Sepsis is one of the leading causes of mortality and critical illness worldwide (van der Poll et al., 2021) and was considered a major cause of health loss (Cecconi et al., 2018), with nearly 50 million cases globally per year (Rudd et al., 2020). It is reported that 18% of intensive care unit (ICU) patients are suspected of infection (Vincent et al., 2020). At present, sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Peritonitis is the inflammation of the peritoneum, usually due to a bacterial or fungal infection. Fecal peritonitis (FP) and subsequent sepsis are major problems when admitted to the ICU (Athanazio, 2003). The mortality rate with peritonitis and peritonitis-related sepsis at 28 days was 19.1%, increasing to 31.6% at 6 months (Tridente et al., 2014). Despite the decades of intensive research and more than 200 randomized controlled trials, the underlying mechanisms are still not comprehensively understood and, therefore, there is still no entirely functional treatment that could save lives in patients with sepsis (Ngamsri et al., 2021). Measures remain largely supportive, such as timely antibiotics, source control, resuscitation, and supportive care for organ dysfunction (Evans et al., 2021).

Poria cocos are mainly native to East Asia and Southeast Asia as a kind of medicinal edible fungus, known as “Fu Ling” in Chinese, which has been used in traditional Chinese medicine for more than 2000 years (Wang et al., 2013) and is contained in approximately 15% of traditional Chinese medicine preparations in the Chinese pharmacopeia (Zhu et al., 2018). The bioactive components in Poria cocos include polysaccharides, triterpenoids, fatty acids, sterols, and enzymes (Li et al., 2022). Poria cocos are found in plant polysaccharides, Poria cocos polysaccharides (PCPs), which shows that it accounts for 84% by weight among all constituents in the dried sclerotium (Li et al., 2019), and it is also the main bioactive component in Poria cocos that has extensive biological activities, such as ameliorating phlegm and edema, relieving nephrosis and chronic gastritis, improving uneasiness of minds (Kobira et al., 2012; Wang et al., 2012; Nie et al., 2020), antitumor (Wang et al., 2015; Li et al., 2020), hepatoprotection (Kim et al., 2019; Wu et al., 2019), immunomodulation (Pu et al., 2019), anti-inflammation (Lee et al., 2017), antioxidation (Wang et al., 2016), antibacterial (Wang et al., 2019), and invigorating the splenic functions for immune regulation (Shen et al., 2005). Studies showed that PCP could elicit strong immune activity and a protective effect against immunosuppression (Zhang et al., 2017; Guo et al., 2018), with few side effects and rich resources (Nie et al., 2020).

There are few effective therapeutic measures for sepsis, and refer to the pharmacological properties of PCP, we predicted that PCP can enhance host defenses and may be effective for the prevention of sepsis.

Notably, many Chinese and East-Southeast Asia residents take traditional medicinal materials, such as “Poria Cocos polysaccharide,” daily to strengthen the body's resistance (Wu et al., 2018). We verified the protective effect of PCP on FP-induced sepsis, which has important practical significance for East-Southeast Asia residents. This study found that the mice fed with PCP could better resist the feces and improve mice's clinical score and survival rate. A further study of this effect showed that the inflammation in mice was lower and there were fewer cytokines promoting inflammation when exposed to fecal. Meanwhile, pretreatment with PCPs, the level of oxidative stress was controlled well, and reactive oxygen species (ROS) were reduced. Then, the splenic injury was gentle, the apoptosis of splenocytes was less, the expression of Treg was reduced, and the splenic function to resist damage-associated molecular patterns (DAMPs) injury was maintained. This study provides a new concept for relieving FP-induced sepsis, given the feeble pretreatment regimen for sepsis based on the anti-inflammatory and protective effects of PCP. As a previous report, this study also compared different dose concentrations of PCP to verify the efficacy of different concentrations and suggested that the efficacy of 400 mg/kg/day PCP was better than that of 200 mg/kg/day.

