H_2S对高糖诱导的人脐静脉内皮细胞损伤的保护作用及其机制
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摘要
研究背景与研究目的
近年来糖尿病患病率急剧增加,大血管和微血管病变是其主要并发症,具有发生率高、致残和致死率高等特点。深入探讨糖尿病血管并发症发生发展的机制,寻求干预措施对于降低糖尿病致死率、致残率,改善糖尿病患者预后具有极其重要的意义。目前认为血管内皮功能损伤是糖尿病血管病变发生的始动因素和主要病理生理学基础,甚至在尚未出现慢性血管并发症的糖尿病患者已出现内皮功能明显降低。高血糖可以通过多种机制损伤血管内皮细胞功能,如炎症、氧化应激、蛋白质非酶糖基化、继发性脂代谢紊乱等。上述途径可在一定程度上解释糖尿病血管病变的发生机制,为其防治提供重要的靶点,对糖尿病血管并发症的防治具有重要意义。
硫化氢(H2S)是继NO和CO之后的第三种气体信号分子,在体内具有广泛的生物学效应,对多种疾病的发生发展有重要的病理生理意义,也逐渐显示出极大的治疗性应用前景。越来越多的研究表明,内源性H2S或其供体如Na2S、NaHS可降低血压、对抗心肌缺血缺氧或缺血再灌注损伤、减轻动脉粥样硬化病变、改善心力衰竭症状等,发挥广泛的心血管保护效应。如NaHS可使ApoE(-/-)小鼠血浆H2S水平明显升高,动脉粥样硬化斑块体积显著缩小,血管超微结构异常均得到明显改善,并抑制Ox-LDL诱导的巨噬细胞转变为泡沫细胞
哺乳动物体内的内源性H2S主要来自半胱氨酸的代谢产物,其中最主要的途径是在胱硫醚-β-合成酶(cystathionine-β-synthase, CBS)、胱硫醚-γ-裂解酶(cystathionine-γ-Iyase, CSE)和半胱氨酸转移酶的催化下生成H2S。CSE主要存在于心血管系统及其他非中枢神经系统的组织中,且CSE在心血管系统中的表达不仅限于平滑肌细胞,还可表达在内皮细胞上。有学者甚至认为,H2S是除NO外的另一种内皮源性舒张因子。
H2S对血管内皮的作用也得到了越来越多的关注。迄今已发现H2S在体外可以促进内皮细胞的生长、迁移及损伤的自我修复,在体内可以促进小鼠新生血管的形成；H2S可抑制肺动脉血管平滑肌细胞增殖,促进凋亡,从而延缓肺动脉高压的进程。H2S也可能通过血管内皮生长因子信号通路促进血管内皮细胞增殖、移行和管腔结构的形成,从而促进血管再生成。H2S也可以通过PI3K-Akt途径促进内皮细胞的生长、迁移,并可以促进小鼠新生血管的形成。H2S还可以使得apoE基因敲除小鼠和TNFα诱导的人脐静脉内皮细胞上血管细胞粘附分子和细胞间粘附分子基因表达下调,从而减弱TNFα引起的血管内皮细胞功能紊乱,对血管炎症性疾病的治疗具有积极的意义。
上述研究结果表明,H2S参与多种原因引起的血管结构紊乱和功能障碍,有可能成为相关疾病治疗的新的靶点。但H2S对糖尿病血管病变作用的研究较少。本研究对体外培养的人脐静脉内皮细胞进行高浓度葡萄糖处理,模拟糖尿病体内环境,通过检测细胞凋亡率来观察内皮细胞的损伤情况及H2S供体NaHS对受损内皮细胞的保护作用,并从H2S生成量、凋亡与抗凋亡、氧化与抗氧化、内皮素等多个角度探讨其作用机制。
研究方法
1.细胞来源及培养：原代培养,采用0.125%胰酶和0.01%EDTA灌注法获得。原代培养的HUVECs置于含10%胎牛血清的M199培养基中,放于37℃、5%C20的细胞培养箱中培养,第2、3代细胞用于实验。
2.原代HUVECs细胞鉴定：HUVECs密度达到70%左右时在倒置显微镜下摄片观察细胞形态,用兔抗人八因子相关抗原(Ⅷ因子-RAg)多克隆抗体进行。
3.分组：HUVECs细胞生长至70%-80%融合时,分为以下四组：①A组：正常糖浓度组(5.5mmol/L葡萄糖)；②B组：高糖组(25mmol/L葡萄糖)；③C组：高糖(25mmol/L葡萄糖)+硫氢化钠(50μmol/L)组；④D组：正常糖浓度(5.5mmol/L葡萄糖)+硫氢化钠(50μmol/L)。处理48h后用于后续实验。
4.MTT法检测各组细胞的存活率、Hoechst33258荧光染色检测细胞凋亡
5.吸光法测定细胞内H2S的含量。
6.Real-time PCR方法检测CSE mRNA表达情况；Western blot方法检测CSE蛋白水平的变化。
7.Western Blot方法检测细胞凋亡相关蛋白Bax、Bcl-2和Cleaved caspase-3的表达。
8.ELISA法检测ET-1水平。
9.氧化应激水平检测：包括ROS、MDA的生成量检测和SOD活性检测。
研究结果
1.一般形态及细胞鉴定：倒置显微镜下可见,HUVECs单层贴壁生长,第1天呈小团状聚集,3-5d后呈铺路石样生长,细胞呈短梭形或多角形,融合成单层。
细胞鉴定显示Ⅷ因子阳性,胞浆呈棕黄色,证实为内皮细胞,阴性对照组胞浆未见着色。
2.细胞存活率检测：MTT法结果显示：HUVECs经25mmol/L葡萄糖处理48h后,细胞活力与正常组(5.5mmol/L葡萄糖组)相比明显下降,25mmol/L葡萄糖处理组细胞存活率为(68.5±3.8)%,比5.5mmol/L葡萄糖组下降了31.5%(P＜0.05)。NaHS对HUVECs具有保护作用,能显著抑制高浓度葡萄糖对HUVECs的损伤,加入NaHS后,细胞存活率得到明显改善,50μmol/L NaHS和25mmol/L葡萄糖共同处理组细胞存活率为(97.1±7.4)%,比25mmol/L葡萄糖组上升了28.6%(P＜0.05)。与5.5mmol/L葡萄糖组相比,5.5mmol/L葡萄糖与50μmol/L NaHS共同处理组细胞存活率无明显变化(P>0.05)。
Hoechst33258荧光染色检测细胞凋亡结果显示,5.5mmol/L葡萄糖处理组细胞核较大,呈正常均匀的蓝色,边缘整齐,无凋亡细胞；25mmol/L葡萄糖处理组部分细胞核致密浓染,核固缩,并呈高亮度荧光,即凋亡细胞数明显增多(P<0.05)；加入50μmol/L NaHS后,可见凋亡细胞数明显减少(P＜0.05)。与5.5mmol/L葡萄糖处理组相比,50μmol/L NaHS组与5.5mmol/L葡萄糖共同处理组细胞凋亡率无明显变化(P＞0.05)。
上述结果提示,高浓度葡萄糖可引起HUVECs凋亡增多,存活量减少；外源性补充H2S可抑制凋亡发生,使细胞存活数量增多。这可能是H2S对血管内皮具有保护作用的重要原因。
3.H2S生成量：与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖组的H2S生成量明显减少(12.85±0.53μmol/分钟vs10.26±0.51μmo1/分钟,P＜0.05)；与25mmol/L葡萄糖组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组的H2S生成量明显增多(11.90±0.32μmol/分钟vs10.26±0.51μmol/分钟,P＜0.05)。
4.CSE mRNA与蛋白生成量：与正常HUVEC细胞(5.5mmol/L葡萄糖处理组)相比,高浓度葡萄糖(25mmol/L葡萄糖)对CSE mRNA表达量的影响无显著性差异(P＞0.05)。
