促红细胞生成素/拟似物,衍生物%氨甲酰促红细胞生成素%神经保护剂%糖尿病/并发症%糖尿病视网膜病变/防控%SD大鼠%视网膜神经节细胞%凋亡%异源二聚体受体/CD131受体
促紅細胞生成素/擬似物,衍生物%氨甲酰促紅細胞生成素%神經保護劑%糖尿病/併髮癥%糖尿病視網膜病變/防控%SD大鼠%視網膜神經節細胞%凋亡%異源二聚體受體/CD131受體
촉홍세포생성소/의사물,연생물%안갑선촉홍세포생성소%신경보호제%당뇨병/병발증%당뇨병시망막병변/방공%SD대서%시망막신경절세포%조망%이원이취체수체/CD131수체
Erythropoietin/analog & derivative%Carbamylated erythropoietin%Neuroprotective agent%Diabedic mellitum/complication%Diabetic retinopathy/prevention & control%Rat,Sprague-Dawley%Retinal ganglion cell%Apoptosis%Heterologous dimmers receptor/CD131 receptor
背景 近年来糖尿病视网膜病变(DR)的研究聚焦于药物治疗.促红细胞生成素(EPO)能减少DR神经组织的结构和功能损伤,但具有促进视网膜新生血管形成的潜在危险.氨甲酰EPO(CEPO)具有相同的神经保护作用,且无促进新生血管形成的作用,但其神经保护作用尚未在眼部疾病,尤其是DR中得到证实.目的 比较CEPO与EPO在抗DR的神经成分和血管成分中的作用及机制.方法 用化学合成法制备EPO衍生物CEPO.将60只清洁级雄性SD大鼠按随机数字表法分为正常对照组、DM组、DM+ EPO组和DM+CEPO组,用链脲佐菌素(STZ)60 mg/kg腹腔内注射建立大鼠DM模型,而正常对照组大鼠腹腔内注射等体积枸橼酸-枸橼酸钠缓冲液.每周监测大鼠血糖.造模后4周,DM+EPO组和DM+CEPO组大鼠分别腹腔内注射50 μg/kg EPO和CEPO,正常对照组和DM组大鼠均采用等量双蒸水腹腔内注射.干预后2周对各组大鼠进行视网膜电图(ERG)检查,然后处死大鼠摘除眼球制备视网膜标本,分别进行视网膜组织学检查,采用TUNEL法检测视网膜神经节细胞(RGCs)凋亡情况,采用实时荧光定量PCR和Western blot法分别检测各组大鼠视网膜中异源二聚体受体(CD131)、EPO受体(EPO-R)、胸腺细胞分化抗原-1(Thy-1)、胶质纤维酸性蛋白(GFAP)和血管内皮生长因子(VEGF) mRNA及其蛋白质的表达.结果 合成产物为纯度较高的CEPO.造模后至药物干预后2周,DM组、DM+EPO组和DM+CEPO组大鼠体质量明显低于正常对照组,而血糖水平明显高于正常对照组,4个组间差异均有统计学意义(F=49.39、455.91,均P=0.00);DM组大鼠ERG OPs振幅较正常对照组大鼠明显下降,差异有统计学意义(P=0.03),而DM+ EPO组与DM+CEPO组OPs波振幅与正常对照组大鼠比较差异均无统计学意义(P=0.55、0.49);4个组间大鼠ERG a波、b波振幅的差异均无统计学意义(F=0.30、1.12,P＞0.05).与正常对照组大鼠比较,DM组大鼠视网膜组织厚度变薄,RGCs计数减少,视网膜中TUNEL染色阳性细胞增多,而DM+ EPO组和DM+CEPO组大鼠视网膜全层厚度、内丛状层(IPL)、内核层(INL)厚度均明显薄于DM组大鼠,4个组间差异均有统计学意义(F=61.23、23.35、13.33,均P=0.00),RGCs数目明显多于DM组,4个组间差异有统计学意义(F=15.64,P=0.00).DM组大鼠视网膜中Thy-1 mRNA(2-ΔΔCt)及其蛋白表达量(A值)明显低于正常对照组,DM+EPO组和DM+CEPO组大鼠视网膜中Thy-1 mRNA及其蛋白表达量明显高于DM组,差异均有统计学意义(P＜0.05).DM组大鼠视网膜中GFAP mRNA(2-ΔΔCt)及其蛋白表达量(A值)明显升高于正常对照组大鼠,DM+EPO组和DM+CEPO组大鼠视网膜中GFAP mRNA及其蛋白表达量明显低于DM组,差异均有统计学意义(P＜0.05).DM+CEPO和DM+EPO组间大鼠视网膜Thy-1或GFAP表达量差异均无统计学意义(P＞0.05).DM+EPO组大鼠视网膜中EPOR mRNA及其蛋白表达量明显高于其他各组,而DM+CEPO组大鼠视网膜中CD131 mRNA及其蛋白表达量明显升高,差异均有统计学意义(P＜0.05).在DM组和DM+EPO组大鼠VEGF mRNA及其蛋白表达量均明显高于正常对照组大鼠,DM+CEPO组大鼠视网膜中VEGF mRNA及其蛋白表达量明显低于DM+EPO组,差异均有统计学意义(P＜0.05).结论 CEPO在防治DR患者视网膜神经细胞损伤中的作用与EPO相似,且不存在EPO的促新生血管形成的作用.CEPO可能通过CD131受体发挥视网膜神经保护作用.
