山茱萸对苯扎氯铵诱导的干眼症小鼠视黄醇转运的调控作用
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摘要
目的:观察山茱萸对视黄醇转运的调控作用,阐明山茱萸对干眼症的治疗作用机制。方法:60只BALB/c小鼠随机分为空白对照组,模型组,阳性对照组,低、中和高剂量山茱萸组。采用苯扎氯铵滴眼法制备干眼症小鼠模型。阳性对照组小鼠经口灌服石斛夜光丸(20mL·kg-1体质量),低、中和高剂量山茱萸组小鼠分别经口灌服600、1 200和2 400g·L-1山茱萸水煎剂(20mL·kg-1体质量),空白对照组小鼠不处理,模型组小鼠经口灌服等量生理盐水;各给药组大鼠均每日给药1次,连续4周。泪液测试试纸法检测各组小鼠泪液分泌量,荧光素染色法检测各组小鼠泪膜破裂时间,ELISA法检测各组小鼠血清中前蛋白(PA)、视黄醇结合蛋白(RBP)和肝组织中视黄醇水平,免疫组织化学法和免疫印迹法检测各组小鼠泪腺组织中胞内视黄醇结合蛋白(CRBP)和RBP受体—维甲酸诱导蛋白6 (STRA6)蛋白表达水平。结果:与空白对照组比较,模型组小鼠泪液分泌量明显减少(P<0.01),泪膜破裂时间明显缩短(P<0.01);与模型组比较,阳性对照组和不同剂量山茱萸组小鼠泪液分泌量明显增加(P<0.05或P<0.01),泪膜破裂时间明显延长(P<0.01)。与空白对照组比较,模型组小鼠血清中PA和RBP水平及肝组织中视黄醇水平明显降低(P<0.01),泪腺组织中CRBP和STRA6蛋白表达水平明显降低(P<0.01);与模型组比较,阳性对照组和不同剂量山茱萸组小鼠血清中PA和RBP水平及肝组织中视黄醇水平明显升高(P<0.01),泪腺组织中CRBP和STRA6蛋白表达水平明显升高(P<0.05或P<0.01);与阳性对照组比较,高剂量山茱萸组小鼠血清中PA和RBP水平明显升高(P<0.05或P<0.01),泪腺组织中CRBP和STRA6蛋白表达水平进一步升高(P<0.01)。结论:干眼症发生时,机体伴随视黄醇缺乏和视黄醇转运障碍,而酸味药山茱萸水煎液可以促进视黄醇吸收,改善机体视黄醇缺乏状态,进而调控视黄醇转运过程。
Objective:To observe the regulatory effect of fructus corni on the retinol transport,and to elucidate the mechanism of fructus corni in the treatment of dry eye syndrome.Methods:A totle of 60 BALB/c mice were randomly divided into blank control group,model group,positive control group,low dose of fructus corni group,medium dose of fructus corni group,and high dose of fructus corni group.Benzalkonium chloride eye drop method was used to set up the dry eye syndrome models of the mice.The mice in positive control group were orally administered with Shihu Yeguang Pill(0.2 mL·10 g~(-1) body mass);the mice in low,medium,and high doses of fructus corni groups were orally administered with 600,1 200 and 2 400 g·L~(-1) fructus corni water decoction,respectively(0.2 mL·10 g~(-1) body mass);the mice in model group were orally administered with the same volume of normal saline;the mice in various adminstration groups were administered once a day,and lasted for four weeks.The tear secretion amount and tear film rupture time of the mice in vavious groups were detected by tear test paper method and fluoresein staining method.The levels of serum prealbumin(PA),retinol binding protein(RBP)and the retinol in liver tissue of the mice in various groups were detected by ELISA;the expression levels of cellular retinol binding protein(CRBP)and RBP receptor-stimulated by retinoic acid gene 6(STRA6)in lacrimal gland tissue of the mice in various groups were detected by immunohistochemistry and Western blotting methods.Results:Compared with blank control group,the tear secretion amount and the tear film rupture time of the mice in model group were significantly decreased(P<0.01).Compared with model group,the tear secretion amount and the tear film repture time of the mice in positive control group and different doses of fructus corni groups were significnatly increased(P<0.05 or P<0.01).Compared with blank control group,the levels of serum PA and RBP and the levels of retinol in liver of the mice in model group were significantly decreased(P<0.01);the expression levels of CRBP and STRA6 proteins in lacrimal gland tissue were significantly increased(P<0.05 or P<0.01).Compared with model group,the levels of serum PA and RBP and the levels of retinol in liver tissue of the mice in positive control and different doses of fructus corni groups were significantly increased(P<0.01).Compared with positive control group,the levels of serum PA and RBP of the mice in high dose of fructus corni group were increased(P<0.05 or P<0.01);the expression levels of CRBP and STRA6 proteins in lacrimal gland tissue of the mice were significantly increased(P<0.01).Conclusion:When dry eye syndrome occurs,the body is accompanied by deficiency of retinol and obstacle of retinol transport.The sour drug fructus corni can improve the deficiency of retinol by increasing the absorption of retinol,and then regulate the process of retinol transport.
