The Study On The Mechanism Of Electroacupuncture Regulating Bladder Overactivity Based On Glutamate Metabolism And Its Excitatory Effect In Urination Center

Objective: To investigate the effects of electroacupuncture（EA）on glutamate-glutamine cycle,glutamate receptor activation and glutamate G-aminobutyric acid pathway inhibition in micturition center（PAG,PMC and L₆-S₁）of overactive bladder rats,and to reveal the role of EA in regulating glutamate mediated micturition reflex in overactive bladder which could provide the oretical basis for clinical acupuncture treatment of overactive bladder.Methods: 75 female Wistar rats were random Ly divided into 5 different groups: blank,model,EA,PDC and PDC+EA.Blank group: no modeling treatment;Model group: intraperitoneal injection of cyclophosphamide caused overactive bladder;Electroacupuncture group: After the overactive bladder rats were successfully established,continuous wave acupuncture was applied on ciluo and Huiyang points at the same time,once a day for three consecutive days;PDC group: after the model of overactive bladder was successfully established,specific glutamate receptor blocker（PDC solution）was injected into PAG of rat brain.PDC + EA group: after successful modeling of overactive bladder,PDC solution was injected into PAG area of rat brain,and EA intervention was performed 30 minutes later（the same acupuncture method as EA group）.Urodynamic measurements were performed in all groups,including the first bladder contraction time,urine storage time,first micturition time,bladder micturition pressure and bladder effective capacity;the pathological changes of bladder tissue were observed by HE staining;the distribution and expression of Glu and GABA in PAG,PMC and L₆-S₁ of brain and the concentration of Glu and GABA were detected by HPLC-MS and immunofluorescence The expressions of GLAST,GLT-1,GS,GAD,NMDA,APMA and mGlu5 in PAG,PMC and L₆-S₁ were detected by RT-PCR,Western blot and immunohistochemistry.Results: 1.Model evaluation: after modeling,the urodynamic performance of rats showed that the first urination time was earlier（P < 0.05）,the effective bladder volume was decreased（P < 0.05）, and the urination pressure was decreased（P < 0.05）,which suggested that the model was successful.2.Urodynamic:（1）intra group comparison: there was no difference in the first micturition time,effective bladder capacity and micturition pressure before and after treatment in blank group（P > 0.05）;The first micturition time was earlier in the model group,PDC group and PDC + EA group before and after treatment（P < 0.05）,and the effective bladder capacity was decreased（P < 0.05）.In the electroacupuncture group,the first urination time was delayed（P < 0.05）,and the effective bladder volume was increased（P < 0.05）.The urination pressure of rats in PDC group after treatment was lower than that before treatment（P < 0.05）,and there was no difference in other groups before and after treatment（P > 0.05）.（2）Comparison between groups: compared with the blank group,the model group had earlier first micturition time,decreased bladder effective capacity and decreased micturition pressure（P < 0.05）;Compared with the model group,the electroacupuncture group had later first micturition time,increased bladder effective capacity and increased micturition pressure（P < 0.05）;Compared with the model group,the time of first micturition was earlier and the effective capacity of bladder was decreased in the PDC+EA group（P < 0.05）;Compared with the EA group,the time of first micturition of PDC+EA group was earlier,the effective capacity of bladder was decreased and the micturition pressure was decreased in the PDC + EA group（P < 0.05）;Compared with the PDC group,the time of first micturition of PDC+EA group was delayed and the effective capacity of bladder was increased in the PDC + EA group（P < 0.05）.3.Bladder histopathology: in the blank group,the lamina propria of the bladder tissue was closely arranged,the blood vessels were not congested and dilated,and the structure of each layer was clear;In the model group,compared with the blank group,the lamina propria of the bladder tissue was edematous,a large number of blood vessels were congestion and dilated,the muscle fibers of the muscle layer were loosely arranged,the gap was widened,and the structure of each layer was fuzzy;in the electroacupuncture group,compared with the model group,the bladder tissue structure was tight,and the blood stasis and Compared with the model group,the shape of bladder in the PDC + EA group had no significant difference;compared with the PDC group,the structure of bladder tissue in the PDC + EA group was compact and blood stasis was reduced,which was similar to that in the EA group.4.The expression of glutamate and G-aminobutyric acid in PAG,PMC and L₆-S₁:（1）compared with blank group,the concentration of glutamate in PAG,PMC and L₆-S₁ in model group increased（P < 0.05）and the concentration of G-aminobutyric acid decreased（P < 0.05）; compared with model group,the concentration of glutamate in PAG,PMC and L₆-S₁ in PDC group increased（P < 0.05）;Compared with the model group,the concentration of glutamate in PAG,PMC and L₆-S₁ of EA group decreased（P < 0.05）,while the concentration of Gaminobutyric acid in PAG and L₆-S₁ of EA group increased（P < 0.05）,which was close to that of blank group（P > 0.05）;Compared with EA group,the concentration of glutamate in PAG and PMC of EA group increased（P < 0.05）;There was no significant difference in the concentration of glutamate in PAG and PMC between PDC + EA group and PDC group（P > 0.05）.The concentration of glutamate in L₆-S₁ decreased（P < 0.05）,while the concentration of PAG and G-aminobutyric acid in L₆-S₁ increased（P < 0.05）.