不同浓度多西环素对碱烧伤大鼠角膜中转化生长因子β1和基质金属蛋白酶9表达的影响
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摘要
目的探讨不同浓度多西环素对碱烧伤大鼠角膜中转化生长因子β1(transforming growth factor-β1,TGF-β1)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达的影响及其调节作用。方法 SD大鼠右眼制备成角膜碱烧伤模型,随机分为对照组、0.5 g·L~(-1)多西环素组和1.0 g·L~(-1)多西环素组,每组15只。3组分别于造模后每天给予20μL眼液溶媒、0.5 g·L~(-1)多西环素、1.0 g·L~(-1)多西环素滴眼至观察期末。于碱烧伤后第3天、第7天、第14天对角膜混浊程度进行评分,并行RT-PCR和ELISA检测角膜组织中TGF-β1、MMP-9 mRNA和蛋白的表达情况。结果与对照组相比,碱烧伤后第7天、第14天,0.5 g·L~(-1)多西环素组[(2.80±0.45)分、(2.20±0.45)分]和1.0 g·L~(-1)多西环素组[(2.00±0.00)分、(0.60±0.55)分]角膜混浊程度评分降低,且1.0 g·L~(-1)多西环素组更低,差异均有统计学意义(均为P<0.05)。碱烧伤后第3天、第7天、第14天,0.5 g·L~(-1)多西环素组、1.0 g·L~(-1)多西环素组TGF-β1 mRNA及蛋白表达量均较对照组低,且1.0 g·L~(-1)多西环素组表达更低,差异均有统计学意义(均为P<0.05)。碱烧伤后第3天、第7天,0.5 g·L~(-1)多西环素组、1.0 g·L~(-1)多西环素组MMP-9 mRNA及蛋白表达均较对照组低,且1.0 g·L~(-1)多西环素组表达更低,差异均有统计学意义(均为P<0.05)。结论多西环素可下调碱烧伤大鼠角膜中TGF-β1和MMP-9的表达,促进角膜愈合,并呈一定程度的剂量依赖性。
Objective To investigate the effect of different concentrations of doxycycline on the expression of transforming growth factor-β1(TGF-β1) and matrix metalloproteinase-9(MMP-9) in rat alkali-burnt corneas and its regulation.Methods The right eyes of SD rats were built for a corneal alkali burn model,then randomly divided into control group,0.5 g·L~(-1) doxycycline group and 1.0 g·L~(-1) doxycycline group,with 15 rats in each group.After the model was established,the three groups were respectively given 20 μL of menstruum eye drops,0.5 g·L~(-1) doxycycline and 1.0 g·L~(-1) doxycycline to the right eye every day until the end of the observation.At 3 days,7 days and 14 days after the model was established,the degree of corneal opacity was evaluated,and the expression of TGF-β1 and MMP-9 mRNA and protein in the corneal tissue was detected by RT-PCR and ELISA.Results Compared with the control group,the score of corneal opacity was reduced in the 0.5 g·L~(-1) doxycycline group[(2.80±0.45)points,(2.20±0.45)points ] and 1.0 g·L~(-1) doxycycline group [(2.00±0.00)points,(0.60±0.55)points ] at 7 days and 14 days after injury,and the 1.0 g·L~(-1) doxycycline group was instinctively lower,and the differences were statistically significant(all P<0.05).The results of the RT-PCR and ELISA analyses demonstrated that the amounts of TGF-β1 mRNA and protein in the 0.5 g·L~(-1) doxycycline group and 1.0 g·L~(-1) doxycycline group were lower than those in the control group at 3 days,7 days and 14 days after the model was established,and the 1.0 g·L~(-1) doxycycline group was obviously lower,and the differences were statistically significant(all P<0.05).The amounts of MMP-9 mRNA and protein in the 0.5 g·L~(-1) and 1.0 g·L~(-1) doxycycline group were lower than those in the control group at 3 days and 7 days after the model was established,and the 1.0 g·L~(-1) doxycycline group was significantly lower,and the differences were statistically significant(all P<0.05).Conclusion The effect of doxycycline on the alkali-burnt corneas of rats can downregulate the expression of TGF-β1 and MMP-9,and promote corneal healing in a dose-dependent manner.
