硫化氢影响同型半胱氨酸诱导心肌细胞H9C2基质金属蛋白酶-2的酶原活化作用
摘要
【背景及目的】
至今心房颤动发生和维持的确切机制尚未完全研究清楚,心房组织重构是导致房颤持续发生的主要原因,其突出表现为组织纤维化。胶原代谢异常是组织纤维化病变的重要病理改变。基质金属蛋白酶(Matrix Metalloproteinases, MMPs)是体内调节胶原代谢的主要蛋白酶家族,是心房纤维化的主导因素之一。已有研究表明同型半胱氨酸(Homocysteine, Hcy)参与房颤的发生及发展并增加房颤合并血栓形成的风险,已证实Hcy能调节MMPs的活性,然而Hcy活化MMPs的机制尚未深入,其代谢产物硫化氢(Hydrogen Sulfide, H2S)的相关影响作用未见报道。由此我们通过体外培养大鼠心肌细胞,观察检测不同浓度Hcy(0～100μmol/L)以及H2S(0.1、1.0、10mmol/L)分别对心肌细胞MMP-2分泌或活化的影响,并首次探讨研究两者的联合作用,旨在通过深入研究Hcy-H2S的代谢调节对MMPs的活化作用,启发改善心肌组织纤维化的新思路。
【方法】
1.检测Hcy和H2S对心肌细胞H9C2的细胞活性影响,将不同浓度Hcy(0～1000μmol/L)和H2S(0.1、1.0、10mmol/L)分别作用体外培养的H9C2心肌细胞6h和24h,利用四甲基偶氮唑盐比色法(MTT法)检测H9C2细胞活性。
2.检测Hcy(0～100μmol/L)影响H9C2细胞分泌的MMP-2总浓度和酶活性的调节作用,作用6h和24h,应用酶联免疫吸附法(ELISA)、明胶酶谱分析等技术检测MMP-2的表达和酶活性。
3.以NaHS(0.1、1.0、10mmol/L)为H2S的外源性供体,作用H9C2细胞6h和24h,利用明胶酶谱分析技术检测其对MMP-2酶活性的调节作用。
4. Hcy(0～100μmol/L)和H2S(0.1、1.0、10mmol/L)共同作用H9C2细胞6h和24h,应用MTT和明胶酶谱法检测细胞活性改变和MMP-2活化程度变化情况。
【结果】
1. Hcy在1000μmol/L超高浓度时对H9C2细胞表现显著的毒性作用(P<0.01),而在本试验应用浓度范围(0～100μmol/L)对细胞活性无明显影响;H2S(0.1、1.0、10mmol/L)以及H2S(0.1、1.0、10mmol/L)和Hcy(0～100μmol/L)共同作用均不影响细胞活性,表明本试验药品的应用浓度对H9C2细胞生长活性无明显影响作用。
2. Hcy(0～100μmol/L)浓度依赖性促进H9C2分泌MMP-2,呈浓度依赖性且时间依赖性地上调H9C2细胞MMP-2的酶原活化。
3. H2S(0.1、1.0、10mmol/L)浓度依赖性地促进MMP-2活化。
4. Hcy和H2S共同作用组比较平行Hcy组上调H9C2的MMP-2酶活性,表明H2S增强Hcy的MMP-2酶原活化作用。
【结论】
在0～100μmol/L范围内同型半胱氨酸浓度依赖性上调H9C2细胞MMP-2的合成分泌,浓度依赖性且时间依赖性地促进MMP-2酶原活化。硫化氢(0.1、1.0、10mmol/L)浓度依赖性地上调MMP-2酶原活化,并且协同促进同型半胱氨酸活化MMP-2,提示同型半胱氨酸诱导心肌细胞的MMP-2活化作用可能与本身代谢或者硫化氢有关。
【Backgroud and Objectives】
Precise mechanisms that lead to the onset and persistence of atrial fibrillation have not completely been elucidated. Atrial structural remodeling was identified as the main mechanism for AF persistence, confirming predominant role of atrial fibrosis. The interplay between cardiac matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs), is thought to be critical in atrial fibrosis. Elevated homocysteine has been suggested to play a role in the AF pathogenesis and to exhibit an association with the presence of LA thrombus in AF. In animal models and in vitro human experiments it has been shown that MMPs are activated by Hcy in endothelial cells, arterial media and left ventrical. However, Whether Hcy itself or Hcy-related substances such as hydrogen sulfide (H2S) is the culprit, and what the exact pathogenic mechanism of Hcy-induced MMPs activation could be, remain to be elucidated. Therefore we have analyzed the activation of Hcy and H2S on MMP-2 as well as the effects of H2S on the activity of Hcy-induced MMP-2 in cultured rat cardiomyocytes.
