| Background and objective:Serious air pollution in cities and population aging make gradually lung cancer risk rise in China. The morbidity and mortality of lung cancer rank first in domestic maligant diseases. 80% patients when they were found belongs to late lung cancer, the vast majority have lost the opportunity of operation. Lung cancer tissue types mainly include small cell lung cancer(SCLC) and non-small cell lung cancer(NSCLC). Lung adenocarcinoma is the most common subtype, and accounts for more than 50% NSCLC. As scientific research achievements were used to clinical practice, lung cancer tissue types gradually converts to the molecular types, then the treatments of lung cancer are becoming more and more individualized, less side effective and longer survival. The most commonly therapeutic treatments of lung cancer include surgery, radiotherapy, chemotherapy, intervention, biological therapy(including targeted therapy, adoptive cellular immunotherapy, cytokine therapy, etc), intervention. It should not be overlooked that Chinese medicine played important roles in adjuvant therapy of lung cancer: anti-tumor, sensitizing radiotherapy and chemotherapy, inhibiting drug resistance, improving quality of life and prolonging survival period, and so on. Studies showed that integrative treatment of lung cancer gets better effect. Herbal medicine to strengthen healthy qi is the main Chinese herbal medicine to treatment of lung cancer and other malignancies. Treatment of lung cancer from "renal" has achieved good results: enhancing bodys’ immune system, inhibit the proliferation, metastasis and recurrence of lung cancer, improving the quality of life and prolonging survival period. Previous studies showed that Bu Shen Shu Gan Formula(BSSGF) could have an anti-lung-cancer effect, improve chemotherapy effect, inhibit metastasis and enhance immunity. MTT experiment was used to evaluate the effects of drug-contained serum of BSSGF on cells proliferation; Three-dimensional cell culture was employed to observe and count the number of capillary structue of mimicry angiogenesis for each group; RT-PCR were experiments were used to observe the effects of drug-contained serum of BSSGF on AKT-1, Cyclin D1, NF-κB m RNA expressions of PTEN-PI3K-AKT signaling pathway; Western blot experiments were used to observe the effects of drug-contained serum of BSSGF on PTEN, p-PI3 K, p-AKT protein expressions of PTEN-PI3K-AKT signaling pathway. Aimed to further explore the reasons why drug-contained serum of BSSGF suppresed A549 cells proliferation and metastasis. These experiments aimed to provide theoretical and experimental basis for the clinical reasonable application of Bu Shen Shu gan formula(BSSGF). Methods:120 male SD rats were randomly assigned to 6 groups,20 on average in each group. And the rats weight of each group were detected by homogeneity test of variance. There is no difference, statistically speaking, in each group weight, with an average of about 250 g. Respectively named each group as, saline group(control group, referred to “group S”): the rats were was in tragastric administration with 2m L normal saline two times per day from day 1 to day 5. Cisplatin group(referred to “group P”) : 4m L 8 mg·kg-1 cisplatin were given to one rat once a day by intraperitoneal injection, in day 1, day 3, day 5. Bu Shen Shu Gan Formula low-dose group(referred to “group L”), 2m L 15g· kg-1 BSSGF dose-concentration were given intragastric administration two time from day 1 to day 5; Bu Shen Shu Gan Formula high-dose group(referred to “group H”): 2m L 30g·kg-1 BSSGF dose-concentration were given intragastric administration to rats two times from day 1 to day 5; Low-dose group combined with cisplatin(referred to “group C1”), 2m L 15g·kg-1 BSSGF dose-concentration were given intragastric administration twice from day 1 to day 5, at the same time,the rats also were given 4m L 8mg· kg-1 cisplatin once a day by intraperitoneal injection, in day 1, day 3, day 5. High dose group combined with cisplatin(referred to “group C2”), 2m L 30g·kg-1 BSSGF dose-concentration were given intragastric administration two time from day1 to day 5, at the same time, the rats also were given 4m L 8 mg· kg-1 cisplatin once a day by intraperitoneal injection, in day 1, day 3, day 5. Chinese herbal medicine boiling process: herbal medicine were soak for 30 minutes in an earthenware pot with water, and the level of water high above Chinese herbal medicine about 2-3cm before boiling. Boil in fire then turn to slow fire for 30 minutes. BSSGF low-dose and highdose decoction were respectively achieved by the way that conventional stewed decoction were 5 and 10 times concentrated, in case of traditional Chinese