白肉灵芝通过抑制Axin2/β-catenin信号通路减轻小鼠急性肾损伤

Ganoderma leucocontextum attenuates acute kidney injury in mice via in⁃hibiting Axin2/β-catenin signaling pathway

目的:探究白肉灵芝提取物(Ganoderma leucocontextum extract,GLE)对顺铂诱导的小鼠急性肾损伤(acute kidney injury,AKI)及脂多糖(lipopolysaccharide,LPS)诱导的细胞炎症的作用及其机制。方法:8周龄雄性C57BL/6小鼠随机分为对照组、AKI组、低剂量(100 mg/kg)GLE组、高剂量(300 mg/kg)GLE组和槲皮素(100 mg/kg)阳性对照组,每组6只。腹腔注射20 mg/kg顺铂溶液建立小鼠AKI模型,给予GLE干预3 d,检测小鼠血清肌酐(serum creatinine,SCr)和血尿素氮(blood urea nitrogen,BUN)水平;HE和PAS染色观察肾脏病理情况;免疫组化检测各组β-连环蛋白(β-catenin)及Axin2蛋白的表达;Western blot和RT-qPCR检测各组白细胞介素1β(interleukin-1β,IL-1β)、IL-6、β-catenin及Axin2的表达。2 mg/L LPS刺激TCMK1细胞模拟细胞炎症损伤,GLE(0.2、0.4、0.6和0.8 g/L)处理24 h,检测各组IL-1β、IL-6、β-catenin及Axin2蛋白的表达,并进一步过表达Axin2正向验证上述指标的变化情况。结果:(1)高剂量GLE干预可显著降低小鼠SCr(P<0.01)和BUN(P<0.05)水平;(2)AKI组小鼠出现肾小管扩张和肾小管上皮细胞坏死、空泡化等病理表现,GLE干预后肾脏损害有所改善;(3)免疫组化显示AKI组小鼠肾脏Axin2和β-catenin蛋白表达增加,GLE干预可降低其表达;(4)体内外Western blot和RT-qPCR结果显示GLE干预可显著抑制IL-1β、IL-6、Axin2及β-catenin蛋白表达和mRNA水平(P<0.05);(5)细胞过表达Axin2后,GLE对IL-1β、IL-6、Axin2及β-catenin的下调作用被拮抗,IL-1β、IL-6、Axin2及β-catenin蛋白表达均显著上调(P<0.01)。结论:白肉灵芝可通过抑制Axin2/β-catenin信号通路保护顺铂诱导的小鼠肾损伤,缓解AKI小鼠和LPS所致细胞的炎症反应。

AIM:To investigate the effect of Ganoderma leucocontextum extract(GLE)on mice with cisplatin-induced acute kidney injury(AKI)and lipopolysaccharide(LPS)-induced cellular inflammation.METHODS:Eight-week-old male C57BL/6 mice were randomly divided into control group,AKI group,low-dose(100 mg/kg)GLE group,high-dose(300 mg/kg)GLE group,and quercetin(100 mg/kg)group,with 6 mice in each group.The AKI model was es-tablished by intraperitoneal injection of a 20 mg/kg cisplatin solution.After GLE intervention for 3 d,serum creatinine(SCr)and blood urea nitrogen(BUN)levels were measured.Renal pathology was observed using HE and PAS staining.The expression ofβ-catenin and Axin2 protein in each group was detected by immunohistochemistry.The expression lev-els of interleukin-1β(IL-1β),IL-6,β-catenin and Axin2 in each group were detected by Western blot and RT-qPCR.The TCMK1 cells were stimulated with 2 mg/L LPS to simulate cellular inflammatory injury.After GLE treatment(0.2,0.4,0.6 and 0.8 g/L)for 24 h,the expressions of IL-1β,IL-6,β-catenin and Axin2 in each group were detected.Fur-ther overexpression of Axin2 was used to verify the changes in the above-mentioned indices.RESULTS:High doses of GLE significantly reduced SCr(P<0.01)and BUN(P<0.05)levels compared with the AKI mice.AKI mice showed re-nal tubule dilatation,tubular epithelial cell necrosis,vacuolation,and other pathological manifestations,which were im-proved after GLE intervention.Immunohistochemistry showed increased expression of Axin2 andβ-catenin protein in the kidneys of AKI mice,which was reduced by GLE intervention.Western blot and RT-qPCR results in vitro and in vivo showed that GLE intervention significantly inhibited the expression and mRNA levels of IL-1β,IL-6,Axin2 andβ-catenin(P<0.05).Overexpression of Axin2 antagonized the effect of GLE on IL-1β,IL-6,Axin2 andβ-catenin,resulting in sig-nificantly up-regulated expressions of these proteins and mRNAs(P<0.01).CONCLUSION:GLE significantly allevi-ates the inflammatory response in AKI mice and LPS-induced cells,and protects against cisplatin-induced kidney injury in mice by inhibiting the Axin2/β-catenin signaling pathway.