Mismatched effects of receptor interacting protein kinase-3 on hepatic steatosis and inflammation in non-alcoholic fatty liver disease

Saeed, Waqar Khalid,Jun, Dae Won,Jang, Kiseok,Ahn, Sang Bong,Oh, Ju Hee,Chae, Yeon Ji,Lee, Jai Sun,Kang, Hyeon Tae Baishideng Publishing Group Inc 2018 WORLD JOURNAL OF GASTROENTEROLOGY Vol.24 No.48

<P><B>AIM</B></P><P>To validate the effects of receptor interacting protein kinase-3 (RIP3) deletion in non-alcoholic fatty liver disease (NAFLD) and to clarify the mechanism of action.</P><P><B>METHODS</B></P><P>Wild-type (WT) and RIP3 knockout (KO) mice were fed normal chow and high fat (HF) diets for 12 wk. The body weight was assessed once weekly. After 12 wk, the liver and serum samples were extracted. The liver tissue expression levels of RIP3, microsomal triglyceride transfer protein, protein disulfide isomerase, apolipoprotein-B, X-box binding protein-1, sterol regulatory element-binding protein-1c, fatty acid synthase, cluster of differentiation-36, diglyceride acyltransferase, peroxisome proliferator-activated receptor alpha, tumor necrosis factor-alpha (TNF-α), and interleukin-6 were assessed. Oleic acid treated primary hepatocytes from WT and RIP3KO mice were stained with Nile red. The expression of inflammatory cytokines, including chemokine (C-X-C motif) ligand (CXCL) 1, CXCL2, and TNF-α, in monocytes was evaluated.</P><P><B>RESULTS</B></P><P>RIP3KO HF diet fed mice showed a significant gain in body weight, and liver weight, liver to body weight ratio, and liver triglycerides were increased in HF diet fed RIP3KO mice compared to HF diet fed WT mice. RIP3KO primary hepatocytes also had increased intracellular fat droplets compared to WT primary hepatocytes after oleic acid treatment. RIP3 overexpression decreased hepatic fat content. Quantitative real-time polymerase chain reaction analysis showed that the expression of very-low-density lipoproteins secretion markers (microsomal triglyceride transfer protein, protein disulfide isomerase, and apolipoprotein-B) was significantly suppressed in RIP3KO mice. The overall NAFLD Activity Score was the same between WT and RIP3KO mice; however, RIP3KO mice had increased fatty change and decreased lobular inflammation compared to WT mice. Inflammatory signals (CXCL1/2, TNF-α, and interleukin-6) increased after lipopolysaccharide and pan-caspase inhibitor (necroptotic condition) treatment in monocytes. Neutrophil chemokines (CXCL1, and CXCL2) were decreased, and TNF-α was increased after RIP3 inhibitor treatment in monocytes.</P><P><B>CONCLUSION</B></P><P>RIP3 deletion exacerbates steatosis, and partially inhibits inflammation in the HF diet induced NAFLD model.</P>

Necroptosis: an emerging type of cell death in liver diseases.

Saeed, Waqar Khalid,Jun, Dae Won WJG Press 2014 WORLD JOURNAL OF GASTROENTEROLOGY Vol.20 No.35

<P>Cell death has been extensively evaluated for decades and it is well recognized that pharmacological interventions directed to inhibit cell death can prevent significant cell loss and can thus improve an organ's physiological function. For long, only apoptosis was considered as a sole form of programmed cell death. Recently necroptosis, a RIP1/RIP3-dependent programmed cell death, has been identified as an apoptotic backup cell death mechanism with necrotic morphology. The evidences of necroptosis and protective effects achieved by blocking necroptosis have been extensively reported in recent past. However, only a few studies reported the evidence of necroptosis and protective effects achieved by inhibiting necroptosis in liver related disease conditions. Although the number of necroptosis initiators is increasing; however, interestingly, it is still unclear that what actually triggers necroptosis in different liver diseases or if there is always a different necroptosis initiator in each specific disease condition followed by specific downstream signaling molecules. Understanding the precise mechanism of necroptosis as well as counteracting other cell death pathways in liver diseases could provide a useful insight towards achieving extensive therapeutic significance. By targeting necroptosis and/or other parallel death pathways, a significant cell loss and thus a decrement in an organ's physiological function can be prevented.</P>

