Department of Biochemistry and Molecular Biology, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China.
Department of Grass Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China.
Int J Mol Med. 2019 Feb;43(2):1067-1075. doi: 10.3892/ijmm.2018.4012. Epub 2018 Dec 3.
The present study investigated the mechanisms of apoptosis induced by cryptotanshinone (CT) in human rheumatoid arthritis fibroblast‑like synoviocytes (RA‑FLSs). Cell Counting kit‑8 assay was performed to determine the cytotoxic effects of CT in human RA‑FLSs, including primary RA‑FLS, HFLS‑RA and MH7A cells, and in HFLS cells derived from normal synovial tissue. Annexin V‑FITC/PI staining was used to detect the apoptotic effects of CT in HFLS‑RA and MH7A cells. Flow cytometry was performed to detect the apoptotic and reactive oxygen species (ROS) levels induced by CT in HFLS‑RA cells. Western blotting was used to assess the expression levels of proteins associated with apoptosis and with the mitogen‑activated protein kinase (MAPK), protein kinase B (Akt), and signal transducer and activator of transcription‑3 (STAT3) signaling pathways. The results demonstrated that CT treatment significantly suppressed HFLS‑RA and MH7A cell growth, whereas no clear inhibitory effect was observed in normal HFLS cells. CT exposure downregulated the expression levels of B‑cell lymphoma 2 (Bcl‑2), p‑Akt, p‑extracellular signal‑related kinase and p‑STAT3, while it upregulated the expression levels of Bcl‑2‑associated death promoter (Bad), caspase‑3, poly (ADP‑ribose) polymerase (PARP), p‑p38 and p‑c‑Jun N‑terminal kinase. Following ROS scavenging, the CT‑induced apoptosis and altered expression levels of Bcl‑2, Bad, cleaved caspase‑3 and cleaved PARP were restored. Furthermore, the Akt, MAPK and STAT3 signaling pathways were regulated by intracellular ROS. These results suggest that ROS‑mediated Akt, MAPK and STAT3 signaling pathways serve important roles in the CT‑induced apoptosis of RA‑FLSs.
本研究探讨了隐丹参酮（CT）诱导人类风湿关节炎成纤维样滑膜细胞（RA-FLSs）凋亡的机制。通过细胞计数试剂盒-8 法测定 CT 对人 RA-FLSs（包括原代 RA-FLS、HFLS-RA 和 MH7A 细胞）和正常滑膜组织来源的 HFLS 细胞的细胞毒性作用。采用 Annexin V-FITC/PI 染色法检测 CT 对 HFLS-RA 和 MH7A 细胞凋亡的影响。采用流式细胞术检测 CT 诱导 HFLS-RA 细胞凋亡和活性氧（ROS）水平。采用 Western blot 法检测与凋亡相关以及与丝裂原活化蛋白激酶（MAPK）、蛋白激酶 B（Akt）和信号转导及转录激活因子 3（STAT3）信号通路相关的蛋白表达水平。结果表明，CT 处理显著抑制 HFLS-RA 和 MH7A 细胞生长，而对正常 HFLS 细胞无明显抑制作用。CT 暴露下调 B 细胞淋巴瘤 2（Bcl-2）、p-Akt、p-细胞外信号调节激酶和 p-STAT3 的表达水平，而上调 Bcl-2 相关死亡启动子（Bad）、caspase-3、多聚（ADP-核糖）聚合酶（PARP）、p-p38 和 p-c-Jun N-末端激酶的表达水平。ROS 清除后，CT 诱导的凋亡和 Bcl-2、Bad、cleaved caspase-3 和 cleaved PARP 的表达水平恢复。此外，ROS 调节 Akt、MAPK 和 STAT3 信号通路。这些结果表明，ROS 介导的 Akt、MAPK 和 STAT3 信号通路在 CT 诱导的 RA-FLSs 凋亡中发挥重要作用。
