Rat Cells Research Articles

Maternal protein restriction affects the differentiation of cells in the epididymal epithelium lining of 44-day-old rats.

Maternal protein restriction delays the differentiation of epididymal mesenchymal cells in newborn rats. However, it's unclear if this delay persists until the full differentiation of the epididymal epithelium at 44 days postnatal. Thus, this study aimed to assess the impact of maternal protein reduction on 44-day-old rats' epididymal epithelium differentiation, following up on the observed delay in newborn animals. Pregnant rats were randomly divided into groups receiving normal-protein (NP - 17% protein) or low-protein (LP - 6% protein) diets during gestation and lactation. On postnatal day (PDN) 44, male offspring were euthanized, and the epididymis (NP n=10, LP n=10) was processed according to immunohistochemical techniques for the detection of aquaporin 9 (AQP9), KI-67, TP63, and ATPase. LP rats showed: a decrease in the intensity of the AQP9 reaction, an increase in cellular proliferation in the initial segment and corpus of the epididymis, an increase in basal cells in the caput and corpus epididymis, and an increase in ATPase-positive clear cells in the cauda region. These findings demonstrate that maternal protein restriction impacts cell differentiation in the epididymal epithelium of 44-day-old rats, persisting even with a normal-protein diet after weaning.

A highly sensitive and specific Homo1-based real-time qPCR method for quantification of human umbilical cord mesenchymal stem cells in rats.

Owing to the characteristics of easier access in vitro, low immunogenicity, and high plasticity, human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are considered as a promising cell-based drugs for clinical application. No internationally recognized technology exists to evaluate the pharmacokinetics and distribution of cell-based drugs in vivo. We determined the human-specific gene sequence, Homo1, from differential fragments Homo sapiens mitochondrion and Rattus norvegicus mitochondrion. The expression of Homo1 was utilized to determine the distribution of UC-MSCs in the normal and diabetic nephropathy (DN) rats. We observed a significant correlation between the number of UC-MSCs and the expression level of Homo1. Following intravenous transplantation, the blood levels of UC-MSCs peaked at 30 min. A large amount of intravenously injected MSCs were trapped in the lungs, but the number of them decreased rapidly after 24 h. Additionally, the distribution of UC-MSCs in the kidneys of DN rats was significantly higher than that of normal rats. In this study, we establish a highly sensitive and specific Homo1-based real-time quantitative PCR method to quantify the distribution of human UC-MSCs in rats. The method provides guidelines for the safety research of cells in preclinical stages.

Evaluation of Proliferative Potential of Musa Paradisiaca Stem Juice on the Pancreatic Cells of Wistar Rats

Evaluation of Proliferative Potential of Musa Paradisiaca Stem Juice on the Pancreatic Cells of Wistar Rats

Antitumor Activity of a Lectin Purified from Punica granatum Pulps against Ehrlich Ascites Carcinoma (EAC) Cells.

Lectins are carbohydrate-binding proteins with various pharmacological activities, such as antimicrobial, antidiabetic, antioxidant, and anticancer. Punica granatum fruit extract has traditional uses, however, the anti-cancer activity of purified lectin isolated from P. granatum pulp is yet to be reported. The goals of this study are purification, characterization of the lectin from P. granatum, and examination of the purified lectin's anticancer potential. Diethylaminoethyl (DEAE) ion-exchange chromatography was used to purify the lectin, and SDSPAGE was used to check the purity and homogeneity of the lectin. Spectrometric and chemical analysis were used to characterize the lectin. The anticancer activity of the lectin was examined using in vivo and in vitro functional assays. A lectin, designated as PgL of 28.0 ± 1.0 kDa molecular mass, was isolated and purified from the pulps of P. granatum and the lectin contains 40% sugar. Also, it is a bivalent ion-dependent lectin and lost its 75% activity in the presence of urea (8M). The lectin agglutinated blood cells of humans and rats, and sugar molecules such as 4-nitrophenyl-α-D-manopyranoside and 2- nitrophenyl -β- D-glucopyranoside inhibited PgL's hemagglutination activity. At pH ranges of 6.0-8.0 and temperature ranges of 30°C -80°C, PgL exhibited the highest agglutination activity. In vitro MTT assay showed that PgL inhibited Ehrlich ascites carcinoma (EAC) cell growth in a dose-dependent manner. PgL exhibited 39 % and 58.52 % growth inhibition of EAC cells in the mice model at 1.5 and 3.0 mg/kg/day (i.p.), respectively. In addition, PgL significantly increased the survival time (32.0 % and 49.3 %) of EAC-bearing mice at 1.5 and 3.0 mg/kg/day doses (i.p.), respectively, in comparison to untreated EAC-bearing animals (p < 0.01). Also, PgL reduced the tumor weight of EAC-bearing mice (66.6 versus 39.13%; p < 0.01) at the dose of 3.0 mg/kg/day treatment. Furthermore, supplementation of PgL restored the haematological parameters toward normal levels deteriorated in EAC-bearing animals by the toxicity of EAC cells. The results indicated that the purified lectin has anticancer activity and has the potential to be developed as an effective chemotherapy agent.

