Skip to main content
Download PDF

Lentinan alleviates arsenic-induced hepatotoxicity in mice via downregulation of OX40/IL-17A and activation of Nrf2 signaling

BMC Pharmacology and Toxicology volume 23, Article number: 16 (2022) Cite this article

Abstract

Background

Arsenic, existing ubiquitously in soil, drinking water, or food, is well known to be an environmental pollutants concerned by European Food Safety Authority. Lentinan, a beta-1,6;1,3-glucan extracts from Lentinus edodes, which has the properties of antioxidant and immunomodulation, present study explored the pharmacological effects of Lentinan on arsenic induced hepatotoxicity in mice.

Methods

Mice experiments were performed by sodium arsenite (SA) treatment or Lentinan intervention, then histopathology, ELISA, Flow Cytometry, or Western-Blotting were applied to evaluate hepatic injury, oxidative stress, CD4+ type 17 helper T (Th17) cells, CD4+CD25+Foxp3+ regulatory T cells (Tregs), T cells receptor OX40/CD134, IL-17A, NLRP3, Nrf2, and NQO1.

Results

SA treatment showed hepatic pathological injury and the elevations of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in serum, and induced the increases of malondialdehyde (MDA), Th17 cells, OX40 or IL-17A in liver tissues, which were consistently ameliorated by Lentinan intervention. Further, immunoblotting experiments showed that Lentinan intervention downregulated the levels of OX40, IL-17A, and NLRP3 signals, while elevated the levels of anti-oxidative Nrf2, NQO1 signals compared to arsenic treatment group. For Tregs, Lentinan intervention showed no significant difference from SA treatment group.

Conclusion

Lentinan antagonizes SA-induced hepatotoxicity in mice, may be involved in the downregulations of pro-inflammatory OX40 or IL-17A and the activation of anti-oxidative Nrf2, NQO1 signals.

Peer Review reports

Introduction

Arsenic, existing ubiquitously in environment, which causes the increased risk of lung, skin and bladder cancer. Particularly, the ‘milk and dairy products’, ‘drinking water’ and ‘food for infants’ and rice is an important source of inorganic arsenic, concerned by European Food Safety Authority (EFSA) [1, 2]. Regarding the arsenic-induced hepatic toxicity, the underlying mechanism demonstrated as the imbalance of oxidant-antioxidant, the elevations of pro-inflammatory cytokines, DNA damage, apoptotic cell death, and pyroptosis [3, 4]. Moreover, our study show arsenic induced the increased levels of proinflammatory cytokines IL-1β, IL-6, and NLRP3 inflammasome in mouse liver [5]. The liver is a frontline immune tissue, and liver function is associated closely with immune response, excessive inflammation in the absence of infection leads to sterile liver injury and tissue damage [6]. Further, recent study demonstrated that arsenic interfered with the differentiation of helper T cells (Th) such as Th1/Th2/Th17 subsets in lung and spleen of mice [7]. Mechanistically, nuclear factor erythroid 2 related factor (Nrf2), a master transcription factor, its activation can suppress Th17 immune responses and rectify oxidant-antioxidant imbalance in periphery and brain of BTBR T + tf/J (BTBR) mice [8]. And, the activated Nrf2 can downregulate oxidative stress and inflammation parameters (NF-κB, IL-6, IL-1β) in autistic children, which is responsible for the ameliorative effects in Autism spectrum disorder (ASD) subjects [9], indicating Nrf2 play a significant role by its management of anti-inflammatory and antioxidant genes in arsenic-induced hepatotoxicity.

Lentinan (LNT) is the bioactive polysaccharide of beta-1,6;1,3-glucan, isolated from the mushroom shiitake Lentinus edodes (L. edodes), which has the high capability of antioxidation against cellular oxidative damage and preserving cell activity [10], and showing the anti-inflammatory activity of inhibiting inflammatory cytokine production [11]. For example, LNT alleviates against cisplatin-induced nephrotoxicity by the inhibitions of reactive oxygen species (ROS) and the activation of the Nrf2-antioxidant response element (ARE) signalling pathway in kidney [12]; LNT attenuates IL-1β secretion resulting from Listeria-mediated AIM2 inflammasome activation in myeloid cells [13]. Interestingly, the polysaccharides in Lentinus edodes possess the immunomodulatory activity of activating different immune responses in the host [14]. LNT cancels Th2-dominant condition and decreases significantly the percentages of CD4+IL-4+ T-cell and CD4+ IL-6+ T-cell in patients with digestive cancers [15], and LNT inhibits the activation of Th2 cells and affects the immune microenvironment, leading to the alleviated allergic reactions in allergic mice [16]. These studies indicate LNT’ bioactivity is cross-linked to arsenic toxicity under the pathological status of poisoning and injury, so the present study investigated the pharmacological effects of LNT by a mice model of sodium arsenite (SA) oral administration.

