泄?jié)峤舛�、活血通絡(luò)中藥對糖尿病腎病大鼠腎組織中血管生成素1表達的影響
發(fā)布時間：2018-08-24 07:36
【摘要】：目的：本研究通過高糖高脂飼料飼養(yǎng)加腹腔注射小劑量鏈脲佐菌素(streptozotocin, STZ)建立DM大鼠模型，應(yīng)用泄?jié)峤舛�、活血通絡(luò)中藥、厄貝沙坦以及兩者聯(lián)合應(yīng)用對其進行干預(yù)，通過觀察實驗動物各生化指標(biāo)，腎臟病理形態(tài)學(xué)變化以及腎皮質(zhì)組織中血管生成素1(Angiopoietin-1，Ang-1)的表達水平，來探討Ang-1與DN的關(guān)系，以及泄?jié)峤舛尽⒒钛ńj(luò)中藥對DN大鼠腎臟的保護作用和可能機制，為臨床治療DN提供實驗依據(jù)。 方法：雄性Sprague-Dauley大鼠85只，體重170±10g，適應(yīng)性飼養(yǎng)一周，測定血糖和尿蛋白均為陰性后，隨機選取15只為正常組（NG)，給予普通飼料喂養(yǎng)。其余大鼠給予高糖高脂飼料喂養(yǎng)，12周后給予一次性腹腔注射STZ35mg/kg，正常組大鼠給予一次性腹腔注射相當(dāng)劑量的0.1mol/L枸櫞酸緩沖液。72小時后尾靜脈采血測隨機血糖≥16.7mmol/L為糖尿病造模成功，造模過程中死亡兩只，另有一只大鼠血糖不達標(biāo)，均予以剔除。將成模大鼠隨機分為4組：模型組(MG)、中藥組(ZG)、西藥組(XG)、中藥加西藥組(JG)，中藥加西藥組16只，剩余各組17只。成模后開始給藥，每日一次，治療組每只大鼠給予4ml/d的藥物溶液，正常組與模型組均灌服等體積的生理鹽水，共給藥16周。自由飲水、進食，室溫22-25℃，相對濕度40%-60%，12小時交替照明。在給藥期間的第4周、8周、12周各監(jiān)測體重、血糖及尿蛋白。于給藥第16周末，禁食12小時，不禁水，留取各組大鼠24小時尿液，測定24小時尿蛋白定量，稱量體重，10%水合氯醛麻醉動物，腹主動脈取血，摘取右側(cè)腎臟并稱重，計算肥大指數(shù)，留取腎臟標(biāo)本，光鏡、電鏡下觀察腎臟病理形態(tài)學(xué)變化，提取各組腎皮質(zhì)RNA，用實時熒光定量PCR方法檢測Ang-1mRNA的含量，同時免疫組化染色觀察Ang-1的表達情況。 結(jié)果： 1各組大鼠體重、腎重、肥大指數(shù)（腎重/體重）與正常組比較，各治療組及模型組體重明顯減輕（P0.05）；各治療組及模型組之間體重?zé)o顯著差異（P0.05）。與正常組比較，各治療組及模型組肥大指數(shù)明顯升高（P0.05)；各治療組及模型組之間比較無明顯差別（P0.05)。各組右腎重比較差異均無統(tǒng)計學(xué)意義（P0.05）。 2各組大鼠24小時尿蛋白定量與正常組比較，各治療組及模型組24小時尿蛋白定量（24-hour urinary totalprotein,24hUTP）均升高(P0.05)；與模型組比較，各治療組UTP下降(P0.05)；與中藥組及西藥組比較，中藥加西藥組UTP下降(P0.05)；中藥組UTP與西藥組相比無明顯差異(P0.05)。 3各組大鼠血清總蛋白、白蛋白、血脂及腎功能的比較與正常組相比，模型組及各治療組血清總蛋白（TP）及白蛋白（ALB)均降低（P0.05)；與模型組比較，各治療組TP及ALB均升高（P0.05)；與中藥組及西藥組比較，中藥加西藥組TP及ALB升高（P0.05)；中藥組與西藥組相比無明顯差異（P0.05）。與正常組相比，模型組及各治療組總膽固醇(TC)及甘油三脂(TG)均明顯升高（P0.05）；與模型組比較，，各治療組TC及TG降低（P0.05）；與中藥組及西藥組比較，中藥加西藥組TC及TG降低（P0.05)；中藥組與西藥組相比無明顯差異(P0.05)。正常組、模型組、各治療組血肌酐及尿素氮比較均無統(tǒng)計學(xué)意義(P0.05)。 