Fas相关死亡结构域蛋白基因沉默对帕金森病模型鼠黑质凋亡相关蛋白的影响

Effects of gene silencing of Fas-associated death domain on apoptosis-related proteins in rat models of parkinsonism

目的探讨Fas相关死亡结构域蛋白（FADD）基因沉默对帕金森病（PD）大鼠行为学变化及黑质死亡受体和半胱氨酸蛋白酶8（caspase-8）表达的影响。方法应用化学修饰的干扰RNA及立体定向技术等，在制作PD模型前黑质内注入FADD siRNA、FADD siRNA阳性对照物或FADD siRNA阴性对照物。观察正常对照组、PD组、FADD siRNA组、FADD siRNA阳性对照组和FADD siRNA阴性对照组（每组各15只）大鼠阿朴吗啡诱导的旋转行为的改变；采用Western blot分析和RT-PCR等方法，检测以上5组大鼠黑质FADD、Fas和caspase-8蛋白及mRNA的半定量表达。结果正常对照组大鼠阿朴吗啡诱导下无旋转行为，其余4组旋转次数超过6r／min（持续30min）的大鼠（只）分别为12（12／14）、3（3／13）、4（4／15）、11（11／14），组间比较差异具有统计学意义（χ^2=18．56，P=0．000）；与正常组比较，PD组各个蛋白及mRNA的表达均明显升高；与PD组比较，FADD siRNA组FADD和caspase-8蛋白及mRNA的表达受到明显抑制，差异具有统计学意义，但PD组和阴性对照组间及其余3组间比较差异均无统计学意义。与正常组比较，其余4组Fas蛋白及mRNA的表达均明显升高，差异具有统计学意义，但4组问比较差异无统计学意义。结论死亡受体凋亡通路在PD发病机制中起重要作用，而FADD siRNA能够有效抑制此通路，从而在一定程度上具有神经保护作用。

Objective To investigate the effects of gene silencing of Fas-associated death domain （FADD） with synthetic small interfering RNA （siRNA） on apomorphine-induccd contralateral rotation, and the expression of Fas and caspase-8 in rat models of parkinsonism. Methods Sprague-Dawley rats were randomly divided into 5 groups： control group, Parkinson＇ s disease （PD） group, FADD siRNA group, FADD siRNA positive control group and FADD siRNA negative control group. Synthetic FADD siRNA sequences, siRNA positive sequences or siRNA negative sequences were infused into right suhstantianigra of midbrain using RNA interference and stereotactic techniques before parkinsonian rat model establishment. Apomorphine-induced contralateral rotations of the rats were observed after the injection. The protein and mRNA expression levels of FADD, Fas and caspase-8 were measured by Western blot and RT-PCR. Results In the control group, no rotation was observed after injecting apomorphine; however, in the rest groups, the number of rats respectively was 12 （ 12/14）, 3 （ 3/13 ）, 4 （ 4/15 ） and 11 （ 11/14 ） in apomorphine-induced contralateral rotation, which had significant statistical differences （χ^2 = 18.56,P = 0. 000）. In parkinsonian substantia nigra, marked increases in the protein and mRNA levels of FADD, Fas and caspase-8 were observed, compared with control group. Furthermore, compared with PD group, FADD protein and mRNA levels were strongly suppressed by administration of FADD siRNA in FADD siRNA group. FADD siRNA administration also resulted in great attenuation of 6-hydroxydopamine-induced increases in expression and activation of caspase-8. However, no decrease in expression of Fas was observed in FADD siRNA group and FADD siRNA positive control group, compared with PD group. Conclusion Our results suggest that death receptor signaling pathway plays a critical role in the pathogenesis of PD. FADD siRNA is effective against this pathway and it may, at least in part,provide a potential neuroprotective effect.