| Part One Peripapillary and macular vessel density in eyes with different phases of Thyroid-associated ophthalmopathyPurpose:Coherence tomography angiography(OCTA)was used to analyze the characteristics of vessel density in the optic disc and macular area of patients with different phases of Thyroid-associated ophthalmopathy(TAO),and their correlation with visual function.Methods:This case-control study was conducted at the Department of Ophthalmology of Peking Union Medical College Hospital between June 2019 and September 2019.TAO patients and healthy volunteers were included in the study.Patients with Clinical activity score(CAS)greater than or equal to 3 points were categorized as active TAO.Dysthyroid optic neuropathy(DON)patients with a course less than 6 months were categorized as acute phase of DON,and those more than 6 months were chronic group.Healthy volunteers were included in the control group.In this study,each group included 12 subjects,only right eyes were used for analysis.All participants underwent comprehensive ophthalmic examination including best corrected visual acuity(BCVA),non-contact intraocular pressure(NCT),slit lamp examination,small pupil fundus examination,axial measurement and visual field examination(VF).VF analyzed mean defect(MD).Optical coherence tomography was used to measure the thickness of retinal nerve fiber layers(RNFL)and retinal ganglion cell complex(GCC).Optical coherence tomography angiography(OCTA)was used to measure the parameters of optic disc,peripapillary and macular vessel density.BCVA was subsequently converted to Logarithm of minimum angle of resolution(LogMAR).The differences in the vessel densities in the optic disc and macular area between the groups and their correlation with different factors were analyzed.Analysis of variance(ANOVA),non-parametric Mann-Whitney U test and Spearman coefficient were conducted for statistical analysis.Results:A total of 36 patients were included,with 12 patients in each TAO or DON group.12 participants were enrolled in control group.The age range of the four groups was(45±11)years old.There was no statistically significant difference in age among the four groups of subjects(all P>0.05).There were 6 males and 6 females in each group.The LogMAR of acute DON was 0.1(0,0.2),worse than the control group 0(0,0)(U=114.000,P=0.014).The overall vessel densities of optic disc in the acute DON and chronic DON were significantly lower than that in the control group[(54.70±2.31)%vs.(54.31±3.65)%vs.(62.54±3.91)%,t=3.104,2.636,all P<0.05)].The overall vessel densities of the macular area in the active TAO,acute DON and chronic DON were significantly lower than that of the control group[(46.07±3.06)%vs.(42.26±5.05)%vs.(45.63±3.87)%vs.(49.34±3.08)%],the differences were statistically significant(t=2.614,4.147,2.603,all P<0.05).There was no statistically significant difference in the foveal avascular zone or the density of deep blood vessels in macular area among four groups(P>0.05).In active TAO period,there was no correlation between MD value,RNFL,GCC thickness and the vessel densities of optic disc and macular area(all P>0.05).The vascular density of the whole layer of the optic disc in the acute DON was negatively correlated with MD value(r=-0.591,P=0.043),and positively correlated with RNFL thickness and GCC thickness(r=0.595,0.693;P=0.041,0.013).In the chronic DON,the overall capillary density of the optic disc was negatively correlated with the MD value(r=-0.673,P=0.017);the superficial overall blood vessel density of the macular area was positively correlated with RNFL and GCC(r=0.732,0.712;P=0.007,0.009).Conclusion:BCVA of patients with acute DON was greatly affected by the disease.Only the blood supply to the superficial layer of macular area was decreased in active TAO period.In the acute and chronic phase of DON,the blood supply to the superficial layer of macular area and the optic disc were both reduced,and the smaller the blood vessel density,the more severe the visual field defect,and the thinner the RNFL and GCC.Part Two Effect of NF-?B inhibitor in orbital fibroblasts on Thyroid associated ophthalmopathyPurpose:To examine the effects of dexamethasone,IMD-0354 and Pyrrolidine dithiocarbamic(PDTC)on the NF-kappa B(NF-?B)pathway,to verify the regulation of transcription factors and inflammatory factors related to the NF-?B pathway and to explore whether the NF-?B inhibitor can combine with glucocorticoids in anti-inflammation.Methods:Orbital connective tissue was obtained from patients with restrictive strabismus due to TAO and other strabismus patients,from which primary orbital fibroblasts(OFs)were cultured and isolated.After 3-5 passages of OFs,inflammation was induced by lipopolysaccharide(LPS),and dexamethasone(DEX)with different concentration gradients was incubated.The expression levels of interleukin-6(IL-6),hyaluronic acid synthase 1,2,3(HAS 1,HAS2,HAS3)mRNA in the two groups of OFs were detected by Reverse transcription quantitative real-time PCR(RT-qPCR),respectively.Detected the NF-?B binding capacity in the nucleus of two groups of OFs by Electrophoretic mobility change assay(EMSA).The best DEX concentration was selected by these methods.Drug intervention was performed by adding NF-?B inhibitors IMD-0354 and PDTC,respectively.The expression levels of IL-6,HAS1,HAS2 and HAS3 mRNAs in the two groups of OFs were detected by RT-qPCR,and the expression levels of P65,phosphorylated P65(p-P65),I?B? and phosphorylated I?B?(p-I?B?)were detected by Western Blot.Results:Nine cases of OFs were included in the experimental group and nine cases in the control group.RT-qPCR results showed that the relative expression of IL-6 in the experimental group reached the lowest level after 2 hours of treatment with different concentrations of DEX.The relative expression of IL-6 in group 10-6M after 2 hours of DEX administration was significantly less than that of 10-7M and 10-8M(both P<0.05).The experimental group treated with different concentrations of DEX for 2 hours reduced the relative expression of HAS1,HAS2,and HAS3(all P<0.05).EMSA showed that after 0.5 hours of DEX administration,the capacity of NF-?B binding to target genes was the strongest in both groups of OFs.10-6M was chosen as the optimum dosing concentration of DEX and 2H as the optimum dosing time.RT-qPCR results showed that 5?M IMD-0354,5?M IMD-0354+DEX and 100?M PDTC+DEX could effectively inhibit IL-6 production compared with LPS+DEX group(all P<0.01).50?M IMD-0354 could effectively inhibit the production of HAS2 and HAS3,and the difference were statistically significant(all P<0.05).50?M IMD-0354,1?M IMD-0354+DEX,5?M IMD-0354+DEX and 100?M PDTC of control group could effectively inhibit the production of HAS2 and HAS3,the differences were statistically significant(all P<0.05).Western blot results showed that the OFs in the experimental group could effectively reduce the production of NF-?B P65,p-P65,I?B?,p-I?B? by using 5?M IMD-0354 in combination with DEX,50?M PDTC,100?M PDTC alone or in combination with DEX(all P<0.001).Conclusion:The expression levels of HAS1,HAS2,HAS3 and IL-6 in OFs were significantly increased after LPS stimulation and inhibited by dexamethasone.IMD-0354 and PDTC could combine with DEX to effectively reduce the expression levels of inflammatory and transcription factors in the NF-?B pathway of OFs in TAO patients.IMD-0354 and PDTC can be used as combination drugs for DEX treatment of TAO to increase the anti-inflammatory effect. |
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