| Lung cancer is one of the most threatening malignant tumour to human health. It has a high mortality. It can be seen for a long period of time, the Cancer Prevention and Treatment in China is a very difficult task. A preventive measure to prevent the occurrence of tumors, plays a decisive role in reducing the tumor incidence and mortality. Chinese medicine "treating disease" prevention of diseases thought, its core idea is disease prevention. Modern medicine also raises chemoprevention concept of certain drugs to inhibit the tumor start, it can very effectively reduce the cancer mortality. Prunella vulgaris L. is Labiatae plant, bitter cold, with the efficacy of detoxification, its ingredients include triterpenes, phenolic acids, flavonoids, polysaccharides and other ingredients, is traditional Chinese medicine of exact effect for lung cancer, is widely used clinically.Nrf2/ARE pathway is an important signal pathway for chemoprevention, it against exogenous carcinogens and oxidizing substances in cells, as well as endogenous oxidative stress plays an important role. The idea of Chinese medicine treatment of disease as the guide of our research, We used Prunella as a model drug, research the material basis of its mechanism of the prevention and treatment for lung cancer from the whole animal level, cellular level, molecular protein level, Main elements:1three terpene component enrichment and separationIn this topic research, the triterpenoids were got from resin. Two kinds of lung cancer cell models:A549, SPC-A-1person adenocarcinoma of lung cell in vitro were established for spica prunellea efficacy screening. The consequence indicated that Prunella vulgaris L from Jiangsu had the strongest inhibition to lung cancer cell A549and SPC-A-1, the IC50was278.17and356.73μg/mL.2extracts from Prunella vulgaris chemoprevention of lung cancer model①human lung cancer cell model:A5491ung cancer cell model:establishment of the A549cell model, using the method of MTT, the inhibition of cell proliferation rate as an index, evaluation the Prunella vulgaris extract anti cancer effect, the results show that the IC50of A549and SPC-A-1were278.17,356.73μg/mL. This indicates that this model could effectively been made use of screening. ②The normal human bronchial epithelial cell model:Normal Human bronchial epithelial cells (NHBE) in vitro pharmacodynamic screening system was established by using MTT, and anti-cancer effect of Prunella vulgaris extract was evaluated by the inhibition of cell proliferation rate as an index. The results show that the IC50of NHBE is509.82μg/mL. Prunella inhibition on NHBE cells is less than the lung cancer cells. This shows that the system of biochemical prescreen could select the potential drug.③The effect of cigarette smoke extract induces NHBE cell model:Establishment of cigarette smoke extract induces NHBE cell model, the experimental results show that, NHBE cells in10%cigarette smoke extract for24h, the inhibition rate was30.12±2.7%, the concentration of50,100,200,400μg/mL Prunella vulgaris were joined, the inhibition rate was26.67±1.4%、22.47±2.2%、19.39±23%、21.19±2.5%. CSE has a toxic effect on normal cells, it shows dose-dependent. It shows that Prunella vulgaris could reduse the damage by inducer in this model.④The whole animal model:KM mouse model was established, the results show that Prunella vulgaris could significantly improve II detoxification enzyme NQO1、GST activity and GSH in KM mice,5g/kg/day and10g crude drug/kg/day concentration continuous intragastric week. The enzyme activity of GST、NQO1and the content of GSH was1.20±0.23times,1.75±0.37times,5.67±0.45times compared with the blank control group in KM mice group treated with low dose group of Prunella vulgaris. The enzyme activity of GST、NQO1and the content of GSH was1.38±0.37times，2.42±0.26times,11.13±0.33times respectively compared with the blank control group in KM mice group treated with high dose group of Prunella vulgaris (p<0.05).Nude model and A/J mouse model was established, Prunella vulgaris can significantly reduce the size of tumor in nude mice, tumor inhibition rate of Prunella in high dose group was89%and tumor inhibition rate of Prunella in low dose group was68%;A/J mouse model was established, Prunella vulgaris can significantly reduce A/J mouse lung tumor number, increase A/J mice spleen index. This mice models show that Prunella has an effect on liver cancer prevention. 