【Methods】
H9C2 cells (rat cardiomyoblast cells) incubated with Hcy and NaHS (H2S donor) were analyzed for cell viability. Furthermore, the effects of different homocysteine levels (0～100μmol/L) on MMP-2 production in cultured rat cardiomyocytes were examined using gelatin zymography and ELISA. Then the effects of different H2S concentrations (0.1、1.0、10mmol/L) on MMP-2 activity and the activation of hyperhomocysteinemia (homocysteine concentrations of 10μmol/L～100μmol/L)-induced MMP-2 in cultured rat cardiomyocytes were evaluated by gelatin zymography. All treatments sustained in the serum-free medium for 6h and 24h.
【Results】
1. Exposing H9C2 cardiomyocytes to Hcy (0～1000μmol/L) for up to 24h, at the concentration of 1000μmol/L, Hcy reduced cell viability, assessed by MTT. Incubation with 0～100μmol/L Hcy had no effects on cardiomyocytes viability.
2. Hcy (10μmol/L～100μmol/L) significantly increased the production of MMP-2 as determined by ELISA in concentration-dependent manner. Hcy (2.5μmol/L～100μmol/L) significantly upregulated the activity of MMP-2 in H9C2 for 6h and 24h, in concentration-and time-dependent fashion.
3. H2S (0.1、1.0、10mmol/L) significantly increased the activity of MMP-2 in H9C2 as assessed by zymography in concentration-dependent fashion.
4. The activation of hyperhomocysteinemia (homocysteine concentrations of 10μmol/L～100μmol/L)-induced MMP-2 in cultured rat cardiomyocytes was significantly stronger in the presence of H2S (1.0mmol/L).
【Conclusions】
Homocysteine upregulates the MMP-2 activity in concentration- and time-dependent manner, the effect being stronger in the presence of H2S (1.0mmol/L), suggesting that Hcy may modulate the extracellular matrix remodeling, in part, by generating H2S.
至今心房颤动发生和维持的确切机制尚未完全研究清楚,心房组织重构是导致房颤持续发生的主要原因,其突出表现为组织纤维化。胶原代谢异常是组织纤维化病变的重要病理改变。基质金属蛋白酶(Matrix Metalloproteinases, MMPs)是体内调节胶原代谢的主要蛋白酶家族,是心房纤维化的主导因素之一。已有研究表明同型半胱氨酸(Homocysteine, Hcy)参与房颤的发生及发展并增加房颤合并血栓形成的风险,已证实Hcy能调节MMPs的活性,然而Hcy活化MMPs的机制尚未深入,其代谢产物硫化氢(Hydrogen Sulfide, H2S)的相关影响作用未见报道。由此我们通过体外培养大鼠心肌细胞,观察检测不同浓度Hcy(0～100μmol/L)以及H2S(0.1、1.0、10mmol/L)分别对心肌细胞MMP-2分泌或活化的影响,并首次探讨研究两者的联合作用,旨在通过深入研究Hcy-H2S的代谢调节对MMPs的活化作用,启发改善心肌组织纤维化的新思路。
【方法】
1.检测Hcy和H2S对心肌细胞H9C2的细胞活性影响,将不同浓度Hcy(0～1000μmol/L)和H2S(0.1、1.0、10mmol/L)分别作用体外培养的H9C2心肌细胞6h和24h,利用四甲基偶氮唑盐比色法(MTT法)检测H9C2细胞活性。
2.检测Hcy(0～100μmol/L)影响H9C2细胞分泌的MMP-2总浓度和酶活性的调节作用,作用6h和24h,应用酶联免疫吸附法(ELISA)、明胶酶谱分析等技术检测MMP-2的表达和酶活性。
3.以NaHS(0.1、1.0、10mmol/L)为H2S的外源性供体,作用H9C2细胞6h和24h,利用明胶酶谱分析技术检测其对MMP-2酶活性的调节作用。
4. Hcy(0～100μmol/L)和H2S(0.1、1.0、10mmol/L)共同作用H9C2细胞6h和24h,应用MTT和明胶酶谱法检测细胞活性改变和MMP-2活化程度变化情况。
【结果】
1. Hcy在1000μmol/L超高浓度时对H9C2细胞表现显著的毒性作用(P<0.01),而在本试验应用浓度范围(0～100μmol/L)对细胞活性无明显影响;H2S(0.1、1.0、10mmol/L)以及H2S(0.1、1.0、10mmol/L)和Hcy(0～100μmol/L)共同作用均不影响细胞活性,表明本试验药品的应用浓度对H9C2细胞生长活性无明显影响作用。
2. Hcy(0～100μmol/L)浓度依赖性促进H9C2分泌MMP-2,呈浓度依赖性且时间依赖性地上调H9C2细胞MMP-2的酶原活化。
3. H2S(0.1、1.0、10mmol/L)浓度依赖性地促进MMP-2活化。
4. Hcy和H2S共同作用组比较平行Hcy组上调H9C2的MMP-2酶活性,表明H2S增强Hcy的MMP-2酶原活化作用。
【结论】
