Mulberry fruit polysaccharides alleviate diethylnitrosamine/phenobarbital-induced hepatocarcinogenesis in vivo: the roles of cell apoptosis and inflammation

Figures & data

Figure 1. (a) Detailed experimental design. Physiological assays of rats with DEN/PB-induced HCC that were treated with Mulberry fruit polysaccharides (MFP). (b) The chromatograms of MFP. (c) Final body weight of rats with DEN/PB-induced HCC that was treated with MFP. (d) Liver weight of rats with DEN/PB-induced HCC that was treated with MFP. (e) Relative liver weight of rats. ^## Significant against Con group at P < 0.01. ** Significant against DEN/PB group at P < 0.01. * Significant against DEN/PB group at P < 0.05. ^& Significant against DEN/PB+HMFP group at P < 0.05

Table 1. Sequences of primers used quantitative real-time PCR

Gene	Forward primer	Reverse primer
Bcl-2	5ʹ-ACTTCTCTCGTCGCTACCGTCGC-3’	5ʹ-AGAGCGATGTTGTCCACCAGGG-3’
Bax	5ʹ-CCAGGACGCATCCACCAAGAAG-3’	5ʹ-CCCAGTTGAAGTTGCCGTCTGC-3’
TNF-α	5ʹ-CCACGCTCTTCTGTCTACTG-3’	5ʹ-GCTACGGGCTTGTCACTC-3’
Caspase‑3	5ʹ-GGAGCAGTTTTGTGTGTGTGA-3’	5ʹ-TGTCTCAATACCGCAGTCCA-3’
Caspase‑9	5ʹ-TGGCATACACCCTGGACTC-3’	5ʹ-GCCGTGACCATTTTCTTAGC-3’
NF-κB	5ʹ-AGCACCAAGACCGAAGCAA-3’	5ʹ-TCTCCCGTA ACCGCGTAGTC-3’
IL-1β	5ʹ-GCCGATGGTCCCAATTACAT-3’	5ʹ-ACAAGACCTGCCGGAAGCT-3’
MTH1	5ʹ-GAGCGGCGGTGCAGAACCCAG-3’	5ʹ-AGAAGACATGCACGTCCATGAG-3’
GAPDH	5ʹ-CCCCCAATGTATCCGTTGTG-3’	5ʹ-TAGCCCAGGATGCCCTTTAGT-3’

Table 2. The chemical analysis of polysaccharides from mulberry fruit

Sample	MFP
Mw (kDa)	86.71
Polydispersity (Mw/Mn)	1.15
Carbohydrate content (%)	71.28 ± 1.21
Protein	2.63 ± 0.07
Uronic acid	20.31 ± 0.41
Sulfate	4.01 ± 0.09

Table 3. Effect of MFP on mortality in DEN/PB-induced hepatocellular carcinoma

Group	Con	Con+HMFP	DEN/PB	DEN/PB+LMFP	DEN/PB+HMFP
Number of rats with nodules/number of rats	0/12	0/12	9/9	5/11	2/12
Total number of nodules	0	0	205	92**	36**
Average number of nodules/nodule-bearing liver (nodule multiplicity)	0	0	22.74 ± 1.83	18.80 ± 1.36**	17.06 ± 1.61**
Tumor incidence (%)	0	0	100	45.45**	16.67**
Number of death (n)	0	0	3	1	0
Number of rats (n)	12	12	12	12	12
Mortality (%)	0	0	25	8.33**	0**

** Significant against DEN/PB group at P < 0.01.

Figure 2. Effect of MFP on hepatic function biomarkers in DEN/PB-induced HCC. (a) The levels of serum aspartate aminotransferase (AST), (b) alanine aminotransferase (ALT), (c) alkaline phosphatase (ALP), (d) glutamyl transpeptidase (GGT), (e) total bilirubin, and (f) albumin in different treatment groups were measured. ^## Significant against Con group at P < 0.01. ** Significant against DEN/PB group at P < 0.01. * Significant against DEN/PB group at P < 0.05. ^&& Significant against DEN/PB+HMFP group at P < 0.01

Figure 3. Effect of MFP on hepatic tumor markers in DEN/PB-induced HCC. (a) The levels of serum alpha-fetoprotein (AFP), (b) carcinoembryonic antigen (CEA), and (c) carbohydrate antigen 19.9 (CA19.9) in different treatment groups were measured. (d) Representative images of immunohistochemical staining with GST-p in different groups (scale bar = 100 μm). (e) The percentage of GST-p positive cells in different groups. ^## Significant against Con group at P < 0.01. ** Significant against DEN/PB group at P < 0.01. * Significant against DEN/PB group at P < 0.05. ^& Significant against DEN/PB+HMFP group at P < 0.05. ^&& Significant against DEN/PB+HMFP group at P < 0.01

Figure 4. Effect of MFP on the hepatic apoptosis markers in DEN/PB-induced HCC. (a) The levels of hepatic caspase-3 and (b) caspase-9 in different treatment groups were measured. ^## Significant against Con group at P < 0.01. ** Significant against DEN/PB group at P < 0.01. ^& Significant against DEN/PB+HMFP group at P < 0.05. ^&& Significant against DEN/PB+HMFP group at P < 0.01

Figure 5. Effect of MFP on the hepatic inflammation markers in DEN/PB-induced HCC. (a) The levels of hepatic IL-1β, (b) TNF-α, and (c) NF-κB in different treatment groups were measured. ^## Significant against Con group at P < 0.01. ** Significant against DEN/PB group at P < 0.01. ^& Significant against DEN/PB+HMFP group at P < 0.05. ^&& Significant against DEN/PB+HMFP group at P < 0.01

Figure 6. mRNA expression in rat’s hepatic tissue with DEN/PB-induced HCC. The level of Bcl-2 (a), Bax (b), caspase-3 (c), and caspase-9 (d) in different treatment groups. (e) Representative images of immunohistochemical staining with Bcl-2 in different groups (scale bar = 100 μm). (f) The percentage of Bcl-2 positive cells in different groups. ^## Significant against Con group at P < 0.01. ** Significant against DEN/PB group at P < 0.01. * Significant against DEN/PB group at P < 0.05. ^&& Significant against DEN/PB+HMFP group at P < 0.01

Figure 7. mRNA expression in rat’s hepatic tissue with DEN/PB-induced HCC. The level of IL-1β (a), TNF-α (b), NF-κB (c), and MTH1 (d) in different treatment groups. (e) Representative images of immunohistochemical staining with NF-κB in different groups (scale bar = 100 μm). (f) The percentage of NF-κB positive cells in different groups. ^## Significant against Con group at P < 0.01. ** Significant against DEN/PB group at P < 0.01. * Significant against DEN/PB group at P < 0.05. ^& Significant against DEN/PB+HMFP group at P < 0.05. ^&& Significant against DEN/PB+HMFP group at P < 0.01

Figure 8. Liver tissues stained with H&E of all group rats. Histopathological assessment was performed under the microscope at 100 × . Black arrows indicate inflammation cell infiltration, red arrows indicate hepatocyte necrosis

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.