Research On The Prevention And Treatment Of Experimental Autoimmune Encephalomyelitis By Wuzi Yanzong Pill Based On Endoplasmic Reticulum Stress Pathway

Objective:In this study,Wuzi Yanzong Pill（WYP）was used to interfere with experimental autoimmune encephalomyelitis（EAE）model constructed by female C57BL/6 mice.To observe the therapeutic effect of WYP on EAE and its effect on endoplasmic reticulum stress（ERS）,and to provide theoretical support for the prevention and treatment of multiple sclerosis（MS）with WYP.Methods:Female C57BL/6 mice were randomly divided into normal group,model group and WYP group.MOG_35-55,TB and PTX were used for modeling.From the third day after immunization,drug intervention was performed.Normal group and model group were given normal saline 50m L/（kg·d）,while WYP group was given WYP liquid 16g/（kg·d）,twice a day for 25 days.The neural function scores of each group of mice were recorded daily.The mice were sacrificed 28 days after immunization,and spinal cord samples were collected.The infiltration of inflammatory cells and the degree of demyelination in spinal cord were observed by hematoxylin-eosin staining and Luxol fast blue staining.The protein expressions of GRP78,GRP94,p-PERK,p-IRE1α,p-e IF2α,ATF4,XBP1,CHOP,ASK1,p-JNK,Caspase-12,Caspase-9,Caspase-3 in the ERS pathway of spinal cord were detected by Western blot.The m RNA expressions of ATF6,ATF6αand ASK1 in the ERS pathway of spinal cord were detected by fluorescence PCR.Results:1.The effect of WYP on therapeutic effect of EAE mice1.1 The effect of WYP on neurological function of EAE mice.Compared with the normal group,the mean onset time,mean maximum neurological score and mean cumulative neurological score were significantly increased in the model group（P<0.01）.Compared with model group,mean onset time,mean maximum neurological score and mean cumulative neurological score in WYP group were all decreased（P<0.05）.1.2 The effect of WYP on inflammatory cell infiltration in spinal cord tissue of EAE miceCompared with the normal group,the percentage of inflammatory cell infiltration area in the white matter of spinal cord in the model group was increased（P<0.01）.Compared with the model group,the percentage in the WYP group was decreased（P<0.05）.1.3 The effect of WYP on demyelination of spinal cord tissue in EAE miceCompared with the normal group,the percentage of spinal white matter demyelination area in the model group to the total spinal white matter area was increased（P<0.01）.Compared with model group,the percentage in WYP group was decreased（P<0.01）.2.The effect of WYP on ERS in EAE mice2.1 The effect of WYP on molecular chaperones of ERS signaling pathway in EAE miceCompared with the normal group,the protein expression of GRP78 that triggers ERS was increased in the model group（P<0.01）,the protein expression of GRP94 was increased（P<0.05）.Compared with the model group,the protein expressions of GRP78and GRP94 in WYP group were decreased（P<0.05）.2.2 The effect of WYP on ERS-triggered UPR signaling pathway in EAE miceCompared with the normal group,the protein expressions of p-PERK,p-IRE1α,p-e IF2α,ATF4 and XBP1 in the model group were increased（P<0.05,P<0.01）,the m RNA expression of ATF6 was not significantly changed（P>0.05）,but the m RNA expression of ATF6αin the model group was increased（P<0.05）.Compared with the model group,the protein expressions of p-PERK,p-IRE1α,p-e IF2α,ATF4 and XBP1 in WYP group were decreased（P<0.05,P<0.01）,the m RNA expressions of ATF6 and ATF6αwere decreased（P<0.01）.2.3 The effect of WYP on the pro-apoptotic pathway triggered by ERS in EAE miceCompared with the normal group,the protein expressions of Caspase-12,CHOP,Caspase-9,p-JNK and Caspase-3 in the model group were increased（P<0.01,P<0.05）,and the protein and m RNA levels of ASK1 were not significantly changed（P>0.05）.Compared with the model group,the protein expressions of Caspase-12,CHOP,Caspase-9,p-JNK and Caspase-3 in the WYP group were decreased（P<0.01,P<0.05）,and the protein and m RNA expressions of ASK1 in the WYP group were decreased（P<0.01,P<0.05）.Conclusion:1.WYP could improve the neurological function of EAE mice,relieve the clinical symptoms,delay the onset,reduce the inflammatory cell infiltration in the spinal cord tissue of the central nervous system of EAE mice and demyelination in the white matter of the spinal cord,and reduce the spinal cord tissue damage.2.WYP may reduce the level of sustained ERS response in the spinal cord by down-regulating the PERK,IRE1 and ATF6 signaling pathways in the spinal cord to reduce the triggering of ERS-mediated JNK,CHOP and caspase-12 pro-apoptotic pathways,thus helping to reduce the damage to the spinal cord.