Anti-inflammatory effects of enokine AHCC

What is inflammation

Chemical stimuli, harmful stimuli such as heat, and infections with pathogens such as bacteria and viruses can cause damage to cells and tissues, triggering a specific response called inflammation.
Celsus, a Roman physician, describes the local inflammation in his book as follows [1] : Notae vero sunt quattuor, rubor et tumor, cum calore et dolore. (Inflammation has four symptoms: redness and swelling accompanied by heat and pain.)

Inflammation is a defensive response to harmful stimuli, facilitating the repair process of damaged tissue, and producing biologically active substances to remove pathogens.
As bioactive substances, pain-inducing substances such as bradykinin and inflammatory mediators play a role.
So-called “inflammatory cells” (macrophages, neutrophils, and lymphocytes) produce a variety of inflammatory mediators, namely, nitric oxide (NO), prostaglandins, inflammatory cytokines, and chemokines.
Among them, NO is produced by stimulated hepatocytes and Kupffer cells (macrophages in the liver) and removes these pathogens by killing bacteria and inhibiting viral proliferation [2].
In other words, hepatocytes and Kupfer cells are involved in the inflammatory response of innate immunity.
The level of induction of inflammatory mediators was almost correlated with the level of inflammatory response.

Systemic inflammation may follow acute inflammation, with sepsis following bacterial infection being the typical case.
In the liver, Kupffer cells were stimulated by Endotoxin of gram-negative bacteria, producing not only NO but also interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) [3] (Figure 1).
The subsequent secretion of IL-1β stimulates the production of NO and TNF-α by hepatocytes.
If excessive production of NO causes systemic vasodilation, leading to septic shock [4].
Excessive TNF-α induces the production of various inflammatory cytokines and chemokines, a state also known as cytokine storm.
Overproduction of these NO and TNF-α leads to multiple organ failure as seen in fulminant hepatitis [5].
Therefore, the main component of bacterial endotoxin lipopolysaccharide (LPS) is often used to induce the production of experimental mouse or rat septicemia model (liver failure model) [3,6].
In addition, IL-1β-treated hepatocytes [2] or LPS-treated macrophages [7,8] have often been used to evaluate the anti-inflammatory effects of drugs and functional foods.

Inflammation causes disease, and disease causes inflammation.
Chronic inflammation is associated with many diseases, such as autoimmune diseases such as rheumatoid arthritis, metabolic diseases such as diabetes, atherosclerosis, chronic obstructive pulmonary disease (COPD), or cancer.
That is, inflammation is closely related to the pathophysiology, and sometimes the etiology, of these diseases.

If the inflammatory response is excessive or prolonged, it is not good for the body.
Therefore, anti-inflammatory drugs are used to suppress undesirable inflammation and restore health.
In order to reduce inflammatory mediators such as NO, prostaglandins, inflammatory cytokines, and chemokines to physiological levels, anti-inflammatory drugs and certain functional foods are required.
Therefore, inflammatory mediators are the best biomarkers for evaluating the effects of drugs and functional foods in cells, animals and humans.

Anti-inflammatory effects of AHCC

1. Bioactive substances contained in AHCC

Functional food AHCC is extracted from the mycelium culture of Lentinula edodes [9,10].
AHCC is mainly composed of carbohydrates, of which α-glucan is the main component.
The content of α-glucan in freeze-dried AHCC was 28.9% (by weight), while the content of β-glucan was very low [9,11].
Among α-glucans, oligosaccharides containing partially acylated α-1, 4-glucans are considered to be active components of AHCC’s biological effects, such as antioxidant effects [12,13].
However, the anti-inflammatory effects of the molecules contained in AHCC have not been identified.

Active molecules with pharmacological activity in AHCC have been explored using hepatocyte models of liver disease in vitro [2].
Because NO is produced in response to IL-1β, primary cultured rat hepatocytes are often used as models for inducible nitric oxide synthase (iNOS or NOS 2) production.
Matsui et al. [14] reported that adding AHCC to the medium could inhibit the increase of NO production induced by IL-1β, and reduce the production of iNOS protein and messenger RNA(mRNA) [14].

In addition, carbohydrates in AHCC were further studied.
In rat hepatocytes, the sugar component of AHCC inhibits NO production and reduces the expression of iNOS gene, which inhibits the induction of type I IL-1 receptor (IL1R1) expression [15].
On the other hand, glucose and dextrin are not known to significantly inhibit the induction of NO production by IL-1β in hepatocytes [16].
In addition, the sugar component of AHCC had no effect on the activation of the transcription factor nuclear factor-kappa B (NF-κB) and the degradation of inhibitor α (IκB-α), which is an inhibitor of NF-κB.
NF-κB is a key transcription factor for the expression of inflammation-related genes, such as iNOS and inflammatory cytokines [17].

