Objective To investigate optimal method of establishing hypoxia/reoxygenation(H/R) model of H9c2 cell by using hypoxia/anoxic workstation under different conditions in hypoxia.Methods H9c2 cell was placed into hypoxia/anoxic workstation and simultaneously cultured with complete medium, glucose-free DMEM and acidic hypoxic solution for 1,2,4,6 and 8 h respectively, and then reoxygenated with complete medium for 1 h in normoxic incubator.The level of ROS was measured by flow cytometry.The cell viability was detected by MTT assay.The cellular morphology was observed by inverted microscope.Results With the extension of cell hypoxia time, there were no significant differences in the ROS level and cell viability in complete medium-and glucose-free DMEM-treated H/R groups compared with control group(P<0.05).There was no obvious morphologic change observed with inverted microscope, either.Nevertheless, when H9c2 cells were treated with acidic hypoxic solution in hypoxia, the ROS level continuously increased and the cell viability decreased with the extension of cell hypoxia time ( P<0.01 ).Since H:1 h/R:1 h, some of the cells shrunk and a few necrotic cells floated in the media under the inverted microscope , and the damage was aggravated with the extension of hypoxia time.After the cells were exposed in hypoxia for 8 h, they wrinkled to be round and a large number of floating necrotic cells were observed.When the cells were reoxygenated for 1 h, the cytomembrane was not smooth and there were still a few necrotic cells floating in culture dish .Conclusion The H9c2 cell H/R model with good repeatability can be established successfully by using hypoxia/anoxic workstation combining with acidic hypoxic solution.

OBJECTIVE：To study the improvement effect of Shenling baizhu p owder on irritable bowel syndrome with diarrhea（IBS-D）model mice with anxiety ，and to elucidate its mechanism from the point of view of intestinal microecology. METHODS：C57 BL/6 mice were randomly divided into blank control group ，model group ，Shenling baizhu powder group （3.6 g/kg），with 8 mice in each group. Except for blank control group ，IBS-D model with anxiety was established in model group and Shenling baizhu powder group by giving corticosterone subcutaneously combined with intragastric administration of Folium Sennae decoction and chronic restraint treatment. After modeling ， blank control group and model group were given intragastric administration of normal saline ，and Shenling baizhu powder group was given relevant medicine intragastrically ，for consecutive 4 weeks. After last medication ，loose stools rate ，diarrhea index ，body weight ，sugar water preference percentage ，the times of crosssing open field center area and minimum pain threshold as well as the levels of BDNF in hippocampal tissue and 5-HT in serum were detected in each group. The cecal contents of mice in each group were extracted for microbial DNA extraction and sequencing； the abundance and diversity of intestinal microorganisms were analyzed by Alpha and Beta diversity analysis. RESULTS ： Compared with blank control group ，body weight ，sugar water preference percentage ，the times of crossing open field center area and minimum pain threshold as well as the levels of BDNF in hippocampal tissue ，relative abundance of Firmicutes phylum microorganism in intestine ， relative abundance of Lachnospiraceae_NK4A136_group genus microorganism were decreased significantly （P＜0.05）；loose stools rate，diarrhea index ，serum level of 5-HT，relative abundance of Verrucomicrobia phylum microorganism and relative abundance of Ackermann phylum microorganism were increased significantly （P＜0.05），and there were great differences in the types of intestinal microorganisms. Compared with model group ，body weight ，sugar water preference percentage ，the times of crossing open field center area ，minimum pain threshold ，BDNF level of hippocampus ，relative abundance of Firmicutes phylum microorganism，relative abundance of Lachnospiraceae_NK4A136_group genus microorganism were increased significantly （P＜ 0.05 or P＜0.01）；loose stools rate ，diarrhea index ，serum level of 5-HT，relative abundance of Verrucomicrobia phylum microorganism and relative abundance of Ackermann phylum microorganism were decreased significantly （P＜0.05），and and there were great differences in the types of intestinal microorganisms. CONCLUSION ：Shenling baizhu powder can improve the diarrhea and anxiety behavior of IBS-D model mice with anxiety ，increase the level of BDNF in hippocampus and decrease serum level of 5-HT. Its mechanism may be related to decreasing relative abundance of Verrucomicrobia phylum microorganism and Ackermann phylum microorganism ，increasing the relative abundance of Firmicutes phylum and Lachnospiraceae_NK4A136_group genus microorganism.