Corilagin attenuates morphine-induced BV2 microglial activation and inflammation via regulating TLR2-mediated endoplasmic reticulum stress

Abstract

Morphine-induced microglia activation and neuroinflammation have been considered as the contributors of morphine tolerance. Corilagin (Cori) has been reported to exhibit strong anti-inflammatory property. The present study aims to investigate whether and how Cori alleviates morphine-induced neuroinflammation and microglia activation. Mouse BV-2 cells were exposed to different concentrations of Cori (0.1, 1 and 10 μM) prior to morphine stimulation (200 μM). Minocycline (10 μM) acted as the positive control. Cell viability was determined by CCK-8 assay and trypan blue assay. The levels of inflammatory cytokines were determined using ELISA. IBA-1 level was examined via immunofluorescence. TLR2 expression level was examined by quantitative real-time PCR and western blot. The expression levels of corresponding proteins were measured by western blot. It was found that Cori was non-toxic to BV-2 cells but greatly inhibited morphine-induced IBA-1 expression, overproduction of pro-inflammatory cytokines, activation of NLRP3 inflammasome and endoplasmic reticulum stress (ERS), and upregulation of COX-2 and iNOS. TLR2 was negatively regulated by Cori, and could promote the activation of ERS. A high affinity between Cori and TLR2 protein was confirmed via Molecular docking investigation. Moreover, TLR2 overexpression or tunicamycin (TM), an agonist of ERS, partly abolished the inhibitory effects of Cori on morphine-induced alternations on neuroinflammation and microglial activation in BV-2 cells as above. In summary, our study suggested that Cori effectively alleviated morphine-induced neuroinflammation and microglia activation through inhibiting TLR2-mediated ERS in BV-2 cells, providing a novel potential drug to overcome morphine tolerance.

INTRODUCTION

As a classic opioid analgesic, morphine has been widely recognized as a golden standard for clinical treatment of severe pain. However, the occurrence of physiological tolerance and dependence as well as the adverse effects along with the long-term use of morphine greatly limits its clinical application (Zhang et al., 2019). In recent decades, extensive research has been focused on the molecular mechanism of morphine tolerance. Accumulating evidence has confirmed that morphine can trigger neuroinflammation, which is featured by the activation of microglia and increased production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), Interleukin (IL)-6 and IL-1β, which in turn aggravates neuroinflammation, consequently leading to a reduced analgesic efficacy of morphine (Eidson and Murphy, 2013; Ferrini et al., 2013). A recent study has disclosed that minocycline, a microglial inhibitor, can inhibit microglia-mediated neuroinflammation and alleviate morphine tolerance (Seki et al., 2013). Our previous article has suggested dihydroartemisinin as a potential clinical drug for attenuating morphine tolerance attributed to its protection against morphine-induced microglia activation and inflammation (Guan et al., 2021). Thus, controlling microglia activation and inflammation may be an effective approach to overcome morphine tolerance.

Corilagin (Cori; C₂₇H₂₂O₁₈), a water-soluble ellagitannin, is an active ingredient in many Chinese herbal plants, such as Terminalia catappa L., Euphorbia longana Lam., Phyllanthus emblica Linn., and Geranium sibiricum L (Hau et al., 2009; Sudjaroen et al., 2012; Li et al., 2018). Previous studies have shown that Cori possesses antioxidant, anticancer, anti-inflammatory, hepatoprotective and other biological activities, and plays a protective role in numerous diseases, including acute lung injury, cancer, liver failure and atherosclerosis, making it a promising medicinal herbal agent (Guo et al., 2017; Qiu et al., 2019; Lv et al., 2019). Of note, Tong et al. (2016) have disclosed a potential therapeutic role of Cori against radiation-induced brain injury where Cori could inhibit microglial activation and the expressions of inflammatory cytokines through inactivating NF-κB signaling. Given that the activation of microglia and the inflammatory response are important pathological processes during morphine tolerance, whether Cori can play its role in repressing microglial activation and inflammation to overcome morphine tolerance is worthy of further investigation.

Therefore, in this study, we hypothesized that Cori could be a potential therapeutic agent for overcoming morphine tolerance. To verify this hypothesis, we attempted to examine the impacts of Cori on morphine-induced microglia activation and inflammation, and explore the underlying mechanism.

