Abstract
Chronic pain, along with comorbid psychiatric disorders, is a common problem worldwide. A growing number of studies have focused on non-opioid-based medicines, and billions of funds have been put into digging new analgesic mechanisms. Peripheral inflammation is one of the critical causes of chronic pain, and drugs with anti-inflammatory effects usually alleviate pain hypersensitivity. Sophoridine (SRI), one of the most abundant alkaloids in Chinese herbs, has been proved to exert antitumor, antivirus and anti-inflammation effects. Here, we evaluated the analgesic effect of SRI in an inflammatory pain mouse model induced by complete Freund’s adjuvant (CFA) injection. SRI treatment significantly decreased pro-inflammatory factors release after LPS stimuli in microglia. Three days of SRI treatment relieved CFA-induced mechanical hypersensitivity and anxiety-like behavior, and recovered abnormal neuroplasticity in the anterior cingulate cortex of mice. Therefore, SRI may be a candidate compound for the treatment of chronic inflammatory pain and may serve as a structural basis for the development of new drugs.
Keywords: Chronic pain, anxiety, anterior cingulate cortex, sophoridine, inflammation
Graphical Abstract.
Introduction
Chronic pain, a common disease characterized by persistent nociceptive hypersensitivity, affects 30% of worldwide, bringing enormous personal and economic burden. 1 Moreover, chronic pain is frequently comorbid with mood disorders, such as anxiety and depression, which enhance the perception of pain in patients. Currently, analgesic drugs, including opioids, steroidal, nonsteroidal anti-inflammatory drugs and serotonin-norepinephrine reuptake inhibitors, may have significant adverse side effects.2,3 Thus, it is urgent to develop novel and safe drugs treating chronic pain.
The anterior cingulate cortex (ACC) plays a crucial role in sensory perception and long-term enhancement and development of negative affect in inflammatory pain conditions.4,5,6 Mounting efforts have revealed that changes of synaptic plasticity in ACC are related to pain maintenance, and restoring the status of synaptic plasticity has positive effects in pain relieving and affective disorders treatment. 7 Several key molecules, like α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, N-methyl-D-aspartate (NMDA) receptors, are involved in neuroplasticity formation and required for pain persistence, and some analgesic drugs could significantly modulate these molecules.8,9,10
Inflammation is one of the most causes of chronic pain, local or systemic inflammation participates in the occurrence and development of pain. Pro-inflammatory cytokines, like TNF-α, IL-1β and IL-6, released during inflammatory process act on neurons and cause neuroplasticity change, which is the common performance for all types of inflammatory pain. 11 Microglia is considered as the resident immune cell of the central nervous system, 12 exerting the main source of cytokines at the beginning of tissue damage. A growing number of studies have proved the important role of microglia in pain development, and inhibiting its activation and pro-inflammatory cytokines release could be an effective way to relieve nociceptive hypersensitivity and associated affective symptoms in inflammatory pain.13,14,15
Chinese traditional medicines have long been considered as safe method of treatment and used in various diseases. A number of studies have demonstrated the potential application of substance products from these herbs in inflammatory pain treatment. Sophoridine (SRI) is one of the most abundant alkaloids in Chinese herbs, and has been approved in tumor suppression. 16 SRI, containing quinolicidine structure (Figure 1), has a wide range of pharmacological activities, including antitumor, antivirus, hepatoprotection, neuroprotection and anti-inflammation. It is still unclear whether SRI could alleviate inflammatory pain and pain-related affective disorders.
Figure 1.
The chemical structure of SRI.
In this study, we investigated the possible role of SRI in CFA-induced chronic pain and anxiety. SRI reduced pro-inflammatory cytokines release from microglia in vitro and that in serum of model mice. SRI administration relieved elevated neuroplasticity proteins, including phosphorylated GluA1, GluN2A and GluN2B. In vivo studies showed that SRI could significantly alleviate inflammatory pain and anxiety-like behavior in CFA-injection model mice. Our results suggest that SRI may be a candidate compound for the treatment of inflammatory pain.
Materials and methods
Cell culture and treatment
Immortalized mouse BV2 microglial cell lines line was. purchased from the China Center for Type Cultuer Collection (GDC0311, CHN). BV2 microglial cell lines were cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 95% air and 5% CO2, and the medium was changed every two days. The cells were split with 0.25% trypsin at 80% confluence and sub-cultured for additional passages. Sophoridine was purchased from the Meilunbio (MB7130, USA), LPS was purchased from the Sigma–Aldrich (L2630, USA). Cells with or without LPS (1 μg/mL) process 24 h, at the same time, different concentrations of SRI (0.1, 1, 10 μm) with 24 h.
