The opioid peptide biphalin modulates human corneal epithelial wound healing in vitro

The opioid peptide biphalin modulates human corneal epithelial wound healing in vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article The opioid peptide biphalin modulates human corneal epithelial wound healing in vitro Ozgun Melike Gedar Totuk, Erdost YILDIZ, Adriano MOLLICA, Kerem KABADAYI, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.11471/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background: Analgesic drugs, including nonselective opioids and non-steroidal anti-inflammatory drugs, should be used with great precautions to relieve pain after physical damage of the corneal epithelium because of their unfavorable effects on the wound-healing process. Biphalin is a synthetic opioid peptide that has been demonstrated to possesses a strong analgesic effect on rodents. The purpose of this study is to investigate the effects of biphalin on human corneal epithelium wound healing. Methods: An immortalized human corneal epithelial cell (HCEC) culture was used to test the effects of biphalin on wound healing. The toxicity of biphalin in various concentrations was measured with the MTT assay. The effect of 1 µM and 10 µM biphalin were tested on wound closure in an in vitro scratch assay of HCECs and for cell migration and proliferation separately. Naloxone, a non-selective competitive antagonist of opioid receptors, was also used to inhibit the effects of biphalin in all experiments. Results: Biphalin did not cause any toxic effect on HCECs in concentrations lower than 100 µM at various incubation time points. Biphalin increased the wound closure process significantly at 1 µM concentration in an in vitro scratch assay of HCECs (p < 0.05). It also increased the migration of HCECs significantly (p < 0.01). There was no significant difference between biphalin and control groups of HCECs in the Ki67 proliferation assay. Conclusion: Biphalin, a synthetic opioid peptide, has a potential role as a novel topical analgesic agent that promotes corneal epithelial wound healing. This role should be evaluated in further in vivo and clinical studies. Ophthalmology synthetic opioid peptide corneal wound healing opioid receptors human corneal epithelial cell culture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Ocular pain is an important symptom of inflammatory or traumatic disorders affecting anterior segment structures, such as the cornea, sclera, conjunctiva, and uvea. It is commonly relieved with ophthalmic topical anesthetic agents, such as tetracaine, procaine, benoxinate, and proxymetacaine (proparacaine), most of which have toxic side effects to the corneal epithelium, such as corneal epithelial erosion and delayed wound healing. [1, 2] Opioids are considered potent and appropriate analgesics for moderate to severe acute and chronic pain and have been used to treat pain since early ages. They are used in ophthalmology by a systemic or periocular (sub-Tenon’s block, peribulbar, retrobulbar) administration route. [3, 4] In addition to their analgesic effect, opioids show cytoprotective, neuroendocrine regulatory, immunomodulatory, and behavioral-modification effects by affecting opioid receptors. [5] Opioids interact with three receptor classes of seven heterotrimeric inhibitory transmembrane G-protein-coupled opioid receptors (GCPRs): delta opioid (DOR), kappa-opioid (KOR), and mu-opioid (MOR). [6] Biphalin is a dimeric nonspecific opioid analog [(Tyr-D-Ala-Gly-Phe-NH–)2], which mainly activates DORs and MORs, resulting in a proven complete analgesic response and neuroprotective effect in the central nervous system. [7] Biphalin exerts less dependence and tolerance compared to morphine, since DOR- and MOR-induced side effects are believed to be prevented by activation of KOR. [8] To date, biphalin has been shown to be one of the most potent peptide-based opioid analgesics and has been recently investigated as a potential treatment for abdominal pain associated with inflammatory bowel disease. [9] The analgesic potency of biphalin is seven times greater than that of etorphine and three orders of magnitude greater than morphine after intracerebroventricular administration. [10] Since common analgesic drugs used to relieve pain after physical damage to the corneal epithelium have unfavorable effects on the wound-healing process, the search for an analgesic that has no adverse effect on corneal wound healing continues. To the best of our knowledge, the role of synthetic opioid peptides on human corneal epithelial cells has not been investigated in the published English literature. Since biphalin is a nonspecific opioid analog, this study aimed to evaluate the effects of biphalin on human corneal epithelial cells, particularly on wound healing, cell migration, and cell proliferation in vitro. Methods Synthesis of the Opioid Agonist Biphalin was synthesized by Adriano Mollica at his laboratory in Università degli Studi G. d’Annunzio Chieti e Pescara, Department of Pharmacy, Chieti, Italy. The peptide’s chemical properties were in full agreement with those already reported in the literature (Fig. 1). [11] Corneal Epithelial Cell Culture Human immortalized corneal epithelial cells (HCECs) were a generous gift from Dr. James Jester (Irvine, CA, USA). The HCECs were cultured in a keratinocyte serum-free medium (KSFM; Gibco, NY, USA) supplemented with bovine pituitary extract (BPE; 25 μg/mL), epidermal growth factor (EGF; 50 ng/mL), penicillin (100 IU/mL), and streptomycin (100 μg/mL). The cells were maintained in 75 cm 2 flasks until experimentation. The HCECs did not differentiate in the keratinocyte serum-free medium. To differentiate HCECs, we followed the protocol described by Sahin et al. [12] We changed the medium with DMEM with 10% FBS. In this way, the HCECs were differentiated and stratified. Cytotoxicity Assay The HCECs were treated with different concentrations of biphalin (from 1 pM to 100 µM) in 96-well culture dishes (Corning, NY, USA) for 24 hours. The cytotoxicity of exposure was measured with the MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay (Thermo Fisher Scientific, MA, USA). The color of the MTT tetrazole salt was measured with a spectrophotometer at a wavelength of 570 nm. In Vitro Scratch Assay The HCECs were grown to confluence on 12-well culture dishes (Corning, NY, USA). On reaching confluence, the cells were rinsed with a phosphate-buffered saline solution (PBS) and exposed to a differentiation medium consisting of Dulbecco’s Modified Eagle Medium (DMEM; Gibco, NY, USA) with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL) for one day. Two perpendicular linear scratches were made using a sterile 200 µL pipette tip, and the wells were washed with PBS. Immediately after the scratch, all groups were incubated in KSFM. Biphalin (in two different concentrations; 1 µM [10 -6 M] and 10 µM [10 -5 M]) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS) was added to the cultures. Naloxone, which is a selective opioid receptor antagonist, was added to inhibit the opioid receptor-related effects of biphalin. The scratch area was captured hourly for 24 hours using live-cell microscopy (DMi 8; Leica, Wetzlar, Germany). The relative wound area (RWA) was measured using ImageJ software (National Institutes of Health [NIH], MD, USA) (Fig. 2). Transwell Migration Assay After wounding, the cells were used to do a Transwell Migration Assay. This is because during confluency, gene expressions of cells are different from the wound-healing situation. By creating a wound, we simulated the wound process in epithelial cells. Immediately after the wounding process was complete, as described above, the HCECs were trypsinized, washed, and plated (2.5×10 5 cells per insert) in 8.0 µm pore size Transwell inserts (Corning, NY, USA) in KSFM. The lower compartment was filled with DMEM with 10% FBS or DMEM with 10% FBS plus either biphalin (in two different concentrations; 1 µM and 10 µM) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS). After 24 hours, the cells on the upper side of the insert were removed by scraping, and the cells that had migrated through were fixed on the lower side of the membrane with 4% paraformaldehyde, then stained with hematoxylin-eosin and quantified by counting the number of cells in 10 separate fields. The data were expressed as the number of migrated cells per micrograph field for each sample well. Ki67 Proliferation Assay The effect of biphalin on in vitro proliferation was assessed by immunofluorescence staining for Ki67. The Ki67 protein is present during the G1, S, G2, and M phases of the cell cycle and is strictly associated with cell proliferation. 