Knockdown of PHLDA1 Alleviates Necrotizing Enterocolitis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis Through Enhancing Nrf2 Signaling

ABSTRACTObjective Pleckstrin homology-like domain family A member 1 (PHLDA1) is involved in the progression of intestine-related diseases, but its role and related mechanisms in Necrotizing enterocolitis (NEC) are unclear. The aim of this study was to better understand the function of PHLDA1 in NEC and the underlying mechanisms.Methods A neonatal mouse model of NEC was established by hypoxic hypothermia, and sh-PHLDA1 was transfected into mice to observe the mortality of each group within 4 days. The levels of IL-1β, IL-6, IL-18 and TNF-α were measured by PCR and ELISA. ROS, MDA, SOD, and GSH-Px levels were detected by Dihydroethidium (DHE) method and kit; expression of pyroptosis-related factors including NLRP3, ASC, cleaved-caspase1, GSDMD-N, IL-1β, IL-18, and Nrf2 were detected by western-blot; mechanistically, the effects of transfection of sh-PHLDA1 and ML385 (Nrf2 inhibitor) were investigated, and the expression of pyroptosis-related factors was detected again.Results PHLDA1 was highly expressed in the intestinal tissues of NEC mice, and transfection of sh-PHLDA1 improved the survival rate, alleviated intestinal lesions, improved intestinal inflammation, oxidative stress and cellular scorching in NEC. In addition, sh-PHLDA1 was able to inhibit NLRP3 activation and pyroptosis by activating Nrf2.Conclusion Knockdown of PHLDA1 attenuated necrotizing small intestinal colitis by enhancing Nrf2 expression to inhibit NLRP3 inflammasome activation and pyroptosis.KEYWORDS: NECNLRP3Nrf2PHLDA1pyroptosis Disclosure statementNo potential conflict of interest was reported by the authors.Author’s contributionsAll authors contributed to the study conception and design. Material preparation and the experiments were performed by Xin Yang. Data collection and analysis were performed by Xihong Li and Chan Wu. The first draft of the manuscript was written by Feng Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.Availability of data and materialsAll data generated or analyzed during this study are included in this published article.Ethics approvalEthical approval was obtained from the Ethics Committee of Chengdu Medical College.Additional informationFundingThis work was supported by the Sichuan Province Medical (Youth Innovation) Research Project (Grant No.Q21026) and the Chengdu Medical Research Project (Grant No.2022328).