Electroacupuncture Alleviates Pain Responses and Inflammation in Collagen-Induced Arthritis Rats via Suppressing the TLR2/4-MyD88-NF-κB Signaling Pathway

Results EA intervention and OxPAPC injection could relieve mechanical allodynia and thermal hyperalgesia caused by CIA. Paw edema and pathological damage of synovium were significantly ameliorated after EA intervention and OxPAPC injection. Furthermore, EA intervention and OxPAPC injection markedly reduced the contents of serum TNF-α, IL-1β, and IL-6, as well as the protein expression levels of synovial TLR2, TLR4, MyD88, and NF-κB p-p65. In particular, the expression of TLR2 and TLR4 on synovial fibroblasts and macrophages in synovium was significantly reduced by EA intervention. Conclusions Repeated EA stimulation at ST36 and SP6 can effectively relieve joint pain and synovial inflammation caused by RA in CIA rats. The analgesic and anti-inflammatory effect of EA may be closely related to the inhibition of innate immune responses driven by the TLR2/4-MyD88-NF-κB signaling pathway in the synovium.


Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune polyarticular disease that can afect all ages, genders, and ethnicities [1]. RA has a worldwide prevalence of 0.5%-1% and is clinically characterized by marked synovial infammation, joint swelling, joint pain, bone erosion, and progressive disability [2]. It severely degrades the suferers' quality of life and is even life-threatening [3]. Existing antirheumatic drugs, including nonsteroidal anti-infammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), or bio-agents, only partially halt the RA progression and have many adverse efects [4,5]. Growing evidence suggests that acupuncture, one of the nonpharmacological alternative therapies, is efective on RA [6][7][8]. It was reported that acupuncture signifcantly improved disease activity scores, pain and overall mobility, joint swelling, and health-related quality of life in patients with RA [9][10][11]. Although it is generally accepted that EA can be used as adjuvant therapy for the treatment of RA, its precise mechanisms remain to be elucidated.
RA is the consequence of dysregulation of the immune system and is characterized by an excessive autoimmune response to synovial membrane. Te process involves the activation of synoviocytes, infammatory cells infltration in synovium, and the increase of pro-infammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6), leading to chronic localized synovial and systemic infammation, joints pain, as well as cartilage and bone erosion [12]. At present, it is widely accepted that the cytokine network plays a vital role in RA pathogenesis [13], and anticytokines therapy in patients with RA has proven to be efective [14]. Moreover, EA has been previously reported to reduce the serum levels of pro-infammatory cytokines in RA patients [15] and RA animal models [16,17], which suggests the possible immunomodulatory mechanism of EA in alleviating RA.
Toll-like receptors (TLRs) are functionally key pattern recognition receptors (PRRs) in the innate immune system, which are mainly expressed in the innate immune cells and play a critical role in the initiation of infammatory responses [18]. Numerous studies have demonstrated that TLRs and their downstream signaling molecules are involved in the development of autoimmune diseases such as RA [19,20]. Te human TLRs family includes 11 members, mainly distributing in cell membranous structures. Among them, TLR2 and TLR4 are closely associated with the pathogenesis of RA [18,21]. Additionally, growing in vivo and in vitro studies have emphasized that TLR2 and TLR4 signaling play pivotal roles in the onset and maintenance of synovial infammatory responses in RA [22][23][24][25]. However, it remains unknown whether the TLR2/4 signaling is the regulatory target through which EA exerts its antiarthritic efects on RA.
In the present study, we established a collagen-induced arthritis (CIA) model in rats to investigate the ameliorative efects of EA on RA synovitis and arthralgia through TLR2/4 and their downstream signaling cascades. Our fndings highlighted the importance of the synovial TLR2/4-MyD88-NF-κB signaling pathway in the analgesic and antiinfammatory efects of EA intervention for RA.

Animals.
Animal care and experimental protocols were approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (ethical approval number: D2017-08-16-1). All experimental animals were cared according to the guidelines provided by the U.S. National Institutes of Health for the Care and Use of Laboratory Animals. 200-250 g male Sprague-Dawley (SD) rats were purchased from Beijing Union Medical College and were housed (up to 5/cage) in standard plastic cages with water and standard mouse feed under controlled temperature (22-24°C), humidity (55 ± 5%), and 12-hr alternating light/dark cycle (lights were turned on between 7:00 a.m. and 7:00 p.m.).

