Triptolide inhibits migration and proliferation of fibroblasts from ileocolonic anastomosis of patients with Crohn's disease via regulating the miR-16-1/HSP70 pathway

Anastomotic fibrosis is highly likely to lead to reoperation in Crohn's disease (CD) patients. Triptolide (TPL) is considered to have anti-inflammatory and antifibrotic effects in a variety of autoimmune diseases, including CD. The present study aimed to investigate the effects of TPL on fibroblasts from strictured ileocolonic anastomosis of patients with CD and its underlying mechanism. Primary fibroblasts were obtained from strictured anastomosis tissue (SAT) samples and matched anastomosis-adjacent normal tissue (NT) samples which were collected from 10 CD patients who underwent reoperation because of anastomotic stricture. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure miR-16-1 and heat shock protein 70 (HSP70) levels. Western blotting was conducted to determine expression of HSP70, collagen I (Col-I), collagen III (Col-III) and α-smooth muscle actin (α-SMA) proteins. Agomir-16-1 and antagomir-16-1 were used to up and downregulate the expression of miR-16-1, respectively. Small interfering RNA (siRNA) was employed to inhibit the expression of HSP70. A wound healing assay was performed to measure the migration of fibroblasts. Cell proliferation was evaluated by MTT and 5-bromo-2-deoxyrudidine assays. Cell apoptosis was determined by caspase-3 activity and TUNEL assays. The results demonstrated that the levels of Col-I, Col-III and α-SMA were all significantly upregulated in SAT compared with NT. miR-16-1 levels in the SAT group were significantly compared with the NT group; conversely, the expression levels of HSP70 mRNA and protein in the SAT group were significantly lower compared with the NT group. Next, fibroblasts were treated with TPL to examine its effect on the miR-16-1/HSP70 pathway. The results demonstrated that the elevated expression of miR-16-1 in the SAT group was effectively inhibited by TPL treatment. Compared with the NT group, both the mRNA and protein levels of HSP70 were significantly downregulated in the SAT group cells, while TPL exhibited a strong promoting effect on HSP70 synthesis. Furthermore, upregulation of miR-16-1 reversed the effect of TPL on the miR-16-1/HSP70 pathway in fibroblasts from SAT. Overexpression of miR-16-1 significantly reversed the inhibitory effects of TPL treatment on migration, proliferation and extracellular matrix (ECM)-associated protein expression of fibroblasts from SAT. Finally, downregulation of miR-16-1 caused similar effects to the fibroblasts as the TPL treatment; however, the inhibitory effects on cell biological functions induced by antagomir-16-1 were all significantly reversed by HSP70 silencing. The present findings indicated that TPL may be a potential therapeutic option for postoperative anastomosis fibrosis of patients with CD. The miR-16-1/HSP70 pathway had a substantial role in the inhibitory effects of TPL on migration, proliferation and ECM synthesis rate of fibroblasts from strictured anastomosis tissues.