Natural Amino Acid-Bearing Carbamate Prodrugs of Daidzein Increase Water Solubility and Improve Phase II Metabolic Stability for Enhanced Oral Bioavailability.

Daidzein (DAN) is an isoflavone, and it is often found in its natural form in soybean and food supplements. DAN has poor bioavailability owing to its extremely low water solubility and first-pass metabolism. Herein, we hypothesized that a bioactivatable natural amino acid-bearing carbamate prodrug strategy could increase the water solubility and metabolic stability of DAN. To test our hypothesis, nine amino acid prodrugs of DAN were designed and synthesized. Compared with DAN, the optimal prodrug (daidzein-4'-O-CO-N-isoleucine, D-4'-I) demonstrated enhanced water solubility and improved phase II metabolic stability and activation to DAN in plasma. In addition, unlike the passive transport of DAN, D-4'-I maintained high permeability via organic anion-transporting polypeptide 2B1 (OATP2B1)-mediated transport. Importantly, D-4'-I increased the oral bioavailability by 15.5-fold, reduced the gender difference, and extended the linear absorption capacity in the pharmacokinetics of DAN in rats. Furthermore, D-4'-I exhibited dose-dependent protection against liver injury. Thus, the natural amino acid-bearing carbamate prodrug strategy shows potential in increasing water solubility and improving phase II metabolic stability to enhance the oral bioavailability of DAN.

A Highly Sensitive and Rapid Colloidal Gold Immunoassay for Puerarin Detection.

Radix Puerariae is a traditional Chinese medicinal material with a rich history of use in East and Southeast Asia. Puerarin, a unique component of the Pueraria genus, serves as a quality control marker for herbal medicines like Pueraria lobata and Pueraria thomsonii in China, displaying diverse pharmacological properties. This study developed puerarin colloidal gold immunoassay dipsticks utilizing an anti-puerarin monoclonal antibody, resulting in a fast and sensitive detection method with a limit of 500-1000 ng·mL-1. Evaluation using tap water-extracted P. lobata and P. thomsonii samples showed consistent results compared to LC-MS analysis. Cross-reactivity assessments of puerarin analogs revealed minimal interference, affirming the dipstick's reliability for distinguishing between the two species.

Genistin: A Novel Estrogen Analogue Targeting ERß to Alleviate Thrombocytopenia.

Thrombocytopenia, a prevalent hematologic challenge, correlates directly with the mortality of numerous ailments. Current therapeutic avenues for thrombocytopenia are not without limitations. Here, we identify genistin, an estrogen analogue, as a promising candidate for thrombocytopenia intervention, discovered through AI-driven compound library screening. While estrogen's involvement in diverse biological processes is recognized, its role in thrombopoiesis remains underexplored. Our findings elucidate genistin's ability to enhance megakaryocyte differentiation, thereby augmenting platelet formation and production. In vivo assessments further underscore genistin's remedial potential against radiation-induced thrombocytopenia. Mechanistically, genistin's efficacy is attributed to its direct interaction with estrogen receptor ß (ERß), with subsequent activation of both ERK1/2 and the Akt signaling pathways membrane ERß. Collectively, our study positions genistin as a prospective therapeutic strategy for thrombocytopenia, shedding light on novel interplays between platelet production and ERß.

Genistein effect in cultured ovine ovarian granulosa cells.

Genistein, an isoflavone has the potential to mimic, augment, or dysregulate the steroid hormone production pathways. We hypothesized that genistein affects the granulosa cell (GCs) functions through a series of biochemical, molecular, and genomic cascades. The present study was conducted to evaluate the impact of genistein exposure on GCs viability, apoptosis, and steroidogenesis. The present study involved 3/5 days of exposure to genistein on GCs collected from abattoir-derived ovine ovaries at doses of 0, 1, 10, 25, 50, and 100 µM. The harvested GCs were used for growth, cytotoxicity, and gene expression studies related to apoptosis, growth, and steroidogenesis. We observed that genistein had both stimulatory at 10 and 25 µM levels as well as inhibitory effects at 50 and 100 µM levels on the growth and proliferation of GCs. Genistein significantly decreased the levels of 17ß-estradiol at higher exposure (50 and 100 µM), whereas the progesterone level increased significantly as the genistein exposure increased. Additionally, genistein could also alter the mRNA expression of the steroidogenic receptor, enzymes, proteins, and growth-related genes suggesting that genistein could potentially alter the steroidogenic pathways. We conclude that genistein can interfere with cell survival and steroidogenesis by exhibiting a dose-dependent biphasic response on the viability, growth-related parameters, and the synthesis of 17ß-estradiol in the cultured GCs.

