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1.
LI Bo  ZHENG Zhi  CHEN Peng-yuan 《园艺学报》2019,35(8):1526-1531
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.  相似文献   

2.
AIM: To observe the expression of microRNA-126-5p during myocardial injury and its role in myocardial cell injury induced by adriamycin (also called doxorubicin, DOX). METHODS: The BALB/c mouse model of DOX-induced acute and chronic myocardial injury was established via intraperitoneal injection of DOX. HE staining was applied to observe the morphological changes of myocardial tissues. Lactate dehydrogenase (LDH) in serum was detected and PowerLab system was used to detect the influence of DOX on the changes of ±dp/dtmax. The expression of microRNA-126-5p in injured myocardial tissues and the H9c2 cells exposed to DOX was detected by real-time PCR. Gain-and loss-of-function experiments were conducted to detect the role of microRNA-126-5p in H9c2 cells treated with DOX on LDH release and caspase-3 activation. RESULTS: In acute and chronic DOX myocardial damage models in mice, HE staining showed disarranged myocardial fibers, dissolved myofibril and inflammatory cell infiltration. Higher serum LDH level and lower ±dp/dtmax in DOX-treated mice than those in normal mice were found. Compared with the normal mice, the expression level of microRNA-126-5p was significant increased in the myocardium with DOX-induced injury. Similarly, the expression level of microRNA-126-5p was significant increased in the H9c2 cells treated with DOX. In addition, over-expression of microRNA-126-5p decreased cell viability and promoted apoptosis, while microRNA-126-5p ablation promoted the viability and inhibited the apoptosis of H9c2 cells. CONCLUSION: The microRNA-126-5p expression is up-regulated in myocardial injury induced by DOX, and microRNA-126-5p inhibits cell viability and promotes apoptosis induced by DOX.  相似文献   

3.
AIM: To investigate the effect and mechanism of osthole on increasing the cytotoxicity of doxorubicin (DOX) to prostate cancer cells. METHODS: MTT assay was performed to evaluate the viability of LNCaP cells treated with osthole and DOX. The protein expression of silent information regulator 1 (SIRT1), p53, acetylated p53 and Puma, as well as release of cytochrome C and activation of caspase-9 and caspase-3 in the LNCaP cells treated with osthole and DOX were determined by Western blot. The apoptosis of the LNCaP cells treated with osthole and DOX was analyzed by flow cytometry. RESULTS: Osthole significantly increased the cytotoxicity of DOX against p53-wildtype prostate cancer cell line LNCaP. Osthole significantly inhibited the expression of SIRT1 in the LNCaP cells. Transfection with SIRT1 plasmid decreased the cytotoxicity of osthole and DOX co-treatment against LNCaP cells. Combination with osthole and DOX significantly induced the over-expression and acetylation of p53. Transfection with p53 siRNA significantly decreased the synergistic effect of osthole on cytotoxicity of DOX-treated LNCaP cells. Combination with osthole and DOX significantly induced the release of cytochrome C into the cytoplasm from mitochondria, followed by activation of caspase-9 and its downstream molecule caspase-3, thus leading to cell apoptosis in the LNCaP cells. CONCLUSION: Osthole promotes the p53-dependent apoptosis in DOX-treated prostate cancer LNCaP cells by down-regulating the expression of SIRT1.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.  相似文献   

