α-SMA在代谢综合征大鼠模型肾脏的表达及其干预研究

发布时间：2018-05-05 17:23

本文选题：代谢综合征 + 早期肾损害　；参考：《广州医学院》2011年硕士论文

【摘要】：研究目的: 1.构建代谢综合征大鼠模型。 2.探讨代谢综合征大鼠模型早期肾脏α-SMA的表达。 3.RAAS拮抗药物及降脂药物分别干预代谢综合征大鼠模型后早期肾脏α-SMA的表达变化。研究方法: 1.动物模型的构建选择3周龄SPF级雄性Wistar大鼠36只,随机分为正常饲料(含0.5%NaCl)喂养组(normal sodium, NS,n=12)、高盐高脂饲料(4%NaCl+49%Fat)喂养组(high sodium and fat, HSF,n=24)。饲养12周时,每组随机取6只处死(为NS-1组和HSF-1组),称体重,测量颈总动脉有创血压、血脂、血糖及血胰岛素浓度;根据稳态胰岛素抵抗指数(HOMA-IR index:fasting insulin X fasting glucose/22.5 [1])评价是否存在胰岛素抵抗,根据代谢综合征的诊断标准及统计学方法进行组间比较评价模型是否构建成功。 2.药物干预 NS组(n=6)继续以正常饲料喂养,为NS-2组。HSF组随机分为3组:一组继续单以高盐高脂饲料喂养(HSF-2组,n=6),其余动物在喂养高盐高脂饲料同时分别加入RAAS拮抗药替米沙坦(HSF+T,n=6,10mg/kg/d)及降脂药物辛伐他汀(HSF+S,n=6,10mg/kg/d)干预。至30周时结束实验,观察药物干预情况下体重、颈总动脉有创血压、血脂、血糖、血胰岛素浓度、计算稳态胰岛素抵抗指数。 3.代谢综合征大鼠肾组织形态学观察及免疫组化a-SMA表达建立以高盐高脂诱导的代谢综合征动物模型,饲养12周处死的大鼠及继续饲养18周到30周后的大鼠分离出肾脏组织,HE染色、Masson染色(20×10倍)使用Leica DM4000显微镜自带的显微成像系统在相同的条件下采集数码图片。免疫组化切片在Leica DM4000显微镜下按统一条件采集,每个组取3个连续切片,每个切片在10×20倍视野下随机取5个非重叠视野,避开切片边缘和组织破损区,拍照,用Image Pro Plus 6.0软件分析每张照片中阳性染色面积,选择常用的参数阳性面积(Area)的百分比代表a-SMA表达量。5个比值的平均值作为每个切片肾组织区域α-SMA的阳性率(%)。研究结果: 1.体重变化 1.1高盐高脂致体重明显增加:实验12周,HSF-1组比NS-1组体重明显增加(326.45±6.84g比303.3±9.13g ,P0.01);但实验30周时,NS-2组(434.73±10.19g),HSF-2组(458.83±12.85g),两组比较,P0.05。结果提示高盐高脂饮食可在短期内增加体重。 1.2 RAAS拮抗药物对体重改变不明显:使用替米沙坦干预18周后测量体重,HSF+T组与NS-2及HSF-2组差异无统计学意义(P0.05),结果提示:使用RAAS拮抗药物对体重无影响。 1.3辛伐他汀对体重改变不明显:使用辛伐他汀治疗18周后,HSF+S体重与NS-2及HSF-2组无差异(P0.05)。结果提示:使用辛伐他汀干预对体重无影响。 2.颈总动脉有创血压 2.1高盐高脂导致收缩压升高:实验12周,HSF-1组(189.43±14.65mmHg)明显高于NS-1组(153.65±20.53mmHg ,P0.01),同样,实验30周时HSF-2组(190.03±14.48mmHg)收缩压明显高于NS-2组(161.00±19.49mmHg,P0.05)。结果提示长期高盐高脂饲料喂养正常Wistar大鼠可诱导形成高血压模型。 2.2替米沙坦有效降低收缩压:给予替米沙坦干预18周后检测收缩压发现,HSF+T组收缩压(161.95±15.62mmHg)较HSF-2组低(P0.05);HSF+T组与NS-2组间无差异(P0.05)。结果提示RAAS拮抗药能有效地降低由高盐高脂诱导的高血压。 2.3辛伐他汀对血压影响不明显:给予辛伐他汀干预18周后检测收缩压发现,HSF+S组收缩压(180.36±9.94mmHg)与HSF-2组比较无明显变化(P0.05),均高于NS-2组(P0.05)。 3.血脂改变 3.1实验12周,TG、TC及LDL在HSF-1组分别为0.97±0.54mmol/L、1.51±0.24mmol/L、0.90±0.23mmol/L,明显高于NS-1组(TG、TC、LDL分别为0.31±0.17mmol/L、0.88±0.12mmol/L、0.53±0.23mmol/L,P0.05);两组HDL相近(0.83±0.17mmol/L对0.89±0.13mmol/L,P0.05)。 3.2实验30周: 3.2.1 HSF-2组TG及LDL高于NS-2组(TG 2.96±0.56mmol/L对1.10±0.24mmol/L,LDL 2.60±0.51mmol/L对0.81±0.10mmol/L,P0.05)。 3.2.2替米沙坦干预后血脂改变不明显:HSF+T组TG及LDL与HSF-2组比较无差异,P0.05;HSF+T组高于NS-2组,P0.05。 