Effects Of Manual Acupuncture On Bowel Motility In Knockout Mice And Its Underlying Peirpheral Neural Mechanism Research

Object: Acupuncture has been tried against various diseases, includinggastrointestinal disorders. A large amount of experimental and clinicalevidence indicates the effectiveness of acupuncture in treating gastro-intestinal diseases. Though there were vast researches on the acupuncturetreatment of bowel diseases, previous studies have demonstrated theeffects of acupuncture stimulation on gastric motility in anesthetized rats,the evidence about the role of acupuncture on bowel motility of rodents innormal state, especially for the knockout mice, did not abound.Nevertheless, rare experiments were carried out to record the motility ofjejunum and colon of rodents in vivo, and even less studies relating to theeffects of acupuncture on that kind of bowel motility were reported athome. So the purpose of this is to investigate the effects of manual acupuncture (MA) on bowel motility in normal and autonomic nervescorrelation factor knockout mice and the mechanisms.MateriaI and Methods：1．Experimental protocols：Fufty adult mice (20mice of C57BL/6, and each10mice of TRPV1-/-mice,ASIC3-/-mice and M2/3-/-) were anaesthetized with10%urethane(intraperitoneally,1-1.2g/kg).A miniaturized solid-state pressuretransducer catheter with a water capsule (dia.3mm) in the end wasinserted into the lumen of the jejunum through the incision on theintestinal surface. Another catheter of the same type was inserted into thedistal colon through the anus. The capsules were placed1.5cm and1cmabove the incision of jejunum and the anus, respectively. This output wasacquired via a Micro1401interface and recorded using Spike2version7.03data acquisition software.2．Acupuncture procedure in anesthetized miceQuchi (LI11); located in the midpoint between the lateral end of thetransverse cubical crease and the lateral epicondyle of the humerus;Needles were inserted to a depth of5mm into the skin and underlyingmuscles at Shangjuxu (ST37) points. ST37is located at5mm lateral andlower from the anterior tubercle of the tibia in mice. Tianshu (ST25) islocated20mm above the symphysis pubis and5mm lateral from themidline in mice. Dachangshu (BL25): in the waist, under the fourthlumbar spines,5mm lateral to posterior midline. Acupuncture needleswere inserted unilaterally into acupoints and were twisted, lifted andthrust manually at a frequency of2Hz for60s in each type of mice. 3．Data acquisition and analysisEach miniature pressure transducer catheter was connected to atransducer control unit, whose output signal was subsequently amplifiedfurther using a transducer amplifier in differential mode. This output wasacquired via a Micro1401interface and recorded using Spike2version7.03data acquisition software. All the data in the present study wereexpressed as means±standard deviation (SD), and analyzed bytwo-tailed Student’s t-test or one-way ANOVA with SPSS software17.0.P-values<0.05were considered significant difference. P-values<0.01were considered very significant difference.RESULTS:1. The effect of manual acupuncture at different acupoint on bowelmotility in C57BL/6and TRPV1KO mice.1.1The effect of manual acupuncture at different acupoint on bowelmotility in C57BL/6mice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) ofC57BL/6mice (P＜0.05). MA at ST25significantly inhibited themotility of jejunum (manifested as changed frequency and intestinalpressure, precisely decreased average amplitude, mean area under thecontractile curve, and the frequency) of C57BL/6mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased amplitude and mean area underthe contractile curve) of C57BL/6mice (P＜0.05). MA at BL25has notsignificantly effect at the motility of jejunum (manifested as changed frequency and intestinal pressure, precisely not significantly changedaverage amplitude, mean area under the contractile curve, and thefrequency) of C57BL/6mice (P＞0.05), while significantly increased thedistal colonic motility (manifested as changed intestinal pressure,precisely increased amplitude and mean area under the contractile curve)of C57BL/6mice (P＜0.05).1.2The effect of manual acupuncture at different acupoint on bowelmotility in TRPV1KO mice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) of TRPV1KO mice (P＜0.05). MA at ST25significantly inhibited the motility ofjejunum (manifested as changed frequency and intestinal pressure,precisely decreased average amplitude, mean area under the contractilecurve, and the frequency) of TRPV1KO mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased amplitude and mean area underthe contractile curve) of TRPV1KO mice (P＜0.05). MA at BL25hasnot