内障丸加减方对晶状体氧化损伤的细胞凋亡及相关基因表达的影响
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摘要
目的
研究内障丸加减方对临床早、中期老年性白内障(SC)患者晶状体混浊度、视功能及脾气虚弱,肝肾不足的虚证证侯的影响;评价内障丸加减方与内障丸方(障眼明片)临床疗效的异同,探讨内障丸加减方治疗早中期SC可能的作用机理。研究内障丸加减方对体外培养的晶状体上皮细胞氧化损伤的干预作用,探讨中药单体与含药血清干预氧化损伤晶状体上皮细胞凋亡作用的异同。试图阐述内障丸加减方抑制晶状体上皮细胞的凋亡的作用机理。
方法
一.临床研究为前瞻性研究,以早、中期老年性白内障(SC)患者96例192眼为观察对象,随机分为内障丸加减方观察组,障眼明片对照组,白内停眼水对照组,以内障丸加减方或障眼明片内服、以白内停眼药水点眼,每3周为1疗程,共用药3个疗程。观察内障丸加减方对SC患者的视力、晶状体混浊度、脾气虚弱,肝肾不足型虚证证侯的影响,通过对组内治疗前后对照,及组间疗效的比较,探讨该方的临床疗效及可能的作用机理。
二.实验研究为体外动物实验。以体外培养的SD大鼠晶状体及其上皮细胞作为研究对象,以含有内障丸加减方活性成分的动物血清作为观察组,以空白组、模型组、槲皮素组和空白血清组作为对照组。研究取透明SD大鼠晶状体分组作体外培养,以Medium199培养液为培养基,加入终浓度300μmol/L的H_2O_2,采用Fenton反应复制SD大鼠晶状体氧化损伤模型。含药血清是以内障丸加减方煎剂给SD大鼠灌胃,腹主动脉采血离心后制备而成。离体晶状体孵育24小时后,对各指标进行观察。1.拍摄晶状体照片,将晶状体的混浊程度分5个等级,比较各组晶状体混浊的异同。2.以晶状体局部的灰度值,比较各组晶状体混浊度的异同。3.以透射电镜观察晶状体上皮细胞形态。4.TUNEL法检测LEC凋亡细胞,计算凋亡率,并作统计学分析。5.SAB法检测LEC凋亡相关基因Bcl-2和Bax表达,按不同表达率分5个等级,并作统计学分析。
结果
1.临床研究显示内障丸加减方能提高SC患者的视力,有效率及显效率分别为64%及8%,与障眼明组的疗效无统计学差异,而与白内停组比较,疗效有显著差异。内障丸加减方能改善SC患者的脾气虚弱、肝肾不足的虚证证侯,且优于障眼明组。内障丸加减方组及障眼明组的证侯改善程度明显优于白内停滴眼液组。三组的晶状体混浊程度在治疗后无明显变化。
2.正常组晶状体在孵育过程中保持透明状态;其余各组随着孵育时间的延长,晶状体逐渐出现不同程度的皮质混浊。模型组与空白血清组混浊程度明显较用药组严重,而含药血清组与槲皮素组比较,含药血清组晶状体呈现更好的透明度。
3.除了空白对照组,其余各组上皮细胞在氧化损伤后均能观察到不同程度的细胞凋亡形态。模型组及空白血清组的细胞形态明显破坏,细胞膜溶解破碎,细胞质及细胞器肿胀,部分胞浆溶解,核膜明显扩张,可见空泡样改变,细胞核固缩,核仁消失,异染色质增多,聚集于核膜边缘;槲皮素组及中药血清组细胞形态较模型组及空白血清组完整,细胞间的间隙增宽,细胞膜部分溶解,细胞质及细胞器出现肿胀,核周间隙增宽,细胞核形态仍呈椭圆形,核内异染色质堆积于核膜下,但异染色质较模型组等少,中药血清组的细胞形态结构优于槲皮素组。电镜下所见的总体印象显示,中药血清组LECs形态结构好于其它各对照组,认为内障丸加减方能更好地保护晶状体上皮细胞的结构,延缓细胞的变性。
4.光镜下观察LECs可见细胞核呈黑蓝色颗粒的凋亡阳性细胞。阴性细胞呈浅紫红色。光镜下显示空白组凋亡细胞数量极少,紫红色非凋亡细胞的排列紧密,模型组及空白血清组的凋亡细胞较多,且细胞间距离增宽;槲皮素组及中药血清组的光镜下所见,凋亡细胞的数量介于空白组与模型组之间,细胞密度亦优于模型组。空白组的LECs偶见凋亡阳性细胞,其凋亡细胞凋亡的百分率为0～8%,模型组、空白血清组的LECs凋亡率为46%～80%,槲皮素组与中药血清组的LECs凋亡率介于空白组与模型组之间,槲皮素组凋亡率为27%～49%,含药血清组凋亡率为13～36%,空白血清组的LECs凋亡率为36%～83%。含药血清组LECs凋亡率与各对照组比较有统计学意义。
