Research On Inflammatory Injury Of Rat Pulmonary Microvascular Endothelial Cells Induced By LPS Of PAF And Regulation Of The Process By GRKs

Objective: To investigate the pathogenesis of rat acute lung injury (ALI) induced by LPS and PAF and acquaint GRKs' roles in the injury process. Following problems will be studied:(l)The changes of permeability coefficient(Kf) and filamentous actin(F-actin) on inflammatory injury in RPMVEC induced by LPS and PAF (2) The role of F-actin on inflammatory injury of RPMVEC.(3) The corrlations among GRKs' changes, the Kf and F-actin in RPMVEC induced by LPS and PAF. Methods: RPMVEC from Wistar rat was isolated and cultured in vitro, the permeability of RPMVEC monolayer was examined with microfilter., The changes of F-actin in RPMVEC were evaluated by flow cytometry. Western blot was also used to observe the changes of GRK2 . On the foundation, we observe: (l)morphological changes of RPMVEC after stimulating by LPS and PAF with different concentrations and different time. (2)effects on the Kf of RPMVEC monolayer and F-actin distribution and variation in RPMVEC induced by LPS and PAF. (3)effects on the expression of GRK2 in RPMVEC induced by LPS and PAF.. (4)the interferring action on those above through anisodamine, phorbol ester and breviscapine.Results: (1) We succeeded in isolating and culturing RPMVEC , and after the treatment with LPS at lug/ml for 48h,10ug/ml for 12h, 100ug/ml for 8h, PAF at 100ng/ml for 48h, 1ug/ml for 24h , 10ug/ml for 8h , 50ug/ml for 30min ,the cells were broken or shedded off. We also found the cells number decreased and morphological changes happened through HE dyes. The morphological changes of RPMVEC which induced by LPS were suppressed with anisodamine and phorbol ester; and which induced by PAF weresuppressed with anisodamine, phorbol ester and breviscapine. (2) After treatment with LPS and PAF for 30min, 60min, 90min and 120min, the Kf were increased significantly in different degree. The changes of Kf were marked inhibited by anisodamine and phorbol ester, but breviscapine could only suppress the changes which induced by PAF. (3) the F-actin of RPMVEC reduced obviously after treatment with LPS and PAF,. F-actin decrease which induced by LPS were suppressed with phorbol ester, but weren't with anisodamine and breviscapine. On the contrary, F-actin decrease which induced by lug/ml LPS were suppressed with anisodamine and breviscapine, and which induced by PAF were suppressed with anisodamine, phorbol ester and breviscapine. (4) The GRK2 contents of LPS group and PAF group for 30min, 60min and 90min were significantly increased than those in normal control group by the methods of Western-blot analysis. The GRK2 contents of LPS + phorbol ester group for 30min and 90min were significantly higher than LPS group, while the contents of PAF+ phorbol ester group and PAF+breviscapine group for 90min were significantly higher than PAF group. But anisodamine had no influence on the GRK2 contents of LPS group and PAF group for 90min.Conclusion: (1) We succeeded in isolating and culturing RPMVEC from Wistar rat and identified that was RPMVEC, we also found that the cells would be shed off and morphological changes would be taken place after treatment with LPS and PAF. The changes induced by LPS were suppressed with anisodamine and phorbol ester, and the changes induced by PAF were suppressed, with anisodamine, phorbol ester and breviscapine. (2) The permeability of RPMVEC monolayer. were increased and the F-actin of RPMVEC reduced obviously after treatment with LPS and PAF. (3) The expression of GRK2 were significantly increased when RPMVEC was induced by LPS and PAF at different time. (4) The increased permeability of RPMVEC monolayer induced by LPS and PAF and decrease of F-actin in RPMVEC induced by PAF were suppressed with anisodamine. The decrease of F-actin induced by lug/ml LPS couldbe suppressed with anisodamine. Anisodamine had no influence on the expression of GRK2 in RPMVEC which induced by LPS and PAF. (5) Small dosage of phorbol ester could suppress the increased permeability of RPMVEC monolayer and the decrease of F-actin in RPMVEC which induced by LPS and PAF. The expression...