Beneficial effects of dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter 2 inhibitors on nonalcoholic fatty liver disease
- 주제(키워드) Nonalcoholic fatty liver disease , inflammation , fibrosis , Dipeptidyl peptidase-4 inhibitors , Sodium-glucose cotransporter 2 inhibitors
- 주제(DDC) 610
- 발행기관 아주대학교
- 지도교수 김혜진
- 발행년도 2022
- 학위수여년월 2022. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031912
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Nonalcoholic fatty liver disease (NAFLD) is a heterogeneous liver disease characterized by hepatic steatosis, inflammation, and fibrosis, with its increasing prevalence adding to the global public health burden. NAFLD is currently understood as a chronic inflammatory disease primarily caused by free fatty acid accumulation and insulin resistance. Chronic inflammation is associated with the progression of fibrosis, which increases the incidence of hepatocellular carcinoma and mortality. Several drugs, including pioglitazone and vitamin E, have been tested for NAFLD treatment; however, no definitive agent is yet available to improve or protect from the progression of NAFLD. Dipeptidyl peptidase-4 (DPP4) inhibitors and sodium–glucose cotransporter 2 (SGLT2) inhibitors are commonly prescribed as anti-diabetic agents for type 2 diabetes patients, as they display different mechanisms of action, and their combination is considered a complementary treatment option. DPP4 inhibitors and SGLT2 inhibitors could reduce insulin resistance, which is one of the important triggers in the pathogenesis of NAFLD. Moreover, these inhibitors have been recently shown to exert anti-inflammatory as well as hypoglycemic effects. Thus, in this study, we evaluated the therapeutic effects of DPP4 inhibitors and SGLT2 inhibitors with respect to the progression of NAFLD and identified the signaling pathways associated with the molecular mechanism for their action. Seven-week-old C57BL/6J male mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 7 weeks to induce NAFLD, while another group was fed a standard chow as a negative control. From the third week of feeding, CDAHFD-fed mice were randomized into four groups based on the tested drug: dimethyl sulfoxide (positive control), gemigliptin (DPP4i), empagliflozin (SGLT2i), and the combination of gemigliptin and empagliflozin (DPP4i + SGLT2i), and administered the corresponding agent for 5 weeks. Thereafter, glucose tolerance, triglyceride content, lipid redox, pro-inflammatory cytokines and chemokines, and extent of fibrosis were analyzed in the serum and liver. In addition, RAW 264.7 macrophage cells were subject to co-treatment with these drugs and lipopolysaccharide (LPS). The resultant changes in oxidative stress, inflammatory response, and intracellular signaling pathways were then investigated to help understand the molecular mechanism of action of these inhibitors. The DPP4 inhibitor and SGLT2 inhibitor tested ameliorated hepatic TG accumulation, mRNA expression of inflammatory cytokines and chemokines, and the profibrogenic IL-33/galectin-3/TGF-β axis in the CDAHFD-induced NAFLD mouse model. In addition, we confirmed this anti-inflammatory effect in LPS-stimulated RAW 264.7 cells on administration of these drugs, and finally demonstrated that these drugs exert anti-inflammatory effects via downregulation of the IKK/NF-kB, MKK4/JNK, MKK7/JNK, JAK2/STAT1, and JAK2/STAT3 pathways. Thus, our results show that DPP4 inhibitors and SGLT2 inhibitors, alone or in combination, improve the development of NAFLD across hepatic steatosis, oxidative stress, inflammation, and fibrosis through diverse cell signaling pathways.
