雷公藤多苷联合小檗碱预防和治疗2型糖尿病肾小管间质病变的作用机制研究

The onset of the tubulointerstitial lesions is characterized by the infiltration of inflammatory cell, disorder of cytokine expression and deposition of extracellular matrix.Infiltration of inflammatory cell and epithelial mesenchymal transdifferentiation of renal tubular play a key role. High glucose can increase the expression of TLR4 in renal tubules, which can activate the NF-KB. TLR4/NF- κ B pathway may play an important role in in renal tubulointerstitial lesions. Previous studies have shown that tripterygium wilfordii polyglycoside(TWP) can inhibit the production of pro-inflammatory cytokine, reduce the local inflammation of kidney, combination of TWP and berberine can both improve glomerular sclerosis and reduce tubulointerstitial lesions. Our group propose a hypothesis that the combination of TWP and berberine (the effective insulinotropic alkaloid) may prevent or delay tubulointerstitial lesions by inhibiting the TLR4/NF-κB inflammatory signaling pathway. To test this hypothesis, we plan to investigate the effects of TWP and berberine on TLR4/NF-κB inflammatory pathways and Smads signaling pathway in both type 2 diabetes rat model and tubular epithelial cells, with light microscopy,immunoelectronmicroscope, immunohistochemistry, real time PCR and western blot. This work may provide a new therapeutic strategy for the treatment and prevention of tubulointerstitial lesions in the early stage. It may also demonstrate the theory of "treat before sick" in Chinese traditional medicine.

肾小管间质病变以炎性细胞浸润、细胞因子表达失调、细胞外基质沉积为特征，炎性细胞浸润和肾小管上皮细胞转分化是其中心环节。高糖状态下肾小管TLR4表达增加，进而激活NF-KB，促进肾间质炎症的进展。TLR4/NF-κB通路在肾小管间质病变中可能起重要作用。前期研究发现：雷公藤多甙能抑制促炎细胞因子产生，减轻肾脏局部炎症，雷公藤多甙联合小檗碱在改善糖尿病大鼠肾小球硬化的同时，能减轻肾小管间质病变。据此我们提出雷公藤多甙联合小檗碱通过抑制TLR4/NF-κB炎症通路延缓糖尿病肾病肾小管间质病变的假说。本研究以自发2型糖尿病大鼠和肾小管上皮细胞为研究对象，以TLR4/NF-κB炎症通路及Smads通路为切入点，通过光镜、电镜、免疫组化、RT-PCR、Western blot等方法探讨雷公藤多甙联合小檗碱早期干预对糖尿病肾小管-间质病变的预防和治疗作用。为中医"未病先防""既病防变"提供理论依据。

肾小管间质病变以炎性细胞浸润、细胞因子表达失调、细胞外基质积聚为特征。TLR4/NF-KB通路在肾小管间质病变中可能其重要作用。前期研究发现，雷公藤多苷联合小檗碱在改善糖尿病大鼠肾小球硬化的同时，能减轻肾小管间质病变。我们发现雷公藤多苷的作用明显强于小檗碱的作用，因此我们应用雷公藤多苷进行后续研究。本研究从临床研究和基础实验两方面观察雷公藤多苷对糖尿病肾小管间质病变的保护作用并探讨其可能的机制。本研究以2型糖尿病肾病大鼠模型和肾小管上皮细胞为研究对象，应用实时荧光定量PCR、Western-Blot法、免疫组化法，探讨雷公藤多苷对糖尿病肾小管间质病变的保护作用并探讨其可能的机制，为中医“治未病”防治糖尿病肾病提供理论依据。.研究结果显示，雷公藤多苷干预后肾小管上皮细胞空泡及颗粒变性减轻，肾小管间质炎细胞浸润明显减少，肾小管扩张、萎缩改善，肾小管基底膜增厚减轻，且中、高剂量干预效果好于低剂量治疗。免疫组化、Westren blot 蛋白检测和 qRT-PCR 结果显示 DM 组大鼠肾小管上皮细胞 E-cadherin 蛋白表达下降，肾小管间质中 α-SMA 和 vimentin 和Ⅳ型胶原蛋白表达增多，大鼠肾小管间质中TLR4、MyD88、NF-κB表达明显高于正常对照组，提示TLR-MyD88-NF-κB炎症通路参与了糖尿病肾小管间质病变的发生。 雷公藤多苷干预后大鼠肾组织中E-cadherin 水平呈剂量依赖性升高，α-SMA，vimentin 和Ⅳ型胶原表达呈剂量依赖性减少，说明雷公藤多苷干预减轻糖尿病肾小管间质病变，可能与其拮抗肾小管上皮细胞转分化有关。雷公藤多苷干预组大鼠肾小管间质中TLR4，MyD88，NF-κB，IL-1β，TGF-β1，TNF-α和VCAM-1表达均显著降低，提示雷公藤多苷减轻肾小管间质损伤的的作用可能是通过抑制TLR4-MyD88-NF-κB炎症通路，最终导致炎症因子表达减少有关。细胞实验与动物实验结果一致。