| Dermatomyositis is an autoimmune connective tissue disease that mainly affects the skin and muscles.DM has a high mortality rate and many harmful complications.Glucocorticoid and immunosuppressive drugs are the main clinical treatments for DM;The western medical therapy has strong dependency with limited efficacy and significant toxicity.Traditional Chinese medicine(TCM)has advantages in syndrome differentiation and treatment.Common symptoms of DM include liver depression,spleen deficiency,and blood heat,according to the therapeutic principles of soothing the liver,strengthening the spleen,and cooling the blood,the compound of Jiaweixiaoyao pills from xue ji's "internal medicine abstract"in the Ming dynasty was selected.The saikosaponin,ferulic acid,paeoniflorin,atractylon,paeonol and other monomers in compound have anti-inflammatory,anti-allergic and immunomodulatory effects.Monkshood,who has a good effect in treating autoimmune diseases,is the rhizome of the anemone in the ranunculaceae.Based on the previous work,this study explored the pharmacodynamics and pharmacology of Diwu and jiaweixiaoyao pills in inhibiting inhanced immune system of DM by down-regulating macrophage JAK2-STAT3 pathway.1.A novel pharmacodynamic rat model of dermatomyositisObjective:To develop a pharmacodynamic rat model for DMMethods:The DM model was developed with allogenic membrane antigen-induced autoimmune,following with toxin-induced cutaneous calciphylaxis.Antigen immunized animals in five groups were administrated with membrane antigen to induce autoimmune injuries(once weekly from d01 to d28 for sensitization,and from d42 to d63 for challenge).At d77,the rats in four groups with antigen-immunization were administrated with dihydrotestosterone(DHT),Fe-dextrin(Fe-Dex),polymyxin(PMX),or the integration of three toxins,to mimic cutaneous calciphylaxis,respectively.The score of clinic DM manifestations,ratios of infiltrated lymphocytes in skeletal muscles or arteriole calcified nodules in subcutaneous tissues,and serum levels of special antibodies and cytokines were detected.Results:Among the five groups with antigen immunization,the rats dealt with toxin showed significant clinic DM scores and pathological changes.Among the four groups dealt with toxin,the series of DM severity ranged as the rats dealt with integration of three toxins,PMX,DHT,or Fe-Dex,which was evaluated as DM clinic scores,T lymphocyte infiltration,cutaneous calciphylaxis,and autoimmune injuries.The mechanism of DM was demonstrated as increased levels of anti-Jo-1,anti-MDA5,TNF-?,and IFN-? in sera.Conclution:A novel pharmacodynamic model of DM in rats had been developed asthe autoimmunity from membrane antigen immxmization and the cutaneous calciphylaxis from toxins.2.The Pharmacodynamics of the compatible enhancement of Diwu to Jiaweixiaoyao pills fordermatomyositis in ratsObjective:To study The dose-efifect relationship of the compatible enhancement of Diwu to Jiaweixiaoyao pills in treating rats dermatomyositis(DM).Methods:108 SD rats were randomly divided into 6 groups,the rats in five groups were administrated with membrane antigen(once weekly from d00 to d07 for sensitization,and twice weely from d28 to d38for challenge),at d63,the rats were administrated with dihydrotestosterone(DHT),Fe-dextrin(Fe-Dex),polymyxin(PMX)to establish the rat DM model.At d21,The rats were treated with gradient dosages of FK506(0.0056?176.71mg·kg-1·d-1,k=3.16),Jiaweixiaoyao pillsincluding Diwu(1.83?18173.79mg·kg-1·d-1,k=3.16),Anemone flaccid(0.28?2795.97mg·kg-1·d-1,k=3.16),the Jiaweixiaoyao pills including Anemone flaccid(2.11?20969.76mg·kg-1·d-1,k=3.16),respectively.The pathological features were observed after hematoxylin-eosin staining.The volume ratio of infiltrated lymphocytes in skeletal muscles or arteriole calcified nodules in subcutaneous tissues were analyzed with morphometry.The expression of foxp3,IL-17RAJAK2 and STAT3 were detected by IHC.The ED50 was calculated by non-linear regression with various slope using Prism-5.0 software.Results:The effects of Jiaweixiaoyao pills and Jiaweixiaoyao pillsincluding Anemone flaccid on DM showed a dose-dependent tendency.The dose-response curves showed "S"shape.The efficacy of Jiaweixiaoyao pills on rat DM was the most significant among the three formulas.The ED50 of Jiaweixiaoyao pillsincluding Anemone flaccid regressed by Jiaweixiaoyao pills dose was 0.63-fold for decreasing the T lymphocytes infiltration,0.72-fold for decreasing the arteriole calcified nodules,0,71-fold for inhibiting the expression of IL-17RA,0.68-fold for increasing the expression of foxp3.The ED50 of Jiaweixiaoyao pillsincluding Anemone flaccid regressed by Anemone flaccid dose was 0.70-fold,0.44-fold,0.74-fold,and 0.81-fold in T lymphocytes infiltration,the arteriole calcified nodules,the expression of IL-17RA and the foxp3.Conclusion:Jiaweixiaoyao pills can prevent and treat rat DM effectively.Anemone flaccid has obvious compatibility synergy in the whole formula of enhancing the therapeutic effect and reducing toxicity.3.Pharmacology of Jiaweixiaoyao pills for dermatomyositis in ratsObjective:To confirm JAK2-STAT3 pathway involving in Jiaweixiaoyao pills for treating dermatomyositis.Methods:60 SD rats were randomly divided into 6 groups,the rats in five groups were administrated with membrane antigen(once weekly from d00 to d07 for sensitization,and twice weely from d28 to d38for challenge),at d63,the rats were administrated with dihydrotestosterone(DHT),Fe-dextrin(Fe-Dex),polymyxin(PMX)to establish the rat DM model.At d21,The rats were treated with FK506(3.12mg·kg-1·d-1),the compound of Jiaweixiaoyao pillsincluding Diwu with Low(664.56mg·kg-1·d-1),Middle(2100.00mg·kg-1·d-1),high(6636.00mg·kg-1·d-1)doses,respectively.The ratios of infiltrated lymphocytes in skeletal muscles or arteriole calcified nodules in subcutaneous tissues were evaluated.Serum TNF-?,IL-1?levels were measured.Expression of JAK2,STAT3,were determined with immunohistochemical staining,real time RT-PCR and Western blot,pSTAT3 entering into nuclear was investigated by immunohistochemical staining,pSTAT3 expression was determined with Western blot.Results:(1)Phranacodynamic effect:?General observation:Compared with the model group,the effect was shown to relieve redness and swelling of rat limbs and myasthenia;?Pathological observation:Compared with the model group,mucle atrophy and necrosis of myoblasts were reduced by the effect of Jiaweixiaoyao pills,T lymphocyte infiltration was significantly reduced.?Calcified nodules:Compared with the model group,the calcified nodules volume was reduced in each administration group;?TNF-?:Compared with the model group,serum TNF-? levels were decreased in each administration group;?IL-1?:Compared with the model group,serum IL-1?levels were decreased in each administration group;(2)pharmacological mechanism:?CCR2:Compared with the model group,the expression of CCR2 dectected by IHC was reduced in each administration group;?JAK2:Compared with the model group,the gene and protein expressions of JAK2 were reduced in each administration group;?STAT3:Compared with the model group,the gene and protein expressions of STAT3 were reduced in each administration group;? pSTAT3 nuclear translocation:Jiaweixiaoyao pills decreased the ratio of pSTAT3 nuclear translocation. |
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