A Gnotobiotic Mouse Model with Divergent Equol-Producing Phenotypes: Potential for Determining Microbial-Driven Health Impacts of Soy Isoflavone Daidzein.

The implications of soy consumption on human health have been a subject of debate, largely due to the mixed evidence regarding its benefits and potential risks. The variability in responses to soy has been partly attributed to differences in the metabolism of soy isoflavones, compounds with structural similarities to estrogen. Approximately one-third of humans possess gut bacteria capable of converting soy isoflavone daidzein into equol, a metabolite produced exclusively by gut microbiota with significant estrogenic potency. In contrast, lab-raised rodents are efficient equol producers, except for those raised germ-free. This discrepancy raises concerns about the applicability of traditional rodent models to humans. Herein, we designed a gnotobiotic mouse model to differentiate between equol producers and non-producers by introducing synthetic bacterial communities with and without the equol-producing capacity into female and male germ-free mice. These gnotobiotic mice display equol-producing phenotypes consistent with the capacity of the gut microbiota received. Our findings confirm the model's efficacy in mimicking human equol production capacity, offering a promising tool for future studies to explore the relationship between endogenous equol production and health outcomes like cardiometabolic health and fertility. This approach aims to refine dietary guidelines by considering individual microbiome differences.

Exploring simple ways to avoid collecting highly 137Cs-contaminated Aralia elata buds for the revival of local wild vegetable cultures.

Collection and cooking of wild vegetables have provided seasonal enjoyments for Japanese local people as provisioning and cultural ecosystem services. However, the Fukushima Daiichi Nuclear Power Plant accident in March 2011 caused extensive radiocesium contamination of wild vegetables. Restrictions on commercial shipments of wild vegetables have been in place for the last 10 years. Some species, including buds of Aralia elata, are currently showing radiocesium concentrations both above and below the Japanese reference level for food (100 Bq/kg), implying that there are factors decreasing and increasing the 137Cs concentration. Here, we evaluated easy-to-measure environmental variables (dose rate at the soil surface, organic soil layer thickness, slope steepness, and presence/absence of decontamination practices) and the 137Cs concentrations of 40 A. elata buds at 38 locations in Fukushima Prefecture to provide helpful information on avoiding collecting highly contaminated buds. The 137Cs concentrations in A. elata buds ranged from 1 to 6,280 Bq/kg fresh weight and increased significantly with increases in the dose rate at the soil surface (0.10-6.50 µSv/h). Meanwhile, the 137Cs concentration in A. elata buds were not reduced by decontamination practices. These findings suggest that measuring the latest dose rate at the soil surface at the base of A. elata plants is a helpful way to avoid collecting buds with higher 137Cs concentrations and aid in the management of species in polluted regions.

Profiling of Metabolites in a Fermented Soy Dietary Supplement Reinforces its Role in the Management of Intestinal Inflammation.

