Molecular Mechanism Of Salvianolic Acid B Activating Akt/GSK3β Signal Pathway Preventing Oxidative Stress Injury Of Intestinal Mucosa

Objective:A variety of major clinical diseases can cause oxidative stress damage to the small intestinal mucosa,leading to intestinal dysfunction,intestinal bacterial translocation,and even severe infection and multiple organ failure,with serious consequences.At present,there is a lack of specific drug treatment for this common injury of the small intestine.At present,prevention and symptomatic treatment are still the main clinical treatment,so it is urgent to find protective drugs for oxidative stress injury of the small intestine.There are many studies on the protective mechanism of salvianolic acid B（Sal-B）in antioxidant stress of various cells,but there are few studies on the protective mechanism of small intestinal mucosal injury,especially in the protection of mitochondrial membrane potential,function and related cellular pathways of small intestinal mucosal cells.This study intends to reveal the molecular mechanism of Salvianolic acid B activating Akt/GSK3βsignal pathway,protecting intestinal mucosa from oxidative stress injury.Compare the Sal-B with several other known natural drugs with antioxidant stress effect to test its antioxidant effect and preliminarily evaluate the protective effect of Sal-B.Materials and methods:IEC-6 intestinal mucosal cells used in this study were cultured by cell culture.The survival and growth of IEC-6 intestinal mucosal cells were tested by MTT method,and Sal-B was compared with acetylcysteine,salidroside,procyanidin and lycopene on preliminary efficacy.The cellular reactive oxygen species of IEC-6 intestinal mucosal cells were detected by the corresponding kit.The transmembrane resistance and permeability of IEC-6 intestinal mucosal cells were tested by Transwell chamber and corresponding kit.The apoptosis of IEC-6 intestinal mucosal cells was detected by flow cytometry,the mitochondrial membrane potential was detected by flow cytometry and immunofluorescence.Mitochondrial membrane potential（ΔΨm）was represented by the proportion of low membrane potential cells in all cells,and the comparative statistical analysis among multiple groups was carried out by one-way ANOVA.P<0.05 indicates that the difference is statistically significant.Akt and GSK-3βwere detected by Western blot,one-way ANOVA were used to analyze the expression of isoprotein and mitochondrial pathway apoptotic protein.In animal experiments,the rat model of small intestinal mucosal ischemia-reperfusion injury was established.The injury group was compared with Sal-B protection group.The villus height and recess depth of intestinal mucosal cells were detected by immunohistochemistry,the proliferation（Brd U）and apoptosis（cleared caspase-3）indexes of small intestinal mucosa were detected,and expression of isoprotein Akt and GSK-3βwere detected by Western blot.On the verification of signal pathway and Sal-B action mechanism,rescue experiment was designed,and LY294002 was used to test the inhibitory effect of Akt/GSK-3βsiganal pathway;TDZD-8 was used to test the activating effect of Akt/GSK-3βsiganal pathway.The expression of P-AKT and P-GSK-3βwas detected semi quantitatively.The mitochondrial membrane potential and the expression of functional related proteins Cyt-C,caspase-3,Bax and Bcl-2 were detected.All results were compared among groups,and the statistical analysis among multiple groups was carried out by one-way ANOVA.P<0.05 indicates that the difference is statistically significant.Results:The protection effect of Sal-B was more obvious than that of acetylcysteine（P<0.05）.Among natural drugs,Sal-B has more significant protection effect than salidroside,procyanidin and lycopene（P<0.05,P<0.05,P<0.05）.Sal-B reduced H₂O₂induced apoptosis（P<0.05）,mitochondrial membrane potential depolarization（P<0.05）,ROS production（P<0.05）,restore the cell transmembrane resistance（P<0.05）and cell permeability（P<0.05）,and had a protective effect on IEC-6 cells damaged by oxidative stress.LY294002 down regulated the phosphorylation level of Akt（P<0.05）and GSK-3β（P<0.05）,weakened the protective effect of Sal-B on IEC-6 cells（H₂O₂injury）;TDZD-8upregulated the level of phosphorylation GSK-3β（P<0.05）,enhanced the protective effect of Sal-B on IEC-6 cells（H₂O₂injury）.When IEC-6 cells were damaged by oxidative stress,the treatment of Sal-B activated Akt/GSK-3βsignal transduction,upregulated Akt（P<0.05）and the level of phosphorylation GSK-3β（P<0.05）in PI3K/Akt signaling pathway,and then protected mitochondrial function and reduced mitochondrial related apoptosis by restoring mitochondrial membrane potential（P<0.05）.Animal experiments confirmed that Sal-B has a significant protective effect on small intestinal mucosa after ischemia-reperfusion injury.This protective effect is through the activation of Akt/GSK-3βsignal pathway.Conclusion:1.Sal-B activates Akt/GSK-3βsignal,and then restore mitochondrial membrane potential,protect mitochondrial function,reduce mitochondrial pathway apoptosis,protect small intestinal mucosal cells and reduce their oxidative stress damage.2.Sal-B may be a natural monomer drug that can effectively protect intestinal mucosa and resist oxidative stress injury.Its mechanism is related to the activation of Akt/GSK-3βsignal and protection of mitochondrial function.