与5.5mmol/L葡萄糖组相比,以25mmol/L葡萄糖处理48h后能使CSE蛋白的表达下降,而25mmol/L葡萄糖+50μmol/L NaHS则能改善这种变化,CSE蛋白的表达比25mmol/L葡萄糖组增强；CSE蛋白半定量分析显示25mmol/L葡萄糖处理HUVECs48h后,能使CSE蛋白的表达下降53.7%(P＜0.05),50μmol/L NaHS和25mmol/L葡萄糖共处理组使CSE的表达较25mmol/L葡萄糖组上升19%(P＜0.05)。
5.细胞凋亡：Western Blot检测结果显示,与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖处理48h后可使Bax表达显著升高(P＜0.05),Bcl-2的表达显著降低(P＜0.05)而Caspase-3活性明显增强(P＜0.05),Bax/Bcl-2比值显著升高(P＜0.05)；与25mmol/L葡萄糖处理组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组Bax水平显著降低(P＜0.05),Bcl-2水平显著升高(P＜0.05),Caspase-3活性显著下降(P＜0.05),Bax/Bcl-2比值显著降低(P＜0.05)；50μmol/L NaHS和5.5mmol/L葡萄糖共同处理组的上述结果与5.5mmol/L葡萄糖处理组无显著性差异(P＞0.05)。
以上结果说明高浓度葡萄糖可以增加促进凋亡蛋白Bax和Cleaved caspase-3的表达,同时减少抑制凋亡蛋白Bcl-2的表达；而NaHS可以抑制高糖的促凋亡作用。
6.内皮素水平：与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖处理48h后可使细胞上清液中内皮素分泌量显著增加(P＜0.05)；与25mmol/L葡萄糖处理组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组内皮素分泌量显著减少(P＜0.05)：50μmol/L NaHS和5.5mmol/L葡萄糖共同处理组的内皮素分泌量与5.5mmol/L葡萄糖处理组无显著性差异(P＞0.05)。
7.氧化应激：与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖处理48h后可使ROS生成量显著增加(P＜0.05),MDA的生成量也显著增加(P＜0.05),而抗氧化酶SOD活性显著降低(P＜0.05)；与25mmol/L葡萄糖处理组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组ROS和MDA的生成量均显著减少(P＜0.05),而抗氧化酶SOD活性显著增强(P＜0.05)；50umol/L NaHS和5.5mmol/L葡萄糖共同处理组的上述结果与5.5mmol/L葡萄糖处理组无显著性差异(P＞0.05)。
上述结果说明,高浓度葡萄糖可以增加氧化酶的活性,降低抗氧化酶的活性,并使得最终氧化应激产物的生成量增加；而NaHS可以抑制高糖的促氧化应激作用。
结论
1.高浓度葡萄糖可使体外培养的人脐静脉内皮细胞凋亡增多、存活减少,该作用与抑制CSE酶活性,减少H2S生成有关；外源性补充H2S可抑制细胞凋亡,使细胞存活增多。
2.高浓度葡萄糖抑制H2S生成后造成细胞存活减少的机制包括：促进凋亡相关蛋白表达、氧化应激反应加强、内皮素分泌增多。
3.外源性补充H2S可通过抑制凋亡相关蛋白Bax、Cleaved caspase-3表达,促进Bcl-2蛋白表达,增强抗氧化酶SOD活性,减少氧化应激产物ROS和MDA生成,抑制ET-1释放等多种途径发挥内皮细胞保护作用。
Backgrounds and aims
Recently the morbidity of diabetes has increased rapidly. Vascular complications are the common in diabetes patients, with the characteristics of high prevalence, disability and mortality. Thus it is pivotal to investigate the mechanism of vascular complications of diabetes. Nowadays it is well known that endothelium dysfunction is an early manifestation in the development of diabetic vascular complications. High concentration of glucose may impair the endothelial function via inflammation, oxidative stress, advanced glycosylation endproducts, and subsequent dyslipidemia, and so on.
Hydrogen sulfide (H2S), a colorless gas, has been considered as the third gasotransmitter except for nitric oxide (NO) and carbon monoxide (CO). H2S has been shown to possess important roles in the physiology and pathophysiology of several biological systems, especially in cardiovascular system. Endogenous H2S or H2S donor may decrease blood pressure, ameliorate myocardial ischemia and hypoxia or ischemia-reperfusion injury, reduce atherosclerosis lesions and improve the symptoms of heart failure, and so on. For example, administration of exogenous H2S (NaHS) has been shown to limit infarct size and preserve ventricular functioning in a myocardial ischemia-reperfusion mouse model. In apoE(-/-) mice, NaHS may increase plasma H(2)S level, decrease the size of atherosclerotic plaque and plasma and aortic ICAM-1levels.
