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目的探讨莱菔硫烷(sulforaphane,SFN)通过诱导核转录因子NF-E2相关因子2(nuclear factor erthroid 2-related factor 2,Nrf2)通路激活对汞所致肝脏氧化损伤的拮抗作用及其机制。方法将60只健康成年SPF级Wistar大鼠按体重随机分成6组,分别为对照(生理盐水)组,DMSO对照组,0.6、1.2、2.4 mg/kg氯化汞染毒组及SFN预处理(2.4 mg/kg氯化汞+2 mg/kg SFN)组,每组10只,雌雄各半。采用皮下注射SFN溶液,其余各组皮下注射生理盐水,DMSO对照组皮下注射DMSO;2 h后,腹腔注射氯化汞溶液,染毒容量均为5 ml/kg,每日1次,连续染毒3 d。测定血清乳酸脱氢酶(lactate dehydrogenase,LDH)及丙氨酸氨基转移酶(alanine transaminase,ALT)活力和肝细胞ROS水平以及肝组织Nrf2、血红素单加氧酶-1(HO-1)、γ-谷氨酰半胱氨酸合成酶催化亚基(γ-GCSh)的m RNA及蛋白的表达水平。结果与对照组比较,各剂量氯化汞染毒组大鼠血清LDH、ALT活力(除0.6 mg/kg氯化汞染毒组ALT活力外)和肝细胞ROS水平以及1.2 mg/kg氯化汞染毒组大鼠肝组织HO-1 m RNA的表达水平和2.4 mg/kg氯化汞染毒组大鼠肝组织Nrf2、HO-1、γ-GCSh m RNA的表达水平及1.2、2.4 mg/kg氯化汞染毒组大鼠肝组织Nrf2、HO-1、γ-GCSh蛋白的表达水平均升高,差异均有统计学意义(P<0.05,P<0.01);且随着氯化汞染毒剂量的升高,大鼠血清LDH、ALT活力和肝细胞ROS水平及肝组织Nrf2、HO-1、γ-GCSh蛋白和m RNA的表达水平均呈逐渐上升的趋势。与2.4 mg/kg氯化汞染毒组比较,SFN预处理组大鼠血清LDH、ALT的活力和肝细胞ROS水平均较低,而肝组织Nrf2、HO-1、γ-GCSh蛋白和m RNA的表达水平均较高,差异均有统计学意义(P<0.05,P<0.01)。结论 SFN可通过诱导Nrf2-ARE通路激活,促使肝细胞Nrf2及其下游抗氧化酶HO-1、γ-GCSh表达,进而清除细胞内过量的ROS,从而拮抗汞所致肝脏氧化应激损伤。
Objective To investigate the antagonistic effect of sulforaphane (SFN) on liver oxidative damage induced by mercury by inducing nuclear factor erthroid 2-related factor 2 (Nrf2) pathway activation and its mechanism. Methods Sixty healthy adult Wistar rats were randomly divided into six groups according to body weight: control (saline) group, DMSO control group, 0.6,1.2,2.4 mg / kg mercuric chloride treatment group and SFN pretreatment group 2.4 mg / kg mercuric chloride + 2 mg / kg SFN) group, 10 in each group, male and female. Subcutaneous injection of SFN solution was used, and the rest groups were injected with normal saline subcutaneously, and DMSO control group was subcutaneously injected with DMSO. After 2 hours, intraperitoneal injection of mercuric chloride solution was administered with a volume of 5 ml / kg once a day 3 d. Serum levels of lactate dehydrogenase (LDH), alanine transaminase (ALT) and the level of ROS in hepatocytes were measured. The levels of Nrf2, HO-1, The mRNA and protein expression levels of γ-glutamylcysteine synthase catalytic subunit (γ-GCSh). Results Compared with the control group, the levels of serum LDH and ALT (except ALT activity in 0.6 mg / kg mercuric chloride group) and the level of ROS in hepatocytes were significantly increased at doses of 1.2 mg / kg The expression level of HO-1 m RNA in liver tissue and the levels of Nrf2, HO-1, γ-GCSh m RNA in liver tissue of 2.4 mg / kg HgCl2-treated rats and 1.2, 2.4 mg / (P <0.05, P <0.01). And with the mercuric chloride treatment group, the expression levels of Nrf2, HO-1 and γ-GCSh in liver tissue were increased The levels of serum LDH, ALT and ROS in hepatocytes and the expressions of Nrf2, HO-1, γ-GCSh proteins and m RNA in liver tissue all increased gradually with the increase of exposure dose. Compared with the 2.4 mg / kg mercuric chloride treatment group, the activity of LDH and ALT in sera of SFN pretreatment group and the level of ROS in hepatocytes were lower than those in the pretreated group, while the levels of Nrf2, HO-1, γ-GCSh and m RNA The expression levels were higher, the differences were statistically significant (P <0.05, P <0.01). Conclusion SFN can induce liver Nrf2 and its downstream antioxidant enzymes HO-1 and γ-GCSh by inducing the activation of Nrf2-ARE pathway, thereby clearing excess intracellular ROS and thereby antagonizing mercury-induced liver oxidative stress injury.