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Objective To investigate the effects of curcumin derivatives (C66) on proliferation and expressions of α-smooth muscle (α SMA) and Collagen Ⅰ in rat hepatic stellate cells (HSC) induced by transforming growth factor-β (TGF-β) in vitro and the relationship with cannabinoid receptor type 1 (CB1).Methods To determine the optimum time and concentration of C66,HSC-T6 cell line was cultured in vitro and divided into control group and groups with different doses of C66 (1 μmol/L,2 μmol/L,5 μmol/L,10 μmol/L,20 μmol/L).Cell proliferation was detected by Cell Counting Kit-8 assay.Then,according to the time and concentration of C66 above,cells were divided into 5 groups including control group,TGF-β only group,TGF-β combined with CB1 antagonist group,TGF-β combined with C66 group and TGF β combined with CB1 antagonist plus C66 group.Quantitative realtime polymerase chain reaction and western blot were used to assess the expressions of α SMA,Collagen Ⅰ,CB1,JNK and phosphorylation of JNK (p-JNK).The variance homogeneity of multiple samples was compared by LSD method.The variance was compared with Dunnett T3 test.One-way analysis of variance was performed to compare the mean values among the groups.Results The inhibitory effect of C66 on HSC-T6 proliferation was dose and time dependent.The optimum time and concentration were 48h and 10 μmol/L,respectively,with the inhibition rate of 54％.Compared with control group,expressions of α-SMA,collagen Ⅰ and CB1 were significantly elevated in TGF-β group (t=6.188,3.48 and 20.64,respectively,all P＜0.05).TGF-β1 could increase the relative mRNA expressions of CB1,collagen Ⅰ and α-SMA with significant differences (t =4.705,9.492 and 38.27,respectively,all P＜ 0.05).Compared with control group,p-JNK expression was significantly elevated in TGF-β group (t=9.567,P＜0.05).Conclusions C66 could inhibit the proliferation and collagen synthesis in HSC-T6 induced by TGF-β and the effect is strengthened when combined with CB1 antagonist,which may involve JNK phosphorylation.Our study provides a better understanding on the mechanism and a new target for treatment of liver fibrosis.
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Objective To investigate the therapeutic effect and possible mechanism of adenosine A2A receptor agonist (CGS21680) combined with bone marrow mesenchymal stem cells (BMMSC) transplantation in acute liver failure (ALF).Methods Fifty male C57BL/6 mice, 6-8 weeks old, were fed with standard diet for 1 week and randomly divided into 5 groups according to random number table: healthy control group (n=6), model group (n=11), BMMSC group (n=11), CGS21680/BMMSC group (n=11) and CGS21680 group (n=11).Except healthy control group, the other mice were injected with D-GalN and lipopolysaccharide (LPS) to establish ALF model.Ten hours later, CGS21680/BMMSC group and CGS21680 group were injected intraperitoneally with adenosine A2A receptor agonist CGS21680 (2.1 mg/kg).In addition, the BMMSC group and CGS21680/BMMSC group were injected BMMSC (1×10.6) through tail vein.After 24 hours, pathological changes of liver tissue was observed by hematoxylin and eosin staining.The change of proportion of mouse splenic Treg among CD4+T lymphocytes was detected by flow cytometry.Toll-like receptor (TLR)4 and nuclear factor (NF)-κB expression levels in liver tissue were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blot.One-way analysis of variance (one-way ANOVA) and SNK-q test was conducted for data analysis.Results Serum IL-6 levels were (23.67±2.97) pg/mL in healthy control group, (151.47±6.03) pg/mL in model control group, (72.10±3.74) pg/mL in BMMSC group, (53.35±2.50) pg/mL in CGS21680/BMMSC group and (84.85±3.25) pg/mL in CGS21680 group.The differences between healthy control group and the other 4 groups were all statistically significant (t=46.02, 25.51, 19.58 and 34.03, respectively, all P<0.01).Serum TNF-ɑ levels were (24.62±3.19) pg/mL in healthy control group, (102.25±2.10) pg/mL in model control group, (54.71±2.23) pg/mL in BMMSC group, (42.20±4.72) pg/mL in CGS21680/BMMSC group and (81.76±3.50) pg/mL in CGS21680 group.The differences between healthy control group and the other 4 groups were all statistically significant (t=46.49, 19.97, 7.72 and 29.57, respectively, all P<0.01).The differences of spleen Treg proportion in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=51.67, 12.22, 5.91 and 18.21, respectively, all P<0.01).The differences of TLR4 mRNA levels of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=26.31, 21.33, 13.24 and 27.14, respectively, all P<0.05).The differences of NF-κB mRNA level of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=16.56, 16.34, 7.83 and 13.11, respectively, all P<0.05).The differences of TLR4 protein level in liver tissue of healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=35.60, 10.38, 6.05 and 18.02, respectively, all P<0.05).The differences of liver NF-κB protein level in the healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=10.80, 7.30, 4.61 and 13.24, respectively, all P<0.05).Conclusions Adenosine A2A receptor agonist combined with BMMSC can significantly up-regulate the proportion of Treg cells in acute liver failure mice and inhibit the TLR4/NF-κB pathway activation, with both coordinated regulation, and further inhibit the liver inflammation.
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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 ofα-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 150μg/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-αin 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 moreα-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.