Objective To investigate the effect of different concentrations of doxycycline on the expression of transforming growth factor-β1(TGF-β1) and matrix metalloproteinase-9(MMP-9) in rat alkali-burnt corneas and its regulation.Methods The right eyes of SD rats were built for a corneal alkali burn model,then randomly divided into control group,0.5 g·L~(-1) doxycycline group and 1.0 g·L~(-1) doxycycline group,with 15 rats in each group.After the model was established,the three groups were respectively given 20 μL of menstruum eye drops,0.5 g·L~(-1) doxycycline and 1.0 g·L~(-1) doxycycline to the right eye every day until the end of the observation.At 3 days,7 days and 14 days after the model was established,the degree of corneal opacity was evaluated,and the expression of TGF-β1 and MMP-9 mRNA and protein in the corneal tissue was detected by RT-PCR and ELISA.Results Compared with the control group,the score of corneal opacity was reduced in the 0.5 g·L~(-1) doxycycline group[(2.80±0.45)points,(2.20±0.45)points ] and 1.0 g·L~(-1) doxycycline group [(2.00±0.00)points,(0.60±0.55)points ] at 7 days and 14 days after injury,and the 1.0 g·L~(-1) doxycycline group was instinctively lower,and the differences were statistically significant(all P<0.05).The results of the RT-PCR and ELISA analyses demonstrated that the amounts of TGF-β1 mRNA and protein in the 0.5 g·L~(-1) doxycycline group and 1.0 g·L~(-1) doxycycline group were lower than those in the control group at 3 days,7 days and 14 days after the model was established,and the 1.0 g·L~(-1) doxycycline group was obviously lower,and the differences were statistically significant(all P<0.05).The amounts of MMP-9 mRNA and protein in the 0.5 g·L~(-1) and 1.0 g·L~(-1) doxycycline group were lower than those in the control group at 3 days and 7 days after the model was established,and the 1.0 g·L~(-1) doxycycline group was significantly lower,and the differences were statistically significant(all P<0.05).Conclusion The effect of doxycycline on the alkali-burnt corneas of rats can downregulate the expression of TGF-β1 and MMP-9,and promote corneal healing in a dose-dependent manner.
引文
[1] CLARE G,SULEMAN H,BUNCE C,DUA H.Amniotic membrane transplantation for acute ocular burns[J].Cochrane Database Syst Rev,2012(9):CD009379.
[2] SALMAN I A,GUNDOGDU C.Epithelial healing in experimental corneal alkali wounds with nondiluted autologous serum eye drops[J].Cutan Ocul Toxicol,2010,29(2):116-121.
[3] SAIKA S.TGF-beta signal transduction in corneal wound healing as a therapeutic target[J].Cornea,2004,23(8 Suppl):S25-30.
[4] KOBAYASHI T,HATTORI S,SHINKAI H.Matrix metalloproteinases-2 and -9 are secreted from human fibroblasts[J].Acta Derm Venereol,2003,83(2):105-107.
[5] ZHAO J B,ZOU W J,FU X Y.Doxycycline inhibits the infiltration of inflammatory cells in rat alkali burn corneas[J].Int Eye Sci,2013,13(6):1090-1092.赵静博,邹文进,付馨余.多西环素对碱烧伤大鼠角膜炎症细胞浸润的抑制作用[J].国际眼科杂志,2013,13(6):1090-1092.
[6] ZOU W J,LIU Z G,LIU M L,FU X Y,ZHAO J B,WANG S,et al.Comparison of inhibitory effects of Doxycycline and Dexamethasone on corneal neovascularization in rats with corneal alkali burn[J].Rec Adv Ophthalmol,2013,33(8):701-704.邹文进,刘祖国,刘曼丽,付馨余,赵静博,王松,等.多西环素与地塞米松抑制碱烧伤大鼠角膜新生血管的对比研究[J].眼科新进展,2013,33(8):701-704.
