Background
Rheumatoid arthritis (RA) is a serious autoimmune disease and affects 0.5–1.0% of adults worldwide [
1]. Increasing evidence indicates that the autonomic nervous system (ANS) plays an important role in the regulation of an abnormal immune response and inhibition of inflammation [
2,
3]. The ANS modulates cytokine production mainly through the cholinergic anti-inflammatory pathway [
4] in which the efferent vagus nerve, the neurotransmitter ACh, and its receptors (especially α7 nicotinic ACh receptor, α7 nAChR) are indispensable [
5‐
7].
Curcumin has been used historically as a spice and medicinal herb in India and China [
8]. Considerable evidence has suggested that curcumin possesses diverse bioactivities [
9‐
12]. In recent years, numerous studies have shown that oral administration of curcumin significantly ameliorated collagen-induced arthritis (CIA) [
13,
14]. A clinical trial has shown that curcumin is a safe and effective agent for RA patients [
15]. However, pharmacokinetic studies have shown that its bioavailability is very poor which raise questions about how curcumin produces an anti-inflammatory effect [
16‐
18].
Previous studies in our laboratory have suggested that curcumin exerts such effects in a gut-dependent manner [
19]. The gut, as a sensory organ, senses luminal content including ingestion of food, microorganisms, gastrointestinal secretions, and pharmaceuticals. The signals arising from the lumen are transmitted though enteric and vagal pathways to the central nervous system, which in turn sends efferent signals to peripheral tissues or organs [
20]. Previous studies strongly suggest that the vagus nerve mediates the direct gut-brain-peripheral communication [
21,
22]. Anti-inflammatory responses initiated by vagal nerve activation were followed by release of ACh. The released ACh subsequently led to activation of the α7 nAChR on macrophages or immune cells, which are located in the RA joint or in the spleen [
21‐
24]. It has been reported that curcumin could improve cholinergic system dysfunction by downregulating AChE activity and increasing ChAT activity, subsequently exerting anti-inflammatory and neuroprotective effects in the brain [
25‐
27]. Based on these previous reports, we hypothesized that increasing cholinergic tone might be a potential anti-arthritic mechanism of curcumin. The present study demonstrated that oral administration of curcumin increased heart rate variability, enhanced ACh biosynthesis and release in the gut, brain, and synovium, and increased excitability of nodose ganglion neurons. These findings indicate that curcumin produces anti-CIA effects through the “gut-brain axis” and reveal a novel approach for the treatment of patients with RA and with other immune-mediated inflammatory diseases such as inflammatory bowel disease (IBD). The present study also provides an intriguing paradigm for mechanistic studies of anti-inflammatory compounds with low oral absorption and bioavailability.
Methods
Animals
Female Wistar rats, 6–8 weeks, weighing 150 ± 20 g, were purchased from Shanghai Super B&K Laboratory Animal Corp. Ltd. (Shanghai, China) and Charles River (MA, USA). They were maintained in standard laboratory chow with tap water ad libitum and under climate-controlled environment. All in vivo studies were carried out in compliance with China Pharmaceutical University Animal Care and Use Committee guidelines. All in vitro studies were carried out in compliance with the Drexel University College of Medicine Animal Care and Use Committee guidelines.
Collagen II (CII)-induced arthritis (CIA) in rats and curcumin treatment
Arthritis was induced in rats by immunization with an emulsion of chicken type II collagen (CII, Sigma-Aldrich Co., St. Louis, MO, USA) and complete Freund’s adjuvant (CFA, Becton Drive Co., NJ, USA) as previously reported [
28]. Briefly, rats were intradermally injected with 200 μl of this emulsion at the base of the tail on day 0. Seven days after the primary immunization, rats were boosted with an emulsion of CII and incomplete Freund’s adjuvant (IFA, Becton Drive). On day 14, rats were randomly assigned to normal group, model group, and curcumin group (100 mg/kg, Nanjing Zelang Medical Technology Co., Nanjing, China). Curcumin was suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) and orally administered daily 2 weeks. Normal and model group rats were orally administered vehicle according to the same schedule.
Unilateral cervical vagotomy (VGX) and induction of CIA in rats
A VGX operation was performed 4 days before the induction of CIA [
29]. In brief, rats were anesthetized and a ventral cervical midline incision was made to expose the left cervical vagus trunk, which was next ligated with 4–0 silk sutures and divided. The skin was closed with three sutures. The left nerve trunk of sham rats was exposed and isolated from surrounding tissue without being transected. After 4 days, CIA was induced as described above and the day of immunization was marked as day 0. Seven days later, rats were boosted with the second injection. On day 14, sham rats were divided into the following groups: sham group, model group, curcumin (100 mg/kg) group, leflunomide (2 mg/kg, Suzhou Changzheng-Xinkai Pharmaceutical Co., Suzhou, China) group, and nicotine (300 μg/kg, Nanjing Zelang Medical Technology) group. VGX rats were divided into VGX group, V-model group, and V-curcumin (100 mg/kg) group. Curcumin and leflunomide were orally administered, and nicotine was intraperitoneally injected for consecutive 2 weeks. Other groups were given an equal volume of vehicle with the same schedule and route of administration.
