Discussion
DSS-induced colitis results in dysfunction of colon, including severe Intestinal barrier dysfunction, inflammatory infiltration, rectal bleeding, diarrhea, and gut microbiota changes (Dai et al., 2015; Rohwer et al., 2019; Wirtz et al., 2017), which is similar to clinical and histological manifestations of human ulcerative colitis (Li et al., 2020). Several studies have demonstrated the antioxidant agents can be against colitis, through effects on reducing inflammation and rebalancing gut microbiota (Liu et al., 2019; Peng et al., 2019). In this study, we adopted in vivo acute and chronic colitis mouse models induced by DSS (the concentration of 3% and 5%).
Diosmetin has already been demonstrated that exert protective effects against colon cancer and LPS induced acute lung injury (Koosha, Mohamed, Sinniah & Alshawsh, 2019; Liu, Ci, Wen & Peng, 2018). In this study, we illustrated that diosmetin can protect against LPS-induced injury to Caco2 and IEC-6 cells in vitro and against DSS-induced colitis in mice in vivo shown by alleviating histopathological changes and restoring the length of the colon. These data suggested that diosmetin can inhibit DSS-induced colitis.
The disruption of the intestinal epithelial barrier has been found to be relevant to the colitis (Su et al., 2009) which can lead to pathogenic antigen invasion. Thus, new ways of repairing mucosal barrier, improving mucosal healing and reducing mucosal permeability are considered to be potential methods of treating colitis. Among many proteins in mucosal barrier, ZO-1 is a major tightly linked scaffold protein, which associates with the epithelial integrity (Furuse et al., 1994). Occludin is essential for barrier function and tight junction stability (Saitou et al., 2000). Claudin-1 is a transmembrane protein and a component of tight junction strands (Furuse, Fujita, Hiiragi, Fujimoto & Tsukita, 1998). Transmembrane proteins Claudin, Occludin and perimembrane protein ZO form a closed complex. The complex formation and breakage of ZO-1, Claudin-1 and Occludin in intestinal mucosal epithelial cells can affect intestinal epithelial barrier function greatly (Morini, Babini, Barbieri, Baiocco & Ottolenghi, 2017). In our study, the expression levels of Claudin-1 and Occludin can be significantly increased by diosmetin in vitro andin vivo compared to DSS group. The expression of ZO-1 in immunofluorescence also significantly increased by diosmetin in vitro and in vivo compared to DSS group. These datas showed that diosmetin could inhibit DSS-induced intestinal epithelial barrier dysfunction.
It has been pointed out that the processes of oxidative stress and inflammation be relevant to DSS-induced colitis (Macias-Ceja et al., 2017; Zhao et al., 2019). The MDA is natural products of lipid hydroperoxide of organisms. Oxidative stress causes some fatty acids in cells to be oxidized and broken down into a series of compounds, including MDA, which has been frequently used as an indicator of oxidative stress (Qian et al., 2010). SOD has been pointed out that it can catalyze the disproportionation of superoxide anions to generate hydrogen peroxide (H2O2) and oxygen (O2), which is an important antioxidant enzyme in the body (Cao et al., 2015). GSH and GSH-Px can catalyze hydrogen peroxide and other peroxides to produce water and organic alcohol (Maejima, Kuroda, Matsushima, Ago & Sadoshima, 2011). In our study, we found DSS promoted oxidative stress shown by the low levels of GSH, GSH-Px and SOD and high level of MDA in colon tissue of mice, which were reversed by diosmetin. In addition, diosmetin significantly reduced ROS levels and mitochondrial ROS in Caco2 and IEC-6 cells caused by DSS. Therefore, inhibition of oxidative stress may be one mechanism by which diosmetin protects against DSS -induced colitis.
