"Intensity-Response" Effects Of Acupuncture And Local Moxibustion-Like Stimuli On Gastric Motility And Its Underlying Afferent And Efferent Receptor Mechanisms

"Intensity-Response" Effects of acupuncture and Local Moxibustion-LikeStimuli on Gastric Motility and Its Underlying Receptor MechanismsAccording to the Miraculous Pivot and other medical classics in China, intensity of acupuncture and moxibustion stimuli is critical for the generation of "De-qi" feelings and the therapeutic effects. The "intensity-response" relationship is one of the most important properties in acupuncture and moxibustion treatment, which has great instructional meaning for clinical practice.Acupuncture and moxibustion have good regulatory effects on the motility and myoelectricity of gastric smooth muscle. Different acupoints induced distinct impact on gastric motility via relevant "somato-autonomic nerve" reflexes. It is shown that acupuncture stimulation to the acupoints on face, forelimbs and hindlinbs, which are distant to the region of gastric innervation, produces a facilitative response of gastric motility with increased activities of vagus. While the acupoints on lower-chest, middle-dorsum and whole abdomen, which are homo-segmental to the region of gastric innervation, induces an inhibitory response of gastric motility with increased activities of sympathetic nerves. Although the regulatory effects of acupuncture and moxibustion on gastric motility has been preliminary proved, the possible "intensity-response" relationship is still elusive. Primary afferent fibers innervated local acupoints site play important roles in the transduction of acupuncture and moxibustion signals, and severing the relevant afferent fibers can eliminate the modulatory effects of acupoints. The primary affernt fibers are classified into three broad groups:Aβ,Aδ and C fibers. Aβ fibers are myelinated (large diameter) and responsible for light touch sensation, lightly myelinated (medium diameter) Aδ and unmyelinated (small diameter) C fibers are respond to noxious mechanical, thermal and chemical stimuli. The smaller diameter afferent fiber has higher threshold. Therefore, the distinct regulatory effects on gastric motility induced by different acupuncture and moxibustion stimuli with different intensities are likely to be mediated by the activation of different afferents.Besides the mechanical and thermal properties, acupuncture and moxibustion stimuli elicit releasing of bioactivators in local tissue, such as H+, histamine and bradykinin. These bioactivators can be detected by some ion channels expressed in afferent fiber terminals. TRPV1receptor is expressed on Aδ and C fibers, which is responsible for the noxious mechanical and heat stimuli(≥43℃); ASIC3is mainly expressed in A fibers and related to non-nociceptive mechanical detection. In addition, TRPV1and ASIC3can be activated by those bioactivator above. Based on the functions of the two channels, it is conceivable that both TRPV1and ASIC3are contribute to the detection of acupuncture and moxibustion signals.The sympathetic and parasympathetic nerve are the efferent limbs underlying acupuncture and moxibustion-modulated gastric motility. Using blockers of sympathetic and vagal nerve, it was shown that the muscarinic and β-adrenergic receptor play critical role in this effect. However, due to the less sensitivity of those blockers, which subtypes of muscarinic and β-adrenergic receptor were contributive is still unclear.1ObjectiveFocusing on the affernet and efferent processes in acupuncture and moxibusiton induced gastric motility responses, the "intensity-response" relationship was investigated by observing the effects of electroacupuncture (EA) and local moxibustion-like stimuli (LMS) with diiferent intensities on gastric