Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Inhibitory effect of acetylcholine on apoptosis of myocardial H9c2 cells induced by norepinephrine

AIM: To investigate the inhibitory effect of acetylcholine (ACh) on the apoptosis of myocardial H9c2 cells induced by norepinephrine (NE). METHODS: The apoptosis model of myocardial H9c2 cells was established by treating the cells with NE at different concentrations, and ACh was used to observed the protective effect. The cell viability was measured by MTT assay and the optimal doses of NE and ACh were selected. Four blockers related to different signaling pathways were also used. The apoptotic rate was analyzed by flow cytometry with Annexin V-FITC/PI staining. JC-1 staining was used to observe the changes of mitochondrial membrane potential of the H9c2 cells. DCFH-DA staining was used to observe intracellular reactive oxygen species (ROS) production. RESULTS: The viability of H9c2 cells was decreased by treatment with NE at 10 μmol/L. The membrane potential of mitochondria was decreased, while ROS production and apoptotic rate were increased significantly (P<0.05). Pretreatment with ACh at 10 mmol/L resulted in the increase in cell viability and decrease in the ROS production, and inhibited the loss of mitochondrial membrane potential and cell apoptosis (P<0.05). After treatment with the 4 signaling pathway blockers before ACh, the protective effect of ACh was significantly reduced only by PDTC. CONCLUSION: ACh inhibits the apoptosis of H9c2 cells induced by NE, and its mechanism may be related to NF-κB signaling pathway.     

Genipin inhibits oxidative stress and apoptotic damage in high glucose-induced H9c2 cardiomyocytes

AIM:To explore the effects of genipin (GEN) on high glucose (HG)-induced oxidative stress injury and apoptosis in H9c2 cardiomyocytes.METHODS:H9c2 cells were cultured in vitro and HG-induced injury model was established. H9c2 cells were divided into 4 groups:normal control (NC) group (glucose at 5.6 mmol/L), HG group (glucose at 50 mmol/L), NG+GEN group and HG+GEN group. The concentration of genipin was used at 10 μmol/L. The viability of the H9c2 cells was measured by CCK-8 assay. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by enzyme labeling and WST-1 methods, respectively. The activity of lactate dehydrogenase (LDH) in the cell culture supernatant was detected by microplate method. Fluorescent probe DCF was used to detect intracellular levels of reactive oxygen species (ROS). Nucleosome fragments was measured to evaluate cell apoptosis by ELISA. The intracellular mitochondrial membrane potential was detected by JC-1 method. The protein levels of Mn-SOD, cytochrome C (Cyt C), Bax and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with HG group, the cell viability in HG+GEN group was increased significantly (P<0.05), the levels of MDA and LDH were decreased (P<0.05), SOD activity was increased (P<0.05), the levels of ROS and nucleosome fragments in HG+GEN group were decreased (P<0.05), and the mitochondrial membranes potential was notably increased (P<0.05). Compared with NG group, the activation of Mn-SOD was decreased, but the protein levels of Cyt C, Bax and cleaved caspase-3 were increased in HG group (P<0.05). Compared with HG group, the activation of Mn-SOD was increased, and the protein levels of Cyt C, Bax and cleaved caspase-3 were decreased in HG+GEN group (P<0.05).CONCLUSION:Genipin protects HG-induced H9c2 cardiomyocytes against oxidative stress injury and apoptosis.     

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.     