4各組大鼠糖化血紅蛋白（HbA1c)的比較與正常組比較，模型組及各治療組HbA1c均明顯升高（P0.05）；與模型組及西藥組相比，中藥加西藥組及中藥組HbA1c均明顯降低（P0.05）；西藥組HbA1c比模型組降低但無統(tǒng)計學(xué)意義(P0.05)；中藥加西藥組HbA1c比中藥組降低但無統(tǒng)計學(xué)意義(P0.05)。 5腎臟病理形態(tài)學(xué)觀察 5.1光鏡觀察正常組大鼠腎臟腎小球正常大小，結(jié)構(gòu)清晰，未見腎小球肥大或萎縮，基底膜無增厚，系膜基質(zhì)分布面積正常。模型組大鼠腎臟腎小球明顯肥大，基底膜增厚，系膜區(qū)顯著增寬。各治療組以上病理改變較模型組為輕，各治療組間無明顯差異。 5.2電鏡觀察正常組腎小球內(nèi)皮細胞未見異常，基底膜厚度均勻，結(jié)構(gòu)完整，足突無融合、缺如等異常改變；模型組腎小球內(nèi)皮細胞明顯增生、突出，基底膜明顯增厚，伴有足細胞足突廣泛融合；各治療組也均有不同程度的病理改變，但較模型組輕，各組間差異不明顯。 6各組大鼠腎皮質(zhì)Ang-1的表達 6.1Real-Time PCR檢測腎組織Ang-1mRNA的表達與正常組相比，模型組及各治療組Ang-1mRNA表達量均明顯升高（P0.05）；與模型組比較，各治療組Ang-1mRNA表達量均明顯升高(P0.05)；與西藥組和中藥組比較，中藥加西藥組Ang-1mRNA表達量明顯升高(P0.05)；西藥組與中藥組相比差異無統(tǒng)計學(xué)意義(P0.05)。 6.2免疫組織化學(xué)法檢測腎組織Ang-1的表達正常組Ang-1在腎小球足細胞少量表達；與正常組相比，各造模組大鼠腎組織中Ang-1表達明顯增加(P0.05)，突出表達于足細胞；與模型組比較，各治療組Ang-1表達明顯增加(P0.05)；各治療組之間比較，中藥加西藥組腎組織Ang-1表達最強，與中藥組和西藥組相比有明顯差異(P0.05)；中藥組與西藥組相比Ang-1的表達無明顯差別(P0.05)。 結(jié)論： 1泄?jié)峤舛�、活血通絡(luò)中藥可減少尿蛋白的排泄，升高血清總蛋白、白蛋白，降低血糖、血脂，延緩DN的進展。 2泄?jié)峤舛�、活血通絡(luò)中藥可以有效抑制系膜基質(zhì)的增生、基底膜的增厚及內(nèi)皮細胞的增生和足細胞的融合，延緩腎臟病理進展。 3泄?jié)峤舛�、活血通絡(luò)中藥能夠上調(diào)DN大鼠腎組織Ang-1的表達，提示該方可以減輕血管的滲漏并促進血管的成熟和穩(wěn)定，減少尿蛋白的排泄，延緩DN進展從而達到對DN大鼠腎臟保護作用。 4泄?jié)峤舛尽⒒钛ńj(luò)中藥和厄貝沙坦聯(lián)合應(yīng)用在減少蛋白尿，升高血清蛋白，降低血脂，調(diào)節(jié)Ang-1表達等方面比單純用中藥或西藥作用均強。
[Abstract]:Objective: To establish DM rat model by feeding high-sugar and high-fat diet and intraperitoneal injection of low-dose streptozotocin (STZ), and to observe the biochemical indexes, pathological morphological changes of kidney and kidney of experimental animals. The expression of Angiopoietin-1 (Ang-1) in cortical tissue was studied to explore the relationship between Ang-1 and DN, and the protective effect and possible mechanism of Chinese herbs on kidney of DN rats.
