乌索酸对高糖诱导人肾小球系膜细胞增殖肥大及细胞外基质分泌的影响

Effect of ursolic acid on hypertrophy and proliferation of renal glomerular mesangial cells and extracellular matrix production accumulation induced by high glucose

目的探讨乌索酸(UA)对高糖(HG)所致人系膜细胞(RMC)损伤的保护作用,保护机制是否通过抑制系膜细胞增殖、肥大及细胞外基质分泌而完成。方法取对数生长期细胞,分为空白对照组、模型组、对照组、实验A、B、C组。空白对照组予以5.5 mmol·L^(-1)葡萄糖培养;模型组予以30.0 mmol·L^(-1)葡萄糖培养;对照组予以5.5 mmol·L^(-1)葡萄糖+24.5 mmol·L^(-1)甘露醇培养;实验A、B、C组分别予以30.0 mmol·L^(-1)葡萄糖+0.5,1.0,2.0μmol·L^(-1)乌索酸培养。6组均给药48 h。用噻唑蓝(MTT)法检测细胞增殖,用PI单染流式细胞仪检测细胞周期及细胞肥大,用逆转录聚合酶链式反应法及免疫印迹法检测转化生长因子-β_1(TGF-β_1)、纤维连接蛋白(FN)mRNA及蛋白表达。结果给药48 h后,空白对照组、模型组、对照组、实验A、B、C组的细胞增殖分别为(3.66±0.32),(4.33±0.23),(3.50±0.22),(3.93±0.11),(3.59±0.06)和(0.67±0.16)。模型组和空白对照组的细胞增殖率比较,差异有统计学意义(P<0.05);实验B组、实验C组与模型组的细胞增殖率比较,差异均有统计学意义(均P<0.05);实验A组与空白对照组及对照组的细胞增殖率比较,差异均无统计学意义(均P>0.05);实验C组大部分系膜细胞已经死亡,设定实验B组为后续实验最终实验组。空白对照组、模型组、对照组、实验组的TGF-β_1mRNA分别(24.80±0.90),(29.89±1.06),(23.72±0.87)和(25.06±0.11),FN mRNA分别为(42.03±0.57),(54.55±1.20),(42.72±0.40)和(45.32±0.51),TGF-β_1蛋白分别为(54.92±3.28),(125.93±3.58),(25.11±0.51)和(101.23±7.55),FN蛋白分别为(46.44±7.90),(66.13±13.10),(34.98±5.52)和(28.70±5.70);模型组的上述指标与空白对照组及实验组比较,差异均有统计学意义(均P<0.05)。结论乌索酸通过抑制系膜细胞增殖、肥大及细胞外基质分泌,从而减轻高糖诱导的系膜细胞损伤。

Objective To explore the protective role of ursolic acid（ UA） on hypertrophy and proliferation of renal glomerrular mesangial cells and extracellular matrix production accumulation induced by high glucose was investigated. Methods The logaritumic growth plase cells were divided into blank control group（ 5. 5 mmol · L^-1 glucose）,model group（ 30. 0 mmol · L^-1 glucose）,control group（ 5. 5 mmol · L^-1 glucose ＋ 24. 5 mmol·L^-1 mannitol）,test A group（ 30. 0 mmol·L^-1 glucose ＋ 0. 5 μmol · L^-1 ursolic acid）, test B group（ 30. 0 mmol·L^-1 glucose ＋ 1. 0 μmol ·L^-1 ursolic acid）,and test C group（ 30. 0 mmol·L^-1 glucose ＋ 2. 0 μmol · L^-1 ursolic acid）,and treated for 48 h. Mesangial cell proliferation was detected by MTT method. Cell cycle and cell hypertrophy were tested by PI single dye flow cytometry. Reverse transcription-polymerase chain reaction and Western Blot were used to evaluate transforming growth factor-β1（ TGF-β1）,fibronectin（ FN） mRNA and protein expression. Results After 48 h,the cell proliferation rates of blank control group,model group,control group,test A group,test B group,and test C group were（ 3. 66 ± 0. 32）,（ 4. 33 ± 0. 23）,（ 3. 50 ± 0. 22）,（ 3. 93 ± 0. 11）,（ 3. 59 ± 0. 06） and（ 0. 67 ± 0. 16）. The comparison between the model group and the blank control group was statistically significant（ P〈0. 05）; the comparisons between the test B,C groups and the model group were statistically significant（ all P〈0. 05）; and there were no statistically significant differences between the test A group and the blank control and control groups（ all P〈0. 05）. Most of the cells in the test C group were dead,and the test B group was set up to be the final test group. The main indexes in blank control group,model group, control group and test group were compared： TGF-β1 mRNA were（ 24. 80 ± 0. 90）,（ 29. 89 ± 1. 06）,（ 23. 72 ± 0. 87） and（ 25. 06 ± 0. 11）,FN mRNA were（ 42. 03 ± 0. 57）,（ 54. 55 ± 1. 20）,（ 42. 72 ± 0. 40） and（ 45. 32 ± 0. 51）,TGF-β1 protein expression were（ 54. 92 ± 3. 28）,（ 125. 93 ± 3. 58）,（ 25. 11 ± 0. 51） and（ 101. 23 ± 7. 55）,FN protein expression were（ 46. 44 ± 7. 90）,（ 66. 13 ± 13. 10）,（ 34. 98 ± 5. 52） and（ 28. 70 ± 5. 70）. The comparisons between the model group and the blank control and test groups were statistically significant（ all P〈0. 05）. Conclusion UA can alleviate the injury ofmesangial cell induced by high glucose by inhibiting mesangial cell proliferation,hypertrophy and extracellular matrix deposition.