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Brain Research Bulletin

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Research report

Effects of nanoparticle-encapsulated curcumin on HIV-gp120-associatedneuropathic pain induced by the P2X3 receptor in dorsal root ganglia

Shanhong Zhaoa,b,1, Jinpu Yangc,1, Xinyao Hand, Yingxin Gongd, Shenqiang Raoa,b, Bing Wua,b,Zhihua Yia,b,e, Lifang Zoua,b, Tianyu Jiaa,b, Lin Lia,b, Huilong Yuana,b, Liran Shia,b,Chunping Zhangb,f, Yun Gaoa,b, Guilin Lia,b, Shuangmei Liua,b, Hong Xua,b, Hui Liua,b,Shangdong Lianga,b,⁎

a Department of Physiology, Medical School of Nanchang University, Nanchang, Jiangxi 330006, People’s Republic of Chinab Jiangxi Provincial Key Laboratory of Autonomic Nervous Function and Disease, Nanchang, Jiangxi 330006, People’s Republic of Chinac Queen Mary School, Medical College of Nanchang University Nanchang, Jiangxi 330006, People’s Republic of Chinad Undergraduate Student of the First Clinical Department, Medical School of Nanchang University, Nanchang, Jiangxi 330006, People’s Republic of Chinae Nursing College, Medical School of Nanchang University, Nanchang, Jiangxi 330006, People’s Republic of Chinaf Department of Cell Biology, Medical School of Nanchang University, Nanchang, Jiangxi 330006, People’s Republic of China

A R T I C L E I N F O

Keywords:HIV-gp120-associated neuropathic painP2X3 receptorDorsal root gangliaNanoparticle-encapsulated curcumin

A B S T R A C T

HIV-1 envelope glycoprotein (Glycoprotein 120, gp120) can directly stimulate primary sensory afferent neuronsand cause chronic neuropathic pain. The P2X3 receptor in the dorsal root ganglia (DRG) is associated with thetransmission of neuropathic pain. Curcumin isolated from the herb Curcuma rhizome has anti-inflammatory andanti-tumor effects. The water solubility, targeting and bioavailability of curcumin can be improved by nano-particle encapsulation. In this study, we sought to explore the effects of nanoparticle-encapsulated curcumin(nano curcumin) on HIV-gp120-induced neuropathic pain mediated by the P2X3 receptor in DRG neurons. Theresults showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared tothose in the control group. The expression levels of P2X3 mRNA and protein in rats treated with gp120 werehigher than those in the control group. Nano curcumin treatment decreased mechanical hyperalgesia andthermal hyperalgesia and upregulated the expression levels of P2X3 mRNA and protein in rats treated withgp120. Nano curcumin treatment also reduced the ERK1/2 phosphorylation levels in gp120-treated rat DRG. Inaddition, P2X3 agonist α,β-methylene ATP (α,β-meATP)-induced currents in DRG neurons cultured with gp120significantly decreased after co-treatment with nano curcumin. Therefore, nano curcumin treatment may inhibitP2X3 activation, decrease the sensitizing DRG primary afferents and relieve mechanical hyperalgesia andthermal hyperalgesia in gp120-treated rats.

1. Introduction

Peripheral neuropathy in patients with human immunodeficiencyvirus (HIV) has become the most common neurological complication.HIV-associated chronic pain is a common neurological disease and itsincidence in HIV infection is as high as 30%. The mechanism of HIV-associated neuropathic pain is not currently clear. The HIV virus doesnot directly infect neurons (Nasirinezhad et al., 2015), but the HIVenvelope glycoprotein, gp120, contributes to HIV-associated painfulneuropathy (Hao, 2013). HIV-1 proteins are capable of producing painsignaling through direct actions on the excitability and the survival ofdorsal root ganglion (DRG) neurons (Hao, 2013). DRG afferent fibers

are distributed to both central and peripheral terminals and transmitnoxious stimuli from the periphery to the central nervous system(Basbaum et al., 2009). The peripheral administration of gp120 fa-cilitated thermal hyperalgesia and mechanical allodynia in rat models(Milligan et al., 2000; Herzberg and Sagen, 2001; Wallace et al., 2007;Maratou et al., 2009; Kamerman et al., 2012; Hao, 2013).

ATP as a neurotransmitter widely exists in the peripheral and cen-tral nervous system and is closely associated with pain. The extra-cellular ATP released from injured cells and inflammatory tissues canactivate P2X and P2Y receptors in primary afferent neurons. The P2X3receptor is selectively expressed in primary afferent sensory neurons inthe DRG (Chen et al., 1995; Burnstock, 2000) and plays an important

http://dx.doi.org/10.1016/j.brainresbull.2017.09.011Received 2 May 2017; Received in revised form 14 September 2017; Accepted 22 September 2017

⁎ Corresponding author at: Department of Physiology, Medical School of Nanchang University, Nanchang, Jiangxi 330006, People’s Republic of China.

1 These authors contributed equally to this work.E-mail address: liangsd@hotmail.com (S. Liang).

Brain Research Bulletin 135 (2017) 53–61

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role in the generation and maintenance of chronic neuropathic pain andinflammatory pain (Burnstock, 2000; Gao et al., 2011a; Burnstock,2013). The interaction of the HIV gp120 with macrophages stimulatesATP release and P2X receptors are necessary for HIV entry into mac-rophages (Hazleton et al., 2011; Lee et al., 2012). ATP signaling is as-sociated with the regulation of inflammatory responses during acuteviral infection (Lee et al., 2012). Blocking P2X receptors leads to asignificant reduction in HIV replication in macrophages (Hazletonet al., 2011; Lee et al., 2012).

