乳双歧杆菌V9对高脂饮食诱导的NAFLD大鼠的改善作用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0630

摘要/Abstract

摘要：

研究乳双歧杆菌V9（V9）对高脂饮食诱导的非酒精性脂肪肝疾病（NAFLD）大鼠脂代谢、肠道损伤的改善作用,并探讨其作用机制。雄性Wistar大鼠随机分为5组：空白对照组（Control）、高脂饮食模型组（HFD）、黄连素阳性药物组（Berberine）、乳双歧杆菌V9治疗组（HFD/V9）和乳双歧杆菌V9对照组（V9）。除Control和V9给予普通维持饲料外,其他给予高脂饮食6周构建NAFLD模型。6周后,将高脂饮食替换为普通维持饲料,大鼠灌胃给予乳双歧杆菌V9（1×10⁹CFU/mL）或黄连素（50 mg/kg）。4周后,收集血液、肝脏以及结肠组织用于后续试验检测。与Control组相比,HFD组中肝脏非酯化脂肪酸（NEFA）、甘油三酯（TG）、肝脏脂肪酸合成酶（FAS）和肝脏脂肪酸结合蛋白1（Fabp1）mRNA的表达明显增高（P<0.01）;同时,肠道细胞因子白介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、Toll-样受体2（TLR2）和Toll-样受体9（TLR9）的mRNA表达在HFD模型组中也明显升高（P<0.01）;相反地,紧密连接蛋白ZO-1和occludin mRNA的表达在HFD模型组中明显降低（P<0.01）。在给予乳双歧杆菌V9治疗以后,肝脏NEFA和TG含量明显降低（P<0.01）;肝脏FAS和Fabp1的mRNA表达也显著降低（P<0.01）。乳双歧杆菌V9和黄连素治疗后都降低了肠道IL-1β、TNF-α、TLR2和TLR9的转录水平（P<0.01）。但是,提高了肠道紧密连接蛋白ZO-1和occludin mRNA的表达。Control组和V9组二者效应无显著差异（P<0.01）。HE染色显示,以上各组的结肠病理学无明显差异。益生乳双歧杆菌 V9 可以通过缓解脂代谢紊乱、降低肠道炎症反应并改善肠道黏膜屏障结构,从而改善高脂饮食诱导的 NAFLD。

关键词: 肠道菌群, 非酒精性脂肪肝病, 益生菌, 高脂饮食

Abstract:

This work aims to study the effect of probiotic Bifidobacterium lactis V9（V9）on lipid metabolism and intestinal injury in non-alcoholic fatty liver disease（NAFLD）rats induced by high-fat diet,and to explore its mechanism. Male Wistar rats were randomly divided into 5 groups（n=8）：blank control group（Control）,high-fat diet model group（HFD）,berberine-positive drug group（Berberine）,the probiotic B. lactis V9 treatment group（HFD/V9）,and the probiotic B. lactis V9 control group（V9）. Except Control and V9 were given ordinary maintenance feed,others were given a high-fat diet for 6 weeks to construct a NAFLD model. After 6 weeks,the high-fat diet was replaced with ordinary maintenance feed,and the rats were given probiotic B. lactis V9（1×10 ⁹CFU/mL）or berberine（50 mg/kg）by gavage. After 4 weeks,blood,liver and colon tissues were collected for follow-up testing. Compared with the Control group,the mRNA expressions of liver non-esterified fatty acids（NEFA）,triglycerides（TG）,liver fatty acid synthase（FAS）and liver fatty acid binding protein 1（Fabp1）in the HFD group significantly increased（P<0.01）. At the same time,mRNA expressions of the intestinal cytokine interleukin 1β（IL-1β）,tumor necrosis factor α（TNF-α）,Toll-like receptor 2（TLR2）and Toll-like receptor 9（TLR9）in the HFD model group also significantly increased（P<0.01）. On the contrary,the expression of tight junction protein ZO-1 and occludin mRNA in the HFD model group significantly decreased（P<0.01）. After treatment with probiotic B. lactis V9,the contents of NEFA and TG in the liver significantly reduced（P<0.01）,the mRNA expressions of FAS and Fabp1 in the liver also significantly reduced（P<0.01）. Both probiotic B. lactis V9 and berberine treatment reduced the transcription levels of intestinal IL-1β,TNF-α,TLR2 and TLR9（P<0.01）,which increased the expressions of intestinal tight junction proteins ZO-1 and occludin mRNA. HE staining showed that there was no significant difference in colonic pathology among the above groups. Probiotic Probiotics B. lactis V9 can improve NAFLD induced by high-fat diet by alleviating lipid metabolism disorders,reducing intestinal inflammation and improving mulosal barrier.

