第一部分　心血管健康评分对衰老及前列腺疾病的预测价值及氧化应激生物标记物在前列腺增生中的中介作用

目的：本研究旨在探究Life’s Critical 9（LC9）心血管健康评分与生物学衰老及前列腺疾病，包括前列腺癌及前列腺增生（Benign Prostatic Hyperplasia，BPH）之间的相关性，并阐明氧化应激生物标记物在其中的中介作用。

方法：本研究基于两个全国性研究队列，即美国National Health and Nutrition Examination Survey（NHANES）队列和英国UK Biobank队列，选择1秒用力呼气量（FEV1）、收缩压、白蛋白浓度、碱性磷酸酶、血尿素氮、肌酐、C反应蛋白、糖化血红蛋白和总胆固醇等指标用于计算KDMAge；选择实际年龄、白蛋白、碱性磷酸酶、肌酐、葡萄糖、C反应蛋白、淋巴细胞百分比（即白细胞中淋巴细胞的比例）、平均红细胞体积、红细胞分布宽度和白细胞计数等指标来计算PhenoAge。同时评估了LC9评分与生物学衰老及各疾病之间的剂量-反应关系。随后选择γ-谷氨酰转移酶（GGT）、总胆红素（TBIL）和尿酸作为氧化应激生物标记物，探究三者在LC9心血管健康评分与生物学衰老及BPH之间的中介作用。

结果：在NHANES队列和UK Biobank队列中，使用PhenoAge和KDMAge计算生物学衰老后，LC9评分均与两者成负相关。在RCS曲线分析中，本研究发现LC9与生物学衰老之间呈倒“L型”的非线性关系。且在UK Biobank队列中，LC9得分为87.8时出现拐点（非线性P值＜0.001）。且LC9与尿路上皮癌及前列腺增生间存在相关性，而与前列腺癌之间无明显相关性。在中介分析中，所有3个生物标志物显著介导了LC9与前列腺增生之间的关系。γ-谷氨酰转移酶（GGT）、总胆红素（TBIL）和尿酸分别解释了总关联的33.26%、29.24%和6.99%（P＜0.001）。

结论：LC9评分与生物学衰老、尿路上皮癌及BPH关系密切，而氧化应激可能在其与BPH间起到关键中介作用，但其与BPH的关系仍有待进一步明确。

第二部分 氧化应激相关基因NOTCH1蛋白在前列腺移行带与外周带间的表达差异及在良性前列腺增生中的意义

目的：本研究旨在探讨氧化应激相关基因NOTCH1在前列腺增生中的治疗价值。

方法：本研究对GEO数据库中BPH患者移行带与外周带患者单细胞测序结果分析，明确NOTCH1表达差异。随后研究者对来自临床患者的前列腺不同组织进行蛋白质组测序，分析差异基因表达及NOTCH1差异情况。最后，使用免疫组化的方法，使用临床患者样本进行验证。

结果：在单细胞数据分析中，本研究发现BPH患者相较于外周带，移行带中Club和Hillock细胞更为丰富，而Luminal和Basal细胞的比例较低，且NOTCH通路明显激活。在GSE132714转录组分析中，发现NOTCH1和NOTCH3在前列腺增生患者组织中高表达。在BPH患者移行带与外周带的蛋白组分析中，本研究仅发现NOTCH1表达存在统计学差异（p＜0.05）。在免疫组化分析验证中，本研究同样发现氧化应激相关基因NOTCH1在移行带中高表达。

结论：氧化应激与BPH存在相关性，且氧化应激相关基因NOTCH1可能是前列腺增生好发于移行带的关键因素。

第三部分 NOTCH1影响前列腺增生的机制研究

目的：本研究旨在探讨NOTCH1在前列腺增生中的潜在治疗作用，并探究其调控的下游机制。

方法：使用siRNA降低NOTCH1表达，CCK8和EdU评估细胞增殖情况，流式细胞术检测细胞凋亡变化。使用RNA-seq技术，分析NOTCH1表达下调后基因转录及下游通路变化情况。降低NOTCH1表达后，使用Western Blot、免疫荧光、线粒体、溶酶体示踪技术以及透射电镜（Transmission electron microscope，TEM）检测蛋白和线粒体自噬的变化情况。采用挽救实验评估ATF3在NOTCH1和线粒体功能之间的关系。在动物试验中，对前列腺增生模型大鼠使用NOTCH1抑制剂DAPT治疗，H&E染色和前列腺/体重指数评估治疗效果。