Curcumin is the active ingredient extracted from the natural med-icine turmeric rhizome (Goel et al., 2008; Aggarwal and Harikumar,2009). Studies have shown that curcumin has anti-tumor, anti-in-flammatory, antioxidant and antimicrobial properties (Larsen et al.,2007; López-Lázaro, 2008). Although curcumin is non-toxic and hassmall side effects (Dickinson et al., 1992; Chu et al., 2013), the use ofcurcumin is limited due to its poor metabolism stability in vivo and lowbioavailability (Anand et al., 2007; Goel et al., 2008). Nanoscale drugdelivery systems formulated from biocompatible and biodegradablepolymers constitute an evolving approach to drug delivery (Bala et al.,2005; Lamprecht et al., 2005). Nanoparticle encapsulation of drugs canimprove the drug targeting, the drug bioavailability and reduce drugtoxicity side effects (Lamprecht et al., 2005; Chu et al., 2013), so themedicinal properties of nanoparticle-encapsulated curcumin can befully utilized (Chu et al., 2013; Yang et al., 2015). In this study, weinvestigated the effects of nanoparticle-encapsulated curcumin ongp120-induced neuropathic pain mediated by the P2X3 receptor in ratprimary afferent neurons.

2. Materials and methods

2.1. Animals and surgical methods

Healthy Male Sprague-Dawley (SD) rats weighed in at 180 g–250 g.They were provided by the Center of Laboratory Animal Science ofNanchang University. The procedures were approved by the AnimalCare and Use Committee of Nanchang University Medical School. Aquiet environment was provided for animal husbandry, including goodindoor ventilation and an air filtration system. The room was main-tained at 22 °C with 50% humidity, a light illumination cycle of12 h:12 h, free access to food, and frequently changed cages and bed-ding. The rats were randomly divided into five groups (with 8 rats ineach group): the control group (ctrl group); the sham operation group(sham group); the HIV-gp120 group (gp120 group); the HIV-gp120 ratstreated with the nanoparticle-encapsulated curcumin group(gp120 + nano curcumin, 4 mg/ml group); and the HIV-gp120 ratstreated with the nanoparticle encapsulation carrier group(gp120 + nano carrier group).

A previously described technique was used for perineural HIV-gp120 administration (Yi et al., 2017). Briefly, under 10% chloral hy-drate anesthesia (3 ml/kg, i.p., supplemented as necessary), the leftsciatic nerve of the SD rats was exposed in the popliteal fossa, withoutdamaging the nerve construction. A 2 × 6 mm strip of oxidized re-generated cellulose was previously soaked in 250 μl of a 0.1% rat serumalbumin (RSA) saline solution containing 200 ng of gp120 (Sigma) or0.1% RSA in saline for the sham surgery. A 3–4 mm length of the sciaticnerve proximal to the trifurcation was wrapped loosely with the soakedcellulose to avoid any nerve constriction and was left in situ. The in-cision was closed with 4-0 sutures (Wallace et al., 2007; Yi et al., 2017).At 7 days, 10 days, and 13 days after surgery, nanoparticle-en-capsulated curcumin (4 mg/ml dose of curcumin) in the gp120 + nanocurcumin group and nanoparticle encapsulation carrier dissolved insaline was administered into the sublingual vein.

2.2. Synthesis of poly-PEGMA-DMAEMA-MAO nano macromolecule

Biological nanocarriers can not only improve the solubility and

bioavailability of a drug, but can also control drug release and attenuatethe toxic side effects (Bala et al., 2005; Bisht et al., 2007). Reversibleaddition fragmentation chain transfer (RAFT) radical polymerizationhas developed into an extremely versatile controlled/living free radicalpolymerization technique with respect to the reaction conditions andthe wide range of applicable monomers (Liu et al., 2010). A typicalprotocol for the synthesis of a amphiphilic polymer macromolecule(PEGMA-DMAEMA- MAO) was conducted through RAFT solutionpolymerization (Chiefari et al., 1998; Byard et al., 2017). PEGMAserved as the hydrophilic segment, MAO served as hydrophobic blocks,and DMAEMA served as a segment to bind DNA. Simply, CTA(0.5 mmol), AIBN (0.05 mmol), DMAEMA (5 mmol), PEGMA(10 mmol), and MAO (10 mmol) were weighed into a 50 ml round-bottomed flask. Methylbenzene (20 ml) was added to produce homo-geneity of the mixture solution, which was purged with nitrogen for30 min. The sealed flask was immersed in an oil bath set at 55 °C for24 h under nitrogen conditions. Then, the polymerization was subse-quently dried under vacuum rotary steam. The block copolymer waspurified by dialysis against distilled water/ethanol for 48 h (MWCO3500) and recovered by freeze-drying. The purified PEGMA-DMAEMA-MAO was obtained as a yellow solid.

2.3. The synthesis of curcumin loaded poly-PEGMA-DMAEMA-MAOmicrospheres

To combine the advantages of biological nanocarriers and the meritsof curcumin, curcumin-polybutylcyanoacrylate nanoparticles had beenprepared using anionic emulsion polymerization (Gaspar et al., 1991;Bisht et al., 2007; Shaikh et al., 2009). A typical oil-in-water (O/W)solvent evaporation method was used to prepare the microspheres(Almaaieh and Flanagan, 2001). Curcumin (50 mg) was dissolved in16 ml of absolute ethyl alcohol. Poly-PEGMA-DMAEMA-MAO (60 mg)was dissolved in 6 ml of acetone. Then, the two solutions were mixed toobtain two-phase organic solvents. The organic solvents were droppedin distilled water (100 ml, pH 5.0) at 2 ml/min under an ice bath. Andorganic phase was emulsified with a homogenizer into the aqueousphase. The dispersion was then evaporated under ambient temperatureand pressure to harden the microspheres. The microspheres were thenseparated by filtration, vacuum dried, weighed and stored in a vacuumdessicator.

