Serum levels of TGFß, IL-10, IL-17, and IL-23 cytokines in ß-thalassemia major patients: the impact of silymarin therapy.

Abstract Several immunological abnormalities have been characterized in ß-thalassemia, many of which are linked to or identified with cytokines. In this study, we investigated the serum levels of TGF-ß, IL-10, IL-17 and IL-23 in ß-thalassemia major patients in comparison with healthy controls. The immunomodulatory effect of silymarin (a flavonoid complex obtained from Silybum marinum) on the serum levels of cytokines was further evaluated in thalassemia patients receiving silymarin (420 mg/day) and compared with patients treated with placebo for 6-month. Serum cytokines levels were measured by enzyme linked immunosorbent assay (ELISA). The results showed a significant higher concentration of TGF-ß and IL-23 in the patient group than control group. Among studied cytokines, a significant reduction in serum IL-10 levels was found in patients treated with silymarin when compared with IL-10 values at baseline. However, no significant difference was observed between baseline values of cytokine compared with end values in placebo group. Our data suggest the presence of imbalanced immune condition involving inflammation and immunosuppression in thalassemia patients, which could be modulated to a more effective immune response by silymarin.

A randomized double-blind, placebo-controlled study of therapeutic effects of silymarin in ß-thalassemia major patients receiving desferrioxamine.

OBJECTIVE: Thalassemia is one of the most common genetic disorders worldwide. Chronic blood transfusions treat the underlying anemia but may lead to iron toxicity. Effective iron chelation remains one of the main targets of clinical management of thalassemia major. In this study, iron-chelating activity of silymarin, a flavonolignan isolated from silybum marianum, was examined in ß-thalassemia major. METHODS: Patients were treated with the combination of desferrioxamine and silymarin (Legalon(®) ; n = 49) or desferrioxamine plus placebo (n = 48) for 9 months. The serum levels of ferritin, iron, total iron-binding capacity (TIBC), soluble transferrin receptor, and hepcidin were determined at the baseline and after 9-month therapy. Liver function test was performed before and after treatment in both groups. RESULTS: Serum ferritin levels decreased significantly from the beginning to the end of silymarin treatment (3028.8 ± 2002.6 vs. 1972.2 ± 1250.6 ng/mL); however, no significant change in serum ferritin was observed in the patients receiving placebo (2249.0 ± 1304.2 vs. 2015.6 ± 1146.8). Moreover, serum iron and TIBC levels were significantly reduced in silymarin group compared with placebo. Patients on silymarin therapy also exhibited a significant decrease in serum levels of hepcidin and soluble transferrin receptor after 9-month treatment period. A significant improvement in liver function test was observed in silymarin group in comparison with placebo. CONCLUSION: This study shows that silymarin is effective at reducing iron overload in patients when used in conjunction with desferrioxamine. Therapeutic effects of silymarin on a background of desferrioxamine suggest the potential effectiveness of silymarin alone in reducing body iron burden.

Elevated serum levels of cell death circulating biomarkers, M30 and M65, in patients with ß-thalassemia major.

Deposition of iron in visceral organs, mainly in the liver, causes tissue damage in ß-thalassemia major (ß-TM) patients. Keratin 18 (K18) represents one of the major caspase substrates during apoptosis of hepatocytes. To better characterize the hepatic apoptosis and/or necrosis in ß-thal patients, the circulating levels of M65 (soluble intact K18) and M30 (the caspases-generated K18 fragment) were measured in 40 ß-TM patients and compared with 40 healthy controls. The ratio of M30/M65 (caspase-cleaved to total K18) was also determined in thalassemic and normal subjects. Results of the ELISA assays revealed that the serum levels of hepatocyte death markers, M65 and M30, were significantly increased in ß-thal patients compared to healthy controls (p <0.0001). M30 serum levels were also positively correlated with the serum levels of liver transaminases including aspartate aminotransferase (AST) (r = 0.337, p = 0.047) and alanine aminotransferase (ALT) (r =0.391, p = 0.02).

Therapeutic potential of silymarin as a natural iron-chelating agent in ß-thalassemia intermedia.

Abnormal iron accumulation in vital organs is one of the major complications of ß-thalassemia intermedia (ß-TI). Silymarin, a flavonolignan isolated from Silybum marianum, significantly decreases the serum ferritin levels of ß-TI patients. This finding suggests silymarin as a safe and effective natural iron-chelating agent for the treatment of iron-overloaded conditions.

Significant immunomodulatory and hepatoprotective impacts of Silymarin in MS patients: A double-blind placebo-controlled clinicaltrial.

