产品介绍
酪酰胺信号放大技术（Tyramide Signal Amplification, TSA）主要是利用酪胺的过氧化物酶反应。酪胺非活性荧光素底物在HRP和过氧化氢的作用下，会被激活产生活化荧光底物，同时形成共价键结合位点，共价结合在蛋白抗原表面或附近的酪氨酸残基上，抗原和抗体的结合部位就会有大量的酪胺荧光素沉积，使抗原位点处的荧光信号增强。
酪胺荧光素底物-抗原酪氨酸共价稳定结合，故TSA信号不会受微波影响，可用热修复法清除第一轮与抗原非共价结合的抗体复合物(冰冻切片，细胞爬片样本请试用抗体洗脱液洗脱法清除)，并能在抗体去除后保留与抗原相关的荧光信号。然后，再用第二种一抗进行第二轮孵育，同时更换另一种酪胺荧光素底物，多次循环反复，不同的酪胺荧光素进行标记就可实现多重免疫组化染色。
TSA原理示意图
产品组成成分
单标双色 | 10T | 50T | 100T | 保存条件 |
TSA单标荧光染料 | 0.5 ml | 2.5 ml | 5 ml | -20℃，避光 |
内源性过氧化物酶阻断剂 | 0.5 ml | 2.5 ml | 5 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 0.5 ml | 2.5 ml | 5 ml | 4℃，避光 |
双标三色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 4 ml | 15 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 2 ml | 10 ml | 4℃，避光 |
三标四色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 5 ml | 25 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 3 ml | 15 ml | 4℃，避光 |
四标五色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-620 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 6 ml | 30 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 4 ml | 20ml | 4℃，避光 |
五标六色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-620 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-780 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 8 ml | 35 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 5 ml | 25 ml | 4℃，避光 |
六标七色 | 20T | 100T | 保存条件 |
TSA-480 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-620 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-780 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 10 ml | 45ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 6 ml | 30 ml | 4℃，避光 |
保存条件
1、荧光染料（TSA-480/520/570/620/690/780）： -20℃避光保存，12个月
2、内源性过氧化物酶阻断剂、超敏酶标山羊抗小鼠/兔 IgG 聚合物 ：4℃避光保存，有效期 12 个月。
3、抗体洗脱液 ：RT保存，有效期 12 个月。
实验前材料准备
1、石蜡或冰冻切片，细胞爬片。建议配合 Abiowell 带有 IF-T 应用的一抗使用。
石蜡切片：建议一抗应用中注明 IHC-P/IF-T；
冰冻切片：建议一抗应用中注明 IHC-F/IF-T；
细胞爬片：建议一抗应用中注明 IF/ICC/IF-C；
2、缓冲液与修复液：PBS 缓冲液（货号：AWC0215）、柠檬酸钠抗原修复液（货号：AWI0206）（或其他适配修复液）。
3、DAPI 染色液（推荐浓度：5μg/mL 货号：AWC0293）
4、其他辅助材料：组化笔、避光湿盒、慢速摇床、抗荧光淬灭封片剂（货号：AWI0197）等。