2.4. Measurement of the mechanical withdrawal threshold (MWT)

Mechanical hyperalgesia in rats was measured every other day afteroperation until day 14 after surgery. Determination of the MWT wasperformed at 8:00–12:00 using a BME-404 electronic mechanical sti-mulator (Institute of Biomedical Engineering, Chinese Academy ofMedical Sciences, Tianjin, China). The main technical parameters ofthis equipment were as follows: end face diameter of the test needle,0.6 mm; pressure measurement range, 0.1–50 g; and a pressure mea-surement resolution of 0.05 g. An organic glass box (22 × 22 × 12 cm)was placed on the sieve of the metal frame. The rat was placed into thebox for 30 min of adaptation. The left hind paws were touched with thetest needle until escaping behavior was observed. The pressure valuewas automatically recorded. Measurements were performed 5 times foreach rat (interval, ≥5 min) and the mean value was calculated as MWTfor this measurement (Lin et al., 2010; Nasirinezhad et al., 2015; Yiet al., 2017).

2.5. Measurement of the thermal withdrawal latency (TWL)

Thermal hyperalgesia in rats was measured every other day afteroperation until day 14 after surgery. The latency to hind paw with-drawal from a thermal stimulus was determined by exposing the plantarsurface of the hind paw to radiant heat using the Thermal PawStimulation System (BME-410C, Tianjin) (Lin et al., 2010; Nasirinezhad

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et al., 2015; Yi et al., 2017). Rats were placed in a transparent, square,bottomless acrylic box (22 cm× 12 cm× 22 cm) on a glass plate witha light located underneath. After a 30-min habituation period, theplantar surface of the paw was exposed to a beam of radiant heat ap-plied through the glass floor. Activation of the bulb simultaneouslyactivated a timer and both were immediately turned off by paw with-drawal or at the 25 s cut-off time. The hind paws were tested by ablinded observer in triplicate at 5 min intervals.

2.6. Real-time PCR

The DRG samples were detected at day 14 postoperatively. The ratsin the five groups were anesthetized by 10% chloral hydrate (3 ml/kg,i.p.). The L4–6 DRGs were isolated immediately and flushed with ice-cold PBS. The total RNA samples were prepared from the L4–6 DRGs ofeach group using TRIzol Total RNA Reagent (Beijing Tiangen BiotechCo.). cDNA synthesis was performed with 2 μg of total RNA using theRevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas,Burlington, Ontario, Canada). The primers were designed with PrimerExpress 3.0 software (Applied Biosystems), and the sequences were asfollows: P2X3, forward 5′-GAGTCCGAGGCAATCTAATG -3′; reverse 5′-CTGTGATCCCAACAAAGGTC-3′; β-actin, forward 5′-TAAAGACCTCTATGCCAACACAGT -3, reverse 5′-GGGGTGTTGAAGGTCTCAAA -3′.Quantitative PCR was performed using the SYBR® Green MasterMix inan ABI PRISM® 7500 Sequence Detection System (Applied BiosystemsInc., Foster City, CA). The quantification of gene expression was per-formed using the ΔΔCT calculation with CT as the threshold cycle. Therelative levels of the target genes, normalized to the sample with thelowest CT, were given as 2−ΔΔCT (Tu et al., 2013; Yi et al., 2017).

2.7. Immunohistochemistry

Immunohistochemical staining was performed using a SP-9001 kit(Beijing Zhongshan Biotech Co.) according to the manufacturer’s in-structions. Briefly, the animals were anesthetized with penthiobarbitalsodium and the DRGs were dissected (Lin et al., 2010; Gao et al., 2011a;Gao et al., 2011b; Yi et al., 2017). The DRGs isolated from rats werewashed with phosphate-buffered saline (PBS). After fixation with 4%paraformaldehyde (PFA) for 24 h, the ganglia were dehydrated with20% sucrose overnight at 4 °C and the ganglia were then cut into 20-μm-thick sections using a cryostat. After three washes with PBS, thesections were incubated in 3% H2O2 for 10 min to block endogenousperoxidase activity and then with 10% goat serum for 30 min at roomtemperature to block non-specific antigens. After several rinses andwashes in PBS, the blocked sections were incubated with rabbit anti-P2X3 (1:2500 diluted in PBS; CHEMICON International, Inc., USA)overnight at 4 °C. After 3 rinses in PBS, the sections were incubatedwith a biotinylated goat anti-rabbit secondary antibody (BeijingZhongshan Biotech Co.) for 1 h at room temperature. The preparationswere washed in PBS and streptavidin-horseradish peroxidase (BeijingZhongshan Biotech Co) was added for 30 min. The diaminobenzidinechromogen was developed for 2 min and the slides were washed withdistilled water and coverslipped. Following immunohistochemistry, animage scanning analysis system (Image-Pro Plus software, Media Cy-bernetics, Silver Spring, USA) was used to analyze the changes in theintensity values (integrated optical density) of the P2X3 stain in theganglia. The immunohistochemical signal was assessed by estimatingthe area of the positive expression objects and the optical intensity perobject, as the integrated optical density (IOD). The IOD value reflectsthe amount of protein expression. All the photos for analysis are ac-quired under the same shooting condition to avoid background differ-ences. We can get a set of data from different photos of the same group.So we calculate the mean and standard deviation of the groups. Andthen we compare the IOD values between different groups by using oneway analysis of variance (ANOVA).