揹景 近年來糖尿病視網膜病變(DR)的研究聚焦于藥物治療.促紅細胞生成素(EPO)能減少DR神經組織的結構和功能損傷,但具有促進視網膜新生血管形成的潛在危險.氨甲酰EPO(CEPO)具有相同的神經保護作用,且無促進新生血管形成的作用,但其神經保護作用尚未在眼部疾病,尤其是DR中得到證實.目的 比較CEPO與EPO在抗DR的神經成分和血管成分中的作用及機製.方法 用化學閤成法製備EPO衍生物CEPO.將60隻清潔級雄性SD大鼠按隨機數字錶法分為正常對照組、DM組、DM+ EPO組和DM+CEPO組,用鏈脲佐菌素(STZ)60 mg/kg腹腔內註射建立大鼠DM模型,而正常對照組大鼠腹腔內註射等體積枸櫞痠-枸櫞痠鈉緩遲液.每週鑑測大鼠血糖.造模後4週,DM+EPO組和DM+CEPO組大鼠分彆腹腔內註射50 μg/kg EPO和CEPO,正常對照組和DM組大鼠均採用等量雙蒸水腹腔內註射.榦預後2週對各組大鼠進行視網膜電圖(ERG)檢查,然後處死大鼠摘除眼毬製備視網膜標本,分彆進行視網膜組織學檢查,採用TUNEL法檢測視網膜神經節細胞(RGCs)凋亡情況,採用實時熒光定量PCR和Western blot法分彆檢測各組大鼠視網膜中異源二聚體受體(CD131)、EPO受體(EPO-R)、胸腺細胞分化抗原-1(Thy-1)、膠質纖維痠性蛋白(GFAP)和血管內皮生長因子(VEGF) mRNA及其蛋白質的錶達.結果 閤成產物為純度較高的CEPO.造模後至藥物榦預後2週,DM組、DM+EPO組和DM+CEPO組大鼠體質量明顯低于正常對照組,而血糖水平明顯高于正常對照組,4箇組間差異均有統計學意義(F=49.39、455.91,均P=0.00);DM組大鼠ERG OPs振幅較正常對照組大鼠明顯下降,差異有統計學意義(P=0.03),而DM+ EPO組與DM+CEPO組OPs波振幅與正常對照組大鼠比較差異均無統計學意義(P=0.55、0.49);4箇組間大鼠ERG a波、b波振幅的差異均無統計學意義(F=0.30、1.12,P＞0.05).與正常對照組大鼠比較,DM組大鼠視網膜組織厚度變薄,RGCs計數減少,視網膜中TUNEL染色暘性細胞增多,而DM+ EPO組和DM+CEPO組大鼠視網膜全層厚度、內叢狀層(IPL)、內覈層(INL)厚度均明顯薄于DM組大鼠,4箇組間差異均有統計學意義(F=61.23、23.35、13.33,均P=0.00),RGCs數目明顯多于DM組,4箇組間差異有統計學意義(F=15.64,P=0.00).DM組大鼠視網膜中Thy-1 mRNA(2-ΔΔCt)及其蛋白錶達量(A值)明顯低于正常對照組,DM+EPO組和DM+CEPO組大鼠視網膜中Thy-1 mRNA及其蛋白錶達量明顯高于DM組,差異均有統計學意義(P＜0.05).DM組大鼠視網膜中GFAP mRNA(2-ΔΔCt)及其蛋白錶達量(A值)明顯升高于正常對照組大鼠,DM+EPO組和DM+CEPO組大鼠視網膜中GFAP mRNA及其蛋白錶達量明顯低于DM組,差異均有統計學意義(P＜0.05).DM+CEPO和DM+EPO組間大鼠視網膜Thy-1或GFAP錶達量差異均無統計學意義(P＞0.05).DM+EPO組大鼠視網膜中EPOR mRNA及其蛋白錶達量明顯高于其他各組,而DM+CEPO組大鼠視網膜中CD131 mRNA及其蛋白錶達量明顯升高,差異均有統計學意義(P＜0.05).在DM組和DM+EPO組大鼠VEGF mRNA及其蛋白錶達量均明顯高于正常對照組大鼠,DM+CEPO組大鼠視網膜中VEGF mRNA及其蛋白錶達量明顯低于DM+EPO組,差異均有統計學意義(P＜0.05).結論 CEPO在防治DR患者視網膜神經細胞損傷中的作用與EPO相似,且不存在EPO的促新生血管形成的作用.CEPO可能通過CD131受體髮揮視網膜神經保護作用.