5、封闭山羊血清（货号：AWI0115）
操作步骤
（一）操作前注意事项
1、TSA 染色灵敏度高于常规的荧光显色，建议开展正式实验前，进行单色预实验，选择最适合的一抗稀释比例；
2、在进行多重染色时，通常建议先标记预实验中阳性丰度低的抗体。
3、针对四标染色，推荐的染色顺序为：TSA-520 → TSA-570 → TSA-690 → TSA-620。
4、由于 TSA-570 与 TSA-620 染料的发射波长较为接近，使用宽带通成像设备易出现信号串色，若需同时使用，需注意：
*建议将 TSA-570 与 TSA-620 搭配弱阳性一抗使用；
*尽量避免将其用于同一细胞中共同表达的靶标（例如 CD3 与 CD4 的共染组合）；
*图像采集时，请务必使用配备窄带通滤光片的成像设备，以有效区分相邻通道信号，确保成像质量。
5、加入荧光染料及后续步骤需进行避光操作。
6、操作过程中需保持载玻片组织的湿润，如出现干片情况，会导致非特异性的染色结果。
7、持续加热修复的过程中，只需中小火维持沸腾，切勿高火加热让容器内试剂蒸发导致干片，且高火易导致组织脱片。
8、使用小鼠组织实验时，建议选用兔源一抗搭配 HRP 标记的抗兔二抗，可减少非特异性染色。
9、对富含脂肪的组织（如肝脏、乳腺等）染色时，建议在第 11 步 DAPI 染色前增加苏丹黑处理步骤，可有效消除脂肪的自发荧光及非特异性着色，避免干扰目标抗原信号检测，提升目标信号清晰度与检测准确性。
（二）石蜡切片操作步骤（四标为例）
1、脱蜡复水：
(1)将石蜡玻片在 60 ℃ 烘烤 1-2 小时。
(2)依次将切片放入二甲苯Ⅰ 15 分钟→二甲苯Ⅱ 15 分钟→二甲苯Ⅱ 15 分钟→无水乙醇Ⅰ 10分钟→无水乙醇Ⅱ 10分钟→ 95% 乙醇 5 分钟→ 85% 乙醇 5 分钟→ 75% 乙醇 5 分钟→蒸馏水洗。
2、抗原修复：
(1)向容器中加入约 2/3 体积的柠檬酸修复液或 EDTA 修复液，加盖后用微波炉高火加热至沸腾（修复液及条件需根据组织、抗原类型调整）；
(2)取出容器，待液体停止沸腾后，将切片间隔插入切片架并放入容器；
(3)容器放回微波炉，低火加热 20 分钟，加热结束后在微波炉内静置 20 分钟后，取出自然冷却；
(4)将玻片转移至 PH7.4 的 PBS 中，用慢速摇床洗涤 3 次，每次 5 分钟；
(5)擦干组织周围水分（保持组织湿润），用组化笔在组织周围画封闭圈。
3、内源性过氧化物酶阻断：
向组织滴加内源性过氧化物酶阻断剂，在湿盒内室温孵育 10-15 分钟；随后将切片放入 PBS中，用慢速摇床洗涤 3 次，每次 3 分钟。
4、封闭：
用正常山羊血清均匀覆盖组织，室温封闭30min。
5、一抗孵育：
沥干玻片（约几秒），稀释一抗后滴加至完全覆盖组织；室温或 37 ℃ 孵育 3-4 小时，或 4 ℃ 湿盒过夜后 37 ℃ 复温 1-2 小时；最后用 PBST 冲洗 3 次，每次 2分钟。
6、二抗孵育：
沥干玻片，滴加 50-100μL 超敏酶标山羊抗小鼠/兔 IgG聚合物（以覆盖组织为宜），室温孵育 30 分钟；用 PBST 冲洗 3 次，每次 2 分钟。
7、TSA 反应：
再次沥干玻片，滴加 50-100μL TSA-520 荧光染料（覆盖组织即可），孵育 5-15 分钟；PBST冲洗 3 次，每次 2分钟。
8、抗体洗脱：
将切片间隔插入切片架，放入含煮沸修复液的容器（煮沸修复液需能没过组织）；微波炉低火加热 20 分钟后，在微波炉内静置 20 分钟，取出自然冷却；将玻片转移至 PH7.4 的 PBS 中，慢速摇床洗涤 3 次，每次 5 分钟。
9、第二轮染色：
重复步骤 4-8，一抗更换为第二种目标一抗，TSA荧光染料换为 TSA-570。
10、第三轮染色：
重复步骤 4-8，一抗更换为第三种目标一抗，TSA荧光染料换为 TSA-690。
11、第四轮染色：
重复步骤 4-7（无需抗体洗脱），一抗更换为第四种目标一抗，TSA荧光染料换为 TSA-620。
（注：多重免疫荧光染色的最后一轮均无需抗体洗脱）
12、DAPI 复染细胞核：
将玻片放入 PH7.4 的 PBS 中，慢速摇床洗涤 3 次，每次 5分钟；稍甩干后，向封闭圈内滴加DAPI 染液，避光室温孵育 10 分钟。
13、封片：
PBS 洗涤 3 次，每次 5 分钟；稍甩干切片，用抗荧光淬灭封片剂封片。
14、镜检拍照：
在荧光显微镜、共聚焦显微镜、多通道荧光扫描仪或多光谱成像系统下观察并采集图像。
（三）细胞爬片/冰冻切片使用步骤（四标为例）
1、固定（选做）：
冰冻切片：复温至室温后，滴加 4% 多聚甲醛（试剂盒未提供）孵育 10-15 分钟，室温晾干后用 PBS 浸泡脱胶（3 缸，每缸 10 分钟）；若 OCT 包埋前未固定，建议必做此步骤。
