Fig. 4

The molecular mechanism of risedronate efficacy in ameliorating the airway obstruction and inflammation response of COPD. A Recombination of KRAS into pEGFP-N1 plasmid to construct the pEGFP-KRAS fusion plasmid and validating it by sanger sequencing. B Transfected the pEGFP-KRAS fusion plasmid into ASMCs cells and the change in green fluorescence after incubation of risedronate for 1 h and 2 h. Scale 40x. C Extraction of the membrane protein and detection of the membrane KRAS in ASMC cells treated with 100 µM risedronate. D The pGFP-RhoA plasmid transfected into ASMCs cells and the change in green fluorescence after incubation of risedronate for 1 h and 2 h. Scale 40×. E The expression of ERK, p-ERK, MLCK, p-MLCK, RhoA, ROCK1, MLCP, MLC20, and p-MLC20 detected by western blot in ASMCs of rats treated with 0, 6.25, 12.5, 25, 50, 100 µM risedronate. The red frame and arrow represented RAS and RhoA-mediated two pathways of ERK-MLCK-pMLC20 and ROCK1-MLCP-MLC20, respectively. F The mRNA level of MLCK and MLCP in ASMCs of rats treated with 0, 6.25, 12.5, 25, 50, and 100 µM risedronate. G The activity of PDE in NR8383 cells treated with different concentrations of risedronate. H Left: Testing the activity of cAMP in NR8383 cells treated with different concentrations of risedronate. Right: cAMP inhibitor, Rp-cAMP could reverse the increase in cAMP-induced by risedronate in NR8383 cells. I: The expression of NF-κB, p-NF-κB, and TNF-α stimulated by 100 ng/ml LPS and its change after accepting the 100 µM risedronate incubation in NR8383 cells. *: p < 0.05, **: p < 0.01; mKRAS*, membrane KRAS; Ris, risedronate