SCOPE: Gastro-AD (GAD) is a soy flour derived product that undergoes an industrial fermentation with Lactobacillus delbrueckii R0187 and has demonstrated clinical effects in gastroesophageal reflux and peptic ulcer symptom resolution. The aim of this study is to describe and link GAD's metabolomic profile to plausible mechanisms that manifest and explain the documented clinical outcomes. METHODS AND RESULTS: 1H NMR spectroscopy with multivariate statistical analysis is used to characterize the prefermented soy flour and GAD products. The acquired spectra are screened using various resources and the molecular assignments are confirmed using total correlation spectroscopy (TOCSY). Peaks corresponding to different metabolites are integrated and compared between the two products for relative changes. HPLC and GC are used to quantify some specific molecules. NMR analyses demonstrate significant changes in the composition of various assigned bioactive moieties. HPLC and GC analysis demonstrate deglycation of isoflavones after fermentation, resulting in estrogenically active secondary metabolites that have been previously shown to help to reduce inflammation. CONCLUSION: The identification of bioactive molecules, such as genistein and SCFAs, capable of modulating anti-inflammatory signaling cascades in the stomach's gastric and neuroendocrine tissues can explain the reported biological effects in GAD and is supported by in vivo data.

Calycosin alleviates titanium particle-induced osteolysis by modulating macrophage polarization and subsequent osteogenic differentiation.

Periprosthetic osteolysis (PPO) caused by wear particles is one of the leading causes of implant failure after arthroplasty. Macrophage polarization imbalance and subsequent osteogenic inhibition play a crucial role in PPO. Calycosin (CA) is a compound with anti-inflammatory and osteoprotective properties. This study aimed to evaluate the effects of CA on titanium (Ti) particle-induced osteolysis, Ti particle-induced macrophage polarization and subsequent osteogenic deficits, and explore the associated signalling pathways in a Ti particle-stimulated calvarial osteolysis mouse model using micro-CT, ELISA, qRT-PCR, immunofluorescence and western blot techniques. The results showed that CA alleviated inflammation, osteogenic inhibition and osteolysis in the Ti particle-induced calvarial osteolysis mouse model in vivo. In vitro experiments showed that CA suppressed Ti-induced M1 macrophage polarization, promoted M2 macrophage polarization and ultimately enhanced osteogenic differentiation of MC3T3-E1 cells. In addition, CA alleviated osteogenic deficits by regulating macrophage polarization homeostasis via the NF-κB signalling pathway both in vivo and in vitro. All these findings suggest that CA may prove to be an effective therapeutic agent for wear particle-induced osteolysis.

Constructing Protein-Scaffolded Multienzyme Assembly Enhances the Coupling Efficiency of the P450 System for Efficient Daidzein Biosynthesis from (2S)-Naringenin.

Daidzein is a major isoflavone compound with an immense pharmaceutical value. This study applied a novel P450 CYP82D26 which can biosynthesize daidzein from (2S)-naringenin. However, the recombinant P450 systems often suffer from low coupling efficiency, leading to an electron transfer efficiency decrease and harmful reactive oxygen species release, thereby compromising their stability and catalytic efficiency. To address these challenges, the SH3-GBD-PDZ (SGP) protein scaffold was applied to assemble a multienzyme system comprising CYP82D26, P450 reductase, and NADP+-dependent aldehyde reductase in desired stoichiometric ratios. Results showed that the coupling efficiency of the P450 system was significantly increased, primarily attributed to the channeling effect of NADPH resulting from the proximity of tethered enzymes and the electrostatic interactions between NADPH and SGP. Assembling this SGP-scaffolded assembly system in Escherichia coli yielded a titer of 240.5 mg/L daidzein with an 86% (2S)-naringenin conversion rate, which showed a 9-fold increase over the free enzymes of the P450 system. These results underscore the potential application of the SGP-scaffolded multienzyme system in enhancing the coupling and catalytic efficiency of the P450 system.

The effect of soy isoflavones on non-alcoholic fatty liver disease and the level of fibroblast growth factor-21 and fetuin A.

