氨基葡萄糖对小鼠实验性过敏性结膜炎的治疗作用
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摘要
目的评价氨基葡萄糖对小鼠实验性过敏性结膜炎的治疗作用。方法取6～8周龄雌性Balb/c小鼠40只(40眼),随机分为5组:空白对照组、过敏性结膜炎组、氨基葡萄糖腹腔注射组、氨基葡萄糖滴眼组、地塞米松治疗组,每组8只。从造模开始第0天和第7天,将100μg卵清蛋白与200μL氢氧化铝佐剂按照体积比1:2的比例完全混合,除空白对照组外,其余各组用混合液进行腹腔注射;第15～18天,给予250μg的卵清蛋白滴眼激发。空白对照组在致敏和激发阶段均用等量生理盐水替代。于造模开始后第15～18天,每次滴眼激发后30 min,开始进行药物干预治疗连续4 d。末次激发后30 min内,裂隙灯显微镜下观察小鼠眼部临床症状。用100 g·L-1水合氯醛麻醉后,眼球取血,提取血清用于ELISA检测;取下小鼠结膜用于制作病理切片并进行HE染色;无菌条件下取小鼠脾脏,分离脾细胞进行培养,检测上清液中炎症因子含量。结果末次激发后30 min,观察小鼠临床症状。空白对照组无明显的过敏症状。过敏性结膜炎组小鼠出现明显的过敏症状,眼睑严重充血水肿,睁眼困难,结膜严重充血、水肿,流泪不止,眼部分泌物较多。氨基葡萄糖腹腔注射组、氨基葡萄糖滴眼组和地塞米松治疗组小鼠均能观察到较轻的眼睑肿胀、结膜少量充血水肿、流泪和少量眼部分泌物。高倍镜下5组小鼠结膜嗜酸性粒细胞计数,总体差异有统计学意义(P=0. 000)。各组间两两比较,氨基葡萄糖滴眼组嗜酸性粒细胞数与地塞米松治疗组相比,差异无统计学意义(P> 0. 05);氨基葡萄糖腹腔注射组与氨基葡萄糖滴眼组相比,差异无统计学意义(P> 0. 05);其余各组间两两比较,差异均有统计学意义(均为P <0. 01)。过敏性结膜炎组嗜酸性粒细胞数均高于其他组。各组小鼠血清Ig E含量,总体差异有统计学意义(P=0. 000)。各组间两两比较差异均有统计学意义(均为P <0. 05)。过敏性结膜炎组血清Ig E含量高于其他各组,氨基葡萄糖滴眼组血清Ig E含量低于地塞米松治疗组,高于氨基葡萄糖腹腔注射组,差异均有统计学意义(均为P <0. 05)。各组小鼠脾细胞培养液上清液中的IL-4、IL-5和IFN-γ的表达水平,总体差异均有统计学意义(均为P=0. 000)。各组间两两比较,氨基葡萄糖腹腔注射组上清液IL-5水平与地塞米松治疗组相比差异无统计学意义(P> 0. 05);氨基葡萄糖腹腔注射组、氨基葡萄糖滴眼组、地塞米松治疗组及过敏性结膜炎组IFN-γ水平两两比较差异均无统计学意义(均为P> 0. 05);过敏性结膜炎组IL-4、IL-5表达水平均高于其余各组;氨基葡萄糖滴眼组IL-4、IL-5表达水平均高于氨基葡萄糖腹腔注射组;空白对照组IFN-γ表达水平均高于其余各组,差异均有统计学意义(均为P <0. 05)。结论氨基葡萄糖全身及局部用药均能改善小鼠实验性过敏性结膜炎的临床症状,减轻嗜酸性粒细胞聚集,降低血清中Ig E含量,局部治疗优于地塞米松治疗,全身用药在降低血清Ig E含量方面优于局部用药。
Objective To evaluate the therapeutic effect of glucosamine on experimental allergic conjunctivitis on mice.Methods Forty female Balb/c mice(40 eyes) aged 6-8 weeks were allocated randomly into 5 groups with 8 mice in each of them:blank control group,allergic conjunctivitis group,glucosamine intraperitoneal injection group,glucosamine eye drops group and dexamethasone treatment group.From the first day to the 7 th day of the modeling,ovalbumin(OVA,100 μg) and aluminum hydroxide adjuvant(200 μL) were completely mixed at a volume ratio of 1:2,except for the blank control group,other groups were intraperitoneally injected with the mixture.On the 15 th to the 18 th day,mice were activated by OVA drops(250 μg) onto the conjunctival sac.The blank control group was replaced by equal amount of sterile saline during the sensitization and challenge phases.On the 15 th to 18 th day after the modeling,we started drug intervention after activation with eye drops for 30 min.The clinical symptoms of the mice' eyes were observed under a light microscope within 30 min after the last stimulation.After the anesthesia with 100 g·L~(-1) chloral hydrate,we took the serum for ELISA.Then we took mice' conjunctiva to make the hematoxylin-eosin(HE) staining.The spleen was taken to separate the cells under sterile condition and then the content of the supernatant inflammatory factors were measured.Results The clinical symptoms of mice were observed within 30 min after the last stimulation.The blank control group had no obvious allergic symptoms.While the allergic conjunctivitis group had obvious allergic symptoms,such as lid edema,chemosis,conjunctival hyperemia,hard to blink,constant tearing and eye secretions.The mice in the glucosamine intraperitoneal injection group,the glucosamine eye drops group and the dexamethasone treatment group had rather slight allergic symptoms.The general difference in eosinophilia of the conjunctiva in these 5 groups were statistically significant(P=0.000).Through pairwise comparison,the difference in the number of eosinophilia had no statistical significance between glucosamine eye drops group and dexamethasone treatment group(P>0.05).And the difference between glucosamine intraperitoneal injection group and glucosamine eye drops group also had no statistical significance in the number of eosinophils(P>0.05).In the pairwise comparison between the other groups,the differences in the number of eosinophilia all had statistically significance(all P<0.01).The