Acupuncture stimulation attenuates TNF-α production via vagal modulation in the concanavalin A model of hepatitis

Abstract

Background:

A growing body of evidence shows that neuronal activity is involved in modulating the efficacy of acupuncture therapy. However, it has been seldom investigated whether neuronal activity following acupuncture stimulation is effective at regulating hepatic inflammation.

Objective:

Using the concanavalin A (ConA) model of hepatitis, we investigated the regulation of inflammatory cytokine tumor necrosis factor (TNF)-α in the liver tissue and the blood after acupuncture stimulation at ST36.

Methods:

Mice were subjected to ConA injection, acupuncture stimulation at ST36 by manual acupuncture (MA) or electroacupuncture (EA) procedures, and vagotomy (VNX). Liver tissue and blood were collected for TNF-α analysis. TNF-α mRNA was analyzed by real-time polymerase chain reaction (PCR), and TNF-α, CD11b, CD68, and Erk1/2 proteins were analyzed by Western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay.

Results:

TNF-α mRNA and protein were induced in CD11b-positive hepatic cells and the plasma at 6–24 h after ConA injection. The application of MA or EA was very effective at attenuating the production of TNF-α. Anti-inflammatory effects of acupuncture were greatly suppressed by VNX in ConA-injected animals, suggesting the requirement of vagus nerve activity in acupuncture-mediated anti-inflammatory responses. Electrical stimulation of the sciatic nerve (SNS) resulted in an anti-inflammatory effect similar to acupuncture stimulation. In parallel with TNF-α, production of phospho-Erk1/2, which was induced in the liver tissue, was downregulated by MA and EA in liver cells.

Conclusion:

The regulatory effects of acupuncture stimulation on inflammatory responses in the liver may be modulated through the activation of the vagus nerve pathway.

Keywords

acupuncture concanavalin A liver tumor necrosis factor alpha vagus nerve

Introduction

Acupuncture therapy is widely used for the treatment of diverse body symptoms and disorders. While the scope of the therapeutic intervention of acupuncture is broad and may impact all body organs and systems, the theory explaining its effects is descriptive and thus is not quantitative. Recent experimental studies have begun to explore the principles of acupuncture therapy and have already provided insights to partially explain its biological basis. Particularly notable is the finding that acupuncture stimulation induces the activation of parasympathetic vagal pathways and leads to the regulation of inflammatory responses in target organs.^1
–3 It has been well documented that electrical stimulation of the vagus nerve acts on the α7 nicotinic acetylcholine receptor in macrophages and activates signal transducer and activator of transcription 3 (STAT3) signaling pathway leading to the inhibition of tumor necrosis factor (TNF)-α production.^4,5 Ever since the initial findings on the principle of cholinergic anti-inflammatory vagus nerve activity in splenic macrophages, similar anti-inflammatory regulation by vagus nerve stimulation (VNS) has been demonstrated in several experimental studies showing the efficacy of VNS in diseases such as sepsis, hemorrhagic shock, ischemia/reperfusion injury, pancreatitis, and arthritis.⁶ In addition, the cholinergic anti-inflammatory pathway was shown to play a role in modulating chronic hepatic disease.⁷

Concanavalin A (ConA) is a carbohydrate-binding protein that is known to stimulate T lymphocytes leading to hepatic inflammation.^8,9 T lymphocytes transmit TNF-α-mediated death signals in the liver tissue,¹⁰ and activated Kupffer cells and macrophages are known to be involved in hepatic inflammation through chemokine signaling.^11,12 In addition, ConA treatment augments the duration of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the dorsal motor neurons of the vagus nerve upon stimulation of the nucleus tractus solitarius.¹³

Recent experimental studies of acupuncture have used diverse animal models of diseases targeting the gastrointestinal tract, lung, spinal cord, and brain in addition to lipopolysaccharide (LPS)-mediated systemic inflammation and have begun to provide a mechanistic basis for its effects.¹⁴ Yet, to our knowledge, there has been no report investigating the effect of acupuncture stimulation on hepatic inflammation using ConA-injected animals. Considering the pathophysiological significance of liver function for the homeostatic regulation of digestive, immune, and renal function, it is of great importance to develop therapeutic strategies for hepatic diseases. Here, using the ConA model of hepatic inflammation, we investigated whether acupuncture stimulation had any effects on the production of TNF-α as an indicator of acute inflammation in the liver, and we also sought to understand the functional involvement of vagus nerve activity.

