Protective Effect And Mechanism Of Rheum Officinale Bail On Stress Related Mucosal Disease

ObjectiveTo observe the clinical therapeutic effect of Rheum officinale on patients with stress gastric mucosal injury,and evaluate the therapeutic effect of rhubarb.To investigate the protective effect and molecular mechanism of Emodin,the main active component of rhubarb,on acute stress related mucosal disease.Methods1.Clinical ResearchIn this study,40 patients with acute gastric mucosal injury will be included in the experiment according to different treatment methods,including the treatment group and the control group.According to the condition,the two groups of patients were given the diagnosis of etiology,anti-infection,improvement of microcirculation,gastric protection,early nutritional support and other conventional treatments.The treatment group was combined with rhubarb on the basis of omeprazole treatment,and was given rhubarb 10g、per/day at 9:00 am every day,for a treatment cycle of 7 days.At one cycle of treatment,observe two groups of patients:①abdominal pressure,Acute gastrointestinal injury grading(AGI);② Sequential organ failure assessment(SOFA)score,Procalcitonin(PCT),plasma interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),TNF-α)in serum.③ Change of length of ICU stay,total length of stay and 28d mortality.2.In vitro animal test① Male Sprague-Dawley SD rats aged 8-10 weeks,weighing 180-200g,were selected for animal experiment.The animal experiment was divided into 3 parts.The first part was animal experiment group(40 rats,randomly divided into 4 groups,10 rats in each group),which were normal control group:Normal feeding,no water immersion and restraint stress(WIRS)7 days after death;WIRS-1d group:The WIRS model was established by water immersion for 16h,and the rats were sacrificed 1 day later.WIRS-3d group:WIRS model was established by water immersion and was sacrificed 3 days later.WIRS-7d group:WIRS model was established by water immersion and was sacrificed 7 days later.The second part was animal experimental grouping(50 rats,randomly divided into 5 groups,10 rats in each group),which were normal control group.Emodin group:normal fed rats were intraperitoneally injected with emodin(20mg/kg);Stress gastric mucosa injury group(WIRS group):WIRS model was established by water immersion and binding for 16h,and 200μL normal saline was intraperitoneally injected 0.5h after model establishment.WIRS+emodin low-dose group:intraperitoneal injection of emodin(20mg/kg)0.5h after WIRS model establishment;WIRS+emodin high-dose group:0.5h after WIRS model establishment,emodin(40mg/kg)was intraperitoneally injected,and the neck was severed 3 days later.The third part was animal experiment grouping(80 rats were randomly divided into 4 groups with 20 rats in each group),which were normal control group:normal feeding,no water immersion and restraint stress WIRS;Stress gastric mucosa injury group(WIRS group):WIRS model was established by water immersion,and 200μL normal saline was intraperitoneally injected 0.5h after model establishment.WIRS+emodin group:intraperitoneal injection of emodin(40mg/kg)0.5h after WIRS model establishment;WIRS+emodin+LY294002 group:0.5h after the establishment of WIRS model,emodin(40mg/kg)and LY294002(20mg/kg)were intraperitoneally injected,and the neck was severed at the corresponding time.② The third part of the rat model was cut off at the corresponding treatment time,the abdominal cavity was opened,the gastric tissue was free,and the pH value of gastric juice was determined.The stomach was removed and fixed in 10%neutral formaldehyde solution for 10 minutes.Then,the gastric cavity was fully exposed.The gastric mucosa was extended as far as possible,and the morphology and changes of the stomach were observed.Gastric mucosa tissue samples were collected and pathological sections were taken to observe the changes of gastric mucosa tissue structure.③ The expression levels of plasma interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in gastric mucosa tissue were determined by ELISA.To observe the effect of emodin on the expression level of inflammatory factors.