Protective effects of mangiferin against myocardial cell damage and its effects on the expression of NFATc4 / 中国药房

OBJECTIVE To study the protective effects of mangiferin against oxidative stress injury of myocardial cells induced by hydrogen peroxide （H2O2）， and its effects on the expression of nuclear factor of activated T cell cytoplasmic 4（NFATc4）. METHODS H9c2 myocardial cells were cultured in vitro and divided into blank group， H2O2 group， and 50， 100， 150 μmol/L mangiferin groups. Mangiferin groups were treated with different concentrations of mangiferin for 12 h， and then were subjected to H2O2 （200 μmol/L） stimulation for 12 hours together with the H2O2 group； relative survival rate was detected in each group， and the levels of superoxide dismutase （SOD）， catalase （CAT） and malondialdehyde （MDA） in cell supernatant and reactive oxygen species （ROS） in H9c2 cells were measured. Meanwhile， the expressions of apoptosis-related proteins [B cell lymphoma 2 （Bcl- 2）， Bcl-2 associated X protein （Bax）， cleaved caspase-3] and nuclear protein NFATc4 were determined. Furthermore， the NFATc4 interference sequence was transfected， and the effects of NFATc4 on oxidant stress indexes and apoptosis-related proteins in H2O2- induced myocardial cells were investigated. RESULTS Compared with blank group， relative cell viability， the levels of SOD and CAT， relative expression of Bcl-2 were decreased significantly， while the levels of MDA and ROS， relative expressions of Bax， cleaved caspase-3 and nuclear protein NFATc4 were increased significantly （P＜0.05 or P＜0.01）. Compared with the H2O2 group， the above indexes of 100 and 150 μmol/L mangiferin groups were reversed significantly （P＜0.05）. After the transfection of the NFATc4 interference sequence， the expression of nuclear protein NFATc4 was down-regulated significantly； the levels of MDA and SOD， the protein expressions of Bax and cleaved caspase-3 were all decreased/down-regulated significantly， while the levels of SOD and CAT， and the protein expression of Bcl-2 were all increased/up-regulated significantly， compared with H2O2 group （P＜ 0.05）. CONCLUSIONS Mangiferin can relieve H2O2-induced oxidative stress of H9c2 cells， reduce the apoptosis and inhibit the nuclear translocation of NFATc4， thereby alleviating myocardial cell damage； reducing the nuclear level of NFATc4 protein is related to reducing H2O2-induced oxidative stress and cell apoptosis.

Molecular mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion injury in H9c2 cardiomyocytes based on molecular docking / 中国中药杂志

The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.

Antioxidant activities of 10 active compounds from Clematis filamentosa Dunn. and their protective effects on H / 中国药理学通报

Aim To investigate the protective effects of the 10 compounds from Clematis filamentosa Dunn, on H

Investigation of Host-guest Complexation Between 4-Sulfonylcalix 6 Arene and 15 Alkaloids from Traditional Chinese Medicines and Inhibitory Effect of Binding System on Proliferation of HepG2 and H9c2 Cells / 中国实验方剂学杂志

Objective:To investigate bonding ability between 4-sulfonylcalix ［6］ arene （SCA6） and 15 alkaloids （matrine， allomatrine， dauricine， daurisoline， quinidine， quinine， crotaline， vincristine， gelsemine， koumine， tetrandrine， aloperine， oxymatrine， sophocarpine and sinomenine）， and to evaluate viability<italic> in vitro</italic> of HepG2 and H9c2 cells with 12 alkaloids/SCA6 bonding systems （except allomatrine， oxymatrine， sinomenine）. Method:Fluorescence competitive titration was used to determine the binding constants of alkaloids and SCA6， the inhibitory effect of alkaloid/SCA6 complex on proliferation of HepG2 and H9c2 cells was investigated by cell counting kit-8 （CCK-8）. Result:All the 15 alkaloids had good bonding with SCA6 at the ratio of 1∶1 （the binding constants >1×10⁵ mol·L^-1， <italic>R</italic>²>0.98）， the aloperine （quinolizidine alkaloids） and SCA6 had the biggest binding constant （20.55×10⁶ mol·L^-1）. In addition to gelsemine， crotaline， matrine and sophocarpine， 8 alkaloids （including aloperine， tetrandrine， dauricine， daurisoline， quinidine， quinine， vincristine and koumine） exhibited significant anti-tumor effects on HepG2 cells. Except for daurisoline， the anti-proliferation effect of the other 11 alkaloids before and after binding with SCA6 had no difference in HepG2 cells. In addition to gelsemine， crotaline， matrine and sophocarpine， the anti-proliferation effect of the other 8 alkaloids before and after binding with SCA6 had no difference in H9c2 cells. Conclusion:SCA6 shows intense binding ability with bisbenzylisoquinoline， quinolizidine and indole alkaloids. It can improve the solubility of alkaloids without affecting their anti-tumor activity， which provides a reference for subsequent related applications of SCA6 as a drug delivery carrier.

