目的探讨烙灸治疗早期激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)的作用机制。方法将新西兰兔按随机数表法分为空白组、模型组、烙灸组、抑制剂组,每组6只,采用内毒素联合激素的方法制备SONFH模型。...目的探讨烙灸治疗早期激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)的作用机制。方法将新西兰兔按随机数表法分为空白组、模型组、烙灸组、抑制剂组,每组6只,采用内毒素联合激素的方法制备SONFH模型。烙灸组进行4周的烙灸治疗,抑制剂组给予IκBα磷酸化抑制剂Bay11-7082(1 mg·kg-1)腹腔注射3周。干预前后分别观察实验兔一般状况变化;HE染色观察各组实验兔股骨头组织病理学变化;Western blot和RT-qPCR分别检测股骨头组织中IKKβ、磷酸化IκBα(p-IκBα)蛋白及mRNA的表达变化;ELISA检测血清中核因子κB(nuclear factor kappa-B,NF-κB)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)水平。结果与空白组比较,模型组实验兔逐渐出现精神萎靡、毛发暗淡等情况,股骨头组织骨小梁出现断裂、稀疏等改变,IKKβ、p-IκBα蛋白水平,IKKβmRNA水平及血清中NF-κB、TNF-α、IL-6水平均升高(P均<0.05);与模型组比较,烙灸组股骨头组织病理学改善,IKKβ、p-IκBα蛋白水平,IKKβmRNA水平及血清中NF-κB、TNF-α、IL-6水平均降低(P均<0.05),而IκBα蛋白及mRNA表达均升高(P均<0.05)。结论烙灸可改善早期SONFH骨代谢,缓解骨坏死,其机制与抑制TLR4/NF-κB通路有关。展开更多
The von Hippel-Lindau tumor suppressor protein(VHL),an E3 ubiquitin ligase,functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors.Recent evidence suggests that mammalia...The von Hippel-Lindau tumor suppressor protein(VHL),an E3 ubiquitin ligase,functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors.Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway,although the specific molecular mechanisms remain unclear.Herein,the roles of mandarin fish(Siniperca chuatsi)VHL(scVHL)in the NF-κB signaling pathway and mandarin fish ranavirus(MRV)replication were explored.The transcription of scVHL was induced by immune stimulation and MRV infection,indicating a potential role in innate immunity.Dual-luciferase reporter gene assays and reverse transcription quantitative PCR(RT-qPCR)results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway.Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα,scIKKβ,scIκBα,or scp65.Co-immunoprecipitation(Co-IP)analysis identified scIκBαas a novel target protein of scVHL.Moreover,scVHL targeted scIκBαto catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway.Following MRV infection,NF-κB signaling remained activated,which,in turn,promoted MRV replication.These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication.This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.展开更多
前期研究结果表明,乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)具有促进细胞增殖的作用.为了进一步阐明其分子机制,观察了HBXIP对核因子κB(NF-κB)转录活性的影响.实验中通过基因共转染将NF-κB报告基...前期研究结果表明,乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)具有促进细胞增殖的作用.为了进一步阐明其分子机制,观察了HBXIP对核因子κB(NF-κB)转录活性的影响.实验中通过基因共转染将NF-κB报告基因质粒pNF-κB-Luc和HBXIP真核表达载体pcDNA3-hbxip导入人肝癌H7402细胞系中,进行荧光素酶活性分析.结果显示:H7402细胞过表达HBXIP后NF-κB的转录活性明显增强;此外,基因转染后经免疫印迹检测显示,与NF-κB二聚体结合的抑制亚基IκBα的磷酸化水平明显增加;同时,提取H7402细胞的核蛋白,然后应用免疫印迹检测细胞核中p65/NF-κB的水平.结果显示,H7402细胞中HBXIP过表达后细胞核中p65/NF-κB的水平明显增加.当应用RNA干扰技术抑制了细胞内源性的HBXIP基因表达后,则出现与上述结果相反的效果.上述结果提示,HBXIP可增加核内p65/NF-κB蛋白水平,进而发挥NF-κB促转录调控的作用.因此,HBXIP可通过调控NF-κB信号途径而促进细胞增殖.展开更多