2.8. Western blotting

The DRG samples were detected at day 14 postoperatively. Theanimals were anesthetized and tissue collection was performed as de-scribed above, except that the tissues were snap-frozen in tubes on dryice during the collection (Lin et al., 2010; Yi et al., 2017). Briefly, on the14th day after the operation, the animals were anesthetized withchloral hydrate and the L4–6 DRGs neurons were dissected. The DRGswere isolated immediately and rinsed in ice-cold phosphate-bufferedsaline (PBS). The ganglia were homogenized by mechanical disruptionin a lysis buffer containing the following: 50 mM Tris-Cl, pH 8.0,150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40,0.02% sodium deoxycholate, 100 μg/ml phenylmethylsulfonyl fluoride,and 1 μg/ml Aprotinin. The cells were incubated on ice for 50 min. Thehomogenates were then centrifuged at 12,000 rpm for 10 min and thesupernatants were collected. The quantity of total proteins in the su-pernatants was determined using the Lowry method. After dilution withloading buffer (250 mM Tris-Cl, 200 mM Dithiothreitol, 10% sodiumdodecyl sulfate (SDS), 0.5% Bromophenol Blue, and 50% Glycerol) andheating to 95 °C for 5 min, the samples containing equal amounts ofprotein (20 μg) were separated by 10% SDS–polyacrylamide gel elec-trophoresis using a Bio-Rad system. The proteins were then transferredonto PVDF membranes by electrophoretic transfer using the samesystem. The membrane was blocked with 5% non-fat dry milk in 25 mMTris buffered saline, pH 7.2, plus 0.05% Tween 20 (TBST) for 2 h atroom temperature followed by incubation with a rabbit anti-P2X3(1:200 dilutions, Alomone Labs, Israel) and mouse monoclonal anti-β-actin antibody (1:800 dilutions, Beijing Zhongshan Biotech Co., China)at 4 °C overnight. The membranes were washed three times with TBSTand incubated (1 h, room temperature) with a horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG or goat anti-rabbitIgG (1:2000, Beijing Zhongshan Biotech Co.) in blocking buffer. Afteranother wash cycle, the labeled proteins were visualized by enhancedchemiluminescence (ECL) on a high-performance film (Shanghai PufeiBiotech Co.). The chemiluminescent signals were collected on auto-radiography film and the band intensity was quantified using Image ProPlus software. The relative band intensities of the target proteins werenormalized against the intensity of the respective β-actin internalcontrol.

2.9. Isolation of DRG neurons and culture

DRG neurons from adult Sprague-Dawley rats were prepared usingthe methods described previously with slight modifications (Oh et al.,2001). Briefly, adult rats were anesthetized and decapitated and theDRGs, together with the dorsal and ventral roots and the attachedspinal nerves, were removed from the inner side of each half of thedissected vertebrae with fine dissecting forceps. After the removal ofthe attached nerves and the surrounding connective tissue, the DRGswere incubated with trypsin (2.5 mg/ml; type III, Sigma), collagenase(1.0 mg/ml; type IA, Sigma) and DNase (0.1 mg/ml; type IV, sigma) at37 °C in a shaking bath for 15 min. Then, 10% fetal bovine serum (FBS)was added to stop the enzymatic digestion. After centrifugation (5 min,1000 rpm), the remaining ganglia were dissociated into single cells bytrituration via heat-polished Pasteur pipettes and then passed through anylon mesh with a pore diameter size of 100 μm. Isolated cells werethen suspended in neurobasal media (Gibco by Life Technologies)supplemented with 2% B27, 50 ng/ml nerve growth factor (NGF),2 mM L-glutamine (Invitrogen by Life Technologies), and 1% peni-cillin/streptomycin and seeded onto uncoated 35-mm dishes at 37 °Cfor 2 h. Non-adherent neuronal cells were dislodged from the dishes bygentle pipetting. Then, the cells were plated on other dishes at 37 °Cwith 5% CO2 for up to 10 days. Half of the media was changed every3 days. Before electrophysiological recording, DRG neurons for controland gp120 treatment groups were cultured with or without HIV gpl20(200 pmol/L) for 24 h. DRG neurons in nano curcumin treatment group

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were cultured with HIV gpl20 plus nano curcumin (0.2 μg/ml). Theexperiments were performed at room temperature (20–30 °C) (Liuet al., 2014).

2.10. Electrophysiological recordings

Electrophysiological recording was carried out using a patch/wholecell clamp amplifier (Axopatch 200B) (Liu et al., 2014). The micro-pipette was filled with an internal solution containing (in mM) 140 KCl,2 MgCl2, 10 HEPES, 11 EGTA, and 5 ATP. The osmolarity of the internalsolution was adjusted to 340 mOsmol/kg with sucrose and the pH wasadjusted to 7.4 with KOH. The external solution contained (in mM) 150NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose. Theosmolarity of the external solution was adjusted to 340 mOsm withsucrose and the pH was adjusted to 7.4 with NaOH. The resistance ofthe recording electrodes was within the range of 1–4 MΩ, with 3 MΩ asthe best. A small patch of membrane underneath the tip of the pipettewas aspirated to form a seal (1–10 GΩ) and then a more negativepressure was applied to rupture it to establish the whole-cell mode. Theholding potential (HP) was set at −60 mV. The drugs [α,β-methyleneATP (α,βme-ATP) or A-317491, Sigma] were dissolved in external so-lution and delivered by gravity flow from an array of tubules (500 μmO.D., 200 μm I.D.) connected to a series of independent reservoirs. Thedistance from the tubule mouth to the examined cell was approximately100 μm. Rapid solution exchange was achieved by shifting the tubuleshorizontally with a micromanipulator.

2.11. Molecular docking

Molecular docking computations were performed using AutoDock4.2 (Rao et al., 2017). Molecular docking is a computer simulation toolthat attempts to predict the binding mode of a ligand in the active siteof a protein. Molecular docking studies mimic the natural interaction ofa ligand with the protein. The technique of docking is to position theligand in different orientations and conformations within the bindingsite to calculate optimal binding geometries and energies. Therefore,after the docking procedure the proper conformation of ligand in theactive site of protein is obtained and used for calculation of moleculardescriptors. For each ligand, a number of configurations called posesare generated and scored (Rao et al., 2017). The score can be calculatedas either a free energy of binding, which takes into account salvationand entropy, or the enthalpic term of the free energy of binding, or aqualitative shaped-based numerical measure. The final top-scoringposes, along with their scores and conformation energies, are written toa database where they are ready for further analysis.