细胞爬片：直接加入 4% 多聚甲醛固定 10-15 分钟，PBS 洗涤 3 次，每次 5 分钟。
2、破膜（选做）：
用 0.1-0.3% Triton X-100（需自备，浓度不建议超过 0.3%）室温通透 20 分钟；免疫原在胞外段的一抗可省略此步骤；后续用 PBS 洗涤 3 次，每次 5 分钟。
3、内源性过氧化物酶阻断：
(1)擦干组织周围水分（保持组织湿润），用组化笔在组织周围画封闭圈。
(2)向组织滴加内源性过氧化物酶阻断剂，在湿盒内室温孵育 10-15 分钟；随后将切片放入PBS中，用慢速摇床洗涤 3 次，每次 3 分钟。
4、封闭：
用正常山羊血清均匀覆盖组织，室温封闭30min。
5、一抗孵育：
沥干玻片（约几秒），稀释一抗后滴加至完全覆盖组织；室温或 37 ℃ 孵育 3-4 小时，或 4 ℃ 湿盒过夜后 37 ℃ 复温 1-2 小时；最后用 PBST 冲洗 3 次，每次 2 分钟。
6、二抗孵育：
沥干玻片，滴加 50-100μL 超敏酶标山羊抗小鼠/兔 IgG聚合物（以覆盖组织为宜），室温孵育 30 分钟；用 PBST 冲洗 3 次，每次 2 分钟。
7、TSA 反应：
滴加 50-100μL TSA-520 荧光染料（覆盖组织即可），孵育 5-15 分钟 PBST 冲洗 3 次，每次 2 分钟。
8、抗体洗脱：
滴加适量 37 ℃ 预热的抗体洗脱液覆盖组织，37 ℃ 放置 5-20 分钟，甩干后无需洗涤，再次滴加适量抗体洗脱液覆盖组织 37 ℃ 放置 5-20 分钟(通常两次洗脱合计20分钟），PBST 洗三次，每次 5 分钟。
9、第二轮染色：
重复步骤 4-8，一抗更换为第二种目标一抗，TSA荧光染料换为 TSA-570。
10、第三轮染色：
重复步骤 4-8，一抗更换为第三种目标一抗，TSA荧光染料换为 TSA-690。
11、第四轮染色：
重复步骤 4-7（无需抗体洗脱），一抗更换为第四种目标一抗，TSA荧光染料换为 TSA-620。
（注：多重免疫荧光染色的最后一轮均无需抗体洗脱）
12、DAPI 复染细胞核：
将玻片放入 PH7.4 的 PBS 中，慢速摇床洗涤 3 次，每次 5分钟；稍甩干后，向封闭圈内滴加 DAPI 染液，避光室温孵育 10 分钟。
13、封片：
PBS 洗涤 3 次，每次 5 分钟；稍甩干切片，用抗荧光淬灭封片剂封片。
14、镜检拍照：
在荧光显微镜、共聚焦显微镜、多通道荧光扫描仪或多光谱成像系统下观察并采集图像。
荧光染料参数表：
染料 | 激发波长 | 发射波长 | 荧光强度 |
DAPI | 350nm | 420nm | - |
TSA-480 | 450nm | 480nm | + |
TSA-520 | 490nm | 520nm | ++ |
TSA-570 | 550nm | 570nm | +++ |
TSA-620 | 590nm | 620nm | + |
TSA-690 | 630nm | 690nm | +++ |
TSA-780 | 750nm | 780nm | ++++ |
常见问题
1、染色过深：一抗浓度过高，时间过长；TSA孵育时间过长，导致非特异性结合。
2、染色过浅或无染色：一抗浓度过低，时间过短；抗原修复不够；HRP是否失效。
3、无特异性染色：切片脱蜡不彻底。可适当延长烤片时间。
4、有明显串色现象：上一支一抗未洗脱干净。
5、抗体洗脱液使用三轮及以上易致组织 / 细胞核损伤，为降低核破碎风险，前两轮染色后的一抗洗脱可采用以下方法：将切片间隔置于切片架，浸入 50-60 ℃热水中浸泡 5-20 分钟，重复 2-3 次；若一抗残留未净，可少量使用抗体洗脱液并缩短孵育时长。第三轮开始使用抗体洗脱液正常洗脱。
注意事项
1、试剂初次使用前请置于4℃解冻，解冻后于4℃短期保存，避免反复冻融，请尽快使用。
2、为了您的安全和健康，请穿好实验服并佩戴一次性手套和口罩操作。
3、本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品。
相关产品
货号 | 名称 |
AWI0709 | 荧光单标信号放大试剂(TSA-480) |
AWI0688 | 荧光单标信号放大试剂(TSA-520) |
AWI0689 | 荧光单标信号放大试剂(TSA-570) |
AWI0690 | 荧光单标信号放大试剂(TSA-620) |
AWI0691 | 荧光单标信号放大试剂(TSA-690) |
AWI0708 | 荧光单标信号放大试剂(TSA-780) |
AWI0692 | 双标多重免疫荧光试剂盒(石蜡切片) |
AWI0693 | 三标多重免疫荧光试剂盒(石蜡切片) |
