Lingling Yang,1 Guohu Di,1 Xia Qi,1 Mingli Qu,1 Yao Wang,1 Haoyun Duan,1 Patrik Danielson,2

Lixin Xie,1 and Qingjun Zhou1

Substance P Promotes DiabeticCorneal Epithelial WoundHealing Through MolecularMechanisms Mediated via theNeurokinin-1 ReceptorDiabetes 2014;63:4262–4274 | DOI: 10.2337/db14-0163

Substance P (SP) is a neuropeptide, predominantlyreleased from sensory nerve fibers, with a potentiallyprotective role in diabetic corneal epithelial woundhealing. However, the molecular mechanism remainsunclear. We investigated the protective mechanism ofSP against hyperglycemia-induced corneal epithelialwound healing defects, using type 1 diabetic mice andhigh glucose–treated corneal epithelial cells. Hypergly-cemia induced delayed corneal epithelial wound heal-ing, accompanied by attenuated corneal sensation,mitochondrial dysfunction, and impairments of Akt, epi-dermal growth factor receptor (EGFR), and Sirt1 activa-tion, as well as decreased reactive oxygen species(ROS) scavenging capacity. However, SP applicationpromoted epithelial wound healing, recovery of cornealsensation, improvement of mitochondrial function, andreactivation of Akt, EGFR, and Sirt1, as well as in-creased ROS scavenging capacity, in both diabeticmouse corneal epithelium and high glucose–treatedcorneal epithelial cells. The promotion of SP on diabeticcorneal epithelial healing was completely abolished bya neurokinin-1 (NK-1) receptor antagonist. Moreover,the subconjunctival injection of NK-1 receptor antago-nist also caused diabetic corneal pathological changesin normal mice. In conclusion, the results suggest thatSP-NK-1 receptor signaling plays a critical role in themaintenance of corneal epithelium homeostasis, andthat SP signaling through the NK-1 receptor contributes

to the promotion of diabetic corneal epithelial woundhealing by rescued activation of Akt, EGFR, and Sirt1,improvement of mitochondrial function, and increasedROS scavenging capacity.

Among various pathological conditions by diabetes, ocularcomplications have been a leading cause of blindness inthe world, including diabetic retinopathy, cataract, andvarious ocular surface disorders (1). The most recognizeddiabetic changes in the cornea, i.e., clinical diabetic kera-topathy, include impaired corneal sensation, superficialpunctate keratitis, and persistent corneal epitheliumdefects (2). The uncontrolled impairment of cornealwound healing increases the susceptibility of corneal ul-cer, microbial keratitis, and even perforation. Diabeticcorneal pathology always exhibits epithelial basementmembrane abnormalities, reduced hemidesmosome den-sity, and delayed wound healing (3,4). However, hypergly-cemia also directly impairs the cellular metabolism andcauses abnormal changes of corneal epithelium, such asAkt-, epidermal growth factor receptor (EGFR)–, andSirt1-mediated cell responses to environmental challenges(5–7). Moreover, excess oxidative stress, resulting fromenhanced accumulation of reactive oxygen species (ROS)and impaired antioxidant capabilities in response to hy-perglycemia, has been postulated as an important

1State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory ofOphthalmology, Shandong Eye Institute, Shandong Academy of Medical Sci-ences, Qingdao, China2Department of Integrative Medical Biology, Umeå University, Umeå, Sweden

Corresponding authors: Qingjun Zhou, qjzhou2000@hotmail.com, and Lixin Xie,lixin_xie@hotmail.com.

Received 29 January 2014 and accepted 26 June 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0163/-/DC1.

L.Y. and G.D. contributed equally to this study.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

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pathological mechanism, whereas the reduction of ROSattenuated the progression of various diabetes complica-tions (8–13), including in the cornea (5,14).

Substance P (SP), released predominantly by peripheralterminal, is an 11–amino acid neuropeptide that acts as aneurotransmitter mediating nociceptive transmission. Itmainly functions through the interaction with neurokininreceptors, members of the tachykinin subfamily of G-protein–coupled receptors, among which the neurokinin-1(NK-1) receptor shows a preferential affinity for SP. SP iscurrently known as a neuroimmunomodulatory regulatorof the immune system (15). In the cornea, SP has beendetected in the nerve fibers of naïve cornea (16,17) and inantigen-presenting cells in herpetic stromal keratitis (18).Moreover, SP causes the mobilization of bone marrow–derived stem cells to participate in corneal wound healing(19,20). Interestingly, recent evidences have shown thatSP activates the EGFR, mitogen-activated protein kinases(MAPKs), extracellular signal–regulated kinases (ERKs),and phosphoinositide 3-kinase–Akt (PI3K-Akt) signalingpathways, as well as promotes the healing of inflamedcolonic epithelium (21) and possesses antiapoptoticeffects in colonocytes (22), dendritic cells (23), tenocytes(24), neutrophils (25), and bone marrow–derived stemcells (26).