MATERIALS AND METHODS

Cell culture

Mouse BV-2 cells were obtained from BeNa Culture Collection (Beijing, China) and were cultured in Dulbecco’s modified Eagle medium (DMEM; Hyclone) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM glutamine (Gibco BRL, Grand Island, NY, USA). The cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

Cell administration

For treatment, BV-2 cells were exposed to different concentrations of Cori (0, 0.1, 1 and 10 μM) for 24 hr. For the last 6 hr, all groups except the control group were stimulated by morphine (200 μM) (Han et al., 2014; Qu et al., 2017), so as to trigger microglia activation and neuroinflammation, which were considered as the features of morphine tolerance in vitro. Actually, this high concentration of morphine (200 µM) was demonstrated to induce robust activation in BV-2 cells in this study. Minocycline (10 μM), an inhibitor of microglia activation, acted as the positive control.

Cell transfection

TLR2 overexpression vector (oe-TLR2) and its negative control (oe-NC) were synthesized by GenePharma (Shanghai, China). BV-2 cells received transfection with the above vectors by using Lipofectamine 3000 reagent (Takara, Kusatsu, Japan) strictly in line with the manufacturer’s instructions. 48 hr post transfection, BV-2 cells were collected for subsequent experiments.

Cell counting kit-8 (CCK-8) assay

Cell viability was assessed using CCK-8 assay. In brief, BV-2 cells were seeded into 96-well plates at a density of 5 × 10³ cells/well and treated with various concentrations of Cori for 24 hr. 10 μL of CCK-8 reagent (Dojindo Molecular Technologies, Inc., Japan) was added to each well and the cells were incubated at 37°C with 5% CO₂ for another 2 hr. The absorbance at 450 nm of each well was detected using a microplate reader (Bio-Rad Laboratories, France).

Trypan blue assay

The living cell rate was detected using trypan blue cell counting method. Briefly, BV-2 cells were seeded into 6-well plates and treated with various concentrations of Cori for 24 hr. Thereafter, cells were digested, resuspended and mixed with 0.4% trypan blue solution, and the cells, including living cells and dead cells, were counted under a light microscope. The living cell rate (%) was calculated according to the ratio of living cells and total cells.

Enzyme-linked immunosorbent assay (ELISA)

The levels of TNF-α, IL-6 and IL-1β in cell supernatant of different groups were measured using Mouse TNF-α ELISA Kit, Mouse IL-6 ELISA Kit and Mouse IL-1β ELISA Kit (Shanghai Enzyme Link Biotechnology Co., Ltd., Shanghai, China), respectively, in agreement with manufacturer’s protocol.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from BV-2 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s guidelines. After determining the RNA quantity and quality using NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, USA), cDNA synthesis was conducted adopting PrimeScript RT reagent Kit (Takara Bio Inc, Japan). Subsequently, qRT-PCR was carried out using SYBR Green Reaction Master Mix (TaKaRa Biotechnology Co. Ltd., Dalian, China) on a 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) in accordance with the manufacturer’s instructions. The 2^-∆∆Ct method was used for data analysis.

Western blot

Total protein was extracted from BV-2 cells using radioimmunoprecipitation assay (RIPA) lysis buffer, followed by the adoption of a Bicinchoninic Acid (BCA) kit (Boster Biological Technology) to determine protein concentration. Subsequently, the proteins (30 μg) were separated by 12% SDS-PAGE gel and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore). After being blocked with non-fat milk at room temperature for 2 hr, the membranes were probed with primary antibodies at 4°C overnight. On the following day, horseradish peroxidase (HRP)-conjugated secondary antibodies were applied to incubate the membranes for another 2 hr at room temperature. The signals were developed by an enhanced chemiluminescence reagent (Tanon, Shanghai, China) and were quantified by ImageJ software (NIH, USA).

Immunofluorescence

Following the indicated treatment, BV-2 cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeated by 0.1% Triton X-100. After being blocked with 1% bovine serum albumin (BSA), BV-2 cells were incubated with anti-IBA-1 antibody (ab178847, Abcam) at 4°C overnight. On the following day, the cells were incubated with Goat Anti-Rabbit IgG (Alexa Fluor® 488) secondary antibody (ab150077, Abcam) at room temperature for 1 hr. Following washing with PBS for three times, the cells were stained with 4’,6-Diamidino-2-phenylindole (DAPI) for 10 min at room temperature. The fluorescent images were captured using a laser confocal microscope (Olympus, Japan).