Study on the Anti-apoptotic Effect of Curcumin on H 2O2-induced H 9c2 Cardiomyocyte Injury and the Regulation of NF-κB Signaling Pathway / 中国药房

OBJECTIVE：To investigate the anti-apoptotic effect of curcumin on H 2O2-induced H 9c2 cardiomyocyte injury and the regulation of NF-κB signaling pathway. METHODS：H9c2 cardiomyocyte were randomly divided into normal control group ， injury model group ，curcumin low-dose ，medium-dose and high-dose groups （25，50，100 μmol/L）. Normal control group didn ’t received any intervention. The cells in injury model group were induced with 50 μmol/L H2O2 for 12 h to establish the injury model. The cells in curcumin groups were treated with relevant concentration of drugs for 24 h，and then induced with 50 μmol/L H2O2 for 12 h. After cultured for 24 h，survival rate and apoptotic rate of cells were measured by MTT method and TUNEL method ；SOD activity and MDA content were determined by WTS- 8 assay and color test ；relative fluorescence intensity of LC 3 positive expression was detected by immunofluorescence method ；mRNA expression of NF-κB p65 in cells was detected by real-time PCR ； Western blotting assay was used to detect the protein expression of NF-κB p65 and p-NF-κB p65 in cells. RESULTS ：Compared with normal control group ，survival rate and SOD activity were decreased significantly in injury model group ，while apoptotic rate ， MDA content ，relative fluorescence intensity of LC 3 positive expression ，mRNA expression of NF-κB，protein expression of NF-κ B p 65 and p-NF-κB p65 as well as p-NF-κB/NF-κB were increased significantly（P＜0.05）. Compared with injury model group ， survival rates and SOD activities were increased significantly in curcumin groups ，while apoptotic rates ，MDA contents ，relative fluorescence intensities of LC 3 positive expression ，mRNA expression of LC 3 positive cells ，protein expression of NF-κB p65 and p-NF-κ B p65 as well as p-NF-κ B p65/NF-κ B p65 were decreased significantly （P＜0.05）. CONCLUSIONS ：Curcumin can increase the survival rate of H 2O2-induced H 9c2 cardiomyocyte injury ，decrease its apoptotic rate ，increase SOD activity and decrease MDA content in cardiomyocytes. Above effects may be related to the regulation of NF-κB signaling pathway.

In-vitro cardiovascular protective activity of a new achillinoside from Achillea alpina

Abstract Achillinoside was isolated from methanol extract of Achillea alpina L., Asteraceae. The structure of the compound was characterized based on various spectrum data, including IR, HR-ESI-MS, 1D and 2D NMR. The cardiovascular protective effect of achillinoside was tested on H2O2-induced H9c2 cells. In our research, achillinoside could increase the cell viability dose-dependently in H2O2-induced H9c2 cells. In addition, the levels of caspase-3/9 cells were significantly decreased in H2O2 and achillinoside incubated H9c2 cells.

