Mechanism Of Acupuncture Regulating Bladder Excitability Based On Sensory Afferent Pathway By Urothelial Mechanical Stimulus

Objective:Overactive bladder（OAB）rats were used as study objects to investigate the regulatory mechanism of acupuncture on bladder（mechanical stimulus）sensory function.This study utilized transient receptor potential vanilloid（TRPV）receptor 4 and Piezo1mechanical stimulus ion channels as cut-ins to explore the release action pathway of acupuncture on Ca²⁺concentration and adenosine triphosphate（ATP）in bladder urothelium cells and provide a scientific basis for the mechanism of acupuncture regulating the sensitivity of bladder sensory function.Methods:SPF female Wistar rats were randomly divided into sham operation group,model group,acupuncture group,TRPV4 antagonist group,TRPV4 antagonist+acupuncture group（referred to as T antagonist+acupuncture group）,Piezo1 antagonist,and Piezo1antagonist+acupuncture group（referred to as P antagonist+acupuncture group）.Intraperitoneal injection of cyclophosphamide prepared the OAB model,and the treatments were as follows:（1）Sham operation group:Modeling treatment-free;（2）Model group:OAB modeling only;（3）Acupuncture group:After successful modeling,an acupuncture treatment was given.The Ciliao and Huiyang points were selected for acupuncture,ipsilateral acupoints were connected to an electro-acupuncture apparatus,and continuous waves were discharged for 20 min at a frequency of 30 Hz,and current intensity of 1 m A;（4）TRPV4 antagonist group:After successful modeling,intravesical TRPV4 receptor antagonist HC-067047 was given;（5）T antagonist+acupuncture group:After successful modeling,intravesical TRPV4 receptor antagonist HC-067047was given,followed by acupuncture treatment;（6）Piezo1 antagonist group:After successful modeling,intravesical Piezo1 receptor antagonist Gs MTx4 was given;（7）P antagonist+acupuncture group:After successful modeling,intravesical Piezo1 receptor antagonist Gs MTx4 was administered,followed by acupuncture treatment.Observation index:（1）Urine storage time,bladder effective capacity,urination pressure,number of urinations within 3 h,and total urination volume within 24 h;（2）Bladder urothelial tissue morphology;（3）Distribution of TRPV4 and Piezo1 channels in bladder urothelium and bladder urothelium cells;（4）mRNA and protein expression of TRPV4and Piezo1 channels in bladder urothelium;and（5）Ca²⁺concentration and ATP content in bladder urothelium cells.Results:1.Model evaluation:After modeling,the urodynamics of rats exhibited a shortened urine storage time,a reduced bladder effective capacity,and a decreased urination pressure（P<0.05）,indicating the successful preparation of the OAB rat model.2.Changes in bladder function:（1）Intra-group comparison:There was no difference in urine storage time,bladder effective capacity,and urination pressure before and after treatment in the sham operation model group（P>0.05）;Following treatment,rats in the acupuncture group,the antagonist group,and the antagonist+acupuncture group exhibited a prolonged urine storage time,an increased effective bladder capacity and an increased urination pressure（P<0.05）.（2）Inter-group comparison:Compared with the sham group,rats in the model group showed shorter urine storage time,reduced effective bladder capacity,decreased voiding pressure,increased frequency of urination,and reduced voiding volume（P<0.05）.Compared with the model group,in the acupuncture group,each antagonist group and each antagonist+acupuncture group,the urine storage time was prolonged,the effective bladder capacity was increased,the urination pressure was increased,the number of urinations was reduced,and the urine volume was increased（P<0.05）.Rats in the Piezo1 antagonist group had an increased urine output compared with the TRPV4 antagonist group（P>0.05）.3.Morphological changes of the bladder urinary tract’s epithelial tissue are as follows:The bladder tissue morphology of rats in the model group exhibited abnormalities,including the thinning of the cortex on the urinary tract,showing urothelial cell detachment with extensive edema of the lamina propria,and the tissue structure was loose.The bladder tissue