L-arginine对高糖诱导的人脐静脉内皮细胞衰老的作用及机制研究
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摘要
现今,糖尿病已成为严重危害人类健康的全球流行性疾病,其血管并发症是患者致残、致死的主要原因。与非糖尿病人群相比,糖尿病人群中动脉粥样硬化性疾病的患病率高、发病年龄轻、病情进展较快,多脏器同时受累较多。一些研究指出,高血糖可以加速细胞的衰老,而内皮细胞衰老是动脉粥样硬化的早期病理改变,因此,研究高糖致内皮细胞衰老的机制,从而发展有效阻断药物,是防治糖尿病大血管病变的有效途径。
内皮细胞是糖尿病血管病变的关键靶组织。它不仅在抗凝血因子形成过程中起着保护屏障作用,而且其自身可分泌因子调节血管管腔和保持内环境稳态的作用。正常生理状态下,内皮细胞的更新率很低,分裂增殖能力有限,但因损伤(如高糖刺激)导致细胞不断的分裂将引起端粒缩短,细胞进入衰老期,表现为SA-β-gal活性增强、细胞停滞在细胞周期的G0／G1期、衰老相关的基因和蛋白表达阳性以及端粒酶活性降低等；同时,血管内皮细胞衰老过程中,分泌的各种生物学活性因子也在发生变化,如NO的合成下降、炎性因子合成增加、粘附因子表达增多等。这些变化都将造成内皮细胞功能障碍,最终导致动脉粥样硬化的发生。可见,衰老与糖尿病相关的动脉粥样硬化存在密切关系。
端粒钟学说是衰老发生机制的学说之一。端粒是真核细胞染色体末端的DNA序列,随着细胞的每次分裂,端粒不断缩短,当缩短到一个阂值时,便不能维持染色体的稳定,使细胞失去分裂增殖能力而进入了衰老阶段。因此,端粒缩短被认为是触发细胞衰老的分子钟,可作为衰老的生物标记。端粒酶是一种核糖核蛋白逆转录酶,可以在缩短的端粒末端加上新的重复序列,保持端粒长度,在调节寿命与细胞增殖方面扮演着重要的角色。端粒酶活性的调控是多因素的,PI3K/Akt通路正是其中之一。对黑色素细胞瘤细胞系SK-MEL28的研究发现,在无血清培养24小时后肿瘤细胞Akt活性升高,端粒酶的活性增加2倍,而端粒酶逆转录酶催化亚基(Human Telomerase Reverse Transcriptase Subunit, hTERT)的两个XXRXRXXSXX序列也相应出现了磷酸化；应用PI3K抑制剂可以降低肿瘤细胞端粒酶的活性并抑制hTERT的磷酸化。因此,Akt必然成为端粒酶十分重要的上游调节点。
氧化应激的增加与NO生物利用度的减少在细胞衰老中起着重要的作用。有研究显示高糖可以增加细胞内ROS的产生,而后者可通过抑制Src家族激酶的活性而缩短端粒的长度,加快衰老的进程。NO能与过量的ROS反应而减轻细胞氧化应激的程度。NO被认为是最主要的内皮源性自分泌因子,它可以调节血管管腔大小,抑制血小板聚集、抗凋亡、抑制平滑肌细胞增殖和白细胞粘附,因而在抗动脉粥样硬化过程中发挥着重要的作用。很多研究发现内皮型NOS(eNOS)的表达和NO的合成随年龄的增加而下降,虽然其机制目前还不明确,但有研究表明PI3K/Akt可以介导eNOS的磷酸化,亦有研究显示2型糖尿病患者内皮功能紊乱与PI3K/Akt/eNOS通路受到抑制有关。因此,在内皮细胞中上调PI3K/Akt信号系统应是防治糖尿病大血管病变的一个重要靶点。
L-Arg是NO合成的前体物质,在1992年Creager和Dubois-Rarde两组学者首次断定,补充eNOS作用的底物L-Arg是增加内皮产生生物活性NO的一种方法。L-Arg改善内皮细胞功能的机制相对多样化,尚未阐明,这种作用可能与增加NO的合成和eNOS蛋白表达,减少ROS的形成有关。
综上所述,本实验在体外模拟高糖状态,观察高血糖是否加速人脐静脉内皮细胞衰老,其对NO、eNOS蛋白表达及转录水平的影响,以及与PI3K/Akt信号通路的关系,而L-Arg是否可以延缓高糖加速的内皮细胞衰老的进程,其作用机制是否与PI3K/Akt通路有关,为进一步探讨糖尿病大血管病变发病机制及其防治手段提供实验依据。
材料与方法
1、人脐静脉内皮细胞株培养,不同浓度的葡萄糖溶液孵育细胞、高糖(33 mM)和／或不同浓度L-arginine共同孵育细胞,以及同一浓度孵育不同的时间,收集内皮细胞及细胞培养液；分别用PI3K/Akt特异性抑制剂LY294002、eNOS特异性抑制剂L-NAME进行预处理2h,然后于上述因素孵育细胞,收集内皮细胞及细胞培养液,用于指标检测。
2、衰老相关的β-半乳糖苷酶活性检测是通过染色计算阳性细胞的百分比来确定,后者是指1000个细胞中被染成蓝绿色细胞的百分比。
