葛根素联用阿格列汀对糖尿病大鼠胰岛素抵抗、心肌纤维化的影响
发布时间：2018-08-05 15:12
【摘要】：目的:观察葛根素与阿格列汀联合用药对高糖高脂饲料+STZ腹腔注射诱导的糖尿病大鼠空腹血糖、血脂、胰岛素抵抗等的影响,并探讨其对心肌的保护作用及其可能机制。方法:SPF清洁级SD雄性大鼠,随机选取8只作为正常组(NC),其余大鼠进行造模。将造模成功的42只大鼠随机分组:糖尿病模型组(蒸馏水,n=8),阿格列汀治疗组(5mg·kg-1·d-1,n=9),葛根素治疗组(80mg·kg-1·d-1,n=8),葛根素联用阿格列汀治疗组(40mg·kg-1·d-1+2.5mg·kg-1·d-1,n=9),阿格列汀联用二甲双胍治疗组(2.5mg·kg-1·d-1+50mg·kg-1·d-1,n=8),分别以相应药物给药,正常组和模型组则注射生理盐水。每间隔两周测大鼠的体重(W)、空腹血糖(FBG)。经持续给药六周后,腹主动脉取血,并分离心脏,测左心室湿重(LVWW)、全心湿重(HWW),计算左心室重量指数(LVWI)、全心重量指数(HWI);ELISA法测血清中总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)及低密度脂蛋白胆固醇(LDL-C)含量水平;比色法测糖化血红蛋白(Hb A1c);ELISA法测空腹血清胰岛素(FINS),并计算胰岛素抵抗指数(HOMA-IR)和胰岛素敏感指数(HOMA-ISI)(HOMA-ISI=ln1/(FBG×FINS);HOMA-IR=FBG×FINS÷22.5);ELISA法测血清中血管紧张素Ⅱ(AngⅡ)和肿瘤坏死因子(TNF-α)含量;HE染色法观察心肌病理损伤;MASSON染色法观察心肌胶原纤维变化,计算心肌胶原容积分数(CVF,%);Western blot法检测各组大鼠心肌TGF-β1、Smad3、Smad7、CTGF蛋白的表达;免疫组织化学法测TGF-β1蛋白平均光密度值(IOD/Area)。结果:(1)与正常对照组相比,DM模型组及各治疗组大鼠的体重均显著下降(P0.01),而FBG、Hb Alc、FINS、TC、TG、LDLC则显著上升(P0.01或P0.05),表现出明显的胰岛素抵抗,且HDLC显著下降(P0.01);血清中AngⅡ、TNF-α含量升高(P0.01或P0.05);HE染色表现为心肌细胞核深染,且间隙明显增大,心肌增生、紊乱排列,产生明显的病理损伤;Masson染色出现大量蓝色胶原纤维沉积,CVF显著升高(P0.01);大鼠的脏器指数HWI、LVWI明显升高(P0.01),出现心肌肥厚,且心肌组织中的蛋白TGF-β1、Smad3、CTGF表达升高(P0.05或P0.01),Smad7蛋白表达降低(P0.05或P0.01);免疫组化法结果显示糖尿病大鼠TGF-β1蛋白棕黄色阳性颗粒增多(P0.01)。(2)与DM模型组比较,各治疗组的体重有所上升(P0.01或0.05),FBG、Hb Alc、FINS、TC、TG、LDLC有不同程度的下降(P0.01或P0.05),胰岛素敏感指数有所改善(P0.01或0.05),除阿格列汀组外,各用药组HDLC上升(P0.01),且葛根素联用阿格列汀效果优于单药治疗组(P0.01或0.05);血清中AngⅡ、TNF-α水平有不同程度的降低(P0.01或P0.05),葛根素联用阿格列汀组降低程度更显著(P0.01或P0.05);HE染色结果显示各用药组大鼠心肌细胞排列紊乱均有不同程度的改善,纤维排列趋于整齐规则化,葛根素联用阿格列汀组改善作用优于单独用药组;Masson染色结果显示用药组蓝色胶原纤维沉积减少,心肌细胞排列紊乱情况有所改善,CVF显著降低(P0.01),葛根素联用阿格列汀组改善作用更明显;脏器指数HWI、LVWI降低(P0.01),心肌肥厚程度有不同程度的改善,葛根素联用阿格列汀效果优于单独用药组(P0.05),且在改善LVWI指标方面效果比阿格列汀联用二甲双胍效果要好,P0.05。心肌组织中TGF-β1、Smad3蛋白含量降低(P0.01);除葛根素组外,各治疗组CTGF表达降低(P0.01);Smad7蛋白表达升高(P0.05或P0.01);免疫组化法结果显示各组TGF-β1蛋白阳性产物表达降低(P0.01)。(3)葛根素联用阿格列汀改善效果显著优于单药治疗组(P0.05或P0.01),且在降低TGF-β1、Smad3,升高Smad7水平方面效果优于阿格列汀联用二甲双胍组(P0.05),显著改善心肌纤维化。结论:(1)葛根素与阿格列汀联合用药对高糖高脂饲料+STZ腹腔注射诱导的糖尿病大鼠有降低空腹血糖、糖化血红蛋白水平,降低血清胰岛素含量改善胰岛素抵抗,改善血脂代谢异常的作用,且效果优于单药治疗组。(2)葛根素与阿格列汀联合用药可降低高糖高脂饲料+STZ腹腔注射诱导的糖尿病大鼠的AngⅡ、TNF-α水平,且效果优于单药治疗组。(3)葛根素与阿格列汀联合用药可改善高糖高脂饲料+STZ腹腔注射诱导的糖尿病大鼠心肌胶原容积分数、左心室肥厚和心肌纤维化程度,效果优于单药治疗和阿格列汀联用二甲双胍组,其机制可能与下调TGF-β1、Smad3、CTGF蛋白表达,上调Smad7有关。
[Abstract]:Objective: To observe the effect of puerarin and A Glenn Dean on the fasting blood glucose, blood lipid and insulin resistance induced by +STZ intraperitoneal injection of high glucose and high fat diet, and to explore the protective effect and possible mechanism of it on the myocardium. Methods: SPF clean SD male rats were randomly selected as the normal group (NC), and the other rats were entered. 42 rats were randomly divided into two groups: diabetic model group (distilled water, n=8), Agger column (5mg. Kg-1. D-1, n=9), puerarin group (80mg, kg-1. D-1, n=8), and Puerarin Combined with A Glenn Dean treatment group (40mg. Kg-1, d-1+2.5mg. +50mg. Kg-1. D-1, n=8), the normal saline was injected in the normal group and the model group respectively. The rats' body weight (W) and fasting blood glucose (FBG) were measured every two weeks. After six weeks of continuous administration, the abdominal aorta was taken and the heart was separated and the left ventricular wet weight (LVWW) and the whole heart wet weight (HWW) were measured. The left ventricular weight index (LVWI) and the whole heart weight were calculated. Index (HWI); ELISA assay of serum total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C); colorimetric assay of glycosylated hemoglobin (Hb A1c); ELISA method for measuring fasting serum insulin (FINS), and to calculate the insulin resistance index (HOMA-IR) and insulin sensitivity index (HOMA-IS). I) (HOMA-ISI=ln1/ (FBG x FINS); HOMA-IR=FBG x FINS 22.5); ELISA method was used to measure the content of angiotensin II (Ang II) and tumor necrosis factor (TNF- alpha) in serum; HE staining method was used to observe pathological injury of myocardium; MASSON staining method was used to observe the changes of myocardial collagen fiber and cardiac collagen volume fraction (CVF,%). The expression of beta 1, Smad3, Smad7, CTGF protein, and the average optical density of TGF- beta 1 protein (IOD/Area) were measured by immunohistochemistry. Results: (1) the weight of DM model group and the treatment group were significantly decreased (P0.01) compared with the normal control group, while FBG, Hb Alc, FINS, TC, and also showed significant insulin resistance. DLC decreased significantly (P0.01); the content of Ang II and TNF- alpha in serum increased (P0.01 or P0.05); HE staining showed deep staining of the nucleus of the myocardium, and the gap between the myocardium increased obviously, the myocardium hyperplasia, the disorder arrangement and the obvious pathological damage; Masson staining showed a large number of blue collagen fibrils, CVF significantly increased (P0.01), the Viscera index of rats was HWI, LVWI obviously rose. High (P0.01), cardiac hypertrophy, and the expression of protein TGF- beta 1, Smad3, CTGF in myocardium increased (P0.05 or P0.01), Smad7 protein expression decreased (P0.05 or P0.01). Immunohistochemical staining showed that TGF- beta 1 protein brown yellow positive particles increased (P0.01) in diabetic rats. (2) the weight of each treatment group increased (0 or 0). .05), FBG, Hb Alc, FINS, TC, TG, LDLC have different degrees of decline (P0.01 or P0.05), the insulin sensitivity index is improved (P0.01 or 0.05). Except for the A Glenn Dean group, each drug group HDLC rises, and the effect of the Puerarin Combined with A Glenn Dean is superior to that of the single drug treatment group (or 0.05). 01 or P0.05), Puerarin Combined with A Glenn Dean group was more significant (P0.01 or P0.05). HE staining showed that the disorder of cardiac myocytes in each group was improved in varying degrees, and the arrangement of the fibers tended to be regular and regular, and the improvement of the Puerarin Combined with the Agger group was better than that of the single drug group; the result of Masson staining showed that the drug was used. Group blue collagen fibrous deposition decreased, myocardial cell arrangement disorder improved, CVF decreased significantly (P0.01). The effect of Puerarin Combined with Agger column was more obvious; the organ index HWI, LVWI decreased (P0.01), the degree of myocardial hypertrophy was improved in varying degrees. The effect of Puerarin Combined with P0.01 was better than that of the single drug group (P0.05), and the effect of Puerarin was better than that of the single drug group (P0.05). The effect of improving LVWI index was better than that of metformin with Agger. The content of TGF- beta 1 and Smad3 protein in P0.05. myocardium decreased (P0.01). Except puerarin group, the expression of CTGF was decreased (P0.01) and Smad7 protein expression increased (P0.05 or P0.01), and the expression of positive product of TGF- beta 1 protein in each group was reduced. (P0.01) (3) Puerarin Combined with A Glenn Dean improved significantly better than single drug treatment group (P0.05 or P0.01), and the effect of reducing TGF- beta 1, Smad3, and increasing Smad7 level was better than that of metformin group (P0.05) combined with Agger, and significantly improved myocardial fibrosis. Conclusion: (1) the combination of Ge Gensu and A Glenn Dean on high glucose and high fat diet +STZ abdominal