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

Table 1 Composition of the regular and high-fat diet

Ingredients	Ingredient concentration (g/kg)
	Regular diet	High fat diet
Casein	210.0	265.0
L-cystine	3.0	4.0
High amylose corn starch	500.0	-
Maltodextrin	100.0	160.0
Sucrose	39.14	90.0
Anhydrous milkfat	20.0	-
Lard	20.0	310.0
Soybean oil	20.0	30.0
Cellulose	35.0	65.5
Mineral mix, AIN-93G-MX (94046) (g/kg)	35.0	48.0
Calcium phosphate, dibasic	-	3.4
Vitamin mix, AIN-93-VX (94047)	15.0	21.0
Choline bitartrate	2.75	3.0
TBHQ, antioxidant	0.01	-
Protein (% by weight)	18.6	23.5
Carbohydrate (% by weight)	50.6	27.3
Fat (% by weight)	6.2	34.3

Table 2 Primers used in this study

Gene marker	Full name	Primer sequence (5'-3')
β-Actin	Beta-actin	GCCCTGGACTTCGAGCAAGA
		TGCCAGGGTACATGGTGGTG
β-2m	Beta-2 microglobulin	TGCTTGTCTCAC TGACCGGCC
		TGGGGGTGAATTCAGTGTGAGCC
SOD2	Super oxide dismutase 2	ACAACAGGCCTTATTCCGCT
		CCCCAGTCATAGTGCTGCAA
SREBP-1c	Sterol regulatory element Binding protein	CACCCTGTAGGTCACCGTTT
		AGAACTCCCTGTCTCCGTCA
ACC1	Acetyl-CoA	GACAGAGGAAGATGGCGTCC
	Carboxylase	TACAACTTCTGCTCGCTGGG
TNF-α	Tumor necrosis	CGTCGTAGCAAACCACCAAG
	Factor-alpha	AGCAAATCGGCTGACGGTG
CRP	C-Reactive Protein	ACTGTGGGGCCAGATGCAAGC
		GGGGCTGAGTGTCCCACCAAC
CAT	Catalase	TCACTCAGGTGCGGACATTC
		TAGTCAGGGTGGACGTCAGT
IL-6	Interleukin-6	TCTATACCACTTCACAAGTCGGA
		GAATTGCCATTGCACAACTCTTT
Adipo	Adiponectin	GACGACACCAAAAGGGCTCA
		GAGGCCATCTCTGCCATCA
AdipoR2	Adiponectin	CTCTGACAGGATTTGGGGTCAA
	Receptor 2	GTGCCCTTTTCTGAGCCGTA

Table 3 Expression of genes relevant to nonalcoholic fatty liver disease

Gene	RW		HRW		P value
	M	Sd	M	Sd
IL-6	1.10	0.46	1.17	0.26	0.26
TNF-α	1.19a	0.36	0.68b	0.22	0.02
SREBP-1c	1.04	0.19	1.02	0.30	0.87
ACOX	1.04a	0.21	1.31b	0.25	0.04
CD36	1.06a	0.28	0.71b	0.06	0.02
CPT1	1.08	0.37	1.43	0.40	0.14
ACC2	1.12	0.50	1.05	0.23	0.64
ACC1	1.06	0.14	1.13	0.17	0.13
FAS	1.09	0.42	1.42	0.36	0.29
SOD	1.04	0.17	0.96	0.08	0.86
Cat	1.04	0.21	1.12	0.24	0.80
AdipoR2	1.01	0.22	1.14	0.28	0.23
Adipo	0.92a	0.29	1.32b	0.38	0.004

Each value is the average of 8 mice after normalization with keeping genes (β2m, β-actin). Statistically significant difference marked by different letters P < 0.05 (Kruskal-Wallis, Mann Whitney test). Regular water or hydrogen-rich water.

Table 4 Expression of genes relevant to nonalcoholic fatty liver disease from hepatocytes isolated from mice drinking regular water or hydrogen-rich water after palmitate overloading in vitro

	RW		HRW		P value
	M	Sd	M	Sd
TNF-α	9.16a	1.90	5.71b	4.59	0.036
IL-6	3.2a	0.87	2.24b	0.64	0.002
CRP	0.96	0.28	1.20	0.34	0.065
SREBP-1c	0.96	0.12	1.05	0.19	0.196
ACC1	0.84	0.18	0.86	0.14	0.902
SOD	1.07	0.34	1.30	0.56	0.184
AdipoR2	0.80	0.08	0.78	0.18	1.000
Adipo	1.00	0.30	0.79	0.30	0.097

Each value is the average of hepatocytes obtained from 4 different mice after normalization with keeping genes (β2m, β-actin). Statistically significant difference marked by different letters (Kruskal-Wallis, Mann Whitney test).