3、采用PCR-ELISA法检测端粒酶的活性。
4、应用流式细胞术检测活性氧及分析细胞周期。
5、应用RT-PCR技术检测PI3K, Akt、eNOS mRNA的表达。
6、采用Westemblot法检测人脐静脉内皮细胞p-Akt (Ser-473)、Akt、p-eNOS (Ser-1177)、eNOS蛋白的表达。
7、采用硝酸盐还原酶还原硝酸盐为亚硝酸盐,然后通过经典的Griess reagent检测亚硝酸盐,从而测定出总一氧化氮。
结果
1、随着糖浓度的增加(即5.5、11、22、33 mmol/L) SAβ-gal活性逐渐增强(P<0.01),端粒酶活性逐渐减弱(P<0.01),G0／G1期细胞不断增加(P<0.01),S期细胞不断减少(P<0.01)。
2、随着糖浓度的不断增加,细胞内ROS逐渐增多(P<0.01),而NO的产量逐渐减少(P<0.001)。
3、与正常对照组相比,33mmol/L糖浓度组72 h PI3K、Akt、eNOS mRNA的表达明显下降(P<0.05)。
4、与正常对照组相比,33 mmol/L糖浓度组48 h p-Akt (Ser-473)/Akt、p-eNOS (Ser-1177)/eNOS蛋白表达量的比值明显减弱(P<0.01)。
5、应用不同浓度L-arginine (0.4、0.8、1.6mmol/L)与高糖(33 mmol/L)共同干预细胞,不同程度地减弱了SAβ-gal活性(与高糖组比较,P<0.01),增强了端粒酶的活性(P<0.01),G0／G1期细胞较高糖组明显减少(P<0.01),S期细胞明显增多(P<0.01)。而3.2mmol/L的L-arginine出现相反作用,各指标与高糖组比较无统计学差异。
6、与高糖组比较,L-arginine (0.4、0.8、1.6mmol/L)明显减少细胞内的ROS水平(P<0.001,),明显增多NO的合成(P<0.001)。
7、与高糖组比较,L-arginine(0.8 mmol/L)作用内皮细胞72 h使PI3K、Akt、eNOS mRNA的表达明显增强(P<0.01)。
8、与高糖组比较,L-arginine (0.4、0.8、1.6mmol/L)明显增加了p-Akt(Ser-473)/Akt. p-eNOS(Ser-1177)/eNOS蛋白的表达量的比值(P<0.01)。
9、PI3K/Akt特异性抑制剂LY294002及eNOS特异性抑制剂L-NAME可以抑制L-arginine的抗衰老作用。
结论
1、高糖可以明显加快内皮细胞衰老的进程,这种作用呈现糖浓度依赖性及时间依赖性。
2、L-arginine可以明显延缓高糖加速的内皮细胞衰老进程。
3、L-arginine可能通过上调PI3K/Akt路径,增强端粒酶及eNOS的活性,从而发挥抗内皮细胞衰老的作用。
Today, diabetes has become a global epidemic disease to damage human health. Chronic complications are the major cause of disability and death in patients with diabetes. Compared with non-diabetic people, morbidity of atherosclerosis disease is higher, age of incidence is younger and development of illness is fast in patients with diabetes. Some studies have shown that high glucose accelerates cell senescence and vascular aging, thereby accelerates the process of atherosclerosis. Therefore, it is therapeutic agent for prevention and treatment of diabetic vascular complications that study mechanism of high glucose-induced cell senescence and thereby development of effective blocking drug.
