Study On Pharmacological Activity And Mechanism Of ADMC

Objective To find out a kind of coumarin compound that has strong pharmacological activity, as well as the immunosuppressive and anti-inflammatory mechanism of ADMC, we screened immunosuppression, anti-inflammatory and hypoglycemic activity of a series of coumarins. So as to confirm the action target and the relationship between immunosuppression and anti-inflammatory function of ADMC. Furthermore, to explore else pharmacological activity of ADMC, we research the anti-cancer activity of ADMC, providing theoretical basis on new drug exploitation.Methods Firstly, T or B lymphocyte proliferation induced by Con A or LPS tests were used in the present study to screen immunosuppressive effect of 12 coumarin compounds. The IC50 values were calculated according to inhibition rates of gradient concentration of different drugs. Anti-inflammatory property of 12 compounds was determined by detecting NO emission of RAW264.7 stimulated by LPS. The Hep G2 cells glucose consumption models were established to assess the hypoglycemic activity of these drugs. Compared between 12 compounds, ADMC exhibited potent and extensively pharmacological activity. Secondly, to further research the mechanism of immunosuppression and anti-inflammation of ADMC, T or B lymphocyte induced by Con A or LPS, myeloid-derived DC, RAW264.7, a murine macrophage cell line, THP-1, a human monocytic leukemia cell line, were used in the research. T/B cell cycle, apoptosis and phenotype of B cells and DC were analyzed by fluorescence-activated cell sorting(FACS). Cytokines in supernatant of RAW264.7 were assayed by kits. Simultaneously, the expression of TLR4 and i NOS were detected by western blot. Quantify real-time PCR was used for the determination of TLR4, My D88, TRIF, i NOS. Monocyte-to-macrophage differentiation induced PMA of THP-1 was observed bymicroscope. PMA-induced increase in auto-fluorescence, a distinguished feature of macrophage differentiation because of the increased cell size were determined by FACS. Than a LPS/D-galactosamine-(D-Gal N-) induced acute hepatitis mouse model were employed to investigate the efficacy of ADMC in vivo. ALT and AST in serum were detected and hepatic pathology were observed by HE to confirm the effect of ADMC on liver function. And inflammatory cytokines were detected by kits. The effect on T cells, B cells, DC, CD8+T cells were tested by MTT, FACS and immunohistochemical staining. The protein and transcript levels of TLR4 in liver were detected by Western Blot and Real-time PCR, respectively. The expression of JAK2-STAT3 in spleen were detected by western blot. In addition, to verify the efficacy of ADMC again, the acute hepatitis mouse model caused by Con A treatment was establish. Finally, Cell viability of Hela, Hep G2, ASPC-1, BXPC-3 and Mia Pa Ca-2 were assayed by MTT method, calculating IC50 values of ADMC. Besides, the subcutaneous human Hela cancer cell xenografts in nude mouse model was used to assess the anti-tumor liver metastasis of ADMC, detecting survival rate and weight change of nude mice and their liver function, ALT, AST, and the HE results of liver tssues.Results T/ B lymphocyte proliferation was suppressed by inducing cell apoptosis after ADMC treatment. Compared with positive drugs, ADMC exhibits less cytotoxicity on splenic lymphocyte. Inflammatory factor NO and TNF-? secreted by LPS-induced RAW264.7 were inhibited remarkably by ADMC, as well as the m RNA and protein level of TLR4 and i NOS. The PMA-induced monocyte-to-macrophage differentiation in THP-1 cells was not been inhibited by ADMC. Meanwhile, the glucose uptake of Hep G2 cells was increased by ADMC with and without insulin. Notably, ADMC played a great role in protecting autoimmune acute liver injury. The serum level of ALT, AST, as well as the inflammatory infiltration of liver tissue were decreased significantly by ADMC. Effect of ADMC on immune response was suppressed by T/B cells proliferation inhibition, secretory NO and TNF-? reduction, CD8+T cells infiltratinginto liver tissue decrease as well as DC migration and maturity inhibition. The protein expression level of TLR4 were obviously down-regulated in liver and spleen, also the JAK2 and STAT3 in spleen. Furthermore, the efficacy of ADMC was affirmed in Con Ainduced autoimmune acute liver injury model. Ultimately, the cell viability of Hela, Hep G2, ASPC-1, BXPC-3 and Mia Pa Ca-2 were inhibited dramatically by ADMC. And in the subcutaneous human Hela cancer cell xenografts in nude mouse model, ADMC decreased tumor shifting stoves, improved the nude mice weight, enhanced the survival rate and recover the liver function.Conclusion These results suggest that ADMC displays extensive pharmacological activity, including immunosuppression, anti-inflammation, hypoglycemic and antitumor effect. The mechanism of ADMC may correlate with the induction of T/B cells apoptosis, down-regulation of TLR4 and i NOS and up-regulation of GLUT4. ADMC has the potential to protect the autoimmune acute liver injury model, the mechanism of which is probably related to inhibition maturity and function of DC, T/B cells proliferation, the infiltration of CD8+T cells and secretion of inflammatory factor. Above all, TLR4 and JAK2-STAT3 might act as the target sites of ADMC to exert immunosuppression and anti-inflammation.Innovative points 1. This experiment for the first time researches the pharmacological activity and pharmacokinetic characteristics of compound ADMC. 2. This paper first clarify the influences of ADMC in vivo on the function of related the immune cells.