Key words: intestinal microflora, non-alcoholic fatty liver disease, probiotics, high fat diet

钟明月, 刘春妍, 颜妍, 张晓慧, 原海升, 徐国全, 张和平, 王玉珍. 乳双歧杆菌V9对高脂饮食诱导的NAFLD大鼠的改善作用[J]. 生物技术通报, 2022, 38(3): 181-187.

ZHONG Ming-yue, LIU Chun-yan, YAN Yan, ZHANG Xiao-hui, YUAN Hai-sheng, XU Guo-quan, ZHANG He-ping, WANG Yu-zhen. Improvement Effect of Bifidobacterium lactis V9 on NAFLD Rats Induced by High-fat Diet[J]. Biotechnology Bulletin, 2022, 38(3): 181-187.

图/表 7

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	脂肪 Fat/%	蛋白质 Protein/%	碳水化合物 Carbohydrate/%
普通维持饲料 Ordinaryl feed	11.4	27.5	65.8
高脂饲料High-fat diet （HFD）	33.1	19.6	47.1

	脂肪 Fat/%	蛋白质 Protein/%	碳水化合物 Carbohydrate/%
普通维持饲料 Ordinaryl feed	11.4	27.5	65.8
高脂饲料High-fat diet （HFD）	33.1	19.6	47.1

引物名称 Primer name	引物序列 Primer sequence
IL-1β antisense	5'-GAATGCCACCTTTTGACAGTG-3'
IL-1β sense	5'-TGGATGCTCTCATCAGGACAG-3'
TNF-α antisense	5'-TTGATGGTGGTGCATGAGAG-3'
TNF-α sense	5'-AAACACAAGATGCTGGGACA-3'
TLR2 antisense	5'-CTCCTGTGAACTCCTGTCCTT-3'
TLR2 sense	5'AGCTGTCTGGCCAGTCAAC-3'
TLR9 antisense	5'-CCGAAGACCTAGCCAACCT-3'
TLR9 sense	5'-TGATCACAGCGACGGCAATT-3'
Fas antisense	5'-GAGCGTTCGTGAAACCGACA-3'
Fas sense	5'-AGGTTGGTGCACCTCCACTTG-3'
Fabp1 antisense	5'-TTCCCCAGTCATGGTCTCCA-3'
Fabp1 sense	5'-TCATGAAGGCGATGGGTCTG-3'
ZO-1 antisense	5'-CCATCTTTGGACCGATTGCTG-3'
ZO-1 sense	5'-TAATGCCCGAGCTCCGATG-3'
Occludin antisense	5'-TTGGGAGCCTTGACATCTTGTTC-3'
Occludin sense	5'-GCCATACATGTCATTGCTTGGTG-3'
GAPDH antisense	5'-AGGTCGGTGTGTGAACGGATTTG-3'
GAPDH sense	5'-TGTAGACCATGTAGTTGAGGTCA-3'

引物名称 Primer name	引物序列 Primer sequence
IL-1β antisense	5'-GAATGCCACCTTTTGACAGTG-3'
IL-1β sense	5'-TGGATGCTCTCATCAGGACAG-3'
TNF-α antisense	5'-TTGATGGTGGTGCATGAGAG-3'
TNF-α sense	5'-AAACACAAGATGCTGGGACA-3'
TLR2 antisense	5'-CTCCTGTGAACTCCTGTCCTT-3'
TLR2 sense	5'AGCTGTCTGGCCAGTCAAC-3'
TLR9 antisense	5'-CCGAAGACCTAGCCAACCT-3'
TLR9 sense	5'-TGATCACAGCGACGGCAATT-3'
Fas antisense	5'-GAGCGTTCGTGAAACCGACA-3'
Fas sense	5'-AGGTTGGTGCACCTCCACTTG-3'
Fabp1 antisense	5'-TTCCCCAGTCATGGTCTCCA-3'
Fabp1 sense	5'-TCATGAAGGCGATGGGTCTG-3'
ZO-1 antisense	5'-CCATCTTTGGACCGATTGCTG-3'
ZO-1 sense	5'-TAATGCCCGAGCTCCGATG-3'
Occludin antisense	5'-TTGGGAGCCTTGACATCTTGTTC-3'
Occludin sense	5'-GCCATACATGTCATTGCTTGGTG-3'
GAPDH antisense	5'-AGGTCGGTGTGTGAACGGATTTG-3'
GAPDH sense	5'-TGTAGACCATGTAGTTGAGGTCA-3'