number of eosinophilia in the allergic conjunctivitis group was higher than that of the other groups.The overall difference in the IgE content of mice' serum in each group was statistically significant(P=0.000).And the differences between each group through pair-to-pair comparison of IgE content all had statistical significance(all P<0.05).The IgE content in the allergic conjunctivitis group was higher than that in the other groups,and the IgE content in the glucosamine eye drops group was lower than that in the dexamethasone treatment group,but higher than that in the glucosamine intraperitoneal group,and these differences all had statistical significance(all P<0.05).The levels of IL-4,IL-5 and IFN-γ in the supernatant of spleen cell between each group were statistically significant(all P=0.000).Through pairwise comparison,there was no statistically significant difference between the glucosamine intraperitoneal injection group and the dexamethasone treatment group in the level of IL-5(P>0.05).The differences in the IFN-γ levels had no statistical significance in the glucosamine intraperitoneal injection group;the glucosamine eye drops group,the dexamethasone treatment group and the allergic conjunctivitis group(all P>0.05).The levels of IL-4 and IL-5 in the allergic conjunctivitis group were higher than those of the other groups;and the level of IL-4 and IL-5 in the glucosamine eye drops group were higher than that in glucosamine intraperitoneal group.The level of IFN-γ in the blank control group was higher than that in the other groups(all P<0.05).Conclusion Both systemic and topical administration of glucosamine can relieve the clinical sufferings of experimental allergic conjunctivitis in mice,reduce eosinophilia accumulation and serum IgE content.Glucosamine topical administration is better than dexamethasone treatment,and the systemic treatment is better than topical treatment in reducing serum IgE level.
Objective To evaluate the therapeutic effect of glucosamine on experimental allergic conjunctivitis on mice.Methods Forty female Balb/c mice(40 eyes) aged 6-8 weeks were allocated randomly into 5 groups with 8 mice in each of them:blank control group,allergic conjunctivitis group,glucosamine intraperitoneal injection group,glucosamine eye drops group and dexamethasone treatment group.From the first day to the 7 th day of the modeling,ovalbumin(OVA,100 μg) and aluminum hydroxide adjuvant(200 μL) were completely mixed at a volume ratio of 1:2,except for the blank control group,other groups were intraperitoneally injected with the mixture.On the 15 th to the 18 th day,mice were activated by OVA drops(250 μg) onto the conjunctival sac.The blank control group was replaced by equal amount of sterile saline during the sensitization and challenge phases.On the 15 th to 18 th day after the modeling,we started drug intervention after activation with eye drops for 30 min.The clinical symptoms of the mice' eyes were observed under a light microscope within 30 min after the last stimulation.After the anesthesia with 100 g·L~(-1) chloral hydrate,we took the serum for ELISA.Then we took mice' conjunctiva to make the hematoxylin-eosin(HE) staining.The spleen was taken to separate the cells under sterile condition and then the content of the supernatant inflammatory factors were measured.Results The clinical symptoms of mice were observed within 30 min after the last stimulation.The blank control group had no obvious allergic symptoms.While the allergic conjunctivitis group had obvious allergic symptoms,such as lid edema,chemosis,conjunctival hyperemia,hard to blink,constant tearing and eye secretions.The mice in the glucosamine intraperitoneal injection group,the glucosamine eye drops group and the dexamethasone treatment group had rather slight allergic symptoms.The general difference in eosinophilia