Oral treatment with glycyrrhizin inhibits NLRP3 inflammasome activation and promotes microglial M2 polarization after traumatic spinal cord injury

Journal Pre-proof Oral Treatment with Glycyrrhizin Inhibits NLRP3 Inflammasome Activation and Promotes Microglial M2 Polarization after Traumatic Spinal Cord Injury Xiao-Qiang Su (Writing - original draft), Xiang-Yang Wang (Project administration), Fu-Tai Gong (Project administration), Min Feng (Project administration), Jing-Jing Bai, Rui-Rui Zhang (Project administration), Xiao-Qian Dang (Writing - review and editing)

PII:

S0361-9230(19)30887-1

DOI:

https://doi.org/10.1016/j.brainresbull.2020.02.009

Reference:

BRB 9862

To appear in:

Brain Research Bulletin

Received Date:

10 November 2019

Revised Date:

19 January 2020

Accepted Date:

20 February 2020

Please cite this article as: Su X-Qiang, Wang X-Yang, Gong F-Tai, Feng M, Bai J-Jing, Zhang R-Rui, Dang X-Qian, Oral Treatment with Glycyrrhizin Inhibits NLRP3 Inflammasome Activation and Promotes Microglial M2 Polarization after Traumatic Spinal Cord Injury, Brain Research Bulletin (2020), doi: https://doi.org/10.1016/j.brainresbull.2020.02.009

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Title page

Oral Treatment with Glycyrrhizin Inhibits NLRP3 Inflammasome Activation and Promotes Microglial M2 Polarization after Traumatic Spinal Cord Injury Xiao-Qiang Su1, 2, Xiang-Yang Wang2, Fu-Tai Gong2, Min Feng3, Jing-Jing Bai2, Rui-Rui Zhang3 ,XiaoQian Dang*,1 1

The First Department of Orthopedics, the Second Affiliated Hospital of Xi’an Jiaotong University, No.157

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Xiwu Road, Xi'an, Shaanxi, 710004, China; Spine Area of Orthopedics, Xi'an Hospital of Traditional Chinese Medicine, No.69 Fengchengba Road,

Xi'an, Shaanxi Province, 710016, China; 3

Department of Orthopedics, Shaanxi Provincial People’s Hospital, No.256 Youyixi Road, Xi'an, Shaanxi

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Province, 710068, China;

Department of ICU, 521 healthy institute of North Industries; No.12 Zhangbadong Road, Xi’an, Shaanxi,

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710065, China;

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*Corresponding Author. Xiao-Qian Dang; Address: The First Department of Orthopedics, the Second Affiliated Hospital of Xi’an Jiaotong University, No.157 Xiwu Road, Xi'an, Shaanxi, 710004, China; Tel.: +

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86-029-87679292; E-mail: [email protected].

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Highlights

Oral treatment with glycyrrhizin promotes functional recovery after traumatic SCI

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Oral treatment with glycyrrhizin attenuates inflammasome activation in the injured spinal cord

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Glycyrrhizin reduces M1 and enhances the protective M2 polarization of microglia after SCI

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Glycyrrhizin increases the expression of M2 microglia-related markers and functional cytokines after

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traumatic SCI

Abstract

The inflammatory response induced by traumatic spinal cord injury (SCI) involves the activation of NLRP3 inflammasomes, which are closely related to the activation of microglia. Microglial polarization between M1/M2 phenotypes is a pivotal regulatory factor in neuroinflammatory responses to traumatic SCI-induced secondary injuries, and altering this polarization could be beneficial. Glycyrrhizin is a neuroprotective agent with a potent anti-inflammatory property in different neurological disorders and could potentially be useful

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in SCI. In this study, we investigated the potency of oral treatment with glycyrrhizin to reduce inflammation and improve functional recovery after traumatic SCI by inhibiting NLRP3 inflammasome activation and promoting microglial M2 polarization. After inducing traumatic SCI by dropping a 10 g impactor on the T9

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and T10 spinal segments of male Sprague-Dawley rats, the animals were given glycyrrhizin orally

immediately after injury and every 12 h for the next 3 d. Behavioral scores improved in glycyrrhizin-treated

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animals compared to the SCI group. The functional improvement in glycyrrhizin-treated rats paralleled the

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decreased expression of NLRP3 inflammasome components, such as ASC, NLRP3, and cleaved caspase-1, as well as IL-1β and IL-18. At the histopathological level, oral treatment with glycyrrhizin diminished the