9.
LIU Dan  SUN Dian  XU Min  ZHOU Min  WU Xiao-mu  HE Ming 《园艺学报》2012,28(12):2113-2118
AIM: To explore the role of AMP-activated protein kinase α2 subunit (AMPKα2) gene in chloride-mediated anoxia/reoxygenation (A/R) injury by transfection of short-hairpin RNA (shRNA) expression vector targeting to AMPKα2 gene into H9c2 cardiomyocytes. METHODS: Recombinant shRNA expression vector pSuper-AMPKα2 targeting to AMPKα2 gene was constructed and transfected into H9c2 cardiomyocytes. The protein expression of AMPKα2 was determined by Western blotting. The cells were divided into 5 groups: control group, A/R group, Cl--free A/R group, pSuper+Cl--free A/R group and pSuper-AMPKα2 shRNA+Cl--free A/R group. After treatment, the cell viability was detected by MTT assay. LDH activity was analyzed with an automatic biochemical analyzer. The apoptotic rate and the level of intracellular ROS was measured by flow cytometry. The activity of SOD and GSH-Px was analyzed by a colorimetric method. RESULTS: The result of sequencing proved that the recombinant plasmid pSuper-AMPKα2 shRNA was correctly constructed. The protein level of AMPKα2 significantly decreased after the plasmid was transfected into the cardiomyocytes. Compared with A/R group, the cell viability and the activity of SOD and GSH-Px were significantly increased, while the activity of LDH, apoptotic rate and ROS production were significantly decreased in Cl--free A/R group. The protective effect of Cl--free solution on the A/R-induced injury of cardiomyocytes was abolished, and the ROS production was increased and the activity of SOD and GSH-Px was decreased after the cells were transfected with pSuper-AMPKα2 shRNA. CONCLUSION: Recombinant plasmid pSuper-AMPKα2 shRNA is successfully constructed, and silencing of AMPKα2 gene abolishes the protective effect of Cl--free solution on A/R injury.  相似文献   

10.
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.  相似文献   

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12.
AIM: To investigate the effects of silent information regulator 1 (SIRT1) over-expression on the apoptosis and the level of reactive oxygen species (ROS) in high glucose-induced H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were transfected with empty plasmid (pcDNA3.1-NC) and SIRT1 over-expression plasmid (pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3K and AKT protein levels were down-regulated (P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3K and AKT protein levels (P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3K/AKT signaling pathway.  相似文献   

13.
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.  相似文献   

14.
LIU Qi-fang  HUANG Jing  XU Min 《园艺学报》2018,34(7):1214-1221
AIM:To investigate the role and mechanism of microRNA-25 (miR-25) in apoptosis of H9c2 cells induced by hypoxia/reoxygenation. METHODS:The H9c2 cells with over-expression of miR-25 were treated with hypo-xia/reoxygenation. Real-time PCR was used to detect the expression of miR-25 and high mobility group box-1 (HMGB1) mRNA. Western blot was performed to examine the protein expression levels of HMGB1, Bcl-2 and cleaved caspase-3. Flow cytometry was used to analyze the proportion of apoptotic cells and the cell cycle. Dual-luciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9c2 cells. Moreover, the H9c2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation to verify whether HMGB1 participated in the regulation of apoptosis of H9c2 cells. RESULTS:Over-expression of miR-25 significantly reduced the protein expression levels of HMGB1 and cleaved caspase-3, and increased the expression of Bcl-2 and the entrance into S phase in H9c2 cells induced by hypoxia/reoxygenation (P<0.01). The result of dual-luciferase reporter assay showed that compared with the control group, transfection with HMGB1-3' UTR-psi-CHECK2+miR-25 mimic strongly inhibited the luciferase activity (P<0.05). After the H9c2 cells transfected with HMGB1-shRNA was treated with hypoxia/reoxygenation, the expression of Bcl-2 was up-regulated, the expression of cleaved caspase-3 was down-regulated, and the cells in S phase were increased (P<0.05). CONCLUSION:miR-25 reduces apoptosis of H9c2 cells induced by hypoxia/reoxygenation, and its mechanism may be related with the inhibition of HMGB1 expression via interacting with its 3'-UTR.  相似文献   