In mammalian, endogenous H2S is generated by three known enzymes, namely cystathionine-β-synthase (CBS), cystathionine-y-lyase (CSE) and3-mercaptopyruvate sulfur transferase (3MST). The former two enzymes are pyridoxal-5'-phosphate dependent, which utilize L-cysteine and homocysteine as substrates to generate H2S. The latter enzyme utilizes L-cysteine and a-ketoglutarate through the metabolism with cysteine aminotransferase (CAT) to produce H2S. CBS and3MST are the main H2S-forming enzyme in the central nervous system, whereas CSE is the principal H2S-forming enzyme in the vasculature and heart.
Recently more and more attention has been put on the effects of H2S on endothelium. It has been shown that NaHS suppressed ICAM-1expression in tumor necrosis factor (TNF)-alpha-treated HUVECs via the NF-κB/IκB pathway. H2S has also been reported to suppress superoxide formation, NADPH oxidase-1(NOX-1) expression in endothelial cells, and protect ventricular function from oxidative stress to restore normal remodeling.
Therefore, H2S may be involved in the vascular structure disorder and dysfunction. However, up to date, little is known about the role of H2S in the development of diabetic vascular complications. In the present study, we intended to investigate the protective effect of H2S on the injury of human umbilical vein endothelium cells (HUVECs) induced by high glucose levels. Furthermore, we determined the mechanism of H2S on HUVECs from the point of H2S production, apoptosis, oxidative stress and secretion of ET-1.
Materials and methods
1. HUVECs were isolated from umbilical vein cords of normal pregnancies as previously described. Briefly, umbilical veins were rinsed with sterile saline and digested with0.25%trypsin. Harvested cells were cultured in M199medium supplemented with10%fetal bovine serum,100U/mL penicillin-streptomycin and20ng/mL vascular endothelial growth factor in an atmosphere of5%CO2at37℃. The medium was refreshed at intervals of3days at cell confluence. To maintain uniform condition, the cells in passage2to3were used for experiments.
2. After growing to70%confluence, the primary HUVECs were observed under inverted microscope, photoed and identified with Ⅷ factor antigen polyclonal antibody.
3. When growing to approximately80%confluence, HUVECs were then cultured in medium containing either normal glucose (5.5mmol/L) which served as a normal control or high glucose (25mmol/L) for48h. To determine the effects of NaHS, an H2S donor, on endothelial cells, HUVECs were pre-treated with NaHS (50μmol/L) for30min before adding high glucose. After48h, the cells were harvested for following experiments.
4. Cell viability was determined by MTT. At the same time, cell apoptosis was examined according to the morphological changes in cell nuclei using Hoechst33258staining. Cells were washed with cold PBS and fixed with paraformaldehyde for30min. Hoechst33258(10μg/mL) was added and incubated for20min before being detected by fluorescence microscopy. Apoptotic cells appeared as chromatin condensation and multiple chromatin fragments.
