【作者】 何跃；

【导师】 魏强；

【作者基本信息】 四川大学 ， 外科学（泌尿）（专业学位）， 2021， 博士

【摘要】 背景和目的:良性前列腺增生(Benign Prostatic Hyperplasia,BPH)是引起中老年男性排尿困难的常见疾病。调查数据显示年龄60岁以上的男性,超过半数都会出现前列腺增生[1]。我国当前已经进入老龄化社会,老年男性人口日益增多,BPH疾病势必给整个医疗卫生系统带来更为沉重的负担。然而,目前BPH的发病机制尚不十分明确,近年的研究表明:年龄变化相关的代谢紊乱、性激素平衡变化、多种细胞生长因子和慢性组织炎症等因素对BPH的发生发展起着重要的作用[2,3]。其中前列腺组织炎症与BPH的发生发展的关系尤为引人关注,大量临床和基础研究已经证实两者之间存在紧密的联系[4-9]。代谢综合征(Metabolic Syndrome,Met S)是一种代谢紊乱的病理状态,常见的亚组成分有腹型肥胖、高血压、脂代谢异常和血糖增高等[10]。研究表明Met S可以影响BPH的发生发展过程[11]。有Met S及亚组成分的老年男性前列腺体积增大更显著,前列腺增生速度也更快;其中2型糖尿病患者的前列腺年生长率增加了47%,肥胖患者的前列腺年生长率增加了36%,降低的血清高密度脂蛋白患者的前列腺年生长率增加了31%,高血压患者的前列腺年生长率增加了17%[12]。临床荟萃分析证明合并Met S疾病的老年男性患者的前列腺体积明显大于对照组患者[13]。本课题组前期研究发现Met S增加了BPH患者前列腺组织炎症程度;通过高脂饮食可以诱导雄性大鼠前列腺组织炎症的发生[14]。生物钟即昼夜节律,是大多数生物体内细胞的基本调节因子,与细胞增殖、代谢、炎症和DNA损伤应答等密切相关[15,16]。生物钟对人的行为和生理的影响体现在睡眠周期、血压、体温和新陈代谢等方面[17]。细胞核受体Rev-erb是生物钟至关重要的组件,包括Rev-erbα和Rev-erbβ,在维持昼夜节律方面起着重要作用[18,19]。研究证据表明Rev-erb在生物钟和免疫炎症通路之间起着关键的桥梁作用[20],进而有学者提出生物钟分子Rev-erbα在炎症调节中起关键作用的假说[21]。值得注意的是,慢性炎症和包括BPH在内的大多数慢性疾病密切相关[22-24]。为进一步明确生物钟蛋白Rev-erbα能否调节BPH组织的炎症,以及探索Rev-erbα通过调控Met S相关的前列腺组织炎症的机制。本研究通过收集临床资料,分析Rev-erbα的表达与BPH组织炎症程度的关系;通过体内实验Rev-erbα激动剂干预高脂饮食诱导的小鼠前列腺组织炎症模型,探索Rev-erbα调控前列腺组织炎症的可能机制;最后通过体外前列腺细胞实验对该机制进行验证。从而为研究前列腺组织炎症的发生和BPH的发展提供思路,为治疗前列腺组织炎症和BPH的发生发展提供新的靶标。材料和方法:第一章:良性前列腺增生人群中前列腺组织炎症与临床相关指标和生物钟蛋白Rev-erbα表达的相关性分析本研究共收集于2019年4月至2019年8月期间,在四川大学华西医院泌尿外科诊断为BPH并行经尿道前列腺电切术(Transurethral resection of the prostate,TURP)或经尿道前列腺剜除术(Transurethral enucleation of prostate,TUEP)的患者的临床数据和前列腺组织标本。前列腺组织标本进行病理切片苏木精-伊红(Hematoxylin-Eosin,HE)染色,根据国际前列腺炎组织学诊断与分度标准对前列腺组织进行炎症程度分级。通过免疫组化(Immunohistochemical staining,IHC)评估患者前列腺组织中白细胞分化抗原4(Cluster of Differentiation4,CD4)、CD8、白细胞介素-6(Interleukin-6,IL-6)、IL-8、IL-1β和Rev-erbα的表达情况;通过实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,q RT-PCR)检测前列腺组织中IL-1β、IL-6和IL-8和生物钟蛋白Reverbα的表达水平;结合患者的临床数据指标,分析BPH组织炎症程度与患者临床数据指标及Rev-erbα表达强度的相关性。第二章:Rev-erbα激动剂干预高脂饮食诱导小鼠前列腺组织炎症模型的研究将6周龄SPF级C57B/6J健康雄性小鼠随机分为四组实验:普通饮食组(Normal diet),高脂饮食组(High fat diet),高脂饮食并腹腔注射药物SR9009(Rev-erbα激动剂)组(High fat diet+SR9009),高脂饮食并腹腔注射药物溶剂对照组(High fat diet+Control)。在相同的饲养条件下,不限制进食饮水,给予正常清洁饲料或者高脂饲料。通过每周记录小鼠体重,最终检测小鼠外周血胆固醇、甘油三酯等指标,测量小鼠前列腺组织重量,前列腺组织病理切片HE染色了解炎症细胞组织浸润情况评估组织炎症程度。前列腺组织通过转录组测序分析Rev-erbα激动剂调控前列腺组织炎症的相关信号通路。蛋白免疫印迹(Western Blotting,WB)检测小鼠前列腺组织IL-6、IL-8和IL-1β的表达水平,IHC评估小鼠前列腺组织IL-6、IL-8、IL-1β、NF-κB-P65和C-C类趋化因子配体21(C-C Motif Chemokine ligand,CCL21)的表达水平。第三章:Rev-erbα通过NF-κB/NLRP3信号通路调节棕榈酸诱导WPMY-1细胞的炎症反应体外利用棕榈酸(Palmitic Acid,PA)模拟体内高脂微环境,诱导前列腺基质细胞WPMY-1产生炎症。使用q RT-PCR方法检测细胞中IL-6、IL-8的m RNA的表达情况来确定PA刺激细胞的最佳浓度和时间。将实验分为四组:Control(PA的有机溶剂)+DMSO,PA+DMSO,PA+SR9009(Rev-erbα激动剂),PA+SR8278(Rev-erbα拮抗剂)。通过q RT-PCR检测细胞中IL-1β、IL-6和IL-8的m RNA表达情况,酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)检测细胞培养液内IL-1β、IL-6、IL-8和CCL21的浓度,采用WB检测达水平。最后,通过流式细胞周期检测和细胞计数试剂-8(Cell Counting Kit-8,CCK-8)检测细胞炎症反应程度对于细胞增殖水平的影响。结果:第一章:良性前列腺增生人群中前列腺组织炎症与临床相关指标和生物钟蛋白Rev-erbα表达的相关性分析总共纳入84例患者的前列腺增生组织标本,病理组织切片HE染色发现86.9%(73/84)的前列腺组织伴有不同程度的组织炎症,其中35.7%(30/84)为轻度炎症,36.9%(31/84)为中度炎症,14.3%(12/84)为重度炎症。通过IHC检测CD4、CD8、IL-6、IL-8和IL-1β在组织中表达。前列腺组织中不同炎症程度与组织炎症因子(IL-6、IL-8和IL-1β)表达强度一致;组织炎症细胞中,CD4阳性细胞的数量远多于CD8阳性细胞数量。患者的临床指标与前列腺组织炎症程度进行Spearman相关性分析。