Polysaccharide-rich extract of Potentilla anserina ameliorates nonalcoholic fatty liver disease in free fatty acid-induced HepG2 cells and high-fat/sugar diet-fed mice

Potentilla anserina L.(PA)belongs to the Rosaceae family,is a common edible plant in the Qinghai-Tibet Plateau areas of China.This study elucidates the mechanism upon which crude polysaccharide of PA(PAP)on fat accumulation in HepG2 cells stimulated by oleic acid(OA)and high fat high sugar induced mice.The result revealed that PAP inhibited lipid accumulation in obese mice and ameliorated the degree of damage in OA-induced HepG2 cells.Specifically,compared to the control group,the TG and TC levels were decreased in cells and mice serum,the aspartate transaminase and alamine aminotransferase contents were declined in liver of obese mice by PAP treatment.The expressions of adipogenic genes of SREBP-1c,C/EBPα,PPARγ,and FAS were inhibited after PAP treatment.Moreover,PAP increased the mRNA levels of CPT-1 and PPARα,which were involved in fatty acid oxidation.The present results indicated the PAP could alleviate the damage of liver associated with obesity and PAP treatment might provide a dietary therapeutic option for the treatment of hyperlipidemia.

Freeze-dried Si-Ni-San powder can ameliorate high fat diet-induced non-alcoholic fatty liver disease

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is a common chronic liver disease worldwide.However,to date,there is no ideal therapy for this disease.AIM To study the effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice.METHODS Twenty-four male C57BL/6 mice were randomized into three groups of eight.The control group(CON)was allowed ad libitum access to a normal chow diet.The high fat diet group(FAT)and Si-Ni-San group(SNS)were allowed ad libitum access to a high fat diet.The SNS group was intragastrically administered Si-Ni-San freeze-dried powder(5.0 g/kg)once daily,and the CON and FAT groups were intragastrically administered distilled water.After 12 wk,body weight,liver index,visceral fat index,serum alanine aminotransferase(ALT),portal lipopoly-saccharide(LPS),liver tumor necrosis factor(TNF)-αand liver triglycerides were measured.Intestinal microbiota were analyzed using a 16S r DNA sequencing technique.RESULTS Compared with the FAT group,the SNS group exhibited decreased body weight,liver index,visceral fat index,serum ALT,portal LPS,liver TNF-αand liver triglycerides(P<0.05).Intestinal microbiota analysis showed that the SNS group had different bacterial composition and function compared with the FAT group.In particular,Oscillospira genus was a bacterial biomarker of SNS group samples.CONCLUSION The beneficial effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice may be associated with its anti-inflammatory and changing intestinal microbiota effects.

Hypolipidemic Activity of Olive Oil (Olea europaea) against High Fat Diet-Induced Nonalcoholic Fatty Liver Disease (NAFLD) in Mice

Aim: The aim of the present study is to access the effect of olive oil supplementation against high fat diet induced fatty liver disease in mice. Methods: Mice were divided into five groups: Group I (normal diet), Group II (high fat diet), Group III (olive oil), Group IV and V (High Fat Diet along with olive oil). All mice were fed for 16 weeks with weight measurements every 2 weeks and then sacrificed. Biochemical analysis of blood samples was done and mice livers were histologically examined. Results: Group II mice showed significant increase in body weight as compared with Group I (p < 0.05). Group IV and V mice were significantly (p < 0.05) reduced in body weight as compared with Group II. Olive oil groups had significantly decreased triglyceride and low density lipoprotein levels as compared with Group II whereas high density lipoprotein levels were significantly increased (p < 0.05). The liver enzymes were significantly increased in Group II as compared with other groups (p < 0.05). Liver histopathology revealed drastically increased lipid droplets in Group II mice as compared with Group IV & V. Conclusion: Olive oil causes weight reduction, decreases the serum triglycerides, normalizes the liver enzymes and significantly reduces the accumulation of fat in liver. Therefore, olive oil may represent a potential therapeutic alternative for NAFLD and other fatty diseases.

Effects of alkaline-electrolyzed and hydrogen-rich water,in a high-fat-diet nonalcoholic fatty liver disease mouse model

AIM To identify the effect of hydrogen-rich water(HRW) and electrolyzed-alkaline water(EAW) on high-fat-induced non-alcoholic fatty acid disease in mice.METHODS Mice were divided into four groups:(1) Regular diet(RD)/regular water(RW);(2) high-fat diet(HFD)/RW;(3) RD/EAW; and(4) HFD/EAW. Weight and body composition were measured. After twelve weeks, animals were sacrificed, and livers were processed for histology and reverse-transcriptase polymerase chain reaction. A similar experiment was performed using HRW to determine the influence and importance of molecular hydrogen(H2) in EAW. Finally, we compared the response of hepatocytes isolated from mice drinking HRW or RW to palmitate overload.RESULTS EAW had several properties important to the study:(1) pH = 11;(2) oxidation-reduction potential of-495 mV; and(3) H2 = 0.2 mg/L. However, in contrast to other studies, there were no differences between the groups drinking EAW or RW in either the RD or HFD groups. We hypothesized that the null result was due to low H2 concentrations. Therefore, we evaluated the effects of RW and low and high HRW concentrations(L-HRW = 0.3 mg H2/L and H-HRW = 0.8 mg H2/L, respectively) in mice fed an HFD. Compared to RW and L-HRW, H-HRW resulted in a lower increase in fat mass(46% vs 61%), an increase in lean body mass(42% vs 28%), and a decrease in hepatic lipid accumulation(P < 0.01). Lastly, exposure of hepatocytes isolated from mice drinking H-HRW to palmitate overload demonstrated a protective effect from H2 by reducing hepatocyte lipid accumulation in comparison to mice drinking regular water.CONCLUSION H2 is the therapeutic agent in electrolyzed-alkaline water and attenuates HFD-induced nonalcoholic fatty liver disease in mice.