5. The content of H2S in cells was tested by UV spectrophotometer. The H2S concentration of each sample was expressed as nanomoles of H2S per milligram soluble protein.
6. The mRNA and protein expression of CSE in HUVECs were determined by Real-time PCR and Western blot analysis, respectively.
7. The apoptosis-associated proteins expression in HUVECs such as Bax、Bcl-2and Cleaved caspase-3were detected by Western blot analysis.
8. The secretion of ET-1of HUVECs was discovered by ELISA method. Results were normalized to cellular protein content in all experiments.
9. Intracellular ROS generation was measured by the oxidation-sensitive fluorescent probe (DCF-DA). The MDA level was measured to assess lipid peroxidation and the activity of SOD was measured as per kit instructions to estimate the role of anti-oxidation in the cells by commercial reagent kits
Results
1. Morphological features of HUVECs are proper and in line with others. This is validated by Ⅷ factor antibody.
2. Cell viability in the high glucose treated HUVECs (68.5±3.8%) was significantly decreased in comparison with normal cells (P<0.05), which could be ameliorated by NaHS (97.1±7.4%)(P<0.05).
HUVECs treated with high glucose showed obvious apoptotic features including chromatin condensation and multiple chromatin fragments. In contrast, nuclei of cells in the normal control group did not show apoptotic morphology. The percentage of apoptotic cells increased significantly in high glucose-treated cells compared with that of the normal glucose treated cells (P<0.05). NaHS administration reduced the apoptotic cells in high glucose cultured HUVECs significantly (P<0.05).
This indicated that high glucose induced HUVECs apoptosis and reduced cell viability. H2S exerted the protective effect on HUVECs as proved by decreased apoptosis and increased cell viability.
3. Production of H2S was significantly less in25mmol/L glucose treated HUVECs than those in the5.5mmol/L glucose treated cells (10.26±0.51μmol/min vs12.85±0.53μmol/min, P<0.05). The reduced H2S production caused by high glucose was retrieved by50μmol/L NaHS (10.26±0.51μmol/min vs11.90±0.32μmol/min, P<0.05).
4. Although the mRNA expression of CSE in high glucose treated HUVECs was somewhat higher than that in normal glucose treated cells, the difference did not achieve statistical significance.
The CSE protein expression was shown by Western blot analysis to be down-regulated about53.7%(P<0.05) in25mmol/L glucose treated HUVECs, while the addition of50μmol/L NaHS up-regulated the expression by19%(P<0.05).
5. High glucose significantly increased Bax protein expression and decreased Bcl-2protein expression (both P<0.05). The ratio of Bax/Bcl-2was increased about2-fold following exposure to high glucose (P<0.05). Accordingly, the level of cleaved caspase-3, the activated form of caspase-3, was higher in the high glucose group compared with that of control cells (P<0.05).
Compared with the high glucose group, the decrease in Bax protein expression and the increase in Bcl-2protein expression were observed in the group pre-treated with NaHS (both P<0.05). Similarly, NaHS attenuated the elevated Bax/Bcl-2ratio induced by high glucose, and inhibited high glucose-induced caspase-3activation (both P<0.05).
These results suggested that high glucose-induced endothelial cell apoptosis might be corrected by H2S.
6. High glucose resulted a significant elevation of ET-1secretion in HUVECs when compared with normal glucose (P<0.05). The glucose induced increase in ET-1was significantly attenuated by NaHS (P<0.05).
7. The production of ROS was measured using fluorescence microscopy, based on ROS-dependent oxidation of DCF-DA to DCF with green fluorescence. Compared with the high glucose group, NaHS-treated group showed a significant decrease in DCF levels (P<0.01). The results indicated that pre-treatment with NaHS could decrease the intracellular ROS generation.
Compared with control group, SOD activity was decreased in the high glucose group from23.59±1.08U/mg protein to19.53±2.17U/mg protein (P<0.05). Whereas, pre-treatment with NaHS increased SOD activity from19.53±2.17U/mg protein to23.66±1.16U/mg protein (P<0.05).
The amount of MDA was measured to assess the extent of lipid peroxidation. Compared with control group, the high glucose group showed a significant increase in MDA level from1.38±0.33μM/mg protein to2.78±0.59μM/mg protein (P<0.01), which was reversed by pre-treatment with NaHS (from2.78±0.59μM/mg protein to1.32±0.16μM/mg protein, P<0.01).
This indicated that H2S may alleviate the oxidative stress and thus protect HUVECs injury from high glucose.
Conelus ions
1. High glucose induces apoptosis of HUVECs via inhibiting of CSE expression and H2S production, which can be alleviated by H2S donor.
2. The cellular mechanisms of high glucose on HUVECs damage include promotion of apoptosis, increase of oxidative stress and elevation of ET-1secretion.
3. Exogenous H2S protect HUVECs from high glucose induced injury through inhibition of Bax and Cleaved caspase-3expression, increase Bcl-2expression, elevation of SOD activity, reduce of ROS and MDA, and suppression of ET-1.
