益气明目丸对视网膜色素变性大鼠视网膜Fas、FasL蛋白表达的影响
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摘要
目的探讨益气明目丸治疗视网膜色素变性的可能作用机制。方法 24只RCS大鼠随机分为空白组、模型组、益气明目丸组,每组8只。空白组RCS (rdy+/+,p+/+)大鼠(无视网膜色素变性)和模型组RCS (rdy-/,p-/-)大鼠(视网膜色素变性模型)均灌胃生理盐水12 ml/(kg·d),益气明目丸组RCS (rdy-/,p-/-)大鼠灌胃益气明目丸悬浊溶液7. 8 g/(kg·d)。灌胃30天后,HE染色观察各组大鼠视网膜各层结构,采用免疫组化法和Western blot法检测各组大鼠视网膜中Fas、FasL蛋白表达。结果HE染色显示,益气明目丸组大鼠视网膜厚度明显较模型组大鼠视网膜增厚,视网膜色素上皮条带部分可见,部分外核层光感受器感觉纤毛层部分可见,光感受器细胞核数目较模型组多,外从状层、外核层、视杆视锥层较模型组结构清晰且增厚。免疫组化法结果显示,与空白组比较,模型组Fas、FasL蛋白表达均明显升高(P <0. 01);与模型组比较,益气明目丸组Fas、FasL蛋白表达均明显下降(P <0. 05或P <0. 01),且与空白组比较差异均无统计学意义(P> 0. 05)。Western blot法结果显示,与空白组比较,模型组Fas、FasL蛋白表达均明显升高(P <0. 01);与模型组比较,益气明目丸组Fas蛋白表达差异无统计学意义(P>0. 05),FasL蛋白表达明显降低,但仍高于空白组(P <0. 01)。结论益气明目丸对视网膜色素变性大鼠视网膜的超微结构具有保护作用,其机制可能是通过抑制视网膜Fas、FasL蛋白表达,从而减轻视网膜感光细胞的凋亡,达到保护视细胞的目的。
Objective To explore the possible mechanism of Yiqi Mingmu Pills( 益气明目丸) in treatment of retinitis pigmentosa( RP). Methods A total of 24 RCS rats were randomly divided into blank group,model group and Yiqi Mingmu Pills group,with 8 rats in each group. The blank group RCS rats( rdy +/+,p +/+),and the model group RCS rats( rdy-/,p-/-) were given 12 ml/( kg·d) of normal saline by gavage. The Yiqi Mingmu Pills group RCS rats( rdy-/,p-/-) were given 7. 8 g/( kg ·d) of suspension turbid solution of Yiqi Mingmu Pills. After intragastric administration for 30 days,the structure of retina layers was observed by HE staining. Expressions of Fas and FasL in retinal tissues of each group were detected by immunohistochemistry and Western Blot method. Results HE staining showed that the retina thickness of rats in Yiqi Mingmu Pills group was significantly thicker than that of the model group. The retinal pigment epithelial band was partially visible,and part of the outer nuclear layer photoreceptor sensory ciliary layer was visible. The number of photoreceptor cell nuclei was higher than that of the model group. The outer layer,outer core layer,and the visual cone layer were clearer and thicker than the model group. The results of immunohistochemistry showed that the expression of Fas and FasL protein in the model group was significantly higher than that in the blank group( P < 0. 01). Compared with the model group,the expression of Fas and FasL protein in Yiqi Mingmu Pill group decreased significantly( P < 0. 05 or P < 0. 01),and there was no significant difference with the blank group( P > 0. 05). Western blot analysis showed that the expression of Fas and FasL protein in the model group was significantly higher than that in the blank group( P < 0. 01). Compared with the model group,the expression of Fas protein in Yiqi Mingmu Pill group was not statistically significant( P > 0. 05),and the expression of FasL protein was significantly lower,but was still higher than that of the blank group( P < 0. 01).Conclusion Yiqi Mingmu Pills has protective effects on the ultrastructure of RP retina. Yiqi Mingmu Pills can reduce the apoptosis of retinal photoreceptor cells and protect the visual cells by inhibiting the expression of Fas and Fas L on the retina.
