| Background Pancreatic cancer has a fast progressive course and most patients are prone to metastasis and recurrence after surgery or other treatments,leading to a poor prognosis,and the situation is not optimistic.It has been found that QHF formula has the advantages of maintaining tumour stability,improving patients’ clinical symptoms and having less toxic side effects,and has been widely used in clinical practice.As the etiological mechanism of pancreatic cancer is complex,this topic aims to elucidate the anti-tumour mechanism of traditional Chinese medicine,especially the anti-invasive metastasis mechanism of pancreatic cancer,in order to promote the rapid development of traditional Chinese medicine in the field of tumour treatment.Objective To investigate the potential relationship between the QHF formula and pancreatic cancer through network pharmacology,and to explore the mechanism of action of the QHF formula in regulating KRAS/MAPK signaling pathway against pancreatic cancer invasion and metastasis,so as to provide a theoretical basis for clinical pancreatic cancer treatment.Methods 1.Network pharmacology: Screening the potential targets of QHF formula against pancreatic cancer by TCMSP,BATMAN-TCM database,Swiss Target Prediction,Gene Cards,OMIM database;String database to predict the mechanism of action of QHF formula for pancreatic cancer.2.In vitro experiments:(1)MTT assay to observe the effect of QHF formula and gemcitabine on the proliferation of PANC-1 cells,and to screen the drug concentration and duration of action in in vitro assays;(2)cell scratch assay to investigate the effect of QHF formula and gemcitabine on the motility of PANC-1 cells;Transwell assay to investigate the effect of QHF formula and gemcitabine on the migration and invasion of cells;(3)q RT-PCR to detect the expression of KRAS,RAF1,MEK,ERK m RNA;(4)Western-blot to detect the expression of KRAS,RAF1,MEK1/2,ERK1/2,Twist-1,Snail,MMP-9,Vimentin protein expression.3.In vivo experiments: BALB/c-nu mice were used as the study subjects.Eight mice were randomly selected as the normal group and the remaining 50 as the moulding group,and PANC-1 cells were injected subcutaneously under the axilla to establish an axillary tumour model in nude mice.After successful modeling,the rats were randomly grouped into model group,QHF low-dose group,QHF high-dose group,gemcitabine group and KRAS inhibitor group,10 rats in each group.The following experiments were performed:(1)Observing and recording of body weight and measurement of tumour volume of nude mice every other day for 14 days;(2)Remove the nude mouse tumor,weigh the tumor weight,and calculate the tumor inhibition rate;(3)HE staining to observe changes in tumour tissues and liver tissues of nude mice;(4)IHC detection of Snail and MMP-9 protein expression in tumour tissues;(5)Western-blot to detect the protein expression levels of KRAS,RAF1,MEK1/2,ERK1/2,Twist-1 and Vimentin in tumor tissues.Results 1.Network pharmacology: The database screening and construction of "component-target-pathway-disease" network diagram predicted 149 potential targets of QHF formula for pancreatic cancer treatment,such as GAPDH,MAPK1,MMP-9,etc.;GO function and KEGG pathway enrichment analysis revealed that the anti-pancreatic cancer action of QHF formula was related to MAPK pathway cascade regulation.2.In vitro experiments:(1)Compared with the control group,QHF formula and gemcitabine can effectively inhibit the proliferation of PANC-1 cells.(2)The results of scratch assay showed that QHF formula and gemcitabine could inhibit cell motility compared with the control group.Transwell results showed that both QHF formula and gemcitabine could effectively inhibit cell migration and invasion compared with the control group;the anti-invasive effect of QHF formula was statistically different compared with gemcitabine.(3)The q RT-PCR results showed that QHF and gemcitabine significantly down-regulated the expression levels of KRAS,RAF,MEK and ERK m RNA.(4)Western-blot results showed that QHF and gemcitabine reduced the expression of KRAS,RAF,MEK,p-ERK,Twist-1,Snail,MMP-9and Vimentin proteins compared with the control group.3.In vivo experiments:(1)The axillary transplantation tumor model was successfully constructed in nude mice,and the tumorigenic rate was about 80%.(2)The tumour suppression rate was calculated as 26.09%for QHF low-dose,53.48% for QHF high-dose,47.79% for gemcitabine and 37.77% for KRAS inhibitor.(3)HE staining results showed that the tumour tissue of the model group had large deep-stained nuclei of cancer cells without obvious necrosis.After administration,the cancer cells showed vacuolar degeneration and extensive necrosis,among which the high dose of QHF and gemcitabine had a significant effect on tumor killing;the normal liver tissue of nude mice had intact cell structure,while the liver tissue of the model group showed large metastatic foci of cancer tissue,and the QHF formula could inhibit liver metastasis in nude mice,and gemcitabine and KRAS inhibitor also played the same role.(4)The IHC results showed that QHF,KRAS inhibitor and gemcitabine effectively reduced the expression of MMP-9 and Snail protein compared with the model group,and the difference was statistically significant.(5)Western-blot results showed that the expression of KRAS,MEK1/2,RAF1 and ERK1/2 proteins in the tumor tissues of QHF,gemcitabine and KRAS inhibitor groups was reduced compared with that of the model group;the expression of Vimentin and Twist-1was also reduced,and the difference was statistically significant.Conclusion The QHF formula can effectively inhibit the proliferation,migration and invasion of PANC-1 cells,significantly inhibit the growth and liver metastasis of axillary transplanted tumors in nude mice,and regulate the expression of invasion and metastasis-related proteins. |
PDF Full Text Request