IM is a common side effect of chemotherapy, and with the increasing number of cancer diagnoses and the prevalence of chemotherapy drug use, chemotherapy-associated mucositis has become increasingly common. MTX, as an anti-cancer drug widely used for leukemia and other malignancies, is a structural analogue of folic acid that can inhibit the metabolism of folic acid by competitively inhibiting dihydrofolate reductase, thereby inhibiting the de novo synthesis of purines and pyrimidines 26,27. Over the past few decades, MTX has been successfully used alone or in combination with other drugs to treat various cancers and autoimmune diseases 28. Unfortunately, however, due to its multi-organ toxicity, especially in the gastrointestinal system, the therapeutic potential of MTX can be reduced, with the drug typically having to be ceased in response to bone marrow toxicity, cardiotoxicity, nephrotoxicity, and liver toxicity 29,30.
Damage to the gastrointestinal mucosa following MTX treatment in cancer patients includes villus shortening and fusion, epithelial atrophy, crypt loss, inflammatory infiltration of the lamina propria, goblet cell depletion, and barrier dysfunction due to loss of mucosal integrity and reduced nutrient absorption 31. Although various measures to prevent these effects have been adopted, such as reducing the therapeutic dose and duration of treatment to reduce the intestinal damage caused by MTX, there remains no satisfactory therapeutic intervention to prevent MTX-induced small intestinal injury 32,33.
MTX-induced IM not only causes direct damage by DNA copy inhibition but also induces inflammation and the generation of reactive oxygen species (ROS) 34–36. Studies have demonstrated that MPO and MDA levels are increased after treatment with MTX, suggesting the possible participation of neutrophil infiltration and ROS in MTX-induced IM 34,37. In the present study, we found that levels of MPO, a marker of neutrophil accumulation and infiltration during intestinal mucosa damage, were increased in the epithelial mucosa of rats with IM induced by MTX. Kolli et al. also reported that oxidants such as MDA, which is a product of lipid peroxidation, were increased in MTX-induced small IM 38. MPO is also an enzyme with peroxidative damage secreted by neutrophils after neutrophil, and it also causes peroxidative damage to tissues. According to our results, oral administration of DKT can inhibit the inflammatory process and ROS damage, as evidenced by the fact that DKT can reduce the production of MPO and MDA.
In addition to peroxidation-related factors, growth factors secreted from mucosa also played a role in this process. TGF-β1 and HIF-1α were reported to protect intestinal integrity 39,40. There have been articles showing that TGF-β1 is dihedral in the homeostasis of the small intestine, having the effect of promoting both inflammation as well as damage repair 41,42. The mRNA level of TGF-β1 in the DKT-MTX group was higher than that in the control group on day 15, but it was lower in the jejunum and ileum than in the MTX group. This method of mucosal repair was also verified in another experiment of DKT in the treatment of intestinal injury repair in rats 20.
The changes in the level and localization of ZO-1 induced by MTX may lead to disorder of the barrier function, which leads to increased intestinal permeability, resulting in intestinal mucosal barrier dysfunction 43. ZO-1, as a tight junction scaffold protein, has been shown to render structure firmness and impermeability to the junction and functions as a link between occludin and the actin, which are the major elements in the structure of the barrier of the small intestine 44. However, Kuo et al. reported that the tight junction protein ZO-1 was dispensable for the barrier function but critical for intestinal mucosal repair 45. An intravital confocal microscopy study suggested that the redistribution of ZO-1 contributed to the intestinal barrier function and was the earliest cellular event identified as a herald of physiological cell shedding. Furthermore, redistribution of this tight junction function along the lateral plasma membrane sustained the epithelial barrier during cell shedding 46. This evidence suggested that ZO-1 was associated with tight junctions in the small intestinal mucosal epithelium as well as with the physiological renewal and essential function of pathological repair of the small intestinal mucosa. In the present study, an immunohistochemical analysis revealed that ZO-1 was predominantly localized along the apical membrane of the intestinal villi in the control and DKT groups. In contrast, a significant increase in ZO-1 immunostaining was observed along the apical membrane of the intestinal villi in the DKT-MTX group compared with the MTX group. DKT has the potential to promote the expression of ZO-1 in the small intestinal mucosa epithelium to facilitate the repair of tight junctions and improve the mucosal nutrient absorption.
