内源性硫化氢在大鼠黑质氧化损伤中的变化及作用

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

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,Na HS)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)、Na HS预处理组(Na HS+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Na HS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。

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; H2 S donor, sodium hydrosulfide（Na HS） 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 Na HS preconditioning group（deafed with Na HS ＋ 6-OHDA）; with 8 rats in each group. Cystathionine-β-synthase（CBS） activity and H2 S 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 H2 S 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 H2 S through Na HS 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 H2 S content decreased, pretreatment with exogenous H2 S 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.