Silymarin impacts on immune system as an immunomodulator: One key for many locks

International Immunopharmacology 50 (2017) 194–201

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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Review

Silymarin impacts on immune system as an immunomodulator: One key for many locks

MARK

Nafiseh Esmaeila,⁎, Sima Balouchi Anarakib, Marjan Gharagozlooc, Behjat Moayedia a b c

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Department of Immunology, School of Medicine, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Department of Pediatrics, Program of Immunology and Allergology, Medical School, Université de Sherbrooke, Canada

A R T I C L E I N F O

A B S T R A C T

Keywords: Silymarin Immunomodulator Antioxidant Anti-inflammatory

Silymarin is a flavonoid complex extracted from the Silybum marianum plant. It acts as a strong antioxidant and free radical scavenger by different mechanisms. But in addition to antioxidant effects, silymarin/silybin reveals immunomodulatory affects with both immunostimulatory and immunosuppression activities. Different studies have shown that silymarin has the anti-inflammatory effect through the suppression of NF-κB signaling pathway and TNF-α activation. It also has different immunomodulatory activities in a dose and time-dependent manner. As an immunomodulator agent, silymarin inhibits T-lymphocyte function at low doses while stimulates inflammatory processes at high doses. Studies have shown that silymarin has attenuated autoimmune, allergic, preeclampsia, cancer, and immune-mediated liver diseases and also has suppressed oxidative and nitrosative immunotoxicity. Silymarin also has indicated dual effects on proliferation and apoptosis of different cells. In conclusion, based on the current review, silymarin has a broad spectrum of immunomodulatory functions under different conditions. Recognizing the exact mechanisms of silymarin on cellular and molecular pathways would be very valuable for treatment of immune-mediated diseases. Also further studies are needed to assess the utility of silymarin in protection against autoimmune, cancer, allergic and other diseases in human subjects.

1. Introduction Flavonoids are a group of naturally occurring polyphenolic compounds that are commonly found in the plant kingdom. These compounds have a benzo-γ-pyrone structure with at least one hydroxyl group and exhibit spectrum of biochemical activities affecting basic cell functions such as proliferation, differentiation, and apoptosis. Besides, flavonoids have been found to possess antioxidant activities dependent on their functional hydroxyl groups by scavenging free radicals and/or by chelating metal ions [1,2]. The Silybum marianum (milk thistle plant) is a member of the daisy family (Asteraceae) which originates from mountains of the Mediterranean region. The milk thistle plant grows mainly in North Africa, the Mediterranean region and the Middle East (now also grown in the U.S.) [3]. It is used as a source of the drug, for > 2000 years to treat liver and gallbladder disorders such as hepatitis and cirrhosis. It also protects the liver against poisoning from various chemical and environmental toxins such as snake bites, insect stings, toxic mushroom, alcohol, and acetaminophen [4–7]. Silymarin is a unique flavonoid complex extracted from fruits and seeds of Silybum marianum. It

⁎

consists of a family of flavolignans; silybin A, silybin B, isosilybin A, isosilybin B, silychristin, isosilychristin, silydianin and the flavonoid taxifoline. In addition to flavolignana, silymarin extract contains small amounts of flavonoids, and approximately 20% to 35% of fatty acids and polyphenolic compounds. But Silybin (synonymous with silibinin) is the major biologically active component of (70–80%) of the silymarin complex and it is a mixture of silybin A and silybin B (Fig. 1) [8–10]. That is why compounds containing milk thistle constituents showing silybin content in various studies, are used to explain the biological activity of silymarin. Silymarin displays strong antioxidant and free radical scavenging abilities by inducing superoxide dismutase, increasing cellular glutathione content and inhibiting lipid peroxidation. Another antioxidant mechanism of silymarin results from metal ions (iron, cooper, etc.) chelation ability of this compound [11–14]. Apart from hepatoprotective and antioxidant effects, silymarin/silybin has been described to exhibit anticarcinogenic, immunomodulatory and anti-inflammatory activities [15–21]. On the other hand, several studies have suggested that silymarin/ silybin reveals immunomodulatory affects with both

Corresponding author at: Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan 81744-176, Iran. E-mail address: [email protected] (N. Esmaeil).

http://dx.doi.org/10.1016/j.intimp.2017.06.030 Received 20 May 2017; Received in revised form 24 June 2017; Accepted 27 June 2017 Available online 30 June 2017 1567-5769/ © 2017 Elsevier B.V. All rights reserved.

