Genistein transfersome-embedded topical delivery system for skin melanoma treatment: in vitro and ex vivo evaluations.

Skin melanoma is considered the most dangerous form of skin cancer due to its association with high risk of metastasis, high mortality rate and high resistance to different treatment options. Genistein is a natural isoflavonoid with known chemotherapeutic activity. Unfortunately, it has low bioavailability due to its poor aqueous solubility and excessive metabolism. In the current study, genistein was incorporated into transferosomal hydrogel to improve its bioavailability. The prepared transferosomal formulations were characterized regarding: particle size; polydispersity index; zeta potential; encapsulation efficiency; TEM; FTIR; DSC; XRD; in vitro drug release; viscosity; pH; ex vivo anti-tumor activity on 3D skin melanoma spheroids and 1-year stability study at different storage temperatures. The optimized formulation has high encapsulation efficiency with an excellent particle size that will facilitate its penetration through the skin. The transfersomes have a spherical shape with sustained drug release profile. The anti-tumor activity evaluation of genistein transfersome revealed that genistein is a potent chemotherapeutic agent with enhanced penetration ability through the melanoma spheroids when incorporated into transfersomes. Stability study results demonstrate the high physical and chemical stability of our formulations. All these outcomes provide evidence that our genistein transferosomal hydrogel is a promising treatment option for skin melanoma.

Augmented ocular uptake and anti-inflammatory efficacy of decorated Genistein-loaded NLCs incorporated in in situ gel.

Genistein (Gen); a naturally occurring isoflavone, acts as a tyrosine kinase inhibitor and efficiently downregulates inflammatory cytokines, which are pivotal in eye inflammation. Also, Gen suffers from sparse ocular bioavailability due to poor solubility. In this work, nanostructured lipid carriers (NLCs) were successfully fabricated by using solid (stearic acid and compritol) and liquid (oleic acid) lipids. The optimized Gen-loaded NLCs showed a nanosize range of 140-246 nm, ≥ 98 % entrapment efficiency, and controlled release over 48 h. The ζ-potential of NLCs was increased from -27.3 mV to 25-27.4 mV due to surface modification with chitosan (CS) or eudragit RS100 (ERS 100). All NLCs showed prominent biocompatibility with enhanced cellular uptake on corneal stromal fibroblasts. Moreover, the different NLCs were incorporated into a mucoadhesive in situ gel. The optimized in situ gel (G9), containing 20 % poloxamers and 0.5 % hydroxyethyl cellulose, exhibited excellent gelling ability within 10.5 s, gelling temperature at 33.1 ± 0.6 ℃, spreadability diameter of 4.73 ± 0.12 cm, shear-thinning behavior, and 20 min ex vivo mucoadhesion time with drug release for 120 h. The in vivo results showed distinguished permeation and distribution potential for ocular delivery. In vivo anti-inflammatory effects after 3 days of treatment with CS-Gen-NLCs/G9 and ERS-Gen-NLCs/G9 revealed a downregulation of interleukin-6 levels in the cornea and retina compared to the untreated group. Our research highlights the promising anti-inflammatory potential of ERS-Gen-NLCs/G9 as an efficient, non-irritant Gen nanodelivery system for managing anterior and posterior ocular inflammation.

Genistein-Chitosan Derivative Nanoparticles for Targeting and Enhancing the Anti-Breast Cancer Effect of Tamoxifen In Vitro.