Recently, active molecules with inhibitory effect of NO have been purified and isolated from AHCC by combining various chromatographic methods.
Adenosine has been identified as a component of AHCC, which inhibits the expression of iNOS gene and reduces the production of inducible NO (50% inhibition concentration, IC50 value of 56μM) [18].
Like the sugar component of AHCC, adenosine inhibited inductive NF-κB activation and IL1R1 expression.
As a result, the expression of iNOS protein is inhibited, leading to the reduction of NO production [18].
However, due to the low adenosine content in AHCC, it is speculated that there are other NO-producing inhibitory molecules present in AHCC.
In addition, there appear to be anti-inflammatory components that play a role in other tissues such as the gut, and future research in this area is expected.

2. Anti-inflammatory effect of AHCC in liver

In a ten-year cohort study of patients with hepatocellular carcinoma, oral AHCC (3g/ day) improved their prognosis [19].
This study scientifically defined “functional food” and advocated AHCC as a functional food [20].
On the other hand, NO is believed to be involved in the pathogenesis and liver protection of various liver diseases.
As described above, AHCC and its sugar components inhibit NO production and iNOS gene expression in IL-1β-treated rat hepatocytes [14,15].
AHCC inhibits the expression of iNOS gene at the transcriptional level by inhibiting NF-κB activation [14].
AHCC, moreover, can be in the transcription level, reducing by iNOS transcription antisense iNOSmRNA stability, inhibition of iNOS gene expression [14, 21] (reference book Ⅲ – 3-3).
AHCC also reduced the production of inflammatory cytokines and chemokines in rat hepatocytes (Oshan, Cesar.
The data are not published).
Therefore, the inhibitory effect of AHCC on NO in liver cells may be related to the improvement of prognosis of patients with liver cancer after operation by AHCC.

Kim et al. reported that the administration of AHCC in patients with slightly elevated serum liver enzymes caused by alcohol reduced serum liver enzymes and inflammatory cytokines (markers of oxidative stress) [22].
Serum TNF-α and IL-1β levels were significantly reduced after 12 weeks of AHCC1 (1g or 3g/day).
The change rate of serum IL-1β level was positively correlated with the level of liver alanine aminotransferase (ALT) in serum.
On the other hand, levels of adiponectin (adipocytokines) were higher in the AHCC group compared to the placebo group.
The results of this clinical trial suggest that AHCC may have protective liver function and anti-inflammatory effects.

3. Anti-inflammatory effect of AHCC in intestinal tract

The role of AHCC in the intestinal tract of mice was also studied.
Mallet et al. administered AHCC to BALB/c mice for 7 consecutive days at a daily dose of 0.1, 0.5 or 1.0g/kg body weight, and prepared intestinal epithelial cell samples [23].
AHCC increased the number of immunoglobulin A (IgA) positive cells in the intestinal epithelium, and increased the secretion of IgA (SIgA), IL-10 and interferon-γ (INF-γ) in the intestine.
In the intestinal lumen, IgA plays a role in the frontier of antigen-specific immune defense, and SIgA prevents inflammation caused by intestinal bacteria [24].

In experiments with primary cultured intestinal epithelial cells, TLR2 and TLR4 in Toll-like receptors (TLR) were associated with immune responses induced by AHCC [23].
Therefore, AHCC may play a role in maintaining intestinal epithelial cell immune homeostasis through TLR2 and TLR4-mediated signaling pathways.

The anti-inflammatory effect of AHCC on the intestinal tract has been verified in an animal model of ulcerative colitis.
An experimental animal model of ulcerative colitis has been used as a model of human ulcerative colitis, Crohn’s disease and other inflammatory bowel diseases (IBD) [25].
Daddaoua et al., using a rat model of colitis induced by trinitrobenzene sulfate (TNBS), carefully examined the anti-inflammatory effect of AHCC [26].
AHCC (100 or 500mg/kg body weight) was administered daily from 2 days before TNBS administration, and rats were euthanized 6 days after TNBS administration.
The results showed that AHCC reduced intestinal inflammation.
In addition, AHCC not only improved mucosal injury score, necrotic area, and intestinal weight, but also reduced the expression of inflammatory cytokines (IL-1β, IL-1 receptor agonist, and TNF-α), chemokines Ccl2 (also known as MCP-1), and trilobar factor 3 (TFF 3).
The anti-inflammatory effect of AHCC is almost the same as that of salazosulfapyridine, which is used to treat ulcerative colitis [26].