Shenxiong Glucose Injection inhibits H_2O_2-induced apoptosis of H9c2 cells by activating PI3K/AKT pathway / 中国中药杂志

The aim of this paper was to explore the mechanism of Shenxiong Glucose Injection antagonizing apoptosis of H9 c2 cells induced by H_2O_2. H9 c2 cells were pretreated with 1. 7%,3. 4% and 6. 8% Shenxiong Glucose Injection,and then H_2O_2 was introduced to induce apoptosis in vitro. Cell viability was detected by MTS assay,morphological changes of apoptosis were observed by AO/EB fluorescence staining,apoptosis rate was detected by Annexin/PI method,cell expression profile was detected by gene chip technology,the mRNA of PIK3 CA,Bcl-2,Bax,caspase-3 and GAPDH were detected by qRT-PCR,the protein expression levels of PIK3 CA,AKT,P-AKT,Bcl-2,Bax and caspase-3 were detected by Western blot,and the contents of LDH and MDA were detected by kit. The results showed that Shenxiong Glucose Injection of different concentrations significantly increased the viability of H9 c2 cells treated with H_2O_2( P<0. 01),and reversed H_2O_2-induced apoptosis( P< 0. 01). The microarray experiments showed that 138 genes were altered in H9 c2 cells after treatment with Shenxiong Glucose Injection. The differential expression fold of PIK3 CA associated with PI3 K/AKT pathway was 3. 59. The results of qRT-PCR and Western blot showed that Shenxiong Glucose Injection could down-regulate the mRNA and protein expression levels of caspase-3( P<0. 01),up-regulate the mRNA and protein expression level of PIK3 CA and Bcl-2( P<0. 01),and up-regulate the phosphorylation levels of AKT( P<0. 01) in H_2O_2-treated H9 c2 cells. The protective effect of Shenxiong Glucose Injection on H_2O_2 cells injury was significantly inhibited by LY294002,a PI3 K/AKT pathway inhibitor. The results suggested that Shenxiong Glucose Injection may inhibit H_2O_2-induced H9 c2 cells apoptosis by regulating PI3 K/AKT signaling pathway.

Aconitine ameliorates cardiomyocyte hypertrophy induced by angiotensin Ⅱ / 中国中药杂志

This paper was aimed to investigate the inhibitory effect of aconitine(AC) on angiotensin Ⅱ(Ang Ⅱ)-induced H9 c2 cell hypertrophy and explore its mechanism of action. The model of hypertrophy was induced by Ang Ⅱ(1×10-6 mol·L-1),and cardiomyocytes were incubated with different concentrations of AC. Western blot was used to quantify the protein expression levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain(β-MHC),and α-smooth muscle actin(α-SMA). Real-time quantitative PCR(qRT-PCR) was used to quantify the mRNA expression levels of cardiac hypertrophic markers ANP,BNP and β-MHC. In addition,the fluorescence intensity of the F-actin marker,an important component of myofibrils,was detected by using laser confocal microscope. AC could significantly reverse the increase of total protein content in H9 c2 cells induced by Ang Ⅱ; qRT-PCR results showed that AC could significantly inhibit the ANP,BNP and β-MHC mRNA up-regulation induced by AngⅡ. Western blot results showed that AC could significantly inhibit the ANP,BNP and β-MHC protein up-regulation induced by AngⅡ. In addition,F-actin expression induced by Ang Ⅱ could be inhibited by AC,and multiple indicators of cardiomyocyte hypertrophy induced by Ang Ⅱ could be down-regulated,indicating that AC may inhibit cardiac hypertrophy by inhibiting the expression of hypertrophic factors,providing new clues for exploring the cardiovascular protection of AC.

Protective effects of exogenous HS to the recovery of hypoxia post-conditioning on aged H9C2 cells and the underling mechanisms / 中国应用生理学杂志

