| Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of rareacquired disorders of skeletal muscle with acute, subacute or chronic onset. Its clinicalmanifestations are muscle weakness, creatine kinase elevation,non-specifically abnormalelectromyogram and so on. Several visceral organs are often involved and lifethreatening situation may induced in serious conditions. The exact pathogenesis in IIMremains unclear so far. According to the clinical and pathological features,IIM are mainlydivided into polymyositis (PM), dermatomyositis (DM), inclusion body myositis (IBM)and immune-mediated necrotizing myositis (IMNM).Due to the unclear pathogenesis and unsatisfactory therapeutic effect, we intend tomake a study with myositis animal model. At present, the better animal model ismyosin-induced experimental model of autoimmune myositis (EAM), it is similar to IIMin many respects, and provides a good basis for our research. The model used in this research is modified EAM model by changing the injection dose of myosin and injectiontimes, which is characterized by short time, strong repeatability, stable and reliablecondition. We have previously observed EAM model for2months and found that theinflammation gradually aggravated. The most severe inflammation occurred in thefourteen day and inflammation was slightly weakened soon after.28days later, theinflammatory state tended towards stability. So we performed the experiments on0day,14th day and28th day.The large number of reports confirmed that the imbalance between Tregs and Th17cells is related to various kinds of autoimmune diseases. Tregs plays a protective role inautoimmune diseases and Th17cell can enhance the progress of the autoimmune responseby the induction of various proinflammatory response. However, these studies did notclarify how to change between Tregs and Th17cells in the IIM? In this study, we observedthe change of Tregs/Th17cells and its related cytokines in the EAM, investigate thepossible role of these cytokines in IIM and the relevance between the cytokines and twokinds of cells in order to find the key factor regulating the change of two kinds of cells inthe pathogenesis of autoimmune myositis.ObjectivesObserve the change rule of Tregs transcription factor(Foxp3) and relatedcytokines(IL-10,TGF-β) and Th17related cytokines(IL-6,IL-17,IL-23) in the progress ofEAM; investigate the possible role of these cytokines in IIM and the relevance betweenthe cytokines and two kinds of cells in order to find the key factor regulating the change oftwo kinds of cells in the pathogenesis of autoimmune myositis.MethodsHealthy female BALB/c mice(body weight15~18g,5~6weeks old) wererandomly divided into three groups(n=5): the first group are normal control group, thesecond group is lilled at the14th day in the EAM,The third group is killed zt the28th dayin the EAM. Muscle strength and histopathological scores are determined; Tregs transcription factor(Foxp3) and related cytokines(IL-10,TGF-β) and Th17relatedcytokines(IL-6,IL-17,IL-23) mRNA in muscles and spleens of each mouse were detectedby Real-Time PCR; The expressions of IL-6, IL-17, TGF-β1in the serum of each mousewere detected by Luminex.Results1. Muscle strength was more than30minutes in normal control group, muscle strengthwas significantly decreased in the EAM group, muscle strength in the EAM at the14thday group decreased obviously, the three groups were statistically significant (P <0.01);Histopathological scores: normal control group is0, the EAM at the14th day group is3.05±0.190,and the EAM at the28th day group is1.85±0.10. we observed by the lightmicroscope, in the normal control group, muscle fibers were polygonal, basically thesame size, the nucleus was located in the periphery of the muscle fibers, type I(deep-dyed) and type II muscle fibers (light-dyed) clearly visible; in the EAM at the14th day group, muscle fibers was infiltrated with a large number of inflammatorycells, perivascular inflammatory cell infiltration as common, muscle fiber size range,we can see scattered necrosis, regenerated fibers; in the EAM at the28th day group,muscle fibers were decreased of inflammation, but still has a large number ofinflammatory cells infiltration.2. In the mRNA of mouse muscle tissue, the expression of Tregs transcription factorFoxp3is low(0.697±0.322) in normal control group, is a little higher(1.274±0.547) atthe14th day, and is much higher(16.491±7.884) at the28th day; the expression ofIL-10is low(1.010±0.479) in normal control group, is much higher(5.670±2.979) atthe14th day, and is decreased(2.648±0.633) at the28th day; the expression of TGF-βis higher(0.576±0.250)in normal control group, is a little lower(0.358±0.186) at the14th day, and is much lower(0.094±0.075) at the28th day; the variation of Th17related IL-6, IL-17, IL-23continues to rise from0to28th day, and at the28th dayincreases most significantly(IL-6:0.459±0.308,0.636±0.113,1.477±0.224; IL-17:0.718±0.208,2.197±0.295,19.237±2.126; IL-23:0.754±0.296,2.035±1.050, 20.158±11.188).3. In the mRNA of mouse spleen tissue, the expression of Tregs transcription factorFoxp3continues to rise from0to28th day, and at the28th day increases mostsignificantly(1.017±0.080;1.908±0.675;4.992±1.924); Instead, its related cytokineIL-10and TGF-β are falling, at the14th day is decreased significantly, at the28th daycontinues to reduce(IL-10:0.994±0.147,0.223±0.057,0.183±0.043;TGF-β:0.697±0.188,0.063±0.104,0.063±0.058); the expression of Th17relatedIL-6is higher(0.507±0.384) in normal control group, is much lower(0.066±0.163) atthe14th day, and is a little higher(0.167±0.069) the at28th day; the expression ofIL-17is low(1.269±0.293) in normal control group, is a little higher(2.681±0.305) atthe14th day, and is much higher(10.252±1.672) at the28th day; the expression ofIL-23has no significant change.4. In the serum, the expressions of IL-6, IL-17and TGF-β1are all lowest in normalcontrol group,are highest at the14th day, and are a little lower at the28th day(IL-6:0，35.840±3.094,3.740±1.108; IL-17:0,35.130±5.524,10.120±3.579; TGF-β1:2413.100±223.563,3861.600±317.621,2895.800±366.154).Conclusions1. The expressions of Tregs transcription factor(Foxp3) and related cytokines(IL-10,TGF-β) and Th17related cytokines(IL-6,IL-17,IL-23)are significantly differentin the progress of EAM. The general tendency is that Tregs related cytokinesdecreased and Th17related cytokines increased, indicating that they play an importantrole in the pathogenesis of autoimmune myositis.2. The cytokines IL-10and IL-17increased in the14th day in EAM, implying that theyhave a crucial role in early inflammatory response. The cytokines IL-6,IL-17,IL-23still increased in the28th day, meaning that these cytokines may participate incontinuous development of inflammation.3. In the progress of the occurrence and development of IIM, the cytokines IL-6,IL-10and TGF-β in the spleen of mice gradually decreased, implying that the spleen, as the immune organ of body, regulate the distribution of T lymphocytes. It leads to theredistribution of T lymphocytes after recirculation, and makes these cytokines homingto inflammatory sites of muscle to play its immune function.4. In the progress of the occurrence and development of IIM, IL-6gradually increasedand TGF-β gradually decreased, they may achieve the appropriate proportion in the14th day so as to play its biggest role, leading to significantly rapid increase of IL-17after14days and reaching its peak on the28th day. It indicates that the increase ofIL-6may make more initial T cell change into Th17cell, so blocking-up the IL-6mayprovide an ideal therapeutic target for myositis. |
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