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.     

Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.     

Effect of oxidative stress-induced autophagy on proliferation and apoptosis of MSCs

AIM: To investigate whether oxidative stress is able to induce autophagy in mesenchymal stem cells (MSCs), and to explore the effects of autophagy on MSC proliferation and apoptosis under oxidative stress circumstance as well as the underlying mechanism for promoting the therapeutic effects of transplanted MSCs on treating diabetes mellitus erectile dysfunction (DMED). METHODS: Hydrogen peroxide (H₂O₂) was applied to simulate the oxidative stress circumstance. The effects of H₂O₂ at concentration of 0, 50, 100, 200, 400 μmol/L on the viability of MSCs were tested by the method of Trypan blue exclusion and MTT assay respectively . The methods of MTT assay, Western blot and transmission electron microscope (TEM) were used to explore the effects of H₂O₂ on MSC apoptosis and autophagy. RESULTS: The proliferation of MSCs was obviously inhibited by H₂O₂ in a dose-dependent manner (P<0.01) and the 50% inhibiting concentration (IC₅₀) was (384.58±16.89) μmol/L. H₂O₂ induced apoptosis and autophay of MSCs. The proliferation rate of MSCs was suppressed by H₂O₂ significantly (P<0.05), with a further decline by blockade of autophagy (P<0.05) whereas increased by blockade of apoptosis (P<0.05). H₂O₂ induced MSCs apoptosis obviously (P<0.05), with an augment of apoptosis (P<0.05) by blockade of autophagy. Furthermore, the H₂O₂ increased expression of cleaved caspase-3 and cleavage of poly ADP-ribose polymerase 1 (PARP1), Which were decreased by apoptosis blockade whereas were enhanced by blockade of autopahgy. CONCLUSION: Oxidative stress plays a dual role in MSC survival, which induces MSC apoptosis and autophagy. Moreover, blockade of autophagy intensifies MSC apoptosis. Therefore, it is a promising method to ameliorate the effects of stem-cell based therapy on DMED by enhancing protective autophagy to increase the survival rate of transplanted MSCs against oxidative stress circumstance caused by diabetes mellitus.     

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.     

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.     

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.     

Mollugin inhibits viability and collagen synthesis of rat CFSC-2G cells

AIM: To investigate the effects of mollugin on the viability and collagen synthesis of rat hepatic stellate cell line CFSC-2G. METHODS: The activation of CFSC-2G cells was induced with low concentration (10 μmol/L) of hydrogen peroxide (H₂O₂) for 30 min in the experiment. The viability of the CFSC-2G cells after exposed to mollugin at different concentrations (0, 20, 40, 60 and 120 μmol/L) was detected by MTT assay. The mRNA and protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor-κB (NF-κB) p65, Bcl-2, Bcl-xL, Bax, and hepatic stellate cell activation markers α-smooth muscle actin (α-SMA) and collagen type I (Col Ⅰ) were detected by real-time PCR and Western blot. The phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK) was determined by Western blot. RESULTS: Mollugin significantly inhibited the viability and collagen synthesis of activated CSFC-2G cells induced by H₂O₂. The expression of Nrf2, HO-1 and Bax at mRNA and protein levels, and the phosphorylation level of p38 MAPK were promoted, while the levels of NF-κB p65, Bcl-2, Bcl-xL, α-SMA and ColⅠwere inhibited by mollugin (P<0.05). CONCLUSION: Mollugin may inhibit H₂O₂-induced viability and collagen synthesis of the CSFC-2G cells by activating Nrf2 and HO-1, and blocking the NF-κB p65 and Bcl-2 expression.     