2.5 x 10 5 HCECs were plated in equal numbers in 24-well culture dishes (Corning, NY, USA). After reaching confluence, cells were rinsed twice with PBS and exposed to a stratification medium consisting of DMEM with 10% FBS. Two perpendicular linear scratches were made using a sterile 200 µL pipette tip, and the wells were washed three times with PBS and incubated with KSFM without EGF and BPE. Immediately after the scratch, biphalin (in two different concentrations; 1 µM and 10 µM) or biphalin plus naloxone solution (in two different concentrations; 1 µM and 10 µM) or their vehicle (PBS) were added to the cell culture medium. The cells were incubated for 6 hours at 37°C. After the treatment, the cells grown on 24-well culture dishes were fixed in 4% paraformaldehyde for 20 min. After three washes with PBS, the cells were incubated with 0.1% TritonX-100 in PBS for 8 min. The cells were incubated with Superblock (Thermo Fisher Scientific, MA, USA) for 10 min at room temperature and then overnight at 4°C with the rabbit anti-Ki67 primary antibody (Abcam, MA, USA) at optimal dilutions in a blocking solution. After three washes with PBS, the cells were incubated with the FITC-conjugated secondary antibody (Abcam, MA, USA) for 90 min at 37°C, then washed, counterstained with 406-diamidino-2-phenylindole (DAPI), and mounted. Negative controls were stained in a similar fashion (DMi 8; Leica, Wetzlar, Germany). The Ki67 proliferation index is the proportion of Ki67 stained cell nuclei to DAPI stained cell nuclei from 20 different micrograph areas. The Ki67 proliferation index is calculated by dividing the number of Ki67 stained cell nuclei by the number of DAPI stained cell nuclei using ImageJ software (National Institutes of Health [NIH], MD, USA) Gene Expression Analysis of Opioid Receptors with Quantitative Reverse Transcription PCR The presence and expression levels of MOR, DOR, and KOR in the HCECs were measured quantitatively using a real-time polymerase chain reaction (qRT-PCR). The presence of OPRM1, OPRD1, and OPRK1 mRNAs, which are related with mu, delta, and kappa opioid receptor proteins, respectively, was studied in differentiated and undifferentiated HCECs and in SH-SY5Y cell lines. SH-SY5Y cell lines are human neuronal cancer cell lines that have demonstrated expressions of these three opioid receptors in previous literature.[13] To show the effect of the serum on HCECs, HCECs cultured in KSFM and in DMEM+10% FBS were used in qRT-PCR analysis. The qRT-PCR primers for the genes that are responsible for the opioid receptor expressions selected is shown in Table 1. RNA isolation from cells was performed via a Quick-RNA MicroPrep Kit (Zymo Research, Irvine, CA, USA). Extracted RNAs were quantified with Nanodrop 2000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA), and 1000 ng cDNA was prepared using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time expressions of mRNAs were detected and compared with a Light Cycler 480 SYBR Green I Master (Roche, Basel, Switzerland). Statistical Analysis Each experiment was performed at least two times. For blind analysis, collection of images was made by E.Y., and each image was assigned a number. Then images were analyzed anonymously by K.K. Values were displayed as a mean ± standard deviation. For the in vitro scratch assay, we measured time-dependent change in the wound area. For other assays, we collected data only at one time point. Since two-way ANOVA is used to examine the interaction between two independent variables (treatment and time), and one-way ANOVA tests the effect of one independent variable (treatment), statistical analysis was performed using two-way ANOVA for in vitro scratch assay results, and one-way ANOVA with a Tukey’s Honest Significant Difference test was used for other results to determine the degree of significance (R; R-Project, Vienna, Austria). Results were considered statistically significant when the p-value was less than 0.05. Results MTT Toxicity Assay First, we measured the cytotoxic effect of biphalin on HCECs with the MTT toxicity assay. Biphalin has no cytotoxic effects at doses lower than 100 µM after a 24-hour drug incubation (Fig. 3). Because of this, we selected two doses, 1 µM and 10 µM, to apply on HCECs during in vitro experiments. The In Vitro Scratch Assay We observed a statistically significant decrease in RWA in biphalin-treated cells at 1 µM concentration compared to the vehicle group (at the 6 th hour, biphalin RWA = 0.02126±0.02299, biphalin plus naloxone RWA=0.06015±0.06286, vehicle RWA = 0.1552±0.16940, n= 8p < 0.05) (Fig. 4a). However, there was no statistically significant difference in RWA in 10 µM biphalin, biphalin plus naloxone, or the vehicle groups (at the 6 th hour, biphalin RWA = 0.05838±0.06159, biphalin plus naloxone RWA=0.06015±0.06897, vehicle RWA = 0.01507±0.02983, n= 8) (Fig. 4b). It shows the positive wound-healing effect of biphalin in the in vitro wound-healing model of HCECs. But this effect could be because of proliferation or migration. To understand which cell behavior leads to the positive effect, we performed a Transwell Migration Assay and a Ki67 proliferation assay. Transwell Migration Assay We recorded a statistically significant increase (n= 20, p < 0.01) in the number of cells passing through the Transwell membrane at 1 µM concentration of biphalin (82.35±42.96) in respect of vehicle (34.35±7.081) and biphalin plus naloxone (52.75±23.76) (Fig. 5a). However, there was no statistically significant difference between the 10 µM concentration of biphalin (56.80±42.36), biphalin plus naloxone (37.20±13.23), or the vehicle (39.85±12.35) groups in terms of the number of cells passing through the membrane (n= 20) (Fig. 5b). Ki67 Proliferation Assay To examine the proliferative activity of HCECs during wound healing, Ki67 expression was observed in the wound areas (n= 20). After image acquisition, for a more precise comparison of Ki67 expression between the groups, the Ki67 labeling index was calculated. On the Ki67 proliferation index, there was no statistically significant difference between experimental groups in either concentration of biphalin, 1 µM and 10 µM, at 3 and 6 hours (Fig. 6). These findings suggest biphalin has no significant proliferative effect on HCECs. Gene Expression Analysis with qRT-PCR of Opioid Receptors On qRT-PCR, we demonstrated the presence of OPRM1, OPRD1, and OPRK1 mRNAs in both differentiated and undifferentiated HCECs compared to the SH-SY5Y cell lines. MOR, DOR, and KOR mRNA expression was significantly lower (p<0.001) in HCECs than in the SH-SY5Y cell lines (Fig. 7). Discussion In this study, we demonstrated that biphalin, which acts mainly via MORs and DORs, increases cell migration at the wound area and accelerates wound closure without affecting cell proliferation. Partial inhibition of the wound-healing effect of biphalin with naloxone can suggest MOR- and DOR-dependent mechanism of action. Our findings on biphalin are consistent with the wound-healing accelerator effects of MOR and DOR stimulations in cell cultures, which have been described previously. [14, 15] We also demonstrated that all three types of opioid receptors are present in HCECs using the qRT-PCR, which is also in line with previous reports.[16] To the best of our knowledge, this is the first study on the effects of a synthetic dimeric opioid peptide on human corneal epithelium cells in the literature. Opioid receptors are distributed in the anterior segment tissues as well as in various layers of the retina and the optic nerve. They play important roles in the regulation of iris function, accommodation power, aqueous humor dynamics, corneal wound healing, retinal development, inhibition of neuroinflammation, neuroprotection against glaucomatous damage, and reduction of intraocular pressure. [17-20] Although various pharmacological mechanisms were proposed, exact processes underlying the effects induced by opioid receptors have not been clearly defined. Regarding opioid receptor types, both DOR and MOR were determined in the corneal tissues of certain animals. [21] Limited data is available regarding the effects of opioids on corneal wound healing. Peyman et al. examined the analgesic and toxic effects of topical morphine on corneal abrasion in a rabbit corneal abrasion model. [22] They showed that morphine sulphate had a desirable analgesic property without irritating or causing any adverse effect on the cornea. Later, Stiles et al. studied the effect of topical application of a 1% morphine sulfate solution on signs of pain and wound healing in dogs with corneal ulcers and examined normal corneas immunohistochemically for the presence of MORs and DORs. [21] They showed that both MORs and DORs were present in normal canine corneas. They also demonstrated that topical morphine sulfate provided analgesia and did not interfere with normal wound healing. A recent study by Bigliardi et al. studied the effect of DORs in an in vitro scratch wound model, which we also used in our study. [14] They found that migration and wound recovery were enhanced in human keratinocyte monolayers overexpressing DORs in vitro. They concluded that opioid receptors affect intercellular adhesion and wound-healing mechanisms, underlining the importance of a neuroendocrine system in wound healing and homeostasis. In another study, Wang et al. showed the role of delta and mu opioid receptors on wound healing and migration in DOR and MOR knockout mice. [15] Synthetic opioids have been known to mimic the cytoprotective effects of endogenous opioidergic ligands and induce their physiological effects by the activation of DORs, KORs, and MORs, which were further subclassified into delta1 and delta2; kappa1, kappa2, and kappa3; and mu1, mu2, and mu3 opioid