NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.

BACKGROUND & AIMS: NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. METHODS: We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. RESULTS: In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1ß, active caspase-1 and IL-1ß increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1ß, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1ß expression, plasma IL-1ß, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1ß; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1ß, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. CONCLUSION: MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. LAY SUMMARY: Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

Cytochrome P450 1B1 Contributes to the Development of Atherosclerosis and Hypertension in Apolipoprotein E-Deficient Mice.

Cytochrome P450 (CYP) 1B1 contributes to vascular smooth muscle cell growth and hypertension in male mice. This study was conducted to determine the contribution of CYP1B1 to the development of atherosclerosis and hypertension and associated pathogenesis in 8-week-old male apolipoprotein E-deficient (ApoE(-/-)/Cyp1b1(+/+)), and ApoE- and CYP1B1-deficient (ApoE(-/-)/Cyp1b1(-/-)) mice fed a normal or atherogenic diet for 12 weeks. A separate group of ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet was injected every third day with the CYP1B1 inhibitor, 2,3',4,5'-tetramethoxystilbene (300 µg/kg), or its vehicle, dimethyl sulfoxide (30 µL, IP); systolic blood pressure was measured by the tail cuff method. After 12 weeks, mice were euthanized, blood collected for lipid analysis, and aortas harvested for measuring lesions and remodeling, and for infiltration of inflammatory cells by histological and immunohistochemical analysis, respectively, and for reactive oxygen species production. Blood pressure, areas of lipids and collagen deposition, elastin breaks, infiltration of macrophages and T lymphocytes, reactive oxygen species generation in the aorta, and plasma lipid levels were increased in ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet; these changes were minimized in mice given 2,3',4,5'-tetramethoxystilbene, and in ApoE(-/-)/Cyp1b1(-/-) mice on an atherogenic diet; absorption/production of lipids remained unaltered in these mice. These data suggest that aortic lesions, hypertension, and associated pathogenesis in ApoE(-/-)/Cyp1b1(+/+) mice on an atherogenic diet are most likely dependent on CYP1B1-generated oxidative stress and increased plasma lipid levels independent of blood pressure and absorption of lipids. CYP1B1 could serve as a novel target for developing drugs to treat atherosclerosis and hypertension caused by hypercholesterolemia.

Study on the tumor growth and angiogenesis in the tumor of colon cancer xenografts in high fat diet induced metabolic syndrome model in nude mice / 中华消化杂志

Objective To investigate the characteristics of growth and angiogenesis of colon cancer xenograft in nude mice with metabolic syndrome induced by high fat diet.Methods Female BALB/C nude mice were fed with high fat diet (45.0％ from fat,HFD group) or common diet (13.8％ from fat,CD group) for 12 weeks (n=15,respectively).Colon cancer cell line SW480 was marked with green fluorescent protein (GFP) and subcutaneous xenograft model was established.The tumor growth was observed by the in vivo imaging system in small animal at the 4th week.By the end of the experiment,serum glucose and lipid level of the two groups were measured,visceral subcutaneous and visceral adipose tissue,liver and xenograft tumor were dissociated and weighted.The differences of proliferating cell nuclear antigen (PCNA) and CD31 expression in the tumors between groups were analyzed.The t-test or x2 test were performed for group comparison.Results Compared with CD group,the body weight,blood serum glucose level,triglyceride and cholesterol level,adipose content of subcutaneous and visceral of the HFD group significantly increased (t=2.91,4.12,4.43,3.92,3.77 and 4.02,all P＜0.05).Averagedaily energy intake of HFD group was significantly higher than that of CD group (t=2.34,P＜0.05).There was no significant difference between the two groups in liver weight (t=1.02,P＞0.05).However,by HE staining lipid vacuoles in the liver tissue was obvious in HFD group.Average bioluminescent index,tumor volume and weight of xenografts of HFD group were remarkably higher than those of CD group (t=8.84,2.48 and 2.86,all P＜0.05).The immunohistochemical staining results indicated that the strong positive rate of PCNA in xenografts of HFD group was 80.00％ and the microvessel density (MVD) was (25.75±0.96)/per high power field,both of which were higher than those of CD group (14.29％ and (13.33±1.53)/per high power field respectively,x2 =12.52,t=13.35,both P＜0.01).Conclusions The colon cancer xenograft in nude mice with metabolic syndrome induced by high fat diet had a high MVD and grew fast.