The cornea is one of the most densely innervatedtissues in the body, containing nerve fibers derived fromthe trigeminal ganglion. Corneal nerve fibers exertimportant trophic influences and contribute to themaintenance of corneal epithelium homeostasis, whereasthe dysfunction of corneal innervation produces animpairment of corneal epithelial wound healing, knownas neurotrophic keratitis, as caused by for instanceherpetic viral infections, trigeminal nerve damage, ordiabetes (27–29). Although tear fluid impairment is alsoinvolved (30), the neurotrophic deficits may play a majorrole in the pathogenesis of diabetic keratopathy, as thedensity of corneal nerve fibers and corneal sensation de-creased in diabetic patients (31–33). However, althoughbeing the major sensory neurotransmitter and neuropep-tide released from corneal nerve fibers, SP was shown notto be significantly decreased in diabetic cornea (34). Nev-ertheless, paradoxically, topical SP application promotedcorneal epithelial wound healing in diabetic animals andhumans when combined with insulin-like growth factor-1(IGF-1) or epidermal growth factor (EGF) per group (29).The promoting mechanisms of SP on diabetic corneal ep-ithelial wound healing remain elusive. In this study, wesought to demonstrate the protective mechanism of SPagainst diabetic corneal epithelial wound healing usingstreptozotocin-induced type 1 diabetic mice and highglucose–treated corneal epithelial cells.

RESEARCH DESIGN AND METHODS

AnimalsAdult male C57BL/6 mice were purchased from the BeijingHFK Bioscience Co. Ltd. (Beijing, China). All animal

experiments were carried out in accordance with TheAssociation for Research in Vision and OphthalmologyStatement for the Use of Animals in Ophthalmic andVision Research. The mice underwent induction of type 1diabetes with an intraperitoneal injection of 50 mg/kgstreptozotocin (Sigma-Aldrich, St. Louis, MO) in ice-coldcitrate–citric acid buffer (pH 4.5) for 5 days, and controlmice received equal amount of buffer. Blood glucose levelswere monitored with a OneTouch Basic glucometer(LifeScan; Johnson & Johnson, Milpitas, CA). In the cur-rent study, diabetic mice were used after 12 weeks of finalstreptozotocin injection, at which point the HbA1c valueswere 10.83 6 0.74% (94.75 6 7.93 mmol/mol), whereasnormal mice had HbA1c values of 4.25 6 0.1% (22.5 6 1mmol/mol). For topical SP application, 5 mL SP (1 mmol/L;Calbiochem, San Diego, CA) in distilled water was droppedon the corneal surface by a 10 mL tip, six times daily pereye for 4 days (for the measurement of corneal sensitiv-ity) or 4 days prescrape and 3 days postscrape (for themeasurement of corneal epithelial wound healing). ForNK-1 receptor inhibition, NK-1 receptor antagonistL-733,060 (6.6 mg in 5 mL distilled water; Sigma-Aldrich)was injected subconjunctivally 3 days before corneal sen-sitivity measurement and corneal epithelium scrape innormal mice, or 24 h before corneal epithelium scrapein diabetic mice according to our preliminary experi-ments. Control mice were treated with distilled watervehicle.

Corneal SensitivityCorneal sensation was measured bilaterally by usinga Cochet-Bonnet esthesiometer (Luneau Ophtalmologie,Chartres Cedex, France) in unanesthetized control, di-abetic, SP-treated diabetic mice, and the NK-1 receptorantagonist–injected mice before the scrape of cornealepithelium. The testing began with the maximal length(6 cm) of nylon filament and shortened by 0.5 cm eachtime until the corneal touch threshold was found. Thelongest filament length resulting in a positive responsewas considered as the corneal sensitivity threshold, whichwas verified four times.