结果提示代谢综合征、体重指数(Body Mass Index,BMI)、血清甘油三酯(Triglycerides,TG)、血清总胆固醇(Total Cholesterol,TC)、血清低密度脂蛋白(Low-density Lipoprotein,LDL)、国际前列腺症状评分(International Prostate Symptoms Score,IPSS)、高血压与前列腺组织炎症程度呈正相关,其相关系数分别为0.702,0.603,0.634,0.216,0.375,0.374,0.340。前列腺组织中Rev-erbα的表达强度与前列腺组织炎症程度呈负相关,其相关系数为-0.691。通过q RT-PCR检测前列腺组织中IL-1β、IL-6、IL-8和Rev-erbα的m RNA表达量,不同的前列腺组织炎症程度与组织中IL-1β、IL-6、IL-8的表达差异趋势一致,与组织中Rev-erbα的表达差异趋势相反。第二章:Rev-erbα激动剂干预高脂饮食诱导小鼠前列腺组织炎症模型的研究与普通饮食组小鼠相比,高脂饮食组小鼠的体重(33.29±3.33 g vs 28.75±0.88 g,p=0.002)、前列腺重量(129±30.81 mg vs 91±17.00 mg,p=0.008)、血清TG水平(1.46±0.09 mmol/L vs 1.03±0.13 mmol/L,p<0.001)、血清TC水平(5.66±1.01 mmol/L vs 2.73±0.15 mmol/L,p<0.001)均明显升高,差异均有统计学意义;高脂饮食+SR9009组小鼠的体重(29.21±1.62 g vs 32.64±2.72g,p=0.047)、前列腺重量(99±13.00 mg vs 131±28.09 mg,p=0.031)、血清TG水平(0.76±0.15 mmol/L vs 1.49±0.16 mmol/L,p=0.030)、血清TC水平(4.46±0.78 mmol/L vs 5.68±1.22 mmol/L,p<0.001)明显低于高脂饮食+Control组小鼠,差异均有统计学意义。通过对小鼠的前列腺组织进行HE染色,显微镜下发现高脂饮食组小鼠较普通饮食组小鼠前列腺组织内炎性细胞浸润增多,SR9009干预后的高脂饮食小鼠前列腺组织炎性细胞浸润减少。对四组小鼠前列腺组织进行转录组测序,差异基因分别与KEGG和Gene Ontology数据库进行富集分析,发现差异基因在NF-κB信号通路中有统计学意义。通过基因热图分析NF-κB/NLRP3通路中的基因Ccl21a、Ccr7、Tlr4、Myd88、Il6、Il1b、Cxcr2、Nlrp3、Caspase1的m RNA表达水平在高脂饮食组较普通饮食组升高,而其表达水平在高脂饮食+SR9009组较高脂饮食+Control组降低。通过WB检测四组小鼠前列腺组织中的炎症因子IL-6、IL-8和IL-1β的表达量,IHC检测组织中IL-6、IL-8、IL-1β、NF-κBP65和CCL21a的表达强度,其四组结果差异趋势与四组小鼠前列腺组织测序结果的差异趋势一致。第三章:Rev-erbα通过NF-κB/NLRP3信号通路调节棕榈酸诱导WPMY-1细胞的炎症反应利用q RT-PCR检测WPMY-1细胞中IL-6和IL-8的m RNA表达量在PA浓度为500μmol/L,作用时间24 h最高。通过ELISA检测PA+DMSO组细胞培养液中IL-6(p=0.005)、IL-8(p=0.006)、IL-1β(p=0.001)和CCL21(p<0.0001)的浓度较Control+DMSO组显著提高,PA+SR9009组中IL-6(p=0.025)、IL-8(p=0.010)、IL-1β(p=0.026)和CCL21(p=0.025)的浓度较PA+DMSO组低,而PA+SR8278组中IL-6(p=0.017)、IL-8(p=0.030)和IL-1β(p=0.030)的浓度较PA+DMSO组高,差异均有统计学意义。PA+SR8278组中CCL21(p=0.622)的浓度虽较PA+DMSO组高,但差异无统计学意义。通过WB检测发现PA+DMSO组细胞的NLRP3(p<0.0001)、pro-caspase1(p<0.001)和ASC(p<0.001)的表达量较Control+DMSO组升高,PA+SR9009组细胞内NLRP3(p<0.001)、pro-caspase1(p=0.005)和ASC(p=0.048)的表达量较PA+DMSO组降低,PA+SR8278组细胞内NLRP3(p=0.010)、pro-caspase1(p=0.013)和ASC(p=0.002)的表达量较PA+DMSO组升高,差异均有统计学意义。同样地,PA+DMSO组细胞NF-κB-P65(p=0.0039)、IκB(p=0.0014)的磷酸化水平较Control+DMSO组升高,PA+SR9009组细胞的NF-κB-P65(p=0.0408)、IκB(p=0.0035)的磷酸化水平较PA+DMSO组降低,PA+SR8278组细胞的NF-κB-P65(p=0.0406)、IκB(p=0.0103)的磷酸化水平较PA+DMSO组升高,差异均有统计学意义。细胞免疫荧光检测发现PA+DMSO组细胞ASC(p=0.0029)、NF-κB-P65(p<0.0001)的免疫荧光强度均强于Control+DMSO组,PA+SR9009组细胞ASC(p=0.0079)、NF-κB-P65(p<0.0001)的免疫荧光强度均弱于PA+DMSO组,PA+SR8278组细胞ASC(p=0.007)、NF-κB-P65(p=0.043)的免疫荧光强度均强于PA+DMSO组,差异均有统计学意义。CCK-8检测和流式细胞周期检测发现,PA+DMSO组在450nm的吸光度明显强于Control+DMSO组(p<0.001),G1期比率低于Control+DMSO组(p<0.001),说明PA+DMSO组细胞较Control+DMSO组细胞增殖能力提高;PA+SR9009组在450nm的吸光度明显弱于PA+DMSO组(p<0.001),G1期比率高于PA+DMSO组(p=0.038),说明PA+SR9009组细胞较Control+DMSO组细胞增殖能力降低;PA+SR8278组在450nm的吸光度强于PA+DMSO组(p=0.077),G1期比率低于PA+DMSO组(p=0.245),但差异无统计学意义。结论:1.大多数BPH组织中合并有不同程度的组织炎症。Met S、BMI、血清LDL、血清TC、血清TG、高血压、IPSS与前列腺组织炎症程度呈正相关,生物钟蛋白Rev-erbα的表达强度与前列腺组织炎症程度呈负相关。2.高脂饮食可以诱导雄性小鼠前列腺组织炎症的发生,体重和前列腺重量增加。通过SR9009干预后的高脂饮食小鼠,其体重和前列腺重量较单纯高脂饮食小鼠降低,前列腺组织炎症水平降低。生物钟蛋白Rev-erbα可能通过NF-κB/NLRP3通路调节高脂饮食诱导的前列腺组织炎症水平。3.PA通过NF-κB/NLRP3通路使WPMY-1细胞炎症水平升高,细胞炎症因子IL-6、IL-8、IL-1β和CCL21产生增加,使细胞增殖能力提高。Rev-erbα激动剂可能通过NF-κB/NLRP3通路减弱PA刺激WPMY-1细胞的炎症水平,使细胞增殖能力降低;Rev-erbα拮抗剂可能通过NF-κB/NLRP3通路增强PA刺激WPMY-1细胞的炎症水平,使细胞增殖能力一定程度提高。4.生物钟蛋白Rev-erbα可能成为防治前列腺组织炎症和BPH发展的潜在靶点。更多还原