Effect of sodium ferulate on expression of BCl-2 and bax in rat lens epithelial cells

AIM: To investigate the effect of sodium ferulate on expression of apoptosis-related genes, BCl-2 and bax, in rat lens epithelial cells (LEC) injured by oxidation.METHODS: Eyes of SD rats were divided randomly into four groups: control group, hydrogen peroxide group (H₂O₂), pirenoxine sodium group (PS) and sodium ferulate group (SF). Eyes were excised and lenses were separated under operating microscope and sterilized condition. Lenses were incubated in CO₂ incubator for 24 h with 300μmol·L^-1H₂O₂ and with or rithout 5 mmol·L^-1SF. The expression of BCl-2 and Bax protein of LEC were measured and compared by tearing the LEC anterior capsule via immunohistochemical analysis.RESULTS: (1) There were BCl-2 and Bax expression in normal lenses of SD rates, BCl-2 expression was stronger than Bax. (2) BCl-2 expression decreased and Bax expression increased markedly (P<0.01),BCl-2/Bax reduced in H₂O₂ group. (3) There were up regulation of BCl-2 expression and down-regulation down of Bax expression, and BCl-2/Bax increased in SF group compared with H₂O₂ group (P<0.01). (4) The above changes were similar between PS group and SF group, but much stronger in SF group than that in PS group (P<0.01).CONCLUSION: These results show that SF regulates expression of apoptosis-related genes, BCl-2 and bax, which may be the molecular mechanism of LEC apoptosis inhibition by SF.     

Erythropoietin inhibits eryptosis induced by reactive oxygen species

AIM: To observe the influence of erythropoietin (EPO) on eryptosis and production of reactive oxygen species (ROS) in erythrocytes under stimulation of hydrogen peroxide (H₂O₂),and to explore its related mechanism. METHODS: The erythrocyte suspension (1%) was cultured in vitro and divided into 3 groups:control group (C group, the culture medium was PBS), H₂O₂ group (H group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L) and EPO group (E group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L and EPO at final concentration of 2×10⁴ U/L). The erythrocytes were collected at 24 h and 60 h. The eryptosis was detected by flow cytometry with Annexin V staining. The production of ROS and intracellular calcium ion concentration ([Ca²⁺]_i) were also analyzed by flow cytometry. RESULTS: The eryptosis in C group was increased as the incubating time extended. The eryptosis in H group was higher than that in C group (P<0.01), while that in E group was lower than that in H group (P<0.01). Meanwhile, ROS production and[Ca²⁺]_i were higher in H group than those in C group (P<0.01), but those were lower in E group than those in H group (P<0.05 or P<0.01). CONCLUSION: EPO inhibits eryptosis induced by H₂O₂ and its mechanism may be related to antioxidant effect and change of[Ca²⁺]_i.     

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.     

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.     

Lycium barbarum polysaccharides attenuate oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide

AIM: To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide (H₂O₂). METHODS: The EA.hy926 cell model of oxidative stress injury was established by H₂O₂ treatment. The EA.hy926 cells were divided into 5 groups:control group, damage (H₂O₂ at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H₂O₂), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA.hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA.hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide (AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS: LBP at concentration of 100 mg/L significantly attenuated the injury of EA.hy926 cells induced by H₂O₂, as indicated by improved cell viability (P<0.05) and decreased apoptosis (P<0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA.hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA.hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION: LBP has protective effect on H₂O₂ -induced EA.hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.     

Adiponectin attenuates H2O2-induced SH-SY5Y cell injury and tau hyperphosphorylation via activating PP2A

AIM: To study the effects of adiponectin on H₂O₂-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was determined by MTT assay. H₂O₂-induced cell injury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed. The level of tau phosphorylation as well as the activities of protein phosphatase 2A(PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting. RESULTS: Adiponectin significantly attenuated H₂O₂-induced cell injury(P<0.01). Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimulation with H₂O₂ (P<0.01). Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin(P<0.01). Adiponectin increased the phosphorylation level of GSK-3β at Ser9 site under H₂O₂ stimulation(P<0.01). CONCLUSION: Adiponectin protects SH-SY5Y cells against H₂O₂-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.     