结果：在BPH-1和WPMY-1细胞中，NOTCH1表达下降明显抑制细胞增殖，并提高凋亡水平（p＜0.05）。在RNA-seq分析中，ATF3在NOTCH1敲低后明显上调，伴随PINK1表达下降。富集分析中，差异蛋白与线粒体功能关系密切。TMRE显示NOTCH1的表达差异可以明显抑制红色荧光，导致线粒体膜电位丢失，引起线粒体稳态的变化。线粒体、溶酶体示踪技术以及透射电镜表明NOTCH1敲低可以改变线粒体形态，并抑制线粒体自噬，而ATF3在中间起到介导作用。动物实验中，DAPT和非那雄胺均能有效改善大鼠前列腺增生状态（p＜0.05），且二者间无统计学差异。H&E染色显示治疗后，大鼠腺体上皮分布与正常组织类似。

结论：NOTCH1通过ATF3调控线粒体自噬-凋亡平衡，进而影响前列腺增生。抑制NOTCH1表达可以改善前列腺增生，并恢复前列腺正常组织形态。

第四部分 HDAC2通过调节STAT1 K298位点巴豆酰化影响NOTCH1表达

目的：本研究旨在探讨前列腺增生患者移行带与外周带间巴豆酰化差异水平，揭示去巴豆酰化酶HDAC2调控NOTCH1表达的具体机制。

方法：本研究首先对来自临床患者的前列腺不同组织进行蛋白修饰组的泛抗体检测，选择差异最大的巴豆酰化修饰进行测序，明确差异修饰的位点和基因。随后在差异基因中选择NOTCH1转录因子进行后续分析，评估其与各巴豆酰化调节酶之间的关系，结合前部分蛋白组结果联合分析。在各真核生物间评估差异位点的保守性，使用免疫共沉淀方法评估巴豆酰化调节酶与转录因子间是否存在相互作用。随后改变巴豆酰化调节酶表达，评估其对NOTCH1转录及蛋白表达影响。使用Cas9技术敲除转录因子，排除内源性因素影响，然后使用点突变方法模拟巴豆酰化变化，并使用Western Blot和CCK8评估在不同处理组细胞间NOTCH1表达及增殖变化情况。

结果：在乳酸化修饰、三羟基丁酰化修饰、丙二酰化修饰和巴豆酰化修饰中，差异最大的为巴豆酰化修饰，且移行带中共有2264个巴豆酰化位点存在统计学差异，其中1786个巴豆酰化位点存在下调的情况，而仅有478个位点存在上调的情况。通过对临床样本组化检测，同样提示前列腺增生移行带中巴豆酰化下调。差异修饰基因的亚细胞定位分析中，排名前三的分别为细胞质、细胞核与线粒体。NOTCH1的许多转录因子均存在巴豆酰化修饰差异，研究较多的STAT1同样有2个位点存在修饰差异，分别为K209及K298，且两个位点均存在明显的保守性。在各巴豆酰化调节酶中，只有去巴豆酰化酶HDAC2在移行带中高表达，且通过组化验证（p＜0.05）。STAT1两个位点巴豆酰化水平与HDAC2呈负相关。免疫共沉淀显示在BPH-1及WPMY-1细胞中，均存在HDAC2与STAT1直接作用的情况。通过siRNA将BPH-1和WPMY-1细胞系中的HDAC2表达降低，同样也会抑制NOTCH1表达。将STAT1位点突变后，K209突变组NOTCH1表达无差异，且细胞增殖情况同STAT1敲除组类似，野生型细胞NOTCH1表达恢复，且CCK8结果显示细胞增殖能力恢复。而在K298突变组中，NOTCH1表达及细胞增殖情况进一步提高。

结论：巴豆酰化表达差异可能是前列腺增生好发于移行带的重要因素，去巴豆酰化酶HDAC2通过调控STAT1 K298位点巴豆酰化影响NOTCH1表达。

Part I  Predictive Value of Cardiovascular Health Scores in Aging and Prostate diseases: The Mediating Role of Oxidative Stress Biomarkers in Benign Prostatic Hyperplasia

Objective: This study aims to investigate the correlation between the Life’s Critical 9 (LC9) cardiovascular health score and biological aging, as well as prostate-related diseases, including prostate cancer and benign prostatic hyperplasia. Besides, the mediating role of oxidative stress biomarkers is also analyzed.

