| Objective:In this study,rats with detrusor over-activity(DO)model were used as subjects,and the effects of acupuncture were observed dynamically(2hours,24hours,48hours,72hours after acupuncture treatment).And by reverse transcription-polymerase chain reaction(RT-PCR),Western blot(Wb),calcium ion imaging and other techniques,the acupuncture on the pacing current channel of bladder ICCs was observed.The effects of Hyperpolarization activates cyclic nucleotidegated cation channels(HCN)-related subtypes and the effects of intracellular calcium transients in ICCs,combined with changes in urodynamics and muscle strips,reveal needles the spur regulates the myogenic mechanism of detrusor excitability and describe the related effects and mechanisms of acupuncture in regulating detrusor over-activity.Methods:SPF female Wistar rats were randomly divided into sham operation group,model group,acupuncture group and drug group.The model of detrusor over-activity was established by intraperitoneal cyclophosphamide(CYP)injection.Model group:giving detrusor over-activity modeling;-Acupuncture group:after successful DO model,acupuncture Ci Liao(BL32)and Hui Yang(BL35)at the same time,electro-acupuncture continuous wave(frequency 30 Hz)for 20 minutes;daily acupuncture treatment for three consecutive days;Drug group:after successful DO model,Glivec(Imatinib Mesylate),a cell-specific blocker of ICCs,was administered once a day for three consecutive days.Urine kinetics measurements and bladder muscle strips were performed in each of the above groups.In order to observe the acupuncture effect dynamically,each group was randomly divided into 4 sub-groups of observation points(corresponding to different observation points:2hours,24hours,48hours,72hours after acupuncture treatment),and each subgroup was 10 rats.Bladder urinary kinetics was measured by Canadian Laboria urodynamic analyzer;detrusor contraction characteristics were observed by ex vivo bladder muscle strip traction test;HCN1-4 subtype expression of bladder ICCs cells was determined by RT-PCR and Wb method;calcium ion imaging technique the intracellular calcium transients characteristics of bladder ICCs were detected.Results:1.Model evaluation:The DO model was successfully prepared by CYP.The main urodynamics were:involuntary contractile waves in the bladder detrusor during the storage stage,showing a contractile contraction,and the effective capacity of the bladder in the model group was obvious.Shrinking,the storage time is significantly shortened.2.Urodynamic characteristics:At the 2,24,48,and 72 hours observation points,the effective volume of bladder in the model group was significantly smaller than that in the sham operation group(P<0.05),and the urine storage time was shorter,which was consistent with the DO model status;Compared with the model group,the effective volume of the bladder at the four observation points were significantly increased in the acupuncture group and the drug group(P<0.05),and the urine storage time was significantly prolonged(P<0.05),but there was no difference between the acupuncture group and the drug group,indicating that both acupuncture and drugs can effectively improve the detrusor instability;At the same time,it was found at four observation points,the urinary pressure of the 24-hour observation point model group was lower than that of the sham operation group(P<0.05),and the urination pressure of the acupuncture group and the drug group was also lower than that of the sham operation group(P<0.05),which was higher than that of the model group.Trends,but the difference was not statistically significant(P>0.05).There was no significant change in bladder effective volume and urine storage time between the observation points in the sham operation group(P>0.05).There was no significant change in bladder effective volume and urine storage time between the observation points in the model group(P>0.05),indicating that the model is stable;the acupuncture group and the drug group were compared in the group respectively,and the effect of increasing the effective volume of the bladder and prolonging the storage time of the bladder was the best at the 24-hour observation point,but the difference between the observation points was no statistical significance(P>0.05),indicating that the effect of acupuncture and drug(Imatinib Mesylate)on the effective volume of bladder and prolonged storage of urine in model rats was sustained,and maybe the 24-hour observation point model group was the best.3.Contraction characteristics of bladder muscles in vitro:Among the four observation points,the contraction frequency of bladder muscle strips in the model group was higher than that in the sham operation group at 24 hours(P<0.05),and the contraction amplitude was decreased(P<0.05),and