青光安颗粒剂对TGF-β1诱导的HTFs增殖影响的实验研究
|
摘要
目的探讨青光安颗粒剂抑制转化生长因子β1 (TGF-β1)诱导的人类Tenon成纤维细胞(HTFs)增殖的可能机制。方法从接受青光眼滤过手术(GFS)的个体获得人结膜下Tenon胶囊样品。实验设计分为3组:对照组(HTFs未经处理,n=10);TGF-β1诱导组(100 ng/ml TGF-β1诱导HTFs,构建GFS术后细胞模型,n=10);TGF-β1+青光安治疗组(TGF-β1诱导+青光安颗粒剂血清处理HTFs,n=30)。TGF-β1+青光安治疗组按照青光安血清的剂量进一步分为3组:TGF-β1+青光安高剂量组(TGF-β1诱导+青光安颗粒剂血清5 ml处理HTFs);TGF-β1+青光安中剂量组(TGF-β1诱导+青光安颗粒剂血清2.5 ml处理HTFs);TGF-β1+青光安低剂量组(TGF-β1诱导+青光安颗粒剂血清处理1 ml HTFs);每组设10个培养皿。通过CCK-8检测青光安对TGF-β1诱导HTFs增殖的影响;通过流式细胞术评估青光安对细胞周期的影响;通过Cyto-ID免疫细胞化学染色测定青光安颗粒剂对HTFs细胞自噬的影响;采用RT-PCR和Western Blot法测定自噬体形成的必需蛋白质Beclin-1,ATG-5和LC3-Ⅲ基因和蛋白的表达水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果青光安能够抑制TGF-β1诱导HTFs的增殖能力,TGF-β1+青光安高剂量组细胞增殖水平(13.52±1.24)较TGF-β1诱导组(23.42±1.25)降低,差异有统计学意义(F=12.347,P <0.01);TGF-β1诱导G0/G1期的比例(88.29±0.35)降低,而S期的比例(9.04±0.25)升高,而青光安治疗能够导致G0/G1期的比例(91.18±1.04)增加,而S期的比例(5.41±0.59)降低,差异有统计学意义(F=13.857,P <0.01);与TGF-β1诱导组相比,用TGF-β1+青光安治疗组导致荧光染色强度(1.84±0.14)降低,HTFs阳性细胞数目(112.46±12.11)减少,差异有统计学意义(F=12.347,P=18.472);TGF-β1+青光安治疗组能抑制TGF-β1诱导对自噬基因Beclin-1、ATG-5和LC-3ⅢmRNA和蛋白质表达增加(P <0.05)。结论青光安颗粒剂抑制TGF-β1诱导的HTFs增殖,且可能机制为青光安诱导HTFs细胞周期停滞于G0/G1期,而且青光安可减少TGF-β1诱导的HTFs自噬。
Objective To explore the possible mechanism of Qingguang' an granule inhibiting the proliferation of HTFs induced by TGF-β1. Methods Samples of human subconjunctival Tenon capsules were obtained from individuals undergoing GFS surgery. The experimental design was divided into three groups: a control group(HTFs untreated, n = 10); TGF-β1 induction group(100 ng/ml TGF-β1 induced HTFs, constructed cell model after GFS, n = 10); TGF-β1 + Qingguang' an treatment group(TGF-β1 induced + Qingguang' an granule serum treated HTFs, n = 30). The treatment group was further divided into three groups according to the dose of Qingguang' an serum: the high dose group of TGF-β1 + Qingguang' an(TGF-β1 induction + Qingguang' an granule serum 5 ml for HTFs), the medium dose group of TGF-β1 + Qingguang' an granule serum 2.5 ml for HTFs, and the low dose group of TGF-β1 + Qingguang' an granule serum 1 ml for HTFs; There were 10 Petri dishes in each group. The effect of glaucoma on the proliferation of TGF-β1-induced HTFs was detected by CCK-8; the effect of glaucoma on cell cycle was evaluated by flow cytometry; the Cyans granules were determined by Cyto-ID immunocytochemical staining. The effect of autophagic cells; the expression levels of the essential proteins Beclin-1, ATG-5 and LC3-Ⅲ genes and proteins formed by autophagosomes were determined by RT-PCR and Western Blot. Results Qingguang' an inhibited the proliferation of HTFs cells induced by TGF-beta1. The proliferation level of HTFs cells in the high dose group of TGF-β1+Qingguang' an(13.52±1.24)was lower than that in the high dose group of TGF-β1(23.42±1.25), with a statistically significant difference(F = 12.347, P < 0.001); the proportion of G0/G1 phase induced by TGF-β1(88.29±0.35)was lower, while the proportion of S phase(9.04±0.25)was higher. The proportion of G0/G1 phase was increased(91.18±1.04), while that of S phase was decreased(5.41+0.59), with a significant difference(F = 13.857, P = 0.007); compared with TGF-β1 induced group, the intensity of fluorescence staining decreased(1.84+0.14) and the number of HTFs-positive cells was decreased(112.46+12.11), with a significant difference(F = 12.347, P = 18.472). TGF-β1 + Qingguang' an treatment group inhibited the increase of expression of autophagy gene Beclin-1, ATG-5 and LC-3 ⅢmRNA and protein induced by TGF-β1(P < 0.05). Conclusion Qingguang' an granule inhibits the proliferation of HTFs induced by TGF-β1, and the possible mechanism is that Qingguang' an induces HTFs cell cycle to stagnate at G0/G1 phase, and Qingguang' an can reduce the autophagy of HTFs induced by TGF-β1.
