小檗碱调控NLRP3炎症小体对TGF-β1诱导HK-2细胞转分化的影响

摘要：目的:探讨小檗碱（BBR）调控NLRP3炎症小体对转化生长因子-β1（TGF-β1）诱导人肾小管上皮细胞（HK-2）转分化的影响。方法:计算机分子对接预测BBR与NLRP3炎症小体的结合情况;采用CCK8法检测不同浓度的BBR对HK-2细胞增殖的影响;将HK-2细胞分为正常组、模型组和小檗碱低、中、高剂量组(10,25,50μmol·L-1),小檗碱预孵18 h后加入TGF-β1刺激48 h,镜下观察各组细胞形态;采用Western blotting法检测E-钙粘蛋白（E-cadherin）、α-平滑肌肌动蛋白（α-SMA）、NOD样受体蛋白3（NLRP3）及半胱氨酸天冬氨酸蛋白酶-1（caspase-1）的表达; caspase-1活性检测试剂盒检测caspase-1的酶活力;采用酶联免疫吸附测定法（ELISA）检测细胞上清液中白细胞介素-1β（IL-1β）的含量。结果:分子对接显示BBR与NLRP3、pro-caspase-1存在结合,可靠性较高。与正常组比较,BBR对HK-2细胞的增殖呈浓度和时间依赖性抑制（P<0.05）。与模型组比较,BBR(10,25,50μmol·L-1)可改善HK-2细胞形态,使其趋向椭圆形;上调E-cadherin蛋白和下调α-SMA、NLRP3及caspase-1蛋白的表达（P<0.05）;并可降低caspase-1的酶活力、减少IL-1β的分泌（P<0.05）。结论:BBR能改善TGF-β1诱导HK-2细胞的转分化,可能与抑制NLRP3炎症小体活化有关。     

基于NLRP3/GSDMD信号通路研究人参皂苷Rb1对高糖诱导的胰岛β细胞焦亡的影响

目的: 研究人参皂苷Rb1(GRb1)抑制高糖诱导的胰岛β细胞焦亡及对NLRP3/GSDMD信号通路的调节机制。方法: 高糖(50 mmol·L^-1)诱导大鼠胰岛细胞瘤细胞(rat insulinoma cells, INS-1)焦亡模型,并构建NLRP3过表达质粒转染INS-1细胞,采用倒置显微镜观察GRb1对细胞形态变化的影响;采用ELISA法检测GRb1对细胞上清液中乳酸脱氢酶(LDH)、白细胞介素-1β(IL-1β)、胰岛素水平的影响;采用Western blot法和qRT-PCR法检测GRb1对INS-1细胞中核苷酸结合域样受体蛋白3(nucleotide binding domain like receptor protein 3, NLRP3)和切割蛋白D(gasdermin D, GSDMD)表达的影响。结果: 在高糖环境下,GRb1可减轻INS-1细胞形态学改变,显著增加INS-1细胞胰岛素分泌,降低细胞上清液中IL-1β、LDH水平(P＜0.05),且呈剂量依赖性;GRb1呈剂量依赖性抑制高糖诱导的INS-1细胞中GSDMD及上游调节因子NLRP3的表达(P＜0.05);过表达NLRP3后可显著逆转高糖环境下GRb1对INS-1细胞的部分保护性作用(P＜0.05)。结论: GRb1能够改善高糖诱导的INS-1细胞焦亡,减轻炎症反应,其机制可能与抑制NLRP3/GSDMD信号通路有关,从而防治糖尿病及其并发症的发生。     