3.2.3辛伐他汀干预后TG及LDL降低:HSF+S组(TG 1.51±0.26mmol/L,LDL 1.17±0.26mmol/L)低于HSF-2组,P0.05;两组TC及HDL差异无统计学意义,P0.05。 4.血糖(FBG)、胰岛素(INS)及稳态胰岛素抵抗指数(HOMA-IR)的改变:实验12周,HSF-1组FBG和HOMA-IR明显高于NS-1组(8.01±1.52mmol/L对6.86±1.18mmol/L, 2.39±0.36对1.84±0.41,P0.01)。INS水平两组比较差异无统计学意义,P0.05。实验30周,HSF-2组HOMA-IR高于NS-2组,HSF-2组、HSF-T组及HSF-S组两两比较结果无统计学意义。结果表明:以高盐高脂饲料喂养正常Wistar大鼠可以导致胰岛素抵抗。综上,高盐高脂能够诱导大鼠在12周后出现典型的代谢综合征改变:①体重增加:HSF-1组较NS-1组大鼠体重明显增加(BW:326.45±6.84g vs.303.3±9.13g,P0.01); ②高血压:HSF-1组较NS-1组大鼠血压明显增高(BP:189.43±14.65 mmHg vs 153.65±20.53 mmHg,P0.01); ③血脂异常:HSF-1组较NS-1组大鼠血脂明显增高(TC:1.51±0.24mmol/L vs. 0.88±0.12mmol/L, TG:0.97±0.54mmol/L vs. 0.31±0.17mmol/L, LDL-C: 0.90±0.23mmol/L vs. 0.53±0.23mmol/L,三者均P0.05,但HDL-C:0.83±0.17mmol/L vs. 0.89±0.13mmol/L,P0.05); ④胰岛素抵抗:HSF-1组大鼠胰岛素抵抗指数较NS-1组明显升高(HOMA-IR index:2.39±0.36 vs. 1.84±0.41,P0.01)。 5.肾组织形态学检测 5.1 HE染色切片光镜下观察:NS-1、NS-2组无明显病理改变,表现为肾小球结构无改变,肾小管上皮细胞胞质丰富、小管管腔小、间质面积少。HSF-1组可见肾小球系膜轻度节段性增生、基底膜稍增厚,部分肾小管上皮细胞空泡变性、部分管腔扩张。HSF-2组病变较HSF-1组增加。HSF-T组、HSF-S组组织形态学类似HSF-2组。提示HSF组肾脏有轻度病理改变。 5.2 Masson染色光镜下观察:所见病理改变同HE染色,胶原纤维被染成兰色,细胞成红色,NS组Masson染色未见明显改变,肾小管基膜处见线状分布的兰染的胶原组织,HSF组胶原纤维较NS组稍增多。 6.肾脏组织免疫组化检测a-SMA表达: 6.1实验12周,HSF-1组肾脏a-SMA表达面积阳性百分比(0.03±0.005)稍高于NS-1组(0.015±0.008),但两组比较,P0.05;提示12周时HSF组肾脏a-SMA表达增高。 6.2实验30周,HSF-2组(0.101±0.003)、HSF+T组(0.038±0.006)、HSF+S组(0.082±0.003)、NS-2组(0.02±0.004),四组比较,肾脏a-SMA表达面积阳性百分比存在差异(P0.01),组间比较发现HSF-2组、HSF+S组肾脏a-SMA表达面积阳性百分比明显高于HSF+T组、NS-2组,而HSF-2组与HSF+S组,HSF+T组与NS-2组之间未发现明显差异,HSF+T组与HSF-2组比较(0.038±0.006 vs 0.101±0.003),P0.05。提示30周时,HSF组肾脏a-SMA表达增高;替米沙坦治疗能有效减少a-SMA的表达;辛伐他汀对a-SMA表达影响不大。结论: 1.高盐高脂饮食喂养12周大鼠出现代谢综合征,此时肾组织呈轻度肾损害,表现为肾小球系膜轻度节段性增生、基底膜稍增厚,部分肾小管上皮细胞空泡变性、部分管腔扩张;α-SMA在大鼠肾脏的表达增加,并随着饲养时间增加a-SMA的表达增高。 2.RAAS拮抗药物能有效降低代谢综合征大鼠肾脏α-SMA的表达,而降脂药物降低肾脏α-SMA的表达不明显。
[Abstract]:The purpose of the study is:
1. the rat model of metabolic syndrome was constructed.
2. to investigate the expression of renal -SMA in the early stage of metabolic syndrome in rats.
3.RAAS antagonist and lipid-lowering drugs interfere the expression of alpha -SMA in the early stage of metabolic syndrome in rats.
Research methods:
The construction of 1. animal models