Methods

Animals and surgery

Adult mice (BALB/c, male, 8–12 weeks old) were purchased from Dae Han Bio Link (Eumseong, Korea) and acclimatized for 1 week in an animal room adjusted to 22°C and 60% humidity. All protocols related to animal care and maintenance were approved by the Institutional Animal Care and Use Committee (IACUC) at Daejeon University (approval no. DJUARB2019-028) and were closely observed during the current investigation.

A vagotomy (VNX) was performed in mice that had been anesthetized with an intraperitoneal (i.p.) injection of 80 mg/kg of ketamine and 5 mg/kg of xylazine. The left vagus nerve was exposed in the cervical midline and excised as described in our previous report.¹ After suturing, animals were recovered and returned to the animal room. Seven days after vagotomy, mice were subjected to ConA treatment and acupuncture stimulation.

Generation of ConA model of hepatic inflammation in mice

To induce hepatic inflammation, we administered ConA (Sigma-Aldrich, St. Louis, MO, United States) into the mice. Prior to injection of ConA, we elevated the animals’ body temperature by exposing them to an infrared irradiator for 20 min and intravenously injected ConA (3 mg/mL in H₂O, 15 or 30 mg/kg) into the tail vein. Animals were sacrificed 6 or 24 h following ConA injection (ConA-6 h or ConA-24 h). A previous study showed that TNF-α induced in the plasma by LPS was downregulated to baseline levels 4 h after electroacupuncture (EA) stimulation.² Thus, as for the experiments applying acupuncture stimulation (manual acupuncture (MA) or EA) or electrical stimulation of the sciatic nerve (SNS) in ConA-24 h animals, we administered MA, EA, or SNS at 19.5 h after ConA treatment, allowed a 30-min period of acupuncture stimulation (see below) and sacrificed animals 24 h after ConA injection. Similarly, in ConA-6 h animals, EA, MA, or SNS was given 1.5 h after ConA injection and sacrifice took place 4.5 h later.

Acupuncture stimulation

Both MA and EA were performed in mice that had been anesthetized with an i.p. injection of 80 mg/kg of ketamine and 5 mg/kg of xylazine. Acupuncture stimulation was performed according to the procedure described in our previous study.¹ Briefly, an acupuncture needle (0.2 × 7.0 mm, HL Medical, Seoul, Korea) was carefully inserted at ST36 (Zusanli) in the hind limb (coordinates: 3- to 4-mm posterior and 1- to 2-mm lateral to the knee midline with a depth of 2–3 mm) and stimulation was applied bilaterally for 30 min. The needle was rotated slowly at time intervals of 5 min. For EA, the needle was also inserted at ST36 and electrical stimulation (1.0 V, 1 Hz, pulse duration: 2 ms) was delivered using an isolated pulse stimulator (A-M Systems 2100, Sequim, WA, United States) for 30 min. In order to apply SNS, the mouse sciatic nerve was exposed mid-thigh in the hind leg and the nerve was placed on a bipolar hook electrode of nichrome wire. Electrical current (10 mA, 5 Hz, pulse duration: 5 ms, A-M Systems) was delivered for 5 min, as described in our previous report.¹⁵