Correction of mitochondrial dysfunction with trimethoxy-substituted monocarbonyl curcumin analogues in experimental Alzheimer’s disease

Alzheimer’s disease (AD) is a neurodegenerative disease that is a terminal form of dementia with an alarming spread rate. The treatment of AD usually involves symptomatic therapy, but the research field for new medicines to correct AD focus on the pathogenetic keys of the disease, i.e., a mitochondrial dysfunction.The aim of the work was to evaluate the effect of trimethoxy-substituted monocarbonyl curcumin analogues on changes in the mitochondrial function of the hippocampus in AD rats.Materials and methods. AD was modeled in female Wistar rats by the injection of β-amyloid aggregates 1-42 into the CA1 part of the hippocampus. The tested compounds AZBAX4 and AZBAX6 at a dose of 20 mg/kg each, as well as the reference donepezil at a dose of 50 mg/kg, were administered orally for 30 days after the surgery. After the specified time had passed, the changes in the cellular respiration, a citrate synthase activity, cytochrome-c-oxidase, succinate dehydrogenase, and adenosine triphosphate (ATP) concentrations were evaluated in the mitochondrial fraction of the rat hippocampus.Results. During the study, it was shown that the use of AZBAX4 and AZBAX6 compounds contributed to an increase in the intensity of aerobic metabolism by 83.9 (p &lt;0.05) and 35.9% (p &lt;0.05), respectively, while reducing the activity of anaerobic one by 27.7 (p &lt;0.05) and 20.6% (p &lt;0.05), respectively. Against the background of the tested compounds AZBAX4 and AZBAX6 administration, there was also a significant increase in the activity of citrate synthase, succinate dehydrogenase and cytochrome-c-oxidase, as well as the level of ATP in the hippocampal tissue by 112.8 (p &lt;0.05) and 117.1% (p &lt;0.05), respectively. The use of donepezil led to a significant increase in the intensity of aerobic reactions – by 24.0% (p &lt;0.05), a citrate synthase activity– by 80.0% (p &lt;0.05) and the ATP concentration – by 68.5% (p &lt;0.05). Against the background of the use of the analyzed substances, a decrease in the apoptosis-inducing factor and mitochondrial hydrogen peroxide is also worth noting.Conclusion. Based on the obtained data, it can be assumed that the use of AZBAX4 and AZBAX6 compounds contributes to an increase in the functional activity of the mitochondria of hippocampal cells of AD rats, while surpassing the reference donepezil. It is perspective to continue a further study of AZBAX4 and AZBAX6 compounds as possible medicines of a pathogenetic correction of AD.

Suppression of Mast Cell Activation by GPR35: GPR35 Is a Primary Target of Disodium Cromoglycate.

Mast cell stabilizers, including disodium cromoglycate (DSCG), were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists, including DSCG and newly designed compounds, suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly downmodulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice but not in the GPR35-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. SIGNIFICANCE STATEMENT: The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their antiallergic effects were abrogated in the GPR35-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.

Synergistic Effect of a Flavonoid-Rich Cocoa-Carob Blend and Metformin in Preserving Pancreatic Beta Cells in Zucker Diabetic Fatty Rats.

