Mechanisms Of Paclitaxel-induced Macular Edema And Alleviation Effect By Carbonic Anhydrase Inhibitor

Objectives:Paclitaxel(PTX)is a natural phytochemical drug approved by FDAof the United States,which is a high efficiency and low toxicity broad-spectrum anticancer drug.Due to the poor water solubility of PTX,it is necessary to add solvent-increasing polyoxyethylene castor oil and anhydrous ethanol.PTX modified preparations used in clinical practice include paclitaxel injection,docetaxel injection,PTX liposome for injection,paclitaxel for injection(Albumin binding type),etc.Common side reactions of PTX drugs mainly include bone marrow suppression,allergic reaction,gastrointestinal reaction,hand and foot numbness and liver injury.A series of related cases had been reported and raised tremendous clinical attention is high.In recent years,PTX drugs have been reported to cause ocular adverse reactions,such as decreased vision,dry eye,lacrimal duct obstruction,conjunctivitis,glaucoma,optic neuropathy,and blindness caused by cisplatin.However,a latest case of macular edema caused by PTX treatment has been reported one after another,which is a rare ocular complication related to PTX use,which brings great inconvenience to patients’ life.There are few studies on PTXinduced macular edema and the mechanism is unknown.The main pathogenic mechanism is only at the stage of speculation,which is still confirmed and needs further study.The mechanism of macular edema induced by PTX was discussed and the therapeutic effect of CAI on PTX-induced macular edema was elucidated.Methods:1.In vivo experimentsC57BL/6J mice were divided into Control group and PTX group.Mice in Control group were treated with intraperitoneal injection of normal saline.and mice in PTX group were given intraperitoneal injection of PTX(5 mg/kg).Injections are given once a week for four weeks.ERG and HE staining were used to evaluate the effects of drugs on visual electrophysiology and retinal tissue structure.The vascular morphology of mice was observed by FFA,and the retinal blood vessels of each group were analyzed by retinal smear and CD31 staining.2.In vitro experimentsARPE cells and Müller cells were treated with different concentrations of PTX(0 mg/L,0.005mg/L,0.05 mg/L,0.5 mg/L,5 mg/L)for a certain period of time(12 h,24 h,36 h,48 h,72 h).CCK8 assay was used to detect the effects of different concentrations of PTX on proliferation of ARPE cells and Müller cells at a preset time.Flow cytometry was used to determine the effect of drugs on apoptosis of ARPE cells and Müller cells at different concentrations and time points.The morphological changes of ARPE cells and Müller cells were observed by immunofluorescence.The cycle arrest of ARPE cells and Muller cells was detected by flow cytometry.ARPE cells and Müller cells were treated with 0.05 mg/ml PTX for 24 h,and Control group was set at the same time.Using WB and qRT-PCR and other methods were used to detect CAⅩⅣ and VEGF,IL-6,TNF-α,MCP-1.ARPE cells and Müller cells were treated with 0.05 mg/mL PTX for 24 h,and then cultured in normal medium for 24 h,36 h,48 h and 72 h.The expression of inflammatory factors was detected by WB.In addition,after Müller cells were treated with 0.05mg/ml PTX for 24 h,the expression of AQP4 was detected by WB.An external barrier model was established,when ARPE cells polarization was formed,medium containing 0 mg/ mL and 0.05 mg/ml PTX was added into the upper chamber of transwell in the way of fluid exchange,and the changes of transepithelial resistance values were measured at 3 h,6 h,12 h and 24 h after administration,respectively.Barrier function related proteins ZO-1 and occludin were detected by WB and qRT-PCR.ARPE cells and Müller cells were divided into control group,PTX group and PTX+CAI group.The changes of inflammatory factors and AQP4 expression were detected by WB and qRT-PCR.3.In vivo validationC57BL/6J mice were divided into three groups: Control group,PTX group and PTX+CAI group.Mice were injected intraperitoneally with PTX(5 mg/kg),and CAI group was injected with CAI(25 mg/kg)half an hour after PTX injection.Injections are given once a week for four weeks.ERG was used to evaluate the effects of drugs on visual electrophysiology.RNA and protein were extracted from the whole retina of mice,and the expression of inflammatory