Effects of pretreatment with nicotinic ACh receptor (nAChR) antagonists on curcumin-treated CIA rats
CIA was induced as describe above. Two weeks after the first immunization, rats were randomly assigned to the following groups: (1) normal group, model group, curcumin (100 mg/kg) group, mecamylamine (1 mg/kg, Sigma) group, curcumin (100 mg/kg) + mecamylamine (1 mg/kg) group, hexamethonium (4 mg/kg, Sigma) group, curcumin (100 mg/kg) + hexamethonium (4 mg/kg) group, and leflunomide (2 mg/kg) group; (2) normal group, model group, curcumin (100 mg/kg) group, α-bungarotoxin (1 μg/kg, Abcam Inc., Cambridge, UK) group, curcumin (100 mg/kg) + α-bungarotoxin (1 μg/kg) group, and nicotine (300 μg/kg) group. Curcumin and leflunomide were suspended in 0.5% CMC-Na and orally administered daily 2 weeks. Antagonists were dissolved in 0.9% NaCl solution and intraperitoneally injected 10 min before curcumin administration. Nicotine was dissolved in 0.9% NaCl solution and intraperitoneally injected. Normal and model group rats were orally administered 0.5% CMC-Na in the same schedule.
Assessment of CIA
CIA was assessed by body weight, paw swelling, and arthritis index (AI) scores. A plethysmometer was used to measure the volumes of paws. The clinical scores were evaluated blindly as follows: 0 point: no arthritis; 1 point: swelling in one type of joint; 2 points: swelling in two types of joint; 3 points: swelling in three types of joint; and 4 points: swelling of the entire paw. The AI scores was the sum of four paws, with the maximum score of 16 for each rat. The ankles of rats were photographed immediately after rats were sacrificed with ether anesthesia. After continuous treatment for 2 weeks, the hind paws were photographed to evaluate the morphological changes in the ankle of rats. On the same day, the representative radiographs of the hind paws of the CIA rats were obtained to examine the bone erosion and joint destruction.
Histological analysis
The right ankles were fixed in 10% buffered formalin for 48 h. The joints were then decalcified in 10% EDTA, embedded in paraffin, cut into 5-μm serial sections, and stained with hematoxylin and eosin (H&E). The histopathological changes in the joints were examined using an optical microscope and graded on a scale of 0–3 (0 = none changes, 1 = mild changes, 2 = moderate changes, and 3 = severe changes) by a pathologist blinded to the experimental groups [
28].
Heart rate (HR), blood pressure, and heart rate variability (HRV) analysis
Heart rate (HR), blood pressure, and HRV summary variables in time and frequency domain were assessed on day 27. Blood pressure was first measured using BP-6 Animal Non-Invasive Blood Pressure Measuring System (Chengdu Tme Technology Co, Ltd., Chengdu, China). Systolic (SP) and diastolic pressures (DP) were recorded, and mean arterial pressure (MP) was calculated according to the following formula: MP = (SP + 2 × DP)/3. Rats were then anesthetized by intraperitoneal injection of 10% urethane and placed in the supine position on a temperature-controlled cushion and allowed to breathe spontaneously. The mean HR and HRV parameters were obtained from electrocardiogram (ECG) by BL420S biological signal acquisition system (Chengdu Tme Technology Co, Ltd.). The needle electrodes were subcutaneously inserted in rat left forelimb and both hind limbs, respectively. ECG was measured for 10 min before and 1 h after treatment to obtain the HRV parameters. The time domain includes standard deviation of all R-R intervals (SDNN) and square root of the mean of the sum of the squares of differences between adjacent R-R intervals (RMSSD). The frequency domain data collected from each period of 10 min underwent spectral analysis by using a fast Fourier transform algorithm to determine the high-frequency power (HF) and low-frequency power (LF) components. The range for HF was 0.15–0.4 Hz and the range for LF was 0.04–0.15 Hz.
