Effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy-related genes in human Tenon's fibroblasts induced by transforming growth factor beta 1

OBJECTIVE:To explore the effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1(TGF-β1)-activated human Tenon's fibroblasts(HTFs).METHODS:(a)Primary HTFs were stimulated by TGF-β1 and underwent immunohistochemistry,which established a cell model after Glaucoma filtration surgery(GFS).(b)The cell models were divided into 4 group:normal group(normal cells),model group(+TGF-β1),treatment group(+TGF-β1+medicated serum),and positive control group(TGF-β1+rapamycin).Then,Qingguang'an medicated serum with optimum concentration was added to the corresponding group.The autophagy positive cells were identified by the Cyto-ID autophagy detection kits under fluorescent microscope and Cytation 5 multifunctional instrument for cell imaging.And the mean fluorescence intensity of autophagy positive cells was determined by flow cytometry.The expression levels of autophagy related genes—Beclin-1,autophagy related gene 5(ATG-5),and microtubule-associated protein 1 light chain 3(LC-3Ⅱ)were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis.RESULTS:Compared with the normal group and the model group,the relative mRNA expression levels of autophagy-related genes(Beclin-1,ATG-5 and LC-3Ⅱ)in the experimental group were notably increased(P<0.05,P<0.01),and with the extension of treatment time,it had an increasing trend(48 h was more obvious),which showed a certain time dependency;the protein expression levels of autophagy-related genes(Beclin-1,ATG-5,and LC-3Ⅱ)were significantly increased in the experimental group(P<0.05,P<0.01).With the prolongation of treatment time,there was an increasing trend(48 h was relatively obvious),and it revealed a certain time dependency CONCLUSION:The Qingguang'an medicated serum could up-regulate autophagy related genes(Beclin1,ATG5,and LC3Ⅱ)in the TGF-β1-activated HTFs.

青光安有效组分对兔青光眼术后滤过道瘢痕组织中胶原纤维、成纤维细胞特异性蛋白-1、结缔组织生长因子及转化生长因子-β1表达的影响

目的观察青光安4种有效组分对兔青光眼术后滤过道瘢痕组织中胶原纤维、成纤维细胞特异性蛋白-1(FSP-1)、结缔组织生长因子(CTGF)、转化生长因子-β1(TGF-β1)表达的影响,对比青光安4种有效组分及青光安混悬液抗滤过道瘢痕化的效果。方法采用青光眼滤过手术制作动物模型。实验兔按体质量随机分为空白组(A组)、模型组(手术对照组,B组)、丝裂霉素C组(C组)、有效组分1组(D组)、有效组分2组(E组)、有效组分3组(F组)、有效组分4组(G组)和青光安混悬液组(H组)。各给药组给予相应药物。观察各组兔滤过道瘢痕组织中胶原纤维、FSP-1、CTGF、TGF-β1水平。结果术后C、E、H组兔眼压回升缓慢,眼压值较术前和A组差异均有统计学意义(P<0.05);胶原纤维面积比值除B、D、G组外,其他各组间比较差异均有统计学意义(P<0.05);FSP-1表达除B、G组外,其他各组间比较差异均有统计学意义(P<0.05);CTGF表达除B、D、G组外,其他各组间比较差异均有统计学意义(P<0.05);TGF-β1表达除B、G组外,其他各组间比较差异均有统计学意义(P<0.05)。结论青光安有效组分2、丝裂霉素C及青光安混悬液通过抑制胶原纤维、FSP-1、CTGF、TGF-β1的表达,降低肌成纤维细胞和成纤维细胞的增殖,从而减少滤过道瘢痕组织的增生,有明显抗青光眼术后滤过道瘢痕化的作用,青光安有效组分2和青光安混悬液抑制滤过道瘢痕化效果基本相当。

青光安Ⅱ号方及其有效组分对DBA/2J小鼠视网膜Ras、MEK及ERK蛋白表达的影响

目的观察青光安Ⅱ号方及其有效组分对DBA/2J小鼠视网膜细胞中Ras、MEK与ERK蛋白表达的影响,探讨其对视神经保护的作用机制。方法将8只雌性C57BL/6J小鼠作为正常对照组(A组),采用随机数字表法将48只雌性DBA/2J小鼠分为6组:模型组(B组)、阳性对照组(C组)、青光安Ⅱ号方汤剂组(D组)、青光安Ⅱ号方有效组分低剂量组(E组)、青光安Ⅱ号方有效组分中剂量组(F组)、青光安Ⅱ号方有效组分高剂量组(G组),每组8只。A、B组给予12.91 mL/(kg·d)蒸馏水;C组给予0.31 g/(kg·d)益脉康分散片;D组给予9.67 g/(kg·d)青光安Ⅱ号方汤剂;E、F、G组分别给予0.85、1.70、3.40 g/(kg·d)青光安Ⅱ号方有效组分。每4周进行眼压测量以及裂隙灯检查;灌胃4周后取视网膜组织行Western blot检测各组小鼠视网膜Ras、MEK与ERK蛋白的表达水平。结果C57BL/6J小鼠不同时相点的眼压不随年龄增长而变化(P>0.05);DBA/2J小鼠眼压在第18~38周高于C57BL/6J小鼠(P<0.05)。裂隙灯检查C57BL/6J小鼠眼前段均未见明显异常;DBA/2J小鼠随周龄增长逐渐出现角膜钙化、虹膜基质萎缩、瞳孔后粘连等眼前节病变。与A组比较,B组Ras、MEK、ERK蛋白表达水平均降低(P<0.05);与B组比较,C、D、E、F、G组Ras蛋白表达水平均升高(P<0.05),G组MEK、ERK蛋白表达水平均升高(P<0.05);与C组比较,G组Ras蛋白表达水平升高(P<0.05);与D组比较,G组MEK蛋白表达水平升高(P<0.05);与E、F组比较,G组Ras蛋白表达水平升高(P<0.05)。结论青光安Ⅱ号方及其有效组分低、中、高剂量对DBA/2J小鼠视网膜Ras、MEK、ERK蛋白可起到不同程度的上调作用,以青光安Ⅱ号方有效组分高剂量组效果最佳。