Ganoderic Acid A Derived from Reishi Mushroom Ganoderma lucidum Protects against Intestinal Immunity Reduction Due to Oxidative Stress in Rat

Abstract

Reishi (Ganoderma lucidum) is known to enhance intestinal immunity, with ganoderic acid A (GA-A) identified as one of its active constituents. However, the specific role of GA-A in regulating immune components such as immunoglobulin A (IgA) from Peyer’s patches (PPs) and α-defensin 5 from Paneth cells remains unclear. Additionally, the ability of Reishi to counteract oxidative stress-induced intestinal immune suppression has not been fully elucidated. Therefore, in this study, we aimed to examine the effects of Reishi and GA-A on intestinal immunity in a rat model of ischemia–reperfusion (I/R) injury. Oral administration of GA-A increased IgA secretion from PP cells isolated from rat small intestine and upregulated the mRNA expression of rat α-defensin 5 (RD-5) and toll-like receptor 4 (TLR4) in the ileum, similar to Reishi. In contrast, GA-A did not exhibit immunostimulatory effects in TLR4-deficient mice. In the I/R rat model, both Reishi and GA-A significantly restored IgA secretion and RD-5 mRNA expression, mitigating immune suppression. They were also associated with changes in superoxide dismutase 1 (SOD1) and SOD3 mRNA expression under I/R conditions and prevented villus shedding and Paneth cell loss, indicating protection against I/R-induced intestinal immune decline. These results were comparable to those observed with caffeic acid, the positive control. Overall, these findings suggest that Reishi mitigates intestinal immune suppression caused by I/R injury, with GA-A serving as a key active component mediating these protective effects.

INTRODUCTION

The intestinal tract is responsible for digestion and nutrient absorption and also serves as a major immune organ, accounting for approximately 60% of total body immunity.^1,2) Its surface is coated with mucus secreted by the mucosal epithelium, which prevents foreign substances from adhering to epithelial cells.³⁾ Intestinal defense mechanisms also include the resident microbiota, which play a key role in maintaining mucosal immunity.⁴⁾ In recent years, there has been a growing global interest in self-medication and in identifying functional foods that can prevent or ameliorate disease conditions.^5,6) Many countries, including Japan, have shown increasing expectations for the health-promoting potential of dietary components.^7,8) However, few studies have focused on the intestinal tract—where food directly interacts with the immune system—and scientific evidence in this area remains limited.⁹⁾

We previously reported that oral administration of Reishi (Ganoderma lucidum) enhances intestinal immunity.¹⁰⁾ Reishi contains various triterpenoids, among which ganoderic acid A (GA-A) is recognized as an active compound.¹¹⁾ These triterpenes are lanosterol derivatives, with GA-A being the most abundant, typically comprising 0.1–0.2% of total extract. GA-A possesses diverse biological activities, including antinociceptive effects and immunomodulatory properties.^12,13) Therefore, GA-A is presumed to contribute to the intestinal immune-enhancing effects of Reishi. However, the direct impact of orally administered GA-A—at doses equivalent to those present in commonly used Reishi preparations—on intestinal immune factors such as immunoglobulin A (IgA) secreted from Peyer’s patches (PPs) and α-defensin 5 secreted from Paneth cells has not yet been clarified.

IgA plays a critical role in mucosal defense by preventing microbial proliferation and antigen invasion in the intestinal lumen,⁴⁾ whereas α-defensin 5 acts as an antimicrobial peptide, both being essential for maintaining intestinal immunity.¹⁴⁾

Furthermore, compounds capable of stimulating intestinal immunity can mitigate oxidative stress-induced immune suppression and villus damage.¹⁵⁾ Oxidative stress is a pathological condition associated with numerous diseases, and a well-established experimental model for studying it in the intestine is the small intestine ischemia–reperfusion (I/R) model.¹⁶⁾ This model mimics transient ischemia, as seen in small intestine transplantation, ileus, or necrotizing enterocolitis, followed by oxidative stress during reperfusion. The resulting reactive oxygen species trigger inflammation and impair the intestinal mucosal barrier, which can lead to systemic damage via bacterial translocation.^16,17)