In vitro studies have recently reported the use of AHCC to treat intestinal epithelial cell lines (IEC 18 and HT 29) [27].
Different from the hepatocytes treated with IL-1β, the chemokines Ccl2 and Cxcl1 of IEC18 cells can be induced to express only by AHCC treatment, and the secretion of IL-8 of HT29 cells can be increased [27].
These effects in IEC18 cells are due to activation of NF-κB and part of MAPK (mitogen-activated protein kinase), mediated by TLR4 and myeloid differentiation primary response 88 (MyD88).
Daddaoua and colleagues conducted more detailed studies using THP-1 cell lines derived from monocytes, and showed that AHCC is mediated by NF-κB and JNK (c-Jun N-terminal kinase) to promote the secretion of IL-8, IL-1β, and TNF-α [27].
These results suggest three possible roles for AHCC.
First, immune activation and anti-inflammatory effects of AHCC depend on cell type; second, immune enactivation or anti-inflammatory effects are determined by external stimuli such as cytokines or bacterial endotoxins; third, AHCC has both immune-stimulating and anti-inflammatory effects.
Further research in the future will help to better understand the role of AHCC.

Daddaoua et al. also analyzed the colonic flora of rats after AHCC injection [26].
Compared with TNBS-treated rats, AHCC-treated rats had more aerobic bacteria, lactic acid bacteria and bifidobacteria, but fewer Clostridium bacteria.
In addition, AHCC synergistically enhanced the symbiotic effect of B. longum BB 536 strain [28].
Bifidobacterium longum is a good bacterium in the human gastrointestinal tract, and strain BB 536 is expected to promote health as a probiotics.
Rats were given AHCC (100 or 500mg /kg body weight) and Bifidobacterium longum strain BB 536 (5×10 6 CFU/ rat) 28 from 7 days before TNBS treatment to 7 days after TNBS treatment.
By using AHCC and BB 536 strains in colitis model rats, high anti-inflammatory activity was found in the intestinal tract, and TNBS treatment induced weight loss, intestinal weight/length ratio, changes in the expression of myeloperoxidase (MPO) and iNOS produced by neutrophils were inhibited.
In summary, these results suggest that AHCC can be used as a functional food (food ingredient that supports probiotic function) with a prebiotic effect. The combination of AHCC and bifidobacterium strain BB 536 (probiotics) shows the potential to inhibit intestinal inflammatory response in patients with IBD.

As another experimental model of ulcerative colitis, ulcerative colitis induced by T lymphocytes was also used to evaluate the anti-inflammatory effect of AHCC.
Recombination activated gene 1 (Rag1) knockout mice (Rag1-/- mice) have defective mature B and T cells and an underdeveloped mucosal immune system [29].
Mascaraque et al transplanted CD4+ CD62L+ T cells, including juvenile T cells and central memory CD4+ cells, into Ragl-/- mice, causing chronic colitis similar to IBD [30].
RCC1-/- mice were then orally administered with AHCC (75mg/ day) for 12 consecutive days after T cell transplantation.
Primary culture lymphocytes were isolated from mouse mesenteric lymph nodes treated with AHCC, and the MPO activity and alkaline phosphatase activity of cells were reduced after adding AHCC in culture medium, and the secretion of TNF-α and IL-1β was significantly reduced [30].
These results suggest that AHCC has an anti-inflammatory effect in the gut and may help ameliorate the symptoms of T cell-induced ulcerative colitis.

Although the results of many animal experiments have been reported to date, no bioactive substances that reduce intestinal inflammation have been identified.
Further studies are needed to explain the anti-inflammatory role of AHCC in intestinal mucosal immunity.

Future Prospects

Many reports accumulated to date have shown that AHCC exhibits anti-inflammatory effects in a variety of cell types and immune-stimulating effects in certain cell lines (Figure 2).
In addition, AHCC has a prebiotic effect on intestinal flora.
These effects contribute to the use of AHCC as a functional food for the prevention of chronic inflammatory diseases such as IBD.
In this sense, AHCC and its components can be used in complementary and alternative medicine.

In addition, it is necessary to elucidate the role of AHCC by identifying molecules other than adenosine that have pharmacological activity.
If such an active molecule is identified, its anti-inflammatory effect of inhibiting the expression of inflammation-related genes could be useful for the development of therapeutic drugs involved in chronic inflammatory disease.