5.在光学显微镜下观察Bcl-2及Bax单克隆抗体在LEC的免疫组织化学染色均能表现各自特异的胞浆定位,阳性细胞的胞浆显示特异性棕黄色颗粒状着色,棕黄色颗粒在细胞胞浆中均匀分布。Bcl-2及Bax在空白组大鼠LEC中均有表达,Bcl-2的表达明显强于Bax的表达,Bcl-2/Bax比值的数值较高。本实验过氧化氢诱导的大鼠LEC凋亡,模型组和空白血清组,Bax的表达上调明显,而Bcl-2的表达下调。槲皮素及中药血清组的LECs样本显示,与模型组及空白血清组相比,LECs的Bcl-2的表达显著上调,而Bax的表达明显下调,差异具有统计学意义。而槲皮素组与中药血清组样本比较,中药血清组更能有效上调Bcl-2的表达,降低Bax的表达,两组比较,作用的差异具有统计学意义。
结论
注:以下所作的结论均建立在有统计学意义的基础上(P＜0.05)
1.内障丸加减方可以提高SC患者的视力,改善脾气虚弱、肝肾不足的证候,其作用可能是通过有效地清除活性氧,增强晶状体的抗氧化能力,延缓衰老演变过程,改善视细胞功能,遏止或减轻白内障患者的视功能损害。认为对内障丸方的加减化裁没有降低其改善视力的作用;改进复方中药的剂型及给药方式有利于提高患者的用药依从性。
2.H_2O_2诱导的体外培养的大鼠晶状体氧化损伤导致的白内障发生,可作为老年性白内障相关研究的实验动物模型。且H_2O_2诱导的体外培养晶状体氧化损伤模型具有可行性。
3.含药血清能反映中药复方在体内的物质基础,根据实验结果可认为内障丸加减方具有抗凋亡及保护细胞结构的作用。
4.实验结果显示模型组和空白血清组Bcl-2显著下调,提示空白血清不具有上调Bcl-2表达的作用。
5.含药血清和槲皮素均能在一定程度上保护晶状体细胞结构,延缓细胞凋亡过程的作用;含药血清的保护作用更强。而空白血清可以认为对晶状体LEC结构无保护作用。
6.槲皮素及含药血清均能通过上调抑制凋亡的Bcl-2基因表达,下调Bax基因的表达来抑制LEC的凋亡的发生和发展。而结果显示含药血清具有更好的对抗LEC氧化损伤,抑制细胞凋亡的作用。
7.含药血清能在一定程度上保护H_2O_2诱导的氧化损伤对体外培养的SD大鼠LECs的损害,抑制或延缓细胞的凋亡。槲皮素作用低于含药血清,认为复方中药在动物体内经过吸收和代谢,各种活性成分之间起到协同和增效的作用。
Objective
1.Study the effect of Neizhangwanjiajian-decoction on improving lens opacities,the visual function,liver,spleen and kidney deficiency syndrome in senile cataract(SC) patients who belong to early and middle phase in clinical.To evaluate the different efficency between Neizhangwanjiajiandecoction and Zhangyanming- pills.The possible mechemism of Neizhangwanjiajian-decoction for SC in early and middle phase were explored.
2.To research the protect effect of Neizhangwanjiajian-decoction on lens epithelial cells(LEC) impaired by hydrogen peroxide(H_2O_2),rat LEC treated by Chinese medicine single component and Chinese medicine complex compound were explored to detect the apoptosis induced by oxidative damage.We attempt to explain the different mechanism of Chinese medicine of single components and complex compounds to inhibit the apoptosis in LEC.