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Fig. 1. Chemical components of Silybum marianum. These compounds have been distinguished in three milk thistle extracts available commercially as silymarin [10]. Silybin (synonymous with silibinin) is the major active component (50–70%) of the silymarin complex and it is a 1:1 mixture of silybin A and silybin B.

activated by a variety of stimuli, including microbial components, proinflammatory cytokines, phorbol esters, oxidants, phosphatase inhibitors and ultraviolet radiation via triggering of various sensors such as Toll-like receptors (TLRs) what results in degradation of IκB (Fig. 2). Following the degradation of IκB, NF-κB subunits translocate into the nucleus and bind to DNA sequences to induce target genes expression [27]. The important role of NF-κB in the pathogenesis of inflammation suggests that inhibitors of the NF-κB pathway could be effective targets for treatment of chronic inflammatory diseases. Silibinin has been demonstrated to inhibit NF-κB activation through suppression of inhibitory kappa B (IκB) degradation, thereby precludes both translocation of NF-κB into the nucleus and initiation of gene transcription related to the inflammatory response [28]. Kang et al. have shown that silymarin (6.25, 12.5, 25, 50 μg/ml) suppresses LPS (200 ng/ml) induced interleukin-1b (IL-1b), prostaglandin E2 (PGE2) production and COX-2 expression in isolated mouse peritoneal macrophages via suppression of NF-κB [29]. Also, silymarin blocks NF-κB activation induced by phorbol ester, LPS, okadaic acid, and ceramide. The effects of silymarin on NF-κB inhibition are specific, as it has not affected AP-1

immunostimulatory and immunosuppression activities in a dose and time-dependent manner. The present review focuses on the immunomodulatory effects of silymarin, which have been assessed in different studies.

2. The inhibitory effects of silymarin on transcription factors (NFκB, Stat3 and MEK/ERK) 2.1. Anti-inflammatory effects Several studies have indicated the role of nuclear factor-kappaB (NF-κB) inflammatory activations in the development of different diseases [22,23]. Silymarin has a potent anti-inflammatory action throughout the suppression of NF-κB regulated gene products, including cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inflammatory cytokines [15,24,25]. NF-κB is responsible for transcription of genes involved in immune responses, inflammation, and carcinogenesis [26]. In the cytoplasm of the unstimulated cells NF-κB dimer (p50/p65), which is present in inactive form bounds to inhibitory kappa B (IκB) protein. NF-κB is 195

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Fig. 2. Anti-inflammatory and anti-carcinogenic effects of silymarin. Silymarin inhibits NF-κB activation through suppression of inhibitory kappa B (IκB) degradation and suppresses inflammatory response, oxidative stress. Also silymarin by suppression of STAT3 and ERK1/2 signaling pathways, inhibits oncogenesis, cell proliferation, cell migration and iNOS gene expression.