AWI0694 | 四标多重免疫荧光试剂盒(石蜡切片) |
AWI0695 | 五标多重免疫荧光试剂盒(石蜡切片) |
AWI0696 | 六标多重免疫荧光试剂盒(石蜡切片) |
AWI0697 | 双标多重免疫荧光试剂盒(冰冻切片) |
AWI0698 | 三标多重免疫荧光试剂盒(冰冻切片) |
AWI0699 | 四标多重免疫荧光试剂盒(冰冻切片) |
AWI0700 | 五标多重免疫荧光试剂盒(冰冻切片) |
AWI0701 | 六标多重免疫荧光试剂盒(冰冻切片) |
AWI0702 | 双标多重免疫荧光试剂盒(细胞爬片) |
AWI0703 | 三标多重免疫荧光试剂盒(细胞爬片) |
AWI0704 | 四标多重免疫荧光试剂盒(细胞爬片) |
AWI0705 | 五标多重免疫荧光试剂盒(细胞爬片) |
AWI0706 | 六标多重免疫荧光试剂盒(细胞爬片) |
AWC0293 | DAPI染色液(5 μg/mL) |
AWC0215 | 磷酸缓冲盐溶液(1×PBS,无钙镁) |
AWI0206 | 柠檬酸钠抗原修复液(50×) |
AWI0117 | EDTA抗原修复液 |
AWI0197 | 抗荧光淬灭封片剂 |
AWI0603 | 免疫染色通透液(Triton X-100) |
参考文献 (9)
In South and Southeast Asia, the habit of chewing betel nuts is prevalent, which leads to oral submucous fibrosis (OSF). OSF is a well-established precancerous lesion, and a portion of OSF cases eventually progress to oral squamous cell carcinoma (OSCC). However, the specific molecular mechanisms underlying the malignant transformation of OSCC from OSF are poorly understood. In this study, the leading-edge techniques of Spatial Transcriptomics (ST) and Spatial Metabolomics (SM) are integrated to obtain spatial location information of cancer cells, fibroblasts, and immune cells, as well as the transcriptomic and metabolomic landscapes in OSF-derived OSCC tissues. This work reveals for the first time that some OSF-derived OSCC cells undergo partial epithelial–mesenchymal transition (pEMT) within the in situ carcinoma (ISC) region, eventually acquiring fibroblast-like phenotypes and participating in collagen deposition. Complex interactions among epithelial cells, fibroblasts, and immune cells in the tumor microenvironment are demonstrated. Most importantly, significant metabolic reprogramming in OSF-derived OSCC, including abnormal polyamine metabolism, potentially playing a pivotal role in promoting tumorigenesis and immune evasion is discovered. The ST and SM data in this study shed new light on deciphering the mechanisms of OSF-derived OSCC. The work also offers invaluable clues for the prevention and treatment of OSCC.
