局部应用上皮钠通道阻滞剂对干眼兔泪液分泌的影响
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摘要
【目的】旨在通过关闭结膜上皮钠通道,抑制上皮钠离子吸收,促进泪液分泌,以期达到治疗东莨菪碱诱导的干眼。【方法】选取体质量约2.0～2.5 kg的新西兰兔24只,实验组(8只)皮下注射东莨菪碱,一天4次,连续12 d,造干眼症模型;对照组(8只)皮下注射等量溶剂生理盐水;治疗组(8只)先经过12 d的皮下注射东莨菪碱造成干眼症后,右眼局部应用上皮钠通道抑制剂阿米洛利(100 mmol/L),左眼局部应用等量溶剂生理盐水,一天3次,连续9 d。应用荧光定量PCR检测上皮钠通道ENaCα和ENaCγ亚基的表达,短路电流技术检测钠电流开放程度。比较对照组、实验组以及治疗组3组中ENaCα和ENaCγ基因表达差异,角膜荧光素钠染色以及泪液分泌情况。【结果】皮下给药12 d后,对照组泪液分泌实验结果为(17.00±0.37)mm/5 min,实验组泪液分泌实验结果为(4.42±1.34)mm/5 min(P<0.001,n=8),治疗组泪液分泌实验结果(14.25±0.54)mm/5 min(P>0.05,n=8)。短路电流结果显示:对照组中的结膜钠电流大小为(5.72±0.35)μA/cm2,实验组结膜钠电流大小为(12.24±0.54)μA/cm2(P<0.001,n=8);经过阿米洛利点眼治疗后,治疗组兔结膜钠电流大小降至(4.00±0.61)μA/cm2(P>0.05,n=8),与对照组对比无统计学差异。荧光定量PCR结果显示:与对照组相比,在实验组中结膜上皮钠通道ENaCα和ENaCγ亚基以及炎症因子IL-1β表达上调(P<0.01),经过局部应用上皮钠通道阻滞剂阿米洛利点眼后,治疗组的IL-1β、ENaCα、ENaCγ和对照组无统计学差异(P>0.05,n=8)。【结论】局部滴用阿米洛利眼药水,可通过关闭结膜上皮钠离子通道,促进泪液分泌,改善干眼症状,有望成为新一代治疗干眼症的药物。
【Objective】To test the hypothesis that inhibiting sodium absorption via the epithelial sodium channel(ENaC) will increase ocular hydration and cure the rabbit′ s dry eye induced by scopolamine. 【Methods】In the experiment,24 New Zealand rabbits weighing about 2.0-2.5 kg were divided into 3 groups:tested group(8 rabbits),control group(8 rabbits),and treatment group(8 rabbits). For the rabbits in the tested group,they were given subcutaneous injection of scopolamine 4 times a day for 12 consecutive days to induce the dry eye. For the rabbits in the control group,they accepted subcutaneous injection of saline solution. For the rabbits in the treatment group,they were firstly given the same treatment as the rabbits in the tested group. Then,the local regions in their right eyes received the application of 100 mmol/L amiloride(a sodium channel inhibitor). Meanwhile,the local regions in their left eyes received the application of equivalent saline solution. We detected ENAC α and ENAC γ subunits of epithelial sodium channel in conjunctival epithelium by fluorescence quantitative PCR and detected the opening of epithelial sodium channel by shortcircuit current technology. Finally,we compared the ENaC α and ENaC γ gene expression,corneal fluorescein sodium staining and tear secretion among the 3 groups. Fluorescence quantitative PCR was used to detect ENaC α and ENaC γsubunits of epithelial sodium channel in conjunctival epithelium and short-circuit current was used to detect the opening of epithelial sodium channel. The ENaC α and ENaC γ gene expression,corneal fluorescein sodium staining and tear secretion(immerged length)were compared among 3 groups.【Results】 The results showed that the quantity of tear secretion was(17.00 ± 0.37)mm for the control group,(4.42 ± 1.34)mm for the tested group(P<0.001 vs Control,n=8) and(14.25 ± 0.54)mm for the treatment group(P>0.05 vs Control,n=8). The results of short-circuit current detection showed that the sodium current was(5.72 ± 0.35)μA/cm2 in normal model and(12.24 ± 0.54)μA/cm2 in dry eye model(P<0.001). After Amiloride treatment,the sodium current decreased to(4.00 ± 0.61)μA/cm2(P>0.05)and there was no statistical difference compared with the normal group. According to the results of fluorescence quantitative PCR,the expression of ENaC α and ENaC γ subunits and IL compared with that in normal group. After topical amiloride application,there were no statistical differences in inflammatory cytokines IL-1 β,ENaC α,and ENaC γ between the normal group and treatment group(P>0.05,n=8).After treatment,tear secretion increased(P<0.001),and ocular surface staining improved significantly.【Conclusion】Topical application of amiloride increase the quantity of preocular tears owing to inhibition of conjunctival sodium channels and could provide an effective new therapy for chronic dry eye.