compared with the model group,the contraction frequency of bladder muscle strips in the acupuncture group decreased(P<0.05),and the contraction amplitude increased(P<0.05).Only the bladder muscle strip contraction frequency decreased in the drug group(P<0.05).At the 2 hours,48hours,and 72 hours observation points,there was no significant difference in the frequency of contraction and amplitude between the groups(P>0.05).There was no significant change in the frequency and amplitude of muscle strip contraction between the observation points in the sham operation group(P>0.05).There was no significant change in the frequency and amplitude of muscle strip contraction between the observation points in the model group(P>0.05);The contraction frequency of the muscle strips in the acupuncture group was lower than the other three observation points at 24 hours,but the difference between the observation points was not statistically significant(P>0.05),and the contraction amplitude was significantly higher at the 24-hour observation point.At the observation point of 2 hours(P<0.05),the advantage of 24-hour observation point was observed;the frequency of muscle strip contraction in the drug group was lower than that of the other 3 observation points at 24 hours,and the contraction amplitude was at 24 hours compared with the other 3 observation points.High,but the difference between the observation points was not statistically significant(P>0.05),reflecting the 24-hour observation point advantage.4.Changes in HCN subtypes of bladder ICCs:Comparison of the two groups,among the four observation points,only in the 24-hour observation point,the sensor group of rat ICCs HCN1 and HCN2 messenger RNA(mRNA)and protein.The expressions were higher than those in the sham-operated group(P<0.05).Compared with the model group,the mRNA and protein expressions of HCN1 and HCN2 were decreased in the acupuncture group and the drug group(P<0.05),at 2 hours and 48 hours.There was no significant difference in the expression of HCN1 and HCN2 mRNA and protein between the groups at the 72-hour observation point(P>0.05).There was no statistical difference between the HCN3 and HCN4 observation points(P>0.05).There was no significant change in HCN1 and HCN2 mRNA expression groups at each observation point of the sham operation group(P>0.05).There was no significant change in HCN1 and HCN2 mRNA expression groups at each observation point in the model group(P>0.05).There were no significant changes in the HCN1 and HCN2 mRNA expression groups in the observation group(P>0.05).The HCN1 mRNA in the drug group was significantly higher than the 24-hour observation point at 48 hours(P>0.05),and the HCN2 mRNA group was prominent.The difference was not statistically significant(P>0.05).There was no significant difference between the four observation points of HCN3 and HCN4 mRNA in each group(P>0.05).5.The intracellular calcium transients characteristics of bladder ICCs at the 24-hour observation point:the intracellular calcium transients frequency of the ICCs in the model group was significantly higher than that of the sham operation group(P<0.0083),and the amplitude of calcium ion transients was significantly increased(P<0.0083),compared with the model group,both the acupuncture group and the drug group showed a decrease in the frequency of calcium ion transients in the ICCs of the bladder(P<0.0083)and a decrease in the amplitude(P<0.0083),indicating acupuncture and drug treatment.It is effective in reducing the excitability of ICCs.The intracellular calcium wave amplitude of ICCs in the model rat was increased compared with the sham operation group(P<0.05),but there was no significant difference between the acupuncture group and the drug group(P>0.05),indicating acupuncture and the drug has no effect on regulating the intracellular calcium transients amplitude of ICCs.Conclusion:1.At 2 hours,24 hours,48 hours,and 72 hours after acupuncture intervention,the bladder capacity of DO rats increased and the time of bladder storage was prolonged.2.At 24 hours after acupuncture intervention,the contractile frequency of bladder detrusor muscle strips in DO rats decreased significantly and the contraction amplitude increased significantly.It shows that the best effect of acupuncture on the detrusor is reflected 24 hours after acupuncture.3.The expression of HCN1 and HCN2 mRNA and protein in bladder ICCs of DO rats decreased significantly 24 hours after acupuncture,and the frequency and amplitude of intracellular calcium oscillations in bladder ICCs decreased,indicating that acupuncture can inhibit bladder ICCs cells by inhibiting the expression of pacemaker channels in ICCs and the intracellular calcium transients of ICCs. |
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