Receptor Protein Expression Research Articles

Ginsenoside Rg3 treats acute radiation proctitis through the TLR4/MyD88/NF-κB pathway and regulation of intestinal flora.

This study aimed to investigate the protective effect of ginsenoside Rg3 (GRg3) against acute radiation proctitis (ARP) in rats. Wistar rats were randomly divided into control, model, dexamethasone-positive, GRg3 low-dose, GRg3 medium-dose, and GRg3 high-dose groups. The ARP rat model was established by a single 22-Gy irradiation of 6 MV) X-rays. The distribution and function of intestinal flora were detected using 16S rRNA high-throughput sequencing, rectal tissue was observed by hematoxylin and eosin (H&E) staining, the expression of interleukin 1β (IL-1β) and IL-10 inflammatory factors was detected by ELISA, and mRNA and protein expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were detected by RT-qPCR and Western blotting, respectively. GRg3 improved the symptoms of ARP in rats in a dose-dependent manner. The species distribution of intestinal flora in GRg3 rats was significantly different from that in ARP rats. These differences were more significant in the high-dose group, where the numbers of Ruminococcus, Lactobacillus, and other beneficial bacteria were significantly increased, whereas those of Escherichia, Alloprevotella, and other harmful bacteria were decreased. In addition, GRg3 was closely related to amino acid metabolism. After GRg3 treatment, the mRNA and protein expression of TLR4, MyD88, and NF-κB in rectal tissue was significantly down-regulated, and the level of downstream inflammatory factor IL-1β decreased, whereas that of IL-10 increased. Our study indicated GRg3 as a new compound for the treatment of ARP by inhibiting the TLR4/MyD88/NF-κB pathway, down-regulating the expression of proinflammatory factors, thus effectively regulating intestinal flora and reducing inflammatory reactions.

C-Met Receptors Deficiency Was Involved in Absence Seizures Development in WAG/Rij Rats

Background: A variety of receptors may be involved in the pathogenesis of absence seizures. The c-Met receptors have a critical role in modulating the GABAergic interneurons and creating a balance between excitatory and inhibitory neurotransmission, sensorimotor gating, and normal synaptic plasticity. Objectives: This study aimed to assess the changes of the c-Met receptor during the appearance of absence attacks in the experimental model of absence epilepsy. Methods: A total of 48 animals were divided into four groups of two- and six-month-old WAG/Rij and Wistar rats. Epileptic WAG/Rij rats showing SWP in electrocorticogram (ECoG) were included in the epileptic group. The two-month-old WAG/Rij rats as well as two- and six-month-old Wistar rats not exhibiting SWP in ECoG were selected as the non-epileptic. Gene (RT-PCR) and protein expression (western blotting) of c-Met receptors as well as c-Met protein distribution (immunohistochemistry) in the somatosensory cortex and hippocampus were assessed during seizure development of the absence attacks. Results: According to the study findings, a lower c-Met gene and protein expression, as well as a lower protein distribution, were observed in the hippocampus (P < 0.001, P < 0.05, and P < 0.001, respectively) and cortex (P < 0.01, P < 0.001 and P < 0.001, respectively) of the two-month-old WAG/Rij rats compared to the same-age Wistar rats. Moreover, the data revealed a reduction of hippocampal and cortical c-Met protein expression (P < 0.001, for both) in six-month-old WAG/Rij rats compared to two-month-old ones. Six-month-old WAG/Rij rats had a lower cortical c-Met gene (P < 0.05) and protein expression (P < 0.001) as well as lower hippocampal and cortical protein distribution (P < 0.05 and P < 0.001) than the same-age Wistar rats. Conclusions: In sum, the c-Met receptor was found to play a significant role in the development of absence epilepsy. This receptor, therefore, may have been considered as an effective goal for absence seizure inhibition.

Protective effects of genistein on the production performance and lipid metabolism disorders in laying hens with fatty liver hemorrhagic syndrome by activation of the GPER-AMPK signaling pathways.