Methods: This study utilized two large national cohort datasets, namely the NHANES cohort from the United States and the UK Biobank cohort from the United Kingdom. The following biomarkers were selected to calculate KDMAge: forced expiratory volume in 1 second (FEV1), systolic blood pressure, albumin concentration, alkaline phosphatase, blood urea nitrogen, creatinine, C-reactive protein, glycated hemoglobin, and total cholesterol. For the calculation of PhenoAge, actual age, albumin, alkaline phosphatase, creatinine, glucose, C-reactive protein, lymphocyte percentage (i.e., the proportion of lymphocytes among white blood cells), mean corpuscular volume, red cell distribution width, and white blood cell count were included. The study also assessed the dose-response relationship between LC9 scores, biological aging, and three diseases. Subsequently, oxidative stress biomarkers, including γ-glutamyltransferase (GGT), total bilirubin (TBIL), and uric acid, were investigated for their mediating role in the relationship between LC9 cardiovascular health scores, biological aging, and BPH.

Results: In both the NHANES and UK Biobank cohorts, after calculating biological aging using PhenoAge and KDMAge, LC9 scores were negatively correlated with both measures of biological aging. In the restricted cubic spline (RCS) analysis, a nonlinear inverse “L-shaped” relationship was found between LC9 and biological aging. Specifically, a turning point was observed at a LC9 score of 87.8 in the UK Biobank cohort (nonlinear P-value ＜ 0.001). Notably, significant associations were observed between LC9 scores and both urothelial carcinoma and BPH, whereas no statistically significant correlation was identified with prostate cancer (p > 0.05). In subsequent mediation analyses, all three oxidative stress biomarkers significantly mediated the relationship between LC9 and BPH. Specifically, GGT, TBIL, and uric acid explained 33.26%, 29.24%, and6.99% of the total association.

Conclusion: The LC9 score significantly associated with biological aging, urothelial carcinoma, and BPH. Notably, oxidative stress emerged as a critical mediator in the relationship between LC9 and BPH, while the precise mechanisms need further investigation.

Part II  The Expression Difference of Oxidative Stress-Related Gene NOTCH1 Protein Between the Transition Zone and Peripheral Zone of the Prostate and Its Significance in Benign Prostatic Hyperplasia

Objective: This study aims to explore the therapeutic value of the oxidative stress-related gene NOTCH1 in BPH.

Methods: Single-cell RNA sequencing data from the GEO database for prostate tissue in BPH patients were analyzed to determine the expression differences of NOTCH1 between the transitional zone and peripheral zone. Further, proteomic sequencing of prostate tissues from clinical patients was performed to analyze differential gene expression, particularly focusing on NOTCH1 expression. Finally, immunohistochemistry (IHC) was used to validate the results in clinical patient samples.

Results: In the single-cell RNA sequencing analysis, compared to the peripheral zone, the transitional zone in BPH patients showed a higher abundance of Club and Hillock cells, while Luminal and Basal cells were less prevalent, and the NOTCH signaling pathway was markedly activated. Transcriptomic analysis from the GSE132714 dataset revealed that NOTCH1 and NOTCH3 were highly expressed in prostate tissue from BPH patients. Proteomic analysis of the transitional and peripheral zones in BPH patients revealed a statistically significant difference in NOTCH1 expression (p ＜ 0.05). In the IHC validation, we also observed high expression of the oxidative stress-related gene NOTCH1 in the transitional zone.

Conclusion: Oxidative stress is significantly associated with benign prostatic hyperplasia, and the oxidative stress-related gene NOTCH1 may play a key role in the higher incidence of BPH in the transitional zone of the prostate.

Part III  Mechanistic Study of the Role of NOTCH1 in Benign Prostatic Hyperplasia

Objective: This study aims to explore the potential therapeutic role of NOTCH1 in BPH and investigate the mechanism.

Methods: NOTCH1 expression was silenced using siRNA, and cell proliferation was assessed by CCK8 and EdU assays. Flow cytometry was used to analyze apoptotic changes. RNA-seq technology was applied to examine transcriptional changes and downstream pathway alterations following NOTCH1 downregulation. Protein and mitochondrial autophagy changes were assessed using Western blotting, immunofluorescence, mitochondrial and lysosomal tracking, and transmission electron microscopy (TEM). A rescue experiment was conducted to investigate the relationship between ATF3, NOTCH1, and mitochondrial function. In animal models of BPH, rats were treated with the NOTCH1 inhibitor DAPT. H&E staining and prostate/weight index measurements were used to evaluate treatment efficacy.