Methods
1.Clinical research(perspective study):The 96 SC patients(192 eyes) in early and middle phase were randomly divided into 3 groups:Neizhang group(taken orally by Neizhangwanjiajian-decoction);Zhangyanming group(taken orally by Zhangyanming-pills);and Pirenoxine Sodium group(treated by Pirenoxine Sodium eye drop).All patients were treated for 3 course of therapy which three weeks for one course.The evaluation indexes for treatment were including visual acuity(VA),lens turbidity,and the effects of liver disorder,spleen disorder and kidney disorder.The therapeutic effect for Neizhangwanjiajian-decoction and related mechanism were evaluated by comparison of before and after treatment and comparison of different groups.
2.Experimental study(in vitro study):Lens and its epithelial cells were cultured in M199.Lens were divided into 5 groups as follows:1 blank group which treated with medium;2 model group which treated with H_2O_2(final concentration 300μmol/L);3 quercetin group which treated with quercetin and H_2O_2;4 decoction group which treated with rat serum containing Neizhangwanjiajian-decoction substances and H_2O_2;5 blank serum group which treated with rat blank serum and H_2O_2.Oxidative Damages model of rat Lens Epithelial Cell was copied by Fenton response.Rat serum containing Neizhangwanjiajian-decoction substances were prepared with 5 SD rats were taken orally with Neizhangwanjiajian-decoction to prepare drug-conditioned serum,twice a day for 3 days.The other 5 rats were taken orally with the same volume of water.Blood was harvested from the abdominal aortas 2 hours following the last medication.The supernatant was obtained after centrifugalization,and stored at -20℃.All lens cultured for 24h and were detected the indexes as follow:1.Lens opacification were divided into 5 grades according photos and the differences within each group in the degree of lens opacification were compared as an index.2.Lens in the local gray value was compared between each group as another index for lens turbidity. 3.Cell morphology was detected by transmission electron microscopy.4.The expression of apoptosis related gene,such as Bcl-2 and Bax were detected by immunohistochemistry in LEC.5.Apoptosis in LEC were also detected by TUNEL and apoptosis rate.
Results
1.Clinical studies have shown Neizhangwanjiajian decoction could improve vision in patients with SC with efficient and remarkable efficiency of 64% and 8%.There was no significant difference between Zhangyanming-pills and Neizhangwanjiajian-decoction.But there were significant differences in efficacy when compared with Pirenoxine Sodium eye drop. Neizhangwanjiajian-decoction and Zhangyanming-pills were better in improved kidney deficiency syndrome,liver deficiency syndrome and spleen deficiency syndrome in patients with SC than Pirenoxine Sodium eye drop.There was no significant difference in lens opacification when treated with these three drugs.
2.Normal lens were keeping in the transparent state during incubated process:but the lens in other groups gradually appeared cortex turbidity along with the incubation time.Each group did not observe obvious signs of turbidity when incubated for 8h.Lens in model group and blank serum group began to appear cortex localized turbidity and turbidity is the uneven distribution of the site at 16h;Lens in quercetin group and decoction group also appeared decrease of the transparency in cortex at the same time.Each group of lens turbidity increase than before when incubated for 20h,and the central region of the cortex appeared turbidity in lens.When incubation extended for 24h, each group of lens cortex appeared both gray and white opacities.The lens in model group and blank serum group had a higher degree of turbidity than the treatment group,and the effect of decoction group had better performance than quercetin group in lens transparency.Along with the rest of the group to extend the incubation time,the progressive lens cortex with varying degrees of turbidity.
3.In addition to the control group,and the remaining epithelial cells of each group in the oxidative damage can be observed the different degrees of morphological apoptosis.Cells in control group kept in normal morphology, it can be seen cells arrangement closely,membrane integrity,cytoplasmic homogeneous,morphology of mitochondria and rough endoplasmic reticulum also keep normal,membrane-free expansion,clearly visible nucleolus,euchromatin uniformly distributed in the nucleus,no accumulation of heterochromatin;The cell morphology in model group and blank serum group significantly were damaged, cell membrane broken and dissolved,cytoplasm and cell organelle swelling, cytoplasm were dissolved partly,a remarkable expansion of the nuclear membrane,showing vacuolar changes,nuclear condensation,nucleolus disappear, increased heterochromatin gathered on the edge of the nuclear membrane;Cells in quercetin group and decoction group were better than the model group and blank serum group in integrity,and the gap widened between cells,cell membrane were also dissolved partly,cytoplasm and cell organelle swelling, gap widened perinuclear,nucleus still kept in oval form,heterochromatin accumulated under the nuclear membrane,its quantity was less than to the model group.Cell morphology and structure in decoction group was superior to the of quercetin group.Ganerally LECs morphology and structure treated by Neizhangwanjiajian decoction was better than the other control group detected by electron microscope.Neizhangwanjiajian decoction can better protect the structure of lens epithelial cells,slow degeneration of cells.