A two-arm randomized open labeled controlled clinical trial was conducted on 50 patients with non-alcoholic fatty liver disease (NAFLD). Subjects were randomized to either receive two tablets of soy isoflavone (100 mg/day) or placebo. At week 12, the serum levels of alanine amino transferase (ALT), aspartate amino transferase (AST) and controlled attenuation parameter (CAP) score were significantly decreased only in the soy isoflavone group (P < 0.05). A significant decline in the gamma glutamyl transferase (GGT) level was observed only in the placebo group (P = 0.017). A significant increase in the serum level of fetuin A was shown in both groups at the end of the trial with a significantly greater increment in the soy isoflavone group compared to the placebo group (P < 0.05). The changes in the serum level of FGF-21 were not significant in any of the two groups. Steatosis grade significantly improved only in the soy isoflavone group (P = 0.045). There was no significant change in the fibrosis grade in the groups. Soy isoflavone intake led to a decrease in ALT, AST, CAP score, steatosis grade and an increase in the level of fetuin A. However, no significant changes were observed in the fibrosis grade and serum levels of GGT and FGF-21.

[Prunetin inhibits TLR4/MyD88 pathway to attenuate intestinal epithelial inflammatory response and ameliorate mouse Crohn's disease-like colitis].

Objective To investigate the regulatory role of natural plant compound prunetin (PRU) on the intestinal epithelial inflammation and the barrier structure in Crohn's disease-like colitis. Methods A lipopolysaccharide (LPS)-induced inflammatory injury model of colonic organoids and a 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model were established to evaluate the effects of PRU on the intestinal epithelial inflammation and intestinal barrier. In addition, network pharmacological predictions, combined with in vitro and in vivo studies, were used to analyze the molecular mechanisms by which PRU modulates intestinal epithelial inflammation and intestinal barrier in CD-like colitis. Results PRU inhibited the release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß in LPS-induced colonic organoids, and ameliorated the colitis symptoms in TNBS-induced mice, including body mass loss, elevated disease activity index and increased inflammation scores. Meanwhile, PRU promoted the expression of tight junction proteins (ZO-1 and claudin-1) and improved their translocation restoration in LPS-induced colonic organoids and TNBS-induced intestinal epithelial cells, while maintaining the intestinal barrier structure. Mechanistically, PRU targeted the Toll-like receptor 4 (TLR4) and inhibited the activation of the TLR4/myeloid differentiation primary response gene 88 (MyD88) signaling pathway. Conclusion PRU can antagonize TLR4/MyD88 signaling, thereby inhibiting intestinal epithelial inflammation and protecting against intestinal barrier damage, which helps ameliorate Crohn's disease-like colitis.

Multifunctional thiolated chitosan/puerarin composite hydrogels with pH/glutathione dual responsiveness for potential drug carriers.

Puerarin (PUE), a natural and biologically active isoflavone extracted from Chinese medicine Pueraria lobata, can self-assemble to form a hydrogel without other chemical modifications. However, although PUE hydrogel has pH responsivity, but it is difficult to adapt to the changeable pathological environment. Therefore, thiolated chitosan (TCS) is synthesized and hybridized with PUE hydrogel to prepare TCS10/PUE composite hydrogel. The results of rheological measurement showed that the resultant composite hydrogels inherited the low loss performance of TCS hydrogel, which means that they have stronger elasticity. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images displayed that TCS10/PUE composite hydrogel has a fibrous-network structure. X-Ray Diffractometer (XRD) and Fourier transform infrared spectroscopy (FT-IR) proved the existence of hydrogen bonds and disulfide bonds in the formation of composite hydrogel. Degradation experiment showed that TCS10/PUE composite hydrogels have pH and glutathione (pH/GSH) dual sensitivity. Furthermore, TCS10/PUE composite hydrogels exhibited multi-functionality including thixotropy, cytocompatibility, antibacterial and anti-inflammatory properties. Berberine chloride hydrate (BCH) was further used as a model drug for in vitro release study. BCH and PUE could be released cooperatively under pH/GSH dual responsivity. These results indicated that the resultant composite hydrogel has eminent pH/GSH dual responsivity and could act as a potential new intelligent drug carrier.

Exploring functional genes' correlation with (S)-equol concentration and new daidzein racemase identification.