We previously demonstrated that caffeic acid (CA), a dietary antioxidant, suppresses small intestinal damage induced by I/R.¹⁸⁾ However, the effects of small intestinal I/R on intestinal immunity remain poorly understood. Bowser et al. highlighted the importance of addressing oxidative stress–related intestinal inflammation to maintain mucosal integrity and improve patient outcomes.¹⁹⁾ Therefore, in this study, we aimed to investigate the effects of small intestinal I/R, an oxidative stress model, on intestinal immunity, and to examine the protective roles of Reishi, its active component GA-A, and CA (as a positive control).

MATERIALS AND METHODS

Chemicals

GA-A was purchased from Tokiwa Phytochemical Co. Ltd. (Chiba, Japan). Reishi powder (Lot. No.: H101GLS-2) was supplied by Hokkaido Reishi Co. Ltd. (Sapporo, Japan); details of its composition and preparation have been reported previously.⁹⁾ CA was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Lipopolysaccharide (LPS) from Escherichia coli O111 (produced by phenol extraction) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All other reagents were of analytical grade and used without further purification.

Animals

Male Wistar rats (190–220 g, 7 weeks old) and C3H/HeJ, HeN mice (20–25 g, 7 weeks old) were obtained from Hokudo (Sapporo, Japan). Animals were housed in plastic cages under standard laboratory conditions (23 ± 2°C; 12 : 12 h light–dark cycle) with ad libitum access to water and standard rodent chow. All procedures followed the Guidelines for the Care and Use of Laboratory Animals of Hokkaido University (Approval No. 17-0005). The administered doses were as follows: GA-A (10 or 100 μg/kg), Reishi powder (5 mg/kg), and CA (50 mg/kg body weight). Each agent was orally administered using a sonde. Animals were euthanized 24 h after administration.

Preparation of PP Cells and Measurement of IgA

PP cell isolation and IgA quantification were performed as described previously.¹⁰⁾ Briefly, PPs were excised from the small intestines of euthanized animals under sevoflurane anesthesia and placed in ice-cold complete medium. The tissues were digested with collagenase to obtain single-cell suspensions, which were filtered and washed. PP cells (2 × 10⁶ cells/mL) were cultured in 24-well plates with or without 100 μg/mL LPS. IgA concentrations in the culture supernatants were measured using rat and mouse IgA enzyme-linked immunosorbent assay (ELISA) kit (Life Diagnostics, Inc., West Chester, PA, U.S.A., and Immunology Consultants Laboratory, Inc., Atlanta, GA, U.S.A., respectively) according to the manufacturer’s protocols.

Quantitative Real-Time PCR (qPCR)

qPCR was performed using a LightCycler 480 ΙΙ System (Roche Diagnostics GmbH, Mannheim, Germany) with a KAPA SYBR Green Fast qPCR kit (KAPA Biosystems, Boston, MA, U.S.A.) as previously described.¹⁰⁾ Primer sequences are shown in Supplementary Table 1. PCR was performed with the following conditions: 40 cycles at 95°C for 3 s, 95°C for 10 s, 58–59°C for 20 s, and 72°C for 1 s. Levels of PCR products were normalized to actin.

Intestinal I/R Treatment

The intestinal I/R model was established as described previously.¹⁵⁾ Briefly, ischemia was induced by clamping the superior mesenteric artery for 30 min, followed by reperfusion for 1 h. Sham-operated animals underwent the same procedure without clamping.

Histological Examination

Ileal samples collected after reperfusion were washed with phosphate-buffered saline and fixed in 10% neutral-buffered formalin. Fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin–eosin (H&E). Morphological changes were evaluated under a light microscope.

