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Notoginsenoside R1 attenuates post-ischemic pyroptosis: a novel CKLF1-endoplasmic reticulum stress pathway-mediated
Hanlong Wang1, Yang Sun2, Shasha Liu3
1Hunan Engineering Technology Center of Standardization and Function of Chinese Herbal Decoction Pieces, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, China; School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangdong, 510006, China.
Background:
Ischemic stroke induces neuronal pyroptosis driven by excessive endoplasmic reticulum (ER) stress, which exacerbates secondary brain injury. Notoginsenoside R1 (NGR1) has demonstrated neuroprotective properties; however, whether its mechanism involves the modulation of chemokine-like factor 1 (CKLF1)-mediated ER stress remains unclear.
Objective:
This study aims to determine whether NGR1 regulates CKLF1 to suppress the CCR5/ER stress/NLRP3 axis and thereby attenuate neuronal pyroptosis following ischemic injury.
Methods:
In vivo experiments were performed using a rat model of middle cerebral artery occlusion (MCAO), while in vitro studies employed oxygen-glucose deprivation (OGD) in PC12 cells and primary neurons. CKLF1-knockout rats, the CKLF1 agonist C27, and pharmacological inhibitors (TAK-799, 4-PBA, and MCC950) were utilized to dissect the pathway. Pyroptosis was assessed via lactate dehydrogenase (LDH) release, TUNEL staining, immunofluorescence, Annexin V/PI flow cytometry, and immunoblotting of NLRP3, cleaved caspase-1, and GSDMD-N. Molecular interactions were examined using co-immunoprecipitation (Co-IP), drug affinity responsive target stability (DARTS), and cellular thermal shift assay (CETSA).
Results:
NGR1 significantly reduced infarct volume, cerebral edema, and neurological deficits in MCAO rats, effects that were abolished in CKLF1-knockout animals. In OGD-treated PC12 cells, NGR1 improved viability, decreased LDH release, and reduced the proportion of Annexin V/PI-positive cells. Similarly, in OGD-treated primary neurons, NGR1 diminished TUNEL-positive cells and reduced immunofluorescence intensity of pyroptotic markers. In both cellular models, NGR1 downregulated NLRP3, cleaved caspase-1, and GSDMD-N expression. Co-IP confirmed interactions between CKLF1 and both NLRP3 and CCR5. C27-induced CKLF1 activation promoted ER stress and pyroptosis, both of which were blocked by TAK-799. 4-PBA suppressed NLRP3 activation, whereas MCC950 did not affect ER stress, confirming that ER stress acts upstream of NLRP3 inflammasome activation. DARTS and CETSA verified direct binding of NGR1 to CKLF1.
Conclusion:
NGR1 directly binds CKLF1, thereby suppressing the CCR5/ER stress/NLRP3 cascade and attenuating neuronal pyroptosis. These findings establish a mechanistic framework supporting the therapeutic potential of NGR1 in ischemic stroke.