【Objective】To test the hypothesis that inhibiting sodium absorption via the epithelial sodium channel(ENaC) will increase ocular hydration and cure the rabbit′ s dry eye induced by scopolamine. 【Methods】In the experiment,24 New Zealand rabbits weighing about 2.0-2.5 kg were divided into 3 groups:tested group(8 rabbits),control group(8 rabbits),and treatment group(8 rabbits). For the rabbits in the tested group,they were given subcutaneous injection of scopolamine 4 times a day for 12 consecutive days to induce the dry eye. For the rabbits in the control group,they accepted subcutaneous injection of saline solution. For the rabbits in the treatment group,they were firstly given the same treatment as the rabbits in the tested group. Then,the local regions in their right eyes received the application of 100 mmol/L amiloride(a sodium channel inhibitor). Meanwhile,the local regions in their left eyes received the application of equivalent saline solution. We detected ENAC α and ENAC γ subunits of epithelial sodium channel in conjunctival epithelium by fluorescence quantitative PCR and detected the opening of epithelial sodium channel by shortcircuit current technology. Finally,we compared the ENaC α and ENaC γ gene expression,corneal fluorescein sodium staining and tear secretion among the 3 groups. Fluorescence quantitative PCR was used to detect ENaC α and ENaC γsubunits of epithelial sodium channel in conjunctival epithelium and short-circuit current was used to detect the opening of epithelial sodium channel. The ENaC α and ENaC γ gene expression,corneal fluorescein sodium staining and tear secretion(immerged length)were compared among 3 groups.【Results】 The results showed that the quantity of tear secretion was(17.00 ± 0.37)mm for the control group,(4.42 ± 1.34)mm for the tested group(P<0.001 vs Control,n=8) and(14.25 ± 0.54)mm for the treatment group(P>0.05 vs Control,n=8). The results of short-circuit current detection showed that the sodium current was(5.72 ± 0.35)μA/cm2 in normal model and(12.24 ± 0.54)μA/cm2 in dry eye model(P<0.001). After Amiloride treatment,the sodium current decreased to(4.00 ± 0.61)μA/cm2(P>0.05)and there was no statistical difference compared with the normal group. According to the results of fluorescence quantitative PCR,the expression of ENaC α and ENaC γ subunits and IL compared with that in normal group. After topical amiloride application,there were no statistical differences in inflammatory cytokines IL-1 β,ENaC α,and ENaC γ between the normal group and treatment group(P>0.05,n=8).After treatment,tear secretion increased(P<0.001),and ocular surface staining improved significantly.【Conclusion】Topical application of amiloride increase the quantity of preocular tears owing to inhibition of conjunctival sodium channels and could provide an effective new therapy for chronic dry eye.
引文
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[2] Braun RJ,King-Smith PE,Begley CG,et al.Dynamics and function of the tear film in relation tothe blink cycle[J]. Prog Retin Eye Res,2015,45(10):132-164.
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[6] Li L, Wang NL, Gong JY, et al. Infantilecholestasis caused by CFTR mutation:case reportand literature review[J]. Zhonghua Er Ke Za Zhi,2016,54(11):851-855.
[7] Feraille E,Dizin E. Coordinated control of ENaCand Na+,K+-ATPase in renal collecting duct[J]. JAm Soc Nephrol,2016,27(9):2554-2563.
[8] Pitk?nen O. Lung epithelial ion transport in neonatallung disease[J]. Biol Neonate,2001,80(Suppl 1):14-17.
[9] Morris LM, DeGagne JM, Kempton JB,et al.Mouse middle ear ion homeostasis channels andintercellular junctions[J]. PLoS One,2012,7(6):e39004.
[10]Li Y,Marcoux MO,Gineste M,et al. Expression ofwater and ion transporters in tracheal aspirates fromneonates with respiratory distress[J]. Acta Paediatr,2009,98(11):1729-1737.