Objective:To observe the regulatory effect of fructus corni on the retinol transport,and to elucidate the mechanism of fructus corni in the treatment of dry eye syndrome.Methods:A totle of 60 BALB/c mice were randomly divided into blank control group,model group,positive control group,low dose of fructus corni group,medium dose of fructus corni group,and high dose of fructus corni group.Benzalkonium chloride eye drop method was used to set up the dry eye syndrome models of the mice.The mice in positive control group were orally administered with Shihu Yeguang Pill(0.2 mL·10 g~(-1) body mass);the mice in low,medium,and high doses of fructus corni groups were orally administered with 600,1 200 and 2 400 g·L~(-1) fructus corni water decoction,respectively(0.2 mL·10 g~(-1) body mass);the mice in model group were orally administered with the same volume of normal saline;the mice in various adminstration groups were administered once a day,and lasted for four weeks.The tear secretion amount and tear film rupture time of the mice in vavious groups were detected by tear test paper method and fluoresein staining method.The levels of serum prealbumin(PA),retinol binding protein(RBP)and the retinol in liver tissue of the mice in various groups were detected by ELISA;the expression levels of cellular retinol binding protein(CRBP)and RBP receptor-stimulated by retinoic acid gene 6(STRA6)in lacrimal gland tissue of the mice in various groups were detected by immunohistochemistry and Western blotting methods.Results:Compared with blank control group,the tear secretion amount and the tear film rupture time of the mice in model group were significantly decreased(P<0.01).Compared with model group,the tear secretion amount and the tear film repture time of the mice in positive control group and different doses of fructus corni groups were significnatly increased(P<0.05 or P<0.01).Compared with blank control group,the levels of serum PA and RBP and the levels of retinol in liver of the mice in model group were significantly decreased(P<0.01);the expression levels of CRBP and STRA6 proteins in lacrimal gland tissue were significantly increased(P<0.05 or P<0.01).Compared with model group,the levels of serum PA and RBP and the levels of retinol in liver tissue of the mice in positive control and different doses of fructus corni groups were significantly increased(P<0.01).Compared with positive control group,the levels of serum PA and RBP of the mice in high dose of fructus corni group were increased(P<0.05 or P<0.01);the expression levels of CRBP and STRA6 proteins in lacrimal gland tissue of the mice were significantly increased(P<0.01).Conclusion:When dry eye syndrome occurs,the body is accompanied by deficiency of retinol and obstacle of retinol transport.The sour drug fructus corni can improve the deficiency of retinol by increasing the absorption of retinol,and then regulate the process of retinol transport.
引文
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[3]何媛,洪薇薇,赵海雁,等.维生素A棕榈酸酯眼用凝胶联合羧甲基纤维素钠对白内障术后干眼的预防[J].国际眼科杂志,2018,18(7):1264-1267.
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[8]马轶群,王琇,杨青,等.维生素A缺乏干眼症模型兔泪液分泌及泪膜稳定性的改变[J].眼科新进展,2006,26(7):517-520.
[9]马轶群,王传富,王青.维生素A缺乏干眼症兔泪腺凋亡及相关基因的表达[J].眼科新进展,2003,23(6):406-408.
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[11]LIN Z R,LIU X C,ZHOU T,et al.A mouse dry eye model induced by topical administration of benzalkonium chloride[J].Mol Vis,2011,17:257-264.
[12]RU Y S,HUANG Y,LIU H J,et al.α-Melanocytestimulating hormone amelio-rates ocular surface dysfunctions and lesions in a scopolamine-induced dry eye model via PKA-CREB and MEK-Erk pathways[J].Sci Rep,2016,5:18619.
[13]王朝晖.羟糖苷滴眼液联合维生素A棕榈酸酯凝胶治疗视网膜脱离术后干眼的疗效观察[J].临床眼科杂志,2018,26(3):243-245.
[14]丛越,王万梅.维生素A缺乏干眼病角膜组织上皮细胞Fas、FasL、Bax和bcl-2的表达与细胞凋亡的关系研究[J].中国实验诊断学,2017,21(10):1820-1823.
[15]NOECKER R J,HERRYQERS L A,ANWARUDDIN R.Coreal and cenjunceivial changes caused by commondly used glancoma medications[J].Corneal,2004,23(5):490-496.
[16]BREEN C J,MARTIN D S,MA H,et al.Production of functional human vitamin A transporter/RBP receptor(STRA6)for structure determination[J].PLoS One,2015,10(3):e0122293.