The loss of functional beta-cell mass in diabetes is directly linked to the development of diabetic complications. Although dietary flavonoids have demonstrated antidiabetic properties, their potential effects on pancreatic beta-cell preservation and their synergistic benefits with antidiabetic drugs remain underexplored. We have developed a potential functional food enriched in flavonoids by combining cocoa powder and carob flour (CCB), which has shown antidiabetic effects. Here, we investigated the ability of the CCB, alone or in combination with metformin, to preserve pancreatic beta cells in an established diabetic context and their potential synergistic effect. Zucker diabetic fatty rats (ZDF) were fed a CCB-rich diet or a control diet, with or without metformin, for 12 weeks. Markers of pancreatic oxidative stress and inflammation, as well as relative beta-cell mass and beta-cell apoptosis, were analyzed. Results demonstrated that CCB feeding counteracted pancreatic oxidative stress by enhancing the antioxidant defense and reducing reactive oxygen species. Moreover, the CCB suppressed islet inflammation by preventing macrophage infiltration into islets and overproduction of pro-inflammatory cytokines, along with the inactivation of nuclear factor kappa B (NFκB). As a result, the CCB supplementation prevented beta-cell apoptosis and the loss of beta cells in ZDF diabetic animals. The observed additive effect when combining the CCB with metformin underscores its potential as an adjuvant therapy to delay the progression of type 2 diabetes.

Alteration of zeta potential and cell viability in rat-derived L6 skeletal muscle cells and H9c2 cardiomyocytes: A study with submicron polystyrene particles.

Microand nanoplastics pollution can cause substantial damage to ecosystems. Since scientists have focused mainly on their impact on aquatic environments, less attention has been paid to the accumulation of polymer particles in terrestrial organisms. We checked if submicron (<5 mm) polystyrene (PS) particles, which can accumulate in living organisms, lead to changes in the physicochemical properties of mammalian cell membranes. The influence of submicron PS particles on the properties of rat-derived L6 myocytes and H9c2 cardiomyocytes was analyzed. Non-functionalized and amine-functionalized PS particles of 100 nm and 200 nm in diameter were used. The MTT assay was performed to evaluate the viability of the polymers-treated cells. The effect of short (6 h) and prolonged (48 h) incubation with different concentrations of PS particles on the cell's zeta (ζ) potential was examined with the electrophoretic light scattering technique (ELS). Polystyrene particles' physicochemical characteristics (size and stability) were performed using dynamic light scattering (DLS) and electrophoretic light scattering methods. The results show that submicron PS particles affect cell viability and cause changes in the physiochemical parameters of rat cell membranes. Differences were observed depending on the origin of the cells. We observed doseand time-dependent alterations in the studied parameters after submicron PS particle incubation in L6 myotubes and H9c2 cardiomyocytes. The size and modification of PS particle surfaces determine the extent to which they affect the analyzed properties of rat cardiomyocytes and myocytes membranes.

Slightly acidic electrolyzed water inhibits inflammation induced by membrane vesicles of Staphylococcus aureus.

Staphylococcus aureus grows in the skin of patients with atopic dermatitis and the associated symptoms are induced by membrane vesicles (MVs). This study explored the effects of slightly acidic electrolyzed water (SAEW) on the expression of virulence factors of S. aureus and MV-induced inflammation to uncover the potential of SAEW as a new treatment method for atopic dermatitis. Expression levels of genes related to virulence factors in S. aureus was assessed and S. aureus-derived MVs were characterized. Moreover, expression level of MV-induced Type I allergic reaction-related genes in RBL2H3 cells was also assessed. Significantly decreased staphylococcal enterotoxin A production and decreased virulence factor-related gene expression were observed after culturing S. aureus in broth supplemented with SAEW at ratios of 1, 2, and 5 per broth. MVs prepared by culturing S. aureus in SAEW-supplemented broth exhibited altered particle size and markedly reduced staphylococcal enterotoxin A content under all addition conditions; moreover, those obtained at a ratio of 1:5 (broth:SAEW) exhibited a reduction in the expression of several proteins associated with hemolytic activity and free iron uptake. The MVs prepared in SAEW-supplemented broth also exhibited remarkably reduced allergy-related gene expression levels in rat cell lines derived from basophilic leukemia-2H3 cells. Overall, SAEW is expected to suppress atopic dermatitis symptoms through the alteration of the properties of S. aureus-derived MVs.