The aim of this study was to evaluate the beneficial effects and potential mechanisms of genistein (GEN) on production performance impairments and lipid metabolism disorders in laying hens fed a high-energy and low-protein (HELP) diet. A total of 120 Hy-line Brown laying hens were fed with the standard diet and HELP diet supplemented with 0, 50, 100, and 200 mg/kg GEN for 80 d. The results showed that the declines in laying rate (P < 0.01), average egg weight (P < 0.01), and egg yield (P < 0.01), and the increase of the ratio of feed to egg (P < 0.01) induced by HELP diet were markedly improved by 100 and 200 mg/kg of GEN treatment in laying hens (P < 0.05). Moreover, the hepatic steatosis and increases of lipid contents (P < 0.01) in serum and liver caused by HELP diet were significantly alleviated by treatment with 100 and 200 mg/kg of GEN in laying hens (P < 0.05). The liver index and abdominal fat index of laying hens in the HELP group were higher than subjects in the control group (P < 0.01), which were evidently attenuated by dietary 50 to 200 mg/kg of GEN supplementation (P < 0.05). Dietary 100 and 200 mg/kg of GEN supplementation significantly reduced the upregulations of genes related to fatty acid transport and synthesis (P < 0.01) but enhanced the downregulations of genes associated with fatty acid oxidation (P < 0.01) caused by HELP in the liver of laying hens (P < 0.05). Importantly, 100 and 200 mg/kg of GEN supplementation markedly increased G protein-coupled estrogen receptor (GPER) mRNA and protein expression levels and activated the AMP-activated protein kinase (AMPK) signaling pathway in the liver of laying hens fed a HELP diet (P < 0.05). These data indicated that the protective effects of GEN against the decline of production performance and lipid metabolism disorders caused by HELP diet in laying hens may be related to the activation of the GPER-AMPK signaling pathways. These data not only provide compelling evidence for the protective effect of GEN against fatty liver hemorrhagic syndrome in laying hens but also provide the theoretical basis for GEN as an additive to alleviate metabolic disorders in poultry.

Sensory Receptor, Inflammatory, and Apoptotic Protein Expression in the Bladder Urothelium of Patients with Different Subtypes of Interstitial Cystitis/Bladder Pain Syndrome.

The aim of this study was to investigate the expression levels of sensory receptors, inflammatory proteins, and pro-apoptotic proteins in the urothelium of non-Hunner's interstitial cystitis (NHIC) bladders of patients with different clinical and cystoscopic phenotypes. The urothelia from the bladders of 52 NHIC patients were harvested. The expression of sensory receptors, including TRPV1, TRPV4, TRPA1, H1-receptors, and sigma-1 receptors; the inflammatory proteins p38 and tryptase; and the pro-apoptotic proteins, such as caspase-3, BAD, and BAX in the urothelium, were investigated using immunohistochemistry and Western blotting. We compared the expression levels of these proteins in NHIC subtypes according to IC symptom scores, visual analog scores of bladder pain, maximal bladder capacity, glomerulation grades, and combined maximal bladder capacity and glomerulations after cystoscopic hydrodistention. The expression levels of TRPV1, TRPV4, sigma-1, P38, tryptase, caspase-3, and BAD were significantly increased in the urothelium of IC/BPS patients compared with the expression levels in the controls. TRPV1 was significantly associated with IC symptom severity. However, no significant differences in sensory receptor expression in the IC/BPS bladders with different bladder conditions were detected. Inflammatory and pro-apoptotic protein expression levels in the urothelium were similar among the IC/BPS subgroups. This study concluded that IC/BPS patients with frequency and bladder pain complaints have higher levels of urothelial sensory receptors, and inflammatory and pro-apoptotic proteins. The expression levels of these sensory receptors, inflammatory proteins, and pro-apoptotic proteins are not significantly different among IC/BPS bladders with different conditions.

High-Fat Diet Promotes Adipogenesis in Offspring Female Rats Induced by Perinatal Exposure to 4-Nonylphenol.

Both high-fat diet (HFD) and 4-nonylphenol (4-NP) could affect fat formation in adipose tissue individually. We investigated whether HFD promote abnormal adipose tissue formation caused by early exposure to 4-NP in life and preliminarily explore the possible mechanisms involved. The first-generation rats were treated with HFD on postnatal day after pregnant rats exposure to 5 ug/kg/day 4-NP. Then, the second generation rats started to only receive normal diet without 4-NP or HFD. We analyzed organ coefficient and histopathology of fat tissues, biochemical index, and gene level involved in lipid metabolism in female offspring rats. HFD and 4-NP interaction synergistically increased birth weight, body weight, and organ coefficients of adipose tissue in offspring female rats. HFD accelerately aggravated abnormal lipid metabolism and increased the adipocyte mean areas around the uterus of the offspring female rats induced by prenatal exposure to 4-NP. HFD also facilitate the regulation of gene expression involved lipid metabolism in offspring female rats induced by perinatal exposure to 4-NP, even passed on to the second generation of female rats. Moreover, HFD and 4-NP interaction synergistically declined the gene and protein expression of estrogen receptor (ER) in the adipose tissue of second-generation female rats. HFD and 4-NP synergistically regulate the expression of lipid metabolism genes in adipose tissue of F2 female rats and promote adipose tissue generation, leading to obesity in offspring rats, which is closely related to low expression of ER. Therefore, ER genes and proteins may be involved in the synergistic effect of HFD and 4-NP.

