|
Purpose of the endocannabinoid system is a ubiquitous lipid signaling system in all vertebrates plays an important regulatory function, mainly by cannabinoid receptor (cannabinoid receptor, CBR), endogenous cannabinoid (endocannabinoid, EC) and the synthesis and degradation of endocannabinoids enzyme composition. Now clear cloned CBR including type 1 cannabinoid receptor (cannabinoid 1 receptor, CB1R) and type 2 cannabinoid receptor (cannabinoid 2 receptor, CB2R) two subtypes. CBR with G protein-coupled, adjust certain signaling pathways, such as inhibition of adenylate cyclase (adenylyl cyclase, AC), regulating ion transport, activation of focal adhesion kinase, mitogen-activated protein kinases and cytosolic Phospholipase A 2 , promote NO production, etc., and with the occurrence of apoptosis are closely related. In this experiment, rat skeletal muscle contusion model based on the application of immunohistochemistry and Western blot, the healing process in rat skeletal muscle contusion phosphorylated CB1R (phospho-cannabinoid 1 receptor, p-CB1R) dynamic changes The time variation of its expression at the time of injury infer applicability are discussed. Materials and methods healthy adult male Sprague-Dawley (SD) rats were 50, weighing 220 ~ 250g, adaptive feeding one week later, were randomly divided into 10 groups of five, nine groups for the experimental group, a group of normal control group. Ether inhalation anesthesia, the rat hind limb placed extensor, ankle dorsiflexion of the 90 ° position, with the free-fall blunt force impactor to 3m / s speed and the indentation depth control lever beyond the closure screw clasp 7mm length ( ie deformation), one against rat right hind talocalcaneal 2cm at the right lower part, causing skeletal muscle contusion. Respectively after injury 3h, 6h, 12h, 1d, 3d, 5d, 7d, 10d and 14d rats died of cervical off, take the right leg gastrocnemius. Normal control rats without a fight, take the same parts of the same size as the gastrocnemius, to prepare for HE staining, immunohistochemistry and Western blot applications. Using immunohistochemistry and Western blot detection of 50 cases of rat gastrocnemius muscle contusion after each time period p-CB1R changes in non gastrocnemius contusion rats as control × 400 times under a microscope, in the surrounding area contusion contusion areas and randomly selected 10 fields, counting multinucleated granulocytes, mononuclear cells and fibroblasts cells total number of positive cells and the positive cell rate. Application Motic Images Advanced 3.2 software testing each fragment average gray value, the measured data are expressed as mean ± standard deviation ((?) ± s) expressed. Using SPSS13.0 forWindows single factor analysis of variance, with P <0.05 was considered statistically significant. Results normal control rats no p-CB1R expression in skeletal muscle. Contusion after each time period rat skeletal no p-CB1R expression, mainly in mononuclear cells and fibroblasts were positive. After injury 3h ~ 6h, skeletal muscle interstitium were few polynuclear neutrophils and mononuclear cell infiltration, polynuclear neutrophils no p-CB1R expression in mononuclear cells visible only to the individual p-CB1R positive; injury after 12h, a large number of multi-core tablets cell infiltration of mononuclear cells and a small amount, p-CB1R in mononuclear cells were positive; injury 1d, along with the increase of mononuclear cells, p-CB1R positive cells also increases; injury 3d ~ 5d, single number of nucleated cells gradually decreased, the number of fibroblasts increased gradually, p-CB1R positive cells in mononuclear cells and fibroblasts mainly; injury 7d ~ 10d, mononuclear cells was significantly reduced, p-CB1R positive reaction mainly seen fibroblasts, and reached the peak at 7d; injury 14d, reduce the number of fibroblasts, p-CB1R expression is also reduced. Western blot analysis showed that the experimental group at each time period p-CB1R expression there are certain rules after injury 3h ~ 12h, p-CB1R expression less; injury 1d ~ 3d, p-CB1R expression gradually increased; injury 5d, p -CB1R decline; injury 7d, p-CB1R peaked; injury 10d, with time, p-CB1R expression decreased gradually; injury 14d, p-CB1R expression levels decline further. Each group average gray value differences were statistically significant (P <0.05). Conclusion 1, the normal control group, no p-CB1R skeletal muscle expression. 2, contusion after each time period skeletal muscle showed no p-CB1R expression. Skeletal muscle interstitial infiltration multicore granulocytes no p-CB1R expression, mononuclear cells and fibroblasts seen p-CB1R expression, suggesting that p-CB1R may be involved in muscle contusion healing process.
|