motility. Using TRPV1and ASIC3gene knockout (TRPV1-/-, ASIC3-/-)mice, the receptor mechanisms underlying the detection of acupuncture and moxibustion signals was addressed. In addition, M2/M3gene knockout (M2/M3-/-) and β1/β2gene knockout mice (β1/β2-/-) were also employed in the present study to reveal the receptor mechanisms mediated the efferent processes in acupuncture modulated gastric motiltiy.2MethodsMale Sprague-Dawley (SD) rats (n=36), weighing250～300g, as well as71 mice, weighing20-25g were used, which include C57BL/6(n=23), ASIC3-/-(n=16), TRPV1-/-(n=16), M2/M3-/-(n=8) and β1/β2-/-(n=8) mice. Gastric motility was recorded by a intrapyloric balloon inserted from a incision on the duodenum. The effects of EA (0.5,1,3,5,7and9mA) and LMS (41,43and45℃) at ST36(Zusanli) or CV12(Zhongwan) with different intensities were observed. After surgery, the balloon was filled with about0.2ml and0.05ml warm water in rats and mice respectively, to keep pressures at about100mmH20. The first EA stimulus was applied when gastric motility wave maintained stable, usually at about30minutes after the surgical procedure. The latter stimulus can only be applied when the gastric motility recovered to control state. The gastric motility induced by EA and LMS was compared with the background activity in terms of average amplitude (the average difference between the cyclic maxima and minima in the selected cycles), integral (returns the integral of the selection, calculated as the sum of the data points multiplied by the sample interval) and frequency (per minute) of gastric contraction waves.3Results3.1"Intensity-response" relationship of EA with different intensities on gastric motility and its underlying peripheral receptor mechanim3.1.1Gastric motility under resting conditionGastric motility wave maintained stable usually at about30minutes after the surgical procedure. The stable gastric motility can last5h and3h in rats and mice, respectively. The average amplitude of gastric motility in rats and mice was about2cm and1cm, respectively. The frequecy of gastric motiltiy was about4-6cycles per minute (cpm).3.1.2The effects of EA with different intensities at ST36on gastric motility0.5mA and1mA EA at ST36have no obvious impact on gastric motility in rats. However,3,5,7and9mA EA have significant stimulatory effect on gastric motility. The amplitude of gastric motility was increased from1.97±0.13cmH2O to2.68±0.20cmH2O (increase rate was35.82±6.54%, P<0.01) by3mA EA, increased from1.93±0.12cmH20to2.79±0.22cmH2O(increase rate was43.78±3.74%, P<0.001)by5mA EA,increased from1.76±0.20cmH20to2.64±0.25cmH20(increase rate was52.33±4.45%,(P<0.001)by7mA EA and increased from1.72±0.14cmH20to2.63±0.23cmH20(increase rate was53.16士5.43%,P<0.001)by9mA EA at ST36.Similarly,the integral of gastric motility was increased by47.52±3.26%(P<0.001),55.28±5.37%(P<0.001),64.53±5.63%妒<0.001)and64.37±6.28%(P<0.001)by3.5.7and9mA EA at ST36.The intensity of EA at ST36produced50%of the maximal regulatory effects on gastric motility was about2.3mA.The stimulatory of EA at ST36on gastric motility was maximized when the intensity reached at5mA.Besides,3,5,7and9mA EA at ST36increase the frequecy of gastric motility by0.44±0.17cpm(P<0.05),0.67±0.16cpm(P<0.01),0.67±0.23cpm(P<0.05) and0.67±0.23cpm(P<0.05),respectively.3.1.3The effects of EA with different intensities at CV12on gastric motilityEA at CV12with each intensity can inhibit the amplitude and integral of gastric motility.The amplitude of gastric motility was decreased from2.01±0.10cm H20to1.81±0.09cm H2O(decrease rate was11.15±2.73%,P<0.05)by0.5mA EA, decreased from1.82±0.14cm H20to1.47±0.13cm H2O(decrease rate was18.86±3.23%,P<0.01)by1mA EA,decreased from2.19±O.23cm H20to1.02±0.27cm H2O(decrease rate was42.58±5.56%,P<0.01) by3mA EA,decreased