Rougan Tongluo Decoction Initiates Neuroprotection Against Cerebral Ischemia by Activating the Endogenous SLC6A8-Creatine-EARS2 Mitochondrial Pathway.

OBJECTIVE

The traditional Chinese medicine formula Rougan Tongluo Decoction (RGTL) was widely used to treat neurological injury after cerebral ischemia, though its specific underlying mechanisms remain unknown. This study investigates the mechanisms via which RGTL helps alleviate cerebral ischemic injury to provide theoretical support for its application in cerebral ischemia treatment.

METHODS

A middle cerebral artery occlusion reperfusion (MCAO/R) rat model was established and treated with RGTL, N-acetylcysteine (NAC), creatine, or sh-EARS2. Network pharmacology and metabolomics were conducted to analyze the key efficacy-related metabolites in the hippocampal tissue. An OGD/R cell model was constructed using PC12 cells and treated with creatine, RGTL-containing serum, sh-SLC6A8, and sh-EARS2. Neuronal damage in the hippocampal tissues was assessed using HE and Nissl staining. Neuronal cell viability, mitochondrial membrane potential, and ROS levels were measured using CCK8, JC-1, and DCFH-DA assays. Mitochondrial damage was determined using transmission electron microscopy. The expression of SLC6A8/EARS2 axis and mitochondrial-related proteins (cytochrome c [Cyt c]) was examined using RT-qPCR and Western blot.

RESULTS

RGTL treatment reduced TNF-α, IL-6, and ROS levels while increasing ATP and JC-1 in brain tissues of MCAO/R rats, thereby alleviating mitochondrial damage. The neuroprotective effects of RGTL were more pronounced than those of NAC. Succinic acid and creatine were identified as active drug ingredients and differential metabolites that may mediate RGTL's therapeutic effects via MMP3, GAMT, SLC6A8, and CASP3. Silencing SLC6A8 abolished the protective effects of creatine against OGD/R-induced neuronal cell apoptosis and mitochondrial damage. Creatine could bind to the EARS2 protein. Cell and animal experiments demonstrated that silencing EARS2 blocked the therapeutic effects of creatine in OGD/R and MCAO/R models, reversing its inhibition of neuronal apoptosis and mitochondrial damage.

CONCLUSION

Creatine mediates the neuroprotective effects of RGTL by binding to EARS2, thus inhibiting mitochondrial damage and neuronal apoptosis to improve ischemic brain injury.