more초록/요약
비알코올성 지방간 질환 (nonalcoholic fatty liver disease, NAFLD)은 간 지방증, 염증 및 섬유화를 특징으로 하는 이질적인 질환으로, 전 세계적으로 공중 보건 분야의 부담과 함께 그 유병률이 크게 증가하고 있다. NAFLD는 현재 주로 유리 지방산 축적과 인슐린 저항성에 의해 유발되는 만성 염증 질환으로 여겨지고 있다. 이 만성 염증은 간세포암종의 발병률과 사망률을 증가시키는 간 섬유화의 진행과 관련이 있다. 피오글리타존 및 비타민 E와 같은 여러 약물이 NAFLD 환자를 치료하기 위해 시도되었지만 NAFLD의 진행을 개선하거나 보호하는 결정적인 약제는 없다. 디펩티딜 펩티다제-4 (dipeptidyl peptidase-4, DPP4) 억제제와 나트륨-포도당 공동수송체 (sodium-glucose cotransporter, SGLT) 2 억제제는 작용 기전이 다르기 때문에 제2형 당뇨 환자에서 흔히 처방되는 혈당강하제로 이들의 조합은 보완적 치료 옵션으로 간주된다. DPP4 억제제와 SGLT2 억제제는 NAFLD의 발병 기전에 중요한 시발점인 인슐린 저항성을 감소시킬 수 있다. 게다가 최근 이러한 억제제의 혈당강하뿐 아니라 항염증효과도 알려졌다. 따라서 본 연구에서는 NAFLD 진행과 관련하여 DPP4 억제제와 SGLT2 억제제의 치료 효과를 평가하고 이의 기전에 관여하는 신호 전달 경로를 확인하고자 한다. 7주령의 C57BL/6J 수컷 마우스에 7주 동안 콜린 결핍 및 L-아미노산으로 한정된 고지방 식이 (choline-deficient, L-amino acid-defined, high-fat diet, CDAHFD)를 먹여 NAFLD를 유도하고, 음성 대조군으로 배정한 마우스에는 표준 식이를 먹였다. 식이 3주차부터 CDAHFD를 먹인 마우스를 디메틸 설폭사이드 (양성 대조군), 제미글립틴 (DPP4 억제제군), 엠파글리플로진 (SGLT2 엑제제군), 그리고 제미글립틴과 엠파글리플로진 (DPP4 억제제 + SGLT2 억제제군)의 4개 군으로 무작위 배정하고, 5주 동안 해당 약제를 투여하였다. 그 후, 혈청 및 간 조직에서 내당능, 중성지방 함량, 지질 산화환원, 전염증성 사이토카인 및 케모카인, 그리고 섬유화 정도를 분석하였다. 또한, RAW 264.7 대식세포에도 이 억제제들과 지질다당류 (lipopolysaccharide, LPS) 를 함께 처리하였다. 그리고 산화 스트레스, 염증 반응 및 세포내 신호전달경로의 변화를 조사하여 이들 억제제의 분자적 작용 기전을 분석하였다. DPP4 억제제와 SGLT2 억제제는 CDAHFD로 유도된 NAFLD 마우스 모델에서 간의 트리글리세리드 축적, 염증성 사이토카인 및 케모카인의 mRNA 발현, 그리고 섬유화와 관련된 IL-33/Gal-3/TGF-β 축을 개선하였다. 또한, LPS로 자극된 RAW 264.7 세포에 DPP4 억제제와 SGLT2 억제제를 처리하였을 때도 이러한 항염증 효과를 확인하였고, 이는 IKK/NF-kB, MKK4/JNK, MKK7/JNK, JAK2/STAT1, 그리고 JAK2/STAT3 신호 경로의 하향 조절을 통해 항염증 효과가 있다는 것을 확인하였다. 이 연구는 DPP4 억제제와 SGLT2 억제제를 단독 또는 병용 투여 시 다양한 세포신호전달경로를 통해 간 지방증, 산화 스트레스, 염증 및 섬유화 전반에 걸쳐 NAFLD의 발달을 개선함을 보여주었다.