of the conjunctiva in these 5 groups were statistically significant(P=0.000).Through pairwise comparison,the difference in the number of eosinophilia had no statistical significance between glucosamine eye drops group and dexamethasone treatment group(P>0.05).And the difference between glucosamine intraperitoneal injection group and glucosamine eye drops group also had no statistical significance in the number of eosinophils(P>0.05).In the pairwise comparison between the other groups,the differences in the number of eosinophilia all had statistically significance(all P<0.01).The number of eosinophilia in the allergic conjunctivitis group was higher than that of the other groups.The overall difference in the IgE content of mice' serum in each group was statistically significant(P=0.000).And the differences between each group through pair-to-pair comparison of IgE content all had statistical significance(all P<0.05).The IgE content in the allergic conjunctivitis group was higher than that in the other groups,and the IgE content in the glucosamine eye drops group was lower than that in the dexamethasone treatment group,but higher than that in the glucosamine intraperitoneal group,and these differences all had statistical significance(all P<0.05).The levels of IL-4,IL-5 and IFN-γ in the supernatant of spleen cell between each group were statistically significant(all P=0.000).Through pairwise comparison,there was no statistically significant difference between the glucosamine intraperitoneal injection group and the dexamethasone treatment group in the level of IL-5(P>0.05).The differences in the IFN-γ levels had no statistical significance in the glucosamine intraperitoneal injection group;the glucosamine eye drops group,the dexamethasone treatment group and the allergic conjunctivitis group(all P>0.05).The levels of IL-4 and IL-5 in the allergic conjunctivitis group were higher than those of the other groups;and the level of IL-4 and IL-5 in the glucosamine eye drops group were higher than that in glucosamine intraperitoneal group.The level of IFN-γ in the blank control group was higher than that in the other groups(all P<0.05).Conclusion Both systemic and topical administration of glucosamine can relieve the clinical sufferings of experimental allergic conjunctivitis in mice,reduce eosinophilia accumulation and serum IgE content.Glucosamine topical administration is better than dexamethasone treatment,and the systemic treatment is better than topical treatment in reducing serum IgE level.
引文
[1] GE J.Ophthalmology[M].Beijing:People’s Medical Publishing House,2010:163-164. 葛坚.眼科学[M].北京:人民卫生出版社,2010:163-164.
[2] RIDOLO E,MONTAGNI M,CAMINATI M,SENNA G,INCORVAIA C,CANONICA G W,et al.Emerging drugs for allergic conjunctivitis[J].Expert Opin Emerg Drugs,2014,19(2):291-302.
[3] ZHU J J,LI B.Effects of artemisinin on early-phase response of allergic conjunctivitis in a murine model induced by pollen[J].Int Eye Sci,2016,16(6):1032-1035. 朱敬敬,李兵.青蒿素在小鼠花粉过敏性结膜炎早期反应相中的作用[J].国际眼科杂志,2016,16(6):1032-1035.
[4] CHEN D F,YU K Y.Research progress on immunological mechanism of allergic conjunctivitis[J].Chin Med Herald,2014,11(33):162-165. 陈大复,于坤瑛.过敏性结膜炎的免疫学研究进展[J].中国医药导报,2014,11(33):162-165.
[5] JUNG A Y,HEO M J,KIM Y H.Glucosamine has an antiallergic effect in mice with allergic asthma and rhinitis[J].Int Forum Allergy Rhinol,2017,7(8):763-769.
[6] LEE H J,KIM B M,SHIN S,KIM T Y,CHUNG S H.Superoxide dismutase 3 attenuates experimental Th2-driven allergic conjunctivitis[J].Clin Immunol,2017,176:49-54.
[7] LEE H S,KWON J Y,JOO C K.Topical administration of β-1,3-glucan to modulate allergic conjunctivitis in a murine model[J].Invest Ophthalmol Vis Sci,2016,57(3):1352-1360.
[8] TALLIA A F,CARDONE D A.Asthma exacerbation associated with glucosamine-chondroitin supplement[J].J Am Board Fam Pract,2002,15(6):481-484.