Objective To explore the possible mechanism of Yiqi Mingmu Pills( 益气明目丸) in treatment of retinitis pigmentosa( RP). Methods A total of 24 RCS rats were randomly divided into blank group,model group and Yiqi Mingmu Pills group,with 8 rats in each group. The blank group RCS rats( rdy +/+,p +/+),and the model group RCS rats( rdy-/,p-/-) were given 12 ml/( kg·d) of normal saline by gavage. The Yiqi Mingmu Pills group RCS rats( rdy-/,p-/-) were given 7. 8 g/( kg ·d) of suspension turbid solution of Yiqi Mingmu Pills. After intragastric administration for 30 days,the structure of retina layers was observed by HE staining. Expressions of Fas and FasL in retinal tissues of each group were detected by immunohistochemistry and Western Blot method. Results HE staining showed that the retina thickness of rats in Yiqi Mingmu Pills group was significantly thicker than that of the model group. The retinal pigment epithelial band was partially visible,and part of the outer nuclear layer photoreceptor sensory ciliary layer was visible. The number of photoreceptor cell nuclei was higher than that of the model group. The outer layer,outer core layer,and the visual cone layer were clearer and thicker than the model group. The results of immunohistochemistry showed that the expression of Fas and FasL protein in the model group was significantly higher than that in the blank group( P < 0. 01). Compared with the model group,the expression of Fas and FasL protein in Yiqi Mingmu Pill group decreased significantly( P < 0. 05 or P < 0. 01),and there was no significant difference with the blank group( P > 0. 05). Western blot analysis showed that the expression of Fas and FasL protein in the model group was significantly higher than that in the blank group( P < 0. 01). Compared with the model group,the expression of Fas protein in Yiqi Mingmu Pill group was not statistically significant( P > 0. 05),and the expression of FasL protein was significantly lower,but was still higher than that of the blank group( P < 0. 01).Conclusion Yiqi Mingmu Pills has protective effects on the ultrastructure of RP retina. Yiqi Mingmu Pills can reduce the apoptosis of retinal photoreceptor cells and protect the visual cells by inhibiting the expression of Fas and Fas L on the retina.
引文
[1]JONES BW,PFEIFFER RL,FERRELL WD,et al. Retinal remodeling in human retinitis pigmentosa[J]. Exp Eye Res,2016,150:149-165. doi:10. 1016/j. exer. 2016. 03.018.
[2]WEISZ HUBSHMAN M,BROEKMAN S,VAN WIJK E,et al. Whole-exome sequencing reveals POC5 as a novel gene associated with autosomal recessive retinitis pigmentosa[J]. Hum Mol Genet,2018,27(4):614-624.
[3]PAWLYK BS,BULGAKOV OV,SUN X,et al. Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa[J]. Gene Ther,2016,23(2):196-204.
[4]VERBAKEL SK,VAN HUET RAC,BOON CJF,et al.Non-syndromic retinitis pigmentosa[J]. Prog Retin Eye Research 2018,66(9):157-186.
[5]FAHIM AT,DAIGER SP,WELEBER RG. Nonsyndromic retinitis pigmentosa overview[M]. Seattle:University of Washington,2017:1993-1995.
[6]VAN SCHIL K,KARLSTETTER M,ASLANIDIS A,et al. Autosomal recessive retinitis pigmentosa with homozygous rhodopsin mutation E150K and non-coding cis-regulatory variants in CRX-binding regions of SAMD7[J]. Sci Rep,2016,6:21307. doi:10. 1038/srep21307.
[7]DAIGER SP,SULLIVAN LS,BOWNE SJ. Genes and mutations causing retinitis pigmentosa[J]. Clin Genet,2013,84(2):132-141.
[8]胡小凤,黎晓新.视网膜色素变性的自然动物模型及人工造模[J].眼科研究,2007,25(2):157-160.
[9]罗永煌.药理学实验教程[M].北京:科学出版社,2015:9.
[10]彭清华,李传课.视网膜色素变性虚中夹瘀的机理研究小结[J].中国医药学报,1993,8(6):7-10,61.
[11]彭清华,罗萍,江晓芬,等.视网膜色素变性血淤机理的初步研究[J].国医论坛,1993(1):37-40.
[12]侯晓敏,秦小江.葛根素对大鼠离体冠状动脉血管环的舒张作用及其机制研究[J].中国药物与临床,2014,14(1):36-37.
[13]ISHIKAWA M,SAWADA Y,YOSHITOMI T. Structure and function of the interphotoreceptor matrix surrounding retinal photoreceptor cells[J]. Exp Eye Res,2015,133:3-18. doi:10. 1016/j. exer. 2015. 02. 017.
[14]MEIER P,FINCH A,EVAN G. Apoptosis in development[J]. Nature,2000,407(6805):796-801.