Nrf-2 modulates excitability of hippocampal neurons by regulating ferroptosis and neuroinflammation after subarachnoid hemorrhage in rats

Excitability of hippocampal neurons in subarachnoid hemorrhage (SAH) rats has not been well studied. The rat SAH model was applied in this study to explore the role of nuclear factor E2-related factor (Nrf-2) in the early brain injury of SAH. The neural excitability of CA1 pyramidal cells (PCs) in SAH rats was evaluated by using electrophysiology experiments. Ferroptosis and neuroinflammation were measured by ELISA, transmission electron microscopy and western blotting. Our results indicated that SAH induced neurological deficits, brain edema, ferroptosis, neuroinflammation and neural excitability in rats. Ferrostatin-1 treatment significantly decreased the expression and distribution of IL-1β, IL-6, IL-10, TGF-β and TNF-α. Inhibiting ferroptosis by ferrostatin-1 can attenuate neural excitability, neurological deficits, brain edema and neuroinflammation in SAH rats. Inhibiting the expression of Nrf-2 significantly increased the neural excitability and the levels of IL-1β, IL-6, IL-10, TGF-β and TNF-α in Fer-1-treated SAH rats. Taken together, inhibiting the Nrf-2 induces early brain injury, brain edema and the inflammatory response with increasing of neural excitability in Fer-1-treated SAH rats. These results have indicated that inhibiting ferroptosis, neuroinflammation and neural excitability attenuates early brain injury after SAH by regulating the Nrf-2.

Artemisinin Attenuates Isoproterenol-induced Cardiac Hypertrophy via the ERK1/2 and p38 MAPK Signaling Pathways.

Artemisinin (ART) is mainly derived from Artemisia annua, a traditional Chinese medicinal plant, and has been found to affect cellular biochemical processes, such as proliferation, angiogenesis, and apoptosis, in addition to its antimalarial properties. However, its effect on cardiac hypertrophy and the underlying mechanisms remain unclear. This study aimed to investigate the effect of ART on cardiac hypertrophy and explore its possible mechanisms. A rat model was established by intraperitoneal injection of isoproterenol (ISO) for 3 days, and the degree of myocardial hypertrophy was compared among 5 groups: a control (CON) group, an ISO group, and groups treated with different doses of ART (7 mg/kg/d, 35 mg/kg/d, and 75 mg/kg/d). Echocardiography was used to evaluate cardiac function and structure. The cross-sectional area of cardiomyocytes was measured by hematoxylin and eosin (H&E) staining. The heart weight (HW), body weight (BW), and tail length were measured, and the HW/tail length ratio and the HW/BW ratio were calculated. H9c2 rat cardiomyocytes were cultured, and different amounts of ART were added 2 hours before ISO stimulation. Phalloidin staining was used to evaluate the degree of cell hypertrophy. The levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were quantified in rat plasma and cell supernatant using enzyme-linked immunosorbent assay (ELISA), while the expression levels of p- ERK1/2, p-JNK, and p-p38 MAPK were assessed in the myocardium and H9c2 cells via western blot analysis. Intragastric administration of ART at a dosage of 35 mg/kg/d or over mitigated the early-stage cardiac hypertrophy induced by ISO in rats led to a reduction in left ventricular posterior wall diastolic thickness, interventricular septal thickness at diastole, lowered ANP and BNP levels, as well as a decrease in HW/tail length and HW/BW ratio. In vitro studies demonstrated that ART at a concentration of 100 μM inhibited ISO-mediated hypertrophy of H9c2 cells. The ISO group showed a higher p-ERK/GAPDH ratio and p-p38 MAPK/GAPDH ratio than the control group both in vivo and in vitro. Although the p-JNK/GAPDH ratio was increased in the ISO group, there was no statistical difference. The p-ERK/GAPDH and p-p38/GAPDH ratios were significantly lower in the ART group than in the ISO group. The mechanism of ART against cardiac hypertrophy was related to inhibition of the ERK1/2 and p38 MAPK signaling pathways.

