载锌氧化镁纳米颗粒在谷氨酸兴奋性毒性青光眼模型中的视网膜保护作用

Retinal protective effects of zinc-loaded magnesium oxide nanoparticles in a glutamate-excitotoxicity glaucoma model

摘要

目的 青光眼的病理特征主要表现为视网膜神经节细胞(retinal ganglion cells，RGCs)的进行性丧失。目前针对青光眼尚缺乏有效的RGCs保护策略，纳米材料是有潜力的药物递送载体。本研究旨在探索载锌氧化镁纳米颗粒(MgO-Zn²⁺ nanoparticles，MgO-Zn NPs)对谷氨酸诱导RGCs损伤的作用，并评价其体内外生物相容性及神经保护潜能。 方法 制备MgO-Zn NPs。通过透射电子显微镜及能谱分析等对MgO-Zn NPs进行表征。体外实验以大鼠视网膜前体细胞系R28为研究对象，采用细胞计数试剂盒8(cell counting kit-8，CCK-8)法系统评估MgO-Zn NPs的细胞毒性。体内实验以C57/BL小鼠为研究对象，通过玻璃体内注射N-甲基-D-天冬氨酸(N-methyl-D-aspartate，NMDA)建立小鼠视网膜兴奋毒性模型，并用MgO-Zn NPs干预。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling，TUNEL)染色评估小鼠视网膜RGCs的数量及凋亡情况。采用苏木精-伊红(hematoxylin and eosin，HE)染色检查小鼠的视网膜层结构。采用闪光视觉诱发电位(flash visual evoked potential，F-VEP)评价RGCs视觉传导功能。对小鼠视网膜组织进行RNA测序，分析差异表达基因的通路及功能，并进一步验证小鼠视网膜组织相关蛋白质表达水平的差异。 结果 透射电子显微镜及能谱分析成功证实了MgO-Zn NPs的形貌及成分特征，确认复合物制备成功。CCK-8检测结果表明：75 µg/mL MgO-Zn NPs对R28细胞无毒性。体内实验发现：小鼠玻璃体腔内注射MgO-Zn NPs溶液后，其眼表和角膜均未出现明显不良反应，显示其良好的眼部耐受性。TUNEL染色结果显示：视网膜兴奋毒性模型小鼠的RGCs数量较正常小鼠显著减少(P<0.05)，表明模型建立成功；而MgO-Zn NPs干预后显著减少了NMDA诱导的RGCs凋亡(P<0.05)。HE染色表明：MgO-Zn NPs干预后模型小鼠视网膜层结构得到部分恢复(P<0.05)。F-VEP测量结果显示：模型小鼠的P2波潜伏期增长、振幅降低(均P<0.001)，接受MgO-Zn NPs干预的模型小鼠P2波的潜伏期和振幅均得到一定程度的恢复(均P<0.05)。RNA测序结果表明：MgO-Zn NPs改善了NMDA诱导的视网膜转录组异常，差异表达基因主要与磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase，PI3K)-蛋白激酶B(protein kinase B，Akt)通路和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)信号通路相关。免疫荧光染色结果显示：MgO-Zn NPs干预显著降低了模型小鼠视网膜组织的p-Akt和p-mTOR的表达水平(均P<0.01)。 结论 MgO-Zn NPs既可保护视网膜神经元，又可作为眼内药物递送载体，有望作为具有双重功能的青光眼治疗候选药物。

