| Objective:Lung cancer is one of the most common cancers worldwide.Its treatment results were the worst among all tumor types,with a 5-year survival rate of 10%to 20%.Whose survival rate is affected by the diagnostic stage ranging from 92%in the early stage to 0%in the late stage,so the mortality rate of lung cancer ranks the first among all tumors and the new incidence rate ranks the second.Therefore,development of novel therapeutic agents is urgently needed to treat it.Proliferation and migration of lung cancer cells through multiple pathways,and apoptosis is an important intracellular physiological activity that controls proliferation and growth of lung cancer cells and plays an important role in the occurrence and development of lung cancer.In recent years,the research of traditional Chinese medicine has made a series of achievements in the research of anticancer drugs and become a hot spot of oncology research.Marsdenia tenacissima also known as Wuguteng or Tongguangsan belongs to the family Asclepiadaceae,Containing C21 steroidal saponins,flavonoids,alkaloids,resins and so on.It has been wildly used to treat lung cancer with great curative effect,mild side.In order to better development and utilization of Marsdenia tenacissima,based on previous studies,network pharmacology of Marsdenia tenacissima was carried out,to clarify the possible mechanism of Marsdenia tenacissima inhibiting lung cancer.In order to verify the mechanism of apoptosis induced by Marsdenia tenacissima,then experiments were conducted to explore the mechanism of promoting apoptosis of lung cancer cells in vivo and in vitro.Methods:(1)The components of Marsdenia tenacissima were collected through the PubMed and CNKI databases,for drug-like and pharmacokinetic analysis whose potential targets were predicted by SwissTargetPrediction,BATMAN-TCM and PharmMapper,then,compared with disease targets from OMIM,TTD and DiGSeE.obtained the target of Marsdenia tenacissima against lung cancer.The compound-target-pathway network model of Marsdenia tenacissima was construct by using the Cytoscape based on the target enrichment analysis of GO functional annotations and KEGG pathways and protein function were analyzed by Metascape.(2)Through literature review and laboratory platform,A549 cells and LLC cells were identified as the vectors for in vitro verification of the inhibition of different parts native to Marsdenia tenacissima on A549 and LLC cells was illustrated using CCK-8;Colony formation experiment and cell staining were used to further observe whether Marsdenia tenacissima promoted apoptosis on A549 and LLC cells.The apoptosis distribution of A549 and LLC cells were detected by flow cytometry.(3)Successful tumor-bearing mice were randomly grouped(n=12 each group):model group,total extract group,petroleum ether group,ethyl acetate group,Xiaoaiping injection group,selumetinib group and cisplatin group,another blank control group(no tumor bearing).Drug intervention for 14 days respectively,use conventional methods to strip tumor tissues,weigh and photo them.Mice were terminated after 14 consecutive days of treatment and tumor tissues were collected.Hematoxylin-eosin staining(HE)was used to observe morphological changes in tumor tissues.TUNEL staining was used to detect the apoptosis of lung cancer cells in situ,and the number of apoptotic cells was counted under the microscope to calculate the apoptosis rate.The expression of Calmodulin,CaMKII,p-CaMKII,MEK1/2,p-MEK1/2,ERK,p-ERK in tumor tissues were detected by Western blot.Results:(1)According to network pharmacology,6 active components and 27 target sites in Marsdenia tenacissima are related to the treatment of lung cancer.(2)The inhibitory effect of petroleum ether and ethyl acetate regions on lung cancer cells was better than other extraction regions.The IC50 values of LLC cells were 0.35±0.04 mg/mL,0.29±0.02 mg/mL,and the IC50 values of A549 cells were 0.56±0.05 mg/mL and'0.85±0.04 mg/mL,respectively,with concentration and time-dependent manner.Compared with the normal control group,the cells of A549 and LLC in the administration groups showed typical morphological changes of apoptosis,and the apoptosis rate through flow cytometry was significantly increased.At the same time,intracellular calcium concentration increased.(3)The weight of tumor-bearing mice in Marsdenia tenacissima groups was increased compared with that in the model group,with significant difference(P<0.05).TUNEL staining found that the apoptosis rate of cells in tumor tissues was significantly increased in each group.HE staining showed that tumor tissue presenting high heterogeneity,low differentiation and high malignancy.According to the Western blot results compared with model group,Marsdenia tenacissima dose groups to reduce Calmodulin,CaMKII,p-CaMKII,p-MEK1/2,p-ERK.Conclusions:(1)The network pharmacology approach presented here provided.important clues for understanding the molecular mechanism of Marsdenia tenacissima treatment in lung cancer by interacting multiple active components with multiple targets to stimulate PI3K-Akt/Ca2+/MAPK pathways.(2)Marsdenia tenacissima inducing apoptosis alike regardless of in vitro or in vivo,non-polar parts are especially effective via increasing i[Ca2+]with less calmodulin lead to deficiently express among CaMKII,p-CaMKII,p-MEK1/2 and p-ERK activate the apoptotic cascade. |
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