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After physical or surgical damage of corneal epithelium, most of analgesic drugs, like non-selective opioid agonists and non-steroid anti-inflammatory drugs, cannot be used because of their negative effects on wound healing process. Biphalin is selective µ and Δ opioid receptor agonist which has proven analgesic effects on rodents. Our purpose of study is finding effects of biphalin on wound healing of corneal epithelium. We used primary culture of human corneal epithelial cells (HCECs) for examining effects of biphalin on wound healing. Firstly, we measured toxicity of Biphalin in various concentrations with MTT assay and we showed biphalin has no toxic effects on HCECs in lower concentrations than 100 µM in various incubation times. After MTT assay, we administered 1 µM and 10 µM biphalin at in vitro scratch assay of HCECs, biphalin increased wound closure process significantly at 1 µM concentration (p < 0.05). Then we tested effects of biphalin on cell migration and proliferation separately. Bifalin increased migration of HCECs significantly (p < 0.01) at transwell migration assay. But we did not observe any significant difference between groups in Ki67 proliferation assay. In all these experiments, we also used naloxone to inhibiting effects of biphalin. In biphalin plus naloxone groups, effects of biphalin decrease partially. Our study results suggest, biphalin has positive effects on epithelial wound healing via opioid receptors. This effect because of increased migration of HCECs under influence of biphalin. With these findings, we propose biphalin as a new analgesic agent for post-surgical and post-traumatic care of corneal epithelial …