[11]Shi R, Xiao ZT, Zheng YJ, et al. Naringeninregulates CFTR activation and expression in airwayepithelial cells[J]. Cell Physiol Biochem,2017,44(3):1146-1160.
[12]Cao X, Huang J, Zhang G, et al. Functionalexpression of G protein-coupled receptor 30 inimmature rat epididymal epithelium[J]. Cell BiolInt,2017,41(2):134-146.
[13]Sun Q,Huang J,Yue YJ,et al. Hydrogen sulfidefacilitates vaginal lubrication by activation ofepithelial ATP-sensitive K+channels and cysticfibrosis transmembrane conductance regulator[J]. JSex Med,2016,13(5):798-807.
[14]Zhou H,Liu J,Zhou C,et al. In vivo simultaneoustranscriptional activation of multiple genes in thebrain using CRISPR-dCas9-activator transgenicmice[J]. Nat Neurosci,2018,21(3):440-446.
[15]Viau S,Maire MA,Pasquis B,et al. Time courseof ocular surface and lacrimal gland changes in anew scopolamine-induced dry eye model[J].Graefes Arch Clin Exp Ophthalmol,2008,246(6):857-867.
[16]Chu YY,Hua N,Ru YS,et al. The protection ofhydrogen-rich saline on a rat dry eye model inducedby scopolamine hydrobromide[J]. Zhonghua YanKe Za Zhi,2017,53(5):363-372.
[17]Henriksson JT,De Paiva CS,Farley W,et al.Morphologic alterations of the palpebral conjunctivalepithelium in a dry eye model[J]. Cornea,2013,32(4):483-490.
[18]Epstein SP, Ahdoot M, Marcus E, et al.Comparative toxicity of preservatives onimmortalized corneal and conjunctival epithelial cells[J]. J Ocul Pharmacol Ther,2009,25(2):113-119.
[19]Ayaki M,Yaguchi S,Shin KS,et al. Cytotoxicity ofophthalmic solutions with and without preservativesto human corneal endothelial cells,epithelial cellsand conjunctival epithelial cells[J]. Clin ExpOphthalmol,2008,36(6):553-559.
[20]Oh JY, Kim MK, Shin KS,et al. Efficientcryopreservative conditions for cultivated limbal andconjunctival epithelial cells[J]. Cornea,2007,26(7):840-846.
[21]Zhang H,Wu H,Yang J,et al. Sodium perbarateand benzalkonium chloride induce DNA damage inChang conjunctival epithelial cells[J]. Cutan OculToxicol,2017,36(4):336-342.
[22]Milner MS, Beckman KA, Luchs JI,et al.Dysfunctional tear syndrome:dry eye disease andassociated tear film disorders-new strategies fordiagnosis and treatment[J]. Curr Opin Ophthalmol,2017,27(Suppl1):3-47.
[23]Ono M, Takamura E, Shinozaki K, et al.Therapeutic effect of cevimeline on dry eye inpatients with Sj?gren′s syndrome:a randomized,double-blind clinical study[J]. Am J Ophthalmol,2004,138(1):6-17.
[24]Spaniol K,Holtmann C,Geerling G,et al. Newapproaches to ocular surface reconstruction beyondthe cornea[J]. Ophthalmologe,2017,114(4):307-317.
[25]Huang P, Gilmore E, Kultgen P,et al. Localregulation of cystic fibrosis transmembrane regulatorand epithelial sodium channel in airway epithelium[J]. Proc Am Thorac Soc,2004,1(1):33-37.
[2] Braun RJ,King-Smith PE,Begley CG,et al.Dynamics and function of the tear film in relation tothe blink cycle[J]. Prog Retin Eye Res,2015,45(10):132-164.
[3] Baudouin C. A new approach for bettercomprehension of diseases of the ocular surface[J].Ophtalmol,2007,30(3):239-246.
[4] Messmer EM. The pathophysiology,diagnosis,andtreatment of dry eye disease[J]. Dtsch Arztebl Int,2015,112(5):71-81.
[5] Stevenson W,Pugazhendhi S,Wang M. Is the mainlacrimal gland indispensable? contributions of thecorneal and conjunctival epithelia[J]. SurvOphthalmol,2016,61(5):616-627.
