Inhibition of GluN2B pathway is involved in the neuroprotective effect of silibinin on streptozotocin-induced Alzheimer's disease models.

BACKGROUND: Over-activation of N-methyl-D-aspartate receptors (NMDARs) is involved in sporadic Alzheimer's disease. Silibinin, a natural flavonoid gained from the seeds of Silybum marianum, exerts neuroprotective effects on sporadic AD models, but its impacts on NMDARs remain unknown. PURPOSE: To study silibinin's regulatory effects on NMDARs pathway in sporadic AD models. METHODS: MTT assay, western blotting, confocal microscopy, flow cytometry, RT-PCR, and siRNA transfection etc. were used for cellular and molecular studies. The direct interactions between silibinin and NMDAR subunits were evaluated by computational molecular docking, drug affinity responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA). Y maze test, novel objects recognition test and Morris water maze test were conducted to examine the learning and memory ability of rats. RESULTS: An in vitro AD model was established by treating HT22 murine hippocampal neurons with streptozotocin (STZ), as evidenced by the amyloid ß (Aß) deposition and hyperphosphorylation of tau proteins. Silibinin shows protection of neurons against STZ-induced cell damage. It is noteworthy that STZ-induced cellular calcium influx is inhibited by silibinin-treatment, indicating the possible modulation of calcium channels. Studies on NMDARs, the most widely distributed calcium channel, by using molecular docking, DARTS and CESTA, reveal that the GluN2B subunit, but not GluN2A, is the potential target of silibinin. Further studies using the pharmacological agonist (NMDA) and the GluN2B-specific inhibitor (Ifenprodil) or siRNA, indicate that the protection by silibinin treatment from STZ-induced cytotoxicity is medicated through interference with GluN2B-containing NMDARs, followed by the upregulation of CaMKIIα/ BDNF/ TrkB signaling pathway and improved levels of synaptic proteins (SYP and PSD-95). The results in vivo using rats intracerebroventricularly injected with STZ (ICV-STZ), a well-established sporadic AD model, confirm that silibinin improves learning and memory ability in association with modulation of the GluN2B/CaMKIIα/ BDNF/TrkB signaling pathway. CONCLUSION: Inhibiting over-activation of GluN2B-containing NMDARs is involved in the neuroprotective effect of silibinin on STZ-induced sporadic AD models.

Protective effects of silibinin against ethanol- or acetaldehyde-caused damage in liver cell lines involve the repression of mitochondrial fission.

Silibinin is a natural polyphenolic flavonoid, isolated from the seeds of the milk thistle of Silybum marianum (L.) Gaertn. Silibinin has been widely used clinically as a traditional medicine for liver diseases. This study investigated the protective role of silibinin in ethanol- or acetaldehyde-induced apoptosis in human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells, focusing on elucidation of the underlying mechanism in vitro. The toxicity of ethanol or acetaldehyde was evaluated by MTT assay. Apoptosis-related proteins, mitochondrial fission-associated proteins and mitochondrial fusion-associated proteins were analyzed by western blotting and immunofluorescence microscopy. Present experimental results demonstrated that silibinin improved cell viability, reduced the enzyme activities of AST/ALT and ALDH/ADH, inhibited apoptosis and recovered mitochondrial function in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. Silibinin reduced the expression of mitochondrial fission-associated proteins, dynamin-related protein 1 (DRP1), but increased mitochondrial fusion-associated proteins, optic atrophy 1 (OPA1) and mitofusin 1 (MFN1). Accordingly, inhibition of DRP1 activity with its pharmacological inhibitor or siDRP1 efficiently attenuated ethanol- or acetaldehyde-induced apoptosis, whereas activation of DRP1 by using staurosporine (STS) further increased apoptosis in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. The results show that silibinin protects cells against ethanol- or acetaldehyde-induced mitochondrial fission that results in apoptosis.

Oral Administration of Silibinin Ameliorates Cognitive Deficits of Parkinson's Disease Mouse Model by Restoring Mitochondrial Disorders in Hippocampus.

