| Cholinergic anti-inflammatory pathway (CAP) has been discovered in recent years. Based on efferent vagus nerve, this pathway aims at suppressing inflammatory response. The pathway can be activated by stimulating vagus nerve directly. Then it releases acetylcholine, and suppresses inflammatory cytokines, and control inflammation.Auricular branch of vagus nerve innervates auricular concha(AC), and its afferent branch projects to nucleus of solitary tract (NTS) and dorsal motor nucleus of vagus (DMV). Neurons in NTS and DMV can be activated by acupuncturing auricular concha. We assume that auricular acupuncture (AA) may activate the cholinergic anti-inflammation pathway via stimulating auricular branch of vagus nerve. This study will focus on systemic inflammatory factors, NF-?B pathway, cytokine expression in organ, and organic pathology in endotoxemia. It aims at reveal mechanism on auricular acupuncture induced CAP.1. Auricular acupuncture regulates serum cytokines in endotoxeamia rats.Male SD rats.280-300g, were housed in standard conditions with access to regular chow and water. The permission number is SCXK-(army) 2007-004.Endotoxemia in animals was induced by administering lipopolysaccharide (LPS, Escherichia coli 0111:B4:Sigma.5mg/kg. i.v.).1.1 Experiment 1 (2h)Group A:Control:animals were treated with sterile saline, i.v.Group B:Model:animals were treated with LPS, i.v.Group C:Simple AA:animals were treated with sterile saline, i.v.1.5h later, proceed AA.Group D:AA+LPS:animals were treated with LPS. i.v.1.5h later, proceed AA.Group E:VNS+LPS:animals were treated with LPS, i.v.1.5h later, treated with vagus nerve stimulation.Group F:ST36+LPS:animals were treated with LPS, i.v.1.5h later, treated with electroacupuncture at "ST36". 2h after injection, all animals were taken blood. Test serum TNF-?, IL-6, IL-1?and IL-10 with ELISA.Results:Compared with the control group, serum TNF-?,IL-6, IL-1?and IL-10 contents in the model group were increased significantly (P<0.01). In comparison with the model group, serum TNF-?and IL-10 contents in the AA+LPS, VNS+LPS and ZST36+LPS groups, and IL-6,IL-1?in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group, serum IL-6, IL-1?and IL-10 contents in the AA+LPS, and TNF-?, IL-6, IL-1?and IL-10 levels in the ZST36+LPS group were significantly different (P<0.05, P<0.01).1.2 Experiment 2 (4h)Group G:Control:animals were treated with sterile saline, i.v.Group H:Model:animals were treated with LPS, i.v.Group I:AA+LPS:animals were treated with LPS, i.v.1.5h later, proceed AA.Group J:ST36+LPS:animals were treated with LPS. i.v.1.5h later, treated with electroacupuncture at "ST36" 4h after injection, all animals were taken blood. Test serum TNF-?, IL-6, IL-1?and IL-10 with ELISA kit.Results:Compared with the control group, serum TNF-?, IL-6, IL-1?and IL-10 contents in the model group were increased significantly (P<0.01). In comparison with the model group, serum TNF-?, IL-6, IL-1?contents in the AA+LPS group, and IL-1?in the ST36+LPS were down-regulated considerably (P<0.05).1.3 Experiment 3 (6h)Group K:Control:animals were treated with sterile saline, i.v.Group L:Model:animals were treated with LPS. i.v.Group M:AA+LPS:animals were treated with LPS. i.v.1.5h later, proceed AA.Group N:ST36+LPS:animals were treated with LPS. i.v.1.5h later, treated with electroacupuncture at "ST36". 6h after injection, all animals were taken blood. Test serum TNF-?, IL-6, IL-1?and IL-10 with ELISA kit. Results:Compared with the control group, serum TNF-?, IL-6. IL-1?contents in the model group were increased significantly (P<0.05, P<0.01). In comparison with the model group, serum IL-6,IL-1?contents in the AA+LPS group,and TNF-?,IL-1?in the ST36+LPS group were down-regulated considerably (P<0.05):serum IL-10 content in AA+LPS group was increased significantly (P<0.05).1.4 Experiment 4 (vagus nerve block)Group A:Control:animals were treated with sterile saline, i.v.Group B:Model:animals were treated with LPS, i.v.Group O:vagotomy+AA+LPS:animals were treated with LPS, i.v. at the same time apply vagotomy 1.5h later, proceed AA.Group P:vagotomy +ST36+LPS:animals were treated with LPS. i.v. at the same time apply vagotomy.1.5h later, treated with electroacupuncture at "ST36"GroupQ:Hexamethonium+AA+LPS:animals were injected with Hexamethonium, i.v. then treated with LPS.1.5h later, proceed AA.Group R:Hexamethonium-ST36+LPS:animals were injected with Hexamethonium. i.v.. then treated with LPS.1.5h later, stimulate ST36.Group S:a-BGT+AA+LPS:animals were injected with a-BGT. i.v.. then treated with LPS,1.5h later, proceed AA.Group T:a-BGT-ST36+LPS:animals were injected with a-BGT. i.v.. then treated with LPS,1.5h later, stimulate ST36. 