Establishment of the CIA Model.
Te CIA model was established according to reference [26][27][28]. Briefy, bovine type II collagen (CII; Chondrex, Redmond, WA, USA) (dissolved in 0.1 M acetic acid) was emulsifed 1 : 1 (v: v) with complete Freund's adjuvant (CFA; Sigma-Aldrich, Darmstadt, Germany) at a fnal concentration of 1 mg/mL. Rats were immunized with intradermal injection of the 300 μl CII emulsion at the base of the tail on day 0, followed by the same booster injection 7 days later. Te animals with no signs of swelling in joints were removed. Controls were handled in the same way except that they were injected with 150 μl of 0.1 M acetic acid.

Experiment Design
To determine the analgesic and antiinfammatory efects of EA treatment on the CIA rats, we randomly divided 36 SD rats into three groups: control (normal), CIA (model only), and CIA + EA (model with EA treatment at ST36 and SP6 acupoints) (n � 12 each group).

Experiment 2.
To assess the role of the TLR2/TLR4 signaling pathway in the maintenance of infammation and joint pain in CIA rats, further 16 rats were randomly assigned to two groups after inducing the CIA model: the CIA + vehicle group (CIA rats treated with normal saline) and the CIA + OxPAPC group (CIA rats treated with the TLR2/TLR4 inhibitor OxPAPC) (n � 8 each group).

Experiment 3.
To determine the expression of TLR2 and TLR4 in synovial macrophages and synovial fbroblasts in the synovium tissue, we evenly separated 12 SD rats into the same three groups as in Experiment 1. Te rats in the CIA + EA group received EA intervention as in Experiment 1 for two weeks. Experiment 1. Rats in the CIA + EA group received EA intervention at ST36 (located about 5 mm inferior to the capitulum fbulae and posterolateral to the hindlimb knee joint) and SP6 (located at the medial side of the hind leg, 10 mm directly above the tip of the medial malleolus) on day 14 after the initial immunization. Briefy, rats were lightly anesthetized with isofurane (1.5% in oxygen) delivered via an anesthesia unit (Matrix Company, Midmark Animal Health, Versailles, OH, USA) and underwent EA stimulation by inserting needles (0.5 × 32 mm, Suzhou, China) at ST36 and SP6 in a depth of about 5 mm and 3 mm, respectively. Te inserted needles were further connected to an electronic acupuncture treatment instrument (Hans-100A, Nanjing Jisheng Medical Technology, Co., Ltd., China). Te EA parameters were alternating frequency of 2 Hz/100 Hz, a pulse width of 0.2-0.6 ms, and an intensity of 1 mA. Te stimulation procedure was performed for 30 min every day for 28 consecutive days ( Figure 1). Rats in the control group and the CIA group were anesthetized in the same manner as those in the CIA + EA group but did not receive EA stimulation.

Electroacupuncture (EA) Intervention and Sampling in
Upon completion of EA intervention, all rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) for blood collection and then sacrifced through decapitation (on day 42 after the frst immunization). Blood (n � 12 per group) was taken via the tail vein and collected into procoagulation tubes (BD Vacutainer, Franklin Lakes, New Jersey, USA) for the separation of serum. Ten, it was divided into aliquots and kept under −80°C for serological determinations. Te ankle joints of hind paws (n � 6 per group) were separated for further histopathological analysis. Te ankle synovial membrane tissues of hind paws (n � 6 per group) were isolated and kept under −80°C for further western blotting analysis.

Paw Edema Analysis.
Te paw edema of each rat's bilateral hind limbs (up to the ankle joint) was examined once a week from day 0 to day 42 ( Figure 1) with a water displacement plethysmometer (Ugo Basile, Comerio, Varese, Italy). Te mean values of water displacement volume were calculated from 3 measurements to judge the extent of paw edema [29].

