Evidence for oral agmatine sulfate safety – A 95-day high dosage pilot study with rats

Agmatine, decarboxylated arginine, exerts beneficial effects in various experimental disease models. Clinical trials indicate the safety and effectiveness of short-term (up to 21 days) high dose regimens of oral agmatine sulfate, but longer term studies are lacking. This pilot study undertook to assess the safety of a longer term high dosage oral agmatine sulfate in laboratory rats. Adult Wistar rats consumed 5.3 g/l agmatine sulfate in their drinking water for 95 days, a regimen estimated to result in a daily dosage of absorbed agmatine of about 100 mg/kg. Animals’ body weight, water consumption and blood pressure were periodically measured, and general cage behavior, fur appearance, urination and feces appearance monitored. These parameters were also determined at 20 days after treatment cessation (day 115). On days 95 and 115, animals were euthanized for gross necropsy assessment. Agmatine-treated rats showed slight, but significant reductions in body weight and blood pressure, and reduced water consumption during treatment, which recovered completely within 20 days after treatment cessation. Otherwise, no abnormal behaviors or organ pathologies were observed. These findings are first to suggest apparent safety of sub-chronic high dosage dietary agmatine sulfate in laboratory rats, thus lending further support to the therapeutic applications of agmatine.     

Fluvastatin increases tyrosinase synthesis induced by α-melanocyte-stimulating hormone in B16F10 melanoma cells

The aim of this study was to evaluate the effect of fluvastatin on the α-melanocyte-stimulating hormone-mediated increase in tyrosinase activity in the melanoma B16F10 cell line and to establish whether Akt and extracellular signal-regulated kinase (Erk) inhibition is involved in tyrosinase synthesis after fluvastatin administration. Fluvastatin modulates α-melanocyte-stimulating hormone induced melanogenesis by increasing tyrosinase mRNA production, as shown by real time PCR, or tyrosinase protein synthesis, as presented by western blot technique. The stimulatory effect of fluvastatin on melanogenesis was, in part, induced by modulation of cell proliferation (decreased melanoma cell proliferation in G2/M phase) and possibly decrease of Akt. These findings indicate that fluvastatin increases tyrosinase synthesis induced by α-melanocyte-stimulating hormone in B16F10 cells and reveal an unknown effect of statin use: their influence on melanin production.     

Phloridzin-induced melanogenesis is mediated by the cAMP signaling pathway

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role in protection against skin photocarcinogenesis. Phloridzin is a phloretin 2′-glucoside that is found in many parts of the apple tree that reportedly increases tyrosinase activity and melanin contents through inhibition of protein kinase C (PKC) activity in B16 melanoma cells. In this study, we attempted to accurately determine the effects and mechanisms of action of phloridzin on melanogenesis. Specifically, we observed that phloridzin-induced a dose-dependent increase in tyrosinase activity and melanin contents, and that these changes were accompanied by an increase in the levels of tyrosinase and the tyrosinase-related proteins, TRP-1 and TRP-2. Furthermore, the cAMP-dependent protein kinase A (PKA) inhibitor H89 impaired the response of the tyrosinase activity and melanin synthesis to phloridzin. Additionally, phloridzin stimulated cAMP production and phosphorylation of the cAMP-response element binding protein (CREB). Taken together, the results of this study indicate that phloridzin increases tyrosinase gene expression through the cAMP signaling pathway, thereby leading to the stimulation of melanogenesis.     

Influence of agmatine on the protective action of numerous antiepileptic drugs against pentetrazole-induced seizures in miceA

The aim of this study was to assess the influence of agmatine (an endogenous neuromodulator/neurotransmitter in the brain) on the protective action of numerous classical and second-generation antiepileptic drugs (clonazepam, ethosuximide, gabapentin, phenobarbital, tiagabine, vigabatrin, and valproate) in the mouse pentetrazole-induced clonic seizure model.The results indicate that agmatine (up to 100 mg/kg, ip, 45 min before the test) did not alter the threshold for pentetrazole-induced clonic seizures in mice. However, agmatine (100 mg/kg, ip) significantly attenuated the anticonvulsant effects of vigabatrin against pentetrazole-induced clonic seizures by elevating the ED₅₀ value of vigabatrin from 517.5 to 790.3 mg/kg (p < 0.01). In contrast, agmatine at a dose of 50 mg/kg did not significantly affect the anticonvulsant action of vigabatrin, although a reduction in the ED₅₀ value of the antiepileptic drug from 517.5 to 629.1 mg/kg was documented. Moreover, agmatine at doses of 50 and 100 mg/kg (ip) had no significant impact on the anticonvulsant action of clonazepam, ethosuximide, gabapentin, phenobarbital, tiagabine, or valproate in pentetrazole-induced seizures in mice.In conclusion, the combination of agmatine with vigabatrin seems to be unfavorable due to the reduction of the anticonvulsant effect of vigabatrin after concomitant administration of agmatine in the pentetrazole-induced seizure model. Therefore, the utmost caution is advised when combining agmatine with vigabatrin in further clinical settings.     

