Hypertensive retinopathy is a microvascular complication caused by systemic hypertension and can lead to severe visual impairment. At present, the molecular mechanisms of this disease remain incompletely understood, particularly the global expression profiles and regulatory networks of non-coding RNA (ncRNA). This study aims to comprehensively analyze the genome-wide differential expression profiles of messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) in retinal tissues from a spontaneously hypertensive rat (SHR) model of hypertensive retinopathy, and to observe transcriptomic changes after treatment with the calcium channel blocker lacidipine, thereby revealing their potential roles in disease pathogenesis and identifying possible therapeutic targets. SHRs were used as a model of hypertensive retinopathy, and normotensive Wistar-Kyoto (WKY) rats were used as controls. Rats were divided into 3 groups: A control group (WKY), a model group (SHR), and a treatment group (SHR+lacidipine). Rats in the treatment group received lacidipine by gavage at 0.5 mg/(kg·d) for 8 consecutive weeks. At the end of the experiment, retinal tissues were collected for histopathological examination by hematoxylin and eosin (HE) staining and for high-throughput sequencing. The lncRNA library was used to analyze mRNA, lncRNA, and circRNA expression profiles, and the small RNA (sRNA) library was used to analyze miRNA expression profiles. Differential expression analysis was performed using DESeq2. The screening criteria were |log2 fold change (FC)|≥1 and false discovery rate (FDR) significance criteria for mRNAs and lncRNAs, and |log2FC|≥1 and P<0.05 for miRNAs and circRNAs as exploratory candidates. Differentially expressed RNAs were subjected to Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Competing endogenous RNA (ceRNA) regulatory networks involving lncRNA/circRNA-miRNA-mRNA were constructed through target prediction and co‑expression analysis. Ten mRNAs whose expression changes were reversed after treatment were selected for validation by real-time reverse transcription polymerase chain reaction (real-time RT-PCR; n=6 per group). Comparisons among multiple groups were performed using one-way analysis of variance and post hoc multiple-comparison tests. 1) Verification of the animal model: Blood pressure was significantly higher in the model group than in the control group. The model group exhibited edema of the retinal nerve fiber layer, dilation of vascular lumens, and increased retinal thickness. Lacidipine treatment reduced blood pressure and alleviated these pathological changes. 2) Differential expression of mRNAs: Compared with the control group, 870 mRNAs were significantly downregulated and 1 018 mRNAs were significantly upregulated in the model group. Compared with the model group, 2 644 mRNAs were significantly downregulated and 186 mRNAs were significantly upregulated in the treatment group. GO analysis showed that upregulated mRNAs in the model group were enriched in immune-inflammatory processes, such as leukocyte activation, chemotaxis, defense response, and regulation of tumor necrosis factor (TNF) production, whereas downregulated mRNAs were enriched in G protein-coupled receptor (GPCR) signaling, glycoprotein synthesis, ion channel activity, and related processes. KEGG analysis revealed that upregulated mRNAs in the model group were enriched in interleukin-17 (IL-17), TNF, oxidative stress, and lipid inflammatory mediator metabolic pathways, whereas downregulated mRNAs were enriched in phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling, vascular smooth muscle contraction, extracellular matrix (ECM)-receptor interaction, and related pathways. In the treatment group, the downregulated mRNAs were mainly enriched in pathways related to immune-inflammatory activation, such as mast cell degranulation and leukocyte degranulation. 