| Purpose:In this study,the effects of ursolic acid on apoptosis and autophagy of gastric cancer cells in vivo and in vitro were studied by means of cytology,cell biology,molecular biology and xenograft tumor experiment in nude mice,To provide a rich experimental basis for the anti gastric cancer treatment of ursolic acid,and provide theoretical support for the traditional Chinese medicine treatment of gastric cancer.Materials and methods:1.Ursolic acid interferes with PI3K / AKT / mTOR signaling pathway to induce apoptosis and autophagic death of gastric cancer xenograftsThe first part of the experiment was divided into two groups: control group and ursolic acid intervention group.Human gastric cancer cell line MGC-803 was cultured and resuscitated: the cells were taken out from liquid nitrogen in a water bath box,quickly shaken to melt completely,and then transferred into a centrifuge tube,and the supernatant was centrifuged and discarded.The culture medium was added and cultured.Cell passage: take out the resuscitated cells,discard the original culture medium,add PBS to clean the cells,add trypsin digestion,carry out cell passage according to the appropriate proportion,store and culture in the incubator.Cell cryopreservation: after mixing the cell suspension,the cell suspension was divided into cryopreservation tubes,which were frozen at-80 ℃ for 24 h in the refrigerator,and then the cryopreservation tubes were frozen in liquid nitrogen for long term storage for use in subsequent experiments.Animal feeding and establishment of transplanted tumor animal model.Animal feeding: laboratory requirements: SPF level.Temperature(21-23)℃,humidity(40-50)%,12 h light / day.The animal feed water supply is sufficient and the cage can move freely.Change the disinfectant pad,feed and drinking water twice a week.Gastric cancer cells were inoculated 2 days after adaptive feeding.In the process of nude mice transplanted tumor modeling,all instruments and articles were strictly disinfected in accordance with the principle of aseptic operation,and completed in the ultra clean workbench.When transplanting,make sure the speed is fast and complete the transplanting work within 2 hours as far as possible to avoid contamination and infection.The cells in logarithmic growth phase were prepared into suspension and inoculated on the axillary back with loose skin,good blood supply and easy to observe and measure.Strengthen postoperative care and keep the articles used in nude mice clean.After 7 days,all nude mice had subcutaneous tumor,and the model was established successfully.Group: control group and ursolic acid intervention group.Six rats in each group were given intervention on the same day.Control group,normal saline,QD,intraperitoneal injection.In the ursolic acid intervention group,the body weight of ursolic acid was 0.05mg/g,dissolved in 0.2ml normal saline,QD,intraperitoneal injection.The inhibitory effect of ursolic acid on the proliferation of gastric cancer cells in vivo was evaluated by detecting the volume and weight of gastric cancer xenografts,Ki67 and PCNA by immunohistochemistry.He staining section and immunohistochemical staining section of the production of cancer tissue samples fixed,placed in the embedding box marked number,put into the dehydrator overnight,dehydrator to complete the fixation,dehydration,wax and cleaning steps.After embedding,the tissue was put into the embedding mold with tweezers,and the wax block was put on the cooling table for cooling.The wax block was fixed on the slicer and sliced with a thickness of 3-5μm.Take out the slide.Hematoxylin eosin staining was carried out by automatic dyeing machine,and he slices were made as quality control slices.Seal with neutral gum,cover with glass slide and dry in the air.After removing the flakes,bake them at 65 ℃ for 60 min.After adding the corresponding primary antibody(Ki67,PCNA,Cleaved-caspase 3,Cleaved-caspase 9,Bax,Bcl-2,Beclin-1,P62,LC3,PI3K,p-AKT,p-mTOR,ULK1)and other related reagents into the automatic immunohistochemical staining machine,the staining machine was started.After about 4 hours,the slides were taken out and cleaned.Dehydrated,transparent,sealed with central gum,covered with glass slide and dried in the air.The results were observed and recorded under microscope.The expression of apoptosis related proteins Cleaved-caspase 3,Cleaved-caspase 9,Bax and Bcl-2 was detected by immunohistochemical staining,and the expression of apoptosis related proteins Cleaved-caspase 3,Cleaved-caspase 9,Bax and Bcl-2was detected by WB method.On this basis,the expression of autophagy related proteins LC3,Beclin-1 and P62 was detected by immunohistochemistry.The expression of autophagy related proteins LC3,Beclin-1 and P62 were detected by WB.The expressions of PI3K,p-AKT,p-mTOR were detected by immunohistochemistry,and the