Statistical Analysis

Comparisons between two groups were performed using Student’s t test, whereas multiple-group comparisons were performed using Tukey’s test or Dunnett’s test for post hoc analysis. Student’s t test was used for Figs. 1C and 1D, Tukey’s test was used for Figs. 2–4, and Dunnett’s test was used for Figs. 1A and 1B. Analyses were performed using R software version 4.3.1 (R Foundation for Statistical Computing, Vienna, Austria), and differences were considered statistically significant at p < 0.05. Data are presented as mean ± standard deviation (S.D.), with representative results from more than two independent experiments presented.

Fig. 1. Effects of GA-A on IgA Production in Cultured Rat PP Cells and RD-5 and TLR4 mRNA Levels in the Rat Ileum

Rats received a single oral administration of GA-A (10, 100 μg/kg), and PP cells were cultured (A) without or (B) with LPS (100 μg/mL) for 72 h. Statistical comparisons were made between the Control group and other groups using Dunnett’s test. Data are presented as mean with S.D., n = 3 per group for more than two independent experiments. (C) RD-5 and (D) TLR4 mRNA levels in the rat ileum were normalized to actin. The expression level of the Control group was arbitrarily set at 1.0. Statistical comparisons were made between the Control group and GA-A group using Student’s t test. Data are presented as mean with S.D.; n = 5 per group for more than 2 independent experiments. GA-A: ganoderic acid A; IgA: immunoglobulin A; PP: Peyer’s patches; RD-5: rat α-defensin 5; TLR4: toll-like receptor 4; LPS: lipopolysaccharide; S.D.: standard deviation.

Fig. 2. Effects of GA-A on IgA Production in Cultured C3H/HeN and C3H/HeJ Mouse PP Cells

(A) C3H/HeN and (B) C3H/HeJ mice received a single oral administration of GA-A (10 μg/kg), and PP cells were cultured with or without LPS (100 μg/mL) for 72 h. Statistical comparisons were made between the LPS (−) and GA-A (−) group and other groups, and between the LPS (+) and GA-A (−) group and the LPS (+) and GA-A (+) group using Tukey’s test. Data are presented as mean with S.D.; n = 3 per group for more than 2 independent experiments.

RESULTS

GA-A Stimulated Intestinal Immunity

We previously demonstrated that oral administration of Reishi enhances intestinal immunity by increasing IgA secretion from PPs upon LPS stimulation and upregulating rat α-defensin 5 (RD-5) and Toll-like receptor 4 (TLR4) mRNA expression in the ileum.¹⁰⁾ To determine whether GA-A exerts similar effects, rats were orally administered GA-A, and intestinal immune markers were assessed.

GA-A significantly increased IgA secretion from primary cultured PP cells in the LPS-stimulated group (Figs. 1A and 1B). Additionally, GA-A significantly upregulated RD-5 and TLR4 mRNA expression in the ileum (Figs. 1C and 1D), showing a response pattern comparable to that of Reishi administration.⁹⁾ These findings suggest that GA-A is one of the key active components mediating the intestinal immunostimulatory effects of Reishi.

The Intestinal Immunostimulatory Effect of GA-A is Absent in TLR4-Deficient Mice

To clarify the role of TLR4 in the intestinal immunostimulatory action of GA-A, IgA secretion was measured in PP cells from C3H/HeN (wild-type TLR4) and C3H/HeJ (TLR4-mutant) mice. In C3H/HeN mice, oral administration of GA-A significantly increased IgA levels in LPS-stimulated PP cell supernatants (Fig. 2A). However, this effect was absent in TLR4-mutant C3H/HeJ mice (Fig. 2B). These results are consistent with our previous findings on Reishi administration in C3H/HeJ mice,¹⁰⁾ suggesting that the intestinal immunostimulatory effects of GA-A are mediated through TLR4 signaling.

Reishi and GA-A Suppress the Decline in Intestinal Immunity Induced by I/R

Small intestinal I/R significantly reduced IgA secretion from PP cells compared with the sham group (Fig. 3A). Oral administration of Reishi or GA-A 24 h prior to I/R significantly restored IgA secretion. Consistent with our previous report that CA, an antioxidant, ameliorates I/R-induced intestinal injury,¹⁸⁾ CA served as a positive control and exhibited similar restorative effects.