from2.59±0.27cm H20to1.09±0.27cm H20(decrease rate was56.72±10.36%, P<0.01)by5mA EA,decreased from2.01±0.10cm H20to1.81±0.09cm H20(decrease rate was55.36±10.25%,P<0.01)by7mA EA and decreased from2.56±0.30cm H20to1.13±0.19cm H20(decrease rate was55.36±7.95%,P<0.01)by9mA EA.Similarly,the integral of gastric motility was decreased by17.34±3.26%(P<0.01),34.72±2.33%(P<0.001),50.16±3.06%(P<0.001),64.32±3.26%(P<0.001),63.89±3.77%(P<0.001)and63.47±2.58%(P<0.001)by0.5,1,3,5,7and9mA EA respectively.The intensity of EA at CV12produced50%of the maximal inhibitory effects on gastric motility was about2.8mA.The inhibition of EA at ST36on gastric motility was maximized when the intensity reached at5mA. Besides,3,5,7and9mA EA at CV12inhibited the frequecy of gastric motility by2.11±0.23cpm(P<0.001),3.16±0.52cpm(P<0.001),3.11±O.45cpm (P<0.001)and3.22±0.43cpm(P<0.001),respectively.3.1.4Comparison of the effects of1mA EA at ST36on gastric motility between C57BL/6,ASIC3-/-and TRPV1-/-mice1mA EA at ST36produced marked stimulatory impact on gastric motility in C57BL/6,ASIC3-/-and TRPV1-/-mice.The amplitude of gastric motility in C57BL/6mice was increased from1.78±0.17cm H2O to2.61±0.28cm H2O (increase rate was45.8±1.78%,P<0.001)by1mA EA,increased by42.13±2.57%(P<0.001)and20.6±2.17%(P<0.01)in ASIC3-/-and TRPV1-/-mice, respectively.The integral of gastric motility was increased by57.24±3.16%(P<0.001),51.93±6.62%(P<0.001)and34.65±3.22%(P<0.01)in C57BL/6, ASIC3-/-and TRPV1-/-mice,respectively.The frequency of gastric motility was increased by57.24±3.16%(P<0.001),51.93±6.62%(P<0.001)and34.65±3.22%(P<0.01)in C57BL/6,ASIC3-/-and TRPV1-/-mice,respectively.The stimulatory effects of1mA EA at ST36on gastric motility in TRPV1-/-mice was significantly smaller than those of in C57BL/6mice(P<0.001,P<0.05).3.1.5Comparison of the effects of1mA EA at CV12on gastric motility between C57BL/6,ASIC3-/-and TRPV1-/-mice1mA EA at CV12produced marked inhibition on gastric motility in C57BL/6, ASIC3-/-and TRPV1-/-mice.The amplitude of gastric motility in C57BL/6mice was decreased from2.35±0.20cm H2O to1.41±0.14cm H2O(decrease rate was40.66±2.74%,P<0.001)by1mA EA,decreased by35.26±1.88%(P<0.001)and20.94±1.70%(P<0.05)in ASIC3-/-and TRPV1-/-mice,respectively.The integral of gastric motility was decreased by58.74±2.36%(P<0.001),55.59±2.37%(P<0.001)and28.76±5.46%(P<0.01)in C57BL/6,ASIC3-/-and TRPV1-/-mice, respectively.The frequency of gastric motility was decreased by31.46±5.33%(P<0.001),21.58±5.11%(P<0.001)in C57BL/6,ASIC3-/-mice,respectively.The inhibitory effects of1mA EA at CV12on gastric motility in TRPV1-/-mice was significantly smaller than those of in C57BL/6mice(P<0.001,P<0.05). 3.2The effects of LMS with different intensities on gastric motility and its underlying peripheral receptor mechanim3.2.1The effects of LMS with different intensities at ST36on gastric motility in rats41℃LMS at ST36had no obvious impact on gastric motility. From the second60s,43℃and45℃LMS at ST36significantly increased the amplitude and integral of gastric motility. In the second and third60s of43℃LMS, the amplitude of gastric motility was increased from2.42±0.20cmH2O to2.77±0.23cmH20and2.81±0.25cmH2O, and the increase rate was15.32±3.64%(P<0.05) and16.75±9.82%(P<0.05) respectively. In the second and third60s of45℃LMS, the increase rate was26.36±3.17%(P<0.001) and32.58±3.13%(P<0.001) respectively. In the second and third60s of43℃LMS, the integral of gastric motility was increased by15.63±4.27%(P<0.05) and17.75±5.52%(P<0.05) respectively. In the second and third60s of45℃LMS, the increase rate was25.68±2.17%(P<0.001) and33.43±1.98%(P<0.001) respectively.43℃LMS at ST36has no marked impact on the frequency of gastric motility. However,45℃LMS significantly increased the frequency in the second and third60s. The