15.
LIU Fang  LIU Ji  ZHANG Ying 《园艺学报》2018,34(8):1376-1382
AIM: This study aims to explore the effect of abietic acid (AA) on advanced glycosylation end products (AGEs)-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes. METHODS: H9c2 cells were divided into 5 groups. The cells in control group were treated with saline for 24 h. The cells in AGEs treatment group were treated with AGEs (100 mg/L) for 24 h. The cells in AGEs+AA (10, 25 and 50 μmol/L) groups were simulta-neously treated with AGEs (100 mg/L) and AA (10, 25 and 50 μmol/L) for 24 h. The cell viability was measured by MTT assay. The protein levels of myoglobin (Mb), creatine kinase MB isoenzyme (CK-MB), cardiac troponin I (cTnI), C/EBP homologous protein (CHOP), cleaved caspase-12, GADD34, BiP, LC3, P62 and beclin 1 were determined by Western blot. The levels of lactate dehydrogenase (LDH) were measured by ELASA. The apoptosis was analyzed by flow cytometry. RESULTS: The low concentration (<50 μmol/L) of abietic acid had no obvious effect on the viability of H9c2 cells. The high concentration (>50 μmol/L) of abietic acid decreased the viability of H9c2 cells. The levels of Mb, CK-MB, cTnI and LDH in AGEs group were higher than those in control group (P<0.05). Compared with AGEs group, the levels of Mb, CK-MB, cTnI and LDH in AGEs+AA (10, 25 and 50 μmol/L) groups were obviously reduced (P<0.05). Abietic acid at concentrations of 10, 25 and 50 μmol/L inhibited AGEs-induced apoptosis, elevated the protein levels of CHOP and cleaved caspase-12, and attenuated expression of GADD34 and BiP (P<0.05). Moreover, abietic acid at concentrations of 10, 25 and 50 μmol/L suppressed AGEs-induced decreased ratio of LC3-Ⅱ/LC3-Ⅰ and expression of beclin 1, and enhanced the expression of P62 (P<0.05). 3-Methyladenine, an inhibitor of autophagy, reversed the effect of abietic acid on the protein levels of LC3, Mb, cleaved caspase-12 and BiP (P<0.05). CONCLUSION: Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes via inducing autophagy.  相似文献   

16.
AIM: To investigate the effect of tanshinone ⅡA on doxorubicin-induced rat H9c2 cardiomyocyte injury. METHODS: The H9c2 cardiomyocytes were treated with tanshinone ⅡA and/or doxorubicin with or without AMPK inhibitor dorsomorphin. The cell viability was measured by CCK-8 assay, the LDH release was examined for evaluating the cell injury, autophagy was analyzed by immunofluorescence staining, and AMPK activation was determined by Western blot. RESULTS: Compared with control group, the viability of H9c2 cells was decreased, the release of LDH was increased, the autophagy degree was increased, and AMPK activation was inhibited after treatment with doxorubicin (P<0.05). Compared with doxorubicin group, the treatment with tanshinone ⅡA restored the cell viability, reduced the release of LDH, further increased autophagy degree, and activated AMPK in the H9c2 cells (P<0.05). AMPK inhibitor dorsomorphin attenuated the abilities of tanshinone ⅡA to restore the cell viability, reduce the release of LDH, and increase autophagy degree in the H9c2 cells (P<0.05). CONCLUSION: Tanshinone ⅡA attenuates doxorubicin-induced injury in H9c2 cardiomyocytes, and its mechanism might be related to AMPK-mediated autophagy, which provides experimental and theoretical basis for the clinical application of tanshinone ⅡA in the prevention and treatment of doxorubicin-induced cardiomyocyte injury.  相似文献   

