Fas／FasL途径在砷致大鼠神经细胞凋亡过程中的作用

The role of Fas/FasL pathway in apoptosis of rat nerve cells induced by arsenic exposure

目的观察慢性砷暴露大鼠大脑皮质中Fas、FasL、FADD、含半胱氨酸的天冬氨酸蛋白水解酶（caspase）8、easpase3表达，探讨砷暴露致神经细胞凋亡的可能机制。方法选取40只SD大鼠，按体质量采用随机数字表法分为4组：对照组（饮用蒸馏水），低、中、高剂量染砷组[分别饮用含5、10、50mg／L亚砷酸钠（NaAsO2）的溶液]，每组10只。染砷3个月后，股动脉采血处死大鼠，取大鼠大脑皮质，采用TUNEL法检测凋亡阳性细胞率，利用实时荧光定量PCR法（real—timePCR）检测Fas、FasL、FADDmRNA表达，免疫组化法、蛋白质免疫印迹法（Westernblot）检测蛋白表达，采用caspase试剂盒检测easpase8和caspase3的活性。结果①凋亡指数：对照组，低、中、高剂量染砷组分别为（15．50±9．85）％、（16．75±10．01）％、（36．75±14．73）％和（50．50±19．16）％，低、中、高剂量染砷组与对照组相比，差异有统计学意义（P均〈0．05）。②mRNA表达：对照组，低、中、高剂量染砷组FasmRNA表达分别为0．88±0．09、1.00±0．22、1．04±0．21和1．24±0．34，中、高剂量染砷组与对照组比较，差异有统计学意义（P均〈0．01）；FasLmRNA表达分别为0．83±0．17、0．89±0．37、0．86±0．42和0．91±0．25．组间比较差异无统计学意义（F=2．018，P〉0．05）；FADDmRNA表达分别为0．77±0．23、0．80±0．13、0．97±0．15和0．98±0．25，中、高剂量染砷组与对照组比较，差异有统计学意义（P均〈0．05）。③Western blot检测结果：对照组，低、中、高剂量染砷组Fas蛋白表达分别为8．22±1．40、10．27±2．16、10．33±1．55和11．71±1．74，中、高剂量染砷组与对照组比较，差异有统计学意义（P均〈0．01）；FasL蛋白表达分别为4．28±1．59、3．91±1．99、3．85±1．05和3．62±1．52，组间比较差异无统计学意义（F=0．181，P〉0．05）；FADD蛋白表达分别为5．01±1．0HD、7．10±1．84、6．04±1．51和7．36±1．32，低、高剂量染砷组与对照组比较，差异有统计学意义（P均〈0．05）。④免疫组化法检测蛋白表达阳性细胞密度：对照组，低、中、高剂量染砷组Fas分别为（8．25±2．99）％、（18．00±0．82）％、（19．00±5．42）％和（23．75±5．19）％，低、中、高剂量染砷组与对照组比较，差异有统计学意义（P均〈0．01）；FasL分别为（13．07±3．95）％、（13．54±4．64）％、（17．10±6．17）％和（19．03±2．79）％，低、中、高剂量染砷组与对照组比较，差异无统计学意义（P均〉0．05）；FADD分别为（17．03±7．34）％、（17．27±5．03）％、（18．68±6．56）％和（24．13±7．25）％，高剂量染砷组与对照组比较，差异有统计学意义（P〈0．05）。⑤easpase活性：对照组，低、中、高剂量染砷组easpase8活性分别为（3．07±0．69）、（4．39±1．32）、（4．51±0．83）、（4．67±1-36）U／g，低、中、高剂量染砷组与对照组比较，差异有统计学意义（P均〈0．05）：easpase3活性分别为（2．84±0．36）、（3．65±1．38）、（4．23±0．99）、（4．41±1．02）U／g，中、高剂量染砷组与对照组比较，差异有统计学意义（P均〈0．05）。结论慢性砷暴露大鼠大脑皮质中Fas、FADD表达及caspase8、caspase3活性异常增高，提示砷可以通过Fas／FasL信号转导途径诱导大鼠神经细胞发生凋亡。