(4)Western Blot was used to determine the levels of Akt,pAkt,Occludin,Zonula Occluden-1 and B-cell lymphoma-2 in gastric mucosal tissues.To investigate the effect of emodin on wIRs-induced stress gastric mucosal injury in rats and the role of PI3K/Akt signaling pathway in WIRSinduced stress gastric mucosal injury.3.In vitro cell test①The use of Lipopolysaccharide(LPS)(0,2,5,10,20 pg/mL)to treat human Gastric epithelial cell-1(GES-1)cells for 24,48,72 h.GES-1 cells were treated with different concentrations of emodin(0,5,10,20 and 40μM)for 24,48 and 72 h,and the effects of LPS and emodin on the proliferation activity of GES-1 cells were determined by Cell Counting Kit-8(CCK-8)assay,and the optimal concentration of emodin was determined.The optimal concentration of emodin was 40μM for subsequent experiments.The experiment was continued to be designed.Cells were grouped as follows:control group:GES-1 cells were cultured normally without any treatment;LPS group:10 μg/mL LPS treatment;LPS+emodin group:100μg/mL LPS and 40μM emodin were added;LPS+emodin+LY294002 group:100μg/mL LPS,40μM emodin and 15μM PI3K/Akt cell signaling pathway inhibitor LY294002 were added.The cells were treated for 24,48 and 72 h,respectively,and the proliferation activity was detected by CCK-8.② Experimental groups:Control group:GES-1 cells were not treated;LPS group:GES1 cells were given 10μg/mL LPS;LPS+emodin group:10μg/mL LPS and 40μM emodin were added.LPS+emodin+LY294002 group:1Oμg/ml LPS+40μM emodin and 15μM LY294002 were added for 48h,and the migration ability of GES-1 cells in each group was detected by cell scratched test and trans-well assay.The expression levels of Akt,pAkt,Occludin,ZO-l,Bax and Bcl2 proteins were detected by western blot and immunofluorescence assay,respectively,to clarify the activation of emodin on PI3K/Akt signaling pathway,and verify the protective effect and mechanism of emodin on the integrity of gastric mucosal epithelial cells.Cell necrosis was observed under light microscope by FDA-PI staining to explore the effect of emodin on LPSinduced necrosis and apoptosis of GES-1 cells,and to clarify the protective effect and molecular mechanism of PI3K/Akt signaling pathway.Results1.Clinical Research① Compared with the control group,the improvement of SOFA score after rhubarb treatment was significantly higher than that of the control group,and the difference was statistically significant(P<0.05).② Compared with the control group,the length of ICU stay,total length of stay and 28-day mortality of patients before and after treatment had no significant difference.③ After one week of treatment,AGI grade of patients in the treatment group was improved and intra-abdominal pressure was significantly decreased compared with that before treatment,and the difference was statistically significant(P<0.05);④ The expression of IL-6 and TNF-α in serum of 2 groups were significantly increased before treatment.After 1 week of treatment,the expressions of IL-6 and TNF-α in serum of patients in both the treatment group and the control group decreased compared with those before treatment,and the decrease in the treatment group was more significant,the difference was statistically significant(P<0.05).2.In vitro animal test① The gross morphology of gastric mucosa and HE staining showed that the gastric mucosa tissue bleeding was obvious,the gastric mucosa epithelial tissue was seriously damaged,the integrity of the epithelial tissue was impaired,and the gastric mucosa injury index increased in the WIRS group.With the development of modeling time,gastric mucosal bleeding and gastric mucosal tissue structure did not improve.However,after emodin intervention,gastric mucosal bleeding was reduced in WIRS+emodin group,and gastric mucosal epithelial tissue integrity was better.High-dose emodin significantly promoted gastric mucosal tissue repair and reduced gastric mucosal epithelial tissue damage.However,LY294002 inhibitor could inhibit the protective effect of emodin,and the integrity of gastric mucosa epithelial tissue was similar to that of WIRS group without significant improvement.ELSIA test results showed that compared with the control