Endothelial cell is the key target tissue to diabetic vascular disease. It not only plays a protective function in the formation of anti-coagulation factor, but also it can secret some cellular factor to regulate blood vessel lumen and maintain internal environment. In normal physiological state, the update rate of endothelial cells is very low, and constant cells cleavage induced by injury (For example high glucose) will lead to telomere shortening. So the cells enter growth arrest period, which is aging period. In senescent state, the cells showed some characteristics, for example, increased SA-β-gal activity, the cells stuck in the cell cycle G0/G1 phase, aging-related gene and protein overexpression, as well as telomere shortening, decreased telomerase activity and so on. Simultaneously, in vascular endothelial cell senescence process, a variety of secretion biologically active factors are also changing, such as decreased the formation of endothelium-dependent vascular relaxing factor, increased synthesis of inflammatory factors and adhesion molecule expression. These changes will result in endothelial cell dysfunction, and eventually lead to the occurrence of atherosclerosis. Therefore, there was a close relationship between aging and diabetes-associated atherosclerosis, namely diabetic macrovascular disease.
Telomere clock theory is one of the doctrine mechanisms of aging. Telomere is the DNA sequence of chromosome ends in eukaryotic cells. Telomere shortens with each cell cleavage. But chromosome stability can't be maintained when telomere reduced to a threshold value. So it will lead to cells lose their proliferative ability and cells enter into the aging phase. Thus, telomere shortening is considered as biomarkers of cell senescence and it enable the molecular clock to start. Telomerase is a ribonucleoprotein reverse transcriptase, can add new repeat sequences to shortened telomere ends so that maintain telomere length. It plays an important role in the regulation of life and cell proliferation, is a multifactor, PI3K/Akt pathway is one of the multifactors in regulating telomerase activity.
Study found that Akt can enhance telomerase activity. On the melanoma cell line SK-MEL28 study found that Akt activity in tumor increased after incubation with the absence of serum for 24 h, and telomerase activity increased 2-fold. Two sequences XXRXRXXSXX of human telomerase reverse transcriptase (hTERT) correspondingly emerged phosphorylation. Application of PI3K inhibitor reduced telomerase activity in tumor cells and inhibited the hTERT phosphorylation. Thus, Akt is become a very important upstream regulator of telomerase.
Increased oxidative stress and reduced NO bioavailability play an important role in cell senescence. Studies showed that high glucose can increase ROS and latter may shorten telomere length through inhibited Src Family kinase activity to accelerate cell senescence process. NO and excess ROS generated response so that attenuated oxidative stress in cell. NO is considered as the most important endothelium-derived aotocrine factor which L-arginine (L-Arg) is catalyzed by nitric oxide synthase (NOS).The effect of NO is extremely broad, which can adjust vascular tube cavity size, inhibit platelet aggregation, anti-apoptosis, inhibit smooth muscle cell proliferation and leukocyte adhesion, which plays an important role in the anti-atherosclerosis. Many studies found that endothelial NOS (eNOS) expression and NO production decrease with age, although the mechanism is not yet clear, studies have shown that phosphatidylinositol 3-kinase(PI3K)/Akt may mediate the phosphorylation of eNOS. Some studies have shown that entothelial dysfunction is related to inhibit PI3K/Akt/eNOS pathway in patients with type 2 diabetes. Thus, upregulation PI3K/Akt/eNOS signaling system should be an important target for prevention and treatment of diabetic vascular disease in endothelial cells.
