Analysis Of The Adenosine And Adenosine Receptor In The Muscle Tissue Of Idiopathic Inflammatory Myopathy

Background and purposeIdiopathic inflammatory myopathy (IIMs) is non-infectious inflammatory myopathy, with the main pathological features of the infiltration of inflammatory cells in the skeletal muscle and muscle fiber atrophy and necrosis, the pathogenetic mechanism is still unclear. The most prevalent subtypes are multiple polymyositis (PM), dermatomyositis (DM) and inclusion body myositis (IBM).The adenosine is the signaling molecule of autocrine or paracrine pathway, and plays an important role in maintaining the internal environment stability. Adenosine can inhibit oxidative burst and tumor necrosis factor-a and other inflammatory cytokines, and is concerned with the regulation of immune and inflammatory responses; it was seen that the extracellular adenosine level was enhanced in patients with sepsis and animal models with Crohn’s disease. Adenosine produces its function mainly through binding to its receptor, and adenosine receptor is the key part to detect the anti-inflammatory and immunomodulatory effects of adenosine.Adenosine receptors (AR) are divided into four types:A1AR, A2AAR, A2BAR, A3AR, and they are all G protein-coupled seven transmembrane receptors. Lynge J et al. used immunohistochemical and western-blotting to detect the distribution of adenosine receptors A1AR, A2AAR, A2BAR in human skeletal muscle, and the result showed that skeletal muscle only expressed A2AAR and A2BAR, both in muscle fiber membrane and cytoplasm, while A1AR was not expressed. In1993, Sajjadi FG et al. used RT-PCR to clone and sequence analysis of A3AR, and found that A3AR was almost not expressed in human skeletal muscle tissue.Extracellular adenosine Source:a) Adenosine monophosphate (AMP) was dephosphorylated to produce adenosine by the extracellular5’-nucleotidase (CD73). AMP was generated by the extracellular adenosine tri-phosphate (ATP) and adenosine diphosphate (ADP) by nucleoside triphosphates dephosphorylation enzymes (NTPDase). There are several types of extracellular NTPDases, CD39is a major extracellular adenylate dephosphorylation enzyme and CD39L1is also a common extracellular NTPDase. b) The inflammatory cells releasing adenosine is also an important source of the extracellular adenosine.Therefore, we detected the expression of A2AAR, A2BAR and A3AR in human skeletal muscle fibers, and muscle tissue adenosine receptor expression in the patients with the IIMs, as well as the expression change of extracellular adenosine generation related enzymes, to explore new directions for the treatment of IIMs by exploring the mechanisms of adenosine via its receptor-mediated muscle fibers in inflammatory myopathies state.Objects of study and methodsIn this study, we selected17cases of the patients with the clinical symptoms of myasthenia gravis but no neuromuscular damage performance in muscle biopsy,14cases of the limbs skeletal muscle and3cases of erector spinae was used to detect the AR expression. We collected skeletal muscle biopsy specimens from30IIMs patients, including15patients with dermatomyositis (DM),11patients with polymyositis (PM),4patients with inclusion body myositis (IBM),14cases of normal control (biopsy parts are all limbs skeletal muscle).We treated the muscle biopsies with western blot analysis and the immunohistochemistry to compare the difference of A2AAR, A2BAR, A3AR and extracellular adenosine generation enzyme CD73, CD39, CD39L1of IMs patients and normal control.Data were described as mean±standard deviation (x±s) and were analyzed using the SPSS17.0statistical software. Comparison between groups were conducted by student t test;classified information was analyzed by χ2test, and Fisher’s exact probabilistic method while frequency<1.P value less than0.05was considered statistically significant.Results1. Western blot analysis of slight of muscle tissue showed the A2AAR, and A3AR straps.2. The skeletal muscle A2AAR, A2BAR and A3AR antibody immunofluorescence staining were positive:A2AAR antibody immune fluorescence staining positive was only found in the muscle fiber membrane, cytoplasm had no obvious coloring, mesenchyme and vascular had no obvious coloring; A2BAR antibody immune fluorescence staining positive was mainly seen in type1muscle fiber cytoplasm; A3AR antibody immune fluorescence staining positive was mainly seen in type2muscle fibers cytoplasm and vascular smooth muscle, the fluorescence intensity of type2muscle fibers cytoplasm:+, the fluorescence intensity of vascular smooth muscle:+++, muscle fiber membrane had no significantly coloring.3. Western blot analysis showed that the corresponding straps of erector spinae3cases of A2AAR, A2BAR and A3AR were wider than of the limb skeletal muscles.4. The A2A AR, A2B AR and A3AR antibody immunofluorescence staining in non-necrotic fibers of IIMs patients was enhanced staining compared with normal control; A2AAR, A2BAR straps gray value was increased compared to the normal control, while A3AR strap gray value has no significant difference compared with normal control. A3AR antibody immunofluorescence staining showed strong fluorescence reaction in some necrotic muscle fibers.5. Extracellular adenosine generation related enzyme CD73antibody staining was seen at fascia musculares and endomysium of the inflammation site and part normal muscle fiber intima, non-inflammation muscle interstitial sites were also deeply stained; CD39and CD39L1antibody staining had no coloring in muscle fibers, while CD39-positive was found in vascular endothelial cells, and CD39L1positive was observed around the intramuscular nerve endings and in the muscle spindle. Conclusions1. Western blot analysis of trace amounts of muscle tissue confirmed that human skeletal muscle expressed A2A, A2B and A3type adenosine receptors.2. A2AAR was expressed in the muscle fiber membrane;A2BAR was mainly expressed in the cytoplasm of type1muscle fibers;A3AR was expressed in the cytoplasm and vascular smooth muscle of type2muscle fibers.3. The expression of erector spinae A2A, A2B and A3adenosine receptors was higher than that in limb skeletal muscle.4. Western blot showed that the expression of A2AAR and A2BAR was increased in IIMs patients, while A3AR expression was not increased; the antibody immunofluorescence staining showed that the expression of A2AAR, A2BAR and A3AR was all enhanced in non-necrotic fibers of IIMs patients, which might be concerned with the fact that inflammation injury and the application glucocorticoid hormone lead to the decrease of type2muscle fibers.5. The expression of A3AR was increased in non-necrotic muscle fibers and some necrotic muscle fibers were highly expressed, which prompted that A3AR might has the two-way adjustment to cell repair and death.6. CD73was mainly expressed in fascia musculares, endomysium and vascular wall of the inflammation site, and was enhanced in small vessel wall; it was also expressed in fascia musculares and endomysium of non-inflammation site, and there were a small amount of CD73expressed in large endangium and adventitia in mesenchymal of normal control, CD73has the effect to assist diagnosis for IIMs.7. CD39was expressed in the endothelial cells of the muscle tissues, CD39L1was expressed around the intramuscular nerve endings and in the muscle spindle, but both of them were not expressed in the surface and the cytoplasm of the muscle fibers, non-inflammation sites and inflammation sites had no significant differences.