生酮饮食诱导db/db小鼠肝脏脂肪沉积

doi:10.16138/j.1673-6087.2023.01.018

摘要/Abstract

摘要：

目的：研究生酮饮食（ketogenic diet，KD）对db/db小鼠肝脏脂质沉积的影响及其机制，探讨KD治疗db/db小鼠的安全性。方法：选用8周龄db/db雄性小鼠20只作为肥胖2型糖尿病（type 2 diabetes mellitus，T2DM）动物模型，适应性喂养3周后，最终18只纳入研究，随机数字表法分为正常喂养（ND）组、KD组、75%热量限制（calorie restriction，CR）组，每组6只。另将8周龄C57BL/6雄性小鼠6只作为正常对照（C）组，以标准饲料喂养。C组、ND组自由进食标准饲料，KD组自由进食生酮饲料，CR组作为阳性对照组，每日摄入ND组75%的标准饲料。干预4周后，由于实验过程中KD组及CR组分别有2只及1只小鼠不明原因死亡，按随机数字表法每组纳入3只小鼠进行统计分析。检测各组小鼠空腹甘油三酯（triglyceride，TG）、总胆固醇（total cholesterol，TC）及低密度脂蛋白胆固醇（low density lipoprotein cholesterol，LDL-C）水平；观察小鼠肝脏形态和结构及肝脏组织中脂滴大小和数量；定量聚合酶链反应（quantitative polymerase chain reaction，qPCR）法检测肝脏组织固醇调节元件结合蛋白1C（sterol regulatory element-binding protein 1C，SREBP1C）、硬脂酰辅酶A去饱和酶-1（stearoyl-CoA desaturase 1，SCD1）、酰基辅酶A氧化酶1（acyl-CoA oxidase 1，ACOX1）、过氧化物酶体增殖物激活受体α（peroxisome proliferator-activated receptor α，PPARα）、白细胞分化抗原36（cluster of differentiation 36，CD36）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白介素-1β（interleukin-1β，IL-1β）、基质金属蛋白酶13（matrix metalloproteinase 13，MMP13）、组织金属蛋白酶抑制物1（tissue inhibitor of metalloproteinase 1，TIMP1）、Ⅲ型胶原a1（type Ⅲ collagen a1，Col3a1）及Ⅰ型胶原a1（type Ⅰ collagen a1，Col1a1）等相关因子的表达；免疫组化及蛋白质印迹法检测肝脏组织中CD36的表达水平。结果：与ND组相比，KD组小鼠TG、TC及LDL-C水平无明显改善，CR组小鼠TG水平显著降低（P<0.05）。KD组较ND及CR组肝脏空泡变性增加，脂质沉积增多。qPCR结果显示，与ND组相比，KD组PPARα、ACOX1等脂质分解代谢基因差异无统计学意义；CD36表达明显升高（P<0.05）；IL-1β、TNF-α等炎症因子表达明显升高（P<0.05）；MMP13表达显著下降（P<0.05），其余肝纤维化相关基因表达无明显变化。与ND组相比，KD组CD36蛋白表达水平显著增加（P<0.05）。结论：KD诱导db/db小鼠肝脏脂质沉积，加重肝脏炎症水平。因此，在使用KD治疗肥胖及肥胖相关疾病时，要密切关注肝功能的变化，优化KD方案，预防其不良反应。

关键词: 2型糖尿病, 肥胖, 生酮饮食, 肝脏, 脂肪沉积

Abstract:

Objectives To study the effect of ketogenic diet （KD） on liver lipid deposition in db/db mice, and to investigate the safety of KD treatment and molecular mechanism in db/db mice. Methods Twenty eight-week-old db/db male mice of obesity type 2 diabetes mellitus(T2DM) were selected. After 3 weeks of adaptive feeding, 18 mice were enrolled and randomly divided into 3 groups: normal feeding (ND) group (n=6), KD group (n=6) and 75% caloric restriction (CR) group(n=6). In addition, 6 eight-week-old C57BL/6 male mice were fed with standard control diet and considered as normal (C) group. C and ND groups were free to get the standard feed, KD group was free to get the ketogenic feed, and CR group was the positive control group, which consumed 75% of standard feed calories of ND group each day. After 4 weeks of diet treatment, 2 mice in KD group and 1 mouse in CR group died. Three mice in each group were randomly included for statistical analysis, and the levels of fasting triglyceride(TG), total cholesterol(TC) and low-density lipoprotein cholesterol(LDL-C) in each group were detected and compared. The morphology and structure of liver were observed by hematoxylin and eosin(HE) staining. The size and number of lipid droplets in liver tissue of mice were observed by oil red O staining. The quantitative polymerase chain reaction (qPCR) was used to detect the expression of sterol regulatory element-binding protein 1C (SREBP1C), stearoyl-CoA desaturase 1 (SCD1), acyl-CoA oxidase 1 (ACOX1), peroxisome proliferator-activated receptor α (PPARα), cluster of differentiation 36 (CD36), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), matrix metalloproteinase-13 (MMP13), matrix metalloproteinase-inhibitor 1(TIMP1), collagen type Ⅲ alpha 1 (COL3a1) and collagen type Ⅰ alpha 1 (COL1a1) in liver. Immunohistochemistry (IHC) and western blot (WB) were used to detect the expression of fatty acid translocase (CD36) in liver tissues. Results After 4 weeks of dietary intervention, compared with ND group, the level of TG, TC and LDL-C in KD group was not improved, but the level of TG in CR group was significantly decreased (P<0.05). HE and oil red O staining showed that liver vacuolar degeneration and lipid deposition increased in KD group compared with ND and CR groups. qPCR showed that compared with ND group, lipid-catabolism genes such as PPARα and ACOX1 showed decreased trend in KD group without significant difference; the expression of CD36, IL-1β and TNF-α was significantly increased (P<0.05). The expression of MMP13 was significantly decreased(P<0.05), while the expression of other hepatic fibrosis related genes was not changed. IHC showed that compared with ND group, the expression level of CD36 protein in KD group was significantly increased(P<0.05). Conclusions KD could induce liver lipid deposition, aggravate liver inflammation in db/db mice. Therefore, in the treatment of obesity with KD, the changes of liver function should be closely observed to prevent the occurrence of adverse reactions.

