The Effects And Mechanism Of Emodin On Experimental Autoimmune Encephalomyelitis(EAE) Mice

Objective1.To explore the effects of different dose of emodin in experimental autoimmune encephalomyelitis(EAE)mice and choose the best dose to have further investigation.2.To investigate the effects of emodin on microglia activation and inflammatory cytokine in EAE.3.To investigate whether emodin has effects on EAE via Myd88/PI3K/Akt signalling pathway.Methods1.36 female C57BL/6 mice were randomly divided into normal group(NC),model group(EAE),low-dose emodin group(Emodin-L)(30 mg/kg emodin suspension by daily gavage),and high-dose emodin group(Emodin-H)(60 mg/kg emodin suspension by daily gavage)according to the random number table method,with 9 mice in each group.After 2 weeks of adaptive feeding,the EAE was induced with myelin oligodendrocyte glycoprotein(MOG)35-55 peptide.The mice in low-dose emodin group were gavaged with 30 mg/kg/d until 21 day post immunization(dpi).The mice in high-dose emodin group were gavaged with 60 mg/kg/d until 21 dpi.The clinical scores were measured every day until 21 dpi.The rotarod test was performed every week until the 21 dpi.The mice were sacrificed on 21 dpi.The mouse brain and spinal cord tissues were quickly removed for hematoxylineosin staining and immunohistochemical determination of MBP and BDNF expression.2.Using the network pharmacology approach,we searched for potential targets associated with multiple sclerosis using GeneCard,DisGeNET,OMIM and Drugbank,and then took potential targets associated with emodin from SwissTargetPrediction,ETCM,Pharmmapper and HERB databases.The main targets were analyzed using STRING and Cytoscape to perform Protein-Protein Interaction(PPI)and Molecular Complex Module Analysis tool(MCODE).The major signaling pathways were explored by KEGG and GO analysis,and molecular docking between emodin and the main targets was performed by Autodock Tool to determine the docking ability.3.27 female C57BL/6 mice were randomly divided into normal group(NC),model group(EAE),and high-dose emodin group(Emodin-H)(gavage of 60 mg/kg/d),with 9 mice in each group.The induction of EAE was described above.The mouse brain and spinal cord tissues were quickly removed for by testing CD86/Ibal and CD206/Iba1 in brain tissue to determine the effects of emodin on microglia activation by immunofluorescence staining on 21 dpi,while the frontal cortex was used to test the mRNA expression of inflammatory cytokine such as IL-6,IL-17,TGF-β and RORyt by qPCR.4.27 female C57BL/6 mice were randomly divided into normal group(NC),model group(EAE),and high-dose emodin group(Emodin-H),with 9 mice in each group.The induction of EAE and method of gavage is same as above.The frontal cortex was quickly removed on 21 dpi to verify the protein expression of markers of PI3K/Akt signaling pathway by Western blot.Meanwhile,the mRNA and protein expression related to Myd88 signaling pathway in mouse frontal cortex were also detected by qPCR and western blot.Results1.Except for the normal group,the model group,the low-dose emodin group,and the high-dose emodin group(Emodin-H),mice in all groups started to have clinical symptoms on the 11 dpi,with gradually increasing clinical scores on following days.Subsequently,symptoms of neurological deficits of different degrees appeared one after another.Compared with mice in the normal group,mice in the model group showed a significant increase in clinical scores from 13 to 21 dpi(P<0.05),suggesting that our model group had significant symptoms.Meanwhile,the clinical scores of both the low-dose and high-dose emodin groups were lower than those of the model group compared with the model group mice.and the difference between the high-dose emodin group and the model group was statistically significant(P<0.05)from 19 to 21 dpi.The daily clinical scores in the highdose emodin group were lower than those in the low-dose emodin group,but there was no significant difference(P>0.05).The cumulative clinical scores were seen to be lower in both the low-dose and high-dose emodin groups than in the model group,while the highdose emodin group showed a significant reduction from the model group(P<0.05).In the rotarod test,both the low-dose and high-dose emodin groups had increased latency to fall and speeds at 2 and 3 week compared with the model group,and the high-dose emodin group had significantly increased latency to fall and speed on rotarod at week 3 compared with the model group(P<0.05).The hematoxylin-eosin staining indicated that there was inflammatory cell infiltration in brain and spinal cord tissues in the model,low-dose and high-dose emodin groups,and in heavy cases,significant inflammatory cell infiltration around blood vessels and damage to myelin was seen.The inflammatory infiltration was significantly decreased in the high-dose emodin group compared with the model group(P<0.05).In addition,immunohistochemistry revealed that MBP and BDNF were significantly decreased in the model group,while high-dose emodin significantly increased the expression of MBP and BDNF(P<0.05).2.Using the network pharmacology approach,there were 137 potential targets for emodin and 1762 potential targets associated with multiple sclerosis through GeneCard,DisGeNET,OMIM,Drugbank,SwissTargetPrediction,ETCM,Pharmmapper,and HERB databases.There were 42 potential therapeutic targets in common as potential core targets.AKT1 was a potential core target.Module 2 was related to the inflammatory response including AKT1 and NFKB1.KEGG enrichment found there were 85 signaling pathways involving toll-like receptor and NF-κB signaling pathways.PI3K/Akt signaling pathway was the main signaling pathway which was involved in the inflammatory response and Myd88 signaling pathway.Toll-like receptor and NF-κB signaling pathways were also related to Myd88 signaling pathway.3.The activation of microglia by emodin and the secretion of inflammatory cytokines were detected by immunofluorescence and qPCR.Emodin could inhibit expression of CD86+/Iba1+ and CD206+/Iba1+ in the brain tissue of EAE mice(P<0.05),while emodin could inhibit the expression of CD86+/Iba1+ and CD206+/Iba1+(P<0.05).In addition,It revealed that mRNA expression of IL-6,IL-17A,TGF-β and RORyt were increased in the cortical tissue of EAE mice,while emodin significantly decreased the mRNA expression of IL-6,IL-17A,TGF-β and RORyt in the frontal cortex of EAE mice.4.Western Blot was used to verify the protein expression of PI3K/Akt signaling pathway,and it was found that protein expression of p-PI3K and p-Akt in the frontal cortex of EAE mice was increased,while emodin inhibited the expression of p-PI3K and p-Akt(P<0.05).The protein expression of Myd88 and NF-κB p50 in the frontal cortex of EAE mice were increased(P<0.05).Emodin significantly inhibited the expression of Myd88 and NF-κB p50(P<0.05).In addition,the mRNA expression of TLR4,Myd88,Ticaml,and NF-κB p50 in the cortical tissue of EAE mice were significantly increased(P<0.05).Emodin decreased the mRNA expression of TLR4,Myd88,Ticaml and NF-κB p50 in the frontal cortex of EAE mice(P<0.05).ConclusionsBased on the perspective of“If pathogenic evils were eliminate,then human were recovered”,the therapeutic effects,microglia activation and anti-infflammatory effects of emodin on acute EAE mice were investigated by network pharmacology analysis and in vivo experiment,and the molecular mechanism was tentatively explored.The study demonstrated as following:1.Emodin reduced the neurological deficits and showed the neuroprotective effects in EAE mice.2.Through network pharmacology,emodin acted in MS through multiple targets and multiple signaling pathways.AKT1 was the main target of action,PI3K/Akt signaling pathway is the main signaling pathway related to Myd88 signaling pathway.3.Emodin inhibited the microglial activation in central nervous system and reduced the inflammatory response,which may be mediated by Myd88/PI3K/Akt signaling pathway.