same cytokines from splenocytes. It also increased intrahepatic and splenocytes IL-10 levels, and hepatocyte apoptosis [40]. These effects are concentration-dependent and silibinin at higher concentrations has been needed to inhibit IL-4 and induce IL-10 production, as compared to its inhibitory effect on TNF, IFN-γ, and IL-2 production. Silymarin also inhibits LPS and mycotoxin-induced TNF-α expression in the liver, which suggests a potential mechanism for its hepatoprotective effect [41,42]. Silymarin dose dependently displays anti-inflammatory actions via inhibition of NF-κB and CXCL-8 (IL-8) transcription in cultured human hepatoma Huh7 cells and directs antiviral effects against HCV infection without inhibiting cell growth. Silymarin also inhibited expression of TNF-α in anti-CD3 stimulated human PBMCs [38]. Studies have shown that silymarin dose-dependently inhibits T cell proliferation and production of TNF-α, IFN-γ and IL-12 by anti-CD3-stimulated PBMC from both HCV-infected and uninfected subjects. Furthermore, silymarin is able to inhibit proliferation, NF-κB transcriptional activation and IL-2 secretion by T cell receptor mediated stimulation of Jurkat T cell line [39]. Silymarin enhanced hepatic glutathione (GSH) by elevating cysteine synthesis while inhibiting its catabolism to taurine, which may subsequently contribute to the antioxidant defense [43]. A mouse study indicated that ethanol significantly reduced the content of GSH and the activity of superoxide desmotase (SOD), catalase, glutathione peroxidase, and glutathione reductase. Treatment with silymarin has normalized the altered parameters and also has reduced levels of IL-10, TNF-α, IFN-γ, vascular endothelial growth factor-A, and TGF-1β. The expression of cytokines in mouse liver was investigated following treatment with silymarin. Treatment caused significant increases in the expressions of TGF-β and c-myc in the liver, while no significant difference was detected in the expression of HGF, IFN-γ, TNF-α and class II

transcription [24]. In the central nervous system (CNS), inflammatory responses mediated by activated microglia (the resident macrophages in CNS) contribute to the development of neurodegenerative diseases such as Parkinson disease, Alzheimer disease and multiple sclerosis [30]. Surprisingly Wang et al. have studied the neuroprotective effect of silymarin against lipopolysaccharide (LPS)-induced neurotoxicity in mesencephalic mixed neuron-glia cultures. They have shown that silymarin protects dopaminergic neurons against lipopolysaccharide (LPS)-induced neurotoxicity by inhibiting microglia activation. Their results have indicated that silymarin inhibits the production of inflammatory mediators, such as TNF-α and nitric oxide (NO), and in this way reduces the LPS stimulated damage to dopaminergic neurons. In addition, their finding of the significant inhibitory function of silymarin in LPS-induced activation of microglia and reduction of nitric oxide synthase (iNOS) production, protein levels, superoxide generation and NF-κB activation, suggests that inhibitory effects of silymarin on microglia activation could be mediated through inhibition of NF-κB pathways activation [31]. Furthermore, several studies have shown that silymarin inhibits the ERK-1/2 pathway, a pathway also involved in NFκB-mediated transcription of inducible nitric oxide synthase (iNOS) (Fig. 2). So it seems that silymarin by affecting of NF-κB signaling is inhibited both inflammation and oxidative stress [16,24,32]. In the chronic liver disease such as chronic hepatitis C, hepatic inflammation is evaluated by the extent of lymphocyte infiltration and production of inflammatory cytokines and chemokines, which are often induced by transcription factor NF-κB, resulting in the pathogenesis of HCV-induced liver diseases including fibrosis and antiviral resistance [33–39]. In a murine ConA-induced T cell-dependent hepatitis model, pre-treatment with silibinin (25 mg/kg) significantly has inhibited intrahepatic mRNA levels of IL-2, IL-4, IFN-γ, TNF-α and secretion of the 196

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MMP-9 expression through inhibition of the MEK/ERK pathway in gastric cancer cells [59]. Interestingly, ERK has been reported to be involved in NF-κB-mediated transcription of iNOS, so inhibition of ERK and the transactivation capacity of NF-κB by silymarin suppress iNOS gene expression [60]. Taken together silymarin probably mediates its anticarcinogenic effects through the suppression of NF-κB-dependent gene expression, the inhibition of Stat3, MEK/ERK signaling and the inhibition of TNFαinduced activation of NF-κB.

major histocompatibility complex. Overall studies have reported on the effect of silymarin and its components on lymphocyte function and its immunosuppressive and anti-inflammatory effects on hepatocytes, which suggest that silymarin could potentially result in control of hepatic inflammation in chronic liver diseases [44]. Preeclampsia (PE) is a serious complication of human pregnancy associated with an intense inflammatory response involving leukocyte activation, as well as the elevated production of pro-inflammatory cytokines. There are increased endogenous activation of NF-κB as well as TNF-α and IL-1β release by PBMC in the PE subjects. A positive correlation between NF-κB activity and cytokine production has also observed in the PE. Bannwart-casro et al. assessed the silibinin (5 μM and 50 μM) effects on isolated PBMCs from women with PE, normotensive (NT) pregnant women, and nonpregnant (NP) women. They found that silibinin has capable of reducing the levels of NF-κB and cytokines TNFα and IL-1β in PE women [45]. So we can conclude that silibinin exhibits potent anti-inflammatory activity on preeclamptic women by downmodulation of NF-κB activation and inflammatory cytokine production.