Effect of electroacupuncture at different frequencies on brain insulin signaling transduction pathway in Alzheimer's disease mice

To observe the effect of electroacupuncture (EA) at different frequencies on learning and memory functions, as well as the relevant proteins of brain insulin signal transduction pathway in Alzheimer's disease (AD) mice and explore the effect mechanism of EA in treatment of AD. Seventy-two SPF Kunming male mice were randomized into a blank group, a sham-operation group, a model group, a 2 Hz EA group, a 15 Hz EA group and a 30 Hz EA group, 12 mice in each one. In the model group and each EA group, AD model were established by the injection with streptozotocin (ST2) solution (8 mg/kg) into the left lateral ventricles. In the sham-operation group, 0.9% sodium chloride solution of the same volume was injected into the left lateral ventricles. After successful modeling, in each EA group, EA was applied at "Baihui" (GV 20), "Dazhui" (GV 14) and "Shenshu" (BL 23) with corresponding frequencies, once daily. One course of EA intervention consisted of 7 treatments and 2 courses were given totally at interval of 1 day. After modeling and intervention, Morris water maze test was conducted for the mice of each group. Using immunohistochemistry and Western blot method, the protein expression of insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3K) was detected in the hippocampal of the mice after intervention. Compared with the blank group, in the model group, the 2 Hz, 15 Hz and 30 Hz EA groups, the escape latency and the first time of crossing the platform were all extended (P<0.01), and the number of crossing the platform was reduced (P<0.01) after modeling. When compared with the blank group, the escape latency and the first time of crossing the platform were all extended (P<0.01), and the number of crossing the platform was reduced (P<0.01) in the model group after intervention. In the 2 Hz, 15 Hz and 30 Hz EA groups, the escape latency and the first time of crossing the platform were all shortened (P<0.01), and the number of crossing the platform was increased (P<0.05, P<0.01) after intervention when compared with the model group. The escape latency and the first time of crossing the platform were all shortened (P<0.01, P<0.05), and the number of crossing the platform was increased (P<0.05) in the 15 Hz and 30 Hz EA groups in comparison with the 2 Hz EA group. The protein expression levels of IR, IRS-1 and PI3K were reduced in the model group when compared with those of the blank group (P<0.01, P<0.05); and these protein expression levels were increased in the 15 Hz and 30 Hz EA groups compared with the model group (P<0.05, P<0.01). Compared with the 2 Hz EA group, the protein expression levels of IR, IRS-1 and PI3K were all elevated in the 15 Hz and 30 Hz EA groups (P<0.05). The learning and memory function of AD mice may be improved through regulating brain insulin signaling transconduction pathway with electroacupuncture, and electroacupuncture at 15 Hz and 30 Hz obtains the overall better effect compared with the intervention at 2 Hz.

Abstract C015: Characterization of HER2-low breast tumors among a cohort of Colombian women

Abstract Introduction: HER2-low expression tumors, either with weak or incomplete membrane expression (immunohistochemistry (IHC) 1+ or 2+ with negative in situ hybridization (ISH) result), are considered and treated as HER2-negative tumors. However, recent clinical trials have shown promising results in the application of antibody-drug conjugates (ADCs) for patients with HER2-low breast tumors, highlighting the need to characterize the clinical phenotype of this group. This is the first study in Colombia aiming to explore the clinical-pathological features and survival of women affected with HER2-low breast cancer, compared with HER2-negative (0+) and HER2-positive groups (2+ with ISH+ or 3+). Methods: We included 516 breast cancer patients from different health institutions in Colombia, diagnosed at clinical stages that ranged between I-III. Protein expression of HER2, Ki67, and hormone receptors (estrogen (ER) and progesterone (PR) receptor) was evaluated by IHC in a paraffin block with tumoral tissue by a single pathologist. We defined HER2 expression as positive (3+ or 2+ with positive ISH test), low (1+/2+ with negative ISH result), and negative (0+). We applied a Chi-square or Fisher’s exact test to evaluate clinical-pathological features for HER2-low cases, compared with HER2-negative (low vs. negative) and HER2-positive tumors (low vs. positive). Differences in overall survival (OS) and risk of mortality were assessed using the Kaplan-Meier and log-rang test, and a Cox proportional hazard model. Results: 325 cases (63%) were classified as HER2-negative, 97 (18.8%) as HER2-low, and 94 (18.2%) as HER2-positive. We encountered more clinical-pathological significant differences between HER2-low and HER2-positive tumors than with HER2-negative cases. A higher proportion of patients diagnosed after the age of 50 (79.4% vs. 62.4%, p=0.018), with better-differentiated tumors (Bloom Richardson II: 58.8 vs. 41.2%, p=0.025), and a lower proliferation index (Ki67 &amp;lt;20%: 50.5 % vs. 21.2%, p&amp;lt;0.001) was observed for HER2-low tumors, compared to the HER2-positive group; these characteristics were not significantly different between HER2-low and HER2-negative tumors. The proportion of ER-positivity was significantly higher in the HER2-low group, compared to both HER2-positive (89.7% vs. 62.4%, p&amp;lt;0.001) and negative groups (89.7% vs. 79.7%, p=0.039). Moreover, although longer OS rates were found in HER2-low patients, compared to the negative group (5.5 vs. 5.0 years, p=0.0032), the multivariate Cox model adjusted by ER status showed no statistically significant differences between these groups (HR=0.62, 95% CI, 0.34 – 1.13, p=0.122). Conclusions: HER2-low tumors represent an important proportion of all breast cancer cases diagnosed in Colombian women. Our results are consistent with other reports that show differences in ER expression between HER2-low tumors and both HER2-positive and negative subtypes, although it seems that the prognosis of HER2-low patients is not different than of the other groups. Citation Format: Laura Rey Vargas, Lina Maria Bejarano, Juan Carlos Mejia Henao, Diego Felipe Ballen Lozano, Maria Carolina Sanabria Salas, Silvia Juliana Serrano Gomez. Characterization of HER2-low breast tumors among a cohort of Colombian women [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C015.