Materials and methods

Animal experimental protocol

The methodology of the present study was reviewed, and approved by Ethics Committee of Guizhou Medical University (no. 1900222). Male C57BL/6 mice weighed 25.5 ± 1.0 g, age range from 9 to 10 weeks, were obtained from the Slack King of Laboratory Animal Co., Ltd. (certificate of conformity: SCXK2019–0004, Changsha, China). Thirty-two mice were randomly allocated into four groups (n = 8/group). (i) Control group: mice mice were administrated orally by deionized water (0.25 ml/time, once every other day for 14 days); (ii) SA treatment group: mice were administrated by gavage sodium arsenite (NaAsO2) (CAS.7784-46-5, Sigma-Aldrich, USA) solution (dissolved in deionized water by 1.0 mg/ml, dose: 10.0 mg/kg.bw, 0.25 ml/time), once every other day for 14 days, referred to Bashir et al. methods [3, 17]; (iii) LNT treatment group: mice were administered by intramuscular injection LNT (CAS.37339–90-5, Shanghai Duma Biotechnology Co., Ltd.; dissolved in deionized water, dose: 1.0 mg/kg.bw), once every other day for 14 days, referred to Mao et al. studies [18, 19]; (iv) LNT + SA treatment group: mice were treated by LNT plus SA, LNT was administered 8 h ahead of the SA treatment, referred to the methods of (ii) and (iii). When the experiment was complete, the mice were euthanized by intraperitoneal injection of sodium pentobarbital, then blood samples were collected and liver tissues were immediately isolated, one part of liver tissue from each mouse was fixed in 4% phosphate-buffered formaldehyde for hematoxylin-eosin (HE) staining, and the rest of liver tissue was placed in liquid nitrogen for further analysis.

Liver function, HE staining for histopathological evaluation

Blood samples were centrifuged at 4 °C and 400×g for 5 min, and the serum supernatants were collected for measuring the levels of ALT (MLGR-E21533), AST (MLGR-E21534) according to the instructions of ELISA assay kit (Shanghai Mlbio Biotechnology Co., Ltd.). For the evaluation of pathological liver injury, liver tissues were fixed with formaldehyde for 72 h, and then embedded in paraffin for preparing 5 μm pathological sections. HE staining was performed for evaluating the histopathological characteristics of liver tissue sections under Motic microscope (DMB5-2231P1).

Measurement of Th17, Tregs in liver tissues by flow Cytometry

Determination of Th17, Tregs referred to Kim MH, et al. method [20]. Briefly, liver tissues were homogenated to collect the constituent cells, and were washed twice with phosphate buffer saline (PBS) for obtaining cell suspension, then were added Cell Stimulation Cocktail 500 × (00–4975-93, eBioscience), at 37 °C incubator for 6 h, and the collected cells were treated by Fixation/Permeabilization Concentrate 4× under 4 °C for 30 min. Cells were re-suspended, and the cells suspensions were incubated with anti-CD4 (11–0041-82, eBioscience) and anti-IL-17 (45–7177-82, eBioscience) antibodies at 4 °C for 30 min for evaluating the content of Th17 cells. Alternatively, the cells suspensions were incubated with the anti-CD4 (11–0041-82, eBioscience), anti-CD25 (17–0251-82, eBioscience) and anti-Foxp3 (12–5773-82) antibodies (ebioscience, USA) at 4 °C for 30 min for evaluating the content of Tregs, according to standard procedures. After two washes with binding buffer, the collected cells were analyzed using the AQUIOS CL Flow Cytometry System (Beckman Coulter, Inc., San Jose, Brea). For gating controls in flow cytometry, FcR Blocking Reagent (130–092-575, Miltenyi) and the isotype control for each antibody, were used during flow cytometry, respectively.

Detection of oxidative stress indices in liver tissues

Briefly, liver tissues were dissected out, and were rinsed with PBS to remove excess blood. Then tissues were homogenized in PBS (10% w/v) by Dounce Homogenizer (BioVision) and were incubated with RPMI media containing 0.05% collagenase type II (C2-BIOC, Sigma-Aldrich) at 37 °C for 30 min. Then the homogenates were done by three repeated freeze-thaw cycles of lysing cells, subsequently centrifuged at 4 °C, 1500×g for 10 min, the final supernatants were collected for detecting the content of MDA (MLGR-E20206, Shanghai Mlbio Biotechnology Co., Ltd.) or Glutathione (GSH) (MLGR-E20324, Shanghai Mlbio Biotechnology Co., Ltd.) by ELISA method. The OD values were measured by Microplate reader (BioTek800 TS, USA) at wavelength of 450 nm, and the measured results were converted from OD values to the nanomolar concentration (nmol/g wet tissue) according to the standard curve and dilution ratio.

Measurement of OX40, IL-17A in liver mononuclear cells

Liver mononuclear cells (LMC) referred to Zhang J et al. methods of digestion and filteration [21]. Briefly, the dissociated liver tissues were homogenized and digested by 0.05% collagenase type II for 30 min at 37 °C. The cells mixture was then filtered through a 70-μm nylon cell strainer and centrifuged at 500×g for 5 min. Further, the supernatants were collected for quantitative analysis of T cells membrane receptor OX40/CD134 (EK0998, Wuhan Boster Biotechnology Co., Ltd.) or inflmmatory cytokine IL-17A (MLGR-E20171, Shanghai Mlbio Biotechnology Co., Ltd.) by ELISA method according to the manufacturer’s instructions, respectively.