Tamoxifen (TAM) is a classical anti-estrogenic drug that antagonizes estrogen by competitively binding to estrogen receptor α (ERα). However, drug resistance to TAM remains a significant challenge in breast cancer treatment. In this study, we aimed to design an actively targeted drug delivery system to enhance the proliferation inhibitory effects of TAM on ER positive breast cancer cells. Herein, chitosan (CS) was modified with genistein (GEN) to obtain the actively targeted GEN-CS. The TAM-loaded nanoparticles (TAM-GEN-CS-NPs) were constructed using an ionic-crosslinking method, with GEN-CS as the carrier material and sodium tripolyphosphate (TPP) as the crosslinking agent. As a result, TAM-GEN-CS-NPs exhibited a spherical morphology with an average size of 299.8 nm. The encapsulation efficiency and drug loading content were 85.77% and 14.13 µg/mg, respectively. Compared with free TAM, TAM-GEN-CS-NPs displayed obvious slow-release performance. In vitro cellular assays demonstrated that TAM-GEN-CS-NPs had active targeting and proliferation inhibitory effects on MCF-7 cells. The IC50 of TAM and TAM-GEN-CS-NPs were 10.25 µg/mL and 7.22 µg/mL, respectively. More importantly, the combination index (CI) value of TAM and GEN was less than 1, indicating synergistic effects. Therefore, TAM-GEN-CS-NPs hold the potential to enhance TAM therapy for breast cancer through active targeting and synergistic treatment strategies.

Synergistic effect of genistein and adiponectin reduces fat deposition in chicken hepatocytes by activating the ERß-mediated SIRT1-AMPK signaling pathway.

Dietary supplementation with bioactive substances that can regulate lipid metabolism is an effective approach for reducing excessive fat deposition in chickens. Genistein (GEN) has the potential to alleviate fat deposition; however, the underlying mechanism of GEN's fat-reduction action in chickens remains unclear. Therefore, the present study aimed to explore the underlying mechanism of GEN on the reduction of fat deposition from a novel perspective: intercellular transmission of adipokine between adipocytes and hepatocytes. The findings showed that GEN enhanced the secretion of adiponectin (APN) in chicken adipocytes, and the enhancement effect of GEN was completely blocked when the cells were pretreated with inhibitors targeting estrogen receptor ß (ERß) or proliferator-activated receptor γ (PPARγ) signals, respectively. Furthermore, the results demonstrated that both co-treatment with GEN and APN or treatment with the medium supernatant (Med SUP) derived from chicken adipocytes treated with GEN significantly decreased the content of triglyceride and increased the protein levels of ERß, Sirtuin 1 (SIRT1) and phosphor-AMP-activated protein kinase (p-AMPK) in chicken hepatocytes compared to the cells treated with GEN or APN alone. Moreover, the increase in the protein levels of SIRT1 and p-AMPK induced by GEN and APN co-treatment or Med SUP treatment were blocked in chicken hepatocytes pretreated with the inhibitor of ERß signals. Importantly, the up-regulatory effect of GEN and APN co-treatment or Med SUP treatment on the protein level of p-AMPK was also blocked in chicken hepatocytes pretreated with a SIRT1 inhibitor; however, the increase in the protein level of SIRT1 induced by GEN and APN co-treatment or Med SUP treatment was not reversed when the hepatocytes were pretreated with an AMPK inhibitor. In conclusion, the present study demonstrated that GEN enhanced APN secretion by activating the ERß-Erk-PPARγ signaling pathway in chicken adipocytes. Subsequently, adipocyte-derived APN synergized with GEN to activate the ERß-mediated SIRT1-AMPK signaling pathway in chicken hepatocytes, ultimately reducing fat deposition. These findings provide substantial evidence from a novel perspective, supporting the potential use of GEN as a dietary supplement to prevent excessive fat deposition in poultry.

Long-term supplementation of genistein improves immune homeostasis in the aged gut and extends the laying cycle of aged laying hens.

Aging is associated with alterations in gut function, including intestinal inflammation, leaky gut, and impaired epithelial regeneration. Rejuvenating the aged gut is imperative to extend the laying cycle of aged laying hens. Genistein is known to have beneficial effects on age-related diseases, but its precise role in homeostasis of the aged gut of laying hens remains to be elucidated. In this study, 160 45-wk-old Hyline Brown laying hens were continuously fed a basal diet or a diet supplemented with 40 mg/kg genistein until they reached 100 wk of age. The results revealed that long-term genistein supplementation led to an improvement in the egg production rate and feed conversion ratio, as well as an increase in egg quality. Moreover, the expression levels of senescence markers, such as ß-galactosidase, P16, and P21, were decreased in the gut of genistein-treated aged laying hens. Furthermore, genistein ameliorated gut dysfunctions, such as intestinal inflammation, leaky gut, and impaired epithelial regeneration. Treg cell-derived IL-10 plays a crucial role in the genistein-induced regulation of age-related intestinal inflammation. This study demonstrates that long-term consumption of genistein improves homeostasis in the aged gut and extends the laying cycle of aged laying hens. Moreover, the link between genistein and Treg cells provides a rationale for dietary intervention against age-associated gut dysfunction.

