Melatonin alleviates intervertebral disc degeneration by disrupting the IL-1β/NF

Fan Chen,Guowei Jiang,Hui Liu,Zemin Li,Yuxin Pei,Hua Wang,Hehai Pan,Haowen Cui,Jun Long,Jianru Wang and Zhaomin Zheng,3

INTRODUCTION

Low back pain(LBP),one of the most common health problems,is a leading cause of disability worldwide and resultsin an enormous global burden to public health and the social economy,and~84%of people experience LBPsome point in their lifetime.1–6LBPis a multifactorial disease,7and the disorder is strongly associated with intervertebral disc degeneration(IVDD),which is characterized by a homeostatic imbalance between anabolism and catabolism,including extracellular matrix(ECM)degradation8–9or nucleus pulposus(NP)cell survival.10–12The intervertebral disc(IVD)is a special organ that consists of an outer fibrocartilaginous annulus fibrosus(AF)and an inner gel-like NP.13–14In flammatory responses,which are induced by in flammatory cytokine overexpression,are a primary and important cause of IVDD.Recent studieshave demonstrated that in flammatory cytokines,including tumor necrosis factor(TNF)-α and interleukin(IL)-1β,are strongly correlated with ECM degradation or NP cell survival.15–16Therefore,a more profound understanding of the molecular mechanisms underlying in flammatory cytokine secretion might provide new therapeutic targets for IVDD.

The NLR pyrin domain containing 3(NLRP3)in flammasome,a primary and crucial source of the highly in flammatory cytokines IL-1βand IL-18,is a canonical multimeric in flammasome complex that is composed of the adaptor apoptosis-associated speck-like protein containing a CARD(ASC)and the effector pro-caspase-1.17–18When exposed to exogenous or endogenous stimuli,the NLRP3 in flammasome becomes activated and drives caspase-1 activation,which results in the cleavage and maturation of IL-1βand IL-18.19Dysregulated NLRP3 in flammasome activation is involved in diverse diseases,including neurodegenerative diseases,20–21osteoarthritis,22cancer,23–24and in flammatory diseases.25–26Few studies have investigated the relationship between NLRP3 in flammasome activation and IVDD.However,numerous studies have con firmed that IL-1βis an important cause of IVDD,16,27and so we hypothesized that the NLRP3 in flammasome activation/IL-1βin flammatory response axis may play an important role in IVDD progression and that eliminating stimuli that activate the NLRP3 in flammasome may alleviate this progression.

Melatonin(N-acetyl-5-methoxytryptamine),which is synthesized by the pineal gland and many other organs,is a neuroendocrine hormone28–29that is involved in a wide range of physiological functions,including anti-in flammatory,30–31antidegenerative,32–33antioxidant,34–35immunomodulatory,36–37circadian rhythm regulation,38and cancer prevention activities.39–42Notably,recent studies have demonstrated that melatonin attenuates the in flammatory response by inhibiting NLRP3 in flammasome activation during the progression of atherosclerosis43and brain,44liver45and lung diseases.46Moreover,melatonin plays crucial roles in the IVDD process,including regulating NP cell proliferation,remodeling the ECM,47protecting vertebral endplate chondrocytes against apoptosis and calci fication,48and preventing oxidative stress-induced NPcell apoptosis,49indicating a strong correlation between melatonin and IVDD.Although these published studies have indicated that melatonin participates in the IVDD process by regulating NLRP3 in flammasome activation in NPcells,thishypothesishasnot been experimentally examined.

Based on these observations,the objective of this study was to explore the molecular mechanisms underlying a novel activation model of the NLRP3 in flammasome in IVDD.We also aimed to investigate whether exogenous melatonin administration prevents IVDDby regulating NLRP3 in flammasome activation in vitro and in vivo.

