5-methoxytryptophan alleviates liver fibrosis by modulating FOXO3a/miR-21/ATG5 signaling pathway mediated autophagy

Figures & data

Table 1. Primer sequences for quantitative PCR

Gene	Forward sequence	Reverse sequence
α-SMA	5ʹ-TATCCCCGGGACTAAGACGGG-3’	5ʹ-CAGAGCCCAGAGCCATTGTC-3’
Fibronectin	5ʹ-GACGTCCATAGCTCCTCTCC-3’	5ʹ-GAGGCCTGACAGTGGGTAAT-3’
Collagen I	5ʹ-GATTCCCTGGACCTAAAGGTGC-3’	5ʹ-AGCCTCTCCATCTTTGCCAGCA-3’
Collagen III	5ʹ-TGGTCTGCAAGGAATGCCTGGA-3’	5ʹ-TCTTTCCCTGGGACACCATCAG-3’
FOXO3a	5ʹ-GATCCGATGATGTCCTTTGC-3’	5ʹ-TGCATAGACTGGCTGACAGG-3’
miR-21	5ʹ-CGCGCTAGCTTATCAGACTGA-3’	5ʹ-GTGCAGGGTCCGAGGT-3’
ATG5	5ʹ-AAGACCTTCTGCACTGTCCA-3’	5ʹ-GAGTTTCCGATTGATGGCCC-3’
U6	5ʹ-CTCGCTTCGGCAGCACA-3’	5ʹ-AACGCTTCACGAATTTGCGT-3’
GAPDH	5ʹ-CTGACTTCAACAGCGACACC-3’	5ʹ-GTGGTCCAGGGGTCTTACTC-3’

Figure 1. 5-MTP inhibited the proliferation and fibrosis of LX-2 cells

LX-2 cells were treated with control, TGF-β1, or TGF-β1 combined with 5-MTP. (a) Represent images of LX-2 cells under bright light. Cell viability was measured by MTT assay (b). Cell proliferation ability was determined by BrdU staining (c). The mRNA and protein levels of fibrosis marker genes (α-SMA, fibronectin, collagenI and collagen III) were detected by qPCR (d) and western blot (e). The expression of LC3 was detected by Immunofluorescent staining (f) and western blot (g). N= 3, *P< 0.05, **P < 0.01, ***P < 0.001.

Figure 2.. 5-MTP repressed the proliferation and fibrosis of LX-2 cells by inducing autophagy

The expression of LC3 was detected by Immunofluorescent staining (a) and western blot (b). Cell viability measured by MTT assay (c). Cell proliferation was determined by BrdU staining (d). The levels of fibrosis marker genes (α-SMA, fibronectin, collagen I and collagen III) were detected by Immunofluorescent staining (e) and western blot (f). N = 3, *P < 0.05, **P< 0.01, ***P< 0.001.

Figure 3. FOXO3a was upregulated by 5-MTP and suppressed the transcription of miR-21

The mRNA and protein levels of FOXO3a were detected by qPCR (a) and western blot (b). Putative FOXO3a binding sites in the promoter region of miR-21 (c). The direct binding of FOXO3a in the promoter region of miR-21 was determined by ChIP assay (d) and Dual-luciferase reporter assays (e). Levels of FOXO3a and miR-21 were detected by qPCR (f) and western blot (g). N = 3, *P< 0.05,**P< 0.01.

Figure 4. miR-21 directly targeted ATG5 and inhibited its expression

The prediction of binding sites between miR-21 and ATG5 using the biological prediction website http://starbase.sysu.edu.cn. (a) The miR-21 mimic and luciferase reporter plasmids with wild-type or mutant ATG5 3ʹ-UTR, were co-transfected into LX-2 cells. Dual luciferase reporter gene assay was performed to verify the direct binding relationship between miR-21 and ATG5 (b). Cells were transfected with NC mimic and miR-21 mimic, and then qPCR was taken to evaluate relative expression levels of miR-21 and ATG5 (c). The protein expression of ATG5 was analyzed by western blot (d). N = 3, P< 0.05, **P < 0.01, **P< 0.001.

Figure 5.. 5-MTP suppressed the proliferation and fibrosis of LX-2 cells by modulating FOXO3a/miR-21/ATG5 signaling pathway mediated autophagy

Expression of autophagy-related proteins was measured by western blot (a). Cell viability was measured by MTT assay (b). Cell proliferation ability was detected by BrdU staining (c). The levels of fibrosis marker genes were analyzed by immunofluorescent staining (d) and western blot (e). N = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.. 5-MTP attenuated CCl4-induced hepatic fibrosis in vivo.

Expression of autophagy-related proteins from liver tissues was measured by western blot (a). Histopathological changes of liver tissues were detected by HE staining (b). Liver fibrosis was evaluated by sirius red staining (c). The levels of fibrosis marker genes were analyzed by immunohistochemistry (d) and western blot (e). N = 3, *P < 0.05, **P < 0.01, ***P < 0.001.