medicine(TCM) volume is too big, so that we could not effectively give all of the required quantity into the rats’ stomach cavity. Each rat given traditional Chinese medicine was in intragastric administration 2m L, twice a day, a total of consecutive 5 days; Saline group’ were given the same volume of saline twice a day. After 2 hours at the end time of intragastric administration and abdominal cavity injection, rats were given anesthesia by intraperitoneal injection of chloral hydrate. Separated abdominal aorta under the condition of sterile and collected blood with the scalp acupuncture, centrifuged to achieve serum, inactivated serum in 56℃ 30 min, then 0.22μm filtered and removed bacteria, preserved-20℃. MTT experiment was used to evaluate the effects of drug-contained serum of BSSGF on A549 cell proliferation; Three-dimensional cell culture was employed to observe and count the number of mimicry angiogenesis forming capillary structue for each group; RT-PCR experiments were used to observe the effects of drug-contained serum of BSSGF on A549 cells PTEN-PI3K-AKT signaling pathway’ and downstream signaling pathway’ AKT-1, Cyclin D1, NF-κB m RNA expressions; Western blot experiments were applied to observe the effects of drug-contained serum on A549 cells PTEN, p-PI3 K, p-AKT protein expressions in PTEN-PI3K-AKT signaling pathway. Results:1.The inhibition rates of low-dose Bu Shen Shu Gan group and low-dose group combined with cisplatin in 48 h, 72 h, 96 h were higher than those in other experment- al groups(p<0.05, p<0.01); The inhibition rate of low-dose group combined with cisplatin in 24 h, 48 h, 72 h, 96 h, was higher than those in the other experimental groups(p<0.05);While multiple comparisons of those in three groups showed no significant differences, cisplatin group, high-dose Bu Shen Shu Gan formula group, high-dose group combined with cisplatin in 24 h, 48 h, 72 h, 96h(p> 0.05).2.The number of mimicry angiogenesis forming capillary structue in all experimental groups was markedly less than those in control group(p<0.05). The number of mimicry angiogenesis forming capillary structue of low-dose Bu Shen Shu Gan Formula group and low-dose group combined with cisplatin were less than those in other groups(p<0.05, p<0.01); The number of vasculogenic mimicry tuber-shaped structure of low-dose group combined with cisplatin was less than those in other groups; While multiple comparisons of those in three groups showed no significant differences, cisplatin group, high-dose Bu Shen Shu Gan formula group, high-dose group combined with cisplatin(p > 0.05).3. The AKT1, Cyclin D1, NF-κB m RNA relative expressions in all experimental groups were markedly less than those in control group(p<0.05); The AKT1, Cyclin D1, NF-κB m RNA relative expressions of low-dose Bu Shen Shu Gan Formula group and low-dose group combined with cisplatin were lower than those in other groups(p<0.05, p<0.01); The AKT1, Cyclin D1, NF-κB m RNA relative expressions of low-dose group combined with cisplatin were lower than those in low-dose Bu Shen Shu Gan Formula group(p<0.01); While multiple comparisons of those in the three groups showed no significant differences, cisplatin group, high-dose Bu Shen Shu Gan Formula group, high-dose group combined with cisplatin(p > 0.05).4.The PTEN, p-PI3 K, p-AKT protein relative expressions in experimental group were markedly different with those in control group(p<0.05). The PTEN protein relative expression of the low-dose Bu Shen Shu Gan Formula group and low-dose group combined with cisplatin were higher than those in the other groups(p<0.05, p<0.01); The p-PI3 K, p-AKT protein relative expression of the low-dose Bu Shen Shu Gan Formula group and low-dose group combined with cisplatin were lower than those in the other groups(p<0.05, p<0.01); The PTEN, p-PI3 K, p-AKT protein relative expressions of low-dose group combined with cisplatin were markedly different with those in the other groups(p<0.05). While multiple comparisons of PTEN, p-PI3 K, p-AKT protein relative expressions in three groups, showed no significant differences, cisplatin group, high-dose Bu Shen Shu Gan Formula group, high-dose group combined with cisplatin(p>0.05). Conclusion:Low-dose and high-dose drug-contained Serum of Bu Shen Shu Gan Formula could inhibit both A549 cells proliferation and mimicry angiogenesis forming; Bu Shen Shu Gan Formula may inhibit cells proliferation and metastasis by PTEN-PI3K-AKT signaling pathway, and had a synergistic sensitization effect with cisplatin to some extent. But there was no dose-effect relationship between dose and effect of Bu Shen Shu Gan Formula, in some dose range. |
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