RESULTS

Melatonin ameliorates the progression of IVDD and LBPin vivo First,we established a rat IVDD model to determine whether melatonin exerts a protective effect during the progression of IVDD in vivo.Melatonin was intraperitoneally injected into rats with AFpuncture-induced IVDD.MRIimages were obtained 4 or 8 weeks post operation,and the IVDs from the rats treated with melatonin displayed a signi ficantly higher signal intensity than those without melatonin treatment(Fig.1b,c).Histologically,hematoxylin and eosin(H&E)and Safranin-O staining showed that the amounts of gelatinous NPtissue and the disc height in the melatonin-treated rats were larger than those in the AF puncture rats,while the histologic score of the AFpuncture rats was higher than that of the melatonin-treated rats(Fig.1d,e).IHC results showed that the expression of Aggrecan and Collagen II was decreased signi ficantly in the AF puncture group,and the levels of Aggrecan and Collagen II were increased in rats that were treated with melatonin(Fig.1f,g).Subsequently,the behavioral study results showed that melatonin signi ficantly decreased mechanical hyperalgesia and thermal hyperalgesia compared to those of the AF puncture group(Fig.1h).Therefore,these resultsindicated that melatonin alleviates the progression of IVDD and LBP.

Melatonin alleviates IVDD by inhibiting NLRP3 in flammasome priming and activation in vivo

Next,we further investigated the correlation between NLRP3 in flammasome priming and activation and IVDD in vivo.First,human discs with different grades of degeneration are shown as representative MRI images in Fig.2a(grades I–V).H&E staining showed that the number of NP cells was decreased in severely degenerated human discs(Fig.2b).Moreover,the expression of NLRP3,p20,and IL-1βwas increased in severely degenerated human discs compared with that in mildly degenerative human discs,as shown by IHC staining(Fig.2c,d).Furthermore,the expression of NLRP3,p20,and IL-1βwas increased in the IVDD rat model group compared with that of the control group,and melatonin administration reduced NLRP3,p20,and IL-1βexpression in the IVDD rat model(Fig.2e,f).Then,to examine the mechanism by which melatonin alleviatesthe progression of IVDD,we examined Aggrecan and Collagen IIlevels in the rat model.The expression of Aggrecan and Collagen II was signi ficantly decreased in the AF puncture with melatonin and LPSgroups(Fig.2g,h).These results demonstrated that NLRP3 in flammasome priming and activation were involved in the process of IVDDand that melatonin alleviated IVDD by inhibiting NLRP3 in flammasome priming and activation in vivo.

Melatonin suppresses NLRP3 in flammasome priming and activation in vitro

First,we elucidated the effect by which melatonin affects NPcell viability.Compared with the cytotoxicity in the control group,the groups treated with melatonin at concentrations below 4 mM for either 24 or 48 h did not show any obvious cytotoxic effects(Fig.3a,b).Subsequently,while investigating the effect of melatonin on NLRP3 in flammasome priming and activation in NP cells,we found that NLRP3 and p20 expression were decreased in NP cells treated with different melatonin doses,with the lowest measured levels occurring at a dose of 1 mM(Fig.3c,d).Furthermore,the expression levels of NLRP3 and p20 started to decrease in NP cells treated with melatonin for different lengths of time and were signi ficantly reduced at 24 h(Fig.3e,f).The RT-qPCR results for NLRP3 were in agreement with the western blot analysis results(Fig.3g,h).Furthermore,IF analysis also showed that melatonin suppressed NLRP3 in flammasome activation in NPcells(Fig.3i).These resultsshowed that melatonin suppresses NLRP3 in flammasome activation in vitro.

IL-1βinduces NLRP3 in flammasome priming and activation in vitro

Then,we investigated whether NLRP3 in flammasome priming and activation were induced in an IVDD cell model.As described in previous studies,IL-1β and TNF-α are classical cytokines that are used to establish an IVDD cell model.IL-1βis also produced by NLRP3 in flammasome activation.LPSis a classical stimulator of the NLRP3 in flammasome.Therefore,we selected IL-1β,TNF-α,and LPS to stimulate NP cells.First,NLRP3 and p20 expression was signi ficantly increased in NPcells treated with IL-1βor LPSbut only slightly increased in NP cells treated with TNF-α(Fig.4a,b).Furthermore,the levels of NLRP3 and p20 gradually increased in NP cells treated with different doses of IL-1βand peaked at a dose of 50 ng·mL-1(Fig.4c,d).After IL-1β treatment for different lengths of time,the expression of NLRP3 and p20 started to increase at 12h and exhibited an obvious increase at 24h(Fig.4e,f).The RT-qPCR results for NLRP3 expression were in agreement with the western blot analysis results(Fig.4g–i).In addition,IFstaining also showed that IL-1βtreatment increased NLRP3 expression in NPcells(Fig.4j).These results suggest that IL-1βenhances NLRP3 in flammasome activation in vitro.