3) Treatment-reversed mRNAs: A total of 67 genes that were upregulated in the model group and downregulated after treatment were identified; these genes were mainly enriched in endoplasmic reticulum stress-associated apoptotic signaling, mitophagy, ubiquitin ligase activity, and related processes. In addition, 54 genes that were downregulated in the model group and upregulated after treatment were identified; these genes were mainly enriched in amino acid transport, integrin complex, focal adhesion, glutamate transport activity, and related processes. Real-time RT-PCR validated the expression changes of Med22, Rmt1, Sytl3, Itgb7, and Slc1a3 which were decreased in the model group and increased after treatment, as well as Rnf183, Lrrc29, Lat2, Hist1h4m, and Dpm3, which were increased in the model group and decreased after treatment. These findings were consistent with the sequencing results. 4) Differential expression of miRNAs: Compared with the control group, 30 miRNAs were downregulated and 26 miRNAs were upregulated in the model group. Compared with the model group, 14 miRNAs were downregulated and 53 miRNAs were upregulated in the treatment group. Treatment-reversed miRNAs were identified: Rno-miR-1-3p, novel_miR_1203, and novel_miR_1417 were upregulated in the model group and downregulated after treatment, whereas novel_miR_107 and novel_miR_905 were downregulated in the model group and upregulated after treatment. Their target genes were enriched in regulation of retinal cone/rod cell differentiation, ECM remodeling, the Notch pathway, fatty acid synthesis, and related processes. 5) Differential expression of lncRNAs: Compared with the control group, 786 lncRNAs were downregulated and 764 lncRNAs were upregulated in the model group. Compared with the model group, 217 lncRNAs were downregulated and 230 lncRNAs were upregulated in the treatment group. A total of 55 lncRNAs that were upregulated in the model group and downregulated after treatment, and 76 lncRNAs that were downregulated in the model group and upregulated after treatment, were identified. Their target genes were enriched in immune regulation, wound healing, vascular remodeling, retinol metabolism related to rod-mediated scotopic vision, glutamatergic neuron differentiation, the forkhead box O (FoxO) pathway, retinoic acid-inducible gene I (RIG-I)-like receptor signaling, and related processes. 6) Differential expression of circRNAs: Compared with the control group, 45 circRNAs were downregulated and 47 circRNAs were upregulated in the model group. Compared with the model group, 25 circRNAs were downregulated and 22 circRNAs were upregulated in the treatment group. Eleven circRNAs that were downregulated in the model group and upregulated after treatment, and 10 circRNAs that were upregulated in the model group and downregulated after treatment, were identified. Their host genes were enriched in regulation of synaptic neural signaling, second messenger transmission, calcium signaling, and vascular endothelial growth factor (VEGF) signaling pathways. 