OBJECTIVE@#To investigate the recovery of protective effects of exogenous hydrogen sulfide (HS) on hypoxia post-conditioning in aged H9C2 cells and its mechanism.@*METHODS@#H9C2 cells (cardiomyocytes line) were treated with 30 μmol/L hydrogen peroxide (HO) for 2 hours, then cultured for 3 days in order to induce cellular aging. Aged H9C2 cells were randomly divided into 5 groups (=8):Control group (Control), hypoxia/reoxygenation group (H/R), H/R + NaHS group, hypoxia post-conditioning (PC) group, PC+NaHS group. H/R model:the cells were exposed to hypoxic culture medium (serum and sugar free medium, pH=6.8) for 3 hours and then cultured at normal condition for 6 hours. PC model:at the end of hypoxia for 3 hours, the cells were exposed to normoxic culture solution for 5 minutes, then the cells were placed in hypoxic solution for 5 minutes, the cycle above-mentioned was repeated 3 times and followed by reoxygenation for 6 hours. Advanced glycation end products (AGEs) content and caspase-3 activity were detected by ELISA. The cell viability was observed by cell counting kit-8 (CCK-8). The reactive oxygen species (ROS) levels were analyzed using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The apoptotic rate was determined through Hoechst 33342 staining. The mRNA levels of relative gene expression were detected by real-time PCR.@*RESULTS@#Thirty μmol/L HO induced H9C2 cell senescence while did not lead to apoptosis. Compared with control group, cell viability was decreased, the apoptotic rate、levels of ROS and the mRNA of caspase-3, caspase-9 and Bcl-2 were increased in H/R and PC groups (<0.01). There were no differences in the above indexes between PC group and H/R group. Supplementation of NaHS increased cell viability and decreased apoptotic rate and oxidative stress. The effects of PC + NaHS on the above indexes were better than those of H/R+NaHS group.@*CONCLUSIONS@#Exogenous HS can restore the protective effect of PC on the aged H9C2 cells, and its mechanism is related to the inhibition of oxidative stress and apoptosis.

Protective Effects of Aqueous Extract from Descurainia sophia on Doxorubicin-Induced Cardiomyocyte Injuryvia Suppressing Apoptosis and Oxidative Stress / 中国药学杂志

OBJECTIVE: To study the protective effects and mechanisms of aqueous extract from Descurainia sophia on doxorubicin(Dox)-induced cardiomyocyte injury.METHODS: The Dox induced H9c2 cell apoptotic model was established, then the cells were divided into normal group (NC), model group (Dox), positive group (resveratrol,RSV), and different doses of aqueous extract of Descurainia sophia (DS). The viability of H9c2 cells was detected by MTT assay. The apoptosis rate, mitochondrial membrane potential and reactive oxygen species (ROS) level were detected by flow cytometry. The levels of T-SOD, LDH, MDA and GSH-PX were measured. And the protein expression levels of caspase-3, Bcl-2, Bax and p53 were detected by western blot, HPLC-MS identification of aqueous extract from Descurainia sophia.RESULTS: After treating with DS products, the survival rate of H9c2 was increased (P<0.01), the apoptosis rate was significantly decreased (P<0.01),mitochondrial membrane potential was significantly increased (P<0.01), the level of ROS was significantly decreased (P<0.05), T-SOD and GSH-PX activities were significantly increased (P<0.01), the levels of LDH and MDA content were significantly decreased(P<0.01). Moreover, DS reduced the expression of caspase-3 (P<0.01), regulated the expression of Bax/Bcl-2 (P<0.01), decreased the expression of p53 (P<0.05). Seven components were identified from DS by HPLC/MS analysis.CONCLUSION: DS can effectively protect cardiomyocyte, and its mechanism is probably associated with correcting functional disorders of oxidative stress of cardiomyocytes, and inhibit mitochondrial apoptotic pathway.

Inhibitory effects of Pin1 silencing by siRNA on apoptosis of H9c2 cardiomyo-cytes induced by hypoxia/reoxygenation / 中国免疫学杂志

Objective:Pin1 plays an important role in the pathogenesis of cardiovascular disease,our study aims to investigate the effects of Pin1 silencing by siRNA on H9c2 apoptosis induced by hypoxia/reoxygenation.Methods:H9c2 cells were cultured and subjected to a hypoxia/reoxygenation (H/R) condition in vitro,mimicking ischemic/reperfusion injury in vivo.The mRNA and protein expression of Pin1 were detected by RT-qPCR and Western blot.H9c2 cells were divided into control group,H/R group,H/R+Pin1 siRNA group,H/R+scramble siRNA group.MTT and flow cytometry with Annexin V-FITC/PI staining were respectively performed to detect cell viability and apoptosis.The expression of Bax and Bcl-2 were measured by Western blot.The activity of Caspase-3 was detected by automatic biochemistry analytic instrument.Results:The mRNA and protein levels of Pin1 were highly expressed in the cells of H/R group.Transfection with Pin1 siRNA strikingly inhibited the expression of Pin1.Compared with H/R group,Pin1 siRNA markedly increased cell viability,decreased the cell apoptosis and the Caspase-3 activity.Furthermore,the increased Bcl-2,decreased Bax and the ratio of Bcl-2 to Bax were observed in Pin1 siRNA group (P<0.05) compared with H/R group.Conclusion:Downregulation of Pin1 protects hypoxia/reoxygenation-injured H9c2 cells from apoptosis,which is possibly through the upregulation of Bcl-2 and downregulation of Bax and Caspase-3 activity.

