基于醛固酮活化/SGK-1/ NF-kB通路诱导细胞增殖探讨"浊毒致瘀"的机制及化瘀解毒中药的调控作用

Cell proliferation plays the important role in renal interstitial fibrosis and inflammatory lesion has the key effect to induce cell proliferation . Results from previous studies which Huayujiedu herbs inhibit TEMT by regulation of Adrenomedullin have been equivocal regarding the association between inflammatory injury and cell proliferation in renal interstitial fibrosis. We speculated cell proliferation induced by aldosterone activation of inflammatory lesions after the kidney damage plays an important role in it. Based on later stage of kidney disease "renal deficiency of spleen deficiency in wet cloud-poison evil connotation" and "long illness into complex", the hypothesis of " stasis induced by poison" was presented by us. In this study, we will research the activated aldosterone induce cell proliferation with inflammatory factors by signal transduction pathway then overproduce extracellular matrix use for explore the mechanism of "stasis" from " poison" . The animal model of renal interstitial fibrosis are used in Wistar rats and Adrenomedullin knockout mice with unilateral ureteral obstruction (UUO). Activated aldosterone induce over-expression of inflammatory mediators then activate the NF-κB by phosphorylation of protein kinase. Cell proliferation was induced and over- production of extracellular matrix to increase process of renal interstitial fibrosis. We want to understand the signal transduction pathway of fibrosis (stasis) induced by inflammatory mediators (poison) with cell proliferation. The protective effect of ADM on cell proliferation and target of Huayujiedu Traditional Chinese Medical herbs also discussion in this study. The study will give the important truths for theory of "stasis induced by poison" and clinic study.

炎性损伤诱导细胞增殖在肾间质纤维化进展中起着重要作用。在前期研究化瘀涤痰通络中药调控肾上腺髓质素（ADM）抑制细胞表型转换的过程中，发现梗阻性肾病存在明显的炎性损伤和细胞增殖，推测肾脏受损后醛固酮活化诱导的炎性损伤是介导细胞增殖的重要因素，基于肾病后期"脾肾亏虚，湿浊毒邪内蕴"及"久病入络"的病机，提出"浊毒致瘀"的假说。本研究以输尿管梗阻诱导醛固酮活化-炎性介质高表达-信号转录-细胞增殖-细胞外基质积聚为路径，结扎大鼠和ADM基因敲除小鼠单侧输尿管，观察梗阻因素诱导醛固酮活化及炎性介质高表达，通过蛋白激酶的磷酸化，激活核转录因子，刺激细胞增殖并分泌过多细胞外基质形成纤维化的病理改变，探讨炎性介质（浊毒）诱导细胞增殖形成纤维化（瘀）的机制和信号传导途径。同时观察ADM对细胞增殖的影响以及化瘀解毒中药的调控靶点，为"浊毒致瘀"假说及化瘀解毒中药的临床应用提供实验依据。

慢性肾脏病成为世界性的公共问题，研究其发生、发展机制尤其是肾脏纤维化机制有着重要的意义。本研究分别采用Wistar雄性大鼠及肾上腺髓质素基因敲除小鼠，结扎单侧输尿管制备肾间质纤维化实验动物模型，观察细胞增殖对肾脏纤维化的影响以及SGK-1信号通路在其中的调控作用，基于中医“浊毒致瘀”病机学说，给以化瘀解毒中药以及醛固酮受体阻断剂干预，观察其调控肾上腺髓质素抑制细胞增殖的作用。结果显示梗阻性肾病实验动物肾脏病理呈现纤维化改变，细胞外基质沉积显著增加，伴有不同程度的肾功能损伤，细胞增殖标记物PCNA阳性细胞表达明显增多，与纤维化程度正相关，敲除肾上腺髓质素基因，细胞增殖明显加重，SGK-1表达明显增强，同时盐皮质激素受体NR3C2入核活化，激活核转录因子NF-κB导致一系列病理生理改变，给以醛固酮受体阻断剂依普利酮可以下调SGK-1的表达，抑制细胞增殖，减轻肾脏纤维化程度。同时观察化瘀解毒中药对实验动物细胞焦亡和自噬的影响，结果显示中药通过调控NLRP3炎症小体信号通路抑制细胞焦亡；通过p-mTOR/Atg5通路途径抑制细胞的自噬。体外研究证实肾上腺髓质素及化瘀解毒中药血清可以下调SGK-1的表达抑制醛固酮诱导的肾小管上皮细胞的过度自噬，从而减轻组织和细胞损伤。在研究中我们证实了盐皮质激素受体阻断剂可以抑制醛固酮诱导的细胞增殖，但醛固酮在体外未能诱导肾小管上皮细胞的增殖。本研究验证了炎性介质等浊毒物质在体内诱导肾脏纤维化的部分机制，为临床治疗提供了实验依据。