The protection of composite salviae dropping pills against HUVECs injure induced by H2O2

AIM: To observe the protective effect of composite salviae dropping pills against human umbilical vein endothelial cells (HUVECs) injure induced by hydrogen dioxide (H2O2) and discuss the mechanism of its therapeutic action on cardiac and encephalic diseases. METHODS: HUVECs were injured by 0.1 mmol/L H2O2 and then different final concentrations of composite salviae dropping pills (0.5 g/L, 0.25 g/L, 0.1 g/L) were added before and after the injury. Cell viability was measured by MTT assay. TBA method was used to detect the intracellular malonaldehyde. Nitric oxide (NO) in the culture medium was detected by using nitrate reductase assay, and immunocytochemisty was used to observe the expression of NOS2, NOS3 and NF-κB. Morphologic observation was also performed. RESULTS: HUVECs were injured by 0.1 mmol/L H2O2. Composite salviae dropping pills increased the cell viability apparently, inhibited the production of MDA induced by H2O2, regulated the generation of NO bilaterally and influenced the expression of NOS3 and NF-κB. CONCLUSIONS: Composite salviae dropping pills at concentrations of 0.5 g/L, 0.25 g/L and 0.1 g/L protects HUVECs from injury by H2O2, no matter it is be added before or affter the injury. The possible mechanism is associated with its regulating the expression of NOS2, NOS3 and NF-κB.     

NRF2 attenuates oxidative stress and lysosomal dysfunction in doxorubicin-induced H9C2 cells

AIM:To study the effect of nuclear factor E2-related factor 2 (NRF2) on oxidative stress injury and lysosomal dysfunction in doxorubicin (DOX)-induced rat myocardial H9C2 cells. METHODS:The H9C2 cells were treated with DOX. The expression of NRF2 at mRNA and protein levels was determined by real-time PCR and Western blot. The H9C2 cells stably over-expressing NRF2 were established by lentiviral infection. Real-time PCR and Western blot were used to identify the efficiency of over-expression. After DOX treatment, the cell viability was measured by CCK-8 assay, the activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the content of malondialdehyde (MDA) in the cell supernatant were detected. FITC-dextran was used to analyze lysosomal pH, and the protein expression of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B was determined by Western blot.RESULTS:The expression of NRF2 at mRNA and protein levels in DOX-treated H9C2 cells was significantly decreased (P<0.05). Over-expression of NRF2 significantly up-regulated the mRNA and protein expression of NRF2 in DOX-treated H9C2 cells (P<0.05). After DOX treatment, the cell viability was decreased, and LDH activity was increased. The activity of SOD, GSH-Px and CAT was decreased, and the content of MDA was increased (P<0.05). The lysosomal pH was increased, and the protein expression of LAMP1 and cathepsin B decreased (P<0.05). Over-expression of NRF2 increased the cell viability, decreased LDH activity, increased the activity of SOD, GSH-Px and CAT, and decreased the content of MDA in cell supernatant (P<0.05). Over-expression of NRF2 also decreased the lysosomal pH, and increased the protein expression of LAMP1 and cathepsin B (P<0.05). CONCLUSION:DOX inhibits the expression of NRF2 in the myocardial H9C2 cells. Over-expression of NRF2 attenuates oxidative stress and lysosomal dysfunction in the H9C2 cells induced by DOX.     

Hesperetin attenuates hypoxia/reoxygenation-induced apoptosis in H9c2 cells via inhibition of calcium overload

AIM: To investigate the effect of hesperetin on hypoxia/reoxygenation (H/R)-induced apoptosis in the H9c2 cells and to clarify the underlying mechanism. METHODS: The H/R model was established and the H9c2 cells were pretreated with hesperetin for 4 h. The cell viability and cell damage were measured by CCK-8 assay and lactate dehydrogenase (LDH) detection. The apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. The intracellular calcium fluorescence intensity was measured by fluorescence microscopy and flow cytometry. The calcium-ATPase activity and the level of adenosine triphosphate (ATP) were measured by ELISA. The mitochondrial membrane potential was measured by JC-1 staining. The protein expression levels of Bcl-2, Bax and cytochrome C (Cyt-C) were determined by Western blot. RESULTS: Hesperetin reduced the apoptosis of the H9c2 cells induced by H/R, decreased intracellular Ca²⁺ fluorescence intensity, elevated Ca²⁺-ATPase activity, inhibited the mitochondrial membrane potential depolarization and increased the level of ATP (P<0.05). In addition, hesperetin significantly reduced the release of Cyt-C protein from mitochondria to cytoplasma and increased the Bcl-2/Bax ratio (P<0.05). After using the calcium ion inhibitor nimodipine, the percentage of the cells with mitochondrial membrane depolarization was decreased, the ATP level was increased and the protein expression of mitochondrion-related apoptosis molecules were decreased (P<0.05). CONCLUSION: Hesperetin reduces the apoptosis of the H9c2 cells induced by H/R, which may be related to inhibition of calcium overload and improvement of mitochondrial function.     

