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Triptolide exerts protective effects against fibrosis following ileocolonic anastomosis by mechanisms involving the miR-16-1/HSP70 pathway in IL-10-deficient mice.

ABSTRACT: Surgeries, particularly ileocecal resection (ICR), are often required in the treatment of Crohn's disease (CD). However, recurrences are common for patients who undergo ICR, and anastomotic fibrosis is the main cause of re-operation. The present study aimed to investigate the therapeutic effects of triptolide (TPL) in ameliorating fibrosis following ileocolonic anastomosis. A model of IL‑10‑/‑ mice undergoing ICR was used to study postsurgical inflammation and fibrosis of anastomosis. For this purpsose, interleukin (IL)‑10‑/‑ mice were randomly divided into 3 groups as follows: the control group, the saline‑treated group subjected to ICR (ST‑ICR) and the TPL‑treated group subjected to ICR (TT‑ICR). Wild‑type (WT) mice of matching ages were assigned to the WT group. The effects of TPL treatment on ileocolonic anastomosis were determined by histopathological evaluation, western blot analysis and ELISA. The analysis of the effects of TPL treatment on microRNA‑16‑1 (miR‑16‑1) and heat shock protein 70 (HSP70) expression was carried out by RT‑qPCR and western blot analysis. Compared with the control group, significantly higher inflammation scores following anastomosis were observed in the ST‑ICR group (P<0.05), although reversion was observed in the TT‑ICR group, which was consistent with changes in the area of CD4+ cell infiltration. The elevated fibrosis scores and the overexpression of procollagen I and III in the ST‑ICR group were all inhibited by TPL. With an increase in the severity of inflammation and fibrosis, the levels of IL‑6, tumor necrosis factor‑α (TNF‑α) and transforming growth factor‑β1 (TGF‑β1) increased; however, a significant decrease in these levels was observed following treatment with TPL (P<0.05). The results of RT‑qPCR revealed that the upregulated miR‑16‑1 levels in the ST‑ICR group were significantly reduced by TPL. HSP70, which can be inhibited by miR-16-1, ameliorates anastomotic inflammation and fibrosis. Thus, the present study demonstrates that TPL exerts a protective effect against fibrosis following anastomosis in CD. The miR‑16‑1/HSP70 signaling pathway, which can be regulated by TPL, may thus represent a novel therapeutic option in CD that deserves further investigation.

SUBMITTER: Hou HW

PROVIDER: S-EPMC5505014 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Background: The mechanisms underlying ozone (O3)-induced pulmonary inflammation remain unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators. Objectives: The current study investigated the molecular mechanisms underlying IL-10-mediated attenuation of O3-induced pulmonary inflammation in mice. Methods: Il10-deficient (Il10-/-) and wild type (Il10+/+) mice were exposed to 0.3-ppm O3 or filtered air for 24, 48 or 72 hr. Immediately following exposure, differential cell counts, and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF). mRNA and protein levels of cellular mediators were determined from lung homogenates. We also utilized global mRNA expression analyses of lung tissue with Ingenuity Pathway Analyses (IPA) to identify patterns of gene expression through which IL-10 modifies O3-induced inflammation. Results: Mean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10-/- mice than in Il10+/+ mice after exposure to O3 at all time points tested. O3-enhanced nuclear NF-kB translocation was elevated in the lungs of Il10-/- compared to Il10+/+ mice. Gene expression analyses revealed several key IL-10 and O3-dependent mediators, including IL-6, MIP-2, IL-1 and CD86. Conclusions: Results indicated that IL-10 protects against O3-induced pulmonary neutrophilic inflammation and cell proliferation. Moreover, gene expression analyses identified three response pathways and several novel genetic targets (e.g. Ccr1, Socs3, Il33, Hat1, and Gale) through which IL10 may modulate the innate and adaptive immune response. These novel mechanisms of protection against the pathogenesis of O3-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals. PARALLEL study design with 26 samples. Biological replicates: 2 to 3 replicates per group with wild type air exposed animals as controls for each time point (24, 48, 72 hours). Time-Course, Dose-Response, Strain comparison

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Triptolide exerts protective effects against fibrosis following ileocolonic anastomosis by mechanisms involving the miR-16-1/HSP70 pathway in IL-10-deficient mice.

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