Research On The Inhibitory Effect Of Strengthening Spleen And Harmonizing Stomach Method On Esophageal Carcinoma EC-9706 Cell’s Survival Based On STAT3 Signal

Objective To study the regulating effect of ethyl acetate extract from Liujunzi decoction and Qige powder on the proliferation, apoptosis, secretion of IL-6/TGF-β1/ VEGF, STAT3 signal of human esophageal carcinoma Eca9706 cell by experiments in vitro, in order to give a preliminary discussion on the mechanism of strengthening spleen and harmonizing stomach method acting on EC-9706 cell’s survival, as well as provide theoretical basis for experimental application.Methods Liujunzi decoction and Qige powder were extracted with 70% ethanol, ethyl acetate n-butanol and water in turn. By using different concentration(125,250,500,1000μg/ml) acting on EC-9706 cell as a preliminary screening, ethyl acetate parts were chosen as the effective components. Ethyl acetate extractions of Liujunzi decoction(6.25~200μg/ ml) and Qige powder(12.5~200 μg/ml) were applied to EC-9706 cells culture last 48 h. Growth inhibition of tumor cells was detected by MTT method, cell apoptosis was tested by FCM, apoptosis body was observed with laser scanning confocal microscope, the level of IL-6, TGF-β1 and VEGF was measured by ELISA, and STAT3/p-STAT3 protein was examined by Western Blot.Results1. Compared with 70% ethanol, n-butanol and water parts, ethyl acetate extractions from Liujunzi decoction and Qige powder had remarkble anti-tumor activity.2. Ethyl acetate extractions of Liujunzi decoction and Qige powder acted on EC-9706 cells for 48 h, both groups at different concentrations could reduce the OD values of cell lines. With the increase of drug concentration, the inhibition rate grew gradually.(P<0.05).3. Results of apoptosis from FCM show that, compared with control group, ethyl acetate extractions of Liujunzi decoction and Qige powder with low, medium and high three dose levels, apoptosis rates of EC-9706 cells significantly increased(P<0.01).4. Results of cytokine from ELISA show that, supernatant of control group expressed the highest level of 3 cytokines. Both ethyl acetate extractions of Liujunzi decoction and Qige powder with low, medium dose levels could inhibit the secretion of them. Ethyl acetate extractions of Liujunzi decoction with low and medium dose levels reduced the content of IL-6/TGF-β1/ VEGF(P<0.01). While Qige powder with low and medium dose levels reduced the content of IL-6/TGF-β1(P<0.01). Qige powder with medium dose levels could reduce the content of VEGF(P<0.01), but no statistical significance difference was obtained between the low dose level and control group(P>0.05).At the same concentration, the inhibition on IL-6 of Qige powder with low and medium dose levels was superior to Liujunzi decoction(P<0.01).While the inhibition on VEGF of Qige powder with medium dose levels was superior to Liujunzi decoction(P<0.01), but no statistical significance difference was obtained between the low dose level(P>0.05). There was no significant difference of inhibition on TGF-β1 between Liujunzi decoction and Qige powder.5. From the analysis of western blot, ethyl acetate extractions of Liujunzi decoction and Qige powder acted on EC-9706 cells for 48 h, both groups at low and medium dose levels could down-regulated expression of STAT3 and p-STAT3 protein.Conclusion1. Ethyl acetate extractions of Liujunzi decoction and Qige powder inhibited EC-9706 proliferation with a dose-dependent manner in vitro.2. Ethyl acetate extractions of Liujunzi decoction and Qige powder could inhibit the secretion of IL-6/TGF-β1/ VEGF.3. Ethyl acetate extractions of Liujunzi decoction and Qige powder could influence the cell survival of EC-9706, its mechanism may be related with the decrease of IL-6/TGF-β1/ VEGF and STAT3/p-STAT3 expression, furthermore, inhibition of STAT3 signal transportation.