Immunofluorescence staining

Mice were anesthetized with 80 mg/kg of ketamine and 5 mg/kg of xylazine and transcardially perfused with 0.05-M phosphate-buffered saline (PBS) buffer followed by 4% paraformaldehyde (PFA). The liver was removed, post-fixed overnight in cold 4% PFA, and then cryoprotected in 30% sucrose solution. Sections (15-μm thickness) of mouse liver tissue were prepared using a cryostat (Leica, Germany) for immunofluorescence staining, which was performed as described previously.¹ Briefly, sections following the pre-treatment steps of tissue fixation, permeabilization, and blocking were subjected to reaction with primary antibodies for 4 h at room temperature, followed by secondary antibody reaction for 2 h in the dark. Fluorescein-labeled sections were analyzed using a fluorescence microscope (Nikon, Japan) and captured digital images were further analyzed by Adobe Photoshop. We used anti-TNF-α (rabbit polyclonal, Abcam, 1:400, Cambridge, England), anti-CD11b (mouse monoclonal, 1:400, Santa Cruz Biotechnology, Inc., Dallas, TX, United States), anti-CD68 (rabbit polyclonal, 1:1000, Santa Cruz Biotechnology, Inc.), and anti-phospho-Erk1/2 (rabbit polyclonal, 1:400, Cell Signaling Technology, Danvers, MA, United States) primary antibodies and isotype control antibodies (rabbit IgG, mouse IgG, 1:400, Santa Cruz Biotechnology, Inc.). We also used fluorescein-goat anti-mouse IgG (H + L) (1:400, Invitrogen, Eugene, OR, United States) and rhodamine goat anti-rabbit (1:400, Invitrogen, Carlsbad, CA, United States) secondary antibodies.

Western blot analysis and enzyme-linked immunosorbent assay

Animals were sacrificed by cervical dislocation, and the liver tissue was immediately removed. Western blot analysis was carried out according to procedures described previously.¹ Briefly, liver tissue was suspended in triton lysis buffer (iNtRON, Seongnam, Korea) and protein was extracted by sonication. The supernatant from the cell lysate was collected by centrifugation, and protein (15 μg) was used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoblotting. Primary antibodies used were total Erk1/2 (rabbit polyclonal, 1:1000, Cell Signaling Technology), anti-phospho-Erk1/2 (rabbit polyclonal, 1:1250, Cell Signaling Technology), anti-TNF-α (rabbit polyclonal, 1:1250, Sigma), and anti-actin (mouse monoclonal, 1:10,000, MP Biomedicals, Santa Ana, CA, United States) antibodies, and secondary antibodies were goat anti-rabbit (1:1000, Santa Cruz Biotechnology, Inc.) and sheep anti-mouse (1:10,000, Amersham Biosciences, Buckinghamshire, United Kingdom) horseradish peroxidase (HRP)-conjugated antibodies. To perform enzyme-linked immunosorbent assay (ELISA) for TNF-α, blood plasma from the mice was collected by centrifugation (800g, 10 min). ELISAs were carried out according to the manufacturer’s instructions (mouse TNF-α ELISA, eBioscience Inc., San Diego, CA, United States) and as described in our previous report.¹

Real-time reverse transcription polymerase chain reaction

Liver tissue was homogenized using Easy-BLUE reagents (iNtRON, Seongnam, Korea). Experimental procedures for the synthesis of cDNA through reverse transcription (RT) reactions using total RNA and subsequent quantitative real-time polymerase chain reaction (qRT-PCR) were essentially the same as described in our previous report.¹ The reaction of cDNA synthesis was performed using reagents from Promega (Madison, WI, United States), and all reagents and a standard protocol for real-time PCR were obtained from Applied Biosystems (Foster City, CA, United States). TNF-α primer sequences were 5′-CATCTTCTCAAAATTCGAGTGACAA-3′ (forward) and 5′-TGGGAGTAGACAAGGTACAACCC-3′ (reverse). Expression levels of TNF-α mRNA in reference to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were normalized using a converting program (Applied Biosystems).