expressions of PI3K,p-AKT,p-mTOR and ULK1 were detected by WB.Objective to analyze the effects of ursolic acid on apoptosis,autophagy and signal transduction pathway of gastric cancer cells.2.Ursolic acid interferes with PI3K / AKT / mTOR signaling pathway and induces apoptosis of gastric cancer cellsIn the second part,human gastric cancer cell line MGC-803 was selected for culture,and the cells were resuscitated,subcultured and cryopreserved in strict accordance with the experimental procedures.CCK8 method was used to detect cell proliferation: the standard curve was made: the cell count plate was selected to calculate the cell number in the cell suspension,and the cells were inoculated.The cells were diluted with medium to obtain the concentration gradient.After inoculation,the cells were cultured for adherence.After adding CCK8 reagent for 4 h,the OD value was determined.Draw the standard curve.Abscissa: cell number;ordinate: OD value.To evaluate the number of cells in subsequent experiments.Detection of cell proliferation: the concentration was 0,20,40,60,80(μmol/L),and the time was 0,12,24,36,48,72(h).CCK 8 solution was added to each group.The absorbance was determined by enzyme-linked immunosorbent assay.The OD value of each measurement was recorded,and the average value was calculated after 3 measurements of each sample.The optimal dosage and time of ursolic acid were determined.The optimal concentration of ursolic acid was 40μmol/L and 48 h.The second part of the experiment was divided into three groups:blank control group,40μmol/LUA,40μmol/LUA + Z-VAD-FMK group.The time was set to48 hours.The cell growth was observed by inverted phase contrast microscope: the cells were cultured according to the experimental requirements,and the ursolic acid medium was added according to the experimental concentration after the cells were completely adherent to the wall,and the number and morphological changes of cells in each group were observed by inverted phase contrast microscope after 48 hours of continuous culture.The apoptosis level of each group was detected by flow cytometry: after inoculating cells,different concentrations of ursolic acid were added according to the experimental groups.The apoptosis of each group was detected and analyzed by flow cytometry through trypsin digestion,buffer washing,centrifugation,re washing,re suspension,repeating the above steps twice,fixation,centrifugation,adding rnaseaa,PI,and avoiding light overnight.The expressions of Cleaved-caspase 3,Cleaved-caspase 9,Bax,Bcl-2 PI3K,p-AKT,p-mTOR were detected by western blot method.Protein extraction: after 48 hours of cell culture,the culture medium was discarded,the culture bottle was inverted,and the culture medium was dried with absorbent paper.Add PBS buffer and shake gently to wash,then pour out the washing liquid.Wash 3times and place on ice.Add protein lysate,operate on ice,centrifugate with protein lysate,and transfer the supernatant to centrifuge tube.Detection of protein content: the working solution of BCA was prepared according to the instructions of BCA protein concentration determination kit.The absorbance value of each hole was recorded.The standard curve was drawn with protein content as abscissa and absorbance value as ordinate.According to the absorbance value of the sample,find out the corresponding protein content on the standard curve,divide it by the volume of the sample,and calculate the actual concentration of the sample.Preparation of gel: reference guide is used to separate separating glue and concentrated glue,then wrap it with wet gauze,seal it and put it overnight at 4 ℃.Electrophoresis: put the glue into the installed electrophoresis tank.The electrophoresis conditions were determined.The concentration of gel was 80 V for 15 min.After the electrophoresis of concentrated gel,the separation gel,120 V,40 min,marker runs to the bottom of gel.Membrane transfer: the condition of membrane transfer: 80 m A,3 h.Clean the membrane with skim milk powder sealing solution.Sealing: after the completion of membrane transfer,the membrane was placed in the sealing liquid and vibrated in water bath at 37 ℃ for 1 h.Incubation: including primary antibody incubation and secondary antibody incubation.Imaging: according to the instruction manual,a,B and other volumes are prepared,and then wrapped with tin foil.After the film is placed on the exposure plate,appropriate amount of luminous liquid is dripped.F3 automatic fluorescence and visible light gel imaging analysis system was used to observe the results.Objective to investigate the mechanism of ursolic acid induced apoptosis in gastric cancer cells.3.Ursolic acid interferes with PI3K / AKT / mTOR signaling pathway and induces autophagic death of gastric cancer cellsThe third part of the experimental group: blank control group,40 μmol / LUA,40 μmol /LUA + 3-MA group.The time was set to 48 hours.The occurrence of autophagy was detected by MDC method.After 48 hours of culture,the culture medium was discarded and digested into single cells with trypsin.After centrifugation,the supernatant was discarded,the cells were collected,washed with buffer,and the supernatant was discarded.Buffer was added to make the cell concentration reach the standard.Then the cells were aspirated and MDC Stein was added into the centrifuge tube for mixing.Wash with buffer and discard the supernatant.The cells were resuspended with collectin buffer.The cells were observed under fluorescence microscope and photographed.Autophagy was detected by double fluorescent m RFP-e GFP-LC3 plasmid transfection.Prepare the working solution of transfection,take EP tube,add serum-free medium,add shuttle plasmid and packaging plasmid containing the target sequence,and mix well.Take EP tube,add serum free medium,and then add RNAi mate to mix well.After placing at room temperature,mix the two tubes.When the cells were cultured in the culture bottle to reach the appropriate fusion degree,the cells were transfected.Discard the medium PBS buffer,wash the cells twice,wash away the residual medium,add serum-free medium,and then add the transfection working solution drop by drop.After shaking and mixing,incubate in the incubator,discard the transfection working solution and add it to the complete medium incubator for incubation.The changes of red and green LC 3bright spots in the cells were observed under fluorescence microscope,and photos were taken.The expression of LC3,Beclin-1,P62,PI3K,p-AKT,p-mTOR and ULK1 protein in each group were observed by WB method and F3 automatic fluorescence and visible gel imaging analysis system.Immunofluorescence cytochemistry was used to stain and seal.The fluorescence signals of PI3K,p-AKT,p-mTOR and ULK1 were detected by confocal laser scanning microscope.Results:1.Ursolic acid interferes with PI3K / AKT / mTOR signaling pathway to induce apoptosis and autophagic death of gastric cancer xenograftsIn the first part,gastric cancer cell line MGC-803 was used to establish subcutaneous tumor model in nude mice.Ursolic acid could significantly inhibit the growth of gastric cancer xenografts.The weight and volume of gastric cancer xenografts in ursolic acid intervention group were significantly reduced.Compared with the control group,the tumor volume of ursolic acid intervention group was significantly reduced(P<0.05).Compared with the control group,the weight of transplanted tumor in ursolic acid group was significantly reduced(P<0.05).PCNA and Ki67 are tumor cell proliferation factors.Compared with the control group,the expression of PCNA protein in ursolic acid intervention group was significantly lower.Compared with the control group,Ki67 protein expression in ursolic acid intervention group was significantly lower.The expression of apoptosis related proteins was detected by immunohistochemistry: compared with the control group,the expression levels of cleaved caspase 3 and cleaved caspase 9 in the ursolic acid intervention group were significantly increased,the expression level of Bax protein was also increased significantly,and the expression level of Bcl-2 protein was significantly reduced.Results: compared with the control group,the expression level of cleaved caspase 3 and cleaved caspase 9 in the ursolic acid intervention group was significantly increased,the expression level of Bax protein was also significantly increased,the expression level of Bcl-2 protein was significantly reduced,the difference was statistically significant(P < 0.05).The results of immunohistochemistry for autophagy related protein were compared with the control group,LC3 Ⅱ protein expression,beclin-1 protein expression increased and P62 protein expression decreased in ursolic acid intervention group.Results: compared with the control group,LC3Ⅱ protein expression level was significantly increased in ursolic acid intervention group,and the expression level of beclin-1 protein was also significantly increased,and the expression level of P62 protein was significantly reduced,and the difference was statistically significant(P < 0.05).The results of immunohistochemistry were used to detect the signal transduction pathway related protein of PI3K / AKT / mTOR: compared with the control group,the expression of PI3K,p-AKT and p-mTOR in the ursolic acid intervention group was decreased,and the expression of ULK1 increased.The results of the detection of PI3K / AKT / mTOR signal pathway related proteins by Western blot: compared with the control group,the expression levels of PI3K,p-AKT and p-mTOR in the ursolic acid intervention group were significantly reduced,and the expression level of ULK1 was significantly increased,and the difference was statistically significant(P < 