Experimental Study Of The Traditional Chinese Medicine Intervention In EAE Rats And Observing Th1Related Cytokines

Objective：To observe the impact of kidney-tonifying, detoxifying and collaterals-dredgingformula via the experiment on interfering experimental autoimmune encephalomyelitis ratsabout the morbidity in acute stage and remittent stage, neurologic symptoms, tumor necrosisfactor-α（TNF-α）, as well as the interleukin-12（IL-12）content changes, discuss thetherapeutical effect, as well as the possible anti-inflammatory mechanism of the Chinesemedicine on experimental autoimmune encephalomyelitis rats in acute stage and remittentstage, which will provide experimental basis for Chinese medicine in treating multiplesclerosis in clinical.Methods:1. The establishment of the EAE model: Established the EAE model by regardingthe guinea pig spinal cord homogenate (GPSCH) and Freund’s adjuvant as the antigen andregarding the bordetella pertussis as the induction immune Wistar rats and observed themorbidity and clinical symptoms of rats.2. Experimental grouping and drug administration:90rats were divided into sixgroups randomly: blank group, model group, steroid group, low-dose group of Chinesemedicine, middle-dose group of Chinese medicine, high-dose group of Chinese medicine.After the morbidity occurring of rats, with the prednisolone acetate as the contrast group,gave different dose of Chinese medicine intervention for Chinese medicine groupsrespectively and observed the morbidity situation of rats.3. Sample collection: had blood sampling at the vena ophthalmica of rats14days(fastigium) and21days (remittent stage) respectively after the model building of rats andplaced it at the EP tube centrifuging for20min and then took the serum for measuring. Thebrain and spinal cord by heart perfusion was placed within the4%paraformaldehyde andfixed, had the paraffin embedded and cut.4. Sample determination: dyeing with the hematoxylin-eosin (HE) methodobserving the pathologic change of brain and pathologic change and with the ELISA methodmeasuring the content of TNF-α and IL-12within the serum in different time.Results:1. This experiment has made make the EAE model of rats successfully. There is nosignificant difference on morbidity and disease time between groups. Neurological scores ofrats attacking at the peak onset and remittent stage in groups of high and middle dose usedkidney-tonifying, detoxifying and collaterals-dredging formula is significantly lower than themodel group（P<0.05）. The neurological score of low-dose group of Chinese medicine at thefastigium attcking stage has zero difference(P>0.05) compared with the model group and thescore at remittent stage is significantly lower than the model group（P<0.05）.2. Through the microscopic observation, at the peak onset and remittent stage,compared with the model group at the same time, the number of rats CNS inflammatorylesions within Chinese medicines group and steroid group has improved markedly.3. The serum TNF-α content in high and middle dose groups of kidney-tonifying, detoxifying and collaterals-dredging formula at peak onset and remittent stage issignificantly reduced compared with the model group (P<0.05) and has zero difference(P>0.05) compared with the steroid group; there is no significant difference （P>0.05）between low-dose group of Chinese medicine. The serum TNF-α content of high-dose groupin peak onset stage and middle-dose group and low-dose group in remittent stage hassignificantly reduced （P<0.05） compared with EAE group and has significant difference(P<0.05) compared with steroid group. High and Low-dose group of Chinese medicine issignificantly higher than middle-dose groups of Chinese medicine（P<0.05）.4. The serum IL-12content in every groups of kidney-tonifying, detoxifying andcollaterals-dredging formula at peak onset stage is significantly reduced compared with themodel group (P<0.05); Compared with the steroid group, there is no significant difference（P>0.05）with middle dose groups of Chinese medicine,and there is significant differencewith high and low-dose groups of Chinese medicine (P<0.05); in the remittent stage, theserum IL-12content in middle dose group of Chinese medicine has significantly reducedcompared with the model group（P<0.05） and has zero difference (P>0.05) compared withthe steroid group; high and low dose groups of Chinese medicine has no significantdifference (P>0.05) compared with the model group.Conclusion:(1) The kidney-tonifying, detoxifying and collaterals-dredging formula cansignificantly reduce EAE rats symptom in the acute phase of disease, reduce nerve damagepeak onset of rats and promote the recovery of clinical symptoms of rats.(2) Through the HE dyeing microscopic observation, the kidney-tonifying,detoxifying and collaterals-dredging formula can significantly reduce EAE ratsinflammatory damage of the central nervous system (CNS), reduce the number ofinflammatory lesions and alleviate nerve dysfunction in rats.(3) The kidney-tonifying, detoxifying and collaterals-dredging formula caneffectively reduce the number of TNF-α and IL-12content within the serum in EAE ratsacute and remission stages and find that the EAE rats neurologic deficit degree has positivecorrelation with the TNF-α and IL-12content. We can deduce that the partial role ofkidney-tonifying, detoxifying and collaterals-dredging formula may be lowering theexpression of TNF-α and IL-12content within the serum in the body, that inhibiting Th0toTh1cells, thereby adjust the balance of Th1/Th2system in order to control thedisorganization of inflammatory factor to achieve the recovery of symptoms.