Angong Niuhuang Pill Regulates MEKK1/JNK/c-Jun Signaling Pathway In Gut To Improve Neurological Function Of Intracerebral Hemorrhage Mice

Background: Stroke is the second leading cause of death in the world and the leading cause of death in China.Stroke includes hemorrhagic stroke and ischemic stroke.Hemorrhagic stroke is also called intracerebral hemorrhage(ICH)in clinic.The gut-brain axis is a twoway communication between the gastrointestinal and the central nervous system.Brain diseases can affect intestinal function,and changes in gastrointestinal microenvironment may also affect the brain.Angong Niuhuang pill(ANP)is often used in clinical treatment of stroke,coma,viral encephalitis,and other diseases.However,in the past,the research on ANP is mainly focused on the brain,whether it can play a role in ICH through the gut-brain axis and the possible mechanism is rarely reported.Objective: To investigate whether ANP can improve neurological function by attenuating intestinal dysfunction.Method: 1.There were 5 groups and 8 mice in each group: sham,model,and ANP(0.1,0.2and 0.4 g/kg).Mice were treated intragastrically with ANP at 24 h after the ICH.Garcia test,foot fault test,and open field test were performed at 24 h after ANP treatment.Brain edema,intestinal mucosal barrier permeability,expression of occludin and ZO-1 and the levels of VIP in intestine,the levels of lipopolysaccharides(LPS)norepinephrine dopamine,5-hydroxytryptami,TNF-α,IL-6,and the effect of ANP on the MEKK1/JNK/c-Jun signaling pathway were evaluated.2.There were 6 groups with 8 mice in each: control,and ANP 0.5 h,1 h,2 h,4 h,6 h,12 h,24 h.Mice were treated intragastrically with ANP(0.2 g/kg/kg).At 0.5 h,1 h,2 h,4 h,6 h,12 h,or 24 h after ANP treatment,serum containing ANP was harvested.Caco-2 cells were incubated with serum containing ANP(10%).24 h later,LPS(100 ?g/ml)was added.At 24 h after LPS exposure,the cell viability,TEER,FD4 flux,the expression of occludin and ZO-1 were assayed,the levels of TNF-α,IL-6 and the effect of serum containing ANP on the MEKK1/JNK/c-Jun signaling pathway were evaluated.Results: 1.Compared with the sham group,the model group showed worse performances in the Garcia test(P < 0.01),the percent of foot fault increased significantly(P < 0.01),the exercise total distance of significantly reduced(P < 0.01),the brain water content was higher(P < 0.01),an increase of accumulation of Evans blue in brain(P < 0.01),the norepinephrine,dopamine,5-hydroxytryptamin concentration was significantly elevated(P < 0.01,P <0.05),the intestinal mucosal barrier permeability,and the LPS concentration were increased(P < 0.01),the expression of occludin and ZO-1 were decreased(P < 0.05 or P < 0.01),the vasoactive intestinal peptide level of intestinal tissue was significantly decreased(P < 0.01),the levels of TNF-α and IL-6 were significantly augmented(P < 0.01),and the levels of phosphorylation of MEKK1,JNK,and c-Jun were increased(P < 0.05 or P < 0.01).ANP could reduce the percent of foot fault,the brain water and Evans blue content,intestinal mucosal barrier permeability,the levels of LPS,norepinephrine,dopamine,5-hydroxytryptamin,TNF-α,IL-6 and the expression of p-MEKK1 p-JNK and p-c-Jun in ICH mice,and increase the Garcia test score,exercise total distance,vasoactive intestinal peptide content,occludin and ZO-1 expression in ICH mice(P < 0.05,P < 0.01).2.Compared with the control group,the serum containing the ANP had no effect on the cell viability(P > 0.05),the TEER decreased and permeability increased(P < 0.05,P < 0.01),the expression of occludin and ZO-1 significantly decreased(P < 0.05 or P < 0.01),the levels of TNF-α and IL-6 were significantly increased(P < 0.01),the levels of phosphorylation of MEKK1,JNK,and c-Jun were increased(P < 0.05 or P < 0.01).The serum containing ANP decreased the permeability,the levels of TNF-α,IL-6 and the expression of p-MEKK1,p-JNK and p-c-Jun,and increased the TEER,the expression of occludin and ZO-1 in Caco-2 cells stimulated by LPS(P < 0.05,P < 0.01).Conclusion: ANP can improve neurological function and reduce brain injury in ICH mice;ANP can improve intestinal mucosal barrier dysfunction caused by ICH and inhibit intestinal and provincial inflammatory reaction;the mechanism of ANP is related to its regulation of intestinal MEKK1/JNK/c-Jun signal pathway.Innovation: Previous research on ANP mainly focused on its effect on the brain after stroke,this study made it clear for the first time that ANP can improve neurological function by reducing intestinal dysfunction caused by cerebral hemorrhage.