Methods: Eighty-five Sprague-Dauley male rats weighing 170 + 10g were fed adaptively for one week. After the blood glucose and urinary protein were tested negative, 15 rats were randomly selected as normal group (NG) and fed with normal diet. The other rats were fed with high glucose and high fat diet, and the other rats were injected with STZ 35mg/kg by abdominal cavity for 12 weeks. The rats were randomly divided into four groups: model group (MG), Chinese medicine group (ZG), western medicine group (XG), Chinese medicine plus Western medicine (TCM) and Chinese medicine plus Western medicine (WM). Group JG, Chinese medicine plus Western medicine group 16, the remaining 17 in each group. After modeling, the rats in the treatment group were given 4 ml/d of the drug solution once a day. The normal group and the model group were given the same volume of physiological saline, a total of 16 weeks. Free drinking water, eating, room temperature 22-25 C, relative humidity 40% - 60%, 12 hours alternate lighting. At the end of the sixteenth week, fasting for twelve hours, water was not forbidden. 24-hour urine was taken from rats in each group. 24-hour urine protein was measured. Weight was weighed, 10% chloral hydrate was anesthetized, abdominal aorta was taken for blood, right kidney was taken for weighing, hypertrophy index was calculated, kidney samples were taken, light microscope and electricity were used. The pathological morphological changes of kidney were observed under microscope. RNA was extracted from renal cortex of each group. The content of Ang-1 mRNA was detected by real-time fluorescence quantitative PCR and the expression of Ang-1 was observed by immunohistochemical staining.
Result:
1 Compared with the normal group, the body weight, kidney weight and hypertrophy index (kidney weight/body weight) of rats in each treatment group and model group decreased significantly (P 0.05). There was no significant difference in body weight between each treatment group and model group (P 0.05). Compared with the normal group, the hypertrophy index of each treatment group and model group increased significantly (P 0.05). There was no significant difference (P0.05). There was no significant difference in right kidney weight between each group (P0.05).
Compared with the normal group, the 24-hour urinary total protein (24-hour UTP) in the treatment group and the model group increased (P 0.05); compared with the model group, the UTP in the treatment group decreased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, the UTP in the traditional Chinese medicine plus Western medicine group decreased (P 0.05); There was no significant difference between the two groups (P0.05).
Compared with the normal group, the serum total protein (TP) and albumin (ALB) in the model group and the treatment group decreased (P 0.05); compared with the model group, TP and ALB in the treatment group increased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TP and ALB in the traditional Chinese medicine plus Western medicine group increased (P 0.05); Compared with the normal group, the total cholesterol (TC) and triglyceride (TG) in the model group and each treatment group were significantly higher (P 0.05); compared with the model group, TC and TG in each treatment group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG in the traditional Chinese medicine plus Western Medicine group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG were lower (P 0.05). Compared with no significant difference (P 0.05). Normal group, model group, the treatment group serum creatinine and urea nitrogen were not statistically significant (P 0.05).
Compared with normal group, HbA1c in model group and treatment group increased significantly (P 0.05); compared with model group and Western medicine group, HbA1c in traditional Chinese medicine plus Western medicine group and traditional Chinese medicine group decreased significantly (P 0.05); HbA1c in western medicine group was lower than that in model group, but there was no statistical significance (P 0.05); HbA1c in traditional Chinese medicine plus Western medicine group was lower than that in model group. Compared with the Chinese medicine group, there was no statistical significance (P0.05).
5 pathological observation of kidney
5.1 The glomeruli of normal rats were normal size, clear structure, no glomerular hypertrophy or atrophy, no basement membrane thickening and normal distribution area of mesangial matrix were observed under light microscope. There was no significant difference.
5.2 Electron microscopic observation showed that there were no abnormal changes in the glomerular endothelial cells in the normal group, such as the thickness of basement membrane was uniform, the structure was intact, the foot process did not fuse, and the absence of abnormal changes; the glomerular endothelial cells in the model group were obviously hyperplasia, prominent, the basement membrane was obviously thickened, and the foot process of podocytes fused extensively; the treatment groups also had different degrees of Patholog There was no significant difference between groups.
6 the expression of Ang-1 in renal cortex of rats in each group.
