Regulation of PMA-induced MUC5AC expression by heparin in human bronchial epithelial cells.

Mucus hypersecretion is a major pathophysiologic feature in chronic inflammatory airway diseases. Oxidative stress plays a pivotal role in this process. Recent studies have found that heparin has antioxidant effects which can reduce free radical damage. Here, we hypothesized that heparin has some influence on the expression of mucin 5AC (MUC5AC) induced by phorbol myristate acetate (PMA) in a bronchial epithelial cell line (HBE16), also we have investigated the potential mechanism involved in the process. We found that ROS, the mRNA of Duox1, EGFR and MUC5AC, as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC in the PMA group were significantly increased when compared with the control group (all P < 0.01). After pretreatment with heparin however, there was a significant decrease in ROS levels, the mRNA of Duox1, EGFR, and MUC5AC, and the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with the PMA group (all P < 0.01). MUC5AC protein in the supernatant was inhibited in a dose-dependent manner by heparin. Pretreatment with DMTU resulted in a significant decrease in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC when compared with the PMA group (all P < 0.01). When cells were pretreated with both heparin and DMTU, there was a further reduction in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with either the PMA group, heparin group, or DMTU group (all P < 0.01). Our results show that PMA can induce MUC5AC expression by activation of the Duox1-ROS-TACE-TGF-α-EGFR signaling pathway. Heparin can decrease the level of Duox1, ROS production and block the PMA-induced activation of EGFR, thus inhibiting the overexpression of mucin MUC5AC in a dose-dependent manner. In addition to reducing ROS production, heparin may also inhibit the expression of MUC5AC through other signal mechanisms.

Study on the interactions of kaempferol and quercetin with intravenous immunoglobulin by fluorescence quenching, fourier transformation infrared spectroscopy and circular dichroism spectroscopy.

The interactions of kaempferol and quercetin with intravenous immunoglobulin (IVIG) were studied in vitro by spectroscopic methods including fluorescence spectra, Fourier transformation infrared (FT-IR) spectra and circular dichroism (CD) spectra. The binding parameters for the reactions calculated according to the Sips equation suggested that the bindings of IVIG to kaempferol and quercetin were characterized by two binding sites with the average affinity constants K(o) at 1.032 x 10(4) M(-1) and 1.849 x 10(4) M(-1), respectively. The binding of IVIG with quercetin is stronger than that of IVIG with kaempferol. They were of non-specific and weak drug-protein interactions. Docking was used to calculate the interaction modes between kaempferol and quercetin with IVIG. The secondary structural compositions of free IVIG and its kaempferol, quercetin complexes were calculated by the FT-IR difference spectra, self-deconvolution, second derivative resolution enhancement and the curve-fitting procedures of amide I band respectively, which are in good agreement with the analyses of CD spectra. The effect of 3'-OH substituent in quercetin is distinct between the interactions of IVIG with kaempferol and quercetin for the secondary structure of the protein. The observed spectral changes indicate a partial unfolding of the protein structure, but the typical beta structural conformation of IVIG is still retentive in the presence of both drugs in aqueous solution. The average binding distances between the chromophores of IVIG with kaempferol (4.30 nm) and quercetin (4.35 nm) were obtained on the basis of the theory of Förster energy transfer. IVIG can serve as transport protein (carrier) for kaempferol and quercetin.