Corneal Epithelial Wound HealingNormal, diabetic, and SP-treated diabetic mice wereanesthetized by an intraperitoneal injection of xylazineand ketamine followed by topical application of 2%xylocaine. The entire corneal epithelium including limbalregion (marked with 3 mm trephine) was scraped withAlger Brush II corneal rust ring remover (Alger Co., LagoVista, TX) and subsequently applied with ofoxacin eyedrops to avoid infection. Usually, one eye was wounded ata time in each animal. The defects of corneal epitheliumwere visualized at 24, 48, and 72 h by instilling 0.25%fluorescein sodium and photographed under slit lamp(BQ 900; Haag-Streit, Bern, Switzerland). The stainingarea was analyzed by using ImageJ software and calcu-lated as the percentage of residual epithelial defect.

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Corneal Epithelial Cell Culture and TreatmentMouse corneal epithelial cell line (TKE2) was provided byDr. Tetsuya Kawakita of Keio University (Tokyo, Japan)(35). Human corneal tissues were handled according tothe tenets of the Declaration of Helsinki. Primary humancorneal epithelial cells (HCECs) were established fromlimbal explants of donor corneas according to a previousreport (36). For the analysis of Akt, EGFR, and Sirt1signaling activation, and the staining of ROS, glutathione(GSH), and mitochondria, both cells were starved over-night in bovine pituitary extract–free keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) and subsequentlyincubated in 30 mmol/L glucose or mannose (osmoticcontrol) for 3 days with or without 1 mmol/L SP.

Cell Proliferation and Migration AnalysisFor the proliferation analysis, the TKE2 cells were starvedovernight in bovine pituitary extract–free keratinocyteserum-free medium, treated with high glucose for 3 daysin the absence or presence of 1 mmol/L SP, and measuredusing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay. For the migration anal-ysis, the cells were cultured in high glucose until conflu-ence, subsequently wounded with a micropipette tip, andincubated with or without SP for 24 h. Digital images ofwound closure were used for quantitative assessment ofmigration by using ImageJ software. Each assay was con-ducted in at least triplicate.

Immunofluorescence StainingEyeballs were snap frozen in Tissue-Tek optimum cuttingtemperature compound (Sakura Finetek, Tokyo, Japan).Frozen corneal sections were fixed in 4% paraformalde-hyde for 15 min, permeabilized with 0.1% Triton X-100for 10 min, and blocked with normal serum for 1 h atroom temperature. The samples were stained withprimary antibodies overnight at 4°C, washed, and incu-bated with fluorescein-conjugated secondary antibodies at37°C for 1 h (antibody information as listed in Supple-mentary Table 1). All staining was observed under a con-focal microscope or an Eclipse TE2000-U microscope(Nikon, Tokyo, Japan) after counterstaining with DAPI.

Reverse Transcription Quantitative PCRTotal RNA was extracted from mouse corneal epitheliumusing NucleoSpin RNA kits (BD Biosciences, Palo Alto, CA).cDNAs were synthesized using the PrimeScript First StrandcDNA Synthesis Kit (TaKaRa, Dalian, China). Real-timePCR was carried out using SYBR Green reagents and theApplied Biosystems 7500 Real-Time PCR System (AppliedBiosystems, Foster City, CA). The specific primers used arelisted in Supplementary Table 2. The cycling conditionswere 10 s at 95°C followed by 45 two-step cycles (15 s at95°C and 1 min at 60°C). The quantification data wereanalyzed with the Sequence Detection System software(Applied Biosystems) using GAPDH as an internal control.

Western Blot AnalysisTotal protein was extracted from the lysed samples ofmouse corneal epithelium, cultured TKE2 cells, and HCECs

in RIPA buffer. Samples (total protein concentration: 40 mgfor mouse corneal epithelium and 45 mg for cultured cells)were run on 12% SDS-PAGE gels and then transferred toa polyvinylidene fluoride membrane (Millipore, Billerica,MA). The blots were blocked by nonfat dry milk for at least1 h and incubated with primary antibodies (SupplementaryTable 1) in Tris-buffered saline with Tween for 1 h at roomtemperature. The blots were washed three times and in-cubated with a horseradish peroxidase–conjugated second-ary antibody (Amersham Biosciences, Piscataway, NJ).Finally, the blots were visualized via enzyme-linked chemi-luminescence using the ECL kit (Chemicon, Temecula, CA).

