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外源性硫化氢减轻D-半乳糖诱导的亚急性衰老大鼠血管老化

乔伟丽, 杨文学, 刘磊, 石玥, 崔洁, 柳红, 闫长栋

徐州医学院生理学教研室；心血管研究所；病理学教研室；江苏省麻醉学重点实验室，徐州 221004

摘要

本文旨在观察亚急性衰老大鼠血管壁结构和功能的改变，探讨硫化氢(hydrogen sulfide, H2S)对血管老化的影响。雄性SD大鼠40只，随机分为溶剂组、D-半乳糖组、NaHS (H2S的供体)低、中、高剂量组，每组8只。D-半乳糖组每日背部皮下注射125 mg/kg D-半乳糖，连续8周；溶剂组每日背部皮下注射等体积生理盐水；NaHS低、中、高剂量组每日背部皮下注射125 mg/kg D-半乳糖，同时分别按1、10、100 μmol/kg腹腔注射NaHS，连续8周。HE和Masson染色法观察各组大鼠主动脉形态学改变；硫代巴比妥酸(TBA)法和黄嘌呤氧化酶法检测大鼠主动脉超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量以及抗超氧阴离子含量；生化方法测定大鼠血清 H2S 浓度的变化；酶联免疫法(ELISA)检测血浆血管紧张素II (AngII)浓度变化；免疫印迹法测定大鼠主动脉血管紧张素II型1类受体(AT1R)蛋白的表达。结果显示，与溶剂组相比，D-半乳糖组大鼠血清H2S的浓度降低(P < 0.05)；而给予NaHS处理后，NaHS各剂量组大鼠血清H2S的浓度较D-半乳糖组明显升高(P < 0.05)。血清H2S浓度的增高能够改善D-半乳糖引起的亚急性衰老大鼠主动脉形态学的变化。HE染色结果中，溶剂组主动脉内膜较薄，结构完整，中膜平滑肌排列规则；D-半乳糖组主动脉内膜增厚、隆起，内皮细胞部分脱落，中膜明显增厚，平滑肌增生、紊乱；与D-半乳糖组相比，NaHS各剂量组内皮细胞脱落减少，平滑肌细胞增生减少。Masson染色结果表明：与溶剂组相比，D-半乳糖组胶原纤维增多，平滑肌细胞增生(P < 0.05)；与D-半乳糖组相比，NaHS各剂量组胶原纤维减少，平滑肌细胞增生减少(P < 0.05)。ELISA检测结果显示：与溶剂组相比，D-半乳糖组大鼠血浆AngII浓度明显升高(P < 0.05)；而给予NaHS处理后，NaHS各剂量组大鼠血浆AngII浓度较D-半乳糖组明显降低(P < 0.05)。与溶剂组相比，D-半乳糖组大鼠主动脉SOD活性下降、MDA含量升高、抗超氧阴离子含量降低(P < 0.05)；而给予NaHS处理后，血管组织氧化应激水平得到明显改善：与D-半乳糖组相比，NaHS各剂量组主动脉SOD活性升高、MDA含量降低、抗超氧阴离子含量升高(P < 0.05)。免疫印迹法测定显示，与溶剂组相比，D-半乳糖组大鼠主动脉血管AT1R蛋白表达上调(P < 0.05)，给予NaHS处理能下调主动脉AT1R蛋白的表达。以上结果提示，外源性H2S能够改善亚急性衰老大鼠主动脉形态学变化，降低血浆AngII的浓度、下调主动脉AT1R蛋白的表达，提高血管组织抗氧化应激的能力，延缓血管老化的发生。

关键词： 硫化氢; D-半乳糖; 主动脉; 老化; 血管紧张素Ⅱ; 血管紧张素Ⅱ型1类受体

分类号：R339.3+8

[Exogenous hydrogen sulfide reduces vascular aging in D-galactose-induced subacute aging rats.] [Article in Chinese]

QIAO Wei-Li, YANG Wen-Xue, LIU Lei, SHI Yue, CUI Jie, LIU Hong, YAN Chang-Dong

Department of Physiology； Institute of Cardiovascular Disease； Department of Pathology； Jiangsu Province Key Laboratory of Anesthesiology； Xuzhou Medical College, Xuzhou 221004, China

Abstract

The present study was aimed to observe the protective effect of exogenous hydrogen sulfide (H2S) on vascular structural and functional changes of aorta in D-galactose-induced subacute aging rats. Adult male SD rats were randomly divided to five groups: the vehicle group, the D-galactose (D-gal) group, and the three NaHS groups treated with low (1 μmol·kg^-1·d^-1), middle (10 μmol·kg^-1·d^-1) or high (100 μmol·kg^-1·d^-1) dose of NaHS respectively. The D-gal group rats were given subcutaneously injection of 125 mg/kg D-gal per day for eight weeks to induce subacute aging model. In the NaHS group, D-gal was administered as above but with NaHS intraperitoneally injected at a dosage of 1, 10, 100 μmol·kg^-1·d^-1 respectively. Equivalent volumes of saline were administered per day for eight weeks in vehicle group. Morphological changes of aorta were observed by HE and Masson staining. The level of H2S in serum, the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA), as well as anti-superoxide anions in vascular tissue were determined by spectrophotometry. Angiotensin II (AngII) levels in plasma were measured using competitive enzyme immunoassay. The expression of angiotensin II type 1 receptor (AT1R) in aorta was determined by Western blot. The results showed that the aging aortic morphologic changes in model rats were ameliorated in NaHS groups. Decreased vascular endothelial exfoliative cells and vascular smooth muscle cell (SMC) proliferation were shown in NaHS groups by HE staining. Masson staining analysis showed reduced relative contents of collagen fibers (P < 0.05) and SMC (P < 0.05) in NaHS groups. Compared to vehicle group, serum concentration of H2S in D-gal group was decreased, while it was increased in NaHS groups after treatment with NaHS (P < 0.05). In the D-gal group, the concentration of AngII in plasma was significantly increased compared with that in vehicle group, while it was decreased in NaHS groups (P < 0.05). Moreover, levels of vascular tissue anti-superoxide anion and the activity of SOD were obviously higher, MDA was significantly lower in all NaHS treated groups than those in the D-gal group respectively (P < 0.05). Western blot analysis showed that the expression of AT1R was increased in D-gal group compared with that in vehicle group, while it was decreased after treatment with NaHS compared with that in D-gal group (P < 0.05). These results suggest that exogenous H2S can ameliorate the age-related changes of aortic morphology, decrease the concentration of AngII in plasma, down-regulate the expression of AT1R in vascular tissue, and mitigate the level of oxidative stress. These changes delay the vascular aging in aging rats ultimately.

Key words: hydrogen sulfide; D-galactose; aorta; aging; angiotensin II; angiotensin II receptor

收稿日期：2013-09-27　　录用日期：2014-01-24

通讯作者：闫长栋　　E-mail: yancd55@163.com

引用本文：

乔伟丽, 杨文学, 刘磊, 石玥, 崔洁, 柳红, 闫长栋. 外源性硫化氢减轻D-半乳糖诱导的亚急性衰老大鼠血管老化[J]. 生理学报 2014; 66 (3): 276-282.

QIAO Wei-Li, YANG Wen-Xue, LIU Lei, SHI Yue, CUI Jie, LIU Hong, YAN Chang-Dong. [Exogenous hydrogen sulfide reduces vascular aging in D-galactose-induced subacute aging rats.] [Article in Chinese]. Acta Physiol Sin 2014; 66 (3): 276-282 (in Chinese with English abstract).