摘要
目的：探讨临床中自拟方通窍明目4号对青光眼性视神经损伤的保护作用及分子学作用机制,为中药方剂对神经元保护提供直接证据。
材料和方法：腹腔注射10%水合氯醛(3.8ml/kg)麻醉小鼠,并辅以0.4%盐酸奥布卡因滴眼液局部麻醉,并用复方托比卡胺眼药水术眼散瞳。采用单侧前房注射复方卡波姆溶液的方法在成年SD大鼠上建立慢性高眼压模型。造模成功后,对高眼压—周的大鼠模型,给予通窍明目4号按照低剂量、中剂量、高剂量进行治疗两周,并用维生素B1、B12作为西药对照组治疗。以大鼠慢性高眼压模型视网膜内信号传导与转录激活因子3(stat3)、视网膜神经节细胞、视网膜抗凋亡基因(Bcl-x1)为干预靶点,采用免疫组织化学、免疫印迹、病理组织学观察和细胞超微结构检测等方法,从整体及细胞、基因等多个层次系统地研究通窍明目4号对青光眼的视神经保护作用及分子学作用机制,定量分析比较通窍明目4号的视神经保护作用。两组之间采用独立样本t检验,多组之间比较采用单因素方差分析,p<0.05被认为有差异性,具有统计学意义,p<0.01为具有及其显著的差异性。
结果：1.光镜下空白组视网膜十层结构清晰,视网膜神经节细胞清晰可见,无水肿。高眼压后的视网膜早期变厚晚期变薄,隔层结构不清,排列紊乱,空泡状结构增多,节细胞数量减少,炎细胞浸润。经过通窍明目4号治疗后的视网膜损伤明显轻于模型组,视网膜各层结构稍紊乱,节细胞排列松散,可见空泡样变,节细胞数量较模型组增多,网膜病变程度减轻。2.电镜下空白组节细胞及神经胶质细胞结构正常,视细胞内节中线粒体丰富,外节形态结构正常,膜盘排列紧密。高眼压模型节细胞结构模糊,细胞器消失,线粒体肿胀,胞浆空泡变性,突触融合,膜盘溶解。经过药物治疗后空泡变性数量较模型组减少,节细胞膜清晰,胞浆内可见结构基本正常的粗面内质网、线粒体。用药后线粒体水肿减轻,突触结构相对清晰,膜盘融合程度减轻。3.免疫组化及免疫印迹提示：空白组的大鼠视网膜stat3、 Bcl-x1呈低表达,高眼压模型呈现出1W表达轻度上升,3W则表达回落,而经过药物治疗后,表达则明显上升,提示通窍明目4号激活了视神经保护通道。
结论：通窍明目4号可以减轻高眼压造成的视网膜病理改变,纠正可逆的变性细胞,减轻高眼压状态下RGCs的凋亡,激活stat3神经通路,上调抗凋亡基因,发挥神经保护作用,这条通路通过整合包括诱导和调节免疫反应,DNA修复、线粒体的修复在内的多种方式发挥作用。因此通窍明目4号对青光眼性视神经、视网膜的损伤有修复作用,改善RGCs的生存微环境,保护未受损的细胞,延缓或阻止部分损伤的细胞继续变性。
Objective:To investigate the clinical through self orifices TongqiaoMingmu No.4on glaucomatous optic neuropathy protective effects and molecular mechanism, Provide direct evi-dence for traditional Chinese medicine prescription to protect neurons.
Materials and methods:Intraperitoneal injection of10%chloral hydrate anesthesia in mice (3.8ml/kg), And with Aobukayinkayin hydrochloride eye drops in local anesthesia,and dilated with mydrinp eye drops.Using unilateral anterior chamber injection method of the compound carbomer solution on adult SD rats to establish chronic high intraocular pressure model. After the success of modeling, The high IOP after one week in rats, Gave orifices Tong qiaoMingmu No.4in low dose, middle dose, high dose were treated for two weeks, and use vitamin B1, B12as the control group of Western medicinetherapy. Signal transduction and transcription in the retina of rats with chronic intraocular hyper-tension in activating factor3(STAT3)、RGCs、 Bcl-xl for interventions targeting, the histopathological observation、 immuno-histochemical、Western blot and cell ultrastructure of detection method, from the whole and cells、genes levels systematically orifices Tongqiao Mingmu No.4on glaucoma optic effect and molecular mechanism of neural protection,quantitative analysis of optic nerve protective effe-ct about the orifices Tongqiao Mingmu No.4Between the two groups using indep-endent sample T test,it used single factor analysis of variance comparison between groups, p<0.05is considered to have differences, statistically significant, p<0.01was significant difference.
Result:1.The blank group retinal ten layerstructure is clear under light microscope,retinal ganglion cells were clearly visible, no edema. The retina became thinning at first and became thick after high intraocular pressure, the interlayer structure is not clear, arranged disorder, increased vesicular structure, the number of ganglion cells reduced, inflammatory cell infiltration. retinal damage was obviously lighter than the model group after Tong Qiao ming mu No4treatment, the retinal structure of each layer was somewhat disorder, the ganglion cell arranged loosely, visible vacuole, cell number increased in comparison with the model group, to reduce the degreeof retinaledema.2.The structure of glial cells and thenerve ganglion cells was normal in the blank group under the Electron microscopic, the cells mitochondria was rich, Outer segment structure is normal, the membrane discarranged closely. Structure set of ganglion cells high intraocular pressurefuzzy, organelle disappear, mitochondria swelling, cytoplasmvacuolar degeneration, synaptic integration, lap dissolve. After drug treatment and vacuolar degeneration compared with model control group, reduce the number of ganglion cells, clear membrane, endoplasmic reticulum, mitochondria structure basically normal cytoplasm. After treatment of mitochondria edema, the synapticstructure is relatively clear, reduce the degree of disc membrane fusion.3.Immunohistochemistry and Western blot indicated:blank group of retinal STAT3, Bcl-xl rats showed low expression, high intraocular pressure model showing expression of mild upward trend, after drug treatment, the expression increased significantly,show tong qiao ming mu No4activation of the optic pathway protection channel.
Conclusion:Tong qiao ming mu No4on glaucomatous optic nerve, retina damage,repair the exact, improve the RGCs microenvironment, the protection of intact cells,delay or prevent part damage celldegeneration. The changes of retinal pathological.It can reduceand delay caused by high intraocular pressure, deformation correcting reversible cell. It can reduce the apoptosis of high intraocular pressure RGCs. It can activate STAT3pathway upregulation of the antiapoptotic gene, plays a neuroprotective role of this pathway, including induction and regulation of immune responses, through the integration of DNA repair, mitochondrial repair, a variety of ways to play the role, sothe early application of STAT3agonists can delay or avoidacute light damage or mutation neuron s tune induction of apoptosis.Therefore it can effectively protect the retina,optic nerve, activation of the STAT3 pathway,and upregulation of the antiapoptotic gene, effectively protect the optic nerve and retina.
引文
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