4.Apoptosis-positive LEC which nuclear emerge black and blue particle could be detected by light microscope.If apoptosis was not happened in LEC, light-purple color would be appeared.Light microscope showed that the number of apoptotic cells in the blank group was few,purple non-apoptotic cells were arranged closely.More apoptotic cells were emerged in model group and blank serum group,and widened the distance between cells as much.The number of apoptotic cells in quercetin group and decoction group were between the control group and model group,the cell density was also better than that in the model group.Apoptosis positive LECs could be detect in blank group occasionally, the apoptosis percentage was 0～8%,the apoptosis rate in model group and blank serum group was 46%～80%,apoptosis rate in quercetin group and decoction group were lower than in model group but higher than in control.Apoptosis rate was 27%～49%in quercetin group,13～36%in decoction group and 36%～83%in blank-serum group.Apoptosis rate of decoction group had statistically significant compared with the otber control groups.
5.Immunohistochemical staining was explored to detect the expression of Bcl-2 and Bax with optical microscope.Specified brown granular uniformly distributed in cytoplasm would be thought to be positive cells.Both Bcl-2 and Bax were expressed in LEC of control group.Bcl-2 expression was significantly stronger than Bax,naturally values of Bcl-2/Bax ratio was higher.In this study Bax expression increases significantly,while the expression of Bcl-2 down when induced apoptosis in rat LECs with hydrogen peroxide as model group and treated with blank rat serum.But when treated with Quercetin and decoction,Bcl-2 expression in LECs increases significantly, while Bax expression was significantly reduced compared with the model group and blank serum group.The decoction group had a better effect in increase of Bcl-2 expression and reduction of Bax expression when compared with quercetin.The difference was significant statistically.
Conclusion
Note:The following conclusions are based on the basis of statistical significance(P＜0.05)
1.Neizhangwanjiajian decoction can improve vision in patients with SC, and improve the liver,spleen and kidney deficiency syndrome.The mechanism maybe related with its role to remove reactive oxygen effectively,and enhance the antioxidant capacity of the lens.As a result,the aging evolution was delayed,the visual cell function was improved,and the damage of visual function in cataract patients was to stop or mitigate.Neizhangwanjiajian decoction can improve eyesight in patients with SC.To improve the way of Traditional Chinese medicine administration would help to improve the of medication compliance in patients.
2.H_2O_2-induced rat lens oxidative damage in vitro can lead to cataract. This model can be used as the relevant experimental animal study for senile cataract.And H_2O_2-induced oxidative damage in vitro model of lens is feasible.
3.Drug-containing serum can reflect the effect of traditional Chinese medicine compound in the body.Neizhangwanjiajian decoction have the effect of anti-apoptotic and protection of role of cell structure in rat LEC.
4.The expression of Bcl-2 in model group and blank serum group significantly reduced,suggesting that blank rat serum do not have raised Bcl-2 expression on lens.
5.Neizhangwanjiajian decoction and quercetin can protect the structure of lens cells;retard the process of apoptosis;with stronger role in the former. Blank serum can be considered non-protective effect of LEC structures.
6.Quercetin and Neizhangwanjiajian decoction can inhibit the occurrence and development of apoptosis by up regulating Bcl-2 gene expression which inhibit the apoptosis,reduced Bax gene expression.The results showed that Neizhangwanjiajian decoction had a better effect against oxidative damage and inhibit cell apoptosis.
7.Neizhangwanjiajian decoction can protect LEC in vitro from H_2O_2-induced oxidative damage,and can inhibit or delay cell apoptosis.Neizhangwanjiajian decoction has a better effect than quercetin.The mechanism may relate that combination of various active ingredients plays the role of synergy and efficiency after absorption and metabolism in the body.
