Citation: Huang, S.; Lin, L.; Wang, S.; Ding, W.; Zhang, C.; Shaukat, A.; Xu, B.; Yue, K.; Zhang, C.; Liu, F. Total Flavonoids of Rhizoma Drynariae Mitigates Aflatoxin B1-Induced Liver Toxicity in Chickens via Microbiota- Gut-Liver Axis Interaction Mechanisms. Antioxidants 2023, 12, 819. https://doi.org/10.3390/ antiox12040819 Academic Editors: Fengna Li, Jie Yin, Dan Wang and Sung Woo Kim Received: 12 February 2023 Revised: 21 March 2023 Accepted: 24 March 2023 Published: 28 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Total Flavonoids of Rhizoma Drynariae Mitigates Aflatoxin B1-Induced Liver Toxicity in Chickens via Microbiota-Gut-Liver Axis Interaction Mechanisms Shucheng Huang 1 , Luxi Lin 1 , Shiqiong Wang 2 , Wenli Ding 1 , Chaodong Zhang 1 , Aftab Shaukat 3 , Bowen Xu 1 , Ke Yue 1 , Cai Zhang 4 and Fang Liu 1, * 1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; huang.sc@henau.edu.cn (S.H.); linmo0726@163.com (L.L.) 2 College of Food Science and Technology, Henan Agricultural University, Zhengzhou 450002, China 3 National Center for International Research on Animal Genetics, Breeding and Reproduction (NCIRAGBR), Huazhong Agricultural University, Wuhan 430070, China 4 Laboratory of Environment and Livestock Products, Henan University of Science and Technology, Luoyang 471023, China * Correspondence: liufang.vet@henau.edu.cn; Tel.: +86-139-0383-1328 Abstract: Aflatoxin B1 (AFB1) is a common mycotoxin that widely occurs in feed and has severe hepatotoxic effects both in humans and animals. Total flavonoids of Rhizoma Drynaria (TFRD), a traditional Chinese medicinal herb, have multiple biological activities and potential hepatoprotective activity. This study investigated the protective effects and potential mechanisms of TFRD against AFB1-induced liver injury. The results revealed that supplementation with TFRD markedly lessened broiler intestinal permeability by increasing the expression of intestinal tight junction proteins, as well as correcting the changes in gut microbiota and liver damage induced by AFB1. Metabolomics analysis revealed that the alterations in plasma metabolites, especially taurolithocholic acid, were significantly improved by TFRD treatment in AFB1-exposed chickens. In addition, these metabolites were closely associated with [Ruminococcus], ACC, and GPX1, indicating that AFB1 may cause liver injury by inducing bile acid metabolism involving the microbiota–gut–liver axis. We further found that TFRD treatment markedly suppressed oxidative stress and hepatic lipid deposition, increased plasma glutathione (GSH) concentrations, and reversed hepatic ferroptosis gene expression. Collectively, these findings indicate that ferroptosis might contribute to the hepatotoxicity of AFB1- exposed chickens through the microbiota–gut–liver axis interaction mechanisms; furthermore, TFRD was confirmed as an herbal extract that could potentially antagonize mycotoxins detrimental effects. Keywords: aflatoxin B1; antioxidation; bile acid; Chinese medicinal herb; ferroptosis; gut microbiota 1. Introduction Aflatoxin B1 (AFB1) is the largest proportion of known aflatoxins, with the highest degree of mycotoxin contamination [1]. The International Agency for Research on Cancer (IARC) evaluated AFB1 as one of the most toxic chemical carcinogens known to disrupt the normal function of multiple organs and tissues [2,3]. It has been reported that poultry might be most susceptible to AFB1 toxicity [4]. AFB1 exposure significantly impaired poultry production performance and reproductive performance, such as meat quality, feed intake, body weight, and egg production, which caused great economic losses to the poultry breeding industry [4,5]. Recently, AFB1 was reported to cause liver oxidative stress, inflammation, ferroptosis, and eventually liver cancer [6,7]. Thus, it is important to find some green harmless substances that can prevent or treat AFB1 poisoning. The gastrointestinal tract is the first place that encounters mycotoxins, as the absorp- tion rate of aflatoxin is approximately 90% in poultry [8]. The intestinal mucosal barrier, a semipermeable structure, consists of biological, physical, chemical, and immune barriers Antioxidants 2023, 12, 819. https://doi.org/10.3390/antiox12040819 https://www.mdpi.com/journal/antioxidants