CAJ | 학술논문

目的观察免疫抑制剂环孢素A(CsA)对糖尿病大鼠肾脏基质金属蛋白酶(MMP)表达的影响.方法健康雄性SD大鼠74只,平均体重250 g,采用数字表法随机分为正常对照组(n=10)、单纯糖尿病组(n=8)、胰岛素治疗组(n=9)、造模前低剂量CsA治疗组(n=9;造模前1周皮下注射1μg·g-1·d-1 CsA)、造模前中剂量CsA治疗组(n=8;造模前1周皮下注射4μg·g-1·d-1CsA)、造模前高剂量CsA治疗组(n=8;造模前1周皮下注射8μg·g-1·d-1CsA)、造模后低剂量CsA治疗组(n=8;造模后1周皮下注射1μg·g-1·d-1CsA)、造模后中剂量CsA治疗组(n=7;造模后1周皮下注射4μg·g-1·d-1 CsA)和造模后高剂量CsA治疗组(n=7;造模后1周皮下注射8μg·g-1·d-1 CsA).8周后处死动物,采用免疫组织化学法、逆转录-聚合酶链反应和Western blot检测肾脏MMP-2、MMP-9 mRNA和蛋白表达水平.采用单因素方差分析和直线相关分析进行统计学分析.结果 8周时,单纯糖尿病组24 h尿微量蛋白显著高于正常对照组[分别为(5.80±3.23)、(1.24±0.21)mg/24 h,F=4.229,P＜0.01];各CsA治疗组24 h尿微量蛋白不同程度降低,与正常对照组比较差异无统计学意义.单纯糖尿病组肾小管上皮细胞和肾小球系膜基质MMP-2、MMP-9mRNA(分别为2.22±0.08、2.55±1.10)和蛋白表达(分别为3.1±1.5、2.8±1.0)显著高于正常对照组(MMP-2 mRNA:0.70±0.26,F=6.031;MMP-9 mRNA:0.37±0.24,F=5.193;MMP-2蛋白:1.0±0.0,F=7.532;MMP-9蛋白:1.0±0.0,F=6.100;均P＜0.01).胰岛素治疗对其表达无影响,但CsA干预可下调MMP-2、MMP-9 mRNA和蛋白的异常表达.MMP-2 mRNA与蛋白表达呈显著正相关(r=0.618,P＜0.01),而MMP-9 mRNA与蛋白表达未见相关性(r=0.420,P＞0.05).结论 CsA可减少糖尿病大鼠肾脏MMP-2和MMP-9基因转录和蛋白表达,改善细胞外基质代谢紊乱,可能具有延缓糖尿病肾病发生的作用.
목적 관찰면역억제제배포소A(CsA)대당뇨병대서신장기질금속단백매(MMP)표체적영향.방법 건강웅성SD대서74지,평균체중250 g,채용수자표법수궤분위정상대조조(n=10)、단순당뇨병조(n=8)、이도소치료조(n=9)、조모전저제량CsA치료조(n=9;조모전1주피하주사1μg·g-1·d-1 CsA)、조모전중제량CsA치료조(n=8;조모전1주피하주사4μg·g-1·d-1CsA)、조모전고제량CsA치료조(n=8;조모전1주피하주사8μg·g-1·d-1CsA)、조모후저제량CsA치료조(n=8;조모후1주피하주사1μg·g-1·d-1CsA)、조모후중제량CsA치료조(n=7;조모후1주피하주사4μg·g-1·d-1 CsA)화조모후고제량CsA치료조(n=7;조모후1주피하주사8μg·g-1·d-1 CsA).8주후처사동물,채용면역조직화학법、역전록-취합매련반응화Western blot검측신장MMP-2、MMP-9 mRNA화단백표체수평.채용단인소방차분석화직선상관분석진행통계학분석.결과 8주시,단순당뇨병조24 h뇨미량단백현저고우정상대조조[분별위(5.80±3.23)、(1.24±0.21)mg/24 h,F=4.229,P＜0.01];각CsA치료조24 h뇨미량단백불동정도강저,여정상대조조비교차이무통계학의의.단순당뇨병조신소관상피세포화신소구계막기질MMP-2、MMP-9mRNA(분별위2.22±0.08、2.55±1.10)화단백표체(분별위3.1±1.5、2.8±1.0)현저고우정상대조조(MMP-2 mRNA:0.70±0.26,F=6.031;MMP-9 mRNA:0.37±0.24,F=5.193;MMP-2단백:1.0±0.0,F=7.532;MMP-9단백:1.0±0.0,F=6.100;균P＜0.01).이도소치료대기표체무영향,단CsA간예가하조MMP-2、MMP-9 mRNA화단백적이상표체.MMP-2 mRNA여단백표체정현저정상관(r=0.618,P＜0.01),이MMP-9 mRNA여단백표체미견상관성(r=0.420,P＞0.05).결론 CsA가감소당뇨병대서신장MMP-2화MMP-9기인전록화단백표체,개선세포외기질대사문란,가능구유연완당뇨병신병발생적작용.
Objective To investigate the expression of matrix metalloproteinase ( MMP)-2 and MMP-9 in the kidney of streptozocin (STZ)-induced diabetic rats and the effects of cyclosporine A (CsA)on their expression. Methods STZ-induced diabetic rats were randomly assigned to the non-treatment group (n =8), insulin intervention group (n=9), 1 μg · g-1 · d-1 CsA before modeling group(n=9) ,4 μg ·g-1 · d-1 CsA before modeling group(n=8) ,8 μg · g-1 · d-1 CsA before modeling group(n=8) ,1 μg ·g-1· d-1 CsA post modeling group ( n=8 ), 4 μg · g · d-1 CsA post modeling group ( n = 7 ), or 8 μg ·g-1 · d-1 CsA post modeling group (n=7). Another 10 normal rats were used as controls. The renal expression of MMP-2 and MMP-9 mRNA and protein were determined by immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The data were analyzed by one-factor analysis of variance and rectilinear correlation analysis. Results At 8 weeks, the 24-h urine microalbuminuria in the diabetic group was significantly higher than that in the normal control group ( (5. 80±3.23) and ( 1.24 ± 0. 21 ) mg/24 h, respectively; F = 4. 229, P ＜ 0. 01 ). The 24-h urine microalbuminuria was decreased after the treatment of CsA. The expression of MMP-2 and MMP-9 mRNA (MMP-2: 2.22±0.08; MMP-9:2.55 ±1.10) and protein (MMP-2: 3. 1 ±1.5; MMP-9:2.8 ±1.0) in the mesengium and epithelium of the renal tubular of the diabetic group was significantly higher than that of the normal control group (MMP-2 mRNA: 0.70 ±0.26, F=6.031; MMP-9 mRNA: 0.37 ±0.24, F=5. 193; MMP-2 protein: 1.0±0.0, F=7.532; MMP-9 protein: 1.0±0.0, F=6. 100; all P＜0.01).There was no preventive effect of insulin on the expression of MMP-2 and MMP-9. However, following the intervention of CsA, all of the abnormal expression could be downregulated. There was a positive relationship between the mRNA and protein expression of MMP-2 (r =0. 618, P ＜0. 01 ), while no relationship existed between those of MMP-9 (r = 0. 420, P ＞ 0. 05). Conclusion The abnormal expression of MMP-2 and MMP-9 in the diabetic kidney could be downregulated by CsA. Diabetic nephropahty might be effectively prevented by immunosuppressive agents.