Western-blotting evaluation of inflammatory or anti-oxidative signaling

Following liver tissues were routinely dissociated and digested by collagenase type II, the hepatic cells suspensions were collected for extracting total proteins by Total Protein Extraction Kit (Shanghai Sangon Biotech, China) according to the manufacturer’s instructions, then the concentration of proteins was determined using the Bradford reagent (Sigma-Aldrich, USA). The expressions of OX40, Nrf2, NQO1, and inflammatory signaling NOD-like receptor family pyrin domain-containing 3 (NLRP3) were evaluated by the standard steps of Western-blotting (WB) experiment. The primary antibodies were provided as the followings: Anti-CD134/OX40L receptor antibody (ab229021, 1:800, Abcam), anti-Nrf2 antibody (ab92946, 1:1000, Abcam), anti-NQO1 antibody (sc-32,793, 1:800, Santa Cruz Biotechnology), or anti-NLRP3 antibody (ab263899, 1:1000, Abcam). Anti-rabbit IgG-HRP antibody (LS-C86382, LSBio) was used as the secondary antibody. WB results were pictured by enhanced chemiluminescence reagent (Beyotime, China), and then the integrated absorbance (IA) of the bands were analyzed quantitatively using LI-COR Odyssey Imaging System. Anti-GAPDH Rabbit Polyclonal antibody (BM1623, Dingguo Biotech Co. Ltd) were used as an internal control for normalizing the values in equal samples, the relative levels of target proteins were evaluated by the ratio of target protein vs internal control (IA/IA).

Statistical analysis

Data were expressed as mean ± standard deviation (SD), and were analyzed by SPSS version 23.0 (IBM Corp). One-way analysis of variance (ANOVA) test and independent sample’s t-test were applied to evaluate the statistical difference between groups. P<0.05 was considered as a significant difference between groups.

Results

LNT intervention alleviated hepatic damage induced by arsenic

As shown in Fig. 1A and B, HE staining sections showed hepatic injury in SA treatment group, such as hepatocytes edema, degeneration, nucleus shrinkage, and even some hepatocytes disappear, hepatocyte cord disconnection, and hepatocytes boundaries unclear. Interestingly, the intervention with LNT showed an amelioration of liver pathological injury, characterized as the alleviated hepatocytes edema, the decreased expansion of hepatic sinusoids compared with that in SA treatment group. LNT intervention showed also the significant antagonism against the high levels of ALT or AST in SA treatment group (ALT: F value as 0.82, t value as 6.43, P < 0.05; AST: F value as 4.61, t value as 2.85, P < 0.05). The results suggest the improvement of liver dysfunction and a beneficial role of LNT intervention, details were shown as Table S1 (Supplementary material).

Fig. 1
figure 1

Characterisrics of hepatic histopathology or liver function in mice. A Representative histopathological images (HE, × 200) in liver. (I) Control: Liver histology was normal, as shown by the arrow, liver cell nucleus uniform, the nuclear membrane smooth, the chromatin stained lightly, the cytoplasm uniformly pink, hepatocyte cord visible, and hepatic sinus no dilation and arrangement regular; (II) SA treatment group: Mice were administrated orally by SA at 10.0 mg/kg.bw once every other day for 14 days, as shown by the arrow, hepatocyte degeneration or even disappear, hepatocyte edema and vacuolar degeneration, accompanied by the disconnected hepatocyte cord and the unclear boundaries of hepatocytes; also, some places showd the different sizes of nucleus and the nucleus shrinkage or even disappear; (III) LNT + SA treatment: Mice were injected intramuscular with LNT at 1.0 mg/kg.bw and subsequent oral administration of SA at 10.0 mg/kg.bw once every other day for 14 days, as shown by the arrow, hepatocyte cord was visible, hepatic sinusoids expansion mild, less severe than SA treatment group, hepatocytes shoed mild to moderate edema, the sizes of dense staining nuclear varied, and nuclear membrane mild thicken; (IV) LNT control: The normal histology of liver tissues were similar to control group, as shown by the arrow, hepatocyte showed normal without edema and the normal ratio of nuclear to plasma, the uniform nucleus size, the smooth nuclear membrane, the lightly stained chromatin, hepatic sinusoids showed normal shape and no dilatation. B Histogram of ALT or AST content in serum after SA treatment or Lentinan intervention. SA treatment showed the increased levels of serum ALT or AST, which was downregulated significantly after the intervention with Lentinan; Data were expressed as mean ± SD; n = 5. Levels of ALT, AST in serum were quantified by the the convertion from OD values to ng values of per milliliter according to the standard curve; Independent sample’s t-test followed ANOVA was performed for analyzing the difference between groups. *P < 0.05 compared with control group; #P < 0.05 compared with SA treatment group. Abbreviations: SA, sodium arsenite; HE, hematoxylin-eosin; LNT, Lentinan; ALT, alanine aminotransferase; AST, aspartate aminotransferase

Full size image

LNT intervention downregulated Th17 levels in liver against arsenic treatment

As shown in Fig. 2A, B, C, SA treatment showed a significant increase of Th17 lymphocytes in liver tissues compared to control group (F value as 3.47, t value as 31.32, P < 0.05). And, SA induced a significant downregulation of Th17 lymphocytes in liver tissues was revealed after LNT intervention compared with that of SA treatment group (F value as 0.04, t value as 9.41, P < 0.05). For the Tregs, SA treatment showed the elevated level of Tregs in liver tissues compared to control group (F value as 4.28, t value as 6.80, P < 0.05), while the intervention with LNT showed no significant difference from SA treatment group (F value as 1.99, t value as 0.79, P > 0.05), details were shown as Table S2 (Supplemental material).