The inflammatory cytokines of IL-1β, IL-6, COX-2 and IFN-γ play leading roles in the formation of colitis (Biasi, Astegiano, Maina, Leonarduzzi & Poli, 2011). In addition, these pro-inflammatory cytokines can stimulate the activation of NF-κB transcription (Cao et al., 2015). Meanwhile, activation of NF-κB pathway further releases proinflammatory cytokines including TNF-α, IL-1β and IL-6 in DSS-induced colitis (Kim, Shin, Chung, Lee, Baek & Lee, 2019). IL-6 can stimulate the secretion of electrolytes from intestinal epithelial cells, result in increased permeability of endothelial cells, prompt the aggregation of neutrophils to the inflammation site and cause IBD (Zhang et al., 2011). Yamamoto (Yamamoto-Furusho, Santiago-Hernández, Pérez-Hernández, Ramírez-Fuentes, Fragoso & Vargas-Alarcón, 2011) have demonstrated that the expression of IL-1β in the lesion site of ulcerative colitis was significantly higher than that in the normal site, and also found that the gene polymorphisms of IL-1β and IL-1RN are relevant to genetic susceptibility and hormone dependence of ulcerative colitis. COX-2 plays a dominant role in the regulation of the inflammatory state by stimulating the biosynthesis of prostaglandins (PG) (Cianciulli, Calvello, Cavallo, Dragone, Carofiglio & Panaro, 2012). IFN-γ is a mediator of the development of DSS-induced colitis (An, Li, Bhang, Song & Youn, 2020). Thus, suppression of these inflammatory cytokine is an important target for the treatment of IBD. Our studies found, diosmetin significantly downregulated the mRNA levels of IL-1β, IL-6, COX-2 and IFN-γ, which suppress the inflammation signalling pathways, it may be another pathway of diosmetin against DSS-induced colitis.
Many studies have shown that gut microbiota is a key factor in the pathogenesis of IBD (Ni, Wu, Albenberg & Tomov, 2017; Zuo, Kamm, Colombel & Ng, 2018). Robert W. Li’s study of porcine colitis model demonstrated that mechanistic insights into the attenuation of intestinal inflammation and modulation of the gut microbiome by krill oil (Liu et al., 2020b). In the present work, we investigated the differences in gut microbiota of mice among control groups, DSS group and DSS with diosmetin group. Alpha diversity index dilution curve of each sample tend to be flat, indicating that the sequencing result was credible. Alpha diversity index has indicated that microbial diversity in DSS group significantly decreased compared with control group, after diosmetin treatment, the diversity was increased and the community composition was changed. From the level at phylum, DSS group increased the ratio of Firmicutes to Bacteroidetes due to the relative abundance decreased in Bacteroidetes and the relative abundance increased in Firmicutes compared with the control group. The similar phenomena has also been found in other studies (Jeffery et al., 2012; Peng et al., 2019). However, DSS with diosmetin group increased the relative abundance of Bacteroidetes and decreased the abundance of Firmicutes compared with the DSS group.
The LEfSe analysis at OTU level exhibited different dominant bacteria in different groups. Herein, Eggerthella lenta is kind of bacterial genera typical for inflammation (Soltys et al., 2020), and it was repressed in the DSS with diosmetin group. Previous studies have found that Pelomonas(25%) and Flavobacterium (13%) dominated the bacterial composition of patients with a high Crohn’s disease activity index, while Bacteroidetes had a relatively low abundance (4%). And in this study, diosmetin treatment observably decreased the bacteria abundance of Flavobacterium . Our research also found that diosmetin significantly increase bacteria abundance of Odoribacteraceae, prevotella, Rikenellaceae, Ruminococcus, Coprococcus, Roseburia, Oscillospira, Anaeroplasma and Synergistales compared with DSS group. These bacteria may become potential biomarkers of colitis in the future. Thus, these data demonstrated that diosmetin can modulate the community of gut microbiota, especially certain specific microbiome. These specific bacteria can be benefit for treating colitis.
Recently, circular RNA has emerged as a regulator of many biological processes through the regulation of the circular RNA-miRNA-mRNA network (Chen & Yang, 2015). Circ-Sirt1 (hsa_circ_0093887), one member of the Sirt1 circRNA, directly binded to miR-132/212 and interferes with Sirt1 mRNA (Kong et al., 2019). In this study, the levels of circ-Sirt1 were observably increased by diosmetin in vitro and in vivo, accompanied by up-regulated expression of Sirt1. Subsequently, the acetylation of NF-κB was inhibited and the Nrf2 pathway was activated. Of course, the mechanism of circ-Sirt1 in colitis still needs to be further demonstrated, and our research provides potential research directions for drug development
In summary, our studies have demonstrated that diosmetin exerted the therapeutic effects in DSS-induced colitis through several pathways, including reducing the expression levels of pro-inflammatory cytokines (IL-6, IL-1β, IFN-γ and COX2) and oxidative stress, increase expression of tight junction proteins of claudin-1, Occludin and ZO-1 and modulate gut microbiota. These pathways are partially mediated by diosmetin through the circ-Sirt1/Sirt1 axis. Diosmetin can potentially use to treat Colitis or become a lead compound for further optimization.