益氣養(yǎng)陰清熱通絡(luò)方治療小鼠糖尿病足的作用機制研究
發(fā)布時間：2018-07-22 20:55
【摘要】：糖尿病足是全身性疾病,足部潰瘍持續(xù)時間越長,潰瘍并發(fā)感染的可能性越大,導(dǎo)致患者截肢的風(fēng)險越高,因此,如何有效地促進糖尿病足潰瘍的愈合是治療中至關(guān)重要的環(huán)節(jié)。本文以絡(luò)病理論為指導(dǎo),提出該病由脾失健運,氣陰兩傷,瘀熱阻絡(luò)所致,絡(luò)虛與絡(luò)瘀并存,主要病位在絡(luò)脈、肌肉,以"絡(luò)以通為用"為總治療原則。益氣養(yǎng)陰清熱通絡(luò)方是依據(jù)本法為組方原則而創(chuàng)立。本論文通過理論梳理和實驗研究,評價該方治療糖尿病足的療效,并探討其可能的作用機理,提升其臨床應(yīng)用價值。目的:觀察益氣養(yǎng)陰清熱通絡(luò)方對2型糖尿病所致糖尿病足小鼠模型的治療作用,探討糖尿病足可能的發(fā)病機制,揭示益氣養(yǎng)陰清熱通絡(luò)方治療糖尿病足的作用機理,為防治該病提供新思路,為臨床推廣提供科學(xué)依據(jù)。方法:選用SPF級C57BL/6J雄性小鼠70只,隨機抽取10只為正常對照組,其余小鼠采用高糖高脂飲食喂養(yǎng)聯(lián)合一次性腹腔快速注射鏈脲佐菌素(Streptozotocin,STZ)(120 mg/kg)、結(jié)扎下肢血管的方法,成功構(gòu)建了 2型糖尿病并發(fā)糖尿病足的小鼠模型。按隨機數(shù)字表將符合標準的小鼠分為6組:模型對照組、二甲雙胍聯(lián)合西洛他唑組(西藥對照組)、通塞脈片組、益氣養(yǎng)陰清熱通絡(luò)方高、中、低劑量組。運用生物化學(xué)、光學(xué)顯微鏡、免疫組織化學(xué)、三維定量體視學(xué)等檢測手段及方法,動態(tài)觀察益氣養(yǎng)陰清熱通絡(luò)方對糖尿病足小鼠的一般狀態(tài)、體重、空腹血糖(Fasting blood glucose,FBG)、創(chuàng)面大小、炎癥因子、血管內(nèi)皮功能、血管新生指標、氧化應(yīng)激指標、空腹胰島素(Fasting insulin,FINS)、空腹 C-肽(Fasting C-Peptide,FC-P)、胰島素樣生長因子-1(Insulin-like growth factor 1,IGF-1)、胰島細胞形態(tài)與功能等的影響。結(jié)果:(1)益氣養(yǎng)陰清熱通絡(luò)方高劑量組、西藥對照組小鼠體重均較模型對照組增加(P0.05),增加趨勢接近正常對照組小鼠的體重。(2)西藥對照組、通塞脈片組、益氣養(yǎng)陰清熱通絡(luò)方高、中、低劑量組可持續(xù)降低FBG,明顯低于模型對照組(P0.001),并且低、中、高劑量組之間的血糖有依次下降趨勢。(3)西藥對照組、益氣養(yǎng)陰清熱通絡(luò)方高、中、低劑量組經(jīng)治療后,潰瘍面積均較模型對照組顯著縮小,結(jié)痂天數(shù)明顯縮短,下肢潰瘍紅腫逐漸消退,跛行、觸覺過敏皆有所改善(P0.001,P0.01或P0.05)。(4)益氣養(yǎng)陰清熱通絡(luò)方高、中、低劑量組的超敏C反應(yīng)蛋白(High sensitive C reaction protein,hs-CRP)含量明顯降低,高劑量組的白介素-6(Interleukin-6,IL-6)、腫瘤壞死因子-a(Tumor necrosis factor-a,TNF-a)、血清血栓素 B2(Thromboxane B2,TXB2)、內(nèi)皮素-1(Endothelin-1,ET-1)含量明顯下降(P0.001,或P0.01),高劑量組的6-酮-前列腺素F-1α(6-Keto-PGF-1α,6-K-PGF-1α)含量明顯升高(P0.01)。(5)與模型對照組比較,益氣養(yǎng)陰清熱通絡(luò)方高劑量組血管內(nèi)皮生長因子(Vascular endothelial growth factor,VEGF)含量明顯增加(P0.05)。