receptors, respectively. [6] Biphalin (PubChem CID: 5487663) was first synthesized by Lipkowski et al. in 1982 and then resynthesized by Mollica et al. with a modification in which two identical enkephalin-like tetrapeptides (Tyr-DAla-GlyPhe) were connected “head to head” by a hydrazide bridge (Fig. 1). [11, 23] In this modification, the natural residues of the dimeric opioid peptide biphalin were replaced by the corresponding homo- β 3 amino acids. The derivative 1 containing h β 3 Phe in place of Phe showed good MOR and DOR affinities and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma. Biphalin has been shown to be one of the most potent peptide-based opioid analgesics. It crosses the blood-brain-barrier, resists enzymatic degradation, and exerts high metabolic stability in serum and the brain with half-lives of 87 and 193 min, respectively. [10] Biphalin exerts a higher binding affinity to DOR and MOR, greater analgesic potency than morphine and etorphine, and less dependence, excitatory hypersensitivity, and tolerance compared to morphine during chronic use. These DOR- and MOR-induced side effects of morphine are believed to be prevented by activation of KOR, and biphalin shows reduced MOR-related side effects due to the high potential of simultaneous MOR and DOR interactions and thus synergistic effects. [7, 8, 24] Although the potential mechanisms of biphalin on promoting migration and accelerating wound healing in the corneal epithelium have not been evaluated in this study, inhibition of the influx of Ca+2, inhibition of PKC-dependent Na-K-Cl cotransporter, activation of MAP kinases, and inhibition of reactive oxygen species production or DORs may play a role in the mechanisms of biphalin-induced effects. [19; 25-27] The antinociceptive effect of biphalin has been suggested to have a potential role in the treatment of cancer pain, abdominal pain associated with inflammatory bowel disease, and in neuropathic pain via different routes of administration, such as subcutaneous, intravenous, intrathecal, intracerebroventricular, and intraperitoneal. [9,28,29] Furthermore, recent studies have revealed the beneficial role of biphalin on cell viability and neuroprotection against excitotoxic and ischemic damage by inhibiting protein kinase C-dependent sodium potassium-chloride cotransporter expression in focal brain ischemia by activating downstream survival mitogen-activated protein kinases, and by inhibiting reactive oxygen species production in an opioid receptor-dependent manner, which was challenged by naltrexone. [19,25,30] Biphalin also acts as an immunomodulatory agent by stimulating human T cell proliferation, natural killer cell cytotoxicity in vitro, and interleukin-2 production and diminishes pro- and anti-inflammatory factors in lipopolysaccharide-treated microglial cells. [28, 31] We proposed that the lack of a significant effect on proliferation of biphalin may be the result of its failure in binding to opioid growth factor receptors (OGFR), since OGFRs bear no resemblance to classical opioid receptors, and blockade of OGFR interaction with naltrexone accelerates cell growth. In the biphalin plus naloxone groups, both proliferation and wound healing were partially increased. We believe this effect was due to the binding of naloxone to OGFRs as a nonselective competitive opioid antagonist. [32] In our study, there was no statistically significant difference between biphalin-treated cells at 10 µM concentration and control groups on wound healing and cell migration. Biphalin shows approximately 100 times lower affinity to KOR than MOR and DOR. [33] Therefore, it is possible that at higher concentrations of biphalin, KORs are also activated and reverse the healing and migration process. Many studies have demonstrated the cytoprotective and reformative effects of DORs and MORs separately, and their synergistic activation may have strengthened their positive effects. [34] In further experiments, the exact effects of specific opioid receptor subgroup activation on human corneal epithelial cells should be explored with specific opioid peptides. Additionally, the effect of biphalin on the wound-healing process in the cornea and changes in keratinocytes, neural fibers, dendritic cells, the basement membrane, and extracellular matrix elements and their interactions should also be investigated. The main limitation of the present study was the lack of data on the effect of biphalin on the intracellular signaling mechanisms and cell metabolism. In future experiments, we plan to examine the effects of biphalin and its derivatives on corneal healing in experimental animals, which will allow us to determine receptor-down signaling pathway interactions. A second limitation of our study that must be addressed is the lack of an in vivo animal model. We plan to conduct studies in transgenic mice in order to shed light on the effects of opioids in corneal wound healing. Conclusion In the corneal epithelium, biphalin accelerates wound healing and cell migration without affecting cell proliferation. Since the analgesic and antinociceptive effects of biphalin have been demonstrated in previous studies, it can be used in the near future as an analgesic after corneal traumas and surgeries to modulate corneal epithelial wound healing. We also propose that biphalin may become a new treatment alternative not only for corneal traumas but also for other neuropathic pain syndromes affecting the ocular surface, such as herpetic or diabetic neuropathy. List of Abbreviations bovine pituitary extract: BPE; delta opioid receptor: DOR; 406-diamidino-2-phenylindole: DAPI; 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide: MTT; Dulbecco modified eagle medium: DMEM; epidermal growth factor: EGF; fetal bovine serum: FBS; G-protein-coupled opioid receptors: GCPRs; human immortalized corneal epithelial cells: HCECs; kappa-opioid receptor: KOR; keratinocyte serum-free medium: KSFM; mu-opioid receptor: MOR; National Institutes of Health: NIH; phosphate-buffered saline solution: opioid growth factor receptors: OGFR; PBS; real-time polymerase chain reaction:qRT-PCR; relative wound area: RWA. Declarations Ethics approval and consent to participate : Since the study was a laboratory- based study not using patients or patient material, ethical approval was not required. Consent for publication : Not applicable Availability of data and material: Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Competing interests: The authors declare that they have no competing interests. Funding: The design of the study and collection, analysis, and interpretation of data and in writing the manuscript was not funded. Author Contributions: All authors have made substantial contributions to all of the following: (1) the conception and design of the study: OMGT, AS, EY (2) Performed the experiments: EY, KK (3) Analysis and interpretation of data: EY, KK (4) Contributed reagents/materials/analysis tools: AM, AS, EY (5) drafting the article or revising it critically for important intellectual content: OMGT, AS, EY, AM (3) Final approval of the version to be submitted: OMGT, AS, EY, AM, KK. All authors read and approved the final manuscript. Acknowledgement: The authors thank Koç University Research Center for Translational Medicine (KUTTAM), funded by the Republic of Turkey Ministry of Development, for allowing us to use their services and facilities. The content of the manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the Ministry of Development. This study was presented in part at 2 nd International Cell Death Research Congress 2018, Izmir, Turkey, 1-4 November 2018, and published as congress proceedings MDPI Proceedings 2018,2(25),1552. doi: 10.3390/proceedings2251552 References Ramselaar JAM, Boot JP, Van Haeringen NJ, Van Best JA, Oosterhuis JA: Corneal epithelial permeability after instillation of ophthalmic solutions containing local anaesthetics and preservatives . Current eye research 1988, 7 (9):947-950. Carney LG, O'Leary DJ, Millodot M: Effect of topical anaesthesia on corneal epithelial fragility . International Ophthalmology 1984, 7 (2):71-73. 