厄洛替尼对非增生型糖尿病视网膜病变的治疗作用及机制的实验研究

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

目的探讨表皮生长因子受体(EGFR)抑制剂厄洛替尼对非增生型糖尿病视网膜病变(NPDR)的治疗作用及机制。方法实验研究。人视网膜Müller细胞(RMC)为Moorfields眼科医院和伦敦大学学院眼科学研究所-Müller 1(MIO-M1)细胞。将MIO-M1细胞分为正常对照、高渗、高糖、高糖+二甲基亚砜(DMSO)、高糖+厄洛替尼0.5 mmol/L、高糖+厄洛替尼1 mmol/L和高糖+厄洛替尼2 mmol/L组。5-乙炔-2′-脱氧尿苷(EdU)掺入实验检测厄洛替尼对高糖条件下MIO-M1细胞增殖的影响;Western印迹检测厄洛替尼对高糖条件下MIO-M1细胞活化标记胶质纤维酸性蛋白(GFAP)和谷氨酰胺合成酶(GS)蛋白水平的影响。Western印迹检测厄洛替尼对高糖条件下RMC中p75神经生长因子受体(p75NTR)、波形蛋白和细胞视黄醛结合蛋白(CRALBP)蛋白水平的影响。将MIO-M1细胞分为正常对照、高糖、高糖+DMSO和高糖+厄洛替尼1 mmol/L组,采用细胞免疫荧光染色检测厄洛替尼对高糖条件下EGFR核转位的影响;免疫共沉淀检测厄洛替尼对高糖条件下MIO-M1细胞中EGFR与转录中间因子2(TIF2)之间相互作用的影响。将MIO-M1细胞分为正常对照、高糖、高糖+DMSO、高糖+Myc-DDK空载体、高糖+厄洛替尼、高糖+厄洛替尼+人双调蛋白、高糖+厄洛替尼+TIF2质粒和高糖+厄洛替尼+人双调蛋白+TIF2质粒组,采用细胞免疫荧光染色检测厄洛替尼通过EGFR/TIF2轴对高糖条件下MIO-M1细胞中EGFR与TIF2结合的影响。采用定量实时逆转录聚合酶链反应(qRT-PCR)检测通过影响高糖条件下MIO-M1细胞中EGFR与TIF2结合对细胞周期蛋白D1(Cyclin D1)转录的调节作用。构建小鼠糖尿病视网膜病变(DR)模型,采用随机数字表法,按照不同喂养方式,分为正常对照、DR、DR+DMSO、DR+厄洛替尼0.25 mg·kg^(-1)·d^(-1)、DR+厄洛替尼0.5 mg·kg^(-1)·d^(-1)和DR+厄洛替尼1 mg·kg^(-1)·d^(-1)组,共25只小鼠,每组5只。组织免疫荧光染色检测小鼠RMC活化标记GFAP的表达水平;FITC-葡聚糖注射实验检测厄洛替尼对小鼠DR模型中视网膜血管渗漏的影响。结果与正常对照组(32.4%±3.0%)相比,高糖组(59.2%±3.8%)RMC中EdU阳性细胞的比例增多(P<0.001)。与高糖组(59.2%±3.8%)相比,高糖+厄洛替尼1 mmol/L组(37.6%±4.4%)中EdU阳性细胞比例(P<0.001)下降。与正常对照组相比,高糖组RMC中GFAP表达增高(正常对照组为1,高糖组为2.27±0.11,P<0.001),GS表达降低(正常对照组为1,高糖组为0.32±0.03,P<0.001)。厄洛替尼1 mmol/L处理降低高糖条件下RMC中GFAP的表达(分别为1.32±0.13和2.27±0.11,P<0.001),升高GS的表达(分别为0.71±0.06和0.32±0.03,P<0.001)。高糖+厄洛替尼1 mmol/L组RMC中EGFR与4′,6-二氨基-2-苯基吲哚(DAPI)的共定位比例低于高糖组(分别为52.2%±4.1%和76.4%±5.7%,P<0.001)。用EGF或TIF2抗体沉淀TIF2或EGFR,两者在高糖条件下相对于正常对照组均升高(正常对照组为1,高糖组TIF2为2.27±0.20,EGFR为2.17±0.21;均P<0.05),并且高糖+厄洛替尼组TIF2(1.38±0.10)或EGFR(1.32±0.13)表达低于高糖组TIF2(2.27±0.20)和高糖组EGFR(2.17±0.21)(均P<0.05)。高糖+厄洛替尼组RMC中EGFR与TIF2共定位比例(17.2%±3.9%)及Cyclin D1 mRNA水平(1.32±0.16)均低于高糖组(EGFR与TIF2共定位比例为54.6%±3.7%,Cyclin D1 mRNA水平为2.58±0.19,均P<0.05),高糖+厄洛替尼+AREG组、高糖+厄洛替尼+Myc-DDK-TIF2质粒组和高糖+厄洛替尼+AREG+Myc-DDK-TIF2质粒组EGFR与TIF2共定位比例(分别为24.1%±1.9%、26.0%±2.3%、35.3%±2.5%)及TIF2 mRNA水平(分别为1.71±0.16、1.72±0.18、2.20±0.18)均高于高糖+厄洛替尼组(均P<0.05)。相对于正常对照组,小鼠视网膜组织中GFAP表达在DR组中增高(正常对照组为1,DR组为3.07±0.19,P<0.001),厄洛替尼0.5 mg·kg^(-1)·d^(-1)处理可(1.73±0.30)显著降低DR组小鼠视网膜中GFAP的表达(P<0.05)。相对于正常对照组(3.97±0.47),DR组(23.13±2.15)荧光素渗漏增加,DR+厄洛替尼组(11.66±1.45)与DR组相比,渗漏显著降低(均P<0.05)。结论厄洛替尼抑制高糖诱导的人RMC增殖和活化,抑制RMC中EGFR入细胞核,抑制RMC中EGFR与TIF2结合,并且通过抑制EGFR与TIF2相互作用,降低RMC中Cyclin D1的转录。厄洛替尼抑制小鼠DR模型中RMC的增殖和活化,改善小鼠视网膜血管渗漏。厄洛替尼通过下调高糖条件下RMC中的EGFR/TIF2/Cyclin D1通路,抑制RMC的活化和增殖,从而缓解NPDR的进展。