Effects of paeoniflorin on insulin resistance and adipose tissue TNFαand GLUT4 expressions in obese rats fed with high-fat food / 中国医师杂志

Objective To observe the effect of paeoniflorin on insulin resistance in rats fed with high-fat diet and to investigate the possible mechanisms.Methods Male Sprague Dawley (SD) rats were randomized into 4 groups:normal control,high-fat diet,high-dosage paeoniflorin (HDP group),and lowdosage paeoniflorin (LDP group).The control group was fed with ordinary diet,while the others with highfat diet,paeoniflorin intervention groups were given low-or high-dosage paeoniflorin by intraperitoneal injection.After 6 weeks,fasting serum triacylglycerol (TG),total cholesterol (TC),free fatty acids (FFA),fasting blood glucose (FBG),and insulin were determined.Insulin sensitivity index (ISI) were calculated and then the animals were sacrificed to acquire epididymal fat mass.The tumor necrosis factor alpha (TNFα) and glucose transporter 4 (Glut4) expressions in adipose tissue were detected by quantitative realtime polymerase chain reaction(PCR).Results Compared with high-fat fed group,HDP group had lower epididymal fat pad weight,reduced level of FBG,insulin and FFA (P ＜0.05) and improved ISI(-5.84 ± 0.24 vs-6.44 ± 0.25,P ＜ 0.05).LDP group had similar trends.In adipose tissue,the TNFα expression in LDP and HDP group was lower,Glut4 expression in HDP group was higher than that of high-fat fed group (P ＜ 0.05).Conclusion Paeoniflorin can reduce visceral adipose content,inhibit TNFα expression and increase Glut4 expression in adipose tissue,eventually lower glucose,and improve insulin resistance caused by high-fat diet.

Grape extract and α-Tocopherol effect in cardiovascular disease model of Apo E -/- Mice / Efeito do extrato de uva e α-Tocoferol em camundongos Apo E -/-, modelo de doença cardiovascular

PURPOSE: To verify the effect of consumption of grape extract isolated or combined with α-tocopherol supplementation on atherosclerosis model with Apo E -/- mice. METHODS: After six weeks of atherogenic diet, Apo E -/- mice were divided into the following groups: Control, Grape, Tocopherol and Grape plus Tocopherol. The treatment progressed for 11 weeks when animals were submitted to euthanasia. RESULTS: All the treatments presented hypocholesterolemic effect with reduction of serum and liver cholesterol levels. This effect was parallel to an increase in the fecal excretion of cholesterol. There was also a higher fecal excretion of saturated fatty acids in groups receiving grape extract or α-tocopherol. All the groups treated presented a tendency to show higher levels of vitamin E. The fatty acid profile showed a tendency for monounsaturated fatty acid preservation after grape extract and α-tocopherol consumption. Morphological analysis revealed a lower degree of evolution of the atherosclerotic plaque of the animals that were fed α-tocopherol combined with grape extract, even when no difference was found in the size of the largest lesion. CONCLUSION: A synergistic effect between the polyphenols and α-tocopherol was observed, resulting in diminished evolution of atherosclerosis and a greater beneficial effect on atherosclerosis than the isolated consumption of antioxidants.

