| Objective: To investigate the mechanism of the decoction of Rhizome Zedoariae and Fructus Trichosanthis in the induction of apoptosis of human lung carcinoma cell line (PGLH7) by serologic pharmacological method and provide theory basis for the decoction of Rhizome Zedoariae and Fructus Trichosanthis to popularize and apply in clinic practice.Methods: According to the basic Traditional Chinese Medicine theory, the pathologic characteristic of lung carcinoma is stagnation of phlegm and turbid qi. The human lung carcinoma PGLH7 cell line was adopted as target cell. After incubating PGLH7 cell and Chinese drug, mainly composed of Rhizome Zedoariae and Fructus Trichosanthis, borne rabbit serum at different dose with serologic pharmacological method. It was divided into 5 groups: low dose serum of the decoction of Rhizome Zedoariae and Fructus Trichosanthis, middle dose serum of the decoction of Rhizome Zedoariae and Fructus Trichosanthis, high dose serum of the decoction of Rhizome Zedoariae and Fructus Trichosanthis, western medicine serum of RA ,the control serum given saline solution. MTT assay is used to calculate the inhibitory rate. The apoptosis morphological changes were observed under the inverted microscope , light microscope and fluorescence microscope in vitro .The target cells were harvested to analyze the cell cycles using flow cytometry (FCM) and to estimate the apoptosis ratio by staining with Annexin V & PI.Result:(l)The drug-bearing serums can inhibit the growth of PGLH7 cell significantly. There was remarkable difference in the inhibitory rate between 10% high dose drug-bearing serum and the control serum ( P < 0.05 ) .A time-dependendent proliferation inhibition was demonstrated in PGLH7 cell.(2)Cell cycle analysis of PGLH7 cell showed that there was significant difference in S phase between 10% high, middle dose drug-bearing serum and the control serum by flow cytometric (middle dose drug-bearing serum P<0.05, high dose drug-bearing serum P<0.01). A time-dependendent S phase inhibition was demonstrated in PGLH7 cell.(3)With the inverted microscope , after being treated with drug-bearing serum, PGLH7 cell grew against the wall and then peeled off gradually, which showed the characters of cells apoptosis like chromatin condensation. With HE stain and light microscope, after being treated with drug-bearing serum, PGLH7 cell showed the typical apoptosis morphological changes, such as shrinkage, crescent shape concentrated chromatin and fragmentation.(4)Fluorescence microscope and staining with Annexin V & PI are used to observe the typically early and final phase changes of PGLH7 cell apoptosis. In the early phrase of cells apoptosis, the membranes are green and the nucleus are not red and in the middle and final phrases of cells apoptosis, the membranes are green and the nucleus are red; the nucleus broke into pieces and cells split into apoptosis bodies in different sizes.(5)The PGLH7 cells apoptosis rate treated with 10% high dose drug-bearing serum at 24 h, was significantly higher than that of the control serum (P<0.05) by flow cytometric assay. The PGLH7 cells apoptosis rates treated with 10% middle dose and high dose drug-bearing serum at 48 h, were significantly higher than that of the control serum (middle dose drug-bearing serum P<0.05, high dose drug-bearing serum P<0.01) by flow cytometric assay. The effect was dependent on both time and dose.Conclusion: It demonstrates that the decoction of Rhizome Zedoariae and Fructus Trichosanthis could be effective in lung carcinoma which was dependent on both time and dose. The above results suggest that the antineoplastic molecular mechanisms of the decoction of Rhizome Zedoariae and Fructus Trichosanthis may be its effect on inhibiting DNA synthesis to restrain PGLH7 cells proliferation and inducing the apoptosis of PGLH7 cells by serologic pharmacological method. It provides theory basis for the decoction of Rhizome Zedoariae and Fructus Trichosanthis to popularize and apply in clinic practice. |
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