Genistein interferes with antitumor effects of cisplatin in an ovariectomized breast cancer xenograft tumor model.

Genistein (GEN) has been demonstrated to interfere with antitumor effects of cisplatin (CIS) in vitro. To analyze whether these findings are also relevant in vivo, we examined the effects of combined GEN and CIS treatment in an ovariectomized nude mouse breast cancer xenograft model. Tumor growth and markers for antitumor activity were determined after three weeks of treatment. Furthermore, the concentrations of GEN metabolites were measured in serum, liver, and xenograft tumor tissues using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three weeks' oral exposure to GEN at a dose of 5 mg kg-1·d-1 resulted in an average concentration of total GEN metabolite equivalent as high as 0.2729 nmol g-1 wet weight in xenograft tumor tissues. At this dosage, GEN significantly antagonized the antitumor effects of CIS. Mechanistically, GEN blocked both the inhibition of cell proliferation and induction of apoptosis triggered by CIS. Moreover, GEN concentrations in xenograft tumor tissues were found to be significantly higher than in serum and liver. In conclusion, our findings suggested that oral GEN exposure at a level comparable to dietary exposure in humans could interfere with CIS chemotherapy.

Developmental exposure to phytoestrogens found in soy: New findings and clinical implications.

Exposure to naturally derived estrogen receptor activators, such as the phytoestrogen genistein, can occur at physiologically relevant concentrations in the human diet. Soy-based infant formulas are of particular concern because infants consuming these products have serum genistein levels almost 20 times greater than those seen in vegetarian adults. Comparable exposures in animal studies have adverse physiologic effects. The timing of exposure is particularly concerning because infants undergo a steroid hormone-sensitive period termed "minipuberty" during which estrogenic chemical exposure may alter normal reproductive tissue patterning and function. The delay between genistein exposure and reproductive outcomes poses a unique challenge to collecting epidemiological data. In 2010, the U.S. National Toxicology Program monograph on the safety of the use of soy formula stated that the use of soy-based infant formula posed minimal concern and emphasized a lack of data from human subjects. Since then, several new human and animal studies have advanced our epidemiological and mechanistic understanding of the risks and benefits of phytoestrogen exposure. Here we aim to identify clinically relevant findings regarding phytoestrogen exposure and female reproductive outcomes from the past 10 years, with a focus on the phytoestrogen genistein, and explore the implications of these findings for soy infant formula recommendations. Research presented in this review will inform clinical practice and dietary recommendations for infants based on evidence from both clinical epidemiology and basic research advances in endocrinology and developmental biology from mechanistic in vitro and animal studies.

Biochanin A as an α-hemolysin inhibitor for combating methicillin-resistant Staphylococcus aureus infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. In S. aureus, α-hemolysin is an important virulence factor as it contributes to the capacity of the bacteria to infect the host. Here, we showed that biochanin A (bioA), an isoflavone present in red clover, cabbage and alfalfa, effectively inhibited hemolytic activity at a dose as low as 32 µg/mL. Further, western blot and RT-qPCR data showed that bioA reduced the production and expression of MRSA hemolysin in a dose-dependent manner. In addition, when different concentrations of bioA were added to a coculture system of A549 cells and S. aureus, it could significantly decrease cell injury. Importantly, the in vivo study showed that bioA could protect mice from pneumonia caused by a lethal dose of MRSA, as evidenced by improving their survival and reducing the number of bacterial colonies in lung tissues, the secretion of hemolysin into alveolar lavage fluid and the degree of pulmonary edema. In conclusion, biochanin A protected the host from MRSA infection by inhibiting the expression of the hemolysin of MRSA, which may provide experimental evidence for its development to a potential anti-MRSA drug.