frequency was increased from3.67±0.17cpm to4.11±0.20cpm and4.22±0.22cpm, and the increase rate was12.94±.27%(P<0.05) and15.73±4.16%(P<0.05).3.2.2The effects of LMS with different intensities at CV12on gastric motility in rats41℃LMS at CV12had no obvious impact on gastric motility. From the second60s,43℃and45℃LMS at CV12significantly inhibited the amplitude and integral of gastric motility. In the second and third60s of43℃LMS, the amplitude of gastric motility was decrease from2.47±0.25cmH2O to2.07±0.28cmH2O and2.03±0.30cmH2O, and the decrease rate was18.24±4.25%(P<0.01) and20.55±4.97%(P<0.01) respectively. In the second and third60s of45℃LMS, the decrease rate was31.57±2.58%(P<0.001) and39.96±2.39%(P<0.001) respectively.In the second and third60s of43℃LMS,the integral of gastric motility was decrease by20.73±4.52%(P<0.05)and21.47±5.43%(P<0.05) respectively.In the second and third60s of45℃LMS,the decrease rate was28.28±3.76%(P<0.001)and38.62±3.51%(P<0.001)respectively.In the second and third60s of43℃LMS,the frequency of gastric motility was decrease by12.95±3.26%(P<0.05)and12.44±4.17%(P<0.05)respectively.In the second and third60s of45℃LMS,the decrease rate was17.68±4.59%妒<0.01)and18.54±4.36%妒<0.05)respectively.3.2.3Comparison of the effects of LMS with different intensities at ST36on gastric motilitybetween C57BL/6.ASIC3-/-and TRPV1-/-mice41℃LMS at ST36had no influence on gastric motility among the three groups of mice.From the second60s,43℃LMS at ST36significantly increased gastric motility.While the stimulatory effects of45℃LMS at ST36appeared from the first60s.In the second and third60s of43℃LMS,the amplitude of gastric motility in C57BL,6mice was increased from2.19±0.32cm H20to2.52±0.30cm H20and2.46±0.27cm H20,and the increase rate was19.36±7.53%妒<0.05)and17.69±7.81%(P<0.05)respectively.The increase rate in ASIC3-/-mice was20.17±7.53%(P<0.05)and25.53±8.26%(P<0.05)respectively.Similarly,the increase rate0f integral induced by43℃LMS at ST36was18.16±6.95%(P<0.05)and18.33±7.35%(P<0.05)in C57BL/6mice,was17.63±5.42%(P<0.05)and22.69±7.72%俨<0.05)in ASIC3-/-mice.However,43℃LMS at ST36has no impact on the frequency of gastric motility.From the first60s of45℃LMS,the amplitude of gastric motility in C57BL/6mice was increased from1.82±0.22cm H20to1.99±0.23cm H2O,2.56±0.23cm H20and2.36±O.23cm H20,and the increase rate was10.37±3.82%(P<0.05),25.96±3.41%(P<0.001)and32.28±4.52%(P<0.001)respectively.The increase rate was10.76±2.52%(P<0.05),27.45±3.99%(P<0.001)and28.36±4.15%(P<0.001)in ASIC3-/-mice.In the second and third60s of45℃LMS,the increase rate of the amplitude in TRPV1/-mice was13.96±3.58%(P<0.01)and12.16± 3.77%(P<0.05)respectively.The increase rate of integral induced by45℃LMS at ST36was10.13±3.25%(P<0.05),22.36±5.52%(P<0.01)and29.67±5.51%(P<0.001)in C57BL/6mice,and was10.43±2.75%(P<0.05),23.28±3.25%P<0.01)and26.33±2.61%(P<0.001)in ASIC3-/-mice.In the second and third60s of45℃LMS,the increase rate of the integral in TRPV1-/-mice was15.08±4.17%(P<0.01)and11.52±4.56%(P<0.05)respectively.Compared with the same time course,the stimulatory of45℃LMS at ST36on amplitude and integral in TRPV1-/-mice was significantly smaller than that of in C57BL/6mice(P<0.05).The stimulatory effects of45℃LMS at ST36on the frequency of gastric motiltiy were appeared in the second and third60s in C57BL/6mice,while emerged only in the third60s in TRPV1-/-mice.The increase rate of the frequency induced by45℃LMS at ST36was15.06±4.43%(P<0.05)and18.58±5.96%(P<0.05)in C57BL/6mice,and was15.26±3.55%(P<0.05)and11.98±3.53%(P<0.05)in ASIC3-/-mice,and was11.52±3.41%(P<0.05)in TRPV1-/-mice.3.2.4Comparison of the effects of LMS with different intensities at CV12on gastric motility between C57BL/6.ASIC3-/-and TRPV1-/-mice41℃LMS at CV12had no influence on gastric motility among the three