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. 2020 Oct 15;10(1):17486.
doi: 10.1038/s41598-020-74324-1.

Hepatic stem cell Numb gene is a potential target of Huang Qi Decoction against cholestatic liver fibrosis

Wen Xu  1   2 , Yan-Nan Xu  1   2 , Xu Zhang  1   2 , Ying Xu  1   2 , Xun Jian  1   2 , Jia-Mei Chen  1   2 , Gao-Feng Chen  1   2 , Hua Zhang  1   2 , Ping Liu  3   4   5 , Yong-Ping Mu  6   7
Affiliations

Hepatic stem cell Numb gene is a potential target of Huang Qi Decoction against cholestatic liver fibrosis

Wen Xu et al. Sci Rep. .

Abstract

Numb is a negative regulator of Notch signal pathway. Previous study has demonstrated that Notch signal pathway activation is required for hepatic progenitor cell (HPC) differentiating into cholangiocytes in cholestatic liver fibrosis (CLF), and Huang Qi Decoction (HQD) could prevent CLF through inhibition of the Notch signal pathway. However, the role of Numb in HQD against CLF is yet unclear. Thus, CLF rats transplanted into rat bone marrow-derived mesenchymal stem cells with knocked down Numb gene (BMSCNumb-KD) were treated with HQD. Simultaneously, Numb gene knockdown was also performed in WB-F344 cell line and then treated with refined HQD in vitro. In vivo study revealed that liver fibrosis was inhibited by HQD plus BMSCNumb-KD treatment, while Hyp content in liver tissue, the gene and protein expression of α-SMA, gene expression of Col I, TNF-α, and TGF-β1 were increased compared to that in HQD group. Furthermore, Notch signal pathway was inhibited by HQD plus BMSCNumb-KD, while the protein expression of Numb was decreased and RBP-Jκ and Hes1 was increased compared to that in HQD group. In vitro, HQD reduced the differentiation of WB-F344 cells into cholangiocyte phenotype, while this effect was attenuated after Numb-knockdown. This study highlights that the absence of hepatic stem cell Numb gene decreases effect of HQD against CLF, which give rise the conclusion that Numb might be a potential target for HQD against CLF.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Lentivirus-mediated knockdown of the BMSC Numb gene. (a) Flow cytometry analysis of CD10, CD14, CD29, CD34, CD45, and CD90. (b) Flow cytometry analysis of BMSC proliferative capacity. (c) Osteogenic induction (×200). (d) Adipogenic induction (×200). (e) Lentivirus transfected BMSCs. (f) The relative Numb mRNA level determined by qRT-PCR, mRNA level were normalized by GAPDH (n = 3 per group). (g) Immunobloting for Numb, and (h) The gray-level score indicates the histogram of immunoblotting for Numb (n = 3 per group). *P < 0.05, **P < 0.01. BMSC, bone marrow-derived mesenchymal stem cell group; NC, empty virusgroup; Numb-KD, Numb knockdown group.
Figure 2
Figure 2
BMSCNumb-KD transplantation reduces the anti-fibrotic effect of HQD. (a) H&E staining (×100). (b) Sirius Red staining (×100). (c) α-SMA immunostaining (×200). (d) Positive area percentage of Sirius Red staining (n = 5 per group). (e) Hyp content in liver tissue (n = 5 per group). (f) Immunoblotting for α-SMA, and (g) The gray-level score indicates the histogram of immunoblotting for α-SMA (n = 5 per group). (h) Relative mRNA levels of α-SMA, ColI, ColIV, TGF-β1 and TNF-α measured by qRT-PCR, mRNA levels were normalized by GAPDH (n = 5 per group). *P < 0.05, **P < 0.01. Sham, sham group; BDL, bile duct ligation group; HQD, Huang Qi decoction group; DAPT, DAPT group; BMSCNC, BMSC negative control group; BMSCNumb-KD, BMSC Numb knockdown group; and HQD + BMSCNumb-KD, HQD plus BMSCNumb-KD group.
