三七总皂苷干预脊髓损伤大鼠诱导型一氧化氮合酶及半胱氨酸天冬氨酸蛋白酶3表达的变化

Effect of panax notoginseng saponin injection on expressions of inducible nitricoxide synthase and Caspase-3 in rats with spinal cord injury

目的:观察三七总皂苷注射液对大鼠脊髓损伤后诱导型一氧化氮合酶及半胱氨酸天冬氨酸蛋白酶3表达的作用机制。方法:实验于2005-06/11在华中科技大学同济医学院附属协和医院骨科实验室完成。将64只SD大鼠数字表法随机分为损伤组和三七总皂苷组(n=32),2组按存活时间分为1,3,7,14d4个时间点,每个时间点8只。所有大鼠采用Allen’s法制备脊髓中度损伤模型(致伤能量为40g·cm),三七总皂苷组伤后30min腹腔注射50mg/L三七总皂苷100mg/kg,伤后2h及4h再次给予50mg/kg,以后1次/d,剂量200mg/kg,连用12d。损伤组相同时间点腹腔注射等体积生理盐水。2组均于预定时间点处死大鼠,取出伤段脊髓(以损伤处为中心,长约10mm),用苏木精-伊红染色观察损伤脊髓组织病理变化,免疫组化染色检测诱导型一氧化氮合酶及半胱氨酸天冬氨酸蛋白酶3阳性细胞率。结果:64只大鼠全部进入结果分析。①苏木精-伊红染色光镜下发现脊髓组织病理学改变三七总皂苷组明显轻于损伤组。②诱导型一氧化氮合酶阳性细胞率:三七总皂苷组在伤后1,3,7,14d均低于损伤组[(21.38±3.69)%,(36.24±3.27)%,(30.56±2.89)%,(21.64±5.31)%;(39.48±3.78)%,(58.69±5.67)%,(68.97±5.34)%,(40.35±4.36)%,t=9.692,9.701,17.893,3.171,P<0.05]。③半胱氨酸天冬氨酸蛋白酶3阳性细胞率:三七总皂苷组在伤后1,3,7,14d均低于损伤组[(26.29±3.21)%,(35.42±2.25)%,(48.58±2.64)%,(23.72±3.26)%;(32.43±2.69)%,(46.18±3.07)%,(60.31±5.35)%,(31.98±4.80)%,t=4.147,7.996,5.561,11.231,P<0.05]。结论:三七总皂苷注射液能抑制脊髓损伤后诱导型一氧化氮合酶及半胱氨酸天冬氨酸蛋白酶3表达,从而减轻细胞损伤和凋亡。

AIM： To investigate the effect of panax notoginseng saponin （PNS） injection on expressions of inducible nitricoxide synthase （iNOS） and Caspase-3 in rats with spinal cord injury. METHODS ：The experiment was conducted in the Laboratory of Orthopedics, Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from June to November 2005. Sixtyfour adult SD rats were randomly divided into injury group and PNS group with 32 rats in each group, and each group was subdivided into four subgroups according to 1,3,7 and 14 days of survival with 8 rats in each time-polnt. Allen＇s technique was adopted to establish rat models of spinal cord injury with the injuring powder of 40 （g·cm）. Rats in the PNS group were given intraperitoneal injection of 50 mg/L PNS （100 mg/kg） as well as 50 mg/kg respectively at 2 hours and 4 hours after injury once a day for 12 continuoos days at the dose of 200 mg/kg. Rats in the injury group were intraperitoneally injected with normal saline at the same volume and the same time-point. The rats were sacrificed at given time to obtain the injured cord （centered as the injury with a length of 10 ram）, and hematoxylin and eosln staining was adopted to study the pathological changes of tissues in injured spinal cord. The iNOS and the positive cell rate of Caspase-3 were examined with immunohistochemical staining. RESULTS：A total of 64 rats were involved in the analysis of results.①The histopathological changes in spinal cord tissue after HE staining under light microscope was lighter in the PNS group than the injury group.②The positive cell rate of iNOS： those in the PNS group at 1, 3, 7 and 14 days after injury were obviously lower than the injury group [（21.38±3.69）%, （36.24 ±3.27）%, （30.56 ±2.89）%, （21.64 ±.5.31 ）% ; （39.48 ±3.78）%, （58.69±5.67）%, （68.97±5.34）%, （40.35±4.36）% ,t=9.692,9.701,17.893, 3.171, P 〈 0.05]. ③The positive cell rate of Caspase-3： those in the PNS group at 1,3,7 and 14 days after injury were remarkably lower than the injury group [（26.29 ±3.21）% , （35.42±2.25）%, （48.58 ±2.64）%, （23.72 ±3.26）% ; （32.43 ±2.69）% , （46.18 ±3.07）% , （60.31 ±5.35）% , （31.98 ±4.80）% ,t=4.147,7.996,5,561,11.231, P 〈 0.05]. CONCLUSION：PNS injection can inhibit the expressions of iNOS and caspase-3 in rats with spinal cord injury, and accordingly ameliorate cell injury and apoptosis.