Xueliankoufuye Suppresses Microglial Activation with Inflammatory Pain by Blocking NF-κB Signaling Pathway

Xuelian, as a traditional Chinese ethnodrug, plays an important role in anti-inflammation, immunoregulation, promoting blood circulation, and other physiological functions. It has been prepared into different traditional Chinese medicine preparations for clinical use, with xuelian koufuye (XL) being widely used to treat rheumatoid arthritis. However, whether XL can relieve inflammatory pain and its analgesic molecular mechanism are still unknown. The present study explored the palliative effect of XL on inflammatory pain and its analgesic molecular mechanism. In complete Freund's adjuvant (CFA)-induced inflammatory joint pain, oral XL dose-dependently improved the mechanical withdrawal threshold of inflammatory pain from an average value of 17.8 g to 26.6 g (P < 0.05) and high doses of XL significantly reduced inflammation-induced ankle swelling from an average value of 3.1 cm to 2.3 cm compared to the model group (P < 0.05). In addition, in carrageenan-induced inflammatory muscle pain rat models, oral XL dose-dependently improved the mechanical withdrawal threshold of inflammatory pain from an average value of 34.3 g to 40.8 g (P < 0.05). The phosphorylated p65 was inhibited in LPS-induced BV-2 microglia and spinal cord of mice in CFA-induced inflammatory joint pain within a value of 75% (P < 0.001) and 52% reduction (P < 0.05) on average, respectively. In addition, the results showed that XL could effectively inhibit the expression and secretion of IL-6 from an average value of 2.5 ng/ml to 0.5 ng/ml (P < 0.001) and TNF-α from 3.6 mg/ml to 1.8 ng/ml with IC50 value of 20.15 μg/mL and 112 μg/mL respectively, by activating the NF-κB signaling pathway in BV-2 microglia (P < 0.001). The above-given results provide a clear understanding of the analgesic activity and mechanism of action not found in XL. Considering the significant effects of XL, it can be evaluated as a novel drug candidate for inflammatory pain, which establishes a new experimental basis for expanding the indications of XL in clinical treatment and suggests a feasible strategy to develop natural analgesic drugs.


Introduction
Infammatory pain is a pain response caused by infammation due to peripheral tissue injury [1]. Nociceptive signals generated after peripheral tissue injury are transmitted to peripheral nerve-ending pain receptors, leading to central sensitization induced by microglia activation [2]. Long-term repeated noxious stimuli maintain the microglia in an activated state, resulting in chronic pain [3,4]. In response to noxious stimuli, an important molecular event that activates microglia is the activation of the nuclear transcription factor-κB (NF-κB) [5]. It regulates infammatory signals, responds to extracellular stimulation signals, and induces a large number of proinfammatory cytokines, eventually leading to central sensitization to mediate the upward transmission of infammatory pain signals [6]. Previous studies confrmed that the expression of proinfammatory factors, including IL-6, TNF-α, and IL-1β in microglia signifcantly decreased the mechanical withdrawal threshold of CFA-induced infammatory pain in rats [7]. In addition, blocking NF-κB activation alleviated microglial activation and allodynia in CFA-induced infammatory pain [8]. Terefore, inhibition of microglial activation is expected to be a potential strategy for relieving infammatory pain.
Te mechanism of action of analgesic drugs is generally categorized into central analgesia and peripheral analgesia, such as the central analgesic efect produced by the binding of phytocannabinoids to cannabinoid receptors, morphine to opioid receptors, and capsaicin to transient receptor potential channels (TRP channels), and the peripheral analgesia induced by nonsteroidal anti-infammatory drugs (NSAIDs). However, the long-term use of the above drugs is associated with adverse reactions such as mental addiction or gastrointestinal disorders. Due to the vast diversity and low adverse efects, a series of natural products derived from Chinese herbal medicines have always been the focus and hotspot for drug research and development, especially in managing chronic infammatory diseases. Ethnic medicine is a treasure of traditional Chinese medicine, with a good safety profle and promising bioactive molecules, which is a strategy to treat infammatory pain [9,10]. Bioactive compounds that perform well in controlled clinical trials are also emerging. As a representative of Xinjiang ethnodrug (Uyghur drugs), xuelian has anti-infammatory and immune regulation and has been made into diferent traditional Chinese medicines to treat clinical diseases [11]. Xuelian koufuye (XL) is extensively used for treating rheumatoid arthritis and dysmenorrhea caused by insufcient "kidney yang," "cold dampness," and "stasis." However, the possible mechanism by which XL exerts analgesic efects remains unclear; i.e., whether it inhibits pain through anti-infammatory action must be determined.
In this study, two in vivo infammatory pain models were established to evaluate the analgesic efect and explore underlying mechanisms. We found that XL, as an antiinfammatory analgesic, can inhibit the NF-κB signaling pathway to block hyperalgesia caused by excessive activation of microglia.

