益气养阴清热通络方治疗小鼠糖尿病足的作用机制研究
发布时间：2018-07-22 20:55
【摘要】：糖尿病足是全身性疾病,足部溃疡持续时间越长,溃疡并发感染的可能性越大,导致患者截肢的风险越高,因此,如何有效地促进糖尿病足溃疡的愈合是治疗中至关重要的环节。本文以络病理论为指导,提出该病由脾失健运,气阴两伤,瘀热阻络所致,络虚与络瘀并存,主要病位在络脉、肌肉,以"络以通为用"为总治疗原则。益气养阴清热通络方是依据本法为组方原则而创立。本论文通过理论梳理和实验研究,评价该方治疗糖尿病足的疗效,并探讨其可能的作用机理,提升其临床应用价值。目的:观察益气养阴清热通络方对2型糖尿病所致糖尿病足小鼠模型的治疗作用,探讨糖尿病足可能的发病机制,揭示益气养阴清热通络方治疗糖尿病足的作用机理,为防治该病提供新思路,为临床推广提供科学依据。方法:选用SPF级C57BL/6J雄性小鼠70只,随机抽取10只为正常对照组,其余小鼠采用高糖高脂饮食喂养联合一次性腹腔快速注射链脲佐菌素(Streptozotocin,STZ)(120 mg/kg)、结扎下肢血管的方法,成功构建了 2型糖尿病并发糖尿病足的小鼠模型。按随机数字表将符合标准的小鼠分为6组:模型对照组、二甲双胍联合西洛他唑组(西药对照组)、通塞脉片组、益气养阴清热通络方高、中、低剂量组。运用生物化学、光学显微镜、免疫组织化学、三维定量体视学等检测手段及方法,动态观察益气养阴清热通络方对糖尿病足小鼠的一般状态、体重、空腹血糖(Fasting blood glucose,FBG)、创面大小、炎症因子、血管内皮功能、血管新生指标、氧化应激指标、空腹胰岛素(Fasting insulin,FINS)、空腹 C-肽(Fasting C-Peptide,FC-P)、胰岛素样生长因子-1(Insulin-like growth factor 1,IGF-1)、胰岛细胞形态与功能等的影响。结果:(1)益气养阴清热通络方高剂量组、西药对照组小鼠体重均较模型对照组增加(P0.05),增加趋势接近正常对照组小鼠的体重。(2)西药对照组、通塞脉片组、益气养阴清热通络方高、中、低剂量组可持续降低FBG,明显低于模型对照组(P0.001),并且低、中、高剂量组之间的血糖有依次下降趋势。(3)西药对照组、益气养阴清热通络方高、中、低剂量组经治疗后,溃疡面积均较模型对照组显著缩小,结痂天数明显缩短,下肢溃疡红肿逐渐消退,跛行、触觉过敏皆有所改善(P0.001,P0.01或P0.05)。(4)益气养阴清热通络方高、中、低剂量组的超敏C反应蛋白(High sensitive C reaction protein,hs-CRP)含量明显降低,高剂量组的白介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-a(Tumor necrosis factor-a,TNF-a)、血清血栓素 B2(Thromboxane B2,TXB2)、内皮素-1(Endothelin-1,ET-1)含量明显下降(P0.001,或P0.01),高剂量组的6-酮-前列腺素F-1α(6-Keto-PGF-1α,6-K-PGF-1α)含量明显升高(P0.01)。(5)与模型对照组比较,益气养阴清热通络方高剂量组血管内皮生长因子(Vascular endothelial growth factor,VEGF)含量明显增加(P0.05)。(6)西药对照组、通塞脉片组、益气养阴清热通络方高、中剂量组的小鼠胰腺超氧化物歧化酶(Superoxide dismutase,SOD)、还原型谷胱甘肽(Glutathione,GSH)含量明显高于模型对照组(P0.05,P0.01,或P0.001),丙二醛(Malondialdehyde,MDA)含量明显低于模型对照组(P0.01)。(7)与模型对照组比较,除益气养阴清热通络方低剂量组外,其余各治疗组的FC-P含量明显上升(P0.05或P0.001)。与益气养阴清热通络方低剂量组比较,通塞脉片组、益气养阴清热通络方高剂量组FC-P含量增加(P0.01,或P0.001)。(8)与模型对照组比较,西药对照组、益气养阴清热通络方高剂量组IGF-1含量明显上升(P0.05)。西药治疗组的FINS水平明显高于模型对照组(P0.001)。与益气养阴清热通络方低剂量组比较,西药对照组、益气养阴清热通络方高、中剂量组的FINS含量增加(P0.05,或P0.001)。(9)与模型对照组比较,各治疗组的胰岛素抵抗指数(Homeostasis model assessment of insulin resistance index,HOMA-IR)水平明显下降(P0.001)。(10)苏木精-伊红(Hematoxylin-eosin,HE)染色、免疫组化染色、三维体视学测量显示:益气养阴清热通络方能延缓小鼠胰岛萎缩,促进胰岛结构及胰岛β细胞的修复,减轻胰岛细胞的病理损伤,调节小鼠全身状态,改善糖尿病足的症状,从而加快足部溃疡的愈合。结论:(1)益气养阴清热通络方可以调节血糖水平,抑制体重持续下降,良性调节血管内皮功能、抑制炎症因子释放,改善组织缺血缺氧状态,从而促进疮口肉芽组织的生长,缩短溃疡愈合时间,尤其是高剂量组的各项指标的综合改善最为明显。(2)高剂量益气养阴清热通络方可以调节血清VEGF浓度,激活细胞增殖和肉芽组织的生长,促进血管生成,加速足部溃疡愈合;增强胰腺抗氧化应激能力,阻止或延缓胰腺功能的损害。(3)高剂量益气养阴清热通络方能降低胰岛素抵抗,提高胰岛素敏感性,促进胰岛结构及胰岛β细胞的修复,改善胰岛素抵抗和胰岛β细胞凋亡、功能紊乱状态,并且高剂量组的改善情况优于中、低剂量组。(4)高剂量益气养阴清热通络方降糖、缩小溃疡面积、减轻胰岛素抵抗的作用优于通塞脉片,综合改善血管内皮功能、降低炎症因子水平、减轻胰岛体积萎缩、改善小鼠整体状态等方面均优于西药、通塞脉片。益气养阴清热通络方不仅可以从整体上增强糖尿病足小鼠抗氧化应激的能力,降低胰岛素抵抗,还能从局部上减轻溃疡氧化炎症损伤,改善疮口微环境,共同发挥对糖尿病足的治疗作用,尤以高剂量益气养阴清热通络方的作用为佳。
[Abstract]:Diabetic foot is a systemic disease. The longer the duration of foot ulcers, the more likely the ulcer complicated with infection, the higher the risk of amputation. Therefore, how to effectively promote the healing of diabetic foot ulcers is a vital link in the treatment. This paper, guided by the theory of collateral disease, suggests that the disease is caused by spleen loss, two injuries of Qi Yin, and blood stasis. The main disease position is collaterals and collaterals, the main diseases are collaterals and blood stasis, the main diseases are collaterals, muscles, and the "collaterals are used" as the general treatment principle. The prescription of Yiqi nourishing yin and clearing heat and collaterals is founded according to the principle of this method. This paper evaluates the therapeutic effect of the prescription on the treatment of diabetic foot by theoretical carding and experimental research, and discusses its possible mechanism of action and promotes it. Objective: To observe the therapeutic effect of Yiqi Yangyin qinluo Tongluo Recipe on diabetic foot mice induced by type 2 diabetes, explore the possible pathogenesis of diabetic foot, and reveal the mechanism of the treatment of diabetic foot by clearing Qi and nourishing yin and clearing heat and dredging collaterals, providing new ideas for prevention and treatment of the disease and providing a scientific basis for clinical popularization. 