Fig. 3. Effects of Reishi, GA-A, and CA on IgA Production in Cultured Rat PP Cells and RD-5 mRNA Levels in the Rat Ileum

(A) Rats received a single oral administration of Reishi (5 mg/kg), GA-A (10 μg/kg), and CA (50 mg/kg) and PP cells were cultured with LPS (100 μg/mL) for 72 h. (B) RD-5 mRNA levels in the rat ileum were normalized to actin. The expression level of the Sham group was arbitrarily set at 1.0. Statistical comparisons were made between the Sham group and other groups, and between the I/R group and the I/R with each reagent group using Tukey’s test. Data are presented as mean with S.D.; n = 5 per group for more than two independent experiments. CA: caffeic acid; I/R: ischemia–reperfusion.

I/R also significantly reduced RD-5 mRNA expression in the ileum (Fig. 3B). Pretreatment with Reishi, GA-A, or CA tended to restore RD-5 mRNA expression to near-sham levels. These findings indicate that Reishi and GA-A protect against intestinal immune suppression induced by small intestinal I/R.

Superoxide Dismutase (SOD) Contributes to the Protective Mechanism

Because small intestinal I/R induces oxidative stress, we next examined the expression of SODs. Compared with the sham group, the I/R group showed a significant reduction in SOD mRNA expression in the ileum (Figs. 4A–4C). In addition, CA significantly restored SOD1–3 mRNA expression compared with the I/R group. Reishi and GA-A tended to attenuate the I/R-induced decrease in SOD1 and SOD3 mRNA expression, whereas SOD2 mRNA expression was not significantly affected. These findings suggest that the protective effects of Reishi and GA-A against I/R-induced intestinal immune suppression are accompanied by changes in SOD1 and SOD3 mRNA expression.

Fig. 4. Effects of Reishi, GA-A, and CA on SOD 1–3 mRNA Levels in the Rat Ileum

Rats received a single oral administration of Reishi (5 mg/kg), GA-A (10 μg/kg), and CA (50 mg/kg) and (A) SOD1, (B) SOD2, and (C) SOD3 mRNA levels in the rat ileum were normalized to actin. The expression level of the Sham group was arbitrarily set at 1.0. Statistical comparisons were made between the Sham group and other groups, between the I/R group and the I/R + each reagent group, and between the I/R + CA group and the I/R + Reishi or GA-A group using Tukey’s test. Data are presented as mean with S.D.; n = 5 per group for more than two independent experiments. SOD: superoxide dismutase.

Reishi and GA-A Prevent Villus Loss Induced by Small Intestinal I/R

Given these results, we investigated whether Reishi and GA-A could protect intestinal morphology from I/R-induced damage. Histological examination of H&E-stained ileal sections revealed that oral administration of Reishi or GA-A appeared to attenuate villus shedding during I/R (Fig. 5). Additionally, both treatments prevented the loss of Paneth cells at the crypt base. Similar protective effects against villus shedding were observed with CA administration.

Fig. 5. Microscopic Appearance of Intestinal Tissue after I/R

Hematoxylin and eosin staining of sham-operated ileum, I/R-treated ileum, and I/R-treated ileum pretreated with Reishi (5 mg/kg), GA-A (10 μg/kg), and CA (50 mg/kg) via oral administration. Images represent typical data.

Overall, these findings indicate that Reishi and its active constituent GA-A protect against oxidative stress–induced intestinal immune suppression and structural damage in the small intestine, supporting GA-A as a major bioactive compound underlying Reishi’s beneficial effects.

DISCUSSION

In this study, oral administration of GA-A significantly increased IgA secretion from PP cells, but only in the group treated with LPS, a known TLR4 ligand.⁴⁾ GA-A also upregulated RD-5 mRNA expression, a key antimicrobial peptide that supports intestinal defense and symbiosis with gut microbiota.²⁰⁾ These results are consistent with our previous findings showing that oral administration of Reishi enhanced intestinal immunity,¹⁰⁾ suggesting that GA-A is a principal bioactive component responsible for this effect.