L-Arg is the precursor of NO synthesis. In 1992, Creager and Dubois-Rarde, for the first time, concluded that complementary role of L-Arg which is the substrate of eNOS, is to increase biological activity of endothelium-generated NO. L-Arg to improve the mechanism of endothelial cell function is relatively diversified. This effect may be relate to increase NO synthesis and eNOS protein expression, reducing reactive oxygen species (ROS) formation.
In summary, this experiment, under high glucose in vitro, aimed to study whether high glucose may accelerate human umbilical vein endothelial cell senescence and its effect on NO, eNOS protein expression and transcription level, as well as the relationship between it and the PI3K/Akt signaling pathway. To study whether L-Arg may delay high glucose-induced cell senescence process and its mechanism is related to inhibit PI3K/Akt/eNOS pathway due to provide experimental evidence for pathogenesis mechanism, prevention and treatment for diabetic macroangiopathy.
Materials and methods
1. We cultured human umbilical vein endothelial cell with different concentration of glucose solution, and high glucose (33mmol/L) and/or different concentration of L-arginine co-incubated cells, as well as the same concentration incubated in different time point, then we collected cells and cell culture medium. Respectively with PI3K/Akt inhibitor LY294002 or eNOS inhibitor L-NAME pretreated for 2 h, then incubated cells with the above-mentioned factors, and collected cells and cell culture medium to used for target detection.
2. Senescence-associated (3-galactosidase (SA P-gal) activity was determined by the percentage of SAβ-gal staining positive cells. The latter was determined by counting, the number of blue-green cells within a sample of 1000 cells.
3. We used PCR-ELISA assay to detect the telomerase activity.
4. ROS and analysis of the cell cycle were determined by flow cytometry assay.
5. Applied RT-PCR to detect the mRNA expression of PI3K, Akt and eNOS.
6. Used Westernblot to detect the p-Akt (Ser-473), Akt, p-eNOS (Ser-1177) and eNOS protein expression in HUVEC.
7. Restored the nitrate to nitrite by nitrate reductase, and then detected the nitrite through the classic Griess reagent, which measured a total nitric oxide.
Results
1. With the glucose concentration increasing, that is 5.5,11,22,33 mmol/L, SA P-gal activity gradually increased (P<0.01), telomerase activity gradually decreased (P<0.01), the percentage of G0/G1 phase cells increased (<0.01), S phase cells decreased (P<0.01) and cell proliferation ability gradually weakened.
2. With the glucose concentration increasing, intracellular ROS gradually increased (P＜0.001), whereas NO production gradually reduced (P<0.001).
3. Compared with normal control group, the mRNA expression of PI3K, Akt and eNOS was significantly decreased in 33 mmol/L glucose concentration group at 72 h (P＜0.05).
4. Compared with normal control group, the ratio of p-Akt (Ser-473) to Akt and p-eNOS (Ser-1177) to eNOS protein expression were significantly reduced (P<0 .01).
5. Co-incubation with different concentration of L-arginine (0.4 mmol/L,0.8 mmol/L, 1.6mmol/L) and high glucose (33 mmol/L) in HUVEC, SAβ-gal activity weakened in varying degrees (compared with high glucose group, P＜0.01), and telomerase activity obviously increased (compared with high glucose group, P<0.01), and G0/G1 phase cells significantly reduced, however, S phase cells increased (compared with high glucose group, P＜0.01), so cell proliferation was improved. But increasing L-arginine to 3.2 mmol/L exerted the opposite effect, it was no statistical difference compared with high glucose group.
6. L-arginine (0.4 mmol/L,0.8 mmol/L,1.6mmol/L) reduced intracellular ROS (P＜0.001), and increased NO formation (P＜0.001).
7. Compared with high glucose group, L-arginine (0.8 mmol/L) increased the mRNA expression of PI3K, Akt and eNOS (P＜0.01).
8. Compared with high glucose group, L-arginine (0.4 mmol/L,0.8 mmol/L, 1.6mmol/L) significantly increased the ratio of p-Akt (Ser-473) to Akt and p-eNOS (Ser-1177) to eNOS protein expression (P＜0.01).
9. PI3K/Akt inhibitor LY294002 or eNOS inhibitor L-NAME can eliminate the anti-senescence effect of L-arginine.
Conclusion
1. High glucose can significantly accelerate the cell senescent process, which presents the role of glucose concentration-dependent and time-dependent.