Objective To explore the possible mechanism of Qingguang' an granule inhibiting the proliferation of HTFs induced by TGF-β1. Methods Samples of human subconjunctival Tenon capsules were obtained from individuals undergoing GFS surgery. The experimental design was divided into three groups: a control group(HTFs untreated, n = 10); TGF-β1 induction group(100 ng/ml TGF-β1 induced HTFs, constructed cell model after GFS, n = 10); TGF-β1 + Qingguang' an treatment group(TGF-β1 induced + Qingguang' an granule serum treated HTFs, n = 30). The treatment group was further divided into three groups according to the dose of Qingguang' an serum: the high dose group of TGF-β1 + Qingguang' an(TGF-β1 induction + Qingguang' an granule serum 5 ml for HTFs), the medium dose group of TGF-β1 + Qingguang' an granule serum 2.5 ml for HTFs, and the low dose group of TGF-β1 + Qingguang' an granule serum 1 ml for HTFs; There were 10 Petri dishes in each group. The effect of glaucoma on the proliferation of TGF-β1-induced HTFs was detected by CCK-8; the effect of glaucoma on cell cycle was evaluated by flow cytometry; the Cyans granules were determined by Cyto-ID immunocytochemical staining. The effect of autophagic cells; the expression levels of the essential proteins Beclin-1, ATG-5 and LC3-Ⅲ genes and proteins formed by autophagosomes were determined by RT-PCR and Western Blot. Results Qingguang' an inhibited the proliferation of HTFs cells induced by TGF-beta1. The proliferation level of HTFs cells in the high dose group of TGF-β1+Qingguang' an(13.52±1.24)was lower than that in the high dose group of TGF-β1(23.42±1.25), with a statistically significant difference(F = 12.347, P < 0.001); the proportion of G0/G1 phase induced by TGF-β1(88.29±0.35)was lower, while the proportion of S phase(9.04±0.25)was higher. The proportion of G0/G1 phase was increased(91.18±1.04), while that of S phase was decreased(5.41+0.59), with a significant difference(F = 13.857, P = 0.007); compared with TGF-β1 induced group, the intensity of fluorescence staining decreased(1.84+0.14) and the number of HTFs-positive cells was decreased(112.46+12.11), with a significant difference(F = 12.347, P = 18.472). TGF-β1 + Qingguang' an treatment group inhibited the increase of expression of autophagy gene Beclin-1, ATG-5 and LC-3 ⅢmRNA and protein induced by TGF-β1(P < 0.05). Conclusion Qingguang' an granule inhibits the proliferation of HTFs induced by TGF-β1, and the possible mechanism is that Qingguang' an induces HTFs cell cycle to stagnate at G0/G1 phase, and Qingguang' an can reduce the autophagy of HTFs induced by TGF-β1.
引文
1谭涵宇,彭清华,李文娟,等.青光安颗粒剂有效组分对兔眼滤过术后滤过道瘢痕组织TGF-β1和Smad3表达的影响[J].中华中医药杂志,2016,(10):3977-3980.
