胰激肽原酶对慢性高眼压大鼠视网膜神经节细胞的保护作用及机制

The protection and mechanism of pancreatic kininogenase on RGCs in chronic high intraocular pressure rats

目的研究注射用胰激肽原酶对慢性高眼压大鼠眼压及视网膜神经节细胞(RGCs)的保护作用。方法SD大鼠30只,按随机数字表法分为空白组、模型组、治疗组,每组10只。模型组和治疗组用巩膜静脉烧灼法制作慢性高眼压大鼠模型,造模成功1周后,治疗组给予胰激肽原酶(0.8 U)治疗,而空白组、模型组给予腹腔注射等剂量0.9%氯化钠溶液(0.2 mL),均每天1次,连续2周。监测大鼠眼压的变化并记录数据。苏木精-伊红(HE)染色观察RGCs形态,DNA断裂的原位末端标记法(TUNEL)测定RGCs凋亡情况,免疫组织化学染色法测定细胞凋亡相关基因B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的含量。结果造模前眼压各组无明显变化(F=0.152,P>0.05),造模后模型组[术后1周:(32.85±2.06)mmHg,术后2周:(33.27±2.24)mmHg,术后3周(33.00±1.09)mmHg]与治疗组[术后1周:(32.77±2.07)mmHg,术后2周:(31.68±2.20)mmHg,术后3周(30.09±2.52)mmHg]眼压均升高,与空白组[术后1周:(15.44±1.02)mmHg,术后2周:(15.21±1.41)mmHg,术后3周(14.90±1.32)mmHg]比较差异有统计学意义(F=355.429,F=427.714,F=315.063;均P<0.001)。HE染色示空白组视网膜各层结构清晰,模型组RGCs排列不规整,细胞缺失,有空泡形成。治疗组细胞排列稍不规整,细胞少量缺失。TUNEL示空白组偶见凋亡细胞,模型组凋亡细胞增多,治疗组凋亡细胞减少,组间比较差异有统计学意义(F=796.179,P<0.001)。免疫组化示模型组Bcl-2、Bax表达均有所增强,而治疗组较模型组Bcl-2表达增强,Bax表达明显减弱,各组间比较差异有统计学意义(F=389.728,F=525.331;均P<0.01)。结论胰激肽原酶可以缓慢降低眼压,还可能通过升高Bcl-2,降低Bax的表达抑制RGCs凋亡,达到保护视神经的目的。

Objective To study the protective effect of pancreatic kininogenase on intraocular pressure and retinal ganglion cells(RGCs)in rats with chronic ocular hypertension.Methods Thirty SD rats were assigned into blank group,model group and treatment group according to random number table method,10 rats in each group.The model group and the treatment group used scleral vein cauterization to make a chronic ocular hypertension rat model.After 1 week of successful modeling,the treatment group was given pancreatic kininogenase(0.8 U),while the blank group and the model group were given intraperitoneal injection of 0.9% sodium chloride solution(0.2 mL),once a day for 2 consecutive weeks.Changes in rat intraocular pressure were monitored and recorded.Hematoxylin-eosin(HE)staining was used to observe the morphology of RGCs,TdT-mediated dUTP nick end labeling(TUNEL)of DNA fragmentation was used to determine the apoptosis of RGCs,and immunohistochemical staining was used to determine the contents of apoptosis related genes Bcl-2 and Bax.Results The pressure had no significant change before modeling(F=0.152,P>0.05),After modeling,the model group[1 week after operation:(32.85±2.06)mmHg,2 weeks after operation:(33.27±2.24)mmHg,3 weeks after operation(33.00±1.09)mmHg]and the treatment group[1 week after operation:(32.77±2.07)mmHg,2 weeks after operation:(31.68±2.20)mmHg,3 weeks after operation(30.09±2.52)mmHg]both had increased intraocular pressures,which was significantly different from that of the blank group[1 week after operation:(15.44±1.02)mmHg,2 weeks after operation:(15.21±1.41)mmHg,3 weeks after operation(14.90±1.32)mmHg](F=355.429,F=427.714,F=315.063,respectively;all P<0.001).HE staining showed clear structure of the retinal in the blank group.But poorly arranged in the model group,the cells were absent,and the vacuoles were formed.In the treatment group,the cells were slightly irregular and decreased.TUNEL showed sporadic apoptotic cells in the blank group,increased apoptotic cells in the model group,and decreased apoptotic cells in the treatment group;there were significant differences among the 3 groups(F=796.179,P<0.001).Immunohistochemical staining showed increased expressions of Bcl-2 and Bax in the model group,and the expression of Bcl-2 was higher and the expression of Bax was significantly lower in the treatment group than in the model group;the differences among the groups were statistically significant(F=389.728,F=525.331;all P<0.01).Conclusion The pancreatic kininogenase can slow down the intraocular pressure gradually,and inhibit the apoptosis of the retinal ganglion cells to protect the optic nerve by increasing the expression of Bcl-2 and reducing the expression of Bax.