Fig. 2
figure 2

Characteristics of Th17 or Tregs in liver after arsenic exposure or Lentinan intervention. A Characteristics of Th17 or Tregs in liver tissues after SA exposure or Lentinan intervention. SA treatment showed the upregulated levels of Th17 or Tregs in liver tissues compared to control, and the intervention with Lentinan led to a significant downregulation of Th17 compared to SA treatment group (P < 0.05), while the change of Tregs was no significant (P > 0.05); B Representative images of Th17 in liver tissues by Flow cytometry analysis; C Representative images of Tregs in liver tissues by Flow cytometry analysis. Data were showed as mean ± SD; n = 5. Th17, Tregs were presented as percentage (%), which was analyzed by ANOVA and independent sample’s t-test; *P < 0.05 indicates a significant difference compared with control group; #P < 0.05 indicates a significant difference compared with SA treatment group. Abbreviations: Th17, CD4+ type 17 helper T cells; Tregs, CD4+CD25+Foxp3+ regulatory T cells

Full size image

LNT downregulated MDA and inflammatory signals during arsenic toxicity

As shown in Fig. 3A, compared to control group, SA treatment group showed an increase of oxidative stress index MDA and a reduce of antioxidant GSH in liver tissues (MDA: F value as 0.38, t value as 3.80, P < 0.05; GSH: F value as 0.27, t value as 11.17, P < 0.05), indicating the occurrence of hepatic oxidative damage after SA exposure. After the pretreatment intervention with LNT, a significant downregulation in MDA and the rebound of GSH were found as a comparison with SA treatment group (MDA: F value as 0.05, t value as 2.50, P < 0.05; GSH: F value as 0.47, t value as 3.53, P < 0.05), suggesting an alleviation of hepatic oxidative stress elicited by LNT intervention.

Fig. 3
figure 3

Lentinan antagonized oxidative stress and inflammatory signals in liver. A. Histogram of oxidative stress indicators MDA, GSH after SA treatment or Lentinan intervention. An increased MDA and a reduced GSH in liver were found in SA treatment group, which were antagonized consistently by Lentinan intervention; B Characteristics of inflammatory signals OX40, IL-17A after SA treatment or Lentinan intervention. SA induced the upregulations of proinflammatory signals OX40, IL-17A, while was downregulated significantly after Lentinan intervention. Data were expressed as mean ± SD; n = 5. The levels of MDA, GSH, OX40, and IL-17A in liver tissues were quantified by the convertion of OD values to nanomole or picogram values of per gram liver tissue (nmol or pg/g tissue) according to the standard curve and dilution ratio. Significant difference was obtained by independent sample’s t-test followed ANOVA. *P < 0.05 indicates a significant difference compared with control; #P < 0.05 indicates a significant difference compared with SA treatment. Abbreviations: IL, Interleukin; MDA, malondialdehyde; GSH, glutathione; OX40 (CD134), Tumor necrosis factor receptor superfamily member 4

Full size image

As shown in Fig. 3B, compared to control group, a significant increase of T lymphocytes immunity associated inflammatory signals OX40, IL-17A in liver were revealed in SA treatment group (OX40: F value as 1.17, t value as 7.60, P < 0.05; IL-17A: F value as 0.38, t value as 7.76, P < 0.05). Compared to SA treatment group, the intervention with LNT showed a significant downregulation against the high levels of OX40, IL-17A (OX40: F value as 0.11, t value as 2.76, P < 0.05; IL-17A: F value as 0.13, t value as 6.09, P < 0.05). The results indicate an amelioration of CD4+ or CD8+ T cell mediated inflammation in liver, more details were shown in Table S3 (Supplemental material).

LNT inhibited inflammatory signaling and activated Nrf2 in liver

As shown in Fig. 4A, the expressions of OX40, IL-17A and NLRP3 inflammatory signals showed a significant upregulation in SA treatment group as compared to control group (OX40: F value as 0.46, t value as 15.58, P < 0.05; IL-17A: F value as 0.86, t value as 54.26, P < 0.05; NLRP3: F value as 11.39, t value as 7.92, P < 0.05). While the intervention with LNT showed a significant downregulation in OX40, IL-17A and NLRP3 signals compared to SA treatment group (OX40: F value as 6.16, t value as 7.68, P < 0.05; IL-17A: F value as 0.64, t value as 18.13, P < 0.05; NLRP3: F value as 0.27, t value as 6.49, P < 0.05). Therefore, the WB experiments further demonstrated the intervention with LNT led to an amelioration against the immunity-related hepatic inflammation induced by SA,

Fig. 4
figure 4

Lentinan downregulated arsenic-induced inflammatory signals and upregulated anti-oxidative signals in liver by western-blotting analysis. A Characteristics of OX40, IL-17A, and NLRP3 inflammatory signaling after SA exposure or Lentinan intervention. SA induced a significant upregulations of OX40, IL-17A, and NLRP3, which were antagonized significantly after the intervention with Lentinan (P < 0.05); B Characteristics of Nrf2, NQO1 after SA exposure or Lentinan intervention. SA induced the upregulations of Nrf2 and NQO1, while the intervention with Lentinan showed the further upregulations of Nrf2 and NQO1 compared to SA treatment group (P < 0.05). Data were expressed as mean ± SD; n = 5. The levels of proteins expression were evaluated by the measurement of relative IA levels, quantified by the ratio of detected protein vs. internal control (β-actin) in each group (IA/IA). The significant difference between groups was obtained by independent sample’s t-test followed ANOVA. *P < 0.05 indicates a significant difference compared with control group; #P < 0.05 indicates a significant difference compared with SA treatment group. Abbreviations: NLRP3, NOD-like receptor family pyrin domain-containing 3; Nrf2, NF-E2 p45-related factor 2; NQO1, NAD(P)H quinone dehydrogenase 1; IA, integrated absorbance