Background Hepatocellular carcinoma (HCC) ranks among the most aggressive malignancies worldwide, with poor outcomes attributed to delayed diagnosis and therapeutic limitations. Emerging evidence suggests that de novo lipogenesis (DNL) plays a crucial role in HCC progression and its interaction with the immune microenvironment. Methods We systematically analyzed DNL-related gene expression profiles from TCGA, GEO, ICGC-LIRI datasets, and our Xiangya HCC cohort ( n = 106) to construct a prognostic risk model. Through LASSO-Cox regression analysis, we identified six signature genes (G6PD, LCAT, SERPINE1, SOAT2, CYP2C9, and UGT1A10) that effectively stratified patients into distinct risk groups. We evaluated clinical characteristics, immune cell infiltration patterns, and differential therapeutic responses between high-risk and low-risk groups. Comprehensive validation included immunohistochemical analysis and Western blotting to assess expression levels of key model genes, along with multiplex immunofluorescence staining and single-cell RNA sequencing(scRNA-seq) to characterize immune microenvironmental differences between risk groups. Results We successfully established a robust six-gene prognostic signature (G6PD, LCAT, SERPINE1, SOAT2, CYP2C9, and UGT1A10) based on de novo lipogenesis pathways, which demonstrated excellent predictive performance (AUC: 0.78–0.82). The model revealed significant differences in immune infiltration patterns between risk groups, with the high-risk group exhibiting immunosuppressive characteristics characterized by increased Treg cell infiltration, while the low-risk group showed greater NK cell retention. Integrated scRNA-seq and our cohort validation further demonstrated that high-risk scores were associated with poorer response to immunotherapy but greater sensitivity to targeted therapies. These findings suggest that de novo lipogenesis-mediated immune evasion contributes to therapy resistance and worse prognosis in high-risk HCC patients, whereas low-risk HCC patients maintain an immunologically active microenvironment more amenable to immunotherapy. Conclusions This study provided a novel prognostic model for HCC, incorporating 6 representative DNLs. The model demonstrated the potential for predicting HCC prognosis and highlighted the involvement of immune cell infiltration and the association between risk scores and clinical therapy. Validation of model genes further supported the association between de novo lipogenesis and HCC development.
Ovarian cancer (OC) is a significant health challenge, yet the mechanisms driving its progression remain unclear. This study explored the role of hexokinase domain-containing protein 1 (HKDC1) in OC, focusing on tumor growth, lipid metabolism, and immune evasion. Human OC cell lines (SKOV3 and HEY) and the murine OC cell line (ID8) were used to knock down and overexpress HKDC1 . An ID8-based epithelial OC mouse model was established to validate the in vitro findings. Our results demonstrated that HKDC1 was upregulated in OC and promoted cell proliferation, migration, and invasion. HKDC1 enhanced lipid accumulation by elevating levels of free fatty acids (FFA), triglycerides, phospholipids, cholesterol, and neutral lipid, while upregulating key enzymes (ACC1, FASN, SCD1, HMGCS1, and HMGCR). It promoted immune escape through PD-L1 upregulation, inhibiting T cell proliferation and reducing IFN-γ, granzyme B, and perforin levels while increasing PD-1 levels. HKDC1 knockdown reversed these effects, which were restored by adding FFA. Mechanistically, HKDC1 interacted with and stabilized glucose-6-phosphatase catalytic subunits (G6PC/G6PC2), supporting its tumor-promoting functions. These findings were confirmed in an OC mouse model, highlighting HKDC1 as a key driver of OC progression through lipid biosynthesis and immune suppression, offering potential therapeutic targets.