We previously observed that Reishi increased RD-5 mRNA levels in a dose-dependent manner in both the jejunum and ileum, with the strongest effect in the ileum.¹⁰⁾ Because Paneth cells, which secrete α-defensin 5, are more abundant in the ileum,²¹⁾ the pronounced response in this region likely reflects their distribution. These findings indicate that GA-A enhances intestinal immunity by upregulating IgA and RD-5, both essential for mucosal defense against external pathogens.

TLR4 is a critical mediator of intestinal immune activation, regulating both IgA secretion and RD-5 expression through dendritic cell and T-cell signaling.^22,23) In this study, GA-A significantly increased TLR4 mRNA levels in the ileum compared with the control group. While excessive TLR4 activation can trigger inflammation,²⁴⁾ our previous work showed that Reishi-induced TLR4 upregulation did not elevate the inflammatory cytokine tumor necrosis factor-α levels,¹⁰⁾ suggesting that GA-A activates intestinal immunity without provoking inflammatory responses. The Reishi extract used in the present study was derived from the same production lot as that used in our previous study,¹⁰⁾ in which its effects on IgA secretion and RD-5 expression were evaluated under comparable experimental conditions.

The GA-A doses used in this study were selected based on the equivalent Reishi dose (5 mg/kg) known to activate intestinal immunity, corresponding to approximately 10 μg/kg GA-A (0.1–0.2% content in Reishi).¹¹⁾ A higher dose (100 μg/kg) was also tested to confirm dose dependence. The estimated human-equivalent dose (≈1 mg/kg) falls within the range of commonly consumed Reishi supplements, which have demonstrated safety in clinical trials administering 3 g daily for 16 weeks.²⁵⁾ Therefore, GA-A may represent a safe and promising candidate for self-medication or dietary supplementation aimed at supporting intestinal immune function. Future studies should establish scientific evidence regarding its safety, including potential food-drug interactions.

Using TLR4-deficient mice (C3H/HeJ), we demonstrated that GA-A-induced IgA secretion and RD-5 upregulation depend on TLR4 signaling. In wild-type C3H/HeN mice, LPS treatment significantly increased IgA secretion from PP cells, whereas in TLR4-deficient mice, no increase in secretion was observed. C3H/HeJ mice are known to have high resistance to LPS, an endotoxin.²⁶⁾ Additionally, HeJ mice have a mutation at proline 712 in the gene encoding TLR4, which is replaced by histidine, leading to a functional deficiency in TLR4.²⁷⁾ These results indicate that GA-A enhances intestinal immune responses in a TLR4-dependent manner, as assessed in PP assays, potentially involving downstream nuclear factor-κB activation.²⁸⁾ However, the present data do not allow conclusions regarding whether this signaling pathway contributes to the regulation of oxidative stress–related genes, including SODs, in the intestinal I/R model.

I/R of the small intestine is a well-established oxidative stress model.¹⁶⁾ We have previously reported that CA suppresses damage caused by small intestine I/R.¹⁸⁾ However, the impact of small intestine I/R-induced damage on intestinal immunity remains insufficiently elucidated. Reishi polysaccharides affect small intestine I/R,²⁹⁾ and ganoderic acids, including GA-A, have been studied in relation to I/R damage in some organs such as the kidney³⁰⁾; however, the effects of GA-A on the decline of intestinal immunity due to small intestine I/R remained unclear. This study demonstrated that both Reishi and its active component GA-A have intestinal immune-enhancing effects and act to suppress the decline in intestinal immunity induced by small intestine I/R.¹⁰⁾ This protection appears linked to the modulation of SODs, enzymes that eliminate reactive oxygen species and mitigate oxidative stress–induced tissue damage.^31,32) SOD2 is a mitochondrial isoform, whereas SOD1 and SOD3 are localized in the cytosol and extracellular space, respectively. Differences in subcellular localization and regulatory mechanisms may account for the distinct responses of these SOD isoforms under intestinal I/R conditions. These results support previous findings that Reishi exhibits antioxidant effects in vitro and that oxidative stress is involved in GA-A’s antitumor effects.^28,33) Additionally, tissue staining results showed that pre-administration of Reishi suppressed villus loss induced by small intestine I/R and suppressed the decrease in Paneth cells, which are located at the bottom of the intestinal crypts. Similarly, CA, used as a positive control in this study, also demonstrated intestinal immune-enhancing effects and suppressed the decline in intestinal immunity due to small intestine I/R, like Reishi and GA-A.

