Transcutaneous auricular vagus nerve stimulation protects endotoxemic rat from lipopolysaccharide-induced inflammation

Abstract

Background. Transcutaneous auricular vagus nerve stimulation (ta-VNS) could evoke parasympathetic activities via activating the brainstem autonomic nuclei, similar to the effects that are produced after vagus nerve stimulation (VNS). VNS modulates immune function through activating the cholinergic anti-inflammatory pathway. Methods. VNS, ta-VNS, or transcutaneous electrical acupoint stimulation (TEAS) on ST36 was performed to modulate the inflammatory response. The concentration of serum proinflammatory cytokines and tissue NF-kappa B p65 (NF-κB p65) were detected in endotoxaemia affected anesthetized rats. Results. Similar to the effect of VNS, ta-VNS suppressed the serum proinflammatory cytokines levels, such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) as well as NF-kappa B p65 expressions of lung tissues. ST36 stimulation also decreases LPS-induced high TNF-α level and NF-κB signal, but it did not restrain proinflammatory cytokine IL-1β and IL-6. Neither ta-VNS nor ST36 stimulation could suppress LPS-induced TNF-α and NF-κB after vagotomy or with α7nAChR antagonist injection. Conclusions. The present paper demonstrated that ta-VNS could be utilized to suppress LPS-induced inflammatory responses via α7nAChR-mediated cholinergic anti-inflammatory pathway.

Figures

Figure 1

The time flow chart indicates the precise time for various operations in the present study, from the time point of anesthetic injection to the time point for sampling. (a) Twenty minutes after anesthesia, rats were injected intravenously with LPS or NS. One and a half hour after modeling, treatment (ta-VNS, VNS, or TEAS on ST36) was performed for twenty minutes. Two hours after LPS injection, rats were killed, and samples were collected. (b) Ten minutes after anesthesia, administration of α-BGT or vagotomy was performed. The rest of the operations were the same with the time flow in (a).

Figure 2

Vagus nerve stimulation (VNS) or transcutaneous auricular vagus nerve stimulation (ta-VNS) attenuates the LPS-induced serum cytokine (TNF-α, IL-1β, and IL-6) response. TEAS on ST36 inhibited TNF-α level significantly. Serum TNF-α (a), IL-1β (b), and IL-6 (c) contents were measured by ELISA. The columns represent mean ± SEM for 12 animals in each group. ∆∆P < 0.01 versus the normal saline (NS) group; *P < 0.05 versus LPS group (LPS); **P < 0.01 versus LPS group (LPS); #P < 0.05 versus LPS+VNS group; ##P < 0.01 versus LPS+VNS group.

Figure 3

ta-VNS or TEAS on ST36 with α-bungarotoxin (α-BGT) administration fails to inhibit the LPS-induced serum TNF-α response. Serum TNF-α concentrations were measured by ELISA. Data are expressed as mean ± SEM (n = 12 per group). ∆∆P < 0.01 versus the normal saline (NS) group.

Figure 4

ta-VNS or ST36 stimulation with bilateral cervical vagotomy (VGX) fails to inhibit the LPS-induced serum TNF-α response. TNF-α amounts were measured by ELISA. Data are expressed as mean ± SEM (n = 12 per group). ∆∆P < 0.01 versus the normal saline (NS) group.

Figure 5

VNS or ta-VNS suppresses LPS-induced NF-κB expression; ST36 stimulation did not affect NF-κB in endotoxemia animals significantly. ta-VNS did not significantly affect pulmonary NF-κB expression with normal saline administration. NF-κB expressions were measured by western blot technique. Data are shown by mean ± SEM (n = 12 per group). ∆∆P < 0.01 versus normal saline (NS) group; **P < 0.01 versus LPS group (LPS); ##P < 0.01 versus LPS+VNS group; ▲P < 0.05 versus LPS+ ta-VNS.

Figure 6

Immunohistochemical staining with anti-NF-κB antibodies reveals significant decrease in LPS-induced NF-κB immunoreactivity evoked by interventions as of VNS, ta-VNS, and TEAS on ST36. Data are expressed as mean ± SEM (n = 12 per group). ∆∆P < 0.01 versus the normal saline (NS) group; **P < 0.01 versus LPS group (LPS); #P < 0.05 versus LPS+VNS group. Original magnification: ×400.

Figure 7

ta-VNS or ST36 stimulation with bilateral cervical vagotomy (VGX) fails to inhibit the LPS-induced overexpression of NF-κB. NF-κB distribution was measured by immunohistochemical staining. Data are shown as mean ± SEM (n = 12 per group). ∆∆P < 0.01 versus the normal saline (NS) group. Original magnification: ×400.

Figure 8

The anti-inflammatory mechanisms of the three interventions used in the present study might be as follows: (1) VNS directly activates the cholinergic anti-inflammatory pathway via stimulating efferent vagus nerve. (2) ta-VNS evoked the activity of the auricular branch of vagus nerve (ABVN). The activated signals ascending within afferent vagus nerve are transmitted to the nucleus tractus solitarius. The integrated output is carried by the efferent vagus nerve to inhibit inflammatory responses. (3) TEAS on ST36 activates the somatic fiber endings of the skin around ST36 point, sending signals to the spinal cord via somatic sensory nerve fibers. The nerve impulses were relayed and integrated by NTS by the secondary order neurons in the spinal cord, and the cholinergic anti-inflammatory pathways are activated by the increased efferent vagal output.