The most commonly used method to assess intestinal mucosal barrier function is the measurement of intestinal mucosal permeability with fluorescein-dextran 4,000 (FITC-dextran 4,000, FD-4), D-lactate, or diamine oxidase (DAO). FD-4 is an organic fluorescent pigment commonly used in immunofluorescence and flow cytometry to bind to different antibodies with the help of isothiocyanate reactive groups. D-lactate is a metabolite of many bacteria in the gastrointestinal tract. Mammals lack D-lactate dehydrogenase and cannot metabolize it rapidly, so the amount of D-lactate in the body under normal condition is small and relatively stable. When the intestinal mucosal barrier is disrupted, more D-lactate enters the circulation through the damaged intestinal mucosa and the blood D-lactate level increases significantly.

DAO is an intracellular enzyme in mammalian intestinal mucosal cells. When the mucosal cells are damaged, the intracellular DAO is released into the blood and the serum DAO is increased. The higher serum DAO, the severer mucosal damage as well as higher permeability of the intestinal mucosa (Xun et al., 2015). In addition, it has been reported that the level of endotoxin in circulation is positively associated with the degree of permeability of the intestinal mucosa (Tornai et al., 2017).

Lu et al. reported that FD4 in the colon of the colitis rats were increased as six times as that of normal rats and QCS could effectively decrease FD4 in the colon and promote ulcer healing of the colitis rats (Lu and Xie, 2010). Previous studies showed that the levels of DAO in serum were increased in the intestinal ischemia/reperfusion (IIR) rats, suggesting IIR could damage the mucosal integrity (Peng et al., 2018). GRb1 has antioxidant and antiapoptotic effects (Zheng et al., 2017). Chen et al. occluded the superior mesenteric artery for 75 min and re-perfused for 3 h to establish an IIR-intestinal epithelial injury model. Compared with the model group, GRb1 treatment group and pretreatment group could decrease the serum DAO and the expression of Akt and p-Akt. The protection of GRb1 could be eliminated by PI3K inhibitor, suggesting that GRb1 could decrease the intestinal permeability via PI3K/Akt pathway (Chen et al., 2019) (Figure 1).

The disruption of intestinal TJs play an important role in the pathogenesis of UC (Tan et al., 2019). The decreased expression of ZO-1 and occludin proteins in UC patients leads to increased intestinal permeability and abundant pro-inflammatory factors invading the lamina propria of the colon, which triggers an intestinal immune response (Kim et al., 2018). Zhou et al. found QCS could increase the expressions of ZO-1 and occludin which were inhibited in the colitis mice induced by DSS (Zhou G. et al., 2021). Cai et al. found that LPS could suppress the expression of IPEC-J2 cells while this suppression could be reversed when the cells were pretreated by GA, an ingredient of gallnut (Liu et al., 2019; Cai et al., 2022).

Zhang et al. found purslane extract could not only decrease p-ERK, p-eIF2 α, Beclin1, and LC3II protein expression in IL-10^−/− model mice. But also reduce the damage of IECs by decreasing endoplasmic reticulum stress through the pERK-eIF2α/Beclin1-LC3II pathway (Zhang et al., 2022). Ginsenosides were found to significantly enhance colonic mucosal repair and promote intestinal mucosal healing in a TNBS-induced rat colitis model by activating the ERK and Rho-dependent pathways (Toyokawa et al., 2019). Indigo could increase IL-10 and IL-22 expression in isolated LP monocytes from colitis rats induced by TNBS and DSS. But this effect was not present in AhR^−/− mice. Moreover, indigo and indirubin could increase IL-10-producing CD4⁺ T cells and IL-22-producing CD3⁺⁻RORγt cells instead of CD4⁺Foxp3⁺Treg cells (Dudakov et al., 2015; Kawai et al., 2017; Wang et al., 2017).

Makoto et al. carried out a multicenter randomized controlled trial to investigate the safety and efficacy of IN in UC patients. A total of 86 patients were enrolled and given IN 0.5, 1.0, and 2.0 g or placebo for 8 weeks. The rates of mucosal healing were 13.6% in the placebo group, 56.5% in the 0.5 g IN group, 60.0% in the 1.0 g IN group, and 47.6% in the 2.0 g IN group (p = 0.0278 compared with placebo) (Naganuma et al., 2018). The post hoc analysis of this trial also demonstrated that the MH rate of IN was significantly higher than that of the placebo in the patients with steroid-dependent disease (p = 0.009) (Naganuma et al., 2020).

Intestinal EP is determined by the epithelia as well as the mucosal micro-vascular endothelia. The pathological micro-vascular endothelia cannot supply sufficient oxygen to the intestinal epithelia, destroy vasular function and accelerate the infiltration of inflammatory cells (Thornton and Solomon, 2002). Increased mucosal VP and decreased and slow blood flow also could lead to epithelia hypoxia (Taylor and Colgan, 2007), which induces increased pro-inflammatory cytokines resulting in intestinal epithelial cell injury and dissolution of TJs between goblet cells (Rezaie et al., 2007). Therefore, VP elevation may be the initial episode, earlier than increased EP, in UC occurrence and recurrence (Tolstanova et al., 2012), and VEGF inhibition can decrease VP and treat UC (Tolstanova et al., 2009). Wang S. et al. (2018) intervened DSS-colitis mice with QCS and found that QCS could improve colonic hypoxia and reduce colonic VP via VEGF/HIF-1a signaling pathway to improve vascular endothelial barrier function.

