Effects Of Bu Shen Yi Sui Formula And Its Decomposed Formulas On Axonal Injury Repair And Neurotrophic Signal In Mice With Experimental Autoimmune Encephalomyelitis

Objective Multiple sclerosis(MS)is an autoimmune disease of the central nervous system(CNS).Previous studies have indicated that Bu Shen Yi Sui(BSYS)formula with the action of tonifying kidney and resolving phlegm and promoting blood circulation could reduce the axonal injury and promote the regeneration and repair in mice with experimental autoimmune encephalomyelitis(EAE),an animal model of MS,but its mechanism is not clear.Therefore,the effect and molecular mechanism of axonal injury and repair on BSYS formula and its decomposed formulas was discussed based on previous studies,combining with the research on the mechanism of axon regeneration and signal transduction in EAE mice,using by of immunohistochemical,immunofluorescence and real-time fluorescence quantitative(qRT)-PCR and Western Blot(WB)techniques,focusing on the axon regeneration neurotrophic factors,to reveal the modern connotation of tonifying kidney and resolving phlegm and promoting blood circulation.It will provide a scientific basis for MS and related mysterious illness based on syndrome differentiation of traditional Chinese medicine.Methods140 female C57BL/6 mice were randomly divided into seven groups,namely normal control(NC),model(MO),prednisone acetate(PA,5 mg/kg),catapol(CA,40 mg/kg),BSYS(3.02 g crude drug/kg),Bu Shen(BS,1.44 g crude drug/kg),Hua Tan Huo Xue(HTHX,1.57 g crude drug/kg)groups.Twenty mice were placed in each group.The mice were injected with myelin oligodendrocyte glycoprotein(MOG)35-55 to establish EAE model.From the Day 0 to Day 40,mice in NC and MO groups were received daily gavage administration with distilled water(0.2 ml),and mice in treatment groups were treated with the corresponding drugs(0.2 ml)once a day.The mice were monitored daily for the body weight change,the neurological score,incidence and death rate.The brain and spinal cord were harvested on Day 20(the acute stage)and Day 40(the remission stage)after post-immunization(PI).The pathological changes and the injuries of axon and myelin in the brains and spinal cords of mice were observed with hematoxylin eosin(H&E)staining and transmission electron microscopy(TEM)respectively;P-tau and beta-tubulin were measured by immunohistochemistry(IHC).The expression of mRNA and protein on BDNF,TrkB,PI3 K and AKT were detected by qRT-PCR and WB.Results 1 Neuroprotective effects of BSYS and its decomposed formulas in EAE mice 1.1 Effects of BSYS and its decomposed formulas BS and HTHX on the incidences in EAE miceThere was no significant difference in body weight among the seven groups before modeling(P>0.05),the body weight of mice increased slowly after modeling,and at the beginning of the day 11,the weight of the other model mice began to decrease significantly compared with the NC group(P<0.05),had a peak in day 19,and then a slow recovery.But day 19 to day 40,the weight of in model groups was significantly lower than that of NC group(P<0.01).In day 21 to day 40,the weight of mice in BSYS and BS group was significantly higher than that in PA group(P<0.05 or P<0.01).1.2 Effects of BSYS and its decomposed formulas BS and HTHX on the pathological changes in EAE miceIn MO group,there was inflammatory cell infiltration around the white matter,and the nuclear pyknosis was seen.In PA,CA,BS,HTHX and BSYS groups,the inflammatory cells infiltrated around the cerebral white matter,and some of them were pyknosis.Pathological degree was lower than MO group.The pathological changes of the spinal cord were similar to those of the brain.1.3 Effects of BSYS and its decomposed formulas BS and HTHX on the axonal injury and repair in EAE miceOn days 20 and 40,the expression of p-Tau was and the expression of beta-tubulin was down regulated in the model group.BSYS,BS and HTHX formulas can be down regulated the expression of p-Tau(P<0.05,P<0.01),up-regulated the expression of beta-tubulin(P<0.05,P<0.01).2 The mechanisms BSYS,BS and HTHX formulas on the axonal repair in EAE miceThe results of WB and qRT-PCR found the mRNA and protein expressions of BDNF,TrkB,AKT and PI3 K in the brain and spinal cord of EAE mice were significantly up-regulated by BSYS formulas and its decomposed BS formulas and HTHX formulas,compared with the EAE mice that on days 20 and 40(P<0.05).The effect of BSYS formulas trend on the above indexes was better than that of the decomposed BS formulas and HTHX formulas,but there was no statistical significance(P>0.05).ConclusionBSYS and its decomposed BS and HTHX formulas had the neuroprotective effects on reducing weight loss and scores,inflammatory cell infiltration,axonal injury and enhancing the repair of axon in EAE mice.Especially,the effect of BSYS and its decomposed BS and HTHX on promoting axonal repair effect might be related to enhance the mRNA expression of BDNF/TrkB and the signal pathway of PI3K/AKT,BSYS was more significant trend.These results provide some scientific basis for the treatment of MS with BSYS formula.