研究内障丸加减方对临床早、中期老年性白内障(SC)患者晶状体混浊度、视功能及脾气虚弱,肝肾不足的虚证证侯的影响;评价内障丸加减方与内障丸方(障眼明片)临床疗效的异同,探讨内障丸加减方治疗早中期SC可能的作用机理。研究内障丸加减方对体外培养的晶状体上皮细胞氧化损伤的干预作用,探讨中药单体与含药血清干预氧化损伤晶状体上皮细胞凋亡作用的异同。试图阐述内障丸加减方抑制晶状体上皮细胞的凋亡的作用机理。
方法
一.临床研究为前瞻性研究,以早、中期老年性白内障(SC)患者96例192眼为观察对象,随机分为内障丸加减方观察组,障眼明片对照组,白内停眼水对照组,以内障丸加减方或障眼明片内服、以白内停眼药水点眼,每3周为1疗程,共用药3个疗程。观察内障丸加减方对SC患者的视力、晶状体混浊度、脾气虚弱,肝肾不足型虚证证侯的影响,通过对组内治疗前后对照,及组间疗效的比较,探讨该方的临床疗效及可能的作用机理。
二.实验研究为体外动物实验。以体外培养的SD大鼠晶状体及其上皮细胞作为研究对象,以含有内障丸加减方活性成分的动物血清作为观察组,以空白组、模型组、槲皮素组和空白血清组作为对照组。研究取透明SD大鼠晶状体分组作体外培养,以Medium199培养液为培养基,加入终浓度300μmol/L的H_2O_2,采用Fenton反应复制SD大鼠晶状体氧化损伤模型。含药血清是以内障丸加减方煎剂给SD大鼠灌胃,腹主动脉采血离心后制备而成。离体晶状体孵育24小时后,对各指标进行观察。1.拍摄晶状体照片,将晶状体的混浊程度分5个等级,比较各组晶状体混浊的异同。2.以晶状体局部的灰度值,比较各组晶状体混浊度的异同。3.以透射电镜观察晶状体上皮细胞形态。4.TUNEL法检测LEC凋亡细胞,计算凋亡率,并作统计学分析。5.SAB法检测LEC凋亡相关基因Bcl-2和Bax表达,按不同表达率分5个等级,并作统计学分析。
结果
1.临床研究显示内障丸加减方能提高SC患者的视力,有效率及显效率分别为64%及8%,与障眼明组的疗效无统计学差异,而与白内停组比较,疗效有显著差异。内障丸加减方能改善SC患者的脾气虚弱、肝肾不足的虚证证侯,且优于障眼明组。内障丸加减方组及障眼明组的证侯改善程度明显优于白内停滴眼液组。三组的晶状体混浊程度在治疗后无明显变化。
2.正常组晶状体在孵育过程中保持透明状态;其余各组随着孵育时间的延长,晶状体逐渐出现不同程度的皮质混浊。模型组与空白血清组混浊程度明显较用药组严重,而含药血清组与槲皮素组比较,含药血清组晶状体呈现更好的透明度。
3.除了空白对照组,其余各组上皮细胞在氧化损伤后均能观察到不同程度的细胞凋亡形态。模型组及空白血清组的细胞形态明显破坏,细胞膜溶解破碎,细胞质及细胞器肿胀,部分胞浆溶解,核膜明显扩张,可见空泡样改变,细胞核固缩,核仁消失,异染色质增多,聚集于核膜边缘;槲皮素组及中药血清组细胞形态较模型组及空白血清组完整,细胞间的间隙增宽,细胞膜部分溶解,细胞质及细胞器出现肿胀,核周间隙增宽,细胞核形态仍呈椭圆形,核内异染色质堆积于核膜下,但异染色质较模型组等少,中药血清组的细胞形态结构优于槲皮素组。电镜下所见的总体印象显示,中药血清组LECs形态结构好于其它各对照组,认为内障丸加减方能更好地保护晶状体上皮细胞的结构,延缓细胞的变性。
4.光镜下观察LECs可见细胞核呈黑蓝色颗粒的凋亡阳性细胞。阴性细胞呈浅紫红色。光镜下显示空白组凋亡细胞数量极少,紫红色非凋亡细胞的排列紧密,模型组及空白血清组的凋亡细胞较多,且细胞间距离增宽;槲皮素组及中药血清组的光镜下所见,凋亡细胞的数量介于空白组与模型组之间,细胞密度亦优于模型组。空白组的LECs偶见凋亡阳性细胞,其凋亡细胞凋亡的百分率为0～8%,模型组、空白血清组的LECs凋亡率为46%～80%,槲皮素组与中药血清组的LECs凋亡率介于空白组与模型组之间,槲皮素组凋亡率为27%～49%,含药血清组凋亡率为13～36%,空白血清组的LECs凋亡率为36%～83%。含药血清组LECs凋亡率与各对照组比较有统计学意义。
