Efficacy Evaluation And Preliminary Mechanism Exploration Of Pien Tze Huang (PZH) On Animal Model Of Multiple Sclerosis

Objective:In this study,we aimed to investigate the possible therapeutic effects of Pien Tze Huang(PZH)on experimental autoimmune encephalomyelitis(EAE),an animal model widely utilized to multiple sclerosis(MS),and explore the underlying mechanisms of action of PZH on EAE mice,providing the experimental basis for the clinical treatment of MS.Methods:1.Male Lewis rats were immunized with myelin basic protein(MBP)peptide to establish EAE model.Rats were then randomly divided into normal group,model group,PZH low dose group(PZH-L,0.054 g/kg/d),PZH middle dose group(PZH-M,0.162 g/kg/d),PZH high dose group(PZH-H,0.486 g/kg/d)and prednisone acetate group(PA,5 mg/kg/d),and orally given drugs for 21 days from the day of disease onset(on day 10th)after immunization.We observed the general status and recorded weight of mice as well as nerve functional scores every day during the drug treatment.Tissues of brain,brainstem and spinal cords were harvested for hematoxylin-eosin(HE)and luxol fast blue(LFB)staining;Sera was collected for detecting IL-17A,IL-23,CCL3 and CCL5 level by ELISA.2.Female SJL mice were injected with proteolipid protein 139-151(PLP139-151)peptide to establish relapsing remitting EAE(RR-EAE)model.Mice were then randomly divided into six groups including normal group,model group,PZH low dose group(PZH-L,0.078 g/kg/d),PZH middle dose group(PZH-M,0.234 g/kg/d),PZH high dose group(PZH-H,0.702 g/kg/d)and prednisone acetate group(PA,7 mg/kg/d).All agents were intragastric administered in a volume of 0.2 ml/20 g for 60 days from the day of disease onset(on day 10th)after immunization.HE and LFB staining was used for assessment of histopathological changes and demyelination.Sera was collected for detecting IFN-?,IL-17A,IL-1? and TNF-a levels by ELISA.Oligodendrocyte transcription factor 2(olig2)and MBP expression in brain were quantified by immunohistochemistry.The differentiation of Thl and Th17 subsets both in central nervous system(CNS)and spleen were detected by flow cytometry.The signaling pathways like STAT and NF-?B including levels of IKBa,P65 and phosphorylation forms of P65,IKB?,STAT1,STAT3 and STAT4 in brain were measured by western blot.MRNA relative expression of transcription factors of ROR-?t,T-bet and their downstream pro-inflammatory of IL-17A,IFN-?,IL-12a and IL-22 in brain were determined by real-time PCR.Results:1.The effect of PZH on EAE rat results:The results showed that compared with model group,PZH ameliorated clinical disease severity of EAE rats to some extent.There were obvious pathological changes with prominent vascular cuffing infiltration of inflammatory cells in the model group,PZH remarkably reduced inflammatory cell infiltration in the brain,brainstem and spinal cord of EAE rats.Furthermore,the levels of IL-17A,IL-23,CCL3 and CCL5 in serum were significantly decreased by PZH treatment compared to model group(P<0.05 or P<0.01).2.The effect of PZH on EAE mice results:Treatment of mice with the PZH effectively alleviated desease severity in clinical symptoms and neurological scores compared with EAE group(P<0.05 or P<0.01).The histopathological results showed that PZH also remarkably reduced inflammatory cell infiltration and myelin damage in the CNS of EAE mice(P<0.05 or P<0.01).Immunohistochemistry results showed the expression of olig2 and MBP in brain of PZH group was significantly increased compared with EAE group(P<0.05 or P<0.01).ELISA results indicated that PZH could down-regulated markedly the levels of cytokines of IL-17A,IL-1?,IFN-? and TNF-? in serum(P<0.05 or P<0.01).3.Study on the mechanism of PZH treatment results:Flow cytometry results indicated that PZH prevented Th1 and Th17 cells differentiation in the CNS and spleen compared with model group(P<0.05 or P<0.01).Western blot results showed PZH inhibited STAT and NF-?B signaling pathways by reducing the the phosphorylation levels of STAT1,STAT3,STAT4,P65 and IKB? compared to model group(P<0.05 or P<0.01).Real-time PCR results demonstrated PZH inhibited expression of transcription factors of ROR-?t,T-bet and their downstream pro-inflammatory of IL-17A,IFN-?,IL-12a and IL-22 in the mRNA level compared to model group(P<0.05 or P<0.01).Conclusions:1.Pien Tze Huang had a therapeutic effect on active EAE rats by preventing the inflammation in the CNS.2.PZH had a therapeutic effect on RR-EAE mice by reducing inflammation and demyelination;3.Therapeutic effect of PZH on EAE mice might be associated with relieving the differentiation and activity of Thl and Th17 cells.