| [Objective]:The intestine is the body’s digestive organ and the largest immune organ.A complete intestinal barrier prevents harmful substances such as toxins and bacteria from entering the blood through the intestinal epithelial cells while absorbing water and nutrients.When the body is in infection,inflammation,trauma,stress and other pathological conditions,this balance will be broken,the integrity of the intestinal barrier will be destroyed,and the permeability will increase,making the intestine represented by lipopolysaccharide(LPS).The endotoxins and bacteria of Negative bacteria enter the systemic circulation through the blood vessel or lymphatic system,induce oxidative stress response,produce excessive reactive oxygen species(ROS),aggravate the damage of the intestinal barrier function,inhibit the regeneration of the intestinal mucosa,and affect the clinical outcome.Glutathione and its related enzymes play an important role in eliminating ROS and maintaining the dynamic balance of intracellular redox.In this study,LPS-treated mice and mouse small intestinal epithelial cells(MODE-K cells)induced inflammation and oxidative stress to construct an intestinal barrier injury model.The purpose is to study whether glutathione can improve the intestinal barrier damage induced by lipopolysaccharide by regulating the expression of ROS,and provide a theoretical basis for the treatment of intestinal barrier damage by glutathione.[Methods]:Animal experiment:C57/BL-6 mice were randomly divided into 3 groups,Control group(n=10),LPS group(n=10),LPS+GSH group(n=10).The control group was given continuous intraperitoneal injection of normal saline(volume 0.2ml)for 6 days,the LPS group and LPS+GSH group were intraperitoneally injected LPS(5mg/kg)for 3 days to induce sepsis in mice,and then the LPS group was injected normal saline(volume 0.2 ml),the LPS+GSH group was intraperitoneally injected with GSH(800mM,volume 0.2ml)for 3 days.The weight changes of the mice were measured every 3 days,and on the seventh day,the mice were given intragastrically with fluorescein isothiocyanate dextran(FD-4).After 6 hours,the mice were sacrificed,and the mice were weighed before sacrifice.During the experiment,observe the ocular inflammation and defecation of the mice.Eyeball blood sampling uses FD-4 technology to detect the integrity of the mouse intestinal barrier,compare the length,thickness,and whether there are bleeding points in each group to observe the anatomy of the small intestine,detect the spleen coefficient of the mouse to evaluate the inflammation status of the mouse’s whole body,and compare the observation with HE staining The morphological integrity of the intestinal mucosa of each group of mice was used to evaluate the LPS-induced mouse injury model and the protective effect of glutathione.Cell experiment:Use mouse normal small intestine cells MODE-K to simulate normal intestinal epithelium,and use different concentrations of LPS and GSH to act on the cells to obtain the most suitable experimental concentration.The cells were divided into Control group,LPS group,and LPS+GSH group.After 24 hours of intervention,the CCK8 experiment was used to detect cell viability,immunofluorescence was used to detect changes in intracellular reactive oxygen species(ROS),and flow cytometry was used to detect intracellular ROS and cell apoptosis.RT-qPCR technology was used to observe the expression of tight junction protein ZO-1 and Occludin-1 genes.[Results]Animal experiment results:mice treated by intraperitoneal injection of LPS(5mg/kg)showed a large amount of inflammatory secretions and diarrhea symptoms around the eyes.After GSH intervention,mice around the eyes reduced inflammatory secretions and reduced diarrhea;GSH intervention can improve The weight loss of mice caused by LPS(P<0.01);the dissection revealed that the mouse gastrointestinal tract became shorter in the GSH intervention mice(P<0.05),thinning and bleeding decreased;the mouse spleen was completely dissected and LPS was seen The spleen coefficient of the mice in the+GSH group was lower than that in the LPS group(P<0.01);the FD-4 experiment showed that the peripheral blood fluorescence of the LPS group was increased compared with the control group mice(P<0.01),while the GSH group mice were compared with LPS Compared with the LPS group,the fluorescence of peripheral blood was reduced in the group first(P<0.01);the intestinal mucosal injury of the mice in the GSH group was alleviated compared with the LPS group after staining the tissue section(P<0.05).Cell test results:The most suitable experimental concentration of LPS on MODE-K cells is 300mg/l through CCK-8 experiment;GSH alone has no effect on cell growth;GSH intervention 4h in advance can improve LPS-induced cells Damage(P<0.001);Observing the number of cells under an inverted microscope shows that the number of cells in the LPS group is less than that in the Control group(P<0.01),while the number of cells after GSH intervention has increased(P<0.05).At the same time,observe GSH can improve cell morphology changes caused by LPS;cell cloning experiments show that the number of cells after LPS intervention is less than that of Conreol group(P<0.001),while the number of cells in LPS+GSH group added with GSH intervention is less than that of Control group(P<0.01)but increased compared with the LPS group(P<0.05);in the apoptosis experiment,it can be seen that the cell apoptosis after LPS intervention increased(P<0.01),and the LPS+GSH group after GSH intervention had higher cell apoptosis than LPS Group remission(P<0.05);cellular immunofluorescence experiments and cell flow cytometry showed that the intracellular ROS in LPS intervention increased significantly(P<0.001),while the ROS in the LPS+GSH group after GSH intervention intersects with the LPS group in remission(P<0.001).Using RT-qPCR technology,it can be seen that GSH improves the expression of connexin ZO-1 and Occludin-1 in intestinal epithelial cells caused by LPS(P<0.01).[Conclusions]1.Glutathione improves systemic inflammation and intestinal barrier damage in mice caused by LPS2.Glutathione inhibits intestinal epithelial cell damage caused by LPS by reducing intracellular ROS,reverses the expression of intestinal epithelial cell connexin,and improves intestinal barrier damage... |
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