Nociceptive Behavioral Tests.
Te pain responses of each rat's bilateral hind paws, including mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), were measured once a week from day 0 to day 42 ( Figure 1). MWT was examined by electronic Von Frey (38450, Ugo Basile, Comerio, Varese, Italy). TWL was examined by a thermal plantar analgesia instrument (37370, Ugo Basile, Comerio, Varese, Italy) [30], with a heat intensity of 50 units and a cut-of time of 30 s. Mean MWT and TWL were calculated from 3 individual tests with a 5-min interval to represent the mechanical allodynia and thermal hyperalgesia.

Radiographic and Histopathological Analysis of Ankle
Joints. For radiographic analysis, the hind limbs were imaged on a multimodality in vivo imaging system (Kodak Company, Rochester, NY, USA) shortly before the rats were sacrifced. Te exposure time was 10 s, and the f-stop factor was 2.5.
For histopathological analysis, the protocol was performed as previously described [31]. Briefy, the fresh ankle joints were fxed with 4% paraformaldehyde (Servicebio, Wuhan, China) and then decalcifed with 10% ethylene diamine tetraacetic acid (EDTA, Servicebio, Wuhan, China) at room temperature for 40 days. Te processed joints were dehydrated, embedded, and sectioned at 4 μm thickness in a sagittal plane. Sections were then stained with hematoxylin-eosin (HE) for assessing EA's efect on the histopathological damage in joints. Images were acquired and observed under an upright light microscope (Nikon Inc., Tokyo, Japan). HE staining score was evaluated by an investigator who was blinded to the experimental protocol. Te following morphological criteria were considered as follows [32]: score 0, no damage; score 1, edema; score 2, presence of infammatory cells; score 3, bone resorption.

Serum Cytokine Levels Detection.
Serum TNF-α, IL-1β, and IL-6 were measured by Meso Scale Discovery (MSD) electrochemiluminescence technology with a V-PLEX custom rat cytokine kit (K153AOH-1, MSD, Rockville, Maryland, USA) according to the manufacturer's instructions [33]. In short, the 96-well plate was washed 3 times with 150 μl wash bufer. After the washing, wells were incubated with 50 μl serum sample for 2 h at room temperature with shaking. Twenty-fveμl of detection antibody solution was then added and incubated for 2 h at room temperature with shaking. After adding 150 μl of 2 × read bufer T to each well, the 96-well plate was detected with MESO QuickPlex SQ 120 machine (MSD, Rockville, Maryland, USA), and data were analyzed via MSD Discovery Workbench software version 4.0.12.

Western Blotting Analysis.
For western blotting analysis, total proteins were obtained from the ankle synovium tissues with RIPA lysis bufer (Beyotime Biotechnology, Shanghai, China), containing 1% PMSF. Protein concentrations were quantitatively determined with a BCA protein assay kit

Statistical
Analysis. Data were reported as mean-± standard deviation (SD) and were examined by one-way ANOVA, followed by the least signifcant diference (LSD) test for comparisons among multiple groups. Te LSD test was run only if F-value achieved P < 0.05, and there was no signifcant variance in homogeneity. P < 0.05 was considered statistically signifcant. All analyses were performed using Statistical Package for the Social Sciences (SPSS) version 23.0 (SPSS Inc., Chicago, Illinois, USA).

EA Intervention Reduced CIA-Induced Hind Paw Edema.
Te hind paw edema was employed for the assessment of EA's efect on RA progression. As expected, the hind paw edema in the CIA group obviously increased from day 14 after the frst immunization and lasted to day 42 compared with that in the control group (Figure 2, p < 0.01). EA stimulation at ST36 and SP6 showed marked alleviation of paw edema in the CIA + EA group after two weeks of EA intervention compared with that in the CIA group ( Figure 2, p < 0.05).

EA Intervention Attenuated Allodynia and Hyperalgesia in CIA.
To explore the analgesic efects of EA, MWT and TWL were detected in all groups of rats. After successful induction of CIA (day 14 after the frst immunization), MWT in the CIA group was much less than that in the control group with marked diferences (Figure 3(a), p < 0.01). However, in comparison with the CIA group, MWT started to prominently recover in the CIA + EA group after two weeks of EA stimulation at ST36 and SP6 (Figure 3(a), p < 0.05). Similar results were corroborated in TWL detection (Figure 3(b), p < 0.01, p < 0.05). Tese results implicated that the long-term EA intervention may attenuate the arthritic infammatory hyperalgesia in CIA rats.