近年来糖尿病患病率急剧增加,大血管和微血管病变是其主要并发症,具有发生率高、致残和致死率高等特点。深入探讨糖尿病血管并发症发生发展的机制,寻求干预措施对于降低糖尿病致死率、致残率,改善糖尿病患者预后具有极其重要的意义。目前认为血管内皮功能损伤是糖尿病血管病变发生的始动因素和主要病理生理学基础,甚至在尚未出现慢性血管并发症的糖尿病患者已出现内皮功能明显降低。高血糖可以通过多种机制损伤血管内皮细胞功能,如炎症、氧化应激、蛋白质非酶糖基化、继发性脂代谢紊乱等。上述途径可在一定程度上解释糖尿病血管病变的发生机制,为其防治提供重要的靶点,对糖尿病血管并发症的防治具有重要意义。
硫化氢(H2S)是继NO和CO之后的第三种气体信号分子,在体内具有广泛的生物学效应,对多种疾病的发生发展有重要的病理生理意义,也逐渐显示出极大的治疗性应用前景。越来越多的研究表明,内源性H2S或其供体如Na2S、NaHS可降低血压、对抗心肌缺血缺氧或缺血再灌注损伤、减轻动脉粥样硬化病变、改善心力衰竭症状等,发挥广泛的心血管保护效应。如NaHS可使ApoE(-/-)小鼠血浆H2S水平明显升高,动脉粥样硬化斑块体积显著缩小,血管超微结构异常均得到明显改善,并抑制Ox-LDL诱导的巨噬细胞转变为泡沫细胞
哺乳动物体内的内源性H2S主要来自半胱氨酸的代谢产物,其中最主要的途径是在胱硫醚-β-合成酶(cystathionine-β-synthase, CBS)、胱硫醚-γ-裂解酶(cystathionine-γ-Iyase, CSE)和半胱氨酸转移酶的催化下生成H2S。CSE主要存在于心血管系统及其他非中枢神经系统的组织中,且CSE在心血管系统中的表达不仅限于平滑肌细胞,还可表达在内皮细胞上。有学者甚至认为,H2S是除NO外的另一种内皮源性舒张因子。
H2S对血管内皮的作用也得到了越来越多的关注。迄今已发现H2S在体外可以促进内皮细胞的生长、迁移及损伤的自我修复,在体内可以促进小鼠新生血管的形成；H2S可抑制肺动脉血管平滑肌细胞增殖,促进凋亡,从而延缓肺动脉高压的进程。H2S也可能通过血管内皮生长因子信号通路促进血管内皮细胞增殖、移行和管腔结构的形成,从而促进血管再生成。H2S也可以通过PI3K-Akt途径促进内皮细胞的生长、迁移,并可以促进小鼠新生血管的形成。H2S还可以使得apoE基因敲除小鼠和TNFα诱导的人脐静脉内皮细胞上血管细胞粘附分子和细胞间粘附分子基因表达下调,从而减弱TNFα引起的血管内皮细胞功能紊乱,对血管炎症性疾病的治疗具有积极的意义。
上述研究结果表明,H2S参与多种原因引起的血管结构紊乱和功能障碍,有可能成为相关疾病治疗的新的靶点。但H2S对糖尿病血管病变作用的研究较少。本研究对体外培养的人脐静脉内皮细胞进行高浓度葡萄糖处理,模拟糖尿病体内环境,通过检测细胞凋亡率来观察内皮细胞的损伤情况及H2S供体NaHS对受损内皮细胞的保护作用,并从H2S生成量、凋亡与抗凋亡、氧化与抗氧化、内皮素等多个角度探讨其作用机制。
研究方法
1.细胞来源及培养：原代培养,采用0.125%胰酶和0.01%EDTA灌注法获得。原代培养的HUVECs置于含10%胎牛血清的M199培养基中,放于37℃、5%C20的细胞培养箱中培养,第2、3代细胞用于实验。
2.原代HUVECs细胞鉴定：HUVECs密度达到70%左右时在倒置显微镜下摄片观察细胞形态,用兔抗人八因子相关抗原(Ⅷ因子-RAg)多克隆抗体进行。
3.分组：HUVECs细胞生长至70%-80%融合时,分为以下四组：①A组：正常糖浓度组(5.5mmol/L葡萄糖)；②B组：高糖组(25mmol/L葡萄糖)；③C组：高糖(25mmol/L葡萄糖)+硫氢化钠(50μmol/L)组；④D组：正常糖浓度(5.5mmol/L葡萄糖)+硫氢化钠(50μmol/L)。处理48h后用于后续实验。
4.MTT法检测各组细胞的存活率、Hoechst33258荧光染色检测细胞凋亡
5.吸光法测定细胞内H2S的含量。
6.Real-time PCR方法检测CSE mRNA表达情况；Western blot方法检测CSE蛋白水平的变化。
7.Western Blot方法检测细胞凋亡相关蛋白Bax、Bcl-2和Cleaved caspase-3的表达。
8.ELISA法检测ET-1水平。
9.氧化应激水平检测：包括ROS、MDA的生成量检测和SOD活性检测。
研究结果
1.一般形态及细胞鉴定：倒置显微镜下可见,HUVECs单层贴壁生长,第1天呈小团状聚集,3-5d后呈铺路石样生长,细胞呈短梭形或多角形,融合成单层。
细胞鉴定显示Ⅷ因子阳性,胞浆呈棕黄色,证实为内皮细胞,阴性对照组胞浆未见着色。
2.细胞存活率检测：MTT法结果显示：HUVECs经25mmol/L葡萄糖处理48h后,细胞活力与正常组(5.5mmol/L葡萄糖组)相比明显下降,25mmol/L葡萄糖处理组细胞存活率为(68.5±3.8)%,比5.5mmol/L葡萄糖组下降了31.5%(P＜0.05)。NaHS对HUVECs具有保护作用,能显著抑制高浓度葡萄糖对HUVECs的损伤,加入NaHS后,细胞存活率得到明显改善,50μmol/L NaHS和25mmol/L葡萄糖共同处理组细胞存活率为(97.1±7.4)%,比25mmol/L葡萄糖组上升了28.6%(P＜0.05)。与5.5mmol/L葡萄糖组相比,5.5mmol/L葡萄糖与50μmol/L NaHS共同处理组细胞存活率无明显变化(P>0.05)。
Hoechst33258荧光染色检测细胞凋亡结果显示,5.5mmol/L葡萄糖处理组细胞核较大,呈正常均匀的蓝色,边缘整齐,无凋亡细胞；25mmol/L葡萄糖处理组部分细胞核致密浓染,核固缩,并呈高亮度荧光,即凋亡细胞数明显增多(P<0.05)；加入50μmol/L NaHS后,可见凋亡细胞数明显减少(P＜0.05)。与5.5mmol/L葡萄糖处理组相比,50μmol/L NaHS组与5.5mmol/L葡萄糖共同处理组细胞凋亡率无明显变化(P＞0.05)。