2. L-arginine can significantly delay high glucose-accelerated cell senescent process.
3. L-arginine maybe exerts anti-senescence effect through upregulation PI3K/Akt pathway to increase telomerase and eNOS activity.
内皮细胞是糖尿病血管病变的关键靶组织。它不仅在抗凝血因子形成过程中起着保护屏障作用,而且其自身可分泌因子调节血管管腔和保持内环境稳态的作用。正常生理状态下,内皮细胞的更新率很低,分裂增殖能力有限,但因损伤(如高糖刺激)导致细胞不断的分裂将引起端粒缩短,细胞进入衰老期,表现为SA-β-gal活性增强、细胞停滞在细胞周期的G0／G1期、衰老相关的基因和蛋白表达阳性以及端粒酶活性降低等；同时,血管内皮细胞衰老过程中,分泌的各种生物学活性因子也在发生变化,如NO的合成下降、炎性因子合成增加、粘附因子表达增多等。这些变化都将造成内皮细胞功能障碍,最终导致动脉粥样硬化的发生。可见,衰老与糖尿病相关的动脉粥样硬化存在密切关系。
端粒钟学说是衰老发生机制的学说之一。端粒是真核细胞染色体末端的DNA序列,随着细胞的每次分裂,端粒不断缩短,当缩短到一个阂值时,便不能维持染色体的稳定,使细胞失去分裂增殖能力而进入了衰老阶段。因此,端粒缩短被认为是触发细胞衰老的分子钟,可作为衰老的生物标记。端粒酶是一种核糖核蛋白逆转录酶,可以在缩短的端粒末端加上新的重复序列,保持端粒长度,在调节寿命与细胞增殖方面扮演着重要的角色。端粒酶活性的调控是多因素的,PI3K/Akt通路正是其中之一。对黑色素细胞瘤细胞系SK-MEL28的研究发现,在无血清培养24小时后肿瘤细胞Akt活性升高,端粒酶的活性增加2倍,而端粒酶逆转录酶催化亚基(Human Telomerase Reverse Transcriptase Subunit, hTERT)的两个XXRXRXXSXX序列也相应出现了磷酸化；应用PI3K抑制剂可以降低肿瘤细胞端粒酶的活性并抑制hTERT的磷酸化。因此,Akt必然成为端粒酶十分重要的上游调节点。
氧化应激的增加与NO生物利用度的减少在细胞衰老中起着重要的作用。有研究显示高糖可以增加细胞内ROS的产生,而后者可通过抑制Src家族激酶的活性而缩短端粒的长度,加快衰老的进程。NO能与过量的ROS反应而减轻细胞氧化应激的程度。NO被认为是最主要的内皮源性自分泌因子,它可以调节血管管腔大小,抑制血小板聚集、抗凋亡、抑制平滑肌细胞增殖和白细胞粘附,因而在抗动脉粥样硬化过程中发挥着重要的作用。很多研究发现内皮型NOS(eNOS)的表达和NO的合成随年龄的增加而下降,虽然其机制目前还不明确,但有研究表明PI3K/Akt可以介导eNOS的磷酸化,亦有研究显示2型糖尿病患者内皮功能紊乱与PI3K/Akt/eNOS通路受到抑制有关。因此,在内皮细胞中上调PI3K/Akt信号系统应是防治糖尿病大血管病变的一个重要靶点。
L-Arg是NO合成的前体物质,在1992年Creager和Dubois-Rarde两组学者首次断定,补充eNOS作用的底物L-Arg是增加内皮产生生物活性NO的一种方法。L-Arg改善内皮细胞功能的机制相对多样化,尚未阐明,这种作用可能与增加NO的合成和eNOS蛋白表达,减少ROS的形成有关。
综上所述,本实验在体外模拟高糖状态,观察高血糖是否加速人脐静脉内皮细胞衰老,其对NO、eNOS蛋白表达及转录水平的影响,以及与PI3K/Akt信号通路的关系,而L-Arg是否可以延缓高糖加速的内皮细胞衰老的进程,其作用机制是否与PI3K/Akt通路有关,为进一步探讨糖尿病大血管病变发病机制及其防治手段提供实验依据。
材料与方法
1、人脐静脉内皮细胞株培养,不同浓度的葡萄糖溶液孵育细胞、高糖(33 mM)和／或不同浓度L-arginine共同孵育细胞,以及同一浓度孵育不同的时间,收集内皮细胞及细胞培养液；分别用PI3K/Akt特异性抑制剂LY294002、eNOS特异性抑制剂L-NAME进行预处理2h,然后于上述因素孵育细胞,收集内皮细胞及细胞培养液,用于指标检测。
2、衰老相关的β-半乳糖苷酶活性检测是通过染色计算阳性细胞的百分比来确定,后者是指1000个细胞中被染成蓝绿色细胞的百分比。
3、采用PCR-ELISA法检测端粒酶的活性。