With its estrogenic activity, (S)-equol plays an important role in maintaining host health and preventing estrogen-related diseases. Exclusive production occurs through the transformation of soy isoflavones by intestinal bacteria, but the reasons for variations in (S)-equol production among different individuals and species remain unclear. Here, fecal samples from humans, pigs, chickens, mice, and rats were used as research objects. The concentrations of (S)-equol, along with the genetic homology and evolutionary relationships of (S)-equol production-related genes [daidzein reductase (DZNR), daidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR)], were analyzed. Additionally, in vitro functional verification of the newly identified DDRC gene was conducted. It was found that approximately 40% of human samples contained (S)-equol, whereas 100% of samples from other species contained (S)-equol. However, there were significant variations in (S)-equol content among the different species: rats > pigs > chickens > mice > humans. The distributions of the four genes displayed species-specific patterns. High detection rates across various species were exhibited by DHDR, THDR, and DDRC. In contrast, substantial variations in detection rates among different species and individuals were observed with respect to DZNR. It appears that various types of DZNR may be associated with different concentrations of (S)-equol, which potentially correspond to the regulatory role during (S)-equol synthesis. This enhances our understanding of individual variations in (S)-equol production and their connection with functional genes in vitro. Moreover, the newly identified DDRC exhibits higher potential for (S)-equol synthesis compared to the known DDRC, providing valuable resources for advancing in vitro (S)-equol production. IMPORTANCE: (S)-equol ((S)-EQ) plays a crucial role in maintaining human health, along with its known capacity to prevent and treat various diseases, including cardiovascular diseases, metabolic syndromes, osteoporosis, diabetes, brain-related diseases, high blood pressure, hyperlipidemia, obesity, and inflammation. However, factors affecting individual variations in (S)-EQ production and the underlying regulatory mechanisms remain elusive. This study examines the association between functional genes and (S)-EQ production, highlighting a potential correlation between the DZNR gene and (S)-EQ content. Various types of DZNR may be linked to the regulation of (S)-EQ synthesis. Furthermore, the identification of a new DDRC gene offers promising prospects for enhancing in vitro (S)-EQ production.

Comparison of the five different definitions of equol producers: The relationship between blood and urine equol concentrations and blood parameters in 466 healthy men and women.

BACKGROUND AND AIMS: No studies have compared various definitions of "equol producers" until now. Therefore, we aimed to explore the accuracy of five different definitions of equol producers (EQP) and their associations with health benefits. METHODS: This is a cross-sectional study of 466 healthy Japanese men and women aged between 22 and 88 years. Equol producer proportions were calculated from their serum and urine isoflavone concentrations using five commonly used definitions. We then examined their accuracy, and associations with the blood parameters. RESULTS: Proportions of equol ranged from 29 % in the most stringent definition to 47.6 % in the most sensitive definition. EQP identified under all definitions had significantly low serum PSA1 levels compared to nonequol producers (NEQP). The most stringent definition, which is defined as the urinary equol level of 1.0 µM and above, corresponded to the highest median serum equol level and was associated with better health outcomes. Male EQP identified by this definition seemed to have reduced risk of LDL2-hypercholesterolemia by 50 %, and female EQP identified by this definition seemed to have lower risk of high hs-CRP,3 compared to NEQP. Both the first and second stringent definition, which is defined as the serum equol level of 1.0 ng/mL and above, was associated with lower thyroid stimulating hormone level. CONCLUSIONS: More stringent definitions were associated with better parameters in general. Combined with the dietary inquires, a reliable definition for equol producer is crucial to evaluate the health benefits of equol.

Perspective: Observational Studies Involving Low-Soy Intake Populations Have Limited Ability for Providing Insight into the Health Effects of Soybean Isoflavones.