Predominantly, sepsis is considered to be an acute inflammatory state mediated by the activation of the innate immune system caused by infection (Grondman et al., 2020). Molecules derived from pathogens, also known as pathogen-associated molecular patterns (PAMPs), can bind to pattern recognition receptors (PRRs) to activate innate immune cells to protect the host. The activation of PRRs on innate or non-immune cells can trigger intracellular signal cascades, thus activating transcription factors (Wang and Wang, 2013). Intracellular signaling cascades initiate multiple cellular processes, including cytokine production. There are many different PRRs subsets on immune cells (mainly monocytes/macrophages) and non-immune cells that recognize different classes of microorganisms, leading to immune activation (Mahla et al., 2013). Early stage sepsis, the initial hyperinflammatory phase, also known as the “cytokine storm,” is ‘described by the overwhelming release of inflammatory molecules by the innate immune system (Chaturvedi et al., 2021). Hyperinflammation is to induce the initial cause of cell impairment, causing necrotic cells and tissue injury from the original insult and inflammatory reaction (Gustine and Jones, 2021). The TNF-α is an endogenous pyrogen involved in the induction of fever, apoptotic cell death, and inflammation. IL-6 is an important mediator that acts as a pro-inflammatory cytokine (Lonsdale et al., 2020). Meanwhile, the body promotes the secretion level of inflammatory factors (TNF-α, IL-6, and IL-1β) and decreases the secretion level of inflammatory factors, making the body's immune system in a state of immune hyperactivity and cope with the pathogens (London et al., 2010). While this state of affairs continued, the body has been high pro-inflammation factors, and the body's immune cells appear to reduce the number of cells and loss of function to make the body's immune chaos and paralysis, accelerating the death of patients (van der Poll et al., 2021). Therefore, controlling the excessive inflammation in the early stage is significant in sepsis (Bosmann and Ward, 2013). After 14 days of feeding with PCP, mice were intraperitoneally injected with excrement and sacrificed 24 h later. Compared with the mice without feeding PCP, the secretion levels of promoting inflammatory factors were reduced. It indicates that daily administration of PCP can resist the FP and has a good protective effect.

Oxidative stress is a result of an imbalance between the formation of reactive oxidizing species, such as superoxide, hydrogen peroxide, and hydroxyl ions as free radicals, and the removal of antioxidant scavengers, such as catalase, SOD, vitamin C, vitamin E, and reduced GSH (Pizzino et al., 2017; Usmani et al., 2021). In septic conditions, the body needs more energy to sustain life activities than it normally does. Mitochondria generate a large number of oxygen free radicals while providing energy through the electron transport chain (Ramirez-Zuniga et al., 2021). In a physiological state, mitochondria can avoid the damage of ROS by the antioxidant enzymes, such as SOD and GSH. As one of the most important oxygen free radicals in the ROS family, MDA is the main degradation product of lipid peroxidation. By attacking the unsaturated fatty acids in the cell membrane, MDA inhibits the function of membrane protein and leads to the destruction of the integrity of cell membrane and mitochondrial membrane, thus increasing the permeability of cell membrane and leading to cell edema and necrosis. MPO is a member of the heme peroxidase superfamily (Denning et al., 2019; Liu et al., 2020). Its main function is to kill microorganisms in phagocytes and form oxidizing free radicals to participate in many processes of regulating inflammatory responses. When inflammation occurs in the body, the production of MPO increases, resulting in cell damage (Ward and Fattahi, 2019), while higher levels of MPO, on day 1, were able to predict 90-day mortality (Bonaventura et al., 2020). After 14 days of feeding PCP, the MDA and MPO levels were maintained at low levels, and the expressions of GSH and SOD were higher.