2 Want A,Gillespie SR,Wang Z,et al.Autophagy and mitochondrial dysfunction in tenon fibroblasts from exfoliation glaucoma patients[J].PLoS One,2016,11(7):e0157404.
3 Noh SM,Abdul Kadir SH,Crowston JG,et al.Effects of ranibizumab on TGF-β1 and TGF-β2 production by human Tenon's fibroblasts:An in vitro study[J].Mol Vis,2015,21:1191-1200.
4 Na JH,Sung KR,Shin JA,et al.Antifibrotic effects of pirfenidone on Tenon's fibroblasts in glaucomatous eyes:comparison with mitomycin C and 5-fluorouracil[J].Graefes Arch Clin Exp Ophthalmol,2015,253(9):1537-1545.
5龙达,周亚莎,李萍,等.青光安颗粒剂对自发性青光眼模型小鼠的降眼压作用及房水动力学的影响[J].中华实验眼科杂志,2017,35(12):1079-1084.
6鲁丹,王康,王艳玲,等.后Tenon囊下注射曲安奈德辅助全视网膜光凝治疗糖尿病视网膜病变[J].眼科新进展,2013,33(5):439-442.
7 Shi HM,Zhang YY,Fu SH,et al.Angiotensin II as a morphogenic cytokine stimulating fibrogenesis of human tenon's capsule fibroblasts[J].Invest Ophthalmol Vis Sci,2015,56(2):855-864.
8 Su Y,Jiang CG,Zhang L,et al.Arsenic trioxide inhibits proliferation of rabbit tenon's capsule fibroblasts after trabeculectomy by downregulating expression of extracellular matrix proteins[J].Invest Ophthalmol Vis Sci,2015,56(11):6663-6670.
9李苑碧,彭清华,黄学思,等.青光安对抗青光眼术后滤过道瘢痕组织中弹性纤维,MMP-7,TIMP-1的实验研究[J].国际眼科杂志,2015,15(1):20-25.
10刘艳,彭清华.青光安有效组份对兔眼滤过术后滤过道瘢痕组织成纤维细胞和Ⅰ型胶原蛋白的影响[J].国际眼科杂志,2013,13(5):845-849.
11 Liu S,Wang J,Zou H,et al.Effect of apigenin on gap junctional intercellular communication in human tenon's capsule fibroblasts[J].Eye Sci,2013,28(2):62-67.
12 Wang Y,Wang J,Wei L J,et al.Biological function and mechanism of lncRNA-MEG3 in Tenon's capsule fibroblasts proliferation:By MEG3-Nrf2 protein interaction[J].Biomed Pharmacother,2017,87(5):548-554.
13陈侠,李磊,鲜光军,等.壳聚糖纳米微粒对靶向转化生子因子-βⅡ型受体核酸适配子的缓释作用及其安全性研究[J].中华实验眼科杂志,2013,31(4):352-357.
14 Fu SH,Sun L,Zhang XY,et al.5-Aza-2'-deoxycytidine induces human Tenon's capsule fibroblasts differentiation and fibrosis by up-regulating TGF-beta type I receptor[J].Exp Eye Res,2017,165:47-58.
15 Bao H,Jiang K,Meng K,et al.TGF-β2 induces proliferation and inhibits apoptosis of human Tenon capsule fibroblast by miR-26 and its targeting of CTGF[J].Biomed Pharmacother,2018,104(8):558-565.
16 Fu S,Wang H,Zhang J,et al.Overexpression of ALK5 induces human tenon's capsule fibroblasts transdifferentiation and fibrosis in vitro[J].Curr Eye Res,2017,42(7):1.
17卢素素,刘姗姗,范晓军,等.氯化锂对人眼Tenon囊成纤维细胞TGF-β及CTGF的影响[J].国际眼科杂志,2017,17(9):1639-1642.
18 Ciftci S,Dogan E,Dag U,et al.Removal of tenon fortified by conjunctival-limbal autograft in treatment of pterygium[J].Int Ophthalmol,2017,37(4):813-818.
19 Wang X,Fan YZ,Yao L,et al.Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts[J].Int J Ophthalmol,2016,9(5):669-676.
20 Xing Y,Cui L,Kang Q.Silencing of ILK attenuates the abnormal proliferation and migration of human Tenon's capsule fibroblasts induced by TGF-β2[J].Int J Mol Med,2016,38(2):407-416.