Molecular docking

A molecular docking investigation was conducted to confirm the binding sites on Cori and TLR2. The crystal structure of the ligand Cori was prepared in Auto Dock Vina software via adding hydrogen atoms and modifying charges to obtain PDBQT format file. The target proteins were prepared via removing crystal water, hydrogenation and removing co-crystal ligand molecules occupying the binding sites. All of the docking models were generated by Auto Dock Vina software, and eventually nine favored binding conformations were calculated. Meanwhile, the binding energy between each ligand was calculated to reflect the binding affinity. The conformation was visualized using PyMoL software.

Statistical analysis

All data were presented as mean ± standard deviation (SD) and analyzed by one-way ANOVA followed by Tukey’s post hoc test using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered to indicate a statistically significant difference.

RESULTS

Cori retards morphine-mediated microglial activation in BV-2 cells

To evaluate the pharmacological activity of Cori in morphine-induced BV-2 cells, the cytotoxicity of Cori in BV-2 cells was firstly examined. As exhibited in Fig. 1A, BV-2 cells were treated with increasing concentrations of Cori (0.1, 1 and 10 μM) for 24 hr, and the CCK-8 assay revealed that Cori treatment did not cause significant impacts on cell viability, suggesting that 0.1, 1 and 10 μM of Cori were nontoxic to BV-2 cells. Subsequently, BV-2 cells were treated with Cori or minocycline prior to morphine induction. It was found from trypan blue assay that there was no significant difference among different groups, suggesting that Cori/minocycline/morphine did not influence the viability of BV-2 cells (Fig. 1B). IBA-1 is a classical hallmark of microglial activation in spinal cord, and its expression level reflects the degree of microglial activation. As displayed in Fig. 1C, the IBA-1 level in morphine group was much higher than that in control group, proving that morphine induced microglial activation. However, treatment with Cori, as well as minocycline, remarkably weakened IBA-1 level, indicating that Cori could suppress morphine-mediated microglial activation.

Fig. 1

Cori retards morphine-mediated microglial activation in BV-2 cells. (A) BV-2 cells were treated with increasing concentrations of Cori (0.1, 1 and 10 μM) for 24 hr, and the CCK-8 assay was performed to examine cell viability. (B) BV-2 cells were treated with Cori or minocycline prior to morphine induction, and the trypan blue assay was performed to examine living cell rate. (C) The IBA-1 level in each group was examined using immunofluorescence.

Cori inhibits overproduction of pro-inflammatory cytokines and inactivates NLRP3 inflammasome in morphine-treated BV-2 cells

Subsequently, the effects of Cori on morphine-triggered neuroinflammation were explored. It was observed from Fig. 2A-C that morphine caused severe overproduction of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6; however, the elevated levels of these cytokines were repressed following Cori treatment in a concentration-dependent manner, as well as minocycline treatment. Consistently, the inhibitory effects of Cori on the protein expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in morphine-challenged BV-2 cells further confirmed the anti-inflammatory activity of Cori (Fig. 2D). Furthermore, morphine led to a remarkable elevation of the protein expression of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and cleaved-caspase1 in BV-2 cells, indicating that NLRP3 inflammasome was activated by morphine, while Cori obviously inhibited the activation of NLRP3 inflammasome by downregulating the protein expression of NLRP3, ASC and cleaved-caspase1 (Fig. 2E).

Fig. 2

Cori inhibits overproduction of pro-inflammatory cytokines and inactivates NLRP3 inflammasome in morphine-treated BV-2 cells. BV-2 cells were treated with Cori (0.1, 1 and 10 μM) or minocycline (10 μM) prior to morphine induction. (A-C) The production of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, was measured by ELISA. (D) The protein expression of COX-2 and iNOS was detected by western blot. (E) The protein expression of NLRP3, ASC and cleaved-caspase1 and caspase1 was detected by western blot. ***p < 0.05 vs Control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs morphine.

Cori alleviates morphine-mediated endoplasmic reticulum stress (ERS) via inhibiting TLR2 in BV-2 cells