OBJETIVO: Verificar o efeito do consumo de extrato de uva isolada ou combinada com a suplementação de α-tocoferol em modelo de aterosclerose, utilizando camundongos Apo E -/-. MÉTODOS: Os camundongos Apo E -/- foram tratados com dieta aterogênica por seis semanas e foram divididos em quatro grupos: Controle, Uva, Tocoferol e Uva e Tocoferol. Após 11 semanas de tratamento os animais foram submetidos à eutanasia. RESULTADOS: Todos os tratamentos apresentaram efeito hipocolesterolêmico, com redução de colesterol plasmático e hepático. Este efeito foi acompanhado de um aumento na excreção fecal de colesterol. Houve também uma maior excreção fecal de ácidos graxos saturados nos grupos que receberam extrato de uva ou de α-tocoferol. Todos os grupos apresentaram uma tendência a apresentar níveis mais elevados de vitamina E. O perfil de ácidos graxos mostrou uma tendência para a preservação de ácidos graxos monoinsaturados, após consumo de extrato de uva e α-tocoferol. A análise morfológica revelou um menor grau de evolução da placa aterosclerótica dos animais que foram alimentados com α-tocoferol combinado com extrato de uva, mesmo quando não houve diferença no tamanho da lesão. CONCLUSÃO: Foi observado um efeito sinergístico entre os polifenóis e α-tocoferol, resultando na redução na evolução da aterosclerose e um maior de efeito benéfico na aterosclerose do que o consumo isolado de antioxidantes sobre a aterosclerose do que o consumo isolado de antioxidantes.

Effects of Taizhian treatment on the macrophages in the plague ofaotic intima and the levels of blood lipid in hypercholesterolemic rabbits / 中华老年医学杂志

Objective To observe the effects of Taizhian treatment on macrophages expression in the plaque of aotic intima and blood lipid levels in hypercholesterolemic rabbits. Methods Sixteen male New Zealand rabbits were given 1%high-cholesterol diet. After 8 weeks,these macrophages expression in aotic intima was detected by immunohistochemistry. Results Compared with normal rabbits,Taizhian treatment decreased the expression of macrophages in aortic intima by 25%and reduced serum levels of low density lipoprotein-cholesterol by 21%in hypercholesterolemic rabbits(both P<0.05). Conclusions Taizhian may significantly decrease blood lipid level and reduce atherosclerosis in hypercholesterolemic rabbits.

Change of CD36 expression in minipigs fed high fat/high cholesterol diet / 中国病理生理杂志

AIM: In order to investigate the change of CD36 expression in atherosclerosis. METHODS: Chinese minipigs were fed a normal control diet (CD) or a high fat/high cholesterol diet (HFHC) for 12 months after common carotid artery injury induced by balloon denudation. Plasma total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were determined by commercially enzymatic methods. CD36 mRNA and protein levels were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry, respectively. RESULTS: After HFHC for 12 months, plasma total cholesterol, HDL cholesterol and triglyceride in HFHC minipigs were increased compared with the control. CD36 expression and aorta PPAR? in HFHC minipigs were upregulated. CONCLUSION: HFHC may induce hyper cholesterolemia, hypertriglyceridemia and upregulation of CD36 and aortic PPAR? expression.

Chronological effects of atherogenic diets on the aorta, liver and spleen of rabbits

To investigate the temporal progression of atherogenesis on the aorta and involvement of the monocyte-macrophage system in the liver and spleen, we fed 74 rabbits with high fat (14 or 7 gm+ACU-) and cholesterol (2 and 1+ACU-) diets for 4 to over 24 weeks. Using both light and electron microscopies, we found that the bro-fatty areas on the luminal surface of aortas was spread over along the eding time dependently. The fat deposits also in the liver and spleen worsened pending on the time of feeding the atherogenic diets. Not only nocyte-derived foam cells, but also parenchymatous cells in the liver and leen involved become fat-laden cells. According to these results, we propose at there are three stages: 1) the primary seeding, 2) the intermediate turing and 3) the advanced periods. These periods may play very important les in designing the management and treatment of atherosclerotic patients.

Chronological effects of atherogenic diets on the aorta, liver and spleen of rabbits

To investigate the temporal progression of atherogenesis on the aorta and involvement of the monocyte-macrophage system in the liver and spleen, we fed 74 rabbits with high fat (14 or 7 gm+ACU-) and cholesterol (2 and 1+ACU-) diets for 4 to over 24 weeks. Using both light and electron microscopies, we found that the bro-fatty areas on the luminal surface of aortas was spread over along the eding time dependently. The fat deposits also in the liver and spleen worsened pending on the time of feeding the atherogenic diets. Not only nocyte-derived foam cells, but also parenchymatous cells in the liver and leen involved become fat-laden cells. According to these results, we propose at there are three stages: 1) the primary seeding, 2) the intermediate turing and 3) the advanced periods. These periods may play very important les in designing the management and treatment of atherosclerotic patients.