An aqueous extract of Limoniastrum guyonianum gall induces anti-tumor effects in melanoma-injected mice via modulation of the immune response

The objectives of this study were to evaluate the in vitro and in vivo anti-tumor potential of the aqueous gall extract (G extract) from Limoniastrum guyonianum and to elucidate its immunological mechanisms, in part, by assessing its effects on the growth of transplanted tumors and the immune response in these tumor-bearing mice. Here, mice were inoculated with B16F10 mouse tumor cells and then treated intraperitoneally with G extract at 25 or 50 mg extract/kg BW for 7, 14, or 21 days. At each timepoint, effects of the extract on the tumor growth, splenocytes proliferation, NK cell activity, and CTL activity among splenocytes isolated from the mice were measured. G extract-induced tumor growth inhibition was associated with characteristic apoptotic changes in the tumor cells, like nuclear condensation. In addition, the extract inhibited melanin synthesis and tyrosinase activity among melanoma cells in a concentration-related manner. G extract did not only significantly inhibit the growth of the transplantable tumor, but also remarkably increased splenocytes proliferation and both NK and CTL activities in tumor-bearing mice. The extract was also seen to have promoted lysosomal activity of host macrophages and gave rise to enhanced cellular anti-oxidant activity in several cell types in mice.     

Agmatine reduces ultrasonic vocalization deficits in female rat pups exposed neonatally to ethanol

Rat pups, in isolation, produce ultrasonic vocalizations (USVs). These USVs have been used as a diagnostic tool for developmental toxicity. We have shown that neonatal ethanol (ETOH) exposure produces deficits in this behavior. The current study was designed to examine whether agmatine (AG), which binds to imidazoline receptors and modulates n-methyl-d-aspartate receptors (NMDAR), could reduce these deficits. In addition, this study examined critical periods for ETOH's effects on USVs by administering ETOH during either the 1st or 2nd postnatal week. Neonatal rats received intragastric intubations of either ETOH (6 g/kg/day), ETOH and AG (6 g/kg/day and 20 mg/kg/day), AG (20 mg/kg/day), or maltose on postnatal days (PND) 1–7 or 8–14. A non-intubated control was also included. Subjects were tested on PND 15. Neonatal ETOH exposure significantly increased the latency to vocalize for females and reduced the rate of USVs in both males and females exposed to ETOH on PND 1–7. Agmatine reduced these deficits, in female but not male pups. Subjects exposed to ETOH on PND 8–14 showed no evidence of abnormal USVs. These findings suggest that there may be gender differences in response to AG following neonatal ETOH exposure and also provide further support that the first neonatal week is a particularly sensitive time for the developmentally toxic effects of ETOH in rodents.     

Nonlethal aluminum maltolate can reduce brain-derived neurotrophic factor-induced Arc expression through interrupting the ERK signaling in SH-SY5Y neuroblastoma cells

Although many studies have demonstrated that aluminum (Al) exposure impairs learning and memory, its underlying mechanism is still uncertain. Long-lasting forms of synaptic plasticity that underlie memory are dependent on new protein synthesis. In particular, activity-regulated cytoskeleton-associated protein (Arc) has a versatile role in synaptic plasticity, and its synthesis can be induced by brain-derived neurotrophic factor (BDNF). BDNF-induced Arc expression has been suggested to play a fundamental role in the stabilization of synaptic plasticity. In the present study, the pretreatment of Al(malt)₃ at nonlethal level (200 μM, 24 h) significantly reduced BDNF (10 ng/ml, 1 h)-induced Arc expression in SH-SY5Y human neuroblastoma cells. BDNF-induced activation of ERK but not PI3K signaling pathway was interfered with the Al(malt)₃ pretreatment, resulting in the subsequent reduction of BDNF-induced phosphorylation of 4EBP1, p70S6K, and eIF4E. Reduced phospho-4EBP1 and phospho-eIF4E hindered the initiation step of translation, which may lead to a reduction in BDNF-induced Arc expression. However, reduced phospho-p70S6K did not influence the phosphorylation of eEF2K and eEF2, indicating no significant effect on BDNF-enhanced translation elongation. Therefore, even at nonlethal level, Al(malt)₃ pretreatment reduced BDNF-induced Arc expression, which was caused by interrupting the ERK signaling pathway as well as the subsequent translation initiation.     