上述结果提示,高浓度葡萄糖可引起HUVECs凋亡增多,存活量减少；外源性补充H2S可抑制凋亡发生,使细胞存活数量增多。这可能是H2S对血管内皮具有保护作用的重要原因。
3.H2S生成量：与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖组的H2S生成量明显减少(12.85±0.53μmol/分钟vs10.26±0.51μmo1/分钟,P＜0.05)；与25mmol/L葡萄糖组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组的H2S生成量明显增多(11.90±0.32μmol/分钟vs10.26±0.51μmol/分钟,P＜0.05)。
4.CSE mRNA与蛋白生成量：与正常HUVEC细胞(5.5mmol/L葡萄糖处理组)相比,高浓度葡萄糖(25mmol/L葡萄糖)对CSE mRNA表达量的影响无显著性差异(P＞0.05)。
与5.5mmol/L葡萄糖组相比,以25mmol/L葡萄糖处理48h后能使CSE蛋白的表达下降,而25mmol/L葡萄糖+50μmol/L NaHS则能改善这种变化,CSE蛋白的表达比25mmol/L葡萄糖组增强；CSE蛋白半定量分析显示25mmol/L葡萄糖处理HUVECs48h后,能使CSE蛋白的表达下降53.7%(P＜0.05),50μmol/L NaHS和25mmol/L葡萄糖共处理组使CSE的表达较25mmol/L葡萄糖组上升19%(P＜0.05)。
5.细胞凋亡：Western Blot检测结果显示,与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖处理48h后可使Bax表达显著升高(P＜0.05),Bcl-2的表达显著降低(P＜0.05)而Caspase-3活性明显增强(P＜0.05),Bax/Bcl-2比值显著升高(P＜0.05)；与25mmol/L葡萄糖处理组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组Bax水平显著降低(P＜0.05),Bcl-2水平显著升高(P＜0.05),Caspase-3活性显著下降(P＜0.05),Bax/Bcl-2比值显著降低(P＜0.05)；50μmol/L NaHS和5.5mmol/L葡萄糖共同处理组的上述结果与5.5mmol/L葡萄糖处理组无显著性差异(P＞0.05)。
以上结果说明高浓度葡萄糖可以增加促进凋亡蛋白Bax和Cleaved caspase-3的表达,同时减少抑制凋亡蛋白Bcl-2的表达；而NaHS可以抑制高糖的促凋亡作用。
6.内皮素水平：与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖处理48h后可使细胞上清液中内皮素分泌量显著增加(P＜0.05)；与25mmol/L葡萄糖处理组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组内皮素分泌量显著减少(P＜0.05)：50μmol/L NaHS和5.5mmol/L葡萄糖共同处理组的内皮素分泌量与5.5mmol/L葡萄糖处理组无显著性差异(P＞0.05)。
7.氧化应激：与5.5mmol/L葡萄糖组相比,25mmol/L葡萄糖处理48h后可使ROS生成量显著增加(P＜0.05),MDA的生成量也显著增加(P＜0.05),而抗氧化酶SOD活性显著降低(P＜0.05)；与25mmol/L葡萄糖处理组相比,50μmol/L NaHS和25mmol/L葡萄糖共同处理组ROS和MDA的生成量均显著减少(P＜0.05),而抗氧化酶SOD活性显著增强(P＜0.05)；50umol/L NaHS和5.5mmol/L葡萄糖共同处理组的上述结果与5.5mmol/L葡萄糖处理组无显著性差异(P＞0.05)。
上述结果说明,高浓度葡萄糖可以增加氧化酶的活性,降低抗氧化酶的活性,并使得最终氧化应激产物的生成量增加；而NaHS可以抑制高糖的促氧化应激作用。
结论
1.高浓度葡萄糖可使体外培养的人脐静脉内皮细胞凋亡增多、存活减少,该作用与抑制CSE酶活性,减少H2S生成有关；外源性补充H2S可抑制细胞凋亡,使细胞存活增多。
2.高浓度葡萄糖抑制H2S生成后造成细胞存活减少的机制包括：促进凋亡相关蛋白表达、氧化应激反应加强、内皮素分泌增多。
3.外源性补充H2S可通过抑制凋亡相关蛋白Bax、Cleaved caspase-3表达,促进Bcl-2蛋白表达,增强抗氧化酶SOD活性,减少氧化应激产物ROS和MDA生成,抑制ET-1释放等多种途径发挥内皮细胞保护作用。
Backgrounds and aims
Recently the morbidity of diabetes has increased rapidly. Vascular complications are the common in diabetes patients, with the characteristics of high prevalence, disability and mortality. Thus it is pivotal to investigate the mechanism of vascular complications of diabetes. Nowadays it is well known that endothelium dysfunction is an early manifestation in the development of diabetic vascular complications. High concentration of glucose may impair the endothelial function via inflammation, oxidative stress, advanced glycosylation endproducts, and subsequent dyslipidemia, and so on.
