Probiotics improve animal health and nutrition by improving feed value and enzymatic effects, and play a very important role in improving animal health, nutrition and activating immune response(Dawood et al., 2016). Zaineldin et al. reported that that supplementation of Bacillus subtilis in the diet can significantly improve growth performance (FBW, WG and SGR) (Zaineldin et al., 2018). Hg can activate energy-consuming detoxification processes, which consumes a lot of energy in the fish, resulting in a decrease in energy, which is not conducive to the growth of the fish (Sfakianakis et al., 2015). In this study, although the Se-rich B. subtilis group did not show an increase in WGR and SGR compared to the control group, it was found to alleviate the growth performance of common carp affected by Hg.
Detection of autoantibodies may detect damage after metal exposure (El-Fawal et al., 1999). Studies have found that exposure to Hg changes and increases IgM levels (Osuna et al., 2014; Queiroz et al., 1994) However, in this study, we observed a decrease in IgM and LZM levels, which may be due to excessive accumulation of Hg in the blood. However, compared with the Hg group Hg plus Se-enriched B. subtilis group, the levels of IgM and LZM increased significantly. It has been shown that dietary supplementation with nano-Se significantly increased IgM levels and enhanced immune function in chickens (Cai et al., 2012; Kumar et al., 2013). In addition, it has been reported that the addition of Bacillus licheniformis to the carp diet increased LZM levels and enhanced disease resistance (Kumar et al., 2013). The secondary metabolites of probiotics may have beneficial effects on the host (Dennis-Wall et al., 2017). Secondary metabolites from probiotics are transported to host organs via blood circulation (Eloe-Fadrosh et al., 2015).
Environmental pollutants such as heavy metals can affect the body's immune system and cause a decline in immunity. Cytokines, including, TNFs, and chemokines (Hawley et al., 2009; Savan and Sakai, 2006). IL-1β, IL-8 and TNF-α are crucial pro-inflammatory cytokines that regulate the inflammatory process and are considered good markers of the inflammatory response (Chen et al., 2017). IL-1β mainly regulates the body's immunity against pathogens, and it regulates the activation of immune cells and non-immune cells in infected sites and organs; IL-8 promotes the migration of neutrophils to fight pathogens and can increase the expression of other cytokines(Tomalka and Hise, 2015; Zhang et al., 2012). TNF-α is a multifunctional inflammatory cytokine, which can induce a variety of responses, including cell proliferation and apoptosis(Arnett et al., 2001). Cytokines can be regulated by nuclear factor kappa B (NF-κB) signaling. In the present study, Hg exposure upregulated IL-1β, IL-8 and TNF-α mRNA expression in the liver and spleen, consistent with results in zebrafish larvae (Zhang et al., 2016). This indicates that Hg exposure triggered an inflammatory response. The changes in the expression of these genes may be due to the accumulation of Hg in 30 days, which triggers a pro-inflammatory immune response and up-regulate IL-1β, IL-8, and TNF-α. However, treatment with Se-rich B. subtilis attenuated the up-regulation of IL-1β, IL-8, and TNF-α. Studies have shown that Se can up-regulate chicken immune cytokines (ie IL-10 gene) (Xu et al., 2015). These results may indicate that anti-inflammatory cytokines effectively suppressed the pro-inflammatory immune response, which is consistent with the up-regulation of IL-10 observed in this study. In addition, the up-regulation of IL-10 in the liver may represent an aspect of the homeostatic mechanism that controls the Hg-induced inflammatory response. Gao et al. reported that the reduction of TGF-β will aggravate the inflammatory damage of liver tissue, but the lack of Se will inhibit the expression of TGF-β and promote the production of TNF-α, IL-1β and IL-6, which may cause carp liver tissue Inflammation, but Se supplementation can prevent the decrease of TGF-β (Gao et al., 2019).The intake of Se-rich B. subtilis will not only increase the Se content in the body, but also B. subtilis will absorb Hg and alleviate the damage of the fish (Shang et al., 2021). In this study, there may be such a mechanism. Hg intake reduced the expression of TGF-β, while the Se-rich B. subtilis plus Hg group alleviated the decrease of TGF-β. The transcription factor NF-κB controls the expression of inflammatory cytokine genes (Taro and Shizuo, 2007). It controls the expression of