| Objective:Traditional Chinese medicine plays an indispensable role in the treatment of cancer. It achieves the purpose of suvival with cancer by extending the survival time and improving the quality of life. Through long-time clinical practice, Yiqi Chutan Decoction is proved effective.This research was designed to further explore the mechanism of its antitumor effect. Two aspects were argued from the effect of the immune functions of patients and the effect of the proliferation and anoikis of lung cancer cell in vitro, which would provide the basis for the status of Chinese medicine in cancer therapy.Methods:1 The effect on immune function of herbs serum of Yiqi Chutan DecoctionUse prospective and self-controlled research methods,38 qualified patients were collected,who were divided into four groups according to differentiation of symptoms and signs:Lung-qi stagnation and phlegm stagnation type, phlegm-dampness due to spleen-deficiency type,yin-dificiency and phlegm-heat type, and deficiency of qi and yin type. They all accepted first-line chemotherapy of NSCLC combining with taking Chinese medicine based on Yiqi Chutan Decoction.Before treatment and 21-28 days later collect peripheral blood to measure the T lymphocyte subsets and NK cell activity with Flow cytometry(FCM), and compare the changes of the proportion of each group in T lymphocyte subsets and Nk cell activity before and after treatment2 The effect of herbs serum of Yiqi Chutan Decoction on proliferation and anoikis of lung adenocarcinoma cell A549(1) Preparation of herbs serum of Yiqi Chutan Decoction and control serum:SD rat (150-180g), divided into two groups:one was control serum group, the other was herbs serum of Yiqi Chutan Decoction (Administration: 10 times of isoeffect dosage). After five days intragastric administration, get blood from abdominal aorta an hour after the last administration. Conservation of serum:-20℃frige.(2) The suitable Medication level of herbs serum of Yiqi Chutan Decoction and control serum:Use MTT assay to detect the sensitivity of herbs serum of Yiqi Chutan Decoction and control serum on A549 cell, with the results to decide the suitable medication level.(3) The effect of herbs serum of Yiqi Chutan Decoction on proliferation of lung adenocarcinoma cell A549:Soft agar colony formation assay was designed to observe the effect of herbs serum of Yiqi Chutan Decoction on proliferation of A549 with Anoikis conditions.(4) The effect of herbs serum of Yiqi Chutan Decoction on anoikis of lung adenocarcinoma cell A 549:Collect A549 cells cultured in poly-Hema coated dish with different treatments. Agarose gel electrophoresis was disigned to analyze the role of apoptosis in A549 cells of Herbs Serum of Yiqi Chutan Decoction from the view of biochemical characteristics; PI single staining and AnnexinV-FITC/PI dual-staining were used to quantitatively detect t rate of apoptosis of each experimental group.(5) By Western blot hybridization, Explore the mechanism of Yiqi Chutan Decoction inducing anoikisis to make sure if it can change the expression of P-akt and P-p38 related with the two signaling pathway:PI3K/akt and MAPK.Results: 1 The effect on immune function of herbs serum of Yiqi Chutan Decoction:The percentage of CD3+, CD4+and NK were significantly higher after treatment than before treatment (P<0.05); CD4/CD8 ratio increased after medication than before treatment, the difference was statistically significant (P<0.05);There was no significant difference in CD8+before and after treatment (P>0.05).2 The effect of herbs serum of Yiqi Chutan Decoction on proliferation and anoikis of lung adenocarcinoma cell A549:(1) The preparation of rat serum and the addition of herbs serum of Yiqi Chutan Decoction:After five days intragastric administration, get blood from abdominal aorta an hour after the last administration. Conservation of serum:-20℃frige. MTT assay showed that A549 cell activity was not significantly different between control group and negative group (P>0.05),which can exclude the interference of rat serum on the growth of A549 cell, and it changed with the change of herb serum concentration. The herb serum of Yiqi Chutan Decoction can inhibit the proliferation of A549 cells in a dose-dependent;but when the final concentration was more than 25%, the state of cell growth was poor with vacuolar changes. According to comprehensive analysis, the final three concentrations of serum for the experimental dose were decided, which were 10%(low dose),20%(medium dose), and 30%(high dose).(2) The effect of herbs serum of Yiqi Chutan Decoction on proliferation of lung adenocarcinoma cell A549:The result of soft agar colony formation assay showed that A549 cells can form clear colonies in 10 days. In the number of colonies, the negative group was less than the herb serum groups and positive group significantly (P<0.05), and there was signifcant difference between the four groups (P<0.05).which showed that Yiqi Chutan Decoction can inhibit A549 cells proliferatiom.(3)The effect of herbs serum of Yiqi Chutan Decoction on anoikis of lung adenocarcinoma cell A 549:l)Agarose gel electrophoresis results showed that:all strips with 30% herb serum group and the positive group show typical apoptosis DNA Ladder bands, while only single-strip with the other groups, which illustrated that Yiqi Chutan Decoction can induce A549 cell apoptosis.2)Whether PI single-stained or double staining method, the results of FCM showed:the herb serum of Yiqi Chutan Decoction can induce A549 cell apoptosis.The apoptotic rate of the low.medium and high dose group:PI single-stained apoptosis rate 8.40±1.67%,15.80±2.95%,27.4±0.96%(P<0.05, compared with negative group;P<0.05, compared between two of them;but P<0.05, compared with the positive group); double staining method 10.23±1.46%,16.30±0.96%,27.10±1.22%(P<0.05, compared with negative group;P<0.05, compared between two of them;but P<0.05, compared with the positive group).(4) The effect of herbs serum of Yiqi Chutan Decoction on the expression of P-akt and P-p38 related with the two signaling pathway:PI3K/akt and MAPK:The results showed that the P-akt protein expression of low-dose group, middle dose group and high dose group were 0.77,0.57,0.54relatively, which were significantly lower than the negative group. This showed that Yiqi Chutan Decoction can lower the expression of P-akt of A549 to affect the signal transduction pathway PI3K/akt. But the expression of P-p38 protein were 1.02,1.07,1.09 relatively, with no significant change compared with negative group, which can not prove that Yiqi Chutan Decoction can affect the expression of P-p38 protein in MAPK signaling pathway.Conclusion:This research observed that Yiqi Chutan Decoction can enhance the immunity of anti-tumor effect by adjusting the proportion of T lymphocyte subsets, increasing CD3+, CD4+values and CD4+/CD8+ratio, and enhancing the NK cell activity;and the herb serum of Yiqi Chutan Decoction can inhibit A549 cell proliferation and induce anoikis.The mechanism is probably related with lowering the expression of P-akt protein in the PI3K/akt signaling pathway. The study provides the evidence for its antitumor effect, however, the more in-depth research has yet to be continued.
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