白藜芦醇对H2O2 及TGF-β2诱导人小梁网细胞的影响及机制探讨

摘要： 目的 观察过氧化氢 （H2O2 ） 及转化生长因子 （TGF） -β2 诱导人小梁网细胞 （HTMCs） 后对纤维连接蛋白（FN）、 胶原蛋白 1 型 （COL1）、 核因子 （NF） -κB P65 蛋白和白细胞介素 （IL） -1β基因表达的影响及白藜芦醇 （RSV） 的干预作用。方法 （1） 选取汇合度 70%～80%的 HTMCs 分为 5 组。实验组于无血清培养基中分别加入浓度为 150、 300、 450、 800 μmol/L 的 H2O2 处理, 对照组的培养基中不加 H2O2。Western blot 法检测各组 FN、 COL1、 NF-κB P65、 NF-κB P65 磷酸化 （P-NF-κB P65） 蛋白的表达, 实时定量 PCR 法检测 IL-1β基因的表达。（2） HTMCs 细胞分为 3 组。对照组以不含 H2O2 及 RSV 的无血清培基处理, H2O2 组以 300 μmol/L 的 H2O2 处理, H2O2+RSV 组同时加入 300 μmol/L 的 H2O2 及 25 μmol/L 的 RSV 处理。检测各组上述蛋白和基因的表达情况。免疫荧光检测各组 NF-κB P65 在 HTMCs 中的定位。（3） HTMCs 细胞分为 3 组。对照组以不含 TGF-β2 及 RSV 的无血清培基处理, TGF-β2 组以 5 μg/L 的 TGF-β2 处理, TGF-β2+RSV 组为同时加入 5 μg/L 的 TGF-β2 及 25 μmol/L 的 RSV 处理。检测各组上述蛋白和基因的表达情况。结果 （1） 与对照组比较, 150、 300、 450、 800 μmol/L 组 FN 和 P-NF-κB P65 蛋白表达水平均增高, 300、 450、 800 μmol/L 组 COL1 蛋白和 IL-1β基因表达水平增高 （P < 0.05）, 其他指标比较差异均无统计学意义。（2） H2O2 组较对照组 FN、 COL1、 P-NF-κB P65 蛋白和 IL-1β基因表达水平均增高, 而 H2O2+RSV 组较 H2O2 组上述指标均降低, H2O2+RSV 组较对照组仅 IL-1β降低 （P < 0.05）。对照组仅细胞质表达 NF-κB P65, H2O2 组细胞胞质及核中均有 NF-κB P65 表达, 且核中表达较多; H2O2+RSV 组细胞胞质中表达 NF-κB P65 较核中多。（3） TGF-β2 组较对照组 FN、 COL1、 P-NF-κB P65 的蛋白和 IL-1β基因水平表达均增高 （P < 0.05）, TGF-β2+RSV 组较 TGF-β2 组上述指标均降低 （P < 0.05）。 结论 H2O2 和 TGF-β2 能上调 HTMCs 的 FN、 COL1、 P-NF-κB P65 蛋白及 IL-1β基因的表达, 可能参与青光眼的发生发展过程。RSV 能抑制H2O2和 TGF-β2对HTMCs 的影响, 对青光眼发挥一定的保护作用。     

益气养阴化浊通络方对高糖诱导小鼠肾足细胞自噬ATG5、Beclin-1及BNIP3表达的影响

目的:观察益气养阴化浊通络方对高糖诱导的小鼠肾足细胞自噬相关蛋白5(autophagy-related protein 5,ATG5)、B细胞淋巴瘤-2蛋白相互作用中心卷曲螺旋蛋白1(B-cell lymphoma-2-interacting myosin-like coiled-coil protein 1,Beclin-1)及B淋巴细胞瘤-2基因/腺病毒E1B相互作用蛋白3(B-cell lymphoma-2/adenovirus E1 B interacting protein 3,BNIP3)表达的影响,探讨其对自噬的作用。方法:选取Wistar大鼠,分别灌胃20,40,80 g·kg^-1益气养阴化浊通络方及生理盐水,制备低、中、高浓度含药血清和空白血清。体外培养小鼠肾足细胞,分为正常对照组、高糖组、益气养阴化浊通络方低、中、高剂量组和雷帕霉素组。CCK-8法检测细胞增殖,细胞划痕检测细胞迁移能力,qRT-PCR和Western blot检测微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3...     

去肾交感神经对2型糖尿病大鼠血管内皮细胞自噬及NLRP3活化的影响

目的 探讨去肾交感神经（RDN）对2型糖尿病（T2DM）大鼠血管内皮细胞自噬及核苷酸结合寡聚化结构域受体蛋白3（NLRP3）炎症小体活化的影响。方法 采用高脂饮食联合小剂量链脲佐菌素（STZ）腹腔注射制备T2DM大鼠模型。采用随机数字表法分为对照组（CON组）、糖尿病对照组（T2DM组）、双侧假手术组（Sham组）、双...     

