A new recombinant MS-superoxide dismutase alleviates 5-fluorouracil-induced intestinal mucositis in mice

Abstract

Intestinal mucositis is a common side effect of anticancer regimens that exerts a negative impact on chemotherapy. Superoxide dismutase (SOD) is a potential therapy for mucositis but efficient product is not available because the enzyme is degraded following oral administration or induces an immune reaction after intravascular infusion. Multi-modified Stable Anti-Oxidant Enzymes^® (MS-AOE^®) is a new recombinant SOD with better resistance to pepsin and trypsin. We referred it as MS-SOD to distinguish from other SODs. In this study we investigated its potential to alleviate 5-FU-induced intestinal injury and the mechanisms. An intestinal mucositis model was established in C57/BL6 mice by 5-day administration of 5-FU (50 mg/kg every day, ip). MS-SOD (800 IU/10 g, ig) was given once daily for 9 days. 5-FU caused severe mucositis with intestinal morphological damage, bodyweight loss and diarrhea; MS-SOD significantly decreased the severity. 5-FU markedly increased reactive oxygen species (ROS) and inflammatory cytokines in the intestine which were ameliorated by MS-SOD. Furthermore, MS-SOD modified intestinal microbes, particularly reduced Verrucomicrobia, compared with the 5-FU group. In Caco2 cells, MS-SOD (250-1000 U/mL) dose-dependently decreased tBHP-induced ROS generation. In RAW264.7 cells, MS-SOD (500 U/mL) had no effect on LPS-induced inflammatory cytokines, but inhibited iNOS expression. These results demonstrate that MS-SOD can scavenge ROS at the initial stage of injury, thus play an indirect role in anti-inflammatory and barrier protein protection. In conclusion, MS-SOD attenuates 5-FU-induced intestinal mucositis by suppressing oxidative stress and inflammation, and influencing microbes. MS-SOD may exert beneficial effect in prevention of intestinal mucositis during chemotherapy in clinic.

Keywords: 5-fluorouracil; chemotherapy; cytokines; diarrhea; intestinal microbes; intestinal mucositis; manganese superoxide dismutase; oxidative stress.

Fig. 1

Schedule for the administration and effects of MS-SOD on 5-FU-induced changes in body weight, diarrhea and fecal blood score in mice. MS-SOD (800 IU/10 g, i.g.) or vehicle (diluted water, i.g.) was administered from day 1 to day 9, and 5-FU (50 mg/kg, i.p.) or vehicle (saline, i.p.) was administered from day 1 to day 5. Animals were sacrificed on days 4, 6, 8 and 10 (n = 6/group) (a). Overview of the study setup. The body weight of mice treated with vehicle or MS-SOD was recorded daily (b), and diarrhea and fecal blood scores (c, d) were recorded from day 4. Significant body weight loss induced by 5-FU was observed on day 3, and MS-SOD significantly reduced this loss on days 5, 8 and 9. Diarrhea and the fecal blood score were worsened daily in the vehicle group, but MS-SOD treatment reduced the score each day. The data are presented as the means ± SEM (n = 6); *P < 0.05, **P < 0.01, and ***P < 0.001 according to unpaired t-tests

Fig. 2

Histological changes and shortening of the small intestine and colon after 5-FU administration and MS-SOD treatment. Five days after 5-FU challenge, the small intestine (a) and colon (b) from control, 5-FU, and 5-FU + MS-SOD mice were sectioned and prepared for H&E staining (×200), and shortening of the small intestine (c) and colon (d) was assessed to evaluate the severity of mucositis. Villus height and crypt depth reduction in the small intestine occurred in the 5-FU group, accompanied by mucosal erosion and subacute inflammation in the colon. Additionally, the intestinal length was decreased due to atrophy induced by 5-FU, but MS-SOD treatment improved the symptoms. The data are presented as the means ± SEM (n = 6); *P < 0.05 according to unpaired t-tests

