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Baicalein alleviates pyroptosis and inflammation in hyperlipidemic pancreatitis by inhibiting NLRP3/Caspase-1 pathway through the miR-192-5p/txnip axis

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INTERNATIONAL IMMUNOPHARMACOLOGY 2021年第PartB期101卷 108315-108315页

作者： Wang, Xiangyang Cai, Hua Chen, Zhiyuan Zhang, Yu Wu, Minghao Xu, Xiaoping Yang, Li Hunan Normal Univ Hunan Prov Peoples Hosp Dept Gastroenterol Affiliated Hosp 1 61 Jiefang West Rd Changsha 410005 Hunan Peoples R China

Objective: Hyperlipidemia is a main reason of pancreatitis. Baicalein can ameliorate the pathological manifestations of pancreatitis. This study evaluated underlying molecular mechanism of baicalein in hyperlipidemic pancreatitis (HP). Methods: HP rat model was successfully established and treated with baicalein. Amylase (AMY) activity and concentrations of triglyceride (TG) and total cholesterol (TC) were detected. Levels of pyroptosis-related proteins (GSDMD, IL-1 ss, IL-18) were detected by Western blot. Expressions of inflammatory factors (IL-6, TNF-alpha, IL-4) were detected by ELISA. Toxicity of baicalein on pancreatic acinar cells (PACs) was detected by MTT assay. HP cell model was established by 0.1 mM palmitic acid and CCK-8 stimulation. Target relation of miR-192-5p and txnip was predicted and verified by RNA22 v2 database and dual-luciferase reporter assay. Expressions of miR-192-5p and txnip were detected by RT-qPCR. Pyroptosis and inflammation in PACs were detected after baicalein treatment combined with silencing miR-192-5p or txnip overexpression. Protein levels of NLRP3/ Caspase-1 pathway in vivo and vitro were detected. Results: Baicalein reduced concentrations of TG and TC, AMY activity, and pathological scores in HP rat model, reduced LDH activity, pyroptosis and alleviated inflammation in vivo and in vitro. Mechanically, miR-192-5p targeted txnip, and baicalein inhibited pyroptosis and inflammation by up-regulating miR-192-5p and downregulating txnip. Silencing miR-192-5p or txnip overexpression partially abolished the anti-pyroptosis and anti-inflammatory effect of baicalein on PACs. Baicalein attenuated HP by inhibiting the NLRP3/Caspase-1 pathway. Conclusion: Baicalein alleviated pyroptosis and inflammation in HP by inhibiting the NLRP3/Caspase-1 pathway through miR-192-5p upregulation and txnip inhibition.

关键词： Hyperlipidemic pancreatitis Baicalein miR-192-5p txnip Pyroptosis Inflammation NLRP3 Caspase-1

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Inhibition of histone acetyltransferase by naringenin and hesperetin suppresses txnip expression and protects pancreatic β cells in diabetic mice

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PHYTOMEDICINE 2021年 88卷 153454-153454页

作者： Wang, Si-Wei Sheng, Hao Bai, Yong-Feng Weng, Yuan-Yuan Fan, Xue-Yu Zheng, Fang Fu, Jing-Qi Zhang, Feng Zhejiang Univ Quzhou Hosp Core Facil Sch Med Quzhou 324000 Peoples R China Zhejiang Univ Sch Med Hangzhou 310058 Peoples R China Zhejiang Univ Quzhou Hosp Dept Clin Lab Sch Med Quzhou 324000 Peoples R China China Med Univ Sch Publ Hlth 77 Puhe Rd Shenyang 110122 Liaoning Peoples R China