5.在光学显微镜下观察Bcl-2及Bax单克隆抗体在LEC的免疫组织化学染色均能表现各自特异的胞浆定位,阳性细胞的胞浆显示特异性棕黄色颗粒状着色,棕黄色颗粒在细胞胞浆中均匀分布。Bcl-2及Bax在空白组大鼠LEC中均有表达,Bcl-2的表达明显强于Bax的表达,Bcl-2/Bax比值的数值较高。本实验过氧化氢诱导的大鼠LEC凋亡,模型组和空白血清组,Bax的表达上调明显,而Bcl-2的表达下调。槲皮素及中药血清组的LECs样本显示,与模型组及空白血清组相比,LECs的Bcl-2的表达显著上调,而Bax的表达明显下调,差异具有统计学意义。而槲皮素组与中药血清组样本比较,中药血清组更能有效上调Bcl-2的表达,降低Bax的表达,两组比较,作用的差异具有统计学意义。
结论
注:以下所作的结论均建立在有统计学意义的基础上(P＜0.05)
1.内障丸加减方可以提高SC患者的视力,改善脾气虚弱、肝肾不足的证候,其作用可能是通过有效地清除活性氧,增强晶状体的抗氧化能力,延缓衰老演变过程,改善视细胞功能,遏止或减轻白内障患者的视功能损害。认为对内障丸方的加减化裁没有降低其改善视力的作用;改进复方中药的剂型及给药方式有利于提高患者的用药依从性。
2.H_2O_2诱导的体外培养的大鼠晶状体氧化损伤导致的白内障发生,可作为老年性白内障相关研究的实验动物模型。且H_2O_2诱导的体外培养晶状体氧化损伤模型具有可行性。
3.含药血清能反映中药复方在体内的物质基础,根据实验结果可认为内障丸加减方具有抗凋亡及保护细胞结构的作用。
4.实验结果显示模型组和空白血清组Bcl-2显著下调,提示空白血清不具有上调Bcl-2表达的作用。
5.含药血清和槲皮素均能在一定程度上保护晶状体细胞结构,延缓细胞凋亡过程的作用;含药血清的保护作用更强。而空白血清可以认为对晶状体LEC结构无保护作用。
6.槲皮素及含药血清均能通过上调抑制凋亡的Bcl-2基因表达,下调Bax基因的表达来抑制LEC的凋亡的发生和发展。而结果显示含药血清具有更好的对抗LEC氧化损伤,抑制细胞凋亡的作用。
7.含药血清能在一定程度上保护H_2O_2诱导的氧化损伤对体外培养的SD大鼠LECs的损害,抑制或延缓细胞的凋亡。槲皮素作用低于含药血清,认为复方中药在动物体内经过吸收和代谢,各种活性成分之间起到协同和增效的作用。
Objective
1.Study the effect of Neizhangwanjiajian-decoction on improving lens opacities,the visual function,liver,spleen and kidney deficiency syndrome in senile cataract(SC) patients who belong to early and middle phase in clinical.To evaluate the different efficency between Neizhangwanjiajiandecoction and Zhangyanming- pills.The possible mechemism of Neizhangwanjiajian-decoction for SC in early and middle phase were explored.
2.To research the protect effect of Neizhangwanjiajian-decoction on lens epithelial cells(LEC) impaired by hydrogen peroxide(H_2O_2),rat LEC treated by Chinese medicine single component and Chinese medicine complex compound were explored to detect the apoptosis induced by oxidative damage.We attempt to explain the different mechanism of Chinese medicine of single components and complex compounds to inhibit the apoptosis in LEC.