Figure 3
Figure 3
BMSCNumb-KD transplantation reduces the inhibitory effect of HQD on bile duct proliferation. (a) Immunostaining of CK7 (×200). (b) Immunostaining of CK19 (×200). (c) Immunostaining of HNF4α (×200). (d) Positive area percentage of CK7 and CK19 immunostaining (n = 5 per group). (e) Immunoblotting of CK7 and CK19, and (f) The gray-level score indicates the histogram of immunoblotting for CK7 and CK19 (n = 5 per group). (g) Relative CK19, CK7 and HNF4α expression levels measured by qRT-PCR, mRNA levels were normalized by GAPDH (n = 5 per group). (h) Double immune-staining of CK7 (red) and EGFP (green) (×400). (i) Double immune-staining of CK19 (red) and EGFP (green) (×400). *P < 0.05, **P < 0.01. Sham, sham group; BDL, bile duct ligation group; HQD, Huang Qi decoction group; DAPT, DAPT group; BMSCNC, BMSC negative control group; BMSCNumb-KD, BMSC Numb knockdown group; and HQD + BMSCNumb-KD, HQD plus BMSCNumb-KD group.
Figure 4
Figure 4
BMSCNumb-KD transplantation reduces the inhibitory effect of HQD on Notch signaling activation. (a) Immunostaining of Numb (×200). (b) Immunostaining of RBP-Jκ (×200). (c) Immunostaining of Hes1 (×200). (d) Immunoblotting for Numb, RBP-Jκ and Hes1, and (e) The gray-level score indicates the histogram of immunoblotting for Numb, RBP-Jκ and Hes1 (n = 5 per group). (f) Relative mRNA levels of Numb, RBP-Jκ and Hes1. (g) Relative mRNA levels of Notch-1, -2, -3 and -4. (h) Relative mRNA levels of DLL1, DLL4, Jag1 and Jag2. (i) Relative mRNA levels of LNX1, LNX2 and ITCH. All mRNA levels were normalized by GAPDH (n = 5 per group). *P < 0.05, **P < 0.01.Sham, sham group; BDL, bile duct ligation group; HQD, Huang Qi decoction group; DAPT, DAPT group; BMSCNC, BMSC negative control group; BMSCNumb-KD, BMSC Numb knockdown group; and HQD + BMSCNumb-KD, HQD plus BMSCNumb-KD group.
Figure 5
Figure 5
Numb knockdown reduces the inhibitory effect of HQD on WB-F344 differentiation into cholangiocyte phenotype. (a) Lentivirus transfected WB-F344. (b) Relative Numb level measured by qRT-PCR. (c) Relative CK19 level measured by qRT-PCR. All mRNA levels were normalized by GAPDH (n = 3 per group). (d) Immunostaining of CK19 (×600). *P < 0.05, **P < 0.01. N, normal group; SB, sodium butyrate group; HQD, SB plus Huang Qi Decoction group; DAPT, SB with DAPT group; NC, empty virus with SB group; Numb-KD, Numb knockdown with SB group; and HQD + Numb-KD, Numb knockdown and SB with Huang Qi Decoction group.
Figure 6
Figure 6
Numb knockdown reduces the inhibitory effect of HQD on Notch signal pathway activation in WB-F344. (a) Immunostaining of Numb (×600). (b) Immunostaining of RBP-Jκ (×600). (c) Immunostaining of Hes1 (×600). (d) Immunoblotting for Numb, RBP-Jκ and Hes1, and (e) The gray-level score indicates the histogram of immunoblotting for Numb, RBP-Jκ and Hes1 (n = 3 per group). (f) Relative levels of Numb, RBP-Jκ and Hes1 measured by qRT-PCR, and mRNA levels were normalized by GAPDH (n = 3 per group). *P < 0.05, **P < 0.01. N, normal group; SB, sodium butyrate group; HQD, SB plus Huang Qi decoction group; DAPT, SB with DAPT group; NC, empty virus with SB group; Numb-KD, Numb knockdown with SB group; and HQD + Numb-KD, Numb knock down and SB with Huang Qi Decoction group.
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