The Expression And Activity Of Phosphodiesterase In Rat Pulmonary Microvascular Endothelial Cells And The Effect Of Related Heat-clearing Herbs On Its Activity

Pulmonary Microvascular endothelial cells(PMVEC)as the main part of air-blood barrier have an important function such as regulating stabilization of pulmonary internal environment, keeping the physiological and immunological function, mediating disease of respiratory system happening, developing and turning over and so on.CAMP in PMVECs was the key factor in regulating barrier function of endothelial cells. Phosphodiesterase (PDE) as the exclusive hydrolytic enzyme of cylic nucleotides regulates a series of organismal physiological pathological change through adjusting the level of cAMP and cGMP.This study was to know the distribution of PDE hypotypes, the activity of basal enzyme and the changes of PDE activity after irritating by inflammatory factor and effecting by heat-clearing herbs through detecting the mRNA expression of 18 PDE hypotypes by RT-PCR and the changes of cylic nucleotides after irritating PMVEC by inflammatory factor and heat-clearing herbs with HPLC, These data was offered as a foundation to choose specific herbal PDE- inhibitors through regarding PMVEC as a target and to detect the effect of Chinese herbs on its activity which can be used as a significance on the treatment of respiratory system disease and it also can enrich anti-inflammatory mechanism of Chinese herbs medicine at molecular level. The contents of the study were as follows:The PMVECwas dissociated and cultured useing tissue-sticking method, which was identified by cell immunostainin with VIII and CD31.MRNA expression of PDE was detected by RT-PCR in PMVEC. The result showed there were 17 PDE subtypes including PDE1A, 1C, 2A, 3A, 3B, 4A, 4B, 4C, 4D, 5A, 7A, 7B, 8A, 9A, 10A, 11A.PDE activity was examined with HPLC, the result showed that there was high activity of PDE in PMVEC which can hydrolysis both cAMP and cGMP, in which cAMP-PDE, PDE4 was the mian subtype.The activity of cAMP-PDE in PMVEC which was irrititated by IL-1βand IL-6 of different concentration on different time was detected. The result showed that cAMP-PDE activity was dose dependent with IL-1βand IL-6 in some extent, the level of cAMP-PDE activity tendenty became stationary 60min later, but still beyond the normal level.The effect of 17 kinds of heat-clearing durgs going to lung meridian on PDE activity was examined with HPLC. The result disclosed that Huangqin was the most effective, its inhibition rate was 75.37%. and then was Huangyaozi, the inhibition rate was 72%. The inhibition rate of Qinghao was 61.26%. These three herbs also had strong inhibition to PMVEC,The result above showed that there were many kinds of PDE which have high activity in PMVEC, its activity was elevated abnormally after being irrititated by IL-1βand IL-6 and inhibited by the heat-clearing herbs especially Huangqin, Huangyaozi and Qinghao. It indicated that It was nended to deeper research to investigate meridian distribution of chinese herbs through regarding the activity of cAMP-PDE as a target and choose specific inhibition of PDE.