7) Construction of ceRNA networks: Based on expression patterns and target prediction, multiple lncRNA/circRNA-miRNA-mRNA regulatory networks were constructed. For example, the networks composed of rno_circ_Rims2_10, rno-miR-1-3p, and related mRNAs, such as Mmp14 and Fasn, as well as the novel_miR_1417 related network, may be involved in retinal stress responses, inflammatory regulation, and neurovascular remodeling. This study is the first to integrate and analyze retinal transcriptomic profiles of mRNAs, miRNAs, lncRNAs, and circRNAs in an SHR model of hypertensive retinopathy, and it reveals RNA expression changes associated with lacidipine treatment. The results suggest that immune-inflammatory activation, neurovascular dysfunction, and ncRNA-mediated ceRNA regulatory networks may participate in the pathogenesis of hypertensive retinopathy. The identified differentially expressed molecules and predicted regulatory axes provide a candidate molecular basis for further mechanistic validation and screening of potential therapeutic targets. 目的: 高血压视网膜病变是全身性高血压引起的微血管并发症，可导致严重的视力损害。目前对该病的分子机制认识尚不全面，尤其是非编码RNA(non-coding RNA，ncRNA)在其中的整体表达谱及调控网络缺乏系统研究。本研究旨在全面分析自发性高血压大鼠(spontaneously hypertensive rat，SHR)高血压视网膜病变模型视网膜组织中信使RNA(messenger RNA，mRNA)、微RNA(microRNA，miRNA)、长链非编码RNA(long non-coding RNA，lncRNA)和环状RNA(circular RNA，circRNA)的全基因组差异表达谱，并观察钙通道阻滞剂拉西地平治疗后的转录组变化，以揭示其参与疾病发病机制的潜在作用，并识别可能的治疗靶点。方法: 采用SHR作为高血压视网膜病变模型，正常血压Wistar-Kyoto(WKY)大鼠作为对照组。将大鼠分为3组:对照组(WKY)、模型组(SHR)和治疗组(SHR+拉西地平)。治疗组给予拉西地平0.5 mg/(kg·d)灌胃，连续8周。实验结束时收集视网膜组织，分别进行苏木精-伊红(hematoxylin and eosin，HE)染色、病理组织学检查和高通量测序。lncRNA文库用于分析mRNA、lncRNA和circRNA表达谱，小RNA(small RNA，sRNA)文库用于分析miRNA表达谱。使用DESeq2进行差异表达分析，筛选标准:mRNA和lncRNA以|log2倍数变化(fold change，FC)|≥1且错误发现率(false discovery rate，FDR)<0.05;miRNA和circRNA以|log2FC|≥1且P<0.05为探索性候选。对差异表达RNA进行基因本体(Gene Ontology，GO)功能注释和京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes，KEGG)信号通路富集分析。通过靶标预测和共表达分析构建lncRNA/circRNA-miRNA-mRNA的竞争性内源RNA(competing endogenous RNA，ceRNA)调控网络。选取10个治疗后逆转的mRNA进行实时反转录聚合酶链反应(real-time reverse transcription polymerase chain reaction，real-time RT-PCR)验证(每组6只)。多组间比较采用单因素方差分析及事后多重比较检验。结果: 1)动物模型验证:模型组血压显著高于对照组，视网膜神经纤维层水肿、血管管腔扩张、视网膜厚度增加;拉西地平治疗后血压降低并减轻了上述病理改变。2)mRNA差异表达:与对照组相比，模型组有870个mRNA表达显著下调、1 018个mRNA表达显著上调;与模型组相比，治疗组有2 644个mRNA表达显著下调、186个mRNA表达显著上调。GO分析显示模型组上调mRNA富集于免疫炎症过程[如白细胞活化、趋化性、防御反应，肿瘤坏死因子(tumor necrosis factor，TNF)产生调节]，下调mRNA富集于G蛋白偶联受体(G protein-coupled receptor，GPCR)信号、糖蛋白合成、离子通道活性等。KEGG分析显示模型组上调mRNA富集于白细胞介素-17(interleukin-17，IL-17)、TNF、氧化应激及脂质炎症介质代谢通路;下调mRNA富集于磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase，PI3K)-蛋白激酶B(protein kinase B，AKT)信号、血管平滑肌收缩、细胞外基质(extracellular matrix，ECM)-受体相互作用等通路。治疗组中下调的mRNA主要富集于免疫炎症激活相关通路(如肥大细胞脱颗粒、白细胞脱颗粒)。3)治疗逆转的mRNA:鉴定出67个在模型组上调、治疗后下调的基因(主要富集于内质网应激凋亡信号、线粒体自噬、泛素连接酶活性等)，以及54个在模型组下调、治疗后上调的基因(主要富集于氨基酸转运、整合素复合物、黏着斑、谷氨酸转运活性等)。Real-time RT-PCR验证了Med22、Rmt1、Sytl3、Itgb7、Slc1a3(模型组降低，治疗后升高)及Rnf183、Lrrc29、Lat2、Hist1h4m、Dpm3(模型组升高，治疗后降低)的表达变化，与测序结果一致。4)miRNA差异表达:与对照组相比，模型组有30个miRNA下调、26个miRNA上调;与模型组相比，治疗组有14个miRNA下调、53个miRNA上调。筛选出治疗逆转的miRNA:Rno-miR-1-3p、novel_miR_1203、novel_miR_1417在模型组上调、治疗后下调;novel_miR_107、novel_miR_905在模型组下调、治疗后上调。其靶基因富集于视网膜视锥/视杆细胞分化调节、ECM重塑、Notch通路及脂肪酸合成等。5)lncRNA差异表达:与对照组相比，模型组有786个lncRNA下调、764个上调;与模型组相比，治疗组有217个lncRNA下调、230个上调。筛选出55个模型组上调、治疗后下调的lncRNA和76个模型组下调、治疗后上调的lncRNA。其靶基因富集于免疫调节、伤口愈合、血管重塑、视黄醇代谢(视杆细胞暗视觉)、谷氨酸能神经元分化、叉头框蛋白O(forkhead box O，FoxO)通路、维A酸诱导基因I样受体信号等。6)circRNA差异表达:与对照组相比，模型组有45个circRNA下调、47个上调;与模型组相比，治疗组有25个circRNA下调、22个上调。筛选出11个模型下调/治疗后上调的circRNA和10个模型上调/治疗后下调的circRNA。其宿主基因富集于突触神经信号调节、第二信使传递、钙信号和血管内皮生长因子(vascular endothelial growth factor，VEGF)信号通路。7)ceRNA网络构建:基于表达模式和靶标预测，构建了多个lncRNA/circRNA-miRNA-mRNA调控网络。例如，rno_circ_Rims2_10与rno-miR-1-3p及相关mRNA(如Mmp14、Fasn)组成的网络，以及novel_miR_1417相关网络，可能参与视网膜应激反应、炎症调控和神经血管重塑。结论: 本研究在SHR高血压视网膜病变模型中整合分析了mRNA、miRNA、lncRNA和circRNA的视网膜转录组图谱，并揭示了拉西地平治疗相关的RNA表达变化。结果提示，免疫炎症激活、神经血管功能紊乱及ncRNA介导的ceRNA调控网络可能参与高血压视网膜病变的发病机制。鉴定出的差异表达分子及预测的调控轴为进一步的机制验证和潜在治疗靶点筛选提供了候选分子基础。.
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