Background: The damage of pancreatic beta cells is a major pathogenesis of the development and progression of type 2 diabetes and there is still no effective therapy to protect pancreatic beta cells clinically. In our previous study, we found that Quzhou Fructus Aurantii (QFA), which is rich in flavanones, had the protective effect of pancreatic beta cells in diabetic mice. However, the underlying mechanism is still unclear. Purpose: In the current study, we administered naringenin and hesperetin, two major active components of QFA, to protect pancreatic beta cells and to investigate the underlying molecular mechanism focusing on the epigenetic modifications. Methods: We used diabetic db/db mouse and INS-1 pancreatic beta cell line as in vivo and in vitro models to investigate the protective effect of naringenin and hesperetin on pancreatic beta cells under high glucose environment and the related mechanism. The phenotypic changes were evaluatedby immunostaining and the measurement of biochemical indexes. The molecular mechanism was explored by biological techniques such as western blotting, qPCR, ChIP-seq and ChIP-qPCR, flow cytometry and lentivirus infection. Results: We found that naringenin and hesperetin had an inhibitory effect on histone acetylation. We showed that naringenin and hesperetin protected pancreatic beta cells in vivo and in vitro, and this effect was independent of their direct antioxidant capacity. The further study found that the inhibition of thioredoxin-interacting protein (txnip) expression regulated by histone acetylation was critical for the protective role of naringenin and hesperetin. Mechanistically, the histone acetylation inhibition by naringenin and hesperetin was achieved through regulating AMPK-mediated p300 inactivation. Conclusion: These findings highlight flavanones and the phytomedicine rich in flavanones as important dietary supplements in protecting pancreatic beta cells in advanced diabetes. In addition, targeting histone ac

关键词： naringenin hesperetin histone acetylation txnip p300 AMPK

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Sub-chronic exposure to realgar induces liver injury via upregulating the txnip/NLRP3 pathway and disturbing bile acid homeostasis in mice

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JOURNAL OF ETHNOPHARMACOLOGY 2021年 281卷 114584-114584页

作者： Li, Aihong Wu, Xinyu Yang, Jing Li, Jian Guo, Haoqi Zhang, Yuwei Jiang, Hong Huo, Taoguang China Med Univ Sch Publ Hlth Dept Hlth Lab Technol Shenyang 110122 Peoples R China China Med Univ Sch Publ Hlth Key Lab Arsen Related Biol Effects & Prevent & Tr Shenyang 110122 Peoples R China

Ethnopharmacological relevance: Realgar is a traditional Chinese medicine used in China for a long history. Longtime or excessive use of realgar causes liver injury. However, its underlying mechanism is not fully clarified. Aim of the study: In this study, we investigated the toxic effect of sub-chronic exposure to realgar on mice liver, and further revealed its underlying mechanism focused on the txnip/NLRP3 pathway and bile acid homeostasis. Material and methods: Mice were divided into control and different doses of sub-chronic realgar exposed groups. Total arsenic levels in the blood and liver were determined by atomic fluorescence spectrometry. The effect of realgar on liver function was evaluated by biochemical analysis and histopathological examination. Assay kits were applied for the measurement of oxidative stress indexes, MPO and plasma inflammatory cytokines. The mRNA and proteins involved in the txnip/NLRP3 and NF-Kappa B pathways were determined by RT-qPCR, western blot, Immunofluorescence and Immunohistochemistry. UHPLC/MS/MS was used for the quantitative analysis of bile acids (BAs) in mice plasma, liver and urine. The genes related to BAs metabolism were measured by RTqPCR. Results: Sub-chronic exposure to realgar led to arsenic accumulation and caused oxidative damage and inflammatory infiltration in mouse liver, finally resulting in liver injury. Realgar treatment activated the NF-Kappa B pathway and significantly upregulated the txnip/NLRP3 pathway in mouse liver. Realgar altered the metabolic balance of BAs, which is related to the abnormal expression of BAs transporters and enzymes. Conclusion: Sub-chronic exposure to realgar caused liver injury in mouse, and the mechanism may involve the upregulation of the txnip/NLRP3 pathway and disordered BAs homeostasis.

关键词： Realgar Liver injury txnip NLRP3 pathway Bile acid

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长链非编码RNA PWARSN通过txnip诱导糖尿病近端肾小管上皮细胞焦亡的机制研究

长链非编码RNA PWARSN通过TXNIP诱导糖尿病近端肾小管上皮细胞焦...