Genistein alleviates H2O2-induced senescence of human umbilical vein endothelial cells via regulating the TXNIP/NLRP3 axis.

CONTEXT: Genistein (Gen) has shown protective effects against ageing process. OBJECTIVE: To explore the role of Gen on the senescence of H2O2-induced human umbilical vein endothelial cells (HUVECs) and investigate the possible mechanism. MATERIALS AND METHODS: HUVECs were treated with different concentrations of H2O2 (50, 100, 200 and 400 µmol/L) for 1 h or Gen administration (20, 40, 80 and 160 µg/mL) for 24 h. Functional experiments (cell counting kit-8, ß-galactosidase staining and flow cytometry) were used to detect the effect of Gen on H2O2-induced HUVECs. After HUVECs were transfected with TXNIP overexpression plasmids, the expression of p16, p21, thioredoxin-interacting protein (TXNIP), nucleotide-binding and oligomerization domain-like receptor 3 (NLRP3), cleaved caspase-3 and cleaved caspase-1 in HUVECs were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. RESULTS: H2O2 (200 and 400 µmol/L) inhibited the proliferation of HUVECs. At concentrations of >50 µmol/L, H2O2 induced the cell cycle progression arrests in G1 phase and promoted cell senescence of HUVECs. Gen had no obvious cytotoxicity to HUVECs below 160 µg/mL. H2O2-induced HUVEC senescence and the expression of TXNIP and NLRP3 in HUVECs were down-regulated by Gen (40 and 80 µg/mL). Expressions of TXNIP and NLRP3 in HUVECs were up-regulated by H2O2 but down-regulated by Gen. Overexpressed TXNIP partially reversed the suppressive effect of Gen on H2O2-induced senescence and apoptosis of HUVECs. Expressions of p16, p21, TXNIP, NLRP3, cleaved caspase-3 and cleaved caspase-1 in H2O2-treated HUVECs were inhibited by Gen, while the inhibition as such was partially reversed by overexpressed TXNIP. DISCUSSION AND CONCLUSIONS: H2O2-induced HUVEC senescence was alleviated by Gen via suppressing the TXNIP/NLRP3 axis, which may offer a potential therapeutic approach for improving HUVEC senescence and provide a new direction for the treatment of cardiovascular disease.

Synergism effect of swimming exercise and genistein on the inflammation, oxidative stress, and VEGF expression in the retina of diabetic-ovariectomized rats.

AIMS: Retinal neovascularization is one of the visual disorders during the postmenopausal period or types two diabetes. Physical activities and also phytoestrogens with powerful antioxidant features have been widely considered to improve nervous system diseases. Therefore, this study investigated the effects of genistein, swimming exercise, and their co-treatment on retina angiogenesis, oxidative stress, and inflammation in diabetic-ovariectomized rats. MAIN METHODS: Wistar rats were randomly divided into six groups (n = 8 per group): sham, ovariectomized group (OVX), OVX + diabetes (OVX.D), OVX.D+ genistein (1 mg/kg, eight weeks; daily SC), OVX.D + exercise (eight weeks), and OVX.D+ genistein+exercise (eight weeks). At the end of 8 weeks, the retina was removed under anesthesia. The assessed effects of treatment were by measuring MiR-146a and miR-132 expression via RT-PCR, the protein levels of ERK, MMP-2, VEGF, and NF-κB via western blotting, inflammation, and oxidative stress markers levels via the Eliza. KEY FINDINGS: The results showed miR-132, miR-146b, and MMP-2, NF-κB, ERK, VEGF, TNF-α, IL-1ß proteins, and MDA factor in the OVX.D group were increased, but glutathione (GSH) was decreased in comparison with the sham and OVX groups. Both exercise and genistein treatment has reversed the disorder caused by diabetes. However, the combination of exercise and genistein was more effective than each treatment alone. SIGNIFICANCE: It can be concluded that the interaction of exercise and genistein on microRNAs and their target protein was affected in the inflammation, stress oxidative, and extracellular matrix metalloproteinase pathways, can leading to a decrease in impairment of retinal neovascularization of the ovariectomized diabetic rats.

