Dental pulp stem cells overexpressing hepatocyte growth factor facilitate the repair of DSS-induced ulcerative colitis

Abstract

Background: Ulcerative colitis (UC) is a chronic and recurrent disease without satisfactory treatment strategies. Dental pulp stem cell (DPSC) transplantation has been proposed as a potential therapy for UC. This study aimed to investigate the therapeutic effects of the rat hepatocyte growth factor (HGF) gene transduced into DPSCs for UC.

Methods: The therapeutic effects of HGF-DPSCs transplanted intravenously into a rat model of UC induced by 5% dextran sulphate sodium (DSS) were compared with the other treatment groups (LV-HGF group, DPSCs group and GFP-DPSCs group). Immunofluorescence and immunohistochemistry were used to observe the localization and proliferation of HGF-DPSCs at the site of colon injury. The expression levels of inflammatory factors were detected by real-time quantitative PCR (RT-PCR) and western blotting. The oxidative stress markers were detected by ELISA. DAI scores and body weight changes were used to macroscopically evaluate the treatment of rats in each group.

Results: Immunofluorescence and immunohistochemistry assays showed that HGF-DPSCs homed to colon injury sites and colocalized with intestinal stem cell (ISC) markers (Bmi1, Musashi1 and Sox9) and significantly promoted protein expression (Bmi1, Musashi1, Sox9 and PCNA). Anti-inflammatory cytokine (TGF-β and IL-10) expression was the highest in the HGF-DPSCs group compared with the other treatment groups, while the expression of pro-inflammatory cytokines (TNF-α and INF-γ) was the lowest. Additionally, the oxidative stress response results showed that malondialdehyde (MDA) and myeloperoxidase (MPO) expression decreased while superoxide dismutase (SOD) expression increased, especially in the HGF-DPSCs group. The DAI scores showed a downward trend with time in the five treatment groups, whereas body weight increased, and the changes were most prominent in the HGF-DPSCs group.

Conclusions: The study indicated that HGF-DPSCs can alleviate injuries to the intestinal mucosa by transdifferentiating into ISC-like cells, promoting ISC-like cell proliferation, suppressing inflammatory responses and reducing oxidative stress damage, which provides new ideas for the clinical treatment of UC.

Keywords: Dental pulp stem cells; Hepatocyte growth factor; Ulcerative colitis.

Fig. 1

DSS-induced UC model in rats. a Rats had bloody stools after 5% DSS was administered for 14 days. b, c The colon and rectum lengths of the control and UC groups were compared, and changes after Evans blue staining were observed. d Comparison of HE staining between the control and UC groups (n = 5, scale bars = 50 μm). e Quantitative analysis of the colon and rectum length showed that the UC group was significantly shorter than the control group. Data are shown as the means ± SD (n = 5, ***p < 0.001). f Comparison of body weight changes between the control and UC group rats. Data are shown as the means ± SD (n = 5, **p < 0.01). g DAI scores were obtained by monitoring the body weight changes, stool consistency and bloody stool extent from the rats in the control and UC groups. Data are shown as the means ± SD (n = 5, **p < 0.01)

Fig. 2

Virus-transduced DPSCs exhibited mesenchymal stem cell antigenic markers. a–c Morphology of the DPSCs. d–e Osteogenic and adipogenic differentiation of DPSCs. f Construction of the lentiviral vector to overexpress the rat HGF gene. g Relationship between the transduction efficiency and MOI of the virus. h HGF-DPSCs expressed green fluorescence under a microscope. Scale bar = 50 μm in all panels. i Western blotting analysis of DPSCs overexpressing HGF. j Specific antigenic markers of DPSCs were detected by flow cytometry (n = 3)

Fig. 3

Transplanted DPSCs homed to injured colons and transdifferentiated into intestinal stem cell-like cells. a, b GFP-DPSCs and HGF-DPSCs expressing green fluorescence were colocalized with Bmi1, Musashi1, Sox9 and PCNA (n = 5). c Few positive cells were found in other organs of the rats (liver, spleen, kidney and lung tissues, n = 5). Scale bar = 50 μm in all panels. d Statistical comparison of the percentages of double-stained (GFP/DAPI) cells between the GFP-DPSCs and HGF-DPSCs groups. Data are shown as the means ± SD (n = 5; ***p < 0.001). e, f The comparison of Pearson’s correlation and the overlap coefficient of colon sections costained with Bmi1, Musashi1, Sox9 and PCNA between the GFP-DPSCs and HGF-DPSCs groups (n = 5; p > 0.05)

Fig. 4

Transplanted DPSCs promoted ISC-like cell proliferation. a Increases in Bmi1-, Musashi1-, Sox9- and PCNA-positive cells were detected by immunohistochemical analysis. Scale bars = 50 μm. b The negative control result was staining without primary antibody. Scale bars = 50 μm. c Statistical comparison of the Bmi1-, Musashi1-, Sox9- and PCNA-positive cells in different groups. Data are shown as the means ± SD (n = 5; ^△△p < 0.01, ^△△△p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001 vs the UC group; ^##p < 0.01, ^###p < 0.001)

Fig. 5

Transplantation of DPSCs promoted injured colon tissue repair and suppressed intestinal inflammatory responses at the mRNA and protein levels. a Transplanted DPSCs promoted the repair of damaged tissues. b The melting curves of TNF-α, IFN-γ, TGF-β and IL-10. c Statistical analysis of TNF-α, IFN-γ, TGF-β and IL-10 mRNA expression in rat colon tissues in different groups using RT-PCR. β-actin served as a reference. Data are shown as the means ± SD (n = 5; ^△△△p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001 vs the UC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001). d Immunoblotting analysis of TNF-α, IFN-γ, TGF-β and IL-10 protein expression in rat colon tissues in different groups. e Quantitative analysis of TNF-α, IFN-γ, TGF-β and IL-10 expression. β-actin served as a reference. Data are shown as the means ± SD (n = 5; ^△△△p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001 vs the UC group; ^##p < 0.01, ^###p < 0.001)

Fig. 6

HGF-DPSCs suppressed oxidative stress responses and ameliorated DSS-induced disease activity. a–c Immunological detection of MPO, MDA and SOD in rat colon tissues from different groups. Data are shown as the means ± SD (n = 5; ^△△△p < 0.001; ***p < 0.001 vs the UC group; ^###p < 0.001). d–f Changes in the DAI and body weights of rats in different groups with prolonged time. Data are shown as the means ± SD (n = 5; ^△△p < 0.01 vs the UC group; **p < 0.01 vs the UC group; ^##p < 0.01 vs the HGF-DPSCs group)