Background Heparin reduces myocardial ischemia-reperfusion (I/R) injury, which is associated with pyroptosis. As a derivative of heparin, non-anticoagulant heparin (NAH) is rarely researched in this field. This study aims to explore the mechanisms of NAH in myocardial I/R injury and pyroptosis. Methods Cardiomyocytes (H9C2) were exposed to hypoxia/reoxygenation (H/R) to simulate myocardial I/R injury in vitro . Cells were treated with NAH, ov-gasdermin D (GSDMD), ov-caspase 11, H 2 O 2 , N -acetyl-L-cysteine (NAC), and recombinant HMGB1 (rHMGB1). The binding of NAH to HMGB1 was detected by molecular docking and DARTS. For in vivo validation, C57BL/6 J male mice underwent myocardial I/R surgery and received NAH and rHMGB1 treatment. Results NAH inhibited H/R-induced pyroptosis of H9C2 cells as evidenced by decreased caspase 11/GSDMD activation, decreased IL-18/IL-1β/LDH release, and increased ATP yields. These effects were attenuated by caspase 11 or GSDMD-N overexpression. Similar to NAC, NAH inhibited H/R and H 2 O 2 -induced oxidative stress. Moreover, NAH reversed the promoting effects of rHMGB1 on cell pyroptosis and oxidative stress. Mechanistically, NAH bound to HMGB1, blocking HMGB1/RAGE interaction. In mice, NAH alleviated myocardial infarction, injury, fibrosis, pyroptosis, and oxidative stress. These effects were reversed by rHMGB1. Conclusions NAH protects against myocardial I/R injury by inhibiting GSDMD-mediated pyroptosis via the HMGB1/RAGE pathway. NAH may serve as a potential drug for treating myocardial I/R injury.
Background Lung cancer exhibits high mortality and incidence rates, with tumor-associated macrophages (TAMs) serving as critical contributors to cancer progression. This study investigates the unexplored mechanistic role of HRD1—an E3 ubiquitin ligase implicated in cancer — in orchestrating TAM polarization to affect lung cancer pathogenesis. Methods HRD1 expression in lung cancer using TCGA database and validated its impact via IHC. THP-1 cells and macrophages isolated from murine tumor tissues via magnetic bead sorting were transfected with the oe-HRD1 plasmid, followed by flow cytometry, ELISA, and RT-qPCR assays to investigate HRD1's regulatory effects on macrophage polarization and function. Co-IP was employed to investigate interactions between USP7 and HRD1/PD-L1, while Immunofluorescence elucidated underlying mechanisms. Results HRD1 was highly expressed in lung cancer and promotes tumor growth in tumor-bearing mice and proliferation in THP-1 cells. Strikingly, both in vivo and in vitro overexpression of HRD1 drove macrophage M2 polarization. Mechanistically, USP7 interacted independently with HRD1 and PD-L1, while HRD1 binding to USP7 facilitated PD-L1 ubiquitination. Furthermore, HRD1 overexpression upregulated USP7 expression, thereby enhancing M2 polarization. Conclusion HRD1 promotes lung cancer progression by regulating TAM M2 polarization via USP7, offering novel therapeutic targets and diagnostic perspectives for early-stage lung cancer intervention.