36 SPF grade male Wistar rats of 3 weeks old were randomly divided into normal feed (0.5%NaCl) feeding group (normal sodium, NS, n=12), high salt and high fat feed (4%NaCl+49%Fat) feeding group (high sodium and fat). After 12 weeks, each group was randomly selected and weighed to measure the blood pressure and blood lipid of the common carotid artery. The blood glucose and blood insulin concentration; the evaluation of insulin resistance based on the HOMA-IR index:fasting insulin X fasting glucose/22.5 [1], and the success of a comparative evaluation model based on the diagnostic criteria and statistical methods of metabolic syndrome.
2. drug intervention
Group NS (n=6) continued to feed in normal diet for group NS-2.HSF randomly divided into 3 groups: one group continued to feed with high salt and high fat diet (HSF-2 group, n=6), and the other animals were fed with RAAS antagonist telmisartan (HSF+T, n=6,10mg/kg/d) and lipid-lowering drug simvastatin (HSF+S, n=6,10mg/kg/d) at the same time. To 30 weeks, the other animals were fed with high salt and high fat diet. At the end of the experiment, the body weight, blood pressure, blood fat, blood sugar and blood insulin concentration of the common carotid artery were observed, and the steady-state insulin resistance index was calculated.
The renal histomorphology of 3. metabolic syndrome rats and the expression of immunohistochemical a-SMA were established with high salt and high fat induced metabolic syndrome animal model. The rats were fed for 12 weeks and the rats were kept for 18 weeks to 30 weeks. The rats were separated from the kidney tissue, HE staining, and Masson staining (20 x 10 times) using the Leica DM4000 microscope self-contained microscopic imaging. The system collected digital pictures under the same conditions. The immunohistochemical slices were collected under the unified conditions under the Leica DM4000 microscope. Each group was taken 3 consecutive slices. Each slice was randomly selected from 10 x 20 times to take 5 non overlapping fields of vision, avoiding the edge of the slice and the damaged area of the tissue, taking photos and analyzing each photo with Image Pro Plus 6 software. The percentage of the positive area (Area) of the commonly used parameters represented the average value of the.5 ratio of the a-SMA expression as the positive rate (%) of the alpha -SMA in each section of the renal tissue.
