Study On The Mechanism Of Inducing Apoptosis Of Weijing Decoction And Its Decomposed Formulas To Human Lung Adenocarcinoma A549Cells Based On EGFR、STAT3Signal Paths

ObjectiveIn order to provide theoretical basis for clinical application of Weijing decoctionto study the effects of Weijing decoction and its disassembled prescriptions to cellactivity, apoptosis, and expression of related signal pathways epidermal growth factorreceptor (EGFR) and signal transducer and activator of transcription3(STAT3) genesof human lung adenocarcinoma A549Cells by experiments in vitro.MethodsDetecting effects of inhibiting growth of human lung adenocarcinoma A549Cells for Weijing decoction and its disassembled prescriptions QingfeihuatanFormula(Weijing), Shenshihuatan Formula(Yiyiren＋Guaban), Huoxuehuayu Formula(Taoren) of different dose group(low dose group, middle dose group and high dose group) by MTT; Observing morphology influence of Weijing decoctionand its disassembled prescriptions to A549cells by inverted microscope; Observing apoptosis morphology influence of Weijing decoction and its disassembled prescriptions to A549cells by fluorescence microscope combined with AO∕EB and DAPI fluorescence staining; Detecting the cells’ cycle and apoptosis rate by FCM; Detecting gene expression of EGFR and STAT3protein Apoptosisof A549cells by Western blot and real-time fluorescence quantitative RT-PCR.Results1Results of MTT show that OD of A549cells inordinately decline andCell inhibition rate change with time and dose after different dose group (lowdose group, middle dose group and high dose group) Weijing decoction and its disassembled prescriptions effecting A549cells for24h,48h and72h comparing with negative control group; After Weijing decoction and its disassembledprescriptions of the same dose effecting A549cells for the same time, inhibiting of Qingfeihuatan Formula(Weijing) to cells is the most obvious, followed by Huoxuehuayu Formula(Taoren), and inhibiting of Shenshihuatan Formula(Yiyir en＋Guaban) is the most unconspicuous, and Weijing decoction is superior to its disassembled prescriptions.2Results from inverted microscope show that after Weijing decoction andits disassembled prescriptions of middle dose group (2.5g·L-1) effecting A549cells for48h, morphological changes of A549cells happen In different degrees,effects of Qingfeihuatan Formula(Weijing) to cells is the most obvious, followed by Huoxuehuayu Formula(Taoren), and effects of Shenshihuatan Formula(Yiyiren＋Guaban) is the most unconspicuous, and Weijing decoction is superior to its disassembled prescriptions.3Results of AO/EB fluorescent dye show, comparing with negative control group, after Weijing decoction and its disassembled prescriptions of middle dose group (2.5g·L-1) effecting A549cells for48h, the number of A549cells reduces, brightness enhances, there are even a lot of orange, pyknosis of apoptotic cells; Effects of Qingfeihuatan Formula(Weijing) to cells is the most obvious, followed by Huoxuehuayu Formula(Taoren), and effects of Shenshihuatan Formula(Yiyiren＋Guaban) is the most unconspicuous, and Weijing decoction is superior to its disassembled prescriptions.4Results of DAPI fluorescent dye show that, comparing with negative control group, after Weijing decoction and its disassembled prescriptions of middle dose group (2.5g·L-1) effecting A549cells for48h, the brightness of A549cells enhances, Chromatin gathered and marginalized; Effects of Qingfeihuatan Formula(Weijing) to cells is the most obvious, followed by Huoxuehuayu Formula(Taoren), and effects of Shenshihuatan Formula(Yiyiren＋Guaban) is the mostunconspicuous, and Weijing decoction is superior to its disassembled prescriptions.5Results of cell cycle from FCM show that, comparing with negative control group, after Weijing decoction and its disassembled prescriptions of middle dose group (2.5g·L-1) effecting A549cells for48h, the number of A549cells in G0/G1phase observably increases (P＜0.01)，and the number of A549cells in G2/M phase reduces.6Results of apoptosis from FCM show that, comparing with negative controlgroup, after Weijing decoction and its disassembled prescriptions of middle dosegroup (2.5g·L-1) effecting A549cells for48h, apoptosis rates of A549cells are higherthan negative control group, besides Shenshihuatan Formula(Yiyiren＋Guaban)(P＜0.05).7Results from Western Blot show that, after Weijing decoction and its disassembled prescriptions of middle dose group (2.5g·L-1) effecting A549cells for48h, the level of expression of apoptosis-related protein EGFR and STAT3from A549cells apoptosis inordinately decline, effects of Qingfeihuatan Formula(Weijing) to cells is the most obvious, followed by Huoxuehuayu Formula(Taoren), and effects of Shenshihuatan Formula(Yiyiren＋Guaban) is the most unconspicuous, and Weijing decoction is superior to its disassembled prescriptions.8Results from real-time fluorescence quantitative RT-PCR show that, after Weijing decoction and its disassembled prescriptions of middle dose group (2.5g·L-1) effecting A549cells for48h, the expression of apoptosis-related gene EGFR and STAT3from A549cells inordinately inhibited, effects of Qingfeihuatan Formula(Weijing) to cells is the most obvious, followed by Huoxuehuayu Formula(Taoren), and effects of Shenshihuatan Formula(Yiyiren＋Guaban) is themost unconspicuous, and Weijing decoction is superior to its disassembled prescriptions.Conclusion1Weijing decoction and its disassembled prescriptions all can inhibit growth of human lung adenocarcinoma A549Cells, make their morphologies change in different levels, and inhibition rate to A549cells change with time and dose; Inhibiting of Qingfeihuatan Formula(Weijing) to cells is the most obvious,followed by Huoxuehuayu Formula(Taoren), and inhibiting of Shenshihuatan Formula(Yiyiren＋Guaban) is the most unconspicuous, and Weijing decoction issuperior to its disassembled prescriptions.2Weijing decoction and its disassembled prescriptions may can inhibit theapoptosis of A549cells, make their apoptosis morphologies change in different levels, and make them stay at G0/G1phase apoptosis by effecting the expression of EGFR and STAT3proteins and genes; Apoptosis, Cell cycle, EGFR andSTAT3are all the important targets inhibiting the growth of A549cells forWeijing decoction and its disassembled prescriptions.3Weijing is the most obvious in inhibiting lung cancer in Weijing decoction；Weijing decoction may mainly can clear lung and eliminat phlegm, and also excret dampness and eliminat phlegm, activate blood circulation, if using itsdisassembled prescriptions at the same time, curative effects inhibiting lung cancer will be better.