| Purpose: Diabetes Mellitus(DM)has become a public health problem in China that cannot be ignored because of its increasing incidence year by year.Diabetic Bladder Dysfunction(DBD)is a common complication of DM.early symptoms of DBD are mild,and when clinically detected,most of the symptoms are in the late stage of DBD,when bladder function damage is irreversible,and clinical treatment is mostly symptomatic but ineffective.Sacral nerve magnetic stimulation generates induced current by applying pulsed magnetic field to change the action potential of nerve cells,affect the electrophysiological activity of sacral nerve and improve the bladder function.It has the therapeutic advantages of being less stimulating and non-invasive.However,the target and mechanism of action of sacral nerve magnetic stimulation are still in doubt.In this study,we investigated the mechanism of the effect of sacral nerve magnetic stimulation on P2X3 receptors and NGF expression in rat bladder tissue by observing the improvement of bladder function after sacral nerve magnetic stimulation and using rats with early DBD as a model.This study provides a theoretical basis for the clinical use of sacral nerve magnetic stimulation in the treatment of early DBD,and lays the foundation for further in-depth research.Experimental methods: 30 female SD rats were fed with normal chow and randomly grouped after one week of adaptation to the environment.The normal group was 10 rats and the modeling group was 20 rats,and the modeling group was divided into sacral nerve magnetic stimulation intervention group and control group.Afterwards,the rats in the modeling group were fasted without water for 18 h and then injected intraperitoneally with a dose of 40 mg/kg streptozotocin(STZ),while the rats in the normal group were injected intraperitoneally with an equal volume of citric acid-sodium citrate buffer solution.After 72 hours,blood glucose was measured in the tail vein of the modeled group,and rats with random blood glucose >16.7mmol/L and 24-h urine output ≥150 ml and with excessive drinking and eating were considered to be diabetic rats.The changes of body weight,blood glucose and 24 h urine output were observed and recorded weekly.At the end of the intervention,the rats were anesthetized by intraperitoneal injection of 25% uratan,and a catheter was inserted into the bladder through the urethra and the bladder was perfused at a rate of 12 ml/h.The urodynamic changes of the rats in each group were detected and recorded.Subsequently,the bladder was cut by opening the abdominal cavity and the wet weight of the bladder was measured.After routine dehydration,paraffin embedding and sectioning,bladder tissues were stained in HZ and then observed histopathologically by light microscopy.Finally,P2X3 receptor as well as NGF protein expression were detected and recorded by western blot technique.Results: In the modeling group,blood glucose was measured in the tail vein after 72 hours,and blood glucose ≥ 16.7 mmol/L in the tail vein and the presence of excessive drinking,eating and urination(24h urine volume ≥ 150 ml)were the signs of successful modeling in the diabetic rats.In the modeling group,one rat died and two rats had blood glucose ≤ 16.7 mmol/L when blood glucose was measured at the sixth week,and the modeling success rate was 85%.At the end of the experiment,a total of 25 rats completed the experiment,accounting for 83%of the total.Among them,10 rats were in the normal group and 15 rats were in the modeling group(including 7 rats in the control group and 8 rats in the intervention group).The normal rats had uniform and firm fur,white and lustrous fur,stable increase in body weight,stable and normal water consumption and urine output,and stable blood sugar.The rats in the modeling group had sparse fur,dull yellow fur,lax eyes,a thin body posture that could obviously touch the rib cage,small limbs,increased urine output,and high blood glucose level all the time.In the intervention group,after 4 weeks of sacral nerve magnetic stimulation,there was no significant effect on the overall condition of the rats,which showed sparse fur and disoriented eyes.The rest of the results were as follows.1.body weight: the body weight of the rats in the normal group showed a stable increasing trend,and the body weight of the rats in the modeling group did not increase significantly and the weight of individual rats decreased,and there was no statistical difference(p>0.05)in the change of body weight after sacral nerve magnetic stimulation in the intervention group.2.blood glucose: compared with the normal group,the blood glucose level in the modeled group was significantly higher(p<0.05),and was greater than 16.7 mmol/L.There was no statistical difference between the control group and the intervention group(p>0.05);compared within the group,there was no statistical difference in the change of blood glucose after the intervention of sacral nerve magnetic stimulation(p>0.05).3.24 h urine output: The 24 h urine output of the rats in the modeled group was significantly higher compared with the normal group(p<0.05),and there was no statistical difference in the24 h urine output before and after stimulation in the intervention group(p>0.05).4.bladder wet weight: bladder wet weight was significantly higher in the modeling group compared with the normal group(p<0.05),and there was no statistically significant change in bladder wet weight in the intervention group compared with the control group(p>0.05).5.Pathological examination: The bladder tissue of each group was observed under light microscope after HE staining,and it was found that the bladder tissue of the normal group was morphologically intact;the forced urinary muscle layer of the modeling group was thickened,and the inflammatory infiltration of forced urinary muscle cells appeared,and the arrangement of muscle fibers was disordered;the forced urinary muscle layer of the intervention group was thickened,and the inflammatory infiltration of cells was slightly improved,and the arrangement of muscle fibers was not neat.6.Urodynamics: the maximum bladder volume,maximum bladder pressure,leak point pressure,and bladder compliance in the modeling group showed statistical differences compared with the normal group(p < 0.05),and the maximum bladder volume decreased in the intervention group compared with the control group(p < 0.05),the leak point pressure decreased(p < 0.05),and the bladder compliance decreased(p < 0.05),but the maximum bladder pressure did not improve significantly(p > 0.05).8.western blot: the expression level of P2X3 receptors in the bladder tissue of control rats was significantly elevated compared to the normal group(p < 0.005),and the expression level of P2X3 receptors in the intervention group was significantly decreased compared to the control group(p<0.005);the expression of NGF in the bladder tissue of control rats was significantly decreased compared to the control group(p<0.005),and the expression of NGF in the intervention group was increased compared to the control group(p<0.05).Conclusion: The 4-week sacral nerve magnetic stimulation intervention improved the expression of P2X3 and NGF proteins in the bladder tissue of DBD rats and reduced the histopathological changes in the bladder,which contributed to the improvement of bladder function in early DBD,but had no significant effect on the improvement of diabetic symptoms,which may suggest the need for long-term treatment or combined treatment with drugs. |
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