2 Want A,Gillespie SR,Wang Z,et al.Autophagy and mitochondrial dysfunction in tenon fibroblasts from exfoliation glaucoma patients[J].PLoS One,2016,11(7):e0157404.
3 Noh SM,Abdul Kadir SH,Crowston JG,et al.Effects of ranibizumab on TGF-β1 and TGF-β2 production by human Tenon's fibroblasts:An in vitro study[J].Mol Vis,2015,21:1191-1200.
4 Na JH,Sung KR,Shin JA,et al.Antifibrotic effects of pirfenidone on Tenon's fibroblasts in glaucomatous eyes:comparison with mitomycin C and 5-fluorouracil[J].Graefes Arch Clin Exp Ophthalmol,2015,253(9):1537-1545.
5龙达,周亚莎,李萍,等.青光安颗粒剂对自发性青光眼模型小鼠的降眼压作用及房水动力学的影响[J].中华实验眼科杂志,2017,35(12):1079-1084.
6鲁丹,王康,王艳玲,等.后Tenon囊下注射曲安奈德辅助全视网膜光凝治疗糖尿病视网膜病变[J].眼科新进展,2013,33(5):439-442.
7 Shi HM,Zhang YY,Fu SH,et al.Angiotensin II as a morphogenic cytokine stimulating fibrogenesis of human tenon's capsule fibroblasts[J].Invest Ophthalmol Vis Sci,2015,56(2):855-864.
8 Su Y,Jiang CG,Zhang L,et al.Arsenic trioxide inhibits proliferation of rabbit tenon's capsule fibroblasts after trabeculectomy by downregulating expression of extracellular matrix proteins[J].Invest Ophthalmol Vis Sci,2015,56(11):6663-6670.
9李苑碧,彭清华,黄学思,等.青光安对抗青光眼术后滤过道瘢痕组织中弹性纤维,MMP-7,TIMP-1的实验研究[J].国际眼科杂志,2015,15(1):20-25.
10刘艳,彭清华.青光安有效组份对兔眼滤过术后滤过道瘢痕组织成纤维细胞和Ⅰ型胶原蛋白的影响[J].国际眼科杂志,2013,13(5):845-849.
11 Liu S,Wang J,Zou H,et al.Effect of apigenin on gap junctional intercellular communication in human tenon's capsule fibroblasts[J].Eye Sci,2013,28(2):62-67.
12 Wang Y,Wang J,Wei L J,et al.Biological function and mechanism of lncRNA-MEG3 in Tenon's capsule fibroblasts proliferation:By MEG3-Nrf2 protein interaction[J].Biomed Pharmacother,2017,87(5):548-554.
13陈侠,李磊,鲜光军,等.壳聚糖纳米微粒对靶向转化生子因子-βⅡ型受体核酸适配子的缓释作用及其安全性研究[J].中华实验眼科杂志,2013,31(4):352-357.
14 Fu SH,Sun L,Zhang XY,et al.5-Aza-2'-deoxycytidine induces human Tenon's capsule fibroblasts differentiation and fibrosis by up-regulating TGF-beta type I receptor[J].Exp Eye Res,2017,165:47-58.
15 Bao H,Jiang K,Meng K,et al.TGF-β2 induces proliferation and inhibits apoptosis of human Tenon capsule fibroblast by miR-26 and its targeting of CTGF[J].Biomed Pharmacother,2018,104(8):558-565.
16 Fu S,Wang H,Zhang J,et al.Overexpression of ALK5 induces human tenon's capsule fibroblasts transdifferentiation and fibrosis in vitro[J].Curr Eye Res,2017,42(7):1.
17卢素素,刘姗姗,范晓军,等.氯化锂对人眼Tenon囊成纤维细胞TGF-β及CTGF的影响[J].国际眼科杂志,2017,17(9):1639-1642.
18 Ciftci S,Dogan E,Dag U,et al.Removal of tenon fortified by conjunctival-limbal autograft in treatment of pterygium[J].Int Ophthalmol,2017,37(4):813-818.
19 Wang X,Fan YZ,Yao L,et al.Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts[J].Int J Ophthalmol,2016,9(5):669-676.
20 Xing Y,Cui L,Kang Q.Silencing of ILK attenuates the abnormal proliferation and migration of human Tenon's capsule fibroblasts induced by TGF-β2[J].Int J Mol Med,2016,38(2):407-416.