内障丸加减方对氧化损伤大鼠晶状体上皮细胞中Bcl-2及Bax表达的影响

目的观察中药单体与含药血清对氧化损伤大鼠晶状体上皮细胞(LECs)中Bcl-2及Bax表达的干预作用异同,探讨内障丸加减方预防或延缓白内障发生发展的作用机制。方法取3只8周龄大鼠,予内障丸加减方45 g/(kg.次)灌胃,每日2次,连续3 d,末次灌胃后1 h,制备含药血清。以Medium199培养液30 mL+青霉素注射液20万U/L+链霉素注射液25万U/L制备成培养液5份,分别为空白对照组、模型组、槲皮素组、含药血清组及空白血清组,除空白对照组外,其余各组培养液以30%过氧化氢(H2O2)溶液诱导Fenton反应。然后含药血清组及空白血清组分别加入相应的血清,将各培养液置于无菌6孔培养板中,每孔含培养液3 mL。另取15只4～5周龄大鼠随机分为5组,分别为空白对照组、模型组、槲皮素组、含药血清组及空白血清组,每组各3只。无菌操作提取晶状体,将晶状体放入含有相应培养液的培养孔中,每孔3枚。经24 h后,取囊膜铺片,以免疫组化法检测LECs的凋亡相关基因Bcl-2及Bax表达,在光学显微镜下观察Bcl-2及Bax的表达情况。结果空白对照组、槲皮素组及含药血清组Bcl-2及Bax表达与模型组比较差异有统计学意义(P<0.05),空白血清组与模型组Bcl-2及Bax表达无统计学意义(P>0.05)。空白对照组、槲皮素组及空白血清组Bcl-2及Bax表达与含药血清组比较差异有统计学意义(P<0.05)。过氧化氢诱导的大鼠LECs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。槲皮素及含药血清组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。含药血清组比槲皮素组更能有效上调Bcl-2的表达,降低Bax的表达。结论内障丸加减方的血清活性成分能有效保护LECs的细胞结构,上调Bcl-2基因的表达,抑制细胞凋亡。

Effect of Neizhang pill on Bcl - 2 and Bax expression in lens epithelial cells of rats with oxidative damage

Objective To observe the intervention of the similarities and differences of Chinese medicine on Bcl-2 and Bax expression in lens epithelial cells（LECs） of rats with oxidative damage,explore action mechanism of Neizhang pill on prevention or delay of cataract development.Methods Rat serum containing Neizhangwanjiajian-decoction substances were prepared with 5 SD rats were taken orally with Neizhangwanjiajian-decoction to prepare drug-conditioned serum.Lens and its epithelial cells were cultured in M199 for 24 hours and were divided into five groups,control group,model group,Quercetin group,serum containing drug group and serum group.Oxidative Damages model of rat Lens Epithelial Cell was copied by Fenton response.Immunohistochemical staining was explored to detect the expression of Bcl-2 and Bax in LECs with optical microscope.Results The expression of Bcl-2 and Bax in control group,Quercetin group and serum containing drug group was different from that in model group（P〈0.05）.There was no difference between serum group and model group on the expression of Bcl-2 and Bax（P〉0.05）.There was obvious difference between control group,Quercetin group,serum group and serum containing drug group on the expression of Bcl-2 and Bax（P〈0.05）.In hydrogen peroxide-induced apoptosis of rat LECs Bax expression significantly increased and the expression of Bcl-2 down in model group and serum group.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 Serum active ingredients of Neizhang pill can effectively protect the cell structure of the LECs to increase the expression of Bcl-2 gene and inhibit apoptosis.