黄芪对白介素1β诱导的大鼠系膜细胞增殖和细胞外基质产生的影响

Effect of Astragalus Membranaceus on Interleukin-1βinduced mesangial cells proliferation and cellular matrix production

目的:观察黄芪(Astragalus Membranaceus,AM)注射剂对5/6肾切除大鼠模型的疗效,及体外对系膜细胞(Mesangial cells,MCs)增殖和细胞外基质(Intracellular Matrix,ECM)产生的影响。方法:建立5/6肾切除大鼠模型后,将大鼠分为四组:对照组、未治疗组、低剂量/高剂量AM治疗组。观察比较16周后各组大鼠肾功能和尿蛋白排泄量情况。体外以白介素1β(IL-1β)作为刺激物,诱导MCs增殖,给予不同剂量AM干预。二苯基四氮唑溴盐(MTT)法检测MCs增殖情况,流式细胞仪检测细胞周期,酶联免疫吸附(ELISA)法检测细胞上清中纤连蛋白(fibronectin)含量,RT-PCR检测细胞p38 mRNA表达,Western免疫印迹法检测胶原Ⅳ、总p38MAPK、磷酸化p38MAPK、磷酸化MKK3/MKK6、磷酸化MKK4等蛋白水平。结果:在体外AM可显著抑制IL-1β诱导的MCs增殖和阻止细胞进入合成周期,显著降低fibronectin、胶原Ⅳ产生,磷酸化p38蛋白和磷酸化MKK3/MKK6的表达显著受抑;在体内AM治疗能显著改善5/6肾切除大鼠的肾功能情况,降低蛋白尿排泄量,延缓肾功能恶化。结论:AM治疗可通过抑制磷酸化MKK3/6和磷酸化p38 MAPK蛋白的表达,发挥抑制IL-1β诱导的MCs增殖作用,能抑制MCs的ECM积聚,有效的治疗进展性肾脏病,为慢性肾纤维化提供了有前景的治疗策略。

Objective： To investigate therapeutic effects ofAstragalus Membranaceus （AM） on 5/6 nephrectomized rats in vivo, and to investigate AM on IL-1β induced rat mesangial cells （MCs） and on their production of Intracellular Matrix （ECM） in vitro. Methods： After successful establishment of 5/6 nephrectomized rats＇ model, we assigned them to 4 groups： sham operation group （normal con- trol）; renal fibrosis group （untreated）; low-dose/high-dose and AM-treated group. After 16 weeks＇ survey, urine protein excretion （UPE） and renal function parameters were assessed. IL-1β was selected as stimulus for cultured MCs in vitro, AM diluted in different concentration was added thereafter. Methyl thiazoleterazolium assay was performed for detecting cell proliferation and flow cytometry for cell-cycle analysis. Fibronectin in cell supernatant and p38 mRNA was quantified by enzyme linked immunosorbant assay （ELISA） and real-time PCR, respectively. Western immunoblotting analysis was performed to evaluate the expression levels of collagen IV, total p38 MAPK, phospho-p38 MAPK, phospho-MKK3/MKK6 and phospho-MKK4 in addition. Results： Results showed that AM significantly suppressed IL-1β induced MCs proliferation and arrested the synthesis cell-cycle progress in vitro. Fibronectin and collagen IV production were markedly reduced by AM intervention. Protein levels of p-p38, P-MKK3/6 was significantly decreased after AM treatment. For the 5/6 nephrectomized rats in vivo, AM therapy significantly improved rats＇ renal function, as evidenced by decreased blood urea nitro- gen, plasma creatinine and 24-hour urine protein excretion. Conclusion： AM treatment ameliorates IL-1β induced MCs proliferation and ECM production by inhibiting activation of P-MKK3/6 and p38, it can retard progressing renal disease effectively, which might suggest a novel and prospective remedy for chronic renal fibrosis.