Molecular mechanism underlining ethanol-induced chloride currents in nasopharyngeal carcinoma cells

AIM：To study the effects and mechanisms of ethanol on chloride channels in poorly differentiated nasopharyngeal carcinoma CNE-2Z cells. METHODS：The effect of ethanol on the cell growth was analyzed by MTT assay. The technique of whole-cell patch-clamp was used to detect the chloride current. The characteristics of the chloride current were analyzed by using the chloride channel blockers. The siRNA technique was used to analyze the molecular basis of the ethanol-sensitive chloride channels. RESULTS：Under isotonic conditions, the background current was weak and stable. Ethanol at concentrations of 0.17~170 mmol/L activated a chloride current in a concentration-dependent manner (an inverted U-shape), with a maximum effect at the concentration of 17 mmol/L. The currents showed obviously outward rectification and were susceptible to extracellular hypertonicity and the chloride channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). ClC-3 siRNA obviously decreased the currents activated by ethanol. CONCLUSION：Extracellular ethanol induces chloride currents through activating the ClC-3 chloride channels.     

Volume-activated chloride currents in human acute lymphoblastic leukemia Molt4 cells

AIM: To characterize the chloride current activated by extracellular hypotonic stress in human acute lymphoblastic leukemia Molt4 cells.METHODS: The technique of whole-cell patch clamp recording was used to detect the volume-activated Cl^- current in Molt4 cells. The characteristics of the current were investigated.RESULTS: The background Cl^- current was weak and stable under isotonic condition. However, a large Cl^- current was induced by exposure of the cells to 47% hypotonic solution. The current showed a characteristic of outward rectification. No voltage-dependent inactivation and time-dependent inactivation were observed. The current was sensitive to the change of cell volume and was inhibited by extracellular hypertonic solution. Extracellular tamoxifen, which is one of the chloride channel blockers, significantly inhibited the current. The effects of tamoxifen were almost equal for both inward and outward currents (P>0.05).CONCLUSION: There are volume-activated chloride channels on the cell membrane of Molt4 cells. Exposure of the cells to a hypotonic solution activates the chloride channels and induces a volume-activated chloride current. The volume-activated Cl^- channels are sensitive to tamoxifen in Molt4 cells.     

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.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

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

Effect of 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.     

Role of Cl- in regulatory volume decrease of nasopharyngeal carcinoma cells

AIM:To clarify the role of Cl^- in regulatory volume decrease (RVD) of nasopharyngeal carcinoma cells (CNE-2Z).METHODS:Analysis of living cell images was used to detect the volume changes following exposure to hypotonic solution. Iron replacement and block of iron channels were also applied in the present study. RESULTS:Extracelluar hypotonic treatment made the cells swell and induced RVD. The RVD was correlated positively to the swelling in the range of 160-230 mOsmol/L. Substitution of gluconate for Cl^- in perfusing solutions markedly increased RVD. Depletion of cellular Cl^- abolished, and chloride channel blockers inhibited RVD. CONCLUSION:Cl^- is the key iron to establish the RVD in CNE-2Z cells. Activation of Cl^- channels and Cl^- efflux are the major mechanisms of RVD.     

Effects of FKBP12.6 gene on intracellular Ca2+ concentration in mouse H9c2 (2-1) myocardial cells transfected by ultrasound-mediated destruction of microbubbles

AIM:To investigate the changes of intracellular calcium ion (Ca²⁺) concentration in mouse H9c2 (2-1) cells transfected with or without FK506 binding protein 12.6(FKBP12.6) gene by ultrasound mediated destruction of microbubbles. METHODS:The pcDNA3.1-FKBP12.6 plasmid, mingled with albumin-coated microbubbles agents, was transfected into H9c2 (2-1) cells by ultrasound-mediated destruction of microbubbles. The H9c2 (2-1) cell growth state was investigated by inverted microscope. The changes of intracellular Ca²⁺ concentration was determined by laser scanning confocal microscope. The FKBP12.6 protein expression was checked by immunohistochemistry. RESULTS:As compared with control cells, the H9c2 (2-1) cells, transfected with FKBP12.6 gene, grew better, had higher gross intracellular Ca²⁺ concentration. CONCLUSION:FKBP12.6 gene augments Ca²⁺ concentration in mouse H9c2 (2-1) cells, enhances the contractibility of the myocardial cell, which may be helpful to improve the myocardial dysfunction.     

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.     

Effects of PTEN on apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation

AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on the apoptosis, oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation (A/R). METHODS: The H9c2 cells were used to establish a model of A/R. The H9c2 cells were transfected with PTEN small interfering RNA (siRNA) and negative control. After A/R, the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Xanthine oxidase method was used to determine superoxide dismutase (SOD) activity. The content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The lactate dehydrogenase (LDH) activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in culture supernatant were examined by ELISA. The protein levels of cleaved caspase-3, Bax and FasL in the cells were determined by Western blot. RESULTS: After A/R, the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells (P<0.05). The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA (P<0.05). The viability of H9c2 cells was decreased after A/R, while the apoptotic rate was increased. The protein levels of cleaved caspase-3, Bax and FasL were increased in the cells. The MDA level was elevated, the activity of SOD was decreased, and the levels of LDH, TNF-α, IL-1β and IL-6 in the culture supernatant were increased (P<0.05). Down-regulation of PTEN partly antagonized the effects of A/R on the viability, apoptotic rate, MDA content, SOD activity, and the levels of LDH, TNF-α, IL-1β and IL-6 in culture supernatant. CONCLUSION: Down-regulation of PTEN attenuates oxidative damage induced by A/R, reduces apoptosis and secretion levels of TNF-α, IL-1β and IL-6 in the H9c2 cells.     

