| Objective: The aim of this study was to investigate the effect of weining granule on autophagy of gastric cancer cells by detecting the levels of autophagy and pathway-related proteins and genes,so as to explain the mechanism of weining granule in the treatment of gastric cancer.To provide the experimental basis for treating gastric cancer with weining granule in the future.Methods: 1.Preparation of medicated serum: 26 male SPF rats were divided into normal saline group,weining granule group and western medicine group.The rats in the saline group were given 10 ml/kg/d of normal saline by gavage.The rats in the traditional Chinese medicine group were given weining granules by gavage at 18.38g/kg/d.The rats in the western medicine group were given intraperitoneal injection of 5-fluorouracil(diluted with 9.0 g/l sodium chloride injection),each rat was given 20mg/kg.After 7 days of continuous administration,the abdominal aorta was collected,centrifuged and inactivated for later use.2.Cell intervention culture: Human gastric cancer SGC-7901 and MKN-45 cells were cultured in 1640 basal medium supplemented with fetal bovine serum(FBS)to logarithmic growth stage and then divided into blank group,saline group,weining granule group and western medicine group.The blank group was cultured with fetal bovine serum,and the other three groups were added with corresponding drug-containing serum.Using cell proliferation test(CCK-8),the cells were treated with serum containing10%,15% and 20% of the medicated serum for 12 h,24h and 48 h,respectively,in order to find the best effective concentration and time of the intervention.concentration and time.The changes in cell morphology and green fluorescence of gastric cancer cells after MDC staining were observed under the microscope.To analyze autophagy formation and mean fluorescence intensity between groups.The expression levels of autophagy-related protein LC3 and p62 were detected by Western blotting,meanwhile,the changes of AMPK/m TOR pathway related proteins AMPK,m TOR and their phosphorylated proteins p-AMPK and p-m TOR were detected.The expression of LC3,P62,AMPK and m TOR m RNA in SGC-7901 and MKN-45 cells were detected by real-time fluorescence quantitative polymerase chain reaction assay.Results: 1.The results of the CCK-8 proliferation experiment showed that,compared with the normal saline group,the proliferation of gastric cancer cells in the group of weining granule was significantly inhibited.The inhibitory effect was dose-dependent and independent of time,the best intervention concentration was 20% and the best intervention time was 24h(P<0.01).2.After 24 h intervention with 20% medicated serum,the cells in the blank group and the normal saline group adhered to the wall well,and the cells were well defined and closely arranged.The morphology of the cells in weining granule group and western medicine group was changed,the cell state was poor and the cell density was small.3.MDC staining showed that the autophagy level in the blank group and the saline group was low,and the cytoplasm was uniform light green,while the weining granule group and the western medicine group showed strong fluorescence signal.By calculating the average fluorescence intensity,compared with the normal saline group,the weining granules group and the western medicine group had higher fluorescence intensity(P<0.01);in SGC-7901 cells,the average fluorescence intensity level of the weining granules group was much higher than that of the western medicine group(P<0.01).4.Western blotting showed that weining Granules could promote the transformation of LC3Ⅰ and LC3Ⅱ and down-regulate the protein expression of p62;increase the expression of AMPK protein,and increase the ratio of p-AMPK/AMPK,down-regulate of m TOR protein level,and decrease the ratio of p-m TOR/m TOR(P<0.05).5.The results of real-time fluorescence quantitative polymerase chain reaction showed that compared with the normal saline group,the level of LC3 m RNA in weining granule group was significantly increased,while the level of p62 m RNA was significantly decreased(P<0.01).Compared with normal saline group,AMPK m RNA expression was increased in weining granule group and western medicine group,while m TOR m RNA expression was down-regulated(P<0.05).Conclusions: 1.Weining granule can increase the expression level of LC3 protein and gene,and down-regulate the expression of p62 protein and gene.weining granule can induce autophagy in gastric cancer cells.2.Weining granules can up-regulate the phosphorylation level of AMPK and inhibit the activation of m TOR protein in gastric cancer cells.The mechanism of autophagy may be related to AMPK/mTOR pathway. |
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