Astragalus polysaccharides attenuate endothelial cell injury caused by homocysteine via AMPK pathway

AIM:To investigate the protective effect of Astragalus polysaccharides (APS) on human umbilical vein endothelial cells (HUVECs) injured by homocysteine (Hcy) and its mechanism. METHODS:HUVECs cultured in vitro were divided into 4 groups:control group, APS group[APS (200 mg/L) treatment for 24 h], Hcy group[Hcy (1 mmol/L) treatment for 24 h], and Hcy+APS group[Hcy (1 mmol/L) and APS (200 mg/L) co-treatment for 24 h]. The cell viability were measured by MTT assay. The activity of lactate dehydrogenase (LDH) and superoxidase dismutase (SOD), and the content of malondialdehyde (MDA) in HUVECs were detected by the commercial kits. The mRNA expression of SOD1, catalase (CAT) and NADPH oxidase 2 (NOX2) was detected by RT-qPCR. The protein levels of AMP-activated protein kinase α (AMPKα) and phosphorylated AMPKα (p-AMPKα) were determined by Western blot. RESULTS:Compared with control group, the cell viability, the activity of SOD, and the mRNA expression of SOD1 and CAT in the HUVECs were decreased, but the activity of LDH, the content of MDA, and the mRNA expression of NOX2 were increased significantly in Hcy group(P<0.05). APS inhibited the decrease in cell viability, and the increases in LDH acti-vity and MDA content induced by Hcy. APS increased SOD activity and the mRNA expression of SOD1 and CAT, but reduced the mRNA expression of NOX2. Compound C, an AMPK inhibitor, reduced the protective effect of APS on HUVECs injured by Hcy. CONCLUSION:APS protects HUVECs from Hcy-induced injury via AMPK signaling pathway to regulate intracellular oxidative stress.     

CPPU对番木瓜干旱胁迫的保护作用

干旱胁迫下,番木瓜叶片水势、可溶性蛋白质、叶绿素含量和SOD活性明显下降,而电导率、POD活性、MDA和H2O2含量则显著上升。分别用10、30、50mg/L的CPPU预处理有效地降低了干旱条件下番木瓜叶片细胞的MDA、电导率和H2O2含量的增加程度,并能减缓叶片叶绿素总含量和可溶性蛋白质含量的降低。试验表明CPPU是通过减轻干旱对番木瓜引起的自由基伤害来提高番木瓜的抗旱能力,其中以30mg/L浓度的CPPU处理效果最好。     

Effects of H2O2 on apoptosis of skeletal muscle satellite cell and mitochondrial membrane potential

AIM: To observe the effects of H2O2 on apoptosis of skeletal muscle satellite cells (SMSC)and mitochondrial membrane potential (MMP), and protective effect of erythropoietin(EPO). METHODS: SMSC in vitro were divided into three groups: H2O2 group, H2O2+EPO group and control. Apoptosis rate and the means were obverted by monofluorescence flow cytometry. The morphological change of apoptosis cells were observed under fluorescence microscopy after Hoechst 33258 staining. RESULTS: The cells in H2O2 group show the highest apoptosis rate (22.13±1.79)%. In H2O2+EPO group, apoptosis rate were (16.47±2.53)%, (4.97±0.55)% and (2.93±0.47)% according to the EPO treated levels (10, 20 or 40 kU/L), respectively. MMP level in H2O2 group was the lowest 9.70±0.09. MMP levels in H2O2+EPO group were 12.67±0.32, 27.90±0.66, 44.53±0.93, respectively according to the EPO treated levels (10, 20 or 40 kU/L). In control group, apoptosis rate was 1.93±0.57 and MMP was 51.37±0.64. In H2O2 group and H2O2+ low dosage EPO group, Hoechst 33258 staining showed obvious apoptosis. CONCLUSION: EPO inhibits the apoptosis induced by H2O2 and stabilizes the MMP, which is related to the dosage of EPO.     