Full size image

As shown in Fig. 4B, compared to control group, the high levels of anti-oxidative Nrf2 and its downstream target NQO1 signaling were revealed in SA treatment group (Nrf2: F value as 1.42, t value as 14.31, P < 0.05; NQO1: F value as 1.40, t value as 4.18, P < 0.05). While the intervention with LNT showed the further upregulations of Nrf2, NQO1 compared to SA treatment group (Nrf2: F value as 1.98, t value as 7.21, P < 0.05; NQO1: F value as 0.02, t value as 4.29, P < 0.05). The results indicate the activation Nrf2-ARE signaling pathway maybe as an intracellular stress response during arsenic toxicity, while the further activation of Nrf2-ARE signaling pathway indicated the enhanced hepatic anti-oxidative capability after LNT intervention, details were shown as Fig. S1 ~ 2 & Table S4 (Supplemental material).

Discussion

Th17 cells, as a pro-inflammatory type of T Lymphocyte subtypes originated from the differentiation of precursor T helper cell 0 (Th0), which were developed from naive CD4+ T cell after antigen stimulation. Th17 differentiation can be directed by their master transcription factor retinoic acid receptor-related orphan receptor-γt (RORγt). RORγt as a specific transcript of the RORC gene regulated by transcription factors signal transducer and activator of transcription 3 (STAT3), are involved in mediating inflammatory responses and exacerbating liver injury by inducing the release of pro-inflammatory cytokines, such as IL-17 and tumor necrosis factor-alpha (TNF-α) [22]. IL-17 as the proinflammatory cytokine secreted by Th17 cells, IL-17 binds to its receptor, can induces the activation of NF-κB signaling [23], and the activated NF-κB signaling further up-regulates the NLRP3 inflammasome response [24], while blocking NLRP3 inflammasome reduces liver inflammation and fibrosis in experimental liver disease model in mice [25]. In the present study, arsenic exposure led to hepatic pathological injury and oxidative stress, accompanied by the elevated proportion of Th17 cells, and the upregulations of inflammatory signals IL-17A, NLRP3 in mouse liver. The results indicate that arsenic exposure activated transcription factors RORγt, STAT3. Consequently, the hepatic augment of Th17 cells induced inflammatory signal IL-17A production, and then IL-17A mediated the activation of NF-κB inflammatory signaling pathway, which is responsible for the pathological injury in liver. Interestingly, the intervention with LNT showed an effective antagonism against arsenic-induced the elevations of IL-17A and inflammasomes NLRP3 in mice, indicating the downregulation in IL-17A secreted by Th17 cells, contributing to the amelioration of downstream NF-κB inflammatory signaling in hepatocytes and the attenuation of arsenic-induced hepatotoxicity in mice. Similarly, the previous studies showed that LNT reduces the activation of NF-κB and inhibits the production of pro-inflammatory cytokines IL-1β, TNF-α, IL-8 in chondrocytes [26], LNT intervention improves hepatic cell morphology by the decreased activity of NF-κB signal in rat’s liver with sepsis [27].

Also, the present study showed that LNT intervention leads to the antagonism against arsenic-induced oxidative stress in liver of mice. Oxidative stress as an intracellular pathological state, indicating the reduced antioxidants and the increased ROS in cells, which is considered as the common pathophysiological characteristic during arsenic induced multiple organ injury such as liver, kidney, skin, etc. [28]. Mechanistically, oxidative stress can elicit DNA damage, leads to mitochondrias dysregulation, endoplasmic reticulum stress, and chronic inflammation [29]. Consequently, the inhibition of oxidative stress leads to the upregulations of anti-oxidative Nrf2 signaling pathway [30], and leads to the amelioration of hepatic inflammation or acute liver injury, involved in the downregulations of NF-κB inflammatory signaling [31]. Nadeem A et al. demonstrated that the activated Nrf2 signal antagonizes NF-κB signaling and inhibits inflammatory parameters [9]. Here, despite the activated Nrf2 signaling induced by arsenic exposure in the present study, the intervention with LNT showed that the amplified elevation of Nrf2-ARE signaling pathway. It is speculated that arsenic induced the activations of Nrf2 and its downstream signaling in mice, which is considered as an intracellular stress response in body, as evidenced by Liu D et al’ study that inorganic arsenic induces the activation of the Nrf2 pathway in Chang human hepatocytes [32]. Significantly, LNT intervention induced the amplified elevation of activated Nrf2, NQO1 signals, indicating the effective inhibitions of oxidative stress, inflammatory cytokines production in hepatocyte, which maybe is involved in its immunomodulation bioactivity of T lymphocytes immune related inflammatory cytokines in arsenic-induced hepatic injury. Indeed, the previous studies demonstrated that LNT can suppress Th17 immune responses in BTBR mice [8], and demonstrated that the antioxidant Nrf2 signaling pathway is associated negatively with the regulation of liver inflammation [33]. Mechanistically, the activated Nrf2 promotes itself nuclear translocation, which, in turn, up-regulated phase II/antioxidant enzyme activities, and simultaneously reduces inflammatory response originated from hepatic injury [34]. For example, the Nrf2 activates its anti-oxidative downstream heme oxygenase-1 (HO-1) signaling pathways, which contributes to the amelioration of hepatic inflammation cell infiltration [35].