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SCI-enhanced production of Iba-1+CD86+ cells (M1 microglia) but improved the release of Iba-1+CD206+ cells (M2 microglia). Likewise, oral therapy with glycyrrhizin significantly enriched the protein expression

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levels of M2 microglia-related markers (CD206 and Arg-1) but reduced those of M1 microglia-related

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markers (CD86 and iNOS) in the injured spinal cord. These findings support and extend the knowledge on post-traumatic SCI glycyrrhizin-mediated neuroprotection. Glycyrrhizin’s regulation of NLRP3 inflammasome activation and microglial polarization might be a new approach to understanding the antiinflammatory potency of glycyrrhizin. Keywords: Traumatic Spinal Cord Injury; Glycyrrhizin; Inflammation; NLRP3 Inflammasome; Microglia

Abbreviations: ASC, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain; BBB, Basso, Beattie, and Bresnahan scale ; BDNF, brain-derived neurotrophic factor; CNS, central nerve system; GDNF, glial cell-derived neurotrophic factor; HE, hematoxylin and eosin; HMGB1, high-mobility group box 1; IL, interleukin; iNOS, inducible nitric oxide synthase; NLRP3, nucleotide-binding domain and leucine-rich repeat-containing proteins 3; SCI, spinal cord injury; SCI+GLY,

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traumatic spinal cord injury plus oral glycyrrhizin treatment group; TNF, tumor necrosis factor

Introduction

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Traumatic spinal cord injury (SCI) is a devastating condition that causes massive loss of neural cells within the lesioned area and results in severe functional-behavioral deficits [1, 2]. Besides the primary injury

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caused by the mechanical impact on the injured spinal cord, the ensuing inflammatory processes have a

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crucial influence on the traumatic SCI-incited secondary injury [3]. Indeed, there is evidence that targeting neuroinflammatory responses could improve functional recovery in SCI rats [2, 4, 5].

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Post-SCI neuroinflammation consists of maturation and release of pro-inflammatory cytokines [6, 7] and could be mediated by the nucleotide-binding domain and leucine-rich repeat-containing proteins 3

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(NLRP3) inflammasomes [8, 9] made of an NLRP domain, an apoptosis-associated speck-like protein

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containing a caspase activation and recruitment domain (CARD) (ASC), and caspase-1. Evidence suggests that NLRP3 inflammasome activation occurs in injured spinal cords and plays a significant role in the postSCI neuroinflammatory response [10, 11]. Moreover, the inhibition of NLRP3 inflammasome reportedly attenuates inflammation and improves functional recovery resulting from SCI [10, 12]. The regulatory mechanisms behind NLRP3 inflammasome activation remain unclear but are closely associated with the activation of immune cells, which are the dominant cell type expressing NLRP3 inflammasome [13]. Microglia, the resident macrophage cells of the central nervous system (CNS), play an

important role in the inflammatory processes resulting from SCI [14]. These macrophage cells are recruited to the injury site after a traumatic SCI and commit to the classically activated phenotype (M1 phenotype) that releases pro-inflammatory cytokines (e.g., TNF-α) [15]. However, during the recovery period, the M1 microglia could switch to an M2 state (alternative activation) that promotes neuroprotection and tissue repair by releasing anti-inflammatory factors (e.g., IL-10) [16]. Previous studies have shown that promoted M2 polarization is associated with improvement in tissue reorganization and functional recovery after SCI [17, 18]. Thus, we hypothesized that promoting M2 polarization could protect against inflammation and reduce

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NLRP3 inflammasome activation after traumatic SCI. Glycyrrhizin is the major component isolated from Glycyrrhiza glabra root, which is one of the most commonly prescribed herbs in Eastern traditional medicine [19]. Glycyrrhiza reportedly can reduce

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inflammatory processes in many neurological diseases, including cerebral ischemia/reperfusion injury [20]

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and traumatic brain injury [21]. Glycyrrhizin has a good blood-brain barrier (BBB) penetration [22], making it possible to treat neuroinflammation and memory deficits via oral delivery [23, 24]. The anti-inflammatory

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action of glycyrrhizin is attributed to its direct inhibition of high-mobility group box 1 (HMGB1), which is an alarmin protein that can induce or potentiate proinflammatory responses in the CNS [19]. HMGB1 plays

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a pivotal role in regulating NLRP3 inflammasome activation and microglial priming [25] and is remarkably

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increased in the injured spinal cord and can cause neurotoxic inflammation [26]. It has also been suggested that treatment with glycyrrhizin inhibits traumatic brain injury by reducing HMGB1 activity [21], and

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glycyrrhizin protects against traumatic brain injury by promoting the microglia/macrophage M2 polarization [27]. Therefore, we proposed that oral treatment with glycyrrhizin might reduce inflammation and improve functional recovery after traumatic SCI by inhibiting NLRP3 inflammasome activation and promoting microglial M2 polarization.