Drugs and Reagents. XL was provided by Xinjiang
Tianshan Lotus Pharmaceutical Co., Ltd. It was dissolved in Dulbecco's Modifed Eagle Medium (DMEM) for the experiments. Te cell culture reagents and enzyme-linked immunosorbent assay (ELISA) kits were purchased from the Invitrogen Corporation (Termo Fisher Scientifc, Carlsbad, CA, USA). Lipopolysaccharide (LPS), complete Freund's adjuvant (CFA), and carrageenan were acquired from Sigma-Aldrich (St Louis, MO, USA). p-p65, p65, and β-tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA). Total glucosides of paeony (TGWP) and diclofenac sodium (DS) were purchased as positive drugs, respectively.
Based on the conversion of data from the adult therapeutic dose to the dose administered to rats, we determined the doses administered for the positive drugs TGWP and DS as 100 mg/kg and 10 mg/kg. Te specifc conversion process is as follows: the adult daily dose of TGWP and DS is 1.2 g/70 kg and 110 mg/kg, and the rat dose is 6.17 times higher than the adult dose. Terefore, the rat doses of TGWP and DS are approximately 100 mg/kg and 10 mg/kg.

Experimental Animals.
Female SD rats (weighing 180-200 g) were obtained from Beijing Huafukang Experimental Animal Institute (Beijing, China). Te rats were housed 5 to 6 per cage at room temperature (22 ± 2°C) in specifc pathogen-free conditions under 12/12-h reversed light-dark cycles, with food and water provided ad libitum. Te rats were acclimatized for 3-4 days prior to the experiments and randomly divided into diferent groups.

CFA-Induced Infammatory Joint Pain
Model. CFA (100 μL) was injected into each rat's right paw (ipsilateral paw) to construct the model of infammatory joint pain [12]. Te control rats were injected in the same way with normal saline solution. Te rats were randomly divided into 7 groups (n � 10): control group (sham), model group, three XL-treated groups (250, 500, and 1000 mg/kg), and positive control groups of TGWP (100 mg/kg) and DS (10 mg/kg) treatment. Te mechanical withdrawal threshold of the rats in each group was measured at 0.5, 1, 2, and 4 h after daily oral administration.

Carrageenan-Induced Infammatory Muscle Pain Model.
An infammatory muscle pain model was constructed by injecting 100 μL of 3% carrageenan (pH � 6.0) into the gastrocnemius muscle of the rats [13]. Te control rats were injected with normal saline solution in the same way. Te grouping, administration, and measurement of the mechanical withdrawal threshold of the rats were the same as the experiments in the CFA-induced infammatory joint pain model.

Mechanical Withdrawal Treshold of the Paw.
Te mechanical withdrawal threshold of the rats was measured by the electronic Von Frey withdrawal test (Von Frey flaments, IITC Life Science Inc., California, USA) 30 min, 1 h, 2 h, and 4 h after administration. Te animals were acclimatized in boxes set on an elevated metal mesh foor for at least 30 min. Te behavioral analyses were performed by an investigator blinded to the experimental grouping.
2.6. Cell Culture and Viability Assay. BV-2 microglia were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 and 95% humidity. To assess cell viability, the BV2 cells (2 × 105 cells/mL) were seeded in 96-well plates and incubated at 37°C under 5% CO 2 with LPS (1 μg/mL) stimulation. After the cells were cotreated with XL with diferent concentrations and LPS (1 μg/mL) for 24 hours, cell counting kit 8 (CCK8) reagent was added to each well. After 1 hour of incubation, absorbance levels for formazan were measured at 450 nm using a microplate reader (BioTek, VT, USA).