more목차
Ⅰ. Introduction 1
A. Epidemiology of nonalcoholic fatty liver disease (NAFLD) 1
B. Pathophysiology of NAFLD 1
C. Dipeptidyl peptidase-4 (DPP4) inhibitors 3
D. Sodium-glucose cotransporter (SGLT) 2 inhibitors 3
E. Combination of DPP4 inhibitors and SGLT2 inhibitors 4
F. Aims of the study 5
Ⅱ. Materials and methods 6
A. Animal groups and treatment 6
B. Glucose tolerance test (GTT) 7
C. Histopathological examination and histological scoring 8
D. Biochemical analysis 8
E. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 9
F. Enzyme-linked immunosorbent assay (ELISA) 9
G. Western blotting 9
H. Cell culture and treatment 10
I. Cell viability 11
J. Flow cytometry 11
K. Statistical analysis 11
Ⅲ. Results 16
PART A. Pleiotropic effects of DPP4 inhibitors and SGLT2 inhibitors on NAFLD in a diet-induced mouse model 16
1. DPP4 inhibitors and SGLT2 inhibitors did not alter glucose tolerance and body weight in diet-induced NAFLD mice 16
2. DPP4 inhibitors and SGLT2 inhibitors ameliorate liver histopathologic steatosis, inflammation, and fibrosis in diet-induced NAFLD mice 18
3. DPP4 inhibitors and SGLT2 inhibitors modulate hepatic injury indices and steatosis in diet-induced NAFLD mice 24
4. DPP4 inhibitors and SGLT2 inhibitors protect liver from redox dysregulation–induced damage in diet-induced NAFLD mice 28
5. DPP4 inhibitors and SGLT2 inhibitors reduce pro-inflammatory cytokines and chemokines in diet-induced NAFLD mice 31
6. DPP4 inhibitors and SGLT2 inhibitors improve hepatic fibrosis in diet-induced NAFLD mice 36
7. DPP4 inhibitors and SGLT2 inhibitors inhibit F4/80 mRNA expression in diet-induced NAFLD mice 39
PART B. Mechanism of protection induced by DPP4 inhibitors and SGLT2 inhibitors against NAFLD progression in mouse macrophages 41
1. DPP4 inhibitors and SGLT2 inhibitors exert anti-inflammatory effects without cytotoxicity in RAW 264.7 cells 41
2. DPP4 inhibitors and SGLT2 inhibitors ameliorate pro-inflammatory mediators, cytokines, and chemokines in LPS-activated RAW 264.7 cells 45
3. DPP4 inhibitors and SGLT2 inhibitors protect liver damage from inflammation via inhibition of IKK/NF-κB signaling pathways in RAW 264.7 cells 51
4. DPP4 inhibitors and SGLT2 inhibitors attenuate LPS-induced inflammation via inhibition of JAK/STAT signaling pathways in RAW 264.7 cells 54
5. DPP4 inhibitors and SGLT2 inhibitors alter LPS-induced inflammation via inhibition of MKK/JNK signaling pathways in RAW 264.7 cells 56
Ⅳ. Discussion 58
A. Changes in serum liver enzymes and hepatic TG content on treatment with DPP4 inhibitors and SGLT2 inhibitors in CDAHFD-induced NAFLD mouse model 58
B. Protective effects of DPP4 inhibitors and SGLT2 inhibitors on oxidative stress and chronic inflammation in CDAHFD-induced NAFLD mouse model 59
C. Anti-fibrotic effects of DPP4 inhibitors and SGLT2 inhibitors in CDAHFD-induced NAFLD mouse model 60
D. Determination of major target inflammatory cell populations of DPP4 inhibitors and SGLT2 inhibitors in the liver 62
E. Anti-inflammatory effects of DPP4 inhibitors and SGLT2 inhibitors in LPS-stimulated RAW 264.7 cells 62
F. Downregulation of IKK/NF-kB signaling pathway by DPP4 inhibitors and SGLT2 inhibitors in LPS-stimulated RAW 264.7 cells 63
G. Downregulation of JAK2/STAT1 and JAK2/STAT3 signaling pathway by DPP4 inhibitors and SGLT2 inhibitors in LPS-stimulated RAW 264.7 cells 63
H. Downregulation of MKK4/JNK and MKK7/JNK signaling pathway by DPP4 inhibitors and SGLT2 inhibitors in LPS-stimulated RAW 264.7 cells 64
I. Strengths and limitations 65
J. Summary 66
Ⅴ. Conclusion 68
REFERENCES 69