[6] Li L, Wang NL, Gong JY, et al. Infantilecholestasis caused by CFTR mutation:case reportand literature review[J]. Zhonghua Er Ke Za Zhi,2016,54(11):851-855.
[7] Feraille E,Dizin E. Coordinated control of ENaCand Na+,K+-ATPase in renal collecting duct[J]. JAm Soc Nephrol,2016,27(9):2554-2563.
[8] Pitk?nen O. Lung epithelial ion transport in neonatallung disease[J]. Biol Neonate,2001,80(Suppl 1):14-17.
[9] Morris LM, DeGagne JM, Kempton JB,et al.Mouse middle ear ion homeostasis channels andintercellular junctions[J]. PLoS One,2012,7(6):e39004.
[10]Li Y,Marcoux MO,Gineste M,et al. Expression ofwater and ion transporters in tracheal aspirates fromneonates with respiratory distress[J]. Acta Paediatr,2009,98(11):1729-1737.
[11]Shi R, Xiao ZT, Zheng YJ, et al. Naringeninregulates CFTR activation and expression in airwayepithelial cells[J]. Cell Physiol Biochem,2017,44(3):1146-1160.
[12]Cao X, Huang J, Zhang G, et al. Functionalexpression of G protein-coupled receptor 30 inimmature rat epididymal epithelium[J]. Cell BiolInt,2017,41(2):134-146.
[13]Sun Q,Huang J,Yue YJ,et al. Hydrogen sulfidefacilitates vaginal lubrication by activation ofepithelial ATP-sensitive K+channels and cysticfibrosis transmembrane conductance regulator[J]. JSex Med,2016,13(5):798-807.
[14]Zhou H,Liu J,Zhou C,et al. In vivo simultaneoustranscriptional activation of multiple genes in thebrain using CRISPR-dCas9-activator transgenicmice[J]. Nat Neurosci,2018,21(3):440-446.
[15]Viau S,Maire MA,Pasquis B,et al. Time courseof ocular surface and lacrimal gland changes in anew scopolamine-induced dry eye model[J].Graefes Arch Clin Exp Ophthalmol,2008,246(6):857-867.
[16]Chu YY,Hua N,Ru YS,et al. The protection ofhydrogen-rich saline on a rat dry eye model inducedby scopolamine hydrobromide[J]. Zhonghua YanKe Za Zhi,2017,53(5):363-372.
[17]Henriksson JT,De Paiva CS,Farley W,et al.Morphologic alterations of the palpebral conjunctivalepithelium in a dry eye model[J]. Cornea,2013,32(4):483-490.
[18]Epstein SP, Ahdoot M, Marcus E, et al.Comparative toxicity of preservatives onimmortalized corneal and conjunctival epithelial cells[J]. J Ocul Pharmacol Ther,2009,25(2):113-119.
[19]Ayaki M,Yaguchi S,Shin KS,et al. Cytotoxicity ofophthalmic solutions with and without preservativesto human corneal endothelial cells,epithelial cellsand conjunctival epithelial cells[J]. Clin ExpOphthalmol,2008,36(6):553-559.
[20]Oh JY, Kim MK, Shin KS,et al. Efficientcryopreservative conditions for cultivated limbal andconjunctival epithelial cells[J]. Cornea,2007,26(7):840-846.
[21]Zhang H,Wu H,Yang J,et al. Sodium perbarateand benzalkonium chloride induce DNA damage inChang conjunctival epithelial cells[J]. Cutan OculToxicol,2017,36(4):336-342.
[22]Milner MS, Beckman KA, Luchs JI,et al.Dysfunctional tear syndrome:dry eye disease andassociated tear film disorders-new strategies fordiagnosis and treatment[J]. Curr Opin Ophthalmol,2017,27(Suppl1):3-47.
[23]Ono M, Takamura E, Shinozaki K, et al.Therapeutic effect of cevimeline on dry eye inpatients with Sj?gren′s syndrome:a randomized,double-blind clinical study[J]. Am J Ophthalmol,2004,138(1):6-17.
[24]Spaniol K,Holtmann C,Geerling G,et al. Newapproaches to ocular surface reconstruction beyondthe cornea[J]. Ophthalmologe,2017,114(4):307-317.
[25]Huang P, Gilmore E, Kultgen P,et al. Localregulation of cystic fibrosis transmembrane regulatorand epithelial sodium channel in airway epithelium[J]. Proc Am Thorac Soc,2004,1(1):33-37.