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作者：宋怡郑州大学

学位级别：博士

糖尿病肾脏疾病（Diabetic kidney disease,DKD）是糖尿病患者严重的微血管并发症之一,已成为世界范围内终末期肾脏疾病（End stage renal disease,ESRD）的主要原因。DKD发病机制复杂,肾小管损伤在其中扮演重要角色。糖尿病状态下,血... 详细信息

糖尿病肾脏疾病（Diabetic kidney disease,DKD）是糖尿病患者严重的微血管并发症之一,已成为世界范围内终末期肾脏疾病（End stage renal disease,ESRD）的主要原因。DKD发病机制复杂,肾小管损伤在其中扮演重要角色。糖尿病状态下,血流动力学改变、脂质代谢紊乱、氧化应激以及炎症反应等均可造成近端肾小管上皮细胞（Proximal tubular epithelial cells,PTECs）形态、结构、功能改变及细胞死亡等,最终导致肾小管间质纤维化和蛋白尿形成。然而,DKD中诱发PTECs损伤的病理机制仍不清楚,进一步明确其具体机制有助于探索DKD治疗的新靶点。细胞焦亡（Pyroptosis）是由无菌性炎症诱发的细胞程序性死亡方式之一,主要由Gasdermin家族蛋白介导。研究表明,在氧化应激等信号刺激下,促炎因子NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3,NLRP3）结合接头蛋白ASC（apoptosis-associated speck-like protein containing a CARD,ASC）,募集半胱氨酸天冬氨酸蛋白酶酶原（pro-Caspase-1）激活Caspase-1,切割GasderminD（GSDMD）,促使其与细胞膜特定位点结合形成孔洞,将IL-1β、IL-18等内容物释放到细胞外,诱导细胞焦亡,放大局部和全身炎症反应。细胞焦亡参与多种疾病的炎症损伤过程,但在DKD肾小管损伤中的作用仍需进一步明确。硫氧还蛋白相互作用蛋白（Thioredoxin-interacting protein,txnip）是体内重要的氧化应激传感器,其异常表达可促进细胞内活性氧簇（reactive oxygen species,ROS）累积,诱导炎症反应。研究表明,txnip可以通过NLRP3炎症小体诱导DKD中肾脏固有细胞焦亡。我们在前期研究中亦发现,txnip在高糖培养的PTECs中表达升高。然而,高糖状态下,txnip是否介导PTECs焦亡以及调节txnip表达的上游信号通路,尚需深入研究。近年来,长链非编码RNA（Long non-coding RNA,lncRNA）得到了研究者的广泛关注,其通过募集染色质修饰复合物,与miRNA、mRNA以及RNA蛋白相互作用,参与细胞内表观遗传调控、转录调控和转录后水平调控。已有研究证实,lncRNAs参与多种糖尿病微血管病变的发生,包括糖尿病视网膜病变、糖尿病肾病等。然而,国内外对lncRNAs在DKD肾小管上皮细胞焦亡中的作用鲜有报道,仍需进行广泛的研究以揭示它们在DKD发生发展过程中的生物学功能。在本研究中,我们首先探索txnip是否诱导DKD患者和糖尿病小鼠PTECs中NLRP3炎症小体激活以及细胞焦亡;其次构建并分析转录组RNA芯片,明确高糖培养的PTECs（HK-2 细胞）中lncRNA PWARSN（Prader-Willi/Angelman区域RNA,SNRPN邻居,PWARSN）与txnip表达水平密切相关,是诱导细胞焦亡的潜在驱动因素;进一步肾皮质靶向注射人源性PWARSN构建小鼠肾脏PWARSN过表达模型,探讨PWARSN对小鼠肾脏功能及近端肾小管上皮细胞焦亡的影响;最后利用生物信息学以及RNA结合蛋白免疫共沉淀等实验探索PWARSN调节高糖培养的PTECs焦亡的具体分子机制。第一部分 txnip诱导糖尿病近端肾小管上皮细胞内NLRP3炎症小体激活及细胞焦亡目的观察DKD患者肾小管组织、糖尿病小鼠肾小管组织以及高糖培养的人近端肾小管上皮细胞中txnip、NLRP3炎症小体激活及细胞焦亡的发生,初步探讨txnip与NLRP3炎症小体诱导的肾小管上皮细胞焦亡之间的关系。方法 1.收集临床DKD患者（DKD组）、糖尿病患者（Diabetes mellitus,DM组）肾组织以及行肾癌根治性切除术且糖耐量正常患者远癌肾组织标本（Normal control,NC组）。构建1型糖尿病小鼠模型（DM组）,并以注射柠檬酸缓冲液的C57BL/6J小鼠作为对照（Normal control,NC组）,饲养16周后处死小鼠并收集肾脏组织。常规行肾脏组织病理染色和透射电镜（Transmission electron microscope,TEM）检测,观察肾脏组织病理变化以及肾小管超微结构改变。 2.免疫组织化学染色（Immunohistochemical staining,IHC）观察txnip和NLRP3炎症小体（NLRP3、Caspasel、ASC）的蛋白表达水平。 3.分别用正常糖（糖浓度为5.6 m

