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固本通络方对IgA肾病小鼠Peyer小结B淋巴细胞IgA类别转换的影响
Effect of “Gubentongluo Formula” on the IgA Class Switch Recombination of B Lymphocytes in Peyer’s Patches in Mice with IgA Nephropathy
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摘要: 目的 探讨固本通络方治疗IgA肾病的作用机制。方法 昆明种小鼠经过12周诱导建立IgA肾病动物模型，随机分为模型组（n=15）、中药组（n=15）；另取正常小鼠作为正常对照组（n=15）。12周造模结束时开始，中药组按小鼠体质量予固本通络方1.35 mL/（g·d）灌胃，正常对照组和模型组予等量生理盐水灌胃，每日1次，均连续8周。第0周、12周和20周时，测定各组24 h尿蛋白；第12周和20周观察肾组织病理学改变，第20周时实时荧光定量PCR检测Peyer小结转化生长因子β(TGF-β)、细胞信号转导分子(Smad)3 mRNA表达，免疫组化法检测Peyer小结TGF-β、Smad3蛋白的表达，流式细胞测定Peyer小结（IgA+B淋巴细胞）/B淋巴细胞水平。结果 与正常对照组相比，模型组小鼠24 h尿蛋白升高（12周和20周），系膜区IgA沉积，Peyer小结TGF-β、Smad3 蛋白和mRNA表达升高，（IgA+B淋巴细胞）/B淋巴细胞水平升高，差异均有统计学意义（P均<0.01）。与模型组相比，中药组各项指标均有所改善，差异有统计学意义（P均<0.01）；但与正常对照组相比，中药组各项指标的差异有统计学意义（P均<0.01）。结论 固本通络方能有效改善IgA肾病小鼠蛋白尿及肾小球系膜区IgA沉积，可能与其调节Peyer小结TGF-β/Smad3，进而影响B淋巴细胞IgA类别转换有关。Abstract: Objective To explore the underlying mechanism of “Gubentongluo Formula” in treatment of IgA nephropathy (IgAN). Methods After the IgAN model was successfully induced at week 12, the Kunming mice were randomly divided into three groups: normal control group (n=15), IgAN group (n=15) and Traditional Chinese Medicine (TCM) group. The mice in normal control and IgAN group were intragastriclly administrated with normal saline for 8 weeks; meanwhile, the mice in TCM group were intragastriclly administrated with “Gubentongluo Formula” 1.35 mL/ (g·d). The levels of 24 h urine protein were determined at Week 0, 12 and 20. At week 20, the changes of renal pathology were detected; the mRNA expressions of transforming growth factor-β (TGF-β) and small mothers against decapentaplegic (Smad) 3 in Peyer’s patches (PPs) were detected by fuorescent quantitative reverse transcription-polymerase chain reaction; the protein expressions of TGF-β and Smad 3 in PPs were detected by immunohistochemistry technique; the levels of (IgA+B)/B lymphocytes in PPs were determined by flow cytometry. Results Compared with those results of normal control group, the levels of 24 h urine protein, IgA deposition in glomerular mesangial area, and expressions of protein and mRNA of TGF-β and Smad3 in IgAN group were significantly increased (P<0.01). Besides, the levels of (IgA+B)/B lymphocytes were significantly elevated in IgAN group (P<0.01). All these indicators were improved in TCM group. Compared with IgAN group, the differences were statistically significant (P<0.01). Compared with those results of control group, the levels of (IgA+B)/B lymphocytes showed no significant difference in TCM group (P>0.05), but other indicators showed significant differences (P<0.01). Conclusion “Gubentongluo Formula” could effectively improve proteinuria and suppress IgA deposition in glomerular mesangial area in IgAN mice, due to affect IgA class switch recombination of B lymphocytes in PPs through regulating TGF-β/Smad3 pathway.
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