Effects and mechanisms of electroacupuncture at PC6 on frequency of transient lower esophageal sphincter relaxation in cats

Abstract

Aim: To investigate the effects of electroacupuncture (EA) at neiguan (PC6) on gastric distention-induced transient lower esophageal sphincter relaxations (TLESRs) and discuss the mechanisms of this treatment.

Methods: Protocol I: Twelve healthy cats underwent gastric distention for 60 min on the first day. Electrical acupoint stimulation was applied at the neiguan or a sham point on the hip in randomized order before gastric distention, on the third day and fifth day. Those cats that underwent EA at neiguan on the fifth day were named "Neiguan Group" and the cats that underwent EA at a sham acupoint on the fifth day were named "Sham Group" (control group). During the experiment the frequency of TLESRs and lower esophageal sphincter (LES) pressure were observed by a perfused sleeve assembly. Plasma levels of gastrin (GAS) and motilin (MTL) were determined by radioimmunoassay. Nitrite/nitrate concentration in plasma and tissues were measured by Griess reagent. The nuclei in the brain stem were observed by immunohistochemistry method of c-Fos and NADPH-d dyeing. Protocol II: Thirty six healthy cats were divided into 6 groups randomly. We gave saline (2 mL iv. control group), phaclofen (5 mg/kg iv. GABA-B antagonist), cholecystokinin octapeptide (CCK-8) (1 microg/kg per hour iv.), L-Arginine (200 mg/kg iv.), naloxone (2.5 micromol/kg iv.) and tacrine (5.6 mg/kg ip. cholinesterase inhibitor) respectively before EA at Neiguan and gastric distention. And the frequencies of TLESRs in experimental groups were compared with the control group.

Results: Protocol I: Not only the frequency of gastric distention-induced TLESR in 60 min but also the rate of common cavity during TLESRs were significantly decreased by EA at neiguan compared to that of sham acupoint stimulation. C-Fos immunoreactivity and NOS reactivity in the solitarius (NTS) and dorsal motor nucleus of the vagus (DMV) were significantly decreased by EA at neiguan compared to that of the sham group. However, the positive nuclei of C-Fos and NOS in reticular formation of the medulla (RFM) were increased by EA at neiguan. Protocol II: The inhibited effect of EA at neiguan on TLESR's frequency was completely restored by pretreatment with CCK (23.5/h vs 4.5/h, P < 0.05), L-arginine (17.5/h vs 4.5/h, P < 0.05) and naloxone(12/h vs 4.5/h, P < 0.05). On the contrary, phaclofen (6/h vs 4.5/h, P > 0.05) and tacrine (9.5/h vs 4.5/h, P > 0.05) did not influence it.

Conclusion: Electric acupoint stimulation at Neiguan significantly inhibits the frequency of TLESR and the rate of common cavity during TLESR in cats. This effect appears to act on the brain stem, and may be mediated through nitric oxide (NO), CCK-A receptor and mu-opioid receptors. But the GABAB receptor and acetylcholine may not be involved in it.

Figure 1

The flow chart of protocol I.

Figure 2

The process of protocol II.

Figure 3

EA at neiguan decreased the rate of TLESRs induced by gastric distention, compared with EA at sham acupoint (^bP < 0.01) and baseline distention (^aP < 0.05). Each number represents one cat.

Figure 4

EA at Neiguan decreased the rate of common cavity during TLESRs compared with EA at sham acupoint (P < 0.05).

Figure 5

Electric stimulation at Neiguan didn't influence the LES pressure and intragastric pressure.

Figure 6

Photomicrographs showing C-Fos and NOS positive neurons in nucleus tractus solitarius/dorsal motor nucleus of vagus (NTS/DMV). A: C-Fos immunohistochemistry of neiguan group; B: C-Fos immunohistochemistry of sham acupoint group; C: NADPH-d histochemistry of neiguan group; D: NADPH-d histochemistry of sham acupoint group (× 10).

Figure 7

Photomicrographs showing C-Fos and NOS positive neurons in reticular formation of medulla (RFM). A: C-Fos immunohistochemistry of neiguan group; B: C-Fos immunohistochemistry of sham acupoint group; C: NADPH-d histochemistry of neiguan group; D: NADPH-d histochemistry of sham acupoint group (× 10).

Figure 8

CCK, L-arginine and naloxone significantly increase the frequency of TLESRs compared with control group (^aP < 0.05). But phaclofen and tacrine did not change the frequency of TLESRs compared with control group (P > 0.05). It was indicated that the inhibited effects of EA at neiguan were completely restored by pretreatment with CCK-A, L-arginine and naloxone. But it was not influenced by phaclofen and tacrine.