Efficient Delivery of Recombinant Human Bone Morphogenetic Protein (Rhbmp-2) With Cockle Shell Derived Calcium Carbonate Nanoparticles (CaCO3NPs)

Bone morphogenetic protein-2 (BMP-2) has a significant function in the formation of cartilage and bones. Notably, dosing of only BMP-2 protein intravenously is ineffective. Persistent transportation of the stabilized BMP-2 through a carrier has been seen to be essential for enhancing the osteogenesis im pact of BMP-2. The current research built a new system of drug delivery by utilising cockle shell derived calcium carbonate nanoparticles (CaCO3NPs) and studied the efficacy of the delivery system on the recombinant human bone morphogenetic protein (rhBMP-2). rhBMP-2-CaCO3NPs nanoparticles were synthesised by means of a modest precipitation procedure along with mechanical grinding. Fourier-tran sform infrared spectroscopy, UV–Vis spectrophotometer, scanning electron microscope, X-ray powder diffraction, transmission electron microscope, and zeta potential were u tilised for characterising the conjugated rhBMP-2-CaCO3NPs . Cytotoxicity of rhBMP-2, CaCO3NPs and rhBMP-2-CaCO 3NPs was studied by utilising methylthiazol tetrazolium assay against fibroblast (Rat-1) cells in comparison to rhBMP-2 and CaCO3NPs. The outcomes signified bio-stability of CaCO3NPs and lower toxicity for Rat-1 cells. In summary, CaCO3NPs were prepared by a simp le precipitation process. The ensuing nanoparticles could competently entrap rhBMP-2 and generated stable rhBMP-2-CaCO3NPs. A sustained discharge of rhBMP-2 from t he CaCO3NPs was seen. CaCO3NPs loaded with r hBMP-2 demonstrated reasonable bio-compatibility. The outcomes indicated that CaCO3NPs may have significant ability as carrier of therapeutic proteins within bone tissue en gineering.

Histopathology and ultrastructural alterations in gastric mucus-secreting cells in diabetic model rats

Diabetes mellitus (DM) can cause gastric ulcers (GU), duodenal ulcers (DU), and gastroesophageal reflux disease (GERD). Mucus-secreting cells secrete mucus, which aids in the neutralization of HCl and inhibits bacteria. DM can alter mucus-secreting cells. Due to a lack of mucosal defense, external stimuli such as bacteria or ethanol can lead to the development of GU, DU, and GERD. This research study used a STZ-induced diabetic rat model to examine the short- and long-term histopathology and ultrastructural alterations in mucus-secreting cells in the cardia, body, and pyloric regions of the stomach. Quantitative analysis was also employed in this study to examine the distribution of mucin granules across all three locations. Twenty-four male adult Sprague-Dawley rats were utilized. Rats were divided into the control (n = 12) and DM (n = 12) groups. Each was separated into short-term (4 weeks) and long-term (24 weeks) rats. For DM induction, streptozotocin (STZ) can selectively destroy the beta cells of the pancreas. The DM was injected with STZ in citrate buffer at 60 mg/kg body weight. The control group was injected with citrate buffer. Histopathology was examined by Alcian blue-Periodic Acid Schiff staining under a light microscope. Image analysis was applied to quantify mucin accumulation. The ultrastructure was explored using transmission electron microscopy. In short-term and long-term DM, there was superficial erosion of the gastric epithelium and a significant decrease in the percentage of mucin granule accumulations in both surface mucous cells (SMCs) and mucous neck cells (MNCs). In short-term DM, SMCs were degenerated with vacuolation, disrupted cristae of mitochondria, and dilated rough endoplasmic reticulum (rER). MNCs were swollen with destroyed organelles. In long-term DM, degenerative nuclei and electron-lucent regions with unidentified structures of SMCs were observed. Nuclear chromatin condensation and the disappearance of mucin granules were present in MNCs. In conclusion, under both LM and TEM, STZ-induced diabetic rats demonstrated both short- and long-term damage to the gastric mucosa and gastric gland structures.