Hydrogen sulfide (H2S), a colorless gas, has been considered as the third gasotransmitter except for nitric oxide (NO) and carbon monoxide (CO). H2S has been shown to possess important roles in the physiology and pathophysiology of several biological systems, especially in cardiovascular system. Endogenous H2S or H2S donor may decrease blood pressure, ameliorate myocardial ischemia and hypoxia or ischemia-reperfusion injury, reduce atherosclerosis lesions and improve the symptoms of heart failure, and so on. For example, administration of exogenous H2S (NaHS) has been shown to limit infarct size and preserve ventricular functioning in a myocardial ischemia-reperfusion mouse model. In apoE(-/-) mice, NaHS may increase plasma H(2)S level, decrease the size of atherosclerotic plaque and plasma and aortic ICAM-1levels.
In mammalian, endogenous H2S is generated by three known enzymes, namely cystathionine-β-synthase (CBS), cystathionine-y-lyase (CSE) and3-mercaptopyruvate sulfur transferase (3MST). The former two enzymes are pyridoxal-5'-phosphate dependent, which utilize L-cysteine and homocysteine as substrates to generate H2S. The latter enzyme utilizes L-cysteine and a-ketoglutarate through the metabolism with cysteine aminotransferase (CAT) to produce H2S. CBS and3MST are the main H2S-forming enzyme in the central nervous system, whereas CSE is the principal H2S-forming enzyme in the vasculature and heart.
Recently more and more attention has been put on the effects of H2S on endothelium. It has been shown that NaHS suppressed ICAM-1expression in tumor necrosis factor (TNF)-alpha-treated HUVECs via the NF-κB/IκB pathway. H2S has also been reported to suppress superoxide formation, NADPH oxidase-1(NOX-1) expression in endothelial cells, and protect ventricular function from oxidative stress to restore normal remodeling.
Therefore, H2S may be involved in the vascular structure disorder and dysfunction. However, up to date, little is known about the role of H2S in the development of diabetic vascular complications. In the present study, we intended to investigate the protective effect of H2S on the injury of human umbilical vein endothelium cells (HUVECs) induced by high glucose levels. Furthermore, we determined the mechanism of H2S on HUVECs from the point of H2S production, apoptosis, oxidative stress and secretion of ET-1.
Materials and methods
1. HUVECs were isolated from umbilical vein cords of normal pregnancies as previously described. Briefly, umbilical veins were rinsed with sterile saline and digested with0.25%trypsin. Harvested cells were cultured in M199medium supplemented with10%fetal bovine serum,100U/mL penicillin-streptomycin and20ng/mL vascular endothelial growth factor in an atmosphere of5%CO2at37℃. The medium was refreshed at intervals of3days at cell confluence. To maintain uniform condition, the cells in passage2to3were used for experiments.
2. After growing to70%confluence, the primary HUVECs were observed under inverted microscope, photoed and identified with Ⅷ factor antigen polyclonal antibody.
3. When growing to approximately80%confluence, HUVECs were then cultured in medium containing either normal glucose (5.5mmol/L) which served as a normal control or high glucose (25mmol/L) for48h. To determine the effects of NaHS, an H2S donor, on endothelial cells, HUVECs were pre-treated with NaHS (50μmol/L) for30min before adding high glucose. After48h, the cells were harvested for following experiments.
4. Cell viability was determined by MTT. At the same time, cell apoptosis was examined according to the morphological changes in cell nuclei using Hoechst33258staining. Cells were washed with cold PBS and fixed with paraformaldehyde for30min. Hoechst33258(10μg/mL) was added and incubated for20min before being detected by fluorescence microscopy. Apoptotic cells appeared as chromatin condensation and multiple chromatin fragments.