Mitochondria Superoxide and Membrane PotentialStainingFor the observation of mitochondrial structure, superox-ide generation, and membrane potential, the cells werepreloaded with 100 nmol/L MitoTracker Green (Beyotime,Haimen, China) for 30 min, 5 mmol/L MitoSOX Redreagent (Beyotime), and 5 mg/mL 5,59,6,69-tetrachloro-1,19,3,39-tetraethyl-benzimidazole-carbocyanide iodine(JC-1; Beyotime), respectively, for 15 min at 37°C. Thefluorescence was observed and captured using a Nikonconfocal microscope.

Measurement of Intracellular ROS Generation andGSH ContentFor the observation of intracellular ROS and GSHstaining, fresh corneal cryostat sections and culturedcells were loaded with 10 mmol/L fluorescence probe2,7-dichlorodihydrofluorescein diacetate, acetyl ester(DCHF-DA; Molecular Probes, Eugene, OR) and 50 mmol/Lmonochlorobimane (Sigma-Aldrich), respectively, for 30min at 37°C. The staining was observed and captured usinga Nikon confocal microscope. For the measurement of ROSgeneration, 50,000 cells were harvested and incubated with5 mmol/L DCHF-DA for 20 min at 37°C. For the measure-ment of GSH content, 50,000 cells were harvested andfreeze-thawed with liquid nitrogen and a 37°C water baththree times. The supernatant was collected and mixed withthe provided working buffer and NADPH. The total GSHcontent was quantified by comparison with known GSHstandards according to the manufacturer’s instructions(Beyotime). The ROS and GSH fluorescence intensity was mea-sured using a Multi-Mode Microplate Reader (SpectraMax M2;Molecular Devices, Menlo Park, CA).

Statistical AnalysisData in this study were representative of at least threedifferent experiments and presented as the means 6 SD.Statistical analysis was performed using SPSS 17.0 software(SPSS, Chicago, IL) and one-way ANOVA. Differences wereconsidered statistically significant at P , 0.05.

RESULTS

SP Promotes Corneal Epithelial Wound Healing andSensitivity Recovery in Diabetic MiceTo assess the effects of SP on diabetic corneal epithelialwound healing, entire corneal epithelium was scraped in

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Figure 1—SP promotes corneal epithelial wound healing and restores corneal sensitivity in diabetic mice. Topical SP application for 7 dayswas used to examine the wound healing rate in diabetic corneal epithelium. The corneal epithelial wound was inflicted after 4 days of SPapplication and then stained with fluorescein sodium at 24, 48, and 72 h after the corneal epithelium scrape, with continuous daily SPadministration in the Diabetic+SP group (A). Histogram of residual epithelial defect is presented as the percentage of the original wound (n =6 per group) (B). Corneal sensitivity was measured in unanesthetized control, diabetic, and SP-treated diabetic mice after 4 days of topicalapplication (n = 8 per group) (C). *P < 0.05.

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age-matched normal mice and diabetic mice with orwithout topical SP application for 7 days. Punctatefluorescence staining showed that SP was detected incorneal epithelium after topical application, which sug-gests the applied SP penetrated the apical tight junctionbarrier in diabetic mice (Supplementary Fig. 1). The cor-neal epithelial healing rate exhibited a significant differ-ence from 48 h after corneal epithelium scrape (Fig. 1A).The defect size of corneal epithelium in SP-treated dia-betic mice (48 h: 28.78 6 11.78%; 72 h: 4.44 6 2.29%,n = 6) was significantly improved from that of diabeticmice (48 h: 63.59 6 7.85%; 72 h: 22.73 6 9.85%, n = 6)and reached the equal level of normal mice (48 h: 19.5965.67%; 72 h: 3.08 6 2.17%, n = 6) (Fig. 1B). Moreover,the attenuated corneal sensitivity in diabetic mice wasalso restored by topical SP application, although still beinglower than that of normal mice (n = 8 per group) (Fig. 1C).In addition, compared with diabetic mice, more inflam-matory cell infiltration was found underneath the cornealepithelium margin of SP-treated diabetic mice at 48 h,whereas it was reduced at 72 h after corneal epitheliumscrape (Supplementary Fig. 2). The results suggest thattopical-applied SP penetrates into corneal epitheliumand promotes corneal epithelial wound healing in diabetic

mice, accompanied by recovery of corneal sensitivity, andan early inflammatory and resolution response.

