The Effects Of Interfering The Expression Of CAMP On TNF-α Induced Rat Lung Fibroblast Proliferation

Objective:To study the effect of tumor necrosis factor-alpha (TNF-a) on signal transduction mechanism of Lung fibroblast proliferation in vitro.To probe the mechanism of pulmonary fibrosis in the facet of signal conduct and provide a scientific basis for designing new drugs to treat pneumocomosis.Methods:SD neonatal rats'lung fibroblasts were cultured and passed up to 4-5 generations. These cells were adjusted to concentration 1×105/ml with serum-free Eagles solution,then to be purify-cultivated and stimulate-cultivated. These cells were divided into six groups:the blank control group:adding Eagles solution without fetal calf serum;the TNF-a group:adding 20ng/mlTNF-αinto Eagles solution without fetal calf serum;the FSK group:adding 10-6mol/LFSK into Eagles solution without fetal calf serum; 20ng/ml TNF-a+10-7mol/LFSK group;20ng/ml TNF-a+10-6mol/LFSK group; 20ng/ml TNF-a+10-5mol/LFSK group. Four parallel samples were designed for each group. These cells were stimulate-cultivated in incubator for 4 hours and 24 hours in 5% CO2 at 37℃. Afterwards,The hydroxyproline level in supernatant fluid was observed by means of Chloramine T method; the member Gq protein of Fibroblast cell was observed by means of Western blot;ELISE was adopted to detect the level of cAMP in the fibroblasts; the phospholipase C and protein kinase C of Fibroblasts was observed by means of immunohistochemical fluorescence technique.Results:1.The results of the level of hydroxyproline showed:At the same time,the hydroxyproline contents in the TNF-a group and TNF-a combined with FSK group was higher than the blank control group;the hydroxyproline contents in most groups of TNF-a combined with FSK was lower than the TNF-a group. With the FSK concentration increased,the hydroxyproline contents in the experimental groups depressed. With the extension of the time, the hydroxyproline contents in all the groups increased markerly.All the discrepancy was significant (P<0.05).2. The results of cAMP showed:At same time,the cAMP contents in the experimental groups was higher than the blank control group; the expression of cAMP in TNF-a+10'5mol/LFSK group was higher than the others; the expression of cAMP in the TNF-a group was higher than the FSK group and TNF-α+10-6mol/LFSK group. The discrepancy was significant (P<0.05). At the time of 4h, the cAMP contents in TNF-α+10-7mol/L FSK group was higher than the FSK group and TNF-α+10-6mol/LFSK group. The discrepancy was significant (P<0.05).3. The result of Western blot showed:At same time, the expression of Gq protein in the TNF-αgroup,TNF-αcombined with FSK group was higher than the blank control group; the expression of Gq protein in TNF-α+10-5mol/LFSK group was lower than TNF-αgroup.The discrepancy was significant (P<0.05). Comparison between the results at different time:the expression of Gq protein in the TNF-a group and TNF-a combined with FSK groups was lower than the time of 4h. The discrepancy was significant (P<0.05).4.The PLC results showed:At the time of 4h, the average optical density (AOD) value of the PLC in all the experimental groups was higher than the blank control.The discrepancy was significant (P<0.05).At the time of 24h, the AOD value in the TNF-αgroup and TNF-α+10-7mol/LFSK was higher than the blank control group. The discrepancy was significant (P<0.05).At the same time, the AOD value in TNF-α+10-6mol/LFSK group and TNF-α+10-5mol/LFSK group was lower than the blank control group.The discrepancy was significant (P<0.05).Moreover, with the concentration of FSK increased and extension of the role of time, the AOD value of PLC gradually decreased. The discrepancy was significant (P<0.05).5.The PLC results showed:At the same time, the AOD value of PKC in the FSK group,the TNF-αgroup, TNF-α+10-7mol/LFSK group and TNF-α+10-6mol/LFSK group was higher than the blank control group.The AOD value of PKC in TNF-α+10-6mol/LFSK group and TNF-α+10-5mol/LFSK group was lower than the TNF-αgroup. The discrepancy was significant (P<0.05). Furthermore, with the FSK concentration increased and extension of the role of time, the AOD value of PKC gradually decreased. The discrepancy was significant (P<0.05).Conclusion:1.TNF-a can promote the proliferation of pulmonary fibroblasts.2.Increased expression of cAMP can inhibit fibroblast proliferation induced by TNF-α.3. TNF-αcan up-regulate the expression of membrane Gq protein of lung fibroblasts.4.cAMP can affect PLC and PKC of downstream signaling molecules on Gq-PLC-PKC signal transduction pathway, so cross talk exists in signal transduction pathway between Gs-AC-cAMP and Gq-PLC-PKC.