Infliximab therapy restores adiponectin expression in perivascular adipose tissue and improves endothelial nitric oxide-mediated vasodilation in mice with type 1 diabetes

Increased TNFα-mediated JNK signaling in the perivascular adipose tissue (PVAT) may contribute to the pathogenesis of vascular complications in T1DM by reducing adiponectin (Ad) synthesis and therefore impairing Ad-mediated activity in the contiguous blood vessel system.We evaluated whether in vivo treatment with the TNFα blocking antibody infliximab normalized expression of Ad and Ad receptors in various fat depots, and whether this effect correlated with improved endothelial activity and vasodilator function in streptozotocin (STZ)-induced diabetic mice. STZ mice were studied at 1 and 2 weeks after diabetes onset, and compared to age-matched infliximab-treated diabetic (I-STZ) and control animals (CTRL) (n = 10 each group). In STZ mice, activation of pro-inflammatory JNK signaling was faster in PVAT (P < 0.01) than in visceral (VAT), epididymal (EAT) and subcutaneous (SAT) adipose depots, and associated with decreased Ad synthesis and dysregulated AdipoR1/R2 levels. In parallel, activation of JNK in aortic endothelial cells and mesenteric arteries was associated with decreased expression/phosphorylation of eNOS and impaired ACh-mediated vasodilation (P < 0.05 vs. CTRL). Treatment with infliximab abrogated JNK activation, ameliorated Ad protein expression, and normalized expression of both AdipoR1 and AdipoR2 in PVAT, concomitantly improving eNOS expression and vessel relaxation in mesenteric arteries from I-STZ mice (P < 0.01 vs. STZ).These observations underline the early susceptibility of PVAT to activation of pro-inflammatory JNK signaling, and highlight its potential importance in early vascular changes of T1DM. Further elucidation of the role of PVAT in cardiovascular complications may allow for the design of novel therapeutic strategies directly addressing PVAT pathophysiology.     

Thymoquinone accelerates osteoblast differentiation and activates bone morphogenetic protein-2 and ERK pathway

Thymoquinone (TQ), the active component of Nigella sativa L. is well known for its various beneficial effects against several diseases. However, its detailed effect on bone metabolism has not been studied before. Therefore, the aim of the present study is to evaluate the effect of TQ on the proliferation, differentiation, and mineralization of MC3T3-E1 osteoblast cells. Our data shows that TQ induced the proliferation of MC3T3-E1 cells and proved to be non-toxic for up to 72 h of incubation. TQ induced the mineralization of MC3T3-E1 cells as evidenced by an increase in bone nodule formation 14 days post TQ treatment. qRT-PCR analysis shows that TQ induced the expression levels of differentiation related genes including alkaline phosphatase, osteocalcin, and osteopontin, while no effect was seen on collagen 1a1. TQ also induced the expression levels of bone morphogenetic protein-2 (BMP-2) and upregulated the phosphorylation of ERK signaling pathway. In summary, the present study shows for the first time that TQ has anabolic effects on MC3T3-E1 cells and that this effect is mediated by an increase in the expression of BMP-2 along with the involvement of the ERK signaling pathway. This study also reveals that TQ may be beneficial in inducing osteogenesis.     

Molecular validation of the precision-cut kidney slice (PCKS) model of renal fibrosis through assessment of TGF-β1-induced Smad and p38/ERK signaling

BackgroundThe precision-cut kidney slice (PCKS) model appears to be a useful ex vivo model of renal fibrosis. However, little in-depth molecular investigation on the PCKS model has been performed. Therefore, the aim of this study will be to investigate and validate the molecular validity of this model.MethodsThe PCKS model was constructed in male C57BL/6 mice. To induce renal fibrosis, PCKS were incubated in recombinant human TGF-β1 for 48 h. Protein expression of phosphorylated Smad2 (p-Smad2, cytosolic and nuclear), Smad7, phosphorylated ERK1 (p-ERK1), phosphorylated ERK2 (p-ERK2), and phosphorylated p38 MAPK (p-p38 MAPK) was measured using Western blotting. To assess Smad2/3 heteromeric complex formation and phosphorylated Smad3 (p-Smad3) expression, immunoprecipitation was performed with an anti-Smad2 or an anti-Smad3 antibody, respectively, prior to Western blotting.Resultsp-Smad2 and p-Smad3 were significantly upregulated in the PCKS model relative to control (p < 0.05). However, we found no significant difference in Smad7 expression between the PCKS model and control (p > 0.05). The PCKS model demonstrated significantly greater Smad2/3 complex formation and nuclear translocation relative to control (p < 0.05). The PCKS model showed significantly greater expression of p-ERK1, p-ERK2, and p-p38 MAPK relative to control (p < 0.05).ConclusionsThe PCKS model displays several well-established molecular markers of renal fibrosis. However, the PCKS model failed to display Smad7 downregulation and appears to display “over-activation” of p-Smad2 and p-Smad3 as well as “under-activation” of ERK1/2 and p38 MAPK signaling vis-à-vis the well-established in vivo unilateral ureteric obstruction model of renal fibrosis.