Total Glucosidesof Peony Ameliorated Experimental Autoimmune Myasthenia Gravis By Up-regulated Treg And Increased Th2 Cytokine

Surroundings of the research:Myasthenia gravis(MG)in human is similar to Experimental autoimmune myasthenia gravis(EAMG),which we can found by animals.It is a diease which is antibody-mediated and T-cell dependent.No matter EAMGor MG are caused by autoantibodies against the nicotinic acetylcholine receptor(AChR)in the post-synaptic membrane of the neurom-uscular junction.EAMG is similar to MG in its clinical,electrophysiological and immuno-pathologic manifestations.The rate of MG may high to 80-200 thousand out of 10million,in addition many MG patients have been discoverd.MG is charactered by easier exhausted musules in limited or all over the body.The disease uarally attacks musules surrounding the eyes,arms and legs and so on.Dangerous MG may lead to the musules related to breath can not make effection which can threat lives.EAMG is similar to MG in its clinical,electrophysiological and immunopathologic manifestations.Different ways have been used in the diease such as anticholinesterase,glucocorticoids,immune globulin,thymusectomy andplamapheresis plasma change.Not all of these methods is effecetive to the diease,otherwise,they have many side effects and the high cost can not afforded by the patients.So,if we can do some research on immunosuppressor which can be get easily,lower-cost,lower-side effect,it must benefit all the patients of MG,and great contributions can be made to our society with unvaluable prospect.TGP(total glucosidesof peony,TGP)is the dry root of paeonialactif lora.Chinese medicine think TGP as good includings of rheumatic diease,autoimmune diease hepatitis hepatorcirrhosis medicine which can nourishing blood,dredging collaterals.TGP is noticed by all of the world.After many years of reaserch by international socientists,the useful monosome morphon has been extracted.Mainly include glycoside which is called total glucosidesof peony.Recently,some research has proved that,TGP has anti-infiammatory,antioxidant oxidation resistance,and immunomodulat effects.Jing Li has found TGP and TNF?,IL-6level by the EAMG.Ming Zhao has found that TGP can increase the Fox3 in CD4+cells in the patients of SLE Which can increaseCD4+,CD25+T cells at the meantime.But a retrospective study has presented that TGP has exactly effect in patients of SLE.And it can decrease the dose of methylprednisolone and CTX.Reduce the reappear of this diease and the rate of it can be lower,especially the patients who has oral TGP for 5 or more years.There is no report about TGP is useful on MG or EAMG both in china and abroad.According to all of above,TGP has exact effect on anti-inflammatory action.And TGP has been permited by the SFDA(State food and drug Administration)to treat RA because of its anti-inflammatory andimmunomodulater effects.It is mature that TGP is used totreat RA in clinic,and the security safety of it is high.So,it will be more effective than any other immunosuppressor if it can be used in MG.Materials and Methods:1 ReagentsThe synthetic peptide range from region 97-116 is used as the antigen of the rat acetylcholine receptor subunit(R97-116).2 Producing and evaluation of EAMGRats which are femal,6–8 weeks of age(body weight 140–160 g),were immunized subcutaneously in both hind footpads with 200l inoculums containing 50 g R97-116 and1mg Mycobacterium tuberculosis emulsified in incomplete Freund's adjuvant on day 0 and were boosted on day 11 post immunization(p.i.)with the same peptide in complete Freund's adjuvant(CFA).The rats were observed in a double-blind fashion for signs of muscle weakness characteristic of EAMG before immunization and thereafter every other day until day 49 p.i..Body weight was recorded in parallel.The clinical symptoms were graded between 0 and 4 according to the Lennon criteria.3 Using of TGPTGP(at a dose of 1 mg/kg in a volume of 100 l DMSO)was administered to immunized rats by intraperitoneal injection every other day from day 10 p.i.(the early periods)to day54 p.i.(the peak periods).4 Detection of Foxp3 in the thymuses of rats by immunohistochemistryAll rats in different group were killed on day 54d,and thymuses were excised,then fixed in 10%paraformaldehyde and embedded in paraffin.The transcription factor Foxp3was detected by immunohistochemistry.5 The lymph node mononuclear cell suspensions should be got.Inguinal lymph nodes were excised from all the rats under aseptic condition.Mononuclear cells(MNC)suspensions from inguinal lymph nodes of individual rats were obtained by grinding through cell strainers in medium.The cells were then suspended to 2×106/ml in RPMI 1640(containing 2.05 mM glutamine)supplemented with 1%(v/v)minimum essential medium(MEM)and 50 g/ml gentamicin and 10%(v/v)fetal bovine serum.6 T cell proliferation assayCCK-8 to prolife T cell assay.Triplicate aliquots(200 l)of MNC suspensions containing 4×105 cells were placed in96-well microtiter plates.Ten l aliquots of R97-116 were added into appropriate wells at a final concentration of 10 g/ml.The same volume of CMC-Na was added into control wells.After 64 h incubation with 5%CO2 at 37?,supernatants were collected and stored at-20?to detect cytokines.Then aliquots(10 l)of CCK-8 were added into all the wells.After 4 hours,results were expressed as optical density(OD)at 450 nm.7 Determination of IL-4 in culture supernatants by ELISA.After 64h incubation,the cell culture supernatants in the presence of R97-116 were collected for IL-4 determination.IL-4 was measured by sandwich ELISA kits according to the manufacturer's instructions.8 Calculate the number of anti-R97-116 antibodiesUse the SPSS 20.0 computer programme to make statistical analysis.Results were expressed as means±standard deviation(SD).Sample come from the three groups were analyzed by independent t test.The level of significance was set at p=0.05.Results:1 TGP treatment reduces the severity of EAMG rats.2 TGP up-regulates the number of Foxp3~+cells in the thymuses of EAMG rats.3 OD values of cell proliferation under both CMC-Na and R97-116 condition are lower in TGP-treated group than the control group.Even though there is no statistic significance,but the trend is obvious.4 TGP increases the levels of IL-4 in the culture supernatants of lymph nodes MNC.5 TGP in decreasing the levels of anti-R97-116 IgG antibodies.Conclusion:Lower dose of atorvastatin could ameliorate EAMG by up-regulated Treg and increased Th2cytokine.