平目汤用药大鼠血清介导Graves眼病眼眶脂肪细胞凋亡途径的研究
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摘要
目的:通过原代培养人眼眶前脂肪细胞,诱导分化为成熟脂肪细胞,研究平目汤促进眼眶脂肪细胞凋亡的作用及分子机制,探讨平目汤治疗非活动期Graves眼病发挥药效的可能作用靶点。方法:收集非活动期Graves眼病患者眼眶球后脂肪组织,共4例4只眼。通过组织块法对人眼眶前脂肪细胞进行分离和培养,观察细胞的形态,采用间接免疫荧光染色鉴定。诱导前脂肪细胞分化为成熟脂肪细胞,油红O染色鉴定。制备平目汤含药血清,配成终浓度分别为5%、10%、20%的平目汤低、中、高剂量组。油红O染色观察平目汤对前脂肪细胞诱导分化的影响;MTT法检测平目汤对眼眶前脂肪细胞活力的影响;流式细胞术测定平目汤对眼眶成熟脂肪细胞凋亡率的影响;检测平目汤对眼眶成熟脂肪细胞fas、fas L基因和蛋白表达的影响。结果:(1)采用组织块法培养的细胞呈梭形,增殖旺盛,间接免疫荧光检测证实为前脂肪细胞,可诱导分化为成熟脂肪细胞。(2)MTT结果显示,平目汤各组均可降低前脂肪细胞活力,其中作用显著的是平目汤中剂量组,呈时间依赖性地降低细胞活力。(3)流式细胞术结果表明,与空白对照组比较,平目汤各组细胞凋亡率均升高(P<0.05),其中平目汤中剂量组早期凋亡率和总凋亡率最高,其次为平目汤低剂量组。(4)与空白对照组相比,除地塞米松组外,平目汤各组对眼眶脂肪细胞的fas、fas L基因和蛋白的相对表达量较空白对照组均有一定程度的升高,其中平目汤中剂量组fas、fas L mRNA表达水平与空白对照组比较差异有统计学意义(P<0.05),Fas蛋白表达以平目汤中剂量组最高,其次为平目汤低剂量组,Fas L蛋白表达以平目汤高剂量组最高,其次为平目汤中剂量组,但二者比较差异无统计学意义。结论:平目汤降低眼眶前脂肪细胞的活力,抑制前脂肪细胞向脂肪细胞的分化能力,可能通过死亡受体信号途径诱导fas、fas L活化,促进成熟脂肪细胞凋亡,进而减少眼眶脂肪细胞积聚而发挥疗效。
Objective: To study the effect and molecular mechanism of Pingmu Decoction in promoting the apoptosis of orbital pre-adipocytes and explore the potential target in inactive period of Graves' ophthalmopathy with the treatment of Pingmu Decoction through primarily culturing human orbital pre-adipocytes and inducing them into mature adipocytes. Methods: Orbital adipose tissues with Graves' ophthalmopathy in inactive period including four cases(4 eyes) were collected. Human orbital pre-adipocytes were isolated and cultured through tissue explant method. Cell morphology was observed and identified by indirect immunofluorescent assay(IFA). Orbital pre-adipocytes were induced into mature adipocytes and then stained by oil red O. Medicated serum with Pingmu Decoction was prepared. The final concentrations of Pingmu Decoction were divided into three groups: low dose group(5%), middle dose group(10%) and high dose group(20%). Effect of Pingmu Decoction on differentiation of orbital pre-adipocytes were observed by oil red O. The cell viability were tested by MTT method. The effect of Pingmu Decoction on cell apoptosis rate of orbital matured adipocytes were measured by flow cytometry. The expression of gene fas, fas L and protein were tested. Results:(1)Cells cultured with tissue explant method was spindle-shaped and highly proliferative. Confirmed by indirect immunofluorescent assay(IFA) these cells were pre-adipocytes which could be induced into mature adipocytes.(2)The results of MTT showed that the cell viability of pre-adipocytes of each group could be reduced by Pingmu Decoction. The middle dose group played significant effects on reducing cells viability which was time-dependent.(3)Compared with control group, the results of flow cytometry showed that the cell apoptosis rate of Pingmu Decoction group was increased(P<0.05). The early and total apoptosis rate of middle dose group were the hightest followed by the low dose group.(4)Compared with control group, the relative expression quantity of gene Fas, fas L and protein were increased certainly inaddition to dexamethasone group. Compared middle dose group with the blank control group, the difference of fas and fas L mRNA expression levels were statistically significant(P<0.05). The Fas protein expression level in the middle dose group was the highest followed by low dose group. Fas L protein expression level in the high dose group was the highest followed by middle dose group, but the difference of the two group was not statistically significant. Conclusion: Pingmu Decoction can reduce the cell viability of orbital pre-adipocytes and inhibit them differentiate into adipocytes, and maybe activate Fas and fas L by the death signaling pathway to promote the apoptosis of matured adipocytes and play a therapeutic effect by reducing the accumulation of orbital adipocytes.
