Intestinal mucosal barrier dysfunction participates in the progress of nonalcoholic fatty liver disease

Abstract

Intestinal mucosal barrier dysfunction is closely related to liver diseases, which implies impaired gut-liver axis may play a role in the pathogenesis of NAFLD. In our study, rats were divided into three groups: normal chow diet (NCD) group, high-fat diet (HFD) group and TNBS-induced colitis with high-fat diet (C-HFD) group. Liver tissues were obtained for histological observation and TNF-α, IL-6 mRNA determination and blood samples were collected for liver enzymes and LPS analysis. Ultrastructural changes of jejuna epithelium, SIBO and amounts of CD103(+)MHCII(+)DCs and CD4(+)CD25(+)FoxP3(+)T-regs in terms of percentage in mesenteric lymph nodes (MLN) were observed by electron microscope, bacterial cultivation and flow cytometry, respectively. The results demonstrated the pathological characteristics accorded with nonalcoholic simple fatty liver (NAFL) and NASH in HFD group by week 8 and 12, respectively. Besides, the degree of hepatic steatosis and steatohepatitis was more severe in C-HFD group compared with HFD-group at the same time point. NAFLD activity score (NAS), liver enzymes, concentration of LPS and mRNA expressions of TNF-α, IL-6 were higher significantly in C-HFD group compared with HFD and NCD group at week 4, 8 and 12, respectively. In HFD group, epithelium microvilli atrophy, disruptive tight junctions and SIBO were present, and these changes were more severe in NASH compared with NAFL. The percentage of CD103+MHCII+DCs and CD4+CD25+FoxP3+T-regs decreased significantly in NAFL and NASH compared with NCD group. Our conclusion was that gut-liver axis was impaired in NAFLD, which played crucial role in the pathogenesis of NAFLD.

Keywords: IL-6; Nonalcoholic fatty liver disease; TNF-α; dendritic cell; intestinal mucosal barrier; regulatory T cell.

Figure 1

No abnormalities were observed in the liver of the NCD rats. In HFD group, hepatic microvesicular steatosis was showed at week 4 and macrovesicular steatosis were present in hepatic cells at week 8, which accorded with pathological changes of NAFL. Hepatocyte ballooning, moderate to severe steatosis and hepatic inflammation as well as focal necrosis were showed at week 12 accorded with NASH changes. In C-HFD group, the pathological result showed mixed diffusely hepatic steatosis at week 4. Hepatocyte ballooning, moderate to severe macrovesicular steatosis, lobular inflammation and focal necrosis as well as mild portal zone inflammation were observed at week 8, and an even greater degree of hepatic steatosis, steatohepatitis and lobular inflammation were present at week 12. Pathological changes in the NCD, HFD and C-HFD groups at week 4, 8 and 12 under light microscope (A: HE×100, B: HE×200).

Figure 2

Both in HFD and C-HFD group, the NAS at week 12 was significantly higher than that at week 8 and 4, and the score was significantly higher at week 8 compared with week 4 (*P<0.05 versus 4 wk, #P<0.05 versus 8 wk). The score was significantly higher in C-HFD group compared with HFD group at week 4, 8, 12, respectively (&P<0.05 versus HFD group). NAFLD activity score (NAS) in HFD and C-HFD group at week 4, 8, and 12.

Figure 3

The LPS concentration had no significant difference in NCD group at different times. In HFD and C-HFD group, the LPS level was significantly higher at week 12 compared with week 8 and 4, and the concentration was significantly higher at week 8 compared with week 4 (*P<0.05 versus 4 wk, #P<0.05 versus 8 wk). The LPS concentration was significantly higher in C-HFD group compared with HFD and NCD group at week 4, 8, 12, respectively (&P<0.05 versus NCD group, $P<0.05 versus HFD group). Plasma concentration of LPS in NCD, HFD and C-HFD group at week 4, 8 and 12.

Figure 4

Regular and tight jejunum epithelium cells alignment, rich and normal microvilli and intercellular tight junctions were observed in NCD group. Lodging and irregular epithelial microvilli and slight increased gap of tight junctions were observed in NAFL. The jejunum epithelium cells and their organelle became swollen and disruptive, and microvilli were atrophic and even deficient as well as gap of intercellular tight junctions became significantly larger in NASH. Changes of jejunum epithelium cell and intercellular tight junctions by electron microscope.

Figure 5

The percentage of DCs and T-regs in NASH and NAFL group decreased significantly compared to NCD group (P<0.05). The percentage of DCs in NASH group decreased significantly compared to NAFL group; but the percentage of T-regs had no significant difference between NAFL and NASH group. The amount of CD103⁺MHCII⁺DCs and CD4⁺CD25⁺FoxP3⁺T-regs in MLN in terms of percentage by flow cytometry.