Protein Data Bank entry 5SVK, which is crystal structure of the openstate ATP-gated human P2X3 ion channel in the ATP-bound, was usedas a target protein (Rao et al., 2017). Curcumin, pubchem CID 969516was used as a ligand. Both of them were prepared with AutoDockTools(ADT) and Python scripts named prepare_ligand4.py and pre-pare_receptor4.py, which are associated with the AutoDock4.2 pro-gram. The binding pocket position in target protein was specified withthe ADT molecular viewer. The parameters were kept at their defaultvalues. Finally, the output files were viewed using MGLtools (Rao et al.,2017) and PyMol (http://www.pymol.org/).

2.12. Statistical analysis

The data were analyzed using SPSS 20 software. The numericalvalues are reported as the mean ± SE. Statistical significance wasdetermined by the one-way analysis of variance (ANOVA) followed byFisher’s post hoc test for multiple comparisons. A value of p < 0.05was considered statistically significant.

3. Results

3.1. The effects of nanoparticle-encapsulated curcumin on hyperalgesia ingp120-treated rats

Mechanical hyperalgesia was tested with a mechanical stimulator.No difference was observed in the mechanical withdrawal threshold(MWT) between the sham group and the control group (p > 0.05). At7–14 days after the operation, the MWT in the gp120 group was lowerthan in the control group (p < 0.01). No difference was observed inthe mechanical withdrawal threshold (MWT) between thegp120 + nano carrier group and the gp120 group (p > 0.05). TheMWT in the gp120 + nano curcumin group was higher than the gp120group from days 7–14 (p < 0.01) (Fig. 1A).

Fig. 1. The effects of nano curcumin treatment on the mechanical withdrawal threshold(MWT) or the thermal withdrawal latency (TWL) in gp120-treated rats.A. The MWT in the gp120 group was lower than the control group (p < 0.01). The MWTin the gp120 plus nano curcumin group was higher than the gp120 group (p < 0.01). Nodifference was found between the sham group and the control group (p > 0.05). Eachgroup consisted of eight rats (n = 8 per group). No difference was found between thegp120 plus nano carrier group and the gp120 group (p > 0.05). The data represent themean ± SE. The significant differences are noted as “*” when p < 0.05 compared to thecontrol group, “**” when p < 0.01 compared to the control group, “#” when p < 0.05compared to the control group, and “##” when p < 0.01 compared to the gp120-treatedgroup.B. The TWL in the gp120 group was lower than in the control group (p < 0.01). The TWLin the gp120 plus nano curcumin group was higher than in the gp120 group (p < 0.01).No difference was found between the sham group and the control group (p > 0.05).Each group consisted of eight rats (n = 8 per group). No difference was found betweenthe gp120 plus nano carrier group and the gp120 group (p > 0.05). The data representthe mean ± SE. The significant differences are noted as “*” when p < 0.05 compared tothe control group, “**” when p < 0.01 compared to the control group, “#” whenp < 0.05 compared to the control group, and “##” when p < 0.01 compared to thegp120-treated group.

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http://www.pymol.org/

Thermal hyperalgesia was tested with a Thermal Paw StimulationSystem. No difference was observed in the thermal withdrawal latency(TWL) between the sham group and the control group (p > 0.05). At7–14 days after the operation, the TWL in the gp120 group was lowerthan in the control group (p < 0.01). No difference was observed inthe thermal withdrawal latency (TWL) between the gp120 + nanocarrier group and the gp120 group (p > 0.05). The TWL in thegp120 + nano curcumin group was higher than in the gp120 groupfrom days 7–14 (p < 0.01) (Fig. 1B).

3.2. The effects of nanoparticle-encapsulated curcumin on the expression ofthe P2X3 receptor in the DRG of gp120-treated rats

The expression of P2X3 mRNA in the DRG was measured by real-time RT-PCR. The studies showed that the relative levels of P2X3 mRNA

in the gp120 group were significantly increased compared to the con-trol group (p < 0.01). No difference was found between the shamgroup and the control group (p > 0.05). The expression levels of P2X3mRNA in the gp120 + nano curcumin group were significantly de-creased compared to the gp120 group (p < 0.01) (Fig. 2A). No dif-ference was found between the gp120 + nano carrier group and thegp120 group (p > 0.05).

The expression levels of P2X3 immunoreactivity in the DRG wereanalyzed by immunohistochemistry. The expression levels of P2X3 im-munoreactivity in the gp120 group were significantly enhanced com-pared to the control group (p < 0.01). No difference was found be-tween the sham group and the control group (p > 0.05). The relativelevels of P2X3 protein expression in the gp120 + nano curcumin groupwere lower than the gp120 group (p < 0.01) (Fig. 2B). No differencewas found between the gp120 + nano carrier group and the gp120

