Tanshinone Ⅰ Alleviates Sepsis Associated Acute Kidney Injury in Rats by Regulating Wnt/β-catenin Signaling Pathway
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摘要:
目的 探讨丹参酮Ⅰ（tanshinone Ⅰ，Tan Ⅰ）调节Wnt/β-catenin信号通路对脓毒症相关急性肾损伤（sepsis associated acute kidney injury，SA-AKI）大鼠的作用。 方法 将Sprague Dawley大鼠随机分为Sham、SA-AKI、SA-AKI+5 mg/kg Tan Ⅰ、SA-AKI+10 mg/kg Tan Ⅰ、SA-AKI+15 mg/kg Tan Ⅰ（SA-AKI+Tan Ⅰ）、SA-AKI+盐霉素钠（Wnt信号抑制剂，salinomycin sodium，SS，SA-AKI+SS组）、SA-AKI+SS+Tan Ⅰ、SA-AKI+Wnt信号激活剂（laduviglusib，LG，SA-AKI+LG组）、SA-AKI+LG+Tan Ⅰ，每组8只（含每组2只备用）。利用盲肠结扎穿孔术（cecal ligation and puncture，CLP）诱导大鼠SA-AKI模型，Tan Ⅰ、SS和LG经腹腔注射至大鼠。苏木精-伊红和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法染色观察大鼠肾脏组织病理损伤，酶联免疫吸附实验检测中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin，NGAL）、IL-1β、IL-8、IL-6、TNF-α血清浓度，肌酐（Creatinine，Cre）和血尿素氮（blood urea nitrogen，BUN）试剂盒检测Cre和BUN血清浓度。Western blot和免疫荧光染色检测Wnt1、GSK3β和β-catenin的表达及荧光强度。 结果 10 mg/kg和15 mg/kg的Tan Ⅰ可明显减轻SA-AKI大鼠肾脏损伤（P < 0.05），抑制SA-AKI标志物NGAL、Cre和BUN和炎性细胞因子水平（P < 0.05），减少细胞凋亡，抑制Wnt1、GSK3β并促进β-catenin表达（P < 0.05）。5 mg/kg Tan Ⅰ对大鼠SA-AKI具有一定保护作用，但与Sham组比较，无统计学差异（P > 0.05）。SS处理可减弱CLP诱导的大鼠肾损伤和炎性细胞因子的产生（P < 0.05），LG处理进一步加剧了CLP诱导的大鼠肾损伤（P < 0.05）。Tan Ⅰ可逆转LG对SA-AKI大鼠肾损伤的促进作用（P < 0.05）。 结论 Tan Ⅰ通过抑制Wnt/β-catenin信号通路，对CLP诱导的大鼠SA-AKI具有保护作用。 -
关键词:
- 脓毒症相关急性肾损伤 /
- 丹参酮Ⅰ /
- Wnt信号通路 /
- 肾功能 /
- 炎性细胞因子
Abstract:Objective To investigate the effect of Tan Ⅰ on SA-AKI in rats by mediating Wnt/β-catenin signaling pathway. Methods Sprague-Dawley rats were randomly divided into the following groups （n = 8 per group, including 2 reserve animals per group）: Sham, SA-AKI, SA-AKI + 5 mg/kg Tan I, SA-AKI + 10 mg/kg Tan I, SA-AKI + 15 mg/kg Tan I （SA-AKI+Tan I）, SA-AKI + salinomycin sodium （SS, Wnt signal inhibitor, SA-AKI + SS）, SA-AKI + SS + Tan I, SA-AKI + laduviglusib （LG, Wnt signal activator, SA-AKI + LG）, and SA-AKI + LG + Tan I. Rat SA-AKI model was induced by cecal ligation and puncture （CLP）, with Tan I, SS, and LG administered via intraperitoneal injection. Hematoxylin-eosin and TUNEL staining were used to observe renal tissue pathological damage. Enzyme-linked immunosorbent assay was used to detect serum concentrations of neutrophil gelatinase-associated lipocalin （NGAL）, IL-1β, IL-8, IL-6, and TNF-α. Creatinine （Cre） and blood urea nitrogen （BUN） kit were used to detect serum Cre and BUN concentrations. Western blot and immunofluorescence staining were used to detect the expression and fluorescence intensity of Wnt1, GSK3β, and β-catenin. Results Administration of Tan I at doses of 10 mg/kg and 15 mg/kg significantly attenuated renal injury in rats with SA-AKI （P < 0.05）, suppressed the levels of SA-AKI biomarkers NGAL, Cre, and BUN and pro-inflammatory cytokines （P < 0.05）, reduced apoptosis, and downregulated Wnt1 and GSK3β while upregulating β-catenin expression （P < 0.05）. Although Tan I at 5 mg/kg exhibited a modest protective effect against SA-AKI in rats, no statistically significant difference was observed compared to the sham group （P > 0.05）. SS weakened CLP-induced kidney injury and the production of inflammatory cytokines in rats （P < 0.05）, and LG further aggravated CLP-induced kidney injury in rats （P < 0.05）. Tan Ⅰ reversed the promoting effect of LG on kidney injury in SA-AKI rats （P < 0.05）. Conclusion Tan Ⅰ provides a protective effect on CLP-induced SA-AKI rat by inhibiting Wnt/β-catenin signaling pathway. -
图 1 Tan Ⅰ减轻SA-AKI大鼠肾损伤
A：大鼠肾组织H&E染色（20×）；B：肾组织损伤评分；C~D：试剂盒检测大鼠血清中血Cre和BUN的含量；E~I：ELISA检测大鼠血清中NGAL、IL-1β、IL-8、IL-6、TNF-α浓度；J：大鼠肾组织TUNEL染色（20×）；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 1. Tan Ⅰ attenuates renal injury in SA-AKI rats
图 2 Tan Ⅰ抑制Wnt/β-catenin信号通路蛋白表达
A～B：Western blot检测5 mg/kg、10 mg/kg和15 mg/kg Tan Ⅰ对Wnt1、GSK3β和β-catenin蛋白表达的作用；C：5 mg/kg、10 mg/kg和15 mg/kg Tan Ⅰ处理后Wnt1、GSK3β和β-catenin免疫荧光染色结果（40×）；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 2. Tan Ⅰ represses the proteins expression of Wnt/β-catenin signalling pathway
图 3 Tan Ⅰ抑制Wnt/β-catenin信号通路蛋白表达
A～B：Western blot检测Wnt1、GSK3β和β-catenin蛋白表达；C：Wnt1、GSK3β和β-catenin免疫荧光染色结果（40×）；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 3. Tan Ⅰ represses the expression of Wnt/β-catenin signalling pathway
图 4 Tan Ⅰ通过Wnt/β-catenin信号通路缓解大鼠SA-AKI
A：大鼠肾组织H&E染色（20×）；B：肾脏组织损伤评分；C~D：生化试剂盒检测大鼠血清中血Cre和BUN的含量；E~I：ELISA试剂盒检测大鼠血清中NGAL、IL-1β、IL-8、IL-6、TNF-α浓度；J：大鼠肾组织TUNEL染色（20×）；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 4. Tan Ⅰ alleviates SA-AKI in rats by regulating Wnt/β-catenin signalling pathway
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