Targeting MLKL Component of Necroptosis Pathway Protects against Hepatic Steatosis

( Waqar Khalid Saeed ),( Dae Won Jun ),( Ki-seok Jang ),( Jae Yoon Jeong ),( Sang Bong Ahn ),( Joo Hyun Sohn ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: We aimed to evaluate the protective effects of MLKL deletion in mouse model of high fat (HF) diet induced steatosis. Methods: 8-9 weeks old C57BL/6 wild type (WT) (n=8) and MLKL-KO (n=6) mice were randomly divided into normal chow (NC) and HF diet groups. NC and HF diet was fed for 12 weeks. The body and diet weight were measured weekly. After 12 weeks the animal were sacrificed, serum and liver samples were collected. Liver weight and liver/body weight ratio was calculated. H&E staining was performed and NAS score was evaluated. Epididymal adipose tissue F4/80 IHC was performed. Liver biopsies of NASH patients were evaluated for MLKL expression. Results: HF diet increased the body and liver weight of the mice. There was no significant difference in body weight of WT and MLKL-KO mice fed on NC and HF diet. MLKL-KO-HF mice only in 12^th week had significantly reduced body weights as compared to WT-HF mice. Interestingly, MLKL-KO animals fed on both NC and HF diet had increased diet intake as compared to corresponding WT groups. After 12 weeks, MLKL-KO animals had reduced liver weights as compared to corresponding WT groups. Accordingly, HF diet fed MLKL-KO animals had decreased serum AST, ALT and TG contents. H&E staining showed increased steatosis in HF diet fed animals. However, MLKL-KO-HF mice had reduced steatosis and NAS score. Adipose tissue F4/80 IHC showed HF fed MLKL-KO group had decreased macrophage associated crown-like structures as compared to WT-HF group. Human NASH liver biopsies showed significantly increased MLKL expression as compared to the normal liver, signifying MLKL induction in NASH patients. Conclusions: Targeting MLKL component of necroptosis pathway significantly decreases liver weight, steatosis and the serum markers of hepatic injury without significantly affecting the overall body weight.

RIP3 Inhibition Promotes Steatosis in High Fat Diet Induced NAFLD

( Waqar Khalid Saeed ),( Dae Won Jun ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: The protective effects of RIP3 inhibition have been reported methionine & choline deficient (MCD) diet induced fibrosis and in ethanol induced hepatic injury; however, the effects of RIP3 inhibition on high fat (HF) diet induced hepatic steatosis and fibrosis have not been evaluated. Methods: 8-9 week old, C57BL/6 (WT) and RIP3KO mice were fed normal chow (NC), HF and MCD diets for 12 weeks. The animals were randomly divided into following groups (n=8): 1) WT-NC 2) WT-HF 3) WT-MCD 4) RIP3KO-NC, RIP3KO-HF and RIP3KO-MCD. The body weight of the animals was evaluated weekly. After 12 weeks, the animals were euthanized, and the liver and blood samples were collected. The liver to body weight ratio, H&E, serum AST, ALT and TG levels were assessed. Liver TG contents were evaluated using commercial kit. Western blot analysis for alpha-SMA, CD36, SREBP1, JNK, p-JNK, p-c-jun, p-peif2a, ATF6-alpha, MLKL, and LC3 were performed. Results: The body weight of animals fed with HF diet increased while MCD diet fed animals decreased. The liver and body weight of RIP3KO-HF mice increased significantly as compared to WT-HF group; moreover, the liver and liver/body weight ratio was also increased in RIP3KO animals. The H&E evaluation showed significantly increased steatosis in HF and MCD diet fed groups. However, the extent of steatosis seemed to be more pronounced in RIP3KO-HF diet group. The serum AST & ALT increased in both HF and MCD diets groups; however, AST and ALT were significantly increased in RIP3KO animals in MCD and HF diet fed groups, respectively. The hepatic TG contents were also significantly increased in RIP3KO-HF group as compared to WT and RIP3KO-MCD groups. Conclusions: RIP3 inhibition in HF diet fed animals promotes steatosis and development of NAFLD.