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.     

Tubuloside B rescues the PC12 neuronal cells from H2O2-induced apoptosis

AIM:To investigate the neuroprotective effect of tubuloside B, one of the phenylethanoids isolated from the stems of Cistanche salsa, on H₂O₂-induced injury in PC12 cells.METHODS:PC12 cells were exposed to various doses of tubuloside B for 12 h, then treated with H₂O₂ at concentration of 100 μmol/L for 24 h. The cell viability was observed with MTT assay. Reactive oxygen species and the mitochondrial membrane potential were measured with laser scanning confocal microscopy (LSCM). The DNA content and percentage of apoptosis were assayed by DNA agarose gel electrophoresis and flow cytometry. The activation of caspase-3 was detected with the caspase-3 activity assay kit. RESULTS:Following treatment with H₂O₂ for 24 h, H₂O₂ induced a significant decrease in cell viability; DNA ladder was observed and apoptosis percentage was as high as 48.0%. Accumulation of intracellular ROS, increase in caspase-3 activity and the decrease in mitochondrial membrane potential as indicated with the decrease of red/green ratios (from 5.97 to 0.41) were detected. However, pretreatment with tubuloside B (1, 10 or 100 mg·L^-1) for 12 h exhibited cytoprotective effects in a dose-dependent manner. Tubuloside B obviously enhanced the cell viability, reduced formation of the DNA ladder, and significantly reduced the number of cells labeled with Annexin-V. The percentage of apoptosis/necrosis neurons was significantly decreased to 30.9%, 18.3% and 6.2%, respectively. LSCM showed that the tubuloside B attenuated the accumulation of ROS and the H₂O₂-induced collapse of mitochondrial membrane potential in PC12 cells. The significant decrease in caspase-3 activity was detected, compared to the H₂O₂-treated cells at the same time point. CONCLUSION:Tubuloside B has the neuroprotective capacity to antagonize H₂O₂-induced apoptosis and injury in PC12 cells, indicating it may be useful for treating some neurodegenerative diseases.     

Role of PERK pathway in morphine-induced myocardial protection of H9c2 cells

AIM: To explore whether morphine protects oxidative stress-damaged myocardial cells by inhibiting the PERK pathway to reduce endoplasmic reticulum stress and prevent mitochondrial permeability transition pore (mPTP) opening. METHODS: Rat myocardial H9c2 cells were cultured to establish an oxidative stress model, and then randomly divided into control group, H₂O₂ group, H₂O₂+morphine group, H₂O₂+morphine+PERK pathway inhibitor GSK2656157 group, morphine group and GSK2656157 group. Immunohistochemical method was used to detect the effects of morphine on expression of glucose-regulated protein (GRP) 78 and GRP94 induced by oxidative stress. The protein levels of PERK signaling pathway-related molecules were determined by Western blot. Confocal microscopy was used to observe the effects of morphine on mPTP opening and endoplasmic reticulum induced by oxidative stress. Cellular toxicity was detected by lactate dehydrogenase (LDH) kit and cell viability was measured by MTT assay. RESULTS: Compared with control group, GRP78 and GRP94 proteins in H₂O₂ group were strongly expressed, and the brown-yellow particles were significantly increased, but morphine significantly inhibited this process. Compared with control group, the phosphorylation of PERK was significantly reduced with GSK2656157 treatment at different concentrations, among which 2 μmol/L had the most significant effect (P < 0.05). Oxidative stress significantly increased the protein levels of GRP78, GRP94, p-PERK and CHOP, but significantly decreased p-GSK-3β level. These changes were inhibited by morphine, and the effects of morphine were further enhanced by GSK2656157 (P < 0.05). Compared with control group, oxidative stress significantly reduced the fluorescence intensity of mitochondrial TMRE and ER-Tracker Red. Morphine significantly inhibited this effect even when mitochondrial membrane potential was reduced, mPTP was open, and endoplasmic reticulum was damaged, while GSK2656157 further enhanced the effect of morphine (P < 0.05). Compared with control group, H₂O₂ significantly increased cellular toxicity and decreased the cell viability. Morphine inhibited this effect and GSK2656157 significantly enhanced the effect of morphine (P < 0.05). CONCLUSION: Morphine protects cardiac H9c2 cells under oxidative condition by inhibiting endoplasmic reticulum stress through PERK pathway and preventing the mPTP opening via GSK-3β inactivation.     