Effects of daidzein and genistein on markers of cardiovascular disease risk among women with impaired glucose regulation: a double-blind, randomized, placebo-controlled trial.

BACKGROUND AND OBJECTIVE: soy protein and soy isoflavones have been suggested to be associated with improved cardiovascular risk factors (e.g., lipid profiles and uric acid (UA)), but few studies have been conducted among women with impaired glucose regulation (IGR). This study is aimed to evaluate the effect of isolated daidzein and genistein on lipid profiles, high sensitive C-reactive protein (hs-CRP), and uric acid (UA) among Chinese women with IGR. METHODS AND RESULTS: this randomized, double-blind, and placebo-controlled trial was conducted in 165 Chinese women aged 30-70 years with IGR. Participants were randomly assigned to one of the three groups: 0 mg of daidzein and genistein with 10 g soy protein (placebo group), 50 mg of daidzein with 10 g soy protein (daidzein group), or 50 mg of genistein with 10 g soy protein (genistein group) supplementation for 24 weeks. Fasting serum total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), lipoprotein a (LP (a)), hs-CRP, and UA were assessed at baseline, 12, and 24 weeks after intervention. The results showed no significant differences in the changes (%) of TC, TG, HDL-C, LDL-C, LP (a), hs-CRP, and UA between the three treatment groups at weeks 12 or 24 (all P > 0.05). CONCLUSION: neither isolated daidzein nor genistein had a significant effect on cardiovascular health in Chinese women with IGR.

Fabrication of Chinese Traditional Medicines incorporated collagen biomaterials for human bone marrow mesenchymal stem cells.

Chinese Traditional Medicines (CTMs) are very popular for therapeutic applications to cure several chronic diseases. Many researchers are trying to discover the potential application and actual mechanism of CTMs in order to scientifically prove their effects for commercial use. One of the main functions of CTMs is to aid stem cell regeneration. Since, this study was focused to fabricate CTMs incorporated fish collagen film, which has good biocompatibility in mammalian cell growth and thus investigated the effect on human Mesenchymal stem cells (hMSCs) proliferation and differentiation. In this study, three types of CTMs such as Genistein, Icariin, and Naringin were used for film fabrication. Mechanical properties of collagen films were improved by the addition of CTMs, especially in Collagen-Naringin films. Solubility and In-vitro biodegradation of collagen films were enhanced by the hydrophobicity and chemical interaction of CTMs with collagen. The proliferation rate was accelerated in hMSCs cultured on CTMs incorporated collagen films in a dose- and time-dependent manner. Proliferation biomarkers such as Ki-67 and BrdU levels were higher in hMSCs cultured on CTMs incorporated collagen films. The proliferative and differentiation effect of CTMs was further confirmed by higher gene expression of Collagen I, Runx2, c-Fos, SMAD3 and TGF-ß1 in hMSCs. Overall, this study provides a new insight on novel biomaterial fabrication using CTMs and fish collagen for making a compatible platform for in-vitro stem cell culture.

High dose genistein in Sanfilippo syndrome: A randomised controlled trial.

The aim of this study was to evaluate the efficacy of high dose genistein aglycone in Sanfilippo syndrome (mucopolysaccharidosis type III). High doses of genistein aglycone have been shown to correct neuropathology and hyperactive behaviour in mice, but efficacy in humans is uncertain. This was a single centre, double-blinded, randomised, placebo-controlled study with open-label extension phase. Randomised participants received either 160 mg/kg/day genistein aglycone or placebo for 12 months; subsequently all participants received genistein for 12 months. The primary outcome measure was the change in heparan sulfate concentration in cerebrospinal fluid (CSF), with secondary outcome measures including heparan sulfate in plasma and urine, total glycosaminoglycans in urine, cognitive and adaptive behaviour scores, quality of life measures and actigraphy. Twenty-one participants were randomised and 20 completed the placebo-controlled phase. After 12 months of treatment, the CSF heparan sulfate concentration was 5.5% lower in the genistein group (adjusted for baseline values), but this was not statistically significant (P = .26), and CSF heparan sulfate increased in both groups during the open-label extension phase. Reduction of urinary glycosaminoglycans was significantly greater in the genistein group (32.1% lower than placebo after 12 months, P = .0495). Other biochemical and clinical parameters showed no significant differences between groups. High dose genistein aglycone (160 mg/kg/day) was not associated with clinically meaningful reductions in CSF heparan sulfate and no evidence of clinical efficacy was detected. However, there was a statistically significant reduction in urine glycosaminoglycans. These data do not support the use of genistein aglycone therapy in mucopolysaccharidosis type III. High dose genistein aglycone does not lead to clinically meaningful reductions in biomarkers or improvement in neuropsychological outcomes in mucopolysaccharidosis type III.