Statistical analysis

Statistical analysis was performed with GraphPad Prism v7.00 (GraphPad Software, San Diego, CA, United States). One-way analysis of variance (ANOVA) with Tukey’s post hoc tests was used to examine for differences between experimental groups. p < 0.05, p < 0.01, and p < 0.001 were set as criteria for statistical significance. Data were expressed as mean ± standard error of mean (SEM).

Results

Regulation of TNF-α production by acupuncture in the liver tissue and plasma in ConA-injected mice

To determine whether ConA injection induces an inflammatory reaction in the liver, we analyzed the expression of TNF-α mRNA and protein. The expression level of TNF-α mRNA was markedly increased 6 h after ConA injection and decreased 24 h later (Figure 1(a)). TNF-α protein, which was detected as two isoforms of 26 and 17 kDa, was also induced after ConA injection, but the time course of induction was slower than that of the mRNA, showing a continuous increase up to 24 h after ConA injection (Figure 1(b) and (c)).

To examine the effect of acupuncture stimulation on systemic inflammation in ConA-injected animals, we determined TNF-α levels in the plasma. Lower (15 mg/kg) and higher (30 mg/kg) doses of ConA injection were similarly effective at inducing TNF-α production. Administration of MA and EA at ST36 significantly decreased TNF-α levels in the plasma. While both MA and EA attenuated TNF-α production, greater effect sizes were observed following injection of the lower dose of ConA than the higher dose (Figure 2(a) and (b)), and thus, the lower dose was used for the rest of the present study.

We then analyzed the hepatic regulation of TNF-α by acupuncture stimulation in ConA-injected animals. Immunofluorescence analysis revealed that ConA injection clearly induced TNF-α signals in the liver tissue, and that the signal intensity was reduced by MA or EA (Figure 3(a)). TNF-α signals were mostly co-localized with CD11b protein, displaying a similar induction pattern as TNF-α in animals receiving ConA and acupuncture stimulation (Figure 3(a)). Reaction with isotype antibodies instead of primary antibodies followed by secondary antibody reaction did not show any signals, as demonstrated by immunofluorescence staining of ConA-injected tissues (Figure 3(a)). Immunofluorescence images at higher magnification revealed that both CD11b and TNF-α signals were localized within the perinuclear area (arrowheads in Figure 3(b)). We further found that the signal intensity of CD68, known to be expressed in the Kupffer cells, was also induced by ConA in the liver and downregulated by EA and MA (Figure 3(c)). Immunofluorescence staining of CD68 protein showed that the signals were seen clearly in the ConA-injected group and reduced by MA and EA (Figure 3(c)). Like the immunofluorescence view of CD11b, CD68 signals were clearly seen in the perinuclear zone in the liver tissue (marked by rectangle in ConA group, Figure 3(c)).

Involvement of vagus nerve activity in acupuncture-mediated TNF-α production

To understand whether vagus nerve activity was involved in the acupuncture-mediated anti-inflammatory response, we analyzed plasma levels of TNF-α in vagotomized animals. In ConA-injected animals, MA and EA were very effective at downregulating the production of plasma TNF-α, then the vagotomy (VNX) greatly elevated TNF-α production with a return to a similar level as that in the ConA-injected group (Figure 4). We further found that SNS abolished the production of TNF-α, and that vagotomy increased TNF-α levels. Vagotomy alone in ConA-injected animals did not change TNF-α level in comparison with the non-vagotomized group, suggesting that the vagus nerve pathway modulates the anti-inflammatory effect of acupuncture without affecting ConA-induced inflammation.

