促红细胞生成素对乳鼠心肌细胞肥大及P13K/Akt-eNOS信号转导通路的影响

目的 探讨促红细胞生成素(EPO)对血管紧张素Ⅱ(AngⅡ)诱导的肥大心肌细胞的影响,以及磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸激酶(Akt)-内皮型一氧化氮合酶(eNOS)信号转导通路在其中的作用.方法 分离乳鼠心肌细胞,利用AngⅡ诱导建立心肌细胞肥大模型,以心肌细胞表面积和心钠素(ANF)mRNA表达作为心肌细胞肥大观察指标.观察不同浓度EPO对肥大心肌细胞的影响,并利用PI3K抑制剂LY294002和一氧化氮合酶抑制剂L-NAME对其相关机制进行探讨,I司时对细胞培养液中一氧化氮(NO)浓度进行检测,蛋白免疫印迹法检测磷酸化Akt(p-Akt)、Akt、磷酸化eNOS(p-eNOS)和eNOS蛋白表达情况.结果 20 U/ml EPO能抑制由AngⅡ诱导的心肌细胞肥大,表现为心肌细胞表面积和ANF mRNA表达均减少(P＜0.05).EPO能激活Akt,促进eNOS及p-eNOS表达增加(均P＜0.05),并使NO合成增加(P＜0.01).LY294002和L-NAME能逆转EPO的抗心肌细胞肥大作用,减少NO产最(P＜0.05).蛋白免疫印迹法榆测显示,LY294002能够抑制EPO对p-Akt、p-eNOS和eNOS蛋白表达的促进作用,而L-NAME能抑制eNOS的磷酸化(均P＜0.05).结论 EPO能够抑制AngⅡ诱导的心肌细胞肥大,该作用可能是通过激活P13K/Akt信号转导通路,促进eNOS表达与活化,从而促进NO的合成来实现的.     

促红细胞生成素对新生大鼠心脏成纤维细胞氧化应激时一氧化氮合酶系统的影响

目的:探讨促红细胞生成素(EPO)对心脏成纤维细胞(CFs)氧化应激时一氧化氮合酶(NOS)系统的影响,以及PI3-K/Akt信号途径在其中的作用。方法:应用胰酶和胶原酶双酶法分离培养新生大鼠CFs,EPO、过氧化氢(H2O2)和PI3-K抑制剂LY294002不同因素干预。化学酶法检测CFs培养液中的NO浓度以及总NOS和其亚型的活性。Western blot检测Akt、p-Akt、eNOS、iNOS蛋白的表达。结果:氧化应激使CFs中iNOS的表达显著上调,活性增强;eNOS蛋白的表达明显下降。同时CFs培养液中NO的合成增加显著,达到(25.94±2.57)μmol/L,是对照组的4倍多(P<0.01)。EPO显著抑制CFs氧化应激时iNOS蛋白的表达,同时上调eNOS蛋白,总的NO浓度也显著下降。Akt磷酸化形式p-Akt的表达水平也明显提高。PI3-K抑制剂LY294002能阻断EPO促进eNOS和p-Akt蛋白表达的作用,但EPO抑制iNOS的作用不能完全被阻断。结论:EPO能促进新生大鼠CFs氧化应激时eNOS蛋白的合成,抑制iNOS蛋白的表达。其促进eNOS的表达是通过激活PI3-K/Akt细胞信号途径来实现,抑制iNOS的机制有待进一步探讨。     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

二甲双胍抑制血管紧张素Ⅱ诱导的成年大鼠心脏成纤维细胞增殖

目的　观察二甲双胍对血管紧张素Ⅱ（AngⅡ）诱导的成年大鼠心脏成纤维细胞（CF）增殖的影响，并探讨其可能机制。方法　通过胰酶、胶原酶Ⅱ联合消化法获取成年大鼠CF，AngⅡ（100　nmol/L）刺激CF建立细胞增殖模型，并给予不同浓度（10、50及200　μmol/L）二甲双胍进行干预。采用噻唑蓝法观察CF的增殖情况，EdU掺入法测定CF的DNA合成能力。免疫印迹法检测内皮型一氧化氮合酶（eNOS）、磷酸化内皮型一氧化氮合酶（p-eNOS）蛋白表达情况，硝酸还原酶法测定CF培养上清一氧化氮（NO）含量。结果　AngⅡ刺激48　h能够明显促进成年大鼠CF增殖（P<0.05），二甲双胍呈浓度依赖性抑制AngⅡ诱导的CF增殖，二甲双胍呈浓度依赖性增加CF内eNOS磷酸化水平和NO含量（P<0.05），eNOS抑制剂N-硝基-L-精氨酸甲酯（L-NAME）能阻断二甲双胍抑制AngⅡ诱导的CF增殖的作用（P<0.05）。结论　二甲双胍能抑制AngⅡ诱导的成年大鼠CF增殖，与二甲双胍激活CF内eNOS/NO通路有关。     

