黄芩苷对脂多糖致内皮细胞炎症反应的保护作用及机制研究

目的研究黄芩苷对脂多糖(LPS)致人微血管内皮细胞(HMEC)炎症反应的保护作用及作用机制。方法采用LPS作用HMEC,建立炎症反应模型;以不同浓度的黄芩苷预处理细胞,然后将细胞暴露于LPS,ELISA检测炎症因子ICAM-1、IL-6和MCP-1的含量;荧光观察Ca^2+内流并采用流式细胞仪检测细胞内Ca^2+水平;免疫荧光法检测NF-κB p65入核情况;双荧光素酶报告基因方法检测NF-κB核内转录活性;Western blot检测NF-κB p65、p-NF-κB p65及TLR4的表达。结果HMEC暴露于LPS后,出现了明显的Ca^2+内流,NF-κB p65发生磷酸化,核内转录活性上调,ICAM-1、IL-6及MCP-1表达上调。不同浓度的黄芩苷均能抑制LPS刺激HMEC后产生的Ca^2+内流、NF-κB p65入核及核内转录活性上调,减少ICAM-1、IL-6、MCP-1的表达,下调NF-κB p65、p-NF-κB p65及TLR4的表达,且抑制作用呈现一定的量效关系。结论黄芩苷能抑制LPS诱导的HMEC炎症反应,其机制与抑制Ca^2+内流和NF-κB信号通路的活化,从而降低炎症反应的水平有关。     

基于高内涵分析技术研究山萘酚的人肾近曲小管细胞HK-2毒性及机制

目的 基于高内涵分析技术探究山萘酚的肾细胞毒性作用及机制。方法 人肾近曲小管细胞HK-2分为细胞对照组(二甲亚砜终浓度<0.01%)、山萘酚5,10,20,50和100μmol·L^-1组及多柔比星(Dox)10μmol·L^-1组。Hoechst 33342染色和噻唑蓝(MTT)比色法分别检测活细胞数目和细胞存活率;荧光探针DCFH-DA,Mito-Tracker Red CMXRos和Fluo-4 AM分别检测活性氧(ROS)、线粒体膜电位(MMP)和Ca²⁺内流水平;化学发光法检测细胞ATP含量;AnnexinⅤ/PI双染法检测细胞凋亡率;Hoechst 33342染色检测DNA含量;免疫荧光法检测NF-κB p65入核情况,以及丝裂原活化蛋白激酶(MAPK)通路蛋白[p38MAPK、磷酸化p38 MAPK(p-p38 MAPK)、c-Jun N端激酶(JNK)、p-JNK、细胞外信号调节激酶(ERK)和p-ERK]和酪氨酸激酶2(JAK2)/信号转导与转录激活因子3(STAT3)通路蛋白(p-STAT3和STA...     

Effect of phillygenin on inflammatory response of A549 cells induced by lipopolysaccharide and normal human plasma北大核心CSCD

Aim To investigate the effect of phillygenin ( PHI) on lipopolysacchride ( LPS) and normal human plasma ( NHP) induced inflammatory injury on alveolar type II epithelial A549 cells and the related mechanism. Methods A549 cells were exposured to 1 mg • L -1 of LPS and 5% NHP to build the inflammatory injury model. The effects of PHI on cell viability, cell number, area, morphology, and DNA content of A549 cells were detected by thiazole blue colorimetric (MTT) and Hoechst 33342 staining. The A549 cells were pretreated with PHI for 2 h, and then exposed to 1 mg • L -1 of LPS and 5% NHP. The contents of inflammatory cytokines and NF-κB p65 were determined by ELISA and quantitative real-time PCR. The transfer of NF-κB p65 from cytoplasm to nucleus was detected by cellular immunofluorescence. The protein expres¬sions of NF-κB and MAPK signaling pathways were de¬tected by Western blot. Results The contents of IL-6 and IL-8 and the transfer of NF-κB p65 from cytoplasm to nucleus were significantly up-regulated in LPS combined with NHP co-stimulated group. PHI at 1, 10, 50, and 100 jjimol • L -1decreased the mRNA and protein expressions of IL-6 and IL-8 , inhibited the transfer of NF-κB p65 from cytoplasm to nucleus, reduced the NF-κB p65 mRNA level, down-regulated the phospho-rylation levels of IκBα, NF-κB p65, p38, JNK, and ERK, and up-regulated the protein expression of IκBα in a dose-dependent manner. Conclusions LPS combined with NHP successfully induces inflammatory injury in alveolar type II epithelial A549 cells. PHI could improve the inflammatory response by inhibiting the activation of LPS-TLR4-NF-κB/MAPK signaling pathways. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