Interestingly, the downregulation of Th17 cells-associated inflammatory signals OX40 was found after the intervention with LNT. OX40, named as CD134 or tumor necrosis factor-receptor superfamily member 4 (TNFRSF4), a member of tumor necrosis factor receptor (TNFR) superfamily expressed on activated T cells, which is defined as a biomarker of T cell activation, whose ligand OX40L is found on activated T and B cells [36]. The bounding of OX40-OX40L play a critical role of regulating the activity of T cell and inflammatory cytokines production, associated closely with the proliferation, differentiation, or activation of CD4+ or CD8+ T cells [37, 38]. Recent study found that OX40 expressed in innate immune cells such as neutrophils, contributing to the prolonged neutrophils survival, and promoted proinflammatory cytokines, ROS production, while Ox40 knockout markedly alleviated liver injury [39]. In the present study, the intervention with LNT downregulated significantly the levels of OX40, indicating the decreases of innate immune cells and adaptive immune helper T cells, contributing to the amelioration of inflammation in liver. And, the immunomodulating activity of downregulating CD4(+) lymphocyte subsets-associated cytokines such as TNF-α, IL-2, IL-17 production in mice, which was demonstrated by other organic compounds such as β-1,3-Glucan (BG) with similar structure to LNT [40].

Summarily, the present study revealed that arsenic induced the activation of T cell immunity associated inflammatory signaling in mouse liver, while the intervention with LNT showed a hepatoprotective effects, manifested as the significant downregulations of OX40, IL-17A, and the enlarged elevation of Nrf2-NQO1 signaling, outlined as Fig. 4C. In future, our study should aim to explore the potential mechanism between Th1/2 cell differentiation and Nrf2-AGE signaling pathway by the LNT experiments in vitro, expecting to elucidate the cross-link pathway of LNT-regulated intracellular antioxidative Nrf2 signaling pathway against T lymphocytes-associated inflammatory response during arsenic-induced hepatotoxicity.

Conclusion

Arsenic induces hepatic pathological injury in mouse, while are antagonized significantly by LNT intervention, may be involved in the inhibition of T cell immunity-associated IL-17A or OX40 inflammatory signaling, and the activation of anti-oxidative Nrf2 signaling.

Availability of data and materials

All data needed to support the conclusions are included in this article, supplementary data are present in the supplemental materials. Additional data related to this paper can be requested from the author (yang1977yuan@sina.com).

References

  1. Roychowdhury T, Uchino T, Tokunaga H, Ando M. Survey of arsenic in food composites from an arsenic-affected area of West Bengal, India. Food Chem Toxicol. 2002;40:1611–21.

    Article  CAS  Google Scholar 

  2. Gundert-Remy U, Damm G, Foth H, Freyberger A, Gebel T, Golka K, et al. High exposure to inorganic arsenic by food: the need for risk reduction. Arch Toxicol. 2015;89:2219–27.

    Article  CAS  Google Scholar 

  3. Thangapandiyan S, Ramesh M, Hema T, Miltonprabu S, Uddin MS, Nandhini V, et al. Sulforaphane potentially ameliorates arsenic induced hepatotoxicity in albino Wistar rats: implication of PI3K/Akt/Nrf2 signaling pathway. Cell Physiol Biochem. 2019;52:1203–22.

    Article  CAS  Google Scholar 

  4. Qiu T, Pei P, Yao X, Jiang L, Wei S, Wang Z, et al. Taurine attenuates arsenic-induced pyroptosis and nonalcoholic steatohepatitis by inhibiting the autophagic-inflammasomal pathway. Cell Death Dis. 2018;9:946.

    Article  Google Scholar 

  5. Yang Y, Wang Q, Wang W, Wei S, Zeng Q, Zhang A. Semaphorin 4A antibody alleviates arsenic-induced hepatotoxicity in mice via inhibition of AKT2/NF-κB inflammatory signaling. Toxicol Appl Pharmacol. 2021;410:115364.

    Article  CAS  Google Scholar 

  6. Kubes P, Jenne C. Immune responses in the liver. Annu Rev Immunol. 2018;36:247–77.

    Article  CAS  Google Scholar 

  7. Zhao L, Yang S, Guo Y, Sun G, Li B. Chronic arsenic exposure in drinking water interferes with the balances of T lymphocyte subpopulations as well as stimulates the functions of dendritic cells in vivo. Int Immunopharmacol. 2019;71:115–31.

    Article  CAS  Google Scholar 

  8. Nadeem A, Ahmad SF, Al-Harbi NO, Attia SM, Bakheet SA, Ibrahim KE, et al. Nrf2 activator, sulforaphane ameliorates autism-like symptoms through suppression of Th17 related signaling and rectification of oxidant-antioxidant imbalance in periphery and brain of BTBR T+tf/J mice. Behav Brain Res. 2019;364:213–24.

    Article  CAS  Google Scholar 

  9. Nadeem A, Ahmad SF, Al-Ayadhi LY, Attia SM, Al-Harbi NO, Alzahrani KS, et al. Differential regulation of Nrf2 is linked to elevated inflammation and nitrative stress in monocytes of children with autism. Psychoneuroendocrinology. 2020;113:104554.