Tempol a superoxide dismutase mimic reduces cardiac muscle cell death through the control of oxidative stress in streptozotocin-induced diabetes

ABSTRACT Diabetes mellitus (DM) a metabolic disorder resulting in myocardial injury and diabetic cardiomyopathy (DCM) in association with oxidative stress induction. This study was therefore, investigated the potential protective effect of Tempol (TPL) on oxidative cardiac damage in experimental diabetic rats. Rats were assigned to three equal groups: group (NC); normal control rats received citrate buffer injections. Group (DC): diabetic control rats received a single i.p. STZ injection (60 mg/kg body weight), and Group (D/TPL) diabetic rats treated with TPL (10 mg/kg b.w; by i.p. injection) for four weeks. Our results showed a significant increment in plasma creatine phosphokinase (CPK), lactate dehydrogenase (LDH) activities, cholesterol, triglycerides, Nitric oxide (NO), and lipid peroxidation (LPO), In addition, GSH content and antioxidant enzyme activity were significantly deceased in DC group compared to normal control. While TPL treatment significantly ameliorated the observed biochemical abnormalities. Further, TPL reduced number of dead myocardial cells in diabetic-treated rats and, remarkably improved the histopathological alterations of the aorta. The results proved the protective effect of tempol on STZ-induced diabetic cardiomyopathy by reducing myocardial cell death through reducing oxidative stress and inhibition of lipid peroxidation.

Retracted: Protective Mechanism of Trimetazidine in Myocardial Cells in Myocardial Infarction Rats through ERK Signaling Pathway.

[This retracts the article DOI: 10.1155/2021/9924549.].

First Evidence of Mineralocorticoid Receptor Gene and Protein Expression in Rat and Human Thyroid Tissues and Cell Cultures.

Aldosterone (Aldo) exerts its action through binding with the mineralocorticoid receptor (MR). Clinically, a link between primary aldosteronism (PA) and thyroid diseases has been hypothesised. However, the presence and activity of MR on the thyroid have not yet been demonstrated. We investigated the gene/protein expression and activation of MR in primary thyroid cell cultures (normal rat thyroid [FRTL-5] and human papillary thyroid cancer [PTC] cell lines, BCPAP and K1) through qRT-PCR analysis, immunofluorescence, and confocal microscopy. We also studied the effects of Aldo on thyroid-specific and inflammation genes in vitro. Paired human normal and neoplastic thyroid tissues were also studied. We demonstrated both gene and protein expression and activation of MR in normal rat thyroid and human PTC lines. Incubation with Aldo induced an acute increase in IL-6 expression in both the FRTL-5 and BCPAP lines, which was antagonised by spironolactone, and an acute and late upregulation of thyroid-specific genes in FRTL-5. MR was also expressed at both gene and protein levels in normal human thyroid tissues and in PTC, with a progressive decline during neoplastic tumourigenesis, particularly in more aggressive histotypes. We present the first evidence of MR gene and protein expression in both normal and pathological thyroid cells and tissues. We have shown that MR is present and functionally activated in thyroid tissue. Binding of Aldo to MR induces the expression of inflammatory and thyroid-specific genes, and the thyroid may thus be considered a novel mineralocorticoid target tissue.

Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

Atorvastatin increases autophagic flux and p62/SQSTM1 of kidney cells in hyperglycemic conditions and treatment in combination with insulin improves renal function of streptozotocin (STZ)-induced diabetic rats