N-acetylcysteine protects H9c2 cells against injuries induced by methyl-glyoxal

AIM: To investigate the protective effect of N-acetylcysteine(NAC) on H9c2 cells from injuries induced by methylglyoxal(MG) and the potential mechanism. METHODS: H9c2 cells were divided into control group, MG treatment group, NAC + MG treatment group, SP600125 pretreatment + MG group, NAC group and SP600125 group. The viability of the H9c2 cells was measured by CCK-8 assay. The protein levels of p-JNK and t-JNK were tested by Western blot. The changes of intracellular reactive oxygen species(ROS) were evaluated by 2', 7'-dichlorofluorescein diacetate(DCFH-DA) staining. Mitochondrial membrane potential(MMP) was measured by rhodamine 123(Rh123) staining. The morphological changes in apoptotic cardiomyocytes were detected by Hoechst 33258 staining. RESULTS: Du-ring 100~800μmol/L concentration range, MG caused significantly reduced viability of the H9c2 cells in a dose-dependent manner. NAC had a protective effect on H9c2 cells against the injuries induced by MG during 500~1500μmol/L concentration range through raising cell viability, inhibiting cellular oxidative stress and improving MMP(P<0.01). SP600125, an inhibitor of JNK, showed the protective effect similar to NAC on H9c2 cells against MG-induced injuries, including attenuating oxidative stress, improving MMP and suppressing apoptosis.CONCLUSION: N-acetylcysteine offers obvious protective effect on H9c2 cells against the injuries induced by methylglyoxal. The underlying mechanisms may be associated with decreasing the production of ROS, ameliorating MMP, inhibiting the activation of JNK and suppressing apoptosis.     

铜污染对芹菜生长及生理特性的影响

以芹菜定植苗为试材,通过设CuSO4.5H2O 05、、10、204、0 mg/L营养液水培试验,研究Cu污染对芹菜生长的影响及生理胁迫伤害。结果表明:低浓度CuSO45 mg/L对芹菜生长有促进作用,随浓度递增20~40 mg/L对生长有抑制作用;根系活力在CuSO4浓度为5 mg/L时较对照上升,随浓度递增而下降;叶绿素a含量在低浓度CuSO45 mg/L含量上升,随浓度递增含量下降,对叶绿素a的影响大于叶绿素b及类胡萝卜素;硝酸还原酶在CuSO4浓度为5 mg/L时较对照上升,再随浓度递增而不断下降;POD活性随Cu浓度升高先上升后下降再上升;SOD和CAT活性先上升后下降;叶细胞膜渗透性丙二醛(MDA)及含量相对电导率随CuSO4浓度升高而不断上升。     

Effects of Astragalus polysacharin on fibroblast proliferation and adhesion between HUVECs and white cells

