NRP-1 is an important member of the NRP family and plays an important role in neurodevelopment, angiogenesis, tumor invasion, metastasis and immunity [28–31]. NRP-1 initially has been identified as a coreceptor for Sema3 and growth factors, including vascular endothelial growth factor, transforming growth factor-b, hepatocyte growth factor and platelet-derived growth factor [32–35], and it have been shown to be strongly expressed in immune cells and to regulate immune response [36, 37]. In macrophages, ablation of NRP1 results in decreased tumor growth and metastasis via enhanced infifiltration of tumor-associated macrophages into normoxic tumor regions, which abolishes the proangiogenic and immunosuppressive functions of tumor-associated macrophages . Consistently, Miyauchi et al. reported that NRP1 ablation in glioma-associated microglia and macrophages suppresses glioma progression by promoting M1 macrophage polarization . Recent findings further established the involvement of the Sema3A/Nrp1 axis, as well as M1/M2 macrophages, with respect to tumorigenic processes . However, specific functions of NRP1 in the context of metabolic dysfunction, such as NAFLD, have not been investigated yet. Studies have shown that: NRP1 may play an important role in the process of capturing TGF-β1 and LAP-TGF-β1 by cell membrane, thereby enhancing TGF-β1 signal transduction and promoting TGF-β1 secretion, thus promoting the occurrence and development of EMT . EMT-producing cells may enhance cell hardness and provide fibrotic cytokines. Indirect stimulation of myofibroblasts induces fibrosis . Other studies have shown that NRP-1 plays a novel role in tumor progression by enhancing the autorine HGF/ c-Met pathway, suggesting that NRP-1 may also play a role as a functional receptor for HGF [42, 43]. HGF regulates the survival, proliferation and migration of endothelial cells, matrix deposition and degradation, and the formation of capillary-like structures through c-Met . All the above studies suggest that NRP-1 can influence fibrosis, but its role in HE has not been reported in the literature.
This study is to observe the effect of NRP-1 expression enhancement and inhibition of NRP-1 expression on the formation of HFD-induced obese mouse NAFLD, and to explore its mechanism of action. Long-term high fat diet feeding in mice could cause whole-body energy imbalance, resulting in a series of obesity-associated metabolic disorders. To study the relation about NRP-1 and NAFLD, we established a NAFLD model. The results suggested that elevated NRP-1 expression can significantly increase the body weight and liver mass of NAFLD model mouse, while inhibition of NRP-1 expression can significantly reduce the body weight and liver mass of NAFLD model mouse.
The results also suggest an increase in NRP-1 expression can aggravate the abnormality of blood lipid and liver function of NAFLD model mice, and inhibiting the expression of NRP-1 can significantly alleviate the abnormality of blood lipid and liver function of NAFLD model mouse. In addition, H&E staining results also indicated that the increase of NRP-1 expression may aggravate the degree of liver degeneration of NAFLD model mouse and inhibit the expression of NRP-1. The degree of NAFLD model mouse liver degeneration can be significantly reduced.
Insulin resistance is considered to be the central link in the development of NAFLD and metabolic syndrome. Inflammatory factors affect insulin sensitivity, and inflammatory factors are involved in the development and progression of NAFLD, adipose tissue and liver immune dysfunction in patients with NAFLD, abnormal macrophages. Macrophages cells are important sources of inflammatory factors, and both fat and hepatocytes secrete inflammatory factors such as TNF-α and IL helps to mediate macrophage infiltration [45, 46]. To explore the possible mechanism of NRP1’s effects on NAFLD, we measured mRNA levels of pro-inflammatory makers. Then we found that the inhibition of NRP1 expression significantly reduced the mRNA level of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6, which were increased in HFD fed mouse, but the enhanced NRP1 expression significantly elevated the mRNA level of pro-inflammatory cytokines.
NAFLD is currently considered to be a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility, in which insulin resistance; oxidative stress and inflammatory response play an important role in the development of NAFLD [47–49]. As a key pathway of insulin signaling, PI3K/Akt/mTOR participates in glycolipid metabolism by regulating insulin level . However, PI3K/Akt/mTOR signal transduction disorders caused by various factors can cause insulin resistance (IR), promote the occurrence and development of NAFLD , and also participate in the pathophysiological process of oxidative damage of cells . Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) was an important signal transduction pathway in cells, which was closely related to cell proliferation, apoptosis, angiogenesis and other biological behaviors . PI3K was an important member of the superfamily of growth factor receptor signal transduction process. AKT and mTOR were important downstream genes of PI3K, and PI3K could affect families related to cell apoptosis through AKT, mTOR and effector molecules, thereby affecting cell apoptosis . Inflammation were a major factor in the occurrence and development of chronic liver disease and lead to liver fibrosis and cirrhosis, PI3K and AKT could adjust the key inflammatory cytokine activation of immune cells , PI3K and mTOR limited the activation of immune cells, adjusted in the liver inflammation and fibrosis formation, regulated the proliferation and migration of HSC in the process of liver fibrosis, synthesis and degradation of the ECM by raising the key anti-inflammatory cytokine interleukin-10 and inhibiting proinflammatory cytokines . In addition, in the state of inflammation, the PI3K/AKT/mTOR signaling pathway was over-activated, resulted in excessive transcription of downstream target genes, abnormal cell proliferation, tissue repair disorders, and aggravating tissue damage. Based on the consensus that "IR→abnormal lipid metabolism→liver fat deposition →oxygen stress and lipid peroxidation→NAFLD changes", the PI3K/AKT/mTOR signaling pathway was expected to be the target for improving NAFLD. To explore the possible mechanism of NRP1’s effects on NAFLD, we measured the active levels of PI3K/AKT/mTOR signaling. The expression of PI3K, AKT and mTOR were similar in six groups, but the phosphorylated levels of PI3K, AKT and mTOR increased in HFD fed mouse when compared with that of RD fed mouse. The activation of the PI3K/AKT/mTOR signaling molecules by HFD was reduced by the regulation of NRP1 expression, but elevated through an enhance NRP1 expression. HFD leads to inflammation and weight gain, and the inhibition of NRP1 expression could alleviate the inflammation as well as obesity caused by HFD. Taken together, our data indicate that NRP1 affects the PI3K/AKT/mTOR signaling pathways leading to elevated inflammatory responses induced by HFD.
There are still some limitations in this study. Only the effects of enhancement or inhibition of NRP1 expression on proteins related to PI3K/Akt/mTOR signal transduction pathway were studied, and it was concluded that NRP1 may have an effect on non-alcoholic fatty liver disease by regulating PI3K/Akt/mTOR signal transduction pathway. Further investigation is needed through the use of PI3K/Akt/mTOR signaling pathway agonists or inhibitors for interference. In addition, does NRP1 regulate other signaling pathways besides PI3K/Akt/mTOR signaling pathway to exert influence on non-alcoholic fatty liver disease? This will require further study.