药学学报, 2019, 54(12): 2267-2276
引用本文:
王颖异, 李建萍, 陆静波, 李成曦, 于金高, 张森, 江曙, 郭建明, 段金廒. 黄葵减轻慢性肾病模型大鼠体内尿毒素蓄积的作用及机制研究[J]. 药学学报, 2019, 54(12): 2267-2276.
WANG Ying-yi, LI Jian-ping, LU Jing-bo, LI Cheng-xi, YU Jin-gao, ZHANG Sen, JIANG Shu, GUO Jian-ming, DUAN Jin-ao. Effect and mechanism of Huangkui capsule on reduction of uremic toxin accumulation in an animal model of chronic kidney disease[J]. Acta Pharmaceutica Sinica, 2019, 54(12): 2267-2276.

黄葵减轻慢性肾病模型大鼠体内尿毒素蓄积的作用及机制研究
王颖异, 李建萍, 陆静波, 李成曦, 于金高, 张森, 江曙, 郭建明, 段金廒
南京中医药大学, 江苏省中药资源产业化过程协同创新中心, 江苏 南京 210023
摘要:
尿毒素是慢性肾病(chronic kidney disease,CKD)患者肾功能衰退时在体内大量蓄积的有害物质,会加速CKD疾病进展,现如今缺少经济有效的降低CKD患者体内尿毒素蓄积的治疗方法。黄葵胶囊作为中医临床治疗CKD的有效药物,疗效确切但作用机制未完全明确。本研究考察了黄葵对CKD大鼠体内尿毒素蓄积的影响,并初步探讨了其作用机制。黄葵灌胃CKD模型大鼠,利用UPLC-TQ/MS、16S rDNA测序、HPLC-FLD及肠道细菌体外厌氧培养等手段探讨黄葵对尿毒素及其前体合成的影响及作用机制。动物实验遵循南京中医药大学动物伦理委员会规定。结果显示,黄葵(0.675 g·kg-1)可有效抑制尿毒素类分子硫酸吲哚酚(indoxyl sulfate,IS)在CKD大鼠体内蓄积,大鼠血浆、肝脏及肾脏中IS含量分别降低49.5%、68.9%、40.6%。黄葵不影响IS在宿主肝脏细胞内合成途径,即不影响IS的前体分子吲哚向IS转化的过程。但是,黄葵显著降低模型大鼠肠道中吲哚合成,粪便吲哚含量降低46.4%,提示黄葵干预IS生物合成的作用靶点可能集中在肠道菌群。进一步研究发现,黄葵对CKD模型大鼠肠道内异常升高的肠杆菌科细菌(吲哚合成的主要菌群)的丰度无显著影响,提示黄葵不是通过直接影响吲哚合成相关细菌的丰度来干预吲哚生物合成的。体外实验结果显示,黄葵(4 000、400、40、4 μg·mL-1)剂量依赖性抑制肠道细菌合成吲哚。培养至12 h后,黄葵组细菌色氨酸转运量由83.4 μmol·L-1降低至43.6 μmol·L-1,表明黄葵通过干扰色氨酸向细菌内转运从而抑制吲哚合成。上述研究结果提示,肠道细菌可能是黄葵减轻CKD导致的尿毒素体内蓄积、延缓CKD病情进展的潜在靶点。
关键词:    慢性肾病      尿毒素      硫酸吲哚酚      吲哚      肠道细菌      黄葵     
Effect and mechanism of Huangkui capsule on reduction of uremic toxin accumulation in an animal model of chronic kidney disease
WANG Ying-yi, LI Jian-ping, LU Jing-bo, LI Cheng-xi, YU Jin-gao, ZHANG Sen, JIANG Shu, GUO Jian-ming, DUAN Jin-ao
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources, Nanjing University of Chinese Medicine, Nanjing 210023, China
Abstract:
Uremic toxins are harmful substances that accumulate in the body when the renal function declines in patients with chronic kidney disease (CKD). It is an important factor contributing to accelerated progression of CKD. There is no effective treatment for reducing uremic toxins. As an extensively used medicine for treatment of CKD in the clinic, Huangkui capsule is effective but the mechanism of its action remains unclear. This study investigated the effect of Huangkui on the accumulation of uremic toxins in CKD rats, with the discussion about its mechanism of action. UPLC-TQ/MS was used to detect the accumulation of uremic toxins in CKD rats after oral gavage with Huangkui. 16S rDNA sequencing technology was used to analyze the gut bacteria composition in rats. HPLC-FLD was used to detect the uremic toxins and their molecular precursors in feces. The effect and mechanism of Huangkui on the uremic toxin precursor in gut bacteria were studied by anaerobic culture system in vitro. All procedures were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine. The results showed that Huangkui (0.675 g·kg-1) could effectively inhibit the accumulation of uremic toxin indoxyl sulfate (IS) in CKD rats, with IS concentration in rat's plasma, liver and kidney decreased by 49.5%, 68.9% and 40.6%, respectively. Huangkui didn't affect the metabolic pathway of IS in host liver, didn't intervene the process of the IS precursor molecule indole conversion to IS. Instead, Huangkui significantly decreased the indole content in gut, with the indole in CKD rat's feces decreased by 46.4%, suggesting that the gut bacteria may be a target for intervene IS biosynthesis by Huangkui. Huangkui didn't affect the abundance of enterobacteriaceae bacteria (the main gut flora of indole synthesis) in CKD rats, suggesting that Huangkui didn't interfere with indole biosynthesis by directly affecting the abundance of indole synthesis related bacteria. Huangkui at 4 000, 400, 40, and 4 μg·mL-1 showed a dose-dependent inhibition of the indole production by gut bacteria in vitro. The bacteria tryptophan transport concentration decreased from 83.4 μmol·L-1 to 43.6 μmol·L-1 after co-incubated with Huangkui for 12 h, suggesting that Huangkui inhibited indole production of gut bacteria by interfering with tryptophan transportation. These results indicate that gut bacteria may be a potential target for alleviation of uremic toxin accumulation and for delaying CKD progression.
Key words:    chronic kidney disease    uremic toxin    indoxyl sulfate    indole    gut bacteria    Huangkui capsule   
收稿日期: 2019-04-30
DOI: 10.16438/j.0513-4870.2019-0349
基金项目: 国家自然科学基金资助项目(81473408,81773983);江苏省研究生科研与实践创新计划项目(KYCX18_1626).
通讯作者: 郭建明,Tel/Fax:86-25-85811917,E-mail:njuguo@njucm.edu.cn;段金廒,E-mail:dja@njucm.edu.cn
Email: njuguo@njucm.edu.cn;dja@njucm.edu.cn
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