2h after LPS injection, all animals were taken blood. Test serum TNF-a with ELISA kit.Results:Compared with the control group, serum TNF-a content in the model group were increased significantly (P<0.01). In comparison with the model group, there was non-significant difference between other groups (P<0.05).2. Auricular acupuncture influences NF-?B pathway.2.1 NF-?B p65 protein expression in lung tissue2.1.1 Experiment 1 (2h)Take lung tissue from group A, B, C, D, E and F. Detect NF-?B p65 protein expression from each group. Western Blot procedure. Results:Compared with the normal control group, pulmonary NF-?B p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-?B p65 expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group pulmonary NF-?B p65 expression levels in the ZST36+LPS group were significantly different (P<0.01). In comparison with the AA+LPS group, pulmonary NF-?B p65 expression level in the ZST36+LPS group was significantly different (P<0.05).2.1.2 Experiment 2 (4h)Take lung tissue from group G, H, I, and J. Detect NF-?B p65 protein expression from each group. Use Western Blot procedure.Results:Compared with the normal control group, pulmonary NF-?B p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-?B p65 expression levels in the AA+LPS groups were down-regulated considerably (P<0.05).2.1.3 Experiment 3 (6h)Take lung tissue from group K, L. M and N. Detect NF-?B p65 protein expression from each group. Use Western Blot procedure. Results:Compared with the normal control group, pulmonary NF-?B p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-?B p65 expression levels in the AA+LPS groups were down-regulated considerably (P<0.01).2.2 Pulmonary NF-?B p65 activity2.2.1 Experiment 1 (2h)Take lung tissue from group A. B, D, E and F. Detect pulmonary NF-?B p65 expression from each group. Use Immunohistochemistry technology.Results:Compared with the normal control group, pulmonary NF-KBp65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-?B p65 expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group pulmonary NF-?B p65 expression levels in the ZST36+LPS group were significantly increased (P<0.05).2.2.2 Experiment 2 (4h)Take lung tissue from group G. H, I and J. Detect pulmonary NF-?B p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control group, pulmonary NF-?B p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-?B p65 expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01, P<0.05).2.2.3 Experiment 3 (6h)Take lung tissue from group K. L. M and N. Detect pulmonary NF-?B p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control group, pulmonary NF-?B p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-?B p65 expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.05).2.2.4 Experiment 4 (vagus nerve block)Take lung tissue from group A, B, O and P. Detect pulmonary NF-?B p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control groupm pulmonary NF-?B p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonaiy NF-?B p65 expression levels in the other two groups was non-significant different (P<0.05).3. Auricular acupuncture protects organs in endotoxemia rats.3.1 TNF-a mRNA, IL-10 mRNA expression in liver3.1.1 Experiment 1 (2h)Take liver sample from group A. B. D. E and F. Detect TNF-a mRNA expression from each group with real time PCR. Results:Compared with the normal control group, TNF-a mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-?mRNA expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.05, P<0.01).3.1.2 Experiment 2 (4h)Take liver sample from group G, H, I and J. Detect TNF-?mRNA and IL-10 expression from each group with real time PCR. Results:Compared with the normal control group, TNF-?mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-?mRNA expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01).Compared with the normal control group. IL-10 mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, IL-10 mRNA expression levels in the AA+LPS and ST36+LPS groups were up-regulated considerably (P<0.01, P<0.05).3.1.3 Experiment 3 (6h)Take liver sample from group K, L, M and N. Detect TNF-?mRNA and IL-10 mRNA expression from each group with real time PCR. Results:Compared with the normal control group. TNF-a mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-?mRNA expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01, P<0.05).Compared with the normal control group, IL-10 mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, IL-10 mRNA expression levels in the AA+LPS and ST36+LPS groups were up-regulated considerably (P<0.05).3.2 lung histopathologyTake lung samples from group A,B,D,E and F.Use HE staining technology.Result demonstrated that samples from AA+LPS and ST36+LPS groups were much improved in pathological states of lung. 4. ConclusionThis study research the mechanism on inhibitory effect of auricular acupuncture in cytokine regulation. We focus on systemic inflammatory factors, NF-?B pathway, cytokine expression in organ, and organic pathology in endotoxemia rats. The results demonstrate that auricular acupuncture plays an important role in inflammatory response, and inhibits pro-inflammatory cytokines. Meanwhile, auricular acupuncture suppresses NF-?B p65 expression as well. In absence of vagus nerve, the anti-inflammatory effect mentioned above was destroyed. It suggests that auricular acupuncture may activate cholinergic anti-inflammatory pathway to control inflammatory development. In addition, auricular acupuncture regulates TNF-a mRNA and IL-10 mRNA expression in liver, and this might be the key point to suppress inflammatory development. |
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