2.3. The effects of silymarin on the components of immune system Johnson and colleagues tested silymarin for its impact on differentiation and cell selection in the thymus via alterations in gene expression at doses (10, 50, 250 mg/kg), which may be encountered in normal medicinal use. The absolute numbers of CD4 + and CD8 + T lymphocytes have been increased in male BALB/c mice treated with intraperitoneal administration of silymarin. C-myc proto-oncogene expression which is important in controlling the differentiation and function of thymocytes has also been increased by silymarin in the thymus [61]. In addition, down-regulation of the ERK pathway support positive selection and may increase single positive cells in the thymus [62]. Therefore silymarin treatment possibly induces positive selection in the thymus through down-regulation of the ERK signaling. The study indicated that silymarin inhibits T-lymphocyte function at low doses (10 and 50 mg/kg) while stimulates inflammatory processes at, high dose (250 mg/kg). So suggests that silymarin exhibits immunomodulatory effects in a dose-dependent manner. CD4 + and CD8 + T-cell populations diminish upon silymarin administration, but the effect on the CD4 + population is significant only at the lowest dose (50 mg/kg). Silymarin also enhances PHA-induced T lymphocyte proliferation and LPS-induced B-lymphocyte proliferation. In addition, the expressions of TNF, iNOS, IL-1, and IL-6 mRNA have increased dose dependently. Whereas the expressions of IL-2 and IL-4 have reduced in mice treated with 10 and 50 mg/kg of silymarin [63]. Furthermore, silymarin (50 mg/kg) has suppressed the LPS-induced (200 ng/ml) production of IL-1β in isolated mouse peritoneal macrophages and RAW, 264.7 a murine macrophage-like cell line by completely blocking the expression of IL-1β mRNA [29]. Sakai et al. have indicated that silymarin at 100 mM dose and after 48 h, induces transcriptional factors, enhance the major histocompatibility complex class I (MHCI) promoter and induces MHCI molecules on human neuroblastoma cell lines [64]. In contrast, Gharagozloo et al. have assessed the immunosuppressive effect of silymarin on MAPK signaling pathway and its impact on T cell proliferation and cytokine production, and results show that silymarin has the ability to inhibit T cell proliferation at the100μM concentration (without causing cell death [16]). Silymarin has been shown to inhibit the production of Th1 related cytokines (IL-2, IFN-γ, and TNF-α) by activated-PBMC dose-dependently [15,39]. Morishima et al. have shown that silymarin at 20 μg/ml dose suppresses secretion of TNF-α, IFN-γ, and IL-2 activated-PBMC from HCV-infected and uninfected subjects [39]. Furthermore, silymarin is able to inhibit ERK1/2 and P38 MAP kinase pathway activation in CD4 + T cells stimulated through TCR engagement. In addition, when the effect of silymarin on cell cycle and mTOR (the mammalian target of rapamycin) on activated human T cells was examined, results indicated a significant G1 arrest in the cell cycle of activated T lymphocytes treated with silymarin without causing apoptotic cell death. Silymarin also significantly has reduced the level of phospho-S6 ribosomal protein and mTOR activity in cell lysates of activated T cells [65,66]. Polyak et al. have assessed the suppressor effects of silymarin at 83 μM concentration (a non-toxic dose) by performing transcriptional profiling and metabolomics in human liver and T cell lines. They have found that in prolonged exposure with silymarin (24 h), it suppress multiple pro-inflammatory mRNAs and signaling pathways including nuclear factor kappa B (NF-κB), forkhead box O (FOXO) and