5. The content of H2S in cells was tested by UV spectrophotometer. The H2S concentration of each sample was expressed as nanomoles of H2S per milligram soluble protein.
6. The mRNA and protein expression of CSE in HUVECs were determined by Real-time PCR and Western blot analysis, respectively.
7. The apoptosis-associated proteins expression in HUVECs such as Bax、Bcl-2and Cleaved caspase-3were detected by Western blot analysis.
8. The secretion of ET-1of HUVECs was discovered by ELISA method. Results were normalized to cellular protein content in all experiments.
9. Intracellular ROS generation was measured by the oxidation-sensitive fluorescent probe (DCF-DA). The MDA level was measured to assess lipid peroxidation and the activity of SOD was measured as per kit instructions to estimate the role of anti-oxidation in the cells by commercial reagent kits
Results
1. Morphological features of HUVECs are proper and in line with others. This is validated by Ⅷ factor antibody.
2. Cell viability in the high glucose treated HUVECs (68.5±3.8%) was significantly decreased in comparison with normal cells (P<0.05), which could be ameliorated by NaHS (97.1±7.4%)(P<0.05).
HUVECs treated with high glucose showed obvious apoptotic features including chromatin condensation and multiple chromatin fragments. In contrast, nuclei of cells in the normal control group did not show apoptotic morphology. The percentage of apoptotic cells increased significantly in high glucose-treated cells compared with that of the normal glucose treated cells (P<0.05). NaHS administration reduced the apoptotic cells in high glucose cultured HUVECs significantly (P<0.05).
This indicated that high glucose induced HUVECs apoptosis and reduced cell viability. H2S exerted the protective effect on HUVECs as proved by decreased apoptosis and increased cell viability.
3. Production of H2S was significantly less in25mmol/L glucose treated HUVECs than those in the5.5mmol/L glucose treated cells (10.26±0.51μmol/min vs12.85±0.53μmol/min, P<0.05). The reduced H2S production caused by high glucose was retrieved by50μmol/L NaHS (10.26±0.51μmol/min vs11.90±0.32μmol/min, P<0.05).
4. Although the mRNA expression of CSE in high glucose treated HUVECs was somewhat higher than that in normal glucose treated cells, the difference did not achieve statistical significance.
The CSE protein expression was shown by Western blot analysis to be down-regulated about53.7%(P<0.05) in25mmol/L glucose treated HUVECs, while the addition of50μmol/L NaHS up-regulated the expression by19%(P<0.05).
5. High glucose significantly increased Bax protein expression and decreased Bcl-2protein expression (both P<0.05). The ratio of Bax/Bcl-2was increased about2-fold following exposure to high glucose (P<0.05). Accordingly, the level of cleaved caspase-3, the activated form of caspase-3, was higher in the high glucose group compared with that of control cells (P<0.05).
Compared with the high glucose group, the decrease in Bax protein expression and the increase in Bcl-2protein expression were observed in the group pre-treated with NaHS (both P<0.05). Similarly, NaHS attenuated the elevated Bax/Bcl-2ratio induced by high glucose, and inhibited high glucose-induced caspase-3activation (both P<0.05).
These results suggested that high glucose-induced endothelial cell apoptosis might be corrected by H2S.
6. High glucose resulted a significant elevation of ET-1secretion in HUVECs when compared with normal glucose (P<0.05). The glucose induced increase in ET-1was significantly attenuated by NaHS (P<0.05).
7. The production of ROS was measured using fluorescence microscopy, based on ROS-dependent oxidation of DCF-DA to DCF with green fluorescence. Compared with the high glucose group, NaHS-treated group showed a significant decrease in DCF levels (P<0.01). The results indicated that pre-treatment with NaHS could decrease the intracellular ROS generation.
Compared with control group, SOD activity was decreased in the high glucose group from23.59±1.08U/mg protein to19.53±2.17U/mg protein (P<0.05). Whereas, pre-treatment with NaHS increased SOD activity from19.53±2.17U/mg protein to23.66±1.16U/mg protein (P<0.05).
The amount of MDA was measured to assess the extent of lipid peroxidation. Compared with control group, the high glucose group showed a significant increase in MDA level from1.38±0.33μM/mg protein to2.78±0.59μM/mg protein (P<0.01), which was reversed by pre-treatment with NaHS (from2.78±0.59μM/mg protein to1.32±0.16μM/mg protein, P<0.01).
This indicated that H2S may alleviate the oxidative stress and thus protect HUVECs injury from high glucose.
Conelus ions
1. High glucose induces apoptosis of HUVECs via inhibiting of CSE expression and H2S production, which can be alleviated by H2S donor.
2. The cellular mechanisms of high glucose on HUVECs damage include promotion of apoptosis, increase of oxidative stress and elevation of ET-1secretion.
3. Exogenous H2S protect HUVECs from high glucose induced injury through inhibition of Bax and Cleaved caspase-3expression, increase Bcl-2expression, elevation of SOD activity, reduce of ROS and MDA, and suppression of ET-1.
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9. Kubo S, Kajiwara M, Kawabata A. Dual modulation of the tension of isolated gastric artery and gastric mucosal circulation by hydrogen sulfide in rats. Inflammopharmacology.2007; 15(6):288-292.
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