Astaxanthin from Haematococcus pluvialis prevents high-fat diet-induced hepatic steatosis and oxidative stress in mice by gut-liver axis modulating properties

Scope: Evidence is mounting that astaxanthin (ATX), a xanthophyll carotenoid, used as a nutritional supplement to prevent chronic metabolic diseases. The present study aims to identify the potential function of ATX supplementation in preventing steatohepatitis and hepatic oxidative stress in diet-induced obese mice.

Methods and Results: In this study, ATX as dose of 0.25%, 0.5% and 0.75% have orally administered to mice along with a high-fat diet (HFD) to investigate the role of ATX in regulating liver lipid metabolism and gut microbiota. The study showed that ATX dose-dependently reduces body weight, lipid droplet formation, hepatic triglycerides and ameliorated hepatic steatosis and oxidative stress. 0.75% ATX altered the levels of 34 lipid metabolites related to hepatic cholesterol and fatty acid metabolism which might be associated with downregulation of lipogenesis-related genes and upregulation of bile acid biosynthesis-related genes. The result also revealed that ATX alleviates HFD-induced gut microbiota dysbiosis by significantly inhibiting the growth of obesity-related Parabacteroides and Desulfovibrio while promoting the growth of Allobaculum and Akkermansia.

Conclusion: The study results suggested that dietary ATX may prevent the development of hepatic steatosis and oxidative stress with the risk of metabolic disease by gut-liver axis modulating properties.

Methods
The body weight and food intake were recorded daily for 63 days. To avoid error values, the measurement of weight was repeated three times for each mouse. Total RNA was extracted from liver tissue using TRIzol reagent (Shenggong BBI Life, Shanghai, China) according to the manufacturer’s instructions. Then, cDNA was synthesized from total RNA using the PrimeScript Reverse Transcription reagent kit (Takara, Dalian, China). Quantitative PCR was conducted in triplicate for each group to detect gene expression. The quantitative analysis of AMPK, SREBP1c, ACC, CPT-1, PPARα, PPARγ, LXRα, SCD-1, PGC-1, FAS, CYP27A1, and CYP7A1 mRNA expression in the liver was measured in triplicate for each group by quantitative PCR. According to the SYBR Premix Ex Taq II (Takara, Dalian, China), the thermal cycle of qPCR was reacted on the CFX 96 Real-Time PCR Detection system (BIO-RAD, Hercules, CA, USA) under the following conditions: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Table S2 (supplement files) shows the PCR primer sequences of each gene, and the target genes were normalized to the reference gene GAPDH. The 2−ΔΔCt method was used to calculate relative gene expression.

The caecal contents were sent to Shanghai Personal Biotechnology Co., Ltd. to investigate microbial diversity through 16S rRNA analysis on the Illumina MiSeq platform. When the microbial DNA was isolated, polymerase chain reaction (PCR) of the V3-V4 region of the bacterial 16S rRNA gene was performed using the forwards primer 5′-ACTCCTACGGGAGGCAGCA-3′ and the reverse primer 5′-GGACTACHVGGGTWTCTAAT-3′ according to the manufacturer’s protocol (24-25). A previous study illustrated the analytical conditions and detailed parameters (18).

All experiments were biologically repeated three times, and the data were analysed with Social Sciences (SPSS 16.0) statistical software and are presented as the mean ± SD. Multiple comparisons among treatments were statistically analysed using Duncan’s multiple range test in one-way analysis of variance (ANOVA) (P < 0.05). Origin 9.1 was used to draw charts.

