The Expression And Significance Of M3 Receptor, MLCK And P-MLC In Type 1 Diabetes Mice Bladder Tissues

Objective: Diabetes is a metabolic disease characterized by chronic hyperglycemia and caused by a lack of insulin secretion and?or? the functional defects. Diabetes can appear the cardiovascular system, nervous system and urinary system, multi-system chronic progressive lesions with the progress of the disease process. The most common urologic complication of diabetes is diabetic bladder dysfunction.Diabetic bladder dysfunction?DBD? refers to a group of clinical syndromes, mainly including the storage problems and voiding problems. Clinical manifestations of diabetic bladder dysfunction involves overactive bladder, impairment of bladder sensation, increased post-void residual urine volume and decreased detrusor contractility. Since its pathogenesis is more complex, which has not yet been fully clarified, the diagnosis and treatment of the disease is still relatively difficult. M3 muscarinic receptor binding with acetylcholine directly induced contraction of smooth muscle cells. Myosin light chain kinase?MLCK? is a key protein regulating smooth muscle cell contraction. At present, it is generally believed that the acetylcholine released from parasympathetic nerve connecting with the M3 muscarinic receptor can make a rapid increase of the cytosolic free calcium concentration. Elevated calcium combining with cytosolic free activity of calmodulin?CAM? form the active Ca²⁺-CaM compound and activate intracellular MLCK. Myosin light chain is phosphorylated by MLCK, thereby activate the myosin head Mg²⁺-ATP enzyme. This enzyme may hydrolyze ATP to provide energy for the interaction of actin and myosin, which lead to bladder smooth muscle contraction. There has not been reported whether this mechanism occurs with obstacles in mouse models of diabetes. The model of type 1 diabetes of C57BL/6 mice was established to explore the expression of M3 muscarinic receptor, MLCK and p- MLC in mice bladder tissues, and the pathogenesis of DBD through immunohistochemistry and Western blot.Methods:1 Experimental animal groups: Thirty-six C57BL/6 mice were randomly divided into 6 groups after 1 weeks of feeding, namely the normal control group A1, B1, C1, type 1 diabetes group A2, B2, C2, each group had six mice. The mice were executed for consequent experiments at the 8th week, 20 th week and 24 th week of the course of diabetes.2 The model of type 1 diabetic mice preparation: The mice were weighed after fasting 8 hours. The mice were intraperitoneally injected of STZ according to the dose of 0.2 mg/g. The change of mice blood glucose was monitored through blood glucose meter and blood glucose test strips after 72 hours. If 3 consecutive detection of the fasting blood glucose is more than 18mmol/l, the model of type 1 diabetes is successfully prepared.3 The mice were killed at the 8th week, 20 th week and 24 th week of the course of diabetes with their bladders taken out, and then the bladder tissue was fixed, embedded and sectioned.4 Hematoxylin-eosinstaining to observe the morphological changes of mice bladder tissues.5 The expression of M3 muscarinic receptor, MLCK and p-MLC in mice bladder tissues were measured by the method of immunohistochemical.6 The expression of M3 muscarinic receptor, MLCK and p-MLC in mice bladder tissues were measured by the method of Western blot.Results: 1 The mice were intraperitoneally injected of STZ and the fasting blood glucose were higher than 18mmol/L after 72 hours, so we believe that the model of type 1 diabetic mice was established successfully. We found that the weight of type 1 diabetic group mice were significantly decreased compared with normal control group mice when the mice were weighed in the 8th week?P < 0.05?, 20 th week?P < 0.05? and 24 th week?P < 0.05?. Compared with normal control gruop mice, the glucose of C57BL/6 mice by intraperitoneal injected of STZ significantly increased at the 8th week?P < 0.05?, 20 th week?P < 0.05? and 24 th week?P < 0.05?. 2 Pathological morphology observationCompared with the control group, the bladder urinary tract epithelia are integrated and neatly arranged, without epithelial erosion or ulcer formation; and there is no obvious denaturation, hypertrophy and atrophy in bladder smooth muscle cells. 