Interferon beta (IFN-ß) has successfully been experimented with to treat multiple sclerosis (MS). However, patients sometimes do not respond effectively to treatment, and |adverse effects, including liver toxicity, accompany this therapy. |Accordingly, we decided to treat MS patients simultaneously with Silymarin (SM) as an immunomodulatory and hepatoprotective agent and IFN-ß in a clinical trial study. Complete blood count (CBC), liver enzyme levels, and the serum concentration of inflammatory and anti-inflammatory cytokines were measured. Also, the frequency of immune cells was determined by flow cytometry. Liver enzyme levels were significantly lower in the intervention group (p < 0.05). The percentage of Th17 cells in the intervention group was significantly reduced compared to the placebo group (P < 0.001). Also, the frequency of Treg cells after treatment with SM plus IFN-ß was significantly increased compared to the placebo group (p < 0.05). Furthermore, the IL-17 and IFNγ cytokine levels were significantly reduced in the intervention group (p < 0.05). Moreover, the levels of anti-inflammatory cytokines IL-10 and TGFß were significantly increased in the intervention group (P < 0.05).Overall, the results provide novel and supplementary information on SM's notable immunoregulatory effects on inflammatory response and liver function in MS patients. Clinical Trial Identifier Number: IRCTID: IRCT20171220037977N1.

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

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.

Effects of silymarin on the proliferation and glutathione levels of peripheral blood mononuclear cells from beta-thalassemia major patients.

Iron toxicity in beta-thalassemia major is the main cause of oxidative stress and cell mediated immune deficiencies. Despite indicative signs of severe oxidative deficiencies associated with beta-thalassemia major, such as decreased level of plasma antioxidants and depletion of erythrocyte glutathione, little is known about intracellular redox status of immune cells. Since glutathione is a primary intracellular antioxidant and plays an essential role in several functions in T cells, in this study intracellular glutathione (GSH) levels as well as proliferation of PHA-activated peripheral blood mononuclear cells (PBMC) were investigated in 28 beta-thalassemia major patients and 28 healthy age-matched individuals. Considering the potential benefits of flavonoids in the therapy of oxidative stress, the effects of silymarin on the GSH levels and proliferation of PBMC from normal and thalassemia individuals were further examined. Quantitative determination of intracellular GSH and proliferative response of PBMC to PHA were performed before and after 72 h incubation of PBMC with various concentrations of silymarin (0, 5, 10, or 20 mug/ml). Results demonstrated a significant reduction of GSH and proliferation in beta-thalassemia major cells; however treatment with silymarin led to restoration of both GSH levels and PBMC proliferation in thalassemia patients. Considerably low levels of GSH and depressed proliferative response of PBMC in beta-thalassemia major may be responsible for the cell mediated immune abnormalities in iron overload conditions. Moreover, the GSH restoration and improvement of PBMC growth by silymarin is a possible explanation for its recently reported antioxidant and immunostimulatory activities. These data suggest the benefit of using flavonoids to normalize immune dysfunction in beta-thalassemia major. The immunomodulatory effects of silymarin in beta-thalassemia major are currently under further investigation in a double blind clinical trial.

Preparation of Immunotoxin Herceptin-Botulinum and Killing Effects on Two Breast Cancer Cell Lines.

BACKGROUND: Worldwide, breast cancer is the most common cancer diagnosed among women and a leading cause of cancer deaths. The age of onset in Iran has become reduced by a decade for unknown reasons. Herceptin, a humanized monoclonal antibody, is a target therapy for breast cancer cells with over expression of HER2- neu receptors, but it is an expensive drug with only 20% beneficial rate of survival. This study introduces a novel approach to enhance the efficacy of this drug through immunoconjugation of the antibody to botulinum toxin. Decreasing the cost and adverse effects of the antibody were secondary goals of this study. MATERIALS AND METHODS: Botulinum toxin was conjugated with Herceptin using heterobifunctional cross linkers, succinimidyl acetylthiopropionate (SATP) and sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the supplier's guidelines and tested on two breast cancer cell lines: SK-BR-3 and BT-474. Toxin and Herceptin were also used separately as controls. The cytotoxicity assay was also performed using the new bioconjugate on cultured cells with Alamar blue and a fluorescence plate reader. RESULTS: Herceptin-Toxin bioconjugation significantly improved Herceptin efficacy on both breast cancer cell lines when compared to the control group. CONCLUSIONS: Toxin-Herceptin bioconjugation can be a potential candidate with increased efficiency for treating breast cancer patients with over expression of the HER2 receptor.

The influence of iron loading and iron chelation on the proliferation and telomerase activity of human peripheral blood mononuclear cells.

