PPAR-γ在IgA肾病发生中的作用及其机理研究

发布时间：2018-05-09 14:43

本文选题：IgA肾病 + PPAR-γ　；参考：《复旦大学》2014年硕士论文

【摘要】：目的：PPAR-γ作为配体依赖的核因子,除了经典的调节糖、脂代谢作用外,其抗炎等作用近年受到广泛关注。本研究运用牛血清γ球蛋白(bovine gammaglobulin, BGG)建立IgA肾病大鼠模型,并观察PPAR-γ激动剂在本病发生中的作用及其与替米沙坦的协同治疗效果和PPAR-γ与TLR4的相互关系。方法：77只雄性Lewis大鼠,体重40-55g,饲养温度25℃,12h昼夜轮替,自由饮食,适应性喂养1周后随机分为5组：①对照组(Control, n=18):自由饮用6mmol/l的盐酸酸化水；②IgA肾病组(IgAN,n=28):自由饮用含0.1%BGG的6mmol/l盐酸酸化水,连续9周,其后连续3天尾静脉注射1mg BGG;③吡格列酮组(Pio,n=9)：将吡格列酮药片研磨成粉,每天取1g溶于90m1生理盐水制成悬浊液,用前摇匀,按3m1/只灌胃4周；④吡格列酮+替米沙坦组(Pio+ARB, n=10):将吡格列酮、替米沙坦药片研磨成粉,每天分别取1g和0.3g溶于120m1生理盐水制成悬浊液,用前摇匀,按3m1/只灌胃4周；⑤TLR4抑制剂组(TAK242,n=12)：将配置好的TLR4抑制剂TAK242脂肪乳剂按7.5ml/kg连续8天尾静脉注射。分别于第4周末、第6周末、第9周末测各组大鼠尿白蛋白/肌酐(ACR),显微镜下行尿红细胞计数,腹主动脉采血检测血肌酐和尿素氮,光镜下观察肾脏病理损害,用RT-PCR法检测各组肾组织PPAR-γ mRNA和TLR4 mRNA的表达,Western Blot方法检测肾组织PPAR-γ蛋白、TLR4蛋白和IL-1p蛋白的表达情况,免疫荧光检测肾小球IgA沉积情况。结果：①第9周末造模结束后,模型组尿白蛋白/肌酐明显高于对照组(4.45 ±1.33mg/mmol vs 2.89±0.96mg/mmol, P=0.05)。尿红细胞计数两组无明显差别。与对照组相比,模型组大鼠SCr、BUN、ALT、AST均无明显变化(均P0.05)模型组大鼠与对照组相比,肾脏组织系膜细胞及系膜基质增生明显,肾小球横截面细胞数明显增多(51±4个 vs 41±2个,P0.01),肾小球体积明显增大,少数肾小管上皮细胞肿胀,间质轻度炎性细胞浸润。对照组肾小球免疫荧光未见IgA沉积,模型组肾小球可见亮绿色团块状或絮状IgA沉积。②与对照组相比,IgA肾病组ACR明显升高(1.72±0.41mg/mmol vs 1.27±0.15mg/mmol),差异有统计学意义(P=0.013)；吡格列酮组尿ACR与对照组相比无明显差异(1.13±0.44mg/mmol vs 1.27±0.15mg/mmol,P=0.41)但显著低于IgA肾病组(1.13± 0.44mg/mmol vs 1.72±0.41mg/mmol,P=0.015)；吡格列酮+替米沙坦组ACR与对照组相比无明显差异(1.01±0.45mg/mmol vs 1.27±0.15mg/mmol,P=0.41)但显著低于IgA肾病组(1.01±0.45mg/mmol vs 1.72±0.41mg/mmol,P=0.00)。吡格列酮组与吡格列酮+替米沙坦组ACR无显著差异(1.01mg/mmol±0.45 vs 1.13 ±0.44mg/mmol,P=0.18)。与对照组相比,IgA肾病组肾小球横截面细胞数量明显增多(50±8个 vs 40±6个,P=0.03),肾小球体积明显增大,少数肾小管上皮细胞肿胀,间质轻度炎性细胞浸润；吡格列酮组与对照组相比肾小球横截面细胞数量无明显差异(46±6个vs40±6个,P0.05),吡格列酮组与IgA肾病组相比,肾小球横截面细胞数量减少未达到统计学意义(46±6个vs 50±8个,P=0.23),肾小管基本正常,间质未见明显炎性细胞浸润；吡格列酮+替米沙坦组与对照组相比肾小球横截面细胞数量无明显差异(41±4个 vs 40±6个,P0.05)但显著低于IgA肾病组(41±4个 vs 50±8个,P=0.03)。与吡格列酮组相比,减少未达到统计学差异(41±4个 vs 46±6个,P=0.08),肾小管正常,间质未见明显炎性细胞浸润。与对照组相比,IgA肾病组血清IL-1β水平和肾组织IL-1β蛋白的表达均明显升高(47.45±12.91pg/ml vs 34.49±12.09pg/ml,P=0.01;0.46 ±0.21 vs 0.27±0.10,P=0.04);吡格列酮组与对照组相比血清IL-1β水平和肾组织IL-1β蛋白的表达均无明显差异(39.06±17.92pg/ml vs 34.49±12.09pg/ml,P0.05；0.32±0.15 vs 0.27±0.10,P=0.45),与IgA肾病组相比,血清IL-1β水平和肾组织IL-1β蛋白的表达降低均无统计学差异(39.06±17.92pg/ml vs 47.45 ±12.91pg/ml,P=0.30；0.32±0.15 vs 0.46±0.21,P=0.19);吡格列酮+替米沙坦组与对照组相比,血清IL-1β的水平和肾组织IL-1β蛋白的表达基本相同(34.49 ±14.55pg/ml vs 34.49±12.09pg/ml,P0.05；0.28±0.09 vs 0.27±0.10,P=0.94)但均显著低于IgA肾病组(34.49±14.55pg/ml vs 47.45±12.91pg/ml,P=0.04;0.28 ±0.09 vs 0.46±0.21,P=0.04)。与吡格列酮组相比,吡格列酮+替米沙坦组血清IL-1β和肾组织IL-1β蛋白的表达水平均未达到统计学差异(34.49±14.55pg/ml vs 39.06±17.92pg/ml,P=0.59;0.28±0.09 vs 0.32±0.15,P=0.46)。与对照组相比,IgA肾病组大鼠肾组织PPAR-γ蛋白的表达明显升高(0.64±0.14vs0.42±0.04,P=0.03), PPAR-γmRNA的表达无显著差异(1.20±0.42 vs 0.98±0.03,P=0.39)；吡格列酮组与对照组相比,PPAR-y蛋白和mRNA的表达均明显增加(0.71±0.19vs 0.42±0.04,P=-0.03；1.58±0.20 vs 0.98±0.03,P=-0.001)。与IgA肾病组相比,吡格列酮组PPAR-y蛋白和mRNA的表达升高未达到统计学意义(0.71±0.19 vs0.64±0.14,P=0.44；1.58±0.20 vs 1.20±0.42,P=0.15)；吡格列酮+替米沙坦组与对照组、IgA肾病组相比,PPAR-y蛋白和mRNA的表达均无统计学差异(均P0.05)。与吡格列酮组相比,吡格列酮组+替米沙坦组PPAR-y蛋白和mRNA的表达均明显降低(0.56±0.08 vs 0.71±0.19,P=-0.047；1.02±0.17 vs 1.58±0.20,P=0.005)。③与IgA肾病组相比,TLR4抑制剂组ACR显著降低(1.13± 0.44mg/mmol vs 1.72±0.41mg/mmol, P=0.015),光镜下系膜细胞和系膜基质明显减少,肾小球横截面细胞数明显降低(35±3个 vs 45±3个,P0.01),血清IL-1p和肾组织IL-1p蛋白的表达无统计学意义(30.20±4.93pg/ml vs 32.99±5.64pg/ml,0.56±0.22 vs 0.63±0.17,均P0.05),PPAR-γ的蛋白表达明显增加(0.86±0.20 vs 0.65±0.13,P=0.03),PPAR-γ的mRNA表达无统计学差异(1.00±0.54vs0.87±0.35,P=0.63)。与IgA肾病组相比,吡格列酮组TLR4蛋白的表达明显降低(0.12±0.03 vs 0.21±0.05,P=0.001),TLR4 mRNA的表达降低未达到统计学差异(0.78±0.21vs0.95±0.09,P=0.13)。结论：①用口服BGG酸化水9周,尾静脉注射BGG 3天的方法可以建立轻-中度IgA肾病大鼠模型。②在IgA肾病动物模型中,PPAR-γ激动剂吡格列酮、血管紧张素受体阻断剂替米沙坦均可以降低炎症因子的水平,降低蛋白尿,抑制系膜细胞和系膜基质的增殖,改善IgA肾病。替米沙坦对吡格列酮的协同治疗效果不明显。③TLR4抑制剂TAL242可以改善IgA肾病,降低蛋白尿,改善系膜细胞和系膜基质的增殖,在IgA肾病动物体内,TLR4与PPAR-γ可以相互抑制,相互影响。
