In this study, L. plantarum DLPT4 could maintain viability in artificial gastrointestinal condition, indicating that the strain could reach host colon with high viable bacterial cells. When probiotics pass through gastrointestinal tract with gastric acid, bile salts and pancreatin respectively, and successfully reach and colonize the host colon, their probiotic function can be exhibited (Aslim, 2004, Lye et al., 2009).
Macrophages were activated to phagocytize and digest pathogens, at the same time, macrophages indirectly activated lymphocytes and other immune cells, making the body produce immune responses to inflammatory reactions (Lian et al., 2017, Zheng et al., 2017) and secrete pro-inflammatory cytokines such as IL-6, TNF-α (Andrina et al., 2019). The lipopolysaccharide (LPS) is a compound of the cellular wall of Gram-negative bacteria, could induce inflammatory response in RAW264.7 cells. (Cao et al., 2019) In the study, L. plantarum DLPT4 treatment inhibited the inflammatory response of RAW 264.7 cells exposed to LPS. Cao et al. (2019) (Cao et al., 2019) observed that Punicalagin could reduce the content of NO, IL-8 and TNF-α by LPS-induced cells. It was speculated that that L. plantarum DLPT4 inhibited the pro-inflammatory cytokines production LPS-treated RAW 264.7 cells through NF-κB and MAPK signaling pathway. In the RAW 264.7 cell experiment, the treatment by L. plantarum DLPT4 prior to LPS exhibited higher ameliorating effect on the inflammatory response induced by LPS. Therefor in the subsequent animal experiment, L. plantarum DLPT4 was administered to BABL/c mice for 25d firstly, and then cyclophosphamide was given to the mice for the subsequent 3 days.
The host immune system is composed of immune organs, immune cells and immune reactive substances (J et al., 2013). The dysregulation of immune response may cause inflammation (J et al., 2006) and autoimmune diseases (Garry and John, 2014). CTX as an alkylating agent can inhibit the cellular, and humoral immune response and the mice treated by CTX can be used as an animal model with weakened immune system to validate the immunoregulatory capacity of probiotics. Different probiotics show different immunomodulatory effects (Bahman et al., 2019, M et al., 2019). In this study, we found that L. plantarum DLPT4 oral administration could mitigate the weight loss, reduce the production of pro-inflammatory factors, and regulate the abundance of gut flora to relieve inflammation symptoms of CTX-treated BALB /c mice, indicating that L. plantarum DLPT4 ameliorate the immunosuppression of the BALB /c mice treated by CTX.
CTX is an effective alkylating agent for treating malignant tumors (Ashkan et al., 2009). However, long-term use of CTX can lead to the decline of host immune function, and the destruction of the intestinal mucosal barrier (Gaoxiang et al., 2019). The intestinal mucosal barrier is essential for the host defense system to eliminate invading pathogenic microorganisms (Krajmalnik-Brown et al., 2012, Ren et al., 2019). Intraperitoneal injection of CTX can reduce the diversity of the intestinal flora in mice, and cause intestinal inflammation of the mice (Xu and Zhang, 2015, In and Soo, 2017). In this study, the decrease of the number of Lactiplantibacillus and Bifidobacterium of the mice feces in MG may be attributed to the intraperitoneal injection of CTX, which led to intestinal flora imbalance of the BALB/c mice. While the CTX-treated BALB/c mice receiving L. plantarum DLPT4 showed the increased levels of Lactiplantibacillus and Bifidobacterium in the feces. Enhancement of Lactiplantibacillus and Bifidobacterium levels are reported to ameliorate impaired intestinal immunity (Feng et al., 2018, Yueyue et al., 2019). The CTX-evoked damage to the colon of immunosuppressed BALB/c mice could be ameliorated by regulating gut microbiota such as Lactiplantibacillus, Coriobacteriaceae, Bacteroides (Yujiao et al., 2019), Alloprevotella (Kong et al., 2020), and up-regulating the secretory immunoglobulin A (SIgA) and mucin2 (Yu et al., 2019, Yujiao et al., 2019, Kong et al., 2020).Oral administration of L. plantarum DLPT4 could improve the ratio of Firmicutes / Bacteroides, which may improve the immune functions and relieve weight loss of the CTX-treated mice. Firmicutes and Bacteroides are generally believed to help the body to collect energy (Xin et al., 2018).
