SPP1 and CXCL9 Promote Non-alcoholic Steatohepatitis Progression Based on Bioinformatics Analysis and Experimental Studies

Abstract

Background and AimsNon-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease worldwide, and non-alcoholic steatohepatitis (NASH) is one of its pathological subtypes. The pathogenesis of NASH has not yet been fully elucidated. The purpose of this study was to identify the hub genes and pathways involved in NASH using bioinformatics methods. The hub genes were confirmed in human and animal models.Materials and MethodsThree Gene Expression Omnibus (GEO) datasets (GSE48452, GSE58979, and GSE151158) of NASH patients and healthy controls were included in the study. We used GEO2R to identify differentially expressed genes (DEGs) between NASH patients and healthy controls. Functional enrichment analyses were then performed to explore the potential functions and pathways of the DEGs. In all DEGs, only two genes were highly expressed in NASH patients throughout the three datasets; these two genes, SPP1 and CXCL9, were further studied. Serum and liver tissues from NASH patients and healthy controls were collected. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured in NASH patients and healthy controls. Liver tissues were stained with hematoxylin and eosin. Immunohistochemical staining was used to evaluate the expression levels of the two genes in liver tissues. Male C57BL/6J mice were fed a methionine choline-deficient (MCD) diet for 8 weeks, after which serum ALT and AST levels were measured and liver tissues were stained.ResultsSPP1 and CXCL9 were the hub genes detected in the three datasets. “Lipid metabolism,” “inflammatory response,” and “lymphocyte activation” were the most significant biological functions in GSE48452, GSE58979, and GSE151158, respectively. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the toll-like receptor signaling pathway was significantly enriched in NASH patients. Serum ALT and AST levels were significantly increased in NASH patients compared to healthy controls. Liver tissues had more serious steatosis, hepatocyte ballooning degeneration, and lobular inflammatory infiltration, and the expression of SPP1 and CXCL9 in liver cells was significantly upregulated in NASH patients compared to healthy controls. MCD diet mice were consistent with NASH patients.ConclusionSPP1 and CXCL9 may play important roles in NASH pathogenesis and could be potential therapeutic targets and biomarkers of NASH in the future. Further experimental studies are needed to confirm our results.