金银花含药血清对脂多糖诱导的人牙周膜细胞活性及NLRP3/IL-1β通路的影响

目的 探讨金银花含药血清对脂多糖(LPS)诱导的人牙周膜细胞(HPDLCs)活性及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、白细胞介素1β(IL-1β)通路的影响。方法 将20只大鼠按随机数字表法分为对照组(生理盐水)和金银花组(5.0 g·kg^-1),每组10只,灌胃给药,1次/d,持续14 d。将HPDLCs分为对照组(空白血清培养)、LPS组(10μg·mL^-1 LPS+空白血清培养)、金银花低浓度(HPDLCs+10μg·mL^-1 LPS+75μL空白血清培养+75μL 5%金银花含药血清)、中浓度(HPDLCs+10μg·mL^-1 LPS+75μL空白血清培养+75μL 10%金银花含药血清)、高浓度(HPDLCs+10μg·mL^-1 LPS+75μL空白血清培养+75μL 20%金银花含药血清)组。MTT检测HPDLCs细胞增殖率,Transwell小室检测HPDLCs细胞迁移,流式细胞仪检测HPDLCs细胞凋亡率,PCR检测HPDLCs中IL-1β、NLR...     

基于mTOR/Beclin1/LC3信号轴探讨制萎扶胃丸对胃癌前病变大鼠胃窦组织自噬的影响

目的：探讨制萎扶胃丸（ZWFW）对胃癌前病变（PLGC）大鼠胃窦组织哺乳动物雷帕霉素靶蛋白（mTOR）/自噬关键分子酵母Atg6同系物（Beclin1）/微管相关蛋白1轻链3(LC3)信号轴关键分子表达的影响。方法：SPF级SD大鼠随机分为正常组,模型组,叶酸组,ZWFW低、中、高剂量组,除正常组常规饲养外,模型组、叶酸组、ZWFW低、中、高剂量组,采用N-甲基-N’-硝基-N-亚硝基胍（MNNG）联合饥饱失常、乙醇灌胃、氨水自由饮用以及雷尼替丁饲料喂养五因素复合造模法建立PLGC大鼠模型后,分别用生理盐水、叶酸片水溶液（0.002 g/kg）、ZWFW低、中、高剂量水溶液（0.42,0.84,1.67 g/kg）予以治疗4周后剖腹取胃。采用苏木素-伊红（HE）染色观察大鼠胃窦组织病理学变化,采用实时荧光定量聚合酶链式反应（Real-time PCR）、蛋白免疫印迹法（Western blot）及免疫组化检测大鼠胃窦组织mTOR、Beclin1、微管相关蛋白1轻链3β(LC3B)的mRNA及蛋白表达。结果：与正常组比较,模型组大鼠胃窦组织胀大,胃壁变薄,胃黏膜色泽苍白,皱襞萎缩浅平,走...     

盐酸纳美芬对心肌缺血再灌注大鼠缺氧诱导因子 1α/B细胞淋巴瘤 -2/E1B-19kDa相互作用蛋白 3通路及心肌自噬的影响

目的 探讨盐酸纳美芬对心肌缺血再灌注损伤(MIRI)大鼠缺氧诱导因子1α(Hif-1α)/B细胞淋巴瘤-2(Bcl-2)/E1B-19kDa相互作用蛋白3(BNIP3)通路及心肌自噬的影响.方法 2020年3—4月采用结扎冠状动脉左前降支法复制心肌缺血再灌注损伤(MIRI)大鼠模型,将造模成功的大鼠使用随机数字表法分成模型组、盐酸纳美芬低、中、高(10μg/kg、15μg/kg、20μg/kg)剂量组和氯喹组(阳性对照,0.02 g/kg),每组15只,另取15只假手术组大鼠作为对照.模型组和假手术组给予等剂量生理盐水,其余各组给予相对应剂量药物,每天1次,连续3 d.给药结束24 h后,氯化三苯基四氮唑(TTC)染色法检测心肌梗死面积;苏木精-伊红(HE)染色观察心肌组织病理学变化;二氯荧光素双醋酸盐(DCFH-DA)法检测心肌组织中活性氧水平;蛋白质印迹法(Western blotting)检测心肌组织中自噬相关蛋白Beclin1、LC3-Ⅰ、LC3-Ⅱ及通路蛋白Hif-1α、BNIP3表达水平.结果 假手术组大鼠心肌组织结构完整,无明显病理变化.与假手术组相比,模型组大鼠心肌组织结构异常、心肌细胞坏死、心肌损伤严重,心肌梗死面积、活性氧水平、Beclin1、LC3-Ⅱ、Hif-1α、BNIP3蛋白表达水平和LC3-Ⅱ/Ⅰ比值显著升高(P<0.05),其中Hif-1α和BNIP3蛋白表达水平分别为0.92±0.11和0.96±0.12,显著高于假手术组的0.10±0.02和0.08±0.01;与模型组相比,盐酸纳美芬低、中、高剂量组大鼠心肌组织病理损伤依次减轻,心肌梗死面积、活性氧水平、Beclin1、LC3-Ⅱ、Hif-1α、BNIP3蛋白表达水平和LC3-Ⅱ/Ⅰ比值依次降低(P<0.05),其中盐酸纳美芬高剂量组Hif-1α和BNIP3蛋白表达水平分别为0.17±0.03和0.12±0.03,显著低于盐酸纳美芬中剂量组的0.29±0.04和0.31±0.05,盐酸纳美芬中剂量组显著低于盐酸纳美芬低剂量的0.58±0.07和0.52±0.08.盐酸纳美芬高剂量组与氯喹组大鼠各项指标差异无统计学意义(P>0.05).结论 盐酸纳美芬可能通过抑制Hif-1α/BNIP3通路,减弱MIRI大鼠心肌自噬,缓解心肌损伤.     

