许岩等

[摘要] 目的 观察大鼠黑质氧化损伤后内源性硫化氢（hydrogen sulfide，H2S）的变化及其作用。 方法 将SD雄性大鼠单侧黑质内微量注射6-羟基多巴胺（6-Hydroxydopamine，6-OHDA）作为黑质氧化损伤模型；H2S供体硫氢化钠（sodium hydrosulfide，NaHS）在6-OHDA损伤前连续腹腔注射3周作为预处理；实验分为对照组、6-OHDA损伤后7 d（D7组）、11 d（D11组）、17 d组（D17组）、NaHS预处理组（NaHS+6-OHDA处理），每组各8只；采用亚甲基蓝分光光度计法检测黑质胱硫醚-β-合酶（cystathionine-β-synthase，CBS）活性及H2S的含量；免疫组织化学法检测黑质酪氨酸羟化酶（tyrosine hydroxylase，TH）阳性细胞数；紫外分光光度法测定黑质谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）活性和丙二醛（malondialdehyde，MDA）水平。 结果 与对照组比较，6-OHDA损伤后7、11、17 d黑质CBS酶活性分别下降为[（96.21±8.40）%，P > 0.05]，[（86.48±9.85）%，P < 0.05]和[（75.16±7.45）%，P < 0.01]；内源性H2S含量分别减少为[（90.12±10.03）%，P < 0.05]，[（82.58±9.52）%，P < 0.01]和[（78.16±11.55）%，P < 0.01]。TH阳性细胞与对照组比较，在6-OHDA损伤后7 d即下降为[（84.32±6.06）%，P < 0.05]，同时伴随黑质GSH-Px活性降低及MDA含量升高，差异有统计学意义（P < 0.05）。但早期给予NaHS预处理补充H2S之后，与单纯6-OHDA损伤后7 d比较，TH阳性细胞则增加为[（96.15±5.03）%，P < 0.05]，且黑质GSH-Px的活性升高，MDA的含量降低，差异有统计学意义（P < 0.05）。 结论 6-OHDA氧化损伤导致大鼠黑质CBS酶活性及H2S含量下降，外源性H2S预处理可早期发挥抗黑质氧化损伤的神经元保护作用，这可能与其增加GSH-Px活性及减少MDA含量有关。

[关键词] 硫化氢；胱硫醚-β-合酶；帕金森病；氧化应激；神经保护

[中图分类号] R742.5 [文献标识码] A [文章编号] 1673-7210（2014）12（c）-0016-05

Changes and roles of endogenous hydrogen sulfide in the substantial nigra oxidative damage of rats

XU Yan1 MA Na2 LIU Bo3 WANG Jingying1 LIAO Wenhui3 WANG Sheng2 WANG Jinquan2 MENG Jinlan2

1.The Third Department of Surgery， Southern Medical University TCM-Integrated Hospital， Guangdong Province， Guangzhou 510315， China； 2.Department of Physiology， Guangdong Pharmaceutical University， Guangdong Province， Guangzhou 510006， China； 3.Department of Pharmaceutical， Guangdong Pharmaceutical University， Guangdong Province， Guangzhou 510080， China

[Abstract] Objective To observe the changes and roles of endogenous hydrogen sulfide in the substantia nigra oxidative damage of rats. Methods 6-hydroxydop- amine （6-OHDA） was microinjected in the unilateral substantia nigra of SD rats as the substantia nigra oxidative damage model； H2S donor， sodium hydrosulfide （NaHS） was injected intraperitoneally for three consecutive weeks as a pretreatment before 6-OHDA injury. The experiment were divided into the control group ， 7 days （D7） group， 11 days （D11） group， 17 days （D17） group after 6-OHDA injury and NaHS preconditioning group （deafed with NaHS + 6-OHDA）； with 8 rats in each group. Cystathionine-β-synthase （CBS） activity and H2S production in substantia nigra were detected by methylene blue spectrophot-ometric method. Immunohistochemistry was used to detect tyrosine hydroxylase （tyrosine hydroxylase， TH） positive cells of the substantia nigra； Glutathione peroxidase （GSH-Px） activity and malondialdehyde （MDA） level of the substantia nigra were measured by UV spectrophotometry. Results Compared with the control group， percentage of CBS activity was decreased respectively to[（96.21±8.40）%， P > 0.05]， [（86.48±9.85）%， P < 0.05 ] and [（75.16±7.45）%， P < 0.01] for 7， 11 and 17 d after 6-OHDA injury； Percentage of endogenous H2S content decreased respectively to [（90.12±10.03）%， P < 0.05]，[（82.58±9.52）%， P < 0.01] and [（78.16±11.55）%， P < 0.01] also. Compared with control group， percentage of TH positive cells was reduced to [（84.32±6.06）%， P < 0.05] at 7 d after 6-OHDA damage， accompanied by GSH-Px activity reducing and MDA content increasing， the difference was statistically significant （P < 0.05）. But compared with 7 d after 6-OHDA injury， early supplementary H2S through NaHS pretreatment， percentage of TH positive cells were increased to [（96.15±5.03）%， P < 0.05]， and GSH-Px activity was increased ，the content of MDA was decreased， the difference was statistically significant （P <0.05）. Conclusion Substantia nigra oxidative damage after 6-OHDA injure leads to CBS activity and endogenous H2S content decreased， pretreatment with exogenous H2S early exert neurons protective effect of anti- oxidative damage in substantia nigra， which may be related to GSH-Px activity increasing and MDA content decreasing.

[Key words] Hydrogen sulfide； Cystathionine-β-synthase； Parkinson's disease； Oxidative stress； Neuroprotection

硫化氢（hydrogen sulfide，H2S）目前被公认为是除NO和CO之外另一种新的气体信号分子。体内半胱氨酸等含硫氨基酸在5'-磷酸吡哆醛依赖酶胱硫醚-β-合酶（cystathi-onine-β-synthase，CBS）和胱硫醚-γ-裂解酶（cystathionine-γ-lyase，CSE）等的催化作用下生成内源性H2S。在中枢神经系统中CBS被认为是脑内源性H2S生成的主要酶，当H2S生成浓度过高或过低时与神经系统疾病如老年痴呆（Alzheimer's disease，AD）[1]、脑卒中[2]等的发生密切相关。Hu等[3]在6-羟基多巴胺（6-Hydroxydopamine，6-OHDA）制备的帕金森病（Parkinson's disease，PD）模型鼠中检测到黑质内源性H2S含量减少，酪氨酸羟化酶（tyrosine hydroxylase，TH）阳性细胞数量及其蛋白表达明显降低，提示内源性H2S参与了PD的病理过程。但是，在PD进展过程中内源性H2S生成的动态变化尚未明确。

PD是一种以黑质纹状体多巴胺神经元进行性损失为特征的神经退行性疾病[4]，其发病机制目前仍未完全清楚，但是氧化应激在PD的发生中发挥了重要的作用[5]。研……