4、应用流式细胞术检测活性氧及分析细胞周期。
5、应用RT-PCR技术检测PI3K, Akt、eNOS mRNA的表达。
6、采用Westemblot法检测人脐静脉内皮细胞p-Akt (Ser-473)、Akt、p-eNOS (Ser-1177)、eNOS蛋白的表达。
7、采用硝酸盐还原酶还原硝酸盐为亚硝酸盐,然后通过经典的Griess reagent检测亚硝酸盐,从而测定出总一氧化氮。
结果
1、随着糖浓度的增加(即5.5、11、22、33 mmol/L) SAβ-gal活性逐渐增强(P<0.01),端粒酶活性逐渐减弱(P<0.01),G0／G1期细胞不断增加(P<0.01),S期细胞不断减少(P<0.01)。
2、随着糖浓度的不断增加,细胞内ROS逐渐增多(P<0.01),而NO的产量逐渐减少(P<0.001)。
3、与正常对照组相比,33mmol/L糖浓度组72 h PI3K、Akt、eNOS mRNA的表达明显下降(P<0.05)。
4、与正常对照组相比,33 mmol/L糖浓度组48 h p-Akt (Ser-473)/Akt、p-eNOS (Ser-1177)/eNOS蛋白表达量的比值明显减弱(P<0.01)。
5、应用不同浓度L-arginine (0.4、0.8、1.6mmol/L)与高糖(33 mmol/L)共同干预细胞,不同程度地减弱了SAβ-gal活性(与高糖组比较,P<0.01),增强了端粒酶的活性(P<0.01),G0／G1期细胞较高糖组明显减少(P<0.01),S期细胞明显增多(P<0.01)。而3.2mmol/L的L-arginine出现相反作用,各指标与高糖组比较无统计学差异。
6、与高糖组比较,L-arginine (0.4、0.8、1.6mmol/L)明显减少细胞内的ROS水平(P<0.001,),明显增多NO的合成(P<0.001)。
7、与高糖组比较,L-arginine(0.8 mmol/L)作用内皮细胞72 h使PI3K、Akt、eNOS mRNA的表达明显增强(P<0.01)。
8、与高糖组比较,L-arginine (0.4、0.8、1.6mmol/L)明显增加了p-Akt(Ser-473)/Akt. p-eNOS(Ser-1177)/eNOS蛋白的表达量的比值(P<0.01)。
9、PI3K/Akt特异性抑制剂LY294002及eNOS特异性抑制剂L-NAME可以抑制L-arginine的抗衰老作用。
结论
1、高糖可以明显加快内皮细胞衰老的进程,这种作用呈现糖浓度依赖性及时间依赖性。
2、L-arginine可以明显延缓高糖加速的内皮细胞衰老进程。
3、L-arginine可能通过上调PI3K/Akt路径,增强端粒酶及eNOS的活性,从而发挥抗内皮细胞衰老的作用。
Today, diabetes has become a global epidemic disease to damage human health. Chronic complications are the major cause of disability and death in patients with diabetes. Compared with non-diabetic people, morbidity of atherosclerosis disease is higher, age of incidence is younger and development of illness is fast in patients with diabetes. Some studies have shown that high glucose accelerates cell senescence and vascular aging, thereby accelerates the process of atherosclerosis. Therefore, it is therapeutic agent for prevention and treatment of diabetic vascular complications that study mechanism of high glucose-induced cell senescence and thereby development of effective blocking drug.