Background: Although atorvastatin is commonly used as a hypolipidemic agent, it confers many health benefits in which the underlying mechanisms are not fully understood. We have previously shown that combined treatment of atorvastatin and insulin effectively restored renal function of streptozotocin (STZ)-induced diabetic rats; nevertheless, the underlying mechanism was not known. Objective: To determine whether the reno-protective effect of atorvastatin and insulin is mediated through its impact on autophagy. Materials and methods: Markers of autophagy, LC3, and p62/SQSTM1, in rat kidney tissues and cell lines treated with atorvastatin and/or insulin were determined by Western blot analysis. Results: Levels of both LC3-I and LC3-II proteins in kidney tissues of STZ-diabetic rats treated with atorvastatin and insulin were significantly increased. The autophagic flux was examined in vitro and showed that high glucose culture conditions suppressed the autophagic flux in kidney cells. Treatment with insulin moderately increased the conversion of LC3-I to LC3-II. Interestingly, atorvastatin increased autophagic flux only in the hyperglycemic but not in the normoglycemic condition. p62/SQSTM1 protein level was decreased in response to high glucose treatment but increased with the addition of insulin and/or atorvastatin. Conclusion: This study has demonstrated that atorvastatin may represent a novel regimen in providing prevention and protection for diabetic nephropathy through the underlying mechanisms of inducing autophagy and p62/SQSTM1.

Gossypol Acetic Acid alleviates the Ferroptosis of Chondrocytes in Osteoarthritis by Inhibiting GPX4 Methylation.

Osteoarthritis (OA) is a chronic joint disease, usually accompanied by degeneration of the articular cartilage, fibrosis, bone hyperplasia around the joint, and damage to the entire articular surface. Gossypol is a natural phenolic compound isolated from the seed of cotton plants, and gossypol acetic acid (GAA) is a medicinal form of Gossypol. Recently, various biological activities of GAA, including anti-inflammatory and anti-tumor effects, have been widely reported. However, its effect on chondrocytes in OA has yet to be determined. In this study, we investigated the effect of GAA on ferroptosis in OA chondrocytes. The effect of GAA on the cell viability and cytotoxicity of chondrocytes in rat cells was investigated using CCK8. Western blotting, Reverse-transcription PCR (RT-PCR), and immunofluorescence staining were used to elucidate the molecular mechanisms and signaling pathways of GAA inhibition of ferroptosis in OA chondrocytes. The effect of GAA on reactive oxygen species (ROS) production and lipid peroxidation levels in chondrocytes was examined using dihydroethidium (DHE) staining and fluorescent dye BODIPY581/591 C11. in vivo, micro-CT imaging, hematoxylin and eosin staining, Safranin O-Fast staining, and immunohistochemistry were performed to evaluate the effects of GAA on OA cartilage. The results showed that GAA treatment regulated the expression of chondrocyte extracellular matrix (ECM) related factors, including ADAMTS5, MMP13, SOX9, Aggrecan, and COL1A2 and reduced the ROS and lipid peroxidation levels. Besides, Erastin could reverse the effects of GAA on chondrocytes. Similar to GAA, 5-AZA caused the reduction of ROS and lipid peroxidation levels and reversed the effect of IL-1β on the expression of ECM-related factors in OA chondrocytes. The above results clarified that GAA alleviated the ferroptosis of chondrocytes in OA by inhibiting GPX4 methylation. Our findings revealed that GAA might be developed as a drug for treating OA clinically.

Biodegradable microspheres via orally deliver celastrol with ameliorated neuropathic pain in diabetes rats.

The treatment of peripheral neuropathy resulting from diabetes primarily emphasizes neurotrophic medications. However, a growing body of clinical studies indicates that neuroinflammation plays a significant role in the pathogenesis of neuropathic pain. This has spurred active exploration of treatment strategies leveraging nanomedicine for diseases, aiming for superior therapeutic outcomes. In this context, we have developed biodegradable nanoparticles made of polylactic-co-glycolic acid, loaded with triptolide (pCel), designed to alleviate somatic cell neuropathic pain induced by diabetes. Treatment with pCel notably reduced levels of reactive oxygen species and apoptosis in vitro. Furthermore, the progression of streptozotocin-induced diabetes, characterized by elevated renal function indices (blood urea nitrogen, creatinine), liver function indices (bilirubin, alkaline phosphatase) and decreased levels of albumin and globulin, was mitigated following pCel administration. Importantly, oral treatment with pCel significantly inhibited mechanical allodynia and the activation of the sciatic glial cells in diabetic rats. These findings indicate that this synthetic, biodegradable nanomedicine exhibits excellent stability, biocompatibility and catalytic activity, making it a promising and innovative approach for the management of chronic pain conditions associated with diabetic neuropathy.