Prolonged high-fat-diet feeding promotes non-alcoholic fatty liver disease and alters gut microbiota in mice

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) has become an epidemic largely due to the worldwide increase in obesity. While lifestyle modifications and pharmacotherapies have been used to alleviate NAFLD, successful treatment options are limited. One of the main barriers to finding safe and effective drugs for long-term use in NAFLD is the fast initiation and progression of disease in the available preclinical models. Therefore, we are in need of preclinical models that (1) mimic the human manifestation of NAFLD and (2) have a longer progression time to allow for the design of superior treatments. AIM To characterize a model of prolonged high-fat diet (HFD) feeding for investigation of the long-term progression of NAFLD. METHODS In this study, we utilized prolonged HFD feeding to examine NAFLD features in C57BL/6 male mice. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 80 wk to promote obesity (Old-HFD group, n = 18). A low-fat diet (LFD)(14% fat, 32% protein, and 54% carbohydrate) was administered for the same duration to age-matched mice (Old-LFD group, n = 15). An additional group of mice was maintained on the LFD (Young-LFD, n = 20) for a shorter duration (6 wk) to distinguish between age-dependent and age-independent effects. Liver, colon, adipose tissue, and feces were collected for histological and molecular assessments.RESULTS Prolonged HFD feeding led to obesity and insulin resistance. Histological analysis in the liver of HFD mice demonstrated steatosis, cell injury, portal and lobular inflammation and fibrosis. In addition, molecular analysis for markers of endoplasmic reticulum stress established that the liver tissue of HFD mice have increased phosphorylated Jnk and CHOP. Lastly, we evaluated the gut microbial composition of Old-LFD and Old-HFD. We observed that prolonged HFD feeding in mice increased the relative abundance of the Firmicutes phylum. At the genus level, we observed a significant increase in the abundance of Adercreutzia, Coprococcus, Dorea, and Ruminococcus and decreased relative abundance of Turicibacter and Anaeroplasma in HFD mice. CONCLUSION Overall, these data suggest that chronic HFD consumption in mice can mimic pathophysiological and some microbial events observed in NAFLD patients.

Papaya improves non-alcoholic fatty liver disease in obese rats by attenuating oxidative stress,inflammation and lipogenic gene expression

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is a global health issue that is correlated with obesity and oxidative stress.AIM To evaluate the anti-NAFLD effect of papaya in high fat diet induced obesity in rats.METHODS Four-week-old male Sprague-Dawley rats were divided into four groups after 1 wk of acclimatization:Group 1 was the rats fed a normal diet(C);group 2 was the rats fed a high fat diet(HFD);group 3 was the rats fed a HFD with 0.5 mL of papaya juice/100 g body weight(HFL),and group 4 was the rats fed a HFD with 1 mL of papaya juice/100 g body weight(HFH)for 12 wk.At the end of the treatment,blood and tissue samples were collected for biochemical analyses and histological assessment.RESULTS The results of the HFH group showed significantly reduced body weight(HFH vs HFD,P<0.01),decreased NAFLD score(HFH vs HFD,P<0.05),and reduced hepatic total cholesterol(HFL vs HFD,P<0.01;HFH vs HFD,P<0.001),hepatic triglyceride(HFH vs HFD,P<0.05),malondialdehyde(HFL,HFH vs HFD,P<0.001),tumour necrosis factor-α(HFH vs HFD,P<0.05)and interleukin-6(HFH vs HFD,P<0.05)when compared to the HFD group.However,the liver weight showed no significant difference among the groups.The activities of catalase and superoxide dismutase significantly increased in HFH when compared with the HFD group(P<0.05 and P<0.001,respectively).The suppression of transcriptional factors of hepatic lipogenesis,including sterol regulatory elementbinding protein 1c and fatty acid synthase,were observed in the papaya treated group(HFH vs HFD,P<0.05).These beneficial effects of papaya against HFDinduced NAFLD are through lowering hepatic lipid accumulation,suppressing the lipogenic pathway,improving the balance of antioxidant status,and lowering systemic inflammation.CONCLUSION These current results provide experimental-based evidence suggesting papaya is an efficacious medicinal fruit for use in the prevention or treatment of NAFLD.

mi R-192-5p regulates lipid synthesis in non-alcoholic fatty liver disease through SCD-1