【Abstract】 Background and purpose: Benign prostatic hyperplasia(BPH)which is more common in middle-aged and elderly men,can cause dysuria.According to survey data,BPH occurs in more than 50% of men over age of 60.In the wake of the increase of the aging male population,BPH disease is bound to bring a heavier burden to the whole medical and health system.However,the pathogenesis of BPH is still unclear.It has been shown recently that a variety of factors such as age-related metabolic disorders,sex hormones balance changes,multiple cell growth factors and chronic tissue inflammation play significant roles in the occurrence and development of BPH.Among various factors,the relationship between inflammation of prostate tissue and the occurrence and progression of BPH is particularly interesting,and the two have been proved to be closely linked by a large number of clinical research and basic experiment.Metabolic Syndrome(Met S),including abdominal obesity,hypertension,abnormal lipid metabolism,and high blood glucose,is a collection of complex metabolic disorders that can affect the occurrence and development of BPH.Studies have reported that men with Met S have a more significant prostate enlargement and faster prostate growth every year;Among them,those with type 2 diabetes had a 47% increase in annual prostate growth,a 36% increase in obesity,a 31% increase in decreased serum high-density lipoprotein,and a 17% increase in hypertension.It was proved by Meta analysis that the prostate volume of patients with Met S was significantly larger than that of patients with non-metabolic syndrome.It was found in a previous study of our research group that Met S can increase the inflammation degree of prostate tissue in patients with BPH,and high-fat diets can induce inflammation of prostate tissue in male rats.Biological clock,i.e.,circadian rhythm,is the basic regulator of cells in most organisms,which has a close bearing on cell proliferation,metabolism,inflammation and DNA damage response.The effect of the biological clock on human behavior and physiology is reflected in sleep cycle,blood pressure,body temperature and metabolism.Nuclear receptors Rev-erb is a vital component of the biological clock,including Rev-erbα and Rev-erbβ,which plays an important role in maintaining the circadian rhythm.Research evidence shows that Rev-erb plays a key role as a bridge between the circadian clock and immune-inflammatory pathways,and the hypothesis that circadian clock molecule Rev-erbα plays a key role in inflammation regulation has been proposed.It is worth noting that chronic inflammation has a close bearing on numerous chronic diseases including BPH.In order to further clarify whether Rev-erbα can regulate inflammation in BPH and explore the mechanism by which Rev-erbα regulates Met S-related inflammation in prostate tissue,the relationship between the expression of Rev-erbα and inflammation in BPH tissue was analyzed by collecting clinical data,the mechanism of biological clock protein Rev-erbα regulating tissue inflammation was explored via the intervention of Rev-erbα agonist on prostate tissue inflammation induced by high fat diet in mice,and finally,the mechanism was verified by