The results of the study:
1. body weight change
1.1 high salt and high fat induced weight increased significantly: the body weight of group HSF-1 was significantly increased (326.45 + 6.84g than 303.3 + 9.13g, P0.01) at 12 weeks, but at 30 weeks, group NS-2 (434.73 + 10.19g), HSF-2 group (458.83 + 12.85g), two groups compared, P0.05. results suggested that high salt and high fat diet could increase weight in the short term.
1.2 RAAS antagonists did not have significant weight change: the use of telmisartan after 18 weeks of intervention to measure weight, HSF+T group and NS-2 and HSF-2 group had no statistically significant difference (P0.05), the results showed that the use of RAAS antagonists had no effect on weight.
1.3 simvastatin did not have significant weight change: there was no difference between the HSF+S body weight and the NS-2 and HSF-2 groups after 18 weeks of simvastatin treatment (P0.05). The results showed that the use of simvastatin did not affect the weight.
2. common carotid artery to create blood pressure
2.1 high salt and high fat led to the increase of systolic pressure: 12 weeks in the experiment, group HSF-1 (189.43 + 14.65mmHg) was significantly higher than that in group NS-1 (153.65 + 20.53mmHg, P0.01). Also, the systolic pressure of group HSF-2 (190.03 + 14.48mmHg) was significantly higher than that of NS-2 group (161 + 19.49mmHg, P0.05) at 30 weeks. Hypertension model.
2.2 telmisartan reduced the systolic pressure effectively: after the intervention of telmisartan for 18 weeks, the detection of systolic blood pressure showed that the systolic pressure of HSF+T group (161.95 + 15.62mmHg) was lower than that of the HSF-2 group (P0.05), and there was no difference between group HSF+T and NS-2 (P0.05). The results suggested that the RAAS antagonist could effectively reduce the high blood pressure induced by high salt and high fat.
2.3 the effect of simvastatin on blood pressure was not obvious: after the intervention of simvastatin for 18 weeks, the detection of systolic blood pressure showed that the systolic pressure (180.36 + 9.94mmHg) in group HSF+S was not significantly different from that in the HSF-2 group (P0.05), which was higher than that in the NS-2 group (P0.05).
3. blood lipid changes
3.1 after 12 weeks of experiment, TG, TC and LDL were 0.97 + 0.54mmol/L, 1.51 + 0.24mmol/L and 0.90 + 0.23mmol/L respectively in group HSF-1 (TG, TC, LDL were 0.31 + 0.17mmol/L, 0.88 and 0.53, respectively), and the two groups were close (0.83 + 0.89 +).
3.2 the experiment was 30 weeks.
TG and LDL in group 3.2.1 HSF-2 were higher than those in group NS-2 (TG 2.96 + 0.56mmol/L to 1.10 + 0.24mmol/L, LDL 2.60 + 0.51mmol/L to 0.81 + 0.10mmol/L, P0.05).
After 3.2.2 telmisartan intervention, blood lipid did not change significantly: HSF+T group TG and LDL had no difference compared with HSF-2 group, P0.05 and HSF+T group were higher than NS-2 group, P0.05.
TG and LDL decreased after 3.2.3 simvastatin: group HSF+S (TG 1.51 + 0.26mmol/L, LDL 1.17 + 0.26mmol/L) were lower than group HSF-2, P0.05, and there was no statistical difference between two groups of TC and HDL.
4. blood glucose (FBG), insulin (INS) and homeostasis insulin resistance index (HOMA-IR) changes: 12 weeks of experiment, FBG and HOMA-IR in group HSF-1 were significantly higher than those in group NS-1 (8.01 + 1.52mmol/L against 6.86 + 1.18mmol/L, 2.39 + 0.36 against 1.84 + 0.41, P0.01).INS level two group had no statistical difference, 30 weeks of P0.05. experiment. There was no significant difference between group HSF-T and group HSF-S in 22. The results showed that normal Wistar rats fed with high salt and high fat diet could lead to insulin resistance.