IL-1βupregulates NLRP3 in flammasome priming and activation by increasing NF-κBsignaling and mtROSproduction in vitro

To examine the molecular mechanism by which IL-1βupregulates NLRP3 in flammasome priming and activation,we detected NF-κB signaling and mitochondrial reactive oxygen species(mtROS)production in NP cells.Previous studies have demonstrated that IL-1β is strongly associated with the NF-κBsignaling pathway.50–51In this study,we found that IL-1β activated the NF-κB signaling pathway in NP cells(Fig.S1).Subsequently,to test the role of NF-κBsignaling in NLRP3 in flammasome priming in IL-1β-treated NPcells,a silencing experiment was performed.Pretreatment with SM7368(a speci fic inhibitor of the NF-κB signaling pathway)or SM7368 plus IL-1βsigni ficantly decreased NLRP3 levels in NP cells compared with those of untreated NP cells(Fig.5a–c).

As previously described,mtROS production plays an important role in NLRP3 in flammasome activation.52–53Therefore,we examined whether mtROSproduction was involved in NLRP3 in flammasome activation in IL-1β-treated NP cells.We first detected that SOD2 expression was signi ficantly decreased in the IL-1βtreatment group compared with that of the control group,as measured by western blot and RT-qPCR analyses(Fig.5d–e).MitoSOX Red staining indicated that mtROS production was signi ficantly upregulated in IL-1β-treated NP cells(Fig.5g,h).Taken together,these results con firmed that an IL-1β/NF-κB-NLRP3 in flammasome positive feedback loop is involved in the process of IVDD.

Melatonin disrupts the IL-1β-NLRP3 in flammasome positive feedback loop in vitro

The preceding results indicated that melatonin suppresses NLRP3 in flammasome priming and activation in NP cells.Therefore,we determined whether melatonin reduces the in flammatory response by disrupting the IL-1β-NLRP3 in flammasome positive feedback loop in NPcells.First,western blot analysis showed that melatonin obviously attenuated the IL-1β-induced increase in NLRP3,pro-IL-1β,and p20,while MCC950 (a speci fic inhibitor of NLRP3 in flammasome activation)did not change the IL-1β-induced upregulation of NLRP3 and pro-IL-1β.In addition,a considerable decrease in p20 levels was observed in NPcells treated with melatonin plus IL-1β(Fig.6a,b).The RT-qPCRand western blot analysisresultsof NLRP3 and pro-IL-1βare shown in Fig.6c,d.In addition,IFstaining also con firmed that melatonin signi ficantly attenuated IL-1β-induced NLRP3 in flammasome activation(Fig.6e).These data con firmed that melatonin effectively disrupted the IL-1β-NLRP3 in flammasome positive feedback loop in vitro.

Melatonin disrupts the IL-1β-NLRP3 in flammasome positive feedback loop by downregulating NF-κBsignaling and mtROS production

We next assessed the underlying mechanism by which melatonin disrupts the IL-1β-NLRP3 in flammasome positive feedback loop.First,we demonstrated that melatonin signi ficantly suppressed NF-κB signaling activation(Fig.S2).Subsequently,an siRNA targeting P65(si-P65)was established and veri fied in NP cells(Fig.S3).Consistently,the NLRP3 level signi ficantly decreased when these cells were transfected with si-P65,and melatonin or si-P65 obviously inhibited the IL-1β-induced increase in NLRP3 expression(Fig.7a–c).Furthermore,IF staining also revealed that si-P65 attenuated NLRP3 expression in NP cells treated with si-P65 plus IL-1β(Fig.7d,e).In addition,melatonin prevented the IL-1β-induced decrease in SOD2 expression in NP cells(Fig.7f–h).MitoSOX Red staining showed that mtROS production was signi ficantly reduced in NP cells of the melatonin plus IL-1βtreatment groups(Fig.7i,j).Therefore,these results con firmed that melatonin disrupted the IL-1β positive feedback loop by suppressing NF-κB signaling and mtROSproduction in vitro.