Parkinson's disease (PD) is a neurodegenerative disorder that gets exacerbated by vascular injury. Neural stem cell-derived exosomes (NSC-Exos) display effective neuroprotective properties in PD models. Cell division control protein 42 (CDC42) is connected to angiogenesis, but its effects in PD remain undefined. This research aims to reveal the role of CDC42 in PD. First, we applied 1-methyl-4-phenylpyridinium (MPP + ) to induce the human cerebral microvascular endothelial cells (HCMECs) model and evaluated cell viability and ferroptosis. Then, we characterized NSC-Exos. Next, to appraise the effect of hypoxia-pretreated NSC-Exos (H-NSC-Exos) on the MPP + -induced cells model, we examined angiogenesis and ferroptosis in HCMECs. Moreover, we constructed the PD mice model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and tested the behavioral experiments and vascular injury of mice. Furthermore, we examined cellular ferroptosis and angiogenesis after knockdown of CDC42. Additionally, we investigated the interaction of CDC42 with Acyl-CoA synthetase long-chain family member 4 (ACSL4) and detected cellular ferroptosis and angiogenesis after overexpression of ACSL4. We found that H-NSC-Exos reversed the MPP + -induced decrease in HCMECs viability and migration, lowered lipid-reactive oxygen species (lipid-ROS) levels, suppressed ferroptosis, and facilitated angiogenesis. Moreover, H-NSC-Exos attenuated MPTP-induced PD development, vascular injury, and ferroptosis in mice. H-NSC-Exos with the knockdown of CDC42 reduced cell viability and angiogenesis and raised ferroptosis and lipid-ROS levels, which were reversed by ferrostatin-1 and liproxstatin-1. CDC42 interacted with ACSL4. Furthermore, overexpression of ACSL4 aggravated the above effects of H-NSC-Exos in which CDC42 was knocked down. Our study reveals that H-NSC-Exos-derived CDC42 inhibited ACSL4-related ferroptosis to alleviate vascular injury in PD mice models. CDC42 may serve as a potent therapeutic target for PD treatment.
Background:Chronic heart failure (CHF) is a serious cardiovascular condition. Vascular peroxidase 1 (VPO1) is associated with various cardiovascular diseases, yet its role in CHF remains unclear. This research aims to explore the involvement of VPO1 in CHF.Methods:CHF was induced in rats using adriamycin, and the expression levels of VPO1 and cylindromatosis (CYLD) were assessed. In parallel, the effects of VPO1 on programmed necrosis in H9c2 cells were evaluated through cell viability assays, lactate dehydrogenase (LDH) level measurements, and analysis of receptor-interacting protein kinase 1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein (RIPK1/RIPK3/MLKL) pathway-related proteins. The impact of CYLD on RIPK1 protein stability and ubiquitination was also investigated, along with the interaction between VPO1 and CYLD. Additionally, cardiac structure and function were assessed using echocardiography, Hematoxylin-eosin (HE) staining, Masson staining, and measurements of myocardial injury-related factors, including N-terminal prohormone of brain natriuretic peptide (NT-proBNP), Aspartate aminotransferase (AST), LDH, and creatine kinase-myocardial band (CK-MB).Results:VPO1 expression was upregulated in CHF rats and in H9c2 cells treated with adriamycin. In cellular experiments, VPO1 knockdown improved cell viability, inhibited necrosis and the expression of proteins associated with the RIPK1/RIPK3/MLKL pathway. Mechanistically, VPO1 promoted cardiomyocyte programmed necrosis by interacting with the deubiquitinating enzyme CYLD, which enhanced RIPK1 ubiquitination and degradation, leading to activation of the RIPK1/RIPK3/MLKL signaling pathway. At animal level, overexpression of CYLD counteracted the cardiac failure, cardiac hypertrophy, myocardial injury, myocardial fibrosis, and tissue necrosis caused by VPO1 knockdown.Conclusions:VPO1 exacerbates cardiomyocyte programmed necrosis in CHF rats by upregulating CYLD, which activates the RIPK1/RIPK3/MLKL signaling pathway. Thus, VPO1 may represent a potential therapeutic target for CHF.