Objective: To study the effect and molecular mechanism of Pingmu Decoction in promoting the apoptosis of orbital pre-adipocytes and explore the potential target in inactive period of Graves' ophthalmopathy with the treatment of Pingmu Decoction through primarily culturing human orbital pre-adipocytes and inducing them into mature adipocytes. Methods: Orbital adipose tissues with Graves' ophthalmopathy in inactive period including four cases(4 eyes) were collected. Human orbital pre-adipocytes were isolated and cultured through tissue explant method. Cell morphology was observed and identified by indirect immunofluorescent assay(IFA). Orbital pre-adipocytes were induced into mature adipocytes and then stained by oil red O. Medicated serum with Pingmu Decoction was prepared. The final concentrations of Pingmu Decoction were divided into three groups: low dose group(5%), middle dose group(10%) and high dose group(20%). Effect of Pingmu Decoction on differentiation of orbital pre-adipocytes were observed by oil red O. The cell viability were tested by MTT method. The effect of Pingmu Decoction on cell apoptosis rate of orbital matured adipocytes were measured by flow cytometry. The expression of gene fas, fas L and protein were tested. Results:(1)Cells cultured with tissue explant method was spindle-shaped and highly proliferative. Confirmed by indirect immunofluorescent assay(IFA) these cells were pre-adipocytes which could be induced into mature adipocytes.(2)The results of MTT showed that the cell viability of pre-adipocytes of each group could be reduced by Pingmu Decoction. The middle dose group played significant effects on reducing cells viability which was time-dependent.(3)Compared with control group, the results of flow cytometry showed that the cell apoptosis rate of Pingmu Decoction group was increased(P<0.05). The early and total apoptosis rate of middle dose group were the hightest followed by the low dose group.(4)Compared with control group, the relative expression quantity of gene Fas, fas L and protein were increased certainly inaddition to dexamethasone group. Compared middle dose group with the blank control group, the difference of fas and fas L mRNA expression levels were statistically significant(P<0.05). The Fas protein expression level in the middle dose group was the highest followed by low dose group. Fas L protein expression level in the high dose group was the highest followed by middle dose group, but the difference of the two group was not statistically significant. Conclusion: Pingmu Decoction can reduce the cell viability of orbital pre-adipocytes and inhibit them differentiate into adipocytes, and maybe activate Fas and fas L by the death signaling pathway to promote the apoptosis of matured adipocytes and play a therapeutic effect by reducing the accumulation of orbital adipocytes.
引文
[1] Bahn RS.Graves’ ophthalmopathy[J].N Engl J Med,2010,362(8):726-738.
[2] Iyer S, Bahn R.Immunopathogenesis of Graves’ ophthalmopathy: the role of the TSH receptor[J].Best Pract Res Clin Endocrinol Metab,2012,26(3):281-289.
[3] Dik WA, Virakul S, Van Steensel L.Current perspectives on the role of orbital fibroblasts in the pathogenesis of Graves’ ophthalmopathy[J].Exp Eye Res,2016,142:83-91.
[4] Barrio-Barrio J, Sabater AL, Bonet-Farriol E, et al. Graves’ Ophthalmopathy: VISA versus EUGOGO Classification, Assessment, and Management[J]. J Ophthalmol, 2015,2015:249125.
[5] Potgieser PW, Wiersinga WM, Regensburg NI, et al. Some studies on the natural history of Graves’ orbitopathy: increase in orbital fat is a rather late phenomenon[J]. Eur J Endocrinol, 2015, 173(2): 14953.
[6] Draman MS, Grennan-Jones F, Zhang L, et al.Effects of prostaglandin F(2α) on adipocyte biology relevant to graves’ orbitopathy[J]. Thyroid, 2013,23(12):1600-1608.