Melatonin disrupts the IL-1β/NF-κB-NLRP3 in flammasome positive feedback loop in vivo

IHC staining showed that,in human samples,p-P65 expression was higher in severely degenerated discs than in mildly degenerated discs,while SOD2 expression was reduced(Fig.8a,b).Furthermore,the percentage of p-P65-positive cells decreased signi ficantly,while the percentage of SOD2-positive cells increased in melatonin plus AF puncture rats compared with those of AFpuncture rats(Fig.8c,d).These results con firmed that melatonin suppresses the in flammatory response by disrupting the IL-1β/NF-κB-NLRP3 in flammasome positive feedback loop in vitro and in vivo.In conclusion,these results indicated that melatonin disrupts the IL-1βpositive feedback loop by suppressing NF-κBsignaling and mtROSproduction and attenuating the progression of IVDD,as illustrated in the proposed schematic representation of melatonin-mediated IVDD in vivo and in vitro(Fig.8e).

DISCUSSION

Melatonin is known to have multiple effects,including antioxidant,anti-in flammatory,and antiapoptotic impacts,in different systems.28–29A recent study showed that melatonin downregulates the gene expression of cyclin D1,PCNA,matrix metallopeptidase(MMP)-3,and MMP-9 and upregulates the gene expression of collagen type II alpha 1 chain and aggrecan in NP cells.47Our results were consistent with previous data and showed that the application of melatonin in an IVDD model alleviated the progression of IVDD and LBP.However,whether NLRP3 in flammasome priming and activation are involved in this process is still unknown.Thus,we evaluated the markersof NLRP3 in flammasome priming and activation and found that in the IVDD group,the expression of NLRP3,P20,and IL-1βwas elevated,indicating the priming and activation of the NLRP3 in flammasome.Furthermore,LPS,which is a proven NLRP3 in flammasome activator,abrogated the effect of melatonin on IVDD,indicating that melatonin acts by inhibiting NLRP3 in flammasome priming and activation.Data from human disc samples also showed that in the degenerative disc,NLRP3 in flammasome priming and activation marker expression were signi ficantly increased,which gives us more con fidence that the NLRP3 in flammasome plays an important role in IVDD.

One of the main causes of IVDD is an abnormal in flammatory response,which is induced by the overexpression of in flammatory cytokines,mainly IL-1β,causing NPcell apoptosis,downregulating matrix gene expression or upregulating the expression of collagen-and aggrecan-cleaving enzymes.For the first time,our study showed that IL-1βpromotes its own expression by upregulating NLRP3 in flammasome activation.Furthermore,melatonin,an anti-in flammatory molecule,disrupts the IL-1βpositive feedback loop by suppressing the NF-κB signaling pathway and decreasing mtROSproduction.

First,we found that IL-1βinduced NLRP3 in flammasome priming and activation and thus promoted its own overexpression.IL-1βhas been widely studied in IVDD due to its catabolic effect.Le Maitre et al.reported that IL-1βis synthesized by native disc cells and that treating human disc cells with IL-1βinduces an imbalance between catabolism and anabolism.27These responses represent the changes observed during IVDD.Hence,these findings provide a potential strategy for biological therapy by inhibiting IL-1βto prevent and reverse IVDD.Thus,it isessential to study the expression and regulatory mechanism of IL-1βin IVDD.We first found that in degenerated human NP tissue,the expression of NLRP3,p20,and IL-1βwas elevated.The NLRP3 in flammasome has been reported to be activated by LPS or hyperosmotic stress in different systems.Dolunay et al.showed that the inhibition of NLRP3/ASC/pro-caspase-1 in flammasome formation and activity prevents LPS-induced in flammatory hyperalgesia in mice.54–55However,whether IL-1β activates the NLRP3 in flammasome has seldom been studied.In this study,we showed for the first time that in NP cells,IL-1βtreatment activated the NLRP3 in flammasome in time-and dose-dependent manners.In addition,IL-1βtreatment had an effect similar to that of LPS treatment in upregulating NLRP3 and p20 expression.Interestingly,according to previous studies,the NLRP3 in flammasome is a component of the innate immune system that processes pro-IL-1β into a mature cytokine.17–19Recently,Tang et al.reported that honokiol suppresses activation of the TXNIP-NLRP3 in flammasome in H2O2-stimulated NP cells,thereby inhibiting the activation of downstream in flammatory mediators such as IL-1β.56Therefore,it is reasonable that IL-1βpromotes its own expression through NLRP3 in flammasome activation,forming a positive feedback loop.