Protective effect of PEDF on H9C2 myocardial cells under hypoxia condition / 重庆医学

Objective To investigate the protective effects and possible mechanism of pigment epithelium derived faetor (PEDF) on myocardial cells H9C2 under hypoxia and serum-free condition.Methods H9C2 cells were culture in vitro and performed the hypoxia and serum-free processing.The cells were divided into the control group (H9C2),hypoxia group (hypoxia + H9C2),PEDF group(hypoxia+H9C2 +PEDF) and mitochondrial fission inhibitor(Mdivi-1) group(hypoxia+h9C2+Mivi-1).The apoptotic rate was detected by TUNNEL staining.The proteins levels of dynamin related peptide1 (Drp1) and cleaved-caspase 3 were measured by Western blot.Electron Microscopy and MitoTracker Red were used to detect the mitochondria morphology,the mitochondrial membrane potential was evaluated by cationic dye JC-1.MitoSOXTM was used to detect mitochondrial reactive oxygen species (ROS).Results Hypoxia induced mitochondrial fission(P＜0.05).The hypoxia group (6 h) and control group had statistical difference(P＜0.05).PEDF reduces mitochondrial fission under hypoxia condition(P＜0.05),which had statistical difference between the PEDF group and hypoxia group (6 h)(P＜0.05).PEDF and Mdivi-1 could decrease cell apoptosis under hypoxia condition(24 h),compared with the hypoxia group,the difference was statistically significant(P＜0.05).Conclusion PEDF decrease cell apoptosis by inhibiting H9C2 cells mitochondrial fission under hypoxia condition.

Protective effect of PEDF on H9C2 myocardial cells under hypoxia condition / 重庆医学

Objective To investigate the protective effects and possible mechanism of pigment epithelium derived faetor (PEDF) on myocardial cells H9C2 under hypoxia and serum-free condition.Methods H9C2 cells were culture in vitro and performed the hypoxia and serum-free processing.The cells were divided into the control group (H9C2),hypoxia group (hypoxia + H9C2),PEDF group(hypoxia+H9C2 +PEDF) and mitochondrial fission inhibitor(Mdivi-1) group(hypoxia+h9C2+Mivi-1).The apoptotic rate was detected by TUNNEL staining.The proteins levels of dynamin related peptide1 (Drp1) and cleaved-caspase 3 were measured by Western blot.Electron Microscopy and MitoTracker Red were used to detect the mitochondria morphology,the mitochondrial membrane potential was evaluated by cationic dye JC-1.MitoSOXTM was used to detect mitochondrial reactive oxygen species (ROS).Results Hypoxia induced mitochondrial fission(P＜0.05).The hypoxia group (6 h) and control group had statistical difference(P＜0.05).PEDF reduces mitochondrial fission under hypoxia condition(P＜0.05),which had statistical difference between the PEDF group and hypoxia group (6 h)(P＜0.05).PEDF and Mdivi-1 could decrease cell apoptosis under hypoxia condition(24 h),compared with the hypoxia group,the difference was statistically significant(P＜0.05).Conclusion PEDF decrease cell apoptosis by inhibiting H9C2 cells mitochondrial fission under hypoxia condition.