关键词：糖尿病肾病 lncRNAPWARSN txnip 细胞焦亡近端肾小管上皮细胞

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txnip研究进展概述

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世界最新医学信息文摘 2022年第94期22卷 1-5页

作者：冯立新孙影华北理工大学基础医学院，河北唐山 063210 华北理工大学基础医学院，河北唐山 063210 华北理工大学河北省慢性疾病重点实验室，河北唐山 063210

硫氧还蛋白相互作用蛋白（txnip）是一种α-arrestin蛋白，在多种细胞内都有表达，可与硫氧环蛋白(Thioredoxin, Trx)结合，抑制其抗氧化的功能，促进氧化应激和炎症损伤。txnip可以激活NOD样受体3（NLRP3）炎症小体，进而促进Caspase-... 详细信息

硫氧还蛋白相互作用蛋白（txnip）是一种α-arrestin蛋白，在多种细胞内都有表达，可与硫氧环蛋白(Thioredoxin, Trx)结合，抑制其抗氧化的功能，促进氧化应激和炎症损伤。txnip可以激活NOD样受体3（NLRP3）炎症小体，进而促进Caspase-1的激活裂解和促进IL-1β分泌，诱发炎症反应。在各种病理变化和疾病进程中txnip也发挥了一定作用，它可促进细胞凋亡和焦亡、影响糖尿病肾病和肾脏纤维化、脑血管疾病、肝脏疾病、心血管疾病的进展、抑制癌细胞增殖。

关键词： txnip 炎症

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消渴安糖方对2型糖尿病肾病患者血清Trx、txnip的影响

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中文科技期刊数据库（引文版）医药卫生 2023年第8期 174-177页

作者：杨望荣姜俊玲钟润芬李慧琪周秋利广西中医药大学附属瑞康医院内分泌科广西南宁530011

研究2型 DN (2型 DN)中硫氧还蛋白(Trx)和硫氧还蛋白(txnip)对 DN (2型 DN)中 Trx和 txnip的调控作用。方法选择广西中医学院瑞康人民医院内分泌内科门诊和入院的2型 DN病人69例,根据纳入条件,将其分成2组:普通疗法+止渴安糖方34例,普... 详细信息

研究2型 DN (2型 DN)中硫氧还蛋白(Trx)和硫氧还蛋白(txnip)对 DN (2型 DN)中 Trx和 txnip的调控作用。方法选择广西中医学院瑞康人民医院内分泌内科门诊和入院的2型 DN病人69例,根据纳入条件,将其分成2组:普通疗法+止渴安糖方34例,普通疗法35例。比较两组的疗效。结果两组患者术后 Trx和 txnip均显著高于对照组,差异有统计学意义。结论我们提出“消渴安糖方剂”通过调控 Trx和 txnip表达,提高2型 DN (气阴夹瘀),为中药防治 DN开辟新途径。

关键词：消渴安糖方 2型糖尿病肾病气阴两虚夹瘀证型 Trx txnip

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txnip促进ROS所致的DNA损伤逆转三阴性乳腺癌的化疗耐药

TXNIP促进ROS所致的DNA损伤逆转三阴性乳腺癌的化疗耐药

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作者：陈嫕婷中南大学

学位级别：硕士

背景:三阴性乳腺癌(TNBC)是乳腺癌中恶性程度最高的一种分子类型,缺乏雌激素和孕激素受体(ER/PR)以及人表皮生长因子受体-2(HER2)的表达,化疗是TNBC患者的主要治疗手段,然而临床上的常用化疗药物如阿霉素(DOX)等极易产生耐药。因此,筛... 详细信息