배경 근년래당뇨병시망막병변(DR)적연구취초우약물치료.촉홍세포생성소(EPO)능감소DR신경조직적결구화공능손상,단구유촉진시망막신생혈관형성적잠재위험.안갑선EPO(CEPO)구유상동적신경보호작용,차무촉진신생혈관형성적작용,단기신경보호작용상미재안부질병,우기시DR중득도증실.목적 비교CEPO여EPO재항DR적신경성분화혈관성분중적작용급궤제.방법 용화학합성법제비EPO연생물CEPO.장60지청길급웅성SD대서안수궤수자표법분위정상대조조、DM조、DM+ EPO조화DM+CEPO조,용련뇨좌균소(STZ)60 mg/kg복강내주사건립대서DM모형,이정상대조조대서복강내주사등체적구연산-구연산납완충액.매주감측대서혈당.조모후4주,DM+EPO조화DM+CEPO조대서분별복강내주사50 μg/kg EPO화CEPO,정상대조조화DM조대서균채용등량쌍증수복강내주사.간예후2주대각조대서진행시망막전도(ERG)검사,연후처사대서적제안구제비시망막표본,분별진행시망막조직학검사,채용TUNEL법검측시망막신경절세포(RGCs)조망정황,채용실시형광정량PCR화Western blot법분별검측각조대서시망막중이원이취체수체(CD131)、EPO수체(EPO-R)、흉선세포분화항원-1(Thy-1)、효질섬유산성단백(GFAP)화혈관내피생장인자(VEGF) mRNA급기단백질적표체.결과 합성산물위순도교고적CEPO.조모후지약물간예후2주,DM조、DM+EPO조화DM+CEPO조대서체질량명현저우정상대조조,이혈당수평명현고우정상대조조,4개조간차이균유통계학의의(F=49.39、455.91,균P=0.00);DM조대서ERG OPs진폭교정상대조조대서명현하강,차이유통계학의의(P=0.03),이DM+ EPO조여DM+CEPO조OPs파진폭여정상대조조대서비교차이균무통계학의의(P=0.55、0.49);4개조간대서ERG a파、b파진폭적차이균무통계학의의(F=0.30、1.12,P＞0.05).여정상대조조대서비교,DM조대서시망막조직후도변박,RGCs계수감소,시망막중TUNEL염색양성세포증다,이DM+ EPO조화DM+CEPO조대서시망막전층후도、내총상층(IPL)、내핵층(INL)후도균명현박우DM조대서,4개조간차이균유통계학의의(F=61.23、23.35、13.33,균P=0.00),RGCs수목명현다우DM조,4개조간차이유통계학의의(F=15.64,P=0.00).DM조대서시망막중Thy-1 mRNA(2-ΔΔCt)급기단백표체량(A치)명현저우정상대조조,DM+EPO조화DM+CEPO조대서시망막중Thy-1 mRNA급기단백표체량명현고우DM조,차이균유통계학의의(P＜0.05).DM조대서시망막중GFAP mRNA(2-ΔΔCt)급기단백표체량(A치)명현승고우정상대조조대서,DM+EPO조화DM+CEPO조대서시망막중GFAP mRNA급기단백표체량명현저우DM조,차이균유통계학의의(P＜0.05).DM+CEPO화DM+EPO조간대서시망막Thy-1혹GFAP표체량차이균무통계학의의(P＞0.05).DM+EPO조대서시망막중EPOR mRNA급기단백표체량명현고우기타각조,이DM+CEPO조대서시망막중CD131 mRNA급기단백표체량명현승고,차이균유통계학의의(P＜0.05).재DM조화DM+EPO조대서VEGF mRNA급기단백표체량균명현고우정상대조조대서,DM+CEPO조대서시망막중VEGF mRNA급기단백표체량명현저우DM+EPO조,차이균유통계학의의(P＜0.05).결론 CEPO재방치DR환자시망막신경세포손상중적작용여EPO상사,차불존재EPO적촉신생혈관형성적작용.CEPO가능통과CD131수체발휘시망막신경보호작용.