    Article  CAS  Google Scholar 

  10. Zi Y, Zhang B, Jiang B, Yang X, Liang Z, Liu W, et al. Antioxidant action and protective and reparative effects of lentinan on oxidative damage in HaCaT cells. J Cosmet Dermatol. 2018;17:1108–14.

    Article  Google Scholar 

  11. Zi Y, Jiang B, He C, Liu L. Lentinan inhibits oxidative stress and inflammatory cytokine production induced by benzo(a)pyrene in human keratinocytes. J Cosmet Dermatol. 2020;19:502–7.

    Article  Google Scholar 

  12. Chen Q, Peng H, Dong L, Chen L, Ma X, Peng Y, et al. Activation of the NRF2-ARE signalling pathway by the Lentinula edodes polysaccharose LNT alleviates ROS-mediated cisplatin nephrotoxicity. Int Immunopharmacol. 2016;36:1–8.

    Article  Google Scholar 

  13. Ahn H, Jeon E, Kim JC, Kang SG, Yoon SI, Ko HJ, et al. Lentinan from shiitake selectively attenuates AIM2 and non-canonical inflammasome activation while inducing pro-inflammatory cytokine production. Sci Rep. 2017;7:1314.

    Article  Google Scholar 

  14. Xu X, Yan H, Tang J, Chen J, Zhang X. Polysaccharides in Lentinus edodes: isolation, structure, immunomodulating activity and future prospective. Crit Rev Food Sci Nutr. 2014;54:474–87.

    Article  CAS  Google Scholar 

  15. Yoshino S, Tabata T, Hazama S, Iizuka N, Yamamoto K, Hirayama M, et al. Immunoregulatory effects of the antitumor polysaccharide lentinan on Th1/Th2 balance in patients with digestive cancers. Anticancer Res. 2000;20:4707–11.

    CAS  PubMed  Google Scholar 

  16. Yang S, Chew H, Jiang Y, Cheng L, Guo X, Che H, et al. Lentinan inhibited the activation of Th2 cells in allergic mice by reducing the amplitude of changes in biological rhythm. Int Arch Allergy Immunol. 2021;182:167–81.

    CAS  PubMed  Google Scholar 

  17. Bashir S, Sharma Y, Irshad M, Gupta SD, Dogra TD. Arsenic-induced cell death in liver and brain of experimental rats. Basic Clin Pharmacol Toxicol. 2006;98:38–43.

    Article  CAS  Google Scholar 

  18. Mao X, Hu H, Xiao X, Chen D, Yu B, He J, et al. Lentinan administration relieves gut barrier dysfunction induced by rotavirus in a weaned piglet model. Food Funct. 2019;10:2094–101.

    Article  CAS  Google Scholar 

  19. Zhang Y, Mei H, Shan W, Shi L, Chang X, Zhu Y, et al. Lentinan protects pancreatic β cells from STZ-induced damage. J Cell Mol Med. 2016;20:1803–12.

    Article  CAS  Google Scholar 

  20. Kim MH, Kim HH, Jeong JM, Shim YR, Lee JH, Kim YE, et al. Ginsenoside F2 attenuates chronic-binge ethanol-induced liver injury by increasing regulatory T cells and decreasing Th17 cells. J Ginseng Res. 2020;44:815–22.

    Article  Google Scholar 

  21. Zhang J, Dong Z, Zhou R, Luo D, Wei H, Tian Z. Isolation of lymphocytes and their innate immune characterizations from liver, intestine, lung and uterus. Cell Mol Immunol. 2005;2:271–80.

    PubMed  Google Scholar 

  22. Ciofani M, Madar A, Galan C, Sellars M, Mace K, Pauli F, et al. A validated regulatory network for Th17 cell specification. Cell. 2012;151:289–303.

    Article  CAS  Google Scholar 

  23. Sønder SU, Saret S, Tang W, Sturdevant DE, Porcella SF, Siebenlist U. IL-17-induced NF-kappaB activation via CIKS/Act1: physiologic significance and signaling mechanisms. J Biol Chem. 2011;286:12881–90.

    Article  Google Scholar 

  24. An Y, Zhang H, Wang C, Jiao F, Xu H, Wang X, et al. Activation of ROS/MAPKs/NF-κB/NLRP3 and inhibition of efferocytosis in osteoclast-mediated diabetic osteoporosis. FASEB J. 2019;33:12515–27.

    Article  CAS  Google Scholar 

  25. Mridha AR, Wree A, Robertson AAB, Yeh MM, Johnson CD, Van Rooyen DM, et al. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. J Hepatol. 2017;66:1037–46.

    Article  CAS  Google Scholar 

  26. Zhang Z, Zha Z, Zhao Z, Liu W, Li W. Lentinan inhibits AGE-induced inflammation and the expression of matrix-degrading enzymes in human chondrocytes. Drug Des Dev Ther. 2020;14:2819–29.

    Article  CAS  Google Scholar 

  27. Lv Q, Gu Y, Qi Y, Liu Z, Ma GE. Effects of lentinan on NF-κB activity in the liver of burn rats with sepsis. Exp Ther Med. 2020;20:2279–83.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Jomova K, Jenisova Z, Feszterova M, Baros S, Liska J, Hudecova D, et al. Arsenic: toxicity, oxidative stress and human disease. J Appl Toxicol. 2011;31:95–107.