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European Journal of Pharmaceutical Sciences 2016, 89 :39-49. Popiolek-Barczyk K, Piotrowska A, Makuch W, Mika J: Biphalin, a Dimeric Enkephalin, Alleviates LPS-Induced Activation in Rat Primary Microglial Cultures in Opioid Receptor-Dependent and Receptor-Independent Manners . Neural plasticity 2017, 2017 :1-19. Kawalec M, Kowalczyk JE, Beresewicz M, Lipkowski AW, Zablocka B: Neuroprotective Potential of Biphalin, Multireceptor Opioid Peptide, Against Excitotoxic Injury in Hippocampal Organotypic Culture . Neurochemical Research 2011, 36 (11):2091-2095. Mehrotra S, Prajapati RK, Haq W, Singh VK: Immunomodulation by biphalin, dimeric synthetic opioid peptide, and its analog . Immunopharmacology and Immunotoxicology 2002, 24 (1):83-96. Zagon IS, Jenkins JB, Sassani JW, Wylie JD, Ruth TB, Fry JL, Lang CM, McLaughlin PJ: Naltrexone, an Opioid Antagonist, Facilitates Reepithelialization of the Cornea in Diabetic Rat . Diabetes 2002, 51 (10):3055-3062. Lipkowski AW, Konecka AM, Sroczynska I, Przewlocki R, Stala L, Tam SW: Bivalent opioid peptide analogues with reduced distances between pharmacophores . Life Sciences 1987, 40 (23):2283-2288. He X, Sandhu HK, Yang Y, Hua F, Belser N, Kim DH, Xia Y: Neuroprotection against hypoxia/ischemia: δ-opioid receptor-mediated cellular/molecular events . Cellular and Molecular Life Sciences 2012, 70 (13):2291-2303. Titles and legends for supplementary files Supplement 1. Video of in vitro scratch assay model showing the effect of 1 µM biphalin on relative wound healing area. Supplement 2. Video of in vitro scratch assay model showing the effect of vehicle on relative wound healing area. Table Table 1. The primers of genes used in the study Gene Sequence GAPDH F 5’-ACAACTTTGGTATCGTGGAAGG-3’ R 5’-GCCATCACGCCACAGTTTC-3’ OPRD1 F 5’-CGTCCGGTACACTAAGATGAAGA-3’ R 5’-GCCACGTCTCCATCAGGTA-3’ OPRK1 F 5’-ATCATCACGGCGGTCTACTC-3’ R 5’-ACTCTGAAAGGGCATGGTTGTA-3’ OPRM1 F 5’-CAGTGCTCATCATTACCGTGT-3’ R 5’-CTGGGATTGTAACCAAGGCTTTA-3’ Supplementary Files Supplement2Vehicle.mp4 Supplement1Biphalin.mp4 Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2344","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":447496,"identity":"b1f55a1a-865c-4f10-a5d7-11c246144311","order_by":1,"name":"Ozgun Melike Gedar Totuk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYFAC5oaDDSD6AAPjAyDFw0dYCyNcC7MBSAsbMVoYoVrYJEA0QS267Y2NB2dU1MrxHT/7rPJrjp0MGwPzw0c38GgxO3Ow4eCGM8eNJc+km92W3ZYMdBibsXEOPi03EhsOPmw7lrjhQBrbbcltzEAtPGzSeLXcfwjU8u9Y/Ybzz9iKJbfVE6HlBjDENjbUJBjcSGNj/LjtMBFazgAdNuPYAcOZN54xSzNuO87DxkzIL8cPH/7YU1Mnz3c+jfHjz23V9vzszQ8f49MCBYfBJDMPmCSsHATqwCTjD+JUj4JRMApGwQgDAOwvU9B16CBFAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1863-6501","institution":"Bahcesehir University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ozgun","middleName":"Melike Gedar","lastName":"Totuk","suffix":""},{"id":447497,"identity":"4b86e4a2-8fc0-46ef-8980-72740c20b266","order_by":2,"name":"Erdost YILDIZ","email":"","orcid":"","institution":"Koc Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Erdost","middleName":"","lastName":"YILDIZ","suffix":""},{"id":447498,"identity":"71d67461-8673-4af6-a62a-f0db18d014fc","order_by":3,"name":"Adriano MOLLICA","email":"","orcid":"","institution":"Universita degli Studi Gabriele d'Annunzio Chieti e Pescara Dipartimento di Farmacia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adriano","middleName":"","lastName":"MOLLICA","suffix":""},{"id":447499,"identity":"e278bba0-a973-4581-9526-f47f7941b7af","order_by":4,"name":"Kerem KABADAYI","email":"","orcid":"","institution":"Bahcesehir Universitesi Tip Fakultesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kerem","middleName":"","lastName":"KABADAYI","suffix":""},{"id":447500,"identity":"a4e8b0c8-8651-4cc8-9949-7700d5bf1353","order_by":5,"name":"Afsun SAHIN","email":"","orcid":"","institution":"Koc Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Afsun","middleName":"","lastName":"SAHIN","suffix":""}],"badges":[],"createdAt":"2019-07-12 16:10:44","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.2.11471/v2","doiUrl":"https://doi.org/10.21203/rs.2.11471/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":805718,"identity":"f0b38f22-b3d1-483f-89d9-cc4a3e528a8e","added_by":"auto","created_at":"2020-03-31 16:26:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130276,"visible":true,"origin":"","legend":"Molecular structure of biphalin.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Figure 1.jpg"},{"id":805719,"identity":"5fe1dfc6-9b80-415a-891c-76c4f867a70e","added_by":"auto","created_at":"2020-03-31 16:26:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":500516,"visible":true,"origin":"","legend":"Examples of in vitro live cell migration assays for each experimental group at various time points and example of the relative wound area analysis in ImageJ.","description":"","filename":"2.PNG","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/2.PNG"},{"id":805721,"identity":"422aa87b-10fc-4cc5-8380-b1c5be025530","added_by":"auto","created_at":"2020-03-31 16:26:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":555717,"visible":true,"origin":"","legend":"MTT cytotoxicity assay on HCECs for biphalin at various doses after 24-hour of drug incubation (n=4).","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Figure 3.jpg"},{"id":805722,"identity":"d12a687e-f14a-46e4-a2ce-bc765e525936","added_by":"auto","created_at":"2020-03-31 16:26:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94914,"visible":true,"origin":"","legend":"Effects of 1 µM (a) and 10 µM (b) biphalin and biphalin plus naloxone, and vehicle on in vitro scratch assay model on relative wound healing area (%). Both of the graphs show the results of two independent experiments (n = 8, * p \u003c 0.05).","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Figure4.jpg"},{"id":805723,"identity":"9f94f0ec-2aad-4cc7-bc6c-cd5fee5244a8","added_by":"auto","created_at":"2020-03-31 16:26:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112851,"visible":true,"origin":"","legend":"Effects of 1 µM (a) and 10 µM (b) biphalin and biphalin plus naloxone, and vehicle on the movement of corneal epithelial cells in the transwell migration assay. Both of the graphs show the mean number of migrated cells in ten different micrograph area (3.6 µm2) in two independent experiments (n = 20, ** p \u003c 0.01, *** p \u003c 0.001).","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Figure5.jpg"},{"id":805724,"identity":"6c8ac067-30f8-4f7b-886f-214e17fb5308","added_by":"auto","created_at":"2020-03-31 16:26:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":155779,"visible":true,"origin":"","legend":"Effects of 1 µM biphalin and biphalin plus naloxone, and vehicle at 3 hours (a) and 6 hours (b); and those of 10 µM biphalin and biphalin plus naloxone, and vehicle at 3 hours (c) and 6 hours (d) on cell proliferation in Ki67 proliferation assay. The graphs show the percentage of Ki67-labeled cells to DAPI-labeled cells in ten different micrographs in two independent experiments (n = 20).","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Figure6.jpg"},{"id":805725,"identity":"849ceef9-de6a-441d-a21a-429ad5543e61","added_by":"auto","created_at":"2020-03-31 16:26:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114570,"visible":true,"origin":"","legend":"Gene expression analysis with real-time polymerase chain reaction (qRT-PCR) of OPRM1 (a), OPRD1 (b), and OPRK1 (c) mRNAs, which are related with mu, delta and kappa opioid receptor proteins, in HCECs with KSFM or with serum (10% FBS) and compared with in SH-SY5Y cell lines.","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Figure7.jpg"},{"id":13496166,"identity":"b264b441-b32f-40d8-939a-da319f5bebc1","added_by":"auto","created_at":"2021-09-16 22:48:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1872589,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/bd13e56a-2705-4a1c-bdb8-84607e49ee61.pdf"},{"id":805720,"identity":"6ddbf85e-3689-4b41-867b-1a63fcff428e","added_by":"auto","created_at":"2020-03-31 16:26:07","extension":"mp4","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10304735,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement2Vehicle.mp4","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Supplement 2 - Vehicle.mp4"},{"id":805717,"identity":"e069c43c-ceeb-4e83-8e12-af3d05f7c431","added_by":"auto","created_at":"2020-03-31 16:26:06","extension":"mp4","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11685774,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement1Biphalin.mp4","url":"https://assets-eu.researchsquare.com/files/rs-2344/v2/Supplement 1 - Biphalin.mp4"}],"financialInterests":"","formattedTitle":"The opioid peptide biphalin modulates human corneal epithelial wound healing in vitro","fulltext":[{"header":"Background","content":"\u003cp\u003eOcular pain is an important symptom of inflammatory or traumatic disorders affecting anterior segment structures, such as the cornea, sclera, conjunctiva, and uvea. It is commonly relieved with ophthalmic topical anesthetic agents, such as tetracaine, procaine, benoxinate, and proxymetacaine (proparacaine), most of which have toxic side effects to the corneal epithelium, such as corneal epithelial erosion and delayed wound healing. [1, 2]\u003c/p\u003e\n\u003cp\u003eOpioids are considered potent and appropriate analgesics for moderate to severe acute and chronic pain and have been used to treat pain since early ages. They are used in ophthalmology by a systemic or periocular (sub-Tenon\u0026rsquo;s block, peribulbar, retrobulbar) administration route. [3, 4] In addition to their analgesic effect, opioids show cytoprotective, neuroendocrine regulatory, immunomodulatory, and behavioral-modification effects by affecting opioid receptors. [5] Opioids interact with three receptor classes of seven heterotrimeric inhibitory transmembrane G-protein-coupled opioid receptors (GCPRs): delta opioid (DOR), kappa-opioid (KOR), and mu-opioid (MOR). [6]\u003c/p\u003e\n\u003cp\u003eBiphalin is a dimeric nonspecific opioid analog [(Tyr-D-Ala-Gly-Phe-NH\u0026ndash;)2], which mainly activates DORs and MORs, resulting in a proven complete analgesic response and neuroprotective effect in the central nervous system. [7] Biphalin exerts less dependence and tolerance compared to morphine, since DOR- and MOR-induced side effects are believed to be prevented by activation of KOR. [8] To date, biphalin has been shown to be one of the most potent peptide-based opioid analgesics and has been recently investigated as a potential treatment for abdominal pain associated with inflammatory bowel disease. [9] The analgesic potency of biphalin is seven times greater than that of etorphine and three orders of magnitude greater than morphine after intracerebroventricular administration. [10] Since common analgesic drugs used to relieve pain after physical damage to the corneal epithelium have unfavorable effects on the wound-healing process, the search for an analgesic that has no adverse effect on corneal wound healing continues.