Furthermore,we studied the mechanism by which IL-1β induces NLRP3 in flammasome priming and activation.Our previous studies showed that in NP cells,IL-1β activates NF-κB signaling through p65 phosphorylation.51There are some studies concerning NLRP3 in flammasome regulation,and various pathways,including the Smad,NFAT,NF-κB,and MAPkinase pathways,regulate NLRP3 expression.57–58Yu et al.showed that hepatitis Be antigen suppresses LPS-induced NLRP3 in flammasome activation and IL-1β production by repressing NLRP3 and pro-IL-1β expression through inhibition of NF-κB phosphorylation and by repressing caspase-1 activation and IL-1βmaturation through inhibition of ROSproduction.59Budaiet al.also reported that LPS induces NLRP3 in flammasome regulation through the NF-κB,p38,JNK,and ERKsignaling pathways.60In this study,we found that IL-1β induced NLRP3 expression through NF-κB activation.This conclusion was determined by the application of SM7368,a speci fic NF-κBpathway inhibitor.mtROSare the main activators of the NLRP3 in flammasome;52–53thus,it is important to know whether IL-1βregulates mtROS production.According to our MitoSOX Red staining and western blotting results,IL-1β treatment reduced SOD2 expression and induced mtROSproduction in NPcells.In conclusion,our results indicated that in NPcells,IL-1β activates the NLRP3 in flammasome through NF-κBsignaling activation and mtROSproduction.

Furthermore,we found that melatonin disrupted the IL-1β positive feedback loop and studied the mechanism of this disruption.Melatonin has also been reported to suppress NLRP3 in flammasome activation and IL-1βexpression.Dong et al.showed that melatonin attenuates NLRP3,ASC,cleaved caspase-1,IL-1β,and IL-6 expression.44Consistent with previous reports,our data showed that melatonin suppressed NF-κBsignaling activation and reduced mtROS production to inhibit NLRP3 in flammasome activation and IL-1βexpression.We con firmed these results by applying p65 siRNA and MCC950(a speci fic inhibitor of NLRP3 in flammasome activation).Furthermore,we con firmed our discovery in vivo.Our data showed that in this IVDD model,p-p65 expression wasdecreased and SOD2 expression was increased after melatonin treatment,indicating that melatonin exerted antiin flammatory effectsvia the NF-κB-NLRP3 in flammasome-IL-1β axis.

There were several limitations to this study.First,the number of human IVDtissue samples was relatively small due to the dif ficulty associated with acquiring grade I/II discs in clinical practice.Second,the detailed mechanisms by which melatonin suppresses NF-κB signaling and mtROSproduction were not addressed and might be elucidated in future studies.

In conclusion,we found for the first time that IL-1βforms a positive feedback loop through NLRP3 in flammasome activation in IVDD and that melatonin disrupts this vicious cycle by suppressing NF-κB signaling activation and mtROS production.Our research showed a new mechanism by which IL-1βpromotes the in flammatory response in IVDD,and melatonin may be used as a therapeutic agent for the treatment of in flammatory cytokinerelated IVDD.

MATERIALSAND METHODS

Collection of human IVDs

Before tissue collection,each patient signed an informed consent form,and the Medical Ethics Committee of The First Af filiated Hospital of Sun Yat-sen University(no:[2017]203)approved this study.All studies in this paper were performed according to The Code of Ethics of the World Medical Association(Declaration of Helsinki).61Between March 2016 and April 2018,we collected 25 disc samples(detailed information about the specimensisin Table 1)from patients(male:female,13:12).The degree of disc degeneration was evaluated by P firrmann classi fication.Normal discs were obtained from patients with trauma and deformity,and degenerated discswere obtained from patientswith degenerative spinal diseases(disc herniation,spinal canal stenosis,and degenerative scoliosis).