[9] CóRDOVA C,GUTIéRREZ B,MARTíNEZ -GARCíA C,MARTíN R,GALLEGO-MU?OZ P,HERNáNDEZ M,et al.Oleanolic acid controls allergic and inflammatory responses in experimental allergic conjunctivitis[J].PLoS One,2014,9(4):e91282.
[10] LI S Y.The effect of immunostimulatory CPG ODN on experimental allergic conjunctivitis caused by aspergillus fumigatus[D].Jinan:Jinan University,2011:1-58. 李思媛.CpG ODN防治烟曲霉菌引起的过敏性结膜炎的实验研究[D].济南:济南大学,2011:1-58.
[11] ZHANG J,WANG D L,YAN D M.Effect of Th1/Th2/Th17 balance on development of experimental allergic conjunctivitis[J].Chin J Modern Med,2016,26(15):32-37. 张剑,王冬兰,闫冬梅.Th1/Th2/Th17细胞在实验性过敏性结膜炎中的作用研究[J].中国现代医学杂志,2016,26(15):32-37.
[12] KIM C H,CHOI Y S,CHEONG K A,LEE A Y.Mechanism underlying the effect of combined therapy using glucosamine and low-dose cyclosporine A on the development of atopic dermatitis-like skin lesions in NC/Nga mice[J].Int Immunopharmacol,2013,15(2):424-432.
[13] NIEDRKORN J Y.Immune regulatory mechanisms in allergic conjunctivitis:insights from mouse models[J].Curr Opin Allergy Clin Immunol,2008,8(5):472-476.
[14] STERN M E,SIEMASKO K F,NIEDERKORN J Y.The Th1/Th2 paradigm in ocular allergy[J].Curr Opin Allergy Clin Immunol,2005,5(5):446-450.
[15] LI Y M,TANG M Y,XIA W,LI T.Antitumor effect of D-glucosamine hydrochloride in mice[J].Chin J Nat Med,2003,1(4):237-239. 李运曼,唐明月,夏雯,李特.D-氨基葡萄糖盐酸盐的抗肿瘤作用[J].中国天然药物,2003,1(4):237-239.
[16] CHUNG M J,PARK J K,PARK Y I.Anti-inflammatory effects of low-molecular weight chitosan oligosaccharides in IgE-antigen complex-stimulated RBL-2H3 cells and asthma model mice[J].Int Immunopharmacol,2012,12(2):453-459.
[17] FU C W.Studies on immunostimulating effects of Glucosa-mine and chito-oligosaccharide in mice[D].Qingdao:Ocean University of China,2006:1-72. 付辰炜.氨基葡萄糖及壳寡糖对小鼠免疫功能的影响[D].青岛:中国海洋大学,2006:1-72.
[18] JIN S E,JUNG J,JUN J,JEON D W,KIM H M,JEONG H J,et al.Anti-allergic activity of crystallinity controlled N-acetyl glucosamine[J].Immunopharmacol Immunotoxicol,2012,34(6):991-1000.
[19] COLLINS P D,MARLEAU S,GRIFFITHS-JOHNSON D A,JOSE P J,WILLIAMS T J,et al.Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo[J].J Exp Med,1995,182(4):1169-1174.
[20] KIM C H,CHEONG K A,PARK C D,LEE A Y.Glucosamine improved atopic dermatitis-like skin lesions in NC/Nga mice by inhibition of Th2 cell development[J].Scand J Immunol,2011,73(6):536-545.
[21] MATHEU V,GRACIA BARA M T,PELTA R,VIVAS E,RUBIO M.Immediate-hypersensitivity reaction to glucosamine sulfate[J].Allergy,1999,54(6):643.
[2] RIDOLO E,MONTAGNI M,CAMINATI M,SENNA G,INCORVAIA C,CANONICA G W,et al.Emerging drugs for allergic conjunctivitis[J].Expert Opin Emerg Drugs,2014,19(2):291-302.
[3] ZHU J J,LI B.Effects of artemisinin on early-phase response of allergic conjunctivitis in a murine model induced by pollen[J].Int Eye Sci,2016,16(6):1032-1035. 朱敬敬,李兵.青蒿素在小鼠花粉过敏性结膜炎早期反应相中的作用[J].国际眼科杂志,2016,16(6):1032-1035.
[4] CHEN D F,YU K Y.Research progress on immunological mechanism of allergic conjunctivitis[J].Chin Med Herald,2014,11(33):162-165. 陈大复,于坤瑛.过敏性结膜炎的免疫学研究进展[J].中国医药导报,2014,11(33):162-165.