17.
AIM: To investigate the protective effect of astragaloside IV (ASIV) on angiotensin II (Ang II)- induced apoptosis of H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were treated with different concentrations of Ang II and ASIV. The effects of Ang II and ASIV on the viability of H9c2 cells was measured by CCK-8 assay. The optimum concentration of Ang II was 1 μmol/L and the concentrations of ASIV were 25, 50 and 100 μmol/L. The H9c2 cells was divided into 6 groups:control group, ASIV group, Ang II group, Ang II+ASIV (25 μmol/L) group, Ang II+ASIV 50 (μmol/L) group and Ang II+ASIV (100 μmol/L) group. The morphological changes of the H9c2 cells were observed under inverted phase-contrast microscope. Apoptosis was detected by TUNEL assay. The generation of reactive oxygen species (ROS) was detected by DCFH-DA staining. The protein expression of Bax, Bcl-2, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. H9c2 cardiomyocytes were transfected with negative control shRNA (NC) or Nrf2-shRNA (shRNA), and the cells were divided into 8 groups:NC+control group, NC+AngⅡgroup, NC+ASIV group, NC+AngⅡ+ASIV group, shRNA+control group, shRNA+AngⅡgroup, shRNA+ASIV group and shRNA+AngⅡ+ASIV group. ROS level was detected by ROS detection kit. The protein expression of Nrf2 and HO-1 was determined by Western blot. RESULTS: Ang II decreased the viability of H9c2 cells in a concentration-dependent manner (P<0.05). ASIV reversed the effect of Ang II on the viability of H9c2 cells in a concentration-dependent manner (P<0.05). Compared with control group, the apoptotic rate, the level of ROS and the protein expression of Bax in Ang II group were increased significantly, while the protein expression of Bcl-2, Nrf2 and HO-1 was decreased significantly (P<0.05). Compared with Ang II group, ASIV reversed the increase in apoptotic rate of H9c2 cells induced by Ang II in a concentration-dependent manner, reduced ROS level, down-regulated the protein expression of Bax and up-regulated the protein expression of Bcl-2, Nrf2 and HO-1 (P<0.05). After shRNA transfection, the effects of ASIV decreasing ROS production induced by Ang II and up-regulating the expression of Nrf2 and HO-1 were eliminated. CONCLUSION: ASIV protects H9c2 cardiomyocytes from apoptosis induced by Ang II, which may be related to reducing ROS generation and mediating the Nrf2/HO-1 signaling pathway.  相似文献   

18.
AIM:To investigate the effects of Homer1a over-expression on the apoptosis and AMP-activated protein kinase (AMPK) protein expression in mechanically injured neurons. METHODS:The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1a group. Neuron models with mechanical injury were constructed and infected with the Homer1a over-expression vector. The mRNA expression of Homer1a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase (LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS:Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased (P<0.05), and Homer1a expression at mRNA and protein levels was significantly increased (P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased (P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased (P<0.05). CONCLUSION:Over-expression of Homer1a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.  相似文献   

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20.
AIM:To study the effect of Toll-like receptor 4 (TLR4) on the secretion of inflammatory factors in the pancreatic acinar AR42J cells induced by lipopolysaccharides (LPS). METHODS:The rat pancreatic acinar AR42J cells were treated with LPS. The expression of TLR4 at mRNA and protein levels was determined by real-time PCR and Western blot. The lentivirus carrying TLR4 small interfering RNA (siRNA) was used to infect the AR42J cells. Under LPS stimulation, the interference efficacy was measured by real-time PCR and Western blot. The cell viability was measured by MTT assay, and the leakage rate of lactate dehydrogenase (LDH) was examined by dinitrophenylhydrazine method. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture medium were detected by ELISA, and the malondialdehyed (MDA) content in supernatant was measured by thiobarbituric acid method. The activity of superoxide dismutase (SOD) in the supernatant was determined by xanthine oxidation, and the activity of glutathione peroxidase (GSH-Px) and catalase (CAT) was detected by colorimetry. RESULTS:The expression of TLR4 at mRNA and protein levels in LPS-treated AR42J cells was significantly increased (P<0.05). Infection with TLR4 siRNA-carrying lentivirus significantly inhibited the expression of TLR4 at mRNA and protein levels in the AR42J cells under LPS stimulation(P<0.05). The viability of AR42J cells was decreased after LPS treatment. The leakage rate of LDH was increased, the levels of IL-1β and TNF-α secreted by the AR42J cells were increased, the content of MDA was increased in the supernatant, and the activity of SOD, GSH-Px and CAT was reduced (P<0.05). After knock-down of TLR4 expression, the viability of AR42J cells was increased under LPS stimulation, the LDH leakage rate, secreted levels of IL-1β and TNF-α, and the content of MDA in cell culture medium were decreased, and the SOD, GSH-Px and CAT levels were increased (P<0.05). CONCLUSION:LPS induces the expression of TLR4 in the pancreatic acinar AR42J cells. Knock-down of TLR4 expression reduces the secretion of inflammatory factors IL-1β and TNF-α, and attenuates the oxidative damage in pancreatic acinar AR42J cells induced by LPS.  相似文献   

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