Improving the genistein oral bioavailability via its formulation into the metal-organic framework MIL-100(Fe).

Despite the interesting chemopreventive, antioxidant and antiangiogenic effects of the natural bioflavonoid genistein (GEN), its low aqueous solubility and bioavailability make it necessary to administer it using a suitable drug carrier system. Nanometric porous metal-organic frameworks (nanoMOFs) are appealing systems for drug delivery. Particularly, mesoporous MIL-100(Fe) possesses a variety of interesting features related to its composition and structure, which make it an excellent candidate to be used as a drug nanocarrier (highly porous, biocompatible, can be synthesized as homogenous and stable nanoparticles (NPs), etc.). In this study, GEN was entrapped via simple impregnation in MIL-100 NPs achieving remarkable drug loading (27.1 wt%). A combination of experimental and computing techniques was used to achieve a deep understanding of the encapsulation of GEN in MIL-100 nanoMOF. Subsequently, GEN delivery studies were carried out under simulated physiological conditions, showing on the whole a sustained GEN release for 3 days. Initial pharmacokinetic and biodistribution studies were also carried out upon the oral administration of the GEN@MIL-100 NPs in a mouse model, evidencing a higher bioavailability and showing that this oral nanoformulation appears to be very promising. To the best of our knowledge, the GEN-loaded MIL-100 will be the first antitumor oral formulation based on nanoMOFs studied in vivo, and paves the way to the efficient delivery of nontoxic antitumorals via a convenient oral route.

Thermoresponsive GenisteinNLC-dexamethasone-moxifloxacin multi drug delivery system in lens capsule bag to prevent complications after cataract surgery.

Cataract surgery is the most common intraocular procedure. To decrease postsurgical inflammation, prevent infection and reduce the incidence of secondary cataract, we built a temperature-sensitive drug delivery system carrying dexamethasone, moxifloxacin and genistein nanostructured lipid carrier (GenNLC) modified by mPEG-PLA based on F127/F68 as hydrogel. Characterizations and release profiles of the drug delivery system were studied. In vitro functions were detected by CCK-8 test, immunofluorescence, wound-healing assay, real time-PCR and western blotting. The size of GenNLCs was 39.47 ± 0.69 nm in average with surface charges of - 4.32 ± 0.84 mV. The hydrogel gelation temperature and time were 32 °C, 20 s with a viscosity, hardness, adhesiveness and stringiness of 6.135 Pa.s, 54.0 g, 22.0 g, and 3.24 mm, respectively. Transmittance of the gel-release medium was above 90% (93.44 ± 0.33% to 100%) at range of 430 nm to 800 nm. Moxifloxacin released completely within 10 days. Fifty percent of dexamethasone released at a constant rate in the first week, and then released sustainably with a tapering down rate until day 30. Genistein released slowly but persistently with a cumulative release of 63% at day 40. The thermoresponsive hydrogel inhibited the proliferation, migration and epithelial-mesenchymal transition of SRA 01/04 cells, which were confirmed by testing CCK-8, wound-healing assay, western blot, real time-PCR (RT-PCR) and immunofluorescence. These results support this intracameral thermoresponsive in situ multi-drug delivery system with programmed release amounts and release profiles to cut down the need of eye drops for preventing inflammation or infection and to reduce posterior capsular opacification following cataract surgery.

