1Department of Immunobiology, Institute of Tissue Transplantation and Immunology, Jinan

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

1Department of Immunobiology, Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou, China.

2Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

3Department of Physiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

4Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

5Brain Hormone Healthy Aging Centre, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

6GMH 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: ; 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:

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 p0.05) 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. Ap0.05 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 p0.05) 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’) / A. Temp. (°C)
adiponectin / Forward / TAAGGGTGACCCAGGAGATG / 58
Reverse / GGA ACATTGGGGACAGTGAC
ATGL / Forward / ACCAACACCAGCATCCAA / 54
Reverse / GAAGTCCATCTCGGTAGCC
Bax / Forward / GGTTGCCCTCTTCTACTTTG / 55
Reverse / CAGCCACCCTGGTCTTG
Bcl-2 / Forward / TGGGATACTGGAGATGAAGACT / 55
Reverse / CGACGGTAGCGACGAGA
CAT / Forward / GAGGCAGTGTACTGCAAGTTCC / 58
Reverse / GGGACAGTTCACAGGTATCTGC
GAPDH / Forward / CCTTCATTGACCTCAACTACATGGT / 55
Reverse / TCATTGTCATACCAGGAAATGAGCT
GPx / Forward / TCCACCGTGTATGCCTTCTCC / 58
Reverse / CCTGCTGTATCTGCGCACTGGA
PPARγ2 / Forward / CAGGTTTGGGCGAATG / 55
Reverse / TTTGGTCAGCGGGAAG
SREBP1c / Forward / GGAGCCATGGATTGCACATT / 58
Reverse / GCTTCCAGAGAGGAGCCCAG

A. Temp. = Annealing Temperature.