摘要
目的观察中药复方活血解毒方(HXJD)对糖尿病大鼠视网膜细胞凋亡及凋亡相关因子NF-κB和Caspase-3 mRNA和蛋白表达的影响。方法采用链脲佐菌素(65 mg/kg)一次性腹腔注射的方法,建立糖尿病大鼠模型,将50只SD大鼠随机分为模型组和HXJD高剂量组(15.4 g/kg)、中剂量组(7.70 g/kg)、低剂量组(3.85 g/kg)及导升明组(0.167 g/kg),每组10只,另设10只正常对照组。造模成功后,灌胃给药,20周后处死动物。应用TUNEL法检测细胞凋亡程度,免疫组化和蛋白印迹技术检测视网膜全层中NF-κB和Caspase-3的表达;采用Real-time PCR法检测视网膜NF-κB和Caspase-3 mRNA的表达。同时,体外培养大鼠视网膜神经节细胞株(RGC-5),制备HXJD含药血清。利用55.5 mmol/L葡萄糖浓度模拟高糖环境诱导,将细胞分为5组:空白组、高糖组、含药血清低剂量组、含药血清中剂量组、含药血清高剂量组。采用CCK-8和AnnexinⅤ-FITC法检测不同血清干预高糖诱导下RGC细胞24 h增殖活性及细胞凋亡的影响。结果与正常对照组比较,模型组凋亡细胞增加,大鼠视网膜组织NF-κB和Caspase-3蛋白及mRNA的表达明显升高(P<0.05);与模型组比较,活血解毒方各剂量组和导升明组视网膜细胞凋亡的程度、视网膜NF-κB和Caspase-3蛋白和mRNA的表达量均降低(P<0.05)。与空白组比较,高糖组细胞活性降低,细胞凋亡比例增加(P<0.05)。与高糖组比较,含药血清高、中剂量组细胞活性增高(P<0.05),含药血清各剂量组细胞凋亡比例减少(P<0.05,P<0.01)。结论活血解毒方可能通过改善糖尿病大鼠视网膜中细胞凋亡的相关细胞因子NF-κB和Caspase-3的表达,同时调节视网膜神经节细胞活力,改善高糖条件下RGC细胞的凋亡程度。
Objective To observe the effects of Huoxue Jiedu Recipe(HXJD)on the apoptosis and the expression of NF-κB and Caspase-3 in the reina tissue of early diabetic rats. Methods The diabetic rat model was established by using one single intraperitoneal injection of streptozotocin(STZ, 65 mg/kg). Then the model rats were randomly divided into five groups,i.e.. the model group, the low dose HXJD group(3.85 g/kg), the middle dose HXJD group(7.70 g/kg), the high dose HXJD group(15.40 g/kg) and the Western medicine treatment group(Calcium Dobesilate capsule, 0.167 g/kg), ten rats in each group. A normal control group consisting of ten rats was also set up. The nomal group and model group were given normal water and the other rats were medicated by intragastric administrate for 20 weeks. All rats were sacrificed after 20 weeks. Apoptotic level was detected by TUNEL. Immunological histochemistry assay and Western blot were used to observe the protein expression of NF-κB and Caspase-3. The expressive abundance of NF-κB mRNA and Caspase-3 mRNA in rats′ retina were detected by Real-time quantitative PCR. At the same time, rat retinal ganglion cells(RGC-5) were cultured in vitro, the application of serum pharmacology of HXJD was prepared. Meanwhile, 55.5 mmol/l glucose was used to induce the high glucose environment. The cells were divided into five groups, i.e.. the control group, the high glucose group, HXJD low dose containing serum group, HXJD middle dose containing serum group, HXJD high dose containing serum group. The proliferation activity and apoptotic of RGC were detected by CCK8 and Annexin Ⅴ-FITC methods. Results Compared to the normal group, the number of apoptotic cells in model group was increased obviously, and the expression of NF-κB and Caspase-3 protein and mRNA were also increased(P<0.05). Compared with the model group, the apoptotic level and the expression of NF-κB and Caspase-3 in HXJD groups and the Western medicine treatment group were decreased evidently(P<0.05). Compared with the control group,the proliferation activity of RGC cells in the high glucose group decreased, and the apoptosis ratio of RGC cells was increased(P<0.05). Compared with the high glucose group, the proliferation activity of RGC cells in the high and middle dose HXJD containing serum group significantly improved(P<0.05), the apoptosis ratio of RGC cells in each dose HXJD containing serum group was reduced(P<0.05, P<0.01). Conclusion Huoxue Jiedu Recipe can inhibit the expression of NF-κB and Caspase-3 in retina of diabetic rats, and improve the proliferation and the apoptotic level of RGC cells, so it can inhibit apoptosis in retinal cell and protect the tissue in diabetic retinopathy.
作者
姚青
韩静
芦晓红
张茜
罗明秀
王伟
YAO Qing;HAN Jing;LU Xiao-hong;ZHANG Qian;LUO Ming-xiu;WANG Wei(College of Basic Medicine,Ningxia Medical University,Yinchuan,750004;College of Basic Medicine,Beijing University of Chinese Medical,Beijing,100029)
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2019年第3期310-316,共7页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金资助项目(No.81460666)
