| Objective: Oleanolic acid (OA) is widely used to treat hepatitis in clinic, recent studies found that it had two-way immune modulation effects, anti-inflammatory antiviral and antitumor activity, etc. Its treatment results were good, but its mechanism of action is still not very clear. So MTT and FCM method were adopted to confirm whether OA has inhibition proliferation of A549 cells. At the same time, OA concentration was determined both in the intracellular and extracellular of A549 cells after different treatment time of the same concentration of OA and different OA concentration of the same treatment time by RP-HPLC, which can help us understand the relationship between the dynamic changes of OA in the A549 cells and its action mechanism. Comparative proteomic analysis method has been used to explore the changes of protein before and after the treatment of OA in A549 cells. The results of this study will help us find out the target or the biological molecules mediated the action of OA so as to speculate the possible mechanism of OA, which may provide the theoretical and experimental data for the clinical application.Methods:1. A research model for the proliferation of apoptosis A549 cells by oleanolic acid has been established. MTT method was used to evaluate the inhibitory effect of OA on A549 cells in vitro; FCM was used to observe the cell apoptosis of A549 cells after treatment; and DAPI staining was used to detect the nuclei changes of A549 cells before and after OA's treatment.2. An RP-HPLC method for the determination of intracellular and extracellular OA was established. The chromatographic condition is as follow, stationary phase is ODS (4.6 mm×2.5 mm, 5μm); mobile phase is methanol-water( pH 3.38 )=90:10(V/V); flow rate is 1.0 ml/min; column temperature is 30℃; detected wavelength is 210 nm and the injection volume is 20μl.3. Total proteins of blank cells and treated cells were extracted respectively and separated by two-way gel electrophoresis technology (2 - DE), then stained with silver nitrate, the differentially expressed proteins were analyzed and identified with PDQuest8.0 software at last.The selected protein spots were cut out of 2-DE gels for MALDI-TOF-MS analysis which was under a reflector mode, and then identified with peptide mass fingerprint(PMF) by searching in Mascot software.Results: 1. MTT assay showed that OA restrained the growth of A549 cells in vitro over the concentration of 10-200μg/ml. The inhibition ratio was related with the treatment time and OA concentration, it could be reach up to 80% at 200μg/ml. The IC50 in 24,48,72h was 109.45,80.08,78.21μg/ml. After DAPI dyeing, the abnormal shape of A549 cell's nuclei was observed after OA treatment of 24h. At the concentration of 80μg/ml, the nuclear chromatin pyknosis, a lot of particulate materials formation or fragmentation were observed, the fluorescence intensity increased, apoptosis was initiated. At higher concentration of OA(200μg/ml), apoptosis phenomenon was more significant. FCM analysis showed that the apoptosis ratio was consistent with the inhibition ratio from MTT assay. Each group's total apoptosis ratio (n = 3, x±s) (%) was: 16.37±2.17,40.97±2.72 , 86.14±1.43 respectively with significant differences from those in control group (p < 0.05).2. The results indicated that the linear range of intracellular OA was 1.02～20.4μg/ml (r=0.9996), the average recovery is 97.57%, the RSD of intra-day precision was 3.03%, 2.56%, 1.99% respectively, and that of the inter-day was 2.42%, 1.79%, 2.59%; and the linear range of extracellular OA was 5.1～81.6μg/ml (r=0.9998), the average recovery is 97.37%, the RSD of intra-day precision was 0.63%, 1.99%, 2.36% respectively, and that of the inter-day was 3.52%, 1.19%, 1.21%.3. Four differentially expressed proteins spots were further identified by MALDI-TOF-MS analysis and PMF database searching. TPx-1,Actin, BTF3 was significantly down-regulated, while Hsp27 was significantly up-regulated in treated sample compared with the control sample.Conclusion: The results from the MTT experiment, DAPI dyeing experiments and FCM showed that OA inhibited the proliferation of A549 cells. An HPLC method was established to determinate the concentration of OA both intracellular and extracellular, the dynamic changes of OA in cells maybe helpful to explain its mechanism. Four differentially expressed proteins spots were successfully identified by PMF database searching, which maybe an important contribution to the antitumor mechanism of OA.
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