EA Intervention Improved Histopathological Lesions in the
Ankle Joint of CIA Rats. On day 42 after the frst immunization, the visual redness and swelling of the hind paws in the CIA + EA group showed a great relief compared with those in the CIA group (Figure 4(a)).
As shown in the representative radiographs, bone erosion in the ankle joint was exacerbated in the CIA group compared to the control group whereas EA could distinctly reduce articular bone destruction and reverse the above trend in CIA rats (Figure 4(b)).
Consistent with the clinical behaviors, histopathological observations of ankle joints revealed signs of severest arthritis in the CIA rats. Tere was no evidence of infammatory activity or joint erosion in the control group, while the massive infammatory cell infltration, synovial hyperplasia, and joint erosion were presented in the CIA group. Te histological damage scores were higher in the CIA group (Figure 4(d), p < 0.001). After 28 days of EA intervention, these histopathological features signifcantly ameliorated in the CIA + EA group, and the HE staining score of the CIA + EA group also decreased remarkably (Figures 4(c) and 4(d), p < 0.001).

EA Intervention
Showed the Anti-Infammatory Efect on CIA Rats. It is well documented that pro-infammatory cytokines play a vital role in the maintenance of chronic infammation and tissue damage during RA progression. MSD test was conducted to assess the contents of serum TNF-α, IL-1β, and IL-6. As shown in Figure 5, compared with those in the control group, contents of serum TNF-α, IL-1β, and IL-6 noticeably elevated in the CIA group (5, p < 0.001). On the contrary, the serum levels of these cytokines signifcantly decreased in the CIA + EA group after 28 days' EA intervention ( Figure 5, p < 0.01, p < 0.001).

EA Intervention Inhibited the Activation of TLR2/4-MyD88-NF-Κb Signaling Pathway in CIA.
To further elucidate the underlying molecular mechanisms of analgesic and anti-infammatory efects of EA intervention, we adopted western blotting to detect the expression levels of key proteins in the TLR2/4-NF-κB pathway in the ankle synovium. Te results indicated that expression levels of TLR2, TLR4, MyD88, and NF-κB p-p65 in the CIA group were signifcantly upregulated by diferent degrees in comparison with those in the control group ( Figure 6, p < 0.05, p < 0.01) whereas those in the CIA + EA group were remarkably downregulated compared with those in the CIA group ( Figure 6, p < 0.05, p < 0.01). Tis illustrated that EA at ST36 and SP6 may ameliorate infammation by inhibiting the TLR2/4-MyD88-NF-κB pathway.

Blockade of TLR2/4 Signaling Mimicked the Analgesic and
Anti-Infammatory Efects of EA in CIA Rats. To further assess whether the anti-infammatory and analgesic efects of EA were mediated by the TLR2/4 signaling pathway, a dual TLR2 and TLR4 inhibitor, OxPAPC, was administered to CIA rats. Subsequently, behavioral tests and experiments such as and WB were conducted. Te results indicated that the hind paw edema in the CIA + OxPAPC group decreased from day 28 to day 42 compared with that in the CIA + vehicle group (Figure 7(a), p < 0.05). Rats in the CIA + OxPAPC group had signifcantly increased MWT and TWL compared to rats in the CIA + vehicle group but

EA Intervention Suppressed the Expression of TLR2 and TLR4 on Synovial Fibroblasts and Macrophages in Synovium.
To characterize the TLR2 and TLR4 expressing cells in the joint synovium, tissue sections were double immunofuorescence stained for TLR2 or TLR4 and the macrophages marker CD68 or the fbroblasts marker Vimentin, respectively. Figure 8 shows representative staining patterns of CD68 or Vimentin and TLR2 or TLR4 expression in the synovium tissue. In all three groups, the majority of TLR2 and TLR4 expression was detected on macrophages and synovial fbroblasts in the lining layer and sublining layer of the synovium, and in particular, there were pronounced TLR2 and TLR4 expression in the synovial lining layer. Compared with the control group, the number of positive staining cells for TLR2 and TLR4 on synovial fbroblasts and macrophages in synovial lining and sublining layers was signifcantly increased in CIA rats ( Figure 8). EA intervention signifcantly reduced the number of positive staining cells for TLR2 and TLR4 on synovial fbroblasts and macrophages in synovium tissues (Figure 8).