SP Promotes Corneal Epithelial Cell Migration andProliferation In VitroTo assess the effect of SP on corneal epithelial woundhealing in vitro, mouse corneal epithelial cells weretreated with high glucose with equal concentration ofmannose as osmotic control. Subsequently, the confluentcells were wounded and treated with or without SP foranother 24 h to analyze the migration rate. The resultsshowed that high glucose treatment caused significantdelay of corneal epithelial cell migration, whereas SPaddition improved the migration capacity of high glucose–treated cells to the same level of normal cells (n = 3 pergroup) (Fig. 2A and B). In addition, SP promoted the pro-liferation rate of corneal epithelial cells that was impairedby high glucose treatment for 3 days (n = 3 per group)(Fig. 2C). The experiments were performed three timeswith similar results.

SP Reactivates Akt, EGFR, and Sirt1 Altered byHyperglycemiaTo elucidate the mechanism underlying the promotion ofSP on corneal epithelial wound healing, we investigated

Figure 2—SP promotes the migration and proliferation of corneal epithelial cells impaired by high glucose. Confluent mouse cornealepithelial cells were wounded after treatment with 30 mmol/L glucose or mannose for 3 days. Cell migration was observed with or withoutSP treatment for another 24 h (A), and the migration area was analyzed by ImageJ software (n = 3 per group) (B). Cell proliferation wasmeasured using MTT assay after treatment for 3 days with glucose or mannose in the absence or presence of SP (n = 3 per group) (C ). *P<0.05.

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the effects of SP on the activation of Akt, EGFR, and Sirt1of diabetic corneal epithelium. Representative phosphor-ylated Akt (p-Akt), p-EGFR, and Sirt1 staining is shown inFig. 3A. The expression levels of p-Akt, p-EGFR, and Sirt1were significantly upregulated in diabetic corneal epithe-lium after topical SP application for 4 days (n = 3 pergroup) (Fig. 3B). Furthermore, SP also upregulated theexpression levels of p-Akt, p-EGFR, and Sirt1 in bothmouse TKE2 cells and primary HCECs, which were im-paired by high glucose treatment (Supplementary Fig.3). The results suggest that SP application in both diabeticmice and cultured corneal epithelial cells reactivates Akt,EGFR, and Sirt1 altered by hyperglycemia, which may inpart explain the promoting mechanisms of SP in diabeticcorneal epithelial wound healing.

SP Attenuates Oxidative Stress of Corneal Epitheliumby HyperglycemiaTo evaluate the effect of SP on the regulation ofhyperglycemia-induced oxidative stress in corneal epithe-lium, corneal sections were loaded with fluorescenceprobe DCHF-DA and monochlorobimane for the detectionof intracellular ROS and GSH. Representative resultsshowed that a significant increased ROS and reducedGSH staining were detected in diabetic mouse cornealepithelium than in that of normal mice. However, a weakROS and strong GSH staining of corneal epithelium wasfound after topical SP application in diabetic mice (Fig.4A). Moreover, the expression of major intracellular

free radical scavengers in corneal epithelium, includingmanganese superoxide dismutase (MnSOD), catalase,NAD(P)H: quinone oxidoreductase 1 (NQO1), thiore-doxin (TXN), and heme oxygenase 1 (Hmox1) at themRNA transcription level, were recovered from the di-abetic group after topical SP application (n = 4 pergroup) (Fig. 4B). In addition, immunofluorescence stain-ing and Western blot revealed that the protein levels ofNQO1, catalase, and MnSOD in diabetic corneal epitheliumpartially recovered after topical SP application (n = 4 pergroup) (Fig. 4C and D). The results suggest that topicalSP application attenuates hyperglycemia-induced oxida-tive stress in diabetic corneal epithelium, at least viathe mechanism of reducing ROS accumulation and in-creasing intracellular GSH content and antioxidant geneexpression.

SP Attenuates Mitochondrial Dysfunction Induced byHigh GlucoseMitochondrial dysfunction plays an important role in theprogress of various diabetes complications. To evaluatethe effects of SP on the mitochondrial dysfunction incorneal epithelium induced by hyperglycemia, culturedcorneal epithelial cells were treated with high glucose inthe presence or absence of SP for 3 days. Exposure toelevated glucose caused increased ROS accumulation andreduced GSH content of corneal epithelial cells in vitro(Fig. 5A and B), similar to the diabetic corneal epithelium

Figure 3—SP reactivates Akt, EGFR, and Sirt1 in diabetic corneal epithelium. Topical SP application for 4 days was used to examine thereactivation of Akt, EGFR, and Sirt1 in diabetic corneal epithelium. SP recovered the positive staining (A) and upregulated the protein levelsof phosphorylated Akt (p-Akt), p-EGFR, and Sirt1 in diabetic corneal epithelium, as compared with that of untreated diabetic mice (n = 3 pergroup) (B). *P < 0.05.

