三七皂苷Rg1对大鼠肾缺血再灌注损伤氧化应激水平和外周血Th1/Th2细胞因子调节作用

Regulative effect of notoginsenoside Rg1 on oxidative stress and th1/th2 cytokines in peripheral blood in rats with renal ischemia reperfusion injury

目的探讨三七皂苷Rg1(NRg1)对大鼠肾缺血再灌注损伤(RIRI)的保护作用及其对氧化应激水平和外周血Th1/Th2细胞因子的调节作用。方法健康雄性SD大鼠60只,随机分为假手术组、模型组及NRg12.5、5.0、10.0mg/kg组,每组12只;建立RIRI模型再灌注后24h后处死大鼠,检测尿蛋白及血清肌酐和尿素氮含量,苏木精–伊红(HE)染色和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)染色检测各组大鼠肾脏组织病理损伤和细胞凋亡,酶联免疫吸附实验(ELISA)检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)含量,蛋白印迹法检测凋亡相关蛋白B细胞淋巴瘤因子2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达及核转录相关因子2(Nrf2)、谷胱甘肽–S–转移酶(GST)和醌氧化还原酶1(NQO1)的表达,流式细胞仪检测外周血中Th1(iNOS+)和Th2(IL-10+)细胞因子的含量。结果与模型组比较,NRg12.5、5.0、10.0mg/kg组大鼠尿蛋白含量[分别为(34.1±6.3)、(25.2±4.5)、(11.7±3.4)mg/24h]均减少、血清肌酐含量[分别为(1.04±0.15)、(0.84±0.09)、(0.62±0.12)mg/dL]均减少、尿素氮含量[分别为(28.6±4.6)、(19.4±3.4)、(15.2±3.2)mg/dL]均减少、肾脏细胞凋亡率[分别为(43.6±7.5)%、(32.4±8.1)%、(11.7±6.3)%]均降低、Bcl-2蛋白表达[分别为(0.12±0.03)、(0.07±0.02)、(0.04±0.01)]均减少、Bax蛋白表达[分别为(0.06±0.03)、(0.09±0.04)、(0.16±0.04)]均增加、GSH-Px含量[分别为(55.29±9.31)、(80.14±11.58)、(106.41±14.16)U/mL]均增加、SOD含量[分别为(0.71±0.13)、(1.26±0.15)、(1.50±0.23)U/mL]均增加、Nrf2蛋白表达[分别为(0.04±0.03)、(0.10±0.04)、(0.18±0.05)]均增加、GST蛋白表达[分别为(0.07±0.02)、(0.14±0.03)、(0.22±0.06)]均增加、NQO1蛋白表达[分别为(0.04±0.02)、(0.09±0.04)、(0.14±0.05)]均增加、Th1(iNOS+)含量[分别为(9.13±1.05)%、(5.26±0.84)%、(2.63±0.61)%]均减少、Th2(IL-10+)含量[分别为(0.92±0.34)%、(2.93±0.57)%、(4.41±0.62)%]均增加(均P<0.01)。结论NRg1能通过减轻氧化应激水平和炎性损伤,改善大鼠肾缺血再灌注造成的损伤。

Objective To investigate protective effect of notoginsenoside Rg1 (NRg1) on renal ischemia-reperfusion injury (RIRI) in rats and its regulative effect on oxidative stress level and Th1/Th2 cytokines in peripheral blood. Methods Sixty healthy male Sprague-Dawley (SD) rats were randomly divided into a sham-operated group, a model group and three NRg1 groups with 2.5, 5.0, 10.0 mg/kg gastric gavage (12 rats in each group). All the rats were sacrificed 24 hous after the establishment of RIRI model. The contents of urine protein, serum creatinine and urea nitrogen were measured. Pathological damage and apoptosis of renal tissues were detected with hematoxylin-eosin (HE) and terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. The contents of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected with enzyme-linked immunosorbent assay (ELISA). The expressions of B-cell cll/lymphoma-2 (Bcl-2), associated X protein (Bax), nuclear factor-erythroid 2-related factor-2 (Nrf2), glutathione S-transferase (GST) and NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) were detected with Western blot. The contents of Th1 (inducible nitric oxide synthase positive, iNOS+) and Th2 (interleukin 10 positive, IL-10+) cytokines in peripheral blood were detected with flow cytometry. Results Compared with the model group, the rats with treatment of 2.5, 5.0 and 10.0 mg/kg NRg1 exhibited significantly decreased urinary protein (34.1 ± 6.3, 25.2 ± 4.5 and 11.7 ± 3.4 mg/ 24 hours), serum creatinine (1.04 ± 0.15, 0.84 ± 0.09 and 0.62 ± 0.12 mg/dL), urea nitrogen (28.6 ± 4.6, 19.4 ± 3.4 and 15.2 ± 3.2 mg/dL), renal cell apoptosis rate (43.6 ± 7.5%, 32.4 ± 8.1% and 11.7 ± 6.3%), Bcl-2 protein expression (0.12 ± 0.03, 0.07 ± 0.02 and 0.04 ± 0.01), and Th1 of iNOS+(9.13 ± 1.05 %, 5.26 ± 0.84% and 2.63 ± 0.61%), but increased Bax protein (0.06 ± 0.03, 0.09 ± 0.04 and 0.16 ± 0.04), GSH-Px (55.29 ± 9.31, 80.14 ± 11.58 and 106.41 ± 14.16 U/mL), SOD (0.71 ± 0.13, 1.26 ± 0.15 and 1.50 ± 0.23 U/mL), Nrf2 (0.04 ± 0.03, 0.10 ± 0.04 and 0.18 ± 0.05), GST (0.07 ± 0.02, 0.14 ± 0.03 and 0.22 ± 0.06), GST (0.07 ± 0.02, 0.14 ± 0.03 and 0.22 ± 0.06), NQO1 (0.04 ± 0.02, 0.09 ± 0.04 and 0.14 ± 0.05), and Th2 of IL-10+(0.92 ± 0.34%, 2.93 ± 0.57 and 4.41 ± 0.62%), respectively (all P < 0.01). Conclusion NRg1 can ameliorate renal ischemia-reperfusion injury by reducing oxidative stress and inflammatory injury in rats.