Mice and treatment
The animals used in this study were 6–8 week old male C57BL/6 mice. The animals were randomly divided into groups and placed in six-animal cages, with food and water provided at will. The feeding temperature was 24 ± 2° C, the relative humidity was 50–60%, and the light-dark cycle was 12 h. We fed all the mice a commercial diet and allowed them to acclimate to the laboratory environment for at least a week before the start of the experiment.
Chronic inflammatory pain was induced by an intraplantar injection of CFA (10 μL, 50% in saline, Sigma, St. Louis, MO, USA) into the plantar surface of the left hindpaw of the mice. The sham group was injected with the same amount of saline.
Animals were randomly distributed into five groups: a Sham group, a CFA group, and three groups that received different doses of SRI (2.0, 10.0, 50.0 mg/kg). Each group contained 6 mice. The SRI groups were given intraperitoneal injections of SRI after the CFA injection once a day for 3 consecutive days. The Sham group was injected with an equal volume of saline at the same time.
Cytokine analysis
The levels of TNF-α, IL-6 and IL-1β in serum and culture supernatant were detected by ELISA according to the manufacturer’s instructions. Mouse IL-1β ELISA kit (BMS6002TEN), mouse IL-6 ELISA kit (BMS603-2), mouse TNF-α ELISA kit (BMS607-3) were purchased from Thermo Fisher.
Behavioral tests
Mechanical pain threshold test
The automatic plantar mechanical prickling instrument was purchased from Jiangsu Cylon Biological Technology Co., LTD (SA502) The mice need to adapt to the experimental environment for 3 hours in advance before the test, and the number of grams displayed during the rapid paw withdrawal reaction was used as an index to evaluate the pain threshold of the mice.
Thermal pain threshold test
The cold and hot plate pain measuring instrument was purchased from Nanjing Calvin Biotechnology Co., LTD. (KW-LB), and the mice were allowed to adapt to the experimental environment for 3 hours in advance before measurement. The temperature of the board was set at 55°C, and the time of lifting the feet was recorded as an index to evaluate the pain threshold of the mice.
Open field test (OFT)
All mice were subjected to the open field test (OFT) and the elevated plus maze (EPM) test, which were conducted as described in previous reports. 17
All tests were performed during the dark phase, and the light intensity was controlled under the same conditions. The instrument (VanBi-OF, Shanghai FanBI Intelligent Technology Co., LTD) was conducted in a square box (40 cm × 40 cm × 40 cm) without a lid at 14 d after CFA injection, and the central region was the middle 24 × 24 cm region. For the testing, each mouse was placed in the center of the box and allowed to freely explore for 15 min. The exploratory behaviors of the mice were recorded using a camera fixed above the chamber. Before the test, the mice were acclimated to the experimental environment for 3 h, and the experimental process was kept quiet. Before the next mouse was tested, the instrument was wiped clean with 70% ethanol to avoid the influence of smell on the mice. The tracking of mice was analyzed using a video-tracking system (VanBi Tracking Master V3.0, Shanghai FanBI Intelligent Technology Co., LTD). The OFT was always performed before the EPM, but both tests were conducted on the same day.
Elevated plus-maze testing (EPM)
All tests were performed during the dark phase, and the light intensity was controlled under the same conditions. The instrument (VanBi-OF, Shanghai FanBI Intelligent Technology Co., LTD) consisted of two open arms (25 × 8 × 0.5 cm3) and two closed arms (25 × 8 × 15 cm3) that extended from a common central zone (8 × 8 cm2). For each test, each mouse was placed in the central area facing the open arm and subsequently allowed to explore freely for 5 min while being filmed with a camera fixed above the maze. Before the test, the mice were acclimated to the experimental environment for 3 h, and the experimental process was kept quiet. Before the next mouse was tested, the instrument was wiped clean with 70% ethanol to avoid the influence of smell on the mice. The time spent in and the number of entries into the open and closed arms were analyzed with a video-tracking system (VanBi Tracking Master V3.0, Shanghai FanBI Intelligent Technology Co., LTD).
