Antioxidant effect of carnosine on H9N2 swine influenza virus-induced acute lung injury in mice

AIM:To investigate the antioxidant effect of carnosine on H9N2 swine influenza virus (H9N2-SIV)-induced acute lung injury (ALI). METHODS:One hundred and fifty SPF female BALB/c mice (6 to 8 weeks old) were randomly divided into control group, ALI group and carnosine intervention group with 50 each. The mice in control group were inoculated intranasally with normal allantoic fluid of chick embryos. The mice in ALI group were inoculated intranasally with allantoic fluid containing H9N2-SIV. The mice in carnosine group were treated with H9N2-SIV plus carnosine. On the 2nd, 4th, 6th, 8th and 14th days after treatment, 8 mice in each group were killed to observe the pathological changes of the lung. Meanwhile, the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO), the content of malondialdehyde (MDA) and the wet weight/dry weight ratio (W/D) of the lung tissues were determined. RESULTS:Carnosine alleviated the symptom of the mice induced by H9N2-SIV infection, and increased the viability of the mice. In carnosine intervention group, edema degree of the lung (W/D) was apparently reduced (P<0.05). The pathological changes were alleviated on the 6th and 8th days of the experiment. On the same days, the content of MDA was lower obviously (P<0.05) and the activity of SOD was improved remarkably (P<0.05). On the 4th day of the experiment, the activity of MPO was reduced apparently (P<0.05) and continuously decreased on the 6th and 8th days (P<0.05). CONCLUSION: Carnosine protects the mice from acute lung injury induced by H9N2-SIV infection and increases the viability by reducing the content of MDA, lowering the activity of MPO, increasing the activity of SOD and inhibiting the production of free radicals and lipid peroxidation.     

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.     

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.     

Effects of Fas,NF-κB and caspases on microvascular endothelial cell apoptosis induced by TNFα

AIM:To study the apoptotic effect of TNFα on rat pulmonary microvascular endothelial cells(PMVEC)and the role of Fas, NF-κB in its mechanism. METHODS: Apoptosis of PMVEC was analyzed and quantitated with TUNEL, flow cytometer. Northern blot was applied to assess the influence of TNFα on PMVEC Fas expression. Fas antibody was used to investigate the apoptotic effect of Fas on PMVEC. Activation of caspase-8 was examined by Western blot. Expression of caspase-3 was analyzed with histo-immunochemical staining. RESULTS:Growth curve showed that TNFα suppressed endothelial cell proliferation in a dose-dependent manner. After treatment with 5×10⁶U/LTNFα, apoptotic rate was 14.0%±3.1% detected with TUNEL, and 13.1% with flow cytometer. When the cells were co-cultured with TNFα and APDC, an NF-κB inhibitor, less cells were viable and more cells were positively stained with TUNEL. Fas expression in PMVEC was elevated after TNFα treatment. Co-culturing with Anti-Fas antibody aggravated PMVEC apoptosis. Caspase-8 activity and caspase-3 expression was elevated. Caspase-3 inhibitor significantly ameliorated PMVEC apoptosis. CONCLUSION: Large dose of TNFα(5×10⁶U/L) can induce apoptosis in rat PMVEC. NF-κB has an anti-apoptotic effect in PMVEC. TNFα up-regulates Fas expression in PMVEC, and the latter takes a part in apoptosis. Caspase-8 and caspase-3 are involved in PMVEC apoptosis induced by TNFα.     

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.     

Critical role of cystic fibrosis transmembrane conductance regulator in hypoxia-induced apoptosis of H9c2 cardiomyocytes

AIM:To explore the roles of cystic fibrosis transmembrane conductance regulator (CFTR) in hypoxia-induced apoptosis of H9c2 cardiomyocytes and the underlying mechanisms. METHODS:The rat H9c2 cardiomyocytes were exposed to a 1% hypoxic environment in a hypoxic chamber. After CFTR overexpression, H9c2 cardiomyocytes were cultured in a hypoxic environment. The mRNA and protein levels of CFTR were examined by RT-qPCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptotic rate was determined by Hoechst 33342 and Annexin V-FITC/PI staining, and the production of reactive oxygen species (ROS) was examined by dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. RESULTS:Hypoxic exposure caused the apoptosis of H9c2 cardiomyocytes, which was accompanied by the down-regulation of CFTR at mRNA and protein levels and over-production of ROS (P<0.05). After CFTR overexpression, the apoptotic rate of the H9c2 cardiomyocytes induced by hypoxia was significantly reduced, with a prominent inhibition of ROS production (P<0.05). However, pretreatment with CFTRinh-172, a specific inhibitor of CFTR, reversed the protective effect of CFTR overexpression in H9c2 cardiomyocytes. CONCLUSION:CFTR has a critical role in protecting against hypoxia-induced apoptosis of H9c2 cells, which may be through inhibiting the generation of ROS.     

Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial BEAS-2B cells

AIM: To explore the effect of hexokinase 2 (HK2) on lipopolysaccharide (LPS)-induced apoptosis of human lung epithelial BEAS-2B cells and the underlying mechanisms.METHODS: BEAS-2B cells were treated with LPS to induce cell injury, and the cell viability was examined by CCK-8 assay. Hoechst 33342 staining and Annexin V/PI double staining were used to analyze the apoptosis. The apoptotic pathway was identified by the specific inhibitor for caspase-8 or caspase-9. The releases of key mediators in mitochondrial apoptosis pathway were examined by Western blot. The effects of HK2 in these process were confirmed by HK2 over-expression followed by LPS treatment.RESULTS: CCK-8 assay showed that LPS treatment decreased the viability of BEAS-2B cells in a dose/time-dependent manner (P<0.01). The apoptosis of BEAS-2B cells was manifested by Hoechst 33342 and Annexin V/PI double staining. Pretreatment with z-LEHD-fmk, but not z-IETD-fmk, reversed the decreased cell viability under LPS stimulation. HK2 down-regulation was involved in LPS-induced apoptosis of the BEAS-2B cells. After HK2 over-expression, the cell viability was increased after LPS treatment. Releases of cytochrome C and apoptosis-inducing factor from mitochondrion to cytoplasm during apoptosis were also inhibited by HK2 over-expression.CONCLUSION: Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial cells.     

Effect of EZH2 regulating Wnt/β-catenin signaling pathway on apoptosis of brain glioma cells

AIM: To investigate the effect of enhancer of zeste homolog 2 (EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87, H4 and U251 and normal human astrocytes (NHA) was detected by RT-qPCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway, the apoptosis rate was measured by flow cytometry, and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87, H4 and U251 were significantly higher than those in NHA (P<0.05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells (P<0.05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells, the apoptotic rate was significantly increased, and the activity of caspase-3 was significantly increased in the cells (P<0.05). Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2, and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Preparation of recombinant human soluble TRAIL and its inducing effect on apoptosis of tumor cells by TRAIL combined with bortezomib

AIM: To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to verify the biological activity of TRAIL. METHODS: The prokaryotic expression plasmid pET-28a (+)-TRAIL_114-281 was constructed. Human soluble TRAIL was obtained through optimized inducing protein expression and purification conditions. The biological activity of TRAIL was verified by CCK-8 assay. The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib (Velcade, PS-341) on the tumor cell lines H460(TRAIL-sensitive) and K562(TRAIL-resistance) for 24 h was determined. The apoptotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining. The activities of caspase-8, -9 and -3 in the cells were detected by colorimetric method. The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot. The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry. RESULTS: The recombinant human soluble TRAIL protein with stable bioactivity was successfully acquired, which induced apoptosis in H460 cells and K562 cells. After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL (P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration. Apoptotic rate in combination group was obviously higher than that in single group (P<0.05). In the process of apoptosis, the activities of caspase-8, -9 and -3 in H460 cells and K562 cells were both increased. The expression of Bcl-2 and cFLIP in treatment groups (especially the combination group) was decreased compared with control group. No significant change of the Bax expression level was observed. The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regulated after treated with bortezomib (P<0.05). CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

Effect of adipose differentiation-related protein on proliferation and apoptosis in H9c2 cells

AIM: To construct an adipose differentiation-related protein (ADRP) eukaryotic expression vector and to explore the effect of ADRP on apoptosis of H9c2 cells induced by palmitic acid (PA). METHODS: The ADRP gene obtained by the method of RT-PCR was cloned into pEGFP-C1 plasmid. The recombinant plasmid was transformed into E.coli DH5α for amplification. The recombinant plasmid was extracted from E.coli DH5α and transfected into H9c2 cells by Lipofectamine^TM2000. The stable transformants were selected by G418 screening. Expression of green fluorescent protein was observed under fluorescence microscope and the ADRP expression was identified by RT-qPCR and Western blotting analysis. The effect of PA on the proliferation of H9c2 cells was detected by MTT assay. The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry. RESULTS: The eukaryotic expression vector pEGFP-C1-ADRP was successfully constructed. Green fluorescent was observed in the cells transfected with pEGFP-C1 or pEGFP-C1-ADRP under fluorescence microscope. RT-qPCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of ADRP. After treated with PA at different concentrations, the apoptosis rates and the proliferation inhibition of recombinant cells were both lower than those of the other two cells. CONCLUSION: The transfected H9c2 cells with stable ADRP expression were successfully established. The over-expression of ADRP prevents the cells from apoptosis and inhibition of proliferation caused by PA, indicating that ADRP plays a protective role in H9c2 cells.     