Methods

Animals

All experimental protocols and animal handling procedures were approved by the Ethics Committee for Animal Experimentation of the Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) and were performed per the committee’s Guidelines for Animal Experimentation. Male Sprague-Dawley rats, weighing 250–280 g, were purchased from the Experimental Animal Center of Xi'an Jiaotong

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University College of Medicine (Xi'an, China) and kept in a standard lab housing with a 12-h light/dark cycle at a temperature of 21 ± 2 °C and 60–70% humidity and allowed access to standard diet and water ad

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libitum.

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Spinal cord injury induction

SCI surgery was performed as previously described [28-30]. Rats were anesthetized with 2.0%

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isoflurane in O2 (Abbott, Germany), and a laminectomy was effected at the T9–T10 (spinal T9) levels to expose the spinal cord beneath. The spinous processes from T8 and T11 were then clamped with amendable

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forceps to stabilize the spine. After laminectomy, an injury to the spinal cord was induced per the weightdrop method, by dropping a 10-g impactor (New York University weight-drop device) from a height of 25

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mm onto the exposed spinal cord. Sham-operated animals received a laminectomy without the weight-drop

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injury. After establishing the traumatic SCI, post-SCI animals were given subcutaneous antibiotics (sodium ampicillin, 80 mg/kg body weight), analgesics (carprofen 10 mg/kg) twice daily for 3 days. Bladders were voided manually twice daily until complete recovery of autonomic bladder function.

Experimental groups

Rats were randomly assigned to three groups: (1) sham group (Sham): rats that only received a laminectomy at the T10 level of the spinal cord and were treated orally with 1 ml of saline immediately after

establishing SCI and every 12 h thereafter up to 72 h after injury; (2) Traumatic spinal cord injury group (SCI): rats that were subjected to spinal cord contusion at the T9–T10 levels and were treated orally with 1 ml of saline immediately after establishing SCI and every 12 h thereafter up to 72 h after injury; (3) Traumatic spinal cord injury plus oral glycyrrhizin treatment group (SCI+GLY): rats that were subjected to spinal cord contusion at the T9–T10 levels and were treated orally with 50 mg/kg of glycyrrhizin (Cat# PHL89217, Sigma Aldrich, St. Louis, Missouri, USA) immediately after establishing SCI and every 12 h thereafter up to 72 h after injury. This dose of glycyrrhizin was selected based on a previously published

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study, which demonstrated that oral treatment with 30 mg/kg of glycyrrhizin successfully attenuated the experimental postoperative cognitive dysfunction (POCD) [24]. As for our study, we thought that the dose should be higher when considering the inflammation and tissue damage after traumatic SCI is more intense

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than those in POCD.

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Two separate batches of animals were used in the behavioral [the Basso, Beattie, and Bresnahan (BBB) locomotor test] and histological [the Western blot, hematoxylin and eosin (HE) staining or immunochemical

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analysis] experiments. The first batch of animals (n=7 per group) were subjected to BBB scoring at the 1, 3, 7, 14 and 21 days after traumatic SCI. Among them, two died before the end of experiment (1 in the SCI

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group & 1 in the SCI+GLY group). To achieve a balanced design, additional subjects were folded into the

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experimental groups to ensure still 7 rats per group for the behavioral test. The second batch of experimental rats were assigned into the Sham, SCI, and SCI+GLY groups (n=23 per group; n=5 for Western blot at days

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3 and 7 after SCI respectively; n=5 for immunohistochemistry at days 3 and 7 after SCI respectively; n=3 for HE staining on post-SCI day 7). No animal in this batch died during the experimental period.

Locomotion Testing

The locomotion function of rats was evaluated using the Basso, Beattie, and Bresnahan (BBB) scale [31-33], which is one of the most widely used methods for rating post-SCI motor functions in rats. Scoring is categorized into 21 levels (0 complete paralyses to 21 normal gaits), which include hindlimb movements,

bodyweight support, forelimb to hindlimb coordination, and whole-body movements. All evaluations were performed by two experienced investigators who were blinded to the experimental procedures (n=7/group).

