ABSTRACT
ObjectiveTo observe the effects of Fuzitang （FZT） on the proliferation of MH7A cells， the human rheumatoid arthritis synovial fibroblasts， and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group， high- （25 g·L-1） and low-dose （12.5 g·L-1） FZT groups， and a positive drug group （hydroxychloroquine， 0.006 25 g·L-1）. The cell proliferation was detected by cell counting kit-8（CCK-8） method， and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes， including SH2 domain-containing inositol 5-phosphatase-1（SHIP-1）， protein kinase B 3（Akt3）， and mammalian target of rapamycin（mTOR）， was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of phosphatidylinositol 3-kinase （PI3K）， Akt3， and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group， the FZT groups showed increased proportions of cells in the G2/M phase （P<0.05）， and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase （P<0.05）. The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group， the FZT groups showed down-regulated miR-155 and mTOR mRNA expression （P<0.05）， and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression （P<0.05）. Compared with the blank group， the FZT groups showed reduced protein expression of PI3K， Akt3， and mTOR （P<0.05）. ConclusionFZT can significantly inhibit the proliferation of MH7A cells， and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.
ABSTRACT
Objective To screen the non-nucleoside compounds against HIV-1 reverse transcriptase by molecular modeling and bioactivity assay. Methods Surflex-Dock module of Tripos SYBYL software was used to simulate the binding pattern of 22 000 compounds in SPECS database with the active pocket of HIV-1 reverse transcriptase. Based on the simulation results, the interaction mode between the above compounds and the crystal structure of HIV-1 reverse transcriptase was analyzed. The compounds with higher docking scores and better binding pattern were determined by anti-HIV-1 ac tivities test in vitro. Results The virtual screening results showed that the docking conformation of 1- (4-fluorophenyl) -3- [2- (1H-indol-3-yl) ethyl] thiourea was similar to the embedded ligand in Rilpivirine crystal structure. 1- ( 4-fluorophenyl) -3- [ 2- ( 1H-indol-3-yl) ethyl] thiourea was held together with the key residue Lys101 in docking pocket of HIV-1 reverse transcriptase by hydrogen bonds, and hadπ-πstacking action together with the conservative residue Trp229 and the aromatic residue Tyr181 respectively. The bioassay in vitro results showed that when the proliferation rate of C8166 lymphocyte syncytium infected by HIV-1ⅢB arrived 50% ( EC50) , the concentration of 1- ( 4-fluorophenyl) -3- [ 2- ( 1H-indol-3-yl) ethyl] thiourea was 5.45μg/mL. Conclusion Molecule docking technology is an effective approach to reducing the screening of candidate compounds with micromolecular activity, and can be used to predict the interaction mode between the compound and the target receptor. In the study, active compound 1- (4-fluorophenyl) -3- [2- (1H-indol-3-yl) ethyl] thiourea has been screened out by molecule docking technology.