to protect RGCs from H₂O₂-induced injury by enhancing Bcl-2 expression and inhibiting cell apoptosis.     

Effects of α-MSH on biological activities of LPS

AIM: The present study was undertaken to explore the effects of α-MSH on partial biological activities of LPS. METHODS:Colorimetric method was used for the measurement of hydrogen peroxide(H₂O₂) production in mouse peritoneal macrophages, the apoptosis of polymorphonuclear leukocytes(PMNs) and the binding of LPS to monocytes were studied with flow cytometry. RESULTS: It was found that LPS strongly stimulated macrophages to release H₂O₂. When macrophages were cultured with α-MSH in the presence of LPS, the H₂O₂ release was markedly suppressed (P<0.01). Neither LPS nor α-MSH alone was capable of affecting the apoptosis of PMNs (P>0.05). In the presence of LPS, however, α-MSH significantly promoted the apoptosis of PMNs (P<0.01). α-MSH significantly inhibited the binding of LPS to monocytes as the binding rate of FITC-LPS and the mean surface fluorescence intensity of monocytes ( P<0.05 and P<0.01, respectively). CONCLUSION: In the presence of LPS, α-MSH not only effectively suppressed the release of H₂O₂ from macrophages , promoted the apoptosis of PMNs, but also interfered with the binding of LPS to monocytes. α-MSH may play an important role in the immunomodulation of the body .     

MSC-conditioned medium activates Nrf2/ARE pathway to protect H9c2 cells against oxidative stress

AIM: To investigate the protective effect of mesenchymal stem cell (MSC)-conditioned medium (MSCCM) on myocardial cell line H9c2 and its mechanism. METHODS: Verification of MSC was performed by flow cytometry analysis, followed by MTT assay to determine the optimal incubation time of MSCCM with myocardial cells. The cells were divided into 4 groups: normal (N) group, model (M) group, M+MSCCM group and MSCCM group. The cells in M+MSCCM group and MSCCM group were pre-incubated with MSCCM for 24 h. The cells in M group and M+MSCCM group were treated with 300 μmol/L H₂O₂ for 4 h to imitate oxidative injury of myocardial cells. Mitochondrial membrane potential and apoptotic rate of injured myocardial cells were detected by flow cytometry. The ROS production was measured by fluorescence microscopy. The nuclear translocation of Nrf2 and expression of HO-1 was examined by Western blot. RESULTS: No difference of mitochondrial membrane potential, apoptotic rate or ROS production between MSCCM group and N group was observed (P>0.05). The mitochondrial membrane potential depolarization, apoptotic rate and ROS production in M+MSCCM group were significantly lower than those in M group (P<0.01). The nuclear translocation of Nrf2 and expression of HO-1 in the myocardial cells were increased with MSCCM incubation time prolonged. CONCLUSION: MSCCM protects the myocardial cells against oxidative injury induced by H₂O₂. The anti-oxidative mechanism would be associated with the activation of Nrf2/ARE pathway.     

Effects of rapamycin on hydrogen peroxide-induced vascular endothelial cell senescence

AIM:To investigate the effects of rapamycin (Rapa) on hydrogen peroxide (H₂O₂)-induced vascular endothelial cell senescence and to explore the underlying mechanisms. METHODS:The human umbilical vascular endothelial cells (HUVECs) were divided into 4 groups:control group, senescence group, Rapa+H₂O₂ group and 3-methyladenine (3-MA)+H₂O₂ group. MTT assay was performed to assess the cell viability. Senescence-associated β-ga-lactosidase (SA-β-Gal) staining was performed to measure the senescent cells in each group. The subcellular structures were observed under transmission electron microscope (TEM). The protein levels of phosphorylated Rb (p-Rb), Rb, p21, LC3-Ⅱ and beclin-1 were determined by Western blot. RESULTS:Compared with control group, the cell viability in H₂O₂ group was significantly decreased accompanied with higher rate of SA-β-Gal staining positive cells (P<0.05) and markedly damaged structure. Additionally, the protein levels of p-Rb and p21 in senescence group were increased markedly compared with control group (P<0.05). However, the cells pre-treated with Rapa prior to stimulation with H₂O₂ showed increased viability, decreased number of senescent cells and decreased protein levels of p-Rb and p21 as compared with the cells stimulated with H₂O₂ alone (P<0.05). Moreover, the TEM observation showed that the structure of the cells in Rapa+H₂O₂ group was roughly normal and the autophagosome was captured, and the expression levels of beclin-1 and LC3-Ⅱ were increased (P<0.05). Conversely, pre-treatment with autophagy inhibitor 3-MA resulted in opposite results. The cell viability was decreased significantly, more senescent cells were stained blue, higher protein levels of p-Rb and p21 were detected (P<0.05), poor subcellular structures were captured, and no beclin-1 and LC3-Ⅱ was detected. CONCLUSION:Rapa may retard the senescence of HUVECs induced by H₂O₂, and promoting autophagy may be the underlying mechanism.     

Salvianolic acid B reduces apoptosis of bone marrow stem cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolic acid B (Sal B) on apoptosis of rat bone mesenchymal stem cells(BMSCs) induced by hydrogen peroxide(H₂O₂). METHODS: BMSCs were incubated with Sal B at the concentration of 1, 10 or 100 μmol/L while treated with lethal concentration of H₂O₂ (500 μmol/L). The effect of Sal B at different concentrations on the viability of BMSCs was detected by MTT. Flow cytometry were used to determine the protective role of Sal B in apoptosis of BMSCs. The changes of chromatin distribution in BMSCs were observed by Hoechst 33342 staining. The expression of p-ERK1/2 was detected by Western blotting. RESULTS: Sal B protected the BMSCs against H₂O₂ as the cell viability was increased from (53.60±4.21)% to (85.33±9.08)% or (75.78±6.28)% in a dose-dependent manner. After exposed to H₂O₂, about 50%-65% BMSCs displayed apoptotic morphology. Treatment with Sal B at the concentrations of 10 and 100 μmol/L reduced the cytotoxic effect of H₂O₂ on BMSCs to about 32% and 47%, respectively. The results of flow cytometric analysis confirmed the cytoprotective effect of Sal B. This protective effect was concomitant with significant reduction of ROS generation. Moreover, H₂O₂ time-dependently induced a pronounced increase in ERK1/2 phosphorylation,which was effectively inhibited by Sal B.CONCLUSION: Sal B protects BMSCs against H₂O₂-induced apoptosis. Sal B may exert its protective effect on BMSCs by triggering intracellular anti-apoptosis mechanism as well as reducing the oxidative stress.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling pathway.