cavity Injection induced diabetic rats have the effect of reducing fasting blood sugar, glycosylated hemoglobin level, reducing serum insulin content to improve insulin resistance and improving blood lipid metabolism, and the effect is better than that of single drug treatment group. (2) the combination of Ge Gensu and Agger can reduce high glucose and high fat diet induced diabetic rats induced by intraperitoneal injection of +STZ Ang II, TNF- alpha level, and the effect is better than the single drug treatment group. (3) the combination of Ge Gensu and A Glenn Dean can improve the myocardial collagen volume fraction, left ventricular hypertrophy and myocardial fibrosis induced by +STZ intraperitoneal injection of high glucose and high fat diet, the effect is better than the single drug therapy and the combination of A Glenn Dean combined with metformin group, the mechanism can be improved. It can be related to downregulation of TGF- beta 1, Smad3, CTGF protein expression and up regulation of Smad7.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2
本文编号：2166180
[Abstract]:Objective: To observe the effect of puerarin and A Glenn Dean on the fasting blood glucose, blood lipid and insulin resistance induced by +STZ intraperitoneal injection of high glucose and high fat diet, and to explore the protective effect and possible mechanism of it on the myocardium. Methods: SPF clean SD male rats were randomly selected as the normal group (NC), and the other rats were entered. 42 rats were randomly divided into two groups: diabetic model group (distilled water, n=8), Agger column (5mg. Kg-1. D-1, n=9), puerarin group (80mg, kg-1. D-1, n=8), and Puerarin Combined with A Glenn Dean treatment group (40mg. Kg-1, d-1+2.5mg. +50mg. Kg-1. D-1, n=8), the normal saline was injected in the normal group and the model group respectively. The rats' body weight (W) and fasting blood glucose (FBG) were measured every two weeks. After six weeks of continuous administration, the abdominal aorta was taken and the heart was separated and the left ventricular wet weight (LVWW) and the whole heart wet weight (HWW) were measured. The left ventricular weight index (LVWI) and the whole heart weight were calculated. Index (HWI); ELISA assay of serum total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C); colorimetric assay of glycosylated hemoglobin (Hb A1c); ELISA method for measuring fasting serum insulin (FINS), and to calculate the insulin resistance index (HOMA-IR) and insulin sensitivity index (HOMA-IS). I) (HOMA-ISI=ln1/ (FBG x FINS); HOMA-IR=FBG x FINS 22.5); ELISA method was used to measure the content of angiotensin II (Ang II) and tumor necrosis factor (TNF- alpha) in serum; HE staining method was used to observe pathological injury of myocardium; MASSON staining method was used to observe the changes of myocardial collagen fiber and cardiac collagen volume fraction (CVF,%). The expression of beta 1, Smad3, Smad7, CTGF protein, and the average optical density of TGF- beta 1 protein (IOD/Area) were measured by immunohistochemistry. Results: (1) the weight of DM model group and the treatment group were significantly decreased (P0.01) compared with the normal control group, while FBG, Hb Alc, FINS, TC, and also showed significant insulin resistance. DLC decreased significantly (P0.01); the content of Ang II and TNF- alpha in serum increased (P0.01 or P0.05); HE staining showed deep staining of the nucleus of the myocardium, and the gap between the myocardium increased obviously, the myocardium hyperplasia, the disorder arrangement and the obvious pathological damage; Masson staining showed a large number of blue collagen