Zhilong Huoxue Tongyu capsule (ZHTC) is an effective traditional Chinese medicine compound for the treatment of ischemic stroke, which is widely used in clinical ischemic stroke patients. However, it is uncertain whether ZHTC affects ischemic stroke through gut microbiota and serum metabolites. In this study, a rat model of middle cerebral artery occlusion (MCAO) was prepared. By evaluating motor nerve function score, cerebral infarct size, brain tissue damage and intestinal barrier damage, it was found that ZHTC improved stroke-related symptoms in MCAO rats. Using 16S rRNA gene sequencing, fecal microbial transplantation (FMT), untargeted metabolomics, and spearman correlation analysis of gut microbiota and serum metabolites, we found that ZHTC can regulate the abundance of p_Firmicutes, p_Bacteroidota,p_Proteobacteria, g_Prevotella, and g_Lactobacillus, and regulated 23 differential metabolites. Spearman correlation analysis found that Arginine was positively correlated with p_Firmicutes, o_Clostridiales, c_Clostridia, and negatively correlated with p_Bacteroidetes, c_Bacteroidia,o_Bacteroidales; L-Lysine was negatively correlated with f_Christensenellaceae; L-methionine was positively correlated with o_Lactobacillales, f_Lactobacillaceae, and g_Lactobacillus. Altogether, this study shows for the first time that ZHTC can ameliorate ischemic stroke by modulating gut microbiota and metabolic disturbances. This lays the foundation for further revealing the causal relationship between ZHTC, gut dysbiosis, plasma metabolite levels and ischemic stroke, and provides a scientific explanation for the ameliorating effect of ZHTC on ischemic stroke.

Background: One of the severely debilitating and fatal subtypes of hemorrhagic stroke is intracerebral hemorrhage (ICH), which lacks an adequate cure at present. The Zhilong Huoxue Tongyu (ZLHXTY) capsule has been utilized effectively since last decade to treat ICH, in some provinces of China but the scientific basis for its mechanism is lacking. Purpose: To investigate the neuroprotective role of ZLHXTY capsules for ICH-induced oxidative injury through the regulation of redox imbalance with the Nrf2 signaling pathway. Methods: Autologous blood injection model of ICH in C57BL/6J mice was employed. Three treatment groups received ZLHXTY once daily through oral gavage at doses 0.35 g/kg, 0.7 g/kg, and 1.4 g/kg, started after 2 h and continued for 72 h of ICH induction. The neurological outcome was measured using a balance beam test. Serum was tested for inflammatory markers IL-1β, IL-6, and TNF-α through ELISA, oxidative stress through hydrogen peroxide content assay, and antioxidant status by total antioxidant capacity (T-AOC) assay. Nuclear extract from brain tissue was assayed for Nrf2 transcriptional factor activity. RT-qPCR was performed for Nfe2l2, Sod1, Hmox1, Nqo1, and Mgst1; and Western blotting for determination of protein expression of Nrf2, p62, Pp62, Keap, HO1, and NQO1. Fluoro-jade C staining was also used to examine neuronal damage. Results: ZLHXTY capsule treatment following ICH demonstrated a protective effect against oxidative brain injury. Neurological scoring showed improvement in behavioral outcomes. ELISA-based identification demonstrated a significant decline in the expression of serum inflammatory markers. Hydrogen peroxide content in serum was found to be reduced. The total antioxidant capacity was also reduced in serum, but the ZLHXTY extract showed a concentration-dependent increase in T-AOC speculating at its intrinsic antioxidant potential. Nrf2 transcriptional factor activity, mRNA and protein expression analyses revealed normalization of Nrf2 and its downstream targets, which were previously elevated as a result of oxidative stress induced by ICH. Neuronal damage was also reduced markedly after ZLHXTY treatment as revealed by Fluoro-jade C staining. Conclusion: ZLHXTY capsules possess an intrinsic antioxidant potential that can modulate the ICH-induced redox imbalance in the brain as revealed by the normalization of Nrf2 and its downstream antioxidant targets.