groups of mice.From the second60s,43℃LMS at CV12significantly inhibited gastric motility.While the inhibitory effects of45℃LMS at ST36appeared from the first60s.In the second and third60s of43℃LMS at CV12,the amplitude of gastric motility in C57BL/6mice was decrease from2.41±0.22cm H2O to1.96±0.18cm H2O and1.86±0.15cm H2O,and the decrease rate-was17.65±5.43%(P<0.01) and20.24±7.15%(P<0.05)respectively.The decrease rate in ASIC3-/-mice was17.75±3.82%(P<0.05) and22.26±4.83%(P<0.05)respectively.Similarly,the decrease rate of integral induced by43℃LMS at CV12was15.27±75.33%(P<0.05)and18.66±6.92%(P<0.05)in C57BL/6mice,was15.57±5.73%(P<0.05)and19.63±7.71%(P<0.05)in ASIC3-/-mice.The decrease rate of frequency induced by43℃LMS at CV12was9.13±2.57%(P<0.05)and13.37±3.36%(P<0.05)in C57BL/6mice,was9.62±2.66%(P<0.05)and12.38±2.96%(P<0.05)in ASIC3-/-mice. From the first60s of45℃LMS at CV12,the amplitude of gastric motility in C57BL/6mice was decreased from2.27±0.14cm H2O to2.00±0.08cm H20,1.43±0.06cm H2O and1.26±0.07cm H2O,and the decrease rate was10.25±3.41%(O<0.01),35.92±2.75%(P<0.001)and343.58±3.46%(P<0.001)respectively. The decrease rate was9.87±1.83%(P<0.05),32.37±2.75%(P<0.001)and42.15±2.63%妒<0.001)in ASIC3-/-mice.In the second and third60s of45℃LMS at CV12,the decrease rate of the amplitude in TRPV1-/-mice was12.85±3.45%(P<0.05) and12.65±3.77%(P<0.05)respectively.The decrease rate of integral induced by45℃LMS at CV12was9.65±4.12%(P<0.05),29.85±4.14%(P<0.001)and37.53±3.96%(P<0.001)in C57BL/6mice,and was10.93±1.62%(P<0.05),10.93±1.62%(P<0.001)and38.95±3.17%(P<0.001)in ASIC3-/-mice. In the second and third60s of45℃LMS at CV12,the decrease rate of the integral in TRPV1-/-mice was11.75±2.93%(P<0.05)and12.96±3.78%(P<0.05) respectively.Compared with the same time course,the inhibitory effects of45℃LMS at CV12on amplitude and integral in TRPV1-/-mice was significantly smaller than that of in C57BL/6mice(P<0.01).The inhibitory effects of45℃LMS at CV12on the frequency of gastric motiltiy were appeared in the second and third60s among the three groups of mice. The decrease rate of the frequency induced by45℃LMS at CV12was24.86±4.73%(P<0.01)and32.33±6.38%(P<0.01)in C57BL/6mice,and was19.45±3.36%(P<0.01)and25.83±2.84%妒<0.001)in ASIC3-/-mice,and was10.23±2.95%俨<0.01)and9.68±2.61%俨<0.05)in TRPVl-/-mice.Compared with the same time course,the inhibitory efiects of45℃LMS at CV12on the frequency in TRPV1-/-mice was significantly smaller than that of in C57BL/6mice(P<0.05).3.3The receptor mechanisms mediated in efferent pathway of manual acupuncture modulated gastric motility3.3.1Comparison of the effect of manual acupuncture at ST37on gastric motility among C57BL,6.M2/M3-/-and β1/β2-/-miceThe increase rate of amplitude,integral and frequency of gastric motility induced by manual acupuncture at ST37was25.06±3.42%,25.06±3.42%and 11.25±4.30%in C57BL/6mice,was22.56±2.87%,26.52±1.82%and10.59±7.73%in β1/β2-/-mice,and was12.51±2.95%,19.56±3.48%and7.41±4.90%in M2/M3-/-mice.The stimulatory effects of ST37on gastric amplitude and integral in M2/M3-/-mice were significantly lower than those of in C57BL/6mice(P<0.01, P<0.05).3.3.2Comparison of the effect of manual acupuncture at LI11on gastric motility among C57BL/6.M2/M3-/-and β1/β2-/-miceThe increase rate of amplitude,integral and frequency of gastric motility induced by manual acupuncture at ST37was29.31±3.40%,31.79±2.37%and7.08±3.48%in C57BL/6mice,was29.27±3.44%,27.22±1.35%and7.62±6.44%in β1/β2-/-mice,and was16.19±2.36%,20.08±1.92%and11.58±4.76%in M2/M3-/-mice.The stimulatory effects of ST36on gastric amplitude and integral in M2/M3-/-mice were significantly lower than those of in C57BL/6mice (P<0.01,P<0.01).3.3.3Comparison of the effect of manual acupuncture at ST25on gastric motility among C57BL/6、M2/M3-/-and β1/β2-/-miceThe