\u003c/p\u003e\n\u003cp\u003eTo the best of our knowledge, the role of synthetic opioid peptides on human corneal epithelial cells has not been investigated in the published English literature. Since biphalin is a nonspecific opioid analog, this study aimed to evaluate the effects of biphalin on human corneal epithelial cells, particularly on wound healing, cell migration, and cell proliferation in vitro.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eSynthesis of the Opioid Agonist\u003c/h2\u003e\n\u003cp\u003eBiphalin was synthesized by Adriano Mollica at his laboratory in Universit\u0026agrave; degli Studi G. d\u0026rsquo;Annunzio Chieti e Pescara, Department of Pharmacy, Chieti, Italy. The peptide\u0026rsquo;s chemical properties were in full agreement with those already reported in the literature (Fig. 1). [11]\u003c/p\u003e\n\u003ch2\u003eCorneal Epithelial Cell Culture\u003c/h2\u003e\n\u003cp\u003eHuman immortalized corneal epithelial cells (HCECs) were a generous gift from Dr. James Jester (Irvine, CA, USA). The HCECs were cultured in a keratinocyte serum-free medium (KSFM; Gibco, NY, USA) supplemented with bovine pituitary extract (BPE; 25 \u0026mu;g/mL), epidermal growth factor (EGF; 50 ng/mL), penicillin (100 IU/mL), and streptomycin (100 \u0026mu;g/mL). The cells were maintained in 75 cm\u003csup\u003e2\u003c/sup\u003e flasks until experimentation. The HCECs did not differentiate in the keratinocyte serum-free medium. To differentiate HCECs, we followed the protocol described by Sahin et al. [12] We changed the medium with DMEM with 10% FBS. In this way, the HCECs were differentiated and stratified.\u003c/p\u003e\n\u003ch2\u003eCytotoxicity Assay\u003c/h2\u003e\n\u003cp\u003eThe HCECs were treated with different concentrations of biphalin (from 1 pM to 100 \u0026micro;M) in 96-well culture dishes (Corning, NY, USA) for 24 hours. The cytotoxicity of exposure was measured with the MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay (Thermo Fisher Scientific, MA, USA). The color of the MTT tetrazole salt was measured with a spectrophotometer at a wavelength of 570 nm.\u003c/p\u003e\n\u003ch2\u003eIn Vitro Scratch Assay\u003c/h2\u003e\n\u003cp\u003eThe HCECs were grown to confluence on 12-well culture dishes (Corning, NY, USA). On reaching confluence, the cells were rinsed with a phosphate-buffered saline solution (PBS) and exposed to a differentiation medium consisting of Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco, NY, USA) with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 \u0026mu;g/mL) for one day. Two perpendicular linear scratches were made using a sterile 200 \u0026micro;L pipette tip, and the wells were washed with PBS. Immediately after the scratch, all groups were incubated in KSFM. Biphalin (in two different concentrations; 1 \u0026micro;M [10\u003csup\u003e-6\u003c/sup\u003e M] and 10 \u0026micro;M [10\u003csup\u003e-5\u003c/sup\u003e M]) or biphalin plus naloxone solution (in two different concentrations; 1 \u0026micro;M and 10 \u0026micro;M) or their vehicle (PBS) was added to the cultures. Naloxone, which is a selective opioid receptor antagonist, was added to inhibit the opioid receptor-related effects of biphalin. The scratch area was captured hourly for 24 hours using live-cell microscopy (DMi 8; Leica, Wetzlar, Germany). The relative wound area (RWA) was measured using ImageJ software (National Institutes of Health [NIH], MD, USA) (Fig. 2).\u003c/p\u003e\n\u003ch2\u003eTranswell Migration Assay\u003c/h2\u003e\n\u003cp\u003eAfter wounding, the cells were used to do a Transwell Migration Assay. This is because during confluency, gene expressions of cells are different from the wound-healing situation. By creating a wound, we simulated the wound process in epithelial cells. Immediately after the wounding process was complete, as described above, the HCECs were trypsinized, washed, and plated (2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per insert) in 8.0 \u0026micro;m pore size Transwell inserts (Corning, NY, USA) in KSFM.\u003c/p\u003e\n\u003cp\u003eThe lower compartment was filled with DMEM with 10% FBS or DMEM with 10% FBS plus either biphalin (in two different concentrations; 1 \u0026micro;M and 10 \u0026micro;M) or biphalin plus naloxone solution (in two different concentrations; 1 \u0026micro;M and 10 \u0026micro;M) or their vehicle (PBS). After 24 hours, the cells on the upper side of the insert were removed by scraping, and the cells that had migrated through were fixed on the lower side of the membrane with 4% paraformaldehyde, then stained with hematoxylin-eosin and quantified by counting the number of cells in 10 separate fields. The data were expressed as the number of migrated cells per micrograph field for each sample well.\u003c/p\u003e\n\u003ch2\u003eKi67 Proliferation Assay\u003c/h2\u003e\n\u003cp\u003eThe effect of biphalin on in vitro proliferation was assessed by immunofluorescence staining for Ki67. The Ki67 protein is present during the G1, S, G2, and M phases of the cell cycle and is strictly associated with cell proliferation. 2.5 x 10\u003csup\u003e5 \u003c/sup\u003eHCECs were plated in equal numbers in 24-well culture dishes (Corning, NY, USA). After reaching confluence, cells were rinsed twice with PBS and exposed to a stratification medium consisting of DMEM with 10% FBS. Two perpendicular linear scratches were made using a sterile 200 \u0026micro;L pipette tip, and the wells were washed three times with PBS and incubated with KSFM without EGF and BPE. Immediately after the scratch, biphalin (in two different concentrations; 1 \u0026micro;M and 10 \u0026micro;M) or biphalin plus naloxone solution (in two different concentrations; 1 \u0026micro;M and 10 \u0026micro;M) or their vehicle (PBS) were added to the cell culture medium. The cells were incubated for 6 hours at 37\u0026deg;C. After the treatment, the cells grown on 24-well culture dishes were fixed in 4% paraformaldehyde for 20 min. After three washes with PBS, the cells were incubated with 0.1% TritonX-100 in PBS for 8 min. The cells were incubated with Superblock (Thermo Fisher Scientific, MA, USA) for 10 min at room temperature and then overnight at 4\u0026deg;C with the rabbit anti-Ki67 primary antibody (Abcam, MA, USA) at optimal dilutions in a blocking solution. After three washes with PBS, the cells were incubated with the FITC-conjugated secondary antibody (Abcam, MA, USA) for 90 min at 37\u0026deg;C, then washed, counterstained with 406-diamidino-2-phenylindole (DAPI), and mounted. Negative controls were stained in a similar fashion (DMi 8; Leica, Wetzlar, Germany). The Ki67 proliferation index is the proportion of Ki67 stained cell nuclei to DAPI stained cell nuclei from 20 different micrograph areas. The Ki67 proliferation index is calculated by dividing the number of Ki67 stained cell nuclei by the number of DAPI stained cell nuclei using ImageJ software (National Institutes of Health [NIH], MD, USA)\u003c/p\u003e\n\u003ch2\u003eGene Expression Analysis of Opioid Receptors with Quantitative Reverse Transcription PCR\u003c/h2\u003e\n\u003cp\u003eThe presence and expression levels of MOR, DOR, and KOR in the HCECs were measured quantitatively using a real-time polymerase chain reaction (qRT-PCR). The presence of OPRM1, OPRD1, and OPRK1 mRNAs, which are related with mu, delta, and kappa opioid receptor proteins, respectively, was studied in differentiated and undifferentiated HCECs and in SH-SY5Y cell lines. SH-SY5Y cell lines are human neuronal cancer cell lines that have demonstrated expressions of these three opioid receptors in previous literature.[13] To show the effect of the serum on HCECs, HCECs cultured in KSFM and in DMEM+10% FBS were used in qRT-PCR analysis. The qRT-PCR primers for the genes that are responsible for the opioid receptor expressions selected is shown in Table 1. RNA isolation from cells was performed via a Quick-RNA MicroPrep Kit (Zymo Research, Irvine, CA, USA). Extracted RNAs were quantified with Nanodrop 2000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA), and 1000 ng cDNA was prepared using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time expressions of mRNAs were detected and compared with a Light Cycler 480 SYBR Green I Master (Roche, Basel, Switzerland).