group,the expression levels of IL-6 and TNF-α in gastric mucosa tissues of WIRS group were significantly increased,indicating an enhanced inflammatory response,while the expression levels of IL-6 and TNF-α in gastric mucosa tissues of WIRS+emodin group were significantly decreased compared with that of WIRS group.Compared with WIRS+emodin group,LY294002 treatment inhibited the anti-inflammatory effect of emodin,and the expression levels of IL-6 and TNF-α in gastric mucosa tissues of WIRS+emodin+LY294002 group were significantly increased,but there was no significant difference between WIRS group and WIRS group.The results of Western Blot showed that compared with the control group,the expression levels of pAkt,Occludin,ZO-1 and Bcl2 in gastric mucosa tissues of WIRS rats were significantly decreased,and the expression level of Bax was significantly increased.After emodin treatment,compared with WIRS group,the expression levels of pAkt,Occludin,ZO-1,Bcl2 and other proteins in gastric mucosa were significantly increased,while the expression level of Bax was significantly decreased.However,LY294002 inhibited the protective effect of emodin,and there was no significant difference in the expression levels of pAkt,Occludin,ZO-1,Bcl2 and Bax compared with WIRS group.3.In vitro cell test① CCK-8 assay showed that the longer LPS treatment time,the lower the proliferation activity of GES-1 cells;The higher LPS concentration was,the lower cell proliferation activity was.Different concentrations of emodin had no significant effect on the proliferation activity of GES-1 cells.However,emodin can reduce the damage of LPS on the proliferation of GES-1 cells,and the protective effect of emodin can be blocked by the PI3K/Akt signaling pathway inhibitor LY294002.In the cell scratches and Trans-well experiments,LPS treatment inhibited the migration ability of GES-1 cells and reduced the number of perforated cells,while emoin significantly promoted the migration and perforation number of GES-1 cells compared with LPS group.In contrast,LY294002 inhibited emodin’s ability to promote migration.FDA-PI staining showed that LPS induced the necrosis and apoptosis of GES-1 cells,while emodin treatment reduced the necrosis and apoptosis of GES-1 cells.LY294002 group increased the necrosis and apoptosis of GES-1 cells,inhibiting the anti-apoptotic effect of emodin.Western Blot analysis showed that compared with the control group,the expression levels of tight junction proteins ZO-1 and Occludin,Bcl2 and pAkt were significantly decreased in LPS group,while the expression level of apoptosis-labeled protein Bax was significantly increased.However,emodin treatment group could up-regulate the protein expression levels of pAkt,Occludin,ZO-1 and Bcl2,and reduce the expression of Bax.Moreover,the expression levels of pAkt,Occludin,ZO1,Bcl2 and Bax were concentration-dependent.Compared with emodin group,LY294002 treatment could inhibit the protective effect of emodin,resulting in decreased protein expression levels of pAkt,Occludin,ZO-1 and Bcl2,while the expression level of Bax increased.Immunofluorescence results showed that LPS could destroy tight junctions of GES-1 cells,resulting in decreased expression of tight junctions and Occludin and ZO-l,while increased expression of Occludin and ZO-1 in emodin group.The expression levels of Occludin and ZO-1 in LY294002 group were inhibited.Conclusion① Rhubarb can effectively improve SOFA score,AGI rating and abdominal pressure in patients with stress gastric mucosal injury and reduce the expression of plasma inflammatory indexes..②Emodin can protect gastric mucosal epithelial tissue of WIRS rats by activating PI3K/Akt signaling pathway,maintain the integrity of gastric mucosal epithelial tissue,and reduce gastric mucosal injury caused by gastric mucosal inflammation in WIRS rats,while the inhibitor LY294002 can inhibit the protective effect of Emodin.③Emodin can maintain the integrity of tight junctions of gastric epithelial cells and promote the expression of tight junction proteins Occludin and ZOL-1.The main molecular mechanism of Emodin is through the activation of PI3K/Akt signaling pathway.