Electroacupuncture Promotes Functional Recovery after Facial Nerve Injury in Rats by Regulating Autophagy via GDNF and PI3K/mTOR Signaling Pathway.

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS: EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.

[Effect of electroacupuncture on intestinal epithelial mucosal barrier function in rats with diarrhea-predominant irritable bowel syndrome].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on intestinal epithelial mucosal barrier function in diarrhea-predominant irritable bowel syndrome(IBS-D) rats, so as to explore its mechanisms underlying improvement of IBS-D. METHODS: Forty SD rats (half male and half female) were randomly divided into control, model, EA and medication (Pinaverium Bromide, PB) groups, with 10 rats in each group. The IBS-D model was established by chronic unpredictable mild stress combined with gavage of Senna-leaf solution. EA (2 Hz/15 Hz，0.1-1 mA) was applied to unilateral "Zusanli"(ST36),"Tianshu" (ST25), "Sanyinjiao"(SP6) and "Taichong"(LR3) alternatively for 15 min, once daily for 14 days. Rats of the medication group was treated by gavage of PB (10 mL·kg-1·d-1) for 14 days. The visceral sensitivity (pain) was assessed by using the pressure threshold which the inserted rectal balloon catheter air-inflation (connected to a blood pressure gauge) induced stronger abdominal muscular contraction to force the rat's abdomen to lift the experimental stand surface. The diarrhea index was used to evaluate loose stool grade. The expression of Claudin-1 and Occludin (intestinal epithelial tight junction proteins) of colon tissue was detected by immunohistochemistry. The activity of plasma diamine oxidase (DAO) was assayed by using spectrophotometry. RESULTS: Compared with the control group, the diarrhea index and plasma DAO activity in the model group were significantly increased (P<0.01), while the visceral pain threshold, expression of Claudin-1 and Occludin in the model group were significantly decreased (P<0.01). After the treatment, the diarrhea index and plasma DAO activity were significantly lower in both EA and medication groups than that in the model group (P<0.01), and the visceral pain threshold and expression levels of Claudin-1 and Occludin were obviously increased (P<0.05, P<0.01). No significant differences were found between the EA and medication groups in all the above-mentioned indexes (P>0.05). CONCLUSION: Electroacupuncture can significantly improve abdominal pain and diarrhea in IBS-D model rats, which may be closely associated with its effects in up-regulating the expression of intestinal epithelial tight junction proteins Claudin-1 and Occludin to restore the function of intestinal epithelial mucosal barrier.

[Effect of Electroacupuncture Intervention on Expression of Synaptic Plasticity-related Molecules in Amygdala in Chronic Pain-negative Affection Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of synaptic plasticity-related glutamate N-methyl-D-aspartate (NMDA) receptor subunit NR 1, gamma-aminobutyric acid (GABA) receptor subunits (Aß 2, B 1), etc. in the amygdala in chronic neuropathic pain negative affection (CNPPNA) rats, so as to reveal its mechanism underlying pain relief. METHODS: Male Wistar rats were randomized into normal control, CNPPNA model, EA, and anesthesia+EA (AEA)groups (n=14 in each group, 8 for quantitative RT-PCR and 6 for immunofluorescence staining). The CNPPNA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the hindpaw plantar skin in the pain-paired compartment. EA was applied to bilateral "Zusanli"(ST 36)and "Yanglingquan"(GB 34)for 30 min, once daily for 7 days.Thermal pain threshold (paw withdrawal latency, PWL)of the bilateral paws was measured by using a Tail-Flick Unit. The conditioned place aversion (CPA) was determined by using a CPA-paired compartment. The expression levels of GABAAß 2, GABAB 1, NMDA receptor subunit NR 1, postsynaptic density-95 protein(PSD-95), Piccolo genes in the right amygdala area were determined using quantitative RT-PCR, and the immunoactivity of metabotropic glutamate receptor subunit 1 (mGluR 1) and GABAB 2 in the basolateral amygdala (BLA) nucleus was detected using immunofluorescence staining. RESULTS: After modeling, PWL difference (PWLD) values of the model group were significantly increased (P<0.001),and the time spent in the CPA-paired compartment was considerably decreased compared with the control group (P<0.001).After EA intervention for 3 and 7 days, the PWLD levels of both EA and AEA groups were apparently decreased(P<0.05),and the time spent in the CPA-paired compartment was apparently increased in the EA and AEA groups(P<0.05),suggesting a pain relief and an improvement of the negative affection after EA intervention. Additionally, following EA, the apparently-decreased expression levels of GABAAß 2,GABAB 1,PSD-95,Piccolo genes and the reduced numbers of GABAB 2 positive cells and NMDA-NR 1 mRNA as well as mGluR 1 positive fiber numbers were remarkably increased in the EA group (P<0.05, P<0.001).The expression levels of Piccolo gene, GABAB 2 and mGluR 1 positive cells/fiber numbers were apparently lower in the AEA group than in the EA group (P<0.001). No significant differences were found between the EA and AEA groups in the PWLD, time spent in the CPA-paired compartment, and the expression levels of NMDA-NR 1, GABAAß 2, GABAB 1 and PSD-95 genes (P>0.05). CONCLUSIONS: Repeated EA stimulation of ST 36-GB 34 has a role in relieving both sensory and affection dimensions of chronic pain in CNPPNA rats, which Feb be respectively related to its effects in up-regulating the expression of GABAAß 2, GABAB 1, NMDA-NR 1, PSD-95 and Piccolo genes, and in promoting the expression of mGluR 1 and GABAB 2 proteins and Piccolo gene in the amygdala.

