RESUMEN
Toll-like receptor 2 (TLR2) mediated macrophages regulate the protective immune response to infectious microorganisms, but the aberrant activation of macrophages often leads to pathological inflammation, including tissue damage. In this study, we identified antagonists of TLR2 by screening 2100 natural products and subsequently identified Taspine, an aporphine alkaloid, as an excellent candidate. Furthermore, analysis of the 10 steps chemical synthesis route and structural optimization yielded the Taspine derivative SMU-Y6, which has higher activity, better solubility, and improved drug-feasible property. Mechanistic studies and seq-RNA analysis revealed that SMU-Y6 inhibited TLR2 over other TLRs, hindered the formation of TLR2/MyD88 complex, and blocked the downstream NF-κB and MAPK signaling pathway, thus suppressing the release of inflammatory cytokines. SMU-Y6 could stabilize TLR2 and bind to TLR2 protein with a Kd of 0.18 μmol/L. Additionally, SMU-Y6 could efficiently reverse the M1 phenotype macrophage polarization, reduce the production of cytokines as well as infiltration of neutrophiles and alleviate the local inflammation in mice with acute paw edema and colitis. Collectively, we reported the first aporphine alkaloid derivative that selectively inhibits TLR2 with high binding affinity and superior drug-feasible property, thus providing an urgently-needed molecular probe and potential drug candidate for inflammatory and autoimmune disease therapy.
RESUMEN
Objective To analyze inhibitory effect of taspine derivative TasD1-6 on human liver cancer cells of Hep3B, HepG2, and BEL7402, and to study their preliminary mechanism of anticancer action. Methods Effect of TasD1-6 on cell growth of Hep3B, HepG2, and BEL7402 were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and real-time cellular analysis (RTCA) assay. Effect of TasD1-6 on Hep3B cell morphology was investigated with crystal violet staining. Next, Hep3B cell apoptosis was analyzed by Annexin V FITC/PI and Hoechst 33258 staining. Flow cytometry was used to determine Hep3B cell cycle. Effect of TasD1-6 on cell apoptosis protein expression in Hep3B cell was determined by Western blotting. Results MTT and RTCA assay demonstrated that TasD1-6 significantly inhibited the growth of Hep3B cell and the IC50 was (11.3 ± 1.6) μmol/L. Hep3B cell morphology indicated the shrinkage and obvious morphology changes. And TasD1-6 induced the Hep3B cell apoptosis in dose-dependent manner. Cell was stopped as G1 phase by TasD1-6. Western blotting analysis showed TasD1-6 could upregulate the protein expression of p53 and Bax and downregulate Mcl-1 protein expression (P < 0.05). Conclusion Taspine derivative TasD1-6 shows better inhibitory action on the growth of human liver cancer Hep3B cell than HepG2 and BEL7402 cells. At the same time, TasD1-6 can change cell cycle and induce cell apoptosis, which probably related to upregulation of p53 and Bax protein expression and downregulation of Mcl-1 protein expression.
RESUMEN
Objective To study the effect and mechanism of taspine on wound healing and fibroblast proliferation. Methods The effect of taspine on skin wound was observed in vivo. The different concentration of taspine hydrochloride was added to L929 fibroblast cultivated in vitro, and lactate dehydrogenase was detected and MTT method was applied to observe effect of taspine on fibroblast proliferation. Results The local application of taspine 3 mg/Ml and 1.5 mg/mL accelerated the healing of skin wounded. In vitro, 0.01～0.5 μg/mL of taspine hydrochloride showed no effect on the change of lactate dehydrogenase activity and fibroblast proliferation. Conclusion Taspine is a kind of active alkaloid from leontice robustum which can enhance wound healing, its mechanism on wound healing is not by means of accelerating the proliferation of fibroblast, other mechanisms are necessary for being further studied.