Background Recently the study on diabetic retinopathy (DR) primarily fucoses on the medication research.Erythropoietin (EPO) has the neuroprotective role on retinal neural cells in DR,but it is an important vasculogenesis growth factor.Carbamylated erythropoietin (CEPO) is proved to have the similar neuroprotection to EPO,what is more,CEPO have no the vasculogenesis effect.However,the neural protection of CEPO has not been verified in DR.Objecctive This study was to investigate the effects of CEPO on DR and compare the differences of protective effects between CEPO and EPO in DR and further to elucidate the underlying mechanism.Methods CEPO,the derivation of EPO,was synthesized by chemical synthesis and evaluated by chromatography,enzyme digestion,SDS-PAGE electrophoresis and silver nitrate dye.Sixty eight-week-old clean male Sprague-Dawley rats were assigned to four groups according to the random number table.Diabetic mellitus (DM) models were established by intraperitoneal injection of 60 mg/kg streptozotocin (STZ) in the rats of the DM group,DM+EPO group and DM+CEPO group,and equivate volume of citric acid-sodium citrate buffer solution was used in the same way in the normal control group.Four weeks later,50 μg/kg EPO or CEPO solution was intraperitoneal injection in the rats of the DM +EPO group and DM+CEPO group,respectively,and retinal examinations were performed 2 weeks after intervention.Retinal founction was evaluated by electroretinogran (ERG),and then the rats were sacrificed.Retinal samples were prepared for the histopathological examination.Retinal gangline cells (RGCs) apoptosis was detected by TUNEL assay.Real-time PCR and Western blot were used to assyed the expressions of betacommon receptor (CD131),EPO receptor (EPO-R),Thy-1,glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) in retina in protein and genetic levels.Results Purified CEPO was obtained by chemical synthesis.The body wight was significantly lower and the blood glucose level was higher in the rats of the DM group,DM+EPO group and DM +CEPO group than that in the normal control group 2 weeks after modeling,with significant difference among the four groups (F =49.39,455.91,all at P =0.00).No significant differences were found in a amplitude and b amplitude among the groups(F=0.30,1.12,both at P＞0.05),but the oscillatory potentials (OPs) amplitude was signicanntly reduced in the DM group compared to the normal control group (P=0.03),but the OPs amplitudes were not significantly different between DM + EPO group and DM + CEPO group compared with the normal control group (P =0.55,0.49).Compared with the normal control group,retinas were thiner and RGCs were decreased,and the positive cells for TUNEL assay were significantly increased in the DM group.However,the thicknesses of entire retinas,inner plexiform layer and inner nuclear layer were reduced in comparison with the normal control group,with significant differences among the groups (F=61.23,23.35,13.33,all at P=0.00),but the number of RGCs was significantly larger in the DM+EPO group and DM+CEPO group compared with DM group (F=15.64,P =0.00).The expressions of Thy-1 mRNA (2 ΔΔCt) and protein (A value) in DM group,and the expressing levels were increseased in the DM +EPO group and DM +CEPO group (P＜0.05).The expressions of GFAP mRNA (2-ΔΔCt) and its protein (A) were significantly downregulated in the DM+EPO group and DM+CEPO group compared with DM group (P＜0.05).The EPOR mRNA and protein expressions in the retinas showed the highest levels in DM+EPO group,and those of CD131 mRNA and protein were highest in DM +CEPO group (P＜0.05).The VEGF expressions in the protein and genetic levels were significantly higher in DM group and DM+EPO group that those of DM+CEPO group,showing significant differences among the groups (all at P＜0.05).Conclusions CEPO shows a similar protective effect on retinal neurons in DR without promorting angiogenesis.CEPO might exert its neuroprotective effect through CD131 receptor,which is different from EPO-EPOR mechanism.
