Effect On H.Pylori-infected Mice Gastritis And Cytotoxicity On Human Gastric Epithelial Cells Of Total Alkaloid Of Sophora Alopecuroides

Objective To investigate the possible mechanism of total alkaloid of Sophora alopecuroides (TASA) on H.pylori-infected BALB/c mice gastritis in vivo and observe the cytotoxicity of TASA on human gastric epithelial cell in vitro, and we assumed that the study could provide evidence and suggestions for us to expore the new anti-H.pylori drugs.Methods In vivo BALB/c mice were orally inoculated with H.pylori bacterial liquid, to construct H pylori infection gastritis animal model. After the model were successful generated, the mice were randomly dividedinto 10 groups (n=10), there were negative control group(the normal saline group), bismuth pectin group, omeprazole group, low dosage of TASA group, middle dosage of TASA group, high dosage of TASA group, TASA+bismuth pectin group, TASA+ omeprazole group, bismuth pectin+clarithromycin+metronidazole group, the omeprazole+ clarithromycin+metronidazole group. After corresponding treatment with intervention, HE staining were carried out to observe the morphology changes of mice gastric mucosa. The expression level of interleukin 8 (IL-8), cyclooxygenase 2 (COX-2), and nuclear factor-kappa B (NF-κB) in gastric mucosa tissue were detected by immunohistochemical. In vitro, we cultured human gastric epithelial cell line GES-1 with TASA of different concentrations of TASA (0.2 mg/ml,0.4mg/ml,0.8 mg/ml,1.6mg/ml,3.2mg/ml,6.4 mg/ml) for 24,48 and 72 hours. And then assessed the cell proliferation with a cell counting kit-8 assay, and calculated the inhibition rate of GES-1 cell cultured with different concentrations of TASA, and the median inhibitory concentration of 48 hours time point.After cocultivation with drug in 48 hours, we observed the morphological changes under inverted microscope, flow cytometry was also used to analyse the cell apoptosis and transmission electron microscope was used to determine the changes of cell ultrastructure.Results Firstly, in vivo part of the study, the TASA treatment group conducted gastric mucosa atrophy and decreased the infiltration of inflammatory cells. TASA combined with bismuth or omeprazole group improved gastric mucosa infiltration of inflammatory cells. The expression level of IL-8, COX-2,NF-κB in the gastric mucosa tissue were significantly lower in the low dosage of TASA group, the middle dosage of TASA group, the high dosage of TASA group, TASA combined bismuth pectin group and TASA combined omeprazole group compared with the negative control group(P<0.01), The expression level of IL-8, COX-2, NF-κB in the gastric mucosa tissue were lower in TASA combined bismuth pectin group the TASA combined omeprazole group compared with low dosage of TASA group, middle dosage of TASA group, and high dosage of TASA group(P<0.01,.P<0.05), there were no statistical differences compared with the conventional trimodality therapy(P>0.05). Secondly, in vitro part of the study, our results clearly showed that TASA exhibited inhibitory activity on GES-1, the inhibition rate increased with drug concentrationat each time point (24h,48h,72h), the median inhibitory concentration was determined as 0.42mg/ml at 48 hours. After cells were intervented for 48 hours, observation by inverted microscopy demonstrated that the morphological alterations including cell shrinkage, refractivity increased, suspension, nuclear fragmentation, pyknosis or lysis, and the higher concentration, the more obvious changes above. The early stage of apoptosis rates of different concentrations of TASA(negative control,0.4mg/ml,1.6mg/ml) were 1.28%,48.55% and 68.04%, respectively.The late stage of apoptosis rates of TASA(negative control,0.4mg/ml,1.6mg/ml) were 0.12%,7.85% and 10.65%,respectively. Compared to control cells,TASA destructed cells with structural alterations, microvillus arrangement in disorder, cytoplasm density increased and vacuolization, nuclear morphology irregular and dissociation, and decreasion of nuclear proportion.Conclusions TASA can reduce expression of IL-8,COX-2,and NF-κB in H.pylori-infected mice gastritis mucosa tissue, and relieve inflammation of H.pylori-infected mice gastritis, while combined with bismuth pectin or omeprazole shows the best effect. TASA conducted gastric mucosa atrophy by single treatment, and no significantly effect while combined with bismuth or omeprazole.TASA can inhibit GES-1 cells growth in vitro in a dosage dependent behavior, and promote apoptosis and influence morphology changes, shows the cytotoxicity on GES-1 cells.