Assessment of ChAT and AChE activities
The synovium tissues were weighed, and an adequate amount of 0.9% NaCl solution was added to obtain 10% (w/v) homogenates. Blood samples were collected from rats under ether anesthesia using orbital eye bleeds. Then, after clotting for 20 min at room temperature, blood was centrifuged for 20 min at a speed of 3000 rpm at 4 °C, and the serum was collected. The enzymatic activities of ChAT and AChE were measured by kits according to the manufacturer’s instructions (Bioswap Biotech Co., Wuhan, China). Activities were expressed as units per gram weight of synovium tissue and units per liter of serum, respectively.
Quantitative PCR (qPCR)
Total RNAs from the small intestine, brain, and synovium were isolated using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. RNA was reversely transcribed to cDNA using HiScript RT SuperMix (Vazyme Biotech Co., Nanjing, China) and then analyzed for the expressions of high-affinity choline transporter 1 (CHT1), ChAT, and VAChT by Ace qPCR SYBR Green Master Mix (Vazyme Biotech) using MyiQ2 Detection System (Bio-Rad Laboratories, Hercules, CA) [
19]. The following primers (Sangon Biotech Co., Shanghai, China) were used: for CHT1, 5′-CTACATTCCCCTACGTGGTCC-3′ (sense) and 5′-AGGCCGATGGCATAAGAGAAG-3′ (antisense); for ChAT, 5′-CCAGTTCTTTGTCTTGGATGTT-3′ (sense) and 5′-GGACGCCATTTTGACTATCTTT-3′ (antisense); for VAChT, 5′-GTGCCCATTGTTCCCGACTA-3′ (sense) and 5′-CTTTCTGTGGGGTAGCGAGG-3′ (antisense); and for β-actin, 5′-CCCATCTATGAGGGTTACGC-3′ (sense) and 5′-TTTAATGTCACGCACGATTTC-3′ (antisense). All values were expressed relative to the expression of the reference gene, β-actin, and the relative expression of each gene was determined according to the 2
-ΔΔCt method.
Immunohistochemistry
Small intestines, brains, and synovium tissues were harvested, fixed in 10% formalin, and then embedded in paraffin and sliced into 5-μm-thick sections (Reichert HistoSTAT, USA). The sections were incubated with the first primary antibodies against ChAT and VAChT overnight at 4 °C, and examined with DAB Envision System (Dako, Glostrup, Denmark) according to the manufacturer’s instructions.
Measurement of cytokines in serum
The concentrations of TNF-α, IL-1β, IL-6, IL-17A, IFN-γ, IL-4, IL-10, and TGF-β in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits (Dakewe Biotech Co., Shenzhen, China) according to the manufacturer’s instructions. The absorbance was measured at 450 nm.
Cell culture
Primary nodose ganglion neuronal cultures were prepared from adult female rats as described previously with some modifications [
30]. Briefly, rats were deeply anesthetized with overdose of isoflurane. The nodose ganglia of the vagus nerve were carefully removed and washed in Hank’s balanced saline solution (HBSS) (Invitrogen) (in mM: 137 NaCl, 5.4 KCl, 0.4 KH
2PO4, 1 CaCl
2, 0.5 MgCl
2, 0.4 MgSO
4, 4.2 NaHCO
3, 0.3 Na
2HPO
4, and 5.6 glucose). The ganglia were then incubated on an incubator shaker (New Brunswick Scientific, NJ, USA) for 30 min at a speed of 135 rpm at 37 °C in HBSS containing papain (15 U/ml, Worthington Biochemical, NJ, USA) and collagenase from clostridium histolyticum (5 × 10
−4 g/ml, Sigma), rinsed three times with HBSS, and placed in culture medium containing Neurobasal A (Invitrogen), 2% B-27 (Life Technologies, NY, USA), 2% heat-inactivated horse serum, 2% fetal calf serum, 0.2 mM L-glutamax, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The fragments were mechanically dissociated by gently triturating with a pipette. The dispersed cells were seeded onto 12-mm poly-D-lysine and laminin (Sigma)-coated coverslips at a density of 3000 cells per well. Neurons were cultured at 37 °C in a humidified atmosphere containing 5% CO
2.
Electrophysiological recordings
Standard whole-cell recordings were performed at room temperature using an EPC 10 amplifier and PatchMaster software (HEKA Elektronik, Lambrecht, Germany) as described previously [
31]. Electrode resistances ranged between 3 and 6 MΩ with series resistances of 6–15 MΩ and were compensated to the maximal current amplitude. For current-clamp recordings, the membrane voltage was held at − 65 mV; the bath solution was Tyrode’s solution containing (in mM) 140 NaCl, 5 KCl, 2 CaCl
2, 1MgCl
2, 10 HEPES, and 5.6 glucose, pH adjusted to 7.36 with NaOH (osmolality ~ 292 mmol/kg). The intracellular solution contained (in mM) 140 KMeSO
4, 2MgCl
2, 1 EGTA, 10HEPES, 3 Na
2ATP, and 0.3 Na
2GTP, pH adjusted to 7.4 with KOH (osmolality ~ 292 mmol/kg). Action potentials were recorded in the current-clamp mode at a membrane potential around − 65 mV. A current injection was used to evoke 2 to 3 action potentials and remained constant throughout the recording. Only one neuron was recorded from each coverslip.
