DMOG ameliorates IFN-γ-induced intestinal barrier dysfunction by suppressing PHD2-dependent HIF-1α degradation.

Hypoxia-inducible factor 1α (HIF-1α) has been well established as a protective factor for intestinal barrier function in intestinal epithelial cells. Recently, a study found that increased HIF-1α-induced intestinal barrier dysfunction. We proposed that lymphocyte-derived interferon-gamma (IFN-γ) might be responsible for the intestinal barrier dysfunction caused by increased HIF-1α. HT-29 cell monolayers were grown in the presence or absence of IFN-γ under hypoxia. Then, the transepithelial electrical resistance was measured, and HIF-1α-modulated intestinal barrier protective factors were quantified by polymerase chain reaction (PCR). PCR, western blotting, and chromatin immunoprecipitation of HIF-1α were performed. Dimethyloxalyglycine (DMOG), an inhibitor of prolyl-hydroxylases (PHDs) that stabilizes HIF-1α during normoxia, and RNA interference of PHDs were also used to identify the signal pathway between IFN-γ and HIF-1α. We demonstrated that IFN-γ caused barrier dysfunction in hypoxic HT-29 cell monolayers via suppressing HIF-1α and HIF-1α-modulated intestinal barrier protective factors. We found that IFN-γ decreased HIF-1α protein expression instead of affecting HIF-1α transcription or transcriptional activity. Study also showed that DMOG reversed the IFN-γ-induced decrease in HIF-1α protein expression. Further, we found that PHD2 is the major regulator of IFN-γ-induced HIF-1α degradation by PHD inhibition and RNA interference. We conclude that IFN-γ caused barrier dysfunction by promoting PHD-, especially PHD2-, dependent HIF-1α degradation, and DMOG or PHD2 inhibition reversed this HIF-1α suppression and ameliorated barrier dysfunction. Combined with other studies demonstrating HIF-1α activation in lymphocytes promotes IFN-γ secretion, these findings suggest a mechanism by which increased HIF-1α-induced intestinal barrier dysfunction.

Up-regulation of intestinal epithelial cell derived IL-7 expression by keratinocyte growth factor through STAT1/IRF-1, IRF-2 pathway.

BACKGROUND: Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine. METHODS: Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA). RESULTS: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively. CONCLUSION: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.

Keratinocyte growth factor up-regulates Interleukin-7 expression following intestinal ischemia/reperfusion in vitro and in vivo.

BACKGROUND: Epithelial cell (EC)-derived Interleukin-7 (IL-7) plays a crucial role in control of neighboring intestinal intraepithelial lymphocytes (IEL) development and homeostasis, and IEL derived keratinocyte growth factor (KGF) promotes intestinal epithelial growth, which was regulated by EC-derived IL-7. On this basis, we hypothesize that there is a crosstalk between IELs and ECs, and KGF could regulate the EC-derived IL-7 expression, which should be associated with the protective effects by KGF on intestinal injury. METHODS: Histological evaluation was performed in small intestine tissues of patients with intestinal obstruction and IL-7 expression was detected by immunofluorescence. Intestinal epithelial cells (LoVo) and adult C57BL/6J mice undergoing ischemia/reperfusion injury were treated with recombinant KGF. KGF, KGF receptor(KGFR) and IL-7 expressions were measured with western blot and immunofluorescence analysis. RESULTS: IL-7 expression increased in the mild ischemia while decreased in severe ischemia small intestinal tissues of patients with intestinal obstruction. KGF expression significantly decreased while IL-7 expression increased early after acute intestinal I/R administration in a mouse model. KGF treatment significantly increased the IL-7 expression both in vitro and in vivo, while when the KGFR was blocked, the findings above were absent. In addition, our results showed changes of IL-7 expression at different stages after acute intestinal I/R administration, KGF treatment significantly attenuated the decreasing of IL-7 expression caused by acute intestinal I/R. CONCLUSION: KGF could up-regulate the IL-7 expression both in vitro and in vivo through KGFR pathway, which should have associated with the protective effects of KGF in intestinal injury.

[Value of the laparoscopy combined with double-balloon enteroscopy in diagnosis and treatment of intestinal diseases].

OBJECTIVE: To evaluate the value of laparoscopy combined with double-balloon enteroscopy (DBE) for the diagnosis and treatment of intestinal diseases. METHODS: Clinical data of 69 cases with suspected small bowel diseases undergoing laparoscopic and DBE for the diagnosis and treatment were retrospectively analyzed. RESULTS: The lesions were found in 48 cases by laparoscopy. DBE was required in the remaining 21 patients to identify the underlying condition. All the operations were successfully completed using the laparoscopic approach, including totally laparoscopic bowel resection (n=11), and laparoscopic-assisted bowel resection (n=58). There were no anastomotic leakage, postoperative bleeding, intestinal obstruction, or wound infection. All the patients were discharged within 7 to 9 days after surgery. Postoperative pathological examination showed vascular abnormally (n=10), gastrointestinal stromal tumor (n=20), intestinal adenocarcinoma (n=5), intestinal neurofibroma (n=2), diverticulum (n=5), intestinal mucosal ulceration (n=8), intestinal tuberculosis (n=3), postoperative pouch bleeding (n=1), intestinal polyp (n=6), Crohn's disease (n=5), Meckel diverticulum (n=2), metastatic kidney cancer (n=1), and metastatic lung cancer (n=1). Length of follow up ranged from 3 months to 4 years, during which no re-bleeding occurred, 2 patients with gastrointestinal stromal tumor died of local recurrence and liver metastasis, 1 patient with adenocarcinoma died of local recurrence involving pancreatic head, duodenum, and mesenteric vessels, 2 patients with metastatic disease died of peritoneal recurrence and liver metastasis. CONCLUSION: Laparoscopic combined with DBE has a high detection rate for small intestinal disease with accurate localization, less trauma, and quicker recovery.