The Effect Of Neizhangjiajian-pill On LEC Apoptosis And Its Related Gene Expression Induced By Oxidative Damage Of Lens

Objective1.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(H₂O₂),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.Methods1.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 H₂O₂(final concentration 300μmol/L);3 quercetin group which treated with quercetin and H₂O₂;4 decoction group which treated with rat serum containing Neizhangwanjiajian-decoction substances and H₂O₂;5 blank serum group which treated with rat blank serum and H₂O₂.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.Results1.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.ConclusionNote: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.H₂O₂-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 H₂O₂-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 H₂O₂-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.