肠道菌群对知母皂苷A-Ⅲ的代谢及其药效的影响

Impacts of gut microbiota on metabolism and efficacy of timosaponin A-Ⅲ

知母皂苷A-Ⅲ (timosaponin A-Ⅲ,TA-Ⅲ)腹腔注射给药对于高脂饮食诱导的代谢相关脂肪性肝病(metabolic dysfunction-associated steatotic liver disease,MASLD)具有治疗作用,而口服给药则无效,提示肠道菌群可能影响TA-Ⅲ的口服生物利用度。代谢相关脂肪性肝炎(metabolic dysfunction-associated steatohepatitis,MASH)是MASLD发展的炎症浸润阶段。本文将探讨TA-Ⅲ不同给药方式对MASH小鼠的治疗作用差异及其与肠道菌群代谢药物的关系。本研究利用缺乏胆碱、限定L-氨基酸的高脂饮食(choline-deficient,L-amino acid-defined,high-fat diet CDAHFD)诱导MASH小鼠模型,比较TA-Ⅲ (10 mg·kg^(-1))腹腔注射(intraperitoneal injection,ip)和TA-Ⅲ (100 mg·kg^(-1))灌胃给药(intragastric administration,ig)的治疗效果,并分析两种给药方式下大鼠血清中TA-Ⅲ的浓度。在此基础上,通过小鼠肠道菌群体外代谢TA-Ⅲ实验和体内联合抗生素干预小鼠的药代动力学实验等方法验证小鼠肠道菌群对TA-Ⅲ的代谢作用;最后比较不同抗生素干预条件下,灌胃给药后小鼠血清中TA-Ⅲ的浓度,结合16S rRNA测序分析,发现可能参与TA-Ⅲ代谢的关键菌。本文动物实验过程遵循上海中医药大学动物伦理委员会的规定,动物伦理批准编号为:PZSHUTCM2307030004,PZSHUTCM2310200003。结果显示,TA-Ⅲ腹腔注射给药对MASH小鼠有明确治疗作用,而十倍剂量的口服给药无效;分析发现,TA-Ⅲ灌胃给药(100 mg·kg^(-1),ig)在小鼠血清和肝脏中原型浓度显著低于腹腔注射(10 mg·kg^(-1),ip),提示口服TA-Ⅲ后可能经肠道菌群代谢。链霉素(streptomycin,Str)干预的小鼠血清中TA-Ⅲ浓度高于正常小鼠,结合16S rRNA基因测序分析发现Akkermansia_muciniphila (A.muciniphila)丰度在Str组显著减少,且体外实验表明A.muciniphila可以代谢TA-Ⅲ。上述结果表明,肠道菌群是影响TA-Ⅲ经胃肠道给药药效的重要因素,其中A.muciniphila可能扮演了重要角色。

Intraperitoneal administration of timosaponin A-Ⅲ(TA-Ⅲ)has therapeutic effects on high-fat dietinduced metabolic dysfunction-associated steatotic liver disease(MASLD),but oral administration has no effect.This suggests that gut microbiota may affect the oral bioavailability of TA-Ⅲ.Metabolic dysfunction-associated steatohepatitis(MASH)is an inflammatory subtype of MASLD.To investigate the therapeutic effect of different administration modes of TA-Ⅲon MASH and its relationship with gut microbiota metabolism.In this study,a MASH mouse model was induced by choline-deficient,L-amino acid-defined,high-fat diet(CDAHFD).Comparing the therapeutic effect of intraperitoneal injection(10 mg·kg^(-1),ip)and intragastric administration(100 mg·kg^(-1),ig)of TA-Ⅲ.The concentration of TA-Ⅲin serum of rats under the two administration modes was analyzed.On this basis,the metabolic effect of gut microbiota on TA-Ⅲin mice was verified by the experiment of metabolism of gut microbiota in vitro.The pharmacokinetic experiment of combined antibiotic intervention in mice further verified the metabolism of TA-Ⅲby gut microbiota in mice.Finally,the concentration of TA-Ⅲin serum of mice after the administration of TA-Ⅲby intragastric administration under different antibiotic intervention conditions was compared,and 16S rRNA sequencing analysis was combined to find the key bacteria that may participate in the metabolism of TA-Ⅲ.The animal welfare and experimental procedures in this paper were in accordance with the provisions of the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine.The ethics approval number is PZSHUTCM2307030004 and PZSHUTCM2310200003.The results showed that TA-Ⅲ(10 mg·kg^(-1),ip)had definite therapeutic effect on MASH mice,but TA-Ⅲ(100 mg·kg^(-1),ig)was ineffective.The analysis showed that the prototype concentration of TA-Ⅲin serum and liver of mice in TA-Ⅲ(100 mg·kg^(-1),ig)was significantly lower than TA-Ⅲ(10 mg·kg^(-1),ip),suggesting that the oral administration of TA-Ⅲmay be metabolized by gut microbiota.The concentration of TA-Ⅲin serum of streptomycin(Str)treated mice was higher than normal mice.Combined with 16S rRNA gene sequencing analysis,it was found that the abundance of Akkermansia_muciniphila(A.muciniphila)was significantly reduced in the Str group.In vitro experiments showed that A.muciniphila could metabolize TA-Ⅲ.In conclusion,gut microbiota is an important factor affecting the efficacy of TA-Ⅲadministration through the gastrointestinal tract,in which A.muciniphila may play an important role.