3. Mechanism research of extract from Prunella vulgaris chemoprevention for lung cancer①Influence of Prunella vulgaris on phase Ⅱ detoxification enzyme activity and Nrf2protein expression in NHBE cells:The enzyme activity of GST were100.21±5.11%、125.38±4.42%、136.48±3.96%、156.19±4.88%at concentration of50,100,200,400μg/mL Prunella, enzyme activity of NQO1were100.72±6.13%、113.25±4.92%、139.53±5.35%、146.81±4.27%, GSH was103.37±4.86%、110.52±5.13%、125.94±5.87%、133.68±5.26%in NHBE cells. Western blot display of Prunella vulgaris can significantly improve A549cells in NQO1, GST and Nrf2protein expression. The results showed that spike can improve the normal lung cell NHBE phase Ⅱ detoxification enzyme activity and expression, improve to the outside stimulation to resist.②The siRNA study of Nrf2signal pathway in Prunella vulgaris three terpene cell protection:The successful establishment of Nrf2silencing of NHBE cell, under normal conditions, the GSTPI and NQO1protein expression were significantly lower, join CSE failed to induce GSTPI and NQO1overexpression, addition of selfheal three terpene also failed to have significant cytoprotective effect, show selfheal three terpene through Nrf2signal pathway on cell protection function.③Influence of Prunella vulgaris on phase Ⅱ detoxification enzyme activity and Nrf2protein expression:The results show that the content of GSH in NHBE cells treated with10%CSE group was decreased35.65±6.33%compared with the control group for24h. the activity of GST was about1.75±0.042times more than the control group, and the NQO1activity was about2.14±0.037times more than control group.After added125μg/mL Prunella vulgaris, the content of GSH was increased to72.61±5.29%compared with the control group, the activity of GST was about1.4±0.059times compared with control group, the activity of NQO1was about1.5±0.049times more than control group. p<0.05.④Influence of ursolic acid on phase Ⅱ detoxification enzyme activity and Nrf2protein expression:The results show that the content of GSH in NHBE cells treated with10%CSE and25μmol/L ursolic acid was68.53±5.33%compared with the control group. The activity of GST was about1.32times than control, and the activity of NQO1was about1.55times than control. It could reduse the DNA damage induced by CSE. The results of WB shows ursolic acid could significantly improve the expression of NQO1、GST and Nrf2in A549. It is suggest that ursolic acid is a part of the Prunella vulgaris Triterpenoids.4. The influence of Prunella vulgaris in phase II detoxification enzyme activity and Nrf2protein expression in lung cancer cells:The enzyme activity of NQO1in A549cells were73.21±6.33%、79.34±7.86%、72.38±5.55%、61.92±4.28%after treated with50,100,200,400μg/mL Prunella. And the content of GSH are57.44±4.86%、27.23±5.92%、21.76±5.03%、18.40±4.22%. The enzyme activity of NQO1in SPC-A-lcells were91.09±5.34%、96.42±3.79%、74.57±4.39%、65.78±5.18%、the content of GSH are52.53±3.18%、25.24±4.82%、20.19±4.76%、17.32±3.92%. Prunella vulgaris can significantly reduce A549cells in NQO1, GST and Nrf2protein expression by Western blot. The results showed that spike can reduce lung cancer cell A549in phase II detoxification enzyme activity and expression, reduces to the external stimuli to resist.Conclusion:Different origin, different varieties of Prunella is a medicinal its inherent potential basis exists essential difference. Prunella triterpenoid inhibitory rate of normal cells less than the lung cancer cells. In the case of10%CSE treated for24h, NHBE cell inhibitory rate of about30%, while adding125μg/mL Prunella triterpenoid causes inhibition rate decreased to80%, indicating Prunella cytoprotection effect. Prunella triterpenoids significantly reduce the number of benzopyrene-induced A/J mice lung tumors, inhibit tumor development in a nude mouse model, Prunella triterpenoid triterpenoid Prunella in the KM mice can stimulate II phase detoxification enzyme activity. In lung cancer cell A549and SPC-A-1, Prunella can be reduced the Nrf2expression, GSTPI, and NQO1expression and vitality, GSH content and showed a dose-effect relationship in NHBE cells and vice versa. Ursolic acid can protect the CSE role of NHBE cells, recovery indicators of changes in the cells caused by the CSE, reducing DNA damage, may be one of the reasons Prunella cytoprotection. |
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