Next, the potential mechanism underlying the role of Cori in morphine-induced BV-2 cells was explored. As shown in Fig. 3A-B, morphine induced an upregulation of TLR2 at both mRNA level and protein expression in BV-2 cells, which was concentration-dependently retarded by Cori treatment, indicating that TLR2 might be a downstream protein of Cori. To confirm this binding relationship, a molecular docking investigation was carried out. As shown in Table 1, the affinity of all of the nine favored modes is in the range of -5.4~-4.7 kCal/mol, indicating that TLR2 is highly likely to interact with Cori. Meanwhile, the 3D conformation of the complex exhibited solid area representing the binding area of ligand in TLR2 protein (Fig. 3C). Subsequently, BV-2 cells overexpressing TLR2 were successfully constructed by transfection with Oe-TLR2 (Fig. 3D-E). Then, the transfected or untransfected BV-2 cells were treated with Cori prior to morphine induction, and the western blot assay revealed that the protein expression of GRP78, ATF4, CHOP and p-PERK was obviously upregulated upon morphine induction in BV-2 cells, which was greatly reversed by Cori treatment, reflecting that Cori could weaken morphine-triggered ERS activation (Fig. 3F). Furthermore, the activation of ERS was partly restored again in TLR2-overexpressing BV-2 cells. These data suggested that Cori might inactivate morphine-mediated ERS via inhibiting TLR2 expression in BV-2 cells.

Fig. 3

Cori alleviates morphine-mediated endoplasmic reticulum stress (ERS) via inhibiting TLR2 in BV-2 cells. (A) BV-2 cells were treated with Cori (0.1, 1 and 10 μM) prior to morphine induction. The protein expression of TLR2 was detected using western blot. (B) The mRNA level of TLR2 was detected using qRT-PCR. ***p < 0.05 vs Control; #p < 0.05, ###p < 0.001 vs morphine. (C) Interaction of Cori and TLR2 via molecular docking investigation. (D) BV-2 cells were transfected wit Oe-TLR2 or the negative control (Oe-NC), and the mRNA level of TLR2 was detected using qRT-PCR. (E) The protein expression of TLR2 was detected using western blot. ***p < 0.001 vs Oe-NC. (F) The transfected or untransfected BV-2 cells were treated with Cori prior to morphine induction, and the protein expression of GRP78, ATF4, CHOP and p-PERK and PERK was measure by western blot assay. ***p < 0.05 vs Control; ###p < 0.001 vs morphine; +++p < 0.001 vs morphine+Cori+Oe-NC.

Table 1. Docking scores for TLR2 protein and Corilagin

Mode of ligand	Affinity (kcal/mol)	Distance from rmsd 1.b.	Best mode rmsd u.b.
1	-5.4	0.000	0.000
2	-5.4	3.582	8.886
3	-5.2	5.308	9.154
4	-5.2	1.499	2.158
5	-5.1	1.938	6.326
6	-5.0	1.748	7.267
7	-5.0	1.581	2.104
8	-4.9	2.662	5.539
9	-4.7	3.117	8.117

Activation of ERS abolishes the protective role of Cori against morphine-mediated inflammation and microglia activation in BV-2 cells

Eventually, tunicamycin (TM), an agonist of ERS, was introduced to BV-2 cells 6 hr prior to Cori treatment. As presented in Fig. 4A, the level of IBA-1 was expected to be upregulated in the morphine+Cori+oe-TLR2 group and the morphine+Cori+TM group, compared to the morphine+Cori group. Meanwhile, the inhibitory effects of Cori on the overproduction of TNF-α, IL-1β and IL-6 and the upregulation of COX-2 and iNOS expression were partly weakened by TLR2 overexpression or TM (Fig. 4B-E), suggesting that Cori might alleviate morphine-induced inflammation partially via inhibiting TLR2 and inactivating ERS. Consistently, TLR2 overexpression or TM also abolished the suppressive effect of Cori on morphine-activated NLRP3 inflammasome, evidenced by the restored protein expression of NLRP3, ASC and cleaved-caspase1 following TLR2 overexpression or TM treatment in BV-2 cells (Fig. 4F). Taken together, the inhibitory effects of Cori on morphine-induced inflammation, NLRP3 inflammasome activation and microglial activation were partly weakened by TLR2 overexpression or TM.

Fig. 4

Activation of ERS abolishes the protective role of Cori against morphine-mediated inflammation and microglia activation in BV-2 cells. BV-2 cells were transfected with TLR2-overexpressing plasmids or subjected to treatment with TM prior to Cori treatment and morphine induction. (A) The IBA-1 level in each group was examined using immunofluorescence (B-D) The production of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, was measured by ELISA. (E) The protein expression of COX-2 and iNOS was detected by western blot. (F) The protein expression of NLRP3, ASC and cleaved-caspase1 and caspase1 was detected by western blot. ***p < 0.05 vs Control; ###p < 0.001 vs morphine; +p < 0.05, ++p < 0.01, +++p < 0.001 vs morphine+Cori.