背景:三阴性乳腺癌(TNBC)是乳腺癌中恶性程度最高的一种分子类型,缺乏雌激素和孕激素受体(ER/PR)以及人表皮生长因子受体-2(HER2)的表达,化疗是TNBC患者的主要治疗手段,然而临床上的常用化疗药物如阿霉素(DOX)等极易产生耐药。因此,筛选出化疗耐药相关的分子并研究其具体作用机制,对改善TNBC患者化疗疗效具有重要的临床意义。硫氧还蛋白互作蛋白(txnip)是一种代谢相关蛋白,在肺癌、乳腺癌等多种恶性肿瘤的发生发展中起到抑癌作用,但txnip在TNBC化疗耐药中的具体作用未见报道。研究目的:研究txnip在三阴性乳腺癌耐药中的具体作用及相关机制;探究基于10058-F4和DOX联用对TNBC耐药的化疗治疗作用。研究方法:通过CCK-8、克隆形成、流式细胞术及裸鼠成瘤等体内外实验检测txnip对TNBC化疗耐药细胞增殖及凋亡的影响;通过Western blotting、免疫荧光-激光共聚焦显微镜、DHE荧光探针-流式分析检测txnip介导的TNBC耐药细胞内活性氧(ROS)的表达及DNA损伤程度;通过Ed U、流式细胞术、皮下成瘤等体内外实验及联合用药指数计算分析c-Myc抑制剂10058-F4与DOX联合用药对耐药细胞的毒性作用。研究结果:过表达txnip抑制三阴性乳腺癌耐药细胞增殖和诱导细胞凋亡。上调txnip诱导耐药细胞活性氧的产生并且诱导了DNA损伤,呈时间及剂量依赖性增加了DNA损伤指标分子γ-H2AX的水平,用ROS清除剂预处理可阻断txnip诱导的DNA损伤并部分恢复了TNBC耐药细胞对阿霉素的耐药性。此外,小分子c-Myc抑制剂10058-F4促进了txnip的表达,增加了细胞内活性氧的产生,并与阿霉素联用能在体内、外显著抑制TNBC耐药细胞增殖和裸鼠移植瘤的生长。结论:***通过增加耐药细胞内活性氧水平以及促进DNA损伤的累积,从而逆转TNBC的化疗耐药性;2.小分子抑制剂10058-F4能上调txnip的表达,并与DOX联用增强化疗药物的细胞毒性作用。图34幅,表7个,参考文献71篇

关键词：三阴性乳腺癌化疗耐药性 txnip ROS DNA损伤 10058-F4

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Role and mechanism of txnip in ageing-related renal fibrosis

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MECHANISMS OF AGEING AND DEVELOPMENT 2021年 196卷 111475-111475页

作者： He, Qirui Li, Yang Zhang, Weiwei Chen, Jie Deng, Wenzhen Liu, Qicong Liu, Yongjian Liu, Dongfang Chongqing Med Univ Affiliated Hosp 2 Dept Endocrinol Chongqing 400010 Peoples R China Qianjiang Cent Hosp Chongqing Dept Endocrinol Chongqing 409000 Peoples R China Chongqing Clin Res Ctr Geriatr & Gerontol Chongqing 409000 Peoples R China

Kidney ageing, which is always accompanied by renal fibrosis, drives the progression of renal fibrosis. Thioredoxin-interacting protein (txnip) is an endogenous suppressor of the reactive oxygen species-scavenging protein thioredoxin, which has been implicated in the ageing of some organs and is involved in renal fibrosis. However, the expression of txnip in ageing kidneys has not been examined, and the relationship between txnip and ageing-related renal fibrosis is unclear. We found that txnip expression was upregulated in aged mouse kidneys, and this upregulation was accompanied by ageing-related renal fibrosis phenotypes. We demonstrated that the ageing biomarkers were downregulated in txnip-knockout mice, and these effects resulted in the alleviation of renal fibrosis and impairments in kidney function. txnip overexpression in tubular cells upregulated senescence markers, promoted a profibrotic response and activated STAT3 signalling, and these parameters were inhibited by the silencing of txnip. Similarly, the txnip-mediated profibrotic response was significantly suppressed by a STAT3 inhibitor. By coimmunoprecipitation, we verified that txnip directly bound to STAT3, which suggested that txnip exacerbates renal tubular epithelial fibrosis by activating the STAT3 pathway. In summary, txnip plays an important role in age-related renal fibrosis and might be a therapeutic target for preventing ageing-associated renal fibrosis.