Fig. 2. The effects of nano curcumin treatment on the expression of the P2X3 receptor in L4–6 DRGs.A. The effects of nano curcumin treatment on the expression of P2X3 mRNA in L4–6 DRGs from each group. Real-time RT-PCR tests showed that the expression of P2X3 mRNA in the DRGof the gp120 group increased compared to the control group (p < 0.01). No difference was found between the sham group and the control group (p > 0.05). The expression of P2X3mRNA in the DRG of the gp120 plus nano curcumin group was decreased compared to the gp120 group (p < 0.01). The expression of P2X3 mRNA in the gp120 plus nano carrier groupwas increased compared to the control group (p < 0.01). No difference was found between the gp120 plus nano carrier group and the gp120 group (p > 0.05). The experiment wasperformed in triplicate and repeated eight times (n = 8 per group). The data represent the mean ± SE. **p < 0.01 compared to the control group; ##p < 0.01 compared to thegp120-treated groupB. The effects of nano curcumin treatment on the expression of immunoreactivity in L4–6 DRGs from each group (n = 8 per group). (B1) The P2X3 immunohistochemical images in eachgroup. (B2) The bar histograms show the integrated optical density (IOD) of P2X3 immunoreactivity in each group. The IOD of P2X3 immunoreactivity in the gp120-treated group wasincreased compared to the control group (p < 0.01). No difference was found between the sham group and the control group (p > 0.05). The IOD of P2X3 immunoreactivity in thegp120 plus nano curcumin group was decreased compared to the gp120 group (p < 0.01). The IOD of P2X3 immunoreactivity in the gp120 plus nano carrier group was increasedcompared to the control group (p < 0.01). No difference was found between the gp120 plus nano carrier group and the gp120 group (p > 0.05). (Note: The data are shown as themean ± SE. **p < 0.01 compared to the control group; ##p < 0.01 compared to the gp120 group).C. The effects of the nano curcumin treatment on the expression of P2X3 protein in L4–6 DRGs from each group (n = 8 per group). (C1) The expression bands of P2X3 protein in the DRGwere detected by Western blotting. (C2) The bar histograms show the ratio of the P2X3 protein level to the β-actin level in each group. The expression of the P2X3 protein in the DRG ofthe gp120-treated group was increased compared to the control group (p < 0.01). No difference was found between the sham group and the control group (p > 0.05). The expression ofP2X3 protein in the DRG of the gp120 plus nano curcumin group was decreased compared to the gp120 group (p < 0.01). The expression of P2X3 protein in the gp120 plus nano carriergroup was increased compared to the control group (p < 0.01). No difference was found between the gp120 plus nano carrier group and the gp120 group (p > 0.05). (Note: The dataare shown as the mean ± SE. **p < 0.01 compared to the control group; ##p < 0.01 compared to the gp120 group).

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group (p > 0.05).The expression levels of the P2X3 protein in the DRG were analyzed

by Western blotting. Using image analysis, the P2X3 protein expression(normalized to each β-actin internal control) in the gp120 group wassignificantly enhanced compared to the control group (p < 0.01). Nodifference was found between the sham group and the control group(p > 0.05). The relative levels of P2X3 protein expression in thegp120 + nano curcumin group were lower than the gp120 group(p < 0.01) (Fig. 2C). No difference was found between thegp120 + nano carrier group and the gp120 group (p > 0.05).

3.3. The effects of nanoparticle-encapsulated curcumin on the expressionlevels of ERK1/2 and p-ERK1/2 in the DRG of gp120-treated rats

The activation of ERK1/2 in inflammatory pain is shown as its

phosphorylation. The expression levels of ERK1/2 and p-ERK1/2 in theDRG were analyzed by Western blotting(Fig. 3A). The integrated op-tical density (IOD) ratio of ERK1/2 to β-actin was not significantlydifferent between the gp120 group and the sham group (p > 0.05,Fig. 3B). However, the IOD ratio of p-ERK1/2 to ERK1/2 was higher inthe gp120 group than the control group (p < 0.01, n = 8 for eachgroup) (Fig. 3C). No difference was found between the sham group andthe control group (p > 0.05). The data revealed that the role of ERKphosphorylation in the DRG is related to P2X3 receptor-mediated hy-peralgesia in the gp120-treated rats.

In addition, we examined the effects of nano curcumin on thephosphorylation of ERK in the DRG of the gp120 group. No differencewas found between the sham group and the control group (p > 0.05,Fig. 3C). The IOD ratio of p-ERK1/2 to ERK1/2 in the gp120 + nanocurcumin group was significantly lower than the gp120 group(p < 0.01, n = 8 for each group) (Fig. 3C). No difference was foundbetween the gp120 + nano carrier group and the gp120 group(p > 0.05, Fig. 3C). These results suggest that the effect of nano cur-cumin in the inhibition of hyperalgesia may involve decreasing thephosphorylation of ERK1/2 in the DRG of the gp120-treated rats.

3.4. The effect of nanoparticle-encapsulated nano curcumin on P2X3agonist-activated currents in the DRG neurons cultured with gp120

P2X3 agonist-activated currents in the DRG neurons were recordedby whole-cell patch clamp. The experimental results revealed that theP2X3 agonist α,βme-ATP (10 μM)-activated currents in DRG neuronscultured with gp120 were higher than in the controls (Fig. 4A). Afterthe DRG neurons were co-cultured with gp120 and nano curcumin(0.2 μg/ml) treatment, α,βme-ATP-induced currents in DRG neuronswas significantly decreased compared with the DRG neurons that wereonly cultured with gp120 (Fig. 4A) (n = 7, p < 0.01). The α,βme-ATP-activated currents in DRG neurons cultured with gp120 can beinhibited by P2X3 antagonist specific antagonist A-317491 (100 nM)(n = 7, p < 0.001) (Fig. 4A). The histogram showed the currentdensity with different drugs (Fig. 4B). These results indicated that nano

Fig. 3. The effects of nano curcumin treatment on the expressions of ERK1/2 and p-ERK1/2 in L4–6 DRGs. A. The protein expression bands of ERK1/2 and p-ERK1/2 in theDRG were detected by using Western blotting. B. The histogram shows the integratedoptical density (IOD) ratio of ERK1/2 to β-actin in five groups. There was no differencebetween the five groups (p > 0.05, F(4,25) = 0.835, n = 8 per group). C. The histogramshows the integrated optical density (IOD) ratio of p-ERK1/2 to ERK1/2 in five groups.The IOD ratio in the gp120 group was higher than the control group (p < 0.01, n = 8 foreach group). The IOD ratio in the gp120 + nano carrier group was higher than thecontrol group (p < 0.01, n = 8 for each group). The IOD ratio in the gp120 plus nanocurcumin-treated rats was significantly lower than the gp120 group (p < 0.01, n = 8 foreach group). No difference was found between the sham group and the control group(p > 0.05). The data represent the mean ± SE, n = 8. **p < 0.01 compared to thecontrol group; ##p < 0.01 compared to the gp120 group.