S-07-3: THE TESTOSTERONE-ANDROGEN RECEPTOR PATHWAY IS ASSOCIATED WITH CAROTID ATHEROSCLEROTIC PLAQUE INSTABILITY IN MEN WITH SEVERE CAROTID ATHEROSCLEROSIS

Objective: Carotid atherosclerotic plaques can be stable or unstable; the latter more likely to rupture resulting in strokes. Sex differences exist in carotid plaque morphology and composition: carotid plaques of men tend to be more unstable than of women, who tend to have more fibrous and stable plaques. Yet, women experience greater morbidity and mortality rates post-stroke. Given that sex hormones exert pleiotropic effects on the cell types involved in the atherosclerotic process, and given that estradiol is largely atheroprotective in women, we hypothesize that sex hormones and their receptors may affect plaque instability. Design and Methods: In this cross-sectional study, we measured sex hormones in the circulation and their receptors in the plaque of older men and postmenopausal women undergoing a carotid endarterectomy (n = 160). Using liquid chromatography mass spectrometry, circulating estradiol, testosterone, dehydroepiandrosterone (DHEA), and androstenedione were measured in blood samples. Plaques collected post-operatively were classified into 4 groups (n = 40/group): women with stable, women with unstable plaques, men with stable, and men with unstable plaques. Protein expression of estrogen receptor α (ER-α), estrogen receptor β (ER-β), G protein-coupled estrogen receptor (GPER), and androgen receptor (AR) was quantified using immunohistochemistry and western blot analysis. Plaque sex hormone receptor mRNA expression was assessed using qRT-PCR. Results: Men with unstable plaques demonstrated greater circulating concentrations of testosterone and androstenedione compared to men with stable plaques (P = 0.02 and P = 0.047, respectively). Unstable plaques in men demonstrated significantly less AR and ER-α mRNA expression compared to stable plaques from men (P = 0.0003 and P = 0.042, respectively) and compared to unstable plaques from women (P = 0.0088 and P = 0.002). Yet, AR, ER-α, ER-β, and GPER protein expression was significantly greater in unstable plaques from men compared to stable plaques from men (P = 0.039, P = 0.006, P &lt; 0.0001, and P = 0.0002, respectively) and compared to unstable plaques from women (P = 0.005, P = 0.041, P = 0.028, and P = 0.002, respectively). Expression was observed in macrophages, foam cells, endothelial cells, and smooth muscle cells. Moreover, unstable plaques from men demonstrated greater features of plaque instability compared to unstable plaques from women, including hemorrhage, large lipid core, abundance of inflammatory cells, and fibrous cap infiltration by inflammatory cells (P &lt; 0.05 for all). Conclusion: Our preliminary findings indicate a possible association between androgens and the androgen receptor pathway and plaque instability. With further investigations, our ongoing work will unravel the underlying mechanisms, and may ultimately lead to hormone-specific therapies aimed at stabilizing plaques and reducing the incidence of stroke for men and women.

Pharmacodynamic structure of deer antler base protein and its mammary gland hyperplasia inhibition mechanism by mediating Raf-1/MEK/ERK signaling pathway activation.

