Effect of Cigarette Smoke Extract on the Role of Protein Kinase C in the Proliferation of Passively Sensitized Human Airway Smooth Muscle Cells

摘要

To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A_1 , A_2 and A_3 and the group B was sub-divided into the group of B_1 , B_2 , B_3 , B_4 and B_5. No other agents were added to the group A_1 and B_2 . The cells of group A_2 and B_2 were stimulated with 5 % CSE for 24 h. HASMCs from group A_3 and B_3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5 %) for 24 h. PKC inhibitor Ro-31-8220 (5 μmol/L) was added to the HASMCs of group B_4 for 24 h. The cells from group B_5 were stimulated with Ro-31-8220 (5 μmol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and ~3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of ~3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_1 , B_2 and B_3 were significantly increased compared to those of group A_1 , A_2 and A_3 correspondingly and respectively (P< 0. 01). The proliferation of HASMCs of group A_2 and B_2 stimulated with CSE and group A_3 and B_3 stimulated with CSE and PMA were also significantly enhanced when group A_1, A_2 and A_3 and group B_1 , B_2 and B_3 compared to each other (P<0. 05, P<0. 01, respectively). The percentage of S phase, absorbency (A) value, ~3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_4 treated with Ro-31-8220 and group B_5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B_1 and B_2 correspondingly and respectively (P<0. 05, P<0. 01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.