\u003c/p\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eEach experiment was performed at least two times. For blind analysis, collection of images was made by E.Y., and each image was assigned a number. Then images were analyzed anonymously by K.K. Values were displayed as a mean \u0026plusmn; standard deviation. For the in vitro scratch assay, we measured time-dependent change in the wound area. For other assays, we collected data only at one time point. Since two-way ANOVA is used to examine the interaction between two independent variables (treatment and time), and one-way ANOVA tests the effect of one independent variable (treatment), statistical analysis was performed using two-way ANOVA for in vitro scratch assay results, and one-way ANOVA with a Tukey\u0026rsquo;s Honest Significant Difference test was used for other results to determine the degree of significance (R; R-Project, Vienna, Austria).\u003c/p\u003e\n\u003cp\u003eResults were considered statistically significant when the p-value was less than 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eMTT Toxicity Assay\u003c/h2\u003e\n\u003cp\u003eFirst, we measured the cytotoxic effect of biphalin on HCECs with the MTT toxicity assay. Biphalin has no cytotoxic effects at doses lower than 100 \u0026micro;M after a 24-hour drug incubation (Fig. 3). Because of this, we selected two doses, 1 \u0026micro;M and 10 \u0026micro;M, to apply on HCECs during in vitro experiments.\u003c/p\u003e\n\u003ch2\u003eThe In Vitro Scratch Assay\u003c/h2\u003e\n\u003cp\u003eWe observed a statistically significant decrease in RWA in biphalin-treated cells at 1 \u0026micro;M concentration compared to the vehicle group (at the 6\u003csup\u003eth\u003c/sup\u003e hour, biphalin RWA = 0.02126\u0026plusmn;0.02299, biphalin plus naloxone RWA=0.06015\u0026plusmn;0.06286, vehicle RWA = 0.1552\u0026plusmn;0.16940, n= 8p \u0026lt; 0.05) (Fig. 4a). However, there was no statistically significant difference in RWA in 10 \u0026micro;M biphalin, biphalin plus naloxone, or the vehicle groups (at the 6\u003csup\u003eth\u003c/sup\u003e hour, biphalin RWA = 0.05838\u0026plusmn;0.06159, biphalin plus naloxone RWA=0.06015\u0026plusmn;0.06897, vehicle RWA = 0.01507\u0026plusmn;0.02983, n= 8) (Fig. 4b). It shows the positive wound-healing effect of biphalin in the in vitro wound-healing model of HCECs. But this effect could be because of proliferation or migration. To understand which cell behavior leads to the positive effect, we performed a Transwell Migration Assay and a Ki67 proliferation assay.\u003c/p\u003e\n\u003ch2\u003eTranswell Migration Assay\u003c/h2\u003e\n\u003cp\u003eWe recorded a statistically significant increase (n= 20, p \u0026lt; 0.01) in the number of cells passing through the Transwell membrane at 1 \u0026micro;M concentration of biphalin (82.35\u0026plusmn;42.96) in respect of vehicle (34.35\u0026plusmn;7.081) and biphalin plus naloxone (52.75\u0026plusmn;23.76) (Fig. 5a). However, there was no statistically significant difference between the 10 \u0026micro;M concentration of biphalin (56.80\u0026plusmn;42.36), biphalin plus naloxone (37.20\u0026plusmn;13.23), or the vehicle (39.85\u0026plusmn;12.35) groups in terms of the number of cells passing through the membrane (n= 20) (Fig. 5b).\u003c/p\u003e\n\u003ch2\u003eKi67 Proliferation Assay\u003c/h2\u003e\n\u003cp\u003eTo examine the proliferative activity of HCECs during wound healing, Ki67 expression was observed in the wound areas (n= 20). After image acquisition, for a more precise comparison of Ki67 expression between the groups, the Ki67 labeling index was calculated. On the Ki67 proliferation index, there was no statistically significant difference between experimental groups in either concentration of biphalin, 1 \u0026micro;M and 10 \u0026micro;M, at 3 and 6 hours (Fig. 6). These findings suggest biphalin has no significant proliferative effect on HCECs.\u003c/p\u003e\n\u003ch2\u003eGene Expression Analysis with qRT-PCR of Opioid Receptors\u003c/h2\u003e\n\u003cp\u003eOn qRT-PCR, we demonstrated the presence of OPRM1, OPRD1, and OPRK1 mRNAs in both differentiated and undifferentiated HCECs compared to the SH-SY5Y cell lines. MOR, DOR, and KOR mRNA expression was significantly lower (p\u0026lt;0.001) in HCECs than in the SH-SY5Y cell lines (Fig. 7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that biphalin, which acts mainly via MORs and DORs, increases cell migration at the wound area and accelerates wound closure without affecting cell proliferation. Partial inhibition of the wound-healing effect of biphalin with naloxone can suggest MOR- and DOR-dependent mechanism of action. Our findings on biphalin are consistent with the wound-healing accelerator effects of MOR and DOR stimulations in cell cultures, which have been described previously. [14, 15] We also demonstrated that all three types of opioid receptors are present in HCECs using the qRT-PCR, which is also in line with previous reports.[16] To the best of our knowledge, this is the first study on the effects of a synthetic dimeric opioid peptide on human corneal epithelium cells in the literature.\u003c/p\u003e\n\u003cp\u003eOpioid receptors are distributed in the anterior segment tissues as well as in various layers of the retina and the optic nerve. They play important roles in the regulation of iris function, accommodation power, aqueous humor dynamics, corneal wound healing, retinal development, inhibition of neuroinflammation, neuroprotection against glaucomatous damage, and reduction of intraocular pressure. [17-20] Although various pharmacological mechanisms were proposed, exact processes underlying the effects induced by opioid receptors have not been clearly defined. Regarding opioid receptor types, both DOR and MOR were determined in the corneal tissues of certain animals. [21]\u003c/p\u003e\n\u003cp\u003eLimited data is available regarding the effects of opioids on corneal wound healing. Peyman et al. examined the analgesic and toxic effects of topical morphine on corneal abrasion in a rabbit corneal abrasion model. [22] They showed that morphine sulphate had a desirable analgesic property without irritating or causing any adverse effect on the cornea. Later, Stiles et al. studied the effect of topical application of a 1% morphine sulfate solution on signs of pain and wound healing in dogs with corneal ulcers and examined normal corneas immunohistochemically for the presence of MORs and DORs. [21] They showed that both MORs and DORs were present in normal canine corneas. They also demonstrated that topical morphine sulfate provided analgesia and did not interfere with normal wound healing. A recent study by Bigliardi et al. studied the effect of DORs in an in vitro scratch wound model, which we also used in our study. [14] They found that migration and wound recovery were enhanced in human keratinocyte monolayers overexpressing DORs in vitro. They concluded that opioid receptors affect intercellular adhesion and wound-healing mechanisms, underlining the importance of a neuroendocrine system in wound healing and homeostasis. In another study, Wang et al. showed the role of delta and mu opioid receptors on wound healing and migration in DOR and MOR knockout mice. [15]\u003c/p\u003e\n\u003cp\u003eSynthetic opioids have been known to mimic the cytoprotective effects of endogenous opioidergic ligands and induce their physiological effects by the activation of DORs, KORs, and MORs, which were further subclassified into delta1 and delta2; kappa1, kappa2, and kappa3; and mu1, mu2, and mu3 opioid receptors, respectively. [6] Biphalin (PubChem CID: 5487663) was first synthesized by Lipkowski et al. in 1982 and then resynthesized by Mollica et al. with a modification in which two identical enkephalin-like tetrapeptides (Tyr-DAla-GlyPhe) were connected \u0026ldquo;head to head\u0026rdquo; by a hydrazide bridge (Fig. 1). [11, 23] In this modification, the natural residues of the dimeric opioid peptide biphalin were replaced by the corresponding homo-\u003cem\u003e\u0026beta;\u003c/em\u003e3 amino acids. The derivative 1 containing h\u003cem\u003e\u0026beta;\u003c/em\u003e3 Phe in place of Phe showed good MOR and DOR affinities and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma. Biphalin has been shown to be one of the most potent peptide-based opioid analgesics. It crosses the blood-brain-barrier, resists enzymatic degradation, and exerts high metabolic stability in serum and the brain with half-lives of 87 and 193 min, respectively. [10] Biphalin exerts a higher binding affinity to DOR and MOR, greater analgesic potency than morphine and etorphine, and less dependence, excitatory hypersensitivity, and tolerance compared to morphine during chronic use. These DOR- and MOR-induced side effects of morphine are believed to be prevented by activation of KOR, and biphalin shows reduced MOR-related side effects due to the high potential of simultaneous MOR and DOR interactions and thus synergistic effects. [7, 8, 24]\u003c/p\u003e\n\u003cp\u003eAlthough the potential mechanisms of biphalin on promoting migration and accelerating wound healing in the corneal epithelium have not been evaluated in this study, inhibition of the influx of Ca+2, inhibition of PKC-dependent Na-K-Cl cotransporter, activation of MAP kinases, and inhibition of reactive oxygen species production or DORs may play a role in the mechanisms of biphalin-induced effects. [19; 25-27]\u003c/p\u003e\n\u003cp\u003eThe