Comparative Research Into The Inhibitory Effect Of Matrine On Differentiated Human's Gastric Cancer Cells Based On ERS Pathway

BackgroundGastric cancer is a common malignant tumor of digestive tract,which is hard to be cured.It has poor prognosis and high mortality and it badly threatens to human health.According to our country's cancer statistics in2015,it found that in China,there were about 679000 patients with gastric cancer in all,ranking only second to lung cancer?733000?^[1],and the number of death was about 498000,which ranked the second in the tumor death rate,only lower than lung cancer too.So in the medical and health field in China,the prevention and treatment of gastric cancer is the first problem to be solved.However,the way that patients with gastric cancer are often treated by comprehensive surgery in clinical practice,doesn't have a good effect for patients in the middle and late stage.The one-year transfer rate is 38%,and the five-year 76%^[2].In recent years,people has paid more attention to the diagnosis and treatment of gastric cancer by traditional Chinese medicine.Not only can traditional Chinese medicine reduce the various clinical symptoms of gastric cancer,but also is good for relieving the side effects of chemotherapy,improving the treatment outcomes,and prolonging the time to live.Matrine is an alkaloid extracted from the roots and above ground portions of the Chinese herbal kuh-seng^[3].It is believed that in traditional Chinese medicine,kuh-seng is cold in property,bitter in flavour and goes into the stomach,which has the function of clearing away the heat,detoxifying the body and killing the parasites.So it is usually used to cure eczema,dysentery,sore throat and cough and so on by the public.However,the Western medicine suggests that oxymatrine may be effective in anti-inflammatory,anti-allergic,anti-viral,anti-fibrotic and cardiovascular protective^[4].For the past few years,it have been found in the studies that it is oxymatrine that can inhibit the reproduction of many kinds of tumor cells,including many kinds of gastric cancer cell lines^[5-11],while there are rare studies reported about oxymatrine's differently inhibitory effect on human's differentiated gastric cancer cells.From the first normal gastric mucosal epithelial cells to the final gastric cancer cells undergoing many steps,the development of gastric cancer is a gradual process.To get the differently differentiated levels of human gastric cancer cell line,the researchers used the professional technologytomakeitimmunizationbytakingdifferentclinical differentiation of gastric cancer tissue including tumor tissue of high,medium and low differentiation.Not only did artificial cultivation of the differentiationofgastriccancercelllineskeepthedifferencein morphology and growth rate,but also from microscopic aspect,showed the difference of gene mutation expression of gastric cancer.In the latest years,a lot of research on the anti-tumor effect of oxymatrine has been done by many experts and scholars including my tutor himself,but there aren't any articles about the inhibitory effects of oxymatrine on gastric cancer cells withdifferentdifferentiationlevelsreportedinthenewspaper,so Comparatively researching the inhibitory effect of oxymatrine on low,middle and high differentiated human's gastric cancer cells and the expression of related indicators in endoplasmic reticulum stress pathway was needed.It can not only further prove oxymatrine's inhibitory effect on this there kinds of human gastric cancer cells,but also found the obvious differences in the concentration of oxymatrine drug between the differently differentiated degrees of gastric cancer cell lines.It provides the experimental basis for that oxymatrine can be applied to the later clinical treatment of the different malignant degree of gastric cancer in the individual therapy.ObjectiveComparatively researching the difference between the inhibitory effect of oxymatrine on human differentiated gastric cancer cells through the vitro experiment,the study explores the differences in the drug response of oxymatrine to different differentiation degree of gastric cancer cells and clarifies the molecular biological differences of differentiated gastric cancer cells induced by matrine,which provide a certain experimental basis for the individualized oxymatrine treatment of gastric cancer.Methods1.The human gastric cancer cells of different differentiation levels were cultured,and the cells in good growth were used to do the experiment.The cells in each group are divided into six groups including positive control group?0.5mg/ml fluorouracil?,negative control group?cell culture medium of equal volume,no oxymatrine?,2mg/ml oxymatrine drug group,4mg/ml oxymatrine drug group,6mg/ml oxymatrine drug group,8mg/ml oxymatrine drug group.I detect the proliferation activity of each cell line after12h,24h,36h and 48h intervented of oxymatrine by using MTT method and calculate its proliferation inhibition rate.Then calculate the half inhibitory concentration IC50 about each cell at every time point by using SPSS17.0and select the subsequent experiment concentration.2.After Annexin-V FITC/PI double staining,observing the apoptosis morphology of human differentiated gastric cancer cells after 24h and 48h by using confocal microscopy technique.3.Determinating the cell apoptosis and cell cycle of human differentiated gastric cells at 24h and 48h after oxymatrine injection by using FCM.4.Detect the difference between the expression levels of apoptosis-related mRNA in 24h and 48h after oxymatrine injection by the PCR method.5.Detect the difference in expression of apoptosis-associated proteins at24h and 48h of oxymatrine on human differentiated gastric cancer cells by the WB method.Results1.The experimental results about MTT?1?Matrine has inhibitory effect in vitro on three differently differentiated stages of human gastric cancer cells,and the results is statistically significant compared with negative control group cells?P<0.05?.In addition,the growth inhibition rate of those three cells rise with the increase of time and dose.?2?In general,after the Marine injection if the cells differentiated levels is different,the inhibitory effect is also different,the lower about the degree of cells differentiation,the inhibition effect is stronger.?3?Using SPSS 17.0 to calculate the IC50 of each cell in each time point.Experiment 2 chose the IC50 of SGC7901 cells in 48h as the concentration of following-up experiment.2.Observating the apoptosis morphology below confocal microscopyBelow the confocal microscope MKN28,SGC7901 and MGC803 cells respectively show apoptotic morphology.It is mainly manifested in red staining of the nucleus,green dyeing of the cell membrane,and cells volume solidification tend to be round,the cytoplasm is uneven,and the cell nucleus is marginalized or fragmented,showing a granular shape.3.Detecting the cells apoptosis and cells cycle taking advantages of FCM3.1 The cell apoptosisAfter the same concentration of matrine treatment three groups in 24h and 48h respectively,the apoptosis rate of MGC803 and SGC7901 cells group arehigherthanMKN28,andthedifferencesarestatistically significant?P<0.05?.3.2 The cell cycleAfter the action of matrine,the proportion of cells in G₀/G₁ stage is higher than that in S and G₂/M stage among the MKN28,SGC7901 and MGC803 cell group.4.The results with regard to RT-qPCRThe results show that Caspase-3,Caspase-4 and CHOP mRNA relative expression level increases along with the degree of differentiation decreases,but GRP78 mRNA decreases.They both show significant difference.5.WB resultsThe results reveal that the GRP78 protein relative expression level decreases along with the degree of differentiation decreases,but Caspase-3,Caspase-4 and CHOP protein increases.They both show significant difference.Conclusion1.Oxymatrine both have the inhibitory effect on MKN28,SGC7901 and MGC803 cells,and the inhibition effect has the dose and time dependent relationship.2.The degree of inhibition effect is defferent among there groups.3.The apoptosis effect of oxymatrine on MKN28,SGC7901 and MGC803cell is different,and the effect on MGC803 cells is strongest.Oxymatrine could be inhibited gastric cancer cells in G₀/G₁ phase to induce apoptosis.4.Matrine can induce apoptosis of MKN28,SGC7901 and MGC803 cell,and the effect of increasing Caspase-3,Caspase-4,CHOP and lowering the expression of GRP-78 mRNA and protein are different.