Intestinal microbiota is a complex community (Franzosa et al., 2019). It is also a crucial factor in intestinal mucosal injury (Kostic et al., 2014). The importance of intestinal flora in the occurrence and recurrence of UC has drawn increasing attention from the global scientific community. Increasing evidence has proven that the alteration of intestinal flora is a common feature in UC patients and mice colitis (Wlodarska et al., 2015). It has been generally accepted that UC is related to lower diversity of bacteria (Machiels et al., 2014; Prosberg et al., 2016; Knoll et al., 2017; Lopez-Siles et al., 2017) and decreased abundance of bacteria (Yao et al., 2016; Ricciuto et al., 2020). The damaged intestinal mucosal defense allows a large number of bacteria and their metabolites to enter the blood circulation and triggers a systemic immune response, which plays an extremely important role in the pathogenesis of UC (Kato et al., 2014; Liu et al., 2016). Therefore, the maintenance of intestinal microbial homeostasis is important to prevent the occurrence and recurrence of UC. Wen et al. observed whether QCS could treat UC via regulating colonic microbiota. They divided SD rats into 4 groups: normal group, DSS-induced colitis model group(M), M1 group and M2 group. The rats in M1 group were treated with DSS and filtrate of faeces of colitis rats. The rats in M2 group were treated with DSS and QCS-treated filtrate of faeces of colitis rats. They found the DAI score and the expression of TLR4 and NF-κB of M2 group were significantly lower than those of M group and M1 group. Additionally, QCS were found to improve the proliferation of B. bifidum and B. thetaiotaomicron. These results suggested that QCS could promote colon mucosa repair by improving the growth of Bifidobacterium bifidum and Bacteroides thetaiotaomicron and inhibiting TLR4/NF-kB signaling pathway (Wen et al., 2021). Harmful bacteria like Bacteroidetes and Proteobacteria producing TNF-α, IL-6, and IL-8 were found to increase, while beneficial bacterium like Firmicutes secreting IL-10 decrease in DSS-induced colitis mice (Ma et al., 2018; Liang et al., 2019). 16S rDNA sequence analysis proved that IN could adjust the composition of intestinal flora in colitis mice, especially related to the anaerobic Gram-positive bacteria of Turicibacter and Peptococcus (Liang et al., 2019; Yang et al., 2021). IN treatment could decrease the percentage of the harmful bacteria in colitis mice and restore the microbiota composition, which proves that IN could repair the DSS-induced gut microbiota imbalance (Liang et al., 2019). Indigo might exert its protective effects by increasing butyrate of the microbiota (Sun et al., 2020). The interaction between indigo and portulaca oleracea polysaccharide could promote short-chain fatty acid (SCFA) generation and metabolism (Fu et al., 2022). Wang et al. divided metronidazole-disposed-C57BL/6 mice into control, DSS, and DSS + ginseng pretreatment (28 mg/d/kg) groups. They found that the levels of three fecal endogenous metabolites including lactate, linoleic acid and malic acid of ginseng pretreatment group were significantly lower than those of DSS group, suggesting ginseng might repair the damaged intestinal barrier by regulating the intestinal microbiota (Wang CZ. et al., 2018).

A fundamental experiment demonstrated that the inhibition of JAK/STAT pathway could benefit the integrity of the intestinal mucosa barrier (Chu et al., 2018; Soendergaard et al., 2018). Tofacitinib can inhibit JAK and is used to treat UC (De Vries et al., 2019). Researchers stimulated murine peritoneal macrophages with lipopolysaccharide (LPS), and they found the inhibition of JAK/STAT could decrease the secretion of inflammatory factors like IL-1β and IL-18 (Montoya et al., 2018). QCS and its ingredients, including indigo, ginsenoside R1, and GA, could exert the same effect as the inhibitor of JAK/STAT to alleviate inflammation and protect the intestinal mucosal barrier (Yu et al., 2021). In addition, GA has shown its antibacterial, anti-inflammatory, and antioxidant activity in vitro (Phonsatta et al., 2017; Cai et al., 2022). IgA produced by B cells, is a strong immunoglobulin on goblet, and protect the intestinal mucosal barrier from microorganism invasion (Pabst, 2012). IgA synthesis and secretion is the most recognized characteristic in mucosal immunity (Lycke and Bemark, 2017). Ginsenoside relieves the immune disorder in three ways: in the spleen, it can reverse proinflammatory and anti-inflammatory lymphocyte subsets ratio; in the intestine, it can stimulate CD4⁺ T cells to produce mucosal beneficial cytokines; and on the surface of goblet, it can assist B cells to secrete IgA to help mucin expression and the expression of TJs (Zhou R. et al., 2021). The decreased secretion of mucin by goblet cells weakens the intestinal mucus barrier and further impairs the function of the intestinal barrier (Zheng et al., 2019). GA could ameliorate dimethylhydrazine (DMH)-induced colonic inflammation, mucin depletion and intestinal epithelial cells’ oxidative stress, proliferation, and apoptosis disintegration in Wistar rats by upregulating the glutathione levels and activities of detoxifying enzymes (Shree et al., 2020).