DISCUSSION

Morphine-induced neuroinflammation has been widely discussed in the past years and much attention has been focused on to seek for effective means for controlling microglia activation and inflammation to overcome morphine tolerance. As we know, in physiological conditions, the usual dose of morphine is 2.5-10 mg intravenously in clinical and 20 mg/kg subcutaneously in animals (Pajohanfar et al., 2017; Lugo and Kern, 2002); however, for in vitro experiments, low concentrations of morphine generally enhance the neuroglia-like differentiation, but high concentrations of morphine are required for inducing the robust activation and inflammatory response of microglial cells, as well as inducing robust activation of NLRP3 (Wang et al., 2021a; Zhaleh et al., 2020; Han et al., 2014; Cai et al., 2016). Hence, a high concentration of morphine (200 µM) was adopted to establish an in vitro morphine-induced neuroinflammation cell model. Subsequently, the impacts of Cori on morphine-induced neuroinflammation were explored. It was found that Cori exerted a protective role against morphine-induced neuroinflammation, evidenced by reduced inflammatory response and microglia activation. Importantly, Cori had an inhibitory effect on TLR2 expression, and TLR2-mediated NLRP3 inflammasome and ERS, which might partly account for the regulatory mechanism underlying the protective role of Cori against morphine-induced neuroinflammation.

NLRP3 inflammasome is the well-studied inflammasome that is composed of NLRP3 sensor, ASC and caspase-1. Upon NLRP3 inflammasome activation, matured caspase-1 cleaves precursor forms of inflammatory cytokines such as IL-18 and IL-1β to release their mature forms, triggering inflammatory cascade (Yan et al., 2015). Currently, NLRP3 inflammasome has been widely recognized as a “gatekeeper of inflammation”, and its function is critical in central nervous system injury, especially regulating neuroinflammation of microglia (Dinarello, 2011; de Rivero Vaccari et al., 2014; Gustin et al., 2015). Morphine has been found to activate the NLRP3 inflammasome, while procyanidins attenuate morphine tolerance by inhibiting NLRP3 inflammasome activation and IL-1β maturation (Cai et al., 2016). miR-223 can bind to NLRP3 and reduce the expression of NLRP3, thereby reducing the tolerance to morphine analgesia in rats (Xie et al., 2017). Therefore, inhibiting NLRP3 inflammasome may be an effective way to alleviate neuroinflammation and morphine tolerance. Consistently, it was also found NLRP3 inflammasome was activated following morphine stimulation in BV-2 cells. Cori not only exerted its prominent anti-inflammatory activity by inhibiting the production of TNF-α, IL-1β and IL-6 and the protein expression of COX-2 and iNOS, but also greatly inhibited the activation of NLRP3 inflammasome in morphine-exposed BV-2 cells, which might partly account for the protection of Cori against neuroinflammation.

ERS has been found to be involved in the regulation of NLRP3 inflammasome activation (Grootjans et al., 2016). Thioredoxin interaction protein (TXNIP) is the binding ligand of NLRP3, which can bind to NLRP3 and activate NLRP3 inflammasome, while ERS can induce TXNIP expression (Soczewski et al., 2020; Ke et al., 2020). Under ERS status, cleaved XBP1 binds to TRPC1-secreted signal peptides, followed by activating NLRP3 inflammasome and regulating IL-1β secretion and subsequent immune responses (Overley-Adamson et al., 2014). Therefore, ERS may act as an upstream signal triggering the activation NLRP3 inflammasome, so as to mediate inflammation. In addition, Tauroursodeoxycholic acid (TUDCA), an antagonist of ERS, is reported to delay the progression of morphine tolerance, suggesting that ERS is involved in and regulates morphine tolerance (Dobashi et al., 2010). Therefore, ERS activation, accompanied with subsequent NLRP3 inflammasome activation, may be an important mechanism of morphine tolerance. A newly published article has revealed that Cori can promote tumor cell apoptosis through regulating ERS pathway (Wu et al., 2021). On the contrary, ERS was greatly activated upon morphine stimulation in BV-2 cells, and Cori exhibited an inhibitory effect on the excessive ERS, so as to alleviate microglial activation and NLRP3 inflammasome-mediated inflammatory injury. The further activation of ERS by TM treatment weakened the protective role of Cori, firmly demonstrating that Cori might alleviate morphine-induced neuroinflammation partly through inhibiting the activation of ERS.

