| Background and objectiveIn China,gastric cancer is a very common malignant tumor of the digestive system,and the incidence of this disease shows a rising trend year by year,with a very high mortality rate.As the clinical symptoms of early gastric cancer patients are non-specific,they are mostly manifested as upper abdominal discomfort and feeling of fullness after eating,etc.,which are easily missed diagnosis and misdiagnosed as other diseases,such as chronic gastritis and duodenal ulcer,etc.,delaying diagnosis and treatment.Therefore,most of the patients are in the middle and late stage of treatment,and the treatment means are surgery or combined chemoradiotherapy.However,due to tumor infiltration and metastasis and multi-drug resistance,the treatment effect is poor and the overall survival rate of patients is low.At present,the treatment methods for gastric cancer are as follows:1.Surgical treatment,which has certain indications and limitations due to the influence of tumor size,invasion depth and stage;2.Radiotherapy and chemotherapy,which can prolong the survival time of gastric cancer patients to a certain extent,have relatively large toxic and side effects,and chemotherapy tends to lead to tumor resistance.In recent years,the research focus of gastric cancer has gradually shifted to signal pathway and targeted therapy.Nowadays,many laboratories are conducting in-depth research on some Chinese herbal medicines,which come from natural plants and have anti-tumor effect with less toxic side effects.They have become the focus of cancer treatment research in recent years.Astragalus is a leguminous plant with a variety of biological activities.It has been found that astragalus astragalus has the functions of immune regulation,cardiovascular regulation and anti-cancer,and has made great progress.Formononetin is one of the main active ingredients of astragalus membranaceus,which has certain anti-tumor effect on prostate cancer,breast cancer,bladder cancer,cervical cancer and colorectal cancer,etc.It is a kind of high-efficiency and low-toxicity drug,but there are few reports on gastric cancer.The phosphatidylinositol kinase 3/protein kinase B(PI3K/AKT)signaling pathway is closely related to cell proliferation,differentiation,metabolism,etc.,and this pathway can play an anti-apoptotic role through a variety of pathways,which is dysregulated in nasopharyngeal cancer,ovarian cancer,endometrial cancer and other malignant tumors.Anti-tumor drugs can exert anti-tumor effects by targeting the PI3K/AKT signaling pathway.This study aims to observe the effect of different concentrations of formononetin on proliferation and apoptosis of gastric cancer cell line mdc-803 and explore its possible mechanism,so as to provide new ideas for the clinical treatment of gastric cancer.Methods:1.MTT method was used to observe the proliferation changes of mgc-803 cells treated with formononetin at different concentrations for 24h,48h and 72h,respectively.Experiments in the logarithmic phase of MGC-803 cells,according to the 5×10~3 cells per hole spread in 96-well plates,set up the experimental group and control group,control group with excluding awn anthoxanthin complete medium culture of MGC-803 cells,the experimental group(20 umol/L,40 umol/L,80 umol/L)with complete medium containing mans anthoxanthin handle culture cell,the experimental group cells with the control group are respectively for 24 h,48 h,72 h,after training to the appropriate time,to add 20 ul determined by MTT solution respectively in every hole,The absorbance values of each group were measured at 490nm and the inhibition rate of cell proliferation was calculated.2.Hoechst 33258 staining was used to observe the effects of different concentrations of formononetin on MGC-803 nuclear morphology.In the experiment,MGC-803 cells in logarithmic growth phase were selected,and 4×10~4 cells per well were spread in a 24-well plate with aseptic slide.The experimental group and the control group were set up.3.The apoptosis of MGC-803 cells under different concentration of formononetin was observed by flow cytometryIn the experiment,MGC-803 cells at logarithmic growth stage were placed on6-well plates with 2×10~4 cells per well and incubated in the cell incubator for 12h.The control group and the experimental group were established,using the same method 1.Cells of the experimental group and the control group were cultured for48h,and the apoptosis rate was detected by flow cytometry according to annexin-apc/PI apoptosis kit.4.mRNA expression of BAX and BCL-2 in MGC-803 cells was detected by RT-PCRExperiments in the logarithmic phase of MGC-803 cells,2 x 10~5 cells per hole spread in 6 orifice,set up the control group and experimental group,with method 1,the experimental group cells with the control group were 72 h,and then according to the total RNA extraction box step extraction cell RNA,reverse transcription synthesis of cDNA,polymerase chain reaction(PCR),get the purpose gene mRNA,finally in the RT-PCR amplification,analysis,experimental results are obtained.5.Western blotting was used to detect the expression of p-AKT,AKT,BAX and BCL-2 in MGC-803 cells with different concentrations of formononetin.Experiment in the logarithmic phase of MGC-803 cells,2 x 10~5 cells per hole spread in 6 orifice,set up the control group and experimental group,with method 1,the experimental group cells with the control group were 72 h,and then extract the sample cell protein,BCA quantitative method of protein sample concentration,glue,on sample,electrophoresis,transfer film,closed,hatch a fight,fight,exposure,and finally the protein band grayscale values of Image J software analysis,calculate the Akt,p-Akt,Bax and relative expression of BCL-2 protein.Results:1.Effect of formononein on proliferation of MGC-803 cells in vitroThe results of MTT experiment showed that formononetin could inhibit the proliferation of MGC-803 cells,and the proliferation inhibition rate also showed an upward trend with the increase of formononetin concentration and action time,with statistically significant difference(P<0.05).2.Effect of formononein on nuclear morphological changes of MGC-803 in gastric cancerThe results of Hoechst 33258 staining showed that the cells in the control group(0umol/L)had uniform shape and size,showing light blue fluorescence.In the experimental group(20,40,80umol/L),after 48h of anthoxanthrin action,the cells showed different degrees of apoptosis:the nucleus showed nuclear shrinkage,nuclear dissolution,and nuclear fragmentation,presenting a granular shape or aggregation,and the apoptotic number of MGC-803 cells also increased with the increasing concentration of anthoxanthrin action.3.Formononein promotes the apoptosis of MGC-803 cellsFlow cytometry results showed that MGC-803 cells in the experimental group(20umol/L,40umol/L,80umol/L)showed a gradual increase in apoptosis rate of MGC-803 cells compared with the control group(0umol/L)after 48h of different concentrations of formonin,which was positively correlated with the concentration of formonin and showed a dose dependence.4.Effect of formononeanthin on mRNA expression of BCL-2 and BAX in MGC-803 cellsRT-PCR results showed that after 48h of MGC-803 cells cultured with different drug concentrations of formonanthocyanin,compared with cells of the control group,the relative expression level of BCL-2 mRNA in the experimental group showed a decreasing trend,while the relative expression level of BAX mRNA showed a rising trend,with statistically significant difference(P<0.01).5.Effect of formononetin on the expression of p-AKT,AKT,BAX and BCL-2 in MGC-803 cellsWestern blotting results showed that compared with the control group(0umol/L),the relative expressions of bcl-2 and p-AKT protein in the experimental group(20umol/L,40umol/L and 80umol/L)showed a decreasing trend and the relative expressions of BAX protein showed a rising trend with the increasing of formonin concentration in the experimental group(0umol/L),with statistically significant differences(P<0.01).Conclusion:1.Formononetin can significantly inhibit the proliferation and promote the apoptosis of MGC-803 cells in gastric cancer.2.Formononein can inhibit gastric cancer MGC-803 cells in vitro by up-regulating the expression of BAX and down-regulating the expression of BCL-2and p-AKT. |
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