藏药吉堪明目方对糖尿病视网膜病变大鼠的影响及机制研究

Effect of Tibetan Medicine Jikan Mingmu Formula on Diabetic Retinopathy Rats and Its Mechanism Research

关键词:  吉堪明目方  糖尿病视网膜病变  血管新生  氧化应激  肠道菌群

Key words:  Jikan Mingmu formula  diabetic retinopathy  angiogenesis  oxidative stress  intestinal flora

摘要:

目的 研究藏药吉堪明目方对糖尿病视网膜病变大鼠肠道菌群、氧化应激、血管异常新生的影响，探讨吉堪明目方治疗糖尿病视网膜病变可能的作用机制。方法 采用高脂饲料喂养合并腹腔注射链脲佐菌素（streptozotocin，STZ）50 mg·kg^-1诱导糖尿病视网膜病变大鼠模型，模型大鼠随机分为模型对照组、羟苯磺酸钙组和吉堪明目方低、中、高剂量组，另设空白对照组。进行体质量、空腹血糖值的常规监测；并于造模成功3个月后连续给药8周，而后测定血清超氧化物歧化酶（superoxide dismutase，SOD）、细胞间黏附分子-1（intercellular adhesion molecule-1，ICAM-1）、血管紧张素Ⅱ（angiotensin Ⅱ，ANG Ⅱ）的含量；采用苏木素和伊红染色观察视网膜常规病理改变，消化铺片观察微血管变化；采用免疫荧光法观察血管内皮生长因子（vascular endothelial growth factor，VEGF）、缺氧诱导因子-1α（hypoxia-inducible factor-1α，HIF-1α）在视网膜组织中的表达情况；采用16S rDN A高通量测序技术分析藏药吉堪明目方对大鼠肠道菌群结构的影响。结果 吉堪明目方可减缓模型大鼠体质量的下降趋势，对血糖无明显影响，可减轻视网膜病理改变，改善视网膜血管紊乱形态。吉堪明目方中剂量组可显著降低ICAM-1含量（P<0.05）。吉堪明目方各剂量组可显著降低ANG Ⅱ、VEGF、HIF-1α含量（P<0.05或P<0.01）。吉堪明目方可以有效抑制模型大鼠肠道菌群多样性减少的趋势，以吉堪明目方低剂量组的抑制效果最为显著。与空白对照组相比，模型对照组大鼠肠道菌群在属水平上有显著变化；给药后，糖尿病大鼠的肠道菌群开始恢复正常，其中以吉堪明目方中剂量组对大鼠肠道菌群调节作用最好。主成分分析表明，空白对照组与模型对照组比较存在明显差异，各给药组对模型大鼠的总体表现均有明显的回调作用，以吉堪明目方中、高剂量组效果最佳。结论 吉堪明目方能够减轻糖尿病视网膜病变程度，对糖尿病大鼠视网膜的保护作用机制可能与抑制新生血管的形成、调节肠道菌群有关。

Abstract:

OBJECTIVE To study the effects of Tibetan medicine Jikan Mingmu formula on intestinal flora, oxidative stress and vascular abnormalities in rats with diabetic retinopathy, and to explore the possible mechanism of Jikan Mingmu formula in treating diabetic retinopathy. METHODS Model rats with diabetic retinopathy induced by high-fat diet and intraperitoneal injection of 50 mg·kg^-1 streptozotocin(STZ) were randomly divided into model control group, calcium dobesilate group, and low, medium and high dose of Jikan Mingmu formula groups, and a blank control group was set. The regular monitoring of body weight and fasting blood glucose level was carried. Continuous administration was given for 8 weeks after successful modeling for 3 months, and the level of serum superoxide dismutase(SOD), intercellular adhesion molecule-1(ICAM-1), angiotensin Ⅱ(ANG-Ⅱ) was tested. Routine pathological changes of retina was observed by hematoxylin and eosin staining, microvascular changes was observed by digested patch, the protein expression of vascular endothelial growth factor(VEGF) and hypoxia-inducible factor-1α(HIF-1α) in retinal tissue was observed by immunofluorescence. The effects of Tibetan medicine Jikan Mingmu formula on the structure of rats' intestinal flora was analyzed by 16S rDNA high-throughput sequencing technology. RESULTS Jikan Mingmu formula could slow down the body weight decline of model rats, but it had no significant effect on blood glucose. And it could reduce pathological changes of retina and improve the morphology of retinal vascular disorders. Jikan Mingmu formula medium-dose group could significantly reduce the content of ICAM-1(P<0.05). Jikan Mingmu formula could reduce the content of ANG Ⅱ, VEGF and HIF-1α significantly(P<0.05 or P<0.01). Jikan Mingmu formula could effectively inhibit the trend of intestinal flora diversity in the model rats, and the inhibitory effect of Jikan Mingmu formula low-dose group was the most significant. Compared with the blank control group, the intestinal flora of the model control group had significant changes at the genus level. After administration, the intestinal flora of diabetic rats began to return to normal. Among them, Jikan Mingmu formula medium-dose group had the best regulation effect on rats' intestinal flora. Principal component analysis showed that there was a significant difference between the blank control group and the model control group. The drug-administered group had significant reversal effects on the overall performance of the model rats. The best effect was achieved in the medium and the high dose group of Jikan Mingmu formula. CONCLUSION Jikan Mingmu formula can reduce the degree of diabetic retinopathy. The protective mechanism of the retina in diabetic rats may be related to the inhibition of neovascularization and regulation of intestinal flora.