Objective To investigate the therapeutic effect and mechanism of erlotinib,an epidermal growth factor receptor(EGFR)inhibitor,on non-proliferative diabetic retinopathy(NPDR).Methods An experimental research was conducted.Human retinal Müller cells(RMC)were MIO-M1 cells from Moorfields Ophthalmology Hospital and the Institute of Ophthalmology at London University College.MIO-M1 cells were divided into normal,hypertonic,high glucose,high glucose+dimethyl sulfoxide(DMSO),high glucose+erlotinib 0.5 mmol/L,high glucose+erlotinib 1 mmol/L,and high glucose+erlotinib 2 mmol/L groups using a random number table method.Detection of the effect of erlotinib on the proliferation of MIO-M1 cells under high glucose conditions was performed by 5-ethynyl-2′-deoxyuridine(EdU)method.Western blotting(WB)was used to detect the effect of erlotinib on the activation markers of glial fibrillary acidic protein(GFAP)and glutamine synthetase(GS)protein levels in MIO-M1 cells under high glucose conditions.WB was used to detect the effect of erlotinib on the protein levels of nerve growth factor receptor(p75NTR),vimentin,and cell retinol binding protein(CRALBP)in RMC under high glucose conditions.MIO-M1 cells were divided into normal group,high glucose group,high glucose+DMSO group,and high glucose+erlotinib(1 mmol/L)group using random number table method.The effect of erlotinib on EGFR nuclear translocation under high glucose conditions was detected by cell immunofluorescence staining.Immunoprecipitation was used to detect the effect of erlotinib on the interaction between EGFR and transcription intermediate factor 2(TIF2)in MIO-M1 cells under high glucose conditions.MIO-M1 cells were randomly divided into normal group,high glucose group,high glucose+DMSO group,high glucose+Myc-DDK empty body group,high glucose+erlotinib group,high glucose+erlotinib+human doublet protein group,high glucose+erlotinib+TIF2 plasmid group,and high glucose+erlotinib+human doublet protein+TIF2 plasmid group.Cell immunofluorescence staining was used to detect the effect of erlotinib on the binding of EGFR and TIF2 in MIO-M1 cells under high glucose conditions through the EGFR/TIF2 axis.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the regulatory effect of EGFR and TIF2 binding on cyclin D1 transcription in MIO-M1 cells under high glucose conditions.The mouse model of diabetes retinopathy(DR)was constructed and divided into normal group,DR group,DR+DMSO group,DR+erlotinib 0.25 mg·kg^(-1)·d^(-1)group,DR+erlotinib 0.5 mg·kg^(-1)·d^(-1)group and DR+erlotinib 1 mg·kg^(-1)·d^(-1)group.25 mice in total,5 in each group.Tissue immunofluorescence staining was used to detect the expression of RMC activation marker GFAP.The FITC-dextran injection experiment was used to detect the effect of erlotinib on retinal vascular leakage in a murine DR model.Results Compared with the normal group(32.4%±3.0%),the proportion of EdU positive cells in RMC in the high glucose group(59.2%±3.8%)increased(P<0.001).Compared with the high glucose group(59.2%±3.8%),the proportion of EdU positive cells in the high glucose+1 mmol/L erlotinib group(37.6%±4.4%)decreased(P<0.001).Compared with the normal group,the expression of GFAP in RMC in the high glucose group increased(1 in the normal