姜黄素对小鼠肾脏再灌注损伤的保护作用及其机制研究

目的 探讨姜黄素对小鼠肾脏缺血再灌注(IR)损伤的保护作用及其可能机制.方法 选择健康雄性C57BL/6小鼠20只,随机均分为4组.假手术(SO)组小鼠仅接受腹中线开腹、游离双侧肾蒂及关腹操作；对照组小鼠制成肾脏IR模型,并于肾脏缺血同时经尾静脉注射生理盐水；姜黄素低剂量(CM-L)及高剂量(CM-H)组小鼠建立肾脏IR模型,并经尾静脉分别注射5mg/kg及20mg/kg姜黄素.再灌注6h后检测各组血清肌酐(Scr)、尿素氮(BUN)水平,观察肾脏组织形态学变化；Western blotting检测肾脏组织内Toll样受体4(TLR-4)、高迁移率族蛋白BI(HMGBI)、透明质烷(HA)蛋白的表达,实时荧光定量RT-PCR检测透明质烷合成酶(HAS)1、HAS2、HAS3以及白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)mRNA的表达；免疫组化检测肾组织内巨噬细胞浸润情况；同时采用ELISA法检测血清IL-6、TNF-α水平.结果 与对照组相比,CM-L及CM-H组小鼠再灌注6h血清Scr、BUN水平明显降低(Scr:对照组235.4±28.7μmol/L,CM-L组167.8±17.2μmol/L,CM-H组125.8±13.3μmol/L; BUN:对照组21.6±2.7mmol/L,CM-L组18.1±2.4mmol/L,CM-H组12.5±1.7mmol/L; P＜0.05或P＜0.01),肾小管上皮细胞损伤情况明显改善(Jablonski评分:对照组2.15±0.28分,CM-L组1.72±0.21分,CM-H组1.42±0.15分,P＜0.01)；肾脏组织内TLR-4、HMGB1的mRNA及蛋白水平,HA、HAS1、HAS2、HAS3以及IL-6、TNF-α mRNA水平均明显降低(P＜0.05或P＜0.01)；巨噬细胞浸润减轻(对照组32.4±4.2个/高倍视野,CM-L组24.8±3.6个/高倍视野,CM-H组18.9±3.1个/高倍视野,P＜0.01)；血清IL-6、TNF-α含量亦显著降低(IL-6:对照组1411.2±150.6pg/ml,CM-L组918.7±113.4pg/ml,CM-H组809.6±108.3pg/ml;TNF-α:对照组154.7±21.1pg/ml,CM-L组135.8±15.2pg/ml,CM-H组125.6±14.6pg/ml; P＜0.05或P＜0.01).结论 姜黄素对小鼠肾脏IR损伤具有明显的保护作用,其机制可能与抑制肾脏再灌注后TLR-4信号活化有关.

Curcumin attenuates renal ischemia reperfusion injury in mice by inhibiting the activation of tolllike receptor 4 signaling

Objective To investigate the protective effect of curcumin on renal ischemia reperfusion(IR) injury and its related mechanism in mice.Methods C57BL/6 mice were randomly divided into 4 groups: sham-operated(SO) group,low-dose and high-dose curcumin(CM-L and CM-H) groups(the mice were injected with 5mg/kg or 20mg/kg curcumin just before ischemia induction.) and control group(the mice were injected with equal volume of physiological saline at the same time.).At a 6-hour reperfusion,the serum and renal samples were collected.The serum creatinine(Scr) and blood urea nitrogen(BUN) levels in serum were determined and renal patho-histological changes were also examined.Expressions of Toll-like receptor 4(TLR-4),high mobility group protein B1(HMGB1),hyaluronan(HA),hyaluronan synthase(HAS) 1,HAS2,HAS3 and interleukin-6(IL-6),tumor necrosis factor-α(TNF-α) and TLR-4 protein were assessed using quantitative fluorescence RT-PCR and Western blotting.The infiltration of F4/80-positive macrophage cells was detected using immunohistochemical analysis and the IL-6 and TNF-α levels in serum were determined by enzyme linked immunosorbent assay(ELISA).Results Compared with control group,CM-L and CM-H group exhibited lower levels of Scr and BUN(Scr: Control 235.4±28.7,CM-L 167.8±17.2,CM-H 125.8±13.3μmol/L;BUN: Control 21.6±2.7,CM-L 18.1±2.4,CM-H 12.5±1.7mmol/L;P<0.05 or P<0.01),less pathological changes(Jablonski scores: Control 2.15±0.28,CM-L 1.72±0.21,CM-H 1.42±0.15;P<0.01),lower mRNA levels of TLR-4,HMGB1,HA,HAS1,HAS2,HAS3,IL-6 and TNF-α,lower levels of TLR-4 protein and HMGB1,less infiltration of macrophage Control 32.4±4.2/high power field(HPF),CM-L 24.8±3.6/HPF,CM-H 18.9±3.1/HPF;P<0.01].Moreover,the serum levels of IL-6 and TNF-α were lower in CM-L and CM-H groups than those in control group(IL-6: Control 1411.2±150.6,CM-L 918.7±113.4,CM-H 809.6±108.3pg/ml;TNF-α: Control 154.7±21.1,CM-L 135.8±15.2,CM-H 125.6±14.6pg/ml;P<0.05 or P<0.01).Conclusion Curcumin can ameliorate renal IR injury in mice,which is attributable to inhibition of the activation of Toll-like receptor 4 signaling.