| Cancer is a worldwide medical challenge.In recent years,the mortality rate caused by cancer has increased significantly and ranks second in both sexes,posing a serious threat to human health.At this stage,the clinical treatment of cancer is still based on surgical resection,radiotherapy,and chemotherapy.Surgical resection and radiotherapy are only applicable to local lesions,and most metastatic lesions can only be treated with chemotherapy drugs.Chemotherapeutic drugs used in clinical applications general ly have the disadvantages of large toxic side effects and strong drug resistance.Therefore,developing safer and more efficient anti-cancer drugs is still one of the focuses of cancer treatment.Plant extracts are a good source of natural anti-tumor drugs and have received more and more attention.This thesis took terpene volatile oil of Pinus koraiensis(PVO)as the research object.The volatile oil was separated from the Pinus koraiensis pinecones by salting out-assisted hydrodistillation and analyzed its components.The gastric cancer MGC-803 cells,being most sensitive to PVO,were selected from five human malignant tumor cells with high incidence.The anti-tumor activity and mechanism of PVO in vitro were systematically studied using this cell as a model.The O/W type PVO nanoemulsion was prepared by ultrasonic method.Using MGC-803 tumor-bearing nude mice as a model,the anti-tumor activity and mechanism of PVO and its nanoemulsion in vivo were studied.PVO was separated by salting out-assisted hydrodistillation,and the composition of PVO was analyzed by GC-MS and GC-FID technology.GC-MS results showed that 41 compounds were identi fied from PVO,accounting for95.73% of the total components,among which monoterpenoids such as α-pinene(40.91%),limonene(24.82%)and β-pinene(7.04%)were the main component of PVO.L-menthol was used as internal standard,and the in ternal standard curve method was used to quantify α-pinene and limonene in PVO by GC-FID technique.The contents of α-pinene and limonene in PVO were determined to be 36.82% and 21.29% respectively.The MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT)experiment was used to study the anti-proliferative activity of PVO on five human malignant tumor cells with higher incidence in vitro.The results showed that among the five tumor cells A549,MCF-7,Hep G2,HT29 and MGC-803,PVO had the best anti-proliferation effect on gastric cancer MGC-803 cells,and its EC50 value was the lowest.The anti-tumor activity of PVO in vitro was systematically studied using MGC-803 cells as a model.The results of scratch healing assay and Transwell invasion assay showed that PVO could significantly reduce the migration and invasion abili ty of MGC-803 cells.Through the flow cytometry,it was found that PVO could arrest the MGC-803 cell cycle in G2/M phase,significantly reduce the mitochondrial membrane potential and promote MGC-803 cell apoptosis.RNA sequencing(RNA-seq)combined with bioinformatics analysis were used to explore the pathway of PVO promoting apoptosis in MG C-803 cells.RNA-seq results showed that after 24 hours of treatment with 50 μg/m L PVO,a total of 157 genes were significantly differentia lly expressed in MGC-803 cells,of which 100 genes were significantly up-regulated,57 genes were significantly down-regulated,and PVO can significantly down-regulate the expression of FAT4,STK3,LATS2,YAP1 and AJUBA m RNA in the tumor-related Hippo/YAP1 signaling pathway.Real-time PCR and Western blot were used to detect the m RNA and protein expression levels of FAT4,STK3,LATS2,YAP1 and AJUBA related to Hippo/YAP1 signaling pathway in MGC-803 cells.The results verified the accuracy of RNA-seq results,and demonstrated that PVO can inhibit the proliferation,invasion and migration of tumor cells by activating the Hippo/YAP1 signaling pathway,and promote the apoptosis of MGC-803 cells.The O/W type PVO nanoemulsion was prepared by a ultrasonic method.The optimal formulation and preparation process of PVO nanoemulsion were determined as follows: PVO 10%,mixed surfac tant(Tween-80:1,2-propanediol=5:1)20%,deionized water 70%,ultrasonic power o f 500 W,ultrasonic time of7.5 min.The nanoemulsion was characterized by laser particle size analyzer and transmission electron microscope(TEM),etc.The results showed th at the PVO nanoemulsion was 46.87 nm in size,PDI was 0.121,and uniformly distributed in a spherical shape,with good centrifugal stability and storage stabilit y.The results of simulated gastric and intestinal fluid digestion expe riment showed that PVO nanoemulsion remained intact in simulated gastric juice,and only a small amount of PVO was released into the simulated gastric juice.However,after the digestion of simulated intestinal fluid,the particles in PVO nanoemulsion were completely broken,and the majority of PVO were released into simulated intestinal fluid.MTT test results showed that PVO nanoemu lsion had better anti-tumor activity than PVO suspension,which was closely related to the better dispersion,stabilit and bioavailability of PVO nanoemulsion.MGC-803 tumor-bearing nude mice were used as animal models to study the anti-tumor activity and mechanism of PVO and its nanoemulsion in vivo.The experimental results showed that PVO nanoemulsion high-dose group(100mg/kg)had the best therapeutic effect on tumor-bearing mice,and the relative tumor proliferation rate(30.23%)in PVO nanoemulsion group was significantly lower than that in other groups.The results of organ index and HE staining showed that PVO and its nanoemulsion could improve spleen injury in nude mice with tumor.TUNEL in situ apoptosis assay showed that PVO and its nanoemulsion could promote the apoptosis of tumor cells in nude mice,and the apoptosis rate of tumor cells in the high-dose PVO nanoemulsion group was38.15%,which was significantly higher than that in the other groups.Immunohistochemical results showed that PVO and its nanoemulsion nanoemulsion could down-regulate the expression of effector protein YAP1 by activating the Hippo/YAP signaling pathway,and then down-regulate the expressions of its target proteins CTGF,AREG and GLI2,promote the apoptosis of tumor cells,and finally inhibit the tumor growth of MGC-803tumor-bearing mice,and exert its anti-tumor activity in vivo.In this study,the terpenes volatile oil isolated from Pinus koraiensis of in vitro anti-tumor activity was systematically studied in MGC-803 cells as a model,and its mechanism was revealed.The performace of PVO were improved by preparing O/W type nanoemulsion.The anti-tumor activity and mechanism of PVO nanoemulsion in vivo were studied by MGC-803 tumor-bearing nude mouse model.This study provides theoretical support for the application of plant volatile oil and the development of Pinus koraiensis resources,and also provides a new perspective for the research and development o f anti-tumor drugs. |
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