原矛头蝮蛇毒及其组分对凝血功能的影响

目的研究原矛头蝮蛇毒(PMV)及其组分作用于血液循环系统的部分生物学活性,进一步探讨其毒性机制。方法采用Sephadex G-75凝胶层析方法分离不同分子质量范围的蛋白组分FⅠ,FⅡ和FⅢ。贫血小板血浆调节富血小板血浆使血小板为3×1011L-1,血小板悬液分别与PMV,FⅠ,FⅡ和FⅢ0.03 g·L-1预孵育5 min,血小板聚集仪测定PMV及其组分诱导血小板聚集活性;PMV,FⅠ,FⅡ和FⅢ0.05 g·L-1分别与纤溶酶原0.1 U·L-1预孵育10 min,采用单一时间点测定和酶动力学测定PMV及其组分酶切发色底物S-2251的作用;将大鼠血浆与PMV,FⅠ,FⅡ和FⅢ1.0 g·L-1分别孵育5和30 min,测定凝血酶时间(TT)、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和纤维蛋白原(FIB)水平;PMV,FⅠ,FⅡ和FⅢ10,50和250 mg·L-1处理微血管内皮细胞24 h,倒置相差显微镜观察细胞形态,MTT法检测细胞存活;PMV,FⅠ,FⅡ和FⅢ0.1 g·L-1与含卵磷脂和不含卵磷脂的豚鼠红细胞悬液分别孵育不同时间,计算溶血率。结果与对照组相比,PMV和高分子质量蛋白组分FⅠ(>71 ku)诱导血小板聚集率显著升高〔(61.0±5.8)%和(56.9±5.9)%vs(12.4±4.1)%〕(P<0.01)。酶切发色底物S-2251结果显示,PMV与中分子质量组分FⅡ(18~37 ku)具有酶切发色底物的作用(P<0.01)。PMV和FⅠ可引起血浆凝固。与对照组相比,FⅡ组分和低分子质量蛋白组分FⅢ(<10 ku)明显使TT,APTT和PT的时间延长(P<0.01)。PMV,FⅠ及FⅡ导致内皮细胞解离悬浮,与对照组相比,细胞存活率下降,分别为(56.8±3.6)%,(71.6±3.8)%和(58.2±5.5)%。在无卵磷脂条件下,PMV和FⅡ可缓慢地引起红细胞轻度溶血;在卵磷脂参与下,PMV和FⅡ引起豚鼠红细胞急剧溶血,与正常对照组相比,在0.5 min内溶血率从(17.7±1.0)%分别升高至(81.0±4.0)%和(81.0±1.0)%(P<0.01)。结论 PMV在体外表现出多方面的血循毒性质的活性,不同分子质量蛋白组分活性机制及作用不同。     

补体旁路激活致血管平滑肌细胞增殖活化及干预研究

目的研究补体旁路途径激活诱导人血管平滑肌细胞(VSMC)增殖活化及其干预作用。方法采用CVF特异激活血浆补体旁路途经。将VSMC与补体旁路激活产物共同孵育,采用倒置相差显微镜和MTT法分别检测细胞形态变化和增殖活性,ELISA法检测培养上清中E-selectin、ICAM-1和VCAM-1含量,Western blot检测p-NF-κB p65、IKK和NF-κB p65蛋白表达水平,双荧光素酶报告基因检测试剂盒检测NF-κB p65转录活性,采用PDTC对上述指标的变化进行干预。结果补体旁路激活导致VSMC增殖活化,上调表达E-selectin、ICAM-1和VCAM-1,其中ICAM-1和VCAM-1含量在6 h达到高峰,E-selectin在12 h出现明显上调;VSMC经补体旁路激活产物刺激后,NF-κB p65磷酸化明显增加,NF-κB p65核内转录活性升高,IKK和NF-κB p65蛋白表达上调,PDTC对上述相关指标的变化有明确的干预作用。结论补体旁路激活可导致VSMC增殖活化,其机制与NF-κB信号通路活化有密切关系,且NF-κB抑制剂PDTC对其增殖活化具有明显的干预作用。