cell experiments in vitro,so as to provide ideas for studying the occurrence of prostate tissue inflammation and the development of BPH,and provide a new target for the treatment of prostate tissue inflammation and the occurrence and development of BPH.Materials and Methods: Part I: Correlation analysis between inflammation of prostate tissue and clinical related indexes and the expression of biological clock protein Rev-erbα in patients with BPH In this part,the clinical data and surgically prostate tissue specimens of patients who underwent TUEP or TURP due to the diagnosis of BPH in the Department of Urology in West China Hospital from April 2019 to August 2019 were collected.Prostate tissue specimens were stained with HE,and the degree of prostatic inflammation was graded accprdomg to the international prostatitis histological diagnosis and grading standards.The expression of CD4,CD8,IL-6,IL-8,IL-1β and Rev-erbα in prostate tissue of patients was assessed by IHC;IL-1β,IL-6,IL-8 and Rev-erbα in prostate tissue were detected based by q RT-PCR;and the correlation between the degrees of inflammation in BPH tissues and the clinical data of patients and the expression of Rev-erbα were investigated.Part II: Study on the intervention of Rev-erbα agonist in the model of inflammation of prostate tissue induced by high-fat diet in mice SPF healthy male C57B/6J mice aged 6 weeks were used as the research objects and randomly divided into four groups: normal diet group,high fat diet group,high fat diet with intraperitoneal injection of drug SR9009(Rev-erbα agonist)group(high fat diet + SR9009),and high fat diet with intraperitoneal injection of drug solvent control group(high fat diet + Control).Under the same feeding conditions,normal clean feed or high-fat feed should be given without restricting eating and drinking water.The weight of the prostate tissue of mice was measured,the weight of mice was regularly recorded every week,cholesterol and triglyceride in the peripheral blood of mice were detected.HE staining was performed to evaluate the inflammation of prostate tissue.Transcriptome sequencing of prostate tissue was performed to analyze the signal pathway of Rev-erbα agonist regulating prostate inflammation.Western blot(WB)was adopted to evaluate the expression of IL-6,IL-8 and IL-1β in prostate tissue,The expression levels of IL-6,IL-8,IL-1β,NF-κB-P65 and CCL21 a in the prostate tissue of inflammatory mice were evaluated by IHC.Part III: Rev-erbα regulates palmitic acid-induced inflammatory response in WPMY-1 cells via NF-κB/NLRP3 signaling pathway Palmitic acid was utilized in vitro to simulate the high lipid microenvironment in vivo and induce inflammation of prostate stromal cells(WPMY-1).q RT-PCR was used to detect the m RNA expression of IL-6 and IL-8 in cells,so as to determine the optimal concentration and time of PA stimulation.The experiment was carried out in four groups: PA organic solvent(Control)+ DMSO,PA + DMSO,PA + SR9009(Reverbα agonist),PA + SR8278(Rev-erbα antagonist).The expression differences of IL-1β,IL-6 and IL-8 in cells detected by q RT-PCR,and the protein concentration of IL-6,IL-8,IL-1β and CCL21 in cell