Fig. 3

Effects of MS-SOD on tight junctions and cytokines in the small intestine after 5-FU treatment. qPCR analysis was performed to measure the transcription levels of occludin (a), IL-6, IL-17α, IL-22 (c), CXCL1 and CXCL2 (d). Western blotting was used to measure the translation level of occludin (a). An ELISA was used to measure the IL-6 concentration in serum (c). Both occludin mRNA and protein levels in the small intestine were decreased in mice after 5 days of 5-FU injection but were increased in the MS-SOD group. The mRNA expression levels of the main pro-inflammatory cytokines, IL-6, IL-17α and IL-22 as well as the chemokines CXCL1 ans CXCL2, were upregulated in the 5-FU group, but these cytokines were downregulated in MS-SOD-treated mice. The serum IL-6 concentration was in accordance with the mRNA level. ELISA method was used for MPO content measure. MPO content in intestinal tissue homogenates was significantly increased in the 5-FU group, but did not decrease in MS-SOD group (b). The data are shown as the means ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. In a, the repeated blots in each group indicate three individuals

Fig. 4

Effects of MS-SOD on ROS levels and antioxidant capacity in the small intestine during 5-FU treatment. The ROS content and total antioxidant capacity of the small intestine at different times were detected using commercial kits (a, b). qPCR analysis was performed to measure the transcription level of Nrf2. Western blotting was performed to measure the level of HO-1 and Nrf2 translation (c). Activity of SOD and the content of superoxide were detected via kits (d). MS-SOD reduced the ROS content and increased the total antioxidant capacity in the small intestine during 5-FU injection. MS-SOD increased both Nrf2 and HO-1 expression in the small intestine after 5 days of 5-FU treatment. MS-SOD also increased the activity of SOD and decreased the superoxide content. The data are shown as the means ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. In (c), the repeated blots in each group indicate three individuals

Fig. 5

Effects of MS-SOD on gut microbes after 5-FU treatment. Cecum feces were collected on day 6, and the relative abundance of microbes was analyzed via next-generation sequencing of bacterial 16S DNA. At the phylum level, 5-FU treatment decreased the abundance of Bacteroides but increased the abundance of Lachnospiraceae and Verrucomicrobia compared with the control mice. MS-SOD reversed this situation (a). At the genus level, more detailed changes were observed (b)

Fig. 6

In vitro activity of MS-SOD in tBHP treated Caco-2 cells. Cells were treated with 31.25 µM tert-butyl hydroperoxide (tBHP) for 1 h, and MS-SOD at the indicated concentrations were also added simultaneously. Cells were incubated for 30 min with 25 µM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and harvested for ROS content detection. tBHP induced an increase in ROS generation in Caco2 cells, but MS-SOD significantly decreased tBHP-induced ROS generation in a dose-dependent manner. **P < 0.01, ***P < 0.001

Fig. 7

In vitro activity of MS-SOD in LPS-treated RAW264.7 cells. RAW264.7 cells were pre-incubated with 500 U/mL MS-SOD for 6 h, then stimulated with 10 g/mL LPS and samples were collected 6 h after LPS stimulation, IL-1β, IL-6, TNF-a and iNOS mRNA expression levels were measured with qPCR. LPS induced an increase in ROS generation in RAW264.7 cells, but MS-SOD significantly decreased LPS-induced ROS generation. *P < 0.05, **P < 0.01

Fig. 8

Possible mechanism by which MS-SOD reduces intestinal injury in mice with 5-FU-induced mucositis. The novel SOD product MS-SOD demonstrated efficacy in treatment of 5-FU-induced intestinal mucositis in this study. MS-SOD exerted effects through the following three main aspects. First, MS-SOD reduced ROS levels at the early stage of mucositis, thus contributing to inhibition of oxidative stress in intestinal crypts. Second, MS-SOD inhibited the expression of select cytokines associated with inflammation in the intestine. Third, MS-SOD changed the relative abundance of intestinal flora in the model mice. These three factors are relevant in the pathogenesis of mucositis, and MS-SOD improved these factors, suggesting several possible mechanisms by which MS-SOD can reduce mucositis