2.2. Anti-carcinogenic effects Anti-carcinogenic and chemopreventive effects of silymarin and silibinin have indicated in multiple in vitro and in vivo cancer models [46–49]. Studies have indicated that silymarin suppresses TNFα-induced activation of NF-κB in a dose- and time-dependent manner via inhibition of phosphorylation and degradation of IkBa and also blocks the translocation of p65 to the nucleus without affecting its ability to bind to the DNA. So silymarin is a potent inhibitor of TNFα expression in different cells and tissues [50–53]. Additionally, TNFα-induced cell signaling inhibits by silymarin, in tumors or in the cells exposed to tumor promoters. NF-κB-dependent reporter gene transcription also inhibits by silymarin. Besides, silymarin inhibits the TNFα-induced activation of mitogen-activated protein kinase (MAPK) and c-Jun Nterminal kinase. It abrogates the TNFα-induced cytotoxicity and caspase activation and suppresses the TNFα-induced production of reactive oxygen intermediates and lipid peroxidation. Accordingly, Agarwal et al., treated human prostate carcinoma DU145 cells with different concentration of silibinin (0, 10, 25 and 50 μM) for 12 and 24 h. They have found that, silibinin inhibits TNFα-induced activation of NF-κB following both 12 and 24 h of treatment at 10, 25 and 50 μM doses and the level of inhibition has been 20, 40 and 70% after 12 h of treatment and 20, 50 and 80% following 24 h of treatment, respectively [51]. Thus inhibition of TNFα signaling by silymarin in myeloid leukemia, Jurkat, HeLa and human prostate carcinoma cells suggests a potential mechanism for its protective effect [26,51,54]. Several genes are involved in tumor promotion that is regulated by NF-κB including some growth factors, cyclooxygenase-2, metalloproteases and cell adhesion molecules [55]. In addition, TNFα has the major role in inflammation and tumor promotion [52]. Furthermore, silymarin strongly inhibits activation of Stat3 signaling pathway which, this pathway is activated constitutively in some malignant cells such as prostate cancer (PCA) as self-dependent for their survival. Silymarin by inhibiting of Stat3 signaling in PCA cell line namely DU145 at 50 μM or higher concentrations for 24 or 48 h, induces caspase activation and apoptotic cell death in malignant cells (Fig. 2) [56,57]. Studies have shown that silymarin inhibits MMP9 expression through MEK/ERK pathway in thyroid and breast cancer cells [58,59]. Kim et al. have treated papillary thyroid cancer TPC-1 and breast cancer MCF7 cells with 50 μM silibinin for 60 min prior to treatment with 20 nM 12-Otetradecanoylphorbol-13-acetate (TPA) for 24 h. They have found that silibinin suppresses TPA-induced cell migration and MMP-9 expression through the MEK/ERK-dependent pathway [58]. In the other study, SNU216 and SNU668 gastric cancer cells have treated with 50 or 100 μM silibinin for 60 min prior to treatment with TNF-α and similarly, they have suggested silibinin down-regulates TNF-α-induced 197

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3. Silymarin inhibits oxidative and nitrosative immunotoxicity