AIM: To investigate the effects of Astragalus polysacharin(APS) on human fibroblast and human umbilical vein endothelia cell (HUVEC) proliferation, as well as its acts on adhesion between white cells and HUVECs. METHODS: Human fibroblasts from distal and proximal skin away the ulcer were cultured as normal fibroblasts(NF) and wounded fibroblasts(WF). MTT assay was used for detecting cell proliferation, Rose Bengal staining and fluorescence immunohistology assay were used for examining the adhesion of human polymorpho-nuclear cell(PMN) and TPH-1 to HUVECs. RESULTS: 2.44-156 mg/L APS promoted WF proliferation, and 2.44-39 mg/L APS also promoted NF proliferation, but it did not show any proliferating effect on HUVECs. APS inhibited the adhesion of PMN or TPH-1 to HUVECs induced by tumor necrosis factor(TNF). At 25-100 mg/L, it also inhibited both VCAM-1 and ICAM-1 expression in HUVECs induced by TNF. Treatment with APS for 12 h also inhibited CD44 expression in HUVECs. CONCLUSION: APS shows mitogenic activity on both human normal and wounded fibroblasts. It also exerts anti-inflammation effects by inhibiting adhesion molecule expression and adhesion of white cells to HUVECs.     

Effect of adipokine chemerin on mitochondrial dysfunction in cardiac H9C2 cells with hypertrophy

AIM To study the regulation of adipokine chemerin on mitochondrial function of rat cardiac H9C2 cells with hypertrophy, and to explore its effect on mitochondrial dysfunction in the H9C2 cells. METHODS In vitro cell experiments were performed, and the H9C2 cells were divided into normal group, chemerin group, angiotensin Ⅱ (Ang Ⅱ) model group and Ang Ⅱ+chemerin group. Immunohistochemistry and qPCR were used to identify whether the model was successfully constructed. The morphological changes of mitochondria in the H9C2 cells were observed under electron microscope. The mitochondrial membrane potential and membrane permeability were analyzed by flow cytometry. The activity of cytochrome C oxidase (COX) and succinate dehydrogenase (SDH) was measured by enzyme activity kit. RESULTS Compared with normal group, the mitochondrial structure in Ang Ⅱ group and chemerin group was seriously damaged, the permeability of mitochondrial membrane was significantly increased (P<0.01), and the mitochondrial membrane potential and the activity of COX and SDH were significantly reduced (P<0.01). Meanwhile， the mitochondrial damage in the H9C2 cells was more serious in Ang Ⅱ+chemirin group (P<0.05). CONCLUSION Chemerin stimulation not only induces cardiomyocyte hypertrophy, but also promote the pathological process of mitochondrial dysfunction in the myocardial cells with hypertrophy.     

Celastrol induces apoptosis of multiple myeloma H929 cells

AIM: To investigate the effect of celastrol on the apoptosis of human multiple myeloma H929 cells and its molecular mechanism. METHODS: The H929 cells were cultured in vitro and treated with celastrol at different concentrations (0.5, 1, 5 and 10 mg/L). The viability of H929 cells was analyzed by CCK8 assay. Annexin V-PE/7-AAD staining was used to analyzed the effect of celastrol on apoptosis of H929 cells, and mitochondrial membrane potential was observed by flow cytometry. The effect of celastrol on DNA damage was detected by comet assay. The protein levels of apoptosis-related molecules P53, XIAP, cleaved PARP-1 and cleaved caspase-3, and the release of mitochondrial cytochrome C in the H929 cells treated with celastrol were determined by Western blot. RESULTS: The viability of H929 cells was significantly inhibited by different concentrations of celastrol in a concentration-dependent and time-dependent manner. Apoptosis and decreased mitochondrial membrane potential of H929 cells in a concentration-dependent manner were observed after treatment with celastrol (P<0.05). The results of comet assay showed that celastrol induced DNA damage in the H929 cells. The protein levels of apoptotic molecules P53, cleaved PARP-1 and cleaved caspase-3 were significantly increased and the expression level of anti-apoptotic protein XIAP was significantly decreased in the H929 cells treated with celastrol (P<0.05). Celastrol promoted the release of cytochrome C in mitochondria, and activated caspase-3 in dependence on caspase-9. CONCLUSION: Celastrol has an apoptosis-inducing effect on multiple myeloma H929 cells. Its mechamism may be related to activation of mitochondrial apoptosis pathway by inducing DNA damage.     