AIM To evaluate the levels of mi R-192-5 p in non-alcoholic fatty liver disease(NAFLD) models and demonstrate the role of mi R-192-5 p in lipid accumulation. METHODS Thirty Sprague Dawley rats were randomly divided into three groups, which were given a standard diet, a high-fat diet(HFD), and an HFD with injection of liraglutide. At the end of 16 weeks, hepatic mi R-192-5 p and stearoyl-Co A desaturase 1(SCD-1) levels were measured. Mi R-192-5 p mimic and inhibitor and SCD-1 si RNA were transfected into Huh7 cells exposed to palmitic acid(PA). Lipid accumulation was evaluated by oil red O staining and triglyceride assays. Direct interaction was validated by dual-luciferase reporter gene assays.RESULTS The HFD rats showed a 0.46-fold decrease and a 3.5-fold increase in hepatic mi R-192-5 p and SCD-1 protein levels compared with controls, respectively, which could be reversed after disease remission by liraglutide injection(P < 0.01). The Huh7 cells exposed to PA also showed down-regulation and up-regulation of mi R-192-5 p and SCD-1 protein levels, respectively(P < 0.01). Transfection with mi R-192-5 p mimic and inhibitor in Huh7 cells induced dramatic repression and promotion of SCD-1 protein levels, respectively(P < 0.01). Luciferase activity was suppressed and enhanced by mi R-192-5 p mimic and inhibitor, respectively, in wild-type SCD-1(P < 0.01) but not in mutant SCD-1. Mi R-192-5 p overexpression reduced lipid accumulation significantly in PA-treated Huh7 cells, and SCD-1 si RNA transfection abrogated the lipid deposition aggravated by mi R-192-5 p inhibitor(P < 0.01).CONCLUSION This study demonstrates that mi R-192-5 p has a negative regulatory role in lipid synthesis, which is mediated through its direct regulation of SCD-1.

Upregulation of caveolin-1 and SR-B1 in mice with non-alcoholic fatty liver disease

BACKGROUND:Non-alcoholic fatty liver disease(NAFLD)is one of the most frequent causes of liver diseases,with markedly increased prevalence.However,its mechanisms are not clear.The present study was undertaken to illustrate the role of caveolin-1(cav1)and the scavenger receptor class B type 1(SR-B1)in NAFLD.METHODS:Adult male C57BL/6 mice were fed with a normal diet or high fat and cholesterol(HFC)diet for 14 weeks.The mice were sacrificed to collect plasma and harvest the liver;their plasma lipid concentration was measured.Hepatic cav1and SR-B1 mRNA and protein expression were determined by real-time quantitative polymerase chain reaction(qPCR)and Western blotting,respectively.In order to study cav1 and SR-B1distribution and change in hepatocytes,immunohistochemical analysis was performed.RESULTS:HFC diet increased plasma lipids,induced NAFLD and increased the liver/body weight ratio.Compared to the control mice(n=6),the mRNA and protein levels of cav1 and SR-B1 in liver tissue of the NAFLD mice(n=12)increased significantly(cav1 mRNA:1.536±0.226 vs 0.980±0.272,P【0.05;protein:0.643±0.240 vs 0.100±0.130,P【0.01;SR-B1 mRNA:1.377±0.125 vs 0.956±0.151,P【0.01;protein:2.156±0.507vs 0.211±0.211,P【0.01).Furthermore,both cav1 and SR-B1immunoreactivity increased and their distribution was also changed,mainly in the plasma membrane of hepatocytes,cytoplasm and membrane of lipid droplets and around.CONCLUSION:NAFLD is associated with increased concentration of plasma lipids and upregulation of hepatic cav1 and SR-B1 gene and protein expressions,which indicate that cav1 and SR-B1 might play crucial roles in the pathogenesis of NAFLD.

Effects of different diets on intestinal microbiota and nonalcoholic fatty liver disease development

