In the current study, we used a BALB/c mouse model to investigate the protective effect of commensal probiotic LBS on gut microbiota dysfunction in cisplatin-induced intestinal mucositis.
Cachexia (involuntary loss of weight > 5%), a typical side effect of chemotherapy that decreased survival in oncology patients is a significant health issue that affected cancer patients. Weight loss in cancer patients has also a detrimental effect on malnutrition as a result of anorexia, which can result in infection and life-threatening conditions. Our study is confirmed with those of another study, which reported that cisplatin caused dramatic weight loss and severe anorexia [39]. Additionally, pretreatment with LBS attenuates the loss of weight, improves food intake, and increases stool output. Interestingly, LBS could Improve gastrointestinal function and promotes intestinal health.
Moreover, the thymus and spleen are the main component of the immune organ in the body and play a vital role in nonspecific immunity, in addition, it considered the site of the proliferation of immunological cells [40]. Consequently, the immune organ index is usually used to indicate the growth of immune organs and assess the role of probiotics in immunoregulation [41, 42]. Lactobacillus has been reported previously that have an impact on activating nonspecific immunity [43, 44]. Meng et al. and Li et al. reported that some types of lactobacilli significantly enhanced the immune organ index[45] [46], and that is consistent with our study which revealed the thymus and spleen indexes in three groups of control and with LBS treatment were greater than in CP group. These results indicated that LBS could resist the influence of cisplatin in immunosuppression on immune organs.
Given that, Intestinal epithelial cells (IECs) play an essential role in regulating intestinal homeostasis and are considered part of the immune system by taking part in the transmission of the signal to the intestine through the secretion of cytokines and oxidative stress mediators [47]. Cytokines are low-molecular-weight glycoproteins that are produced by various cells in the body and have a crucial role in the progression of immune response and the pathogenesis of the inflammatory disease [48], the effect of LBS on the production of IL-2, IL-6, and IFN-γ was determined and suggested to play a significant role in intestinal mucositis [49]. Previous studies have reported that pro-inflammatory cytokines are significantly increased in the large intestine of rats following post-treatment with chemotherapy [50]. In addition, several studies have endeavored to target pro-inflammatory cytokines as a precautionary measure for intestinal barrier damage [51–54]. Our findings are confirmed with previous results and showed that (IL-1β, IL-6, and TNF-α) are significantly upregulated in the serum and/or colon tissue at mRNA levels following administration of cisplatin and decreased proinflammatory cytokine levels in mice receiving CP.LBS. This indicated that LBS could reduce the inflammation and therefore enhance those pro-inflammatory mediators involved in the progression of mucositis.
Also, tight junction proteins (TJs) offer a physical barrier to the intestine that contributes to maintaining intestinal barrier function, enhancing GI permeability, and maintaining the intestinal mucosal barrier [55]. TJs are comprised of two protein categories, integral transmembrane proteins that form a connection between neighboring cell membranes like Claudin and Occludin, and peripheral membranes like ZO-1, which connects Claudin and Occludin that may serve to maintain tight junctions intact [56]. Therefore, TJ integrity is dramatically maintained by the strong bond between the integral transmembrane and peripheral membrane protein in addition to the arrangement of the actin cytoskeleton.
Moreover, decreased in TJs levels always revealed an elevated permeability of the intestinal epithelial cell barrier [55–57]. Leocádio et al.[58] and Beutheu Youmba[59] showed that chemotherapeutic agents caused elevation in intestinal permeability that led to damage of the epithelial barrier through lowering protein expression level of TJs. Maintenance of the integrity of TJ suggests an important strategy to prevent and/or treat the pathogenesis of illness and intestinal damage. However, the mucin secreted by the goblet cells in the intestine is also important for creating the intestinal barrier [60]. Intestinal mucus could protect the intestinal epithelium against microbes by removing harmful bacteria [61].
In the CP model group staining results indicated intestinal and mucosal barrier alteration, and reduction in the goblet cells and tight junction proteins. The LBS restores the damaged TJs integrity caused by CP via restoring the goblet cell and improving tight junction stability. In keeping with these observations, an up-regulate level of Occludin has a role in further improving TJ integrity and preventing disorders of the TJ [62].