significantly effect at the motility of jejunum (manifested as changedfrequency and intestinal pressure, precisely not significantly changeaverage amplitude, mean area under the contractile curve, and thefrequency) of TRPV1KO mice (P＞0.05), while significantly increasedthe distal colonic motility (manifested as changed intestinal pressure,precisely increased amplitude and mean area under the contractile curve)of TRPV1KO mice (P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of TRPV1KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased change rate of average amplitude and increased change rate of mean area underthe contractile curve both lower than C57BL/6mice)(P＜0.05). Thestrength of the inhibited motility of jejunum that MA at ST25of TRPV1KO mice was lower than C57BL/6mice (manifested as changedfrequency and intestinal pressure, precisely decreased change rate ofaverage amplitude, decreased change rate of mean area under thecontractile curve, and the frequency all lower than C57BL/6mice)(P＜0.05), while The strength of the increased motility of the distal colonic ofTRPV1KO mice was lower than C57BL/6mice (manifested as changedintestinal pressure, precisely increased change rate of amplitude andincreased change rate of mean area under the contractile curve both lowerthan C57BL/6mice)(P＜0.05). MA at BL25has not significantly effectat the motility of jejunum (manifested as changed frequency andintestinal pressure, precisely no significant change rate of averageamplitude, change rate of mean area under the contractile curve, and thefrequency between TRPV1KO mice and C5BL/6mice) of two type miceboth (P＞0.05), while the strength of the increased motility of distalcolonic of TRPV1KO mice was lower than C57BL/6mice (manifestedas changed intestinal pressure, precisely increased change rate ofamplitude and change rate of mean area under the contractile curve bothlower than C57BL/6mice)(P＜0.05).2．The effect of MA at different acupoint on bowel motility in ASIC3KOmice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) of ASIC3KO mice (P＜0.05). MA at ST25significantly inhibited the motility ofjejunum (manifested as changed frequency and intestinal pressure, precisely decreased average amplitude, mean area under the contractilecurve, and the frequency) of ASIC3KO mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased average amplitude and mean areaunder the contractile curve) of ASIC3KO mice (P＜0.05). MA at BL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged average amplitude, mean area under the contractile curve, andthe frequency) of ASIC3KO mice (P＞0.05), while significantlyincreased the distal colonic motility (manifested as changed intestinalpressure, precisely increased the average amplitude and the mean areaunder the contractile curve) of ASIC3KO mice (P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of ASIC3KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under thecontractile curve both lower than C57BL/6mice)(P＜0.05). The strengthof the inhibited motility of jejunum that MA at ST25of ASIC3KO micewas lower than C57BL/6mice (manifested as changed intestinal pressure,precisely decreased change rate of average amplitude, change rate ofmean area under the contractile curve, and the frequency all lower thanC57BL/6mice)(P＜0.05), while The strength of the increased motilityof the distal colonic of ASIC3KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under thecontractile curve both lower than C57BL/6mice)(P＜0.05). MA at BL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged change rate of average amplitude, change rate of mean area under the contractile curve, and the frequency between ASIC3KO miceand C57BL/6mice) of both two type mice (P＞0.05), while the strengthof the increased motility of distal colonic of ASIC3KO mice was lowerthan C57BL/6mice (manifested as changed intestinal pressure, preciselyincreased change rate of average amplitude and change rate of mean areaunder the contractile curve both lower than C57BL/6mice)(P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of ASIC3KO mice was lower than C57BL/6micebut higher than TRPV1KO mice(manifested as changed intestinalpressure, precisely increased change rate of average amplitude andchange rate of mean area under the contractile curve both lower thanC57BL/6mice but higher than TRPV1KO mice)(P＜0.05). The strengthof the inhibited motility of jejunum that MA at ST25of ASIC3KO micewas lower than C57BL/6mice but higher than TRPV1KO mice(manifested as changed intestinal pressure, precisely decreased changerate of average amplitude, change rate of mean area under the contractilecurve, and the frequency all lower than C57BL/6mice but higher thanTRPV1KO mice)(P＜0.05), while