Abstract

Objective Glaucoma is pathologically characterized by the progressive loss of retinal ganglion cells (RGCs). Currently, effective strategies for protection of RGCs in glaucoma remain lacking, and nanomaterials represent promising drug-delivery carriers. This study aims to investigate the effects of zinc-loaded magnesium oxide nanoparticles (MgO-Zn²⁺ nanoparticles, MgO-Zn NPs) on glutamate-induced RGC injury, and to evaluate their in vivo and in vitro biocompatibility and neuroprotective potential. Methods MgO-Zn NPs were prepared and characterized by transmission electron microscope and energy-dispersive spectroscopy. In vitro cytotoxicity was systematically evaluated in the R28 rat retinal precursor cell line using the cell counting kit-8 (CCK-8) assay. In vivo, an excitotoxic retinal injury model was established in C57/BL mice by intravitreal injection of N-methyl-D-aspartate (NMDA), followed by MgO-Zn NP intervention. RGC numbers and apoptosis were evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Retinal-layer structure was examined by hematoxylin and eosin (HE) staining. Flash visual evoked potential (F-VEP) was used to evaluate RGC visual-conduction function, and RNA sequencing was performed to analyze pathways and functions of differentially expressed genes, with further validation of associated protein-expression differences. Results Transmission electron microscope and energy-dispersive spectroscopy confirmed the morphological and compositional characteristics of MgO-Zn NPs, indicating successful composite synthesis. CCK-8 results showed that MgO-Zn NPs at 75 µg/mL exhibited no cytotoxicity in R28 cells. After intravitreal injection of MgO-Zn NPs in mice, no significant ocular surface or corneal adverse reactions were observed, indicating favorable ocular tolerance. TUNEL staining showed that RGC numbers in the excitotoxic model were significantly lower than those in normal mice (P<0.05), confirming successful model establishment, whereas MgO-Zn NPs significantly reduced NMDA-induced RGC apoptosis (P<0.05). HE staining showed partial structural restoration of retinal layers after MgO-Zn NP intervention (P<0.05). F-VEP measurements showed prolonged P2 latency and decreased amplitude in model mice (both P<0.001), while MgO-Zn NP intervention resulted in partial recovery of P2 latency and amplitude (both P<0.05). RNA sequencing indicated that MgO-Zn NPs alleviated NMDA-induced retinal transcriptome abnormalities, with differentially expressed genes mainly associated with the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway and the mammalian target of rapamycin (mTOR) signaling pathway. Immunofluorescence staining further showed that MgO-Zn NPs significantly decreased retinal p-Akt and p-mTOR expression levels (both P<0.01). Conclusion MgO-Zn NPs may serve as a dual-functional glaucoma treatment candidate, providing retinal-neuron protection while acting as an intraocular drug-delivery carrier.

Graphical abstract

关键词

锌 / 氧化镁纳米颗粒 / 谷氨酸兴奋毒性 / 青光眼 / 视网膜神经节细胞

Key words

zinc / MgO nanoparticles / glutamate excitotoxicity / glaucoma / retinal ganglion cells

视网膜神经节细胞(retinal ganglion cells，RGCs)的不可逆死亡是所有类型青光眼的主要特征^[1-3]。降低眼压是临床唯一有效的青光眼治疗手段^[4-5]。但部分患者即使眼压正常仍会出现视神经损伤^[6]。因此，采用神经保护剂延缓失明是更理想的治疗策略^{[3, 7]}。然而，目前尚无应用于临床的青光眼神经保护药物^[8]。

研究人员^{[7, 9-11]}开发了多种药物以保护RGCs，但这些药物易被代谢、组织穿透性差^[12]。近年来，纳米材料在药物递送方面取得进展^[12-17]。但现有纳米载体仍存在诸多限制：水凝胶眼内注射可能引起视物模糊^[18-19]；高分子纳米颗粒(nanoparticle，NPs)合成复杂、成本较高^[12]；脂质体存在药物负载量低等问题^[20-21]；非离子胶束在高温灭菌下易遭破坏^[22]。这些限制使上述纳米载体在临床转化过程中面临挑战。

无机NPs是青光眼药物递送的理想载体^[23-24]。在使用这类纳米材料时，须精准控制金属离子的释放速率及负载量，以避免短期或长期毒性。锌是视网膜中最丰富的金属离子之一^[25-28]。研究^[29]显示：锌的摄入可增加睫状神经营养因子水平，提示锌离子在视网膜损伤模型中具有保护作用。而锌离子过量亦可引发神经毒性^[30-32]。氧化镁(magnesium oxide，MgO)NPs具备生物可降解、低毒性的特性，为药物递送提供了一个安全且环保的平台^[33-35]，有望作为青光眼的神经保护制剂，将锌离子递送至眼内以保护RGCs。

