Anti-fibrosis Potential Of Pirarubicin Via Inducing Apoptotic And Autophagic Cell Death In Rabbit Conjunctiva

Purpose:This study aimed to create a conjunctival fibro-vascular model in the rabbit and to investigate the potential efficacy of pirarubicin?THP?in modulating rabbit conjunctival fibrosis both in vitro and in vivo and characterized the underlying mechanisms.Methods:This study included three parts.In the first part,limbal stem cell excision?LSCE?in combination with alkali burn was performed on the eyes of 9 New Zealand White rabbits to establish a model of limbal deficiency and conjunctival fibro-vascular invasion.This step also determined the modeling endpoint when the conjunctival fibro-vascular lesion was most similar to human pterygium in morphological and immunohistochemical traits?ie,Vimentin,?-SMA,TGF-?1/2,collagen-I expressions?.In the second part,primary rabbit conjunctival fibroblasts?RCF?were cultured and treated with THP or mitomycin C?MMC?for 5 min.The cells were cultured for another 24 h or 48 h followed by assaying for cell viability,cell cycle distribution,apoptotic and autophagic related pathways.In the third part,both eyes of another 9rabbits were molded.After 3 months,the molded fibro-vascular tissue was excised combined with topical subconjunctival 5-min exposure to THP or DMEM compared with MMC intraoperatively.The postoperative fibrosis recurrence,side-effects of drugs and the expressions of apoptosis and autophagy markers were evaluated by immunohistochemistry.THP toxicity on cornea,heart,liver and kidney was further evaluated by using the C57BL/6 mice model.Results:All modeled rabbits developed conjunctival fibro-vascular lesions,which were similar to human recurrent pterygium?HRP?,especially the 3-month model in terms of the triangle shape,hypervascularity and abundant fibroblasts deposition.THP showed the inhibitory effect on the growth of RCF with an IC₅₀ of 0.01 mg/m L,exhibiting greater efficacy than MMC(IC₅₀value of 0.2 mg/m L).Both THP and MMC inhibited RCF proliferation and arrested cell cycle at the G0/G1 phase.In particular,0.005 mg/m L THP remarkably promoted RCF autophagy by upregulating the levels of Beclin 1,Atg 5/12 conjugate,and LC3B,whereas,0.01 mg/m L THP significantly triggered a cascade of mitochondrial-associated RCF apoptosis.Both THP and MMC inhibited HCECs proliferation,induced cell apoptosis,and arrested cell cycle at the G2/M phase.Similar to 0.2 mg/m L MMC,both 0.005 mg/m L and 0.01 mg/m L THP attenuated postoperative conjunctival fibrosis in the rabbit models compared with the recurrence rate of 44.4%?4/9?in the vehicle group;0.005 mg/m L THP preferentially enhanced autophagy while causing less side effects.During the wound healing process of mice cornea,0.005 mg/m L THP showed significantly smaller healing area than the vehicle in the first day and healed completely in the second day.0.01 mg/m L THP and0.2 mg/m L MMC healed significantly slower than the vehicle in the early four days post-operation and completely healed at the 6-7^th day.All three drug treated groups of0.005 mg/m L THP,0.01 mg/m L THP,and 0.2 mg/m L MMC did not destroy the structure of cornea,heart,liver and kidney,but 0.01 mg/m L THP and 0.2 mg/m L MMC showed infiltration of inflammatory cells in the cornea stroma.Conclusion:Conjunctival fibro-vascular lesions were developed in the rabbit eyes by applying LSCE in combination with alkali burn and this model might be useful in both basic and clinical research of conjunctival fibrosis.THP exerted its anti-fibrotic action by modulating autophagy in RCF,inducing cell cycle arrest and mitochondrial-mediated apoptosis.THP at the dose of 0.005 mg/m L potentially safely prevented postoperative conjunctival fibrosis in animal model.Clinical study is warranted in the future.