Results: In BPH-1 and WPMY-1 cell lines, knockdown NOTCH1 expression significantly inhibited cell proliferation and increased apoptosis (p ＜ 0.05). RNA-seq analysis showed a significant upregulation of ATF3 expression after NOTCH1 knockdown, accompanied with a decreased PINK1 expression. Enrichment analysis revealed a significantly relationship between differentially expressed genes and mitochondrial function. TMRE assay demonstrated that reduced NOTCH1 expression inhibited red fluorescence, leading to a loss of mitochondrial membrane potential and alterations in mitochondrial homeostasis. Mitochondrial and lysosomal tracking analysis and TEM revealed that NOTCH1 knockdown influence mitochondrial morphology and suppresses mitophagy, while ATF3 playing a mediating role. In animal experiment, both DAPT and finasteride effectively improved the BPH in rats (p ＜ 0.05), with no statistical difference between the two groups. H&E staining showed that the glandular epithelium distribution in these two groups was similar to normal tissue.

Conclusion: NOTCH1 regulates the mitophagy-apoptosis balance via influence ATF3 expression, thereby regulating BPH and restore the normalize prostate tissue.

Part IV  HDAC2 Regulates NOTCH1 Expression by Modulating the Crotonylation of STAT1 at K298 in Benign Prostatic Hyperplasia

Objective: This study aims to investigate the differential crotonylation levels between the transitional and peripheral zones in BPH and to elucidate the specific mechanism by which the de-crotonylation enzyme HDAC2 regulates NOTCH1 expression.

Methods: PTMs were obtained from prostate tissue samples of clinical BPH patients after using pan-antibodies. Crotonylation, the most significant difference, was further analyzed to identify differential modification sites and genes. Among these differentially modified genes, the transcription factor NOTCH1 was selected for further analysis to evaluate its relationship with crotonylation regulated enzymes. The conservation of differential modified sites was assessed across different species. The immunohistochemistry was used to evaluate potential interactions between crotonylation regulated enzymes and transcription factor. Then the crotonylation regulated enzymes was knockdown to evaluate their impact on NOTCH1 expression. CRISPER/Cas9 was employed to knock out transcription factor to exclude endogenous effects, followed by site mutation to simulate crotonylation changes. Western blot and CCK 8 analyses were conducted to assess NOTCH1 expression and cell proliferation in different treat groups.

Results: Among lactylation, hydroxylbutyrylation, malonylation, and crotonylation modifications, crotonylation exhibited the most significant differential expression. In the transitional zone, 2,264 crotonylation sites showed statistical differences, with 1,786 sites downregulated and 478 sites upregulated. Immunohistochemical analysis of clinical samples similarly suggested downregulation of crotonylation in the transitional zone of BPH. Subcellular localization analysis of differential modification genes revealed that the top three locations were the cytoplasm, nucleus, and mitochondria. Many transcription factors of NOTCH1 exhibited differential crotonylation, with STAT1 being one of the most studied. Two STAT1 sites, K209 and K298, displayed significant crotonylation differences, with both sites showing notable conservation across species. Among the crotonylation regulators, only the de-crotonylation enzyme HDAC2 was highly expressed in the transitional zone, as confirmed by immunohistochemical validation (p ＜ 0.05). The crotonylation levels at both STAT1 K209 and K298 sites were negatively correlated with HDAC2 expression. Immunoprecipitation analysis revealed direct interaction between HDAC2 and STAT1 in both BPH-1 and WPMY-1 cells. Knockdown of HDAC2 expression by siRNA in these cell lines also inhibited NOTCH1 expression. In STAT1 K209 mutant cells, NOTCH1 expression remained unchanged compared to STAT1 knockout controls, with comparable proliferation rates observed via CCK-8 assays. Conversely, wild-type cells exhibited restored NOTCH1 expression and proliferation capacity. Strikingly, STAT1 K298 mutation further elevated NOTCH1 levels and potentiated cellular proliferation.

Conclusion: Crotonylation may be a key factor contributing to the high incidence of hyperplasia in prostate transitional zone. Besides, HDAC2 regulates NOTCH1 expression by modulating the crotonylation of STAT1 at the K298 site.