Western blotting
On day 14 after CFA injection, Bilateral ACC of mice were quickly separated. The total protein extraction kit is used to lyse the tissues in the lysate buffer, and the protein concentration in the lysate is quantified by BCA. After SDS-PAGE separation, the protein was transferred to the polyvinylidene fluoride membrane and sealed with 5% skimmed milk in Tris HCl buffer salt solution (TBST) for 2 h. After washing with TBST, incubate the membrane with the indicated antibody at 4°C overnight: β-Actin (mouse, 1:10000, NB600-501, Novus), GluN2A (rabbit, 1:1000, ab124913, Abcam), GluN2B (rabbit, 1:3000, ab124913, Abcam), p-GluR1-Ser831(rabbit, 1:5000, ab109464, Abcam), p-GluR1-Ser845(rabbit, 1:5000, ab76321, Abcam), and GluR1(rabbit, ab31232, 1:1000). After washing with TBST, incubate the corresponding secondary antibody at room temperature for 1 hour. Develop with chemiluminescent agent (reagent A: reagent B = 1:1).
Data analysis
Results are expressed as the mean ± standard error of the mean (SEM). Statistical analysis of multiple groups was performed using one-way analysis of variance (ANOVA) in Microsoft Excel and Prism (GraphPad, San Diego, CA, USA). In all cases, p < 0.05 was considered to be statistically significant.
Results
SRI inhibit the release of LPS-induced pro-inflammatory factors in BV2
Since inflammation plays a crucial role in chronic inflammatory pain, we first verified the effect of SRI on LPS-induced inflammatory factors in BV2 cells. The anti-inflammatory effect of SRI was first investigated in BV2 cells, and Elisa results showed that TNF-α, IL-6, and IL-1β secreted by microglia in the LPS group were increased compared with the control group (Figure 2). However, TNFα, IL-6, and IL-1β secreted by microglia in the SRI group were gradually decreased with increasing SRI concentration compared with the LPS group (Figure 2). These results indicate that SRI can inhibit the release of inflammatory factors and has a certain anti-inflammatory effect.
Figure 2.
SRI suppressed pro-inflammatory cytokine levels in LPS reduced BV2 cells. The content of inflammatory factors in BV2 cells. (a) The contents of TNF-α in the BV2 were detected by ELISA. (b) The contents of IL-6 in the BV2 were detected by ELISA. (c) The contents of IL-1β in the BV2 were detected by ELISA. (“*” means compared with the control group at p < 0.05, “**” means compared with the control group at p < 0.01.)
SRI alleviated CFA induced hyperalgesia in mice
After confirming the anti-inflammatory effect of SRI in vitro, we hypothesized that SRI could exert an analgesic effect, which was subsequently tested in C57BL/6 mice in vivo. In the mechanical pain threshold test, mice in the CFA group showed a significant reduction in pain threshold after CFA injection compared to the sham group, and the pain threshold of SRI treated mice was significantly higher than that of CFA treated mice (Figure 3a). In the contralateral mechanical pain threshold test, there was no significant difference in the pain threshold of each group (Figure 3b). In the thermal pain threshold test, after CFA injection, the pain threshold of CFA group mice was significantly reduced compared with the sham group, and the pain threshold of SRI treated mice was significantly higher than that of CFA treated mice (Figure 3c). The ipsilateral hindpaw of mice usually swells after CFA injection. We also measured the thickness of the hindpaw using the vernier caliper. The results showed a significant increase in the thickness of the hindpaw in the CFA group compared to the sham group, as well as redness and swelling. The hindpaw thickness of SRI mice was significantly lower than that of CFA mice (Figure 3d). These results suggest that SRI exerts a potent analgesic effect in a chronic inflammatory pain model and also has a significant effect on inflammatory symptoms.
Figure 3.
SRI alleviates CFA-induced hyperalgesia and hindpaw inflammation. (a) The mechanical pain threshold of ipsilateral in mice receiving CFA injection. (b) The mechanical pain threshold of contralateral in mice receiving CFA injection. (c) The thermal pain threshold in mice receiving CFA injection. (d) The maximum dorsal-ventral thickness of the CFA-injected hindpaw. (n = 6, “**” means compared with the sham group at p < 0.05. “##” means compared with the CFA group at p < 0.01.)