decrease rate of amplitude,integral and frequency of gastric motility induced by manual acupuncture at ST25was33.83±3.50%,37.79±2.75%and15.95±5.47%in C57BL/6mice,was40.18±3.80%,44.47±2.41%and12.60±4.98%in M2/M3-/-mice,and was18.52±3.99%,12.09±4.31%and11.33±3.61%in β1/β2-/-mice.The inhibitory effects of ST25on gastric amplitude, integral and frequency in β1/β2-/-mice were significantly lower than those of in C57BL/6mice(P<0.001,p<0.001,P<0.05).3.3.4Comparison of the effect of manual acupuncture at BL25on gastric motility among C57BL/6、M2/M3-/-and β1/β2-/-miceThe decrease rate of amplitude,integral and frequency of gastric motility induced by manual acupuncture at BL25was21.70±3.02%,29.42±2.75%and5.00±4.54%in C57BL/6mice,was18.64±3.67%,27.39±2.40%and10.65±3.2%in M2/M3-/-mice,and was15.38±2.24%,18.74±1.07%and5.12±5.35%in β1/β2-/-mice.The inhibitory effects of BL25on gastric amplitude and integral in β1/β2-/-mice were significantly lower than those of in C57BL/6mice (P<0.05, P<0.01).3.3.5Comparison of the effect of manual acupuncture at ST37+LI11on gastric motility among C57BL/6M2/M3-/-and β1/β2-/-miceThe increase rate of amplitude, integral and frequency of gastric motility induced by manual acupuncture at ST37+LI11was33.53±2.65%,32.47±2.27%and11.25±5.73%in C57BL/6mice, was36.72±2.19%,34.76±2.28%and15.71±7.25%in β1/β2-/-mice, and was19.66±2.77%,26.68±3.51%and7.71±4.71%in M2/M3-/-mice. The stimulatory effects of ST37+LI11on gastric amplitude and integral in M2/M3-/-mice were significantly lower than those of in C57BL/6mice (P<0.01, P<0.05).3.3.6Comparison of the effect of manual acupuncture at ST37+ST25on gastric motility among C57BL/6、M2M3-/-and β1/β2-/-miceThe decrease rate of amplitude, integral and frequency of gastric motility induced by manual acupuncture at ST37+ST25was24.73±2.24%,29.12±2.02%and9.17±3.49%in C57BL/6mice, was30.02±3.35%,34.87±2.51%and9.25±5.83%in M2/M3-/-mice, and was16.22±0.92%,20.58±2.02%and8.09±3.32%in β1/β2-/-mice. The inhibitory effects of ST37+ST25on gastric amplitude and integral in β1/β2-/-mice were significantly lower than those of in C57BL/6mice (P<0.01,P<0.01).4ConclusionThere existed "intensity-response" relationship between electroacupuncture (EA) stimulation and its effects on gastric motility. The effects of EA on gastric motility was mediated by activation of different afferent fibers. When A8afferent fiber was activated, EA can produce a significant regulation on gastric motility. When C fiber was activated, EA elicited maximal regulatory effects on gastric motiltiy. These results showed that A8and C fibers were contributed to the peripheral neural pathway of EA modulated gastric motility. The modulatory effects of EA on gastric motility was abolished by deleting of TRPV1ion channel, indicating that TRPV1receptor might be critical for the detection of EA signal. The modulatory effects of local moxibustion-like stimuli (LMS) on gastric motility were also closely related to the thermal intensity.≥43℃LMS at ST36and CV12could facilitate and inhibit gastric motility via activating A8and C fibers. Likely, the deletion of TRPV1receptor significantly reduced the modulatory effects of LMS on gastric motility, indicating that TRFV1receptor was also contribute to the response.Muscarinic receptors and β adrenergic receptors are highly expressed in gastrointestinal tract and play important role in modulating gastrointestinal function. The stimulatory effects of manual acupuncture at ST37, LI11and ST37+LI11on gastric motility were significantly reduced in M2/M3-/-mice, while the inhibitory effects of manual acupuncture at ST25, BL25and ST37+ST25were markedly decreased in β1/β2-/-mice. These results demonstrated that M2/M3and β1/β2were more likely be involved in the efferent pathway of acupuncture modulated gastric motility.