70 SPF grade C57BL/6J male mice were selected and 10 were randomly selected as normal control group. The other mice were fed with high glucose and high fat diet combined with Streptozotocin (STZ) (120 mg/kg) and ligated the lower limbs of the lower limbs. The mice model of type 2 diabetes mellitus with diabetic foot was constructed successfully. The digital table was divided into 6 groups: model control group, metformin combined with cilostazol group (Western medicine control group), Tongsai Mai group, Yiqi Yangyin qinluo Tongluo prescription high, middle, low dose group. Using biochemistry, optical microscope, immunohistochemistry, three dimensional quantitative stereology and other detection methods and methods, dynamic observation of Qi Nourishing Yin and nourishing yin. The general state, weight, fasting blood glucose (Fasting blood glucose, FBG), wound size, inflammatory factor, vascular endothelial function, angiogenesis index, oxidative stress index, Fasting insulin (FINS), empty abdominal C- peptide (Fasting C-Peptide, FC-P), insulin-like growth factor -1. Owth factor 1, IGF-1), the effect of islet cell morphology and function. Results: (1) the high dose group of Yiqi Yangyin clearing heat Tongluo recipe, the weight of the mice in the western medicine control group was increased (P0.05), the increase trend was close to the normal control group. (2) the western medicine control group, Tongsai Mai tablet group, Yiqi Yangyin clearing heat dredging collaterals prescription high, middle, low dosage. The volume group continued to reduce FBG, obviously lower than the model control group (P0.001), and low, middle, high dose groups of blood sugar decreased in turn. (3) western medicine control group, Yiqi Yangyin qinluo Tongluo prescription high, middle, low dose group after treatment, the ulcer area is significantly smaller than the model group, the number of scab days obviously shortened, lower limb ulcers redness and swelling gradually Regression, claudication and tactile allergy were improved (P0.001, P0.01 or P0.05). (4) the concentration of the hypersensitive C reactive protein (High sensitive C reaction protein, hs-CRP) in the middle and low dose groups was significantly lower than that in the middle and low dose groups. The content of thromboxane B2 (Thromboxane B2, TXB2) and endothelin -1 (Endothelin-1, ET-1) decreased significantly (P0.001, or P0.01), and the content of 6- ketone prostaglandin F-1 alpha (6-Keto-PGF-1 alpha, alpha) in high dose group increased significantly. (5) the higher dose group was compared with the model control group. The content of othelial growth factor, VEGF) was significantly increased (P0.05). (6) the western medicine control group, Tongsai Mai tablet group, Yiqi Yangyin Tongluo Fang Gao, the medium dose group of mice pancreatic superoxide dismutase (Superoxide dismutase, SOD), and the content of reduced glutathione (Glutathione, GSH) was significantly higher than the model control group (P0.05, P0.01, or VEGF), malondialdehyde The content of (Malondialdehyde, MDA) was significantly lower than that of the model control group (P0.01). (7) compared with the model control group, the FC-P content in the rest of the treatment groups was obviously increased (P0.05 or P0.001) except the low dose group of Yiqi Yangyin clearing heat Tongluo recipe. The high dose of Tongsai Mai tablet group and the high dose of Yiqi Yangyin clearing heat dredging collaterals were compared with that of the group of Yiqi Yangyin qinluo Tongluo Fang Group FC-P content increased (P0.01, or P0.001). (8) compared with the model control group, the IGF-1 content in the high dose group of the western medicine control group was significantly higher (P0.05). The FINS level in the western