艾迪注射液中拮抗斑蝥肾脏毒性的物质基础和作用机制研究

Aidi injection is composed of Chinese blister beetle, Panax ginseng, Astragalus root and Radix acanthopanacis semticos, and widely used for treatment with various tumor in clinical. Chinese blister beetle is monarch drug and plays an essential role for antitumor in this prescription. But Chinese blister beetle has renal toxicity with chronic use. Renal toxicity induced by Chinese blister beetle is a complex phenomenon characterised by an increase in blood urea nitrogen, protein in urine and serum creatinine and severe renal tubular necrosis followed by renal dailure in rats. Chinese blister beetle also inhibited NRK cell viability by apoptosis in vitro. 15 days after administration of Chinese blister beetle, there are morphological changes, including nuclei degenerated, cytoplasm shrinkage, chromatin condensated, ruptured to the nuclear margin and decreases of cell volume were observed in the Chinese blister beetle-treated group under transmission electronic microscope. Chinese blister beetle also induced autophagy in NRK cell. 3-methyladenine (autophagy specificity inhibitors) increased cell viability at the concentration of 5.0-10.0ug/ml Chinese blister beetle, but inhibited cell viability at the concentration of 0.078-2.5 ug/ml. On the other hand, there are 23 chemical compounds from the extract of Aidi injection. Those compounds were confirmed by spectrum data and accumulated sum of 8 chemical compounds.On the base of summarizing the previous results, this research is projected on effective active constitutes and protective mechanisms of kidney toxicity induced by Chinese blister beetle in Aidi injection. The protective effect of Aidi decomposed recipes and 22 compounds were observed on renal toxicity induced by Chinese blister beetle in vivo and in vitro. Human renal cell (HK-2), rat renal cell (NRK) and a rat renal injury model induced by Chinese blister beetle were used to clarify the association between Bcl-2, Bax, caspase3, caspase8, caspase9 expression and Chinese blister beetle-induced nephrotoxicity, the associaton between autophagic markers LC3 and Beclin-1 expression or enzymatic activity or expression of PP1 and PP2A using flow cytometry analysis, transmission eletron microscopy, westblotting and biochemical determination. The present study was to indicate mechanisms of renal injury induced by Chinese blister beetle and principles of formulating prescription in Aidi injection, provide new experimental data for clinical use, and new ideas for the studies of tranditional prescription including chinese blister beetle.

艾迪注射液是由斑蝥、人参、黄芪和刺五加组成的复方中药制剂，广泛用于多种肿瘤的治疗。斑蝥作为君药，起主要作用，但单独使用可引起明显的肾脏毒性，诱导肾细胞凋亡和自噬，而含相同剂量斑蝥的艾迪注射液却未表现出明显的肾脏毒性，提示其中可能存在拮抗斑蝥肾脏毒性的活性成分。在完成艾迪注射液系统化学成分研究的基础上，本项目拟采用体内斑蝥致大鼠肾损伤和体外培养大鼠、人肾细胞损伤模型，探索斑蝥致大鼠肾脏毒性的机制，考察艾迪注射液中拮抗斑蝥肾脏毒性的物质基础。并进一步采用透射电镜、流式细胞仪和western blotting等方法，考察减毒作用与细胞凋亡信号分子Bcl-2、Bax、caspase3、caspase8、caspase9，与自噬信号蛋白LC3和Beclin-1以及PP1和PP2A活性和表达的相关性研究，探索活性组分的减毒机制，阐明艾迪注射液组方的科学内涵，也为含有斑蝥的中药复方研究提供新的研究思路。

斑蝥在临床上有很好的抗肿瘤作用，但其肾脏毒性使其临床应用受到限制。斑蝥素是斑蝥的主要活性成分，其肾脏毒性报道较少以及毒性机制缺乏研究。基于我们课题组前期研究发现斑蝥有致大鼠肾脏毒性作用和艾迪注射液中存在减毒物质基础之上，本研究采用斑蝥素致SD大鼠肾脏损伤模型和HEK-293细胞毒性模型，初步阐明斑蝥素致肾脏毒性机制，同时考察人参皂苷Rb1对斑蝥素致肾脏毒性的保护作用和机制。MTT、细胞流式结果提示人参皂苷Rb1明显促进细胞增殖，降低斑蝥素对HEK-293细胞毒性作用。斑蝥素连续15天腹腔注射诱导大鼠形成严重的肾脏损伤，体重减轻，血清中肌酐(CREA)和尿素氮（UREA）含量明显升高，肾脏指数明显上升，肾脏组织HE染色显示大鼠肾小球出现萎缩，肾远曲小管水肿及肾脏组织炎性浸润；血清中细胞因子TNF-α、IL-6显著减少；透射电镜下，肾脏基底膜厚薄不均匀，足细胞不规律及有脱落现象，血常规发现ENU、MONO、CYM、EOS、WBC显著升高。人参皂苷Rb1可明显改变斑蝥素所致肾损伤大鼠的血清中尿素氮、肌酐含量、大鼠尿量和体重等指标，改善肾小球坏死、肾脏远曲小管水肿状态，减少肾脏组织的炎性浸润；改善肾基底膜和足细胞损伤状况；ENU、MONO、CYM、EOS、WBC及TNF-α、IL-6升高趋于正常大鼠水平。在斑蝥素致SD大鼠肾脏损伤模型上及斑蝥素致HEK-293细胞毒性模型中，PI3K-AKT通路中肾脏组织蛋白PP2A、Bcl-2表达显著下调，而p-AKT显著上调，人参皂苷Rb1预保护后，能显著增强PP2A、Bcl-2的表达及减弱p-AKT的表达。凋亡相关蛋白caspase-3、PARP没有显著改变；炎症相关蛋白COX-2、显著上调，TNF-ɑ显著下调，人参皂苷Rb1能减弱COX-2的表达和增强TNF-ɑ的表达，在体内TNF-ɑ蛋白的变化和体外趋势一致；程序性坏死相关蛋白RIP1显著下调，人参皂苷Rb1能增强RIP1的表达。人参皂苷Rb1在体内外能明显的拮抗斑蝥素的毒性作用，其抑制细胞凋亡和促进细胞增殖机制可能和细胞周期阻滞、PI3K-AKT、MAPK信号通路、NF-κB信号通路、程序性坏死蛋白RIP变化有关；炎性调节机制可能和COX-2、TNF-α蛋白变化、细胞因子TNF-α、IL-6有关。