The strength of the increased motilityof the distal colonic of ASIC3KO mice was lower than C57BL/6micebut higher than TRPV1KO mice (manifested as changed intestinalpressure, precisely increased change rate of average amplitude andchange rate of mean area under the contractile curve both lower thanC57BL/6mice but higher than TRPV1KO mice)(P＜0.05). MA atBL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged change rate of average amplitude, change rate of mean areaunder the contractile curve, and the frequency among TRPV1KO mice、ASIC3KO mice and C57BL/6mice) of all three type mice (P＞0.05),while the strength of the increased motility of distal colonic of ASIC3KO mice was lower than C57BL/6mice but higher than TRPV1KO mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under thecontractile curve both lower than C57BL/6mice but higher than TRPV1KO mice)(P＜0.05).3．The effect of MA at different acupoint on bowel motility in M2&3KOmice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) of M2&3KO mice (P＜0.05). MA at ST25significantly inhibited the motility ofjejunum (manifested as changed frequency and intestinal pressure,precisely decreased average amplitude, mean area under the contractilecurve, and the frequency) of M2&3KO mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased average amplitude and mean areaunder the contractile curve) of M2&3KO mice (P＜0.05). MA at BL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged average amplitude, mean area under the contractile curve, andthe frequency) of M2&3KO mice (P＞0.05), while significantlyincreased the distal colonic motility (manifested as changed intestinalpressure, precisely increased average amplitude and mean area under thecontractile curve) of M2&3KO mice (P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of M2&3KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under the contractile curve both lower than C57BL/6mice)(P＜0.05). MA at ST25has not significantly different effect at the motility of jejunum(manifested as changed frequency and intestinal pressure, precisely notsignificantly different decreased change rate of average amplitude,change rate of mean area under the contractile curve, and the frequencybetween M2&3KO mice and C57BL/6mice) between two type mice (P＞0.05), while The strength of the increased motility of the distal colonicof M2&3KO mice was lower than C57BL/6mice (manifested as changedintestinal pressure, precisely increased average amplitude and mean areaunder the contractile curve both lower than C57BL/6mice)(P＜0.05).MA at BL25has not significantly effect at the motility of jejunum(manifested as not significantly changed frequency and intestinalpressure, precisely not significantly changed change rate of averageamplitude, change rate of mean area under the contractile curve, and thefrequency between M2&3KO mice and C57BL/6mice) of two type mice(P＞0.05), while the strength of the increased motility of distal colonic ofM2&3KO mice was lower than C57BL/6mice (manifested as changedintestinal pressure, precisely increased change rate of average amplitudeand change rate of average mean area under the contractile curve bothlower than C57BL/6mice)(P＜0.05).Conclusion:1．Tianshu (ST25) is the “homotopic point” for jejunum, and MA at ST25could inhibit the motility of jejunum via somato-sympathetic reflexpathway. Thus, Quchi (LI11) Shangjuxu (ST37) are the “heterotopicpoint” for jejunum, and MA at LI11and ST37could promote themotility of jejunum via increased parasympathetic tone.2.Quchi (LI11), Shangjuxu (ST37), Tianshu (ST25) and Dachangshu(BL25) are the “heterotopic point” for distal colon, and MA at LI11,ST37,ST25and BL25could promote the motility of distal colonvia increased parasympathetic tone.3．In TRPV1KO and ASIC3KO mice, MA at LI11, ST37, ST25andBL25have the same regulation of the motility of jejunum and distalcolon with C57BL/6mice. But the size of effect have the apparentdifferent. It points that TRPV1and ASIC3might be participated inthe afferent neural mechanism of MA.4．In M2&3KO mice, MA at LI11, ST37, ST25and BL25have the sameregulation of the motility of jejunum and distal colon with C57BL/6mice. But the size of effect of MA at LI11and ST37has the apparentreduce. It points that M2&3might be participated in the target’sresponse receptor mechanism of MA.So we can observe from this experiment that the effect of MA wasclosely related with acupoint’s inherent character. Both of TRPV1, andASIC3have its action in the the afferent neural mechanism of MA. AndM2&3might be participated in the target’s response receptor mechanismof MA. And why the different effect of MA at distal colon, the intrinsiccontradistinction must be investigated and research further.