AIM To study the effects of different diets on intestinal microbiota and nonalcoholic fatty liver disease(NAFLD) development at the same caloric intake.METHODS Thirty male Sprague-Dawley rats were randomized into five groups(six rats each). The control diet(CON) group and free high-fat diet(FFAT) group were allowed ad libitum access to a normal chow diet and a highfat diet, respectively. The restrictive high-fat diet(RFAT) group, restrictive high-sugar diet(RSUG) group, and high-protein diet(PRO) group were fed a highfat diet, a high-sugar diet, and a high-protein diet, respectively, in an isocaloric way. All rats were killed at 12 wk. Body weight, visceral fat index(visceral fat/body weight), liver index(liver/body weight), insulin resistance, portal lipopolysaccharide(LPS), serum alanine aminotransferase(ALT), serum aspartate aminotransferase(AST), and liver triglycerides were measured. The intestinal microbiota in the different groups of rats was sequenced using high-throughput sequencing technology.RESULTS The FFAT group had higher body weight, visceral fat index, liver index, peripheral insulin resistance, portal LPS, serum ALT, serum AST, and liver triglycerides compared with all other groups(P < 0.05). Taking the same calories, the RFAT and RSUG groups demonstrated increased body weight, visceral fat index, peripheral insulin resistance and liver triglycerides compared with the PRO group(P < 0.05). The RFAT group also showed increased portal LPS compared with the PRO group(P < 0.05). Unweighted Uni Frac principal coordinates analysis of the sequencing data revealed that the intestinal microbiota structures of the CON, FFAT, RSUG and PRO groups were roughly separated away from each other. Taxon-based analysis showed that, compared with the CON group, the FFAT group had an increased abundance of Firmicutes, Roseburia and Oscillospira bacteria, a higher ratio of Firmicutes to Bacteroidetes, and a decreased abundance of Bacteroidetes, Bacteroides and Parabacteroides bacteria(P < 0.05). The RFAT group showed an increased abundance of Firmicutes and decreased abundance of Parabacteroides bacteria(P < 0.05). The RSUG group showed an increased abundance of Bacteroidetes and Sutterella bacteria, higher ratio of Bacteroidetes to Firmicutes, and a decreased abundance of Firmicutes(P < 0.05). The PRO group showed an increased abundance of Bacteroidetes, Prevotella, Oscillospira and Sutterella bacteria, and a decreased abundance of Firmicutes(P < 0.05). Compared with the FFAT group, the RFAT group had an increased abundance of Bacteroidetes, higher ratio of Bacteroidetes to Firmicutes, and decreased abundance of Firmicutes and Oscillospira bacteria(P < 0.05).CONCLUSION Compared with the high-protein diet, the NAFLDinducing effects of high-fat and high-sugar diets are independent from calories, and may be associated with changed intestinal microbiota.

铁过载对不同类型高脂膳食所致肝损伤的影响

目的:探究铁过载联合不同类型高脂膳食对小鼠肝损伤的影响。方法:将48只SPF级雄性C57BL/6J小鼠按体重随机分为6组,每组8只,普通对照组(ND)、高铁对照组(NDFe)给予基础饲料,棕榈油高脂组(PHFD)、棕榈油高脂高铁组(PHFDFe)、大豆油高脂组(SHFD)和大豆油高脂高铁组(SHFDFe)分别给予对应高脂饲料。从第10周开始,NDFe、PHFDFe和SHFDFe组连续8周每周两次肌肉注射右旋糖酐铁100 mg/kg·bw,其余组注射等剂量生理盐水至第17周。麻醉后取血和肝脏,测定小鼠血清和肝脏生化指标、肝脏病理改变及铁代谢和脂代谢相关基因表达。结果:与对应高脂组相比,铁过载联合高脂膳食可使血清总胆固醇(Total cholesterol,TC)、肝脏甘油三酯(Triglyceride,TG)和谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)水平显著下降(P<0.05),血清TG和谷丙转氨酶(Alanine aminotransferase,ALT)水平、肝脏系数、肝脏铁含量和丙二醛(Malondialdehyde,MDA)水平显著升高(P<0.05),SHFDFe组肝脏MDA水平显著高于PHFDFe组(P<0.05)。PHFDFe组二价金属转运蛋白1(Divalentmetal-iontransporter-1,DMT-1)和膜铁转运蛋白(Ferroportin,FPN)mRNA表达量显著高于PHFD组(P<0.05);SHFDFe组FPN mRNA表达量显著高于与NDFe、PHFDFe和SHFD组(P<0.05),乙酰CoA羧化酶1(acetyl-Coenzyme A carboxylase alpha 1,ACC1)和脂肪酸合酶(Fatty Acid Synthase,FASN)mRNA表达量显著低于SHFD组(P<0.05)。结论:铁过载联合高脂膳食会加重脂质代谢紊乱和氧化应激,铁过载联合大豆油高脂饲料喂养导致的肝损伤高于联合棕榈油高脂饲料喂养。

脱氢表雄酮改善高脂饲料诱导的肉鸡肝损伤和炎症反应

目的:探讨脱氢表雄酮(Dehydroepiandrosterone,DHEA)对饲喂高脂饲料肉鸡生长性能、肉质、肝损伤和炎症反应的影响。方法:选择200只15日龄青脚麻鸡,随机分为5组,其中对照组(CK组)饲喂基础饲料,高脂饲料组(HFD组)、DHEA低剂量干预组(L-DHEA组)、DHEA中剂量干预组(M-DHEA组)、DHEA高剂量干预组(H-DHEA组)在饲喂高脂饲料的基础上同时添加0、5、15、45 mg/kg DHEA。试验期间,测定鸡的体质量、生长性能、屠宰率、胸肌pH、肉色、肝脏相对质量、肝脏出血分数及血清ALT、AST、胆碱酯酶(CHE)活性、血清总蛋白(TP)、白蛋白(ALB)、前白蛋白(PA)、尿酸、肌酐、C-反应蛋白(CRP)、白细胞介素-6(IL-6)含量等指标。结果:与对照组相比,高脂饲料能提高肉鸡的体质量、平均日增重、屠宰率、肝脏相对质量(P<0.05)和肝脏出血分数(P<0.05),但降低胸肌b^(*)值;同时,高脂饲料显著提高血清ALT活性(P<0.05)与CRP、IL-6水平。与HFD组相比,添加DHEA后饲喂高脂饲料肉鸡的体质量、日均体增重、料重比有降低趋势,而胸肌a^(*)、b^(*)值则有上升的趋势;同时,DHEA能够提高血清ALB、PA含量,并降低血清ALT、CHE活性和CRP、IL-6水平。结论:高脂饲料可诱导肉鸡发生肝脏损伤、炎症反应和FLHS,在高脂饲料中添加DHEA能够有效改善高脂饲料诱导的肉鸡肝损伤、炎症反应,缓解高脂日粮引起的鸡FLHS,DHEA添加量为15 mg/kg时综合效果更好。

