Lycium barbarum polysaccharides therapeutically improve hepatic
functions in non-alcoholic steatohepatitis rats and cellular steatosis model
Jia Xiao1,2, Feiyue Xing1, Jie Huo1, Man Lung Fung3,5, Emily C. Liong2, Yick Pang
Ching2, Aimin Xu4,5, Raymond Chuen Chong Chang2,5, Kwok Fai So2,5,6 & George L.
Tipoe2,5
1
Department of Immunobiology, Institute of Tissue Transplantation and Immunology,
Jinan University, Guangzhou, China.
2
Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong
Kong, Hong Kong SAR, China.
3
Department of Physiology, Li Ka Shing Faculty of Medicine, The University of
Hong Kong, Hong Kong SAR, China.
4
Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong
Kong, Hong Kong SAR, China.
5
Brain Hormone Healthy Aging Centre, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Hong Kong SAR, China.
6
GMH Institute of Central Nervous System Regeneration, Jinan University,
Guangzhou, China.
Running Title: LBP therapeutically ameliorates NAFLD
Correspondences: Kwok Fai So, Ph.D., GMH Institute of Central Nervous System
Regeneration, Jinan University, Guangzhou, China. Telephone: (86) 20-85228362;
Fax: (86) 20-85223563; E-mail: hrmaskf@hku.hk; George L. Tipoe, M.D., Ph.D.,
Department of Anatomy, The University of Hong Kong, No. 21 Sassoon Road, Hong
Kong, Hong Kong SAR, China. Telephone: (852) 28199185; Fax: (852) 28170857;
E-mail: tgeorge@hkucc.hku.hk
Supplementary Figure 1. NASH was successfully established before the
therapeutic LBP treatment. (a,b) Representative image of H&E staining of the rat
liver with or without 8-week high-fat diet feeding. Yellow arrows indicate typical fat
deposition in the liver. Magnification: ×200. Bar: 20 μm. (c) NAFLD activity score
(NAS) of rat with or without 8-week high-fat diet feeding. Data from each group were
expressed as means ± SEM (n = 3). Statistical comparison between groups was done
using the Kruskal–Wallis test followed by Dunn’s post hoc test to detect differences in
all groups. Different letters (e.g. a and b) mean a statistically significant change (at
least p < 0.05) between each other.
Supplementary Figure 2. LBP improved glucose metabolism and insulin
clearance damaged by NASH induction. Serum insulin level was assessed after the
NASH induction. (a) Rats were i.p. injected with D-glucose (a fixed bolus of 350 mg)
and the serum insulin level was recorded 0, 20, 40, 60, 80, and 100 min later using
ELISA kit. (b) Rats were i.p. injected with recombinant insulin (a fixed bolus of 0.17
IU) and the serum insulin level was recorded 0, 20, 40, 60, 80, and 100 min later
using ELISA kit. Statistical comparison between groups was done using the
Kruskal–Wallis test followed by Dunn’s post hoc test to detect differences in all
groups. A p < 0.05 was considered to be statistically significant. “*” represents
significant change against control and “#” represents significant change against either
LBP co-treatment group or LBP after-treatment group.
Supplementary Figure 3. Optimization of sodium palmitate treatment dosage in
inducing steatosis. (a,b) Cellular viability and Oil Red O read (normalized by MTT
results) in BRL-3A cells received different doses of sodium palmitate incubation for
24 hrs. Data from each group were expressed as means ± SEM (n = 5). Statistical
comparison between groups was done using the Kruskal–Wallis test followed by
Dunn’s post hoc test to detect differences in all groups. Different letters (e.g. a and b)
mean a statistically significant change (at least p < 0.05) between each other.
Supplementary Figure 4. Full length images of Western blot results from all
figures. C: control, N: NASH, L: vehicle-LBP, NL: NASH + LBP treatment
throughout the experiment, TL: vehicle-LBP treatment from the 9th week to the 12th
week with NASH induction, NTL: NASH + LBP treatment from the 9th week to the
12th week with NASH induction.
Supplementary Table 1. Primer sequences and annealing temperatures for
quantitative PCR.
Target gene
Direction
Primer sequence (5’-3’)
Forward
TAAGGGTGACCCAGGAGATG
adiponectin
58
Reverse
GGA ACATTGGGGACAGTGAC
Forward
ACCAACACCAGCATCCAA
ATGL
54
Reverse
GAAGTCCATCTCGGTAGCC
Forward
GGTTGCCCTCTTCTACTTTG
Bax
55
Reverse
CAGCCACCCTGGTCTTG
Forward
TGGGATACTGGAGATGAAGACT
Bcl-2
55
Reverse
CGACGGTAGCGACGAGA
Forward
GAGGCAGTGTACTGCAAGTTCC
CAT
58
Reverse
GGGACAGTTCACAGGTATCTGC
Forward
CCTTCATTGACCTCAACTACATGGT
GAPDH
55
Reverse
TCATTGTCATACCAGGAAATGAGCT
Forward
TCCACCGTGTATGCCTTCTCC
GPx
PPARγ2
A. Temp. (°C)
58
Reverse
CCTGCTGTATCTGCGCACTGGA
Forward
CAGGTTTGGGCGAATG
55
Reverse
TTTGGTCAGCGGGAAG
Forward
GGAGCCATGGATTGCACATT
SREBP1c
58
Reverse
GCTTCCAGAGAGGAGCCCAG
A. Temp. = Annealing Temperature.