中药牛蒡子对晚期氧化蛋白产物致肾小管上皮细胞肥大的影响及机制研究

Renal hypertrophy usually appears in early-stage of chronic kidney disease, and is closely correlated with renal fibrosis in end-stage. Tubular epithelium hypertrophy plays an important role in renal hypertrophy. In our recently study, we found that advanced oxidation protein products (AOPPs） could induce renal tubular epithelial cells hypertrophy with endoplasmic reticulum stress (ERS). But there is no information about the relationships between AOPPs, ERS and hypertorphy. .We have also demonstrated that monomer arctiin, a traditional medicine ,alleviated ERS of podocyte cells induced by AOPPs. Some later studies reported that arctiin could alleviate renal hypertrophy of diabetic nephropathy. The mechanisms are still unclear. Therefore, we hypothesize that AOPPs induce ERS in tubular epithelia then lead to hypertrophy, which can be prevented by Fructus Arctii.In ordre to prove the hypothsis, the following studies will be performed in human tubular epithelium cells. .First, demonstrate the process of ERS and hypertrophy induced by AOPPs. Second, clarify the signal pathways involving the process. Third, investigate if hypertrophy would be reversed by blocking the signal pathway. Finally, we would like to observe the effect of Fructus Arctii on the ERS, signal pathway activity and hypertrophy induced by AOPPs. Our reseach may offer new strategy for early prevention of CKD with traditional chinese medicine.

肾脏肥大是许多慢性肾脏病（CKD）的早期改变，与后期肾脏纤维化的发生发展密切相关。肾小管上皮细胞（TEC）肥大是肾脏肥大的重要原因。本课题组在新近的研究中发现晚期氧化蛋白产物(AOPPs)可直接诱导TEC肥大伴有内质网应激(ERS)。但AOPPs通过何途径引起ERS，ERS是否介导了TEC肥大则均无报导。我们还观察到牛蒡子苷可抑制足细胞的ERS。有报道牛蒡子苷可减轻糖尿病肾病的早期肾脏肥大，机制不清。因此我们推测ERS可能是AOPPs诱导TEC肥大的关键环节，牛蒡子可能通过拮抗ERS来减轻TEC肥大。为验证该推测，我们拟以人肾小管上皮细胞系为研究对象，首先验证AOPPs可诱导细胞ERS及具体信号通路，然后研究阻断该通路后对肥大的影响，进而观察牛蒡子提取物对AOPPs诱导细胞ERS及肥大的影响并探寻可能机制。本研究从新的角度探索CKD的进展机制，为中药牛蒡子对CKD的早期防治提供理论依据。

肾脏纤维化是各种慢性肾脏病（CKD）进行性发展至终末期肾病的共同途径。肾小管上皮细胞肥大和间质转分化（EMT）可能导致肾脏纤维化。晚期氧化蛋白产物（AOPPs）可促进肾脏疾病进展，但其具体机制尚待阐明。近年大量研究表明内质网应激（ERS）参与多种肾脏疾病的发生发展，但其是否介导AOPPs诱导肾小管上皮细胞（HK2细胞）肥大和发生EMT尚未清楚。本项目主要探讨ERS在此过程中的作用及具体机制，并观察牛蒡子提取物对此过程的影响。主要结果结论如下：① AOPPs可上调细胞ERS标志性蛋白GRP78及CHOP的表达，并可上调间充质细胞标志性蛋白α-SMA，及下调上皮细胞标志性蛋白E-cadherin，提示AOPPs可诱导HK2细胞发生ERS及EMT; AOPPs可使细胞内总蛋白含量增高、p27表达增高、停滞于G1期，提示AOPPs可导致细胞肥大。氧化应激拮抗剂DPI和SOD及ERS的阻断剂可显著降低AOPPs诱导的HK2细胞ERS现象，同时伴有细胞肥大及EMT现象受抑制，而ERS的诱导剂可直接诱导细胞发生ERS并伴有细胞肥大及EMT的发生，提示ERS可能介导AOPPs诱导HK2细胞肥大及发生EMT，并与氧化应激有关；② AOPPs可使PERK、eIF2α和IRE1磷酸化，并使cleaved ATF6和XBP1s的表达增高，提示AOPPs可激活未折叠蛋白反应的三条信号通路；但仅ATF6 siRNA转染的HK2细胞中上皮细胞标志性蛋白表达升高，间质细胞标志性蛋白的表达降低，细胞肥大减轻，而PERK siRNA和IRE1 siRNA转染的肾小管上皮细胞上述指标并未发生明显变化，提示仅ATF6信号通路参与了AOPPs诱导HK2细胞肥大及发生EMT的过程；结果还提示Src激酶参与了上述过程的发生，并与ERS有关；③CTGF siRNA可抑制AOPPs下调的上皮细胞标志性蛋白表达及上调的间质细胞标志性蛋白表达，并抑制AOPPs诱导的细胞肥大，提示CTGF参与了AOPPs诱导肾小管上皮细胞肥大和发生EMT的过程；④牛蒡子可逆转AOPPs诱导的ERS、细胞肥大及EMT，提示牛蒡子可能通过抑制ERS从而减轻AOPPs诱导的细胞肥大及EMT。本项目进一步阐明AOPPs促进肾脏疾病进展的机制，且为探索抑制CKD进展提供新干预靶点及为牛蒡子提取物应用于临床治疗CKD提供理论依据。