The Influence Of Tonifying The Kidney,Resolving Phlegm And Activating Blood On The Proliferation And Differentiation Of Oligodendrocyte Precursor Cells In Mice With Experimental Autoimmune Encephalomyelitis

Subject Headings:methods of tonifying the kidney, resolving phlegm and activating blood; multiple sclerosis; Erhuang Capsule; oligodendrocyte precursor cells; oligodendrocyte lineage gene 2; experimental autoimmune encephalomyelitis; repair and regeneration of myelin sheathEXPERIMENTObjectiveThe proliferation and differentiation of OPCs is key to myelin repair and regeneration. Erhuang Capsule is a typical formula based on the TCM theory of tonifying the kidney, resolving phlegm and activating blood. The influence of Erhuang Capsule on OPCs of EAE mice was observed in our study. The indexes related to the proliferation and differentiation of OPCs were detected through immunohistochemistry, ELISA, RealTimePCR, Western Blot. We want to reveal the therapeutic mechanism of Erhuang Capsule on multiple sclerosis (MS). Furthermore, it is also affirmed that treatment based on differentiation of TCM is effective and efficient to MS.MethodsC57BL/6 mice, SPF and female, were randomly divided into five groups:normal group, model group, PA group, Erhuang high-dose group and Erhuang low-dose group. Each group was consisted of twenty-five mice. The mice of the latter four groups were injected subcutaneously over flanks with the antigen which contains MOG35-55 and complete Freund's adjuvant in the same ratio, respectively. The mice of normal group were injected with physiological saline instead. PTX was used to strengthen the immunization. On day 1 post-immunization, normal group and model group were administrated orally with physiological saline while Erhuang high-dose group and Erhuang low-dose group with Erhuang suspension of different concentration. On day 12, PA suspension was given to PA group. Every day the body weight, the incidence, the mortality rate and the neurological score of C57BL/6 mice were observed. On day 23 and 43, blood, the tissue of brain and spinal cord were taken for HE staining, ELISA (which was to detect IFN-γand TNF-a in serum and tissue homogenate), immunohistochemistry (which was to detect Olig2,MBP,GFAP,Caspase3,IGF-1,FGF-2. BrdU), RealTimePCR and Western Blot (which were to detect the gene and protein expression of Olig2).Results1. The mice which were immunized began to fall ill on day 9 post-immunization. The symptoms reached the peak level on day 21-24. The maximum neurological score on average was 1.17±0.83. On day 12, the body weight of PA group and Erhuang high-dose group was significantly increased compared with model group(P<0.05) while Erhuang low-dose group even more significantly(P<0.01). On day 16 and 22, the body weight of Erhuang low-dose group was notably increased compared with normal group, model group and PA group(P< 0.01). HE staining showed that a large number of lymphocytes accumulated around small veins and formed sleeve-like patterns. Microglia was increasing and neurons were degenerating with the karyopykriosis.2. In the region superior to lateral ventricle(LV), the Olig2 expression of Erhuang high-dose group and Erhuang low-dose group was much more than that of normal group, model group and PA group(P<0.01). In the region inferior to LV, the Olig2 expression of Erhuang high-dose group and Erhuang low-dose group was significantly increased compared with PA group(P<0.01). The Olig2 expression of Erhuang high-dose group was more than model group(P<0.05). In the region interior to LV, the Olig2 expression of Erhuang high-dose group was more than PA group(P<0.01). In the region exterior to LV, the Olig2 expression of Erhuang high-dose group was more than normal group, model group and PA group(P<0.01). The Olig2 expression of Erhuang low-dose group was much more than normal group, and PA group(P<0.01) and more than model group(P<0.05). In the ventral part of spinal cord, the Olig2 expression of Erhuang high-dose group and Erhuang low-dose group was much more than that of model group(P<0.01). In the dorsal part of spinal cord, the Olig2 expression of Erhuang high-dose group was much more than normal group, model group and