Protective Effect of Methane-Rich Saline on Acetic Acid-Induced Ulcerative Colitis via Blocking the TLR4/NF- κ B/MAPK Pathway and Promoting IL-10/JAK1/STAT3-Mediated Anti-inflammatory Response

Abstract

Ulcerative colitis (UC) is an inflammation-related disease involved in uncontrolled inflammation and oxidative stress and is characterized by high recurrence and relapse risk. As a rising star in gas medicine, methane owns the properties of anti-inflammation, antioxidation, and antiapoptosis. Based on the possible mechanism, we aimed to investigate the effect of methane on UC. Methane-rich saline (MRS) was introduced here, and UC was induced by acetic acid. All the C57BL/6 mice were allocated into groups as follows: control group, colitis model group, colitis treated with salazosulfapyridine (SASP) group, and colitis treated with MRS (1 or 10 ml/kg) groups. Tissue damage, the degree of inflammation, oxidative stress, and apoptosis were evaluated in the study, as well as the TLR4/NF-κB/MAPK and IL-10/JAK1/STAT3 signaling pathways for further exploration of the potential mechanism. The results showed that MRS (1) alleviated tissue damage caused by acetic acid, (2) controlled acetic acid-induced inflammation, (3) inhibited acetic acid-caused oxidative stress, (4) reduced colonic cell apoptosis due to acetic acid, (5) suppressed the TLR-4/NF-κB/MAPK signaling pathway, and (6) activated IL-10/JAK1/STAT3 anti-inflammatory response to improve the injury induced by acetic acid. We conclude that MRS has a protective effect on acetic acid-induced ulcerative colitis in mice via blocking the TLR4/NF-κB/MAPK signaling pathway and promoting the IL-10/JAK1/STAT3-mediated anti-inflammatory response.

Figure 1

Treatment with MRS mitigated acetic acid-induced tissue damage. Mice were administered with MRS at a dose of 1 or 10 ml/kg by gastric gavage for a week before colitis establishment. SASP was used as a positive control at a dose of 500 mg/kg. Colitis was induced by acetic acid solution (v/v) injection into the lumen of the colon intrarectally with a volume of 1 ml and dose of 5% except for the sham group. The (a) spleen weight, (b) disease activity index (DAI), (c) colon weight/length ratio, (d) ulcer area, and (e) ulcer index were measured. Additionally, colon tissues were collected to evaluate the damage microscopically by (g) H&E staining (200x) and (f) microscopic scores were calculated. Data were expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001.

Figure 2

Treatment with MRS reduced the serum levels of inflammatory cytokines in acetic acid-induced UC. Mice were administered with MRS at a dose of 1 or 10 ml/kg by gastric gavage for a week before colitis establishment. SASP was used as a positive control at a dose of 500 mg/kg. Colitis was induced by acetic acid solution (v/v) injection into the lumen of the colon intrarectally with a volume of 1 ml and dose of 5% except for the sham group. Blood samples were collected to determine the levels of (a) TNF-α and (b) IL-6 in the serum. Data were expressed as means ± SD. ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001.

Figure 3

Treatment with MRS inhibited the expression of inflammatory cytokines at the mRNA level in acetic acid-induced UC. Mice were administered with MRS at a dose of 1 or 10 ml/kg by gastric gavage for a week before colitis establishment. SASP was used as a positive control at a dose of 500 mg/kg. Colitis was induced by acetic acid solution (v/v) injection into the lumen of the colon intrarectally with a volume of 1 ml and dose of 5% except for the sham group. Total RNA was collected from colon specimens, and the expression levels of (a) TNF-α, (b) IL-6, (c) IL-1β, and (d) IL-10 were tested. Data were expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗∗P < 0.0001.

Figure 4

Treatment with MRS suppressed oxidative stress induced by acetic acid. Mice were administered with MRS at a dose of 1 or 10 ml/kg by gastric gavage for a week before colitis establishment. SASP was used as a positive control at a dose of 500 mg/kg. Colitis was induced by acetic acid solution (v/v) injection into the lumen of the colon intrarectally with a volume of 1 ml and dose of 5% except for the sham group. Colon sections were isolated to investigate the markers of oxidative stress including (a) MDA, (b) MPO, (c) SOD, and (d) GSH. Data were expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001.

Figure 5

Treatment with MRS improved colonic cell apoptosis caused by acetic acid. Mice were administered with MRS at a dose of 1 or 10 ml/kg by gastric gavage for a week before colitis establishment. SASP was used as a positive control at a dose of 500 mg/kg. Colitis was induced by acetic acid solution (v/v) injection into the lumen of the colon intrarectally with a volume of 1 ml and dose of 5% except for the sham group. The collected colon slides were subjected to TUNEL staining to assess cell apoptosis, and green spots represented apoptotic cells (400x). And the apoptosis rate was calculated. Data were expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001.

Figure 6

Treatment with MRS downregulated the inflammation-related TLR4/NF-κB/MAPK signaling pathway. Mice were administered with MRS at a dose of 10 ml/kg by gastric gavage for a week before colitis establishment. SASP was used as a positive control at a dose of 500 mg/kg. Colitis was induced by acetic acid solution (v/v) injection into the lumen of the colon intrarectally with a volume of 1 ml and dose of 5% except for the sham group. Colon tissues were collected, and the protein levels of major pathway components, including TLR4, MyD88, ERK, p-JNK, p38, and NF-κB p65, were analyzed by Western blotting. Data were expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001.

Figure 7

Treatment with MRS activated the anti-inflammatory response-related IL-10/JAK1/STAT3 signaling pathway. Mice were administered with MRS at a dose of 10 ml/kg by gastric gavage for a week before colitis establishment. SASP was used as a positive control at a dose of 500 mg/kg. Colitis was induced by acetic acid solution (v/v) injection into the lumen of the colon intrarectally with a volume of 1 ml and dose of 5% except for the sham group. Colon tissues were collected, and the protein levels of IL-10, p-JAK1, JAK1, p-STAT3, and STAT3 were analyzed by Western blotting. Data were expressed as means ± SD. ^∗P < 0.05 and ^∗∗∗∗P < 0.0001.