[7] HOLLAND E J,CHAN C C,WETZIG R P,PALESTINE A G,NUSSENBLATT R B.Clinical and immunohistologic studies of corneal rejection in the rat penetrating keratoplasty model[J].Cornea,1991,10(5):374-380.
[8] SAIKA S,IKEDA K,YAMANAKA O,MIYAMOTO T,OHNISHI Y,SATO M,et al.Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali[J].Am J Pathol,2005,166(5):1405-1418.
[9] CHENG W,XU R,LI D,BORTOLINI C,HE J,DONG M,et al.Artificial extracellular matrix delivers TGF-β1 regulating myofibroblast differentiation[J].RSC Adv,2016,6(26):21922-21928.
[10] TOVEY J,GRONKIEWICZ K M,GIULIANO E A,SIDDIQUI S,SHARMA A,BROOKE J L,et al.Role of cellular kinases and Smads in the modulation of SAHA-mediated corneal fibrosis inhibition[J].Curr Opin Infect Dis,2014,3(5):480-487.
[11] KLEIN T,BISCHOFF R.Physiology and pathophysiology of matrix metalloproteases[J].Amino Acids,2011,41(2):271-290.
[12] NISSJNEN L,K?H?VI V N.Matrix metalloproteinases in inflammation[J].BBA-General Subjects,2014,1840(8):2571-2580.
[13] SLANSKY H H,FREEMAN M I,ITOI M.Collagenolytic activity in bovine corneal epithelium[J].Arch Ophthalmol,1968,80(4):496-498.
[14] BROWN S I,AKIYA S,WELLER C A.Prevention of the ulcers of the alkali-burned cornea.Preliminary studies with collagenase inhibitors[J].Arch Ophthalmol,1969,82(1):95-97.
[15] SIVAK J M,FINI M E.MMPs in the eye:emerging roles for matrix metalloproteinases in ocular physiology[J].Prog Retin Eye Res,2002,21(1):1-14.
[16] YANG H H,KIM J M,CHUM E,VAN BREEMEN C,CHUNG A W.Effectiveness of combination of losartan potassium and doxycycline versus single-drug treatments in the secondary prevention of thoracic aortic aneurysm in Marfan syndrome[J].J Thorac Cardiovasc Surg,2010,140(2):305-312.
[17] PFLUGFELDER S C,FARLEY W,LUO L,CHEN L Z,DE PAIVA C S,OLMOS L C,et al.Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye[J].Am J Pathol,2005,166(1):61-71.
[18] KIM E C,KIM T K,PARK S H,KIM M S.The wound healing effects of vitamin A eye drops after a corneal alkali burn in rats[J].Acta Ophthalmol,2012,90(7):e540-e546.
[19] CORRALES R M,DE PAIVA C S,VILLARREAL A L,FARLEY W J,LI D Q,STERN M E,et al.Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression,MAPK activation in the corneal epithelium in experimental dry eye[J].Exp Eye Res,2006,83(3):526-535.
[20] KIM H S,LUO L,PFLUGFELDER S C,LI D Q.Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells[J].Invest Ophthalmol Vis Sci,2005,46(3):840-848.
[2] SALMAN I A,GUNDOGDU C.Epithelial healing in experimental corneal alkali wounds with nondiluted autologous serum eye drops[J].Cutan Ocul Toxicol,2010,29(2):116-121.
[3] SAIKA S.TGF-beta signal transduction in corneal wound healing as a therapeutic target[J].Cornea,2004,23(8 Suppl):S25-30.
[4] KOBAYASHI T,HATTORI S,SHINKAI H.Matrix metalloproteinases-2 and -9 are secreted from human fibroblasts[J].Acta Derm Venereol,2003,83(2):105-107.