Fig. 4. The effects of nano curcumin treatment on the α,βme-ATP-activated currents bythe P2X3 receptor in DRG neurons from controls, DRG neurons treated with gp120 andDRG neurons co-treated with gp120 plus nano curcumin.A. shows the typical current trace induced by P2X3 agonist α,βme-ATP. α,βme-ATP(10 μM)-activated currents in DRG neurons cultured with gp120 were higher than thosein the controls. P2X3 agonist α,βme-ATP-activated currents in DRG neurons co-treatedwith gp120 plus nano curcumin were significantly inhibited compared with those fromthe DRG neurons that were only cultured with gp120. P2X3 antagonist A-317491 in-hibited α,βme-ATP-induced currents in the DRG neurons cultured with gp120.B The histogram shows the current density with different drugs. The data represent themean ± SE, n = 7 (**p < 0.01 vs. control neurons).

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curcumin treatment inhibited the activation of the P2X3 receptor in theHIV-associated neuropathic pain state.

3.5. Molecular docking of curcumin on a hP2X3 receptor

Molecular docking of curcumin on a hP2X3 protein were generatedby the AutoDock 4.2. Docking score of hP2X3 and curcumin (−7.7,Kcal/mol) showed that curcumin was enabled the perfect fit to interactwith hP2X3 receptor (See Table 1). The perfect match enabled thecurcumin to interact with residues in the ATP-binding site (Fig. 5).

4. Discussion

Neurotoxins and inflammatory mediators can cause hyperexcit-ability of the DRG neurons, leading to spontaneous or persistent firing(Richardson and Vasko, 2002; Basbaum et al., 2009). HIV-1 gp120protein has neurotoxin properties and therefore may directly impact theexcitability of DRG neurons, leading to HIV-1-associated pain (Milliganet al., 2000; Herzberg and Sagen, 2001; Wallace et al., 2007; Maratou

et al., 2009; Kamerman et al., 2012; Hao, 2013). Our study indicatedthat the mechanical withdrawal threshold (MWT) and the thermalwithdrawal latency (TWL) in the peripheral gp120 application modelwere decreased compared to those in the sham group. Meanwhile, ourresults also showed that the enhanced hyperalgesia was associated withthe upregulated expression of P2X3 mRNA and protein in the DRG inthe HIV-1 gp120 treatment group. The P2X3 receptor is preferentiallyexpressed on dorsal root ganglion (DRG) neurons and has been im-plicated in neuropathic pain hypersensitivity (Barclay et al., 2002;Ford, 2012; Burnstock, 2013). The immunoreactivity test in this studyrevealed that the expression levels of the P2X3 immunoreactivity in theDRG neurons was significantly increased in the gp120 group comparedto the sham group. Therefore, the upregulated P2X3 receptor in theDRG neuron participated in the signal transmission of HIV-associatedpain.

Curcumin, a derived product from the common spice turmeric wasformulated into biodegradable nanoparticles, which improved itsbioavailability (Bisht et al., 2007; Shaikh et al., 2009). Our experimentsshowed that nano curcumin decreased the expression levels of the P2X3receptor and relieved the MWT and TWL in the gp120-treated rats,which indicated that the nano curcumin lessened the pathological in-jury mediated by the P2X3 receptor in the DRG neurons in the signaltransmission of HIV-associated pain. Reducing the size into nanoscale indrug carriers has many advantages. Nanoscale in drug carriers improvesthe pharmacokinetics and the biodistribution of therapeutic agents dueto higher ratio of surface area to volume, diminishes toxicity by theirpreferential accumulation at the target site, facilitates intracellulardelivery and prolongs their retention time (Bala et al., 2005; Bisht et al.,2007). The concentration of the nano curcumin in our experiment wasonly 4 mg/ml for one rat, which was significantly lower than thecommon dose of 50 mg/kg–300 mg/kg.

The activation of the ERK pathway is involved in the transmission ofpain signaling by sensitizing primary afferents (Ji et al., 2009; Lai et al.,2011). Therefore, the blockade of ERK activation in the primary sensoryneurons may decrease the mechanical hypersensitivity and the thermalhypersensitivity in inflammatory pain. The activation of the P2X3 re-ceptor follows the activation of the ERK1/2-mediated signal transduc-tion pathways and results in pain (Lai et al., 2011; Liu et al., 2014; Yiet al., 2017). Accompanied with the upregulated expression of the P2X3receptor, our study also showed that the IOD ratio of p-ERK1/2 toERK1/2 in the gp120 group was higher than in the sham group. Thephosphorylation of the MAPK indicates its activation. The IOD ratios ofp-ERK1/2 to ERK1/2 in the gp120 plus nano curcumin-treated ratssignificantly decreased compared to the gp120 group. Our studies in-dicated that the nano curcumin inhibited both the upregulation of theP2X3 receptor and the phosphorylation of ERK1/2 in the DRG of thegp120-treated rats. Therefore, nano curcumin treatment decreased theupregulated expression of the P2X3 receptor and lowered the activationof ERK1/2 in the DRG to reduce the mechanical hyperalgesia andthermal hyperalgesia in gp120-treated rats.

