Study on the Protective Effect of EGCG Against the Cytotoxicity Induced by Topical Anesthetic Proparacaine Hydrochloride

Purpose: To identify the apoptosis caused by Proparacaine hydrochloride (PPC), a topical anesthesia, applied to conjunctival cell lines and determine whether pigallocatechin-gallate (EGCG), has protective effects on. Methods: The conjunctival cell lines were treated with 0.5% of Alcaine$^{(R)}$, 0.5% of PPC and 0.01% of Benzalkonium chloride (BAC) for 15 minutes, respectively in order to investigate the effects of topical anesthesia on cells, and followed by cultured for 12 and 24 hours. The recovery effects were investigated by measuring level of cellular proliferation inhibiting using MTT assay and LDH assay. The conjunctival cell lines were pre-treated with EGCG $10{\mu}M$ for 3 hrs and post-treated with 0.5% PPC for 15 mins in order to investigate whether EGCG has protective effects, flow cytometry were performed in order to observe apoptosis. Results: A result of the additional culture of 12 and 24 hours and again immediately after the treatment for 15 minutes 0.5% of Alcaine$^{(R)}$, 0.5% of PPC, the 0.01% of BAC, cell viability was not increased in all groups (p<0.05). The cell viabilities were higher than in cells 3 hours post-treated with $10{\mu}M$ of EGCG and pre-treated PPC 0.5% (68.2%), compared to cells ($32.2{\pm}2.0%$) treated only with 0.5% of PPC. PPC 0.5% also induced apoptosis in the treated group was reduced by the addition of EGCG. Conclusions: It is considered that the EGCG has cell protective effects when it is added to PPC, a topical anesthesia, by improving cell viability and inhibiting apoptosis.

점안마취제 성분인 Proparacaine Hydrochloride의 세포독성에 대한 Epigallocatechin-Gallate의 효과

목적: 점안마취제 성분인 proparacaine hydrochloride (PPC)에 의한 결막세포주의 세포자연사와 이에 대한 epigallocatechingallate(EGCG)의 보호효과를 알아보고자 하였다. 방법: 동일한 조건으로 배양된 결막세포주에 Alcaine$^{(R)}$ 0.5%, PPC 0.5%, 그리고 0.01% benzalkonium chloride (BAC)을 각각 15분간 처리한 후, 추가적으로 12시간과 24시간 배양한 후 세 군으로 나누어 MTT assay와 LDH assay를 실시하였다. 배양된 결막세포주에 EGCG를 $10{\mu}M$의 농도로 3시간 동안 전 처리한 후 0.5% PPC를 15분간 추가 배양한 다음 유세포분석기를 이용한 세포자연사 정도를 측정하였다. 결과: Alcaine$^{(R)}$ 0.5%, PPC 0.5%, BAC 0.01%를 15분 동안 처리한 직후와 다시 12시간과 24시간 추가 배양한 결과 모든 군에서 세포생존율이 증가하지 않았다(p<0.05). 0.5% PPC 단독처리군($32.2{\pm}2.0%$)에 비해 $10{\mu}M$의 EGCG 3시간 전 처리하고 PPC 0.5% 후 처리한 군(68.2%)에서 세포 생존율은 더 높아졌다. 또한 PPC 0.5% 처리한 군에서 유도된 세포자연사는 EGCG 첨가로 감소되었다. 결론: 점안마취제인 PPC에 EGCG를 전처리함으로써 세포생존율을 증가시키고, 세포자연사를 억제함으로서 EGCG는 세포보호 효과가 있는 것으로 나타났다.