Western Blotting Assay.
After preincubation of BV-2 microglia with diferent concentrations of XL for 1 hour, LPS was used for 24 hours to activate microglia, BV2 cells were washed with phosphate-bufered saline (PBS) solution and treated with 1× cell lysis bufer containing 1× protease and phosphatase inhibitor cocktails. After incubating on ice for 30 minutes, the cells were collected and centrifuged at 12000 rpm for 10 minutes at 4°C. Te spinal dorsal lumbar enlargements were separated from mice after measurement of the mechanical withdrawal threshold and then lysed to extract protein. Te protein concentration in the cell and tissue lysates were determined by BCA Protein Assay Kit (Termo Fisher Scientifc, Carlsbad, CA, USA), and 20 μg of proteins per sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Te bands were transferred to a polyvinylidene fuoride (PVDF) membrane (Millipore Co., Bedford, MA, USA) that was then blocked with 5% bovine serum albumin (BSA) in tris-based saline-Tween 20 (TBST) at room temperature for 1 h. After overnight incubation with the primary antibodies against p-NF-κB p65, NF-κB p65, and β-tubulin (all diluted 1 : 1000) at 4°C, the blots were then incubated with goat antirabbit and goat antimouse secondary antibodies. Te membrane was developed using enhanced chemiluminescence reagents (PerkinElmer, USA), the bands were visualized with Tanon 2000 Imaging system (Beijing, China), and their intensities were quantifed using ImageJ software (NIH, USA).

ELISA Assay.
After preincubating BV-2 microglia with diferent concentrations of XL for 1 hour, microglia were activated with LPS for 24 hours, and then the cell supernatant was collected to detect proinfammatory factors. Te concentrations of proinfammatory cytokines in the culture supernatant were determined using an ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Statistical Analysis.
Te data were expressed as means ± SEM. Statistical signifcance was determined by two-way ANOVA followed by post hoc Tukey analyses P < 0.05 was considered signifcant.

XL Alleviated CFA-Induced Infammatory Joint Pain in
Rats. CFA-induced infammatory joint pain model rats were used to detect the mechanical withdrawal threshold using the electronic Von Frey test to evaluate the efect of XL (Figure 1(a)). Subcutaneous injection of CFA decreased the mechanical withdrawal threshold in the ipsilateral paw from an average value of 53.7 g to 19.2 g, without afecting the contralateral paw (P < 0.001; Figure 1(b)). However, 500 mg/ kg and 1000 mg/kg of XL signifcantly increased the mechanical withdrawal threshold in the ipsilateral paw in a dose-dependent manner from an average value of 17.8 g to 26.6 g compared to the placebo-treated mice (P < 0.05 or P < 0.001; Figure 1(c)). Te analgesic efect of XL peaked within one hour, remaining stable over fve consecutive days (P < 0.05 or P < 0.001; Figure 1(d)). Positive control of TGWP and DS did not exhibit a noticeable analgesic efect.

XL Relieved CFA-Induced Paw Tickening and Ankle
Swelling. CFA injection of rat paws induced a severe infammatory response, with decreased pain threshold and paw swelling. To explore whether XL played an analgesic role through anti-infammatory action, we observed the degree of paw swelling in each group with continuous daily administration of XL. Te results showed that paw thickness was alleviated by the oral administration of XL in the infammatory joint pain rat model compared to the CFA injection group alone (Figure 2(a)). In addition, ankle circumference was measured in each group of rats. High doses of XL signifcantly reduced infammation-induced ankle swelling from an average of 3.1 cm to 2.3 cm compared to the model group (P < 0.05; Figure 2(b)).

XL Alleviated Carrageenan-Induced Infammatory Muscle Pain in Rats.
To explore the efect of XL on infammatory muscle pain, we injected 3% carrageenan into the rats' gastrocnemius muscle to induce muscle infammation (Figure 3(a)). Te electronic Von Frey test was used to detect the rats' mechanical withdrawal threshold. Our data showed that carrageenan signifcantly decreased the mechanical withdrawal threshold in the ipsilateral paw from an average value of 54.2 g to 36.5 g, but not the contralateral paw (P < 0.001; Figure 3(b)). A high dose of XL increased the mechanical withdrawal threshold in the ipsilateral paw from an average value of 34.3 g to 40.8 g compared to the placebo-treated mice (P < 0.001 or P < 0.05; Figure 3(c)). Meanwhile, TGWP and DS also increased the mechanical withdrawal threshold. Te analgesic efect of XL peaked within one hour, remaining stable over fve consecutive days (P < 0.05; Figure 3(d)).

XL Inhibited Painful Response Induced by Microglial
Activation via the NF-κB Signaling Pathway. Neuroinfammation induced by microglial activation is involved in transmitting infammatory pain signals. To explore the anti-infammatory mechanism of XL, we evaluated the efect of XL on LPS-induced NF-κB phosphorylation in BV-2 microglia and the spinal cord. Under LPS stimulation, XL did not afect the viability of BV-2 microglia at a concentration range of 0.03-100 μg/mL (Figure 4(a)). Western blotting results showed that XL inhibited phosphorylation of p65 in BV-2 microglia and spinal cord within the value of 75% and 52% reduction on average, respectively (P < 0.001 or P < 0.05; Figures 4(b) and 4(c)).

