In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-α （PKC-α） in chronic exposure smoke rats, 54 male Wistar rats were randomly divided into 7 groups： control group （C group）, smoke exposure groups （S4w group, S8w group）, puerarin groups （P4w group, P8w group）, propylene glycol control groups （PC4w group, PC8w group）. Rats were exposed to cigarette smoke or air for 4 to 8 weeks. Rats in puerarin groups also received puerarin. To evaluate vascular remodeling, alpha-smooth muscle actin （α-SM-actin） staining was used to count the percentage of completely muscularised vessels to intraacinar pulmonary arteries （CMA/IAPA） which was determined by morphometric analysis of histological sections. Pulmonary artery smooth muscle cell （PASMC） apoptosis was detected by in situ end labeling technique （TUNEL）, and proliferation by proliferating cell nuclear antigen （PCNA） staining. Reverse transcription-polymerase chain reaction （RT-PCR）, immunofluorescence staining and Western blot analysis were done to detect the PKC-α mRNA and protein expression in pulmonary arteries. The results showed that in cigarette smoke-exposed rats the percentage of CMA/IAPA and α-SM-actin expression were increased greatly, PASMC apoptosis was increased and proliferation was markedly increased; Apoptosis indices （AI） and proliferation indices （PI） were higher than in C group; AI and PI were correlated with vascular remodeling indices; The expression of PKC-α mRNA and protein in pulmonary arteries was significantly higher than in C group. In rats treated with puerarin, the percentage of CMA/IAPA and cell proliferation was reduced, whereas PASMC apoptosis was increased; The expression levels of PKC-α mRNA and protein were lower than in smoke exposure rats. There was no difference among all these data between S groups and PC groups. These findings suggested that cigarette smoke-induced pulmonary vascular remodeling was most likely an effect of t
Background Increased proliferation of pulmonary vascular cells and muscularisation of pulmonary vessels are frequently observed in human smokers and in animals exposed to cigarette smoke. To elucidate the molecular mechanisms leading to these changes, we studied the in vitro effect of cigarette smoke extract （CSE） on proliferation of pulmonary artery smooth muscle cells （PASMCs） and activation of protein kinase C （PKC）, an important kinase implicated in cell proliferation. Methods PASMCs cultured from 12 normal Wistar rats were studied in the following conditions： （1） PASMCs were exposed to different concentrations of CSE for 24 hours, then MTT colorimetric assay was used for detection of cell proliferation. Cell viability was assessed by trypan blue exclusion. （2） PASMCs were pre-incubated with phorbol 12-myristate 13-acetate （PMA） for 24 hours or Ro31-8220 for 30 minutes before exposure to 5% CSE for 24 hours. Cell proliferation was examined by MTT colorimetric assay, cell cycle analysis and proliferating cell nuclear antigen （PCNA） immunocytochemical staining. （3） PASMCs were exposed to 5% CSE for 24 hours. Then PKC-a mRNA expression was detected by reverse transcription-polymerase chain reaction （RT- PCR） and protein expression by Western blotting, while PKC-α translocation was observed by immunofluorescence staining and confocal microscopy. （4） PASMCs were transfected with specific antisense oligodeoxynucleotides against PKC-a 6 hours before exposure to 5% CSE for 24 hours. PKC-α protein expression and cell proliferation were detected by methods described previously. Results （1） Low concentration of CSE （5%） increased proliferation of PASMCs, whereas high concentrations （20%, 30%） were inhibitory as a result of cytotoxicity. （2） The value of absorbance （Value A）, proliferation index （PI）, S-phase cell fraction （SPF） and average optical density of PCNA staining in PASMCs from 5% CSE exposure group （0.306 ± 0.033, 0.339 ± 0.033, 0.175