基于生物信息学对比分析高脂饮食和蛋氨酸胆碱缺乏饮食构建的非酒精性脂肪性肝病小鼠模型

目的比较高脂饮食(high-fat diet,HFD)饮食和蛋氨酸胆碱缺乏(methionine-choline-deficient,MCD)饮食对C57小鼠非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)模型的影响,通过生物信息学方法揭示其差异表达基因(GEO-data-based analysis of differentially expressed genes,DEGs)并进行功能分析。方法从GEO数据库中获取GSE78170、GSE123354的表达数据。使用R语言分别比较C57小鼠对照组与MCD饮食组、C57小鼠对照组与HFD饮食组数据获得DEGs。对DEGs进行进一步的差异分析、功能富集分析、构建基因和蛋白互作网络及重叠DEGs对比分析。结果筛选出HFD诱导的上调基因44个,下调基因16个;MCD诱导的上调基因43个,下调基因73个;分析富集分析排名前12位的生物学过程,提示HFD与肝脏单纯脂肪积累相关,MCD则能全方位促进NAFL进一步转变为NASH。结论HFD诱导的基因主要涉及肝脏脂肪积累,而MCD更倾向于全方位促进NAFL向NASH的进展。

不同高脂饲料配方对建立非酒精性脂肪肝大鼠模型的影响

目的:比较3种不同配方高脂饲料构建非酒精性脂肪肝大鼠模型的差异,提高高脂饲料建立非酒精性脂肪肝大鼠模型的成功率,为非酒精性脂肪肝病的研究提供稳定可靠的动物模型。方法:将SPF级雄性SD大鼠随机分为普通饲料对照组、高脂饲料1组(HFD1组)、高脂饲料2组(HFD2组)、高脂饲料3组(HFD3组)。各组大鼠分别给予相应饲料8周。造模期间记录大鼠一般情况、体质量和摄食量。喂养8周后,采用腹部超声、计算机断层扫描(CT)、核磁共振成像(MRI)对大鼠肝脏进行检查。取血和肝脏,检测肝功能指标(丙氨酸氨基转移酶、天门冬氨酸氨基转移酶)、血脂指标(甘油三酯、总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇)和炎症指标(IL-1β、IL-6、TNF-α)的变化。肉眼观察肝脏大体形态,计算其肝指数和Lee’s指数。比较上述指标在各组间的差异,综合评估不同配方高脂饲料对非酒精性脂肪肝大鼠模型的影响。结果:与对照组大鼠相比,HFD1组、HFD2组、HFD3组大鼠精神变差、活动减少、脱毛现象加重、摄食量减少、体质量明显升高,肝指数、Lee’s指数显著提高,肝脏体积增大,边缘较钝,可见脂肪变性和沉积,且以HFD3组大鼠变化最为明显;HFD1组、HFD2组、HFD3组大鼠血清谷丙转氨酶、天门冬氨酸氨基转移酶、甘油三酯、总胆固醇、低密度脂蛋白胆固醇和IL-1β、IL-6、TNF-α水平均显著升高,高密度脂蛋白胆固醇显著降低,且HFD3组更为明显;HFD1组、HFD2组、HFD3组大鼠肝脏肿大,实质回声增强,肝内管状结构显示欠清,肝和脾的CT值比值明显降低,同/反相位图上肝脏实质信号差别明显,且HFD3组对于大鼠影像学变化影响更大。结论:三种高脂饲料喂养SD大鼠8周后,均可建立非酒精性脂肪肝大鼠模型,但HFD3组诱导非酒精性脂肪肝大鼠模型优于其他两组,病变相对严重,预计维持时间也更长,更适于非酒精性脂肪肝病的机制研究和降脂药物的筛选。