辛伐他汀对非酒精性脂肪性肝病大鼠肝组织纤维化的影响及分子机制

目的 探讨辛伐他汀对非酒精性脂肪性肝病(NAFLD)肝组织纤维化模型及肝星状细胞的作用及其分子机制.方法 ①体内实验应用高脂饮食建立NAFLD肝组织纤维化大鼠模型,并用辛伐他汀干预,RT-PCR法和Western印迹检测大鼠肝组织中内皮型一氧化氮合酶(eNOS)、诱导型NOS(iNOS)和Ⅰ型胶原mRNA和蛋白的表达.②体外实验采用促进脂肪细胞分化的培养基诱导人肝星状细胞株LX-2细胞获得静止表型,分别用转化生长因子β1( TGF-β1)、NOS抑制剂亚硝基左旋精氨酸甲酯(L-NAME)、辛伐他汀、TGF-β1+辛伐他汀、L-NAME+辛伐他汀处理静止型LX-2细胞,RT-PCR法和Western印迹检测各组LX-2细胞中eNOS、iNOS、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原mRNA及蛋白的变化.结果 ①随造模时间延长,模型组大鼠肝组织eNOS mRNA和蛋白表达逐渐减少,iNOS和Ⅰ型胶原mRNA和蛋白表达逐渐增加,与正常对照组比较差异有统计学意义(P分别＜0.05和0.01).与24周末模型组相比,辛伐他汀干预组大鼠肝组织eNOS mRNA和蛋白的表达分别增加(0.30±0.02比0.24±0.01和0.45±0.04比0.22±0.02,P值均＜0.05),iNOS mRNA和蛋白的表达分别减少,Ⅰ型胶原mRNA和蛋白表达分别减少(P值均＜0.05).模型组大鼠肝组织中eNOS mRNA和蛋白表达与Ⅰ型胶原mRNA和蛋白表达均呈负相关(P值均＜0.01);iNOS mRNA和蛋白表达与Ⅰ型胶原mRNA和蛋白表达均呈正相关(P值均＜0.01).②体外培养LX-2细胞中,L-NAME能抑制LX-2细胞活化,减少eNOS和iNOS的表达,增加α-SMA和Ⅰ型胶原表达,与TGF-β1作用一致;辛伐他汀能直接增加静止型及活化型LX-2细胞中eNOS的表达,减少iNOS的表达,维持其静止表型,抑制其活化.结论 辛伐他汀通过增加LX-2细胞中eNOS表达,减少iNOS表达,减少α-SMA和Ⅰ型胶原生成,抑制或逆转肝纤维化发生和发展.     

瑞舒伐他汀对TGF-β1诱导的新生大鼠心脏成纤维细胞增殖及其胶原合成与分泌的作用和机制

目的 探讨瑞舒伐他汀对转化生长因子β1 (TGF-β1)诱导的新生大鼠心脏成纤维细胞增殖及其胶原合成与分泌的作用和可能机制.方法 分离、培养新生SD大鼠的心脏成纤维细胞,采用CCK-8比色法测定细胞数目.用RT-PCR检测Ⅰ型胶原蛋白和Ⅲ型胶原蛋白mRNA的表达,用ELISA测定Ⅰ型胶原蛋白和Ⅲ型胶原蛋白含量,用Western blot检测心脏成纤维细胞Akt的蛋白表达.结果 CCK-8比色法显示,瑞舒伐他汀能抑制TGF-β1引起的心脏成纤维细胞增殖;RT-PCR结果显示,瑞舒伐他汀能抑制TGF-β1诱导的Ⅰ型胶原蛋白和Ⅲ型胶原蛋白mRNA的表达;ELISA结果显示,瑞舒伐他汀能抑制TGF-β1诱导的Ⅰ型胶原蛋白和Ⅲ型胶原蛋白分泌;Westernblot结果显示,瑞舒伐他汀能抑制TGF-β1诱导的Akt蛋白表达.结论 瑞舒伐他汀可能通过抑制TGF-β1诱导的新生大鼠心脏成纤维细胞增殖、Ⅰ型和Ⅲ型胶原蛋白的合成与分泌来抑制TGF-β1诱导的心脏纤维化,其分子机制可能与抑制Akt信号通路有关.     