PA group(P<0.01). The Olig2 expression of Erhuang low-dose group was much more than that of model group(P<0.01).In cerebral cortex and CA2, the MBP expression of model group, PA group, Erhuang high-dose group and Erhuang low-dose group was significantly decreased compared with normal group(P<0.01). In white matter, the MBP expression of model group was much less than that of normal group(P<0.01) while Erhuang high-dose group much more than model group(P<0.01). In the peripheral region of LV, the GFAP expression of Erhuang high-dose group was much more than normal group and model group(P<0.01).In white matter, the Caspase3 expression of Erhuang high-dose group and Erhuang low-dose group was much less than that of model group(P<0.01).In white matter, the IGF-1 expression of Erhuang low-dose group was more than that of model group(P<0.05) while Erhuang high-dose group was even more than model group(P< 0.01). In the cornu posterius medullae spinalis, the IGF-1 expression of Erhuang high-dose group and Erhuang low-dose group was much more than that of PA group(P< 0.01).In cerebral cortex, the FGF-2 expression of Erhuang high-dose group and Erhuang low-dose group was much more than that of normal group(P<0.01). In white matter, the FGF-2 expression of Erhuang high-dose group was more than that of normal group(P<0.05) while Erhuang low-dose group was even more than normal group(P<0.01). In the cornu posterius medullae spinalis, the FGF-2 expression of Erhuang high-dose group and Erhuang low-dose group was much more than that of normal group, model group and PA group(P< 0.01).On the exterior wall of LV, the BrdU expression of Erhuang high-dose group, Erhuang low-dose group and model group was more than that of normal group(P<0.05). In the ventral white matter of spinal cord, the BrdU expression of Erhuang low-dose group was more than that of model group(P<0.05). The expression of Erhuang high-dose group was more than that of PA group(P<0.05) while Erhuang low-dose group was even much more than PA group(P<0.01).3. In periodⅠ, the content of serous IFN-γof Erhuang high-dose group was decreased compared with PA group(P<0.05). In periodⅡ, the content of serous TNF-αof PA group, Erhuang high-dose group and Erhuang low-dose group was notably decreased compared with model group(P<0.01).In periodⅠ, IFN-γin the homogenate of spinal cord of model group was increased compared with Erhuang high-dose group(P<0.05). In periodⅡ, TNF-αin the homogenate of spinal cord of PA group, Erhuang high-dose group and Erhuang low-dose group was much less than that of model group(P<0.01).4. In periodⅠ, Olig2 gene expression of Erhuang high-dose group and Erhuang low-dose group was increased compared with normal group(P<0.05). Olig2 gene expression of Erhuang low-dose group was more than that of PA group(P<0.05) while Erhuang high-dose group was even more than PA group(P<0.01). In periodⅡ, Olig2 gene expression of Erhuang low-dose group was more than that of normal group and PA group(P<0.05).In periodⅠ, Olig2/GAPDH of Erhuang low-dose group was much lower than that of model group(P<0.01).In periodⅡ, Olig2/GAPDH of Erhuang high-dose group was lower than that of model group(P<0.05). Olig2/GAPDH of Erhuang low-dose group in periodⅡwas higher than that in periodⅠ(P<0.05). ConclusionEAE mouse model was successfully induced through the evaluation of incidence, clinical manifestation, neurological score, body weight and pathological observation. It was proved to be an ideal model for MS. Erhuang Capsule could effectively improve the clinical symptoms of EAE mice, lower the neurological score and mortality, promote the recovery of neural function, and alleviate the demyelination of CNS. Erhuang Capsule could significantly advance the proliferation of OPCs in the specific region of brain and spinal cord, notably increase the gene and protein expression of Olig2, encourage the differentiation of OPCs, enhance the hyperplasia and activation of astrocytes, decrease the expression of Caspase3, raise the release of several nerve growth factors. It applied good condition to repair and regeneration of myelin. Moreover, Erhuang Capsule could regulate inflammatory factors to alleviate the immune reaction of EAE.