Furthermore, Cori was noticed to inhibit the expression of TLR2, despite the finding that Cori was considered as NF-κB and TNF-α inhibitor (Okabe et al., 2001; Gambari et al., 2012), consistent with the previous report which supported that Cori protected against HSV1 encephalitis through inhibiting the TLR2 signaling pathway (Guo et al., 2015). TLR2 plays an important role in innate immune response. The promoted TLR2 generally combined with the endogenous ligands and leads to recruitment/activation of MyD88, thereby activating the transcription factor and increasing expression of pro-inflammatory cytokines (Zhou et al., 2016). TLR2 is broadly expressed in the central nervous system, especially in microglial cells. It has been demonstrated that TLR2 is indispensable for morphine-induced microglia activation and inflammatory responses, as the main symptoms of morphine withdrawal were significantly attenuated in TLR2-knockout mice, highlighting the critical role of TLR2 in morphine-induced microglia activation, and indicating that inhibition of TLR2 may find application in the development of novel therapeutics to treat opioid dependence and addiction (Zhang et al., 2011). In the present study, TLR2 was confirmed to be upregulated upon morphine stimulation, in agreement with previous studies (Schilling et al., 2021; Wang et al., 2005; Ghosh et al., 2023). Considering that Cori negatively impacted TLR2 expression and TLR2 overexpression partly abolished the suppressed neuroinflammation imposed by Cori, it could be suggested that Cori attenuated morphine-induced microglial activation and inflammation through inhibiting TLR2. Moreover, TLR2-mediated pathways, such as NLRP3 inflammasome and ERS, are closely related to multiple inflammatory reactions (Wang et al., 2021c; Wu et al., 2020). Thus, TLR2/ERS/NLRP3 inflammasome might be the pathway Cori targeted in morphine-induced BV-2 cells (Fig. 5).

Fig. 5

Proposed schematic of the mechanism that Cori alleviated morphine-induced neuroinflammation and microglia activation in BV-2 cells through. TLR2/ERS/NLRP3 inflammasome pathway. Morphine is an important Contributor to neuroinflammation and microglia activation. Cori directly binds to the protein structure of TLR2 to down-regulate the protein level of TLR2, despite the unclear mechanism by which Cori down-regulates the mRNA level of TLR2. The elevated TLR2 caused by morphine is alleviated by Cori treatment. Then, the downregulated TLR2 further restricts ERS/NLRP3 inflammasome pathway, thereby reducing IL-1β and IL-18 secretion.

Last but not least, there are some limitations in the present study. On the one hand, the molecular docking investigation demonstrates a direct binding interaction between Cori and protein TLR2, accounting for the regulation of Cori on TLR2 protein expression, but the molecular mechanism underlying how Cori regulates TLR2 at mRNA level is still uncertain, which is deserved to be further explored in our future work. On the other hand, it is widely recognized that heterogeneous responses and signaling pathways of microglia are usually activated under exposure of various neuro-immunostimulants, and the activated microglia is mainly reflected by the dysregulated inflammation, accompanied with the unimpaired cell viability (Roosen et al., 2021; Wang et al., 2021b), which is in line with the findings in our manuscript. Accordingly, this study focused on the controlling of microglia activation and inflammation, and the inhibitory effects of Cori on NLRP3 inflammasome and ERS might be partly responsible for the protection role of Cori against morphine-induced BV2 microglial activation and inflammation. However, in addition to regulating inflammation, NLRP3 inflammasome and ERS ultimately induce pyroptosis and apoptosis, respectively, which seems to be inconsistent with the unchanged cell viability in this study. The existing evidence revealed that single treatment of morphine was sufficient to induce NLRP3 activation but was not enough to induce pyroptosis (Cai et al., 2016; Liu et al., 2020), which might partly explain this discrepancy. Anyway, whether Cori also influences microglia pyroptosis or apoptosis may be another interesting topic, which is deserved for further exploration.

In summary, our study demonstrated that Cori effectively alleviated the development of morphine tolerance by reducing morphine-induced neuroinflammation and microglia activation in BV-2 cells. Focusing on the molecular mechanism, Cori might exert its protective role through repressing TLR2, which subsequently inactivating ERS and NLRP3 inflammasome. This study might provide a novel potential strategy for overcoming morphine tolerance.

ACKNOWLEDGMENTS

This study was supported by Chengde Science and Technology Research and Development Program (202102A009).

Conflict of interest

The authors declare that there is no conflict of interest.

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