| Hepatic stellate cells(HSCs) play a critical role in the development and maintenance of liver fibrosis, which is well characterized by abnormal accumulation of extracellular matrix(ECM). More and more researches have focus on the interaction between macrophages and HSCs during liver fibrosis. In the process of liver fibrosis, kupffer cells as the resident macrophages have been implicated as key mediators of liver fibrosis through production of various cytokines, such as tumor necrosis factor-?(TNF-?), interleukin-1?(IL-1?) and platelet-derived growth factor-BB(PDGF-BB), which can promote the activation and proliferation of HSCs. And also activated HSCs can secrete CC chemokine ligan 2(CCL2) to promote the blood mononuclear macrophages to recruit to liver, which expands the inflammatory response. So the impact of macrophages on HSCs is very important to investigate the mechanism of liver fibrosis.Tumor necrosis factor-like ligand 1 aberrance(TL1A) that mainly expresses in the endothelial cells, lymphocytes, plasma cells, mononuclear cells and dendritic cells is the protein production of the tumor necrosis factor superfamily member 15(TNFSF15) gene. Our previous studies have demonstrated that TL1A promoted the intestinal fibrosis of chronic experimental colitis mice. So far, the research on TL1A in liver diseases is relatively rare. One research had shown that the expression of TL1A, which was from the serum and macrophages in hepatic tissue, was higher in patients with primary biliary cirrhosis and serum level reduced after ursodesoxycholic acid treatment. TL1A, as the only ligand of TNF binding to death receptors 3(DR3), can promote the activation of macrophages. A lot of studies in vitro had demonstrated that macrophages promoted the activation and proliferation of HSCs, but there is no research about the effects of macrophages with high expression of TL1A on the activation and proliferation of HSCs. Therefore, this study aims to explore the effects of macrophages with high expression of TL1A in vitro on the activation and proliferation of HSCs that may provide new target for the treatment of liver fibrosis.Objective: To explore the effects of macrophages with high expression of TL1A in vitro on the activation and proliferation of HSCs.Methods:(1) FMS-TL1A-GFP-tansgenic mice were identified by RT real-time PCR.(2) Bone marrow-derived macrophages(BMMs) and peritoneal macrophages(PMs) were extracted, differentiated and activated from wild type(WT) and transgenic(Tg) C57BL/6 mice; the fresh WT murine HSCs were isolated with in-situ recirculating perfusion technology and purified by density gradient centrifugation.(3) BMMs and PMs were divided into Control/WT group, LPS+IFN-?/WT group, Control/Tg group, LPS+IFN-?/Tg group, respectively, and the supernatant of macrophages that intervened by LPS and IFN-? as conditional medium(CM) to intervene HSCs were collected. HSCs were grouped as follows: Control group, HSCs with DMEM containing 10% fetal bovine serum(FBS); LPS+IFN-?+M-CSF group, HSCs with DMEM containing 10% FBS and joined LPS, IFN-? and M-CSF(CM from PMs cultivated HSCs without M-SCF); CM WT group, HSCs with CM from WT macrophages containing 10% FBS; CM Tg group: HSCs with CM from Tg macrophages containing 10% FBS.(4) The fresh HSCs were observed by fluorescence microscope; The co-expression of F4/80 and TL1A in macrophages, desmin and ?-smooth muscle actin in HSCs were detected by immunofluorescence; the optimal concentration of promoting the proliferation HSCs was examined by CCK-8; CCK-8 and BrdU were used to detect the proliferation of HSCs; the expression of TL1A mRNA and DR3 mRNA in macrophages and the expression of ?-SMA mRNA at day 2, day 4 and day6 in HSCs were detected by real-time Q-PCR. The expression of TL1A protein and DR3 protein in macrophages was analyzed by Western blot; the concentration of IL-1? and PDGF-BB in supernatant from macrophages was detected by enzyme-linked immune sorbent assay(ELISA).Results:(1) The FMS-TL1A-GFP-transgenic mice were identified by PCR. The TL1A DNA was located at 191 bp in Tg mice, while there is no band at 191 bp in WT mice.(2) BMMs and PMs were successfully isolated, and 90×106~110×106, 10×106~12×106cells were obtained from each mouse, respectively. The survival rate was higher than 95%.