山姜素对脂多糖诱导的结肠NCM460细胞损伤及对VDR/Nrf2/HO-1通路的作用研究

目的 研究山姜素对脂多糖诱导的结肠NCM460细胞损伤修复及对维生素D3受体（VDR）/核因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)通路的调节作用。方法 使用脂多糖建立NCM460细胞损伤模型,分别设置对照组,山姜素低、中、高剂量组及白藜芦醇组,山姜素低、中、高剂量组分别加入终浓度为25、50、100μmol/L的山姜素,白藜芦醇组加入终浓度为120μmol/L的白藜芦醇,继续培养72 h后,CCK-8法测定NCM460细胞增殖率,酶联免疫吸附法（ELISA）测定NCM460细胞上清液肿瘤坏死因子-α(TNF-α)、白细胞介素（IL）-17、IL-8、IL-6、IL-1β及NCM460细胞匀浆液超氧化物歧化酶（SOD）、一氧化氮（NO）、丙二醛（MDA）水平,流式细胞术测定NCM460细胞凋亡率,使用试剂盒测定NCM460细胞线粒体膜电位水平,实时定量聚合酶链式反应（RT-qPCR）测定NCM460细胞VDR、Nrf2及HO-1 m RNA水平,免疫印迹法（Western blotting）测定NCM460细胞VDR、Nrf2及HO-1蛋白水平。结果 与模型组比较,山...     

缬沙坦对颈动脉粥样硬化大鼠AT1R/PI3K/Akt/mTOR通路及血管内皮细胞自噬的影响

目的 探讨缬沙坦对颈动脉粥样硬化大鼠血管紧张素Ⅱ1型受体(AT1R)/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)通路及血管内皮细胞自噬的影响.方法 采用高脂饲料饲喂结合颈动脉球囊损伤法制备颈动脉粥样硬化大鼠模型,随机分为模型组(生理盐水),缬沙坦低、中、高(10、20、30 ...     

The adaptor Lnk (SH2B3): an emerging regulator in vascular cells and a link between immune and inflammatory signaling

A better knowledge of the process by which inflammatory extracellular signals are relayed from the plasma membrane to specific intracellular sites is a key step to understand how inflammation develops and how it is regulated. This review focuses on Lnk (SH2B3) a member, with SH2B1 and SH2B2, of the SH2B family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase and receptor tyrosine kinases. SH2B adaptor proteins contain conserved dimerization, pleckstrin homology, and SH2 domains. Initially described as a regulator of hematopoiesis and lymphocyte differentiation, Lnk now emerges as a key regulator in hematopoeitic and non hematopoeitic cells such as endothelial cells (EC) moderating growth factor and cytokine receptor-mediated signaling. In EC, Lnk is a negative regulator of TNF signaling that reduce proinflammatory phenotype and prevent EC from apoptosis. Lnk is a modulator in integrin signaling and actin cytoskeleton organization in both platelets and EC with an impact on cell adhesion, migration and thrombosis. In this review, we discuss some recent insights proposing Lnk as a key regulator of bone marrow-endothelial progenitor cell kinetics, including the ability to cell growth, endothelial commitment, mobilization, and recruitment for vascular regeneration. Finally, novel findings also provided evidences that mutations in Lnk gene are strongly linked to myeloproliferative disorders but also autoimmune and inflammatory syndromes where both immune and vascular cells display a role. Overall, these studies emphasize the importance of the Lnk adaptor molecule not only as prognostic marker but also as potential therapeutic target.     