XL Inhibited Secretion of Proinfammatory Cytokines Induced by Microglial Activation via the NF-κB Signaling
Pathway. Since XL inhibited painful responses induced by microglial activation via the NF-κB signaling pathway, we fnally investigated the efect of XL on the activation of BV-2 microglia and secretion of proinfammatory cytokines. Compared with the LPS stimulation group alone, XL inhibited the secretion of IL-6 from an average value of 2.5 ng/ml to 0.5 ng/ml (P < 0.001; Figure 5(a)) and TNF-α from an average value of 3.6 mg/ml to 1.8 ng/ml (P < 0.001; Evidence-Based Complementary and Alternative Medicine Figure 5(c)) in the culture supernatant of BV-2 microglia, with IC 50 value of 20.15 μg/mL ( Figure 5(b)) and 112 μg/mL ( Figure 5(d)) in a dose-dependent manner, respectively. Te above-given results indicated that XL inhibited the neuroinfammation induced by LPS.

Discussion
Te search for therapeutic agents with anti-infammatory and analgesic efects in natural products has always been an important direction to relieve infammatory pain, and many active ingredients have been evaluated in a large number of animal pain models in preclinical studies, including infammatory pain induced by acetic acid, carrageenan, and formalin [14][15][16]. Although XL is widely used in the clinic for various diseases, its efcacy in relieving infammatory joint pain remains unclear. Here, our study showed for the frst time that XL could efectively relieve infammatory joint pain induced by CFA and muscle pain induced by carrageenan in a rat model, respectively. Tis gives us a hint that XL has the potential to become a pharmaceutical formulation for the treatment of infammatory pain.
Proinfammatory factors play an essential role in infammatory diseases. Excessive infammatory responses induce peripheral and central pain sensitization, which is also the main pathway for diferent types of infammatory pain [17]. Terefore, natural products with signifcant anti-infammatory efects intervene in central and peripheral pain responses by efectively inhibiting nociceptive sensations and infammatory receptors [18]. Further research indicated that XL inhibited the expression of IL-6 and TNF-α in LPS-induced BV-2 cells. Mechanistically, XL suppressed the phosphorylation of p65 of the NF-κB signaling pathway in activated BV-  Data are expressed as means ± SEM (n � 10 rats in each group). Statistical signifcance was determined by two-way ANOVA followed by post hoc Tukey analyses where * * * P < 0.001 vs. control-ipsilateral paw, ### P < 0.001 vs. control group, * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. model group. 4 Evidence-Based Complementary and Alternative Medicine 2 cells involved in infammatory pain by activating the infammatory signaling. Tus, XL could be a potential therapeutic strategy to alleviate the infammatory pain associated with microglial activation. Neuroinfammation induced by microglial activation mediates the pathological development of infammatory pain [19]. Noxious stimuli acting on microglia can induce microglial activation with a strong phagocytic function and infammatory response. Terefore, many proinfammatory cytokines are produced, including IL-6, TNF-α, and iNOS promoting the infammatory response in the damaged area and aggravating the damage to neurons, leading to the pain nerve signal transmission and sensitization of central pain [20,21]. Neuroprotection by inhibiting the neuroinfammatory response caused by the overactivation of MAPKs and NF-κB signaling pathways is an important strategy to address disorders associated with infammatory and neuropathic pain. In this study, in vitro experiments showed that BV-2 microglia exposed to LPS produced a stronger infammatory response, which can mimic neuroinfammation induced by microglial activation when infammatory pain occurs. However, XL dosedependently inhibited the secretion of IL-6 and TNF-α produced by BV-2 microglia due to the infammatory response. Te above-given fndings demonstrated that XL inhibited LPS-induced microglia activation and neuroinfammation in BV-2 cells.
In an overly aggressive infammation-induced pain response, cytokines cause the activation of multiple signaling pathways, resulting in the continuous conduction of pain signals [22]. A series of natural derivatives inhibit the activation of pain signaling pathways by reducing the secretion of cytokines such as IL-6 and TNF-α, thus achieving analgesic efects [23,24]. Whether the signifcant analgesic efect shown by XL is related to the inhibition of these signaling pathways needs further experimental confrmation. Terefore, we next explored the possible molecular mechanism of XL to inhibit the activation of BV-2 microglia that results in neuroinfammation. An important molecular event in microglial activation is the activation of the NF-κB signaling pathway, which regulates cell proliferation, differentiation, survival, and apoptosis by an infammatory response and the expression of related infammatory cytokines [25,26]. Various bioactive ingredients, such as favonoids and glycosides, can exert