1 材料与方法

1.1 伦理声明

本研究所有实验程序均已获得中南大学实验动物伦理委员会批准(审批号：CSU-2023-0297)，并严格遵循国际视网膜研究协会关于眼科与视觉研究中动物使用的声明进行操作。

1.2 NPs的制备和表征

MgO NPs通过共沉淀法合成^[34]。将50 mg MgO NPs粉末分别加入25 mL、体积浓度为1 mmol/L和10 mmol/L的Zn(NO₃)₂溶液中，室温下搅拌2 h后，以10 000 r/min离心10 min，弃去上清液，收集沉淀，于70 ℃下干燥24 h，得到载锌氧化镁纳米颗粒(MgO-Zn²⁺ nanoparticles，MgO-Zn NPs)。根据Zn(NO₃)₂溶液体积浓度(1 mmol/L和10 mmol/L)，将合成的NPs命名为MgO-1Zn和MgO-10Zn。通过透射电子显微镜及能谱分析对NPs进行表征。

1.3 细胞培养与分组

大鼠视网膜前体细胞系R28由中南大学提供，被广泛用于体外研究RGCs的病理机制^[36-40]。本研究将R28细胞培养于含1 g/L葡萄糖的杜氏改良eagle培养基(Dulbecco’s modified eagle medium，DMEM；购自武汉普诺赛生命科技有限公司)中，并补充10%胎牛血清(fetal bovine serum，FBS；购自美国Gibco公司)，在37 ℃、5% CO₂条件下维持细胞生长。

分别将MgO NPs、MgO-1Zn NPs和MgO-10Zn NPs溶解于PBS中，以80 W功率、工作3 s/间歇1 s的参数超声处理5 min，得到质量-体积浓度为1 mg/mL的NPs溶液，并用PBS进一步稀释，配置为系列浓度梯度溶液。

将R28细胞随机分为4组：对照(Control)组用培养基常规孵育，MgO组、MgO-1Zn组、MgO-10Zn组分别在含不同梯度浓度的MgO NPs、MgO-1Zn NPs和MgO-10Zn NPs的培养液中培养。采用细胞计数试剂盒8(cell counting kit-8，CCK-8；购自上海碧云天生物技术股份有限公司)比较不同组别和浓度下的细胞凋亡情况。

1.4 NPs的生物相容性和眼内分布

C57/BL小鼠(6~8周龄)购自湖南省斯莱克景达实验动物有限公司，在湿度40%~70%，温度(21±1) ℃、12 h昼夜循环的无特定病原体(specific pathogen free，SPF)饲养条件下适应性喂养1周后开展实验。分别向小鼠玻璃体腔内注射1 µL 100 µg/mL的MgO NPs、MgO-1Zn NPs和MgO-10Zn NPs溶液5 d后，在立体视显微镜下观察其眼球和晶状体的变化，并拍照以分析注射NPs的体内生物相容性。

眼内分布实验：取0.12 g的MgO-Zn NPs悬浮于PBS中，加入1 mL 20 µg/mL的Sulfo-Cy3 N-羟基琥珀酰亚胺酯溶液，于室温下搅拌1 h。1 000 r/min离心 4 min，取上清液用PBS稀释100倍得Cy3-MgO-Zn NPs注射液。注射1 µL至小鼠玻璃体腔内，注射后 6、12及24 h处死小鼠，取眼球行冷冻切片。采用荧光显微镜(日本尼康公司)观察NPs眼内分布。

1.5 视网膜兴奋毒性青光眼模型的构建与给药

配置给药溶液：将N-甲基-D-天冬氨酸(N-methyl-D-aspartate，NMDA；购自美国Sigma-Aldrich公司)溶解于磷酸盐缓冲液(phosphate buffered saline，PBS)中，制备成体积浓度为100 mmol/L的NMDA溶液。

体内预实验结果表明：500 µg/mL的MgO-1Zn NPs能够在NMDA诱导的视网膜兴奋毒性青光眼模型小鼠中发挥保护作用，因此后续正式实验选取 500 µg/mL作为干预浓度(附图1、2，https://doi.org/10. 57760/sciencedb.xbyxb.00141)。将小鼠随机分为3组，每组各5只：对照(Control)组于玻璃体腔注射1 µL无菌生理盐水，NMDA组注射1 µL 20 mmol/L的NMDA溶液建立视网膜兴奋毒性青光眼模型，NMDA+MgO-Zn组注射1 µL 500 µg/mL的MgO-1Zn NPs颗粒与20 mmol/L NMDA的混合给药液，同步建模并干预。