松弛素对血管紧张素Ⅱ诱导的大鼠血管外膜成纤维细胞胶原水平的影响

目的:探讨松弛素(RLX)对血管紧张素(Ang)Ⅱ诱导的大鼠血管外膜成纤维细胞胶原水平的影响及机制。方法:体外培养大鼠主动脉外膜成纤维细胞,随机分为对照组、AngⅡ组(10-6 mmol/L),RLX组(100μg/L)及AngⅡ+RLX组。采用波形蛋白染色法鉴定血管外膜成纤维细胞,用RT-PCR检测Ⅰ、Ⅲ型胶原蛋白mRNA表达,用Western blot印迹法检测基质金属蛋白酶(MMP)-2、MMP-9及转化生长因子(TGF)-β1蛋白的表达。结果:AngⅡ可显著增加培养液上清中Ⅰ、Ⅲ型胶原蛋白含量及TGF-β1蛋白表达,而RLX明显抑制AngⅡ的上述作用;AngⅡ可显著下调细胞内MMP-2和MMP-9的蛋白表达,而RLX明显抑制AngⅡ的上述作用(均P<0.05)。结论:AngⅡ可刺激血管外膜成纤维细胞胶原合成增加,而RLX通过上调MMP-2、MMP-9表达和下调TGF-β1表达,抑制AngⅡ刺激的Ⅰ、Ⅲ型胶原蛋白生成,从而发挥抗血管纤维化作用。     

阿司匹林抗高糖诱导内皮细胞衰老过程中对一氧化氮-一氧化氮合酶系统的影响

目的 探讨阿司匹林是否通过影响一氧化氮（NO）-一氧化氮合酶（NOS）系统发挥抗高糖诱导的内皮细胞衰老的作用. 方法 人脐静脉血管内皮细胞（HUVECs）分别于正常糖浓度培养液（5.5 mmol/L）、高糖培养液（33 mmol/L）、含阿司匹林（0.01～1mmol/L）培养液及含L-NAME（300 μmol/L）培养液中培养48 h.采用β-半乳糖苷酶（SA-β-gal）染色鉴定衰老细胞,流式细胞仪检测细胞内活性氧簇（ROS）水平.Griess法检测细胞总NO水平,荧光酶标仪检测细胞内NOS活性.Westernblot分析eNOS蛋白及eNOS人丝氨酸（Ser-1177）蛋白表达. 结果 高糖作用内皮细胞48 h后与正常糖浓度相比,SA-β-gal阳性细胞数明显增多.阿司匹林剂量依赖性地减少SA-β-gal阳性细胞数,降低ROS的水平,升高NO的水平、总NOS活性和eNOS Ser-1177蛋白表达（P＜0.05）.L-NAME（NOS的抑制剂）能完全抑制高糖环境下阿司匹林的上述作用（P＜0.05）.各组间eNOS总蛋白表达差异无统计学意义（P＞0.05）. 结论 高糖环境下阿司匹林通过提高细胞NOS活性而升高NO水平,降低氧化应激反应而发挥抗衰老作用.     