Besides motor disorder, cognitive dysfunction is also common in Parkinson's disease (PD). Essentially no causal therapy for cognitive dysfunction of PD exists at present. In this study, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD was used to analyze the neuroprotective potential of orally administered silibinin, a proverbial hepatoprotective flavonoid derived from the herb milk thistle (Silybum marianum). Results demonstrated that silibinin administration significantly attenuated MPTP-induced cognitive impairment in behavioral tests. Nissl staining results showed that MPTP injection significantly increases the loss of neurons in the hippocampus. However, these mice were protected by oral administration of silibinin, accompanying reduction in the cell apoptosis in the hippocampus. The hippocampal aggregates of α-synuclein (α-syn) appeared in MPTP-injected mice, but were significantly decreased by silibinin treatment. MPTP injection induced oxidative stress, as evidenced by increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD). The oxidative stress was alleviated by silibinin treatment. Mitochondrial disorder including the decline of mitochondrial membrane potential (MMP) was another signature in the hippocampus of MPTP-treated mice, accompanying increased mitochondrial fission and decreased fusion. Silibinin administration restored these mitochondrial disorders, as expected for the protection against MPTP injury. These findings suggest that silibinin has a potential to be further developed as a therapeutic candidate for cognitive dysfunction in PD.

Ginger-Degraded Collagen Hydrolysate Exhibits Antidepressant Activity in Mice.

Collagen is the most abundant protein in animals. Collagen hydrolysate has been found to have multiple functions in the skin, bones, joints, muscles, and blood vessels. Recently, it has been reported that the low molecular weight fraction of collagen hydrolysate exhibited anxiolytic activity, suggesting that collagen peptides affect brain functions. In the present study, we found that oral administration of ginger-degraded collagen hydrolysate (GDCH) significantly decreased depression-like behavior in a forced swim test, suggesting that GDCH exhibited antidepressant activity in mice. The antidepressant activity of GDCH was abolished by pre-treatment with an antagonist of the dopamine receptor, but not treatment with a serotonin receptor antagonist. GDCH significantly increased gene expression of glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the hippocampus, molecules that affect the differentiation and survival of neurons, relative to that in the control condition. Meanwhile, there were no changes in the gene expression of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3, major factors related to depression-like behavior. We also found that GDCH exhibited antidepressant activity in corticosterone-administered mice in a model of stress. In addition, GDCH increased GDNF and CNTF expression in the stressed condition, suggesting that mechanisms of the antidepressant activity of GDCH were the same in unstressed and stressed conditions. These results imply that GDCH exhibits antidepressant activity in unstressed and stressed conditions in mice. The upregulation of neurotrophic genes in the hippocampus may contribute to the reduction of depression-like behavior via a dopamine signal pathway modulated by GDCH.

Silibinin protects rat pancreatic ß-cell through up-regulation of estrogen receptors' signaling against amylin- or Aß1-42 -induced reactive oxygen species/reactive nitrogen species generation.

Amylin and amyloid-ß (Aß) were found to induce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat pancreatic ß-cell line, INS-1 cells, leading to cell death. In this study, we report on reciprocal relationship between the expression of estrogen receptors (ERs) α and ß (ERα and ERß) and generation of ROS/RNS in amylin/Aß1-42 -treated INS-1 cells. That is, pharmacological activation of ERs in INS-1 cells significantly decreases ROS/RNS generation, but blockage of ERs increases ROS/RNS generation. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with phytoestrogen activities, also known as silybin. Treatment with silibinin down-regulated ROS/RNS production induced by treatment with amylin/Aß1-42 in the cells. Silencing ERs expression with siRNAs targeting ERs showed that the protective effect of silibinin was markedly weakened, indicating that silibinin protection is largely attributed to activation of ERs' signaling. The binding of silibinin to ERs implies that the protective effect of silibinin on amylin/Aß1-42 -treated INS-1 cells owes to down-regulation of ROS/RNS through the activation of ERs phosphorylation. Amylin and Aß1-42 cotreatment enhanced furthermore ROS/RNS generation and cytotoxicity through further down-regulation of ERs phosphorylation, and this was reversed by silibinin. Silibinin also protects INS-1 cells from amylin and Aß1-42 cotreatment. These results indicate that protective effect of silibinin is mediated by enhancement of ERs phosphorylation that depresses ROS/RNS generation in amylin/Aß1-42 -treated INS-1 cells.

Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8.