culture medium was detected by ELISA.The expression level of marker protein(P65、p-P65、IκB、p-IκB、NLRP3、pro-caspase1 and ASC)in NF-κB/NLRP3 signaling pathway was detected by WB,and the expression levels of P65 and ASC were detected by cell immunofluorescence.Finally,flow cytometry and CCK-8 were utilized to detect the effect of cell inflammation on cell proliferation.Results: Part I: Correlation analysis between inflammation of prostate tissue and clinical related indexes and the expression of biological clock protein Rev-erbα in patients with BPH A total of 84 patients with benign prostatic hyperplasia tissue specimens were included in the study.HE staining showed that 86.9%(73/84)of prostate tissues were accompanied by different degrees of tissue inflammation,among which 35.7%(30/84)were mild inflammation,36.9%(31/84)were moderate inflammation and 14.3%(12/84)were severe inflammation.According to IHC detection,the expression degree of CD4,CD8,IL-6,IL-8 and IL-1β in tissues was consistent with that of tissue inflammation in HE staining,and the number of CD4 positive cells in inflammatory cells was more than that of CD8 positive cells.In this part,the clinical indexes of patients were statistically analyzed,and Spearman correlation analysis was performed between the clinical indexes and the degree of prostatic inflammation.As indicated by the results,Met S,body mass index,serum triglycerides,serum total cholesterol,serum low density lipoprotein,international prostate symptom score and hypertension were positively correlated with the degree of inflammation of prostate tissue,and the correlation coefficients were 0.702,0.603,0.634,0.216,0.375,0.374 and 0.340,respectively.The expression intensity of Rev-erbα in prostate tissue was negatively correlated with the degree inflammation in prostate tissues,and the correlation coefficient was-0.691.The m RNAs of IL-1β,IL-6,IL-8 and Rev-erbα in prostate tissues were detected by q RTPCR.The trend of differences in the expression of IL-1β,IL-6 and IL-8 was consistent with the trend of the degree of inflammation in prostate tissues,but the trend of differences in the expression of Rev-erbα was opposite to the trend of the degree of inflammation in prostate tissues.Part II: Study on the intervention of Rev-erbα agonist in the model of prostatic inflammation induced by high fat diet in mice The body weight(33.29 ± 3.33 g vs 28.75 ± 0.88 g,p =0.002),prostate weight(129 ± 30.81 mg vs 91 ± 17.00 mg,p = 0.008),serum TG level(1.46 ± 0.09 mmol/L vs 1.03 ± 0.13 mmol/L,p < 0.001)and serum TC level(5.66 ± 1.01 mmol/l vs 2.73 ± 0.15 mmol/l,p < 0.001)of C57B6 J mice fed with high fat diet for 10 weeks were significantly higher than those of normal diet mice,with a statistical difference.The body weight(29.21 ± 1.62 g vs 32.64 ± 2.72 g,p = 0.047),prostate weight(99 ± 13.00 mg vs 131 ± 28.09 mg,p = 0.031),serum TG level(0.76 ± 0.15 mmol/L vs 1.49 ± 0.16 mmol/L,p = 0.030)and serum TC level(4.46 ± 0.78 mmol/L vs 5.68 ± 1.22 mmol/L,p < 0.001)of mice injected intraperitoneally with drug SR9009 for 3 weeks were significantly lower than those in the High fat diet + Control group,with a statistical significance.It was found