Electrophysiological data analysis
Off-line data analysis was processed using PatchMaster (HEKA) and Origin 8.1 software (OriginLab, Northampton, USA).
Statistical analysis
Data were presented as mean ± S.E.M.. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test. An 0.05 (p < 0.05) was considered statistically significant. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology.
Discussion
Rheumatoid arthritis (RA) is a systemic refractory arthropathy that impacts patient quality of life. Up to now, the fundamental pathophysiology of RA has not been fully elucidated [
39,
40]. However, evidence is mounting that the nervous system plays a vital role in the occurrence and development of RA [
3]. The neural-endocrine-immune system constitutes a complex network and participates in maintaining homeostasis in organisms. It is well known that the nervous system is the regulating center of inflammatory and immune responses [
41]. Growing evidence indicates that the immune and nervous system maintain extensive interactions. The bidirectional communication between them could regulate the inflammatory responses in diseases such as RA.
The autonomic nervous system (ANS) is divided into the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS), which act either in synergy or in opposition to control life functions like body temperature, heart rate, blood pressure, and gastrointestinal motility to maintain homeostasis [
42]. A clinical trial has demonstrated that the ANS is imbalanced in RA patients, with an increased sympathetic and reduced parasympathetic tone [
43]. Moreover, recent reports have suggested that the PNS plays an important role in chronic immune-mediated inflammatory diseases. Activation of the PNS by either pharmacological or electrical stimulation of the vagus nerve attenuates inflammatory diseases [
44‐
46]. Pharmacological cholinoceptor antagonists or vagotomy attenuate these cholinoceptor agonist-induced anti-inflammatory effects [
47‐
50]. Hence, it is possible to target the neural pathways for treatments of excessive inflammation and autoimmune conditions.
Clinical studies have shown that curcumin can bring relief to osteoarthritis and RA [
13‐
15]. However, the pharmacokinetic studies have indicated that curcumin has poor bioavailability due to poor absorption, rapid metabolism, and rapid systemic elimination [
16‐
18]. This contradiction between the therapeutic efficacy and poor pharmacokinetics of curcumin has yet to be resolved and required further investigation.
We have previously demonstrated that oral administration of curcumin attenuates adjuvant-induced arthritis through a gut-dependent mechanism [
19]. In the present study, we confirmed the anti-arthritic effects of curcumin and found that curcumin increases the activity of ChAT and regulates the cholinergic system function by reducing sympathetic tone and increasing parasympathetic tone. There is a great body of evidence indicating that the vagus nerve is a vital component of the inflammatory reflex. Once it is damaged, the cholinergic anti-inflammatory pathway cannot function normally [
2,
3,
28,
51,
52].
Our data showed that unilateral cervical vagotomy completely abolished the anti-arthritic effects of curcumin, suggesting a critical role of an intact vagus nerve. Since the vagus nerve contains both afferent and efferent fibers, vagotomy removes both afferents and efferents. Further investigation is needed to elucidate the role of afferent fibers in anti-arthritic effect of curcumin. The electrophysiological results demonstrated that curcumin markedly increased neuronal excitability in nodose ganglion neurons, which further indicates that curcumin may exert its effect by directly activating the vagus nerve, suggesting the anti-arthritic effect of curcumin is mediated by the “gut-brain axis.” We further examined whether ACh receptors are involved in the curcumin-induced modulation of the cholinergic system function. Our results showed that the α7 nAChR antagonist markedly blocked the effects of curcumin on collagen-induced arthritis (CIA) and action potentials, suggesting that α7 nAChR mediates curcumin-induced modulation of neuronal excitability and CIA. Interestingly, recent studies have shown that curcumin exhibits regulatory effects on the gut microbiota in menopausal model, hepatic steatosis, DSS-induced colitis [
53‐
56], and that the gut microbiota is involved in the pathogenesis of arthritis [
57‐
59]. Based on these findings, it is possible that curcumin affects the cholinergic anti-inflammatory pathway through the gut-brain axis via modulation of gut microbiota.
Acknowledgements
We thank Dr. James E. Barrett and Dr. Frances M. Munoz for their critical reading and valuable comments on the manuscript.