鼓槌石斛乙醇提取物对糖尿病大鼠视网膜组织凋亡相关因子表达的影响

Effects of ethanol extract of Dendrobium chrysotoxum Lindl. on expressions of retinal apoptosis related factors in diabetic rats

目的:观察鼓槌石斛乙醇提取物(EDC)对糖尿病大鼠视网膜凋亡相关因子表达的影响。方法:SD大鼠随机分为正常对照组、糖尿病模型组(DM组)及EDC低剂量组(LEDC组)和高剂量组(HEDC组)。除正常对照组外,其余各组大鼠均通过腹腔注射链脲佐菌素(STZ)建立糖尿病模型。8周后,LEDC组、HEDC组分别给予EDC100 mg·kg^(-1)·d^(-1)、300 mg·kg^(-1)·d^(-1)灌胃,正常对照组和DM组予等量0.9%Na Cl溶液灌胃。干预8周后,采用HE染色观察各组大鼠视网膜各层结构,Western blot、免疫组化法检测视网膜组织Bax、Bcl-2、Caspase-3蛋白表达,TUNEL法观察视网膜组织凋亡指数。结果:与正常对照组比较,DM组视网膜内界膜不完整,内、外核层界限模糊,神经纤维层肿胀明显;而EDC不同剂量组与DM组比较,内、外核层界限渐清晰,神经纤维层肿胀减轻。与正常对照组比较,DM组Bax、Caspase-3蛋白表达增加,Bcl-2蛋白表达下降;LEDC组、HEDC组视网膜Bax、Caspase-3蛋白表达明显低于DM组,Bcl-2蛋白表达明显高于DM组(P<0.05)。免疫组化结果显示,正常对照组视网膜中未见Caspase-3阳性表达,DM组Caspase-3明显表达于神经纤维层、内核层、色素上皮层,LEDC、HEDC组少量表达于神经纤维层、色素上皮层,并且HEDC组Caspase-3阳性表达低于LEDC组。TUNEL结果显示,DM组大鼠视网膜细胞凋亡指数明显高于正常对照组(P<0.01),而HEDC、LEDC组凋亡指数明显降低(P<0.01)。结论:EDC可能通过调控糖尿病视网膜病变过程中凋亡相关因子的表达,对糖尿病大鼠视网膜有一定的改善作用。

Objective： To observe the effects of ethanol extract of Dendrobium chrysotoxum Lindl. on the expressions of retinal apoptosis related factors in diabetic rats. Methods： SD rats were randomly divided into the normal control group,diabetes model（ DM）group,ethanol extract groups with low-and high-dose. Except the normal control group,the diabetes model was established in other groups by intraperitoneal injection of streptozotocin（ STZ）. 8 weeks after modeling,the ethanol extract groups were treated with ethanol extract by intragastric administration at 100 mg·kg^（-1）·d^（-1） and 300 mg·kg^（-1）·d^（-1） respectively,and the normal control group and the DM group were treated with 0.9% Na Cl solution at equal volume. After treatment for 8 weeks,the structures of retinal different layers were observed by HE staining. The protein expressions of Bax,Bcl-2 and Caspase-3 were detected by Western blot and immunohistochemistry. The apoptosis index of retina tissue was observed by TUNEL method. Results： Compared with the normal control group,the internal limiting membrane of retina was incomplete,the boundary of inner and outer nuclear layers was obscure,and the nerve fiber layer was obviously swollen in the DM group. While compared with the DM group,the boundary of inner and outer nuclear layers was clearer,and the swelling of nerve fiber layer was alleviated in the ethanol extract groups with low-and high-dose. Compared with the normal control group,the protein expressions of Bax and Caspase-3 were increased and the protein expression of Bcl-2 was decreased in the DM group. The protein expressions of Bax and Caspase-3 in retina in the ethanol extract groups with low-and high-dose were obviously lower than those in the DM group,while the protein expression of Bcl-2 was obviously higher than that in the DM group（ P〈0.05）. The results of immunohistochemistry revealed that no positive expression of Caspase-3 was observed in the retina of normal control group,Caspase-3 in the DM group was mainly expressed in nerve fiber layer,inner nuclear layer and pigment epithelial layer,while a weak expression of Caspase-3 in nerve fiber layer and pigment epithelial layer was observed in the ethanol extract groups with low-and high-dose,and the positive expression of Caspase-3 in the high-dose group was lower than that in the low-dose group. The results of TUNEL showed that the retinal apoptosis index in the DM group was significantly higher than that in the normal control group（ P〈0.01）,while the apoptosis index in the ethanol extract groups was obviously decreased（ P〈0.01）. Conclusion： The ethanol extract of Dendrobium chrysotoxum Lindl. may regulate the expressions of apoptosis related factors in diabetic retinopathy process,and thus exerts certain improvement effects on retina of diabetic rats.