mTOR, which these, in turn, can profoundly affect immune cell functions, cell metabolism, growth and inflammation [66]. The effect of silymarin on cell cycle of T cells in human peripheral blood leukemia T cells (Jurkat cells), shown that silymarin well tolerates by Jurkat cells and caused the G2/M arrest and induces significant apoptosis at higher doses (200–400 μM) with longer treatment times (48–72 h) [67]. In experimental autoimmune encephalomyelitis (EAE), the multiple sclerosis animal models, silymarin has been effective in reducing of histological signs of demyelination and inflammation and has significantly down regulated the secretion of pro-inflammatory Th1 cytokines ex vivo and up-regulated the anti-inflammatory Th2 cytokines in-vitro [68]. It has been reported that Silibinin is a potent inhibitor of phenotypic and functional maturation of LPS exposed dendritic cells (DCs). It significantly suppressed the expression of CD80, CD86, MHC class I, and MHC class II in the DCs, and caused impairment of LPS-induced IL-12 expression in the murine BM-DCs. Silibinin-treated DCs have proved highly efficient with regard to Ag capture via mannose receptor-mediated endocytosis. Silibinin-treated DCs show an impaired capacity to induce a Th1 response, and a normal cell-mediated immune response. Moreover, naive T cells stimulated with silibinin-treated DCs have generated lower levels of IFN-γ, but have not exhibited significantly altered IL-4 generation capabilities. They imply that silibinin impacts on the capacity of DCs for Th1/Th2 polarization through the expression of co-stimulatory molecules and IL-12 secretion. Therefor it has been suggested that silymarin may be especially helpful for the improvement of therapeutic adjuvants for acute and chronic DC-associated diseases [69]. The immunoprotective effects of silymarin, also have been assessed in UVB-exposed and sensitized to 2,4-dinitrofluorobenzene (DNFB) mice by Katiyar et al. They used an adoptive transfer method in which dendritic cells (DCs) from donor mice that had been UVB-exposed and sensitized to 2,4-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice and then the contact hypersensitivity (CHS) response of the recipient mice has measured. Topical application of 1.0 mg silymarin/cm2 skin area has been used in this study. They found that the CHS response in mice receiving DCs from silymarin-treated UVexposed donor mice, associates with increased secretion of Th1-type cytokines and stimulation of T cells. Accordingly, they have suggested that silymarin acts to save DCs from UV radiation-induced DNA damage and improve UVB-induced DNA damage in DCs [70]. Studies have indicated that silymarin attenuates allergic airway inflammation, atopic dermatitis, and allergic rhinitis [71–73]. Activated mast cells produce histamine, as well as inflammatory mediators such as leukotrienes, prostaglandins, proteases and pro-inflammatory and chemotactic cytokines such as TNF-α, IL-6, IL-4, IL-13 and IL-8 [74,75]. Silibinin has reduced mast cell-mediated anaphylaxis-like reactions in allergic diseases and it seems that silybin acts as a stabilizer of mast cell membrane, thus preventing histamine release [74]. BeomRak Kim et al. have measured the secreted protein and mRNA levels of TNF-a, IL-6, and IL-8 as well as the level of histamine in stimulatedhuman mast cells HMC-1 under treatment with silibinin. They have found that silibinin decreases histamine release and reduces the production and mRNA expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-8 [76]. Furthermore they showed that silibinin inhibits the mast cell-derived allergic inflammatory response through inhibition of the NF-κB signaling pathway and according to their finding, they have suggested that silibinin might have inhibitory effects on allergic inflammation through early phase, transition, and late phase reaction [76]. Silibinin also has attenuated anti-dinitrophenyl IgEmediated passive systemic and cutaneous anaphylaxis. Silibinin had no cytotoxic effects on rat peritoneal mast cells (RPMC) and dose-dependently (82 and 91% inhibition at 50 and 100 mM, respectively) has reduced histamine release from RPMC stimulated by compound 48/80 or anti-DNP IgE [74]. According to above findings, silymarin may serve as an effective therapeutic agent for allergic diseases.