关键词： txnip Kidney ageing Renal fibrosis STAT3 signalling pathway

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Estradiol exerts a neuroprotective effect on SH-SY5Y cells through the miR-106b-5p/txnip axis

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JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY 2021年第9期35卷 e22861-e22861页

作者： Pan, Qiong Guo, Ke Xue, Min Tu, Qiuyun Cent South Univ Dept Obstet & Gynecol Xiangya Hosp 3 Changsha Peoples R China Cent South Univ Dept Neurol Xiangya Hosp 3 Changsha Peoples R China Sun Yat Sen Univ Dept Geriatr Affiliated Hosp 5 Zhuhai Peoples R China

Alzheimer's disease (AD) is a neurodegenerative disease. Thioredoxin and thioredoxin-interacting protein (txnip) complexes help sustain cell oxidation/reduction balance. In the present study, we verified the neuroprotective role of estradiol against amyloid-beta 42 in SH-SY5Y cells through inhibiting txnip expression, promoting cell viability and DNA synthesis ability, inhibiting cell apoptosis, and affecting caspase and Bax/Bcl-2 apoptotic signaling. miR-106b-5p could bind to txnip 3 '-untranslated region to inhibit the expression level of txnip. Within SH-SY5Y cells, miR-106b-5p inhibition repressed cell viability and DNA synthesis ability and promoted cell apoptosis through caspase and Bax/Bcl-2 apoptotic signaling, while miR-106b-5p overexpression or txnip knockdown exerted the opposite effects on SH-SY5Y cells;txnip knockdown remarkably attenuated the roles of miR-106b-5p inhibition. In conclusion, estradiol treatment on SH-SY5Y cells downregulates txnip expression and upregulates miR-106b-5p expression. miR-106b-5p exerts a neuroprotective effect on SH-SY5Y cells by promoting cell proliferation and inhibiting cell apoptosis through targeting txnip.

关键词： apoptosis estradiol miR-106b-5p SH-SY5Y cell txnip

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Nrf2通过txnip调控NLRP3/Caspase-1通路在香烟烟雾诱导支气管上皮细胞焦亡中的作用及机制

Nrf2通过TXNIP调控NLRP3/Caspase-1通路在香烟烟雾诱导支气管上皮...

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作者：张梦宇山东大学

学位级别：博士

研究背景:慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease,COPD)是呼吸系统的常见病。吸烟是COPD最主要的危险因素,但其确切的致病机制尚未阐明。细胞焦亡的特征是可以释放大量炎症因子,最终引起强烈的炎症反应。细胞焦亡的... 详细信息