The expression of genes related to the amino acid transport-related proteins EAAT3 and B0 + AT was up-regulated after the administration of DKT in the DKT-MTX group. A prospective study of the effects of DKT on the blood flow in the superior mesenteric artery (SMA) and portal vein (PV) reported that DKT may modulate the SMA and PV blood flows by acting on intestinal microvessels 47. The absorption of nutrients around the ileocecal region was related to the blood flow of the intestine and PV. A prospective open-labeled randomized exploratory study also demonstrated that DKT can improve the perioperative nutritional status of patients with colorectal cancer 48.
DAO is a highly active intracellular enzyme in the upper villi of the human and mammalian small intestinal mucosa that plays a role in the metabolism of histamine and various polyamines, and its activity is closely related to nucleic acid and protein synthesis in mucosal cells, reflecting the integrity and degree of damage to the mechanical barrier of the intestine 49. DAO regulates cell proliferation by degrading polyamines, which are essential substances in the processes of mitosis and meiosis 50,51. Plasma DAO activity was reported to be associated with the degree of small intestinal injury and had potential utility for measuring mucositis during chemotherapy 49,50. A previous study showed that an increase of DAO activity in the intestine of mature rats leads to an increase in the degree of DAO activity in plasma 52. In contrast, intestinal mucosal damage caused by hypertonic sodium sulfate solution or atrophy caused by a low-fiber diet reduces plasma DAO activity 53.
In the present study, with the occurrence of MTX-induced IM, the DAO content in rat plasma decreased, and with the onset of gastrointestinal tract symptoms aggravated and reduced to a lower level. In the DKT-MTX group, as DKT promoted the recovery of rat mucosa, the serum content of DAO also increased. The IHC results also confirmed that the rat small intestinal mucosal epithelial villi of the DKT-MTX group contained higher DAO levels in the mature apical membrane than that in MTX group.
DKT is mainly composed of dried Japanese pepper extract, ginger extract, and ginseng extract. Ginger can reportedly improve ileum damage caused by MTX, shortened villus fusion, inflammatory cell infiltration, and goblet cell depletion 54. The present findings support the efficacy and safety of ginger, possibly due to the antioxidant properties, as was observed in our experiments. As one of the main components of DKT, ginger extract has been verified to have a variety of intestinal effects, including anti-inflammatory and antioxidative effects in ulcerative colitis 55–57. The mechanism underlying the restoration of the intestinal barrier function by ginger extract involves the increased expression of ZO-1 and claudin-1 protein 55. Regarding dried Japanese pepper extract (Zanthoxylum fructus), there have been articles reporting that Zanthoxylum fructus extract inhibits the reduction in mast cell activation by inhibiting sphingosine kinase 1, mainly reducing the release of inflammatory mediators 58. In research on DKT, it is reported that DKT depolarizes the pacemaker potential of Cajal interstitial cells in an internal or external Ca2+-dependent manner by stimulating the 5-HT4 and muscarinic M3 receptors. Its main ingredients are ginseng and ginger root, which help DKT regulate the activity of the intestine and reduce the dysfunction of the small intestine 14. Some researchers have screened cytoprotective agents against MTX-induced cell genotoxicity from among biologically active phytochemicals and found that agents such as Siberian ginseng and curcumin have cytoprotective effects 59. Total ginsenosides was reported to promote intestinal epithelial cell proliferation, presumably via the regulation of the cell cycle and of the expression of proliferation-related proteins by polyamines 60. Specifically, DKT appears to be less effective in reducing rat mortality due to severe mucosal necrosis but shows marked efficacy in another aspect, such as reducing injury and promoting mucosal repair, as well as improving symptoms. Furthermore, DKT may also have some efficacy against chronic mucositis of the small intestine, so a further investigation regarding chronic mucositis in the clinical setting or as a daily disease will be needed in the future, with particular focus on the therapeutic effect of DKT on chronic mucositis of the small intestine.
In conclusion, DKT may be able to protect against MTX-induced acute small intestinal mucosal injury in a rat model via anti-peroxidation, stimulating cell proliferation, and stabilizing the mucosal barrier. Although DKT comprise numerous chemically diverse compounds with multi-target effects, it has been shown to be able to protect and treat intestinal injury, regardless of administration as a single component or a mixture of multiple components.