Western blot analysis

Rats were anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and then sacrificed with CO2 asphyxiation (n=5/group) on days 3 and 7 after SCI. The spinal cords were harvested and lysed in an icecold RIPA buffer (Beyotime, Jiangsu, China) supplemented with protease inhibitors (1 mg/ml aprotinin).

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Cytoplasmic protein was extracted using the Nuclear and Cytoplasmic Extraction kit (Beyotime) following the manufacturer's protocol; the cytosolic protein extracts were used for the Western blotting analysis of cytoplasmic HMGB1. Protein concentration was determined using a BCA assay kit (Boster, Wuhan, China).

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Equal amounts of protein samples (20 μg) were loaded, separated by 12% SDS-PAGE and transferred onto

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polyvinylidene difluoride (Millipore, Bedford, MA, USA) membranes, and detected using the enhanced chemiluminescence method (ECL plus, Pierce Scientific, Waltham, MA, USA). The bands were analyzed

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employing densitometry and normalized to β-actin in the same blot using ImageJ software (free Java software provided by the National Institute of Health, Bethesda, MD, USA).

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The following primary antibodies were used: anti-Arg-1 (Abcam, Cambridge, MA, USA; 1:1000), anti-ASC (Abcam; 1:1000), anti-brain-derived neurotrophic factor (BDNF) (Abcam; 1:400), anti-CD206

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(Lifespan Biosciences, Seattle, WA; 1:400), anti-CD86 (Abclonal technology, Wuhan, China; 1:400), anti-

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Cleaved caspase-1 (Cell Signaling Technology, Beverly, MA; 1:1000), anti-glial cell-derived neurotrophic factor (GDNF) (Abcam; 1:400), anti-HMGB1 (Abcam; 1:2000), anti-interleukin (IL)-18 (Abcam; 1:1000), anti-IL-1β (Abcam; 1:1000), anti-inducible nitric oxide synthase (iNOS) (Abcam; 1:1000), anti-NLRP3 (Abcam; 1:1000), anti-IL-10 (Abcam; 1:1000), anti-tumor necrosis factor (TNF)-α (Abcam; 1:1000), and anti-β-actin (Santa Cruz Biotechnology; 1:400). The experiments were carried out in triplicates (n=5/group).

Immunohistochemistry

For immunohistochemistry, rats were transcardially perfused with 4% iced formaldehyde on post-SCI day 7 (n=5/group), after which the thoracic spinal cord was removed, embedded in paraffin, and cut into 4μm thick serial sections. After heat-induced antigen retrieval, the sections were blocked with normal goat serum for 30 min and then incubated overnight at 4 °C with specific primary antibodies against active caspase-3 (rabbit monoclonal, Cell Signaling Technology, 1:500), CD206 (rabbit monoclonal, LifeSpan BioSciences, 1:1,000), CD86 (rabbit polyclonal, ABclonal Technology, 1:1,000), and Iba-1 (mouse

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monoclonal, Millipore, Billerica, MA, USA, 1:1,000). The following day, for the immunostaining of active caspase-3, a secondary antibody conjugated with Dylight 488 (1:500; goat anti-rabbit; Abcam) was applied

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to the sections for 2 h at room temperature. For the double-staining of Iba-1 with CD86 and CD206, secondary antibodies conjugated to Dylight 488 (1:500; goat anti-mouse; Abcam) or Cy3 (1:500; goat anti-

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rabbit; Abcam) were used. Nuclei were counterstained with DAPI. The quantification of cell numbers was

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performed by manually counting the number of positive cells under an immunofluorescent microscope (Nikon, Optihot-2, Tokyo, Japan) using the Image Pro™ Plus software.

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Hematoxylin and eosin staining

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Tissue preparation for hematoxylin and eosin (HE) staining was executed in the same way as that for immunochemical staining on post-SCI day 7 (n=3/group). Briefly, the T9–T10 segments of the spinal cord

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were dissected and then postfixed in 4% paraformaldehyde overnight. After dehydration the following day, the tissue was embedded in paraffin and cut into sections of 5-μm thickness. The sliced tissue sections were subjected to HE staining using a commercial kit (Shanghai Solarbio Bioscience & Technology, Shanghai, China) following the manufacturer’s protocol.