Feasibility and Stability of Liver Biopsy before Treatment for Preclinical Nonalcoholic Fatty Liver Studies

채연지,전대원,Waqar Khalid Saeed,강현태,오주희,이승민,장기석 대한의학회 2019 Journal of Korean medical science Vol.34 No.2

Background: The heterogeneity of histological findings in preclinical diet-induced nonalcoholic fatty liver disease (NAFLD) animal models is highly challenging. Here, we aimed to evaluate the feasibility and stability of repeated liver biopsy in NAFLD animal models. Methods: Heterogeneity of diet-induced NAFLD was evaluated at different time points in 52 high-fat diet (HFD), 35 methionine choline-deficiency diet (MCD), and 166 western diet (WD) induced NAFLD mice. Serial liver biopsies (left lateral, right medial, and left medial lobes) were performed monthly for up to 3 months. Mortality rates and changes in food intake, body weight, and liver enzymes were assessed. Results: At 12 weeks, of the HFD animals, 14% and 30% did not develop steatosis and lobular inflammation, respectively; of the MCD animals, 7% did not develop lobular inflammation; and of the WD animals, 14% and 51% did not develop steatosis and lobular inflammation, respectively. The mortality rate of repeated liver biopsy was 1.62% (2/123 mice died). Repeated liver biopsy can be used to trace disease progression. Although body weight, food intake, and liver enzymes slightly changed after biopsy, all recovered within a week. Repeated liver biopsy did not affect the degrees of inflammation and steatosis of the other liver lobes. Conclusion: The diet-induced NAFLD models were quite heterogeneous. Our results suggest that the repeated liver biopsy before treatment was applicable and stable in this NAFLD animal study.

Basic, Research : Role of 5-hydroxytryptamine Receptor 2A Antagonist in Hepatic Fibrosis

( Dae Won Jun ),( Waqar Khalid Saeed ),( Tae Yeob Kim ),( Joo Hyun Sohn ),( Kang Nyeong Lee ),( Hang Lak Lee ),( Oh Young Lee ),( Byung Chul Yoon ),( Ho Soon Choi ) 대한간학회 2013 춘·추계 학술대회 (KASL) Vol.2013 No.1

Background: The aim of this study was to determine whether 5-HT2A receptor antagonists affect the activation or apoptosis of HSCs in vitro and/or in vivo. Methods: For the in vitro experiments, the viability, apoptosis and wound healing ability of LX-2 cells were examined after treatment with various 5-HT2A receptor antagonists. Levels of HSC activation markers (procollagen type I, α-SMA, TGF-β and Smad 2/3) were measured. For in vivo experiments, rats were divided into three groups: control group, cirrhosis, 5-HT2A antagonist group. Results: 5-HT2A receptors expression was essentially absent in inactive LX-2 cells but was induced in activated LX-2 cells. Expression of the 5-HT2A and 5-HT2B receptors was significantly decreased by sarpogrelate, with a somewhat greater effect on the 5-HT2A receptor. Similar results were obtained with primary hepatic stellate cells. There was a time and dose dependent decrease in sarpogrelate-treated cell proliferation compared to untreated cells (P<0.05). Ketanserin and ritanserin also had anti-proliferative effects. Ketanserin and sarpogrelate significantly increased HSC apoptosis, with the effect strongest for ketanserin in TUNEL assay. The expression of α-SMA was decreased by sarpogrelate in a dose dependent manner. LX-2 cell migration was significantly suppressed in sarpogrelate or ketanserin- treated cultures and wound healing was delayed compared to cultures treated with only PDGF. There was less severe periportal and septal fibrosis in the rats in the treatment group, but the difference was not statistically significant in Masson`s trichrome stain. But expression of α-SMA was lower in the treatment group than in the cirrhotic group (61.0±7.2% vs 136.7±11.7%, P<0.05), and the treatment group expression was lower than in the disease group (58.9±0.8% vs 118.2±18.2%, P<0.05). Conclusions: 5-HT2A receptor antagonists can reduce inflammation and the activation of HSCs in this cirrhotic model.