(6)西藥對照組、通塞脈片組、益氣養(yǎng)陰清熱通絡(luò)方高、中劑量組的小鼠胰腺超氧化物歧化酶(Superoxide dismutase,SOD)、還原型谷胱甘肽(Glutathione,GSH)含量明顯高于模型對照組(P0.05,P0.01,或P0.001),丙二醛(Malondialdehyde,MDA)含量明顯低于模型對照組(P0.01)。(7)與模型對照組比較,除益氣養(yǎng)陰清熱通絡(luò)方低劑量組外,其余各治療組的FC-P含量明顯上升(P0.05或P0.001)。與益氣養(yǎng)陰清熱通絡(luò)方低劑量組比較,通塞脈片組、益氣養(yǎng)陰清熱通絡(luò)方高劑量組FC-P含量增加(P0.01,或P0.001)。(8)與模型對照組比較,西藥對照組、益氣養(yǎng)陰清熱通絡(luò)方高劑量組IGF-1含量明顯上升(P0.05)。西藥治療組的FINS水平明顯高于模型對照組(P0.001)。與益氣養(yǎng)陰清熱通絡(luò)方低劑量組比較,西藥對照組、益氣養(yǎng)陰清熱通絡(luò)方高、中劑量組的FINS含量增加(P0.05,或P0.001)。(9)與模型對照組比較,各治療組的胰島素抵抗指數(shù)(Homeostasis model assessment of insulin resistance index,HOMA-IR)水平明顯下降(P0.001)。(10)蘇木精-伊紅(Hematoxylin-eosin,HE)染色、免疫組化染色、三維體視學(xué)測量顯示:益氣養(yǎng)陰清熱通絡(luò)方能延緩小鼠胰島萎縮,促進胰島結(jié)構(gòu)及胰島β細胞的修復(fù),減輕胰島細胞的病理損傷,調(diào)節(jié)小鼠全身狀態(tài),改善糖尿病足的癥狀,從而加快足部潰瘍的愈合。結(jié)論:(1)益氣養(yǎng)陰清熱通絡(luò)方可以調(diào)節(jié)血糖水平,抑制體重持續(xù)下降,良性調(diào)節(jié)血管內(nèi)皮功能、抑制炎癥因子釋放,改善組織缺血缺氧狀態(tài),從而促進瘡口肉芽組織的生長,縮短潰瘍愈合時間,尤其是高劑量組的各項指標的綜合改善最為明顯。(2)高劑量益氣養(yǎng)陰清熱通絡(luò)方可以調(diào)節(jié)血清VEGF濃度,激活細胞增殖和肉芽組織的生長,促進血管生成,加速足部潰瘍愈合;增強胰腺抗氧化應(yīng)激能力,阻止或延緩胰腺功能的損害。(3)高劑量益氣養(yǎng)陰清熱通絡(luò)方能降低胰島素抵抗,提高胰島素敏感性,促進胰島結(jié)構(gòu)及胰島β細胞的修復(fù),改善胰島素抵抗和胰島β細胞凋亡、功能紊亂狀態(tài),并且高劑量組的改善情況優(yōu)于中、低劑量組。(4)高劑量益氣養(yǎng)陰清熱通絡(luò)方降糖、縮小潰瘍面積、減輕胰島素抵抗的作用優(yōu)于通塞脈片,綜合改善血管內(nèi)皮功能、降低炎癥因子水平、減輕胰島體積萎縮、改善小鼠整體狀態(tài)等方面均優(yōu)于西藥、通塞脈片。益氣養(yǎng)陰清熱通絡(luò)方不僅可以從整體上增強糖尿病足小鼠抗氧化應(yīng)激的能力,降低胰島素抵抗,還能從局部上減輕潰瘍氧化炎癥損傷,改善瘡口微環(huán)境,共同發(fā)揮對糖尿病足的治療作用,尤以高劑量益氣養(yǎng)陰清熱通絡(luò)方的作用為佳。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285.5
本文編號：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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285.5
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