ATP as a transmitter can be released from injured cells and sensorynerve endings (Sperlágh et al., 1995; Sperlágh et al., 1997; Sperlághet al., 1998; Burnstock, 2013, 2014). ATP can activate the P2X3 re-ceptor in the DRG neurons (Gao et al., 2011a; Xu et al., 2012). In DRGneurons cultured with gp120, P2X3 agonist α,βme-ATP-activated cur-rents were higher than in controls. The peripheral sensitization of pri-mary DRG neurons is the key event in the onset of chronic pain con-ditions (Richardson and Vasko, 2002; Basbaum et al., 2009). HIV-1gp120 protein triggers rapid and sustained enhancement of the excit-ability of rat primary DRG neurons. Peripheral sensitization is enhancedafter exposure to inflammatory mediators such as ATP (Barclay et al.,2002; Richardson and Vasko, 2002; Basbaum et al., 2009; Ford, 2012;Burnstock, 2013). After DRG neurons were co-cultured with gp120 andnano curcumin, α,βme-ATP-induced currents were significantly de-creased compared with those in DRG neurons that were only culturedwith gp120. These results revealed that nano curcumin treatment

Table 1MOE score of hP2X3 protein and curcumin (kcal/mol).

Mode/Rank Affinity (kcal/mol) Dist from rmsba l.b Best mode rmse u.b

1 −7.7 0 02 −7.7 38.013 41.0123 −7 3.345 11.1714 −6.8 58.24 60.2035 −6.6 38.579 41.1586 −6.5 39.677 41.437 −6.4 37.881 39.7858 −6.4 36.248 38.4859 −6.2 22.741 26.944

Explanation: The predicted binding affinity is in kcal/mol (Energy).armsd: RMSD values are calculated relative to the best mode and use only movable heavyatoms. Two variants of RMSD metrics are provided, rmsd/lb (RMSD lower bound) andrmsd/ub (RMSD upper bound), differing in how the atoms are matched in the distancecalculation.

Fig. 5. Molecular docking of curcumin on hP2X3 protein.Simulation modeling of curcumin docking with hP2X3 protein was simulated by com-puter. Figures in A (forward map) and B (top view) showed that the best docking positionbetween curcumin and hP2X3. The docking position was in the outside of the cellmembrane and the entrance of the channel. Figure C showed that the pink stick-likestructure was the B chain of the hP2X3 protein, the blue stick-like structure was the Cchain of the hP2X3 protein. The yellow dotted line was a hydrogen bond between theresidues on the chains and curcumin. The photo indicated that there was strong bindingenergy between curcumin and LYS 63 in B chain, THR 172 in C chain. The results re-vealed that curcumin could interact with the hP2X3 protein.

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reduced the activation of the P2X3 receptor in gp120-treated neurons.Nano curcumin treatment decreased neuronal firing in the DRG neuronsmediated by the P2X3 receptor and relieved pain behaviors in thegp120-treated rats. As shown in Fig. 5, curcumin could interact with thehP2X3. Interaction energies for the docked-complexes were calculatedby AutoDock 4 and showed in Table 1. In Table 1, a higher value ofnegative interaction energy is an indicator of more efficient interactionbetween the hP2X3 and curcumin. Therefore, curcumin may be acted asthe inhibitor of P2X3 receptor.

In conclusion, this study showed that peripheral nerve exposure toHIV gp120 increased mechanical hyperalgesia and thermal hyper-algesia accompanied by upregulated expression of the P2X3 receptor inthe DRG of the gp120-treated model rats. Nano curcumin treatmentdecreased the upregulated expression of the P2X3 receptor in gp120-treated model rats and enhanced the currents of P2X3 activation in DRGneurons treated with gp120. After the inhibition of the P2X3 receptor inthe DRG, nano curcumin treatment decreased the phosphorylation ofERK1/2 in the DRG of gp120-treated rats. Therefore, nano curcumintreatment may inhibit P2X3 activation, decrease the sensitization ofDRG primary afferents and relieve mechanical hyperalgesia andthermal hyperalgesia in gp120-treated rats.

Funding

These studies were supported by grants (№s: 81460200, 31560276,81701114, 81570735, and 81200853) from the National NaturalScience Foundation of China, a grant (№: 20151BBG70250,20151BBG70253) from the Technology Pedestal and SocietyDevelopment Project of Jiangxi Province, a grant (№:20171BAB205025, 20142BAB205028, 20142BAB215027, 20121512040234) from the Natural Science Foundation of Jiangxi Province, andgrants (№s: GJJ13155 and GJJ14319) from the EducationalDepartment of Jiangxi Province.

Competing interests

The authors declare no conflicts of interest.

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Effects of nanoparticle-encapsulated curcumin on HIV-gp120-associated neuropathic pain induced by the P2X3 receptor in dorsal root gangliaIntroductionMaterials and methodsAnimals and surgical methodsSynthesis of poly-PEGMA-DMAEMA-MAO nano macromoleculeThe synthesis of curcumin loaded poly-PEGMA-DMAEMA-MAO microspheresMeasurement of the mechanical withdrawal threshold (MWT)Measurement of the thermal withdrawal latency (TWL)Real-time PCRImmunohistochemistryWestern blottingIsolation of DRG neurons and cultureElectrophysiological recordingsMolecular dockingStatistical analysis

ResultsThe effects of nanoparticle-encapsulated curcumin on hyperalgesia in gp120-treated ratsThe effects of nanoparticle-encapsulated curcumin on the expression of the P2X3 receptor in the DRG of gp120-treated ratsThe effects of nanoparticle-encapsulated curcumin on the expression levels of ERK1/2 and p-ERK1/2 in the DRG of gp120-treated ratsThe effect of nanoparticle-encapsulated nano curcumin on P2X3 agonist-activated currents in the DRG neurons cultured with gp120Molecular docking of curcumin on a hP2X3 receptor

DiscussionFundingCompeting interestsReferences

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