As the prevalence of diabetes mellitus gradually rises, the number of diabetic patients with MASLD has also increased significantly. According to the 2018 MASLD prevention and management guidelines, the prevalence of fatty liver in Chinese diabetic patients is 28% to 70%.19Globally, the prevalence of T2DM complicated by MASLD is increasing year by year, and a recent meta-analysis showed that the global prevalence of the disease is approximately 55.48%, with regional differences, and the prevalence in East Asia is approximately 52.04%.20Patients with type 2 diabetes and MASLD are prone to liver dysfunction and cirrhosis, and at the same time, they are at increased risk of cardiovascular diseases, such as kidney disease and coronary heart disease, which require urgent treatment. Because type 2 diabetes and MASLD share a common pathophysiological mechanism, insulin resistance, both diseases affect the development of each other. Type 2 diabetes can aggravate MASLD by promoting the progression of nonalcoholic hepatic steatosis or fibrosis, while MASLD can cause macrovascular and microvascular complications in type 2 diabetes patients.twenty oneThe overall mortality rate in diabetic patients with MASLD was found to be high at 58.5/103, significantly higher than the mortality rate for hepatitis B and hepatitis C combined.20Furthermore, MASLD is an important cause of cryptogenic cirrhosis and is associated with high liver-related morbidity and mortality.twenty twoTherefore, early and effective treatment of T2DM with MASLD can improve the quality of patient survival and at the same time help reduce the burden on health insurance. Although research on diabetes diagnosis and treatment has increased in recent years, our understanding of the pathogenesis of T2DM with MASLD is still very limited, and targeted drug treatments are lacking. Therefore, it is particularly important to find gold standard prevalence factors for T2DM with MASLD.
In this study, we analyzed the GEO database datasets GSE23343 and GSE49541 on type 2 diabetes and MASLD, and screened 185 co-expressed DEGs to construct a PPI network. 20 hub genes were also screened, and combined with GO and KEGG enrichment analysis, potential biomarkers and biological pathways of T2DM with MASLD were finally identified. Two target genes, namely SPP1 and collagen IV, which may be key genes for pathogenicity, were obtained. This time, we verified the results by real-time fluorescent quantitative PCR experiments in the MASLD rat model with diabetes, and the following results were obtained. The mRNA contents of SPP1 and collagen IV in the MASLD rat group with diabetes were higher than those in the control group, which was consistent with the results of the bioinformatics analysis, again validating the results of the bioconfidence analysis. Meanwhile, KEGG enrichment analysis also found that two target genes, SPP1 and collagen IV, were mainly involved in ECM-receptor interaction, focal adhesion, human papillomavirus infection, PI3K-Akt signaling pathway, Toll-Akt signaling pathway, and Toll-mediated protein expression signaling pathway, Toll-like receptor signaling pathway.
Secreted phosphoprotein 1 (SPP1) is widely expressed in human tissues and organs, and its expression is significantly increased in various tumors.23,24,25Patients with macrophage-positive SPP1-expressing colorectal cancer have shorter progression-free survival26SPP1 is an important extracellular matrix component secreted by a variety of cells, including tumor cells, immune cells, fibroblasts, osteoblasts, smooth muscle cells, lymphocytes, epithelial cells, etc. Upregulation of SPP1 expression in tumor tissues and serum of various tumors correlates with poor patient prognosis.27,28,29.the study30 In lung adenocarcinoma, SPP1 has been shown to control the polarization of macrophages into the M2 type, but the exact mechanism is unclear and it has been less studied in HCC. In this study, SPP1 expression in tumor tissues of HCC HCC patients was higher than that in paracancerous tissues, elevated in patients with advanced BCLC (Barcelona Clinic Liver Cancer) stage, large tumor diameter, and multiple tumor foci in the liver, and was shown to be an independent prognostic factor for HCC patients. The survival rate of patients with high SPP1 expression was significantly lower than that of HCC patients. The survival rate of patients with high SPP1 expression was shorter than that of patients with low SPP1 expression. Meanwhile, SPP1 expression was positively correlated with the number of M2 macrophages, suggesting that SPP1-targeted therapy may be a potential therapeutic option to improve the prognosis of HCC patients.
Expression of spp1 is associated with fibrosis and progression to MASH. In humans and mice, upregulation of myeloid cell-derived spp1 is associated with progression to MASH. However, it is unclear whether increased spp1 in these cells is protective or detrimental. Studies have shown that spp1 accelerates the progression of MASLD.31.