研究背景:慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease,COPD)是呼吸系统的常见病。吸烟是COPD最主要的危险因素,但其确切的致病机制尚未阐明。细胞焦亡的特征是可以释放大量炎症因子,最终引起强烈的炎症反应。细胞焦亡的发生主要由活化的炎症小体介导,同时伴随着GSDMD(Gesdermin D)的切割、白介素(IL)-1β和IL-18的释放。NLRP3作为研究最广泛的炎症小体,已证实在COPD患者肺组织中高表达;敲减NLRP3可抑制香烟烟雾引起的支气管上皮细胞焦亡及炎症反应,发挥对支气管上皮细胞的保护作用。吸烟可以导致肺部氧化应激反应的发生,促进活性氧簇(Reactive Oxygen Species,ROS)的增多从而促进炎症小体的活化。txnip是氧化应激的调节剂,在氧化应激发生时可与NLRP3传感器C末端的富含亮氨酸重复(Leucine-Rich Repeats,LRR)结构域相互作用,促进NLRP3炎症小体的激活。Nrf2是氧化还原敏感性转录因子,可增强抗氧化酶(GST、HO-1和NQO1)的转录从而调节ROS水平。白藜芦醇具有抗氧化特性,体内外研究均已证实它在肺部疾病中的抗炎和抗氧化作用。综合以上,我们推测Nrf2可能通过ROS水平调控txnip的表达,影响炎症小体的活化,参与香烟烟雾引起的支气管上皮细胞焦亡。本研究拟阐明:1.香烟烟雾能否引起支气管上皮细胞焦亡及炎症反应;2.香烟烟雾如何导致支气管上皮细胞焦亡及炎症反应;3.白藜芦醇能否抑制香烟烟雾诱导的支气管上皮细胞焦亡及炎症反应?如果可以,是以何种方式发挥作用的?方法:1.梯度浓度的香烟烟雾提取物(Cigarette Smoke Extract,CSE)处理支气管上皮细胞,RT-qPCR及Western Blotting检测细胞焦亡相关分子的表达;乳酸脱氢酶(Lactate dehydrogenase,LDH)释放实验及碘化丙锭(Propidium Iodide,PI)染色检测细胞焦亡的水平;酶联免疫吸附试验(Enzyme-Linked Immunosorbent Assay,ELISA)检测细胞上清液中炎症因子的表达。此部分拟阐明CSE引起支气管上皮细胞焦亡和炎症反应。2.5%CSE处理支气管上皮细胞24小时,加用VX-765(一种caspase-1抑制剂)后,用上述方法检测细胞焦亡及炎症因子表达,明确caspase-1在CSE引起支气管上皮细胞焦亡中的作用。3.5%CSE处理支气管上皮细胞24小时后,使用小干扰RNA(siRNA)敲减支气管上皮细胞中NLRP3的表达,RT-qPCR及Western Blotting验证siRNA的敲减效率;检测干扰组和干扰对照组细胞中caspase-1的表达;LDH释放实验及PI染色检测si-NLRP3组及si-NC组细胞焦亡水平的变化;ELISA检测si-NLRP3组及si-NC组细胞上清液中炎症因子的表达。明确NLRP3对caspase-1的影响及其在CSE导致的气道上皮细胞焦亡中的作用。4.5%CSE处理支气管上皮细胞24小时,加用N-乙酰半胱氨酸(NAC)(一种ROS清除剂)。活性氧试剂盒检测ROS水平;RT-qPCR检测NLRP3的表达;caspase-1试剂盒检测caspase-1的活性;LDH释放实验及PI染色检测细胞焦亡的变化;ELISA检测细胞上清液中炎症因子的表达。明确ROS水平对NLRP3和caspase-1的影响,及其在CSE导致的气道上皮细胞焦亡中的作用。5.5%CSE处理支气管上皮细胞24小时,RT-qPCR及Western Blotting检测txnip的表达。随后5%CSE处理支气管上皮细胞24小时,使用siRNA敲低支气管上皮细胞中txnip的表达,检测si-txnip组及si-NC组细胞的ROS水平、细胞焦亡相关分子的表达。此部分拟明确txnip对NLRP3表达的影响,及其在CSE诱导的支气管上皮细胞焦亡的作用,同时阐明ROS与txnip的上下游关系。6.5%CSE处理支气管上皮细胞24小时,检测Keap1、Nrf2及依赖Nrf2的抗氧化酶(GST,HO-1和NQO1)的表达。5%CSE处理支气管上皮细胞24小时后加用tBHQ(一种 Nrf2 激活剂),Western Blotting 检测 Nrf2、GST、HO-1 和 NQO1 的表达,txnip、NLRP3及细胞焦亡相关分子的表达;活性氧试剂盒检测ROS水平;RT-qPCR检测txnip、NLRP3及细胞焦亡相关分子的表达;ELISA检测炎症因子的表达。阐明Nrf2对ROS及txnip的影响,明确Nrf2在CSE诱导支气管上皮细胞焦亡中的作用。7.5%CSE处理支气管上皮细胞24小时后加

关键词： COPD Nrf2 txnip NLRP3/Caspase-1 细胞焦亡

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