0.05).2.Ursolic acid interferes with PI3K / AKT / mTOR signaling pathway and induces apoptosis of gastric cancer cells Ursolic acid significantly inhibited the proliferation of gastric cancer cell line MGC-803 in a concentration and time-dependent manner.0-80μmol/L The inhibition rate of ursolic acid on gastric cancer cell proliferation increased with the increase of ursolic acid concentration.The best concentration of ursolic acid is 40 μmol / L.The optimal time of action was 48 hours.The results of inverted phase contrast microscope showed that MGC-803 cells grew vigorously in the blank control group with only culture medium,showing epithelial cell like type,with different sizes,polygonal or slender shape,clear cell outline,large nucleus and tight cell to cell connection.Compared with the blank control group,40μmol/LUA intervention group,the number of gastric cancer cells MGC-803 decreased,the cell proliferation was inhibited,the gap between cells increased,the extensibility was poor,the morphology of some cells changed,and the surface granulation and fragmentation were more obvious,suggesting that the cells may have undergone apoptosis at this time.Flow cytometry showed that: compared with the blank control group,40μmol / LUA intervention group,the apoptosis rate was significantly increased in the treatment group(P<0.05).And compared with the 40μmol / LUA intervention group,the apoptosis rate was significantly decreased in the Z-VAD-FMK + 40μmol / LUA group(P<0.05).WB test results showed that: compared with the blank control group,40μmol / LUA intervention group,the protein expression levels of caspase 3 and caspase 9 were significantly increased(P<0.05),the protein expression level of Bax was also significantly increased(P<0.05),and the protein expression level of Bcl-2was significantly decreased(P<0.05).And compared with the control group,40 μmol / LUA+ Z-VAD-FMK group,the protein expression levels of Cleaved Caspase 3 and Cleaved Caspase 9 were significantly decreased(P<0.05),the protein expression level of Bax was also significantly decreased(P<0.05),and the protein expression level of Bcl-2 was significantly increased(P<0.05).The expression of PI3K / AKT / mTOR signaling pathway related proteins was detected by Western blot.Compared with the control group,40 μ The protein expression levels of PI3K,p-AKT and p-mTOR were significantly decreased in the treatment group(P < 0.05).And 40 μ Compared with the control group,40 μ The protein expression levels of PI3K,p-AKT and p-mTOR were significantly increased in mol / LUA +Z-VAD-FMK group(P<0.05).3.Ursolic acid interferes with PI3K / AKT / mTOR signaling pathway and induces autophagic death of gastric cancer cellsThe results of MDC method showed that in 40μmol / LUA grounp,the number and intensity of dot like fluorescence signal increased in the treatment group.And compared with the control group,40μmol / LUA + 3-MA group,the number and intensity of dot like fluorescence signal decreased.The results showed that 40μmol / LUA grounp,the number of green and red fluorescent spots in the 40μmol / LUA grounp intervention group was significantly increased(P<0.05).And 40μmol / LUA grounp compared with the control group,the number of green and red fluorescent spots decreased significantly in 40μmol / LUA +3-MA group(P<0.05).WB method was used to detect the protein expression of each group μThe expression level of LC3 Ⅱ protein,Beclin-1 protein and P62 protein were significantly increased(P<0.05)and decreased(P<0.05).And compared with the control group,40μmol /LUA grounp,the expression of LC3 Ⅱ,Beclin-1 and P62 were significantly decreased(P<0.05)and increased(P<0.05)in 40μmol / LUA + 3-MA group.WB method was used to detect the protein expression of each group.The protein expression levels of PI3K,p-AKT and p-mTOR were significantly decreased(P<0.05)and the protein expression levels of ULK1 were significantly increased(P<0.05).And compared with the control group,in40μmol/ LUA intervention group,there was no significant difference in PI3K,p-AKT,p-mTOR and ULK1 protein expression levels between the two groups(P>0.05).Immunofluorescence cytochemical staining method was used to detect the protein expression of each group.The expression of ULK1 protein was stronger than that of PI3K,p-AKT and p-mTOR in the treatment group And compared with the control group,40μmol / LUA intervention group,the protein expression of PI3K,p-AKT,p-mTOR and ULK1 had no significant change in 40μmol / LUA + 3-MA group.Conclusion:1.Ursolic acid can inhibit the growth of gastric cancer cells in vitro and in vivo.2.Ursolic acid inhibits the growth of gastric cancer cells by inducing apoptosis and autophagic death.3.Ursolic acid can induce apoptosis and autophagic death of gastric cancer cells by interfering with PI3K / AKT / mTOR signaling pathway. |
PDF Full Text Request