Cell culture and cell viability assay

As previously described,62NPcells were isolated and cultured in DMEM(Invitrogen,CA,USA)containing 10%fetal bovine serum(Invitrogen,CA)and penicillin/streptomycin(Invitrogen,CA)at 37°C in a humidi fied incubator with 5%CO2.We harvested NP cells using solutions containing trypsin(0.25%)and EDTA(1 mM)(Invitrogen,CA)and subcultured the cells in 10cm dishes.NPcells were seeded in six-well plates,grown to~80%con fluence and treated with melatonin(1 mmol·L-1,M5250,Sigma-Aldrich,USA),MCC950(10 nmol·L-1,Selleck,a speci fic inhibitor of NLRP3 in flammasome activation),lipopolysaccharide(LPS,200ng·mL-1,L2880,Sigma-Aldrich,USA),TNF-α (100 ng·mL-1,210-TA-020,R&D Systems,USA),or IL-1β (50ng·mL-1,201-LB-005,R&D Systems,USA)for 24 h for subsequent experiments.The cytotoxic effect of melatonin on NP cells was detected using a cell counting kit(CCK)-8 assay(Dojindo Laboratories,Kumamoto,Japan)according to the manufacturer’s instructions.

Table 1.Information of human disc samples from 25 patients

Small interfering RNA(siRNA)transfection

Rat NPcells were seeded at 5×106per well in a six-well plate and transfected with negative control or siRNAtargeting P65(RiboBio,Guangzhou,Guangdong,China)when the cells reached 60%–70%con fluence.The sequences for the P65-speci fic siRNAs were as follows:1:GCTGCAGTTTGATGATGAA,2:GCCCTATCCCTTTACGTCA,and 3:GGACATATGAGACCTTCAA(10nmol·L-1).Then,we used 250μL of serum-free optical-MEM (Invitrogen,CA,USA)to individually dissolve 5μL of siRNA or 10μL of Lipofectamine 3000(Invitrogen,CA,USA).After mixing them together,the mixture was added to the cells.After treatment,the cells were harvested for protein/RNA extraction.

Western blot analysis

The proteins of treated NPcells were extracted and electrophoretically separated via 10%or 15%SDS-PAGE,as previouslydescribed.63Subsequently,the membranes were blocked with 3%bovine serum albumin and incubated with primary antibodies.The primary antibodies included anti-pro-IL-1β(1:1000,12703,Cell Signaling Technology),anti-IL-1β(1:1 000,ab8320,Abcam),anti-phospho-P65(1:1 000,3033,Cell Signaling Technology),anti-P65(1:1 000,8242,Cell Signaling Technology),anti-phospho-Erk1/2(1:1000,4370,Cell Signaling Technology),anti-Erk1/2(1:1 000,4695,Cell Signaling Technology),anti-phospho-P38(1:1 000,4511,Cell Signaling Technology),anti-P38(1:1 000,8690,Cell Signaling Technology),anti-NLRP3(1:1 000,AG-20B-0014,AdipoGen),anticleaved Caspase-1(p20)(1:1 000,AG-20B-0042,AdipoGen),anti-ASC(1:1 000,AG-25B-0006-C100,AdipoGen),anti-Caspase-1(1:1000,ab1872,Abcam),anti-superoxide dismutase 2(SOD2)(1:1 000,13141,Cell Signaling Technology),and anti-β-actin(1:3 000,4970,Cell Signaling Technology).After washing with PBS,the membranes were incubated with the following secondary antibodies:anti-rabbit IgG(1:5000,7074,Cell Signaling Technology)or anti-mouse IgG(1:5 000,7076,Cell Signaling Technology).Finally,the Western blot bands were detected using enhanced chemiluminescence detection reagents(Invitrogen,CA,USA)and quanti fied using ImageJ software(National Institutes of Health,Bethesda,MD,USA).

Table 2. Speci fic primers

Real-time quantitative polymerase chain reaction(RT-qPCR)

The isolation of total RNA from NP cells was performed using RNAiso Plus(Takara,Japan).Complementary DNA synthesis was performed using a Prime Script RT Master Mix kit(Takara)according to the manufacturer’s instructions.SYBRgreen Premix Ex Taq II(Takara)was used to detect the relative mRNA levels of NLRP3,pro-IL-1β,SOD2,P65,and β-Actin,and the sequences of the primer pairs are listed in Table 2(Sangon Biotech,Shanghai,China).RT-qPCRwas performed on an ABI7900HTFast Real-Time PCR System(Applied Biosystems)for 40 cycles and quanti fied using the 2-ΔΔCtmethod.