甘露寡糖干预采食高脂饲粮罗曼蛋鸡的脂肪肝综合征

【目的】研究日粮添加甘露寡糖(Mannose oligosaccharide,MOS)对罗曼蛋鸡脂肪肝综合征及肠道菌群的影响。【方法】试验选取80只230日龄的罗曼蛋鸡,分为4组,每组20只。分别为普通日粮组(CK组),高脂日粮组(HFD组),高脂日粮+0.1%(w)甘露寡糖组(0.1%MOS组),高脂日粮+0.5%(w)甘露寡糖组(0.5%MOS组)。试验第9周采集蛋鸡血清、肝脏、腹脂及肠道内容物,通过血清指标、肝脏中相关基因表达分析及肠道微生物测序等,探讨甘露寡糖对蛋鸡脂肪肝发生的调控作用。【结果】高脂日粮显著提高了罗曼蛋鸡体质量、肝脏质量、肝脏指数、腹脂质量和腹脂指数,并诱发蛋鸡脂肪肝;甘露寡糖显著降低了高脂日粮引起的蛋鸡体质量、肝脏质量和腹脂质量增加;甘露寡糖显著抑制了高脂日粮引起的蛋鸡血清脂肪含量和肝脏中的脂肪沉积增加,抑制了肝脏中脂肪合成基因ACC、FAS和SCREBP-1c的mRNA表达水平,并缓解了蛋鸡脂肪肝;甘露寡糖显著提高了蛋鸡盲肠中拟杆菌门Bacteroidetes的相对丰度,显著降低了厚壁菌门Firmicutes的相对丰度。【结论】在高脂日粮中添加甘露寡糖能够改善蛋鸡肠道菌群,抑制肝脏中脂肪合成相关基因的mRNA表达水平,缓解高脂日粮引起的蛋鸡脂肪肝。饲粮中添加质量分数为0.5%的甘露寡糖对降低蛋鸡体质量、肝脏质量和腹脂质量的效果更好。

NLRP3基因敲减对高脂高糖饮食诱导的非酒精性脂肪性肝炎小鼠模型的影响

目的探讨NOD样受体热蛋白结构域相关蛋白3(NLRP3)基因敲减对高脂高糖诱导的非酒精性脂肪性肝炎(NASH)小鼠模型的影响。方法将44只小鼠随机分为正常饮食组(CON)20只,高脂高糖造模组(HFHC)24只。造模14周末,随机选取4只HFHC组小鼠进行腺相关病毒9(AAV9)尾静脉注射预实验,4周后验证NLRP3敲减模型是否成功。18周末确认敲减成功后,对剩余40只小鼠进行AAV9一次性尾静脉注射,分为CON+NLRP3敲减阴性对照组(CON+NLRP3-NC)、CON+NLRP3敲减组(CON+NLRP3-KD)、HFHC+NLRP3-NC及HHFHC+NLRP3-KD组,每组10只,继续造模6周。24周末取材观察炎症小体活化效应,检测小鼠体质量、肝质量、肝指数及糖代谢(空腹血糖、空腹胰岛素及HOMA-IR指数)指标;检测小鼠肝脂质含量(肝组织TG及油红O染色)、肝脏炎症(血清ALT活性、HE染色及炎症相关基因)及肝纤维化(天狼星红染色及纤维化相关基因)指标。计量资料多组间比较使用单因素方差分析,进一步两两比较采用LSD-t检验。结果与CON+NLRP3-NC组相比,Western Blot结果提示,HFHC+NLRP3-NC组的NLRP3、pro-Caspase1、Caspase1、ASC及IL-1β蛋白水平均升高,HFHC+NLRP3-KD组均降低(P值均<0.05);HFHC+NLRP3-NC组小鼠体质量、肝质量、肝指数及糖代谢指标均有不同程度升高,HFHC+NLRP3-KD组均显著改善(P值均<0.05);在肝脂肪沉积方面,与CON+NLRP3-NC组相比,HFHC+NLRP3-NC组肝脏TG明显增高,油红O染色显示大量红色脂滴,HFHC+NLRP3-KD组肝脏TG及肝脂滴数量显著减少(P值均<0.01);在肝脏炎症方面,HFHC+NLRP3-NC组血清ALT,非酒精性脂肪性肝病活动度(NAS)评分及炎症相关基因均较CON+NLRP3-NC组明显升高,HFHC+NLRP3-KD组均明显降低(P值均<0.01);在肝纤维化方面,HFHC+NLRP3-NC组肝胶原纤维面积以及纤维化相关基因均较CON+NLRP3-NC组明显升高,HFHC+NLRP3-KD组纤维化相关基因均明显降低(P值均<0.05),胶原纤维面积虽有降低趋势但差异无统计学意义(P>0.05)。结论NLRP3基因敲减可显著改善高脂高糖饮食诱导的NASH小鼠模型肝脂肪沉积及炎症。