Endothelial cell is the key target tissue to diabetic vascular disease. It not only plays a protective function in the formation of anti-coagulation factor, but also it can secret some cellular factor to regulate blood vessel lumen and maintain internal environment. In normal physiological state, the update rate of endothelial cells is very low, and constant cells cleavage induced by injury (For example high glucose) will lead to telomere shortening. So the cells enter growth arrest period, which is aging period. In senescent state, the cells showed some characteristics, for example, increased SA-β-gal activity, the cells stuck in the cell cycle G0/G1 phase, aging-related gene and protein overexpression, as well as telomere shortening, decreased telomerase activity and so on. Simultaneously, in vascular endothelial cell senescence process, a variety of secretion biologically active factors are also changing, such as decreased the formation of endothelium-dependent vascular relaxing factor, increased synthesis of inflammatory factors and adhesion molecule expression. These changes will result in endothelial cell dysfunction, and eventually lead to the occurrence of atherosclerosis. Therefore, there was a close relationship between aging and diabetes-associated atherosclerosis, namely diabetic macrovascular disease.
Telomere clock theory is one of the doctrine mechanisms of aging. Telomere is the DNA sequence of chromosome ends in eukaryotic cells. Telomere shortens with each cell cleavage. But chromosome stability can't be maintained when telomere reduced to a threshold value. So it will lead to cells lose their proliferative ability and cells enter into the aging phase. Thus, telomere shortening is considered as biomarkers of cell senescence and it enable the molecular clock to start. Telomerase is a ribonucleoprotein reverse transcriptase, can add new repeat sequences to shortened telomere ends so that maintain telomere length. It plays an important role in the regulation of life and cell proliferation, is a multifactor, PI3K/Akt pathway is one of the multifactors in regulating telomerase activity.
Study found that Akt can enhance telomerase activity. On the melanoma cell line SK-MEL28 study found that Akt activity in tumor increased after incubation with the absence of serum for 24 h, and telomerase activity increased 2-fold. Two sequences XXRXRXXSXX of human telomerase reverse transcriptase (hTERT) correspondingly emerged phosphorylation. Application of PI3K inhibitor reduced telomerase activity in tumor cells and inhibited the hTERT phosphorylation. Thus, Akt is become a very important upstream regulator of telomerase.
Increased oxidative stress and reduced NO bioavailability play an important role in cell senescence. Studies showed that high glucose can increase ROS and latter may shorten telomere length through inhibited Src Family kinase activity to accelerate cell senescence process. NO and excess ROS generated response so that attenuated oxidative stress in cell. NO is considered as the most important endothelium-derived aotocrine factor which L-arginine (L-Arg) is catalyzed by nitric oxide synthase (NOS).The effect of NO is extremely broad, which can adjust vascular tube cavity size, inhibit platelet aggregation, anti-apoptosis, inhibit smooth muscle cell proliferation and leukocyte adhesion, which plays an important role in the anti-atherosclerosis. Many studies found that endothelial NOS (eNOS) expression and NO production decrease with age, although the mechanism is not yet clear, studies have shown that phosphatidylinositol 3-kinase(PI3K)/Akt may mediate the phosphorylation of eNOS. Some studies have shown that entothelial dysfunction is related to inhibit PI3K/Akt/eNOS pathway in patients with type 2 diabetes. Thus, upregulation PI3K/Akt/eNOS signaling system should be an important target for prevention and treatment of diabetic vascular disease in endothelial cells.
L-Arg is the precursor of NO synthesis. In 1992, Creager and Dubois-Rarde, for the first time, concluded that complementary role of L-Arg which is the substrate of eNOS, is to increase biological activity of endothelium-generated NO. L-Arg to improve the mechanism of endothelial cell function is relatively diversified. This effect may be relate to increase NO synthesis and eNOS protein expression, reducing reactive oxygen species (ROS) formation.
