The Effects Of Aminophylline On The Proliferation And Apoptosis Of Rat Airway Smooth Muscle Cells In Vitro And Its Relationship With The Expression Of C-fos And MMPs

Objective: To investgate the effects of aminophylline on the proliferation and apoptosis of rat airway smooth muscle cells(RASMCs) and their relationship with the expression of MMPs and c-fos, demonstrate the mechanism of treating asthma using aminophylline at cellular and molecular levels . Methods: Normal RASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Cells of passages 4 to 6 were used in our experiments. The cells were cultured with aminophylline of different concentration (0, 50, 100, 200, 300, 400mg/L) for 12 to 72h. The effects on the proliferation of the RASMCs were observed by cell counting and MTT assays. RT-PCR was used to semi-quantify c-fos expression of RASMCs. Gelatin zymography and Western blot were used to detect the activity and the expression of MMP2 and MMP9. Light and fluorescent microsopy were used to observe morphological changes and calculating the percentage of apoptostic cells. DNA ladders of the apoptotic cells were analysed by agarosegel electrophoresis.Results: The cell number and A490nmof RASMCs was decreased after adding 100mg/L (p<0. 05), and with the increase of the dose of aminophylline, the inhibitory "effects enhanced, showing a time- and dose-dependent manner. RT-PCR showed a 371bp band in the agarose gel just as predicted using specific primers for c-fos mRNA. The c-fos expression decreased with the increase of the dose of aminophylline when comparing with the internal control of B-actin. Western blot showed that the expression of MMP2 decreased significantly when comparison with the control (3.70 + 0.28, 3.58 + 0.27, 3.37 ?.25 vs 3.83 + 0.30) (P<0. 05, P<0. 01, P<0. 01, respectly), and the expression of MMP9 decreased in comparison to the control (0.19 + 0.04, 0. 16 + 0. 02, 0. 13 + 0. 02 vs 0. 21 ?. 06) (P<0.05, P<0.01, P<0.01, respectly). Gelatin zymography demonstrated that there were MMP2 and MMP9 activity in the supernatant of DMEM with 10% FBS. After treating with 50, 100, 200mg/L aminophylline for 48h, the area(mm2) of gelatin degradated by MMP2 were decreased in comparison to the control (6.17 + 0.52, 4.70 + 0.30, 3. 98 + 0. 42 vs 6. 58 + 0. 35) (P<0.05, P<0.01, P<0.01, respectly), showing a dose-dependent manner. Meanwhile, the area (mm2) of gelatin degradated by MMP9 were decreased in comparison to the control (0. 23?. 02, 0. 17 + 0.02, 0. 12 + 0. 02 vs 0. 25 + 0. 02) (P<0.05, P<0.01, P<0.01, respectly), also showing a dose-dependent manner. Light microscopy observed that the normal RASMCs appeared fusiform with cytoplasm and nucleus lightpainted, while the apoptotic cells have nuclear contraction, chromation condensation and apoptotic bodies. Most of the cells in the control group were normal cells. After treating with aminophylline, there were more apoptotic cells and less normal cells. Fluorescent microscopic examination after staining with AO/EB showed the same cellular morphology as under light microscopy, but there were green or bright yellow-green flurescence in cytoplasm and nucleus of normal RASMCs and both green and orange flurescence in cytoplasm and nucleus of apoptotic RASMCs, and apoptotic bodies as well. The percentage of apoptotic cells detected by photo capturing and image analysis was increased with the increase of the dose of aminophylline, and reached its peak at 40% after treating by 200mg/L aminophylline for 24h ( P<0. 01 vs control ) . Agrose gel electrophoresis of DNA fragements showed a ladder-like patten while the control group has no ladder-like band.Conclusion: Our experiments shows that aminophylline can inhibit the proliferation of RASMCs in a time- and dose-dependent manner. Further effort demonstrate that aminophylline can supress not only the expression of c-fos mRNA in a dose- dependent manner but also the activity and expression of MMP2 and MMP9 in a dose-dependent manner. All these results and the finding that aminophylline can directly induce apoptosis of RASMCs lead us to such a conclusion that aminophylline can be used in treating airway remodeling by inhibiting the proliferation of ASMCs...