SRI alleviated CFA induced anxiety-like behavior in mice
The effects of SRI on anxiety-like behavior in mice were assessed using OFT (Figure 4a) and EPM (Figure 4e) tests. The results of OFT showed that the central area time of CFA group mice was significantly shorter than that of sham group mice, and compared with the CFA group, the time of central area of mice was significantly increased after SRI treatment (Figure 4b). The CFA group of mice also showed a significant reduction in central distance. SRI treatment increased the distance traveled in the central area of mice (Figure 4c). The total distance traveled showed no remarkable change in any group, suggesting that the mice had no deficit in locomotor activities (Figure 4d). Results from the EMP test showed that the CFA group had significantly less open arms time compared to the sham group, while the CFA group had increased open arms time after SRI therapy (Figure 4f). Compared with the sham group mice, the number of times CFA mice entered the open arms was also significantly reduced, and the number of times CFA mice entered the open arms was increased after SRI treatment (Figure 4g) Compared with the sham group mice, the open arms distance traveled of CFA group mice was also significantly reduced. Similarly, the open arms distance traveled of mice increased after SRI treatment (Figure 4h). These results indicate that CFA does cause anxiety like behavior in mice, and SRI can ameliorate anxiety like behavior in mice.
Figure 4.
SRI alleviates CFA-induced anxiety behaviors. (a) Representative traces of locomotor activity in the OFT. (b)The time spent in the central area of mice in the OFT after CFA injection. (c) The distance traveled in the central area of mice in the OFT after CFA injection. (d) The total distance of mice in the OFT after CFA injection. (e) Representative traces of locomotor activity in the OFT and EPM. (f)The time spent in the open arms of mice in the EPM after CFA injection. (g) The distance traveled in open arms of mice in the EPM after CFA injection. (h) The number of entries into the open arms of mice in the EPM after CFA injection. (n = 6 mice per group, “*” means p < 0.05, “**” means p < 0.01).
SRI inhibit the release of CFA-induced pro-inflammatory factors in the mice
To further test the anti-inflammatory effect of SRI, the serum levels of pro-inflammatory cytokines were also measured. Elisa results showed that the serum secretion of TNF-α, IL-6, and IL-1β was increased in the CFA group compared with the sham group (Figure 5); TNFα, IL-6, and IL-1β secreted by microglia were significantly reduced in the SRI group compared with the CFA group (Figure 5); These results indicate that SRI can also inhibit the release of inflammatory factors and play an anti-inflammatory role in vivo.
Figure 5.
SRI suppressed pro-inflammatory cytokine levels in the serum of CFA-injected mice. (a) The contents of TNF-α in the serum were detected by ELISA. (b) The contents of IL-6 in the serum were detected by ELISA. (c) The contents of IL-1β in the BV2 were detected by ELISA. (n = 6, “*” means p < 0.05, “**” means p < 0.01).
SRI reverses the expression or function of ionized glutamate receptors induced by CFA
Glutamate ionotropic receptors in ACC play an important role in excitatory and inhibitory transmission, which imbalance can lead to chronic pain and anxiety. Hence, we determined the expression alterations in excitatory AMPA and NMDA receptors, which play crucial roles in regulating synaptic neurotransmission and plasticity. The results showed that the expression of GluN2B, p-GluR1-Ser831 and p-GluR1-Ser845 were significantly increased in the CFA group compared to the sham group, and the expression of GluN2A and GluR1 were not significantly different. Protein expression levels of GluN2B, p-GluR1-Ser831 and p-GluR1-Ser845 were significantly reduced after SRI treatment compared to the CFA group (Figure 6). These results suggest that SRI may restore the abnormal expression of glutamate ionotropic receptors in the ACC region of CFA mice, which may play an essential role in the analgesic and anxiolytic effects of SRI.
Figure 6.
Protein expression of glutamate ionized receptors NMDAR and AMPAR in ACC. (a) The expression of protein was analyzed in ACC by Western blot. (b) The expression of GluN2A in each group. (c) The expression of GluN2B in each group. (d) The expression of p-GluR1-Ser831 in each group. (e) The expression of p-GluR1-Ser845 in each group. (f) The expression of GluR1 in each group. (n = 6, “*” means p < 0.05, “**” means p < 0.01).
