NAFLD is the most common type of liver disease that affects a large number of individuals worldwide[21]. Hepatitis, cirrhosis and liver cancer caused by NAFLD are global public health problems[3, 22–25]. The prevalence of NAFLD in our population is increasing year by year, and the proportion of new cases of NAFLD is about 4% each year[26]. There is a significant difference in the prevalence rate of NAFLD in different regions, with the prevalence rate in the economically developed eastern and southern regions being higher than that in the central and western inland regions, and the difference in lifestyles is also an important reason for the difference in prevalence rates[27]. Over the past two decades, the burden of NAFLD has increased significantly with China's booming economy and radical lifestyle changes. The early diagnosis of NAFLD is essential for the treatment of this disease, Unfortunately, there are no diagnostic tools that can allow a prompt diagnose[28, 29].
In our study, machine learning methods were used to find DEGs. We analyzed the GEO dataset and identified 334 DEGs between NAFLD and healthy samples. GO analysis showed that these 334 DEGs were mainly involved in chemical reactions, negative regulation of cell processes, organic material and cell-to-chemical stimulation reactions. KEGG pathway analysis showed that cytokine–cytokine receptor interactions, the interleukin (IL)-17 signaling pathway, the tumor necrosis factor (TNF) signaling pathway, and transcriptional dysregulation were obviously enriched. IL-17 is an inflammatory factor induced by Th17 cells, which can stimulate endothelial cells and epithelial cells to produce other inflammatory factors and chemokines, resulting in tissue damage. Overnutrition mediates DNA damage in hepatocytes through nonclassical prefoldin RPB5 interaction factor. DNA damage induces inflammation and neutrophilc infiltration of white adipose tissue through Th17 and IL-17A, which in turn mediates insulin resistance and fatty acid release. Fatty acids are stored in the liver as triglycerides, leading to the development of NAFLD and NASH. TNF works through two receptors TNFR1 (also known as p55) and TNFR2 (also known as p75). TNFR1 is expressed in most tissues, and interacts with TNF, leading to a classic pro-inflammatory response, most through activation of the NF-κB and c-Jun pathways. Studies have that a high-fat diet increases the expression of TNF-a in the body, which further promotes the activation of NF-κB and TLRs signaling pathway, leading to the increase of inflammatory factors such as IL-6, and ultimately the formation of chronic inflammation. These results suggest that the selected DEGs are actively involved in the inflammatory process and are essential for the progression of NAFLD[30].
To identify potential diagnostic genes for NAFLD, two machine learning algorithms were used to analyze the 334 DEGs, thus four genes (FOSB, GPAT3, RGCC, and RNF43) were identified. FOSB is an early hepatocyte response protein that is essential for lipid metabolism[31]. Studies have shown that the expression of FOSB is significantly downregulated in patients with fatty liver disease[32]. Meanwhile, GPAT3 is the first critical rate-limiting enzyme in the glycerol-3-phosphate synthesis pathway[33]. Therefore, regulating the synthesis of triglycerides in the liver and reducing the accumulation of excess lipids in hepatocytes may be used as a novel therapeutic strategy to improve hepatic steatosis, and preventing and treating NAFLD[34]. Moreover, RGCC, also known as C13, is an important complement response gene that is widely expressed in various tissues[35]. It can regulate cell cycle, promote cell proliferation and differentiation[36], and regulate the immune response[37]. Interestingly, deletion or mutation of RNF43 was found to lead to lipid accumulation and liver inflammation in nonobese mice fed a with normal diet. In humans, mutation of RNF43 increases the risk of liver diseases such as fatty liver disease and liver cancer, as well as reduces the life expectancy of patients[38]. In addition, the ROC method confirmed their strong ability to differentiate NAFLD samples from normal samples. Thus, our findings suggest that FOSB, GPAT3, RGCC, and RNF43 are potential diagnostic biomarkers for NAFLD.
We also examined FOSB, GPAT3, RGCC, and RNF43 expression in patients with hepatitis B, hepatitis C, and AIH. There are some consistency between the mouse liver transcriptome and the human blood transcriptome[39]. To compensate for the difficulty of obtaining human liver samples mouse liver could be used to detect the expression of some genes[40]. The results showed an increased expression of FOSB in patients with hepatitis B, an increased expression of RNF43 in patients with hepatitis C, and an increased expression of RNF43 along with a decreased expression of RGCC in patients with AIH. These results were consistent with the expression profile in patients with NAFLD. Many studies have suggested that FOSB, RNF43 and RGCC are closely related to the inflammatory response of immune cells[41, 42]. Hepatitis B virus and hepatitis C virus infection can stimulate the inflammatory response of activated liver immune cells, thus change the expression of these genes will. However, hepatitis B, hepatitis C and AIH can only be diagnosed by the detection of corresponding virus genes and antibodies. In addition, FOSB is reported to be related to hepatitis C[43], while RNF43 and GPAT3 are related to hepatitis B[44, 45]. Our results show that in the diagnosis of NAFLD, the ROC value of the four gene expressions in blood is close to 1. It is suggested that the simultaneous changes of four genes are closely related to NAFLD. Therefore, combination of these four gene expressions in blood might be an alternative diagnostic method to the gold diagnostic standard (liver biopsy) with high sensitivity, specificity and safety.
