The intestinal epithelial barrier consists of a monolayer of epithelial cells and intercellular junctions between adjacent cells that seal the paracellular space and regulate the barrier permeability (Jin and Blikslager, 2020; Nighot and Ma, 2020; Slifer and Blikslager, 2020). This barrier separates harmful luminal substances, such as microorganisms, toxins, and antigens from the body, and thus plays a critical role in maintaining intestinal homeostasis. Impaired barrier function can lead to gut hyperpermeability and trigger mucosal inflammation (Tabat
The mitogen-activated protein kinases (MAPKs) contain highly conserved serine/threonine protein kinases that play a central role in the regulation of TJ permeability (Xiong
Heme oxygenase-1 (HO-1) is an enzyme specialized in degrading heme and is assembled with biliverdin, carbon monoxide (CO), and free iron (Vijayan
The human colonic adenocarcinoma cell line Caco-2 was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal calf serum (FCS, Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a 5% CO2 atmosphere. Then, TNF-α (100 ng/mL, PeproTech, USA) was added to Caco-2 cells for 24 h. Lentiviral transfection was performed using LipofiterTM (Hanbio Biotechnology, China), according to the manufacturer’s instructions. The FUGW-HO-1 and pLKO.1-sh-HO-1 plasmids were obtained from Hanbio Biotechnology. The sequences used for HO-1 overexpression were as follows: F-CACAGACCGGTATGGAGCGTCCGCAACCCGACAG, R-CACAGGAATTCTCACATGGCATAAAGCCCTACAGC. The sequence used for short hairpin RNA (shRNA) targeting HO-1 was F-CCGGACAGTTGCTGTAGGGCTTTATCTCGAGATAAAGCCCTACAGCAACTGTTTTTTG; R-AATTCAAAAAACAGTTGCTGTAGGGCTTTATCTCGAGATAAAGCCCTACAGCAACTGT.
C57BL/6 male wild type (WT) mice (6-8-week-old and weighing 20-25 g) were obtained from the Laboratory Animal Center of Dalian Medical University (Liaoning, China). Briefly, the mice were administered 2 mL/kg carbon tetrachloride (CCl4, Aladdin Biochemical Technology Co., Ltd, Shanghai, China) via intraperitoneal injection (CCl4:olive oil=1:3) twice a week for 12 weeks to induce barrier loss. The control group was administered olive oil. In the last 2 weeks, according to the groups, all surviving mice were administered cobalt protoporphyrin (CoPP, 5 mg/kg, Sigma-Aldrich, Saint Louis, MO, USA) (Bakhautdin
Freshly dissected ileum biopsies were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) (pH 7.2-7.4), embedded in paraffin, and stained with hematoxylin and eosin (H&E), according to the standard protocol. The slides were examined by an experienced histopathologist who was blinded to the study design. Intestinal mucosal injuries were graded according to the Chiu’s scoring system (Li
Western blot analysis was performed, as described previously (Mishra
The data were representative of three or more independent experiments, each with similar results. The continuous data are shown as mean ± standard deviation. The comparison between two groups were performed using Mann-Whitney U test. The comparisons among multiple groups were made using Kruskal-Wallis H test.
In order to examine the effects of HO-1 on the intestinal epithelial barrier, gain- or loss-of-HO-1-function experiments were conducted using Caco-2 cells transfected with FUGW-HO-1 or pLKO.1-sh-HO-1 plasmid. The cells transfected with empty plasmids served as the control groups. For these in-vitro studies, TNF-α was used to stimulate the Caco-2 epithelial cells as it is one of the most frequently used cell models for mimicking the intestinal epithelial barrier injury, and TNF-α stimulation does not affect the expression of HO-1 (Fig. 1A-1D, all
We used RNA-seq to explore the putative molecular mechanism of HO-1 in barrier disruption, and the results demonstrated that HO-1 regulates the expression of MAPK signaling pathway-related genes in TNF-α-induced Caco-2 cells treated with CoPP or ZnPP (Supplementary Fig. 1A-1D). We also examined the expression of MAPK signaling pathway-related proteins, such as ERK, phosphorylated-ERK (p-ERK), p38, phosphorylated-p38 (p-p38), JNK, and phosphorylated-JNK (p-JNK), by Western blot analysis and found that the ratios of p-ERK/ERK (
To determine the therapeutic effects of CoPP, ZnPP, CORM-2, or iCORM-2, we used CCl4 to induce intestinal epithelial barrier damage. Mice were administered CoPP, ZnPP, CORM-2, iCORM-2, or normal saline after CCl4 administration, and then, serum was collected and intestinal tissues harvested. The disrupted architecture and the disarranged ileal epithelial mucosal villi were observed by H&E staining in the CCl4-treated group (Fig. 3A). CoPP significantly upregulated the level of HO-1 protein in the intestine of the mice model (Supplementary Fig. 2,
To confirm the intestinal epithelial cell-specific function of HO-1 in mediating the barrier loss, we specifically deleted HO-1 from intestinal epithelial cells (VillinCreHmox1floxp/floxp) (Supplementary Fig. 3) by crossing Villin-Cre mice with Hmox1floxp/floxp mice. WT and Hmox1floxp/floxp mice displayed relatively mild mucosal lesion, TJ disruption, and cell apoptosis after CCl4 challenge (Fig. 4A-4D), while VillinCreHmox1floxp/floxp mice showed a significant aggravation of mucosal lesions (Fig. 4A), disruption of TJ proteins (Fig. 4B, 4C), and increase in cell apoptosis (Fig. 4B, 4D). Moreover, the animals presented a significantly elevated MAPK phosphorylation as compared to the WT and Hmox1floxp/floxp mice (Fig. 4B, 4E). In summary, HO-1 on intestinal epithelial cells contributed to the protection of the paracellular leakage pathway, effectuating the repair of barrier disruption. These processes were accompanied by the inhibition of MAPK phosphorylation in a HO-1-dependent manner.
Gut leakiness is a well-characterized phenomenon in patients with many diseases (Shao
HO-1 is the rate-limiting enzyme in the degradation of heme, converting heme to free iron, biliverdin, and CO, which in turn, plays a major role in conferring protection against the different types of damage, such as intestinal diseases (Onyiah
The MAPK signaling pathway (including p38, ERK, and JNK) harbor highly conserved serine/threonine protein kinases that function in various fundamental cellular processes, such as growth/proliferation, differentiation, motility, apoptosis/survival, inflammation, and innate immunity (Hua
In summary, this study indicated that intestinal HO-1 contributes to maintaining the integrity of intestinal epithelial barrier, increasing the intestinal TJs and reducing the apoptosis of intestinal epithelial cells through inhibiting the phosphorylation of the MAPK signaling pathway. Targeting the gut HO-1-MAPK crosstalk would track the clinical management of patients with barrier loss.
This work was supported by the National Natural Science Foundation of China (Grant No. 81670479).
The authors declare that they have no conflicts of interest.