Isoflavones are naturally occurring plant compounds found in uniquely high amounts in soybeans and foods made from this legume. These soybean constituents have been proposed to exert several health benefits and as such they have been the subject of an enormous amount of research. This research includes randomized controlled trials (RCTs) and epidemiologic investigations. Although statistically significant associations between isoflavone intake and a wide range of health outcomes have been identified in cohorts involving low-isoflavone intake populations, we suggest that these associations are unlikely to have a causal basis because exposure is too low for isoflavones to exert physiologic effects. In cohorts involving predominantly non-Asian, non-vegetarian populations, the highest isoflavone intake category is typically ≤3 mg/d, an amount of isoflavones provided by ∼30 mL (2 tablespoons) of soymilk made from whole soybeans. In comparison, mean isoflavone intake in the upper intake categories in observational studies involving high-isoflavone intake populations is typically ≥50 mg/d. In RCTs, intervention doses of isoflavones typically range between 40 and 100 mg/d. Health professionals advising patients and clients about soy food and isoflavone intake need to be aware of the limitations of epidemiologic research involving low-isoflavone intake populations. Intake recommendations are best based on the results of RCTs using clinically relevant doses of isoflavones and epidemiologic studies involving populations for whom soy foods are a habitual part of the diet.

Effect of soy isoflavone supplementation on blood pressure: a meta-analysis of randomized controlled trials.

BACKGROUND: Previous experimental studies have suggested that the consumption of soy isoflavones may have a potential impact on lowering blood pressure. Nevertheless, epidemiological studies have presented conflicting outcomes concerning the correlation between soy isoflavone consumption and blood pressure levels. Consequently, a comprehensive meta-analysis of all eligible randomized controlled trials (RCTs) was conducted to explore the influence of soy isoflavones on systolic blood pressure (SBP) and diastolic blood pressure (DBP) in adults. METHODS: A thorough search of PubMed, Embase, and the Cochrane Library for relevant literature up to April 30, 2023 was conducted. RCTs involving adults that compared soy isoflavone supplementation with a placebo (the same matrix devoid of soy isoflavone) were included. The combined effect size was presented as the weighted mean difference (WMD) along with 95% confidence interval (CI), employing a fixed-effects model. RESULTS: Our meta-analysis included a total of 24 studies involving 1945 participants. The results revealed a significant reduction in both SBP and DBP with soy isoflavone supplementation. Subgroup analyses suggested more pronounced reductions in SBP and DBP for interventions lasting ≥6 months, in individuals receiving mixed-type soy isoflavone, and among patients with metabolic syndrome or prehypertension. However, we did not detect significant nonlinear associations between supplementation dosage and intervention duration concerning both SBP and DBP. The overall quality of evidence was deemed moderate. CONCLUSIONS: The current meta-analysis revealed that supplementation with soy isoflavones alone effectively reduces blood pressure. Additional high-quality studies are required to investigate the efficacy of blood pressure reduction through supplementation with an optimal quantity and proportion of soy isoflavone.

Puerarin Induces Macrophage M2 Polarization to Exert Antinonalcoholic Steatohepatitis Pharmacological Activity via the Activation of Autophagy.

To determine the protective mechanism of puerarin against nonalcoholic steatohepatitis (NASH), the pharmacodynamic effects of puerarin on NASH were evaluated by using zebrafish, cells, and mice. Western blotting, flow cytometry, immunofluorescence, and qRT-PCR were used to detect the effects of puerarin on RAW264.7 autophagy and polarization. Key target interactions between autophagy and polarization were detected using immunoprecipitation. Puerarin regulated the M1/M2 ratio of RAW 264.7 cells induced by LPS + INF-γ. Transcriptomics revealed that PAI-1 is a key target of puerarin in regulating macrophage polarization. PAI-1 knockout reduced the number of M1-type macrophages and increased the number of M2-type macrophages. Puerarin regulated PAI-1 and was associated with macrophage autophagy. It increased p-ULK1 expression in macrophages and activated autophagic flux, reducing the level of PAI-1 expression. Stat3/Hif-1α and PI3K/AKT signaling pathways regulated the number of macrophage polarization phenotypes, reducing liver lipid droplet formation, alleviating liver structural abnormalities, decreasing the number of cytoplasmic vacuoles, and decreasing the area of blue collagen in NASH mice. Puerarin is a promising dietary component for NASH alleviation.

Puerarin attenuates remifentanil­induced postoperative hyperalgesia via targeting PAX6 to regulate the transcription of TRPV1.