anti-infammatory, antibacterial, antiviral, immunomodulatory, and other biological efects by inhibiting the NF-κB signaling pathway [24]. In the present experiment, XL inhibited the phosphorylation of the transcription factor NF-κB and the downstream expression of IL-6 and TNF-α under LPS stimulation. At the same time, whether it is CFA-induced infammatory joint pain or carrageenan-induced infammatory muscle pain model, XL has good analgesic effects. Terefore, the above-given fndings indicated that XL inhibited the infammatory cytokines produced by the  Figure 2: Anti-infammatory efects of XL on complete Freund's adjuvant (CFA)-induced infammatory joint pain in rats. (a) Paw thickness of rats with infammatory joint pain injected by CFA was shown as microscopic evidences. (b) Ankle circumference was measured in each group of rats. Data are expressed as means ± SEM (n � 7 rats in each group). Statistical signifcance was determined by two-way ANOVA followed by post hoc Tukey analyses where ### P < 0.001 vs. control group, * * P < 0.01, * * * P < 0.001 vs. model group.
Evidence-Based Complementary and Alternative Medicine activation of the NF-κB signaling pathway in spinal microglia, which may be the main mechanism of its analgesic efects.
Previous studies have reported that CFA can efectively activate microglia in the spinal cord to secrete of infammatory cytokines, causing mechanical allodynia [27,28]. Tis is due to the fact that CFA can cause an intrinsic immune response dominated by pathogen-associated molecular patterns (PAMPs), which leads to a persistent infammatory environment and microglia activation. It has been suggested that microglial activation plays an important role in infammatory pain. Inhibition of microglial activation can decrease the mechanical hypersensitivity caused by peripheral infammation and nerve damage [29]. Proinfammatory cytokines also play an important role in maintaining pain. Intrathecal injection of IL-6, TNF-α, and IL-1β signifcantly decreased the pain threshold [30,31], blocking proinfammatory cytokines via antibodies or eliminating pain behaviors linked to infammation or neuropathic pain [32][33][34]. Tus, bioactive compounds with signifcant anti-infammatory activity are potential therapeutic agents for relieving infammatory pain and neuralgia. In the present study, XL inhibited the secretion of proinfammatory factors in microglia, such as IL-6 and TNF-α. Te results demonstrated that XL inhibited CFA-induced mechanical allodynia by blocking microglial activation and secretion of proinfammatory cytokines. Tis study provides a new experimental basis for expanding the indications of XL in clinical treatment and suggests a feasible strategy to develop natural analgesic drugs. However, there are still some limitations in this study, frstly, although the infammatory signaling pathway afected by XL in pain response was found, the exact molecular target of action has not been confrmed and further experimental analysis is  Figure 3: Efect of XL on carrageenan-induced infammatory muscle pain in rats. Te analgesic activity evaluation protocol is operated in accordance with (a). (b) After carrageenan injection, the mechanical withdrawal threshold of the ipsilateral paw in the model group was signifcantly reduced, which was statistically diferent from that of the control group, while the contralateral mechanical withdrawal threshold was not afected. (c) After oral administration of XL 30 mins, 1 h, 2 h and 4 h mechanical withdrawal threshold was measured by electronic Von Frey test. (d) On the frst day, the third day, and the ffth day after carrageenan injection, the mechanical withdrawal threshold of rats one hour after oral administration of XL was measured by electronic Von Frey test. Data are expressed as means ± SEM (n � 10 rats in each group). Statistical signifcance was determined by two-way ANOVA followed by post hoc Tukey analyses where * * * P < 0.001 vs. control-ipsilateral paw, ### P < 0.001 vs. control group, * P < 0.05, * * P < 0.01, * * * P < 0.001 vs. model group.  Evidence-Based Complementary and Alternative Medicine needed, and secondly, to expand the prospects of XL in pain clinical treatment, more pain models such as cancer pain and diabetic pain are needed to support its preclinical pharmacological activity evaluation, which is a must for the development of natural analgesic drugs.

Conclusion
In summary, infammatory pain is associated with microglial activation and subsequent NF-κB signaling pathway activation and secretion of infammatory factors in the spinal cord. In the present study, XL improved the CFA-induced infammatory joint pain and carrageenan-induced infammatory muscle pain by raising the mechanical withdrawal threshold of rat paws. Moreover, XL reduces central sensitization in the pain signaling process by inhibiting the expression of infammatory factors mediated via NF-κB activation in microglia ( Figure 6). Taken together, our fndings suggest a new indication of XL for expanding the analgesic efect.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

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