MitoSOXRed and immuno fluorescence(IF)staining

NP cells were treated as described,incubated with 2.0mL of 5 μmol·L-1MitoSOX Red reagent or fixed with 4%paraformaldehyde and then blocked with 5%normal goat serum.Subsequently,the cells were incubated with anti-NLRP3(1:200,ab4207,Abcam)and anti-phospho-P65(1:200,3033,Cell Signaling Technology)antibodies overnight,followed by incubation with Alexa Fluor-488-conjugated anti-rabbit and anti-goat secondary antibodies(Invitrogen,1:2 000).Nuclear staining was performed using DAPI.Finally,the cells were photographed under an Olympus BX63 microscope(Tokyo,Japan)at×400 and ×1 000 magni fications.

Animal model and magnetic resonance imaging(MRI)evaluation As previously described,64the rats(weighing 200–250 g,n=5 per group)were divided into three groups:the blank group received no puncture and was intraperitoneally injected with 0.9%normal saline;the other two groups underwent AFpuncture surgery with a 21-gauge needle inserted 3.0 mm into the L3/4 IVDs for 30 s.At 4 or 8 weeks post operation,MRI(T2-weighted images)signal change examinations were performed on the rats(n=5 per group).In addition,the degree of disc degeneration was evaluated by P firrmann classi fication.

Melatonin was diluted to 20mg·mL-1with normal saline and then intraperitoneally injected into the rats(30 mg·kg-1per week),in addition to LPS(2 mg·kg-1per week),until the day of euthanasia.

Immunohistochemistry(IHC)and histopathological analysis

The specimens were embedded in paraf fin and cut into 5μm sections.Subsequently,the sections were deparaf finized and rehydrated,followed by H&E and Safranin-O staining or antigen retrievalwith 0.01 mol·L-1sodium citrate.The sectionswere blocked with 3%hydrogen peroxide and 5%normal goat serum.Then,the slides were incubated with the following primary antibodies:anti-NLRP3(1:200,AG-20B-0014,AdipoGen),anti-cleaved caspase-1(p20)(1:200,AG-20B-0042,AdipoGen),anti-IL-1β(1:200,ab8320,Abcam),anti-SOD2(1:1000,13141,Cell Signaling Technology),and antiphospho-P65(1:200,3033,Cell Signaling Technology).The sections were incubated with a secondary antibody and then developed with DABsolution.Hematoxylin was used for nuclear staining.Finally,the sections were observed and imaged under an Olympus BX63 microscope at ×10, ×50,and ×400 magni fications,and the percentages of NLRP3+,IL-1β+,p20+,SOD2+,and phospho-P65+cells in the IVD samples were quanti fied using ImageJ software(National Institutes of Health,Bethesda,MD,USA).65The histologic scores were assessed as previously described:normal disc,fi ve;moderately degenerated disc,6–11;and severely degenerated disc,12–14.66–67

Behavioral study

The rats were subjected to behavioral tests as described in our previous study.63Re flex reactions to both mechanical and harmful thermal stimuliapplied to both hind paws were measured in five rats from each group within a week of surgery and every 2 weeks during the following 10 weeks.

Statistical analysis

The differences among the groups were assessed by one-way analysis of variance(ANOVA),which was performed using SPSS software version 19.0 for Windows(IL,USA).If the ANOVA results were statistically signi ficant,the differences between the two groups were examined by using Bonferroni’s post hoc test.The data are presented as the mean±SD.Pvalues＜0.05 were considered statistically signi ficant.

ACKNOWLEDGEMENTS

We thank Qifeng Ou,Zheng Yu,and Haita o Cui(Sun Yat-sen University)for assisting with the established IVDD rat model and immunohistochemical staining.This work was supported by grants from the Natural Science Foundation of Guangdong Province(Grant no:2017A030313670)awarded to J.W.and the National Natural Science Foundation of China(81572175 and 81772386),awarded to Z.Z.

AUTHORCONTRIBUTIONS

Study design:J.W.and Z.Z.Study conduction:F.C.,G.J.,H.L.,and Z.L.Data collection:F.C.,Y.P.,and Z.L.,H.P.,J.L.,and H.C.Data analysis:F.C.,Z.Z.,and J.W.Data interpretation:F.C.,H.L.,Z.Z.,and J.W.Drafting the paper:F.C.and J.W.

ADDITIONAL INFORMATION

The online version of this article (https://doi.org/10.1038/s41413-020-0087-2)contains supplementary material,which is available to authorized users.

Competing interests:The authors declare no competing interests.