medicine treatment group was significantly higher than that of the model control group (P0.001). The western medicine control group was compared with the low dose group of Yiqi Yangyin Tongluo prescription, the western medicine control group, the Yiqi Yangyin clearing heat and the heat clearing. The FINS content in the middle dose group increased (P0.05, or P0.001). (9) compared with the model control group, the insulin resistance index (Homeostasis model assessment of insulin resistance index, HOMA-IR) decreased significantly (P0.001). (10) the hematoxylin eosin staining, immunohistochemical staining, and three-dimensional stereopsis. The study showed that Yiqi Yangyin Qingre Tongluo recipe could postpone pancreatic islet atrophy, promote islet structure and repair of islet beta cells, alleviate the pathological damage of islet cells, regulate the state of the mice, improve the symptoms of diabetic foot, and accelerate the healing of foot ulcer. (1) the level of blood sugar can be adjusted by the prescription of nourishing yin and nourishing yin and clearing heat and dredging collaterals. Inhibiting the continuous decline of body weight, regulating vascular endothelial function, inhibiting the release of inflammatory factors, improving the state of tissue ischemia and hypoxia, thus promoting the growth of the granulation tissue and shortening the healing time of the ulcer, especially in the high dose group, the comprehensive improvement is most obvious. (2) the high dose of Supplementing Qi and nourishing yin and clearing heat and dredging collaterals can regulate the serum V EGF concentration, activating cell proliferation and growth of granulation tissue, promoting angiogenesis, accelerating healing of foot ulcers, enhancing pancreatic antioxidant stress and preventing or postponing pancreatic function damage. (3) high dose of nourishing yin and clearing heat and dredging collaterals can reduce insulin resistance, increase insulin sensitivity, promote islet structure and islet beta cell repair. The improvement of insulin resistance and islet beta cell apoptosis and dysfunction were improved, and the improvement of high dose group was better than that in the middle and low dose group. (4) high dose of Yiqi nourishing yin and clearing heat and dredging collaterals prescription reduced sugar, reduced the area of ulcer, reduced the effect of insulin resistance better than the Tongsai tablet, improved the function of vascular endothelium, reduced the level of inflammatory factors and reduced the level of inflammatory factors. The volume atrophy of the small islets of the pancreas and the improvement of the overall state of the mice are superior to those of the western medicine. Tongsai Tongmai Recipe. It can not only enhance the antioxidant stress ability of the diabetic foot mice, reduce the insulin resistance, but also relieve the injury of the ulcerative oxidizing inflammation, improve the microenvironment of the sores, and make a common use of diabetes. The therapeutic effect of foot disease is better than that of high dosage of Supplementing Qi, nourishing Yin, clearing heat and dredging collaterals.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5
本文编号：2138550
[Abstract]:Diabetic foot is a systemic disease. The longer the duration of foot ulcers, the more likely the ulcer complicated with infection, the higher the risk of amputation. Therefore, how to effectively promote the healing of diabetic foot ulcers is a vital link in the treatment. This paper, guided by the theory of collateral disease, suggests that the disease is caused by spleen loss, two injuries of Qi Yin, and blood stasis. The main disease position is collaterals and collaterals, the main diseases are collaterals and blood stasis, the main diseases are collaterals, muscles, and the "collaterals are used" as the general treatment principle. The prescription