NUP85 alleviates lipid metabolism and inflammation by regulating PI3K/AKT signaling pathway in nonalcoholic fatty liver disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is one of the common causes of chronic liver disease in the world. The problem of NAFLD had become increasingly prominent. However, its pathogenesis is still indistinct. As we all know, NAFLD begins with the accumulation of triglyceride (TG), leading to fatty degeneration, inflammation and other liver tissues damage. Notably, structure of nucleoporin 85 (NUP85) is related to lipid metabolism and inflammation of liver diseases. In this study, the results of researches indicated that NUP85 played a critical role in NAFLD. Firstly, the expression level of NUP85 in methionine-choline-deficient (MCD)-induced mice increased distinctly, as well as the levels of fat disorder and inflammation. On the contrary, knockdown of NUP85 had the opposite effects. In vitro, AML-12 cells were stimulated with 2 mm free fatty acids (FFA) for 24 h. Results also proved that NUP85 significantly increased in model group, and increased lipid accumulation and inflammation level. Besides, NUP85 protein could interact with C-C motif chemokine receptor 2 (CCR2). Furthermore, when NUP85 protein expressed at an extremely low level, the expression level of CCR2 protein also decreased, accompanied with an inhibition of phosphorylation of phosphoinositol-3 kinase (PI3K)-protein kinase B (AKT) signaling pathway. What is more, trans isomer (ISRIB), a targeted inhibitor of NUP85, could alleviate NAFLD. In summary, our findings suggested that NUP85 functions as an important regulator in NAFLD through modulation of CCR2.

Keywords: CCR2; ISRIB; NUP85; Nonalcoholic fatty liver disease; PI3K/AKT.

Figure 1

The expression level of NUP85 is increased in the liver of NAFLD patients and model mice. A. Human liver tissues stained with HE. B. Expression levels of NUP85 in liver of healthy people and NAFLD patients. C-D. RT-qPCR and Western blotting were used to detect the expression level of NUP85. E. Oil Red O staining of NAFLD patients. The percentage of lipid area in liver sections was detected by Oil red O staining. F. HE staining of mice liver tissues. G-I. The levels of ALT, AST and TG in serum. M. Oil Red O staining of mice tissues. The percentage of lipid area in liver sections was detected by Oil red O staining. J-L. Levels of serum IL-1β, IL-6 and TNF-α. N. Immunohistochemical analysis of NUP85 in MCS group and MCD group. O-P. The expression level of NUP85 was detected by RT-qPCR and Western blotting. Measurement metrics are shown in the figure. All experimental results of this study were replicated at least three times. **p<0.01, ***p<0.001 compared with the pair group.

Figure 2

NUP85 silencing alleviates inflammation and lipid accumulation in primary liver cells. A. Image of extracted primary liver cells. B-E. The mRNA and protein expression levels of NUP85, SREBP-1C, PPAR-α, ACOX-1, IL-1β, TNF-α and IL-6 in the MCS group and MCD group. F-I. The mRNA and protein expression levels of NUP85, SREBP-1C, PPAR-α, ACOX-1, IL-1β, IL-6 and TNF-α in control NUP85 knockdown primary liver cells and NUP85 knockdown primary liver cells. All experimental results of this study were replicated at least three times. **p<0.01, ***p<0.001 compared with the control group.

Figure 3

The expression level of NUP85 is raised after FFA treatment in AML-12 cells. A. The expression level of NUP85 protein with different FFA concentrations was detected by Western blotting. B. The expression level of NUP85 protein after 2 mm FFA treatment for 24 h was detected by Western blotting. C-E. NUP85 expression levels were detected by immunofluorescence, Western blotting and RT-qPCR. F-G. RT-qPCR and Western blotting were used to examine the expression level of NUP85 after transfected with NUP85-siRNA. H-I. RT-qPCR and Western blotting were used to examine the level of NUP85 after transfected with pcDNA3.1-3×Flag-c-NUP85. J. Immunofluorescence analysis was used to examine the level of NUP85 after transfected with pcDNA3.1-3×Flag-c-NUP85 and NUP85-siRNA. Measurement metrics are shown in the figure. All experimental results of this study were replicated at least three times. * *p<0.01, ***p<0.001 compared with the pair group.