Mammary gland hyperplasia (MGH) is a common mammary disease whose main pathogenesis is the disruption of estradiol (E2) and progesterone (P) secretion, thereby causing overproliferation of mammary epithelial cells and mammary gland tissue hyperplasia. Deer antler base is a traditional Chinese medicine that has been used for many years to treat MGH. However, its pharmacological mechanism and pharmacodynamic material basis are unclear. In this study, we for the first time used the graded salting method to classify deer antler base protein (CNCP) as CNCP-A, CNCP-B, and CNCP-C and explored the pharmacological mechanism of the anti-MGH properties of CNCP. We found that CNCP could regulate the hormonal levels of E2, P, and follicle stimulating hormone (FSH) and improve the histopathological condition. The potential mechanism might be related to the recombinant C-Raf proto oncogene serine/threonine protein kinase/mitogen-activated protein/extracellular regulated protein kinase (Raf-1/MEK/ERK) signaling pathway. By upregulating the protein expression of the follicle stimulating hormone receptor (FSHR), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) inhibited the activation of the downstream Raf-1/MEK/ERK signaling pathway, which in turn inhibited the proliferation of mammary epithelial cells. We analyzed the physicochemical properties of CNCP-A, CNCP-B, and CNCP-C and obtained CNCP-C-I by column chromatographic purification of the best pharmacophore protein CNCP. Using high-performance liquid gel filtration chromatography (HPGFC), we determined the molecular weight of CNCP-C-I and identified it by high-performance liquid tandem mass spectrometry (LC-MS/MS) to obtain the first match for a high confidence protein KRT1. This study provides a theoretical basis for the development of effective traditional Chinese medicines with low toxicity levels for the prevention and treatment of mammary gland diseases.

Chronic spinal cord compression associated with intervertebral disc degeneration in SPARC-null mice.

Chronic spinal cord compression (CSCC) is induced by disc herniation and other reasons, leading to movement and sensation dysfunction, with a serious impact on quality of life. Spontaneous disc herniation rarely occurs in rodents, and therefore establishing a chronic spinal cord compression (CSCC) animal model is of crucial importance to explore the pathogenesis and treatment of CSCC. The absence of secreted protein, acidic, and rich in cysteine (SPARC) leads to spontaneous intervertebral disc degeneration in mice, which resembles human disc degeneration. In this study, we evaluated whether SPARC-null mice may serve as an animal model for CSCC. We performed rod rotation test, pain threshold test, gait analysis, and Basso Mouse Scale score. Our results showed that the motor function of SPARC-null mice was weakened, and magnetic resonance images revealed compression at different spinal cord levels, particularly in the lumbar segments. Immunofluorescence staining and western blot assay showed that the absence of SPARC induced apoptosis of neurons and oligodendrocytes, activation of microglia/macrophages with M1/M2 phenotype and astrocytes with A1/A2 phenotype; it also activated the expression of the NOD-like receptor protein 3 inflammasome and inhibited brain-derived neurotrophic factor/tyrosine kinase B signaling pathway. Notably, these findings are characteristics of CSCC. Therefore, we propose that SPARC-null mice may be an animal model for studying CSCC caused by disc herniation.

Efficient viral expression of a chemogenetic receptor in the old-world monkey amygdala.

Genetically encoded synthetic receptors, such as the chemogenetic and optogenetic proteins, are powerful tools for functional brain studies in animals. In the primate brain, with its comparatively large, intricate anatomical structures, it can be challenging to express transgenes, such as the hM4Di chemogenetic receptor, in a defined anatomical structure with high penetrance. Here, we compare parameters for lentivirus vector injections in the rhesus monkey amygdala. We find that four injections of 20μl, infused at 0.5μl/min, can achieve neuronal hM4Di expression in 50-100% of neurons within a 60mm3 volume, without observable damage from overexpression. Increasing the number of hM4Di_CFP lentivirus injections to up to 12 sites per hemisphere, resulted in 30%-40% neuronal coverage of the overall amygdala volume, with coverage reaching 60% in some subnuclei. Manganese Chloride was mixed with lentivirus and used as an MRI marker to verify targeting accuracy and correct unsuccessful injections in these experiments. In a separate monkey we visualized, in vivo, viral expression of the hM4Di receptor protein in the amygdala, using Positron Emission Tomography. Together, these data show efficient and verifiable expression of a chemogenetic receptor in old-world monkey amygdala.

Correlation of expression of Major Vault Protein with androgen receptor and immune checkpoint protein B7-H3, and with poor prognosis in prostate cancer.