[Abstract]:Objective: PPAR- gamma, as a ligand dependent nuclear factor, has been widely concerned in recent years, in addition to the classical regulation of sugar and lipid metabolism, and its anti-inflammatory effects have been widely concerned in recent years. This study used bovine serum gamma globulin (bovine gammaglobulin, BGG) to establish a rat model of IgA nephropathy, and observed the role of PPAR- gamma agonist in the pathogenesis of this disease and its effect on telmisartan. Co therapy effect and the relationship between PPAR- gamma and TLR4. Methods: 77 male Lewis rats, weight 40-55g, feeding temperature 25 degrees, 12h circadian rotation, free diet, and free diet after 1 weeks of adaptive feeding, randomly divided into 5 groups: (Control, n=18): free drinking of 6mmol/l in hydrochloric acid water; and IgA nephrotic group (IgAN, n=28): free drinking 0. 1%BGG 6mmol/l hydrochloric acid water for 9 weeks, followed by 3 days of continuous injection of 1mg BGG in the tail vein; 3. Pioglitazone group (Pio, n=9): grind pioglitazone tablets into powder, take 1g dissolved in 90m1 physiological saline daily to make the suspension, shake well before using 3m1/ only for 4 weeks; 4. Pioglitazone + telmisartan group (Pio+ARB, n=10): pioglitazone, tiimi Sartan tablets were grinded into powder, and 1g and 0.3g dissolved in 120m1 saline solution were made every day respectively. Shake well before use and only gavage the stomach for 4 weeks by 3m1/; 5. TLR4 inhibitor group (TAK242, n=12): a configured TLR4 inhibitor, TAK242 fat emulsion, was injected into the tail vein for 8 days by 7.5ml/kg. The rats were measured at the fourth weekend, sixth weekend, and ninth weekend. Urinary albumin / creatinine (ACR), urinary red blood cell count under microscope, blood creatinine and urea nitrogen in abdominal aorta, the pathological damage of kidney was observed under light microscope. The expression of PPAR- gamma mRNA and TLR4 mRNA in renal tissue were detected by RT-PCR. The expression of PPAR- gamma protein, TLR4 protein and IL-1p protein in renal tissue was detected by Western Blot method, and the expression of TLR4 protein and IL-1p protein was detected and immunized. Results: after the end of the ninth weekend model, the urinary albumin / creatinine was significantly higher in the model group than in the control group (4.45 + 1.33mg/mmol vs 2.89 + 0.96mg/mmol, P=0.05). There was no significant difference between the two groups of urine red blood cell count. Compared with the control group, there was no significant change in SCr, BUN, ALT, and AST in the model group (all P0.05) model. Compared with the control group, the proliferation of mesangial cells and mesangial matrix in