发酵虫草菌粉对DEN致大鼠肝损伤的保护作用及TGF-β1/Smads通路的影响

doi:10.12092/j.issn.1009-2501.2018.09.002

中国临床药理学与治疗学 ›› 2018, Vol. 23 ›› Issue (9): 974-982.doi: 10.12092/j.issn.1009-2501.2018.09.002

发酵虫草菌粉对DEN致大鼠肝损伤的保护作用及TGF-β1/Smads通路的影响

何阳¹，宋立莹¹，彭向东¹，方伟进¹，王春江¹，邓珍珍¹，樊志强¹，刘世坤^1,2

¹中南大学湘雅三医院药学部，长沙 410013，湖南；²中南大学湘雅三医院临床药理中心，长沙 410013，湖南

收稿日期:2018-03-13 修回日期:2018-03-29 出版日期:2018-09-26 发布日期:2018-09-26
通讯作者: 刘世坤，男，博士，主任药师，博士生导师，主要从事慢性肝损伤及精准药学服务等相关研究。 Tel：0731-88618496 E-mail：L8618496@126.com
作者简介:何阳，男，硕士研究生，主要从事慢性肝损伤研究。 Tel：13677484808 E-mail：heyang2015@csu.edu.cn
基金资助:
湖南省自然科学基金（2016JJ216）；湖南省卫生计生委科研计划课题项目（B2017029）

Protective effects of Cordyceps sinensis powder on liver injury induced by DEN in rats and the effect of TGF-β1/Smads pathway

HE Yang¹, SONG Liying¹, PENG Xiangdong ¹, FANG Weijin¹, WANG Chunjiang¹, DENG Zhenzhen¹, LIU Shikun ^1,2

¹Department of Pharmacy, the Third Xiangya Hospital, Central South University, Changsha 410013, Hunan, China;² Center of Clinical Pharmacology, the Third Xiangya Hospital, Central South University, Changsha 410013, Hunan, China

Received:2018-03-13 Revised:2018-03-29 Online:2018-09-26 Published:2018-09-26

摘要/Abstract

摘要：

目的: 探讨发酵虫草菌粉（fermented cordyceps sinensis powder，CS）对二乙基亚硝胺（DEN）诱导大鼠肝损伤的保护作用及对TGF-β1/Smads通路的影响。方法: 无特定病原体（SPF级）健康雄性Sprague Dawley（SD）大鼠48只，体质量（180±20） g，随机分为4组：对照组、模型组、CS治疗组与CS溶媒组，每组12只。模型组：灌胃给予10 mg/kg DEN每周5次共8周，构建慢性肝损伤模型；对照组：灌胃给予与模型组相同体积的生理盐水；CS治疗组：在建模4周后灌胃给予80 mg/kg CS每周5次共8周；CS溶媒组：灌胃给予与CS组相同体积的5%甲基纤维素钠（CMC-Na）溶液。实验结束后，麻醉，脊椎脱臼法处死大鼠，收集血液，紫外分光光度法检测各组大鼠血清中AST、ALT以反映肝功能；H&E染色观察肝组织形态变化，Masson染色观察胶原沉积情况；改良Nycodenz法分离各组大鼠原代肝星状细胞（HSC），RT-qPCR检测肝组织与HSC中α-平滑肌肌动蛋白（α-SMA）、转化生长因子（TGF）-β1、Smad3、Smad7 mRNA的表达水平；Western blot检测肝组织Smad3与Smad7蛋白表达水平。结果: 与对照组相比，模型组大鼠血清中AST、ALT含量显著升高，H&E染色结果显示肝脏组织形态显著改变，Masson染色结果显示胶原沉积增多；与模型组相比，CS治疗四周能显著降低肝损伤大鼠血清中升高的AST、ALT水平，改善其肝脏病理损伤程度，减少肝脏中胶原沉积。RT-qPCR结果显示，CS可减少肝损伤大鼠组织及HSC中α-SMA、TGF-β1 mRNA表达水平，增加Smad7 mRNA表达水平；CS溶媒组各项检测指标均与模型组无差异；此外，Western blot结果显示，CS可下调肝损伤大鼠肝组织Smad3蛋白的表达，上调Smad7。结论: CS可显著改善DEN诱导的大鼠肝损伤，该作用可能与CS上调Smad7，并抑制TGF-β1/Smads通路有关。

关键词: 发酵虫草菌粉, 慢性肝损伤, TGF-β1/Smads通路, 肝星状细胞

Abstract:

AIM: To investigate the protective effects of fermented Cordyceps Sinensis powder (CS) on Diethylnitro-samine (DEN) induced liver injury in rats and its effect on TGF-β1/Smads pathway. METHODS: Forty-eight male SD rats［SPF, weighing (180±20) g］ were randomly divided into four groups (12 rats in each group): control group, model group, CS treatment group and CS solvent group; model group were gavaged with DEN (10 mg/kg), 5 times a week for 8 weeks to build a model of chronic liver injury; control group were gavaged with saline with the same volume of the model group; CS treatment group: after 4 weeks of modeling, CS treatment group were gavaged with CS (80 mg/kg), 5 times a week for 8 weeks; CS solvent group were gavaged with 5% sodium carboxymethyl cellulose solution (CMC-Na) with the same volume of the CS group. At the end of the experiment, the rats were sacrificed by spinal dislocation, blood was collected, and the serum AST and ALT were detected by UV spectrophotometry to reflect the liver function; H&E staining was used to observe the morphological changes of liver tissue; Masson staining was used to observe collagen deposition; primary hepatic stellate cells (HSCs) were isolated by modified Nycodenz method; the expression of α-SMA, TGF-β1, Smad3 and Smad7 mRNA in liver tissue and HSC were detected by RT-qPCR; expression of Smad3 and Smad7 protein in liver tissue were detected by Western blot. RESULTS:Compared with the control group, the levels of AST and ALT in the serum of the model group were significantly increased, the morphology of the liver was significantly changed by H&E staining, and Masson staining showed that collagen deposition increased; compared with the model group, CS treatment could significantly reduce serum AST and ALT levels, improve liver pathological damage and reduce collagen deposition in the liver. RT-qPCR results showed that CS could reduce the expression of α-SMA, TGF-β1, Smad3 mRNA and increase expression of Smad7 mRNA in liver tissue and HSC of rats with liver injury. In addition, Western blot results showed that CS could reduce the expression of Smad3 protein and increase Smad7 expression in the liver tissue of rats with liver injury. CONCLUSION:CS can significantly improve DEN induced hepatic injury in rats, which may be related to the up-regulation of Smad7 by CS and the inhibition of TGF-β1/Smads pathway.

Key words: fermented Cordyceps Sinensis powder, chronic liver damage, TGF-β1/Smads pathway, hepatic stellate cell

中图分类号:

R965.2

何阳，宋立莹，彭向东，方伟进，王春江，邓珍珍，樊志强，刘世坤. 发酵虫草菌粉对DEN致大鼠肝损伤的保护作用及TGF-β1/Smads通路的影响[J]. 中国临床药理学与治疗学, 2018, 23(9): 974-982.

HE Yang, SONG Liying, PENG Xiangdong, FANG Weijin, WANG Chunjiang, DENG Zhenzhen, LIU Shikun. Protective effects of Cordyceps sinensis powder on liver injury induced by DEN in rats and the effect of TGF-β1/Smads pathway[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2018, 23(9): 974-982.

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发酵虫草菌粉对DEN致大鼠肝损伤的保护作用及TGF-β1/Smads通路的影响

Protective effects of Cordyceps sinensis powder on liver injury induced by DEN in rats and the effect of TGF-β1/Smads pathway

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