Beneficial Role Of 1, 25-dihydroxyvitamin D₃ In Experimental Autoimmune Myositis

Idiopathic inflammatory myopathies can be classified into four main categories based on clinical and immune histological features. Currently, therapies focus on improving systemic functions by using corticosteroids and immunosuppressive drugs. However, these drugs are partially efficacious while have many serious side effects. Hence, we need to understand thoroughly the pathogenesis of IIM and propose other promising treatments.1,25-Dihydroxyvitamin D₃ is a regulator of mineral absorption as well as its immune-modulatory functions. It has been demonstrated that 1,25（OH）₂D₃ prevents many autoimmune diseases such as type 1 diabetes/IBD/multiple sclerosis through many mechanisms like suppressed Th17 while enhanced Treg function. Experimental autoimmune myositis（EAM） by immunization with partially purified myosin in mice is a primary model which mimics closely polymyositis with showing muscle weakness and inflammation, and inflammation cells infiltrated and so on. Considering the broad physiological relevance, vitamin D may potentially become a treatment method for autoimmune diseases.The aim of this study was to evaluate the efficacy of 1, 25（OH）₂D₃ in the EAM model. Our findings established that the mice conditions intervention with 1, 25（OH）₂D₃ were improved compared to the EAM through its immunology effects. Therefore, 1, 25（OH）₂D₃ could be a promising regulated drug for polymyositis. Objectives:To explore if 1, 25（OH）₂D₃ has the regulatory effect for the EAM model and compare its function with methylprednisolone in the different periods which are severe stage and alleviation period about Th17/Treg cells related cytokines. Methods:There were twenty five healthy female BALB/c mice distributed into five groups randomly: normal group, EAM group, calcitriol group, methylprednisolone group and combined intervention group. Each group had five mice and observed fourteen days. The mice in the normal group kept normal status without any interference. In the EAM group, the mice were given myosin to be the model. Calcitriol group: modeling while giving calcitriol intraperitoneal. Methylprednisolone intervention group: modeling while giving methylprednisolone intraperitoneal, continuous fourteen days. Combined group:modeling while giving methylprednisolone and calcitriol intraperitoneal. Competed the five groups, and used the intervention methods from fifteenth day and observed twenty-eight days.Both in fourteen days and twenty-eight days, there were some contents to be compared among these five groups which were general status and muscle strength and histopathological scores; Tregs transcription factor（Foxp3） and related cytokines（IL-10 and TGF-β） and Th17 related cytokines（IL-6, IL-17 and IL-23） m RNA in muscles and spleens of each mouse were detected by Real-Time PCR; The expressions of IL-6, IL-17, and IL-23 in the serum of each mouse were detected by Luminex. Results:1. Compared with EAM group, mice muscle strength were improved and the degree of inflammatory in muscle were reduced in calcitriol intervention group both in fourteen days and twenty eight days. The order was the calcitriol intervention group 、methylprednisolone group and combined group based on the general status. Although the general status in fourteen days were better in combined group compared with methylprednisolone intervention group, there was no difference. However, in twenty eight days, it was better in combined group than methylprednisolone group.2. Detected m RNA expression levels of cytokines in the muscle and lymph nodes tissue of mice in every group, we found that compared with the EAM group, Th17 cells related cytokines expression levels decreased in varying degrees（P<0.05） in calcitriol group both in fourteen days and twenty-eight days, and decreased more in the methylprednisolone group and combined group. Compared with the EAM group in fourteen days, the expression of foxp3 and IL-10 in muscle were increased in fourteen days while TGF-β decreased. The expressions of IL-10 increased both in the methylprednisolone group and combined group. Moreover, there was difference between the two groups（P<0.05）. Compared with the EAM group in twenty-eight days, the expressions of foxp3 and TGF-β were increased in lymph nodes after intervention. There was difference between methylprednisolone group and combined group about TGF-β（P<0.05）.3. Detected m RNA expression levels in the spleen tissue of mice found that, among fourteen groups, compared with EAM group, IL-17 and IL-23 m RNA expression levels in calcitriol group increased in varying degrees（P<0.05） as well as Tregs related cytokines（P<0.05）. The expression of IL-10 and TGF-β were increased both in methylprednisolone group and combined group（P<0.05）. Among twenty-eight groups, compared with EAM group, IL-17 expression levels had different increased（P <0.05） as well as Treg related cytokines IL-10 and TGF-β; IL-6 and foxp3 decreased among three intervention groups.4. In the serum, we found that the expressions of IL-6, IL-17 and IL-23 are all lowest in normal group, and highest at EAM model group, which were tested by luminex. Compared with the EAM group, the expression levels had different degrees lower in three intervention groups（P<0.05）, especially in the combined group decreased more significantly. Conclusions:1. Calcitriol can improve muscle strength and general status as well as alleviate the inflammation in muscle both in fourteen days and twenty-eight days. Although its function was less than methylprednisolone, the general status in myositis mouse got better after combined intervention.2. Compared with EAM, the expressions of Tregs transcription factor（Foxp3） and related cytokines IL-10 were increased and Th17 related cytokines（IL-6, IL-17 and IL-23） were decreased in intervention group generally. It was indicated that calcitriol play an immune-regulatory role in the pathogenesis of autoimmune myositis.3. The expressions of Tregs related cytokines（IL-10 and TGF-β） were more increased in combined intervention group than methylprednisolone intervention group while there was no difference in Th17 related cytokines. It was indicated that calcitriol may play an important role in enhancing the Treg role when combined with methylprednisolone.