Prostate cancer diagnosis and early stratification is an important aspect to avoid undertreatment of high-risk prostate cancer patients. Major Vault Protein (MVP) has been proposed as a prognostic biomarker in prostate cancer. PTEN and the immune checkpoint protein B7-H3 interact with MVP and are important in prostate cancer progression and therapy response. We evaluated the expression of MVP by immunohistochemistry of tissue microarray samples from a retrospective cohort consisting of 119 prostate cancer patients. We correlated the protein expression of MVP with clinicopathological characteristics, and protein expression of androgen receptor (AR), PTEN, immune checkpoint proteins B7-H3 and PD-L1. We found MVP to be expressed in 53% of prostate tumors, and correlated positively with biochemical recurrence (ρ=0.211/p=0.021). Furthermore, we found positive correlation of MVP expression with expression of AR (ρ=0.244/p=0.009) and the immune checkpoint protein B7-H3 (ρ=0.200/p=0.029), but not with PD-L1 (ρ=0.152/p=0.117) or PTEN expression (ρ=-0.034/p=0.721). Our findings support the notion that expression of MVP is associated with poor prognosis in prostate cancer. The correlation between MVP and immune checkpoint protein B7-H3 in prostate cancer suggests a role for MVP in immunoregulation and drug resistance.

Rhamnetin decreases the expression of HMG-CoA reductase gene and increases LDL receptor in HepG2 cells

Context: Rhamnetin is a naturally occurring methylated derivative of quercetin. This flavonoid is abundant in Syzygium aromaticum, Coriandrum sativum Prunus cerasus, and Rhamnus spp. Aims: To evaluate the effects of rhamnetin on HMG-CoA reductase and low-density lipoprotein receptor (LDLR) gene and protein expressions in the HepG2 hepatoma cell line. Methods: The expression of HMG-CoA reductase and LDLR genes and proteins were studied in HepG2 liver cancer cell line by PCR, Western blot, and indirect ELISA, as well as their antioxidant activity. Results: Rhamnetin was non-toxic up to 200 μM on HepG2 at 24, 48, and 72 h. Rhamnetin (25 µM) upregulated LDLR gene expression by 1.66 folds compared to 3.12 folds exerted by the well-known hypocholesterolemic drug simvastatin. Rhamnetin (100 µM) increased the expression of LDLR protein at the cell membrane, while the other concentrations produced no significant change from the control (vehicle-treated). In HepG2 cell lysate, LDLR was increased by 50 µM of rhamnetin. Also, rhamnetin increased SOD activity significantly by 100.98, 86.28, and 100.98% by the concentrations 25, 50, and 100 µM, respectively. Using the same concentrations, rhamnetin reduced H2O2 levels by 50, 67, and 76.34%, respectively. Conclusions: This study demonstrated for the first time that rhamnetin reduced HMG-CoA reductase gene expression and increased LDLR in HepG2 cells.

The effects of sodium benzoate exposure on learning and neurobehavior during the prepubertal period in rats

Abstract Objectives Exposure to food additives, especially during the critical development period, has been associated with some behavioral disorders and learning difficulties in childhood. In this study, we aimed to evaluate the impact of sodium benzoate (NaB) on learning and neurobehavior in prepubertal rats. Methods A total of male rats (4-week-old) were divided into three groups as the control (CG), experiment 1 (NaB-ADI (acceptable daily intake) dose, Exp1G), and experiment 2 (NaB-NOAEL (no observed adverse effect level) dose, Exp2G) groups and NaB was administered by oral gavage for six weeks. Changes in learning, memory, motor activity and neurobehavior were evaluated by Morris water maze and open field tests. At the end of the experiment, rats’ hippocampi were extracted; NR1, NR2A, NR2B, and nAChRα7 receptor concentrations and mRNA expressions were determined. Results Neurobehavioral tests supported the possibility of increased anxiety and decreased motivation in the Exp1G. Also, molecular tests showed that NR2B receptor protein and mRNA expression levels significantly increased in Exp2G compared to CG. Conclusions Our study showed that NaB administration in the prepubertal period may cause increased anxiety, decreased motivation, and attention with ADI dose. Furthermore, NOAEL dose administration may lead to an increase in NR2B receptor levels. In light of these results, NaB consumption in childhood may affect hippocampal functions related to learning and neurobehavior.

