LCN2 Regulates Pyroptosis To Affect Neuroinflammation After Experimental Intracerebral Hemorrhage In Rats

Objective:Intracerebral hemorrhage(ICH)is a devastating form of stroke that can lead to secondary brain injury.Neuroinflammation is a key host defense response to secondary brain injury after ICH.Pyroptosis is a novel inflammatory cell death process that triggers a cascading inflammatory response and exacerbates secondary brain injury.Lipocalin-2(LCN2)is widely involved in cellular physiological and pathological processes related to inflammation.It plays an important role in various diseases,including inflammation,infection,cancer,and neurological disorders,but its significance in post-ICH neuroinflammation is unknown.This study aims to investigate the changes in LCN2 expression and its intervention on neuroinflammation and pyroptosis in a rat ICH model.Methods:Part 1: The bulk RNA-seq and single-cell RNA-seq data of human brain tissue after ICH were obtained from the Gene Expression Omnibus(GEO)database,and identified changes in neuroinflammation and pyroptosis following ICH and determined the direction of study.Part 2: An ICH model was constructed using adult Sprague-Dawley(SD)rats in this investigation,and differentially expressed genes(DEGs)and target pathways were identified by sequencing with reference transcripts.The levels of key DEGs and pyroptosis-related proteins were detected in brain tissues around the hematoma,and the levels of TNF-α and IL-1βinflammatory factors were analyzed by ELISA in the peripheral blood of rats.HE staining,immunohistochemistry,and TUNEL staining analyses were performed on brain tissue slices,respectively.Part 3: To elucidate the underlying mechanism,lentivirus-mediated sh RNA silencing methods were employed for the knockdown of LCN2 in ICH rats.RT-q PCR,brain water content,HE staining,western blotting,and TUNEL staining were performed.Results:Part 1:A total of 11 brain specimens RNA-seq data were obtained from the GSE24265.The limma package of R was used to perform gene expression differential analysis,and 30 differential genes related to cell scorch death were identified,with NLRP3,CASP1,and GSDMD being upregulated in the ICH group.Proteomaps revealed that the differential genes primarily function in the NOD-like receptor signaling pathway,the TNF signaling pathway,and the MAPK signaling pathway.A total of 9630 cells with 10 cell subtypes were obtained by Single-cell RNA-seq downscaled clustering annotation.The time-series analysis proposed by the Monocle package revealed that pyroptosis and inflammation gradually increased,and the Add Module Score algorithm demonstrated that pyroptosis-related genes expression was higher in microglia after ICH.Part 2: A rat ICH model was constructed with significant hematoma,high brain water content,marked interstitial edema and inflammatory infiltration,and a high number of apoptotic cells in brain tissue.RNA-seq analysis of rat brain tissue revealed 103 DEGs that were significantly up-regulated and 81 that were significantly down-regulated.LCN2,HSPB1,CXCL10,and MEF2 B expressions were increased in ICH rats.The release of IL-1 and TNFwas induced by ICH,as were the pyroptosis-related proteins Caspase-1,GSDMD,and NLRP3.Part 3:With the lentiviral knockdown of LCN2,the degree of pyroptosis in brain tissue is reduced.Conclusion:LCN2 knockdown might alleviate pyroptosis and minimize the inflammatory response following ICH,ameliorate secondary brain injury,and provide directions for targeted ICH treatment.