[7] Fang S, Huang Y, Zhong S,et al.Regulation of Orbital Fibrosis and Adipogenesis by Pathogenic Th17 Cells in Graves Orbitopathy[J].J Clin Endocrinol Metab,2017,102(11):4273-4283.
[8] Garrity JA, Bahn RS. Pathogenesis of graves ophthalmopathy: implications for prediction, prevention,and treatment[J]. Am J Ophthalmol,2006,142:147-153.
[9] Kumar S, Nadeem S, Stan MN, et al.A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves’ ophthalmopathy[J]. J Mol Endocrinol, 2011,46(3):155-163.
[10] Marique L, Senou M, Craps J,et al.Oxidative Stress and Upregulation of Antioxidant Proteins, Including Adiponectin, in Extraocular Muscular Cells, Orbital Adipocytes, and Thyrocytes in Graves’ Disease Associated with Orbitopathy[J].Thyroid, 2015,25(9):1033-1042.
[11] Pawlowski P, Reszec J, Eckstein A,et al.Markers of inflammation and fibrosis in the orbital fat/connective tissue of patients with Graves’ orbitopathy: clinical implications[J]. Mediators Inflamm, 2014,2014:412158.
[12] 张亚利,汪涛,李红,等.平目颗粒治疗非活动期 Graves 眼病阳气亏虚、痰瘀阻滞证的临床观察[J].中华中医药学刊,2014,32(9):2124-2127.
[13] Li H,Wang Y,Xu R.Pingmu decoction enhances apoptosis of orbital adipocytes derived from patients with Graves'ophthalmophathy[J].Mol Med Rep,2012,6(6): 1361-1366.
[14] Bossowski A, Czarnocka B, Stasiak-Barmuta A, et al.Analysis of Fas, FasL and Caspase-8 expression in thyroid gland in young patients with immune and non-immune thyroid diseases[J]. Endokrynol Pol, 2007,58(4):303-313.
[15] Tao W, Ayala-Haedo JA, Field MG, et al. RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy[J]. Invest Ophthalmol Vis Sci, 2017,58(14):6146-6158.
[16] Stan MN, Bahn RS. Risk factors for development or deterioration of Graves’ ophthalmopathy[J]. Thyroid, 2010, 20(7): 777-783.
[17] Yoon JS, Lee HJ, Chae MK,et al.Autophagy is involved in the initiation and progression of Graves’ orbitopathy[J]. Thyroid, 2015,25(4):445-454.
[18] M Comerci, A Elefante,D Strianese, et al.Semiautomatic Regional Segmentation to Measure Orbital Fat Volumes in Thyroid-Associated OphthalmopathyA Validation Study[J]. Neuroradiol J, 2013, 26(4): 373-379.
[19] Potgieser PW, Wiersinga WM, Regensburg NI, et al.Some studies on the natural history of Graves’ orbitopathy: increase in orbital fat is a rather late phenomenon[J]. Eur J Endocrinol, 2015,173(2):149-153.
[20] Kumar S,Coenen MJ,Scherer PE,et al.Evedence for enhanced adipogenesis in the orbits of patients with Graves’ophthalmopathy[J].J Clin Endocrinol Metab,2004,89:930-935.
[21] Cho RI, Elner VM, Nelson CC, et al. The effect of orbital decompression surgery on lid retraction in thyroid eye disease[J]. Ophthal Plast Reconstr Surg 2011, 27(6):436-438.
[22] Kim KA, Kim JH, Wang Y, et al.Pref-1(Preadipocyte Factor 1) activates the MEK/Extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation[J].Mol Cell Biol,2007,27(6): 2294-2308.
[23] Lee K, Villena JA, Moon YS,et al.Inhibition of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1[J].J Clin Invest,2003,111(4): 453-461.
[2] Iyer S, Bahn R.Immunopathogenesis of Graves’ ophthalmopathy: the role of the TSH receptor[J].Best Pract Res Clin Endocrinol Metab,2012,26(3):281-289.
[3] Dik WA, Virakul S, Van Steensel L.Current perspectives on the role of orbital fibroblasts in the pathogenesis of Graves’ ophthalmopathy[J].Exp Eye Res,2016,142:83-91.
[4] Barrio-Barrio J, Sabater AL, Bonet-Farriol E, et al. Graves’ Ophthalmopathy: VISA versus EUGOGO Classification, Assessment, and Management[J]. J Ophthalmol, 2015,2015:249125.