To sum up, high salt and high fat could induce a typical change of metabolic syndrome in rats after 12 weeks: (1) weight gain: the body weight of group HSF-1 was significantly higher than that in group NS-1 (BW:326.45 + 6.84g vs.303.3 + 9.13g, P0.01).
Hypertension: the blood pressure of HSF-1 group was significantly higher than that of NS-1 group (BP:189.43 + 14.65 mmHg vs 153.65 + 20.53 mmHg, P0.01).
Dyslipidemia: the blood lipid of group HSF-1 was significantly higher than that in group NS-1 (TC:1.51 + 0.24mmol/L vs. 0.88 + 0.12mmol/L, TG:0.97 + 0.54mmol/L vs. 0.31 + 0.17mmol/L, LDL-C: 0.90 + 0.23mmol/L 0.53).
Insulin resistance: the insulin resistance index of HSF-1 group was significantly higher than that of NS-1 group (HOMA-IR index:2.39 + 0.36 vs. 1.84 + 0.41, P0.01).
5. renal histomorphological detection
5.1 HE staining section under light microscope: NS-1, group NS-2 had no obvious pathological changes, showing no changes in the glomerular structure, abundant cytoplasm of renal tubular epithelial cells, small tubules and small tubules, low interstitial area in group.HSF-1, which showed mild segmental hyperplasia of mesangial membrane, slightly thickening of basal membrane, vacuolation of some renal tubular epithelial cells, and partial lumen dilatation of.HS In group F-2, the lesions in group.HSF-T were increased compared with those in group HSF-1, while those in group HSF-S were similar to those in group HSF-2, suggesting that there were mild pathological changes in the HSF group.
5.2 Masson staining under light microscope: the pathological changes were stained with HE, the collagen fibers were dyed blue, the cells were red, and the Masson staining in the NS group did not change obviously. The collagenous tissue in the basement membrane of the renal tubule was linearly distributed, and the collagen fibers in the HSF group were slightly more than those in the NS group.
6. the expression of a-SMA in renal tissue was detected by immunohistochemistry.
6.1 after 12 weeks of experiment, the positive percentage of a-SMA expression area in group HSF-1 (0.03 + 0.005) was slightly higher than that in group NS-1 (0.015 + 0.008), but the two groups were compared, P0.05. It was suggested that the expression of a-SMA in the kidney of HSF group was increased at 12 weeks.
6.2 and 30 weeks, group HSF-2 (0.101 + 0.003), group HSF+T (0.038 + 0.006), group HSF+S (0.082 + 0.003), group NS-2 (0.02 + 0.004), compared with group four, the positive percentage of a-SMA expression area in kidney was different (P0.01). The percentage of HSF-2 in HSF-2 group was found in group HSF+S, and the percentage of a-SMA expression area in HSF+S group was significantly higher than that in HSF+T group and NS-2 group. There was no significant difference between group HSF+T and group NS-2. Group HSF+T was compared with group HSF-2 (0.038 + 0.006 vs 0.101 + 0.003), P0.05. suggested that the expression of a-SMA in group HSF increased at 30 weeks, and telmisartan could effectively reduce the expression of a-SMA; simvastatin had little effect on the expression of a-SMA.
Conclusion:
1. the rats fed with high salt and high fat diet for 12 weeks had metabolic syndrome. At this time, the renal tissue showed mild renal damage, characterized by mild segmental hyperplasia of mesangial membrane, slightly thickening of the basement membrane, vacuolation of some tubular epithelial cells and partial dilatation of the lumen. The expression of alpha -SMA in the kidney of rats increased and increased with the increase of the expression of a-SMA in the feeding time. High.
2.RAAS antagonist can effectively reduce the expression of alpha -SMA in the kidney of rats with metabolic syndrome, while the decrease of renal -SMA expression by lipid-lowering drugs is not obvious.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R589;R-332

【参考文献】