Mechanisms of polygalasaponin F against brain ischemia-reperfusion injury by targeting NKCC1

Stroke is a serious threat to human health and current clinical therapies remain unsatisfactory. Elevated expression of Na⁺-K⁺-2Cl^- cotransporter 1 (NKCC1) following stroke can disrupt the blood-brain barrier (BBB) and result in brain edema, indicating that NKCC1 may be a potential therapeutic target for improving stroke outcomes. Polygalasaponin F (PGSF) is a triterpenoid saponin isolated from Polygala japonica Houtt, which has showed neuroprotective effects in previous studies. The present study aimed to assess the protective effects of PGSF on cerebral ischemia-reperfusion injury (CIRI) in vivo and elucidate its underlying mechanism by targeting NKCC1. Experimental results revealed that following CIRI, rats displayed neurological deficits, cerebral infarction and brain edema, concurrent with increased NKCC1 mRNA and protein expression in the cerebral tissue. Notably, the administration of PGSF at both 10 mg/kg and 20 mg/kg effectively mitigated these adverse outcomes. To explore the mechanism of PGSF, pyrosequencing was used to find that CIRI reduces the methylation of the NKCC1 promoter, while PGSF enhances it. It was thereby demonstrated that PGSF could reduce NKCC1 expression in this manner. Simultaneously, we also observed that the protein expression of DNA methyltransferase 1 (DNMT1) in the ischemic penumbra was augmented after CIRI, whereas PGSF reduced the expression of DNMT1, which was contrary to the trend of NKCC1 methylation under the treatment of PGSF. These results imply that the enhancement of NKCC1 methylation by PGSF may not be catalyzed by DNMT1 and that the reduction of NKCC1 methylation level after CIRI may not be related to DNMT1. Finally, we discovered that PGSF can decrease the leakage of the BBB and enhance the expression of the BBB structural proteins occludin and ZO-1. In conclusion, PGSF can target NKCC1 as an epigenetic target and downregulate its expression following CIRI by enhancing DNA methylation of NKCC1, thereby safeguarding the structure and function of brain tissue.