factors,CAⅩⅣ and AQP4 were detected by WB and qRT-PCR.Results:1.The results of ERG in mice showed that PTX treated mice had different degrees of reduction in light adaptation 3.0 ERG,dark adaptation 3.0 ERG and dark-adapted 3.0 oscillation potential(P<0.05).At the same time,no abnormality and leakage of blood vessels were found in fundus angiography,and no abnormality was observed in CD31 staining of retina.Finally,HE results showed that PTX changed the total retinal thickness and the total retinal thickness was slightly decreased,suggesting that PTX caused certain damage to the retina of mice.2.PTX affected the function and morphology of ARPE cells and Müller cells.PTX reduced the proliferation ability of ARPE cells and Müller cells and increased apoptosis,and the proliferation ability and apoptosis were correlated with drug concentration.PTX affected the cell cycle and blocked ARPE cells and Müller cells in G2-M phase.PTX induced significant changes in cell morphology.Normal ARPE cells were fusiform or aberrant,while in the drug group,the number of cells was relatively reduced and the shape tended to be round.Normal Müller cells were elongated fibrous.After drug stimulation,the cells decreased and tended to be round.3.PTX affects the expression of CAⅩⅣ and VEGF.QRT-PCR results showed that PTX significantly promoted the expression of CAⅩⅣ and the expression of VEGF was decreased(P<0.05).WB results also confirmed that the protein expression of CAⅩⅣ was up-regulated and the protein expression of VEGF was down-regulated compared with the Control group(P<0.05).4.PTX induces high expression of inflammatory factors in cells,but this process is transient and can be alleviated by stopping drug stimulation.The addition of CAI also reduced the expression of inflammatory factors.qRT-PCR results showed that PTX could promote the expression of inflammatory factors(IL-6,TNF-α,MCP-1)in ARPE cells and Müller cells(P<0.05).WB also confirmed upregulation of inflammatory cytokine protein expression after dosing stimulation(P<0.05).WB results showed that the expression of inflammatory factors decreased after drug withdrawal compared with that after drug stimulation(P<0.05),about36 h or 48 h after the cessation of drug stimulation,the expression of inflammatory factors tended to control group.When CAI was added without stopping the drug stimulation,qRT-PCR results showed that the expression of inflammatory factors was significantly reduced(P<0.05),WB also confirmed that the protein levels of inflammatory factors were down-regulated and similar to Control group(P<0.05).5.WB results showed that PTX significantly up-regulated AQP4 expression in Müller cells(P<0.05).PTX reduced the transepithelial resistance of ARPE cells,and the degree of reduction change was correlated with drug concentration.WB results showed that PTX reduced the expression of tight junction proteins ZO-1 and Occludin(P<0.05),qRT-PCR results also confirmed.The results of ERG in mice showed that PTX treatment mice had different degrees of reduction in light-adapted 3.0 ERG,dark-adapted 3.0 ERG and dark-adapted 3.0 oscillatory potentials(P<0.05).In PTX+CAI group,these three groups were higher than PTX group(P<0.05).In addition,qRT-PCR results showed that the expression of AQP4 in Müller cells was down-regulated after adding CAI(P<0.05),WB results were the same as qRT-PCR results.In vivo,qRT-PCR results showed that AQP4 and inflammatory factors(IL-6,TNF-α,MCP-1)were increased in PTX group compared with control group(P<0.05),AQP4 and inflammatory factors(IL-6,TNF-α,MCP-1)were down-regulated in PTX+CAI group(P<0.05).Conclusions:This study shows that PTX affects the function and morphology of ARPE cells and Müller cells,and PTX could lead to transient high expression of inflammatory factors.Our in vivo and in vitro experimental results suggested that PTX-induced macular edema was mainly caused by Müller cell swelling.PTX also slightly damages the blood-retinal barrier.CAI regulates water conduction by reducing inflammation,inhibiting AQP4,inhibiting membrane-bound CAexpression,and enhancing subretinal fluid absorption,and causes resultant edema.CAI is a potential treatment for PTX-induced macular edema.The treatment of PTX-induced macular edema by CAI can be alleviated by regulating water channel and enhancing the absorption of subretinal fluid.