在0～100μmol/L范围内同型半胱氨酸浓度依赖性上调H9C2细胞MMP-2的合成分泌,浓度依赖性且时间依赖性地促进MMP-2酶原活化。硫化氢(0.1、1.0、10mmol/L)浓度依赖性地上调MMP-2酶原活化,并且协同促进同型半胱氨酸活化MMP-2,提示同型半胱氨酸诱导心肌细胞的MMP-2活化作用可能与本身代谢或者硫化氢有关。
【Backgroud and Objectives】
Precise mechanisms that lead to the onset and persistence of atrial fibrillation have not completely been elucidated. Atrial structural remodeling was identified as the main mechanism for AF persistence, confirming predominant role of atrial fibrosis. The interplay between cardiac matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs), is thought to be critical in atrial fibrosis. Elevated homocysteine has been suggested to play a role in the AF pathogenesis and to exhibit an association with the presence of LA thrombus in AF. In animal models and in vitro human experiments it has been shown that MMPs are activated by Hcy in endothelial cells, arterial media and left ventrical. However, Whether Hcy itself or Hcy-related substances such as hydrogen sulfide (H2S) is the culprit, and what the exact pathogenic mechanism of Hcy-induced MMPs activation could be, remain to be elucidated. Therefore we have analyzed the activation of Hcy and H2S on MMP-2 as well as the effects of H2S on the activity of Hcy-induced MMP-2 in cultured rat cardiomyocytes.
【Methods】
H9C2 cells (rat cardiomyoblast cells) incubated with Hcy and NaHS (H2S donor) were analyzed for cell viability. Furthermore, the effects of different homocysteine levels (0～100μmol/L) on MMP-2 production in cultured rat cardiomyocytes were examined using gelatin zymography and ELISA. Then the effects of different H2S concentrations (0.1、1.0、10mmol/L) on MMP-2 activity and the activation of hyperhomocysteinemia (homocysteine concentrations of 10μmol/L～100μmol/L)-induced MMP-2 in cultured rat cardiomyocytes were evaluated by gelatin zymography. All treatments sustained in the serum-free medium for 6h and 24h.
【Results】
1. Exposing H9C2 cardiomyocytes to Hcy (0～1000μmol/L) for up to 24h, at the concentration of 1000μmol/L, Hcy reduced cell viability, assessed by MTT. Incubation with 0～100μmol/L Hcy had no effects on cardiomyocytes viability.
2. Hcy (10μmol/L～100μmol/L) significantly increased the production of MMP-2 as determined by ELISA in concentration-dependent manner. Hcy (2.5μmol/L～100μmol/L) significantly upregulated the activity of MMP-2 in H9C2 for 6h and 24h, in concentration-and time-dependent fashion.
3. H2S (0.1、1.0、10mmol/L) significantly increased the activity of MMP-2 in H9C2 as assessed by zymography in concentration-dependent fashion.
4. The activation of hyperhomocysteinemia (homocysteine concentrations of 10μmol/L～100μmol/L)-induced MMP-2 in cultured rat cardiomyocytes was significantly stronger in the presence of H2S (1.0mmol/L).
【Conclusions】
Homocysteine upregulates the MMP-2 activity in concentration- and time-dependent manner, the effect being stronger in the presence of H2S (1.0mmol/L), suggesting that Hcy may modulate the extracellular matrix remodeling, in part, by generating H2S.