Regulation of phospho-Erk1/2 production by acupuncture stimulation in the liver tissue

It was previously reported that activated mitogen-activated protein (MAP) kinases act upstream or downstream of the intracellular signaling pathway of TNF-α.¹⁶ Here, we investigated whether acupuncture stimulation in ConA-injected animals involved regulation of the production of phospho-Erk1/2 in the liver. Phospho-Erk1/2 was induced in the liver tissue 6 h after ConA injection and further increased 24 h later (Figure 5(a) and (b)). Here, administration of MA or EA significantly decreased the production of phospho-Erk1/2. Immunofluorescence staining revealed clear induction of phsopho-Erk1/2 signals by ConA treatment and marked reduction by acupuncture stimulation in the liver tissue. Induced phospho-Erk1/2 signals were highly co-localized with TNF-α in ConA, ConA + MA, and ConA + EA groups (Figure 5(c)). Finally, replacement of the primary antibody reaction with isotype antibodies resulted in no fluorescence signals, indicating the reaction specificity of primary antibodies.

Discussion

The objective of the present study was to determine whether acupuncture stimulation was involved in regulating inflammatory responses in the liver, and if so, what the possible mechanistic basis underlying its effect might be. Our results demonstrate that acupuncture stimulation using either MA or EA was effective at attenuating the level of TNF-α production in the liver tissue and also in the plasma of ConA-injected animals. Our study further showed that the anti-inflammatory effects of acupuncture stimulation were negated to a great extent by vagotomy, suggesting that vagus nerve activity may be involved in the therapeutic effects of acupuncture in the liver.

ConA is a mitogenic lectin known to increase immune activity by stimulating T lymphocytes. T lymphocytes produce lymphokines and also activate antigen-presenting cells such as macrophages.¹⁷ Given that ConA treatment in experimental animals induces systemic inflammation in addition to pathological changes in several types of liver cells, including Kupffer cells and stellate cells,^18–20,21 we assumed that the ConA animal model would be useful to examine the regulatory effects of an acupuncture intervention which has, in theory, regulatory effects on the whole body.²¹ We applied acupuncture stimulation at ST36, which has been shown to have regulatory effects on gastrointestinal and cardiovascular function, as well as pain regulation in relation to cancer,^22,23 and investigated its regulatory effects on the production of TNF-α.

TNF-α is expressed in immune cells, including macrophages, T cells and natural killer (NK) cells, and non-immune cells such as endothelial cells and fibroblasts.²⁴ TNF-α protein is produced as a membrane-bound form (26 kDa) and secreted in a soluble form after proteolytic cleavage (17 kDa).²⁵ Both forms bind to the TNF-α receptor (TNFR) and are biologically active.²⁶ Here, we investigated both membrane-bound and soluble forms of TNF-α using immunofluorescence staining of liver tissue and ELISA, respectively. ConA injection strongly but transiently induced TNF-α mRNA expression in the liver tissue, showing a peak at 6 h, with a return close to the basal level 24 h after ConA injection. Induction of TNF-α protein continued to increase up to 24 h, suggesting that the translational and posttranslational processes in gene expression further elevate protein levels after reaching the peak of mRNA synthesis. Immunofluorescence staining revealed that the majority of, if not all, TNF-α signals were largely localized within CD11b-positive cells. Immunofluorescent views of CD68, a transmembrane protein expressed in hepatic Kupffer cells,²⁷ showed a similar induction pattern as CD11b, a marker protein of activated macrophages and Kupffer cells, suggesting that infiltrated macrophages and Kupffer cells are the principal cells that express TNF-α in the liver of ConA-injected animals.