Glycyrrhizin micelle as a genistein nanocarrier: Synergistically promoting corneal epithelial wound healing through blockage of the HMGB1 signaling pathway in diabetic mice.

The purpose of this study was to explore the feasibility of targeting the HMGB1 signaling pathway to treat diabetic keratopathy with a dipotassium glycyrrhizinate-based micelle ophthalmic solution encapsulating genistein (DG-Gen), and to evaluate whether these dipotassium glycyrrhizinate (DG) micelles could synergistically enhance the therapeutic effect of encapsulated genistein (Gen). An optimized DG-Gen ophthalmic solution was fabricated with a Gen/DG weight of ratio 1:15, and this formulation featured an encapsulation efficiency of 98.96 ± 0.82%, and an average particle size of 29.50 ± 2.05 nm. The DG-Gen ophthalmic solution was observed to have good in vivo ocular tolerance and excellent in vivo corneal permeation, and to remarkably improve in vitro antioxidant activity. Ocular topical application of the DG-Gen ophthalmic solution significantly prompted corneal re-epithelialization and nerve regeneration in diabetic mice, and this efficacy might be due to the inhibition of HMGB1 signaling through down-regulation of HMGB1 and its receptors RAGE and TLR4, as well as inflammatory factor interleukin (IL)-6 and IL-1ß. In conclusion, these data showed that HMGB1 signaling is a potential regulation target for the treatment of diabetic keratopathy, and novel DG-micelle formulation encapsulating active agents such as Gen could synergistically cause blockage of HMGB1 signaling to prompt diabetic corneal and nerve wound healing.

Aggravated behavioral and neurochemical deficits and redox imbalance in mice with enhanced neonatal iron intake: improvement by biochanin A and role of microglial p38 activation.

Objectives: We aim to investigate the joint effect of iron (enhanced neonatal iron intake), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and biochanin A (BA, oral administration) and possible mechanisms for action on behavioral and neurochemical indicators in the mice. Methods: Rotarod test, pole test and swim test were used to evaluate animal behavior. The neurochemical analysis was conducted by HPLC-ECD. Oxidative stress was determined in this study. Further mechanism was investigated through in vitro experiments. Results: Iron and MPTP co-administration significantly induced behavioral deficits and decreased striatal dopamine content in the male and female mice. The co-administration of iron and MPTP also significantly induced redox imbalance in the substantia nigra (SN) of mice. Furthermore, BA significantly improved behavioral deficits and increased striatal dopamine content in the mice co-treated with iron and MPTP. BA also significantly improved redox imbalance in the SN of mice co-administered with iron and MPTP. Finally, we showed that iron and 1-Methyl-4-phenylpyridinium (MPP+) co-treatment significantly increased superoxide production in microglial cultures by inducing p38 mitogen-activated protein kinase (MAPK) activation. BA also significantly decreased superoxide production and p38 MAPK phosphorylation in the cultures co-treated with iron and MPP+. Conclusion: Iron and MPTP co-treatment may result in worsened behavioral and neurochemical deficits and aggravated redox imbalance through inducing microglial p38 MAPK activation. BA may improve behavioral and neurochemical deficits and redox imbalance through repressing microglial p38 MAPK activation.

Genistein nanoformulation promotes selective apoptosis in oral squamous cell carcinoma through repression of 3PK-EZH2 signalling pathway.