Discussion
In the present study, an experimental RA model of the rat was established through immunization with CII collagen, a major component of hyaline cartilage, combined with CFA, which is the most commonly used method for developing an autoimmune model of RA [26]. Our results showed that CIA rats exhibited severe evoked joint pain and paw edema compared with rats in the control group. Radiographic and histopathological analysis confrmed the obvious pathological changes such as degeneration of joint structures, synovial hyperplasia, infammatory cell infltration, and bone erosion in the arthritic joints of CIA rats. Also, the contents of pro-infammatory cytokines TNF-α, IL-1β, and IL-6 in serum were signifcantly elevated, and the expression levels of TLR2, TLR4, MyD88, and NF-κB p-p65 were signifcantly upregulated in the synovium of CIA rats. Tese are consistent with the previous results obtained from patients with RA [34,35] and experimental RA model animals [36][37][38][39], indicating that CIA rats showed signifcant infammatory responses and hyperalgesia at arthritic joints, accompanied by the enhanced activation of the TLR2/4 signaling in the synovium. Acupuncture is one of the oldest therapeutic strategies in the world and now has been widely reported to possess analgesic and anti-infammatory efects in autoimmune diseases [40,41]. In clinical practice, ST36 is the most frequently used acupuncture point for treating RA [42]. And in the studies of acupuncture efects on experimental RA animals, ST36 is also commonly used [43][44][45]. Meanwhile, a systemic review has shown that ST36 alone or combined with other acupoints is benefcial to the clinical status of RA [7]. Terefore, in this study, we selected two acupoints, ST36 and SP6, for EA intervention. Te results showed that EA at ST36 and SP6 had a distinct anti-infammatory and analgesic efect on CIA rats, as evidenced by reduced articular swelling, improved arthro-pathology, decreased levels of pro-infammatory cytokines, and relieved mechanical allodynia and thermal hyperalgesia. Te synovium is the principal target tissue of infammation in RA [46], and persistent chronic infammation of the synovial membrane in RA causes infammatory pain, which is known to be difcult to treat [47]. Emerging studies have indicated that EA has signifcant analgesic efects on infammatory pain in CIA rats, which is related to the central descending pain inhibitory system mediated by adrenergic, cholinergic, and serotonergic receptors [48,49]. However, it has been reported that pro-infammatory cytokines, such as TNF-α, IL-1β, and IL-6, could directly increase the sensitivity and excitability of the primary aferents in the infamed joints, leading to both peripheral and central sensitization [50,51]. Our results also showed a positive correlation between nociceptive behavioral responses and articular infammation in CIA rats. Terefore, inhibiting the secretion of pro-infammatory cytokines in RA can efectively relieve joint pain.
Over the past two decades, increasing data have supported the critical role of the innate immune system in the pathogenesis of RA [52,53]. Specifcally, TLRs, the key recognition structures of the innate immune system, can recognize microbial products and endogenous ligands released upon cell damage and necrosis and are expressed on cells within the RA joint [20,54]. Meanwhile, a variety of endogenous TLR ligands, including heat shock proteins, high mobility group box-1 protein (HMGB1), host DNA, fbrinogen, and tenascin-C, have been identifed in the synovium of patients with RA, predominantly TLR2 and/or TLR4 agonists [55,56]. Te functions of TLR2/4 signaling have been extensively studied in RA. A large body of evidence obtained from in vivo animal models and in vitro human explants has confrmed that the activation of TLR2 and TLR4 by endogenous TLR ligands in arthritic joints can trigger the innate immune response and initiate the production of pro-infammatory cytokines, chemokines, and proteases, which are proposed to be responsible for the perpetuating infammation and joints destruction of RA [22,[57][58][59][60]. Terefore, the pivotal role of TLR2/4 signaling in the pathogenesis of RA had been well established, and our study focused on the modifcation of the TLR2/4 signaling pathway in EA intervention.