Astragalosides inhibit autophagy and apoptosis of rat cardiomyocytes induced by H2O2 through mTOR signaling pathway

AIM: To investigate the effects of astragalosides on autophagy and apoptosis of rat cardiomyocytes induced by hydrogenperoxide (H₂O₂).METHODS: The injury model of H9c2 cells induced by H₂O₂ was established, and the cells in astragalosides group and rapamycin group were treated with 20 mg/L astragalosides and 0.1 mg/L rapamycin, respectively. The apoptotic rate was detected by flow cytometry. The autophagy was observed by acridine orange staining. Western blot was used to detect the protein levels of p-mTOR, P70S6K, LC3 and caspase-3. RESULTS: Compared with H₂O₂ group and rapamycin group, the viability of H9c2 cells in astragalosides group was significantly increased (P<0.05). The shape of the H9c2 cells in astragalosides group was complete, the nuclei were stained with yellow-green fluorescence, and the chromatin was distributed evenly. The protein levels of p-mTOR and P70S6K in the H9c2 cells of astragalosides group were significantly increased (P<0.05), whereas the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H9c2 cells of astragalosides group were decreased significantly (P<0.05). CONCLUSION: Astragalosides enhance the viability, inhibit the apoptosis, increase the protein levels of p-mTOR and P70S6K, and decrease the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H₂O₂-induced rat myocardial H9c2 cells. The mechanism is related to the mTOR signaling pathway.     

Role of 17-AAG in inducing apoptosis and cell cycle arrest of HCT-15 cells

AIM: To investigate the effects of 17-AAG on apoptosis and cell cycle of HCT-15 cells and to clarify the related mechanisms. METHODS: MTT method was employed to evaluate the inhibitory effects of 17-AAG with Aifferent time and different doses on the proliferation of HCT-15 cells. The cells were stained with Annexin V-FITC/propidiumiodide and measured by flow cytometry. The expression of STAT3, cyclin D1, Cyt C, caspase 9 and caspase 3 at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS: Treatment with 17-AAG at concentration of 1.25~20 mg/L for 24 h and 48 h significantly inhibited the activity of HCT-15 cells at both time-and concentration-dependent manners. Treatment with 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h significantly induced apoptosis and cell cycle arrest of HCT-15 cells. The exposure of 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h to the HCT-15 cells significantly down-regulated the expression of STAT3 and cyclin D1 at mRNA and protein levels, but up-regulated Cyt C, caspase 9 and caspase 3 mRNA and protein in a concentration-dependent manner. CONCLUSION: 17-AAG inhibits the cell activity, induces apoptosis and G₁ arrest by down-regulating the expression of cyclin D1, and promoting the mitochondria apoptosis through STAT3 pathway.     

Adeno-associated virus type 9-mediated p65 silencing inhibits angiotensin II-induced apoptosis of H9c2 cells

AIM: To study the effect of p65 gene silencing by adeno-associated virus type 9 (AAV9)-mediated RNA interference on angiotensin Ⅱ (Ang Ⅱ; 10^-6 mol/L for 24 h)-induced apoptosis of rat ventricular H9c2 myocytes, and to elucidate the possible mechanism. METHODS: The H9c2 cells were transfected with rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA at multiplicity of infection (MOI)=4×10⁶ vg/cell. eGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of eGFP positive cells was determined by flow cytometry. The expression of p65 was determined by Western blot. CCK-8 assay was used to measured the viability of transfected H9c2 cells. The apoptosis of the cells transfected with the virus and with Ang Ⅱ stimulation was analyzed by flow cytometry. RESULTS: The cells began to exhibit eGFP expression on the 2nd day after transfection. The fluorescence intensity was increased over the time of transfection. eGFP expression reached the maximum on the 5th day, and the transfection efficiency was (52.7±1.9)% at this time point. Compared with blank control group, no significant effect of AAV9 on the viability of H9c2 cells was observed. In resting state, p65 in the H9c2 cells had a certain activity. After Ang Ⅱ stimulation, the activity of p65 was obviously increased, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited the expression of p65. The apoptosis of H9c2 cells in Ang Ⅱ stimulation group was significantly higher than that in blank control group, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited apoptosis of H9c2 cells. CONCLUSION: Transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibits the expression of p65 gene of NF-κB pathway in the H9c2 cells without causing cell growth inhibition, and reduces the apoptosis induced by Ang Ⅱ.     

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.     