under the microscope that after HE staining of the prostate tissue of mice,the infiltration of inflammatory cells in the prostate tissues of mice fed with high fat diet was higher than that of mice fed with normal diet,while the infiltration of inflammatory cells in the prostate tissue of mice fed with high fat diet after SR9009 intervention was decreased.Transcriptome sequencing was performed on the prostate tissues of four groups of mice.Differential genes were enriched and analyzed with KEGG and Gene Ontology databases respectively,it was found that the differential genes had a statistical significance in the NF-κB signaling pathway.The m RNA expression levels of Ccl21a、Ccr7、Tlr4、Myd88、Il6、Il1b、Cxcr2、Nlrp3、Caspase1in NF-κB/NLRP3 pathway were higher in high fat diet group than those in the normal diet group by heat map,but the level of their expression was lower in the high fat diet group + SR9009 than those in high fat diet + Control group.WB was utilized to detect the expression levels of inflammatory factors IL-6,IL-8 and IL-1β in the prostate tissues of the four groups of mice,and IHC was adopted to detect the expression of IL-6,IL-8,IL-1β,NF-κB-P65 and CCL21 a in the prostate tissues of the four groups of mice.The differences trend of the four goups of results is consistent with the differences trend of the four groups of mouse prostate tissues in the sequencing results.Part III: Rev-erbα regulates palmitic acid-induced inflammatory response in WPMY-1 cells via NF-κB/NLRP3 signaling pathway The q RT-PCR test results show that the highest expression of IL-6 and IL-8 in WPMY-1 cells at the PA concentration of 500 μmol/L and the action time of 24 h.The results detected by the ELISA method showed that the concentrations of IL-6(p = 0.005),IL-8(p = 0.006),IL-1β(p = 0.001)and CCL21(p < 0.0001)in the cell culture medium of the PA + DMSO group were substantially higher than those of the Control + DMSO group.The concentrations of IL-6(p = 0.025),IL-8(p = 0.010),IL-1β(p = 0.026)and CCL21(p = 0.025)in the PA+SR9009 group were lower than those in the PA + DMSO group;the concentrations of IL-6(p = 0.017),IL-8(p = 0.030)and IL-1β(p = 0.030)in the PA + SR8278 group were higher than those in the PA + DMSO group,the differences were statistically significant.While the concentrations of CCL21(p = 0.622)in the PA + SR8278 group was higher than that in the PA + DMSO group,the difference was not statistically significant.WB detection showed that the expressions of NLRP3(p < 0.0001),pro-caspase1(p < 0.001)and ASC(p < 0.001)in the PA + DMSO group were higher than those in the Control + DMSO group,the difference was statistically significant;the expressions of NLRP3(p < 0.001),pro-caspase1(p = 0.005)and ASC(p = 0.048)in the PA + SR9009 group were lower than those in the PA + DMSO group,the difference was statistically significant;the expressions of NLRP3(p = 0.010),pro-caspase1(p = 0.013)and ASC(p = 0.002)in the PA + SR8278 group were higher than those in the PA + DMSO group,the difference was statistically significant.Similarly,the phosphorylation level of NF-κB-P65(p = 0.0039)and IκB(p = 0.0014)in the PA + DMSO group was higher than that in the