We found that acupuncture stimulation at ST36 in ConA-injected animals was very effective at downregulating the production of TNF-α in the plasma, as well as in liver cells. It was noted that anti-inflammatory effect of acupuncture was similar between EA and MA. In previous reports, EA was more effective at attenuating stress than MA²⁸ and the variation of stimulation frequency in EA caused different patterns of responses in brain tissues.²⁹ Possibly, in our experimental system, MA stimulation at ST36 could be effective enough to transmit the signals that modulate target organ pathogenesis through an unknown mediator. When we applied electrical stimulation to the sciatic nerve (SNS), TNF-α production was completely abolished in the plasma of ConA-injected animals. This observation is consistent with a previous study showing the suppression of TNF-α production by SNS in LPS-injected animals.² Afferent fibers in the sciatic nerve may transmit the acupuncture signals from the somatic tissues at ST36 into the spinal cord and the brain. In this regard, it would be worthwhile to discuss the specificity of acupuncture stimulation at ST36 in relation to anti-inflammatory effects. We have previously demonstrated that sham needle stimulation around ST36 can partially induce acupuncture-like responses such as the induction of α6 integrin at the stimulation site or increased expression of GAP-43 and Erk1/2 in the dorsal root ganglion (DRG).^15,30 Transcutaneous electrical stimulation and EA show similar effectiveness in the regulation of gastric motility in humans.³¹ We therefore speculate that acupuncture needle stimulation may have varying anti-inflammatory effects depending on subcutaneous locations (i.e. traditional acupuncture vs non-acupuncture sites). Whether the electrical signaling through the axonal tract properly reflects the intrinsic property of reportedly acupuncture-specific neural sensation and presents it to the target organ requires further investigation.¹⁴

Our data also showed that the vagus nerve may be involved in mediating the transmission of acupuncture signals into target organs. In vagotomized animals, MA, EA and SNS showed no suppressive effects on TNF-α production. The vagus is the major parasympathetic nerve connecting the brain with most of the visceral organs. Ever since the early indications that vagus nerve activity mediates the anti-inflammatory effects of acupuncture stimulation,^21,32 emerging evidence demonstrates that vagus nerve activity indeed modulates acupuncture-induced anti-inflammation in LPS-injected animals and animal models of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.^1
–3 Yet, the connection between vagus nerve fibers and the cells in the target organ has not been clearly identified for some internal organs. For instance, the efferent vagal nerve connection to the spleen is controversial, despite experimental evidence demonstrating that efferent vagus nerve activity regulates splenic inflammation through the celiac connection to T lymphocytes releasing acetylcholine to splenocytes.^33,34 In the efferent hepatic vagal pathway, postganglionic vagus nerve fibers, originating from the ganglia located at the hepatic hilus and the portal space, may innervate cells in the vascular walls and hepatic parenchymal and non-parenchymal cells directly or indirectly.³⁵ Cholinergic inputs into the liver were shown to induce dilation of the liver sinusoids,^36,37 and immunohistochemical studies have identified intrahepatic cholinergic innervation in rats.³⁵ A recent study showed that VNS regulated inflammation in the Kupffer cells via α7 nicotinic acetylcholine receptors.³⁸ Yet, the mechanistic basis underlying the interaction between postganglionic nerve fibers and hepatic Kupffer cells leading to cholinergic anti-inflammation remains largely obscure.

Our data showed that in the liver tissue of ConA-injected animals, phospho-Erk1/2 was induced from TNF-α-positive cells and downregulated by acupuncture stimulation. Extracellular stress such as ethanol treatment was shown to induce TNF-α production in Kupffer cells via the activation of phospho-Erk1/2.³⁹ Erk1/2 can phosphorylate STAT3 at its S727 residue, which negatively modulates STAT3 phosphorylation at Y705, thus inhibiting STAT3 function as a transcriptional activator.⁴⁰ Interestingly, STAT3 phosphorylation at Y705 was reported to be linked to cholinergic anti-inflammation in macrophages given cholinergic stimulation or VNS.⁵ Future studies investigating the involvement of Erk1/2-STAT3 signaling pathway in a system of acupuncture-induced anti-inflammation will be useful experimental approaches to understand the biological basis of acupuncture.

Footnotes

Contributors

L-HD, K-KJ, and J-BG performed the research and analyzed the data. P-JY wrote and revised the manuscript. N-U designed and conducted the study, and wrote and revised the manuscript. All authors approved the final version of the manuscript accepted for publication.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1A6A1A03025221).

ORCID iD

Ji-Yeun Park

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