摘要：目的 评价菊睛丸改善肾阳虚衰老小鼠视网膜损伤的效应部位并探索作用机制。方法 通过联合应用D-半乳糖皮下注射与氢化可的松后肢注射的复合造模方法，建立肾阳虚型衰老小鼠模型，并给予菊睛丸各溶剂提取组分进行干预治疗。通过体征观察、测定血清氧化应激标志物水平、环核苷酸水平评价菊睛丸不同溶剂提取部位对肾阳虚衰老小鼠的改善作用。通过光学相干断层扫描技术（OCT）检测小鼠视网膜情况，HE染色法检测视网膜细胞损伤程度，TUNEL染色法检测视网膜细胞凋亡情况，试剂盒检测氧化应激因子表达，酶联免疫吸附法检测小鼠眼球中睫状神经营养因子（CNTF）、脑源性神经营养因子（BDNF）与神经生长因子（NGF）表达，采用蛋白印迹法（Western blotting，WB）对小鼠视网膜组织内凋亡及神经营养通路相关蛋白的表达水平进行定量分析。结果 与空白组相比，模型组小鼠体重增长缓慢，活动减少，血清中抗氧化因子超氧化物歧化酶（SOD）与谷胱甘肽（GSH）等表达减少（P<0.01），cAMP/cGMP比值降低（P<0.01）。菊睛丸总提取物部位显著提高抗氧化因子SOD与GSH等表达（P<0.01），减少氧化产物丙二醛（MDA）产生（P<0.01），提高cAMP/cGMP比值（P<0.01）；显著增加模型小鼠视网膜厚度（P<0.01），改善视网膜损伤并抑制视网膜细胞凋亡（P<0.01），显著下调Caspase-3与Cleaved Caspase-3凋亡通路蛋白表达（P<0.05），而Cleaved Caspase-3与Caspase-3的比值表达水平则有明显的下降（P<0.01）；提升CNTF、BDNF与NGF表达（P<0.01），并显著上调眼球CREB和ERK的磷酸化水平（P<0.05）。结论 菊睛丸总提取物部位是菊睛丸改善肾阳虚衰老模型小鼠视网膜损伤状况的效应部位，其作用途径与改善氧化应激导致的视网膜凋亡以及提高营养因子表达相关。

Abstract: Objective To evaluate the effective site of Ju Jing Wan in improving retinal injury in aging mice with kidney yang deficiency and explore its mechanism of action. Method A composite modeling method of subcutaneous injection of D-galactose and hind limb injection of hydrocortisone was used to establish a kidney yang deficiency type aging mouse model, and various solvent extracted components of Jujing Pill were given for intervention treatment. Evaluate the improvement effect of different solvent extracts of Jujing Pill on aging mice with kidney yang deficiency by observing physical signs, measuring serum oxidative stress marker levels, and cyclic nucleotide levels. Optical coherence tomography (OCT) was used to detect the condition of the mouse retina, HE staining was used to detect the degree of retinal cell damage, TUNEL staining was used to detect retinal cell apoptosis, assay kit was used to detect oxidative stress factor expression, enzyme-linked immunosorbent assay was used to detect the expression of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) in the mouse eyeball, and Western blotting (WB) was used to quantitatively analyze the expression levels of apoptosis and neurotrophic pathway related proteins in the mouse retina tissue. Results Compared with the blank group, the model group mice showed slow weight gain, reduced activity, decreased expression of antioxidant factors such as superoxide dismutase (SOD) and glutathione (GSH) in serum (P<0.01), and decreased cAMP/cGMP ratio (P<0.01). The total extract of Jujing Pill significantly increased the expression of antioxidant factors such as SOD and GSH (P<0.01), reduced the production of oxidative product malondialdehyde (MDA) (P<0.01), and increased the cAMP/cGMP ratio (P<0.01); Significantly increased the retinal thickness of model mice (P<0.01), improved retinal damage and inhibited retinal cell apoptosis (P<0.01), significantly downregulated the expression of Caspase-3 and Cleaved Caspase-3 apoptosis pathway proteins (P<0.05), and the ratio expression level of Cleaved Caspase-3 to Caspase-3 showed a significant decrease (P<0.01); Enhance the expression of CNTF, BDNF, and NGF (P<0.01), and significantly upregulate the phosphorylation levels of CREB and ERK in the eyeball (P<0.05). Conclusion The total extract of Jujing Pill is the effective site of Jujing Pill in improving retinal damage in mice with kidney yang deficiency aging model. Its mechanism of action is related to improving retinal apoptosis caused by oxidative stress and increasing the expression of nutritional factors.

1. 江苏省高校自然科学研究重大项目（22KJA360008）：基于“肝-眼轴”的菊睛方防治年龄相关性黄斑变性功效物质与生物学机制研究，负责人：朱悦；2. 国家自然科学基金区域创新发展联合基金目重点支持项目（U21A20408）：基于“肝-眼轴”的枸杞子及其复方防治青少年近视与年龄相关性黄斑变性的功效物质与生物学机制研究，负责人：段金廒；3. 南京中医院大学中药学一流学科“引领计划”科学研究专项（ZYXYL2024-015）：面向老龄人口健康需求的若干经典方剂功效物质研究与创新药物发现，负责人：宿树兰。