SPP1 has not been widely studied in diabetes with MASLD. In this study, we found that SPP1 is involved in diabetes with MASLD. In this real-time fluorescent quantitative PCR experiment, the expression of SPP1 was clearly and statistically significantly higher in the diabetes with MASLD group compared with the control group.
Collagen is a large family of glycoprotein molecules that are the main protein components of connective tissues, accounting for approximately 25% of all proteins in the body. All collagens are distributed in the extracellular matrix in a supramolecular structure of triple-helical polypeptide chains. Common proteins are double-helical, and as structural proteins of the extracellular matrix (ECM), collagen is composed of three polypeptide chains, forming a triple-helical structure, or collagen domain. Each of the three polypeptide chains rotates counterclockwise to form a left-handed helical structure, and then is occluded by hydrogen bonds to form a strong right-handed superhelical structure. Reticular collagens include collagen type IV, collagen type VIII, and collagen type X. Collagen type IV is a typical reticular collagen found in basement membranes and plays an important role in molecular filtration. Collagen type VIII is localized in Descemet’s membrane and the subendothelial matrix of blood vessels, while collagen type X is localized in the proliferative zone of growth plate cartilage. The more classical collagens in pancreatic cancer are types COLI, COLIII, and COLIV. Collagen type IV is a member of the collagen family and a major component of cell basement membrane (BM). The main biological behavior of malignant tumors is for cancer cells to break through the BM and invade adjacent or distant sites for metastasis. As a major component of BM, relevant studies have shown that type IV collagen plays an important role in the invasion and metastasis mechanisms of multisystem malignant tumors, as well as in clinical diagnosis and treatment.32Fatty liver is a progressive disease, and 10-25% of patients will develop cirrhosis and death from liver disease within 10 years.
Most researchers believe that excessive deposition of large amounts of ECM in the liver is the underlying cause of the formation of liver fibrosis. Liver fibrosis is a dynamic process in which collagen is the most important component of the ECM, and collagen deposition and degradation lead to disease progression and regression, respectively. Currently, studies have shown that hyperglycemia significantly induces mesangial cell proliferation and extracellular matrix proteins, including the expression of fibronectin and collagen IV.33Furthermore, activation of Nrf2 suppressed high glucose-induced oxidative stress and the expression levels of TGF-β1, fibronectin, and type IV collagen in mesangial cells.34.
Hyperglycemia and TGF-β1 synergistically induce collagen IV and VEGF production in podocytes. Hyperglycemia-induced increases in collagen IV and VEGF protein are mediated by the TGF-β system. By increasing TβRII expression, hyperglycemia increases the responsiveness of podocytes to environmental levels of TGF-β.35.
Collagen IV has not been studied much in diabetes combined with MASLD. In this study, we found that collagen IV is involved in diabetes combined with MASLD. In this real-time fluorescent quantitative PCR experiment, we found an obvious statistically significant higher expression in the MASLD combined with diabetes group compared with the control group, which could be verified by Western blot in the subsequent experiment.
This study has several limitations. First, this study is based on bioinformatics analysis of transcriptome profiles in public databases, which may differ from reality. Second, although the two screened genes have previously been reported to mediate type 2 diabetes and metabolic-related diseases, there is no direct evidence that they control the onset, progression, and prognosis of type 2 diabetes with MASLD. Although animal models can morphologically reproduce some of the characteristics of human diabetes with MASLD, the emergence of more ethical genetically humanized animal models is also expected. Finally, prospective clinical trial cohorts and more detailed molecular biology experiments need to be designed and conducted to further verify the mechanism of action of these two related genes in the onset and progression of type 2 diabetes with MASLD.
The necessity and clinical significance of this study is that, at the current level of medical development, diabetes complicated by MASLD is not yet curable, so it is especially important to consider active prevention of diabetes complicated by MASLD that has not yet developed, to improve the quality of survival of more patients and reduce disability and mortality. Finding the target point of treatment, providing individualized treatment plans, and more precise treatment can reduce the economic pressure on patients, families, and even the whole society.
In summary, the candidate genes SPP1 and collagen IV screened based on bioinformatics analysis may affect the course of type 2 diabetes mellitus complicated with MASLD. Through ECM-receptor interactions, focal adhesions, human papillomavirus infection, PI3K-Akt signaling pathway, and Toll-like receptor signaling pathway, they may play important roles in the course and disease outcomes of type 2 diabetes mellitus complicated with MASLD, and the results of this study provide meaningful clues and directions for clinical prognosis and treatment.