fibrils, CVF significantly increased (P0.01), the Viscera index of rats was HWI, LVWI obviously rose. High (P0.01), cardiac hypertrophy, and the expression of protein TGF- beta 1, Smad3, CTGF in myocardium increased (P0.05 or P0.01), Smad7 protein expression decreased (P0.05 or P0.01). Immunohistochemical staining showed that TGF- beta 1 protein brown yellow positive particles increased (P0.01) in diabetic rats. (2) the weight of each treatment group increased (0 or 0). .05), FBG, Hb Alc, FINS, TC, TG, LDLC have different degrees of decline (P0.01 or P0.05), the insulin sensitivity index is improved (P0.01 or 0.05). Except for the A Glenn Dean group, each drug group HDLC rises, and the effect of the Puerarin Combined with A Glenn Dean is superior to that of the single drug treatment group (or 0.05). 01 or P0.05), Puerarin Combined with A Glenn Dean group was more significant (P0.01 or P0.05). HE staining showed that the disorder of cardiac myocytes in each group was improved in varying degrees, and the arrangement of the fibers tended to be regular and regular, and the improvement of the Puerarin Combined with the Agger group was better than that of the single drug group; the result of Masson staining showed that the drug was used. Group blue collagen fibrous deposition decreased, myocardial cell arrangement disorder improved, CVF decreased significantly (P0.01). The effect of Puerarin Combined with Agger column was more obvious; the organ index HWI, LVWI decreased (P0.01), the degree of myocardial hypertrophy was improved in varying degrees. The effect of Puerarin Combined with P0.01 was better than that of the single drug group (P0.05), and the effect of Puerarin was better than that of the single drug group (P0.05). The effect of improving LVWI index was better than that of metformin with Agger. The content of TGF- beta 1 and Smad3 protein in P0.05. myocardium decreased (P0.01). Except puerarin group, the expression of CTGF was decreased (P0.01) and Smad7 protein expression increased (P0.05 or P0.01), and the expression of positive product of TGF- beta 1 protein in each group was reduced. (P0.01) (3) Puerarin Combined with A Glenn Dean improved significantly better than single drug treatment group (P0.05 or P0.01), and the effect of reducing TGF- beta 1, Smad3, and increasing Smad7 level was better than that of metformin group (P0.05) combined with Agger, and significantly improved myocardial fibrosis. Conclusion: (1) the combination of Ge Gensu and A Glenn Dean on high glucose and high fat diet +STZ abdominal cavity Injection induced diabetic rats have the effect of reducing fasting blood sugar, glycosylated hemoglobin level, reducing serum insulin content to improve insulin resistance and improving blood lipid metabolism, and the effect is better than that of single drug treatment group. (2) the combination of Ge Gensu and Agger can reduce high glucose and high fat diet induced diabetic rats induced by intraperitoneal injection of +STZ Ang II, TNF- alpha level, and the effect is better than the single drug treatment group. (3) the combination of Ge Gensu and A Glenn Dean can improve the myocardial collagen volume fraction, left ventricular hypertrophy and myocardial fibrosis induced by +STZ intraperitoneal injection of high glucose and high fat diet, the effect is better than the single drug therapy and the combination of A Glenn Dean combined with metformin group, the mechanism can be improved. It can be related to downregulation of TGF- beta 1, Smad3, CTGF protein expression and up regulation of Smad7.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2
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