[5] Potgieser PW, Wiersinga WM, Regensburg NI, et al. Some studies on the natural history of Graves’ orbitopathy: increase in orbital fat is a rather late phenomenon[J]. Eur J Endocrinol, 2015, 173(2): 14953.
[6] Draman MS, Grennan-Jones F, Zhang L, et al.Effects of prostaglandin F(2α) on adipocyte biology relevant to graves’ orbitopathy[J]. Thyroid, 2013,23(12):1600-1608.
[7] Fang S, Huang Y, Zhong S,et al.Regulation of Orbital Fibrosis and Adipogenesis by Pathogenic Th17 Cells in Graves Orbitopathy[J].J Clin Endocrinol Metab,2017,102(11):4273-4283.
[8] Garrity JA, Bahn RS. Pathogenesis of graves ophthalmopathy: implications for prediction, prevention,and treatment[J]. Am J Ophthalmol,2006,142:147-153.
[9] Kumar S, Nadeem S, Stan MN, et al.A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves’ ophthalmopathy[J]. J Mol Endocrinol, 2011,46(3):155-163.
[10] Marique L, Senou M, Craps J,et al.Oxidative Stress and Upregulation of Antioxidant Proteins, Including Adiponectin, in Extraocular Muscular Cells, Orbital Adipocytes, and Thyrocytes in Graves’ Disease Associated with Orbitopathy[J].Thyroid, 2015,25(9):1033-1042.
[11] Pawlowski P, Reszec J, Eckstein A,et al.Markers of inflammation and fibrosis in the orbital fat/connective tissue of patients with Graves’ orbitopathy: clinical implications[J]. Mediators Inflamm, 2014,2014:412158.
[12] 张亚利,汪涛,李红,等.平目颗粒治疗非活动期 Graves 眼病阳气亏虚、痰瘀阻滞证的临床观察[J].中华中医药学刊,2014,32(9):2124-2127.
[13] Li H,Wang Y,Xu R.Pingmu decoction enhances apoptosis of orbital adipocytes derived from patients with Graves'ophthalmophathy[J].Mol Med Rep,2012,6(6): 1361-1366.
[14] Bossowski A, Czarnocka B, Stasiak-Barmuta A, et al.Analysis of Fas, FasL and Caspase-8 expression in thyroid gland in young patients with immune and non-immune thyroid diseases[J]. Endokrynol Pol, 2007,58(4):303-313.
[15] Tao W, Ayala-Haedo JA, Field MG, et al. RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy[J]. Invest Ophthalmol Vis Sci, 2017,58(14):6146-6158.
[16] Stan MN, Bahn RS. Risk factors for development or deterioration of Graves’ ophthalmopathy[J]. Thyroid, 2010, 20(7): 777-783.
[17] Yoon JS, Lee HJ, Chae MK,et al.Autophagy is involved in the initiation and progression of Graves’ orbitopathy[J]. Thyroid, 2015,25(4):445-454.
[18] M Comerci, A Elefante,D Strianese, et al.Semiautomatic Regional Segmentation to Measure Orbital Fat Volumes in Thyroid-Associated OphthalmopathyA Validation Study[J]. Neuroradiol J, 2013, 26(4): 373-379.
[19] Potgieser PW, Wiersinga WM, Regensburg NI, et al.Some studies on the natural history of Graves’ orbitopathy: increase in orbital fat is a rather late phenomenon[J]. Eur J Endocrinol, 2015,173(2):149-153.
[20] Kumar S,Coenen MJ,Scherer PE,et al.Evedence for enhanced adipogenesis in the orbits of patients with Graves’ophthalmopathy[J].J Clin Endocrinol Metab,2004,89:930-935.
[21] Cho RI, Elner VM, Nelson CC, et al. The effect of orbital decompression surgery on lid retraction in thyroid eye disease[J]. Ophthal Plast Reconstr Surg 2011, 27(6):436-438.
[22] Kim KA, Kim JH, Wang Y, et al.Pref-1(Preadipocyte Factor 1) activates the MEK/Extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation[J].Mol Cell Biol,2007,27(6): 2294-2308.
[23] Lee K, Villena JA, Moon YS,et al.Inhibition of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1[J].J Clin Invest,2003,111(4): 453-461.