引文
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1. Fiorucci S, Distrutti E, Cirino G, et al. The emerging roles of hydrogen sulfide in the gastrointestinal tract and live.Gastroenterology,2006,131(1):259-271.
2. Wang R. Two’s company, three's a crowd:can H2S be the third endogenous gaseous transmitter?.FASEB,2002;16(13):1792-1798.
3. Chen P, Poddar R, Tipa EV, et al. Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease.Adv Enzyme Regul,1999,39:93-109.
4. Geng B, Yang J, Qi Y, et al. H2S generated by heart in rat and its effects on cardiac function.Biophys Res Commun,2004,313(2):362-368.
5. Zhu YZ, Wang ZJ, Ho P, et al. Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats.J Appl Physiol,2007,102(1):261-268.
6. Johansen D, Ytrehus K, Baxter GF. Exogenous hydrogen sulfide (H2S) protects against regional myocardial ischemia-reperfusion injury—evidence for a role of KATP channels.Basic Res Cardiol,2006,101:53-60.
7. Hu X, Li T, Bi S, et al.Possible role of hydrogen sulfide on the preservation of donor rat hearts.Transplant Proc,2007,39(10):3024-3029.
8. Zhang Z, Huang H, Liu P, et al.Hydrogen sulfide contributes to cardioprotection during ischemia|reperfusion injury by opening KATP channels.Can J Physiol Pharmacol,2007,85(12):1248-1253.
9. Bian JS, Yong QC, Pan TT, et al. Role of hydrogen sulfide in the cardioprotection caused by ischemic preconditioning in the rat heart and cardiac myocytes.J Pharmacol Exp Ther,2006,316(2):670-678.
10. Sivarajah A, McDonald MC, Thiemermann C. The production of hydrogen sulfide limits myocardial ischemia and reperfusion injury and contributes to the cardioprotective effects of preconditioning with endotoxin,but not ischemia in the rat.Shock,2006,26(2):154-161.
11. Pan TT, Feng ZN, Lee SW, et al. Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes.Journal of Molecular and Cellular Cardiology,2006,40:119-130.
12. Hu Y, Chen X, Pan TT, et al. Cardioprotection induced by hydrogen sulfide preconditioning involves activation of ERK and PI3K/Akt pathways.Physiological Change of Voice Eur J Physi,2008,455:607-616.
13. Pan TT, Neo KL, Hu LF, et al. H2S preconditioning|induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes.Am J Physiol Cell Physiol,2008,294(1):C169-177.
14. Rossoni G, Sparatore A, Tazzari V, et al.The hydrogen sulphide|releasing derivative of diclofenac protects against ischemia reperfusion injury in the isolated rabbit heart.Br J Pharmacol,2008,153(1):100-109.
15. Hu LF, Pan TT, Neo KL, et al. Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes.P Flugers Arch,2008,455(2):971-978.
16. Elrod JW, Calvert JW, Morrison J, et al. Hydrogen sulfide attenuates myocardial ischemia reperfusion injury by preservation of mitochondrial function.Proc Natl Acad Sci Proc Natl Acad Sci USA ,2007,104(39):15560-15565.
17. Sodha NR, Clements RT, Feng J, et al. The effects of therapeutic sulfide on myocardial apoptosis in response to ischemia|reperfusion injury.Eur J Cardiothorac Surg,2008,33(5):906-913.
18. Haouzi P, Notet V, Chenuel B, et al. H2S induced hypometabolism in mice is missing in sedated sheep.Respir Physiol Neurobiol,2008,160(1):109-115.
19. Eric B, Mike M, Mark BR. H2S induces a suspended animation|like state in mice.Science,2005,308:518.
20. Gian PV, Robert S, Binglan Y, et al. Inhaled hydrogen sulfide:A rapidly reversible inhibitor of cardiac and metabolic function in the mouse.Anesthesiology,2008,108:659-668.
21. Yong QC, Pan TT, Hu LF, et al. Negative regulation of beta|adrenergic function by hydrogen sulphide in the rat hearts.J Mol Cell Cardiol,2008;44(4):701-710.
22. Geng B, Chang L, Pan C, et al. Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol.Biochem Biophys Res Commun,2004;318(3):756-763.
23. Lin Chang, Bin Geng, Fang Yu, et al.Hydrogen sulfide inhibits myocardial injury induced by homocysteine in rats.Amino Acids,2008,34(4):573-585.