group,2.27±0.11 in the high glucose group,P<0.001),while the expression of GS decreased(1 in the normal group,0.32±0.03 in the high glucose group,P<0.001).1 mmol/L erlotinib treatment reduced the expression of GFAP in RMC under high glucose conditions(1.32±0.13 and 2.27±0.11,respectively;P<0.001),and increased the expression of GS(0.71±0.06 and 0.32±0.03,respectively;P<0.001).The colocalization of EGFR and DAPI in RMC of the high glucose+1 mmol/L erlotinib group was lower than that of the high glucose group(52.2%±4.1%and 76.4%±5.7%,respectively;P<0.001).The expression of TIF2 or EGFR both increased while using EGF or TIF2 antibodies to precipitate TIF2 or EGFR under high glucose conditions compared to the normal group(1 in the normal group,2.27±0.20 in the high glucose group,2.17±0.21 in the EGFR,all P<0.05).And the expression of TIF2(1.38±0.10)or EGFR(1.32±0.13)in the high glucose+erlotinib group was lower than that in the high glucose group(2.27±0.20)and the high glucose group(2.17±0.21)(all P<0.05).The colocalization of EGFR and TIF2(17.2%±3.9%)and the mRNA level of Cyclin D1(1.32±0.16)in the RMC of the high glucose+erlotinib group were lower than those in the high glucose group(54.6%±3.7%of EGFR and TIF2 colocalization ratio,2.58±0.19 of Cyclin D1 mRNA level,all P<0.05).The high glucose+erlotinib+AREG(EGFR agonist)group,high glucose+erlotinib+Myc DDK-TIF2 plasmid group and high sugar+erlotinib+AREG+Myc-DDK-TIF2 plasmid group EGFR colocalization with TIF2(colocalization ratios 24.1%±1.9%,26.0%±2.3%,35.3%±2.5%)and TIF2 mRNA levels(1.71±0.16,1.72±0.18,2.20±0.18).Compared with the high glucose+erlotinib group,The increases were statistically significant(all P<0.05).Compared to the normal group,the expression of GFAP in mouse retina tissue was increased in the DR group(1 in the normal group,3.07±0.19 in the DR group,P<0.001),and 0.5 mg·kg^(-1)·d^(-1)erlotinib(1.73±0.30)significantly reduced the expression of GFAP in the retina of DR group mice(P<0.05).Compared to the normal group(3.97±0.47),the DR group(23.13±2.15)showed an increase in fluorescein leakage,while the DR+erlotinib group(11.66±1.45)showed a significant decrease in leakage compared to the DR group(all P<0.05).Conclusions Erlotinib inhibits the proliferation and activation of RMC induced by high glucose,inhibits the entry of EGFR into the nucleus,inhibits the binding of EGFR to TIF2 in RMC,and reduces the transcription of Cyclin D1 in RMC by inhibiting the interaction between EGFR and TIF2.At the same time,erlotinib inhibits the proliferation and activation of RMC in the mouse DR model,ameliorating retinal vascular leakage in mice.These results suggest that erlotinib inhibits the activation and proliferation of RMC by downregulating the EGFR/TIF2/Cyclin D1 pathway under high glucose conditions,thereby alleviating the progression of NPDR.