APPL1 reduces injury of H9c2 cardiomyocytes induced by LPS

AIM:To study the effect of APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) on H9c2 cardiomyocyte injury induced by lipopolysaccharides (LPS). METHODS:The H9c2 cells were treated with LPS. RT-qPCR and Western blot were used to detect the expression of APPL1 in the H9c2 cells. The recombinant APPL1 lentiviral vector was used to transfect into the H9c2 cells. After LPS treatment, the over-expression efficiency was detected by RT-qPCR and Western blot. The viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein level of activated caspase-3 in the H9c2 cells was determined by Western blot. The content of malonaldehyde (MDA) in the H9c2 cells and the level of lactate dehydrogenase (LDH) in the culture medium were detected. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the level of reative oxygen species (ROS) in the H9c2 cells were also examined. RESULTS:The expression of APPL1 at mRNA and protein levels in LPS-treated H9c2 cells was decreased significantly (P<0.05). Over-expression of APPL1 by transfection of recombinant lentiviral vector significantly increased the level of APPL1 at mRNA and protein levels in the H9c2 cells with LPS treatment (P<0.05). LPS treatment reduced the viability, but increased the apoptotic rate of the H9c2 cells, the protein level of activated caspase-3, the content of MDA and the level of LDH in the culture medium. The activity of SOD and GSH-Px was reduced, while the level of ROS was increased as compared with control group (P<0.05). Over-expression of APPL1 elevated the viability of H9c2 cells treated with LPS, and the apoptotic rate and the protein level of activated caspase-3 were decreased. The content of MDA and the level of LDH in the culture medium were reduced, the activity of SOD and GSH-Px was elevated, and the level of ROS was reduced as compared with the H9c2 cells treated with LPS alone (P<0.05). CONCLUSION:Over-expression of APPL1 reduces oxidative damage and apoptosis of the H9c2 cells induced by LPS.     

CaMKII aggravates hypoxia/reoxygenation injury in H9c2 cells by activating apoptosis

AIM: To investigate the effect of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: H9c2 cells were randomized into 4 groups:control group, KN-93 (an inhibitor of CaMKⅡ; 1 μmol/L) treatment group, H/R group and H/R+KN-93 (1 μmol/L) treatment group. The cells in KN-93 group and KN-93+H/R group were pretreated with KN-93 for 2 h before the other treatment was performed. The viability of H9c2 cells in each group was measured by CCK-8 assay. Lactate dehydrogenase (LDH) activity in the culture medium was detected. The protein levels of phosphorylated CaMKⅡ (p-CaMKⅡ), phosphorylated phospholamban (p-PLN) and cleaved caspase-3 were determined by Western blot. The apoptosis was analyzed by TUNEL staining and the flow cytometry. RESULTS: No significant difference of all indexes tested between control group and KN-93 group was observed. H/R treatment significantly reduced the cell viability, and increased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). KN-93 (1 μmol/L) significantly increased the cell viability, and decreased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). CONCLUSION: CaMKⅡ aggravates hypoxia/reoxygenation injury in the H9c2 cells by activating apoptosis.     

Lentivirus-mediated CCDC80 knock-out inhibits proliferation of ovarian cancer cells by decreasing Aib1 expression

AIM: To explore the role of coiled-coil domain-conaining protein 80 (CCDC80) gene deletion in the proliferation and apoptosis of human ovarian cancer ES-2 cells. METHODS: Lentivirus-mediated CCDC80 deletion in ovarian cancer cells was conducted by CRISPR/Cas9 method. Genomic sequencing was used to detect knock-out efficiency. The proliferation and colony formation of CCDC80 deletion cells were determined by cell growth curve and soft agar assay. The migration of CCDC80 deletion cells was measured by cell scratch assay. The apoptosis and cell cycle were analyzed by Annexin V/PI staining and flow cytometry. The protein levels of p-histone H3 and p-ERK1/2 were determined by Western blot. Nude mouse model was established to detect the tumorigenic capacity of CCDC80 deletion cells in vivo. RESULTS: Genomic sequencing results showed that CCDC80 was efficiently knocked out in ES-2 cells. CCDC80 deletion significantly repressed the proliferation, migration and colony formation of ES-2 cells (P<0.01).CCDC80 deletion increased the apoptosis rate and affected G¹ and S progression (P<0.01).CCDC80 deletion repressed the cell proliferation (P<0.01) in vivo. IHC results showed that CCDC80 deletion increased DNA damage and decreased cell proliferation. Western blot results showed that the protein level of p-histone H3 was decreased, while the protein level of p-ERK1/2 was increased in CCDC80 deletion cells (P<0.01). qPCR results showed that CCDC80 deletion significantly decreased Aib1 mRNA expression (P<0.01). CONCLUSION: Genetically CCDC80 deletion represses ES-2 cell proliferation, migration and colony formation, and promotes cell apoptosis by decreasing Aib1 expression.