Electroacupuncture Reduces the Effects of Acute Noxious Stimulation on the Electrical Activity of Pain-Related Neurons in the Hippocampus of Control and Neuropathic Pain Rats.

To study the effects of acupuncture analgesia on the hippocampus, we observed the effects of electroacupuncture (EA) and mitogen-activated protein kinase (MEK) inhibitor on pain-excited neurons (PENs) and pain-inhibited neurons (PINs) in the hippocampal area CA1 of sham or chronic constrictive injury (CCI) rats. The animals were randomly divided into a control, a CCI, and a U0126 (MEK1/2 inhibitor) group. In all experiments, we briefly (10-second duration) stimulated the sciatic nerve electrically and recorded the firing rates of PENs and PINs. The results showed that in both sham and CCI rats brief sciatic nerve stimulation significantly increased the electrical activity of PENs and markedly decreased the electrical activity of PINs. These effects were significantly greater in CCI rats compared to sham rats. EA treatment reduced the effects of the noxious stimulus on PENs and PINs in both sham and CCI rats. The effects of EA treatment could be inhibited by U0126 in sham-operated rats. The results suggest that EA reduces effects of acute sciatic nerve stimulation on PENs and PINs in the CA1 region of the hippocampus of both sham and CCI rats and that the ERK (extracellular regulated kinase) signaling pathway is involved in the modulation of EA analgesia.

[Effect of Electroacupuncture Intervention on Expression of Pain Sensory and Affective Proce- ssing-related µ-opioid Receptor, etc. in the Amygdala in Chronic Neuropathy Pain Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of pain sensory and affective processing-related µ-opioid receptor (MOR), glutamatergic AMPA receptor subunit GIuA 1, extracellular signal-regulated kinase (ERK 1/2), cAMP response element binding protein(CREB) in the amygdala in chronic constrictive injury (CC) + negative affection(NA) rats, so as to reveal its mechanism underlying pain relief. METHODS: A total of 32 male Wistar rats were randomized into normal control, model, EA, and anesthesia+ EA (AEA) groups (n = 8 in each group). The neuropathic pain NA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the paw-bottom in the pain-paired compartment. EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 7 days. Thermal pain threshold (paw withdrawl latency, PWL) of the bilateral paws was measured by using a Tail-Flick Unit. The expression levels of MOR and p-CREB in the central amygdala (CeA) and those of MOR, GluA 1, p-ERK 1/2 and p-CREB in the right amygdala area were determined using immunofluorescence and Western blot, respectively. RESULTS: in comparison with the normal group, PWL difference (PWLD) values of the model group were significantly increased (P < 0.001), and the time spent in the CPA-paired. compartment was considerably decreased (P < 0.001). After EA, the PWLD levels of both EA and AEA groups were apparently decreased (P < 0.05), showing a pain relief; and the time spent in the CPA-paired compartment was apparently increased in the EA group (P < 0.05) , rather than in the AEA group (P > 0.05). Additionally , compared to the normal group, the expression level of MOR protein in the amygdala was remarkably increased (P < 0.05) and those of GIuA 1, p-ERK 2 and p-CREB proteins were apparently decreased (P < 0.05). After EA intervention for 7 days, the expression levels of these four proteins in the EA group, and those of MOR, p-ERK 2 and p-CREB in the AEA group were significantly up-regulated (P < 0.001, P < 0.01, P < 0.05). The expression level of GIuA 1 was significantly higher in the EA group than in the AEA group (P < 0.05). CONCLUSION: Repeated EA stimulation of ST 36-GB 34 has a definite effect in relieving both sensation and affection dimensions of pain in NA rats, which may be related to its effect in up-regulating the expression of GIuA 1 in the amygdala, but the effects of MOR, p-ERK 2 and p-CREB need being researched further.

Involvement of hippocampal acetylcholinergic receptors in electroacupuncture analgesia in neuropathic pain rats.