1.6 视网膜铺片免疫荧光染色

于玻璃体腔内注射5 d后，用过量戊巴比妥麻醉、处死小鼠并摘取眼球，在4%多聚甲醛中固定30 min后分离视网膜。使用2%牛血清白蛋白和0.5% Triton-X100配制的PBS封闭1 h。采用1꞉100的Brn3a抗体(购自英国Abcam公司)标记RGCs，于4 ℃孵育过夜。次日用0.5% Triton-X100洗涤3次，再次用4 %多聚甲醛固定10 min并清洗。加入1꞉200的Alexa Fluor 488标记的山羊抗兔免疫球蛋白G H&L二抗(购自英国Abcam公司)，在室温下孵育1.5 h，随后用PBS洗涤。使用荧光显微镜拍摄图像，并用ImageJ软件分析RGC数量及荧光强度。

1.7 细胞凋亡检测

采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling，TUNEL)染色检测细胞凋亡情况。将小鼠眼球制备为3 µm厚石蜡切片，常规二甲苯脱蜡、梯度乙醇复水后，用20 μg/mL无DNase的蛋白酶K溶液(P0106免疫组织化学洗涤液配制)于37 ℃孵育30 min。用PBS洗涤3次后，按TUNEL试剂盒(购自上海碧云天生物技术股份有限公司)说明书要求配置检测液。每张切片滴加20 μL TUNEL检测液，在37 ℃下避光孵育60 min；再次用PBS洗涤3次，用4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole，DAPI)复染细胞核5 min，用抗荧光淬灭封片剂封片。使用荧光显微镜采集图像，保持各组拍摄亮度参数一致，统计阳性凋亡细胞比例。

1.8 苏木精-伊红染色

小鼠眼球固定后进行乙醇梯度脱水、石蜡包埋并切片。进行苏木精-伊红(hematoxylin and eosin，HE)染色后，在光学显微镜下观察，并使用CaseViewer软件进行定量分析。

1.9 闪光视觉诱发电位分析

在玻璃体注射药物后5 d，对每组4只小鼠进行戊巴比妥麻醉，开展闪光视觉诱发电位(flash visual evoked potential，F-VEP)测试。暗适应15 min后，将电极分别插入小鼠背部皮下(接地电极)、前囟(负极)和枕骨(正极)。使用厚纱布和锡箔遮盖对侧眼。利用多焦视网膜电图仪(GT-2008V-VI，重庆国特医疗设备有限公司)和Ganzfeld系统记录视觉功能。

1.10 RNA测序及生物信息学分析

采集小鼠视网膜，使用TRIzol试剂盒(购自美国Omega Therapeutics公司)提取视网膜总RNA，采用Agilent 2100 Bioanalyzer系统和RNA Nano 6000试剂盒(购自中国安捷伦科技有限公司)评估RNA的完整性及浓度，对合格样本进行后续测序实验。使用逆转录试剂盒(购自北京全式金生物技术股份有限公司)将视网膜总RNA逆转录为互补DNA后，将样本送至北京诺禾致源科技股份有限公司，在Illumina NovaSeq 6000平台进行测序。使用edgeR筛选差异表达基因，筛选标准为差异倍数≥1.5且错误发现率<0.05。差异基因的功能注释基于京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)和基因本体论(Gene Ontology，GO)数据库。

1.11 视网膜石蜡切片免疫荧光染色

将小鼠眼球石蜡切片用二甲苯、梯度乙醇进行脱蜡。随后用3%过氧化氢(购自武汉赛维生物科技有限公司)封闭内源性过氧化物酶活性5 min。将切片浸入0.01 mmol/L柠檬酸盐抗原修复液(pH为6.0)中进行抗原修复，并在微波炉中高火加热15 min，冷却至室温后，分别加入1꞉100稀释的Brn3a抗体、抗p-Akt抗体和抗p-mTOR抗体(均购自英国Abcam公司)，在4 ℃下孵育过夜。次日加入1꞉200稀释的Alexa Fluor 488标记的山羊抗兔免疫球蛋白G H&L或Alexa Fluor 594标记的山羊抗兔免疫球蛋白G H&L室温孵育2 h。最后经DAPI染色5 min。使用荧光显微镜对切片进行拍照。