Compared with the normal group, the expression of Ang-1 mRNA in the model group and each treatment group was significantly increased (P 0.05); compared with the model group, the expression of Ang-1 mRNA in each treatment group was significantly increased (P 0.05); compared with the western medicine group and the traditional Chinese medicine group, the expression of Ang-1 mRNA in the traditional Chinese medicine plus Western medicine group was significantly increased (P 0.05); There was no significant difference between western medicine group and traditional Chinese medicine group (P0.05).
6.2 Immunohistochemical method was used to detect Ang-1 expression in podocytes of the normal group; compared with the normal group, the expression of Ang-1 in renal tissue of rats in each model group was significantly increased (P 0.05), especially in podocytes; compared with the model group, the expression of Ang-1 in each treatment group was significantly increased (P 0.05); The expression of Ang-1 was strongest in the kidney tissue of Chinese medicine plus Western medicine group, which was significantly different from that of Chinese medicine group and Western medicine group (P 0.05).
Conclusion:
1. Dispelling turbidity and detoxification, promoting blood circulation and dredging collaterals can reduce urinary protein excretion, increase serum total protein, albumin, reduce blood sugar and lipid, and delay the progress of DN.
2. The Chinese herbal medicine of relieving turbidity and detoxification, promoting blood circulation and dredging collaterals can effectively inhibit the proliferation of mesangial matrix, thickening of basement membrane, proliferation of endothelial cells and fusion of podocytes, and delay the progress of renal pathology.
3. The Chinese herbal medicine of eliminating turbidity and detoxification, promoting blood circulation and dredging collaterals can up-regulate the expression of Ang-1 in kidney tissue of DN rats, suggesting that this prescription can reduce the leakage of blood vessels, promote the maturation and stability of blood vessels, reduce the excretion of urinary protein, delay the progress of DN, and thus achieve the renal protection of DN rats.
The combination of Chinese herbal medicine and irbesartan in reducing albuminuria, elevating serum protein, lowering blood lipid and regulating Ang-1 expression is more effective than Chinese herbal medicine or western medicine.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R259;R277.5
本文編號：2200086
[Abstract]:Objective: To establish DM rat model by feeding high-sugar and high-fat diet and intraperitoneal injection of low-dose streptozotocin (STZ), and to observe the biochemical indexes, pathological morphological changes of kidney and kidney of experimental animals. The expression of Angiopoietin-1 (Ang-1) in cortical tissue was studied to explore the relationship between Ang-1 and DN, and the protective effect and possible mechanism of Chinese herbs on kidney of DN rats.
Methods: Eighty-five Sprague-Dauley male rats weighing 170 + 10g were fed adaptively for one week. After the blood glucose and urinary protein were tested negative, 15 rats were randomly selected as normal group (NG) and fed with normal diet. The other rats were fed with high glucose and high fat diet, and the other rats were injected with STZ 35mg/kg by abdominal cavity for 12 weeks. The rats were randomly divided into four groups: model group (MG), Chinese medicine group (ZG), western medicine group (XG), Chinese medicine plus Western medicine (TCM) and Chinese medicine plus Western medicine (WM). Group JG, Chinese medicine plus Western medicine group 16, the remaining 17 in each group. After modeling, the rats in the treatment group were given 4 ml/d of the drug solution once a day. The normal group and the model group were given the same volume of physiological saline, a total of 16 weeks. Free drinking water, eating, room temperature 22-25 C, relative humidity 40% - 60%, 12 hours alternate lighting. At the end of the sixteenth week, fasting for twelve hours, water was not forbidden. 24-hour urine was taken from rats in each group. 24-hour urine protein was measured. Weight was weighed, 10% chloral hydrate was anesthetized, abdominal aorta was taken for blood, right kidney was taken for weighing, hypertrophy index was calculated, kidney samples were taken, light microscope and electricity were used. The pathological morphological changes of kidney were observed under microscope. RNA was extracted from renal cortex of each group. The content of Ang-1 mRNA was detected by real-time fluorescence quantitative PCR and the expression of Ang-1 was observed by immunohistochemical staining.
Result:
1 Compared with the normal group, the body weight, kidney weight and hypertrophy index (kidney weight/body weight) of rats in each treatment group and model group decreased significantly (P 0.05). There was no significant difference in body weight between each treatment group and model group (P 0.05). Compared with the normal group, the hypertrophy index of each treatment group and model group increased significantly (P 0.05). There was no significant difference (P0.05). There was no significant difference in right kidney weight between each group (P0.05).