BACKGROUND: Overexpression of polycomb protein contributes to epigenetic repression in oral squamous cell carcinoma (OSCC) ensuing in poor prognosis and aggressive phenotype. Several plant-based compounds could help prevent epigenome alteration and cancer progression, but their low bioavailability limits their therapeutic activity. HYPOTHESIS: In this study, we have synthesized genistein nanoformulation (GLNPs) and evaluated its epigenetic regulation mechanism for selective apoptosis induction in OSCC. METHODS: Lactalbumin was used to prepare nanoformulation of Genistein. The mechanism of epigenetic regulation and selective apoptosis by Genistein loaded nanoparticles was studied in OSCC cell line JHU011 and fibroblast cell line L929 using immunofluorescence, Western blotting and ChIP-qPCR assay. RESULTS: We have found that GLNPs treatment selectively induced apoptosis in OSCC compared to the normal fibroblast cells. This selective effect in OSCC is achieved through enhanced reactive oxygen species (ROS) generation followed by Bax mitochondrial translocation and caspase 3 activation. Further, GLNPs induced withdrawal of epigenetic transcription repression through concurrent downregulation of the polycomb group proteins (PcG) Bmi 1 and EZH2 along with their successive targets, UbH2AK119 and H3K27me3, which have immense therapeutic implications in the treatment of OSCC. Last, we have established that GLNPs regulate EZH2expression through proteasomal mediated degradation and 3PK inhibition; 3PK protein was found physically linked with EZH2 protein and its promoter region (-1107 to -1002). This event indicates that 3PK might play some crucial role in EZH2 expression and epigenetic control of OSCC. Moreover, the formulation showed improved biodistribution, aqueous dispersibility and enhanced biocompatibility In-vivo. CONCLUSIONS: These results provide evidence that GLNPs may withdraw epigenetic transcriptional repression and selectively induce apoptosis in human oral squamous cell carcinoma.

Neonatal exposure to genistein affects reproductive physiology and behavior in female and male Long-Evans rats.

The present study was designed to examine the effects of neonatal genistein exposure on measures of reproductive physiology and behavior. Approximately 24 h after birth, female and male Long-Evans rat pups were injected daily with genistein (150 µg, subcutaneous; n = 29) or olive oil (n = 23) between postnatal days 1 and 5. After weaning, we examined all subjects daily until they reached puberty (i.e. vaginal opening in female rats and preputial separation in male rats). For all female subjects, we also examined vaginal cytology. After monitoring estrous cyclicity, the female subjects were given the opportunity to interact with a gonadally intact male or a sexually receptive female rat on the day of behavioral estrus to assess sexual motivation (i.e. partner-preference test with and without physical contact), which has never been evaluated before. For all male subjects, we assessed the development of copulatory behavior and sexual motivation (partner-preference test without physical contact). Consistent with previous findings, we found that neonatal exposure to genistein did not affect puberty onset in female or male rats. However, female rats exposed to genistein displayed significantly more irregular estrous cycles than controls. Neonatal genistein exposure also altered the development of male copulatory behavior, as indicated by an increase in mount frequency and intromission frequency and shorter interintromission intervals. We extended previous findings confirming that neither female nor male sexual motivation was affected by neonatal genistein. The results of the present study have important implications for the development of reproductive physiology and behavior in human neonates exposed to genistein in soy-based baby formula.

Genistein Improves Bone Healing via Triggering Estrogen Receptor Alpha-Mediated Expressions of Osteogenesis-Associated Genes and Consequent Maturation of Osteoblasts.

Osteoporosis-associated fractures may cause higher morbidity and mortality. Our previous study showed the effects of genistein, a phytoestrogen, on the induction of estrogen receptor alpha (ERα) gene expression and stimulation of osteoblast mineralization. In this study, rat calvarial osteoblasts and an animal bone defect model were used to investigate the effects of genistein on bone healing. Treatment with genistein caused a time-dependent increase in alkaline phosphatase (ALP) activity in rat osteoblasts. Levels of cytosolic and nuclear ERα significantly augmented following exposure to genistein. Subsequently, genistein elevated levels of ALP mRNA and protein in rat osteoblasts. Moreover, genistein induced other osteogenesis-associated osteocalcin and Runx2 mRNA and protein expressions. Knocking-down ERα using RNA interference concurrently inhibited genistein-induced Runx2, osteocalcin, and ALP mRNA expression. Attractively, administration of ICR mice suffering bone defects with genistein caused significant increases in the callus width, chondrocyte proliferation, and ALP synthesis. Results of microcomputed tomography revealed that administration of genistein increased trabecular bone numbers and improved the bone thickness and volume. This study showed that genistein can improve bone healing via triggering ERα-mediated osteogenesis-associated gene expressions and subsequent osteoblast maturation.