Effects and potential mechanism of bone marrow-derived stem cells on hepatic stellate cells in two contact coculture system

Abstract

Objective To investigate the effects of bone marrow-derived stem cells (BMMSC) on the expressions of inflammatory cytokines and Toll-like receptor (TLR) 4 pathway in primary hepatic stellate cells (HSC) with direct and indirect contact coculture system. Methods Purified HSC were separately treated with LPS in the concentrations of 0, 50, 100 and 150 g/L for 48 h. Proliferation ratio was tested with cell counting kit-8 to determine the optimal concentration. HSC in LPS were divided into three groups, including HSC alone group, cocultured with BMMSC at 1∶1 group and cocultured with transwell group at the optimal concentration. The supernatants were collected to detect the concentrations of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α. Cells were further divided into seven groups, including BMMSC without LPS group, HSC without LPS group, BMMSC with LPS group, HSC with LPS group, BMMSC in transwell system group, HSC in transwell system group, all cells in transwell system group and direct contact system group. The mRNA expressions of α-smooth muscle actin (α-SMA) and collagen I were detected by quantitative real-time PCR, and protein expressions of TLR4, myeloid differentiation factor 88 (MyD88) and nuclear factor-κB (NF-κB) were analyzed by Western blot. Data were analyzed with one-way ANOVA analysis. Results The proliferation rate of HSC in 50, 100 and 150 g/L LPS were (129.77±11.26)%, (162.90±13.15)% and (55.12±11.6)%, respectively compared with HSC without LPS group. The differences were statistically significant (t=5.91, 10.70 and 8.65, respectively, all P<0.05). The concentrations of IL-6 and TNF-α in HSC alone group, directly/indirectly cocultured group were (252.02±30.94) ng/L and (148.00±10.27) ng/L, (88.52±6.61) g/L and (72.63±5.54) ng/L, (103.74±7.14) ng/L and (81.79±6.92) ng/L, respectively. The differences between HSC alone group and directly/indirectly cocultured group were significant (t=12.66 and 15.82, 11.81 and 12.34, respectively, all P<0.05). The directly and indirectly cocultured groups were significantly different (t=3.83 and 2.53, respectively, both P<0.05). The mRNA expressions of α-SMA and collagen I in HSC with LPS were remarkably increased compared with HSC without LPS (t=14.16 and 11.84, respectively, both P<0.05) and reversed by cocultured with BMMSC (t=11.98 and 4.47, respectively, P<0.05). All cells in transwell group expressed more α-SMA and collagen I than the direct contact group (t=3.70 and 3.19, respectively, P<0.05). The TLR pathway associated protein expressions, TLR4, MyD88, and NF-κB in HSC in transwell group were significantly down-regulated compared with HSC with LPS group. And all cells in transwell system had higher level of protein expressions compared with direct contact system (P<0.05). Conclusions BMMSC are effective in inhibiting HSC activation and inflammatory cytokines excretion, which may be modulated through TLR4 pathway and cell to cell contact. Key words: Mesenchymal stem cells; Hepatic stellate cells; Liver cirrhosis; Toll-like receptors; Co-culture system