Statistical analysis

All data are given as means ± standard deviations (SD). Statistical differences between various groups were analyzed using the one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test using GraphPad Prism 5 (GraphPad Software Inc., USA). The BBB test was evaluated using two-way ANOVA.

Results

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Oral treatment with glycyrrhizin promotes functional recovery after traumatic SCI

The BBB scoring was used to evaluate the lower limb motor function on days 1, 3, 7, 14, and 21 after traumatic SCI. The behavioral results showed that the motor function of all the rats subjected to SCI

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exhibited partial recovery from day 3 post-SCI. Rats treated with glycyrrhizin achieved better locomotor

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performance compared to rats in the SCI group (Fig. 1A, F(1, 12)=86.16, P<0.0001, n=7/group). The difference in functional recovery between the SCI and SCI+GLY groups was not evident until day 3 post-

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SCI (Fig. 1A, n=7/group), implying that glycyrrhizin treatment had an impact on the inflammation-related secondary damage but not the primary injury.

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Moreover, we determined the cytoplasmic protein expression of HMGB1, as HMGB1 is a critical

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inflammatory mediator that can translocate from the nucleus to the cytoplasm upon inflammatory stimuli [34], and its activity is reportedly inhibited by glycyrrhizin [19]. The result suggested that traumatic SCI

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induced a marked increase in the cytoplasmic HMGB1 protein expression (Fig. 1B, P<0.01, n=5/group), which was blocked by glycyrrhizin treatment (Fig. 1B, P<0.01, n=5/group). We next examined the histopathological changes with HE staining after traumatic SCI. As seen in Fig.1C, analyses from HE staining showed that hemorrhage and necrotic patches appeared in the spinal cord of all SCI-ravaged rats. However, post-SCI tissue damage was attenuated by glycyrrhizin treatment, as indicated by less necrotic patches and hemorrhage (Figure 1C). To further ascertain the neuroprotective action of glycyrrhizin, we examined apoptosis in the spinal

cord by identifying apoptotic cells with an active caspase-3 antibody on day 7 after traumatic SCI. The result revealed that traumatic SCI caused a significant increase in the number of active caspase-3-positive cells (Fig. 1D, P=0.001, n=5/group). However, the post-SCI increase in active caspase-3-positive cells was attenuated by treatment with glycyrrhizin (Fig. 1D, P=0.0204, n=5/group). Along with the anti-apoptotic effect of glycyrrhizin, we also investigated its influence on the spinal protein expression levels of BDNF and GDNF, as these neurotrophic factors have been associated with neuronal survival and functional recovery after SCI [35]. We observed a slight elevation of spinal BDNF and GDNF levels in SCI-ravaged animals on

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day 7 post-SCI (Fig. 1E, P=0.0029 for BDNF & P=0.0305 for GDNF, n=5/group). Notably, the protein expression levels of BDNF and GDNF were significantly promoted by treatment with glycyrrhizin on day 3 (P=0.0093 for BDNF & P=0.0264 for GDNF) and day 7 (P=0.0003 for BDNF & P=0.0080 for GDNF)

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after SCI (Fig. 1E, n=5/group).

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Oral treatment with glycyrrhizin attenuates inflammasome activation in the spinal cord

Previous studies have demonstrated that NLRP3 inflammasomes are significantly activated in the

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lesion epicenter 72 h after compression-induced SCI [10]. Thus, we determined the effect of glycyrrhizin on inflammasome activation by measuring the levels of ASC, NLRP3, and cleaved caspase-1 [36] in the spinal

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cord on days 3 and 7 after SCI and found that SCI significantly increased the protein expression levels of all

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three factors on both days after SCI (Fig. 2A, P<0.01, n=5/group). However, oral treatment with glycyrrhizin reduced the SCI-induced enhancement of the protein expression levels of ASC, NLRP3, and cleaved caspase-1 (Fig. 2A, P<0.01, n=5/group). IL-1β and IL-18 are both pro-inflammatory cytokines that acquire their active forms after cleavage by the pro-inflammatory cysteine protease caspase-1 [37]. The activation of caspase-1 and its related proteolytic maturation of IL-1β and IL-18 are closely associated with the activation of inflammasomes [38]. Western blot results showed that the spinal protein levels of IL-1β and IL-18 increased significantly by >3

folds on days 3 and 7 post-SCI (Fig. 2B, P<0.01, n=5/group). Importantly though, the post-SCI increase in IL-1β and IL-18 was reduced by oral treatment with glycyrrhizin (Fig. 2B, P<0.01, n=5/group).