antinociceptive effect of biphalin has been suggested to have a potential role in the treatment of cancer pain, abdominal pain associated with inflammatory bowel disease, and in neuropathic pain via different routes of administration, such as subcutaneous, intravenous, intrathecal, intracerebroventricular, and intraperitoneal. [9,28,29] Furthermore, recent studies have revealed the beneficial role of biphalin on cell viability and neuroprotection against excitotoxic and ischemic damage by inhibiting protein kinase C-dependent sodium potassium-chloride cotransporter expression in focal brain ischemia by activating downstream survival mitogen-activated protein kinases, and by inhibiting reactive oxygen species production in an opioid receptor-dependent manner, which was challenged by naltrexone. [19,25,30] Biphalin also acts as an immunomodulatory agent by stimulating human T cell proliferation, natural killer cell cytotoxicity in vitro, and interleukin-2 production and diminishes pro- and anti-inflammatory factors in lipopolysaccharide-treated microglial cells. [28, 31]\u003c/p\u003e\n\u003cp\u003eWe proposed that the lack of a significant effect on proliferation of biphalin may be the result of its failure in binding to opioid growth factor receptors (OGFR), since OGFRs bear no resemblance to classical opioid receptors, and blockade of OGFR interaction with naltrexone accelerates cell growth. In the biphalin plus naloxone groups, both proliferation and wound healing were partially increased. We believe this effect was due to the binding of naloxone to OGFRs as a nonselective competitive opioid antagonist. [32]\u003c/p\u003e\n\u003cp\u003eIn our study, there was no statistically significant difference between biphalin-treated cells at 10 \u0026micro;M concentration and control groups on wound healing and cell migration. Biphalin shows approximately 100 times lower affinity to KOR than MOR and DOR. [33] Therefore, it is possible that at higher concentrations of biphalin, KORs are also activated and reverse the healing and migration process.\u003c/p\u003e\n\u003cp\u003eMany studies have demonstrated the cytoprotective and reformative effects of DORs and MORs separately, and their synergistic activation may have strengthened their positive effects. [34] In further experiments, the exact effects of specific opioid receptor subgroup activation on human corneal epithelial cells should be explored with specific opioid peptides. Additionally, the effect of biphalin on the wound-healing process in the cornea and changes in keratinocytes, neural fibers, dendritic cells, the basement membrane, and extracellular matrix elements and their interactions should also be investigated.\u003c/p\u003e\n\u003cp\u003eThe main limitation of the present study was the lack of data on the effect of biphalin on the intracellular signaling mechanisms and cell metabolism. In future experiments, we plan to examine the effects of biphalin and its derivatives on corneal healing in experimental animals, which will allow us to determine receptor-down signaling pathway interactions. A second limitation of our study that must be addressed is the lack of an in vivo animal model. We plan to conduct studies in transgenic mice in order to shed light on the effects of opioids in corneal wound healing.\u003c/p\u003e"},{"header":"Conclusion ","content":"\u003cp\u003eIn the corneal epithelium, biphalin accelerates wound healing and cell migration without affecting cell proliferation. Since the analgesic and antinociceptive effects of biphalin have been demonstrated in previous studies, it can be used in the near future as an analgesic after corneal traumas and surgeries to modulate corneal epithelial wound healing. We also propose that biphalin may become a new treatment alternative not only for corneal traumas but also for other neuropathic pain syndromes affecting the ocular surface, such as herpetic or diabetic neuropathy.\u003c/p\u003e"},{"header":"List of Abbreviations","content":"\u003cp\u003ebovine pituitary extract: BPE; delta opioid receptor: DOR; 406-diamidino-2-phenylindole: DAPI; 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide: MTT; Dulbecco modified eagle medium: DMEM; epidermal growth factor: EGF; fetal bovine serum: FBS; G-protein-coupled opioid receptors: GCPRs; human immortalized corneal epithelial cells: HCECs; kappa-opioid receptor: KOR; keratinocyte serum-free medium: KSFM; mu-opioid receptor: MOR; National Institutes of Health: NIH; phosphate-buffered saline solution: opioid growth factor receptors: OGFR; PBS; real-time polymerase chain reaction:qRT-PCR; relative wound area: RWA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: Since the study was a laboratory- based study not using patients or patient material, ethical approval was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The design of the study and collection, analysis, and interpretation of data and in writing the manuscript was not funded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions: \u003c/strong\u003eAll authors have made substantial contributions to all of the following: (1) the conception and design of the study: OMGT, AS, EY (2) Performed the experiments: EY, KK (3) Analysis and interpretation of data: EY, KK (4) Contributed reagents/materials/analysis tools: AM, AS, EY (5) drafting the article or revising it critically for important intellectual content: OMGT, AS, EY, AM (3) Final approval of the version to be submitted: OMGT, AS, EY, AM, KK.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e The authors thank Ko\u0026ccedil; University Research Center for Translational Medicine (KUTTAM), funded by the Republic of Turkey Ministry of Development, for allowing us to use their services and facilities. The content of the manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the Ministry of Development.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis study was presented in part at 2\u003csup\u003end\u003c/sup\u003e International Cell Death Research Congress 2018, Izmir, Turkey, 1-4 November 2018, and published as congress proceedings MDPI Proceedings 2018,2(25),1552. doi: 10.3390/proceedings2251552\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRamselaar JAM, Boot JP, Van Haeringen NJ, Van Best JA, Oosterhuis JA: \u003cstrong\u003eCorneal epithelial permeability after instillation of ophthalmic solutions containing local anaesthetics and preservatives\u003c/strong\u003e. \u003cem\u003eCurrent eye research \u003c/em\u003e1988, \u003cstrong\u003e7\u003c/strong\u003e(9):947-950.\u003c/li\u003e\n\u003cli\u003eCarney LG, O'Leary DJ, Millodot M: \u003cstrong\u003eEffect of topical anaesthesia on corneal epithelial fragility\u003c/strong\u003e. \u003cem\u003eInternational Ophthalmology \u003c/em\u003e1984, \u003cstrong\u003e7\u003c/strong\u003e(2):71-73.\u003c/li\u003e\n\u003cli\u003ePereira VB, Garcia R, Torricelli AA, Bechara SJ: \u003cstrong\u003eOpioids for Ocular Pain - A Narrative Review\u003c/strong\u003e. \u003cem\u003ePain Physician \u003c/em\u003e2017, \u003cstrong\u003e20\u003c/strong\u003e(5):429-436.\u003c/li\u003e\n\u003cli\u003eBenyamin R, Trescot AM, Datta S, Buenaventura R, Adlaka R, Sehgal N, Glaser SE, Vallejo R: \u003cstrong\u003eOpioid complications and side effects\u003c/strong\u003e. \u003cem\u003ePain Physician \u003c/em\u003e2008, \u003cstrong\u003e11\u003c/strong\u003e(2 Suppl):S105-120.\u003c/li\u003e\n\u003cli\u003eTamura Y, Monden M, Shintani M, Kawai A, Shiomi H: \u003cstrong\u003eNeuroprotective effects of hibernation-regulating substances against low-temperature-induced cell death in cultured hamster hippocampal neurons\u003c/strong\u003e. \u003cem\u003eBrain Research \u003c/em\u003e2006, \u003cstrong\u003e1108\u003c/strong\u003e(1):107-116.\u003c/li\u003e\n\u003cli\u003eHusain S: \u003cstrong\u003eOpioid Receptors: Methods for Detection and Their Modes of Actions in the Eye\u003c/strong\u003e. 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\u003cem\u003ePeptides \u003c/em\u003e1982, \u003cstrong\u003e3\u003c/strong\u003e(4):697-700.\u003c/li\u003e\n\u003cli\u003eKosson D, Klinowiecka A, Kosson P, Bonney I, Carr DB, Mayzner-Zawadzka E, Lipkowski AW: \u003cstrong\u003eIntrathecal antinociceptive interaction between the NMDA antagonist ketamine and the opioids, morphine and biphalin\u003c/strong\u003e. \u003cem\u003eEuropean Journal of Pain \u003c/em\u003e2008, \u003cstrong\u003e12\u003c/strong\u003e(5):611-616.\u003c/li\u003e\n\u003cli\u003eYang L, Shah K, Wang H, Karamyan VT, Abbruscato TJ: \u003cstrong\u003eCharacterization of Neuroprotective Effects of Biphalin, an Opioid Receptor Agonist, in a Model of Focal Brain Ischemia\u003c/strong\u003e. \u003cem\u003eJournal of Pharmacology and Experimental Therapeutics \u003c/em\u003e2011, \u003cstrong\u003e339\u003c/strong\u003e(2):499-508.\u003c/li\u003e\n\u003cli\u003eYang L, Islam MR, Karamyan VT, Abbruscato TJ. \u003cstrong\u003eIn vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment.\u003c/strong\u003e Brain Research 2015, 1609:1-11.\u003c/li\u003e\n\u003cli\u003eChao D, Bazzy-Asaad A, Balboni G, Xia Y. \u003cstrong\u003edelta-, but not mu-, opioid receptor stabilizes K(+) homeostasis by reducing Ca(2+) influx in the cortex during acute hypoxia.