（2）Compared with the blank group,the average intensity of glutamate in PAG,PMC and L₆-S₁ of the model group increased（P < 0.05）,while the average intensity of G-aminobutyric acid decreased（P < 0.05）;Compared with the model group,the average intensity of PAG,PMC and L₆-S₁ of the electroacupuncture group decreased（P < 0.05）.The average intensity of glutamate in L₆-S₁ decreased（P < 0.05）,the mean intensity of PAG and G-aminobutyric acid in L₆-S₁ increased significantly（P < 0.05）.The mean intensity of glutamate in PAG,PMC and L₆-S₁ increased（P < 0.05）;The mean intensity of glutamate in PAG,PMC and L₆-S₁ increased in the PDC + EA group compared with the PDC group（P > 0.05）;The mean intensity of G-aminobutyric acid in PAG was increased;the mean intensity of glutamate in PAG,PMC and L₆-S₁ increased in the PDC + EA group compared with the EA group（P < 0.05）.5.The expression of glutamate pathway transporters,metabolic enzymes and receptors in PAG,PMC and L₆-S₁ were summarized as follows.（1）Compared with blank group,the average optical density,protein expression and mRNA expression of GLAST and GLT-1 in PAG,PMC and L₆-S₁ in model group were decreased（P < 0.05）;Compared with model group,the average optical density of GLAST and GLT-1 in PAG and PMC of EA group were increased（P < 0.05）;The protein and mRNA expression of GLAST and GLT-1 in PAG,PMC and L₆-S₁ were increased（P < 0.05）;Compared with model group,the average optical density of GLAST and GLT-1 in PMC of PDC group were decreased（P < 0.05）,the average optical density of GLT-1 in PAG was decreased（P < 0.05）;The average optical density of GLAST and GLT-1 in PAG,PMC and L₆-S₁ decreased（P < 0.05）;The expression of GLAST and GLT-1 in PAG,PMC and anterior horn of spinal cord（L₆-S₁）,protein and mRNA in PDC + EA group were lower than those in EA group（P < 0.05）,and there was no significant difference between PDC group and EA group（P > 0.05）.（2）There was no significant difference in the mean optical density,protein and mRNA expression of GS in PAG,PMC and L₆-S₁ among the groups（P > 0.05）.Compared with blank group,the average optical density and mRNA expression of GAD in PAG,PMC and L₆-S₁ of model decreased（P < 0.05）,and the protein expression in PMC and L₆-S₁ decreased（P < 0.05）;compared with model group,the average optical density and mRNA expression of GAD in PAG of EA group were increased（P < 0.05）;The expression of GAD mRNA in L₆-S₁ and PMC increased（P < 0.05）,and the expression of GAD protein in PMC increased（P < 0.05）.（3）Compared with blank group,the expression of AMPA protein,NMDA average optical density and mRNA,mGlu5 average optical density in PAG of model group were increased（P < 0.05）,the expression of AMPA average optical density,NMDA and mGlu5 mRNA and protein in PMC increased（P < 0.05）,and the expression of AMPA and NMDA in L₆-S₁ increased（P < 0.05）Compared with the model group,the protein and mRNA expression of AMPA,NMDA and mGlu5 in PAG of EA group were decreased（P < 0.05）,the expression of NMDA and mGlu5 in PMC of EA group were decreased（P < 0.05）,and the expression of AMPA protein and mRNA in L₆-S₁ of EA group were decreased（P < 0.05）The protein and mRNA expression of NMDA and mGlu5 of EA group were decreased（P < 0.05）.Conclusions: 1.Electroacupuncture could effectively delay the first micturition time of OAB rats,increase the effective bladder capacity,increase the micturition pressure,and improve the bladder tissue morphology of OAB rats.And the effect of Electroacupuncture on improving bladder function was closely related to glutamate glutamine cycle.2.Glutamate glutamine cycle in PAG,PMC and L₆-S₁ was correlated.Electroacupuncture could improve bladder function by increasing the expression of GLAST and GLT-1 in PAG,PMC and L₆-S₁ of OAB rats,regulating glutamate glutamine cycle and reducing the concentration of glutamate in micturition center.3.Electroacupuncture could effectively increase the expression of GAD in PAG,PMC and L₆-S₁ of OAB rats,which furtherly increased the concentration of G-aminobutyric acid in the urination center and improving the bladder function.4.Electroacupuncture could decrease the expression of AMPA,NMDA and mGlu5 in micturition center,and then reduce its excitability,which could improve bladder function.