眶内及眶外针刺对兔眼非动脉炎性前部缺血性视神经病变细胞Bax/Bcl-2及半胱氨酸天冬氨酸蛋白酶3表达的影响

Acupuncture of intraorbital and extraorbital acupoints reduces apoptosis of retinal ganglion by down-regulating expression of Caspase-3 and ratio of Bax/Bcl-2 in rabbits with nonarteritis anterior ischemic optic neuropathy

目的:观察眶内及眶外针刺对兔眼非动脉炎性前部缺血性视神经病变(NAION)组织Bax/Bcl-2及半胱氨酸天冬氨酸蛋白酶3(Caspase-3)表达的影响,探讨针刺抗NAION细胞凋亡的机制,验证选择眶内腧穴治疗的必要性。方法:雌性新西兰大耳白兔随机分为模型组、眶内针刺组、眶外针刺组、眶内+眶外针刺组,每组5只。以右眼为实验眼,光动力法制备兔眼NAION模型,选取模型组5只左眼为正常组。眶内针刺组腧穴选取"睛明""承泣""球后",眶外针刺组腧穴选取"攒竹""鱼腰""窍明",眶内+眶外针刺组腧穴即为以上两组之和。造模成功后次日,3个针刺组开始针刺干预,每次留针30min,每日1次,连续治疗3d。通过TUNEL免疫荧光检测病变视网膜神经节细胞调亡,免疫组织化学染色检测Bax、Bcl-2及Caspase-3的表达。结果:与正常组比较,模型组视网膜神经节的细胞凋亡个数、Bax及Caspase-3阳性表达均显著增加(P<0.01,P<0.05),Bax/Bcl-2值增高(P<0.01),Bcl-2阳性表达显著降低(P<0.01)。与模型组比较,眶内针刺组细胞凋亡个数显著降低(P<0.01),Caspase-3阳性表达降低(P<0.05);眶外针刺组细胞凋亡个数、Bax/Bcl-2值均显著降低(P<0.01),Bcl-2阳性表达显著增加(P<0.01);眶内+眶外针刺组细胞凋亡个数、Bax/Bcl-2值及Caspase-3阳性表达均显著降低(P<0.01),Bcl-2阳性表达显著增加(P<0.01)。与眶内针刺组比较,眶外针刺组及眶内+眶外针刺组Bcl-2阳性表达增高(P<0.05),Bax/Bcl-2值降低(P<0.05)。与眶外针刺组比较,眶内+眶外针刺组Caspase-3阳性表达降低(P<0.05)。结论:针刺对NAION组织Bax及Caspase-3表达有一定的抑制作用,对Bcl-2表达有促进作用,以眶内+眶外针刺为优。

Objective To observe the effect of acupuncture of intraorbital and extraorbital acupoints on apoptosis and expression of Bax,Bcl-2 and Caspase-3 proteins of retinal ganglion tissue in rabbits with nonarteritis anterior ischemic optic neuropathy(NAION),so as to reveal its mechanism underlying improvement of NAION.Methods Female New Zealand rabbits were randomly divided into four groups:model,intraocular needling(ION),extraocular needling(EON),ION+EON groups(n=5 per group),and the 5 healthy eyes of those rabbits in the model group were selected to be used as the control group.The NAION model of the right eye was established by photodynamic stroke method.For ION group,acupoints'Jingming'(BL1),'Chengqi'(ST1)and'Qiuhou'(EX-HN7)were needled,and for EON group,'Cuanzhu'(BL2),'Yuyao'(EX-HN4)and'Qiaoming'(EX)were needled with filiform needles,followed by retaining the needles for 30 min.For ION+EON group,the 6 acupoints were needled simutaneously.The treatment was conducted once daily for 3 days.The apoptosis of retinal ganglion tissue was detected by using TUNEL fluorescence labeling,and the expression of Bax,Bcl-2 and active Caspase-3 in the retinal ganglion were detected by immunohistochemistry.Results Following modeling and compared with the control group,the number of the apoptotic cells,and the expression levels of Bax and Caspase-3 proteins,as well as the ratio of Bax/Bcl-2 were significantly increased in the model group(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01).After intervention,the number of the apoptotic cells in the ION,EON and ION+EON groups,and the ratio of Bax/Bcl-2 in EON and ION+EON groups,and the expression of Caspase-3 proteins in the ION and ION+EON groups were significantly down-regulated(P<0.01,P<0.05),and the expression levels of Bcl-2 in both EON and ION+EON groups were significantly up-regulated in comparison with the model group(P<0.01).No significant changes were found in the expression levels of Bax in the 3 needling groups relevant to the model group(P>0.05).Conclusion Acupuncture of intraorbital and extraorbital acupoints can reduce apoptosis of retinal ganglion in NAION rabbits via inhibiting the activation of Caspase-3 protein and ratio of Bax/Bcl-2,and up-regulating the expression of Bcl-2 protein.