Figure 4

Interference with NUP85 mitigated lipid accumulation, inflammation and apoptosis in FFA-induced AML-12 cells. A-B and E. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of SREBP-1C, PPAR-α and ACOX-1 in AML-12 cells transfected with NUP85-siRNA and pcDNA3.1-3×Flag-c-NUP85. C-D and F. After transfected with NUP85-siRNA and pcDNA3.1-3×Flag-c-NUP85, Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of IL-6, IL-1β and TNF-α. G-H Secretion levels of IL-1β, IL-6 and TNF-α in culture medium. All experimental results of this study were replicated at least three times. * *p<0.01, ***p<0.001 compared with the pair group.

Figure 5

NUP85 interacts with CCR2 in AML-12 cells. A. The result of STRING database. B. CO-IP results showed that NUP85 combined with CCR2. C. Immunofluorescence staining bespeak the colocalization of NUP85 and CCR2. D. immunofluorescence analysis of CCR2. E-G. Western blotting, RT-qPCR and Immunofluorescence results of the expression levels of NUP85 and CCR2 in cells after transfected with NUP85-siRNA. Measurement metrics are shown in the figure. All experimental results of this study were replicated at least three times. * *p<0.01, ***p<0.001 compared with the pair group.

Figure 6

NUP85 disruption attenuates lipid accumulation and inflammation in FFA-treated AML-12 cells by inhibiting the PI3K/AKT signaling pathway. A. Western blotting was used to detect the expression levels of PI3K and p-PI3K in AML-12 cells after transfected with CCR2-siRNA. B-C. Western blotting results of the proteins associated with PI3K/AKT signaling pathways were tested in AML-12 cells after transfected with NUP85-siRNA and pcDNA3.1-3×Flag-c-NUP85. D-E. Western blotting was used to detect the expression levels of related proteins in the PI3K/AKT signaling pathway after transfected and co-cultured with LY294002. F-G. The protein expression levels of ACOX-1, PPAR-α, IL-6 and IL-1β were detected using Western blotting after transfected with NUP85-siRNA and pcDNA3.1-3× Flag-c-NUP85 and co-cultured with LY294002 for 8 h in AML-12 cells. H-I Secretion levels of IL-1β, IL-6 and TNF-α in culture medium. All experimental results of this study were replicated at least three times. **p<0.01, ***p<0.001 compared with the control group.

Figure 7

NUP85 knockdown attenuates liver injury in MCD-fed mice. A and C. The mRNA and protein expression levels of SREBP-1C, ACOX-1 and PPAR-α in mice liver tissues were detected by RT-qPCR and Western blotting. B and C. RT-qPCR and Western blotting were used to detect the expression levels of IL-6, TNF-α and IL-1β in the liver tissues of mice. D. Analysis of small animals imaging. E-F. The mRNA and protein expression levels of NUP85. G. Serum ALT, AST and TG assay. H. HE staining of liver tissues. I. Oil Red O staining in liver tissues. J. Levels of serum IL-1β, IL-6 and TNF-α. K. Western blotting results of the expression levels of CCR2. L-M. IHC analysis of NUP85 and CCR2. N-O. The mRNA and protein expression levels of IL-1β, IL-6, TNF-α, SREBP-1C, PPAR-α and ACOX-1. Measurement metrics are shown in the figure. All experimental results of this study were replicated at least three times. **p<0.01, ***p<0.001 compared with the control group.

Figure 8

ISRIB could target NUP85 to protect NAFLD. A. Binding site of ISRIB to NUP85. B-C. In vitro, CCK8 was used to explore the concentration of ISRIB. D. AML-12 cells were cultivated with or without ISRIB (80 um) for 24 h and CETSA test was performed. E-G. H&E staining and Oil Red O staining of liver sections. H. Immunohistochemistry of liver tissues. I-K. Serum ALT, AST and TG assay. L-N. Levels of serum IL-1β, IL-6 and TNF-α. Measurement metrics are shown in the figure. All experimental results of this study were replicated at least three times. **p<0.01, ***p<0.001 compared with the control group.