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[摘要]
目的 探讨红花黄色素通过调控p38丝裂原活化蛋白激酶(p38MAPK)信号通路对糖尿病(DM)大鼠视网膜神经节细胞(RGC)的保护作用。方法 取50只大鼠,其中40只采用单次ip链脲佐菌素法制备DM大鼠模型,并将其随机分为模型组和红花黄色素低、中、高剂量组,其余10只为对照组;红花黄色素低、中、高剂量组分别ip 20、40、80 mg/kg红花黄色素,对照组和模型组ip等量生理盐水溶液,连续给药6周。观察大鼠一般状态,采用苏木精-伊红(HE)染色法观察RGC形态学变化并计数;采用原位凋亡(TUNEL)法检测并对比RGC凋亡率;采用实时荧光定量聚合酶链反应(qRT-PCR)法和蛋白免疫印迹法检测各组大鼠视网膜组织中p38MAPK、磷酸化p38MAPK(p-p38MAPK)、含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspasae-3)、血管内皮生长因子(VEGF)mRNA和蛋白表达情况,并对比p-p38MAPK与p38MAPK蛋白表达比值。结果 实验期间所有大鼠均存活,对照组大鼠无异常;模型组大鼠血糖较高、饮食量及尿量较大,体质量减轻;红花黄色素低、中、高剂量组大鼠各指标较模型组均有所改善。病理学观察发现,对照组大鼠视网膜各层细胞均无异常;模型组大鼠RGC排列紊乱、细胞核稀疏,双极细胞层及感光细胞层细胞数目减少,排列稀疏;红花黄色素低、中、高剂量组大鼠RGC及外层双极细胞层和感光细胞层异常程度均较模型组减轻,其中红花黄色素高剂量组减轻最为明显。与对照组比较,模型组大鼠RGC数量显著减少(P<0.05),凋亡率显著升高(P<0.05),视网膜中Caspase-3、VEGF mRNA及蛋白表达水平显著升高(P<0.05),p-p38MAPK/p38MAPK值显著升高(P<0.05);与模型组比较,红花黄色素低、中、高剂量组大鼠RGC数量显著增多(P<0.05),RGC凋亡率显著降低(P<0.05),视网膜Caspase-3、VEGF mRNA及蛋白表达水平显著降低(P<0.05),p-p38MAPK/p38MAPK值显著降低(P<0.05),且各剂量组间比较差异显著(P<0.05)。结论 红花黄色素可通过抑制p38MAPK信号通路保护DM大鼠RGC,减少其凋亡,其中80 mg/kg的红花黄色素保护作用最佳。
[Key word]
[Abstract]
Objective To investigate the protective effect of safflower yellow pigment (SYP) on diabetic (DM) retinal ganglion cells (RGC) by regulating p38 mitogen-activated protein kinase (MAPK) signaling pathway. Methods Selected 50 rats and 40 of them were used to establish DM rat model by single intraperitoneal injection of streptozotocin. They were randomly divided into model group and safflower yellow pigment low, medium and high dose groups, and the remaining 10 rats was control group. Safflower yellow pigment low, medium and high dose groups were intraperitoneally injected with 20, 40, and 80 mg/kg of SYP. Model group and control group were intraperitoneally injected with the same amount of physiological saline solution for six weeks. The general state of the rats was observed, and the morphological changes of RGC were observed by hematoxylin-eosin (HE) staining, and the number of RGC cells was counted. RGC apoptosis rate was detected by in situ apoptosis (TUNEL) method. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expressions of p38 mitogen-activated protein kinase (MAPK), phosphorylated p38MAPK (p-p38MAPK) and cysteine-containing aspartate proteolytic enzyme-3 (Caspasae-3), vascular endothelial growth factor polyclonal antibody (VEGF), and protein expression ratio of p-p38/p38MAPK was compared. Results All the rats survived during the experiment. There were no abnormalities in the rats in the control group. The blood glucose in the model group was higher, the diet and urine volume were larger, and the body weight was reduced. The safflower yellow pigment low, medium and high dose groups were better than the model group. Pathological observation showed that there were no abnormalities in the cells of the retina of the control group, while the RGC cells in the model group were disordered arranged and the nucleus were sparse, and the number of cells in the bipolar cell layer and the photoreceptor layer was reduced, and the arrangement was sparse. The abnormalities of RGC cells and outer bipolar cells in the safflower yellow pigment low, medium and high dose groups were significantly lower than those in the model group, and the high dose groups was the most obvious. There was significant difference in the number of RGC between groups (P < 0.05). Compared with the control group, the number of RGC in the model group was significantly decreased (P < 0.05), the apoptotic rate was significantly increased (P < 0.05), the levels of Caspase-3 and VEGF in the retina were significantly increased (P < 0.05), and the values of p-p38MAPK/p38MAPK were significantly increased (P < 0.05). Compared with the model group, the number of RGC was increased significantly (P< 0.05), the apoptotic rate of RGC was decreased significantly (P < 0.05), the expression levels of Caspase-3, VEGF and protein in retina were decreased significantly (P < 0.05), and the value of p-p38MAPK/p38MAPK was decreased significantly (P < 0.05), and the differences among the dose groups were significant (P < 0.05). Conclusion Safflower yellow pigment can protect RGC of DM rats by inhibiting p38MAPK signaling pathway, and reduce RGC apoptosis. The 80 mg/kg of SYP has the best protective effect.
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