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  • Reversing Postcardiopulmonary Bypass Associated Cognitive Dysfunction Using k-Opioid Receptor Agonists to Regulate Microglial Polarization via the NLRP3/Caspase-1 Pathway.

Reversing Postcardiopulmonary Bypass Associated Cognitive Dysfunction Using k-Opioid Receptor Agonists to Regulate Microglial Polarization via the NLRP3/Caspase-1 Pathway.

Journal of healthcare engineering (2021-10-12)
Pei Song, Zhuo Yi, Yiji Fu, Dandan Song, Keyan Chen, Jingjing Zheng, Yingjie Sun, Yugang Diao
ABSTRACT

Cardiopulmonary bypass (CPB) is mainly used during cardiac surgeries that treat ischemic, valvular, or congenital heart disease and aortic dissections. The disorders of central nervous system (CNS) that occur after cardiopulmonary bypass are attracting considerable interest. Postoperative neurocognitive disorders (PND) have been reported as the leading cause of patients' disability and death following CPB. The k-opioid receptor (KOR) agonists (U50488H) have been suggested to be vital in the treatment of surgically induced CNS neuroinflammatory responses. In this article, the transitions between the M1 and M2 microglial polarization state phenotypes were hypothesized to significantly affect the regulatory mechanisms of KOR agonists on postcardiopulmonary bypass (post-CPB) neuroinflammation. We investigated the effects of U50488H on neuroinflammation and microglia polarization in rats exposed to CPB and explored the method of the NLRP3/caspase-1 pathway. Thirty SD rats were randomly divided into three groups: sham operation group, cardiopulmonary bypass model group, and CPB+ k-opioid receptor agonist (U50488H) group, with ten rats in each group. The Morris water maze was used to evaluate the changes in the cognitive function of CPB rats. Hematoxylin and eosin (HE) staining and TUNEL were performed to assess the rats' hippocampal damage. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect changes in brain injury markers and inflammatory factors. Furthermore, immunofluorescence was used to observe the expression of microglia polarization and NLRP3 followed by Western blots to detect the expression of the NLRP3/caspase-1 pathway and microglia polarization-related proteins. Rat microglia were cultured in vitro, with LPS stimulation, and treated with U50488H and a caspase-1 antagonist to evaluate the effects and mechanism of action of U50488H. KORs alleviated hippocampal damage caused by CPB and improved PND. CPB activated the NLRP3 inflammasome and upregulated pro-caspase-1 expression which promoted the expression of pro-IL-lβ and pro-IL-18 and resulted in increased inflammation. However, KORs also inhibited NLRP3 and transformed microglia from the M1 to the M2 state. Caspase-1 inhibitor treatment reduced the microglial polarization induced by KORs. The κ-opioid receptor agonists inhibited the inflammation mediated by microglia and improved PND through the NLRP3/caspase-1 signaling pathway.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
(±)-trans-U-50488 methanesulfonate salt
Sigma-Aldrich
Lipopolysaccharides from Escherichia coli O55:B5, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
Protease Inhibitor Cocktail, for plant cell and tissue extracts, DMSO solution
Millipore
RIPA Lysis Buffer, 10X, 100 mL RIPA Lysis Buffer, 10X for Immunoprecipitation & Western Blotting.
Sigma-Aldrich
Ac-YVAD-cmk, ≥95% (HPLC)
Sigma-Aldrich
Anti-Iba1/AIF1 Antibody, clone 20A12.1, Alexa Fluor 647 Conjugate, clone 20A12.1, from mouse, ALEXA FLUOR 647
Sigma-Aldrich
Phenylmethanesulfonyl fluoride solution, ~0.1 M in ethanol (T)