Discussion
SRI, a quinolizidine-based alkaloid obtained from the stem and leaves of medicinal plants Euchresta japonica Benth and Sophora alopecuroides L., 18 attenuated inflammatory pain and associated anxiety-like behaviors induced by CFA-injection in the hindpaw of mice. The increased lease of pro-inflammatory factors, TNF-α, IL-1β and IL-6, after LPS stimulation in BV-2 cells was inhibited by SRI. SRI administration also reduced these pro-inflammatory factors in the serum of CFA-injected mice. GluN2A- and GluN2B-containing NMDA receptors and phosphorylation levels of GluA1-containing AMPA receptors, which were considered as key molecules responsible for neuroplasticity, were obviously upregulated in the ACC of CFA-induced model mice. While, SRI treatment reversed the expression of above proteins. These data suggest that SRI exerted analgesic and anxiolytic effects by modulating ACC microglia activation, inhibiting inflammation and recovering neuroplasticity.
Inflammation, one of the main causes of chronic pain and affective disorders, is induced by tissue injury, chemical stimuli and autoimmune processes.19,20 The levels of inflammatory factors provide the indicator of therapies, and a growth number of evidence has shown the regulatory effect of analgesic drugs on inflammatory factors. 21 Microglia is considered as the main immune cell in the CNS, supports and affects neurons all the time. Local damage and stimuli activate microglia, which releases high levels of pro-inflammatory factors, acting on neurons and inducing neuroplasticity, enhancing the perception of pain and increasing the possibility of occurrence of negative emotion.22,23 Previous study have shown that SRI could inhibit lung inflammation through reducing the secretion of inflammatory factors in LPS induced acute lung injury mice. 24 The analgesic effects of alkaloids, such as marine 25 and sinomenine, 26 have been proved. Therefore, we assumed SRI may exert analgesia effect through inhibiting inflammation. We performed in vitro studies on BV-2 cells, and found that SRI pretreatment significantly reduced pro-inflammatory factors releasing in medium after LPS stimulation. The anti-inflammatory effects of SRI were also obtained by determining the levels of TNF-α, IL-6 and IL-1β in the serum of CFA-injected mice.
Abnormal neuroplasticity, which is responsible for the onset and progression of some CNS diseases, plays critical role in chronic pain and affective diseases. A large number of studies have proved that neuroplasticity change in ACC participates in the modulation of sensory and emotional responses.27,28 At the induction phase of pain, presynaptic glutamate release increases and transfers to postsynaptic membranes, activity-dependent Ca2 + flux through NMDA receptors activation, leading to AMPAR potentiation, which manifested as high level of phosphorylation of GluA1 subunit and transporting onto postsynaptic membranes. 29 Accumulation of postsynaptic AMPA receptors within ACC is positively associated with behavioral hyperalgesia under chronic pain conditions, and inhibition of ACC activity with ether pharmacological or epigenetic methods could alleviate pain sensitization and improve emotional performance. 30 In the present study, the expressions of GluN2B, p-GluR1-Ser831 and p-GluR1-Ser845 were significantly increased in the CFA group compared with the sham group, while the expressions of GluN2A and GluR1 did not significantly changed. Compared with the CFA group, the protein expression levels of GluN2B, p-GluR1-Ser831 and p-GluR1-Ser845 were significantly decreased after SRI treatment. The results suggest that the abnormal expression or function of glutamate ion receptors is involved in chronic inflammatory pain, and SRI treatment alleviates pain hypersensitivity and anxiety induced by CFA.
In conclusion, the present study showed that SRI relieves inflammatory pain induced by CFA injection in the hindpaw of mouse. It also established that inhibition of proinflammatory factor release and modulation of neuroplasticity attribute to the analgesic and anxiolytic effects of SRI. SRI may be considered as a potential compound to treat chronic pain, but future studies will be need to produce a more thorough understanding of the mechanisms involved.
Footnotes
Author contributions: M-GZ and Y-NL conceived and designed the study. ZR, YC, YQ, C-YC, JZ and L-FL acquired, analyzed, and interpreted the data. LY and XM drafted and edited the manuscript. Y-MW and S-BL made the revision. All the authors have read and agreed to the published version of the manuscript.
The authors declared no potential conflicts of interest with respect to the research, authorship, or publication of this article.
Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by The National Natural Science Foundation of China [grant numbers 31800887, 31972902] and the China Postdoctoral Science Foundation [2020M683750], and partially by Young Talent Fund of University Association for Science and Technology in Shaanxi, China [20200307].
Ethical approval: The animal study was reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Fourth Military Medical University.
Data availability: Original data is available from the corresponding authors upon reasonable request.
ORCID iDs
Le Yang https://orcid.org/0000-0003-1706-5241
Ming-Gao Zhao https://orcid.org/0000-0002-7539-3887
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