獐牙菜苦苷对H2O2诱导大鼠心肌细胞凋亡的保护作用

[目的]观察獐牙菜苦苷对氧化应激引起的大鼠心肌细胞凋亡的保护作用,初步探讨獐牙菜苦苷抗心肌细胞氧化应激的保护作用机制。[方法]用差速贴壁法培养SD乳鼠心肌细胞,检测不同给药组中SD乳鼠细胞心肌细胞存活率以及培养液中超氧化物岐化酶（SOD）、丙二醛（MDA）含量;用免疫细胞化学检测心肌细胞的相关凋亡蛋白bcl-2、Bax表达;用吖啶橙荧光染色检测细胞的凋亡情况。[结果]3种不同浓度獐牙菜苦苷均能提高心肌细胞存活率和细胞上清液中SOD含量,且能降低MDA含量,与H2O2刺激组比较有显著性差异（P〈0.05;P〈0.01）。[结论]獐牙菜苦苷对H2O2诱导损伤的心肌细胞有保护作用,其作用机制可能是通过影响氧化系统以及调控特异性蛋白来抑制心肌细胞凋亡。     

Neonatal rat cardiomyocyte hypertrophy is induced by tumor necrosis factor-α through PI3K-IP3R-Ca2+ pathways

AIM: To investigate the role of PI3K-IP₃R-Ca²⁺ pathways in cardiomyocyte hypertrophy induced by tumor necrosis factor-α (TNF-α). METHODS: Myocardial cells of neonatal rats were cultured in vitro. The hypertrophic model was induced by TNF-α. The protein content was assayed with Lowry's method. The volumes of the cardiomyocytes were detected by computer photograph analysis system. The protein synthesis was determined by the method of[³H]-leucine incorporation.[Ca²⁺]_i transient was measured by Till image system with cell-loading Fura-2/AM. RESULTS: LY294002, a PI3K inhibitor, significantly suppressed the amplitude elevation of the spontaneous[Ca²⁺]_i transients induced by TNF-α in cultured ventricular myocytes from neonatal rats. The effect was similar to that of LY294002+2-APB (P>0.05), but lower than that in LY294002+ryanodine group (P<0.05). LY294002 significantly reduced the enhancements of protein content,[³H]-leucine incorporation and cell size induced by TNF-α. The effect was similar to that in 2-APB+LY294002 group, but higher than that in 2-APB group and lower than that in ryanodine+LY294002 group. CONCLUSION: TNF-α induces cardiac hypertrophy through PI3K-IP₃R-Ca²⁺ pathways.     

Role of ubiquitin E3 ligase TRIM10 in regulating cardiomyocyte hypertrophy

AIM: To explore the role of ubiquitin E3 ligase tripartite motif 10 (TRIM10) in the development of cardiomyocyte hypertrophy. METHODS: Primary cultured neonatal rat cardiomyocytes (NRCMs) were infected with siRNA-TRIM10, siRNA-control, Ad-TRIM10 or Ad-GFP for 24 h respectively, and then stimulated with phenylephrine (PE) for additional 24 h. The protein levels of TRIM10, AKT and ERK1/2 were determined by Western blot. The size of the NRCMs was measured by immunofluorescence staining. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was detected by RT-qPCR. RESULTS: Compared with the control, PE treatment significantly increased the protein expression of TRIM10. Moreover, transfection of NRCMs with siRNA-TRIM10 markedly inhibited cardiomyocyte size, the mRNA expression of ANP and BNP, and the phosphorylation levels of AKT and ERK as compared with siRNA-control after PE treatment. In contrast, overexpression of TRIM10 significantly enhanced PE-induced hypertrophic effect on NRCMs above. CONCLUSION: TRIM10 regulates cardiomyocyte hypertrophy partially through AKT and ERK signaling pathways.     

Effects of atorvastatin on release of endothelial microparticles and myocardial apoptosis in rats with acute myocardial infarction

AIM: To investigate the effect of atorvastatin(AT) on the release of endothelial microparticles(EMP) and myocardial apoptosis in the rats with myocardial infarction. METHODS: SD male rats(n=24) were randomly divided into 3 groups:sham operation(sham) group, myocardial infarction(MI) group and MI+AT group. The rat model of acute myocardial infarction was prepared by coronary artery ligation. At 2 h and 24 h after modeling, the peripheral blood was collected to detect creatine kinase-MB(CK-MB) and cardiac troponin T(cTnT). The circulating levels of EMP were measured by flow cytometry. The myocardial apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay. RESULTS: At 2 h after modeling, the level of CK-MB was significantly increased in MI group compared with sham group, and the level of EMP and the myocardial apoptotic rate were significantly increased in MI group and MI+AT group compared with sham group. At 24 h after modeling, the level of EMP was significantly increased in MI group compared with sham group. The levels of CK-MB, cTnT, EMP and the myocardial apoptotic rate were significantly decreased in MI+AT group compared with MI group. Moreover, the level of CK-MB in MI group was significantly increased at 24 h compared with that at 2 h after modeling. The levels of CK-MB, cTnT and EMP were significantly decreased in MI+AT group at 24 h compared with those at 2 h after modeling. CONCLUSION: Ator-vastatin may reduce the level of EMP and the myocardial apoptotic rate in the rats with acute myocardial infarction, indicating that atorvastatin plays a role in protecting endothelium.     