研究背景：越来越多的证据表明，叶黄素类胡萝卜素虾青素（astaxanthin，ATX）常被用作预防慢性代谢疾病的营养补充剂。本研究旨在探究ATX补充剂在饮食诱导肥胖小鼠中预防脂肪性肝炎与肝脏氧化应激的潜在作用。
方法与结果：本研究中，小鼠在喂食高脂饮食（high-fat diet，HFD）的同时，分别以0.25%、0.5%及0.75%的剂量灌胃给予ATX，以探究ATX调控肝脏脂质代谢与肠道菌群的作用。研究结果显示，ATX可剂量依赖性地降低小鼠体重、减少脂滴形成与肝甘油三酯含量，并改善肝脏脂肪变性与氧化应激。0.75%剂量的ATX可改变34种与肝脏胆固醇及脂肪酸代谢相关的脂质代谢物水平，这一效应可能与脂肪生成相关基因的下调及胆汁酸生物合成相关基因的上调有关。此外，研究还发现ATX可通过显著抑制肥胖相关副杆菌属（Parabacteroides）与脱硫弧菌属（Desulfovibrio）的增殖，同时促进别样杆菌属（Allobaculum）与嗜黏蛋白阿克曼菌（Akkermansia）的生长，从而缓解HFD诱导的肠道菌群失调。
结论：本研究结果表明，膳食ATX可通过调控肠-肝轴（gut-liver axis）的相关特性，预防肝脏脂肪变性与氧化应激，从而降低代谢疾病的发病风险。
方法
每日记录小鼠体重与摄食量，连续记录63天。为避免误差，每只小鼠的体重测量重复3次。
采用TRIzol试剂（Shenggong BBI Life，中国上海）按照产品说明书从肝脏组织中提取总RNA。随后，使用PrimeScript逆转录试剂盒（Takara，中国大连）将总RNA反转录为cDNA。每组设置3个复孔进行定量PCR（qPCR）以检测基因表达水平。通过定量PCR分别检测肝脏中腺苷酸活化蛋白激酶（AMPK）、固醇调节元件结合蛋白1c（SREBP1c）、乙酰辅酶A羧化酶（ACC）、肉碱棕榈酰转移酶1（CPT-1）、过氧化物酶体增殖物激活受体α（PPARα）、过氧化物酶体增殖物激活受体γ（PPARγ）、肝X受体α（LXRα）、硬脂酰辅酶A去饱和酶1（SCD-1）、过氧化物酶体增殖物激活受体γ辅激活因子1（PGC-1）、脂肪酸合成酶（FAS）、胆固醇27-羟化酶（CYP27A1）及胆固醇7α-羟化酶（CYP7A1）的mRNA表达水平，每组同样设置3个复孔。
按照SYBR Premix Ex Taq II试剂盒（Takara，中国大连）的操作说明，在CFX 96实时荧光定量PCR检测系统（BIO-RAD，美国加利福尼亚州赫尔克里士市）上进行qPCR反应，反应程序为：95℃预变性10 min，随后40个循环的95℃变性15 s、60℃退火30 s、72℃延伸30 s。补充材料表S2列出了各基因的PCR引物序列，目的基因均以内参基因甘油醛-3-磷酸脱氢酶（GAPDH）进行标准化校正。采用2^(-ΔΔCt)法计算相对基因表达量。
收集小鼠盲肠内容物送至上海派森诺生物科技股份有限公司（Shanghai Personal Biotechnology Co., Ltd.），通过Illumina MiSeq测序平台进行16S rRNA测序分析以探究菌群多样性。提取微生物DNA后，按照试剂盒操作流程（24-25），以上游引物5′-ACTCCTACGGGAGGCAGCA-3′与下游引物5′-GGACTACHVGGGTWTCTAAT-3′对细菌16S rRNA基因的V3-V4可变区进行聚合酶链式反应（PCR）扩增。此前已有研究详细阐述了该分析的条件与参数（18）。
所有实验均进行3次生物学重复，数据采用社会科学统计软件包16.0（SPSS 16.0）进行统计分析，结果以平均值±标准差（mean ± SD）表示。组间多重比较采用单因素方差分析（ANOVA）结合邓肯多重比较检验，以P < 0.05为差异具有统计学意义。采用Origin 9.1软件绘制统计图。