| Objective: Taking acontractile detrusor rats as the research object,electroacupuncture at Ciliao and Huiyang points was used to observe the effect of electroacupuncture from a dynamics angle(2 hours,24 hours,48 hours and 72 hours after electroacupuncture treatment).Furthermore,by means of PCR,Westernblot,calcium ion imaging and other techniques,the effects of electroacupuncture on various subtypes of bladder ICCs HCN channel and calcium ion oscillation characteristics in ICCs were observed.Combined with urodynamics detection and detrusor muscle strip traction experiment in vitro,the effect target and mechanism of electroacupuncture on acontractile detrusor were revealed.Methods: 160 SPF female Wistar rats were randomly divided into sham operation group,model group,electroacupuncture group and drug group.A rat model of acontractile detrusor was established by liquid nitrogen frostbite.The sham operation group: no modeling was done,only suture was given after laparotomy.Model group:detrusor muscle weakness was not treated after successful modeling.Electroacupuncture group:after successful modeling of acontractile detrusor,electroacupuncture was applied to Ciliao and Huiyang points;Drug group:After successful modeling of acontractile detrusor,0.1% neostigmine 0.02 ml was injected subcutaneously twice daily.In the above groups,the same dose of normal saline was injected subcutaneously into all three groups except the drug group,which received0.1% neostigmine 0.02 ml twice daily.Except the electroacupuncture group,which is treated with electroacupuncture for 20 minutes per day,the other three groups are bound for 20 minutes per day.For urodynamics observation,each group was randomly divided into 4 groups(corresponding to different periods: 2h,24 h,48h and 72 h after treatment).Ten in each group.Urodynamic analysis technique was used to analyze the bladder urodynamics characteristics,detrusor strips in vitro were pulled to analyze the contractility of detrusor strips in vitro,PCR and Westernblot methods were used to analyze the expression changes of HCN channel m RNA and protein in bladder detrusor ICCs,and calcium ion imaging technique was used to observe the changes of calcium ion oscillation characteristics in bladder ICCs.Results:1.Changes in urodynamics:(1)The model of acontractile detrusor was established by liquid nitrogen frostbite.Urodynamics showed that bladder volume and residual urine volume increased(P<0.05)and average urination speed decreased(P<0.05)compared with sham-operated rats,which indicated that the model was successfully established and could be used for related research on acontractile detrusor diseases.(2)After electroacupuncture treatment for 2h,24 h,48h and 72 h,bladder volume and residual urine volume of acontractile detrusor rats were lower than those of model group(P<0.05).After electroacupuncture treatment,the average micturition speed of the rats was higher than that of the model group at 2h and 24h(P<0.05),while at 48 h and 72 h after treatment,electroacupuncture had no effect on the average micturition speed of the acontractile detrusor rats(P>0.05),and the average micturition speed of the rats at 2h after electroacupuncture treatment was higher than that of the other three periods(P<0.05).(3)After subcutaneous injection of neostigmine for 2h,24 h,48h and 72 h,bladder volume and residual urine volume of acontractile detrusor rats were lower than those of model group(P<0.05).After subcutaneous injection of neostigmine for 2h,24 h and 48 h,the average micturition speed of rats was higher than that of model group(P<0.05),while subcutaneous injection of neostigmine for 72 h after treatment had no effect on the average micturition speed of acontractile detrusor rats(P>0.05).2.Contractility changes of detrusor strips in vitro of bladder:(1)Contraction amplitude and frequency of detrusor strips in vitro of rats in detrusor muscle weakness model group are lower than those in sham operation group(P<0.05);(2)2h,24 h and 48 h after electroacupuncture treatment,the contraction amplitude of isolated detrusor strips in rats with acontractile detrusor was higher than that in model group(P<0.05).72 h after treatment,electroacupuncture had no effect on the contraction amplitude of isolated detrusor strips in rats with acontractile detrusor(P>0.05),and the contraction amplitude of isolated detrusor strips at 2h after electroacupuncture treatment was larger than that in other three time periods(P<0.05).2h and 24 h after electroacupuncture treatment,the contraction frequency of isolated detrusor strips in acontractile detrusor rats was higher than that in the model group(P<0.05).48 h and 72 h after treatment,electroacupuncture had no effect on the contraction frequency of isolated detrusor strips in acontractile detrusor rats(P>0.05).