Xiangshao Granules reduce the aggressive behavior and hippocampal injury of premenstrual irritability in rats by regulating JIK/JNK/p38 signal pathway

Ethnopharmacological relevanceAs a typical prescription for soothing the liver, Xiangshao granule has a good effect on the symptoms of irritability and anxiety. Clinical evidence suggests that it has significant efficacy in the treatment of Premenstrual dysphoria disorder (PMDD). However, the underlying mechanism remains unclear. Aim of the studyPMDD is a common disease in women of childbearing age, seriously affecting their family, society, and daily work life. The registered herbal medicine, Xiangshao granules, is used for relieving PMDD dysphoria and irritability symptoms with excellent efficacy in China. This study was focused on the deep intervention mechanism of Xiangshao granules in treating PMDD. Materials and methodsThe vaginal smear and open field test were used to screen rats in nonreception phase of estrus cycle with similar macroscopic behaviors and regular estrus cycle. The rat model of PMDD irritability was established through social isolation and residential invasion, with which, the irritability symptoms of PMDD patients with menstrual cycle dependence was also well simulated. Elevated plus Maze Test and Social interaction activities were used to measure the anxiety-like behavior of rats. TUNEL Staining and Hematoxylin-Eosin staining were used to measure apoptosis of hippocampal neurons. RT-PCR, Western blot and immunofluorescence were used to measure the expression of GR, JIK, p-JIK, p38, P–P38, JNK, caspase 3, and caspase 12. ResultsIn this study, Xiangshao granules showed consistent therapeutic effects similar with those in clinic, significantly reducing aggressive and anxiety-like behaviors with improved social skills in PMDD rats. In mechanism, Xiangshao granules lowered the apoptosis of hippocampal neurons and weakened the morphological damage of the hippocampal brain evidenced by the decreased mRNA and protein expression of glucocorticoid receptor, caspase-3, and caspase-12. In addition, administration of Xiangshao granules led to the decreased expression of JIK in the PMDD irritability rat model which agreed well with the previous studies. The JNK/p38 mitogen-activated protein kinases (MAPKs) signaling pathway is abnormally activated in the hippocampal brain region of PMDD rats, while treated with Xiangshao granules could increase JIK expression and inhibit the abnormal activation of the JNK/p38 MAPK signaling pathway, effectively reducing the stress damage in the hippocampus. ConclusionsXiangshao Granules Reduce the Aggressive Behavior and Hippocampal Injury of Premenstrual Irritability in Rats by Regulating JIK/JNK/p38 Signal Pathway.

Evaluation of toxicity of polystyrene microplastics under realistic exposure levels in human vascular endothelial EA.hy926 cells

Microplastics (MPs) have emerged as a global concern, with a recent study being the first to detect them in the bloodstream of healthy people. However, precise information regarding the toxic effects of MPs on the human vascular system is currently lacking. In this study, we used human vascular endothelial EA. hy926 cells to examine the toxic potential of polystyrene MPs (PSMPs) under realistic blood concentrations. Our findings indicated that PSMPs can cause oxidative stress by reducing the expression of antioxidants, thereby leading to apoptotic cytotoxicity in EA. hy926 cells. Furthermore, the protective potential of heat shock proteins can be reduced by PSMPs. PSMP-induced apoptosis might also lower the expression of rho-associated protein kinase-1 and nuclear factor-κB expression, thus dampening LRR- and pyrin domain-containing protein 3 in EA. hy926 cells. Moreover, we observed that PSMPs induce vascular barrier dysfunction via the depletion of zonula occludens-1 protein. However, although protein expression of the nuclear hormone receptor 77 was inhibited, no significant increase in ectin-like oxidized low-density lipoprotein receptor-1 was noted in PSMP-treated EA. hy926 cells. These results demonstrate that exposure to PSMPs may not sufficiently increase the risk of developing atherosclerosis. Overall, our research signifies that exposure to realistic blood concentrations of PSMPs is associated with low atherosclerotic cardiovascular risk in humans.

Apoptosis Evaluation in Circulating CD34+-Enriched Hematopoietic Stem and Progenitor Cells in Patients with Abnormally Increased Production of Endogenous Glucocorticoids in Course of Cushing's Syndrome.

Abnormalities in hematological parameters of peripheral blood have been noted in patients with endogenous Cushing's Syndrome (CS) in the corticotropin (ACTH)-dependent and ACTH-independent forms. Nevertheless, the exact mechanism of glucocorticoids (GCs) action on human hematopoiesis is still not entirely clear. The aim of the study was to determine whether endogenous excessive production of GCs could affect apoptosis of CD34+ cells enriched in hematopoietic stem and progenitor cells (HSPCs) collected from the peripheral blood of newly diagnosed CS patients. Flow cytometry, Annexin-V enzyme-linked immunosorbent assay, TUNEL assay, real-time quantitative PCR, and microarray RNA/miRNA techniques were used to characterize CS patients' HSPCs. We found that the glucocorticoid receptor (GR) protein expression levels in CS were higher than in healthy controls. A complex analysis of apoptotic status of CS patients' HSPC cells showed that GCs significantly augmented apoptosis in peripheral blood-derived CD34+ cells and results obtained using different methods to detect early and late apoptosis in analyzed cell population were consistent. CS was also associated with significant upregulation in several members of the BCL-2 superfamily and other genes associated with apoptosis control. Furthermore, global gene expression analysis revealed significantly higher expression of genes associated with programmed cell death control in HSPCs from CS patients. These findings suggest that human endogenous GCs have a direct pro-apoptotic activity in hematopoietic CD34+ cells derived from CS subjects before treatment.