银杏叶提取物对AngⅡ诱导的心肌成纤维细胞纤维化的干预作用

目的研究银杏叶提取物对血管紧张素Ⅱ(AngⅡ)诱导的心肌成纤维细胞纤维化的干预作用。方法体外培养心肌成纤维细胞,加入10-7mol AngⅡ模拟大鼠心肌纤维化体外模型。ELISA检测Ⅰ型胶原、Ⅲ型胶原、结缔组织生长因子(CTGF)、转化生长因子-β(TGF-β)含量。结果银杏叶提取物可明显降低AngⅡ引起的心肌成纤维细胞Ⅰ型胶原、Ⅲ型胶原分泌增加,并能降低CTGF、TGF-β表达。结论银杏叶提取物可改善AngⅡ引起的心肌成纤维细胞Ⅰ型胶原分泌增加,其机制可能与CTGF、TGF-β有关。     

氟伐他汀和卡托普利对高血压大鼠肾脏保护机制的研究

目的观察氟伐他汀和卡托普利联用对高血压大鼠肾损害的治疗作用及其机制。方法给40只雄性SD大鼠口服左旋硝基精氨酸甲酯(N(omega)-nitro-L-argininemethylester,L-NAME)50mg·kg-1·d-1连续4周后,其中24只制作高血压肾损害模型,分成3组,各组继续给予L-NAME,氟伐他汀组给予氟伐他汀5mg·kg-1·d-1;卡托普利组给予卡托普利30mg·kg-1·d-1;联用氟伐他汀和卡托普利组给予氟伐他汀和卡托普利,剂量同前。12周后,把大鼠处死,测定血及肾皮质一氧化氮(NO)、血管紧张素Ⅱ(anginotensinⅡ,AngⅡ)的水平。电镜检查肾小球。蛋白印迹法测定肾皮质转化生长因子β1(transforminggrowthfactorβ1,TGF-β1)和Ⅰ型胶原、Ⅳ型胶原等细胞外基质(extracellularmatrix,ECM)的表达。结果口服L-NAME大鼠血及肾皮质中NO水平明显降低(P<0.001),AngⅡ水平明显升高(P<0.05),肾小球基底膜增厚变形以及足突紊乱,肾皮质TGF-β1、Ⅰ型胶原和Ⅳ型胶原表达明显升高;氟伐他汀或卡托普利能缓解口服L-NAME大鼠血及肾皮质中NO水平的降低和AngⅡ水平的升高,两药联用作用更明显。(P<0.001)。结论L-NAME引起的高血压肾损害有血及肾皮质中NO水平降低,AngⅡ水平升高,TGF-β1在肾脏过度表达:Ⅰ型胶原、Ⅳ型胶原的聚集,在高血压肾小球硬化中可能起重要作用;氟伐他汀或卡托普利及两药联用可缓解上述改变。     

促红细胞生成素对心脏成纤维细胞表型转化的影响及其信号机制

目的 观察促红细胞生成素(EPO)对血管紧张素Ⅱ(AngⅡ)诱导培养的乳鼠心脏成纤维细胞(CF)表型转化的影响,以及其可能的信号通路(TGF-β1-TAK1-p38MAPK)在其中的作用.方法 体外分离培养乳鼠心脏成纤维细胞,用10-6 mol/L的AngⅡ诱导培养心脏成纤维细胞表型转化,加入20 kU/L的EPO进行预干预,同时加或不加15 μmol/L的SB203580进行处理,以平滑肌肌动蛋白(SMA)表达作为心脏成纤维细胞表型转化的观察指标,应用免疫组织化学观察心脏成纤维细胞内SMA表达情况;采用实时荧光定量PCR方法检测细胞内信号分子转化生长因子β1(TGF-β1)及p38丝裂原活化蛋白激酶(p38MAPK) mRNA的表达情况;采用蛋白免疫印迹法检测α平滑肌肌动蛋白(α-SMA)、TGF-β1、转化生长因子激活性激酶1(TAK1)、p38MAPK以及磷酸化的TAK1(p-TAK1)和p-p38MAPK的表达情况.结果 20 kU/L的EPO能有效抑制AngⅡ诱导的CF细胞表型转化,减少CF细胞内α-SMA蛋白的沉积,降低CF表型转化相关信号分子TGF-β1、p-TAK1、TAK1、p-p38MAPK和p38MAPK的活化或表达,且加入SB203580后该作用增强.结论 EPO可抑制AngⅡ诱导的乳鼠心脏成纤维细胞表型转化为肌成纤维细胞,减轻心肌纤维化,并可降低相关信号分子mRNA及蛋白的表达,初步考虑EPO抑制心肌纤维化,减轻心室重构的作用是通过TGF-β1-TAK1-p38MAPK进行的.