A Study On The Time-dependent Expression Of Phospho-CB1R During Repair Of Contused Skeletal Muscle In Rats

ObjectiveEndogenous cannabinoid system is a common type of lipid signaling system in all vertebrates and plays an important regulative function.It consists of cannabinoid receptors,endogenous cannabinoids(endocannabinoids) and the enzymes which can synthesize and degrade endocannabinoids.There are two subtypes of cannabinoid receptors has been cloned which were named of cannabinoid 1 receptor and cannabinoid 2 receptor.CBR is G protein-coupled receptor.It can regulate some signal transduction pathways such as inhibition of adenylate cyclase(adenylyl cyclase,AC), regulation of ion transportion,activation of focal adhesion kinase,mitogen-activated protein kinase and cytosolic phospholipase A₂,promotion of NO generation.CB1R is also closely related to the occurrence of apoptosis.To investigating the mechanism of p-CB1R after skeletal muscle contusion and exploring the applicability of p-CB1R to determinate the wound age,the expression of p-CB1R was studied by immunohistochemical,Western blot techniques in skeletal muscle contusion at different posttraumatic interval in rat.Materials and MethodsA total of 50 male,healthy rats,weighting 220g-250g was anesthetized by ether, and received a single impact trauma at the skeletal muscle of the right rear leg.Muscle of right lower limb were taken after the animals being anaesthetized and sacrificed by cervical dislocation at 3h,6h,12h,1d,3d,5d,7d,10d and 14d(5 rats in each group) postwounding.The remaining 5 rats used as control.The level of p-CB1R in rat skeletal muscle contusion was detected by immunohistochemical and western blotting, and the non-contused rat skeletal muscle was used as control.The number and ratio of positive cells were counted and analyzed by microscope and comparative the band AverGray by software of Motic Images Advanced 3.2.The date were analyzed comparatively by the software of SPSS 13.0 for Windows.ResultsThe expression of p-CB1R was neither detected in normal rat skelecal mucle nor. postwounding skelecal mucle.Non-immunoreactivity of p-CB1R was detected in polymorphonuclear cells(PMNs) in the wound specimens.The expression of p-CB1R was detected in mononuclear cells(MNCs) in the wound specimens aged from 3h to 6h. In the wound specimens aged of 12h after injury,p-CB1R were identified in part of infiltrating MNCs.After ld injured,the p-CB1R positive cells increased as the MNCs growing in number.Afterwards from 3d to 5d postwounding,the p-CB1R positive cells were mainly found in MNCs and fibroblastic cells(FBCs) while mainly in FBCs from 7d to 10d postwounding with the peak at the 7d and then decreased at 14d.The obvious bands of 53kD were noticed at 3d and 7d and the expression of p-CB1R revealed a time-dependent pattern.Conclusions1.The expression of p-CB1R was neither detected in normal rat skelecal mucle nor.postwounding skelecal mucle.2.Non-immunoreactivity of p-CB1R was detected in PMNs in the wound specimens.The expression of p-CB1R was detected in the MNCs and FBCs in the injured rats.p-CB1R may be involved in the healing process of skeletal muscle after contusion.