of Yiqi nourishing yin and clearing heat and collaterals is founded according to the principle of this method. This paper evaluates the therapeutic effect of the prescription on the treatment of diabetic foot by theoretical carding and experimental research, and discusses its possible mechanism of action and promotes it. Objective: To observe the therapeutic effect of Yiqi Yangyin qinluo Tongluo Recipe on diabetic foot mice induced by type 2 diabetes, explore the possible pathogenesis of diabetic foot, and reveal the mechanism of the treatment of diabetic foot by clearing Qi and nourishing yin and clearing heat and dredging collaterals, providing new ideas for prevention and treatment of the disease and providing a scientific basis for clinical popularization. 70 SPF grade C57BL/6J male mice were selected and 10 were randomly selected as normal control group. The other mice were fed with high glucose and high fat diet combined with Streptozotocin (STZ) (120 mg/kg) and ligated the lower limbs of the lower limbs. The mice model of type 2 diabetes mellitus with diabetic foot was constructed successfully. The digital table was divided into 6 groups: model control group, metformin combined with cilostazol group (Western medicine control group), Tongsai Mai group, Yiqi Yangyin qinluo Tongluo prescription high, middle, low dose group. Using biochemistry, optical microscope, immunohistochemistry, three dimensional quantitative stereology and other detection methods and methods, dynamic observation of Qi Nourishing Yin and nourishing yin. The general state, weight, fasting blood glucose (Fasting blood glucose, FBG), wound size, inflammatory factor, vascular endothelial function, angiogenesis index, oxidative stress index, Fasting insulin (FINS), empty abdominal C- peptide (Fasting C-Peptide, FC-P), insulin-like growth factor -1. Owth factor 1, IGF-1), the effect of islet cell morphology and function. Results: (1) the high dose group of Yiqi Yangyin clearing heat Tongluo recipe, the weight of the mice in the western medicine control group was increased (P0.05), the increase trend was close to the normal control group. (2) the western medicine control group, Tongsai Mai tablet group, Yiqi Yangyin clearing heat dredging collaterals prescription high, middle, low dosage. The volume group continued to reduce FBG, obviously lower than the model control group (P0.001), and low, middle, high dose groups of blood sugar decreased in turn. (3) western medicine control group, Yiqi Yangyin qinluo Tongluo prescription high, middle, low dose group after treatment, the ulcer area is significantly smaller than the model group, the number of scab days obviously shortened, lower limb ulcers redness and swelling gradually Regression, claudication and tactile allergy were improved (P0.001, P0.01 or P0.05). (4) the concentration of the hypersensitive C reactive protein (High sensitive C reaction protein, hs-CRP) in the middle and low dose groups was significantly lower than that in the middle and low dose groups. The content of thromboxane B2 (Thromboxane B2, TXB2) and endothelin -1 (Endothelin-1, ET-1) decreased significantly (P0.001, or P0.01), and the content of 6- ketone prostaglandin F-1 alpha (6-Keto-PGF-1 alpha, alpha) in high dose group increased significantly. (5) the higher dose group was compared with the model control group. The content of othelial growth factor, VEGF) was significantly increased (P0.05). (6) the western medicine control group, Tongsai Mai tablet group, Yiqi Yangyin Tongluo Fang Gao, the medium dose group of mice pancreatic superoxide dismutase (Superoxide dismutase, SOD), and the content of reduced glutathione (Glutathione, GSH) was significantly higher than the model control group (P0.05, P0.01, or VEGF), malondialdehyde The content of (Malondialdehyde, MDA) was significantly lower than that of the model control group (P0.01). (7) compared with the model control group, the FC-P content in the rest of the treatment groups was obviously increased (P0.05 or P0.001) except the low dose group of Yiqi Yangyin clearing heat Tongluo recipe. The high dose of Tongsai Mai tablet group and the high dose of Yiqi Yangyin clearing heat dredging collaterals were compared with that of the group of Yiqi Yangyin qinluo Tongluo Fang Group FC-P content increased (P0.01, or P0.001). (8) compared with the model control group, the IGF-1 content in the high dose group of the western medicine control group was significantly higher (P0.05). The FINS level in the western medicine treatment group was significantly higher than that of the model control group (P0.001). The western medicine control group was compared with the low dose group of Yiqi Yangyin Tongluo prescription, the western medicine control group, the Yiqi Yangyin clearing heat and the heat clearing. The FINS content in the middle dose group increased (P0.05, or P0.001). (9) compared with the model control group, the insulin resistance index (Homeostasis model assessment of insulin resistance index, HOMA-IR) decreased significantly (P0.001). (10) the hematoxylin eosin staining, immunohistochemical staining, and three-dimensional stereopsis. The study showed that Yiqi Yangyin Qingre Tongluo recipe could postpone pancreatic islet atrophy, promote islet structure and repair of islet beta cells, alleviate the pathological damage of islet cells, regulate the state of the mice, improve the symptoms of diabetic foot, and accelerate the healing of foot ulcer. (1) the level of blood sugar can be adjusted by the prescription of nourishing yin and nourishing yin and clearing heat and dredging collaterals. Inhibiting the continuous decline of body weight, regulating vascular endothelial function, inhibiting the release of inflammatory factors, improving the state of tissue ischemia and hypoxia, thus promoting the growth of the granulation tissue and shortening the healing time of the ulcer, especially in the high dose group, the comprehensive improvement is most obvious. (2) the high dose of Supplementing Qi and nourishing yin and clearing heat and dredging collaterals can regulate the serum V EGF concentration, activating cell proliferation and growth of granulation tissue, promoting angiogenesis, accelerating healing of foot ulcers, enhancing pancreatic antioxidant stress and preventing or postponing pancreatic function damage. (3) high dose of nourishing yin and clearing heat and dredging collaterals can reduce insulin resistance, increase insulin sensitivity, promote islet structure and islet beta cell repair. The improvement of insulin resistance and islet beta cell apoptosis and dysfunction were improved, and the improvement of high dose group was better than that in the middle and low dose group. (4) high dose of Yiqi nourishing yin and clearing heat and dredging collaterals prescription reduced sugar, reduced the area of ulcer, reduced the effect of insulin resistance better than the Tongsai tablet, improved the function of vascular endothelium, reduced the level of inflammatory factors and reduced the level of inflammatory factors. The volume atrophy of the small islets of the pancreas and the improvement of the overall state of the mice are superior to those of the western medicine. Tongsai Tongmai Recipe. It can not only enhance the antioxidant stress ability of the diabetic foot mice, reduce the insulin resistance, but also relieve the injury of the ulcerative oxidizing inflammation, improve the microenvironment of the sores, and make a common use of diabetes. The therapeutic effect of foot disease is better than that of high dosage of Supplementing Qi, nourishing Yin, clearing heat and dredging collaterals.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5
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