1.12 统计学处理

使用统计学软件GraphPad Prism 9.1.1进行统计分析。连续型数据以均数±标准差表示，所有实验重复至少3次。两组间比较使用双尾Student’s t检验，多组间比较使用单因素方差分析，随后进行Tukey多重比较检验。P<0.05表示差异具有统计学意义。

2 结 果

2.1 MgO-Zn NPs的表征

纳米复合材料的合成流程见附图3(https://doi. org/10.57760/sciencedb.xbyxb.00141)。透射电子显微镜下MgO NPs呈板状结构，平均尺寸为33.2 nm(图1A、1B)。而MgO-Zn NPs的纳米结构发生转变(图1C~1F)。通过能谱分析元素图(图1G、1H)可视化MgO-1Zn NPs和MgO-10Zn NPs中Mg、Zn和O元素的分布，结果表明3种元素都均匀分布。

2.2 MgO-Zn NPs的生物相容性和眼内分布

CCK-8检测结果(图2A~2C)显示：相较于Control组，MgO组R28细胞活性差异无统计学意义(P>0.05)，当MgO-1Zn NPs和MgO-10Zn NPs浓度升至100 µg/mL时，R28细胞活性均显著下降(均P<0.01)。

采用立体视显微镜观察小鼠眼球宏观形态，结果(图2D)显示：小鼠玻璃体腔注射MgO-1Zn NPs或MgO-10Zn NPs溶液5 d后未见角膜缺损或溃疡、晶状体混浊等症状。HE染色结果(图2E~F)也未发现角膜和晶状体组织结构损伤。

免疫荧光染色结果表明：MgO-Zn NPs注射6 h后即可在视网膜神经节细胞层(retinal ganglion cell layer，GCL)观察到Cy3红色荧光信号(附图4， https://doi.org/10.57760/sciencedb.xbyxb.00141)。

2.4 MgO-Zn NPs预防NMDA诱导的小鼠视网膜损伤

视网膜铺片免疫荧光染色结果(图3A、3B)显示：与Control组比较，NMDA组RGC密度(绿色荧光)显著下降(P<0.001)；与NMDA组比较，NMDA+MgO Zn组的RGC密度显著增加(P<0.01)。HE染色(图3C~3E)结果显示：与Control组比较，NMDA组的视网膜神经节细胞复合体(ganglion cell complex，GCC)显著变薄(P<0.05)；NMDA+MgO-Zn组GCC厚度较NMDA组有所恢复(P<0.05)。

TUNEL染色结果(图3F)显示：与Control组比较，NMDA组视网膜神经节细胞层(ganglion cell layer，GCL)出现大量绿色荧光标记的阳性凋亡细胞；而NMDA+MgO-Zn组的绿色荧光信号较NMDA组减弱。F-VEP检查结果(图3G)显示：NMDA组较Control组P2波潜伏期延长，振幅下降(均P<0.001)；NMDA+MgO-Zn组较NMDA组P2波潜伏期部分恢复(P<0.001)、振幅增加(P<0.01)。

2.5 MgO-Zn NPs引起视网膜转录组改变

韦恩图显示：2组有14 124个共同表达基因，此外NMDA+MgO-Zn组中检测到513个特异表达基因，NMDA组中检测到391个特异表达基因，表明2组样本在整体转录表达谱上具有较高的一致性，同时仍存在一定程度的组间特异性表达(图4A)。火山图显示：与NMDA组相比，NMDA+MgO-Zn组有973个基因上调，871个基因下调(图4B)，其中包括29个与mTOR信号通路相关的基因(图4C)。