基金supported by the National Key Research and Development Program of China(2022YFE0203900)Guangdong Key Research and Development Program(2021B0202040002 and 2022B1111030001)+4 种基金China Agriculture Research System(CARS-46)Guangdong Basic and Applied Basic Research Foundation(2021A1515010647)Basic and Applied Basic Research Project of Guangzhou Science and Technology Plan Project(202102020299)Science and Technology Planning Project of Guangdong(2023B1212060023)Guangdong Laboratory for Lingnan Modern Agriculture(NZ2021018)。
文摘The von Hippel-Lindau tumor suppressor protein(VHL),an E3 ubiquitin ligase,functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors.Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway,although the specific molecular mechanisms remain unclear.Herein,the roles of mandarin fish(Siniperca chuatsi)VHL(scVHL)in the NF-κB signaling pathway and mandarin fish ranavirus(MRV)replication were explored.The transcription of scVHL was induced by immune stimulation and MRV infection,indicating a potential role in innate immunity.Dual-luciferase reporter gene assays and reverse transcription quantitative PCR(RT-qPCR)results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway.Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα,scIKKβ,scIκBα,or scp65.Co-immunoprecipitation(Co-IP)analysis identified scIκBαas a novel target protein of scVHL.Moreover,scVHL targeted scIκBαto catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway.Following MRV infection,NF-κB signaling remained activated,which,in turn,promoted MRV replication.These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication.This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.
文摘目的研究鳖甲煎丸(鳖甲胶、阿胶、蜂房等)对大鼠肝星状细胞(HSC-T6)中核转录因子-κB(NF-κB)信号通路中NF-κB、p65、p50、IκB及靶基因表达的影响。方法使用鳖甲煎丸药物血清培养HSC-T6细胞24 h后,采用q PCR检测p65、p50、血管内皮生长因子(VEGF)、基质金属蛋白酶抑制因子-1(TIMP-1)m RNA的表达,免疫荧光法检测p65的表达,Western blot检测NF-κB抑制蛋白α(IκBα)、NF-κB抑制蛋白β(IκBβ)及α平滑肌肌动蛋白(α-SMA)的表达。结果与空白对照组及阴性对照组相比,鳖甲煎丸的中、高剂量组及阳性对照组p65、VEGF、TIMP-1 m RNA的表达显著降低,各组间p50 m RNA的表达无显著差异,但免疫荧光显示p65在细胞质中的表达降低。同时,鳖甲煎丸能显著上调IκBα的蛋白表达,并能明显下调α-SMA的表达,具有剂量依赖性,但对IκBβ的表达无显著影响。结论鳖甲煎丸对肝纤维化的治疗作用可能与影响NF-κB信号通路、抑制下游靶基因的表达有关。
文摘前期研究结果表明,乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)具有促进细胞增殖的作用.为了进一步阐明其分子机制,观察了HBXIP对核因子κB(NF-κB)转录活性的影响.实验中通过基因共转染将NF-κB报告基因质粒pNF-κB-Luc和HBXIP真核表达载体pcDNA3-hbxip导入人肝癌H7402细胞系中,进行荧光素酶活性分析.结果显示:H7402细胞过表达HBXIP后NF-κB的转录活性明显增强;此外,基因转染后经免疫印迹检测显示,与NF-κB二聚体结合的抑制亚基IκBα的磷酸化水平明显增加;同时,提取H7402细胞的核蛋白,然后应用免疫印迹检测细胞核中p65/NF-κB的水平.结果显示,H7402细胞中HBXIP过表达后细胞核中p65/NF-κB的水平明显增加.当应用RNA干扰技术抑制了细胞内源性的HBXIP基因表达后,则出现与上述结果相反的效果.上述结果提示,HBXIP可增加核内p65/NF-κB蛋白水平,进而发挥NF-κB促转录调控的作用.因此,HBXIP可通过调控NF-κB信号途径而促进细胞增殖.