Feasibility and Stability of Liver Biopsy before Treatment for Preclinical Nonalcoholic Fatty Liver Studies

Chae, Yeon Ji,Jun, Dae Won,Saeed, Waqar Khalid,Kang, Hyeon Tae,Oh, Ju Hee,Lee, Seung Min,Jang, Kiseok KOREAN ACADEMY OF MEDICAL SCIENCE 2019 JOURNAL OF KOREAN MEDICAL SCIENCE Vol.34 No.2

<P><B>Background</B></P><P>The heterogeneity of histological findings in preclinical diet-induced nonalcoholic fatty liver disease (NAFLD) animal models is highly challenging. Here, we aimed to evaluate the feasibility and stability of repeated liver biopsy in NAFLD animal models.</P><P><B>Methods</B></P><P>Heterogeneity of diet-induced NAFLD was evaluated at different time points in 52 high-fat diet (HFD), 35 methionine choline-deficiency diet (MCD), and 166 western diet (WD) induced NAFLD mice. Serial liver biopsies (left lateral, right medial, and left medial lobes) were performed monthly for up to 3 months. Mortality rates and changes in food intake, body weight, and liver enzymes were assessed.</P><P><B>Results</B></P><P>At 12 weeks, of the HFD animals, 14% and 30% did not develop steatosis and lobular inflammation, respectively; of the MCD animals, 7% did not develop lobular inflammation; and of the WD animals, 14% and 51% did not develop steatosis and lobular inflammation, respectively. The mortality rate of repeated liver biopsy was 1.62% (2/123 mice died). Repeated liver biopsy can be used to trace disease progression. Although body weight, food intake, and liver enzymes slightly changed after biopsy, all recovered within a week. Repeated liver biopsy did not affect the degrees of inflammation and steatosis of the other liver lobes.</P><P><B>Conclusion</B></P><P>The diet-induced NAFLD models were quite heterogeneous. Our results suggest that the repeated liver biopsy before treatment was applicable and stable in this NAFLD animal study.</P>

Hepatocyte Nuclear Factor 4Alpha Attenuates Lipotoxicity and Hepatic Fibrosis in Nonalcoholic Fatty Liver Disease Model

( Jai Sun Lee ),( Dae Won Jun ),( Waqar Khalid Saeed ),( Hyun Tae Kang ),( Yeon Ji Chae ),( Seung Min Lee ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: Hepatocyte nuclear factor 4α (HNF4α) is known as a master regulator of bile acid metabolism and triglyceride synthesis pathway. Role of HNF4α on non-alcohol fatty liver disease (NAFLD) are still debate. Methods: Hepatic HNF4α expression was evaluated in four different liver disease models: high fat (HF), methionine-choline deficient (MCD), common bile duct (CBD) ligation, thioacetamide (TAA). HNF4α over-expressed and down-regulated in palmatic acid induced lipotoxicity model and chenodeoxycholic acid induced bile acid toxicity In vitro model. HNF4α over-expressed and down regulation using adenovirus in methionine choline deficiency (MCD) diet induced NAFLD model. H&E staining, qPCR genes for lipid and bile acid pathways, VLDL assay and sirus red staining were evaluated. Results: HNF4α expression increased in NAFLD models, but decreased in cirrhosis animal models. HNF4α overexpression attenuated palmitic acid induced hepatocyte apoptosis as well as intracellular fat accumulation in In vitro model. HNF4α overexpression increased fatty oxidation (ACOX1, and CPT1A), and VLDL secretion pathway (MTTP, and ApoB) in lipotoxicity In vitro model. Meanwhile, siRNA induced down regulation of HNF4α were protected against bile acid-induced toxicity. HNF4α down-regulation also exhibited decreased mRNA expression of bile acid transporter (NTCP) and bile acid synthesis (CYP7a1) enzymes. HNF4α over-expression using adenovirus decreased serum triglyceride, ALT level in NAFLD model. The expression inflammatory markers (TNF-α, MCP-1 and IL-6) also decreased HNF4a over-expression group as compared to the MCD group. The sirus red staining indicated that liver fibrosis decreased in HNF4a over-expression group as compared to the MCD group. Conclusions: HNF4α decreased hepatic inflammation and fibrosis via protecting lipotoxicity in NAFLD model.