\u003c/strong\u003e Journal of Cell Physiology 2007, 212(1):60-67.\u003c/li\u003e\n\u003cli\u003eLesniak A, Bochynska-Czyz M, Sacharczuk M, Benhye S, Misicka A, Bujalska-Zadrozny M, Lipkowski AW: \u003cstrong\u003eBiphalin preferentially recruits peripheral opioid receptors to facilitate analgesia in a mouse model of cancer pain - A comparison with morphine\u003c/strong\u003e. \u003cem\u003eEuropean Journal of Pharmaceutical Sciences \u003c/em\u003e2016, \u003cstrong\u003e89\u003c/strong\u003e:39-49.\u003c/li\u003e\n\u003cli\u003ePopiolek-Barczyk K, Piotrowska A, Makuch W, Mika J: \u003cstrong\u003eBiphalin, a Dimeric Enkephalin, Alleviates LPS-Induced Activation in Rat Primary Microglial Cultures in Opioid Receptor-Dependent and Receptor-Independent Manners\u003c/strong\u003e. \u003cem\u003eNeural plasticity \u003c/em\u003e2017, \u003cstrong\u003e2017\u003c/strong\u003e:1-19.\u003c/li\u003e\n\u003cli\u003eKawalec M, Kowalczyk JE, Beresewicz M, Lipkowski AW, Zablocka B: \u003cstrong\u003eNeuroprotective Potential of Biphalin, Multireceptor Opioid Peptide, Against Excitotoxic Injury in Hippocampal Organotypic Culture\u003c/strong\u003e. \u003cem\u003eNeurochemical Research \u003c/em\u003e2011, \u003cstrong\u003e36\u003c/strong\u003e(11):2091-2095.\u003c/li\u003e\n\u003cli\u003eMehrotra S, Prajapati RK, Haq W, Singh VK: \u003cstrong\u003eImmunomodulation by biphalin, dimeric synthetic opioid peptide, and its analog\u003c/strong\u003e. \u003cem\u003eImmunopharmacology and Immunotoxicology \u003c/em\u003e2002, \u003cstrong\u003e24\u003c/strong\u003e(1):83-96.\u003c/li\u003e\n\u003cli\u003eZagon IS, Jenkins JB, Sassani JW, Wylie JD, Ruth TB, Fry JL, Lang CM, McLaughlin PJ: \u003cstrong\u003eNaltrexone, an Opioid Antagonist, Facilitates Reepithelialization of the Cornea in Diabetic Rat\u003c/strong\u003e. \u003cem\u003eDiabetes \u003c/em\u003e2002, \u003cstrong\u003e51\u003c/strong\u003e(10):3055-3062.\u003c/li\u003e\n\u003cli\u003eLipkowski AW, Konecka AM, Sroczynska I, Przewlocki R, Stala L, Tam SW: \u003cstrong\u003eBivalent opioid peptide analogues with reduced distances between pharmacophores\u003c/strong\u003e. \u003cem\u003eLife Sciences \u003c/em\u003e1987, \u003cstrong\u003e40\u003c/strong\u003e(23):2283-2288.\u003c/li\u003e\n\u003cli\u003eHe X, Sandhu HK, Yang Y, Hua F, Belser N, Kim DH, Xia Y: \u003cstrong\u003eNeuroprotection against hypoxia/ischemia: \u0026delta;-opioid receptor-mediated cellular/molecular events\u003c/strong\u003e. \u003cem\u003eCellular and Molecular Life Sciences \u003c/em\u003e2012, \u003cstrong\u003e70\u003c/strong\u003e(13):2291-2303.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Titles and legends for supplementary files","content":"\u003cp\u003e\u003cstrong\u003eSupplement 1.\u003c/strong\u003e Video of \u003cem\u003ein vitro\u003c/em\u003e scratch assay model showing the effect of 1 \u0026micro;M biphalin on relative wound healing area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplement 2.\u003c/strong\u003e Video of \u003cem\u003ein vitro\u003c/em\u003e scratch assay model showing the effect of vehicle on relative wound healing area.\u003c/p\u003e"},{"header":"Table","content":"\u003cp style=\"margin-left: .5in; text-indent: -.5in; line-height: 200%;\"\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e The primers of genes used in the study\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\" width=\"616\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 104.65pt; border-top: solid black 2.25pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%;\"\u003eGene\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80.8pt; border-top: solid black 2.25pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%;\"\u003eSequence\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border-top: solid black 2.25pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 104.65pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" rowspan=\"2\" width=\"140\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eGAPDH\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eF\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-ACAACTTTGGTATCGTGGAAGG-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.8pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-GCCATCACGCCACAGTTTC-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 104.65pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" rowspan=\"2\" width=\"140\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eOPRD1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eF\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-CGTCCGGTACACTAAGATGAAGA-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.8pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-GCCACGTCTCCATCAGGTA-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 104.65pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" rowspan=\"2\" width=\"140\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eOPRK1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eF\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-ATCATCACGGCGGTCTACTC-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 80.8pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; border-bottom: solid black 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-ACTCTGAAAGGGCATGGTTGTA-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 4.0pt;\"\u003e\n\u003ctd style=\"width: 104.65pt; border: none; border-bottom: solid black 2.25pt; padding: 0in 5.4pt 0in 5.4pt; height: 4.0pt;\" rowspan=\"2\" width=\"140\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eOPRM1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 4.0pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eF\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 4.0pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-CAGTGCTCATCATTACCGTGT-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 4.0pt;\"\u003e\n\u003ctd style=\"width: 80.8pt; border: none; border-bottom: solid black 2.25pt; padding: 0in 5.4pt 0in 5.4pt; height: 4.0pt;\" width=\"108\"\u003e\n\u003cp style=\"text-align: center; text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003eR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.4pt; border: none; border-bottom: solid black 2.25pt; padding: 0in 5.4pt 0in 5.4pt; height: 4.0pt;\" width=\"369\"\u003e\n\u003cp style=\"text-indent: .25in; line-height: 200%;\"\u003e\u003cspan style=\"line-height: 200%;\"\u003e5\u0026rsquo;-CTGGGATTGTAACCAAGGCTTTA-3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"synthetic opioid peptide, corneal wound healing, opioid receptors, human corneal epithelial cell culture","lastPublishedDoi":"10.21203/rs.2.11471/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.11471/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Analgesic drugs, including nonselective opioids and non-steroidal anti-inflammatory drugs, should be used with great precautions to relieve pain after physical damage of the corneal epithelium because of their unfavorable effects on the wound-healing process. Biphalin is a synthetic opioid peptide that has been demonstrated to possesses a strong analgesic effect on rodents. The purpose of this study is to investigate the effects of biphalin on human corneal epithelium wound healing.\u003c/p\u003e\u003cp\u003eMethods: An immortalized human corneal epithelial cell (HCEC) culture was used to test the effects of biphalin on wound healing. The toxicity of biphalin in various concentrations was measured with the MTT assay. The effect of 1 µM and 10 µM biphalin were tested on wound closure in an in vitro scratch assay of HCECs and for cell migration and proliferation separately. Naloxone, a non-selective competitive antagonist of opioid receptors, was also used to inhibit the effects of biphalin in all experiments.\u003c/p\u003e\u003cp\u003eResults: Biphalin did not cause any toxic effect on HCECs in concentrations lower than 100 µM at various incubation time points. Biphalin increased the wound closure process significantly at 1 µM concentration in an in vitro scratch assay of HCECs (p \u0026lt; 0.05). It also increased the migration of HCECs significantly (p \u0026lt; 0.01). There was no significant difference between biphalin and control groups of HCECs in the Ki67 proliferation assay.\u003c/p\u003e\u003cp\u003eConclusion: Biphalin, a synthetic opioid peptide, has a potential role as a novel topical analgesic agent that promotes corneal epithelial wound healing. This role should be evaluated in further in vivo and clinical studies.\u003c/p\u003e","manuscriptTitle":"The opioid peptide biphalin modulates human corneal epithelial wound healing in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-03-31 16:26:02","doi":"10.21203/rs.2.11471/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2019-07-18 11:17:56","doi":"10.21203/rs.2.11471/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ac669d2-f177-4161-b72e-454fc3ecb4fc","owner":[],"postedDate":"March 31st, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":17280,"name":"Ophthalmology"}],"tags":[],"updatedAt":"2020-07-11T13:46:50+00:00","versionOfRecord":[],"versionCreatedAt":"2020-03-31 16:26:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-2344","version":["v2"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}