[15]叶河江,王莹,张露,等.针刺对感光细胞凋亡大鼠视网膜形态学的影响[J].辽宁中医杂志,2012,39(9):1857-1859.
[16]PARK JB,LEE JK,PARK EY,et al. Fas/Fas L interaction of nucleus pulposus and cancer cells with the activation of caspases[J]. Int Orthop,2008,32(6):835-840.
[17]ZHANG J,JIN P P,GONG M,et al. Roles of Fas/Fas L-mediated apoptosis and inhibin B in the testicular dysfunction of rats with left-side varicocele[J]. Andrologia,2018,50(2). doi:10. 1111/and. 12850.
[18]RISTIC T,DJORDJEVIC VB,DELJANIN-ILIC M,et al.Serum Fas/Fas L levels in dependence on clinical presentations of coronary disease and their relationship with risk factors[J]. Vojnosanit Pregl,2010,67(7):537-542.
[19]CARNEVALE G,CARPINO G,CARDINALE V,et al.Activation of Fas/Fas L pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells[J].Sci Rep,2017,7(1):14419. doi:10. 1038/s41598-017-14838-3.
[20]XIA HL,LI CJ,HOU XF,et al. Interferon-γaffects leukemia cell apoptosis through regulating Fas/Fas L signaling pathway[J]. Eur Rev Med Pharmacol Sci,2017,21(9):2244-2248.
[21]TAO H,LU L,XIA Y,et al. Antitumor effector B cells directly kill tumor cells via the Fas/Fas L pathway and are regulated by IL-10[J]. Eur J Immunol,2015,45(4):999-1009.
[22]HU X,HE D,ZHOU C,et al. Marginatoxin induces human hepatoma BEL-7402 cells apoptosis in vitro and in vivo via activation of Fas/Fas L-mediated apoptotic pathway[J]. Biomed Res,2017,28(3):1242-1246.
[23]ZHANG JF,SHI LL,ZHANG L,et al. MicroRNA-25negatively regulates cerebral ischemia/reperfusion injuryinduced cell apoptosis through Fas/Fas L pathway[J]. J Mol Neurosci,2016,58(4):507-516.
[24]MATSUMOTO H,MURAKAMI Y,KATAOKA K,et al.Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium[J]. Cell Death Dis,2015,6:e1986. doi:10. 1038/cddis. 2015. 334.
[25]徐剑.基于RHO、XBP1、Caspase12表达探讨枸杞、丹参对虚中夹瘀证RP模型大鼠的干预研究[D].长沙:湖南中医药大学,2016.
[26]BOURNE MC,CAMPBELL DA,TANSLEY K. Hereditary degeneration of the rat retina[J]. Br J Ophthalmol,1938,22(10):613-623.
[27]NEUHARDT TH,MAY CA,WILSCH C,et al. Morphological changes of retinal pigment epithelium and choroid in rd-mice[J]. Exp Eye Res,1999,68(1):75-83.
[28]LUE CL. Rod cell activity in retinal degenerative rats[J]. J Formos Med Assoc,1994,93(7):605-610.
[29]STRAUSS O,STUMPFF F,MERGLER S,et al. The Royal College of Surgeons rat:an animal model for inherited retinal degeneration with a still unknown genetic defect[J]. Acta Anat,1998,162(2-3):101-111.
[2]WEISZ HUBSHMAN M,BROEKMAN S,VAN WIJK E,et al. Whole-exome sequencing reveals POC5 as a novel gene associated with autosomal recessive retinitis pigmentosa[J]. Hum Mol Genet,2018,27(4):614-624.
[3]PAWLYK BS,BULGAKOV OV,SUN X,et al. Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa[J]. Gene Ther,2016,23(2):196-204.
[4]VERBAKEL SK,VAN HUET RAC,BOON CJF,et al.Non-syndromic retinitis pigmentosa[J]. Prog Retin Eye Research 2018,66(9):157-186.
[5]FAHIM AT,DAIGER SP,WELEBER RG. Nonsyndromic retinitis pigmentosa overview[M]. Seattle:University of Washington,2017:1993-1995.
[6]VAN SCHIL K,KARLSTETTER M,ASLANIDIS A,et al. Autosomal recessive retinitis pigmentosa with homozygous rhodopsin mutation E150K and non-coding cis-regulatory variants in CRX-binding regions of SAMD7[J]. Sci Rep,2016,6:21307. doi:10. 1038/srep21307.
[7]DAIGER SP,SULLIVAN LS,BOWNE SJ. Genes and mutations causing retinitis pigmentosa[J]. Clin Genet,2013,84(2):132-141.
