葛根素聯(lián)用阿格列汀對糖尿病大鼠胰島素抵抗、心肌纖維化的影響
發(fā)布時間：2018-08-05 15:12
【摘要】：目的:觀察葛根素與阿格列汀聯(lián)合用藥對高糖高脂飼料+STZ腹腔注射誘導的糖尿病大鼠空腹血糖、血脂、胰島素抵抗等的影響,并探討其對心肌的保護作用及其可能機制。方法:SPF清潔級SD雄性大鼠,隨機選取8只作為正常組(NC),其余大鼠進行造模。將造模成功的42只大鼠隨機分組:糖尿病模型組(蒸餾水,n=8),阿格列汀治療組(5mg·kg-1·d-1,n=9),葛根素治療組(80mg·kg-1·d-1,n=8),葛根素聯(lián)用阿格列汀治療組(40mg·kg-1·d-1+2.5mg·kg-1·d-1,n=9),阿格列汀聯(lián)用二甲雙胍治療組(2.5mg·kg-1·d-1+50mg·kg-1·d-1,n=8),分別以相應藥物給藥,正常組和模型組則注射生理鹽水。每間隔兩周測大鼠的體重(W)、空腹血糖(FBG)。經(jīng)持續(xù)給藥六周后,腹主動脈取血,并分離心臟,測左心室濕重(LVWW)、全心濕重(HWW),計算左心室重量指數(shù)(LVWI)、全心重量指數(shù)(HWI);ELISA法測血清中總膽固醇(TC)、甘油三酯(TG)、高密度脂蛋白膽固醇(HDL-C)及低密度脂蛋白膽固醇(LDL-C)含量水平;比色法測糖化血紅蛋白(Hb A1c);ELISA法測空腹血清胰島素(FINS),并計算胰島素抵抗指數(shù)(HOMA-IR)和胰島素敏感指數(shù)(HOMA-ISI)(HOMA-ISI=ln1/(FBG×FINS);HOMA-IR=FBG×FINS÷22.5);ELISA法測血清中血管緊張素Ⅱ(AngⅡ)和腫瘤壞死因子(TNF-α)含量;HE染色法觀察心肌病理損傷;MASSON染色法觀察心肌膠原纖維變化,計算心肌膠原容積分數(shù)(CVF,%);Western blot法檢測各組大鼠心肌TGF-β1、Smad3、Smad7、CTGF蛋白的表達;免疫組織化學法測TGF-β1蛋白平均光密度值(IOD/Area)。結果:(1)與正常對照組相比,DM模型組及各治療組大鼠的體重均顯著下降(P0.01),而FBG、Hb Alc、FINS、TC、TG、LDLC則顯著上升(P0.01或P0.05),表現(xiàn)出明顯的胰島素抵抗,且HDLC顯著下降(P0.01);血清中AngⅡ、TNF-α含量升高(P0.01或P0.05);HE染色表現(xiàn)為心肌細胞核深染,且間隙明顯增大,心肌增生、紊亂排列,產生明顯的病理損傷;Masson染色出現(xiàn)大量藍色膠原纖維沉積,CVF顯著升高(P0.01);大鼠的臟器指數(shù)HWI、LVWI明顯升高(P0.01),出現(xiàn)心肌肥厚,且心肌組織中的蛋白TGF-β1、Smad3、CTGF表達升高(P0.05或P0.01),Smad7蛋白表達降低(P0.05或P0.01);免疫組化法結果顯示糖尿病大鼠TGF-β1蛋白棕黃色陽性顆粒增多(P0.01)。(2)與DM模型組比較,各治療組的體重有所上升(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),胰島素敏感指數(shù)有所改善(P0.01或0.05),除阿格列汀組外,各用藥組HDLC上升(P0.01),且葛根素聯(lián)用阿格列汀效果優(yōu)于單藥治療組(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),葛根素聯(lián)用阿格列汀組降低程度更顯著(P0.01或P0.05);HE染色結果顯示各用藥組大鼠心肌細胞排列紊亂均有不同程度的改善,纖維排列趨于整齊規(guī)則化,葛根素聯(lián)用阿格列汀組改善作用優(yōu)于單獨用藥組;Masson染色結果顯示用藥組藍色膠原纖維沉積減少,心肌細胞排列紊亂情況有所改善,CVF顯著降低(P0.01),葛根素聯(lián)用阿格列汀組改善作用更明顯;臟器指數(shù)HWI、LVWI降低(P0.01),心肌肥厚程度有不同程度的改善,葛根素聯(lián)用阿格列汀效果優(yōu)于單獨用藥組(P0.05),且在改善LVWI指標方面效果比阿格列汀聯(lián)用二甲雙胍效果要好,P0.05。心肌組織中TGF-β1、Smad3蛋白含量降低(P0.01);除葛根素組外,各治療組CTGF表達降低(P0.01);Smad7蛋白表達升高(P0.05或P0.01);免疫組化法結果顯示各組TGF-β1蛋白陽性產物表達降低(P0.01)。(3)葛根素聯(lián)用阿格列汀改善效果顯著優(yōu)于單藥治療組(P0.05或P0.01),且在降低TGF-β1、Smad3,升高Smad7水平方面效果優(yōu)于阿格列汀聯(lián)用二甲雙胍組(P0.05),顯著改善心肌纖維化。結論:(1)葛根素與阿格列汀聯(lián)合用藥對高糖高脂飼料+STZ腹腔注射誘導的糖尿病大鼠有降低空腹血糖、糖化血紅蛋白水平,降低血清胰島素含量改善胰島素抵抗,改善血脂代謝異常的作用,且效果優(yōu)于單藥治療組。(2)葛根素與阿格列汀聯(lián)合用藥可降低高糖高脂飼料+STZ腹腔注射誘導的糖尿病大鼠的AngⅡ、TNF-α水平,且效果優(yōu)于單藥治療組。(3)葛根素與阿格列汀聯(lián)合用藥可改善高糖高脂飼料+STZ腹腔注射誘導的糖尿病大鼠心肌膠原容積分數(shù)、左心室肥厚和心肌纖維化程度,效果優(yōu)于單藥治療和阿格列汀聯(lián)用二甲雙胍組,其機制可能與下調TGF-β1、Smad3、CTGF蛋白表達,上調Smad7有關。
[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.
【學位授予單位】：皖南醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R587.2
本文編號：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.
【學位授予單位】：皖南醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R587.2
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