Glycyrrhizin reduces M1 and enhances the protective M2 polarization of microglia after SCI

Previous studies have demonstrated that microglial polarization plays a vital role in the post-SCI inflammatory response [14]. To assess the effect of glycyrrhizin on microglial polarization, we evaluated the

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outcome of double staining for Iba-1 (a microglial marker) and CD206 (a marker of M2 microglia/macrophage [39]) or CD86 (a cell surface marker of M1 microglia/macrophage [15]) in the spinal cord 7 d after traumatic SCI and found that the number of Iba-1+, Iba-1+CD206+, and Iba-1+CD86+ cells

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increased substantially in the spinal cord of the SCI group rats (Fig. 3 A to E, P<0.01, n=5/group).

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However, oral treatment with glycyrrhizin limited the SCI-induced elevation of Iba-1+ microglia and Iba1+CD86+ cells (Fig. 3 A to E, P<0.01, n=5/group) but enhanced the production of CD206+ microglia (Fig. 3

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A to E, P<0.01, n=5/group) in the spinal cord. These data suggest that oral treatment with glycyrrhizin

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could reduce M1 and enhance protective M2 polarization to promote recovery after SCI.

Glycyrrhizin increases the expression of M2 microglia-related markers and functional cytokines after

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traumatic SCI

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To verify the effect of glycyrrhizin on microglial M2 polarization, we examined the spinal protein expression levels of CD206, Arg-1 [40], and IL-10 (a cytokine specifically released by M2 microglia [41]) on days 3 and 7 after traumatic SCI. We found that the protein expression levels of CD206, Arg-1, and IL10 on days 3 and 7 after traumatic SCI were elevated in the SCI-ravaged animals (Fig. 4A, P<0.01, n=5/group). Moreover, oral treatment with glycyrrhizin significantly enhanced the protein levels of CD206, Arg-1, and IL-10 (Fig. 4A, P<0.01, n=5/group), indicating that oral treatment with glycyrrhizin up-regulates the production of M2 microglia-related markers and functional cytokines after traumatic SCI.

We also assessed the protein expression levels of CD86, iNOS, and TNF-α, which are markers and functional cytokines of M1 microglia [42]. Our findings revealed that the protein expression levels of CD86, iNOS, and TNF-αwere markedly up-regulated in the SCI group when compared with the Sham group (Fig. 4B, P<0.01, n=5/group); however, this increase was blocked by oral treatment with glycyrrhizin (Fig. 4B, P<0.01, n=5/group).

Discussion

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To the best of our knowledge, this is the first study to establish that oral treatment with glycyrrhizin provides neuroprotection against a traumatic SCI-induced secondary injury. This beneficial aspect of glycyrrhizin was manifested by enhanced hindlimb function recovery and decreased apoptosis and

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inflammation in the spinal cord. We also showed that the anti-inflammatory effect of glycyrrhizin is

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associated with fostering microglial M2 polarization and inhibiting NLRP3 Inflammasome activation. The mechanisms of a traumatic SCI-induced secondary injury have been studied extensively by

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researchers who have found inflammation to be a target of significance due to its critical role in exacerbating

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secondary damages caused by traumatic SCI [2-5]. HMGB1 has been identified as a crucial inflammatory mediator in a variety of neurological diseases, such as cerebral ischemic injury [43] and traumatic brain

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injury [44]. Up-regulated HMGB1 in the spinal cord of animals with SCI is closely linked to post-SCI inflammation [26, 45, 46], leading us to speculate that glycyrrhizin, a direct inhibitor of HMGB1, could

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attenuate inflammatory responses to traumatic SCI. Indeed, a previous investigation showed that a single dose of glycyrrhizin administered intravenously (6 mg/kg) suppresses inflammation caused by ischemic spinal cord injury by inhibiting HMGB1 [47]. By using a higher dose orally (50 mg/kg immediately after injury and every 12 h for the next 3 d), we found a similar neuroprotective and anti-inflammatory effect of glycyrrhizin in rats with traumatic SCI. This finding is in agreement with studies showing that oral treatment with glycyrrhizin attenuates neuroinflammation and memory deficits [22, 23], implying that the oral

approach with glycyrrhizin may be slower but is efficient and would be highly productive in clinical settings. Following a traumatic SCI, microglia in the spinal cord become active and infiltrate the injury epicenter to initiate the inflammatory process [7, 48]. These glial cell types in the spinal cord could also serve as targets of glycyrrhizin, as studies have shown that glycyrrhizin alters spinal microglial activation in experimental models of inflammatory pain [49] and autoimmune encephalomyelitis [50] favorably. In the present study, we demonstrated that oral treatment with glycyrrhizin significantly reduced the number of