[8]胡小凤,黎晓新.视网膜色素变性的自然动物模型及人工造模[J].眼科研究,2007,25(2):157-160.
[9]罗永煌.药理学实验教程[M].北京:科学出版社,2015:9.
[10]彭清华,李传课.视网膜色素变性虚中夹瘀的机理研究小结[J].中国医药学报,1993,8(6):7-10,61.
[11]彭清华,罗萍,江晓芬,等.视网膜色素变性血淤机理的初步研究[J].国医论坛,1993(1):37-40.
[12]侯晓敏,秦小江.葛根素对大鼠离体冠状动脉血管环的舒张作用及其机制研究[J].中国药物与临床,2014,14(1):36-37.
[13]ISHIKAWA M,SAWADA Y,YOSHITOMI T. Structure and function of the interphotoreceptor matrix surrounding retinal photoreceptor cells[J]. Exp Eye Res,2015,133:3-18. doi:10. 1016/j. exer. 2015. 02. 017.
[14]MEIER P,FINCH A,EVAN G. Apoptosis in development[J]. Nature,2000,407(6805):796-801.
[15]叶河江,王莹,张露,等.针刺对感光细胞凋亡大鼠视网膜形态学的影响[J].辽宁中医杂志,2012,39(9):1857-1859.
[16]PARK JB,LEE JK,PARK EY,et al. Fas/Fas L interaction of nucleus pulposus and cancer cells with the activation of caspases[J]. Int Orthop,2008,32(6):835-840.
[17]ZHANG J,JIN P P,GONG M,et al. Roles of Fas/Fas L-mediated apoptosis and inhibin B in the testicular dysfunction of rats with left-side varicocele[J]. Andrologia,2018,50(2). doi:10. 1111/and. 12850.
[18]RISTIC T,DJORDJEVIC VB,DELJANIN-ILIC M,et al.Serum Fas/Fas L levels in dependence on clinical presentations of coronary disease and their relationship with risk factors[J]. Vojnosanit Pregl,2010,67(7):537-542.
[19]CARNEVALE G,CARPINO G,CARDINALE V,et al.Activation of Fas/Fas L pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells[J].Sci Rep,2017,7(1):14419. doi:10. 1038/s41598-017-14838-3.
[20]XIA HL,LI CJ,HOU XF,et al. Interferon-γaffects leukemia cell apoptosis through regulating Fas/Fas L signaling pathway[J]. Eur Rev Med Pharmacol Sci,2017,21(9):2244-2248.
[21]TAO H,LU L,XIA Y,et al. Antitumor effector B cells directly kill tumor cells via the Fas/Fas L pathway and are regulated by IL-10[J]. Eur J Immunol,2015,45(4):999-1009.
[22]HU X,HE D,ZHOU C,et al. Marginatoxin induces human hepatoma BEL-7402 cells apoptosis in vitro and in vivo via activation of Fas/Fas L-mediated apoptotic pathway[J]. Biomed Res,2017,28(3):1242-1246.
[23]ZHANG JF,SHI LL,ZHANG L,et al. MicroRNA-25negatively regulates cerebral ischemia/reperfusion injuryinduced cell apoptosis through Fas/Fas L pathway[J]. J Mol Neurosci,2016,58(4):507-516.
[24]MATSUMOTO H,MURAKAMI Y,KATAOKA K,et al.Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium[J]. Cell Death Dis,2015,6:e1986. doi:10. 1038/cddis. 2015. 334.
[25]徐剑.基于RHO、XBP1、Caspase12表达探讨枸杞、丹参对虚中夹瘀证RP模型大鼠的干预研究[D].长沙:湖南中医药大学,2016.
[26]BOURNE MC,CAMPBELL DA,TANSLEY K. Hereditary degeneration of the rat retina[J]. Br J Ophthalmol,1938,22(10):613-623.
[27]NEUHARDT TH,MAY CA,WILSCH C,et al. Morphological changes of retinal pigment epithelium and choroid in rd-mice[J]. Exp Eye Res,1999,68(1):75-83.
[28]LUE CL. Rod cell activity in retinal degenerative rats[J]. J Formos Med Assoc,1994,93(7):605-610.
[29]STRAUSS O,STUMPFF F,MERGLER S,et al. The Royal College of Surgeons rat:an animal model for inherited retinal degeneration with a still unknown genetic defect[J]. Acta Anat,1998,162(2-3):101-111.