The Effect and Mechanism of Zhuang Medicine Shuanglu TongnaoFormula in the Treatment of Cerebral Ischemia/Reperfusion Injury

Introduction: Ischemic stroke (IS), characterized by high rates of disability and mortality, is among the most severe cerebrovascular diseases affecting the elderly. Shuanglu Tongnao Formula (SLT), a traditional Chinese Zhuang medicine, has been proposed as a prospective therapeutic agent for IS. However, the potential mechanism of the treatment of IS by SLT remains largely unknown. This study aimed to evaluate the therapeutic effect of SLT on IS and its possible mechanisms. Methods: The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established to induce focal cerebral ischemia/reperfusion (I/R) injury in male Sprague Dawley rats. Infarct volume was measured by 2,3,5-triphenyltetrazolium chloride staining. Ischemic brain tissue damage was evaluated by Haematoxylin-eosin staining and transmission electron microscopy. The serum levels of inflammation markers (TGF-β1 and TNF-α) and oxidative stress markers (ROS and MDA) were examined. Apoptotic markers (Bad, Cleaved Caspase-3, and Bcl-2), Blood-brain barrier (BBB) proteins (ZO-1, Occludin, and Claudin-5), and the activation of the TLR4/NF-κB signaling pathway were detected by Western blot (WB). The effect of SLT on gene expression of ischemic brain tissue was analyzed by RNA sequencing. Results: SLT attenuated I/R injury as evidenced by the reduction in the cerebral infarct volume and amelioration of the neurological deficits. Moreover, SLT mitigated cerebral histopathological damage, abnormal mitochondrial morphology, inflammation, and oxidative stress after cerebral I/R in rats. Additionally, SLT attenuated apoptosis and recovered BBB integrity in cerebral I/R rats. RNA sequencing revealed that SLT may alleviate cerebral I/R injury by regulating inflammation and oxidative stress signaling pathways in rats. WB results showed that SLT downregulated the protein expression of TLR4, p-NF-κB, and p-IκB after cerebral I/R in rats. Discussion: SLT can alleviate cerebral I/R injury in rats. Mechanistically, SLT may inhibit the activation of the TLR4/NF-κB signaling pathway, thereby limiting downstream inflammatory and oxidative stress cascades. These findings suggested that SLT exerts neuroprotective effects through anti-inflammatory and antioxidant mechanisms Conclusion: SLT could alleviate cerebral I/R injury by inhibiting TLR4/NF-κB signaling pathways. Our study will enrich the theo