morphology of rats in the acupuncture group,each antagonist group,and each antagonist+acupuncture group was improved compared with the model group,the cortical tissue structure on the urinary tract was left unscathed,without significant degeneration and shedding,the lamina propria was slightly edematous,and the tissue structure was arranged more compactly.4.Altered expression of TRPV4 and Piezo1 channels in the bladder urothelium:（1）TRPV4 was mainly expressed in urinary tract epithelium and urinary tract epithelial layer,and there was also a little expression in detrusor layer,while Piezo1 was mainly expressed in bladder urinary tract epithelium and bladder urothelium cell layer,and almost no expression in detrusor layer.（2）compared with the sham operation group,the average fluorescence intensity of TRPV4 and Piezo1,the expression of protein and mRNA in the urinary tract epithelium of the model group were significantly higher than those in the sham operation group（P<0.05）;Compared to the model group,the mean intensity,mRNA expression and protein expression of TRPV4 in the bladder uropath of rats in the acupuncture group,TRPV4 antagonist group and T antagonist+acupuncture group were decreased（P<0.05）;Compared to the model group,the mean intensity value,mRNA expression and protein expression of Piezo1 in the cortex of the urinary bladder were decreased in the acupuncture group,Piezo1 antagonist group and P+acupuncture group（P<0.05）.5.Changes of Ca²⁺concentration and ATP content in bladder urothelium cells:（1）Compared to the sham operation group,the Ca²⁺concentration in bladder urothelium cells of the model group increased by 358.17%（P<0.05）;Compared to the model group,the Ca²⁺concentration in the bladder urothelium cells of the acupuncture group,each antagonist groups and each antagonist+acupuncture groups decreased（P<0.05）;including 48.98%in the acupuncture group,51.49%in the TRPV4 antagonist group,61.47%in the T antagonist+acupuncture group,69.59%in the Piezo1antagonist group,and 70.07%in the P antagonist+acupuncture group;The intracellular Ca²⁺concentration in the T antagonist+acupuncture group decreased by 20.56%compared to the TRPV4 antagonist group.（2）Compared to the sham operation group,the ATP content in bladder urothelium cells of the model group was increased in the isotonic environment（physiological state）（P<0.05）;Compared to the model group,the ATP content of bladder urothelium cells in the acupuncture group,each antagonist group and each antagonist+acupuncture group was decreased in the hypotonic environment（P<0.05）;Compared to the model group,the ATP content of bladder urothelial cells in the acupuncture group,each antagonist group and each antagonist+acupuncture groups was decreased in the hypotonic environment（P<0.05）.（3）Compared to the sham operation group,the ATP ratio of bladder urothelium cells in the model group increased by 290.23%（P<0.05）;Compared to the model group,the ATP ratio of bladder urothelium cells in the acupuncture group,each antagonist group and each antagonist+acupuncture group decreased（P<0.05）,including 40.02%in the acupuncture group,40.77%in the TRPV4 antagonist group,50.89%in the T antagonist+acupuncture group,58.25%in the Piezo1 antagonist group,and 58.12%in the P antagonist+acupuncture group;The intracellular ATP ratio in the T antagonist+acupuncture group decreased by 17.09%compared to the TRPV4 antagonist group.Conclusions:1.Acupuncture can effectively prolong the urine storage time,boost the effective capacity of the bladder,and increase the urination pressure of OAB rats,simultaneously reducing the frequency of urination within 3 h and increasing the total urination volume within 24 h in OAB rats,and improving the bladder urinary tract epithelial tissue morphology;2.Acupuncture can reduce the sensitivity of the bladder to mechanical stimulus in OAB rats through the downregulation of the expression of TRPV4 and Piezo1 channels;3.Acupuncture can reduce the intracellular Ca²⁺concentration in bladder urothelium cells through TRPV4 and Piezo1 channels,which in turn down-regulates ATP release from the bladder urothelium cells,therefore,inhibiting the sensory sensitivity of the bladder.