Objective To study the expression of Fas, FasL, FADD, cysteinyl aspartate specific proteinase （caspase）-8 and caspase-3 in rat brain cortex under chronic arsenic exposure, in order to explore the possible mechanism of nerve cell apoptosis under arsenic exposure. Methods Forty SD rats were randomly assigned to four treatment groups with equal number in each group according to body weight. Control group was given distilled water while low-, medium- and high-dose groups were given sodium arsenite solution in concentrations of 5, 10 and 50 mg/L, respectively. After exposed to sodium arsenite for three months, terminal deoxynucleotidyl transferase （TdT）-mediated dUTP nick end labeling （TUNEL） was used to detect the apoptosis positive nerve cells; real-time PCR was used to detect the mRNA expression level of Fas, FasL and FADD; immunohistochemical and Western blotting were used to test the protein expression level; caspase kits was used to detect the activity of caspase-8 and caspase-3. Results （1）According to the results of TUNEL： for control, low-, medium- and high-dose groups, their apoptotic cell rates were （15.50 ± 9.85）%, （16.75 ± 10.01）%, （36.75 ± 14.73）% and （50.50 ± 19.16）%; low-, medium- and high-dose groups were significantly higher than control group （all P 〈 0.05）. （2）The expression of mRNA： Fas were 0.88±0.09, 1.00 ± 0.22, 1.04 ± 0.21 and 1.24 ± 0.34, respectively, for control, low-, medium- and high-dose groups; medium- and high-dose groups were significantly higher than the control group （all P 〈 0.01）; FasL were 0.83 ± 0.17, 0.89±0.37, 0.86 ± 0.42 and 0.91± 0.25, respectively, for control, low-, medium- and high-dose groups, there was no statistically significant difference in any pair of groups （F = 2.018, P 〉 0.05）; FADD were 0.77 ± 0.23, 0.80 ± 0.13, 0.97 ± 0.15 and 0.98 ± 0.25, respectively, for control, low-, medium- and high-dose groups; a significant rise was found in medium- and high-dose groups than the control group （all P 〈 0.05）. （3）The results of Western blotting： Fas were 8.22± 1.40, 10.27 ± 2.16, 10.33±1.55 and 11.71± 1.74, respectively, for control, low-, medium- and high-dose groups; medium- and high-dose groups were significantly higher than control group （all P 〈 0.01）; FasL were 4.28± 1.59, 3.91 ± 1.99, 3.85 ± 1.05 and 3.62 ± 1.52, respectively, for control, low-, medium- and high-dose groups; no significant difference was found in any pair of groups （F = 0.181, P 〉 0.05）; FADD were 5.01 ± 1.00, 7.10± 1.84, 6.04 ± 1.51 and 7.36 ±1.32, respectively, for control, low-, medium- and high-dose groups; low-, high-dose groups were higher than control group statistically （all P 〈 0.05）. （4）The results of immunohistochemistry： Fas were （8.25±2.99）%, （18.00± 0.82）%, （19.00± 5A2）% and （23.75 ± 5.19）%, respectively, for control, low-, medium- and high-dose groups; low-, medium- and high-dose groups were significantly higher than control group （all P 〈 0.01）; FasL were （13.07±3.95）%, （13.54 ± 4.64）%, （17.10 ± 6.17）% and （19.03 ± 2.79）%, respectively, for control, low-, medium- and high--dose groups; no significant difference was found in any pair of groups （F = 0.119, P 〉 0.05）; FADD were （17.03 ± 7.34）%, （17.27 ± 5.03）%, （18.68 ± 6.56）% and （24.13 ± 7.25）%, respectively, for control, low-, medium- and high-dose groups; high-dose group was significantly higher than control group （P 〈 0.05）. （5）The caspase-8 activity for the four groups were （3.07± 0.69）, （4.39 ± 1.32）, （4.51 ± 0.83）, （4.67± 1.36） U/g, respectively; low-, medium- and high-dose groups were significantly higher than control group （all P 〈 0.05）; the caspase-3 activity of the above four groups were （2.84± 0.36）, （3.65 ± 1.38）, （4.23 ± 0.99）, （4.41±1.02） U/g; medium- and high-dose groups were significantly higher than control group （all P 〈 0.05）. Conclusion The expression of Fas and FADD and activity of caspase-8 and caspase-3 in rat cortex are increased significantly under chronic arsenic exposure, which may be related to the apoptosis of nerve cells due to arsenic exposure through the Fas/FasL signal transduction pathway.