下瘀血汤对非酒精性脂肪性肝病小鼠模型肾损伤的干预作用及其机制

目的探讨高脂饮食(HFD)诱导非酒精性脂肪性肝病(NAFLD)对小鼠肾脏的影响,以及下瘀血汤对NAFLD小鼠模型的保护作用及其机制。方法选取2020年9月—2021年9月在上海中医药大学附属普陀医院健康对照及NAFLD患者各25例,检测总胆固醇(TC)、甘油三酯(TG)、尿素氮(BUN)、肌酐(Cr)及尿酸(UA)水平;将24只雄性C57BL/6小鼠随机分为对照组(LFD组)、高脂组(HFD组)及高脂+下瘀血汤组(XYXD组),每组8只,第13周开始给予下瘀血汤每日1次灌胃至18周末,维持6周。检测各组TC、TG、BUN及Cr水平。HE、油红O染色观察肝脏和肾脏病理变化,免疫组化双染技术检测CD68和α-SMA表达。实时荧光定量PCR检测肾组织固醇调节元件结合蛋白1(SREBP1)、脂肪酸合酶(FASN)、IL-6、TNF-α、结蛋白(Desmin)和α-平滑肌肌动蛋白(α-SMA)的mRNA表达。Western Blot检测SREBP1和TNF-α蛋白水平。计量资料多组间比较采用单因素方差分析,两两比较采用LSD-t检验;两组间比较采用成组t检验。结果与健康对照组患者相比,NAFLD患者TC、TG、BUN、Cr、UA水平均升高(P值均<0.05)。与LFD组小鼠相比,HFD组小鼠体质量、TC、TG、BUN、Cr水平均升高(P值均<0.001);与HFD组相比,XYXD显著抑制NAFLD小鼠TC、TG、BUN、CR表达(P值均<0.001)。肝脏病理学显示:HFD组小鼠肝脂肪变及炎症浸润较LFD组显著增加,而XYXD组病变显著减轻。肾脏病理学显示:与LFD组相比,HFD组肾组织炎性浸润明显,脂肪变显著,肾脏胶原形成明显;与HFD组相比,XYXD显著减轻炎症浸润、抑制脂肪变及肾脏胶原形成。实时荧光定量PCR结果显示:与LFD组相比,HFD组肾组织中SREBP1、FASN、IL-6、TNF-α、Desmin和α-SMA的m RNA相对表达水平均显著升高(P值均<0.001),XYXD组较HFD组均显著降低(P值均<0.001)。Western Blot结果显示:与LFD组比较,HFD组SREBP1及TNF-α蛋白表达水平均明显上调(P值均<0.05);与HFD组比较,XYXD组SREBP1及TNF-α蛋白表达水平均明显下调(P值均<0.05)。免疫组化染色结果显示:HFD组α-SMA和CD68阳性染色及双阳性染色较LFD组均显著升高(P值均<0.05),XYXD组较HFD组均显著下降(P值均<0.05)。结论HFD可诱导肾脏脂肪变、炎症浸润及胶原形成,下瘀血汤可能通过抑制肾组织巨噬细胞与肌成纤维细胞表达发挥肾脏保护作用。

肝细胞DEP结构域蛋白5/哺乳动物雷帕霉素靶蛋白复合物1信号轴在非酒精性脂肪肝形成中的作用

目的建立肝细胞Dishevelled/Egl-10/pleckstrin(DEP)结构域蛋白5(DEPDC5)基因(Depdc5)肝细胞特异性敲除小鼠高脂喂养模型,探讨DEPDC5/哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)信号轴对非酒精性脂肪肝的调控。方法构建肝细胞特异性敲除Depdc5^(flox/flox)模型;Alb-Cre小鼠(LKO),Depdc5^(flox/flox)小鼠(Loxp)作为对照。32只2~3月龄雄性小鼠随机分为高脂LKO组、高脂Loxp对照组、高脂+雷帕霉素LKO组及高脂+雷帕霉素Loxp对照组,每组8只。检测肝脏血清生物化学指标、脂质含量、蛋白、mRNA及病理切片,采用GraphPad Prism 8软件进行统计学分析。结果高脂喂养导致LoxP小鼠肝脏脂肪变性,LKO小鼠肝脏脂肪变性减轻但合并出现肝损伤;雷帕霉素抑制了Depdc5敲除引起的mTORC1通路激活,显著改善Loxp小鼠肝脏脂肪变性,并改善LKO小鼠的肝损伤。结论Depdc5基因敲除能够保护高脂喂养小鼠肝脏脂肪变性,雷帕霉素可以改善DEPDC5缺失诱发的肝损伤。

Nogo-B敲除NAFLD小鼠的肠道微生物群和血清代谢组学变化研究

背景非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是目前全球范围内最常见的慢性肝病,Nogo-B作为其潜在治疗靶点,需确定是否会对肠道微生物群及代谢途径产生影响。目的探讨Nogo-B敲除对NAFLD的保护作用,明确Nogo-B敲除对NAFLD小鼠肠道微生物群和血清代谢产物的影响。方法12只8周龄野生型小鼠随机分为野生正常饮食组(WT-NCD组)和野生高脂饮食组(WT-HFD组)。12只8周龄Nogo-B敲除(Nogo-B^(-/-))小鼠随机分为Nogo-B敲除正常饮食组(Nogo-B^(-/-)-NCD组)和Nogo-B敲除高脂饮食组(Nogo-B^(-/-)-HFD组)。WT-HFD组和Nogo-B^(-/-)-HFD组小鼠通过喂食12周60 kal%的高脂饮食构建NAFLD小鼠模型,WT-NCD组和Nogo-B^(-/-)-NCD组小鼠则同时喂食正常饮食。4组小鼠均测量体质量;酶联免疫吸附测定检测4组小鼠的肝三酰甘油(triglyceride,TG)和总胆固醇(total cholesterol,TC)水平;苏木精-伊红染色评估4组小鼠的肝病理学特征;对WT-HFD组和Nogo-B^(-/-)-HFD组小鼠进行肠道微生物群和血清代谢物检测。结果12周造模结束后,与WT-NCD组小鼠相比,WT-HFD组小鼠的体质量及肝组织中TC、TG水平升高(P<0.01);与Nogo-B^(-/-)-NCD组小鼠相比,Nogo-B^(-/-)-HFD组小鼠的体质量及肝组织中TC、TG水平升高(P<0.01);而当Nogo-B敲除后,与WT-HFD组小鼠相比,Nogo-B^(-/-)-HFD组小鼠的体质量及肝组织中TC、TG水平下降(P<0.01);肠道微生物组学显示Nogo-B敲除后丁酸球菌科成为其肠道微生物中的优势物种;血清代谢组学显示两组间(WT-HFD组、Nogo-B^(-/-)-HFD组)筛选到差异代谢物159种(上调79,下调80种),极其显著富集(P<0.001)的代谢通路为柠檬酸循环/三羧酸循环(tricarboxylic acid cycle,TCA cycle)等,主要富集到的代谢物为柠檬酸、琥珀酸、异柠檬酸盐和苹果酸。结论Nogo-B敲除后的NAFLD小鼠肝脂质积累情况减轻,肠道有益微生物群增加,一定程度上改善代谢紊乱。Nogo-B可能是NAFLD治疗的潜在靶点。