Keratinocyte line cells HaCaT and FEPE1L-8 are used for skin model with type I collagen fibrils (gels). For this purpose, not only differentiation but also regulation of proliferation on type I collagen gels by exogenous calcium concentration is important. When exogenous calcium concentration is low, primary keratinocyte proliferation is repressed and eventually cells are induced to apoptosis on type I collagen gels. The apoptosis induced on type I collagen gels is suppressed by increasing calcium concentration in the medium. That is, higher exogenous calcium concentration is necessary for primary keratinocyte survival on type I collagen gels than for that on dish surface culture. Meanwhile much higher exogenous calcium causes cell differentiation and inhibition of proliferation. The optimal calcium concentrations for proliferation on type I collagen gels have not been clarified in keratinocyte line cells. HaCaT cells have a unique calcium sensitivity in comparison with primary keratinocytes, whereas FEPE1L-8 cells have a similar sensitivity to primary keratinocytes. In this study, we compared the effect of calcium concentrations on proliferation of HaCaT and FEPE1L-8 cells on type I collagen gels. On type I collagen gels, both line cells required higher calcium concentrations for proliferation than on dish surface. HaCaT cells proliferated better in a wider range of calcium concentrations than FEPE1L-8 cells.

Collagen-derived peptides modulate CD4+ T-cell differentiation and suppress allergic responses in mice.

INTRODUCTION: Collagen peptides have been widely used as a food supplement. After ingestion of collagen peptides, oligopeptides containing hydroxyproline (Hyp), which are known to have some physiological activities, are detected in peripheral blood. However, the effects of collagen-peptide administration on immune response are unclear. In the present study, we tested the effects of collagen-peptide ingestion on allergic response and the effects of collagen-derived oligopeptides on CD4+ T-cell differentiation. METHODS: BALB/c mice fed a collagen-peptide diet were immunized with ovalbumin (OVA), and their serum IgE and IgG levels, active cutaneous anaphylaxis, and cytokine secretion by splenocytes were examined. Naive CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of collagen-derived oligopeptides, and the expression of IFN-γ, IL-4, and Foxp3 was analyzed. RESULTS: In an active anaphylaxis model, oral administration of collagen peptides suppressed serum OVA-specific immunoglobulin E (IgE) production and diminished anaphylaxis responses. In this model, the ingestion of collagen peptides skewed the pattern of cytokine production by splenocytes toward T-helper (Th) type 1 and regulatory T (Treg) cells. In vitro T-helper cell differentiation assays showed that Hyp-containing oligopeptides promoted Th1 differentiation by upregulating IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) signaling. These oligopeptides also promoted the development of Foxp3+ Treg cells in response to antigen stimulation in the presence of TGF-ß. CONCLUSIONS: Collagen-peptide ingestion suppresses allergic responses by skewing the balance of CD4+ T cells toward Th1 and Treg cells and seems to be a promising agent for preventing allergies and inflammatory diseases.

Estrogen Receptors Are Involved in the Neuroprotective Effect of Silibinin in Aß1-42-Treated Rats.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by a cascade of pathologic changes. A widely discussed theory indicates that amyloid ß (Aß) peptides are the causative agents of AD. Silibinin, a flavonoid derived from milk thistle, is well known for its hepato-protective activities and we have reported the neuroprotective effects of silibinin. In this study, we investigated the role of estrogen receptors (ERs) in silibinin's neuroprotective effect on Aß1-42-injected rats. Results of Morris water maze and novel object-recognition tests demonstrated that silibinin significantly attenuated Aß1-42-induced memory impairment. Silibinin attenuated ERs and PI3K-Akt pathways, as well as modulated mitogen-activated protein kinases in the hippocampus of Aß1-42-injected rats. Taken together, silibinin is a potential candidate in the treatment of Alzheimer's disease.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

Silibinin ameliorates Aß25-35-induced memory deficits in rats by modulating autophagy and attenuating neuroinflammation as well as oxidative stress.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that inflammatory response, oxidative stress and autophagy are involved in amyloid ß (Aß)-induced memory deficits. Silibinin (silybin), a flavonoid derived from the herb milk thistle, is well known for its hepatoprotective activities. In this study, we investigated the neuroprotective effect of silibinin on Aß25-35-injected rats. Results demonstrated that silibinin significantly attenuated Aß25-35-induced memory deficits in Morris water maze and novel object-recognition tests. Silibinin exerted anxiolytic effect in Aß25-35-injected rats as determined in elevated plus maze test. Silibinin attenuated the inflammatory responses, increased glutathione (GSH) levels and decreased malondialdehyde (MDA) levels, and upregulated autophagy levels in the Aß25-35-injected rats. In conclusion, silibinin is a potential candidate for AD treatment because of its anti-inflammatory, antioxidant and autophagy regulating activities.