BACKGROUND: Iron is an essential trace element in cell proliferation. Several investigations demonstrate that iron deprivation inhibits cell proliferation. However, the impact of iron on telomerase activity of activated lymphocytes remains unexplained to date. OBJECTIVE: In this study, the effect of iron on the proliferation and telomerase activity of lymphocytes stimulated by phytohemagglutinin (PHA) were investigated. METHODS: Iron loading was performed by incubating peripheral blood mononuclear cells in 500microM FeSO4.7H2O for 24 h and iron chelation was done by exposing cells to desferrioxamine, a potent iron chelator. The effects of silymarin, a flavonoid with both antioxidant and iron chelating activities, on the proliferation and telomerase activity of PHA-activated lymphocytes were also compared with desferrioxamine. Proliferation and telomerase activity were assessed using BrdU incorporation assay and Telomeric Repeat Amplification Protocol (TRAP), respectively. RESULTS: The proliferations of lymphocytes were significantly inhibited by 10 and 20 microg/ml desferrioxamine in a dose dependent manner, while iron loading recovered suppressed cell proliferation to the normal level. Silymarin at 20 microg/ml significantly increased the proliferation of lymphocytes in both normal and iron-treated conditions. Telomerase activity of lymphocytes was markedly increased by iron treatment and suppressed by desferrioxamine. Conversely, iron treatment had no effect on the telomerase activity of lymphocytes incubated with silymarin. CONCLUSION: Iron plays a significant role in the proliferation and telomerase activity of lymphocytes. The effects of silymarin on the proliferation and telomerase activity of lymphocytes were completely different from those of desferrioxamine, suggesting that the immunomodulatory effect of silymarin is probably not associated with its iron chelating activity.

Combined therapy of silymarin and desferrioxamine in patients with beta-thalassemia major: a randomized double-blind clinical trial.

Silymarin, a flavonolignan complex isolated from Silybum marianum, has a strong antioxidant, hepatoprotective, and iron chelating activities. The present study was designed to investigate the therapeutic activity of orally administered silymarin in patients with thalassemia major under conventional iron chelation therapy. A 3-month randomized, double-blind, clinical trial was conducted in 59 beta-thalassemia major patients in two well-matched groups. Patients were randomized to receive a silymarin tablet (140 mg) three times a day plus conventional desferrioxamine therapy. The second group received the same therapy but a placebo tablet instead of silymarin. Clinical laboratory tests were assessed at the beginning and the end of the trial, except for serum ferritin level that was assessed at the middle of the trial as well. Results of this study revealed that the combined therapy was well tolerated and more effective than desferrioxamine in reducing serum ferritin level. Significant improvement in liver alkaline phosphatase and glutathione levels of red blood cells was also observed in silymarin-treated beta-thalassemia patients. However, no significant difference in serum ferritin levels was detected between silymarin and placebo groups after 1.5 and 3 months treatment, probably because of insufficient sample size to detect subtle changes in ferritin levels between groups. This is the first report showing the beneficial effects of silymarin in thalassemia patients and suggests that silymarin in combination with desferrioxamine can be safely and effectively used in the treatment of iron-loaded patients.

Premature senescence of T lymphocytes from patients with beta-thalassemia major.

Several researches have demonstrated a suppressed cell mediated immunity in patients with beta-thalassemia major. To know whether the premature aging of T cells is involved in abnormalities of cell mediated immunity, the biomarkers of immunosenescence including telomerase activity, apoptosis, and the expression of CD28 and CD95 were evaluated in T lymphocytes from beta-thalassemia major patients. The ex vivo spontaneous apoptosis in CD4(+) or CD8(+) T cells from patients and healthy subjects was assessed by an in situ TdT mediated dUTP-biotin nick end labelling (TUNEL) assay after 24h incubation in medium. Flow cytometric data revealed that lymphocytes from beta-thalassemia patients were resistant to spontaneous apoptosis compared to the normal lymphocytes. Moreover, the percentages of TUNEL(+)CD4(+) or TUNEL(+)CD8(+) T cells from patients were significantly lower than those control cells. Quantitative determination of telomerase activity in resting and activated T cells was performed using the Telomeric Repeat Amplification Protocol (TRAP). The results showed a decreased telomerase activity of activated T cells in patients with thalassemia major compared to that in healthy controls. However, the percentages of CD8(+)CD28(-) and CD3(+)CD95(+) T lymphocytes were significantly higher in thalassemia patients, indicating the phenotypes associated with senescent T lymphocytes. These data provide evidences for the occurrence of accelerated aging of T cells in beta-thalassemia major; possibly result in abnormal T cell function leading to suppressed cell mediated immunity.