2.6 MgO-Zn NPs调控小鼠视网膜p-Akt和p-mTOR基因表达

KEGG和GO分析结果显示：经MgO-Zn NPs干预后，小鼠视网膜的生物过程、细胞组分及分子功能均发生改变，差异表达基因在生物过程相关功能中富集程度最高(附图5，https://doi.org/10.57760/sciencedb. xbyxb.00141)。基因富集分析结果显示：磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase，PI3K)-蛋白激酶B(protein kinase B，Akt)信号通路和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)信号通路在MgO-Zn NPs处理后的视网膜中发生显著变化。进一步对视网膜切片进行免疫荧光染色，结果显示：NMDA+MgO-Zn组RGCs中的p-Akt和p-mTOR荧光(红色)强度均较NMDA组显著回升(均P<0.05，图5A~5D)。

3 讨 论

锌在维持正常视网膜功能中起着关键作用，但游离锌水平过高会通过兴奋毒性、氧化应激及能量生成障碍等机制促进RGCs死亡^{[26, 41-42]}。这提示需要开发新的锌离子递送载体，以精确调控锌离子在眼内的释放。本研究的体内实验未观察到MgO-Zn NPs干预引起的眼部毒性。但该递送系统的长期安全性，特别是在多次注射条件下的安全性，仍需进一步研究。

玻璃体腔注射所需跨越的屏障比眼药水给药少^[43-44]。已有的研究^[45-46]表明：注射进入玻璃体的药物会受到玻璃体结构的阻碍，尤其是阳离子药物更难迁移。因此，直接注射锌离子难以通过玻璃体到达视网膜。当前关于靶向眼底的纳米药物研究大多集中于药效的持续性，对于药物传递速率的研究却较少^{[44, 47-48]}。本研究构建的MgO-Zn NPs在注射后6 h内到达视网膜的GCL，实现了锌离子的快速递送，并对RGCs产生保护作用。

MgO-Zn NPs在穿过玻璃体后，可能被RGCs吞噬，进入溶酶体并逐步分解，释放出镁离子和锌离子。镁离子是人体重要的微量元素，适合作为药物载体的候选材料^[49-51]。值得注意的是，MgO-Zn NPs的神经保护作用可能与负载的锌离子浓度密切相关。已有大量文献^[52-54]报道锌离子在中枢神经系统中具有双重效应。因此，将锌离子的释放控制在最佳范围内具有重要意义。本研究通过调节NPs表面锌离子的负载比例与释放速率来实现对其生物学效应的精准调控。

本研究结果表明：MgO-Zn NPs缓解了谷氨酸诱导的RGC损伤。眼内注射低浓度锌离子可上调睫状神经营养因子，缓解视网膜神经元的谷氨酸兴奋毒性^{[29-30, 55]}。本研究为进一步探究MgO-Zn NPs保护机制，进行了视网膜RNA测序分析，观察到PI3K-Akt-mTOR信号通路在MgO-Zn NPs处理后的视网膜中发生变化。已有研究^[56-59]证实调控该通路可通过自噬机制保护RGCs。研究还发现Wnt信号通路发生改变，已知该通路在Norrin激活下对RGCs有保护作用^[60-62]；同时丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)通路也表现出富集变化，该通路可控制小胶质细胞活化，缓解视网膜炎症，减少RGCs凋亡^[63-66]。值得注意的是，本研究中MgO-Zn NPs未能使受损的RGCs数量恢复至正常水平。未来研究可考虑在MgO-Zn NPs表面结合有神经保护作用的药物，实现更强的RGC保护效果。

综上，本研究成功构建了MgO-Zn NPs递送载体，证实其在玻璃体内注射给药后具有良好的生物相容性，可在NMDA诱导的视网膜兴奋毒性青光眼模型中缓解RGC死亡。PI3K-Akt-mTOR等信号通路可能是MgO-Zn NPs介导的RGC保护作用的潜在机制。

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基金资助

国家自然科学基金(82101127)

湖南省自然科学基金(2022JJ30076)

湖南省自然科学基金(2023JJ20067┫。This work was supported by the National Natural Science Foundation ┣82101127)

the Natural Science Foundation of Hunan Province(2022JJ30076)

the Natural Science Foundation of Hunan Province(2023JJ20067)

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