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目的:观察小檗皮浸膏对基因突变自然发病型db/db糖尿病小鼠视网膜蛋白激酶C-β(PKC-β),缺氧诱导因子-1α(HIF-1α),血管内皮细胞生长因子(VEGF)的影响,探索该药防治糖尿病视网膜病变的作用机制。方法:选用18周龄db/db糖尿病小鼠,db/m型同性同窝瘦型小鼠为对照。采用随机方法将db/db小鼠分为db/db组,小檗皮高、中、低剂量组(1.50,0.75,0.38 g·kg~(-1)),羟苯磺酸钙组(0.23 g·kg~(-1)),盐酸小檗碱组(0.135 g·kg~(-1))。相关药物连续ig 2个月,并于ig 2个月末处死小鼠。采用免疫组化方法观察3种因子在视网膜结构的表达情况,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白质免疫印迹(Western blot)方法检测视网膜内3种因子的基因与蛋白表达。结果:与db/m组比较,db/db组视网膜组织PKC-β,VEGF免疫组化反应呈阳性,表达显著增加(P<0.01);HIF-1α免疫组化反应呈阳性,表达量有升高趋势,但差异无统计学意义;db/db组三种因子的基因与蛋白表达显著增加(P<0.01)。与db/db组比较,羟苯磺酸钙组视网膜组织PKC-β免疫组化反应呈阳性,表达显著降低(P<0.05),小檗皮高剂量组视网膜组织PKC-β免疫组化反应呈阳性,表达显著降低(P<0.01),其他各给药组较db/db组PKC-β表达有降低趋势,但差异无统计学意义;各给药组视网膜组织HIF-1α免疫组化反应呈阳性,表达有降低趋势,但差异无统计学意义;小檗碱组、小檗皮低剂量组视网膜组织VEGF免疫组化反应呈阳性,表达明显降低(P<0.01),其他各给药组视网膜组织VEGF有降低趋势,但差异无统计学意义。与db/db组比较,羟苯磺酸钙组HIF-1α基因表达明显降低(P<0.05),其他各给药组PKC-β,HIF-1α,VEGF基因表达有降低趋势,但差异无统计学意义。与db/db组比较,羟苯磺酸钙组、盐酸小檗碱组、小檗皮高、低剂量组PKC-β蛋白表达显著降低(P<0.01),小檗皮中剂量组PKC-β蛋白表达有降低趋势,但差异无统计学意义;羟苯磺酸钙组、盐酸小檗碱组、小檗皮高、中剂量组HIF-1α蛋白表达显著降低(P<0.01),小檗皮低剂量组HIF-1α蛋白表达量有降低趋势,但差异无统计学意义;各给药组VEGF蛋白表达显著降低(P<0.01)。结论:小檗皮浸膏防治db/db糖尿病小鼠视网膜病变的作用机制可能与抑制PKC-β,HIF-1α,VEGF表达有关。
OBJECTIVE: To observe the effects of Bombyx Pilobagum extract on the expression of protein kinase C-β (PKC-β), hypoxia-inducible factor-1α (HIF-1α), vascular endothelial cell growth factor VEGF) to explore the mechanism of action of the drug in the prevention and treatment of diabetic retinopathy. Methods: 18 db / db diabetic mice and db / m isoforms of the same type of litter were used as controls. The db / db mice were randomly divided into db / db group, berberine high, medium and low dose groups (1.50,0.75,0.38 g · kg -1), calcium dobesilate group (0.23 g · kg ~ (-1)) and berberine hydrochloride group (0.135 g · kg ~ (-1)). The drug was given ig for 2 months and mice were sacrificed at the end of ig 2 months. Immunohistochemistry was used to observe the expression of three factors in the retina, Real-time PCR and Western blot were used to detect the expression of three factors in the retina . Results: Compared with the db / db group, the immunohistochemical expression of PKC-β and VEGF in the db / db group was significantly increased (P <0.01); the immunohistochemical reaction of HIF-1α was positive and the expression level was increased But the difference was not statistically significant. The gene and protein expression of three factors in db / db group were significantly increased (P <0.01). Compared with the db / db group, PKC-βimmunohistochemical reaction in retinoid calcium calcium group was significantly decreased (P <0.05), PKC-βimmunohistochemical reaction in high dose group was (P <0.01). The expression of PKC-β in db / db group was lower than other groups, but the difference was not statistically significant. The immunohistochemical reaction of HIF-1α in each group (P <0.01). The retinas of other groups showed no significant difference (P> 0.05). The expression of VEGF in the low dose berberine group and the low dose Berberine group was significantly higher than that in the other groups Tissue VEGF decreased trend, but the difference was not statistically significant. Compared with db / db group, the expression of HIF-1α gene in calcium dobesilate group was significantly decreased (P <0.05), and the expression of PKC-β, HIF-1α and VEGF in other groups decreased, but there was no statistical difference Significance of learning. Compared with the db / db group, PKC-βprotein expression was significantly decreased in the calcium dobesilate group, the berberine group, the barberry berry high and low doses groups (P <0.01), and the PKC-β The expression of HIF-1α in calcium dobesilate group, berberine group and Bupleurum palmatum group were significantly decreased (P <0.01) The expression of HIF-1α protein in the low-dose group had a tendency to decrease, but the difference was not statistically significant. The expression of VEGF protein in each group was significantly decreased (P <0.01). Conclusion: The mechanism of action of Bombyx mori extract in preventing and treating diabetic retinopathy in db / db diabetic mice may be related to inhibiting the expression of PKC-β, HIF-1α and VEGF.