外源性H2S通过核因子-κB介导的炎性反应通路减轻大鼠局灶性脑缺血再灌注损伤

Exogenous hydrogen sulfide reduces focal cerebral ischemia/reperfusion injury in rats via nuclear factor-KB-mediated inflammatory response pathway

目的探讨外源性硫化氢（hydrogensulfide，H2S）对脑缺血再灌注后脑损伤和炎性反应的影响。方法48只雄性SD大鼠随机分为假手术组、缺血再灌注（ischemiaJreperfusion，I／R）组、H，S30ppm组和H，S60ppm组（1ppm=1mg／L），每组12只。采用大脑中动脉闭塞法建立大鼠局灶性脑缺血2h再灌注24h模型。再灌注24h后应用胶带剔除实验进行神经功能评价，应用2,3，5-三苯基氯化四氮唑染色法测定脑梗死体积百分比，采用酶联免疫吸附法测定缺血脑组织白细胞介素（interleukin，IL）-1β和IL-6水平，采用蛋白质印迹法测定缺血脑组织诱导型一氧化氮合酶（induciblenitricoxidesynthase，iNOS）和细胞间黏附分子-1（nercellularcelladhesionmolecule-1，ICAM-1）的表达及核因子．KB（nuclearfactor-κB，NF-κB）的转位激活情况。结果与I／R组比较，吸入H2S能以剂量依赖性方式缩短胶带剔除所需的时间[I／R组与H2S30ppm组和H2S 60ppm组比较：180S对130（113～157）S对110（87～138）S；P〈0．05]，缩小脑梗死体积（48．8％±9．1％对23．3％±5．1％对17．3％±3．5％；P〈0．05）]，下调缺血脑组织IL-1p[（39．53±6．02）pg/mg蛋白对（30．17±3．46）pg／mg蛋白对（22．69±6．09）pg／mg蛋白；P〈0．05]和IL-6[（54．65±10．68）pg／mg蛋白对（37．89±4．54）pg／mg蛋白对（27．00±3．08）pg／mg蛋白；P〈0．05]表达水平，显著降低NF—κB胞核／胞质比[4．40±1．05对3．07±0．82对2．30±0．60；P〈0．05）]，抑制iNOS（4．22±0．67对3．14±0．90对2．08±0135；P〈0．05）和ICAM-1（5．45±1．08对3．45±0．67对2．21±0．39；P〈0．05）]表达。结论吸入外源性H2S能以剂量依赖性方式缩小脑缺血再灌注后的脑梗死体积和改善神经功能，其机制可能与抑制NF—κB激活，下调其下游的iNOS和ICAM-1表达以及降低IL-1B和IL-6水平有关。

Objective To investigate the effect of hydrogen sulfide （H2S） on brain injury and inflammatory response after cerebral ischemia/reperfusion in rats. Methods Forty-eight male SD rats were randomly divided into four groups： sham operation group, ischemia/reperfusion （I/R） group, H2S-30 ppm group, and H2S-60 ppm group （n = 12 in each group; 1 ppm= 1 mg/L）. The middle cerebral artery occlusion method was used to induce a model of focal cerebral ischemia for 2 h and reperfusion for 24 h. After reperfusion for 24 h, the tape remove experiment was used to perform the nerve function evaluation. 2,3,5-triphenyl-tetrazolium chloride staining method was used to measure the percentage of cerebral infarction volume. Enzyme-linked immunosorbent assay was used to measure the levels of interleukin （IL） -1β and IL-6. Western blotting was used to detect the expression levels of inducible nitric oxide synthase （iNOS） and intercellular cell adhesion molecule-1 （ICAM-1） , as well as the transposition activation of nuclear factor-KB （NF-KB）. Results Inhalation of H2 S could shorten the time required to remove the tape in a d0se-dependent manner compared with the I/R group （I/R group vs. H2S 30 ppm group and H2S 60 ppm group： 180 s vs. 130 [113-157]s vs. 110 [87-138] s; P〈 0.05）, reduced the cerebral infarct volume （48.8% ± 9. 1% vs. 23.3% ± 5.1% vs. 17. 3%± 3.5% ; P 〈 0.05）, downregulated the expression levels of IL-113 （ 39.53 ± 6. 02 pg/mg protein vs. 30. 17 ± 3.46 pg/mg protein vs. 22.69 ± 6. 09 pg/mg protein; P 〈 0. 05） and IL-6 （54. 65 ±10. 68 pg/mg protein vs. 37. 89 ± 4. 54 pg/mg protein vs. 27.00 ±3.08 pg/mg protein; P 〈 0. 05） in ischemic brain tissue, significantly decreased NF-KB nucleus/ cytoplasm ratio （4. 40 ± 1.05 vs. 3.07 ± 0. 82 vs. 2. 30 ± 0. 60; P 〈 0. 05）, inhibited expressions of iNOS （4. 22 ± 0. 67 vs. 3.14 ±0. 90 vs. 2.08 ±0. 35; P 〈 0. 05）, and ICAM-1 （5.45 ± 1.08 vs. 3.45 ±0. 67 vs. 2.21 ±0. 39; P 〈0. 05）. Conclusions Inhalation of exogenous H2S can reduce cerebral infarct volume after cerebral ischemia/reperfusion in a dose-dependent manner and improve neurological function. Its mechanism may be associated with the inhibition of NF-KB activation, down-regulation of its downstream iNOS and ICAM-1 expression levels, and decrease of IL-1β and IL-6 levels.