[5] ZHAO J B,ZOU W J,FU X Y.Doxycycline inhibits the infiltration of inflammatory cells in rat alkali burn corneas[J].Int Eye Sci,2013,13(6):1090-1092.赵静博,邹文进,付馨余.多西环素对碱烧伤大鼠角膜炎症细胞浸润的抑制作用[J].国际眼科杂志,2013,13(6):1090-1092.
[6] ZOU W J,LIU Z G,LIU M L,FU X Y,ZHAO J B,WANG S,et al.Comparison of inhibitory effects of Doxycycline and Dexamethasone on corneal neovascularization in rats with corneal alkali burn[J].Rec Adv Ophthalmol,2013,33(8):701-704.邹文进,刘祖国,刘曼丽,付馨余,赵静博,王松,等.多西环素与地塞米松抑制碱烧伤大鼠角膜新生血管的对比研究[J].眼科新进展,2013,33(8):701-704.
[7] HOLLAND E J,CHAN C C,WETZIG R P,PALESTINE A G,NUSSENBLATT R B.Clinical and immunohistologic studies of corneal rejection in the rat penetrating keratoplasty model[J].Cornea,1991,10(5):374-380.
[8] SAIKA S,IKEDA K,YAMANAKA O,MIYAMOTO T,OHNISHI Y,SATO M,et al.Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali[J].Am J Pathol,2005,166(5):1405-1418.
[9] CHENG W,XU R,LI D,BORTOLINI C,HE J,DONG M,et al.Artificial extracellular matrix delivers TGF-β1 regulating myofibroblast differentiation[J].RSC Adv,2016,6(26):21922-21928.
[10] TOVEY J,GRONKIEWICZ K M,GIULIANO E A,SIDDIQUI S,SHARMA A,BROOKE J L,et al.Role of cellular kinases and Smads in the modulation of SAHA-mediated corneal fibrosis inhibition[J].Curr Opin Infect Dis,2014,3(5):480-487.
[11] KLEIN T,BISCHOFF R.Physiology and pathophysiology of matrix metalloproteases[J].Amino Acids,2011,41(2):271-290.
[12] NISSJNEN L,K?H?VI V N.Matrix metalloproteinases in inflammation[J].BBA-General Subjects,2014,1840(8):2571-2580.
[13] SLANSKY H H,FREEMAN M I,ITOI M.Collagenolytic activity in bovine corneal epithelium[J].Arch Ophthalmol,1968,80(4):496-498.
[14] BROWN S I,AKIYA S,WELLER C A.Prevention of the ulcers of the alkali-burned cornea.Preliminary studies with collagenase inhibitors[J].Arch Ophthalmol,1969,82(1):95-97.
[15] SIVAK J M,FINI M E.MMPs in the eye:emerging roles for matrix metalloproteinases in ocular physiology[J].Prog Retin Eye Res,2002,21(1):1-14.
[16] YANG H H,KIM J M,CHUM E,VAN BREEMEN C,CHUNG A W.Effectiveness of combination of losartan potassium and doxycycline versus single-drug treatments in the secondary prevention of thoracic aortic aneurysm in Marfan syndrome[J].J Thorac Cardiovasc Surg,2010,140(2):305-312.
[17] PFLUGFELDER S C,FARLEY W,LUO L,CHEN L Z,DE PAIVA C S,OLMOS L C,et al.Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye[J].Am J Pathol,2005,166(1):61-71.
[18] KIM E C,KIM T K,PARK S H,KIM M S.The wound healing effects of vitamin A eye drops after a corneal alkali burn in rats[J].Acta Ophthalmol,2012,90(7):e540-e546.
[19] CORRALES R M,DE PAIVA C S,VILLARREAL A L,FARLEY W J,LI D Q,STERN M E,et al.Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression,MAPK activation in the corneal epithelium in experimental dry eye[J].Exp Eye Res,2006,83(3):526-535.
[20] KIM H S,LUO L,PFLUGFELDER S C,LI D Q.Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells[J].Invest Ophthalmol Vis Sci,2005,46(3):840-848.