Oxidative and nitrosative stress occurs when the antioxidant defense system is devastated by the production of reactive oxygen species (ROS) and nitric oxide (NO) [77]. An in-vitro study has shown that silymarin provides as an alternative approach for treating B(a)P (important prototype of PAHs an environmental contaminants) induced damage and oxidative stress in PBMC. Silymarin possesses substantial protective effect against B(a)Pinduced oxidative stress and biochemical change by restoring redox status, modulating glutathione metabolizing enzymes, hindering the formation of protein oxidant products, inhibiting LPO and further reducing ROS-mediated damages by changing the level of antioxidant enzymes. Results of this study suggest that silymarin could be considered as a potential protective agent for environmental contaminant induced immunotoxicity [78]. In vivo administration of silymarin has attenuated nitric oxide (NO) production by peritoneal macrophages in LPS-treated mice. Silymarin also dose-dependently has suppressed the LPS-induced production of NO in isolated mouse peritoneal macrophages and in RAW 264.7 cell line (a murine macrophage-like cell line). Moreover, iNOS mRNA and its protein expression were completely abrogated in LPS-stimulated RAW 264.7 cells treated with silymarin via the inhibition of LPS-induced NF-κB activation. Silymarin was shown to inhibit NO production and iNOS gene expression by inhibiting NF-κB/ Rel activation, possibly through its radical scavenging activity [79]. It also has attenuated ethanol-induced oxidative stress and acute liver injury induced by restraint stress through suppression of inflammatory response and immunomodulatory activity [44,80]. In addition, silymarin dose-dependently (1–10 μg/kg, i.v.) has a protective effect against cerebral ischemia-reperfusion (CI/R)-induced injury by reducing lipid peroxidation, protein nitrosylation and oxidative stress. It significantly prevented the expression of inflammation-associated proteins (iNOS, COX-2, myeloperoxidase) and reduced the production of pro-inflammatory cytokines (IL-1β, TNF-α) through impeding the activation of transcriptional factors such as NF-κB and STAT1 [81]. Matsuda et al. have shown that silymarin protects pancreatic cells against cytokine-mediated toxicity through the suppression of cytokineinduced NO production [82]. These cytoprotective effects of silymarin on oxidative and nitrosative conditions appear to be mediated through the suppression of transcriptional factors and activators of transcription pathways. Therefore, increasing the antioxidant capacity of cells by silymarin will provide a potential strategy to protect them from oxidative and nitrosative immunotoxicity. 4. Silymarin effects on apoptosis As we mentioned earlier, several studies have indicated that silymarin/silibinin shows multiple anti-cancer activities including induction of apoptosis, inhibition of proliferation, growth, and migration [83,84]. It was found to suppress the growth and induces the apoptosis of human endothelial ECV304 cells, silibinin treatment has caused a significant decrease in the nuclear level of the p65 subunit of NF-κB and it changes the ratio of Bax/Bcl-2 in a manner that supports apoptosis. In addition, it induces cytochrome c release, activation of caspase-3 and caspase-9 and cleavage of RARP [85]. Studies have shown that the rats azoxymethane-induced colon carcinogenesis model treated with silibinin shows down-regulation of the anti-apoptotic protein Bcl-2, upregulation of pro-apoptotic protein Bax and inverting the Bcl-2/Bax ratio < 1. Silibinin treatment also decreased the genetic expression of biomarkers of inflammatory response including IL-1β, TNFα and their downstream target MMP7 which are up-regulated during colon carcinogenesis [86]. The effect of silibinin on UVB-induced apoptosis was examined in human epidermoid carcinoma A 431 cells. Results indicated that silibinin (100–200 μM) treatment before radiation caused a further increase in apoptosis, whereas post-treatment protects against apoptosis. 198

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Fig. 3. Different immune protective effects of silymarin.

molecular pathways would be very valuable for treatment of immunemediated diseases. Also further studies are needed to assess the utility of silymarin in protection against autoimmune, cancer, allergic and other diseases in human subjects.

Differential effects of silibinin on UVB-induced apoptosis involve the modulation of mitochondrial apoptotic machinery (Bcl-2 family members, cytochrome c), caspases activation and mitogen-activated protein kinase (MAPK) signaling [87]. The dual effect of silibinin on apoptosis was also observed in HaCaT (human keratinocytes) to be dose and time dependent manner [87]. At lower doses, silibinin affords strong protection against UVB-induced apoptosis, whereas at a higher dose it increases apoptosis with strong down-regulation of activated protein-1 (AP-1) DNA binding activity. These findings suggest that silibinin could protect normal human skin keratinocytes against sunburn or apoptosis when the damage is moderate. Therefore, when the UVB damage is severe, silibinin can lead to apoptotic cell death, which might be an important advantage in removing DNA damaged cells from cell cycle progression.

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5. Conclusion In conclusion, based on the current review, silymarin has a broad spectrum of immunomodulatory functions under different conditions (Fig. 3). Recognizing the exact mechanisms of silymarin on cellular and 199

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