Background Cerebral ischemia–reperfusion (CI/R) injury, a major complication of ischemic stroke, is characterized by mitochondrial dysfunction and neuronal apoptosis, and understanding its underlying molecular mechanisms is essential for the development of effective therapeutic strategies. This study aimed to investigate the role of ubiquitin-specific protease 7 (USP7) in CI/R injury and elucidate its regulatory mechanisms. Methods A rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) and an in vitro neuronal model subjected to oxygen-glucose deprivation/reperfusion (OGD/R) was used to mimic CI/R injury. USP7 was overexpressed or knocked down, with or without co-treatment, using the autophagy inhibitor 3-methyladenine (3-MA). Neurological function was evaluated using standardized scoring systems, and cerebral infarct volume was quantified by TTC staining. Histopathological alterations in the cortex and hippocampus were assessed using hematoxylin-eosin (HE) and Nissl staining. Neuronal viability and apoptosis were measured by CCK-8 assay, TUNEL staining, and flow cytometry. To assess cellular metabolism and oxidative stress, ATP and LDH levels, along with antioxidant markers including SOD, GSH, and GSH-Px, were analyzed using commercial biochemical kits. Mitochondrial morphology and autophagosome formation were visualized using transmission electron microscopy. Gene and protein expression levels were quantified by qRT-PCR and Western blotting, respectively. Immunofluorescence microscopy was performed to evaluate subcellular localization of target proteins and co-localization with mitochondrial membrane markers. Lastly, protein–protein interactions and ubiquitination modification were analyzed by co-immunoprecipitation assays. Results USP7 overexpression significantly alleviated neurological deficits, reduced infarct volume, attenuated histological damage, and decreased neuronal apoptosis in the MCAO/R model. In parallel, in the OGD/R model, USP7 overexpression markedly enhanced neuronal viability, suppressed apoptosis, restored ATP production, improved antioxidant capacity (as indicated by increased levels of SOD, GSH, and GSH-Px), and reduced LDH release. Mechanistically, USP7 stabilized SIRT1 protein expression through deubiquitination, which in turn activated the PINK1/Parkin pathway and enhanced mitophagy. This activation was demonstrated by an increased LC3II/LC3I ratio, elevated ATG5 expression, enhanced co-localization of Tomm20 and Parkin, and increased autophagosome formation. Moreover, these protective effects could be abolished when either 3-MA treatment was applied or SIRT1/PINK1 expression was knocked down. Conclusion USP7 mitigates CI/R injury by promoting PINK1/Parkin-dependent mitophagy through SIRT1 deubiquitination and stabilization, supporting USP7 as a potential therapeutic target for ischemic stroke.
This study aimed to investigate the role of mesenchymal homeobox 2 (MEOX2) on breast cancer cell metastasis and its underlying mechanism. Overexpression of MEOX2 in human lymphatic endothelial cell (HLEC) lines was established to assess the adhesion and transendothelial migration of MCF7 and MDA-MB-231 cells to the HLEC cells. After being treated with the oxidative stress inducer H 2 O 2 and the antioxidant N-acetylcysteine (NAC), cell viability, reactive oxygen species (ROS) levels, adhesion, and transendothelial migration of MCF7 and MDA-MB-231 cells to HLEC cells were detected. Tumor volume changes were observed in the xenograft model. The expression of C-X-C chemokine receptor type 4 (CXCR4), C–C chemokine receptor type 7 (CCR7), MEOX2, and G protein signal transduction regulator 5 (RGS5) in tumor tissues and ROS levels were detected. MEOX2 was lowly expressed in breast cancer tissues. Upregulated MEOX2 inhibited the proliferation of lymphatic endothelial cells and the adhesion and transendothelial migration of MCF7 and MDA-MB-231 cells to HLEC cells. After MCF7 and MDA-MB-231 cells were treated with oxidative stress inducer H 2 O 2 , ROS levels increased, and cell viability and MEOX2 expression decreased. After NAC or overexpressed MEOX2 treatment, MEOX2 expression increased, ROS and RGS5 levels, adhesion, and transendothelial migration ability decreased in HLEC cells. Overexpression of MEOX2 resulted in smaller tumor volume, lower ROS levels, and lower CXCR4 and CCR7 expression levels. MEOX2 and RGS5 are pivotal in regulating breast cancer metastasis, offering valuable insights into potential therapeutic strategies for breast cancer metastasis.
