Role of visfatin in pathogenesis and progression of non-alcoholic fatty liver disease
- 주제(키워드) visfatin , insulin resistance , inflammation , fibrosis , NAFLD
- 발행기관 아주대학교
- 지도교수 김혜진
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030491
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease affecting people worldwide. It includes a complex array of diseases ranging from simple steatosis to non-alcoholic steatohepatitis and cirrhosis. Insulin resistance and inflammation are primarily associated with the pathogenesis of NAFLD. Visfatin, an adipocytokine, was reported to induce pro-inflammatory cytokines and can be associated with liver fibrosis. This study aimed to investigate the role and related mechanism of action of visfatin in the progression of NAFLD. The objectives of the present study included the determination of the effect of visfatin on: i) insulin resistance and inflammation in hepatocytes; ii) chemokine secretion in macrophages and the activation of hepatic stellate cells (HSCs); and iii) hepatic inflammation and fibrosis in a methionine-choline-deficient (MCD)-diet-induced mouse model of NAFLD. Visfatin significantly decreased the level of phosphorylation in proteins involved in insulin signaling (IR, IRS-1, GSK, and AKT), increased IRS-1 S307 phosphorylation, and enhanced the level of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) in HepG2 cells compared to that of the untreated cells. Interestingly, visfatin led to the activation of the JAK2/STAT3 and IKK/NF-κB but not the JNK, p38, and ERK signaling pathways. NF-κB and STAT3 inhibitor-mediated blockage of signaling pathways significantly decreased the level of pro-inflammatory cytokines leading to the rescue of insulin signaling. Thus, visfatin induced the secretion of pro-inflammatory cytokines and inhibited insulin signaling via the STAT3 and NF-κB pathways in HepG2 cells. Further, in THP-1 cells, a pro-monocytic cell line, visfatin enhanced the expression of both, the mRNA and protein levels of chemokine (C-C motif) ligand 20 (CCL20), an important inflammatory mediator, through the activation of the NF-κB, p38, and MKK3/6 signaling pathway. Co-treatment of cells with visfatin and inhibitors of NF-κB, p38, or MLK3, significantly inhibited the expression of visfatin-induced CCL20. Subsequently, co-culturing of LX-2 cells (human HSCs) with THP-1 cell culture supernatant in the presence or absence of anti-CCL20 neutralizing antibodies resulted in reduced expression of fibrosis markers in LX-2 cells in the presence of anti-CCL20 neutralizing antibodies. Thus, visfatin increased the expression of CCL20 through the NF-κB, p38, and MKK3/6 signaling pathway in macrophages. In addition, visfatin-induced CCL20 expression promoted the expression of fibrosis markers in HSCs. The role of visfatin in hepatic inflammation and fibrosis was investigated in a MCD-diet-induced NAFLD mouse model. Eight-week-old male C57BL/6J mice were randomly assigned to one of the following three groups: (i) saline-injected control diet group (CD/Sal; n = 8); (ii) saline-injected MCD diet group (MCD/Sal; n = 8); and (iii) visfatin-injected MCD diet group (MCD/visfatin; n = 8). At the end of the experiment, their livers were isolated and subjected to histological, biochemical, and molecular analyses. Hepatic steatosis was aggravated and the expression of pro-inflammatory cytokines was enhanced in the MCD/visfatin group, compared to that of the CD/Sal or MCD/Sal groups. Enhanced inflammatory cell infiltration indicated by F4/80, CD68, ly6G, and CD3, and expression of fibrosis markers including cTGF-β, TIMP1, collagen 1α2, collagen 3α2, αSMA, fibronectin, and vimentin were evident in the liver of the MCD/visfatin group. In addition, visfatin upregulated ER stress and ROS, and activated JNK signaling in the liver of MCD/visfatin group, compared to that of the CD/Sal or MCD/Sal groups. Thus, visfatin aggravated hepatic inflammation and fibrosis in the MCD-diet-fed mouse model. In conclusion, visfatin can exacerbate hepatic insulin resistance and aggravate hepatic inflammation and fibrosis. Thus, visfatin can play an important role in the pathogenesis and progression of NAFLD.
more목차
Ⅰ. Introduction 1
A. Progression of Non-alcoholic fatty liver disease (NAFLD) 1
B. Insulin resistance 2
C. Visfatin 2
D. Hepatic inflammation 3
E. Hepatic fibrosis 6
F. Aims of the study 7
Ⅱ. Materials and methods 8
A. Reagents 8
B. HepG2 cell culture 8
C. THP-1 and LX-2 cell culture 8
D. Immunoblotting 9
E. Reverse transcriptase-polymerase chain reaction (RT-PCR) 10
F. Flow cytometric measurement of reactive oxygen species (ROS) 10
G. Immunocytochemistry 11
H. Human cytokine array and image analyses 11
I. Enzyme-linked immunosorbent assay (ELISA) 11
J. Animal studies 12
1. High fat (HF) diet model 12
2. Methionine/Choline Deficient (MCD) diet model 12
K. Isolation and treatment of liver cells 13
L. ROS/RNS assay 13
M. Histopathological analyses 13
N. Statistical analyses 13
Ⅲ. Results 21
PART A. Visfatin induces insulin resistance and inflammation via the NF-κB and STAT3 signaling pathway in hepatocytes 21
1. Effect of visfatin on the expression of lipid metabolism-related genes in HepG2 cells 21
2. Visfatin enhanced ER stress and ROS production in HepG2 cells 23
3. Visfatin induced insulin resistance in a dose-dependent manner in HepG2 cells 26
4. Visfatin induced the production of pro-inflammatory cytokines in HepG2 cells 28
5. Visfatin activated NF-κB/STAT3 signaling and expression of IRS-1 S 307 in HepG2 cells 30
6. JAK/STAT3 and NF-κB inhibitors inhibited visfatin-induced expression of inflammatory cytokines and insulin resistance in HepG2 cells 34
PART B. Visfatin induced the expression of CCL20 in macrophages via the NF-κB and MKK3/6-p38 signaling pathway and activated hepatic stellate cells (HSCs) 37
1. Visfatin treatment promoted differentiation of THP-1 cells to M1 macrophages 37
2. Visfatin upregulated the expression of CCL20 in THP-1 cells 39
3. Visfatin activated NF-κB and MAPK cascade pathways in THP-1 cells 41
4. Inhibition of NF-κB and MAPK cascade pathways attenuated visfatin-induced expression of CCL20 and fibrosis markers in HSCs 44
5. Visfatin-induced CCL20 enhanced the expression of fibrosis genes in LX-2 cells 46
6. Visfatin enhanced the expression of fibrosis markers in LX-2 cells 49
PART C. Visfatin exacerbates hepatic inflammation and fibrosis in an animal model of NAFLD 51
1. Increased hepatic expression of visfatin was associated with hepatic inflammation 51
2. Visfatin induced marked steatosis and liver inflammation in mice on high fat diet (HFD) 54
3. Visfatin aggravated hepatic steatosis and inflammation in the liver of MCD-diet fed mice 56
4. Visfatin induced infiltration of immune cells and production of chemokines in the liver 58
5. Visfatin induced the expression and production of chemokines in the liver 60
6. Visfatin induced ER stress and ROS generation 62
7. Visfatin accelerated the development of liver fibrosis in MCD-diet-fed mice. 64
Ⅳ. Discussion 66
Ⅴ. Conclusion 74
REFERENCES 75
국문요약 87