CA was included in this study as a positive control because its protective effects against small intestinal I/R injury have been previously demonstrated by our group.¹⁸⁾ GA-A and CA differ markedly in their chemical structures, pharmacological properties, and dosage; therefore, their mechanisms of action are not assumed to be identical. The present study was not designed to compare their molecular mechanisms directly, but rather to evaluate whether GA-A can exert protective effects on intestinal immunity that are functionally comparable to those of a known antioxidant compound. Accordingly, the observed similarities in phenotypic outcomes do not necessarily imply shared signaling pathways or antioxidant mechanisms. In addition, because SOD expression was assessed only at the mRNA level, any mechanistic interpretation regarding antioxidant pathways should be made with caution.

SODs are enzymes that remove reactive oxygen species generated inside cells and have a protective effect against oxidative stress models.^31,32,34) I/R significantly reduced ileal SOD1, SOD2, and SOD3 mRNA levels compared with the sham group. In the Reishi or GA-A groups, SOD1 and SOD3 mRNA levels tended to recover compared with the I/R group, whereas SOD2 mRNA levels showed no effect. These results suggest that intracellular SOD1 and extracellular SOD3 may be involved in the suppression of intestinal immune decline caused by intestinal I/R with Reishi and GA-A, followed by enhanced resistance to oxidative stress. While CA administration restored SOD1, SOD2, and SOD3 mRNA levels, the intestinal immune and tissue-protective effects were similar to those observed with GA-A, suggesting that SOD1 and SOD3, rather than SOD2, play a primary role in mitigating I/R-induced damage. These differences may also be related to differences in the localization of SOD isoforms. Future studies should investigate the effectiveness under various oxidative stress-related conditions.

Although this study provides valuable insights, it has some limitations. One key limitation is that different animal species and strains were used across experiments. Ex vivo PP assays were conducted using an inbred C3H mouse strain, whereas the I/R model was established in outbred Wistar rats. Because intestinal immune functions, including IgA responses and TLR reactivity, may vary with species, strain, and genetic background, these differences could have influenced the observed responses. Although PP cell numbers were standardized and experiments were independently repeated to ensure reproducibility, the potential impact of strain- and species-specific variability cannot be fully excluded. Therefore, caution is required when directly comparing results across experimental models, and future studies using a single inbred strain would help further validate the present findings. Reishi is a complex natural product containing multiple bioactive constituents, including various triterpenoids and polysaccharides. Although GA-A is one of the major and well-characterized triterpenoid components, the present study does not exclude the contribution of other constituents to the overall biological effects of Reishi. The observation that GA-A alone elicited effects comparable to those of Reishi suggests that GA-A is a key contributor; however, this does not imply that GA-A is the sole active component. Comprehensive chemical profiling of Reishi extracts and functional evaluation of other constituents will be necessary to fully elucidate their respective contributions.

In conclusion, this study demonstrates that Reishi enhances intestinal immunity and prevents oxidative stress-induced immune decline through its active component GA-A. These findings provide new insights into the mechanisms underlying Reishi’s health benefits and support the potential use of GA-A as a safe, natural agent for managing oxidative stress-related intestinal disorders and for promoting intestinal immune health in functional food applications.

DECLARATIONS

Funding

Kieikai Research Foundation and All Japan Coffee Association supported this work.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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