pro-inflammatory genes and is also a key target for regulating inflammatory diseases (Xu et al., 2005; Yang et al., 2007). Study demonstrated that by catalyzing the degradation of IkBα, NF-κB can be activated by IKK (including IKKα, IKKβ and IKKγ), which plays an important role in regulating human pro-inflammatory cytokines(Jobin and Sartor, 2000; Bollrath and Greten, 2009). In this study, we found that the expression of IkBα in the liver and spleen decreased, and the corresponding NF-κB p65 expression increased, and this phenomenon was alleviated in the Se-rich B. subtilis treatment group. So there may be such a mechanism, Se-rich B. subtilis may be involved in the regulation of the IkBα/NF-κB signaling pathway. When the body consumes too much Hg, it leads to insufficient Se content in the body and triggers the inflammatory response and activates the IkBα/NF-κB signaling pathway. After feeding Se-rich B. subtilis to supplement Se, Se inhibits the up-regulation of pro-inflammatory cytokines in the cells and promotes the expression of anti-inflammatory cytokines, thereby reducing the harm of Hg to the fish
The intestine is a complex ecosystem, and the intestinal flora has an important role in this ecosystem. Intestinal flora can assist the digestion and absorption of food and promote nutrient metabolism (Sommer and Backhed, 2013). Changes in the intestinal flora can lead to disorders of the body’s normal physiological functions, leading to diseases(Nicholson et al., 2012). Through previous studies, we found that Hg significantly reduced the activity of enzymes such as CAT and GSH-PX and triggered inflammation(Shang et al., 2021). This experiment uses Illumina high-throughput sequencing technology to explain how the composition and diversity of carp intestinal microbial communities change under Hg exposure conditions, and provide a theoretical basis for fish intestinal health and normal human growth and development. In this study, the levels of Aeromonas sobria and Aeromonas hydrophila in the intestine of common carp after Hg treatment were higher than those in the control group. Many studies have shown that changes in the diversity of intestinal flora can cause diseases such as enteritis, inflammatory diseases and obesity.(Chassaing and Gewirtz, 2017; Beaz-Hidalgo and Figueras, 2013). Therefore, Hg-induced changes in intestinal flora may affect the health of common carp.
In this study, our results indicate that Verrucomicrobiaceae, Cetobacterium, Pseudorhodobacter, Gemmobacter and Aeromonas are the most important bacterial groups in common carp. The main flora in the intestines after Hg exposure are Verrucomicrobiaceae, Gemmobacter, Cetobacterium, Aeromonas and Pseudomonas. Hg exposure caused changes in the intestinal flora, and it was found that the abundance of Aeromonas in the Hg treatment group was much higher than that of the control group. Aeromonas can colonize and infect the host, and can cause diseases such as sepsis and fungal infections. The extracellular products (hemolysin, lipase and protease) produced by Aeromonas can cause soft tissue, hepatobiliary system, respiratory system and arthritis disease (Elorza et al., 2020; Lian et al., 2020). In this study, Hg exposure increased the proportion of Aeromonas in the intestines of fish. However, in the Se-rich B. subtilis plus Hg group, we found that the abundance of Aeromonas was reduced, which indicates that feeding the Se-rich B. subtilis can change the intestinal microbes of the fish and reduce the abundance of Aeromonas. Aeromonas sobria can cause oxidation in fish bodies to change superoxide dismutase, glutathione peroxidase, and up-regulate immunoglobulins IgM and TNF-α (Harikrishnan et al., 2020).Aeromonas hydrophila can cause Catla catla immune response and increase IL-1β and TNF-α (Harikrishnan et al., 2021). In this study, it was found that Aeromonas sobria and Aeromonas hydrophila were significantly increased, which may be another cause of the disease. Hg induction will change the Aeromonas in the common carp intestine, and increase the Aeromonas sobria and Aeromonas hydrophila in the Aeromonas, which leads to an inflammatory response in the fish. Se-rich B. subtilis through the action of Se and the probiotic B. subtilis, regulates the IKBα/NF-κB signaling pathway and reduces the inflammatory response. The composition of the intestinal flora was detected by 16S rRNA gene sequencing, and this phenomenon may be that the Se-rich B. subtilis improved the intestinal flora and reduced the abundance of Aeromonas, thereby reducing the inflammatory response.