基于内质网应激探讨中药落新妇苷拮抗镉致鸡肾小管上皮细胞凋亡的机制

Smilax glabra Roxb. (SGR) is the Chinese traditional medicine named Tufuling. Astibin, as an element of SGR’s quality control, has extensive pharmacological activities. Astilbin has a protective effect of kidney which was damaged by a variety of reasons. Being an ancient prescription of Chinese traditional medicine, astibin was recognized with the ability to heavy metal detoxification. Kidney seems to be one leading target organ of Cadmium. The mechanism for antagonism effects of astilbin on the cadmium chloride-induced kidney toxic response was unkown. Thus far, it is still unclear about the mechanism of renal toxicity and the antidotal effect of astilbin and the potential relationships between the two aspects. The current research showed that Cadmium-induced Calcium homeostasis, endoplasmic reticulum stress (ERS) lead to apoptosis. Astibin involved in intracellular calcium regulation, which are crucial for cellular homeostasis. It is closely related to the development of renal toxicity by cadmium, but the influence of calcium homeostasis with astilbin on the cadmium chloride-induced kidney toxic apoptosis by ER stress pathway was unclear. This study will establish renal tubular epithelial cells culture model in vitro by astilbin and astilbin phosphate intervention. By the use of light microscopy, flow cytometry, and laser co-focus, real time quantitative PCR, western blot, the morphological observation of renal tissue and renal tubular epithelial cells, and the detection of Calcium homeostasis , ER stress pathway, apoptosis and its related gene expressions, antioxidant, the molecular mechanisms that role of ER stress path way in astilbin detoxifying Cadmium induced apoptosis which should be elucidated. The study will provide a new type of traditional Chinese veterinary medicine clues on the expansion of the application range of SGR as well as the prevention of Cadmium toxicity.

落新妇苷是中药土茯苓的质控成分，具有广泛的药理活性。古方记载土茯苓具有解重金属中毒的作用，但具体作用物质与机制尚未明确。肾脏是镉毒性的重要靶器官之一，但其中毒机制尚未完全清楚，且落新妇苷对其解毒效应仍未见报道。研究发现镉可导致鸡肾细胞钙稳态失衡、内质网应激引起细胞凋亡，且落新妇苷具有调节钙稳态的作用，但其与镉及内质网应激、凋亡的关系尚不清楚。本研究在复制鸡肾小管上皮细胞染镉体外培养模型及落新妇苷和其磷酸盐干预模型的基础上，应用透射电镜、激光共聚焦、实时荧光定量PCR、免疫印迹、流式细胞术等技术，通过对鸡肾小管上皮细胞形态学的观察，以及钙稳态、内质网应激、细胞凋亡及其相关基因、抗氧化功能等指标的检测，阐明落新妇苷拮抗镉致鸡肾小管上皮细胞损伤的机制，为拓展土茯苓的应用提供理论依据，为防治镉中毒提供借鉴。

落新妇苷是中药土茯苓的质控成分，具有广泛的药理活性。现阶段研究证明落新妇苷具有解镉毒、保护肾脏的功效。镉中毒鸡肾脏损伤时，肾组织中发生氧化应激，钙稳态失衡、内质网应激、细胞凋亡。内质网应激通路作为诱发细胞凋亡的主要信号通路，参与镉暴露肾损伤病理生理机制。落新妇苷作为强抗氧化剂可以维持钙稳态且对多种原因引起的肾损伤均具有保护作用。本项目共合成出六种落新妇苷衍生物，在比对落新妇苷、落新妇苷衍生物和同系物（花旗松素、槲皮素）等九种物质抗氧化活性的基础上，选出抗氧化和非抗氧化两条途径的供试药物。在复制鸡肾小管上皮细胞染镉体外培养模型及落新妇苷和其不同抗氧化活性的同系物干预模型的基础上，应用流式细胞术、实时定量PCR法、免疫印迹法等，证实镉暴露可引起鸡肾小管上皮细胞发生氧化应激同时死亡率、凋亡率升高，钙离子通道相关基因（CaM、CNX、CRT）升高，内质网应激相关基因（IRE、CASP12、PERK、ATF4、GRP78）基因表达升高，凋亡基因（Bax、Bcl-2、Caspase-3）紊乱。落新妇苷、落新妇苷苷元（花旗松素）干预均可降低肾小管上皮细胞损伤。落新妇苷可通过降低肾小管上皮细胞氧化应激损伤，调节钙通路、内质网应激相关基因、凋亡基因的变化拮抗镉毒，其具体机制为落新妇苷能够通过 PI3K/Akt/Bcl-2 信号通路降低促凋亡基因 Bax、Caspase3的表达，进而缓解镉暴露引起的的鸡肾小管上皮细胞过度的凋亡，阐明落新妇苷干预镉暴露鸡肾小管上皮细胞损伤的具体机制。本项目的顺利实施，为拓展落新妇苷的应用提供理论依据，为开发新药提供了新的思路和方向，为防治镉中毒提供借鉴。