Enzymatic and non-enzymatic activities of SHIP-1 in signal transduction and cancer

PI3K cascade is a central signaling pathway regulating cell proliferation, growth, differentiation, and survival. Tight regulation of the PI3K signaling pathway is necessary to avoid aberrant cell proliferation and cancer development. Together with SHIP-1, the inositol phosphatases PTEN and SHIP-2 are the gatekeepers of this pathway. In this review, we will focus on SHIP-1 functions. Negative regulation of immune cell activation by SHIP-1 is well characterized. Besides its catalytic activity, SHIP-1 also displays non-enzymatic activity playing role in several immune pathways. Indeed, SHIP-1 exhibits several domains that mediate protein-protein interaction. This review emphasizes the negative regulation of immune cell activation by SHIP-1 that is mediated by its protein-protein interaction.     

Inhibition of PI3K and calcineurin suppresses chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)-dependent responses of Th2 lymphocytes to prostaglandin D(2)

Interaction of prostaglandin D2 (PGD2) with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) triggers chemotaxis and pro-inflammatory cytokine production by Th2 lymphocytes. We have investigated the role of inhibitors of various cell-signalling pathways on the responses of human CRTH2+ CD4+ Th2 cells to PGD2. Phosphatidylinositol 3-kinase (PI3K) and Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) pathways were activated by PGD2 in Th2 cells in a CRTH2-dependent manner. Inhibition of the PI3K pathway with LY294002 significantly reduced both PGD2-induced cell migration and cytokine (interleukin-4, interleukin-5 and interleukin-13) production. The inhibitory effect of LY294002 on cell migration is likely to be related to cytoskeleton reorganization as it showed a similar potency on PGD2-induced actin polymerization. The calcineurin inhibitors, tacrolimus (FK506) and cyclosporin A, had no effect on cell migration but completely blocked both cytokine production and the nuclear translocation of NFATc1 suggesting that Ca2+/calcineurin/NFAT is involved in CRTH2-dependent cytokine production but not chemotaxis. The promotion of NFAT nuclear location by PI3K activation may be mediated by negative regulation of glycogen synthase kinase-3beta (GSK3beta), since the PGD2-stimulated increase in phospho-GSK3beta was down-regulated by LY294002, and inhibition of GSK3beta by SB216763 enhanced PGD2-induced Th2 cytokine production and reversed the inhibitory effect of LY294002. These data suggest that PI3K and Ca2+/calcineurin/NFAT signalling pathways are critically involved in pro-inflammatory responses of Th2 cells to PGD2.     

The on-off action of Forkhead protein O3a in endotoxin tolerance of Kupffer cells depends on the PI3K/AKT pathway

BackgroundThe endotoxin tolerance (ET) of Kupffer cells (KCs) is an important protective mechanism for limiting endotoxin shock. As a key anti-inflammatory molecule, the roles and mechanism of Forkhead protein O3a (Foxo3a) in ET of KCs are not yet well understood.MethodsET and nonendotoxin tolerance (NET) KCs models were established in vitro and in vivo. The levels of cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression and phosphorylation levels were detected by western blotting (WB). Changes in the localization of nuclear factor kappa B (NF-κB) and Foxo3a in KCs were detected by immunofluorescence assays. KCs apoptosis and survival rates were detected by flow cytometry and an automatic cell counter, respectively.ResultsThe activity of NF-κB and the levels of p-Foxo3a and tumor necrosis factor (TNF-α) in the ET group were significantly lower than those in the NET group, while the levels of Foxo3a and interleukin 10 (IL-10) in the ET group were significantly higher than those in the NET group. Overexpression of Foxo3a or the use of a phosphatidylinositol-3-hydroxykinase (PI3K) inhibitor suppressed the activation of NF-κB by decreasing the levels of p-Foxo3a by inhibiting the activity of PI3K/AKT, which improved the tolerance of KCs and mice to endotoxin. In contrast, silencing Foxo3a or the use of a PI3K agonist reduced the tolerance of KCs and mice to endotoxin. The PI3K agonist counteracted the inhibitory effects of Foxo3a overexpression on NF-κB, impairing the tolerance of KCs to endotoxin.ConclusionsThe on-off action of Foxo3a in the ET of KCs depends on the PI3K/AKT pathway.