At present, the development of promising therapeutic strategies for the treatment of RA targeting TLRs is emerging [61]. Several preclinical studies have shown that the blockade of TLR2 signifcantly inhibited the production of proinfammatory cytokines TNF-α, IL-1β, and IL-6 in cultured synoviocytes from RA patients [62]. Inhibition of TLR4 not only alleviated the severity of experimental arthritis and suppressed IL-1β expression in arthritic joints of CIA mice [63] but also ameliorated infammatory symptoms in adjuvant-induced arthritis (AIA) rat model and inhibited the secretion of IL-6 and IL-8 in both serum of the AIA rats and human synovial fbroblasts [64]. In addition, a recent study indicated that blocking TLR4 could efectively attenuate monoiodoacetate-induced arthritis in rats by relieving joint pain and reducing the expression of TNF-α, IL-1β, and matrix metallopeptidase-13 (MMP13) [65]. Similar to the results of these studies, our fndings suggested that the blockade of TLR2/4 signaling by the TLR2/4 inhibitor OxPAPC could efectively alleviate articular infammation and joint pain. Meanwhile, evidence from animal models suggested that EA can also alleviate arthritis by inhibiting TLR2 and/or TLR4 signaling [66,67]. In line with these fndings, our results indicated EA exerted anti-infammatory and analgesic efects by inhibiting the infammatory responses driven by the TLR2/4-MyD88-NF-κB signaling pathway in the synovium of CIA rats.
Te synovium is a connective tissue structure mainly comprised of resident macrophages and synovial fbroblasts [68]. Te resident and infltrating macrophages and the synovial fbroblasts are the principal innate immune efector cells of RA and are mainly activated by TLRs signaling [69,70]. Biological agents targeting the pro-infammatory cytokines TNF-α and IL-1β, predominantly produced by macrophages, have been proven clinically efective in RA [71]. Meanwhile, classical (M1, pro-infammatory phenotype) macrophage activation occurs in the infammatory environment of the RA joint dominated by TLRs signaling [72]. Numerous studies have confrmed that TLR2 and/or TLR4 were highly expressed on the synovial fbroblasts and triggered the production of the pro-infammatory cytokines such as IL-6, chemokines, and tissue destroying mediators leading to infammatory response and joints destruction [23,59,[73][74][75][76]. In view of the important arthritic functions of TLRs signaling, especially TLR2 and TLR4 signaling in macrophages and synovial fbroblasts, we examined the efect of EA intervention on the expression of TLR2 and TLR4 on these two types of synovial cells of CIA rats. Our results showed that EA intervention markedly reduced the expression of TLR2 and TLR4 on macrophages and synovial fbroblasts in the synovial lining and sublining layers of CIA rats. In fact, the cellular and molecular mechanisms underlying the pathogenesis of RA are not fully understood by far, which undoubtedly increases the difculty of elucidating the precise mechanism by which EA alleviates this disease. Inhibition of the innate immune response driven by TLRs signaling in the synovium seems to be the promising mechanism by which EA intervention alleviates the severity of RA, but the precise cellular and molecular mechanisms need to be further studied.

Conclusion
Taken together, the present fndings indicate that EA markedly alleviated the severity of CIA in the rats by reducing paw swelling, serum levels of pro-infammatory cytokines, and relieving joint pain. EA also can suppress the expression of TLR2/4-MyD88-NF-κB signaling pathway-related proteins in the synovium, especially the TLR2 and TLR4 expression on synovial fbroblasts and macrophages, and inhibition of TLR2/4 signaling could mimic the analgesic and anti-infammatory efects of EA. Tese data suggested that the anti-infammatory and analgesic efects of EA treatment on RA are closely related to the inhibition of innate immunity-mediated infammatory response in the macrophages and synovial fbroblasts driven by TLR2/4-MyD88-NF-κB signaling pathway in the synovium.

Data Availability
All data generated or analyzed during this study are included in this article.

Ethical Approval
Animal care and experimental protocols were approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (ethical approval number: D2017-08-16-1).

Conflicts of Interest
All authors declare that they have no conficts of interest.