In summary, this experiment, under high glucose in vitro, aimed to study whether high glucose may accelerate human umbilical vein endothelial cell senescence and its effect on NO, eNOS protein expression and transcription level, as well as the relationship between it and the PI3K/Akt signaling pathway. To study whether L-Arg may delay high glucose-induced cell senescence process and its mechanism is related to inhibit PI3K/Akt/eNOS pathway due to provide experimental evidence for pathogenesis mechanism, prevention and treatment for diabetic macroangiopathy.
Materials and methods
1. We cultured human umbilical vein endothelial cell with different concentration of glucose solution, and high glucose (33mmol/L) and/or different concentration of L-arginine co-incubated cells, as well as the same concentration incubated in different time point, then we collected cells and cell culture medium. Respectively with PI3K/Akt inhibitor LY294002 or eNOS inhibitor L-NAME pretreated for 2 h, then incubated cells with the above-mentioned factors, and collected cells and cell culture medium to used for target detection.
2. Senescence-associated (3-galactosidase (SA P-gal) activity was determined by the percentage of SAβ-gal staining positive cells. The latter was determined by counting, the number of blue-green cells within a sample of 1000 cells.
3. We used PCR-ELISA assay to detect the telomerase activity.
4. ROS and analysis of the cell cycle were determined by flow cytometry assay.
5. Applied RT-PCR to detect the mRNA expression of PI3K, Akt and eNOS.
6. Used Westernblot to detect the p-Akt (Ser-473), Akt, p-eNOS (Ser-1177) and eNOS protein expression in HUVEC.
7. Restored the nitrate to nitrite by nitrate reductase, and then detected the nitrite through the classic Griess reagent, which measured a total nitric oxide.
Results
1. With the glucose concentration increasing, that is 5.5,11,22,33 mmol/L, SA P-gal activity gradually increased (P<0.01), telomerase activity gradually decreased (P<0.01), the percentage of G0/G1 phase cells increased (<0.01), S phase cells decreased (P<0.01) and cell proliferation ability gradually weakened.
2. With the glucose concentration increasing, intracellular ROS gradually increased (P＜0.001), whereas NO production gradually reduced (P<0.001).
3. Compared with normal control group, the mRNA expression of PI3K, Akt and eNOS was significantly decreased in 33 mmol/L glucose concentration group at 72 h (P＜0.05).
4. Compared with normal control group, the ratio of p-Akt (Ser-473) to Akt and p-eNOS (Ser-1177) to eNOS protein expression were significantly reduced (P<0 .01).
5. Co-incubation with different concentration of L-arginine (0.4 mmol/L,0.8 mmol/L, 1.6mmol/L) and high glucose (33 mmol/L) in HUVEC, SAβ-gal activity weakened in varying degrees (compared with high glucose group, P＜0.01), and telomerase activity obviously increased (compared with high glucose group, P<0.01), and G0/G1 phase cells significantly reduced, however, S phase cells increased (compared with high glucose group, P＜0.01), so cell proliferation was improved. But increasing L-arginine to 3.2 mmol/L exerted the opposite effect, it was no statistical difference compared with high glucose group.
6. L-arginine (0.4 mmol/L,0.8 mmol/L,1.6mmol/L) reduced intracellular ROS (P＜0.001), and increased NO formation (P＜0.001).
7. Compared with high glucose group, L-arginine (0.8 mmol/L) increased the mRNA expression of PI3K, Akt and eNOS (P＜0.01).
8. Compared with high glucose group, L-arginine (0.4 mmol/L,0.8 mmol/L, 1.6mmol/L) significantly increased the ratio of p-Akt (Ser-473) to Akt and p-eNOS (Ser-1177) to eNOS protein expression (P＜0.01).
9. PI3K/Akt inhibitor LY294002 or eNOS inhibitor L-NAME can eliminate the anti-senescence effect of L-arginine.
Conclusion
1. High glucose can significantly accelerate the cell senescent process, which presents the role of glucose concentration-dependent and time-dependent.
2. L-arginine can significantly delay high glucose-accelerated cell senescent process.
3. L-arginine maybe exerts anti-senescence effect through upregulation PI3K/Akt pathway to increase telomerase and eNOS activity.
引文
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