酒精及高脂饮食双重诱导下脂肪肝的转录组学变化分析

目的通过酒精灌胃和高脂饮食诱导脂肪肝小鼠模型,观察小鼠肝脏表型和转录组学的改变,分析酒精、高脂饮食和联合饮食对肝脏的危害性。方法实验小鼠分为对照组(PBS)、高脂饲喂组(高脂饲料)、酒精灌胃组(40%酒精)、联合饲喂组(酒精+高脂饲料)。HE染色观察各组小鼠肝脏组织病理形态。提取肝脏RNA进行转录组测序,并针对各组转录组学的变化进行分析,观察在单一风险因素作用下和双重风险因素作用下对肝脏的影响。构建基因表达调控网络锁定网络核心基因,并对其进行验证。结果在病理层面,联合饲喂组肝脏相较于单一风险因素各组表现出更为严重的脂质累积和炎症。转录组学分析结果显示,联合饲喂组的基因表达相较于单纯的高脂饲喂组和酒精灌胃组表现出了更为明显的脂质累积通路的活跃和恶化倾向。对单风险因素和双重风险因素的差异表达基因取交集,筛选出了导致脂肪肝肝损伤的主要风险基因。功能分析结果显示高脂饮酒小鼠肝脏的基因表达变化不仅包括脂肪代谢异常,同时也和药物的不良代谢有关,最终导致肝脏炎症增加。通过网络分析可以确定Acacb,Cyp2b10和Cd36可能是高脂饮群体肝脏基因表达变化的主要靶点。结论饮酒和高脂饮食对肝脏的影响虽然都和脂肪累积引起脂肪肝相关,但并不完全一样,高脂饮食人群饮酒会对肝脏产生更严重的影响。然而关于高脂饮食和饮酒对肝脏的具体作用机制有待于进一步深入探索。

非酒精性脂肪性肝病患者血清HIF-1α、HMGB1和脂联素水平变化及其与颈动脉粥样硬化的关系研究

目的探讨非酒精性脂肪性肝病(NAFLD)患者血清低氧诱导因子-1α(HIF-1α)、高迁移率族蛋白1(HMGB1)和脂联素(APN)水平变化及其与颈动脉粥样硬化(CAS)的关系。方法2018年5月~2023年3月我院诊治的NAFLD患者158例(其中合并CAS者71例),使用Fibrotouch弹性成像仪诊断脂肪肝,使用超声诊断仪检测颈动脉斑块形成。采用ELISA法检测血清HIF-1α、HMGB1和APN水平,应用二元Logistic回归分析NAFLD合并CAS的影响因素,应用受试者工作特征曲线下面积(AUC)评估血清指标预测NAFLD患者合并CAS的效能。结果合并CAS组收缩压为(137.1±10.3)mmHg,显著高于未合并CAS组【(132.9±8.2)mmHg,P<0.05】;合并CAS组血清TC、LDL-C、HIF-1α和HMGB1水平分别为(6.5±2.3)mmol/L、(3.7±0.6)mmol/L、(25.7±6.5)pg/L和(9.4±2.3)ng/ml,显著高于未合并CAS组【分别(5.1±1.7)mmol/L、(2.8±0.3)mmol/L、(17.2±4.1)pg/L和(6.1±1.5)ng/ml,P<0.05】,而血清APN为(7.5±3.0)mg/L,显著低于未合并CAS组【(12.8±4.6)mg/L,P<0.05】;多因素Logistic回归分析显示,TC(OR=1.411,95%CI:1.133~1.757)、LDL-C(OR=1.419,95%CI:1.128~1.785)、HIF-1α(OR=1.504,95%CI:1.182~1.914)、HMGB1(OR=1.520,95%CI:1.206~1.916)和APN(OR=1.530,95%CI:1.226~1.909)均是影响NAFLD患者合并CAS的独立危险因素(P<0.05);ROC曲线分析显示,血清HIF-1α、HMGB1和APN水平联合预测NAFLD患者合并CAS的AUC为0.863,其敏感度为94.5%,特异度为75.0%,优于各指标单独预测(P<0.05)。结论NAFLD患者血清HIF-1α和HMGB1水平升高而血清APN水平降低是发生CAS的危险因素,应及时发现和给予必要的干预。