Effect of adenosine A2A receptor agonist combined with bone marrow mesenchymal stem cells transplantation on the negative immune regulation in mice with acute liver failure

Abstract

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 (1×106) 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. Key words: Mesenchymal stem cells; Liver failure, acute; Receptor, adenosine A2A; TLR4/ NF-κB signal pathway