Cytoprotective role of low-dose sodium nitrite preconditioning against H2O2 induced damage in PC12 cells

AIM: To study the cytoprotective role of NaNO2 preconditioning against H2O2 induced damage in PC12 cells. METHODS: PC12 cells were treated with different concentrations of NaNO2 for 6 h, 12 h, 24 h and 48 h, respectively. The viability of the cells was measured by MTT method and cell counting. The apoptotic rate of PC12 was determined by Hoechst 33258 staining to calculate the ratio of the cells between concentrated and broken nucleus in the total cell count. PC12 cells were pretreated with NaNO2 at concentration of 3 mmol/L for 24 h. The cytoprotective role to the toxicity of H2O2 at concentration of 1.1 mmol/L for 6 h was observed by MTT. The cell apoptosis was measured by flow cytometry and staining with Hoechst 33258 and PI. The activities of catalase (CAT), superoxide dismutase (SOD), and the changes of glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured by the method of colorimetry. RESULTS: The dose-response results showed that the effect of NaNO2 on PC12 cell proliferation was a typical β-shape curve. The maximal stimulatory response was at 24 h, and the concentration of the maximum stimulatory response was 1.4 mmol/L. The maximal stimulation of the concentration-responses was 156% above the control. No observable adverse effect level (NOAEL) was 6 mmol/L. IC50 was 45 mmol/L. When the cells were pretreated by NaNO2 at concentration of 3 mmol/L for 24 h, and then exposed to H2O2 at concentration of 1.1 mmol/L for 6 h, the proliferation rate was increased as compared to the cells treated with H2O2 alone. Under the conditions of treating the cells with NaNO2 at concentration of 3 mmol/L to induce the adaptive response, then exposing the cells to H2O2 at concentration of 1.1 mmol/L, the apoptosis rate in non-preconditioning group was 44.9%, the apoptosis rate in preconditioning group was 19.1%, the difference was significant (P<0.05). The cytoprotective effect of NaNO2 was inhibited by nitric oxide (NO) scavenger ferrohemoglobin. The activities of SOD, CAT and the level of GSH-Px were markedly increased, the content of MDA decreased in preconditioning group. CONCLUSION: Exposure of NaNO2 at concentration of <6 mmol/L induces hormesis on PC12 cells. Low dose of nitrite plays an important role in cytoprotection by reducing nitrite to NO, indicating that decrease in lipid peroxidation and increase in endogenous antioxidants may play a key role in cytoprotection induced by preconditioning with low dose of NaNO2 in PC12 cells.     

Protective effect of procyanidins on PC12 cells exposed to Aβ25-35

AIM: To investigate the protective effect of procyanidins on the PC12 cells exposed to Aβ_25-35 and the mechanisms.METHODS: Aβ_25-35 at 25 μmol/L was used to treat the PC12 cells for 48 h, and the PC12 cells were pretreated with procyanidins at 25, 50 and 100 mg/L for 24 h. The cell vitality was measured by MTT assay. The content of reactive oxygen species (ROS) was detected by DCFH-DA staining. The change of mitochondrial membrane potential was examined by JC-10 staining. The apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of activated caspase-3 was determined by Western blot.RESULTS: Under the exposure of the PC12 cells to Aβ_25-35, procyanidins increased the cell viability, reduced intracellular ROS level, prevented mitochondrial membrane potential decline, attenuated the caspase-3 activation and inhibited the apoptosis of PC12 cells (P<0.05 or P<0.01).CONCLUSION: Procyanidins have a significant protective effect on the PC12 cells exposed to Aβ_25-35. Its mechanism may be related to removing intracellular ROS induced by Aβ_25-35, relieving the damage to the mitochondrial membrane, and thereby inhibiting cell apoptosis.