High glucose induces H9c2 cardiomyocyte hypertrophy through Ca2+-CaN-NFAT3 signaling pathway

AIM: To study the morphological changes of cardiac H9c2 cells during the developmental process of fetal rat. METHODS: Embryonic rat heart-derived H9c2 cells were maintained in DMEM supplemented with 10% fetal bovine serum. The H9c2 cells were plated at a density of 6000 cells/cm and divided into 5 groups: H9c2 cells were treated with 5 mmol/L glucose, 25 mmol/L glucose, 50 mmol/L glucose, Norvasc (25 nmol/L)+25 mmol/L glucose, or Norvasc (25 nmol/L)+50 mmol/L glucose for 48 h. The morphology of H9c2 cells was observed. The cell surface area was measured by Image-Pro Plus 6.1 software. Fluorescence spectrophotometry was used to detect the concentration of intracellular calcium ion ([Ca²⁺]_i)in the cardiomyocytes. The concentration of CaN in the cell was measured by ELISA. The mRNA expression of CaNAβ, NFAT3 and β-MHC in the cells was detected by real-time PCR. The protein levels of CaNAβ, NFAT3 and β-MHC in cultural H9c2 cells were detected by Western blot. RESULTS: The mean area of the cells, the mean fluorescence value of [Ca²⁺]_i and the concentration of CaN in 25 mmol/L glucose group were higher than those in 5 mmol/L glucose group, and those were lower than those in 50 mmol/L glucose group. After treated with Norvasc, those results decreased significantly. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels in 25 mmol/L glucose group was higher than those in 5 mmol/L glucose group, but was lower than those in 50 mmol/L glucose group. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels decreased significantly in Norvasc treatment group. CONCLUSION: Ca²⁺-CaN-NFAT3 signaling pathway is perhaps involved in high glucose-induced H9c2 cardiomyocyte hypertrophy.     

PQS attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibiting endoplasmic reticulum stress

AIM:To study the effect of Panax quinquefoliumsaponin (PQS) on cardiomyocyte apoptosis induced by thapsigargin (TG). METHODS:Primary cultured cardiomyocytes from neonatal SD rats were divided into control group, TG group, PQS (40 mg/L, 80 mg/L and 160 mg/L)+TG group, si-PERK+TG group, and mock+TG group. The cells were treated with 1 μmol/L TG for 24 h to induce apoptosis. The PERKgene in the cardiomyocytes was knocked down by RNAi. The cell viability was detected by CCK-8 assay. Apoptosis was analyzed by flow cytometry. Wes-tern blotting was used to determine the expression of ERS molecules GRP78, CRT, ATF4 and CHOP, anti-apoptosis protein Bcl-2 and pro-apoptosis protein Bax. RESULTS:Compared with control group, TG significantly and the apoptosis, reduced the cell viability (P<0.05), increased the phosphorylation of PERK and eIF2α, increased the expression of GRP78, CRT, ATF4, CHOP and pro-apoptosis protein Bax, and decreased the expression of anti-apoptosis protein Bcl-2 (P<0.05). Compared with TG group, PQS treatment (160 mg/L) significantly reduced the apoptosis and increased the cell viability (P<0.05). All the 3 different concentrations of PQS significantly increased the expression of anti-apoptosis protein Bcl-2 and reduced the expression of pro-apoptosis protein Bax (P<0.05) in a dose-dependent manner. PQS pretreatment and knockdown of PERK both reduced the protein levels of GRP78, CRT, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP and pro-apoptosis protein Bax, and increased the expression of anti-apoptosis protein Bcl-2 (P<0.05). CONCLUSION: PQS at concentration of 160 mg/L attenuated cardiomyocyte apoptosis induced by TG. PQS had the similar effect as PERKknockdown on cardiomyocyte apoptosis. The mechanism may be associated with inhibiting PERK-eIF2α-ATF4-CHOP pathway of ERS-related apoptosis.     

Effects of PPARα activation on AngⅡ-induced cardiomyocyte hypertrophy and interaction of NFATc4 with p65-NFκB