Remifentanil­induced hyperalgesia (RIH) is characterized by the emergence of stimulation­induced pain, including phenomena such as allodynia and thermal hyperalgesia following remifentanil infusion. As a sequence­specific DNA binding transcription factor, PAX6 positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system. It was hypothesized that puerarin could relieve RIH via targeting PAX6 to regulate transcription of transient receptor potential cation channel subfamily V Member 1 (TRPV1). A total of 32 rats were randomly divided into five groups, namely control group, RI group, RI + 10 mg/kg puerarin group (RI + puerarin10), RI + 20 mg/kg puerarin group (RI + puerarin20), and RI + 40 mg/kg puerarin group (RI + puerarin40). Mechanical and thermal hyperalgesia were tested at ­24, 2, 6, 24 and 48 h after remifentanil infusion. Following the sacrifice of rats after the last behavioral test, western blot was used to detect the expression levels of TRPV1 in the tissues; Immunofluorescence staining and western blotting were used to detect the expression of PAX6 in the spinal cord. PharmMapper and JASPAR were used to predict the binding sites of puerarin/PAX6/TRPV1. Chromatin immunoprecipitation­PCR and dual luciferase reporter assay were used to verify the targeting relationship between PAX6 and TRPV1. Immunofluorescence was used to detect the expression levels of TRPV1 and p­NR2B. The results revealed that puerarin (10, 20, 40 mg/kg) dose­dependently reduced thermal and mechanical hyperalgesia from 2 to 48 h after remifentanil infusion. Remifentanil infusion remarkably stimulated the expression of phosphorylated (p­)NR2B. Nevertheless, the increased amount of p­NR2B by RIH was dose­dependently suppressed by puerarin in rats. In conclusion, puerarin was revealed to attenuate postoperative RIH via targeting PAX6 to regulate the transcription of TRPV1.

Formononetin alleviates cerebral ischemia-reperfusion injury in rats by targeting the PARP-1/PARG/Iduna signaling pathway.

Formononetin has been demonstrated to protect against cerebral ischemia-reperfusion injury, however its mechanism has to be further researched. This study examined the effect of formononetin on cerebral ischemia-reperfusion injury in rats using the PARP-1/PARG/Iduna signaling pathway. In male SD rats, a model of cerebral ischemia-reperfusion injury was developed. Animals were randomly assigned to one of eight groups: Sham operation, Sham operation + formononetin, MCAO, MCAO + formononetin, PARP inhibitor (PJ34) + MCAO, formononetin + PJ34 + MCAO, PARG inhibitor (Ethacridine lactate) + MCAO, and ethacridine lactate + formononetin. The neurological deficit test, TTC staining, HE staining, Nissl staining, TUNEL staining, and western blotting were utilized to assess formononetin's protective effects in MCAO rats. The data show that formononetin can effectively alleviate neurological dysfunction and pathological changes in brain tissue in rats with cerebral ischemia-reperfusion injury, reduce the area of cerebral infarction and neuronal apoptosis, decrease the protein levels of PARP-1, PARG, Caspase-3, P53, and AIF in brain tissue, and increase the protein levels of Iduna and p-AKT. As a result, we concluded that formononetin improves brain ischemia-reperfusion injury in rats by modulating the PARP-1/PARG/Iduna signaling pathway.

Formononetin Restrains Tumorigenesis of Breast Tumor by Restraining STING-NF-κB and Interfering with the Activation of PD-L1.

BACKGROUND: Breast cancer (BC), a common tumor in women, has high morbidity and mortality. Formononetin, an active ingredient in red clover and Astragalus membranaceus, has a wide range of pharmacological applications, including as an anticancer agent. Since immunotherapy is a hot topic in the treatment strategy of BC, it was dedicated to appraising the specific mechanism of formononetin in BC immunotherapy in this research. METHODS: Different formononetin concentrations (0, 20, 40, 60, 80, 100 µM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot. RESULTS: Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone. CONCLUSION: Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.