安徽医科大学学报

目的 探讨汉黄芩素(WOG)对脂多糖(LPS)诱导的急性肾损伤的保护作用。方法 用LPS试剂诱导C57BL/6J小鼠构建脓毒症致急性肾损伤模型。以未经处理的C57BL/6小鼠作为常规对照组。24只小鼠被随机分配到四个组别：常规对照(NC)组、WOG(WOG 12.5 mg/kg)组、LPS(LPS 10 mg/kg)组以及LPS+WOG(LPS 10 mg/kg+WOG 12.5 mg/kg)组。检测小鼠血肌酐(CRE)和尿素氮(BUN)水平。聚合酶链式反应(PCR)检测小鼠肾损伤分子1(KIM-1)和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的表达情况。苏木精-伊红(HE)染色和糖原(PAS)染色观察肾脏病理损伤程度。免疫组化检测肾组织的炎性标志物白细胞介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α的表达程度。Western blot法检测肾脏组织核因子κB(NF-κB)信号通路亚基P65和PP65蛋白表达变化的情况。结果 相较于NC组，LPS组CRE和BUN上升(F_CRE=60.90,P<0.001;F_BUN=82.13,P<0.001);相较于LPS组，LPS+WOG组CRE和BUN降低(P<0.001)。PCR检测结果显示，相对于NC组，LPS处理的小鼠其肾脏内的KIM-1和NGAL mRNA表达增加(F_KIM-1=146.3,F_NGAL=161.2,均P<0.001),而在LPS+WOG组中，KIM-1和NGAL mRNA表达下降(均P<0.01)。肾脏组织病理学检查显示，与NC组相比，LPS组小鼠肾组织肾小管扩张明显、炎症细胞浸润；与LPS组相比，LPS+WOG组肾小管扩张数量减少和炎症细胞浸润减少(F_HE=721.4,F_PAS=518.9,P<0.001)。免疫组化染色检测结果显示，相较于NC组，LPS处理的小鼠中IL-1β、IL-6及TNF-α的表达量上升(F_IL-1β=114.6,F_IL-6=108.9,F_TNF-α=251.6,均P<0.001);而对比LPS组，LPS+WOG组中的这些指标则有所减少(均P<0.01)。使用Western blot方法进一步研究表明，相较于NC组，LPS处理的小鼠其NF-κB信号途径已被激活并产生高磷酸化状态(F_PP65=13.02,P<0.01),然而对比LPS组时，LPS+WOG组的该路径却表现出相反的效果，即活性减弱且磷酸化程度降低(P<0.01)。结论 WOG能有效地阻断LPS引发的急性肾损伤模型小鼠的NF-κB信号途径，从而削弱由LPS引起的急性肾损伤小鼠肾脏的炎性应答及其组织损害。

Objective To investigate the protective effect of wogonin on acute kidney injury(AKI) induced by lipopolysaccharide(LPS). Methods The model of septic-induced AKI was established on male C57BL/6J mice by a single intraperitoneal injection of LPS and normal C57BL/6J mice were used as normal control group. Twenty-four male C57BL/6 mice were randomly divided into 4 groups(6 mice in each group): normal control group(NC), normal control+wogonin(NC+WOG 12.5 mg/kg), LPS model group(LPS 10 mg/kg), LPS model+wogonin(LPS 10 mg/kg+WOG 12.5 mg/kg). After LPS intervention for 24 h, serum samples were collected to detect blood creatinine(CRE) and urea nitrogen(BUN) levels. HE staining and PAS staining were performed to observe the degree of renal pathological injury. Immunohistochemistry was performed to detect the degree of expression of inflammatory markers interleukin(IL)-1β, IL-6 and tumor necrosis factor(TNF)-α in renal tissues. PCR was performed to detect the expression of KIM-1 and NGAL in renal tissues. Western blot was performed to detect the changes in protein expression of NF-κB signaling pathway subunits P65 and PP65 in renal tissues. Results Compared with NC group, CRE and BUN levels in LPS group increased(F_CRE=60.90, P<0.001, F_BUN=82.13, P<0.001); compared with LPS group, these indexes decreased in LPS+WOG group(P<0.001). PCR test results showed that compared with the NC group, the expression of KIM-1 and NGAL mRNA was significantly increased in LPS group(F_KIM-1=146.3, P<0.001, F_NGAL=161.2, P<0.001). In contrast, KIM-1 and NGAL mRNA expression was decreased in the LPS+WOG group(P<0.01). Renal histopathological examination showed that compared with the NC group, renal tissues of mice had renal tubular dilatation and inflammatory cell infiltration in LPS group; compared with LPS group, the number of tubular dilatation reduced and inflammatory cell infiltration was reduced in LPS+WOG group(F_HE=721.4, P<0.001; F_PAS=518.9, P<0.001). Immunohistochemical staining showed that the expression of IL-1β, IL-6 and TNF-α was significantly increased in the LPS group; compared with the LPS groups(F_IL-1β=114.6, F_IL-6=108.9, F_TNF-α=251.6, all P<0.001), these indexes decreased in LPS+WOG group(all P<0.01). Further studies using Western blot showed that the NF-κB signaling pathway of LPS-treated mice had been activated and produced a hyperphosphorylated state in comparison to the NC group(F_PP65=13.02, P<0.01), yet this pathway in the LPS+WOG group showed the opposite effect, namely attenuated activity and reduced phosphorylation when the control was LPS(P<0.01). Conclusion WOG effectively blocked the NF-κB signaling pathway in the LPS-induced acute kidney injury model mice, thereby attenuating the inflammatory response and tissue damage in the kidneys of LPS-induced acute kidney injury mice.