    CAS  PubMed  Google Scholar 

  29. Hussain T, Tan B, Yin Y, Blachier F, Tossou MC, Rahu N. Oxidative stress and inflammation: what polyphenols can do for us? Oxidative Med Cell Longev. 2016;2016:7432797.

    Article  Google Scholar 

  30. Xu L, Yu Y, Sang R, Li J, Ge B, Zhang X. Protective effects of Taraxasterol against ethanol-induced liver injury by regulating CYP2E1/Nrf2/HO-1 and NF-κB signaling pathways in mice. Oxidative Med Cell Longev. 2018;2018:8284107.

    Google Scholar 

  31. Ding Y, Li Q, Xu Y, Chen Y, Deng Y, Zhi F, et al. Attenuating oxidative stress by Paeonol protected against acetaminophen-induced hepatotoxicity in mice. PLoS One. 2016;11:e0154375.

    Article  Google Scholar 

  32. Liu D, Duan X, Dong D, Bai C, Li X, Sun G, et al. Activation of the Nrf2 pathway by inorganic arsenic in human hepatocytes and the role of transcriptional repressor Bach1. Oxidative Med Cell Longev. 2013;2013:984546.

    Google Scholar 

  33. Zhao XJ, Yu HW, Yang YZ, Wu WY, Chen TY, Jia KK, et al. Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway. Redox Biol. 2018;18:124–37.

    Article  CAS  Google Scholar 

  34. Lee IC, Kim SH, Baek HS, Moon C, Kang SS, Kim SH, et al. The involvement of Nrf2 in the protective effects of diallyl disulfide on carbon tetrachloride-induced hepatic oxidative damage and inflammatory response in rats. Food Chem Toxicol. 2014;63:174–85.

    Article  CAS  Google Scholar 

  35. Wei X, Wang H, Sun X, Huang X, Xu W, Liang Y, et al. 4-hydroxy-2(3H)-benzoxazolone alleviates acetaminophen-induced hepatic injury by inhibiting NF-κB and activating Nrf2/HO-1 signaling pathways. Am J Transl Res. 2020;12:2169–80.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Eckhard S, et al. Cross-linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells[J]. Immunity. 1995;2:507–21.

    Article  Google Scholar 

  37. Flynn S, Toellner KM, Raykundalia C, Goodall M, Lane P. CD4 T cell cytokine differentiation: the B cell activation molecule, OX40 ligand, instructs CD4 T cells to express interleukin 4 and Upregulates expression of the chemokine receptor, Blr-1[J]. J Exp Med. 1998;188:297–304.

    Article  CAS  Google Scholar 

  38. Linch SN, Kasiewicz MJ, McNamara MJ, Hilgart-Martiszus IF, Farhad M, Redmond WL. Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice. Proc Natl Acad Sci U S A. 2016;113:E319–27.

    Article  CAS  Google Scholar 

  39. Jin H, Zhang C, Sun C, Zhao X, Tian D, Shi W, et al. OX40 expression in neutrophils promotes hepatic ischemia/reperfusion injury. JCI Insight. 2019;4:e129736.

    Article  Google Scholar 

  40. Bakheet SA, Attia SM, Alwetaid MY, Ansari MA, Zoheir KM, Nadeem A, et al. β-1,3-Glucan reverses aflatoxin B1-mediated suppression of immune responses in mice. Life Sci. 2016;152:1–13.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors gratefully thank Well Biological Technology Co., Ltd. for technical assistance in this study.

Funding

Natural Science Foundations of China (grant nos. 81960597), Science and Technology Fund of Guizhou Province [2020]1Y359, and the first-class discipline construction project in Guizhou Province-Public Health and Preventive Medicine (NO.2017[85]).

Author information

Authors and Affiliations

  1. School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, 550025, China

    Yuan Yang & Shuang Song

  2. Dong Medicine Key laboratory of Hunan Province, Department of Laboratory Medicine, Hunan University of Medicine, Huaihua, 418000, China

    Yuan Yang, Yuanyuan Nie, Rong Chen & Peng Chen

  3. Department of Toxicology, School of Public Health, Guilin Medical University, Guilin, 541004, China

    Yuan Yang

Authors
  1. Yuan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuang Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuanyuan Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peng Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y. Y. designed this study and wrote the main manuscript, S. S. and Y. N., R. C. and P. C. performed the experiments and prepared Figs. 1, 2, 3 and 4. All authors reviewed the manuscript. The authors read and approved the final manuscript.

Corresponding author

Correspondence to Yuan Yang.

Ethics declarations

Ethics approval and consent to participate

This study was approved by Ethics Committee of Guizhou Medical University (no. 1900222), all procedures reporting in this study on the animals were carried in accordance with the ARRIVE guidelines, and all methods were carried out in accordance with relevant guidelines and regulations. Informed consent was therefore not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, Y., Song, S., Nie, Y. et al. Lentinan alleviates arsenic-induced hepatotoxicity in mice via downregulation of OX40/IL-17A and activation of Nrf2 signaling. BMC Pharmacol Toxicol 23, 16 (2022). https://doi.org/10.1186/s40360-022-00557-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40360-022-00557-7

Keywords

BMC Pharmacology and Toxicology

ISSN: 2050-6511

Contact us