乌梅丸对慢性高眼压模型大鼠生物节律的干预作用及机制研究

Intervention Effect and Mechanism of Wumei Pill on Chronobiological Rhythm Disorder in Chronic High Intraocular Pressure Model Rats

目的探讨乌梅丸对慢性高眼压模型大鼠生物节律紊乱的干预作用。方法采用巩膜上静脉烧灼法建立慢性高眼压大鼠模型,将30只大鼠随机分为模型组(MG)和乌梅丸组(WM),另将进行假手术的大鼠设为对照组(CG),每组各15只。WM组大鼠每日予乌梅丸水煎剂9 g/kg灌胃,CG组、MG组予等体积0.9%氯化钠注射液灌胃,每周6次,连续给药4周。给药结束次日2:00、8:00、14:00、20:00的4个时间点等距测量眼压来评估24 h眼压节律;取材后采用苏木精-伊红(HE)染色观察大鼠视网膜形态;原位末端转移酶标记技术(TUNEL)检测大鼠视网膜神经节细胞(RGC)凋亡情况;酶联免疫吸附法(ELISA)检测大鼠2:00血清生物节律相关指标,包括褪黑素(MT)、皮质酮(CORT)、促肾上腺皮质激素(ACTH)、促肾上腺皮质激素释放激素(CRH);Western Blot法检测大鼠内在光敏感性视网膜神经节细胞(ipRGC)特异性蛋白—视黑质蛋白表达水平。结果(1)视网膜形态:MG组大鼠视网膜组织结构破坏,内、外核层组织疏松,RGC排列紊乱,数量明显减少;WM组视网膜组织结构较为清晰完整,RGC少量丢失。(2)RGC凋亡率:MG组RGC凋亡率高于CG组(t=16.010,P=0.000),WM组RGC凋亡率低于MG组(t=-12.890,P=0.000),差异均有统计学意义。(3)昼夜眼压波动:MG组大鼠各时间点眼压均高于CG组(t2:00=16.600、t8:00=19.190、t14:00=16.230、t20:00=15.900,均P=0.000);WM组大鼠2:00与8:00眼压低于MG组(t2:00=-2.796,P=0.009;t8:00=-4.166,P=0.000),差异均有统计学意义。大鼠眼压整体峰值出现在凌晨2:00,2:00—8:00眼压呈缓降趋势,8:00—14:00眼压下降明显,14:00为眼压谷值时间。(4)生物节律相关指标:MG组大鼠血清MT表达低于CG组(t=-7.480,P=0.000);WM组大鼠血清MT、CORT表达均高于MG组(tMT=7.136、tCORT=5.390,均P=0.000),CRH表达低于MG组(t=-3.144,P=0.004),差异均有统计学意义。(5)视黑质蛋白:MG组大鼠视网膜视黑质蛋白表达低于CG组(t=-16.127,P=0.000);WM组大鼠视网膜视黑质蛋白表达高于MG组(t=6.390,P=0.000),差异均有统计学意义。结论慢性高眼压模型大鼠28 d可导致视黑质蛋白表达降低,2:00血清MT分泌降低,昼夜眼压波动幅度增大,从而诱导RGC凋亡。厥阴病欲解时主方乌梅丸可能通过介导视黑质蛋白表达,改善慢性高眼压模型大鼠生物节律紊乱,抑制RGC凋亡。

OBJECTIVE To investigate the intervention effect of Wumei Pill on chronobiological rhythm disorder in a chronic high intraocular pressure(IOP)rat model.METHODS A total of 30 rats were used to establish the chronic high IOP rat model using scleral venous occlusion.They were randomly divided into the model group(MG)and Wumei Pill group(WM),with a control group(CG)undergoing sham surgery.The WM group rats were orally administered Wumei Pill decoction 9 g/kg daily,while CG and MG groups were given an equal volume of 0.9%sodium chloride solution orally six times a week for four consecutive weeks.IOP was measured at 2:00,8:00,14:00,and 20:00 on the day after the last administration to evaluate the 24 h IOP rhythm.Retinal morphology was observed using hematoxylin-eosin(HE)staining after tissue collection.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay was used to detect retinal ganglion cell(RGC)apoptosis.Enzyme-linked immunosorbent assay(ELISA)was used to measure serum levels of melatonin(MT),cortisol(CORT),adrenocorticotropic hormone(ACTH),and corticotropin-releasing hormone(CRH)at 2:00.Western Blot was used to assess the expression level of the ipRGC-specific protein,melanopsin,in the retina.RESULTS(1)Retinal morphology:Retinal tissue structure was disrupted in MG group rats,with loose inner and outer nuclear layers and disorganized RGCs and significantly reduced numbers.The retinal tissue structure in the WM group was relatively clear and intact,with a slight loss of RGC.(2)RGC apoptosis rate:The RGC apoptosis rate was higher in the MG group than in the CG group(t=16.010,P=0.000),and lower in the WM group than in the MG group(t=-12.890,P=0.000),with statistically significant differences.(3)Diurnal IOP fluctuation:The IOP at each time point in the MG group rats was higher than that in the CG group(t2:00=16.600,t8:00=19.190,t14:00=16.230,t20:00=15.900,all P=0.000).The IOP at 2:00 and 8:00 was lower in the WM group rats than in the MG group(t2:00=-2.796,P=0.009;t8:00=-4.166,P=0.000),with statistically significant differences.The overall peak of IOP occurred at 2:00 in the rats,with a gradual decrease from 2:00 to 8:00,a significant drop from 8:00 to 14:00,and the lowest IOP at 14:00.(4)Chronobiological rhythm parameters:Serum MT expression was lower in the MG group rats than in the CG group(t=-7.480,P=0.000).Serum MT and CORT expression were higher in the WM group rats than in the MG group rats(tMT=7.136,tCORT=5.390,both P=0.000),while CRH expression was lower in the WM group than in the MG group(t=-3.144,P=0.004),all with statistically significant differences.(5)Melanopsin Protein:Retinal melanopsin protein expression was lower in the MG group rats than in the CG group rats(t=-16.127,P=0.000),and higher in the WM group rats than in the MG group rats(t=6.390,P=0.001),with statistically significant differences.CONCLUSIONS Chronic high IOP in rats for 28 days led to decreased melanopsin protein expression,decreased serum MT secretion at 2:00,increased diurnal IOP fluctuation,and induced RGC apoptosis.Wumei Pill,the principal prescription for alleviating the hypochondriac disease,may improve the chronobiological rhythm disorder in the chronic high IOP rat model and inhibit RGC apoptosis by mediating melanopsin protein expression.