AIM：To investigate the effects of fenofibrate on angiotensin Ⅱ (AngⅡ)-induced cardiomyocyte hypertrophy. METHODS：Primary neonatal cardiomyocytes were pretreated with fenofibrate (10 μmol/L) for 1 h followed by stimulation with AngⅡ (100 nmol/L). The mRNA levels of ANF, BNP and β-MHC were measured by real-time PCR. Western blotting was employed to determine the nuclear translocations of NFATc4 and p65-NFκB. Co-immunoprecipitation was used to investigate the interaction of NFATc4 with p65-NFκB in the nucleus of cardiomyocytes. In addition, the DNA binding activity of NFATc4 on the BNP promoter was determined by EMSA. RESULTS：Fenofibrate significantly inhibited AngⅡ-induced cardiomyocyte hypertrophy. Fenofibrate treatment inhibited the nuclear translocations of NFATc4 and p65-NFκB, as well as the interactions of NFATc4 with p65-NFκB in the nucleus of cardiomyocytes induced by AngⅡ. Fenofibrate inhibited the binding activity of NFATc4 with the BNP promoter, which was strengthened by AngⅡ. CONCLUSION： Fenofibrate enhances the interaction of NFATc4 with PPARα, decreases the interaction of NFATc4 with p65-NFκB in the nucleus of cardiomyocytes, and inhibits the DNA binding activity of NFATc4 induced by AngⅡ, which may be the important mechanisms of fenofibrate on inhibiting cardiac hypertrophy.     

Bio-effects of salusins on isolated rat heart and neonatal cardiomyocytes

AIM: To investigate the bio-effects of salusins on rat heart and cardiomyocytes. METHODS: The cardiac function was determined by multipurpose polygraph in isolated rat heart treated with various concentrations of salusin-α or salusin-β.[⁴⁵Ca²⁺] and[³H]-Leu incorporation were determined in cultured neonatal rat cardiomyocytes with β-liquid scintillation counter. RESULTS: 10^-12-10^-7mol/L salusin-α and salusin-β had no effects on isolated rat cardiac function. However, salusin-α and salusin-β stimulated uptake and[³H]-Leu incorporation. The [⁴⁵Ca²⁺] uptake induced by salusins were inhibited by nicardipine, and were synergistically increased by endothelin-1. The[³H]-Leu incorporation induced by salusin-α and salusin-β was inhibited by nicardipine, FK506 (a special inhibitor of carcineulin), PD₉₈₀₅₉ (inhibitor of MAPK) and chelerthine (inhibitor of PKC). The effects of salusin-β[⁴⁵Ca²⁺] on uptake was stronger than those of salusin-α. But there were no statistical difference in[³H]-Leu incorporation between salusin-α and salusin-β. CONCLUSIONS: Salusin-α and salusin-β did not affect directly cardiac function in rat hearts. But salusins improved calcium uptake and protein synthesize in neonatal rat cardiomyocytes. Those effects of salusins were related with calcium channel, carcinuelin, MAPK and PKC signal pathways. Salusins may be the regulatory factors for myocardium growth and hypertrophy.     

Effects of P38 MAPK signaling pathway on anacardic acid attenuating mouse cardiomyocyte hypertrophy induced by phenylephrine

AIM: To investigate the roles of P38 mitogen-activated protein kinase (P38 MAPK) in the process of anacardic acid (AA) attenuating mouse cardiomyocyte hypertrophy induced by phenylephrine (PE). METHODS: Cardiomyocyte hypertrophy was induced by PE in primary neonatal mouse myocardial cells. According to random number table method, the experiments were designed in 6 groups as following: control group, PE+ DMSO group, PE group, PE+ AA group, PE+ AA+ P38 inhibitor group and PE+ P38 inhibitor group. Mouse myocardial cells were collected after intervention for 48 h. The protein levels of p-P38, acetylated histone H3 at lysine 9 (H3K9ac) and atrial natriuretic peptide (ANP) were determined by Western blot. The interaction between p-P38 and H3K9ac was verified by co-immunoprecipitation (Co-IP). The mRNA expression of myocyte enhancer factor 2C (MEF2C) were tested by RT-qPCR. Mouse myocardial cell surface area was observed by immunofluorescence staining. RESULTS: The results of Western blot showed that the protein levels of p-P38 and H3K9ac in PE group were significantly increased compared with control group (P<0.05), and the levels of MEF2C mRNA and ANP protein in PE group were apparently increased compared with control group (P<0.05). However, P38 inhibitor and histone acetylase inhibitor AA attenuated P38 phosphorylation and H3K9 acetylation induced by PE, and down-regulated the over-expression of MEF2C and ANP in the mouse myocardial cells (P<0.05). The results of immunofluorescence staining showed that PE apparently increased mouse myocardial cell surface area (P<0.05), while P38 inhibitor and AA attenuated cardiomyocyte hypertrophy induced by PE (P<0.05). CONCLUSION: The mechanism of AA attenuating cardiomyocyte hypertrophy induced by PE may be related to the regulation of histone acetylation modification imbalance mediated by P38 MAPK signaling pathway.     

山羊骨髓间充质干细胞生物学性能鉴定及诱导分化为心肌细胞的研究

研究了关中奶山羊骨髓间充质干细胞的分离、培养和生物学特性,并在体外诱导分化为心肌细胞表型。结果表明,山羊MSCs具有很好增殖能力;能够耐受反复冷冻和解冻;表达细胞表面标志CD44、CD105、CD166,弱表达CD34、CD106,而不表达CD14、CD45。该类细胞用5-氮胞苷诱导分化后,能够表达心肌α-actin,并呈PAS染色阳性。