Control + DMSO group,the phosphorylation level of NF-κB-P65(p = 0.0408)and IκB(p = 0.0035)in the PA + SR9009 group was lower than that in the PA + DMSO group,while phosphorylation level of NF-κB-P65(p = 0.0406)and IκB(p = 0.0103)in the PA + SR8278 group was higher than that of the PA + DMSO group,the differences were statistically significant.Cellular immunofluorescence detection revealed that the immunofluorescence intensities of ASC(p = 0.0029)and NF-κB-P65(p <0.0001)of the PA + DMSO group were stronger than those of the Control + DMSO group,ASC(p = 0.0079)and NF-κB-P65(p <0.0001)of the PA + SR9009 group were weaker than the PA + DMSO group,and the immunofluorescence intensities of ASC(p = 0.007)and NF-κB-P65(p = 0.043)of the PA + SR8278 group stronger than the PA + DMSO group,the differences were statistically significant.CCK-8 detection and flow cytometry showed that the absorbance of the PA + DMSO group at 450 nm was significantly stronger than that of the Control + DMSO group(p < 0.001),and the G1 phase ratio of the PA + DMSO group was lower than the Control + DMSO group(p < 0.001),the cell proliferation ability was improved;The absorbance at 450 nm of the PA + SR9009 group was significantly weaker than that of the PA + DMSO group,the G1 phase ration of the PA + SR9009 group was higer than the PA + DMSO group(p = 0.038),the cell proliferation ability was decreased.The absorbance at 450 nm of the PA + SR8278 group was stronger than that of the PA + DMSO group(p = 0.077),and the G1 phase ration of the PA + SR8278 group was lower than the PA + DMSO group(p = 0.245),but the differences were not statistically significant.Conclusion: 1.The majority of BPH tissues are associated with different degrees of inflammation.Met S,BMI,serum LDL,serum TC,serum TG,hypertension and IPSS are positively correlated with the degree of prostatic inflammation,while the expressions of the biological clock protein Rev-erbα is negatively correlated with the degree of prostatic inflammation.2.High fat diet can induce prostatic tissue inflammation in male mice,and increase body weight and prostate weight of mice.After continuous intraperitoneal injection of SR9009,the body weight and prostate weight of mice fed with high fat diet decreased,and the inflammation level of prostate tissue decreased.The circadian clock protein Rev-erbα may regulate the inflammation of prostate tissue induced by high-fat diet through the NF-κB/NLRP3 pathway.3.PA can make the level of inflammation in WPMY-1 cells via the NF-κB/NLRP3 pathway keep high,and produce more inflammatory factors including IL-6,IL-8,IL-1β and CCL21,which improves cell proliferation.The levels of inflammation in WPMY-1 cells stimulated by PA can be attenuated by the intervention of Rev-erbα agonist on the NF-κB/NLRP3 pathway and reduced cell proliferation ability,but can be enhanced by the intervention of Rev-erbα antagonist on the NF-κB/NLRP3 pathway and increased the cell proliferaton ability to a certain extent.4.The biological clock protein Rev-erbα may be a potential target for the prevention and treatment of inflammation of prostate tissue and 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