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in vivo. However, SP treatment significantly reduced theoxidative stress caused by high glucose in corneal epithe-lial cells, as showed by decreased ROS accumulation andincreased GSH content (n = 3 per group) (Fig. 5A and B).The results were also repeated by using the primaryHCECs (Supplementary Fig. 4). Furthermore, comparedwith normal or mannose-treated cells, high glucose–treatedcells assumed apparent mitochondrial superoxide staining(MitoSOX staining in Fig. 5C), accompanied by a significantchange of mitochondrial structure (MitoTracker stainingin Fig. 5C) and loss of mitochondrial membrane potential(red to green fluorescence of JC-1 staining in Fig. 5C).However, the addition of SP attenuated the generationof mitochondrial superoxide and promoted the recoveryof mitochondrial structure and membrane potential that

was impaired by high glucose treatment in corneal epithe-lial cells (Fig. 5C).

NK-1 Receptor Mediates the Promotion of SP onDiabetic Corneal Epithelial Wound HealingTo assess if the NK-1 receptor mediates the improve-ments of SP on diabetic corneal epithelial wound healing,the NK-1 receptor–specific antagonist L-733,060 wasinjected before topical SP application in diabetic mice.In unwounded mouse corneal epithelium, the stainingdensity of p-Akt, p-EGFR, and Sirt1 was attenuated inantagonist-injected SP-treated diabetic mice, as comparedwith the diabetic mice treated with SP alone (Fig. 6A). Incorneal epithelium–scraped mice, the antagonist injectionbefore SP application fully reversed the promotion of SP

Figure 4—SP attenuates oxidative stress of diabetic corneal epithelium. Topical SP application for 4 days was used to examine theattenuation of oxidative stress in diabetic corneal epithelium. SP restored the ROS and GSH staining in diabetic corneal epithelium tosimilar levels as those in normal corneal epithelium (A). SP elevated the mRNA transcript levels of antioxidant genes MnSOD, catalase,NQO1, GCLC, TXN, and Hmox1 (n = 4 per group) (B), as well as the staining density and protein levels of NQO1, catalase, and MnSOD indiabetic corneal epithelium, as compared with that in untreated diabetic corneal epithelium (n = 4 per group) (C and D). *P < 0.05.

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on diabetic corneal epithelial wound healing, with 28.9563.89% epithelial defect in antagonist-injected SP-treatedmice as compared with only 4.44 6 2.29% defect in micetreated with SP alone and with 22.73 6 9.85% defect inuntreated diabetic mice at 72 h (n = 5 per group) (Fig. 6B).Moreover, at 72 h postscrape, a stronger staining intensity

of p-Akt and proliferation marker Ki-67 was found in themigrating area of SP-treated diabetic corneal epitheliumthan in that of either untreated or antagonist-injected di-abetic corneal epithelium (Fig. 6C). The results suggest thatthe NK-1 receptor mediates the reactivation of Akt, EGFR,and Sirt1 by SP, and that the promotion of SP on diabetic

Figure 5—SP improves mitochondrial functions of corneal epithelial cells triggered by high glucose. Mouse corneal epithelial cells weretreated with 30 mmol/L glucose or mannose for 3 days in the presence or absence of SP. SP recovered the staining density and levels ofintracellular ROS and GSH (n = 3 per group) (A and B). SP improved the impaired mitochondrial functions by high glucose (C), including themitochondrial superoxide (MitoSOX staining), mitochondrial structure (MitoTracker staining), and mitochondrial membrane potential (JC-1staining). *P < 0.05.

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Figure 6—NK-1 receptor antagonist blocks the promotion of SP on diabetic corneal epithelial wound healing. NK-1 receptor antagonistL-733,060 was injected subconjunctivally at 24 h before topical SP application in diabetic mice. In the unwounded corneal epithelium, theelevation of p-Akt, p-EGFR, and Sirt1 staining density by SP application was attenuated in antagonist-injected SP-treated diabetic mice(A). In the corneal epithelium 72 h after scrape, the antagonist injection reversed the promotion of SP on diabetic corneal epithelial woundhealing (n = 5 per group) (B) and the staining intensity of p-Akt and the proliferation marker Ki-67 in the regenerated corneal epithelium(C). *P < 0.05.