[17]MONACO H L,RIZZI M,CODA A.Structure of a complex of two plasma proteins:transthyretin and retinol-binding protein[J].Science,1995,268(5213):1039-1041.
[18]金宏.视黄醇转运的分子机制[J].生理科学进展,1997,28(2):166-168.
[19]MALPELI G,STOPPINI M,ZAPPONI M C,et al.Interactions with retinol and retinoids of bovine cellular retinolbinding protein[J].Eur J Biochem,1995,229(2):486-493.
[20]付玉,张盼,张雅文,等.猪视黄醇结合蛋白基因研究进展[J].中国畜牧杂志,2018,54(12):10-13.
[21]ROSS A C.Vitamin A and carotenoids[A]//SHILS M E,SHIKE M,ROSS A C.Modern nutrition in health and disease[M].10th ed.Baltimore:Lippincott Williams&Wilkins,2006:351-371.
[2]薛研,赵耀东,颉瑞萍,等.“进火补”针法治疗气阴两虚型干眼症[J].中国针灸,2018,38(6):609-612.
[3]何媛,洪薇薇,赵海雁,等.维生素A棕榈酸酯眼用凝胶联合羧甲基纤维素钠对白内障术后干眼的预防[J].国际眼科杂志,2018,18(7):1264-1267.
[4]昝云强,赵维杰.针灸配合补充维生素A治疗干眼症的疗效观察[J].天津中医药,2017,34(12):823-825.
[5]姜开运,于凯洋.五味子对维生素A缺乏型干眼症RBP、STRA6蛋白表达的影响[J].辽宁中医药大学学报,2016,18(8):27-29.
[6]姜开运,牛爽,李珂.五味子治疗维生素A缺乏型干眼症疗效及作用机制[J].辽宁中医药大学学报,2016,18(7):33-35.
[7]刘超,耿燕.NFκ-Bp65、NOS2在维生素A缺乏干眼模型兔角膜中的表达及意义[J].眼科研究,2009,27(1):19-22.
[8]马轶群,王琇,杨青,等.维生素A缺乏干眼症模型兔泪液分泌及泪膜稳定性的改变[J].眼科新进展,2006,26(7):517-520.
[9]马轶群,王传富,王青.维生素A缺乏干眼症兔泪腺凋亡及相关基因的表达[J].眼科新进展,2003,23(6):406-408.
[10]BARABINO S,SHEN L L,CHEN L,et al.The controlled-environment chamber:a new mouse model of dry eye[J].Invest Ophthalmol Vis Sci,2005,46(8):2766-2771.
[11]LIN Z R,LIU X C,ZHOU T,et al.A mouse dry eye model induced by topical administration of benzalkonium chloride[J].Mol Vis,2011,17:257-264.
[12]RU Y S,HUANG Y,LIU H J,et al.α-Melanocytestimulating hormone amelio-rates ocular surface dysfunctions and lesions in a scopolamine-induced dry eye model via PKA-CREB and MEK-Erk pathways[J].Sci Rep,2016,5:18619.
[13]王朝晖.羟糖苷滴眼液联合维生素A棕榈酸酯凝胶治疗视网膜脱离术后干眼的疗效观察[J].临床眼科杂志,2018,26(3):243-245.
[14]丛越,王万梅.维生素A缺乏干眼病角膜组织上皮细胞Fas、FasL、Bax和bcl-2的表达与细胞凋亡的关系研究[J].中国实验诊断学,2017,21(10):1820-1823.
[15]NOECKER R J,HERRYQERS L A,ANWARUDDIN R.Coreal and cenjunceivial changes caused by commondly used glancoma medications[J].Corneal,2004,23(5):490-496.
[16]BREEN C J,MARTIN D S,MA H,et al.Production of functional human vitamin A transporter/RBP receptor(STRA6)for structure determination[J].PLoS One,2015,10(3):e0122293.
[17]MONACO H L,RIZZI M,CODA A.Structure of a complex of two plasma proteins:transthyretin and retinol-binding protein[J].Science,1995,268(5213):1039-1041.
[18]金宏.视黄醇转运的分子机制[J].生理科学进展,1997,28(2):166-168.
[19]MALPELI G,STOPPINI M,ZAPPONI M C,et al.Interactions with retinol and retinoids of bovine cellular retinolbinding protein[J].Eur J Biochem,1995,229(2):486-493.
[20]付玉,张盼,张雅文,等.猪视黄醇结合蛋白基因研究进展[J].中国畜牧杂志,2018,54(12):10-13.
[21]ROSS A C.Vitamin A and carotenoids[A]//SHILS M E,SHIKE M,ROSS A C.Modern nutrition in health and disease[M].10th ed.Baltimore:Lippincott Williams&Wilkins,2006:351-371.