Studies On Elavating Antipsoriatic Drugs Efficacy In Th1-type Animal Model And Mechanism Of Chinese Medicine Treatment

Psoriasis is a T-cells-mediated autoimmune disease. A certain amount of literatures indicate Chinese medicine plays an important role in the treatment of psoriasis. It is necessary to find a suitable animal model that can be used in studying the pathogenesis of psoriasis and exploring the mechanism of antipsoriatic drugs, for example, Chinese medicine. T helper cell (Th) 1 lymphocytes and cytokines may be predominatly contributed to the pathogenesis of psoriasis. Clearly, the first problem comes to us is how to find an animal model of psoriasis characterized by these biological features.This study used the animal model which inducing experimental autoimmune thyroiditis (EAT) on rats by thyroglobulin and iodine-rich water, and we assessed the antipsoriatic effects of different drugs and Chinese medicine in vivo by screening Anti-Thyroglobulin antibody (TgAb) levels in the serum. Meanwhile, we investigated the possible molecular mechanisms of these drugs on the Th1/Th2 lymphocyte balance by gene chip technology which has the advantages of high-throughput, high degree of parallelism and high sensitivity from the perspective of the whole genome. In addition, we explore the immune mechanism of Xiaoyinfang on the Th1/Th2 lymphocyte balance, which was effectively used in Changhai Hospital for the treatment of psoriasis with syndrome of heat in blood.Part I Establishment of a Rat Model with Experimental Autoimmune ThyroiditisObjective: To verify the method of establishing rat models with EAT and to observe its impact on Th1/Th2 bias in peripheral blood. Methods: Pig thyroglobulin and iodine-rich water (0.05%) were adopted to induce EAT in Wistar rats. Unimmunized rats were used as control. Seven weeks after immunization, serum of each rat was screened for TgAb and the histological thyroid specimen of EAT were assessed by blind evaluation. The expression of TNF and IL-10 mRNA in peripheral blood mononuclear cells was assessed by Realtime-PCR method.Results: The rats of model group showed about 3 fold increase of the levels of TgAb as high as that of control and there were 7 cases whose score of lymphocytic infiltration index was more than 2 that took up about 87.5% of experimental rats. TNF mRNA expression in peripheral blood mononuclear cells in the model group were elevated, however, IL-10 mRNA expression decreased significantly. Conclusions: EAT can be characterized by high level TgAb and lymphocytic infiltration in the thyroid tissue. From this study, we may conclude both Tg and iodine-rich water could induce EAT successfully. There was a certain degree of Th1 cytokines bias in rats with EAT, which could provide a theoretical basis for psoriasis in vivo animal model.Part II Effects of Different Antipsoriatic Drugs in Rat with Experimental Autoimmune ThyroiditisObjective: To observe the effect of different antipsoriatic drugs in experimental autoimmune thyroiditis .Methods: EAT models were established, which were randomly divided into four groups and the rats were given gavage for 4 weeks, such as model group(normal Saline), cyclosporine A group(25mg/kg/d), tripterygium glycosides group(2.75mg /kg/d), and Xiaoyinfang Group(2g/kg/d). Four weeks after treatment, all rats were killed, whose serum was screened for TgAb and the histological grade of throid specimen were acesssed by blind evaluation.Results: The levels of TgAb in the model group were significantly higher than the other groups (P<0.01), and there was no significant difference among the treatment groups(P>0.05). Severity of inflammatory infiltriation in thyroid was not significantly different among all groups(P>0.05).Conclusions: These results suggested that all of antipsoriatic drugs decreased the levels of TgAb in EAT rats on certain degree. However, there were no improvement of imflamatory infiltration in thyroid among treatment groups and control group. This might be related to the course and the dosage of our administration and irreversible damages in the thyroid cells of some rats. Therefore, we could use cyclosporine A and tripterygium glycosides as positive control drugs to observe contrasting effects of traditional Chinese medicine and the levels of TgAb as standards for determining whether a drug acts. It should be noted that preventive medication and extending course be important in the further study. The effective results of Xiaoyinfang treating EAT supported the idea that treating different diseases with same method in traditional Chinese medicine, which provided additional evidence for exploring new therapeutic drugs or new uses of old drugs for Th1-type diseases.Part III Expression Profile of Gene in Response to Different Antipsoriatic Drugs in Treatment for EATObjective To analysis the difference of Th1-Th2 gene expression profile of peripheral blood mononuclear cells between the pre-treatment and post-treatment with different antipsoriatic drugs for EAT, isolate the differentially expressed genes and look for possible molecular mechanisms of these drugs on the Th1/Th2 lymphocyte balance by gene chip technology.Methods Total RNA of peripheral blood mononuclear cells which were isolated from EAT rats in vivo from each groups were extracted and subjected to reverse transcription into cRNA.The cRNA probes were hybridized with rat Oligo nucleotides gene chips containing 115 Th1-Th2 -related genes. Scan Array Scanner was used to screen the signals of hybridization and GEArray software was applied to analysis and isolate differential expression gene.The result of gene chip was assessed by Realtime-PCR method.Results From all 3 groups, there were 10 common genes in those differentially epressed genes, of which one gene(IL-10) were up-regulated and the remaining (CD28, CD69, Fadd, IL-18, Socs3, Socs6, Stat1, Tnfsf5 and Tnfsf6) were all significantly down-regulated. In Xiaoyinfang group, a total of 13 genes(Ccr4, Cd2, C2ta, Gfi1, Gpr2, Il1r1,Mapk10, Nfatc4, Nfκb, Tyk2, Socs4, Tbx21, and Tnfrsf9) expressed differently, and all 13 genes were down-regulated. The function of those differentially expressed genes was searched in PubMed.Conclusions The result of gene chips suggested that each antipsoriatic drugs could regulate Th1/Th2 lymphocyte balance by up-regulating expression of Th2 cytokine(IL-10)and down-regulating expression of Th1 cytokines, of which such as Socs3, Socs6, Stat1 and Tnfsf6 might be involed in the reulation of the balance. It provided strong evidences for investigating the mechanism of development, progression and prognosis in psoriasis and research and development of drugs targeting to some signal transduction pathways and related genes. In addition to the above mechanisms, the possible mechanisms of Xiaoyinfang treating psoriasis might be involved in many genes such as TNF Ligand and receptor family, the signal transduction pathways related with TNF and Th1/Th2 cell differentiation-specific transcription factor such as Gata-3 and Tbx21, etc.Part IV Clinical Effects and Changes of the Th1/Th2 Lymphocyte Balance in the Peripheral Blood in Xiaoyinfang Treating Psoriasis with Syndrome of Heat in BloodObjective To explore machenisms of Xiaoyinfang in treating psoriasis with syndrome of heat in blood by observing the clinical efficacy and the impact on the cytokines in the serum.Methods Observe the difference of scores of PASI, scores of differentiation of syndrome and level of TNF-αand IL-10 in the serum in 32 psoriatic patients with syndrome of heat in blood between pre-treatment and post-treatment. Results The levels of TNF-αin the serum were significantly lower in pre-treatment group than in post-treatment group (P<0.05), whereas the levels of IL-10 had no significant difference (P>0.05). PASI scores and the secretion of TNF-αlevels in patients with psoriasis were positively correlated (P <0.05).Conclusion These results suggested the efficacy of Xiaoyinfang treating psoriasis maybe by TNF-αpathway.