renal tissue was obvious, and the number of mesangial cross section cells increased significantly (51 + 4 vs 41 + 2, P0.01), the volume of glomeruli was obviously enlarged, a small number of renal tubular epithelial cells were swollen, and the interstitium was mild inflammatory cell infiltration. The glomerular immunofluorescence of the control group was not IgA deposit, and the model group kidney was not found. Compared with the control group, the ACR in the IgA nephropathy group increased significantly (1.72 + 0.41mg/mmol vs 1.27 + 0.15mg/mmol), and the difference was statistically significant (P=0.013). The urine ACR of the pioglitazone group was not significantly different from the control group (1.13 + 0.44mg/mmol vs 1.27 + 0.15mg/mmol, P=0.41) was significantly lower than that of the control group. The disease group (1.13 + 0.44mg/mmol vs 1.72 + 0.41mg/mmol, P=0.015) and pioglitazone + telmisartan group ACR had no significant difference compared with the control group (1.01 + 0.45mg/mmol vs 1.27 + 0.15mg/mmol, P=0.41), but significantly lower than the IgA nephropathy group (1.01 + 0.45mg/mmol vs 1.72 + 0, 0). There was a difference (1.01mg/mmol + 0.45 vs 1.13 + 0.44mg/mmol, P=0.18). Compared with the control group, the number of glomerular cross section cells in the IgA nephropathy group increased significantly (50 + 8 vs 40 + 6, P=0.03), the glomerular volume increased obviously, a few renal tubular epithelial cells were swollen and the interstitium was mild inflammatory cell infiltration, and the pioglitazone group was compared with the control group There was no significant difference in the number of cross section cells (46 + 6 vs40 + 6, P0.05). Compared with the IgA nephropathy group, the number of mesangial cross section cells decreased not statistically (46 + 6 vs 50 + 8, P=0.23). The renal tubules were basically normal and the interstitium was not obviously inflammatory cell infiltration; pioglitazone + telmisartan group was compared with the control group. There was no significant difference in the number of cells in the cross section (41 + 4 vs 40 + 6, P0.05), but significantly lower than the IgA nephropathy group (41 + 4 vs 50 + 8, P=0.03). Compared with the pioglitazone group, the decrease was not statistically significant (41 + 4 vs 46 + 6, P=0.08), renal tubules were normal, and no obvious inflammatory cell infiltration was found in the interstitium. Compared with the control group, the blood of IgA nephropathy group was compared with the control group. The level of IL-1 beta and the expression of IL-1 beta protein in the renal tissue were significantly increased (47.45 + 12.91pg/ml vs 34.49 + 12.09pg/ml, P=0.01; 0.46 + 0.21 vs 0.27 + 0.10, P=0.04), and there was no significant difference between the serum IL-1 beta level and the expression of IL-1 beta protein in the renal tissue compared with the control group (39.06 + 39.06 + 0.10). 