Gut commensal microbes colonize on the intestinal mucosa to produce organic acids and bacteriocins to inhibit the adhesion of pathogenic bacteria presenting as decreasing proinflammatory signaling (Ulla and Airi, 2013, Young et al., 2015). In this study, L. plantarum DLPT4 promote the recovery of intestinal flora of the CTX-treated mice. We speculate that the recovery of the mice intestinal barrier function may be due to the expression of Occludin like intestinal tight junction proteins and the recovery of intestinal flora by L. plantarum DLPT4 oral administration.
Intestinal flora plays an important role in the maintenance of intestinal barrier function. CTX induced intestinal mucosal injury, resulting in intestinal immune disorder. According to the HE stained histopathology images, L. plantarum DLPT4 induced crypt epithelial proliferation and increased goblet cells, indicating that L. plantarum DLPT4 oral administration increase the secretion of tight junction proteins and enhance the intestinal barrier function
The oral administration of L. plantarum DLPT4 stimulated the secretion of IgA, and IgG, and reduced the expression level of pro-inflammatory factors (IL-6, IL-1β and TNF-α) in the CTX- immunosuppressed BALB /c mice. Immunoglobulin plays an important role in the body's humoral immunity, for example, IgA, IgG and IgM have antibacterial and antiviral effects (Feng et al., 2014). The expression of cytokines and immunoglobulins plays important roles in the host immune system (Huang et al., 2016, Gaoxiang et al., 2019). Persistent releases of pro-inflammatory cytokines cause deleterious effects and affect the repair process of damaged tissues at the site of immune reaction (Jose and Kurup, 2017).
Non-digestible carbohydrates reach the cecum and proximal colon, and are metabolized by gut microbes to produce short-chain fatty acids, which is beneficial to host health (A et al., 2005, Yu et al., 2018). The decrease of SCFAs content in intestinal tract can increase intestinal permeability and promote inflammation (V et al., 2014). Conversely, the increase of SCFAs can promote intestinal cell metabolism, promote antibody production by B cells, and regulate intestinal mucosal immunity by influencing various immune cells (Young et al., 2015). Probiotics such as Bifidobacterium (Tian et al., 2019) and Lactiplantibacillus (Yujun et al., 2020) can affect the production of SCFAs in colon of the host. In this study, intraperitoneal injection of CTX reduced the fecal SCFAs contents of the CTX-treated mice. L. plantarum DLPT4 oral administration could stimulate the production of part of SCFAs in the CTX-treated mice but no significant difference. The results were similar to Liu (Wenwei et al., 2021). But L. plantarum DLPT4 oral administration increased the Bifidobacterium and Lactiplantibacillus proportion of the gut flora in the mice.
T cell receptor (TCR) signal transduction plays a key role in the development and function of T cells, while T cells affect the host immunity (Warner et al., 2013). Several studies showed that TCR signal strength is important to Treg and Th17 Cell Differentiation (Hidehiro and E, 2013). Further the Tec family kinase ITL is activated, leading to the activation of MAPK pathway (Berg et al., 2005). L. plantarum DLPT4 activated T cell receptor signaling pathway and MAPK signaling pathway in CTX-immunosuppressed BALB/c mice. It was reported that the immunity of CTX-immunosuppressed mice could be enhanced by regulating the MAPK pathway mediated by TLR2/4 and activating the transcription activities of activator protein-1 and NF-κB (Ting et al., 2017, Bai et al., 2019). It was speculated that oral administration of L. plantarum DLPT4 might activate phosphorylated p38 and AP-1 MAPK signaling pathways to exert immunomodulatory activity.
Overall data suggested that L. plantarum DLPT4 oral administration modulated the intestinal microbiota of the CTX-immunosuppressed BALB/c mice, which affect certain metabolic pathways and immune system of the mice.