To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1β(IL-1β) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1β showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.
Rats ; Animals ; Chondrocytes ; Osteoarthritis, Knee/pathology* ; RNA, Circular/pharmacology* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MicroRNAs/metabolism* ; Apoptosis ; Autophagy/genetics* ; Collagen/metabolism*

The present study was aimed to investigate the effects of circRNA-0028171 on the apoptosis of vascular endothelial cells induced by arsenic trioxide (As₂O₃). Human umbilical vein endothelial cells (HUVECs) were treated with 0-15 μmol/L As₂O₃ for 24 h. Then, cellular viability was measured by MTT assay. The expression levels of circRNA-0028171, Bcl-2 and Bax mRNA were detected by real-time quantitative PCR. Bcl-2/Bax protein ratio was detected by Western blot. Whether circRNA-0028171 was involved in the regulation of HUVECs by As₂O₃ was investigated by transfection with overexpression plasmid of circRNA-0028171 and siRNA. The results showed that compared with the control group, As₂O₃ group showed decreased cellular viability, reduced Bcl-2/Bax mRNA and protein ratios, and significantly lower expression of circRNA-0028171. Overexpression of circRNA-0028171 inhibited apoptosis of HUVECs induced by As₂O₃. Knockdown of circRNA-0028171 by siRNA promoted As₂O₃-induced apoptosis in HUVECs. These results suggest that circRNA-0028171 is involved in the vascular endothelial cell apoptosis induced by As₂O₃.
Humans ; Arsenic Trioxide/pharmacology* ; RNA, Circular ; bcl-2-Associated X Protein/metabolism* ; RNA, Small Interfering/metabolism* ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; RNA, Messenger/metabolism*

OBJECTIVE: To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect. METHODS: We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells. RESULTS: The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5%, P < 0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P < 0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P < 0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P < 0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/ JAK2 and p-STAT3/STAT3 (P < 0.01). CONCLUSION The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.
Arthritis, Rheumatoid/pathology* ; Cells, Cultured ; Diterpenes/pharmacology* ; Epoxy Compounds/pharmacology* ; Fibroblasts/pathology* ; Humans ; Interleukin-17/metabolism* ; Interleukin-4/metabolism* ; Interleukin-6/metabolism* ; Janus Kinase 2/metabolism* ; Leukocytes, Mononuclear/metabolism* ; Phenanthrenes/pharmacology* ; RNA, Circular/metabolism* ; STAT3 Transcription Factor/metabolism* ; Signal Transduction/drug effects* ; Synovial Membrane/pathology*