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corneal epithelial wound healing is also NK-1 receptormediated.

Local Injection of NK-1 Receptor Antagonist CausesDiabetic Pathological Changes in Normal MiceTo assess the effects of SP-NK-1 receptor signalinginhibition on corneal epithelium, L-733,060 was injectedsubconjunctivally in normal mice. After 3 days of antago-nist injection, the unwounded corneal epithelium assumeda similar attenuation of p-Akt, p-EGFR, and Sirt1 stainingas that in diabetic mice (Fig. 7A), accompanied by de-creased corneal sensitivity (n = 5 per group) (Fig. 7B). After72 h of corneal epithelium scrape, the antagonist-injectedmice showed a significant delay of corneal epithelial woundhealing, with 31.776 5.07% epithelial defect in antagonist-injected mice as compared with only 3.08 6 2.17%defect in control mice (n = 5 per group) (Fig. 7C), andattenuated p-Akt and Ki-67 staining in the migrating areaof corneal epithelium as compared with that in normalmice (Fig. 7D), which was similar to the pathologicalchanges in diabetic corneal epithelium (Fig. 7C and D).Similar results were also obtained with the injection ofanother NK-1 receptor antagonist spantide I (data notshown). The results show that local injection of NK-1

receptor antagonist in normal mice causes similar patho-logical changes in corneal epithelial wound healing andcorneal sensitivity as that seen in diabetic mice, suggest-ing that normal activation of SP-NK-1 receptor signalingplays a critical role in the homeostasis of corneal epithe-lium and corneal sensation.

DISCUSSION

The cornea is one of the most densely innervated parts ofthe human body, and as such, its sensory nerves play notonly a prominent role in nociception but also in providingtrophism to the corneal tissue. In diabetes, corneal sen-sitivity, nerve fiber density, and epithelial wound healingare reduced significantly (27,31,32). However, the mecha-nisms are not completely understood. In the current study,we found that SP promoted epithelial wound healing, stim-ulated the reactivation of Akt, EGFR, and Sirt1, as well asattenuated oxidative stress in diabetic corneal epithelium.Furthermore, the study shows that the impairment of SP-NK-1 receptor signaling causes changes of normal cornealepithelium similar to those of diabetic mice. The resultssuggest that SP-NK-1 receptor signaling regulates the acti-vation of multiple signaling pathways that are needed for

Figure 7—Local inhibition of SP-NK-1 receptor signaling causes diabetic-like pathological changes in the cornea of normal mice. NK-1receptor antagonist L-733,060 was injected subconjunctivally in normal mice. In the unwounded corneal epithelium 3 days after antagonistinjection, the staining density of p-Akt, p-EGFR, and Sirt1 (A) and corneal sensitivity (B) (n = 5 per group) were attenuated similarly to that indiabetic mice. In the corneal epithelium 72 h after scrape, the local antagonist injection caused a significant delay of corneal epithelialwound healing (n = 5 per group) (C) as well as attenuated p-Akt and Ki-67 staining density in the regenerated corneal epithelium (D). *P <0.05.

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corneal epithelial wound healing, whereas this regulation isimpaired in diabetic corneal epithelium and rescued by SPvia an autoregulatory mechanism (37,38). Moreover, SP re-stored the corneal sensitivity of diabetic mice close to thesame level of normal mice, whereas local inhibition of SP-NK-1 receptor signaling caused decreased corneal sensitivityin normal mice similar to that in diabetic mice. The resultssuggest that the impairment of SP-NK-1 receptor signalingmay also be involved in the attenuation of corneal sensitiv-ity in diabetes, which is supported by the fact that the NK-1receptor exists in peripheral nerve (39). Taken together, SP,secreted by corneal sensory nerve fibers, may be the keyneurotransmitter and neuropeptide that mediates cornealnociception transmission and provides trophism to the cor-neal epithelium. Furthermore, as for the treatment of di-abetic keratopathy, many growth factors, cytokines, andvarious agents have been evaluated for their capacity toaccelerate corneal wound healing (40). However, SP andits functional derivative (FGLM-amide), with the advan-tages of smaller molecules and higher efficiency, havebeen shown to be effective for the treatment of persistentcorneal epithelial defects in clinical studies (40–42). In ad-dition, we found that SP promoted the regeneration ofnerve fibers in diabetic corneal epithelium and acceleratedtrigeminal neuronal growth in vitro that was impaired byhigh glucose (Supplementary Fig. 5).

