基于“通肾络、益脾肾”治法研究足细胞凋亡及自噬

[目的]基于中医"通肾络、益脾肾"治法,探讨通络益肾方对体外高糖培养的小鼠肾小球足细胞自噬和凋亡蛋白SIRT1、BNIP3、P62、Bax表达的调控影响。[方法]成熟无特定病原体(SPF)级SD雄性大鼠40只,随机分为正常组、中药高、中、低剂量组各10只。按照人与大鼠体表面积折算方法计算出大鼠所需中药灌胃浓度:高剂量浓度4.76 g/mL、中剂量浓度2.38 g/mL、低剂量浓度1.19 g/m L,灌胃10 d取含药血清备用。足细胞分6组,正常组5.6 mmol/L葡萄糖+10%正常大鼠血清、高糖组30mmol/L葡萄糖+10%正常大鼠血清、通络益肾方含药血清高、中、低干预组30 mmol/L葡萄糖+10%高、中、低剂量大鼠血清、高渗组甘露醇24.5 mmol/L+10%正常大鼠血清。细胞培养48 h后收集,Hoechst33342荧光染色,观察各组足细胞凋亡状况及形态变化;流式细胞仪检测足细胞凋亡率;Western Blot检测细胞内自噬标志蛋白SIRT1、P62及促凋亡蛋白BNIP3、Bax表达水平。[结果]流式细胞仪检测结果显示通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率(P0.01或P0.05),高剂量组不能改善凋亡情况(P0.05);Hoechst33342荧光染色观察结果也证实通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率;蛋白印迹结果显示,与高糖组相比,通络益肾方中、低剂量组自噬标志蛋白SIRT1表达升高,自噬标志蛋白P62及促凋亡蛋白BNIP3、Bax蛋白表达下降(P0.05或P0.01),高剂量组SIRT1、BNIP3、P62、Bax蛋白表达未见明显改变(P0.05)。[结论]中剂量、低剂量通络益肾方能够启动细胞自噬减少高糖刺激下体外培养足细胞凋亡,降低细胞凋亡率及凋亡蛋白的表达。     

Necroptotic TNFα-Syndecan 4-TNFα Vicious Cycle as a Therapeutic Target for Preventing Temporomandibular Joint Osteoarthritis

Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative disease for which the underlying mechanism still remains unclear. Compared with apoptosis and autophagy, necroptosis causes greater harm to tissue homeostasis by releasing damage-associated molecular patterns (DAMPs). However, the role of necroptosis and downstream key DAMPs in TMJOA is unknown. Here, rodent models of TMJOA were established by the unilateral anterior crossbite (UAC). Transmission electron microscopy (TEM) and immunohistochemistry of receptor interacting protein kinase 3 (RIPK3)/phosphorylation of mixed lineage kinase domain-like protein (pMLKL) were conducted to evaluate the occurrence of necroptosis in vivo. The therapeutic effects of blocking necroptosis were achieved by intra-articularly injecting RIPK3 or MLKL inhibitors and using RIPK3 or MLKL knockout mice. In vitro necroptosis of condylar chondrocyte was induced by combination of tumor necrosis factor alpha (TNFα), second mitochondria-derived activator of caspases (SMAC) mimetics and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (z-VAD-fmk). The possible DAMPs released by necroptotic chondrocytes were screened by quantitative proteomics and blocked by specific antibody. Translucent cytosol, swollen organelles, and ruptured cell membranes, features of necroptosis, were frequently manifested in chondrocytes at the early stage of condylar cartilage degeneration in TMJOA, which was accompanied by upregulation of RIPK3/pMLKL. Inhibiting or knocking out RIPK3/MLKL significantly prevented cartilage degeneration. DAMPs released by necroptotic condylar chondrocytes, such as syndecan 4 (SDC4) and heat shock protein 90 (HSP90), were verified. Furthermore, blocking the function of SDC4 significantly attenuated the expression of TNFα in cartilage and synovium, and accordingly increased cartilage thickness and reduced synovial inflammation. Thus, the necroptotic vicious cycle of TNFα-SDC4-TNFα contributes to cartilage degeneration and synovitis, and can serve as a potential therapeutic target for treating TMJOA. © 2022 American Society for Bone and Mineral Research (ASBMR).     

构建数字化、可视化教学平台实现妇产科学教学手段多元化

介绍了构建数字化、可视化教学平台,实现妇产科教学手段多元化的做法。内容包括多媒体课件开发制作,利用医学视听教材,建设妇产科图片数据库,建设妇产科标本库(馆),利用模型、模块进行模拟操作,加强实践技能培养和通过临床见习、实习将理论应用于实践等问题。     

乌蛇败毒胶囊对接触性皮炎小鼠血清IL-2、TNF-α的影响

目的探讨乌蛇败毒胶囊治疗湿疹的机理。方法采用变应性接触性皮炎小鼠模型,分为模型组,对照组及大、中、小剂量组和空白组,观察各组治疗前后血清IL-2、TNF-α的变化。结果中药组能明显降低模型小鼠血清中IL-2、TNF-α的含量(P<0.01),降低诱发后鼠耳的肿胀度,减少真皮内单一核及多形核白细胞数目(P<0.01)。结论乌蛇败毒胶囊治疗湿疹的部分机理是抑制小鼠Ⅳ型变态反应、降低血清IL-2,TNF-α含量,减轻炎症损伤。     

金茵利胆胶囊的体外抗炎活性评价及其谱效关系研究

目的 评价金茵利胆胶囊的体外抗炎活性,并研究其抗炎谱效关系。方法 建立LPS诱导的RAW264.7细胞炎症模型,通过检测细胞上清中NO、TNF-α和IL-6等指标的含量来评价金茵利胆胶囊的抗炎活性。在此基础上,采用均匀设计法制备金茵利胆胶囊组成饮片的不同配伍样本,采用高效液相法建立均匀设计样品的指纹图谱,对均匀设计样品进行抗炎活性测定;以细胞上清液中NO、TNF-α和IL-6的抑制率为药效指标,与共有峰峰面积经灰色关联分析构建抗炎谱效关系。结果 金茵利胆胶囊提取物（0.25~1.00 mg/mL）能显著降低LPS诱导的RAW264.7细胞分泌NO、TNF-α和IL-6。经对照品比对指认出其中7个色谱峰,分别为新绿原酸（9号峰）、绿原酸（13号峰）、隐绿原酸（14号峰）、1, 3-二咖啡酰奎宁酸（16号峰）、对羟基苯乙酮（17 号峰）、柚皮苷（21 号峰）、新橙皮苷（23 号峰）。抗炎谱效关系结果表明,共有峰均具有一定的抗炎贡献度,其中共有峰峰21（柚皮苷）、峰23（新橙皮苷）与3个细胞炎症指标的关联度均大于0.74。结论：金茵利胆胶囊发挥抗炎作用是多种成分共同作用的结果,为金茵利胆胶囊抗炎药效物质基础及其质量控制提升提供参考。