Liver biopsy is the current gold standard for the diagnosis of NAFLD. Although it can directly observe the pathological features of the liver to diagnose NAFLD, it is inconvenient for patients to tolerate side effects such as pain and discomfort after puncture due to the trauma. Additionally, the technique of liver puncture biopsy is complicated and many clinicians are not skilled enough resulting in dirty biopsy. However, these patients collected for reasons of safety and financial expense have been diagnosed with NAFLD by ultrasound. Ultrasound is used as the primary screening tool in clinical practice. Recently, a meta-analysis showed that the sensitivity of diagnosing moderate and severe fatty liver with ultrasound was 84.8% and the specificity was 93.6%. However, ultrasound has its drawbacks and its diagnostic sensitivity is only 55% when the fat content of the liver is below 20%. Although ultrasound screening for NAFLD is safer than liver tissue biopsy, it is not diagnostic of early NAFLD due to the clinician's experience. Our results show that the sensitivity of mRNA analysis using blood while reducing the least damage to the body can reach a maximum of 0.985, we compared the expression of these genes in patients with hepatitis B, C, and autoimmune hepatitis and found that these genes were specifically expressed in patients with nonalcoholic fatty liver. Compared to biopsy, our diagnostic method is highly safe, it requires only a small amount of fingertip blood is independent of the physician's skill, and is more acceptable to the patient as it is less harmful to the body. Compared to imaging, the diagnosis by finger blood is not influenced by the physician experience and is more sensitive and specific for NAFLD.
In recent years, multiple studies have indicated that immune cell infiltration plays a crucial role in the occurrence and development of NAFLD. The progression of NAFLD is closely related to macrophages (Kupffer cells)[46], neutrophils[47], dendritic cells[48], and natural killer (NK) T cells[49]. Kupffer cells constitute about 20% of nonparenchymal hepatocytes and are involved in T lymphocyte activation and tolerance. Furthermore, TNF-α is mainly derived from Kupffer cells. In response to high-fat diet stimulation, the TNF-α levels are significantly increased in Kupffer cells, and large amounts of proinflammatory factors such as IL-6 are released to aggravate the inflammatory response[50]; additionally, stimuli such as a high-fat diet also increase the number of Kupffer cells themselves, thereby changing the activation state of NK T cells and inducing cell necrosis and apoptosis[51]. Moreover, increased oxidative stress activates Kupffer cells and the corresponding macrophages as well as it increases the levels of chemokines and cytokines[52]; thus, it plays a pivotal role in the progression of NAFLD. Therefore, it is necessary to evaluate the infiltration of immune cells and to identify the diversity of infiltrating immune cell components to reveal the molecular mechanisms in NAFLD in order to find new immunotherapeutic targets.
In this study, we observed dysregulated levels of CD8+ T cells, resting dendritic cells, γδ T cells, M2 macrophages, resting mast cells, neutrophils, and naive B cells between normal and NAFLD samples. Moreover, the histamine produced during the degranulation of mast cells in the liver of patients with NAFLD induces an inflammatory response that damages the surrounding tissues. In livers from NAFLD mice, mast cells promote the progression of NAFLD to NASH hepatitis by upregulating the aldehydrogenase 1 family member A3 and downregulating microRNA-144-3. In contrast, mast-cell deficient mice exhibit improved the symptoms of NAFLD. The change of M1/M2 polarization may induce hepatic inflammation and hepatocyte injury, thus activating satellite cells. After activation, satellite cells release large numbers of collagen fibers, which promote liver fibrosis. In addition, neutrophils[53], CD4+ T cells[54], CD8+ T cells[55], NK T cells[56], T helper 17 cells[57], and other immune cells have been confirmed to participate in the development of NAFLD. In the liver, these different types of cells regulate each other through the expression and release of inflammatory cytokines, chemokines, interleukins, and other cytokines in autocrine and paracrine modes, thus maintaining liver homeostasis. The abnormal expression of these secreted factors can cause necrosis and apoptosis of hepatocytes, activate satellite cells, and promote the development of NAFLD/NASH. Our data indicate that FOSB, GPAT3, RGCC, and RNF43 may participate in the progression of NAFLD by modulating some immune cells. Nevertheless, further studies are warranted to confirm these findings.