3 Immunohistochemistry 3.1 M3 receptor is mainly expressed in bladder smooth muscle cell cytoplasm and bladder urinary tract epithelial cytoplasm, which is also observed in a number of bladder smooth muscle cell membranes. Immunohistochemical quantitative analysis showed that the expression of M3 receptor in type 1 diabetic mice bladder smooth muscle is higher than the normal control groups?P < 0.05? at the 8th week, 20 th week and 24 th week of the course of diabetes. The expression of M3 receptor in type 1 diabetes mice bladder urothelium is higher than the normal control group?P < 0.05? at the 8th week of the course of diabetes. The expression of M3 receptor in type 1 diabetes mice bladder urothelium showed no significant difference with those of normal control groups at the 8th week, 20 th week and 24 th week of the course of diabetes. 3.2 MLCK is mainly expressed in bladder smooth muscle cell cytoplasm and bladder urinary tract epithelial cell cytoplasm, so is in the capillary endothelia of the lower layer in bladder urothelial cells. Immunohistochemical quantitative analysis showed that the expression of MLCK in type 1 diabetic mice bladder smooth muscle is higher than the normal control groups?P < 0.05? at the 8th week, 20 th week and 24 th week of the course of diabetes. The expression of MLCK in type 1 diabetes mice bladder urothelium showed no significant difference with those of normal control groups at the 8th week, 20 th week and 24 th week of the course of diabetes. 3.3 p-MLC is mainly expressed in bladder smooth muscle cell cytoplasm and bladder urinary tract epithelial cell cytoplasm, as well as the capillary endothelia of the lower layer in bladder urothelial cells. Immunohistochemical quantitative analysis showed that the expression of p-MLC in type 1 diabetic mice bladder smooth muscle is higher than the normal control groups?P < 0.05? at the 8th week, 20 th week and 24 th week of the course of diabetes. The expression of p-MLC in type 1 diabetes mice bladder urothelium showed no significant difference with those of normal control groups at the 8th week, 20 th week and 24 th week of the course of diabetes. 4 Western blot 4.1 The expression of M3 receptor in type 1 diabetic mice bladder tissues is higher than the normal control groups?P < 0.05? at the 8th week, 20 th week and 24 th week of the course of diabetes. 4.2 The expression of MLCK in type 1 diabetic mice bladder tissues is higher than the normal control groups?P < 0.05? at the 8th week, 20 th week and 24 th week of the course of diabetes. 4.3 The expression of p-MLC in type 1 diabetic mice bladder tissues is higher than the normal control groups?P < 0.05? at the 8th week, 20 th week and 24 th week of the course of diabetes.Conclusions:1 The expression of M3 receptor, MLCK and p-MLC in bladder tissues of type 1 diabetic mice is higher than that of the control group at the time points of 8th week, 20 th week and 24 th week, and is mostly elevated in bladder smooth muscle cells, suggesting that the contraction mechanism of bladder smooth muscle cells mediated by M3 receptor can be activated in the processing of diabetes.2 Theoretically, the increasing expression of M3 receptor, MLCK and p-MLC in bladder smooth muscle cells may lead to an enhancing contractility of bladder smooth muscle of type 1 diabetic mice at the 8th week of diabetes course, which might be one of the causes of overactive bladder?OAB? in the early stage of diabetic bladder dysfunction?DBD?.3 The expression of M3 receptor, MLCK and p-MLC was increased in the bladder smooth muscle of type 1 diabetic mice at the course of diabetes 20 weeks and 24 weeks, which is not parallel to the bladder contraction dysfunction in the progression of diabetic bladder dysfunction. We consider the elevated expression of the three factors is compensatory changes of bladder smooth muscle cells. However, DBD is caused by many factors, the bladder smooth muscle contractile function will develop into a loss of compensatory changes in the progression of diabetes eventually. The specific mechanism is needed to be further researched.