Impact of Glyphosate on the Development of Insulin Resistance in Experimental Diabetic Rats: Role of NFκB Signalling Pathways.

Glyphosate, an endocrine disruptor, has an adverse impact on human health through food and also has the potential to produce reactive oxygen species (ROS), which can lead to metabolic diseases. Glyphosate consumption from food has been shown to have a substantial part in insulin resistance, making it a severe concern to those with type 2 diabetes (T2DM). However, minimal evidence exists on how glyphosate impacts insulin-mediated glucose oxidation in the liver. Hence the current study was performed to explore the potential of glyphosate toxicity on insulin signaling in the liver of experimental animals. For 16 weeks, male albino Wistar rats were given 50 mg, 100 mg and 250 mg/kg b. wt. of glyphosate orally. In the current study, glyphosate exposure group was linked to a rise in fasting sugar and insulin as well as a drop in serum testosterone. At the same time, in a dose dependent fashion, glyphosate exposure showed alternations in glucose metabolic enzymes. Glyphosate exposure resulted in a raise in H2O2 formation, LPO and a reduction in antioxidant levels those results in impact on membrane integrity and insulin receptor efficacy in the liver. It also registered a reduced levels of mRNA and protein expression of insulin receptor (IR), glucose transporter-2 (GLUT2) with concomitant increase in the production of proinflammatory factors such as JNK, IKKβ, NFkB, IL-6, IL-1β, and TNF-α as well as transcriptional factors like SREBP1c and PPAR-γ leading to pro-inflammation and cirrhosis in the liver which results in the development of insulin resistance and type 2 diabetes. Our present findings for the first time providing an evidence that exposure of glyphosate develops insulin resistance and type 2 diabetes by aggravating NFkB signaling pathway in liver.

Delivery of Melittin as a Lytic Agent via Graphene Nanoparticles as Carriers to Breast Cancer Cells.

Melittin, as an agent to lyse biological membranes, may be a promising therapeutic agent in the treatment of cancer. However, because of its nonspecific actions, there is a need to use a delivery method. The conducted research determined whether carbon nanoparticles, such as graphene and graphene oxide, could be carriers for melittin to breast cancer cells. The studies included the analysis of intracellular pH, the potential of cell membranes, the type of cellular transport, and the expression of receptor proteins. By measuring the particle size, zeta potential, and FT-IT analysis, we found that the investigated nanoparticles are connected by electrostatic interactions. The level of melittin encapsulation with graphene was 86%, while with graphene oxide it was 78%. A decrease in pHi was observed for all cell lines after administration of melittin and its complex with graphene. The decrease in membrane polarization was demonstrated for all lines treated with melittin and its complex with graphene and after exposure to the complex of melittin with graphene oxide for the MDA-MB-231 and HFFF2 lines. The results showed that the investigated melittin complexes and the melittin itself act differently on different cell lines (MDA-MB-231 and MCF-7). It has been shown that in MDA-MD-231 cells, melittin in a complex with graphene is transported to cells via caveolin-dependent endocytosis. On the other hand, the melittin-graphene oxide complex can reach breast cancer cells through various types of transport. Other differences in protein expression changes were also observed for tumor lines after exposure to melittin and complexes.

Postnatal developmental expression and localization of apelin and apelin receptor protein in the ovary and uterus of mice.

Postnatal ovarian and uterine development is crucial to accomplished female fertility. Thus, the investigations of factors that present in pre-pubertal stages are important as it might be responsible for the regulation of ovarian and uterine function. Apelin, an adipokine and its receptor (APJ) are present in female reproductive organs. However, no study has reported its postnatal expression in uterus and ovary. Thus, we investigated the postnatal developmental changes in expression and localization of apelin and APJ in the ovary and uterus of mice. Postnatal ovary and uterus were collected from postnatal day (PND) 1, 7, 14, 21, 42, 65 and performed western blot analysis and immunohistochemistry. Uterine APJ is elevated in PND14 and PND65, whereas, ovarian APJ elevated in PND7, PND14, and PND65. Apelin expression in both ovary and uterus showed intense staining at PND65 and PND14. Our results showed that apelin and APJ abundance was lower at PND21 in uterus and ovary. In conclusion, apelin and APJ are developmentally regulated in the ovary and uterus, and its localization in the different compartments of ovary and uterus suggest its distribution specific physiological role in the uterus and ovary.