People with asplenia or hyposplenism are at an increased risk of fulminant sepsis which carries a high mortality rate (Kanhutu et al., 2017), and it is proved that splenic function is particularly important for patients with sepsis. Splenic cellular histopathology is one of the direct methods to evaluate the splenic injury, and these outcomes were consistent with the results of improved cytoarchitecture in H&E staining (Suttie, 2006; Coccolini et al., 2019). FoxP3⁺ regulatory T cells (Treg), a mature T cell subset with regulatory functions, play an important role in immune homeostasis, allergic reaction, tumor immunity, and transplantation tolerance (Kurup et al., 2017). Treg is a good candidate owing to explaining the impairment of the CD4⁺ T-cell compartment observed during sepsis due to their highly suppressive function (Qureshi et al., 2011). Moreover, the number of Treg cells has upregulated in early-stage patients with sepsis (Leng et al., 2013). Therefore, Treg cells might be contributed to the higher morbidity and mortality in patients with sepsis (Xu et al., 2020), and the role of Treg cells in the immune response to infection has attracted wide attention (Nascimento et al., 2017). While the first line defenders are combating infection, Treg cells respond to infection by suppressing excessive immune responses elicited by other cells of the adaptive immune system, in turn, further shaped by increased numbers of Treg cells (Huang et al., 2010). In current findings from this study, the expression of Treg cells significantly increased after FP-induced sepsis; nevertheless, decreased FoxP3-labeled cells in FP-induced sepsis and PCP pretreated mice were observed.

Until recently, apoptosis is the best-known form of programmed cell death and resulted in the premature death of cells in living tissue by autolysis (Moreno-Gonzalez et al., 2016); some studies have shown that immune cell apoptosis is directly related to the pathogenesis (Huang et al., 2019; Chen and Wei, 2021) and is the major mechanism of lymphocyte death, which is one of the reasons for the gradual decrease in the number of immune cells in sepsis (Aziz et al., 2014). Thereby, a functionally regulated apoptotic program in thymus and splenic cells may provide a promising strategy for combating FP-induced sepsis. Thereby, we reasoned that the anti-apoptotic biological activity of PCP might be one of the underlying molecular mechanisms of protective effects against FP-induced sepsis.

Taken together, our current findings disclose that PCP exerts a promising immunity protective effect against FP-induced sepsis, suggesting PCP's potential perspective of clinical application. The current findings showed that FP-induced and PCP-pretreated sepsis mice resulted in lower TNF-α, IL-6, and IL-1β contents in plasma, downregulating expression of Treg cells in the spleen. Consequently, we concluded that PCP pretreatment inhibited MPO and MDA release, eventually blocking apoptosis. Furthermore, the underlying molecular mechanisms associated with PCP-mediated cytoprotection warrant should be discussed, respectively.

This study has several limitations. First, the concentration of PCP is the key factor to determine the pharmacological effect in vivo, and we did not determine under the present condition. Second, we only injected with 1 mg/g of feces according to previous reports and did not conduct relevant studies on dose gradient. The number of feces may have imparity pathophysiological. Our results only suggest that PCP 400 mg/kg/day is effective in preventing sepsis caused by 1 mg/g FP-induced sepsis. Finally, although many people in East Asia have been taking PCP orally for a long time, PCP was fed to mice 14 days in advance. Therefore, these results only suggest that PCP has certain anti-inflammatory and protective effects on FIP-induced mice. The relevant studies on the therapeutic effects of PCP on mice with fecal peritonitis-induced sepsis were absent, and we will continue to do relevant studies. Finally, although some evidence has emerged, we have not been able to connect the evidence into a complete pathway to a specific mechanism and block or overexpression of relevant upstream and downstream pathways, and further studies are needed to explore the specific mechanism of these pharmacological effects.

The results in this study suggest that PCPs have pharmacological activity against FP-induced sepsis, by the potential molecular mechanism of inhibiting splenic cell apoptosis, inflammatory stress, and oxidative stress.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Naval Medical University.

YW performed mostly experiments, analyzed the data, and drafted the manuscript. DL and HW analyzed the data and critically revised the manuscript. DL contributed to the data analysis and interpretation. XW conceived and designed the study. All authors substantially contributed to editing, revising, and finalizing the manuscript and read and approved the final manuscript.

This work was supported by the Shanghai Natural Science Foundation (19ZR1456600), the Shanghai Pujiang Program (2020PJD059), the Youth Cultivation Project for Special Basic Medical Research of the First Affiliated Hospital of Naval Medical University (2021JCQN02), and the Incubator Program for Startups of the 980th Hospital of the Joint Logistic Support Force (FYJHMS-04).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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