The NK-1 receptor, the preferred receptor of SP,mediates a variety of physiological and pathophysiologicalresponses (22,23,43). Although previous studies haveconfirmed that SP enhances corneal epithelial migrationwhen combined with IGF-1 or EGF (41,44,45), the exactmechanism remains unclear. Here we show that SP reac-tivates Akt, EGFR, and Sirt1 and promotes ROS scaveng-ing capacity that are impaired by hyperglycemia. Theresults suggest a molecular basis for the synergistic effectsof SP and IGF-1 or EGF on the enhancement of diabeticcorneal epithelial wound healing (46,47). Even moreinterestingly, we found that the local inhibition of theSP-NK-1 receptor signaling in normal mice causes patho-logical changes of the corneal epithelium similar to thoseof diabetic corneal epithelium. These results suggest thatSP-NK-1 receptor signaling may be involved in the main-tenance of corneal epithelium homeostasis and also in theprotection from hyperglycemia stress, whereas the im-pairment of SP-NK-1 receptor signaling may explain thefragility of diabetic corneal epithelium in vivo.

Prolonged hyperglycemia always causes the perturba-tion of catabolic pathways and the overproduction of ROSin the mitochondria, which in turn plays a critical role inthe development of diabetes complications (48), includingdiabetic keratopathy (5). In the current study, we con-firmed that the mitochondrial superoxide level was upreg-ulated in high glucose–treated corneal epithelial cells,accompanied by changes of mitochondria structure andloss of mitochondrial membrane potential. Interestingly,we found that SP attenuates the dysfunction of the mi-tochondria by high glucose. Moreover, SP also elevates the

intracellular GSH level, the main antioxidant in the cells.In addition, although increased Nrf2 expression wasdetected in diabetic corneal epithelium (data not shown),the expressions of Nrf2 downstream antioxidant genes,including MnSOD, catalase, NQO1, TXN, and Hmox1, weredownregulated in diabetic corneal epithelium, whereasthey were upregulated after SP application, which sug-gests that additional regulatory mechanisms may existbetween Nrf2 and its downstream antioxidant geneexpressions in diabetes (49,50). All things considered, theimprovement of oxidative stress by SP via the improvedmitochondrial function, elevated GSH level, and upregulatedexpression of antioxidant genes may also play an importantrole in the protection of corneal epithelium in diabetes.

In conclusion, our study demonstrates, for the firsttime, that SP promotes diabetic corneal epithelial woundhealing while simultaneously triggering the reactivation ofthe Akt, EGFR, and Sirt1 signaling of importance for thathealing, as well as rescuing corneal sensation, improvingmitochondrial function, and decreasing ROS accumula-tion caused by hyperglycemia. Furthermore, we show thatlocal inhibition of SP-NK-1 receptor signaling abolishes thepromotion of SP on corneal epithelial healing in diabeticmice and causes diabetic corneal pathological changes innormal mice. Thus, the SP-NK-1 receptor signaling mayplay a critical role in the maintenance of corneal epitheliumhomeostasis, and SP signaling may, through the NK-1receptor, contribute to the promotion of diabetic cornealepithelial wound healing by the rescued activation of Akt,EGFR, and Sirt1, the improvement of mitochondrialfunction, and the increased ROS scavenging capacity ofcorneal epithelium.

Acknowledgments. The authors thank Yangyang Zhang, Wenjie Sui,Qian Wang, Hua Gao, Suxia Li, and Zhaoli Chen (Shandong Eye Institute) fortheir help with the animal experiments, statistical analysis, and human tissuecollection.Funding. This work was partially supported by the National Basic ResearchProgram of China (2012CB722409) and the National Natural Science Foundationof China (81170816 and 81200665). Q.Z. is partially supported by the ShandongProvincial Excellent Innovation Team Program and Taishan Scholar Program(20081148). P.D. is partially supported by the J.C. Kempe and Seth M. KempeMemorial Foundations, the Swedish Society of Medicine, the Cronqvist and KMAFoundations, and the National Swedish Research Council (521-2013-2612, Q.Z.coapplicant).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. L.Y. and G.D. contributed to sample testing,data analysis, and study design. X.Q., M.Q., Y.W., and H.D. contributed tosample testing and data analysis. P.D., L.X., and Q.Z. contributed to studydesign, data analysis, and manuscript preparation. L.X. and Q.Z. are theguarantors of this work and, as such, had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy ofthe data analysis.

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