(3)After subcutaneous injection of neostigmine,the contraction amplitude of isolated detrusor muscle strips in rats with acontractile detrusor was higher than that in model group(P<0.05),and after 48 hours and 72 hours of treatment,subcutaneous injection of neostigmine had no effect on the contraction amplitude of isolated detrusor muscle strips in rats with acontractile detrusor(P>0.05).After subcutaneous injection of neostigmine for 2h,24 h and 48 h,the contraction frequency of isolated detrusor strips in acontractile detrusor rats was higher than that in model group(P<0.05).After treatment for 48 h and 72 h,subcutaneous injection of neostigmine had no effect on the contraction frequency of isolated detrusor strips in acontractile detrusor rats(P>0.05).3.Changes of calcium oscillation characteristics in bladder ICCs:(1)The amplitude and frequency of calcium oscillation in bladder ICCs in acontractile detrusor model group were lower than those in sham operation group(P<0.05).(2)2 hours after electroacupuncture treatment,the amplitude and frequency of calcium ion oscillation in bladder ICCs of acontractile detrusor rats were higher than those of model group(P<0.05).(3)2 hours after subcutaneous injection of neostigmine,the amplitude and frequency of calcium ion oscillation in bladder ICCs of acontractile detrusor rats were higher than those of model group(P<0.05).4.Changes in m RNA and protein expression of each subtype of ICCs HCN channel:(1)m RNA and protein expression of four subtypes of ICCs HCN channel exist in bladder tissue of rats in sham-operated group,and m RNA and protein expression of four subtypes of iccshcn channel in bladder of rats with acontractile detrusor are lower than those in sham-operated group(P<0.05);(2)Electroacupuncture can increase the expression of ICCs HCN1 and HCN4 m RNA at2 h,24h,48 h and 72 h after treatment(P<0.05),and the expression of hcn4 m RNA at2 h after electroacupuncture treatment is the most obvious improvement(P<0.05).Electroacupuncture had no effect on the expression of ICCs HCN2 and HCN3 m RNA at 2h,24 h,48h and 72 h after treatment(P>0.05).Electroacupuncture can increase the expression of ICCs HCN1 and HCN4 protein 2 hours after treatment(P<0.05).Electroacupuncture has no effect on the expression of ICCs HCN2 and HCN3 protein2 hours after treatment(P>0.05).(3)Subcutaneous injection of neostigmine can increase the expression of ICCs HCN1 and HCN4 m RNA at 2h,24 h,48h and 72 h after treatment(P<0.05),and increase the expression of ICCs HCN2 m RNA at 2h,24 h and 48 h after treatment(P<0.05),and the expression of 2h HCN1 m RNA after subcutaneous injection of neostigmine is the most obvious improvement(P<0.05).Subcutaneous injection of neostigmine had no effect on the expression of ICCs HCN3 m RNA at 2h,24 h,48h and 72 h after treatment(P>0.05).Subcutaneous injection of neostigmine can increase the expression of ICCs HCN1,HCN2 and HCN4 protein 2hours after treatment(P<0.05).subcutaneous injection of neostigmine has no effect on the expression of ICCs HCN3 protein 2 hours after treatment(P>0.05).Conclusion: 1.Electroacupuncture can effectively reduce bladder volume and residual urine volume of acontractile detrusor rats,and improve average urination speed of acontractile detrusor rats and the amplitude and frequency of contraction of isolated detrusor muscle strips.Moreover,the effect of electroacupuncture has the characteristic of urodynamics change,and the improvement effect of average micturition speed and contraction amplitude of isolated detrusor muscle strips in rats with acontractile detrusor 2 hours after treatment is the best.2.Electroacupuncture can up-regulate the expression of ICCs HCN1 and HCN4,and increase the amplitude and frequency of calcium oscillation in bladder ICCs,indicating that electroacupuncture enhanced the expression of ICCs pacing channel HCN and ICCs excitability is one of the myogenic action mechanisms in the treatment of acontractile detrusor. |
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