基于ROS介导的Akt、MAPK信号通路探讨冬虫夏草预防糖尿病大鼠对比剂肾病的作用机制

The incidence of contrast induced nephropathy (CIN) is increasing year after year, and thus to prevent CIN timely is of great social and economic significance. In our previous clinical research, we found that cordyceps sinensis (CS) was a protective factor against CIN in type 2 diabetics with renal insufficiency undergoing coronary angiography. The Traditional Chinese Medicine (TCM) mechanism is that CS can reinforce the kidney, replenish Qi, and correct obvious primary deficiency syndrome. However, the molecular mechanism remains unclear. In our previous animal study, we proved that renal cell apoptosis is the main pathological basis for the development of CIN in type 2 diabetics. First, in this research, CIN model in diabetic rats will be established and the expression level of reactive oxygen species (ROS) in renal tissue will be studied in order to discuss the effect of CS on oxidative stress in diabetic CIN. Second, the expression of renal cell apoptosis related protein will be analyzed to explore the effect of CS on apoptosis in diabetic CIN. Third, by analyzing the expression level of Akt/mTOR/P70S6K and MAPK apoptosis signaling pathways and the expression level of target transcriptional factor FoxO1 in renal tissue, signaling transduction pathways in renal cell apoptosis in diabetic CIN will be explored and the association between these two signaling pathways will be analyzed. In this way, signaling pathways and targets through which renal protective effects of different dose of CS on renal cell apoptosis will be revealed, and hence theory basis and laboratory evidence for TCM prevention and treatment of CIN will be provided.

对比剂肾病(CIN)的发病率逐年上升，及早防治CIN有重要社会经济意义。申请人前期临床试验发现冬虫夏草可以预防糖尿病肾功能不全患者CIN的发生，其中医机制是虫草能起到补气、滋补肾脏、纠正患者本虚之功效，但其发挥作用的分子生物学机制尚不明确。申请人前期动物实验证实肾细胞凋亡是贯穿糖尿病CIN发生发展的主要病理基础。本研究将首先建立糖尿病大鼠CIN模型，通过测定肾组织活性氧(ROS)水平，明确对比剂所致的肾脏氧化应激及虫草对ROS的影响；其次通过分析肾组织凋亡蛋白的表达情况，探讨虫草对糖尿病CIN时细胞凋亡的作用机制；最后通过分析Akt/mTOR/P70S6K、MAPK信号通路及其靶转录因子FoxO1在肾内的表达情况，探讨糖尿病CIN时肾细胞凋亡旳信号转导途径，分析这两条信号通路之间的关联。揭示不同剂量的虫草制剂拮抗肾细胞凋亡所作用的信号通路及靶点，为中医药防治CIN提供理论基础和实验室证据。

近年来随着临床上对比剂的广泛应用，对比剂肾病（CIN）发病率逐渐上升。中医认为CIN属于“癃闭、关格、溺毒”等范畴，对比剂属于“药毒、外邪”等范畴，故而中医对CIN的治疗原则为“扶正祛邪”。冬虫夏草具有补气、滋补肾脏、纠正患者本虚的功效。临床研究发现冬虫夏草能够防预CIN。前期研究证实肾细胞凋亡是糖尿病CIN发生发展的主要病理基础。本研究应用RT-PCR、免疫组化、HE、Tunel及电镜等方法检测相应指标，探讨冬虫夏草调控ROS介导的Akt、MAPK细胞凋亡信号通路预防糖尿病大鼠CIN的机制。研究结果显示，糖尿病CIN大鼠给予冬虫夏草干预，各组大鼠肾功能指标BUN、Scr、KIM-1、NGAL、IL-18、γ-GT含量有所降低；肾组织氧化应激指标NO、NOS、SOD、T-AOC含量升高，O2-、MDA含量均降低；肾组织凋亡相关蛋白Caspase-9、Bax、Fas、Fasl表达降低、Bcl-2及Bcl-2/Bax比值升高，且高剂量组Caspase-3亦降低；凋亡信号通路中p-Akt、p-mTOR、p-P70S6K蛋白水平明显升高，p-ERK、p-JNK蛋白水平降低；关键转录因子NF-κB、FoxO1基因表达降低。上述指标变化呈剂量相关性。高剂量虫草组大鼠病理性损伤明显改善：光镜下肾小管上皮细胞坏死和空泡样改变减少；电镜下肾小球和肾小管超微结构基本正常，肾小球血袢基底膜大部分厚度均匀；肾小管上皮细胞胞质可见少量次级溶酶体，少量空泡，线粒体呈短杆状，板层状嵴，凋亡细胞少见。研究结论：糖尿病CIN发生发展与ROS介导的MAPK、Akt信号通路导致肾小管的凋亡，造成严重肾损伤密不可分；冬虫夏草能够降低氧化应激水平，调控上述信号通路中的相关蛋白表达，从而减少肾小管细胞凋亡，改善肾功能，减少肾损伤，防预糖尿病CIN发生发展。本课题通过探讨冬虫夏草对糖尿病CIN的肾保护作用及其对凋亡信号通路的影响，为中医药防治CIN提供新的理论基础和实验室证据。