0.32 0.15 vs 0.27 + 0.10, P=0.45), compared with the IgA nephropathy group, there was no significant difference in the level of serum IL-1 beta and the expression of IL-1 beta protein in renal tissue (39.06 + 17.92pg/ml vs 47.45 + 12.91pg/ml, P=0.30; 0.32 + 0.15 vs 0.46 + 0.21, P=0.19); and the level of the beta and renal tissue of the pioglitazone + telmisartan group The expression of protein was basically the same (34.49 + 14.55pg/ml vs 34.49 + 12.09pg/ml, P0.05; 0.28 + 0.09 vs 0.27 + 0.10, P=0.94), but significantly lower than that of IgA nephropathy group (34.49 + 14.55pg/ml vs 47.45 + 12.91pg/ml, P=0.04; 0.28 + 0.09 vs 0.46 + 0.21). The expression level of 1 beta protein did not reach statistical difference (34.49 + 14.55pg/ml vs 39.06 + 17.92pg/ml, P=0.59; 0.28 + 0.09 vs 0.32 + 0.15, P=0.46). Compared with the control group, the expression of PPAR- gamma protein in renal tissue of IgA nephropathy rats increased significantly (0.64 + 0.14vs0.42 + 0.04, P= 0.03), PPAR- gamma mRNA was no significant difference (1.20 + 0.42 0.98 0.98) The expression of PPAR-y protein and mRNA increased significantly (0.71 + 0.19vs 0.42 + 0.04, P=-0.03, 1.58 + 0.20 vs 0.98 + 0.03, P=-0.001) in the pioglitazone group (1.58 + 0.20 vs 0.98 +, P=-0.001). The increase of the expression of PPAR-y protein and mRNA in the pioglitazone group was not statistically significant (0.71 + 0.19 vs0.64 + 0.14, P=0.44; 1.58). 0.20 vs 1.20 + 0.42, P=0.15), the expression of PPAR-y protein and mRNA was not significantly different between the pioglitazone + telmisartan group and the control group (P0.05). Compared with the pioglitazone group, the expression of PPAR-y protein and mRNA in the pioglitazone group was significantly decreased (0.56 + 0.08 vs 0.71 + 0.19, P=-0.047; 1.02 + 0.17 vs. 1.58 + 0.20, P=0.005). Compared with IgA nephropathy group, ACR in TLR4 inhibitor group was significantly decreased (1.13 + 0.44mg/mmol vs 1.72 + 0.41mg/mmol, P=0.015), mesangial cells and mesangial matrix decreased significantly under light microscope, and the number of glomerular cross section cells decreased significantly (35 + 3 vs 45 + 3, P0.01). The expression of serum IL-1p and renal tissue IL-1p protein was not statistically significant Significance (30.20 + 4.93pg/ml vs 32.99 + 5.64pg/ml, 0.56 + 0.22 vs 0.63 + 0.17, P0.05), the protein expression of PPAR- gamma increased significantly (0.86 + 0.20 vs 0.65 + 0.13, P=0.03), and PPAR- gamma mRNA expression was not statistically significant (1 + 0.54vs0.87 + 0.35, P=0.63). 0.21 + 0.05, P=0.001), the expression of TLR4 mRNA decreased not statistically (0.78 + 0.21vs0.95 + 0.09, P=0.13). Conclusion: (1) oral BGG acidified water for 9 weeks and 3 days of BGG in the tail vein can establish a rat model of mild to moderate IgA nephropathy. (2) in the animal model of IgA nephropathy, PPAR- gamma agonist, pioglitazone, angiotensin receptor blocker Telmisartan can reduce the level of inflammatory factors, reduce proteinuria, inhibit the proliferation of mesangial cells and mesangial matrix, and improve IgA nephropathy. Telmisartan's synergistic effect on pioglitazone is not obvious. (3) TLR4 inhibitor TAL242 can improve IgA nephropathy, reduce proteinuria, improve the proliferation of mesangial cells and mesangial matrix, in IgA In nephrotic animals, TLR4 and PPAR- gamma can inhibit each other and interact with each other.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

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