BACKGROUND: Cumulating evidence has shown a close correlation between electroacupuncture stimulation (EAS) frequency-specific analgesic effect and central opioid peptides. However, the actions of hippocampal acetylcholinergic receptors have not been determined. This study aims to observe the effect of different frequencies of EAS on the expression of hippocampal muscarinic and nicotinic acetylcholinergic receptors (mAChRs, nAChRs) in neuropathic pain rats for revealing their relationship. METHODS: Forty male Wistar rats were randomly and equally divided into sham, CCI model, 2, 2/15 and 100 HzEA groups. The neuropathic pain model was established by ligature of the left sciatic nerve to induce chronic constriction injury (CCI). EAS was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) for 30 min, once daily for 14 days except weekends. The mechanical pain thresholds (withdrawal latencies, PWLs) of bilateral hindpaws were measured. The expression levels of hippocampal M1 and M2 mAChR, and α4 and ß2 nAChR genes and proteins were detected by quantitative RT-PCR and Western blot, separately. The involvement of mAChR and nAChR in the analgesic effect of EAS was confirmed by intra-hippocampal microinjection of M1mAChR antagonist (Pirenzepine) and α4ß2 nAChR antagonist (dihydro-beta-erythroidine) respectively. RESULTS: Following EAS, the CCI-induced increase of difference values of bilateral PWLs on day 6 and 14 was significantly reduced (P < 0.05), with 2/15 Hz being greater than 100 Hz EAS on day 14 (P < 0.05). After 2 weeks' EAS, the decreased expression levels of M1 mAChR mRNA of both 2 and 2/15 Hz groups and M1 mAChR protein of the three EAS groups, α4 AChR mRNA of the 2/15 Hz group and ß2 nAChR protein of the three EAS groups were considerably increased (P < 0.05), suggesting an involvement of M1 mAChR and ß2 nAChR proteins in EAS-induced pain relief. No significant changes were found in the expression of M2 mAChR mRNA and protein, α4 nAChR protein and ß2 nAChR mRNA after CCI and EAS (P > 0.05). The analgesic effect of EAS was abolished by intra-hippocampal microinjection of M1mAChR and α4ß2 nAChR antagonists respectively. CONCLUSIONS: EAS of ST36-GB34 produces a cumulative analgesic effect in neuropathic pain rats, which is frequency-dependent and probably mediated by hippocampal M1 mAChR and ß2 nAChR proteins.

[Effect of electroacupuncture intervention on expression of pain sensory and affection processing related corticotropin-releasing factor receptor mRNA, etc. in the amygdala in neuropathic pain and negative affection rats].

OBJECTIVE: To observe the effect of electroacupuncture .(EA) stimulation of "Zusanli" (ST 36)-"Yang- lingquan" (GB 34) on expression of pain sensory and affection processing-related corticotropin-releasing factor (CRF) receptor, glutamatergic NMDA receptor and GABA receptor subtype genes in the amygdala in chronic constrictive injury (CCI) of the sciatic nerve rats, so as to reveal its mechanism underlying pain relief. METHODS: Experiments were separately performed in 36 male Wistar rats which were randomized into normal control, CCI model and EA + CCI; normal control, CCI + negative affection (NA) model and CCl+ NA+ EA groups (n =6 in each group). Neuropathic pain model was established by ligature of the left sciatic nerve, and NA model established by ligation of the left sciatic nerve and repeated skin stimulation (acupuncture needle pricking + direct current stimulation) of the paw-bottom, once daily for 3 days. EA (2 Hz/15 Hz, 1 mA) was applied to bilateral ST 36-GB 34 for 30 min, once daily for 7 days. Thermal pain threshold (paw withdrawal latency, PWL) of the bilateral paws was measured by using a Tail-Flick Unit 37360. Expression levels of CRF-1 R, CRF-2 R, NR 2 A,NR 2 B,GABAaR and GABAcR genes in the amygdala were determined using quantitative RT-PCR. RESULTS: In comparison with the normal control groups, PWL difference (PWLD) values of the bilateral paws of CCIl model and CCI+NA model groups were significantly increased (P<0. 05). In comparison with the model group, following 7 days' EA stimulation, PWLD were considerably decreased (P<0. 05), showing a pain relief. RT-PCR results indicated that compared to the normal control group, the expression levels of CRF-1 R, CRF-2 R, NR 2 A and NR 2 B genes were apparently increased in the CCI model group (P<0. 01, P<0. 001), and those of CRF-1 R, CRF-2 R, NR 2 A, NR 2 B, GABAaR and GABAcR genes were remarkably down-regulated in the CCI + NA model group (P<0. 05, P<0. 01, P<0. 001). After EA intervention for 7 days, CRF-2 R, NR 2 A and NR 2 B were significantly down-regulated in the CCI + EA group, and CRF-1 R, CRF-2 R, NR 2 B,GABAaR and GABAcR genes were obviously up-regulated in the CCI + NA + EA group (P<0. 01, P<0. 001). CONCLUSION: Repeated EA stimulation of ST 36-GB 34 has a definite analgesic effect in neuropathic pain and negative affection rats, which may be respectively related to its effects in down-regulating expression of CRF-2 R, NR 2 A and NR 2 B genes, and up-regulating expression of CRF-1 R, CRF-2 R, NR 2 B,GABAaR and GABAcR genes in the amyg- dala.