(3) Immunofluorescence results showed that the co-expression of F4/80 on the macrophages membrane and TL1A in Tg group was significantly higher than in WT group(BMMs: 0.029±0.002 vs 0.011±0.002, P<0.01; PMs: 0.025±0.002 vs 0.010±0.002, P<0.01), indicating that macrophages with high expression of TL1A were extracted successfully. For BMMs, real-time Q-PCR were used to detected TL1A mRNA and the results showed that the TL1A mRNA of macrophages that stimulated by LPS and IFN-? was more notable than the corresponding control groups, and a significantly higher level in LPS+IFN-?/Tg group than LPS+IFN-?/WT group(P<0.01); TL1A expression was detected by western blot and the results showed that there is no different of TL1A expression between LPS+IFN-?/WT group and Control group, the TL1A expression in macrophages from Tg mice was more than macrophages from WT mice(P<0.01), and a significantly higher level in LPS+IFN-?/Tg group than LPS+IFN-?/WT group(P<0.01); the similar tendency was also found in PMs.(4) The expression level of DR3 protein and mRNA showed no obvious changes in both types of cell(P>0.05).(5) The fresh HSCs were successfully isolated with in-situ recirculating perfusion technology, and purified with Percoll by density gradient centrifugation, and 0.8×106~1.0×106 cells were obtained from each mouse. The survival rate and the purity were higher than 90%. The fresh isolated HSCs were round and rich in fat droplet under inverted microscope. The Spontaneous blue fluorescence of fresh isolated HSCs was also detected using fluorescence microscope and observed by fluorescence microscope under 328 nm and desmin was detected at the first day.(6) The proliferation of HSCs was detected by CCK-8 and show a significantly higher expression level which cultured with 60% CM for 24 hours.(7) The results of CCK-8 showed that the proliferation rate of HSCs cultured from BMMs were significantly higher than Control group(100.82%±8.48%) and LPS+IFN-?+M-CSF group(100.54%±8.11%), and more notable in CM Tg group(246.21%±8.34%) than CM WT group(203.24%±10.50%), P<0.01; the similar tendency was also found in HSCs cultured with CM from PMs.(8) The results of Brd U showed that CM from BMMs were able to promote the DNA synthesis of HSCs, and more higher in CM Tg group(182.24%±8.68%) than CM WT group(150.75%±9.12%), P<0.01; the similar tendency was also found in experiments of HSCs cultured with CM from PMs.(9) Immunofluorescence were used to detect ?-SMA in HSCs and the results showed as follows: for HSCs cultured with CM from BMMs group, there was no differernt of the expression of ?-SMA protein in HSCs at 2nd day and 6th day(P>0.05); at the 4th day, there was no significant difference between LPS+IFN-?+M-CSF group and Control group, however, the expression of ?-SMA protein in HSCs intervened by CM showed a significantly higher level than Control group and LPS+IFN-?+M-CSF group, and significantly increased in CM Tg group than CM WT group(P<0.01); the similar tendency was also found in HSCs cultured with CM from PMs. For HSCs cultured with CM from BMMs group, the expression of ?-SMA mRNA in HSCs at 2nd day showed no significant difference(P>0.05); at the 4th day and 6th day, there was no significant difference between Control group and LPS+IFN-?+M-CSF group, however, the expression of ?-SMA mRNA in HSCs intervened by CM showed a significantly higher level than Control group and LPS+IFN-?+M-CSF group, and significantly increased in CM Tg group than CM WT group(P<0.05); the similar tendency was also found in experiments of HSCs cultured with CM from PMs.(10) The results of ELISA showed that the concentration of IL-1? and PDGF-BB from supernatant of BMMs were significant higher in macrophages that were stimulated by LPS and IFN-? than corresponding control groups(P<0.01), and more notable in LPS+IFN-?/Tg group(246.21±8.34) than LPS+IFN-?/WT group(203.24±10.50), P<0.01; except the concentration of PDGF-BB from supernatant of PMs was significant higher in LPS+IFN-?/Tg group than Control group(P<0.05), the other results were consistent with supernatant of BMMs.Conclusions: The macrophages with high expression of TL1A could accelerate the activation and proliferation of HSCs, which may be related to the higher level of IL-1? and PDGF-BB. |
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