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Iba-1+ microglia in the injured spinal cord. However, the situation may be more complex because microglia can polarize into various phenotypes with distinct functions under inflammatory conditions [39, 41, 51]. The most well-studied phenotypes are the classically activated M1 and alternatively activated M2 phenotypes

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that are thought to represent the two major extreme states of microglia [41]. M1 microglia are characterized

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by the inflammatory phenotype, while M2 microglia exert anti-inflammatory effects in tissue repair and debris clearance [51]. Therefore, it stands to reason that during an SCI-induced secondary injury, where

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inflammation is a significant event, promoting microglial M2 polarization would be favorable to improving functional recovery.

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Although numerous previous studies have investigated the involvement of microglia in traumatic SCI

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[14, 52, 53], most of them refer to microglia as mere markers of inflammation (M1 phenotype) and ignore the M2 phenotype. Hence, we tested the effect of traumatic SCI and glycyrrhizin on microglial M2

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polarization in the spinal cord. Our results showed that CD206+ M2 microglia, as well as the protein expression of M2 microglia-related markers and functional cytokines, increased in the spinal cord after traumatic SCI, indicating that M2 polarization might be an anti-inflammatory response to traumatic SCI. Importantly, we observed that the increase in M2 polarization was markedly augmented by treatment with glycyrrhizin, demonstrating a positive effect of glycyrrhizin on microglia M2 polarization after traumatic SCI. Our findings were consistent with those of Gao et al. [27], which showed that treatment with

glycyrrhizin protected against traumatic brain injury by promoting M2 microglia/macrophage polarization. The present study also suggests an inhibitory role for glycyrrhizin against inflammasome stimulation and inflammatory processes after traumatic SCI. Inflammasomes are multiprotein inflammatory complexes that act as intracellular sensors to a variety of exogenous, endogenous, and pathogenic signals [8, 9]. The stimulation of these complexes results in the maturation and release of proinflammatory cytokines IL-1β and IL-18 [37]. The NLRP3 inflammasome has been associated with acute conditions (e.g., traumatic brain injury [54, 55]) and chronic diseases, such as Parkinson’s disease [56] and Alzheimer’s disease [57]. The

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NLRP3 inflammasome is mainly expressed in microglia and macrophages but has also been found to have functions in neurons [10, 58]. A previous study demonstrated how compression-induced SCI led to a significant elevation in the mRNA and protein levels of NLRP3 complexes 72 h after injury [10]. Consistent

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that investigation, we found that traumatic SCI prompted a marked increase in the protein expression levels

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of NLRP3 inflammasome components, such as ASC, NLRP3, and cleaved caspase-1, as well as IL-1β and IL-18 3 and 7 d after injury. These elevations were blocked by oral treatment with glycyrrhizin, suggesting a

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notable inhibitory effect of glycyrrhizin on inflammasome activation after traumatic SCI. It is also worth noting that HMGB1, a well-established target of glycyrrhizin, can be released from

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inflammasomes [59], implying that glycyrrhizin could antagonize NLRP3 inflammasomes upstream of its

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inhibitory effect on HMGB1. As microglia represent the primary source of inflammasomes after SCI [10, 60], IL-1β, and IL-18 [61], we argue that the effect of glycyrrhizin on inflammasomes are related to changes

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in microglia phenotypes. Although the relationship between inflammasome activation and microglia phenotypic changes has not been studied directly, our argument is indirectly justified by the following evidence: First, IL-1β and IL-18 are specifically produced by M1 but not M2 microglia in the CNS [62]; Second, the inhibition of the NLRP3 inflammasome is typically in parallel with microglial M2 polarization in cuprizone-induced demyelination [63] and compression-induced SCI [10] models. In conclusion, the current study shows that oral treatment with glycyrrhizin improves functional

recovery and suppresses inflammation after traumatic SCI. These beneficial effects are related to the promotion of microglial M2 polarization and inhibition of NLRP3 inflammasome activation. Our results allow us to suggest that controlling NLRP3 inflammasome activation and microglial polarization might be a promising strategy to improve outcomes after traumatic SCI. Author statement

Xiao-Qiang Su: Experimental design, Writing - Original Draft, Creation of models;

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Xiang-Yang Wang: Project administration (Western blot); Fu-Tai Gong: Project administration (behavioral tests); Min Feng: Project administration (Immunohistochemistry);

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Jing-Jing Bai: Statistical analysis;

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Rui-Rui Zhang: Project administration (Hematoxylin and eosin staining);

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Xiao-Qian Dang: Experimental design, Writing - Review & Editing

Conflict of interest

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The authors declare no conflict of interest.

