The Effect Of The Tumour Necrosis Factor-alpha (TNF-α) Combining The Oxymatrine On The Signaling Material Of Pulmonary Fibroblast

Objective: Study the effect of the tumour necrosis factor-alpha (TNF-α) combining the on the signaling material of pulmonary fibroblast. To probe the mechanism of pulmonary fibrosis from the facet of signal conduct and provide the evidence for looking for the target of molecular to treat pulmonary fibrosis. Methods: SD neonatal rats' pulmonary fibroblasts were cultured and were subcultured by 4-5 generations. The pulmonary fibroblasts stimulated by the different concentration TNF-αcombining different concentration oxymatrine were cultured for certern time and oberved the effect which TNF-αworked to the pulmonary fibroblasts proliferation by means of MTT. , the expression of PKC of pulmonary fibroblast by means of immunohistochemistry, the expression of NF-κB of pulmonary fibroblasts by means of immunohistochemistry, the expression of G_s protein of pulmonary fibroblast by means of Polyacrylamide gel electrophoresis and Western blot. Results: 1. The results of MTT shows: with different concentration of TNF-αactting on the pulmonary fibroblasts, the OD value of TNF-αgroups was obviously higher than the control group (P<0.05).When actting 4 hours, the OD value of 10ng/ml TNF-αgroup was higher than those of 5ng/ml and 20ng/ml TNF-αgroups significantly (P<0.05). The OD value of 20ng/ml TNF-αgroup was higher than that of 5ng/ml TNF-αgroup significantly (P<0.05). When actting 24 hours, with the dose of TNF-αincresing, the OD value increased obviously and the OD value during the groups was different significantly (P<0.05). The OD value of 10ng/ml and 20ng/ml TNF-αgroups increased significantly with the time lasting. 2. The result of Western blot shows: with different concentration of TNF-αactting on the pulmonary fibroblasts, the expression of G_s protein of all groups had no obvious difference. 3. The results of PKC shows:with different concentration of TNF-αactting on the pulmonary fibroblast, the positive units of TNF-αgroups were higher than the control group significantly (P<0.05) and the positive unit of PKC of 10ng/ml TNF-αgroup was obviously higher than those of 5ng/ml and 20ng/ml TNF-αgroups (P<0.05) .When actting 24 hours, the positive unit of 20ng/ml TNF-αgroup was higher than that of 5ng/ml TNF-αgroup and higher than that of the similar does group when acting 4 hours significantly (P<0.05) .When 10 ng/ml TNF-αcombing different does of OM acting on the pulmonary fibroblast, the positive units of TNF-αgroups were higher than the control group significantly (P<0.05). With the concentration of OM increasing, the positive unit of PKC increased. The positive units of 10ng/ml TNF-αcombing 400ug/ml OM and 10ng/ml TNF-αcombing 800μg/ml OM groups were lower than that of 10ng/ml TNF-αgroup. Besides, the positive unit of 10ng/ml TNF-αcombing 800μg/ml OM group was obviously lower than that of 10ng/ml TNF-αcombing 200μg/ml OM groups (P<0.05) .4. The result of NF-κB shows: When different concentration of TNF-αactting on the pulmonary fibroblast and when different acting time, the positive units of NF-κB of the TNF-αgroups were obviously higher than the control .With the dose of TNF-αincresing, the positive unit of NF-κB increased. When 4 hours acting time, the positive unit of 20ng/ml TNF-αgroup was higher than those of 5ng/ml TNF-αand 10ng/ml TNF-αgroups. When 24 hours acting time, the positive units of 10ng/ml TNF-αand 20ng/ml TNF-αgroups were higher than that of 5ng/ml TNF-αgroup. With the acting time lasting, the positive unit of NF-κB of the TNF-αgroups decreased significantly (P<0.05). When TNF-αcombing OM acting on the pulmonary fibroblast homochronously, the positive units of other groups were higher than the control group significantly (P<0.05). The positive unit of 10ng/ml TNF-αcombing 400μg/ml OM group was lower than those of 10ng/ml TNF-αgroup, 10ng/ml TNF-αcombing 200μg/ml OM and 10ng/ml TNF-αcombing 800μg/ml OM groups significantly (P<0.05).The positive unit of 10ng/ml TNF-αcombing 800μg/ml OM group was higher than those of other groups significantly (P<0.05). Conclusion: 1.TNF-αcan promote the proliferation of pulmonary fibroblasts, this actting may be worked in a dose-effect way at some acting times.2.The G_s protein of pulmonary fibroblasts did not worked when stimulated by TNF-α. 3. TNF-αpromoting the proliferation of pulmonary fibroblasts may be through the PKC—NF-κB pathway.4. OM may interfered in some single material of the PKC—NF-κB pathway to inhibt the proliferation of pulmonary fibroblasts .