慈姑多糖激活Nrf2对异烟肼和利福平联用致肝损伤保护作用的体内外实验研究  

In vivo and in vitro experimental study on the protective effect of Nrf2 activated by Sagittaria Sagittifolia polysaccharides on liver injury caused by isoniazid and rifampicin in combination

作　　者：刘红双 王晶 张园园[3] 张烯 廖艳 LIU Hong-shuang;WANG Jing;ZHANG Yuan-yuan;ZHANG Xi;LIAO Yan(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102488,China;Bai-Zhi-Fang Community Health Service Center,Xicheng District,Beijing 100054,China;School of Chinese Herbal Medicine,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中医学院,北京102488 [2]北京市西城区白纸坊社区卫生服务中心,北京100054 [3]北京中医药大学中药学院,北京102488

出　　处：《中华中医药杂志》2022年第2期1112-1117,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.81503633);北京中医药新奥奖励基金项目(No.1000062720044/004);北京中医药大学校级科研纵向发展基金项目(No.2018-2XF2JJ-030)。

摘　　要：目的:从体内外两方面探讨慈姑多糖(SSP)是否通过激活核因子相关因子2(Nrf2)信号通路实现异烟肼(INH)和利福平(RFP)联用诱导肝损伤的保护作用。方法:将48只雄性BALB/C小鼠分为6组:正常对照组,INH/RFP模型组,水飞蓟素阳性对照组和SSP低、中、高剂量组,每组8只。应用INH与RFP联合灌胃建立肝损伤模型,同时进行SSP灌胃干预30 d后,应用HE染色观察小鼠肝脏损伤情况,并确定SSP最佳给药剂量;免疫组化检测对照组、模型组及SSP最佳剂量组小鼠肝细胞Nrf2、血红素加氧酶-1(HO-1)含量;体外实验应用MTT法检测SSP对HepG2细胞的无毒浓度;ELISA法检测HepG2细胞中Nrf2、Kelch样环氧氯丙烷相关蛋白-1(Keap1)、促凋亡蛋白(Bax)、抑制凋亡蛋白(Bcl-2)的浓度;RT-PCR法检测SSP对转染siRNA细胞中Nrf2 mRNA的表达。结果:HE染色结果显示,与模型组比较,SSP给药组肝脏渗出性病变损伤明显减轻,其中SSP高剂量给药组损伤改善最明显;免疫组化结果显示,与模型组比较,SSP组可显著增加肝细胞中Nrf2及HO-1的表达(P<0.05)。在体外ELISA实验中,与对照组比较,模型组HepG2细胞中Nrf2、Bax、Bcl-2浓度升高,Keap1浓度降低;与模型组比较,SSP给药组能显著提高Nrf2活性,并降低Keap1浓度,激活抗凋亡因子Bcl-2活性,降低促凋亡基因Bax的活性(P<0.05)。转染siRNA细胞后,与模型siRNA组比较,SSP siRNA组可显著促进细胞内Nrf2 mRNA含量(P<0.05)。结论:SSP能有效改善INH和RFP联用导致的小鼠渗出性肝损伤情况,并可能通过激活Nrf2信号通路及调节凋亡因子含量,发挥保护作用。Objective: To explore whether Sagittaria Sagittifolia polysaccharide(SSP) can activate the Nrf2 signaling pathway to achieve the protective effect of isoniazid(INH) and rifampin(RFP) inducing liver injury from both in vivo and in vitro.Methods: Forty-eight male BALB/C mice were divided into 6 groups: control group, INH/RFP model group, Silymarin positive control group and SSP low, medium and high dose groups, each with 8 mice. INH and RFP were combined to establish liver injury model by intragastric administration. After 30 days of intervention with SSP polysaccharide intragastric intervention, HE staining was used to observe the liver damage in mice and determine the optimal dose of SSP;Immunohistochemistry experiment to detect the contents of Nrf2 and HO-1 in the liver cells of mice in control group and model group and the optimal SSP dose group;in vitro experiments used MTT method to detect the non-toxic concentration of SSP on HepG2 cells;ELISA experiment to detect Nrf2, Keap1, Bax, Bcl-2 in HepG2 cells of concentration change;RT-PCR method detect the expression of Nrf2mRNA in SSP transfected siRNA cells. Results: HE staining results showed that compared with the model group, the liver exudative lesions in the SSP administration group were significantly reduced, and the high-dose SSP group had the most significant improvement;the Immunohistochemical results showed that compared with the model group, the SSP administration group can significantly increase the levels of Nrf2 and HO-1 in the nucleus of liver cells(P<0.05). In the in vitro ELISA experiment, compared with the control group, the concentration of Nrf2 and Bcl-2 and Bax in the model group HepG2 cells increased, and the concentration of Keap1 decreased;compared with the model group, the SSP group could significantly increase the activity of Nrf2 and decrease concentration of Keap1 and activates the activity of the anti-apoptotic factor Bcl-2 and reduces the activity of the pro-apoptotic gene Bax(P<0.05). After transfection of siRNA cells, compared wit

关 键 词：慈姑多糖 异烟肼 利福平 肝损伤 HepG2细胞 NRF2 免疫组化 

分 类 号：R285.5[医药卫生—中药学]