Ezetimibe decreased nonalcoholic fatty liver disease activity score but not hepatic steatosis

( Hyo Young Lee ),( Dae Won Jun ),( Hyun Jung Kim ),( Hyunwoo Oh ),( Waqar Khalid Saeed ),( Hyeongsik Ahn ),( Ramsey C. Cheung ),( Mindie H. Nguyen ) 대한내과학회 2019 The Korean Journal of Internal Medicine Vol.34 No.2

Background/Aims: A number of clinical trials reported varying effects of cho-lesterol lowering agents in nonalcoholic fatty liver disease (NAFLD) patients. We, therefore, assessed the changes in hepatic steatosis and NAFLD activity score (NAS) after treatment with cholesterol lowering agents in NAFLD patients by meta-analysis. Methods: The Cochrane Library, the MEDLINE, and the Embase databases were searched until May 2015, without any language restrictions, for randomized con-trolled trials (RCTs) and nonrandomized studies (NRSs). Additional references were obtained from review of bibliography of relevant articles. The quality of ev-idence was assessed using the grading of recommendations assessment, develop-ment and evaluation guidelines. Results: Three RCTs (n = 98) and two NRSs (n = 101) met our study inclusion cri-teria (adult, NAFLD, liver biopsy). Liver biopsy was performed in all five studies, but only the three studies reported NAS. Ezetimibe significantly decreased NAS (standardized mean difference [SMD], -0.30; 95% confidence interval [CI], -0.57 to -0.03) but not hepatic steatosis in RCT (SMD, -0.1; 95% CI, -0.53 to 0.32), while the effect was significant for both NAS and intrahepatic content in NRSs (SMD, -3.0; 95% CI, -6.9 to 0.91). Conclusions: Ezetimibe decreased NAS without improving hepatic steatosis.

Cholesterol-lowering Agents Decreased NAS Score without Intrahepatic Fat Improvement in Patients with Non-alcoholic Fatty Liver Disease: Systematic Review with Meta-analysis

( Hyo Young Lee ),( Dae Won Jun ),( Hyunwoo Oh ),( Waqar Khalid Saeed ),( Jae Yoon Jeong ),( Joo Hyun Sohn ),( Chang Hong Lee ),( Hyun Jung Kim ),( Hyeong Sik Ahn ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: A number of clinical trials reported diverse effects of cholesterol- lowering agents in nonalcoholic fatty liver disease (NAFLD) patients. We, therefore, assessed the changes in non-alcoholic steatohepatitis activity score (NAS) and intrahepatic fat contents after treatment with cholesterol lowering agents in NAFLD patients by performing a meta-analysis. Methods: The Cochrane library, the MEDLINE, and the EMBASE databases were searched until June 2015, without any language restrictions, for randomized controlled trials (RCTs) and non-randomized studies (NRSs); additional references were obtained from reviewed articles. The quality of evidence was assessed using the grading of recommendations assessment, development and evaluation guidelines. Results: Three RCTs and three NRSs (235 participants) met the inclusion criteria for this updated systematic review. Liver biopsy was performed in three RCTs, but only the two studies reported NAS. Ezetimibe showed decreased NAS (Pooled RR: -0.65, 95% CI: -1.15 ~ -0.015). The intrahepatic fat contents were not decreased in RCTs, while they were decreased in NRSs. The baseline cholesterol level in all three RCTs was lower than the three NRSs (191mg/dl vs. 227.5mg/dl). The pre-planned secondary outcomes of AST, ALT, Glucose, HbA1c analysis did not show significant difference after treatment with cholesterol lowering agents. Conclusions: Ezetemibe decreased NAS score without improving intrahepatic fat contents.