ABSTRACT
AIM:To observe the effect of Pingwei capsule on the precancerous lesions of gastric cancer(PLGC)cell model induced by N-methyl-N'-nitro-N-nitrosoguanidine(MNNG),and to preliminarily explore its mecha-nism.METHODS:Blank serum and Pingwei capsule-containing serum were prepared for later use.A PLGC cell model was established by MNNG-induced human gastric mucosal epithelial cell line GES-1.To evaluate the model,cell morpho-logical changes were observed under inverted microscope,and the expression of proliferating cell-related antigen Ki67 was detected by immunofluorescence staining.CCK-8 assay was used to screen the optimal intervention concentration and time of serum containing drugs.Reactive oxygen species(ROS)content in cells was detected using a fluorescent probe DCFH-DA.Malondialdehyde(MDA)content was detected by ELISA,and the activity of superoxide dismutase(SOD)and gluta-thione peroxidase(GSH-Px)was detected using biochemical reagents.A novel fluorescent probe JC-10 was used to detect mitochondrial membrane potential.The mRNA expression levels of Ki67 and melanoma differentiation-associated gene-7(MDA-7)were detected by real-time fluorescence quantitative PCR.The protein expression levels of Ki67,interleukin-6(IL-6)and MDA-7 were detected by Western blot.RESULTS:Compared with normal group,the ROS and MDA levels in model group and blank serum group were significantly increased(P<0.01),while the activity of SOD and GSH-Px was significantly decreased(P<0.01).The mitochondrial membrane potential was significantly decreased(P<0.01).The protein expression levels of Ki67 and IL-6 were significantly increased(P<0.01),while the protein expression level of MDA-7 was significantly decreased(P<0.01).There were no significant differences of the above indicators between model group and blank serum group(P>0.05).Compared with blank serum group,the Pingwei capsule-containing serum group showed significantly decreased ROS and MDA levels(P<0.01),significantly increased activity of SOD and GSH-Px(P<0.05),significantly increased mitochondrial membrane potential(P<0.01),significantly decreased protein expres-sion levels of Ki67 and IL-6(P<0.01),and significantly increased protein and mRNA expression levels of MDA-7(P<0.01).CONCLUSION:Pingwei capsule can significantly alleviate MNNG-induced oxidative damage and inflammatory response,and regulate the expression of oncogenes and tumor suppressor genes,thereby playing a role in prevention and treatment of PLGC.
ABSTRACT
ObjectiveTo explore the possible mechanism of Pingwei Capsules （平胃胶囊） for chronic atrophic gastritis from rapidly accelerated fibrosarcoma / mitogen-activated protein kinase /extracellular-signal-regulated kinase （Raf/MEK/ERK） pathway that influences the activation of fibrosarcoma protein/mitogen. MethodsFifteen SD rats were randomly divided into 5 rats in the blank group and 10 rats in Pingwei Capsules group. The rats in the blank group were given 1 ml/100 g of saline by gavage， and the rats in Pingwei Capsules group were given 0.63 g/（kg·d） of Pingwei Capsule suspension by gavage， and serum was collected for 3 consecutive days. N-methyl-N'-nitro-N-nitrosoguanidine （MNNG） was used to induce human gastric mucosal epithelial cells GES-1 to establish a precancerous lesion cell model. The successful cells were divided into control group （10% fetal bovine serum）， blank serum group （10% fetal bovine serum plus 10% blank serum）， and medication-containing serum group （serum with medication of Pingwei Capsule）， and the volume fraction and time of intervention of Pingwei Capsule-containing serum were screened by CCK-8 assay. Human gastric mucosal epithelial cells GES-1 were divided into normal group， model group， blank serum group， medication-containing serum group， U0126 group， and combined group， with 6 replicate wells in each group. After successful modelling of the cells in all groups except the blank group， an equal volume of fetal bovine serum was added to the normal and model groups， an equal volume of blank serum was added to the blank serum group， a screening volume fraction of Pingwei Capsule-containing serum was added to Pingwei Capsule group， a 10 μmol/L mitogen-activated extracellular signal regulated kinase 1 （MEK1） inhibitor U0126 was administered in the U0126 group， an equal dose of Pingwei Capsule-containing serum plus 10 μmol/L of U0126 was administered to the combined group. After the selected incubation time， the level of interleukin 6 （IL-6） was detected in the cells by ELISA， the expression of IL-6 and MEK1 was detected by immunofluorescence， and the expression of IL-6， Raf， MEK1， and ERK mRNA was detected by RT-qPCR， and the expression of IL-6， Raf， MEK1， and ERK mRNA in the cells was detected by Western blot. ResultsThe 5.35% volume fraction， 48 h intervention of Pingwei Capsule-containing serum was selected for subsequent experiments. Compared with the normal group， the IL-6 content in cell supernatants and the expression of IL-6， Raf， MEK1， ERK mRNA and ERK1/2 proteins in cells increased in the model group and blank serum group （P＜0.01）. Compared with the model group， all of the above indexes were improved in medication-containing serum group， U0126 group， and combined group （P＜0.05 or P＜0.01）. Compared with medication-containing serum group， the expression of IL-6， MEK1 expression， the expression of IL-6， Raf， MEK1 and ERK mRNA， and the expression of IL-6， Raf， MEK1 and ERK1/2 proteins reduced in the cells of combined group （P＜0.05 or P＜0.01）. Compared with the U0126 group， IL-6 expression reduced and IL-6， MEK1 and ERK1/2 protein expression reduced in cells of combined group （P＜0.05 or P＜0.01）. ConclusionThe Pingwei Capsule-containing serum may play a role in the treatment of chronic atrophic gastritis by improving the inflammation-cancer transformation of GES-1 cells through inhibiting the Raf/MEK/ERK pathway.