This drives us to explore further whether there is a difference in the gut microbiome structure or composition involved in the development of mucositis. The protective effect of LBS and restoration of microbiota was examined using microbiota 16S rRNA pyrosequencing. The intestine plays a significant role in homeostasis. The microbiota mainly interacts to enhance barrier integrity. However, diseases associated with metabolic disorders and immune suppression led to an imbalance in the microbial ecology and reduce the diversity and richness of the gut microbiome.
About five to seven of 52 identified bacterial phyla on Earth are known to live in the mammalian gut, Bacteroidetes and Firmicutes typically are the most common and highest relative abundance in the GIT, whereas phyla of Proteobacteria, Actinobacteria, Verrucomicrobia are found the less frequently [63]. Moreover, our experimental groups noted variations in the intestinal microbiota. Indeed, the model group increased the abundance of pathogenic bacteria Bacteroidota and Proteobacteria (mucosa-associated inflammation-promoting bacteria) at the phylum level and decrease beneficial bacteria Firmicutes, when compared with the other three groups, as well as LBS treated groups reversed the alteration induced by cisplatin.
However, in a healthy intestine, the gut flora normally has a minor abundance of phylum Proteobacteria, but the increased appearance of these bacteria in the gut refers to an imbalanced microbial community (dysbiosis) and has been reported to be elevated in chemotherapy-induced mucositis [64–66].
Obligate anaerobic bacteria are responsible to convert many fermentation products into short-chain fatty acids [67, 68]. The gut microbiota of a healthy colon is dominated by obligate anaerobes, whereas dysbiosis is often characterized by an increase of facultative anaerobic bacteria, Thus, in the large intestine, the dominance of obligate anaerobic bacteria maintains gut stability via the production of metabolites. Indeed, our results support this hypothesis that an imbalanced gut microbial community is featured by the enrichment of those facultative anaerobic bacteria. To sum up, we suggested that intestinal inflammatory response caused by cisplatin is associated with the overgrowth of members of facultative anaerobic bacteria such as Proteobacteria, and Actinobacteria while CP.LBS group showed less abundance in those bacteria and was more similar to the Control group.
Given that, a leaky gut is demonstrated by the raising of gram-negative bacteria, Proteobacteria is considered gram-negative bacteria, and their cell wall is mainly composed of lipopolysaccharide. Risk of disease associated with the secretion of LPS which is positively correlated and triggers inflammation [69]. Therefore, it may be hypothesized the microbes that are identified as a potential overgrowth in the CP group may relate to the inflammatory response in the intestine and induced mucositis.
Undoubtedly, at the genus level, the beneficial bacteria include: Lachnospiraceae, Lactobacillus, Alistipes, and Roseburia are observed in LBS groups and decreased dramatically in the cisplatin group. Those bacteria play a key role in the metabolism of undigested carbohydrates[70] and produce butyrate and other SCFAs through hydrolyzing starch and sugars that contribute to increasing the energy extracted from the diet [71–73]. Also, clinical studies have shown that Lachnospiraceae is vital in attenuating intestinal inflammation and repairing intestinal mucosal damage, serving as protective intestinal commensal bacteria [63]. Moreover, previous studies reported that Lactobacillus species upregulate mucin content that inhibits by cisplatin chemotherapy, it is suggested that dysfunction of the mucus barrier may contribute to cisplatin-induced mucositis [74].
We further studied the metabolome functions from 16S rRNA data by using the bioinformatics tools, the dysbiosis has side effects on metabolic and functional pathways and has an impact on the physiological processes of the organism, the host immune system, and nutrient biosynthesis as studied in the KEGG orthologous analysis. Ultimately, in our study, 16S rRNA sequencing analysis and a STAMP analysis were used to investigate the metabolome of the mice’s gut microbiome. We observed that LBS may improve the method of utilizing energy, carbohydrate metabolism, and nutrient absorption. Implying that LBS may act as an immunoprotective agent.
In summary, these findings enhanced that pretreatment with probiotic Lactobacillus rhamnosus protects against cisplatin-induced mucosal barrier damage through maintenance of the barrier integrity and up-regulating of tight junction proteins and anti-inflammatory properties, in addition, enhance positively the microbiome diversity and increasing the beneficial bacteria (Lachnospiraceae and Lactobacillus). In turn, this regulates gut microbiota imbalances caused by cisplatin and provides great potential in mitigating intestinal mucositis as a dietary agent.