[5] JUNG A Y,HEO M J,KIM Y H.Glucosamine has an antiallergic effect in mice with allergic asthma and rhinitis[J].Int Forum Allergy Rhinol,2017,7(8):763-769.
[6] LEE H J,KIM B M,SHIN S,KIM T Y,CHUNG S H.Superoxide dismutase 3 attenuates experimental Th2-driven allergic conjunctivitis[J].Clin Immunol,2017,176:49-54.
[7] LEE H S,KWON J Y,JOO C K.Topical administration of β-1,3-glucan to modulate allergic conjunctivitis in a murine model[J].Invest Ophthalmol Vis Sci,2016,57(3):1352-1360.
[8] TALLIA A F,CARDONE D A.Asthma exacerbation associated with glucosamine-chondroitin supplement[J].J Am Board Fam Pract,2002,15(6):481-484.
[9] CóRDOVA C,GUTIéRREZ B,MARTíNEZ -GARCíA C,MARTíN R,GALLEGO-MU?OZ P,HERNáNDEZ M,et al.Oleanolic acid controls allergic and inflammatory responses in experimental allergic conjunctivitis[J].PLoS One,2014,9(4):e91282.
[10] LI S Y.The effect of immunostimulatory CPG ODN on experimental allergic conjunctivitis caused by aspergillus fumigatus[D].Jinan:Jinan University,2011:1-58. 李思媛.CpG ODN防治烟曲霉菌引起的过敏性结膜炎的实验研究[D].济南:济南大学,2011:1-58.
[11] ZHANG J,WANG D L,YAN D M.Effect of Th1/Th2/Th17 balance on development of experimental allergic conjunctivitis[J].Chin J Modern Med,2016,26(15):32-37. 张剑,王冬兰,闫冬梅.Th1/Th2/Th17细胞在实验性过敏性结膜炎中的作用研究[J].中国现代医学杂志,2016,26(15):32-37.
[12] KIM C H,CHOI Y S,CHEONG K A,LEE A Y.Mechanism underlying the effect of combined therapy using glucosamine and low-dose cyclosporine A on the development of atopic dermatitis-like skin lesions in NC/Nga mice[J].Int Immunopharmacol,2013,15(2):424-432.
[13] NIEDRKORN J Y.Immune regulatory mechanisms in allergic conjunctivitis:insights from mouse models[J].Curr Opin Allergy Clin Immunol,2008,8(5):472-476.
[14] STERN M E,SIEMASKO K F,NIEDERKORN J Y.The Th1/Th2 paradigm in ocular allergy[J].Curr Opin Allergy Clin Immunol,2005,5(5):446-450.
[15] LI Y M,TANG M Y,XIA W,LI T.Antitumor effect of D-glucosamine hydrochloride in mice[J].Chin J Nat Med,2003,1(4):237-239. 李运曼,唐明月,夏雯,李特.D-氨基葡萄糖盐酸盐的抗肿瘤作用[J].中国天然药物,2003,1(4):237-239.
[16] CHUNG M J,PARK J K,PARK Y I.Anti-inflammatory effects of low-molecular weight chitosan oligosaccharides in IgE-antigen complex-stimulated RBL-2H3 cells and asthma model mice[J].Int Immunopharmacol,2012,12(2):453-459.
[17] FU C W.Studies on immunostimulating effects of Glucosa-mine and chito-oligosaccharide in mice[D].Qingdao:Ocean University of China,2006:1-72. 付辰炜.氨基葡萄糖及壳寡糖对小鼠免疫功能的影响[D].青岛:中国海洋大学,2006:1-72.
[18] JIN S E,JUNG J,JUN J,JEON D W,KIM H M,JEONG H J,et al.Anti-allergic activity of crystallinity controlled N-acetyl glucosamine[J].Immunopharmacol Immunotoxicol,2012,34(6):991-1000.
[19] COLLINS P D,MARLEAU S,GRIFFITHS-JOHNSON D A,JOSE P J,WILLIAMS T J,et al.Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo[J].J Exp Med,1995,182(4):1169-1174.
[20] KIM C H,CHEONG K A,PARK C D,LEE A Y.Glucosamine improved atopic dermatitis-like skin lesions in NC/Nga mice by inhibition of Th2 cell development[J].Scand J Immunol,2011,73(6):536-545.
[21] MATHEU V,GRACIA BARA M T,PELTA R,VIVAS E,RUBIO M.Immediate-hypersensitivity reaction to glucosamine sulfate[J].Allergy,1999,54(6):643.
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