Cytotoxicity of ophiopogonin D′ for rat H9c2 cardiomyocytes / 中国药理学与毒理学杂志

OBJECTIVE To study the cardiotoxicity of ophiopogonin D′(OPD′) for rat H9c2 cardio? myocytes. METHODS H9c2 cells were exposed to OPD′ 0.1, 1, 5, 10, 20, 25 and 50 μmol·L-1 for 24 h. Cell viability was examined by MTS assay, and the morphological changes in H9c2 cells were quanti? fied. The cell nucleus injury was examined by high content immune fluorescence screening and the morphological changes were observed under a fluorescence microscope. After treatment with OPD′ 0.1, 1, 5 and 10 μmol·L- 1 for 24 h, the effect on reactive oxygen species (ROS), mitochondrial mem? brane potential(MMP) and apoptosis was detected by flow cytometry. RESULTS The viability was sig? nificantly reduced following exposure to OPD′ 0.1, 1, 5, 10, 20, 25 and 50 μmol·L- 1 (P<0.05,P<0.01). The IC50 value was 9.9 μmol ·L- 1 and cell shrinkage and apoptosis occurred. The levels of ROS and apoptosis rate of H9c2 cells were significantly increased after exposure to OPD′ 0.1, 1, 5 and 10 μmol·L-1 for 24 h (P<0.05,P<0.01) and MMP markedly declined (P<0.05,P<0.01). CONCLUSION OPD′ has significent cytotoxicity on H9c2 cells. It may be related to inducing apopotsis pathways.

Protective effect of ecdysterone on H9c2 cells against oxidative stress / 中国病理生理杂志

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 cardio-myocytes in H 2 O2 group and high , middle and low doses of EDS groups were exposed to H 2 O2 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 colorime-try.The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cy-tometry 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 H 2 O2 , but were decreased by EDS treatment in a dose-dependent man-ner.The levels of SOD and mitochondrial membrane potential of the H 9c2 cells in H2 O2 group were reduced significantly compared with control group , but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mito-chondrial membrane potential in H 2 O2-treated H9c2 cells.The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H2 O2 group showed significant elevation in comparison with control group , and the protein expression of Bcl-2 de-clined in H2 O2 group compared with control group , but high, middle and low doses of ecdysterone treatments down-regula-ted the protein levels of Bax , cleaved caspase-3 and up-regulated the expression of Bcl-2 in H2 O2-treated H9c2 cells. CONCLUSION:Ecdysterone attenuates the effect of H 2O2-induced oxidative stress on H9c2 cardiomyocytes.The mecha-nism may be involved in scavenging oxidative stress products , increasing antioxidant enzyme activity and improving mito-chondrial function .

N-acetylcysteine protects H9c2 cells against injuries induced by methyl-glyoxal / 中国病理生理杂志

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 di-acetate (DCFH-DA) staining.Mitochondrial membrane potential (MMP) was measured by rhodamine 123 (Rh123) stai-ning.The morphological changes in apoptotic cardiomyocytes were detected by Hoechst 33258 staining.RESULTS: Du-ring 100~800 μmol/L concentration range , MG caused significantly reduced viability of the H 9c2 cells in a dose-depend-ent manner.NAC had a protective effect on H9c2 cells against the injuries induced by MG during 500~1 500μmol/L con-centration 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, in-cluding 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 ap-optosis.

Ophiopogonin D attenuates angiotensin Ⅱ-induced myocardial hypertrophy by reducing autophagy / 中国药理学通报

Aim To study whether Ophiopogonin D has an effect inhibitory on myocardial hypertrophy induced by AngiotensinⅡand its possible mechanism. Methods Rat myocardial cell line H9 c2 were cultured in vitro. The effect of Ophiopogonin D on cell vitality was tested by;H9 c2 cells were treated with AngⅡ 1μmol ·L-1 after 24h to induce the cardiac hypertrophy,then it was co-treated with different concentrations of Ophio-pogonin D were added for 24h. After above,the total protein content was detected by BCA method;Quantita-tive real-time PCR ( qRT-PCR ) technique was used to examine the expression of marker genes BNP and β-MHC mRNA ,which representing the function of hear-ing; Western blot was used method to detect the ex-pression of autophagy protein LC3 B and high-through-put screening technology was emptoyed to verify it. In addi-tion, the changes of mitochondrial membrane po-tential in H9c2 myocardial cell were also examined. Results The cell viability results showed that H9 c-2 cells exposed to different concentrations of AngⅡ had no significant effect on vitality compared with the con-trol group after 24 h,but high concentrations of Ophio-pogonin D ( 50 ~100μmol · L-1 ) could obviously in-hibit the cell activity. Ot-her experimental results showed that myocardial cells treated with AngⅡ for 24h could cause myocardial hypertrophy,which appar-ently displayed the growth level of specific hypertrophic gene mRNA expression and the marked increase of the total protein expression. As hypertrophy was activated by AngⅡ, cells autophagy would be significantly en-hanced at the same time, more-over, the mitochondrial membrane potential would be reduced. But the effects of Ophiopogonin D could significantly reverse those pathological changes. Conclusion All above experi-mental results indicate that Ophiopogonin D can in-hibitmyocardial hypertrophy induced by AngⅡand pos-sibly plays a critical role in cardiovascular protection.

