Fibrosis And Spp1 Gene Expression In The Skeletal Muscle Of Mdx Mice

Objective: Duchenne muscular dystrophy(DMD), an X-linked disorder, is the most common muscular dystrophy in children, presenting in early childhood and characterized by progressive, symmetry proximal muscle weakness and muscle atrophy in affected boys. Patients usually become wheelchair-bound by the age of 12 years, and die of respiratory tract infection or cardiorespiratory complications in their twenties.The main pathological features of this disease is muscle fiber degeneration, necrosis and fibrosis.Despite we all knows the etiology of DMD is the lack of dystrophin protein,but the specific mechanism of muscle degeneration and fibrosis remains unclear.Fibrosis is a pathological change that extracellular matrix(extracellular matrix, ECM) deposition and parenchymal cells gradually reduce. It may occurs in many different organs.Continuously progress of fibrosis can cause structural failure, organ function decline and even failure. Dystrophin protein is missing in DMD patients sarcolemma leading the membrane structure changed and integrity destroyed. Respond to mechanical stimulation,an influx of sodium and calcium cannot be compensated for by pumps regulating electrolyte balance. Cellular oedema and calcium overload cause depletion of energy supply. Calcium acts as a second messenger and activates a cascade of inflammatory processes. At the same time, the inflammatory cells release profibrosis factors which promotes fibroblasts proliferation and differentiation into myofibroblast(MFb).Fibroblasts and MFb secrete ECM and ECM remodeling factors leading to imbalances of synthesis and degradation of the ECM.OPN,also known as osteopontin(OPN), is a highly acidic,secreted and glycosylated phosphoprotein.OPN is a Multi-functions glycoprotein and is also expressed in a wide variety of other cells and tissues.OPN is located in ECM and binds to the ECM molecules fibronectin and collagen influcing the ECM remodeling. While OPN plays a important role in fibrosis of wound healing, Obstructive uropathy and Myocardial Infarction,but relationship of spp1 and fibrosis remains unknown.Because mdx mice has the same genetics foundation with human, it has been widely used in the researches of the DMD.Our experiment focuses on the mdx mice skeletal muscle fibrosis and differentially expressed genes Involved in fibrosis in mdx mice at different time points. We are alse interested in the Spp1 genes expressed in MDX mice in different periods and the control mouse to further explore the relationship between Spp1 and fibrosis in DMD.Methods:C57BL/10 Sc Sn-Dmdmdx/JNju mice were used as the experimental animals and C57BL/6Sc Sn mice served as control group. There were four groups: 2 weeks group,4 weeks group,8 weeks group,12 weeks group.Each group included six mice for conventional histochemical staining, gene chip detection and q RT- PCR. After 10% hydration aldehydes(350mg/kg body weight) intraperitoneal anesthesia was injected, the quadriceps femoris musclel of mouse was extracted immediately and frozen in liquid nitrogen or for the embedding frozen processing. Muscle biopsies were done for staining,including HE staining,masson staining to observe skeletal muscle fibrosis and other pathologic changes. We compaired gene expression profile in quadriceps of mdx mouse different periods by gene microarray analysis. Additionally,q RT- PCR were used to detect the Spp1 gene expression in mdx and contral mouse quadriceps at the same time. Data were analysed by spss13.0.Results: 1 Fibrosis in mdx muscle mice at different periodFibrosis in the quadriceps femoris muscle with H&E staining and masson staining: almost normal morphology of quadriceps without connective tissue hyperplasia. Individual degeneration muscle fibers can be saw occasionally in the quadriceps femoris muscle of 2w mdx mouse;at 4w muscle specimens show initial disruption of muscle tissue due to degeneration-regeneration,but muscle fibers were closed,without obvious connective tissue hyperplasia; only partial region with slightly broadening gap between muscle fibers begin to occur at 8w;endomysial connective tissue proliferation fibers in partial region at 12 w.(Fig. 1、2) 2 Gene expression profile in muscle of mdx mouse different periods by gene microarray analysisComparison of gene expression profile of mdx mouse quadriceps femoris muscle at 2 weeks and 12 weeks :P≤0.05,fold-change≥+2 or ≤-2. There are 2928 genes Differentially expressed in Comparison of 2w vs 12 w. Parts of genes associated with fibrosis which selected Through Pathway analysis listed in Table 1.The fold- change of comparison that the expression of Spp1 gene at 2 weeks versus 12 weeks is 15.1354 which means the change is very obvious. 3 The expression of Spp1 gene In the skeletal muscle of mdx mouse and control mouse at different periods 3.1 The expression of Spp1 gene in the skeletal muscle of mdx mouse at different periods:8 weeks group is significantly higher than the 4 weeks group; the expression of Spp1 gene is decree;sed in 12 weeks group compared with the eight weeks group, but still higher than 4 weeks. Differences were statistically significant. 3.2 The expression of Spp1 gene in the skeletal muscle of mdx mouse at different periods compared with control mouse:the expression is significantly higher in mdx mouse than control group at 8weeks and 12 weeks. 3.3 The expression of Spp1 gene in the skeletal muscle of control mouse at different periods:There is no statistically significant between every groups.(Fig. 3)Conclusions:1 Fibrosis is not obvious in the muscle of mdx mice at early stage(2 weeks-4weeks) and then slowly progress.A small amount of connective tissue hyperplasia can be seen in skeletal muscle at the age of 8 weeks.2 The expression of Spp1 gene increased significantly in mature mdx mice(8 weeks-12 weeks),inferencing that Spp1 plays a role in muscle fibrosis of mdx mice.