Study on apoptotic and nonapoptotic injuries induced by hydrogen peroxide in cardiac myocytes

AIM: To study apoptotic injury induced by reactive oxygen species-hydrogen peroxide (H₂O₂) on cardiac myocytes.METHODS:Cultured rat neonatal cardiac myocytes were treated with H₂O₂ of various concentration to observe apoptotic injury of cardiomyocytes by agarose gel electrophoresis, Giemsa-stained smears of cell, and flow cytometry, meanwhile lactate dehydrogenas (LDH) and malondialdehyde(MDA) were determined to assess the effect of H₂O₂ on lipid peroxidation and permeability of the plasma membrane. RESULTS: 5 mmol/L H₂O₂ caused cultured cardiomyocytes apoptotic morphological characteristics, including nucleosomal DNA fragmentation in myocytes by agarose gel electrophoresis (DNA ladder), cell shrinkage, nuclear condensation, and chromatin margin by Giemsa-stained cell smears, and aneuploid peak(AP)-apoptotic bodies occurrence by flow cytometry.CONCLUSIONS: H₂O₂-induced apoptosis in myocytes was a time-and concentration-dependent process. Treatment with low concentration of H₂O₂(＜1 mmol/L) only caused cardiomyocyted early biochemical changes, such as increase of free radicals level and membrane permeability ,which were pro-apoptotic injurious features. High concentration of H₂O₂ (＞10 mmol/L) rapidly induced a necrotic form of death characterized by smeared patterns of DNA digestion on agarose gel electrophoresis and lethal membrane disruption (as measured by LDH release). Exposure of 5~10 mmol/L H₂O₂ induced cardiomyocytes apoptosis concurrently with biochemical changes of LDH and MDA increase in the medium.     

Effects of schisandrin B on apoptosis of lens epithelial cells treated with H2O2

AIM: To investigate the effects of Schisandrin B (Sch B) on apoptosis of lens epithelial cells (LEC) treated with H₂O₂. METHODS: Eyes in SDrats were excised and lenses were separated under operating microscope in sterilized condition. Lenses were divided randomly into four groups with different treatment: control group, hydrogen peroxide group (H₂O₂), pirenoxine sodium group (PS) and schisandrin Bgroup (Sch B). Lenses were incubated in CO₂ incubator for 24 h with 300 μmol·L^-1 H₂O₂ and with or without 0.5 mmol·L^-1 Sch B. LECaoptosis and apoptosis rate were measured by TUNELmethod. Ultrastructure changes and apoptosis bodies of LECwere observed via transmitted electron microscope. RESULTS: (1) Apoptosis rate in H₂O₂ group (92.0±2.6) was significantly higher than that in control group (3.5±1.8). Apoptosis rate in Sch Bgroup (13.8±3.27) was remarkably lower than that in H₂O₂ group and PSgroup. (2) Ultrastructure observation indicated that apoptosis cells occurred in most LEC in H₂O₂ group and the changes were severe presenting different stages. While a few apoptosis cells were observed in Sch Bgroup, the changes were slight and most of them were in early and middle stages. CONCLUSION: These data indicated that Sch Bsignificantly inhibited apoptosis of LECduring experimental oxidative injury, the effects were stronger than PS.     

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     

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 hydrogen peroxide on sodium current in acutely isolated rat hippocampal CA1 neurons

AIM and METHODS: The effects of hydrogen peroxide on Na⁺ currents were studied in freshly dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp techinique. RESULTS: ①H₂O₂ caused a dose-dependent and voltage-dependent increase in the voltage-activated Na⁺ currents. The amplitudes of Na⁺ currents were increased (48.0±4.2)% and (88. 2±5. 1)% (n=10) by H₂O₂ at 10 μmol/L and 100 μmol/L, respectively. ②H₂O₂ (10 μmol/L) did not affect the activation process, but changed the inactivation process significantly. Before and after application of 10 μmol/L of H₂O₂, the half-inactivation voltage was (-64.58±1.22)mV and (-53.55±0.94)mV (n=10, P<0.01), but the slope factor was not changed. CONCLUSION: As a product of oxidation metabolism, H₂O₂ is related to some diseases in the central nervous system.