Structural modification and biological evaluation of monomeric compounds from Aralia elata / 中草药

Objective: To modify the structure of natural monomeric compound based on oleanolic acid-3-O-β-D-glucuronide from medicinal plant Aralia elata and to evaluate the myocardial protection activity of the derivatives. Methods: Taking oleanolic acid or ursolic acid as material, the target compounds were prepared by five steps of reactions and evaluated for myocardial protection effects by H9c2 cells in vitro. Results: Ten derivatives F1-F10 were synthesized. The structures of the target compounds were identified by spectrum. Pharmacological results showed that all of the compounds had the different levels potency of myocardial protection effects in cells. In particular, compounds F3 showed the significant myocardial protection activity compared to lead compound. Conclusion: The new compounds F1-F10 which show the potential of biological activity in myocardial protection, have not been reported in any literature and deserved further research.

Role of RISK signal pathway in reducing cardiomyocytes hypoxia/reoxygenation injury induced by S1 P postconditioning / 中国药理学通报

Aim To study the protective effects of sphingosine 1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation in reperfusion injury salvage kinase ( RISK ) signal path-way. Methods The cultured rat H9c2 cells were ran-domly divided into seven groups: ( 1 ) control group;(2) hypoxia/reoxygenation (H/R) group; (3) S1P group;(4) S1P+LY294002 group(S1P+LY); (5) LY group; ( 6 ) S1 P +PD98059 group ( S1 P +PD );(7) PD group. The viability of H9c2 cells was detec-ted using MTT method. The content of MDA in the cultured medium and the activity of T-SOD and Mn-SOD were measured with colorimetry. The concentra-tion of intracellular free calcium ion was detected by confocal microscopy. The rate of cell apoptosis was de-termined by flow cytometric analysis. Western blot was used to assess phosphorylation of Akt and ERK1/2 in H9c2 cells. Results Compared with the H/R group, S1P significantly increased vaibility of cells, lowered the rate of apoptosis, decreased the content of MDA in the culture medium, increased the activity of T-SOD and Mn-SOD, reduced concentration of intracellular calcium and increased the phosphorylation of Akt and ERK1/2 . When added LY294002 or PD98059 , the effects of S1P above were inhibited. Conclusion S1P protects H9 c2 cells against hypoxia/reoxygenation inju-ry. The protection of S1P was inhibited by LY294002, the inhibitor of PI3 K/Akt and PD98059 , the inhibitor of ERK1/2 . S1 P protects H9 c2 cells against hypoxia/reoxygenation injury via RISK pathway.

Construction of a lentiviral low expression of miR-139-5 p vector and validation of its transduction efficiency in H9 c2 cells / 中国药理学通报

Aim To construct a lentiviral low expres-sion of miR-139-5 p vector and validate its expression efficiency in H9c2 cells. Method Target sequence was designed according to the sequence of rat miR-139-5p. Oligonucleotide duplex was synthesized and cloned into the lentiviral vector pGC-LV. The recombi-nant lentiviral vector, pHelper 1. 0, and pHelper 2. 0 were co-transfected into 293T cells, packaging virus. Then H9 c2 cells were infected with the supernatant containing lentiviral particles, and its infection effi-ciency and miR-139-5 p expression were determined by fluorescent microscope and real-time quantitative PCR, respectively. Results A lentiviral low expression of miR-139-5p vector was successfully constructed. The infection efficiency in H9c2 cells reached over 95%, and the relative expression of miR-139-5p was significantly down-regulated. Conclusion The lentiviral low expression of miR-139-5p vector is successfully constructed, and the expression of miR-139-5p in infected H9c2 cells is inhibited effectively.