Key words: Type 2 diabetes mellitus, Obesity, Ketogenic diet, Liver, Fat deposition

中图分类号:

R587.1

张梦潇, 孙烁烁, 韦晓, 张少红, 陈国芳, 刘超. 生酮饮食诱导db/db小鼠肝脏脂肪沉积[J]. 内科理论与实践, 2023, 18(01): 56-63.

ZHANG Mengxiao, SUN Shuoshuo, WEI Xiao, ZHANG Shaohong, CHEN Guofang, LIU Chao. Ketogenic diet promotes hepatic lipid accumulation in db/db mice[J]. Journal of Internal Medicine Concepts & Practice, 2023, 18(01): 56-63.

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基因	上游引物	下游引物
SREBP1C	CACTTCTGGAGACATCGCAAAC	ATGGTAGACAACAGCCGCATC
SCD1	TTCTTGCGATACACTCTGGTGC	CGGGATTGAATGTTCTTGTCGT
PPARα	TATTCGGCTGAAGCTGGTGTAC	CTGGCATTTGTTCCGGTTCT
ACOX1	CAAGACCCAAGAGTTCATT	TTCAGGTAGCCATTATCCA
CD36	TGGTCAAGCCAGCTAGAAA	CCCAGTCTCATTTAGCCAC
IL-1β	GAAATGCCACCTTTTGACAGTG	TGGATGCTCTCATCAGGACAG
TNF-α	CTGAGGTCAATCTGCCCAAGTAC	CTTCACAGAGCAATGACTCCAAAG
MMP13	CCTTCTGGTCTTCTGGCACAC	GGCTGGGTCACACTTCTCTGG
TIMP1	CGAGACCACCTTATACCAGCG	ATGACTGGGGTGTAGGCGTA
Col3a1	ATTGGTGGTTTTCAGTTCAGC	TGGGGTTTCAGAGAGTTTGGC
Cpl1a1	ACCTGTGTGTTCCCTACTCA	GACTGTTGCCTTCGCCTCTG
GAPDH	TGTGGATGGCCCCTCTGGAA	TGACCTTGCCCACAGCCTTG

项目	C组	ND组	KD组	CR组	F	P
TG(mmol/L)	1.07±0.40	3.01±0.20¹⁾	2.19±0.08	1.54±0.33²⁾	8.999	0.006 1
TC(mg/dL)	76.34±2.03	150.27±5.12¹⁾	27.87±14.40¹⁾	35.45±0.79¹⁾	17.34	0.000 7
LDL-C(mmol/L)	2.19±0.17	5.78±0.10¹⁾	3.70±1.33	4.60±0.33	4.729	0.035 1

项目	C组	ND组	KD组	CR组	F	P
SREBP1C	1	0.37±0.04¹⁾	0.12±0.02¹⁾²⁾	0.49±0.05¹⁾	116.8	<0.000 1
Scd1	1	0.51±0.01¹⁾	0.004±0.002¹⁾²⁾	0.99±0.12²⁾	59.9	<0.000 1
PPARα	1	0.49±0.05¹⁾	0.33±0.04¹⁾	0.72±0.04¹⁾²⁾	59.48	<0.000 1
Acox1	1	0.58±0.02¹⁾	0.38±0.08¹⁾²⁾	0.78±0.03¹⁾²⁾	40.02	<0.000 1

项目	C组	ND组	KD组	CR组	F	P
IL-1β	1	1.14±0.10	2.76±0.53¹⁾²⁾	0.69±0.076²⁾	11.61	0.002 8
TNF-α	1	1.46±0.11¹⁾	3.22±0.46¹⁾²⁾	0.90±0.04²⁾	20.76	0.000 4

项目	C组	ND组	KD组	CR组	F	P
TIMP1	1	1.46±0.09¹⁾	1.72±0.05¹⁾	1.18±0.07	24.80	0.000 2
Col3a1	1	2.33±0.27¹⁾	2.85±0.25¹⁾	1.46±0.15	17.76	0.000 7
Col1a1	1	3.05±0.36¹⁾	4.47±0.74¹⁾	1.59±0.24	13.26	0.001 8
MMP13	1	0.69±0.05¹⁾	0.40±0.07¹⁾²⁾	0.67±0.08¹⁾	18.32	0.000 6