Study Of The Mechinisms Of SQW On OAB Rat Base On TRPV1 Channel

The physiological function of bladder is to store and release urine. Controlled micturition is initiated by bladder afferent nerves and depends on an integration of somatic and autonomic efferent mechanisms that coordinate bladder contraction and sphincter relaxation. Sensory mechanisms are responsible for the initiation of voiding reflexes. Afferent discharge from the bladder is coveyed by a dual innervation of hypogatric and pelvic afferent nerves. Changes of bladder sensory result of LUTS, such as urgency, incontinence, increased daytime frequency and nocturia. Modulation of TRPV1 signaling has been clinically investigated as a novel approach in treating OAB type symptoms. It plays a crucial role in maintaining bladder physiological function stability. Chinese Traditional Prescription Suo Quan Wan (SQW) have been used on clinical dealing with lower urinary tract symptoms (LUTS) for decades, but scientific basis of the efficacy and mechanisms of SQW is still needed. The present study was undertaken to investigate effect of SQW on bladder function and the mechanism of SQW on OAB rat base on TRPV1 channel.Objectivesstudy recreated OAB model rats and resuscitated TRPV1 knockout mouse to study the mechanism of SQW on OAB. Using molecular biology technology combining the traditional and modern theory, to investigate the effect of SQW on micturition and urodynamic parameters of OAB model rats, on the expression of TRPV1 on the bladder of OAB rats, on the function of afferent nerve of OAB rats and TRPV1 knockout mouse, on the TRPV1 signalling change.Methods1, At first, study aimed on obtain an appropriate rat model by observe bladder function of rat after BOO surgery in different period. female Sprague-Dawley rats received sham operations or partial bladder outlet obstruction operations. Two, four and six weeks later, the OAB model groups and control sham operation groups were subjected to urodynamic tests to measure differences in bladder functions.2, Once the appropriate rat model obtain, the next experiment was preformed to reserch the expression of TRPV1 in OAB rat bladder and the relationship with OAB. The rat model would be recreated, Immunofluorescence staining, Real-Time PCR and western blotting was preformed to localize and quantify the expression of TRPV1 in the bladder. On the other hand, TRPV1 knockout mice was enroll to study the effect of trpvl gene in normal bladder function, an wildtype group was used as control. The mice were anesthetized via the administration of urethane, pelvic nerve was comfirm and separate without and damage. The PN firing was recorded by BIOPAC instruments during intravesical instillation with NS, CAP, CPZ.3, In this part, study focus on the effect of SQW on the bladder function of OAB model rat and the expression of TRPV1 in bladder. The rat model would be recreated, six groups were studied and consisted of sham-operated group, OAB model group, drug treated control group, SQW treated low group, SQW treated middle group and SQW treated high group. The rats were assessed by urodynamic. Additionly, the NVC was measured during the filling period and the micturition times and voided volume was recorded after the last urodynamic tests. After urodynamic tests have been done, bladder weight was measured, and the ratio of bladder weight to body weight is received by calculate. The rats were assessed by urodynamic. Immunofluorescence staining, Real-Time PCR and western blotting was preformed to localize and quantify the expression of TRPV1 in the bladder. Next, another experiment perform to research the effect of SQW on the pelvic nerve firing of OAB rat. Rats were anesthetized via the administration of 10% urethane, pelvic nerve was comfirm and separate without and damage. The PN firing was recorded by physiological signal record instruments during intravesical instillation with NS, CAP, CPZ.4, TRPV1 knockout mice was grouping into knockout group, SQW treated low group and SQW treated high group, an wildtype group was used as control. Using powerlab instruments to record the force of in vitro bladder smooth muscle strips response to a,(3-meATP, Carbachol, CAP, KCl. Measured the ATP release was measured by luciferin-luciferase assays. Another part of this experiment, mice was sacrifice and the bladder of each mice was excised at the level of the ureteral orifices,The bladder body was cut open vertically and divided into 2 portions (half for RT-PCR, the rest for western blotting) to study the expression of TRPV1 and P2X3.Results1, Results of this part indicated that OAB model group got weight lost. And, at two and four weeks, the OAB model group exhibited significant differences in urodynamic parameters including bladder leak point pressure(BLPP), maximum voiding pressure(MVP), maximum bladder capacity(MBC) compared to the sham group. At four and six weeks, the OAB model group exhibited significant differences in residual volume(RV), non-voiding contraction frequency. Further more, six weeks OAB model rats have much more RV but less vioding efficiency when compared with 6 weeks sham group or 2-,4-week OAB model group. Indicated that rats undergone BOO were exhibited similarities to the compensated stage before four weeks and might have entered the decompensated state at six weeks. Study carry on with four weeks OAB model would be appropriate.2, Expression of the TRPVl mRNA and proteins in the bladder was significantly greater in the OAB model group than in the control group. Pattern of aferent nerve firing of mice: at first phase in which the pressure increases slowly and the afferent response increases rapidly and a second phase where there is a more rapid increase in intravesical pressure and a slower increase in afferent activity. For TRPV1-/- mice firing record during infusion, results show lower bladder pressure and contraction as well as PN firing of TRPV1-/-mice.3, The unstable bladder in the OAB model group recovery of bladder stability after SQW treatment, and the weight loss and bladder hypertrophy are improved as well as the urodynamic parameters.and the expression and distrbution of TRPV1 in the bladder of BOO-induced rat subsequently decreased significantly with SQW treatment. Aferent nerve firing of rat show similar pattern with TRPV1-/- mice.Increasing firing was show in OAB rats during intravesical instillation with NS compare with sham rats and become stable after SQW treated. CAP increase the PN firing of sham rats but can block by CPZ. However, both CAP and CPZ exhibited treated effect on OAB rats. SQW treated can stable the response to CPZ and CAP of bladder.4, ATP release TRPV1-/- mice from bladder smooth muscle strips was significant reduce as well as the response to α,β-me ATP, KCl and carbachol and no response to CAP. SQW treated can improve the ATP release and the response to α,β-me ATP, KCL and carbachol. TRPV1-/- mice show no TRPV1 expression and lower experssion level of P2X3 in bladder, SQW treated have no effect on TRPV1 & P2X3 expression.ConclusionTRPVl is a pressure sensor in the bladder, mediating stretch detection. TRPVl can also regulate urinary bladder contractions and function of afferent nerve. Drug treatment demonstrated that SQW can modulate the expression of TRPV1 in accordance with recovery of bladder function.