Compared with the normal group, the 24-hour urinary total protein (24-hour UTP) in the treatment group and the model group increased (P 0.05); compared with the model group, the UTP in the treatment group decreased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, the UTP in the traditional Chinese medicine plus Western medicine group decreased (P 0.05); There was no significant difference between the two groups (P0.05).
Compared with the normal group, the serum total protein (TP) and albumin (ALB) in the model group and the treatment group decreased (P 0.05); compared with the model group, TP and ALB in the treatment group increased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TP and ALB in the traditional Chinese medicine plus Western medicine group increased (P 0.05); Compared with the normal group, the total cholesterol (TC) and triglyceride (TG) in the model group and each treatment group were significantly higher (P 0.05); compared with the model group, TC and TG in each treatment group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG in the traditional Chinese medicine plus Western Medicine group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG were lower (P 0.05). Compared with no significant difference (P 0.05). Normal group, model group, the treatment group serum creatinine and urea nitrogen were not statistically significant (P 0.05).
Compared with normal group, HbA1c in model group and treatment group increased significantly (P 0.05); compared with model group and Western medicine group, HbA1c in traditional Chinese medicine plus Western medicine group and traditional Chinese medicine group decreased significantly (P 0.05); HbA1c in western medicine group was lower than that in model group, but there was no statistical significance (P 0.05); HbA1c in traditional Chinese medicine plus Western medicine group was lower than that in model group. Compared with the Chinese medicine group, there was no statistical significance (P0.05).
5 pathological observation of kidney
5.1 The glomeruli of normal rats were normal size, clear structure, no glomerular hypertrophy or atrophy, no basement membrane thickening and normal distribution area of mesangial matrix were observed under light microscope. There was no significant difference.
5.2 Electron microscopic observation showed that there were no abnormal changes in the glomerular endothelial cells in the normal group, such as the thickness of basement membrane was uniform, the structure was intact, the foot process did not fuse, and the absence of abnormal changes; the glomerular endothelial cells in the model group were obviously hyperplasia, prominent, the basement membrane was obviously thickened, and the foot process of podocytes fused extensively; the treatment groups also had different degrees of Patholog There was no significant difference between groups.
6 the expression of Ang-1 in renal cortex of rats in each group.
Compared with the normal group, the expression of Ang-1 mRNA in the model group and each treatment group was significantly increased (P 0.05); compared with the model group, the expression of Ang-1 mRNA in each treatment group was significantly increased (P 0.05); compared with the western medicine group and the traditional Chinese medicine group, the expression of Ang-1 mRNA in the traditional Chinese medicine plus Western medicine group was significantly increased (P 0.05); There was no significant difference between western medicine group and traditional Chinese medicine group (P0.05).
6.2 Immunohistochemical method was used to detect Ang-1 expression in podocytes of the normal group; compared with the normal group, the expression of Ang-1 in renal tissue of rats in each model group was significantly increased (P 0.05), especially in podocytes; compared with the model group, the expression of Ang-1 in each treatment group was significantly increased (P 0.05); The expression of Ang-1 was strongest in the kidney tissue of Chinese medicine plus Western medicine group, which was significantly different from that of Chinese medicine group and Western medicine group (P 0.05).
Conclusion:
1. Dispelling turbidity and detoxification, promoting blood circulation and dredging collaterals can reduce urinary protein excretion, increase serum total protein, albumin, reduce blood sugar and lipid, and delay the progress of DN.
2. The Chinese herbal medicine of relieving turbidity and detoxification, promoting blood circulation and dredging collaterals can effectively inhibit the proliferation of mesangial matrix, thickening of basement membrane, proliferation of endothelial cells and fusion of podocytes, and delay the progress of renal pathology.
3. The Chinese herbal medicine of eliminating turbidity and detoxification, promoting blood circulation and dredging collaterals can up-regulate the expression of Ang-1 in kidney tissue of DN rats, suggesting that this prescription can reduce the leakage of blood vessels, promote the maturation and stability of blood vessels, reduce the excretion of urinary protein, delay the progress of DN, and thus achieve the renal protection of DN rats.
The combination of Chinese herbal medicine and irbesartan in reducing albuminuria, elevating serum protein, lowering blood lipid and regulating Ang-1 expression is more effective than Chinese herbal medicine or western medicine.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R259;R277.5
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