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1. Fiorucci S, Distrutti E, Cirino G, et al. The emerging roles of hydrogen sulfide in the gastrointestinal tract and live.Gastroenterology,2006,131(1):259-271.
2. Wang R. Two’s company, three's a crowd:can H2S be the third endogenous gaseous transmitter?.FASEB,2002;16(13):1792-1798.
3. Chen P, Poddar R, Tipa EV, et al. Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease.Adv Enzyme Regul,1999,39:93-109.
4. Geng B, Yang J, Qi Y, et al. H2S generated by heart in rat and its effects on cardiac function.Biophys Res Commun,2004,313(2):362-368.
5. Zhu YZ, Wang ZJ, Ho P, et al. Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats.J Appl Physiol,2007,102(1):261-268.
6. Johansen D, Ytrehus K, Baxter GF. Exogenous hydrogen sulfide (H2S) protects against regional myocardial ischemia-reperfusion injury—evidence for a role of KATP channels.Basic Res Cardiol,2006,101:53-60.
7. Hu X, Li T, Bi S, et al.Possible role of hydrogen sulfide on the preservation of donor rat hearts.Transplant Proc,2007,39(10):3024-3029.
8. Zhang Z, Huang H, Liu P, et al.Hydrogen sulfide contributes to cardioprotection during ischemia|reperfusion injury by opening KATP channels.Can J Physiol Pharmacol,2007,85(12):1248-1253.
9. Bian JS, Yong QC, Pan TT, et al. Role of hydrogen sulfide in the cardioprotection caused by ischemic preconditioning in the rat heart and cardiac myocytes.J Pharmacol Exp Ther,2006,316(2):670-678.
10. Sivarajah A, McDonald MC, Thiemermann C. The production of hydrogen sulfide limits myocardial ischemia and reperfusion injury and contributes to the cardioprotective effects of preconditioning with endotoxin,but not ischemia in the rat.Shock,2006,26(2):154-161.
11. Pan TT, Feng ZN, Lee SW, et al. Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes.Journal of Molecular and Cellular Cardiology,2006,40:119-130.
12. Hu Y, Chen X, Pan TT, et al. Cardioprotection induced by hydrogen sulfide preconditioning involves activation of ERK and PI3K/Akt pathways.Physiological Change of Voice Eur J Physi,2008,455:607-616.
13. Pan TT, Neo KL, Hu LF, et al. H2S preconditioning|induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes.Am J Physiol Cell Physiol,2008,294(1):C169-177.
14. Rossoni G, Sparatore A, Tazzari V, et al.The hydrogen sulphide|releasing derivative of diclofenac protects against ischemia reperfusion injury in the isolated rabbit heart.Br J Pharmacol,2008,153(1):100-109.
15. Hu LF, Pan TT, Neo KL, et al. Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes.P Flugers Arch,2008,455(2):971-978.
16. Elrod JW, Calvert JW, Morrison J, et al. Hydrogen sulfide attenuates myocardial ischemia reperfusion injury by preservation of mitochondrial function.Proc Natl Acad Sci Proc Natl Acad Sci USA ,2007,104(39):15560-15565.
17. Sodha NR, Clements RT, Feng J, et al. The effects of therapeutic sulfide on myocardial apoptosis in response to ischemia|reperfusion injury.Eur J Cardiothorac Surg,2008,33(5):906-913.
18. Haouzi P, Notet V, Chenuel B, et al. H2S induced hypometabolism in mice is missing in sedated sheep.Respir Physiol Neurobiol,2008,160(1):109-115.
19. Eric B, Mike M, Mark BR. H2S induces a suspended animation|like state in mice.Science,2005,308:518.
20. Gian PV, Robert S, Binglan Y, et al. Inhaled hydrogen sulfide:A rapidly reversible inhibitor of cardiac and metabolic function in the mouse.Anesthesiology,2008,108:659-668.
21. Yong QC, Pan TT, Hu LF, et al. Negative regulation of beta|adrenergic function by hydrogen sulphide in the rat hearts.J Mol Cell Cardiol,2008;44(4):701-710.
22. Geng B, Chang L, Pan C, et al. Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol.Biochem Biophys Res Commun,2004;318(3):756-763.
23. Lin Chang, Bin Geng, Fang Yu, et al.Hydrogen sulfide inhibits myocardial injury induced by homocysteine in rats.Amino Acids,2008,34(4):573-585.