Methods
1.Clinical research(perspective study):The 96 SC patients(192 eyes) in early and middle phase were randomly divided into 3 groups:Neizhang group(taken orally by Neizhangwanjiajian-decoction);Zhangyanming group(taken orally by Zhangyanming-pills);and Pirenoxine Sodium group(treated by Pirenoxine Sodium eye drop).All patients were treated for 3 course of therapy which three weeks for one course.The evaluation indexes for treatment were including visual acuity(VA),lens turbidity,and the effects of liver disorder,spleen disorder and kidney disorder.The therapeutic effect for Neizhangwanjiajian-decoction and related mechanism were evaluated by comparison of before and after treatment and comparison of different groups.
2.Experimental study(in vitro study):Lens and its epithelial cells were cultured in M199.Lens were divided into 5 groups as follows:1 blank group which treated with medium;2 model group which treated with H_2O_2(final concentration 300μmol/L);3 quercetin group which treated with quercetin and H_2O_2;4 decoction group which treated with rat serum containing Neizhangwanjiajian-decoction substances and H_2O_2;5 blank serum group which treated with rat blank serum and H_2O_2.Oxidative Damages model of rat Lens Epithelial Cell was copied by Fenton response.Rat serum containing Neizhangwanjiajian-decoction substances were prepared with 5 SD rats were taken orally with Neizhangwanjiajian-decoction to prepare drug-conditioned serum,twice a day for 3 days.The other 5 rats were taken orally with the same volume of water.Blood was harvested from the abdominal aortas 2 hours following the last medication.The supernatant was obtained after centrifugalization,and stored at -20℃.All lens cultured for 24h and were detected the indexes as follow:1.Lens opacification were divided into 5 grades according photos and the differences within each group in the degree of lens opacification were compared as an index.2.Lens in the local gray value was compared between each group as another index for lens turbidity. 3.Cell morphology was detected by transmission electron microscopy.4.The expression of apoptosis related gene,such as Bcl-2 and Bax were detected by immunohistochemistry in LEC.5.Apoptosis in LEC were also detected by TUNEL and apoptosis rate.
Results
1.Clinical studies have shown Neizhangwanjiajian decoction could improve vision in patients with SC with efficient and remarkable efficiency of 64% and 8%.There was no significant difference between Zhangyanming-pills and Neizhangwanjiajian-decoction.But there were significant differences in efficacy when compared with Pirenoxine Sodium eye drop. Neizhangwanjiajian-decoction and Zhangyanming-pills were better in improved kidney deficiency syndrome,liver deficiency syndrome and spleen deficiency syndrome in patients with SC than Pirenoxine Sodium eye drop.There was no significant difference in lens opacification when treated with these three drugs.
2.Normal lens were keeping in the transparent state during incubated process:but the lens in other groups gradually appeared cortex turbidity along with the incubation time.Each group did not observe obvious signs of turbidity when incubated for 8h.Lens in model group and blank serum group began to appear cortex localized turbidity and turbidity is the uneven distribution of the site at 16h;Lens in quercetin group and decoction group also appeared decrease of the transparency in cortex at the same time.Each group of lens turbidity increase than before when incubated for 20h,and the central region of the cortex appeared turbidity in lens.When incubation extended for 24h, each group of lens cortex appeared both gray and white opacities.The lens in model group and blank serum group had a higher degree of turbidity than the treatment group,and the effect of decoction group had better performance than quercetin group in lens transparency.Along with the rest of the group to extend the incubation time,the progressive lens cortex with varying degrees of turbidity.
3.In addition to the control group,and the remaining epithelial cells of each group in the oxidative damage can be observed the different degrees of morphological apoptosis.Cells in control group kept in normal morphology, it can be seen cells arrangement closely,membrane integrity,cytoplasmic homogeneous,morphology of mitochondria and rough endoplasmic reticulum also keep normal,membrane-free expansion,clearly visible nucleolus,euchromatin uniformly distributed in the nucleus,no accumulation of heterochromatin;The cell morphology in model group and blank serum group significantly were damaged, cell membrane broken and dissolved,cytoplasm and cell organelle swelling, cytoplasm were dissolved partly,a remarkable expansion of the nuclear membrane,showing vacuolar changes,nuclear condensation,nucleolus disappear, increased heterochromatin gathered on the edge of the nuclear membrane;Cells in quercetin group and decoction group were better than the model group and blank serum group in integrity,and the gap widened between cells,cell membrane were also dissolved partly,cytoplasm and cell organelle swelling, gap widened perinuclear,nucleus still kept in oval form,heterochromatin accumulated under the nuclear membrane,its quantity was less than to the model group.Cell morphology and structure in decoction group was superior to the of quercetin group.Ganerally LECs morphology and structure treated by Neizhangwanjiajian decoction was better than the other control group detected by electron microscope.Neizhangwanjiajian decoction can better protect the structure of lens epithelial cells,slow degeneration of cells.