None.

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Acknowledgments

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References

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Figure legends

Fig. 1.

Oral treatment with glycyrrhizin promotes functional recovery from traumatic SCI. (A) Oral treatment with glycyrrhizin improved post-SCI locomotor function, as reflected by the BBB scores. The BBB scores decreased to zero in the SCI and SCI+GLY groups 3 d after SCI, after which they recovered significantly better in the SCI+GLY group compared to the SCI group. Repeated-measures with ANOVA indicated that

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the difference between animals in the SCI and SCI+GLY groups was statistically significant (SCI vs. SCI+GLY, F(1, 12)=86.16, P<0.0001). n=7/group. (B) The effects of glycyrrhizin on the cytoplasmic protein expression level of HMGB1 (n=5/group) 3 and 7 d after SCI. Traumatic SCI elevated, but the oral treatment

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with glycyrrhizin abolished the cytoplasmic protein expression of HMGB1. (C) Representative graphs of the

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HE-stained lesion epicenters on day 7 after SCI. Scale bars=100 μm. n=3/group. (D) The effects of glycyrrhizin on the number of active caspase-3-positive cells (n=5/group) on day 7 after SCI. Scale bars=50

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μm. Apoptotic cells were stained with an anti-active-caspase-3 (green) antibody and counterstained with DAPI (blue). (E) The effects of glycyrrhizin on the protein levels of BDNF and GDNF (n=5/group) on days

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represent means ± SD.

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3 and 7 after SCI. **P < 0.01 compared with the sham group. ##P < 0.01 compared with the SCI group. Data

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Fig. 2.

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Oral treatment with glycyrrhizin attenuates inflammasome activation in the spinal cord. (A) Representative images of Western blot depicting ASC, NLRP3, and cleaved caspase-1 in the spinal cord after traumatic The effects of SCI and glycyrrhizin on the spinal protein expression levels of ASC, NLRP3, and

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SCI.

cleaved caspase-1 (n=5/group) 3 and 7 d after SCI. (B) The effects of glycyrrhizin on the spinal protein

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expression levels of IL-1β and IL-18 (n=5/group) 3 and 7 d after SCI. **P < 0.01 compared with the sham

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group. ##P < 0.01 compared with the SCI group. Data represent means ± SD.

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Fig. 3.

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Glycyrrhizin reduces M1 and enhances the protective M2 polarization of microglia after SCI. (A) Double immunostaining for Iba-1 (red) and CD86 (green) in the spinal cord on day 7 after SCI. The nuclei were

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counterstained with DAPI (blue). Scale bars=50 μm. (B) Double immunostaining for Iba-1 (red) and CD206 (green) in the spinal cord on day 7 after SCI. The nuclei were counterstained with DAPI (blue). Scale

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bars=50 μm. n=5/group. (C) Statistical comparison between Iba+ cells in the spinal cord on day 7 after SCI. n=5/group. (D) Statistical comparison between Iba+CD86+ cells in the spinal cord on day 7 after SCI.

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n=5/group. (E) Statistical comparison between Iba+CD206+ cells in the spinal cord on day 7 after SCI.

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n=5/group. **P < 0.01 compared with the sham group. ##P < 0.01 compared with the SCI group. Data represent means ± SD.

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Fig. 4.

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Glycyrrhizin increases the expression of M2 microglia-related markers and functional cytokines after traumatic SCI. (A) Representative images of Western blot depicting CD206, Arg-1, and IL-10 in the spinal

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cord after traumatic SCI. The effects of SCI and glycyrrhizin on the spinal protein expression levels of CD206, Arg-1, and IL-10 (n=5/group) 3 and 7 d after SCI. (B) The effects of glycyrrhizin on the spinal

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protein expression levels of CD86, iNOS, and TNF-α(n=5/group) 3 and 7 d after SCI. **P < 0.01 compared

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with the sham group. ##P < 0.01 compared with the SCI group. Data represent means ± SD.

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