Lobetyolin alleviates microglial inflammation by activating CK2α/Opa1-mediated mitochondrial fusion in ischemic stroke.

Abstract

BACKGROUND: The inflammatory response triggered by mitochondrial damage is considered one of the key pathogenic mechanisms of ischemic stroke (IS). Lobetyolin (LBT), the main active component of Codonopsis Radix, has pharmacological potential for inhibiting neuroinflammation, but its underlying mechanisms has not been fully defined. PURPOSE: The aim of this study was to investigate the neuroinflammatory inhibitory effect of LBT on mitochondrial disorders in IS and its underlying mechanisms. METHODS: In this study, key targets and biological pathways of IS were determined by RNA sequencing, and network pharmacology was used to predict key drug-disease targets. We established a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model and an oxygen-glucose deprivation/reperfusion (OGD/R) cellular model in vivo and in vitro. 2,3,5-Triphenyltetrazolium chloride (TTC) staining, cerebral blood flow measurements, neurological behavioural scoring and behavioural testing were performed to elucidate the neuroprotective effects of LBT. Moreover, the mitochondrial membrane potential and reactive oxygen species (ROS) levels were measured to assess mitochondrial function. Mechanistically, molecular docking experiments, CETSA, DARTS and inhibitor experiments were performed to elucidate the potential mechanism by which LBT treats IS. RESULTS: LBT inhibited inflammatory responses, maintained mitochondrial function, promoted Opa1-mediated mitochondrial fusion, and exerted neuroprotective effects on IS in vivo and in vitro. The inhibition of Opa1 expression weakened the inhibitory effect of LBT on neuroinflammation in OGD/R-induced Bv2 cells. Mechanistically, the results of molecular docking, CETSA, and DARTS experiments revealed that CK2α is a direct target of LBT and interacts with Jak2, leading to the phosphorylation and activation of the Jak2-Stat3 signalling pathway. The inhibition of CK2α attenuated the phosphorylation of Stat3 and Jak2 by LBT and promoted Opa1-mediated mitochondrial fusion and mitochondrial function. CONCLUSIONS: This study demonstrated that LBT alleviates neuroinflammation by directly targeting CK2α and promoting Opa1-mediated mitochondrial fusion, which is a previously unrecognized mechanism underlying its neuroprotective effects. These findings revealed the potential of LBT as a therapeutic agent targeting CK2α/Opa1 for IS treatment, providing insights for the development of new strategies.