4.Apoptosis-positive LEC which nuclear emerge black and blue particle could be detected by light microscope.If apoptosis was not happened in LEC, light-purple color would be appeared.Light microscope showed that the number of apoptotic cells in the blank group was few,purple non-apoptotic cells were arranged closely.More apoptotic cells were emerged in model group and blank serum group,and widened the distance between cells as much.The number of apoptotic cells in quercetin group and decoction group were between the control group and model group,the cell density was also better than that in the model group.Apoptosis positive LECs could be detect in blank group occasionally, the apoptosis percentage was 0～8%,the apoptosis rate in model group and blank serum group was 46%～80%,apoptosis rate in quercetin group and decoction group were lower than in model group but higher than in control.Apoptosis rate was 27%～49%in quercetin group,13～36%in decoction group and 36%～83%in blank-serum group.Apoptosis rate of decoction group had statistically significant compared with the otber control groups.
5.Immunohistochemical staining was explored to detect the expression of Bcl-2 and Bax with optical microscope.Specified brown granular uniformly distributed in cytoplasm would be thought to be positive cells.Both Bcl-2 and Bax were expressed in LEC of control group.Bcl-2 expression was significantly stronger than Bax,naturally values of Bcl-2/Bax ratio was higher.In this study Bax expression increases significantly,while the expression of Bcl-2 down when induced apoptosis in rat LECs with hydrogen peroxide as model group and treated with blank rat serum.But when treated with Quercetin and decoction,Bcl-2 expression in LECs increases significantly, while Bax expression was significantly reduced compared with the model group and blank serum group.The decoction group had a better effect in increase of Bcl-2 expression and reduction of Bax expression when compared with quercetin.The difference was significant statistically.
Conclusion
Note:The following conclusions are based on the basis of statistical significance(P＜0.05)
1.Neizhangwanjiajian decoction can improve vision in patients with SC, and improve the liver,spleen and kidney deficiency syndrome.The mechanism maybe related with its role to remove reactive oxygen effectively,and enhance the antioxidant capacity of the lens.As a result,the aging evolution was delayed,the visual cell function was improved,and the damage of visual function in cataract patients was to stop or mitigate.Neizhangwanjiajian decoction can improve eyesight in patients with SC.To improve the way of Traditional Chinese medicine administration would help to improve the of medication compliance in patients.
2.H_2O_2-induced rat lens oxidative damage in vitro can lead to cataract. This model can be used as the relevant experimental animal study for senile cataract.And H_2O_2-induced oxidative damage in vitro model of lens is feasible.
3.Drug-containing serum can reflect the effect of traditional Chinese medicine compound in the body.Neizhangwanjiajian decoction have the effect of anti-apoptotic and protection of role of cell structure in rat LEC.
4.The expression of Bcl-2 in model group and blank serum group significantly reduced,suggesting that blank rat serum do not have raised Bcl-2 expression on lens.
5.Neizhangwanjiajian decoction and quercetin can protect the structure of lens cells;retard the process of apoptosis;with stronger role in the former. Blank serum can be considered non-protective effect of LEC structures.
6.Quercetin and Neizhangwanjiajian decoction can inhibit the occurrence and development of apoptosis by up regulating Bcl-2 gene expression which inhibit the apoptosis,reduced Bax gene expression.The results showed that Neizhangwanjiajian decoction had a better effect against oxidative damage and inhibit cell apoptosis.
7.Neizhangwanjiajian decoction can protect LEC in vitro from H_2O_2-induced oxidative damage,and can inhibit or delay cell apoptosis.Neizhangwanjiajian decoction has a better effect than quercetin.The mechanism may relate that combination of various active ingredients plays the role of synergy and efficiency after absorption and metabolism in the body.
引文
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