Protective effects and mechanisms of losartan on apoptosis of H9c2 cells induced by β-adrenergic stimulation in vitro

AIM: To investigate the protective effect of losartan (Los) on apoptosis of H9c2 cells induced by isoprenaline (ISO), and to discover its related mechanism. METHODS: H9c2 cells cultured on plastic plates were divided into control, ISO, ISO+Los, ISO+Los+LY294002 and DMSO groups. Cell apoptosis was evaluated by flow cytometery and agarose gel electrophoresis. The mRNA levels of bax, bcl-2 and caspase-9 were detected by RT-PCR and the expressions of phosphorylated and total Akt (p-Akt and t-Akt) were assessed by Western blotting. The cAMP was measured by radioimmunoassay. RESULTS: ISO at concentration of 10 μmol/L induced apoptosis of H9c2 with an increase in bax/bcl-2, caspase-9 and cAMP. Addition of 10 μmol/L losartan inhibited apoptosis obviously with a decrease in bax/bcl-2, caspase-9 and cAMP. A significant increase in p-Akt was observed, and its protein level was elevated. LY294002 at concentration of 1 μmol/L abolished the protective effects of losartan on ISO-induced apoptosis in H9c2 cells. CONCLUSION: ISO might induce H9c2 cell apoptosis through stimulation of β-adrenergic receptor (β-AR). Los inhibits downstream signaling of β-AR, and promotes the activation of Akt. Subsequently it might attenuate the apoptosis induced by β-adrenergic stimulation of ISO.     

Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells via PI3K/Akt signaling pathway

AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.     

AT1-AA induces autophagy and apoptosis of H9c2 cardiomyocytes through oxidative stress

AIM: To investigate the effects of angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) on oxidative stress, autophagy and apoptosis in H9c2 cells, and to analyze the possible mechanism. METHODS: The rat H9c2 cells were cultured in vitro. The effect of AT1-AA at different concentrations for different time on the cell viability was measured by CCK-8 assay. Upon the optimum concentration (10^-5 mol/L) and time point (24 h) determined in this stu-dy, the experssion levels of autophagy- and apoptosis-related proteins were detected by Western blot, and the levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) were examined by oxidative stress kits. RESULTS: AT1-AA decreased cell viability in a concentration- and time-dependent manner, promoted apoptosis, and up-regulated the levels of autophagy and oxidative stress (P<0.05). The apoptosis of H9c2 cells induced by AT1-AA was decreased after pretreatment with autophagy inhibitor 3-methyladenine (P<0.05). The levels of autophagy and apoptosis in the H9c2 cells pretreated with α-lipoic acid were decreased (P<0.05). Pretreatment with angiotensin Ⅱtype 1 receptor inhibitor telmisartan inhibited oxidative stress, and significantly decreased the levels of autophagy and apoptosis induced by AT1-AA in the H9c2 cells (P<0.05). CONCLUSION: AT1-AA induces autophagy and apoptosis of H9c2 cells through oxidative stress.     

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.     

Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic pro-perties

AIM：To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thyroid follicular epithelial (FRT) cells and its electrophysiologic properties. METHODS：The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression, the quantity and ratio of lipid/DNA complexes were optimized. The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16A protein was associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS：The results of double digestion and sequencing indicated that TMEM16A was cloned into pUB6/V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L. CONCLUSION：The protein expression of TMEM16A in the FRT cells was observed. TMEM16A is the molecular identity of calcium-activated chloride channels.     

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.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

Expression and role of TRPM2 in H9N2 swine influenza virus infection-induced mouse pulmonary microvascular endothelial cell damage

AIM: To investigate the expression and role of transient receptor potential cation channel M2 (TRPM2) in H9N2 swine influenza virus (H9N2-SIV) infection-induced mouse pulmonary microvascular endothelial cell (PMEVC) damage. METHODS: At 24 h and 48 h after PMEVC (3 parallel test in each group) was infected by H9N2-SIV, the superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, reactive oxygen species (ROS) level and nitric oxide synthase (NOS) activity were measured according to the manufacturer's instructions. The ultrastructural changes of PMVEC were observed under electron microscope. Transendothelial electrical resistance (TEER) and the percentage of horse radish peroxidase (HRP) influx was measure by Transwell culture. The expression of TRPM2 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptosis of PMVEC was observed with Annexin V/PI staining. RESULTS: The SOD and GSH levels were decreased significantly in H9N2-SIV-infected PMEVC as compared with control group(P<0.05). However, NOS activity and ROS level were increased dramatically (P<0.01). The organelles of PMVEC infected by H9N2-SIV were decreased sharply, and vacuolization in mitochondria and apoptosis were also observed under electron microscope. The value of TEER was decreased significantly and the percentage of HRP influx was increased dramatically with the H9N2-SIV infection (P<0.01). The expression of TRPM2 at mRNA and protein levels in H9N2-SIV-infected PMVEC was increased dramatically at 24 h and 48 h as compared with control group (P<0.01). Annexin V/PI double staining showed that the cytomembrane was stained by great green fluorescence and some nuclei were stained by red fluorescence in H9N2-SIV-infected PMVEC. However, less fluorescence stained PMVEC were found in control group. CONCLUSION: The expression of TRPM2 at mRNA and protein levels is increased significantly, which is involved in cell damage and apoptosis induced by oxidative stress of H9N2-SIV infection.