Rat Cells Research Articles

Investigating the biochemical response of proton minibeam radiation therapy by means of synchrotron-based infrared microspectroscopy

The biology underlying proton minibeam radiation therapy (pMBRT) is not fully understood. Here we aim to elucidate the biological effects of pMBRT using Fourier Transform Infrared Microspectroscopy (FTIRM). In vitro (CTX-TNA2 astrocytes and F98 glioma rat cell lines) and in vivo (healthy and F98-bearing Fischer rats) irradiations were conducted, with conventional proton radiotherapy and pMBRT. FTIRM measurements were performed at ALBA Synchrotron, and multivariate data analysis methods were employed to assess spectral differences between irradiation configurations and doses. For astrocytes, the spectral regions related to proteins and nucleic acids were highly affected by conventional irradiations and the high-dose regions of pMBRT, suggesting important modifications on these biomolecules. For glioma, pMBRT had a great effect on the nucleic acids and carbohydrates. In animals, conventional radiotherapy had a remarkable impact on the proteins and nucleic acids of healthy rats; analysis of tumour regions in glioma-bearing rats suggested major nucleic acid modifications due to pMBRT.

Balancing Risks versus Benefits: Vitamin C Therapy versus Copper Oxide Nanoparticles Toxicity in Albino Rats' Submandibular Salivary Gland.

This study aimed to examine the suppressive effect of the natural antioxidant vitamin C (VC) against submandibular gland toxicity induced by copper oxide nanoparticles (CuO-NPs). Three groups of 30 mature male albino rats (4 weeks old) weighing between 150 and 200 g were selected. The rats were randomly assigned for 6 weeks to receive: intraperitoneal injection (IP) of vehicle (control group); IP of 2.5 mg/kg body weight (bw) of CuO-NPs (CuO-NPs group); and IP of 2.5 mg/kg bw of CuO-NPs, combined with a daily oral dose of 100 mg/kg bw of VC in drinking water via gavage (CuO-NPs/VC group). The rats were euthanized, and their submandibular glands were dissected for histological evaluation, including hematoxylin and eosin staining and immunohistochemistry for Ki-67 and caspase-3. The area expression for Ki-67 and caspase-3 was statistically analyzed using GraphPad Prism. Following analysis of variance analysis, Tukey's post hoc was used for multiple comparisons. The significance level was set at p < 0.05. CuO-NPs caused significant cytotoxic effects on submandibular salivary gland cells in albino rats. This led to an increase in Ki-67 and caspase-3 levels compared with the control group. VC administration improved tissue histology and reduced Ki-67 and caspase-3 levels in the VC/CuO-NPs group compared with rats treated with CuO-NPs alone. The study revealed significant cytotoxic effects of CuO-NPs on the submandibular salivary gland of albino rats. VC effectively mitigated these toxic effects, suggesting its potential as a readily available antioxidant.

Comparative Analysis of Epigallocatechin-3-Gallate and TNF-Alpha Inhibitors in Mitigating Cisplatin-Induced Pancreatic Damage Through Oxidative Stress and Apoptosis Pathways

Oxidative stress and inflammation caused by cisplatin, which is frequently used in the treatment of many cancers, damage healthy tissues as well as cancer cells. In this study, we aimed to investigate the effect of epigallocatechin-3-gallate (EGCG) and infliximab (INF) administration on pancreatic endocrine cells in rats treated with systemic cisplatin (CDDP). The rats were randomly divided into 6 groups: group 1 (control group), group 2 (EGCG group), group 3 (CDDP group), group 4 (EGCG + CDDP group), group 5 (CDDP + INF group), and group 6 (EGCG + CDDP + INF group). The study’s findings demonstrated that EGCG and INF effectively reduced the cellular damage induced by CDDP in histopathologic investigations of the pancreas. EGCG and INF, whether used individually or in combination, demonstrated a significant reduction in malondialdehyde (MDA) levels and an increase in glutathione (GSH) levels in the rat pancreas compared to the CDDP group. Immunohistochemically, the enhanced presence of insulin and glucagon positivity in the EGCG and INF groups, along with the absence of TUNEL immunopositivity, indicate that both treatments reduced CDDP-induced apoptosis. Furthermore, the observed lack of immunopositivity in TNF-α and 8-OHdG in the groups treated with EGCG and INF, compared to those treated with CDDP, indicates that these substances can inhibit inflammation. EGCG and INF, whether provided alone or together, can potentially reduce the damage caused to pancreatic islet cells by cisplatin. This effect is achieved through their anti-inflammatory and antioxidant properties during the early stages of the condition.

The effect of the AIM2 inflammasome in noise-induced cognitive dysfunction in rats

Objective: To explore the effect of the absent in melanoma 2 (AIM2) -mediated neuroinflammation in noise-induced cognitive dysfunction in rats. Methods: In April 2023, sixteen male Wistar rats were randomly divided into control group and noise group, with 8 rats in each group. The rats in the noise group were placed in 50 cm×50 cm×40 cm transparent boxes and exposed to 100 dB (A) white noise with a sound pressure level of 100 dB (A) (4 h/d for 30 d) . At the same time, rats in the control group were kept in similar boxes with environmental noise less than 60 dB (A) . After 30 days of noise exposure, the Morris water maze experiment was applied to test the learning and memory abilities of the rats; the pathological morphology of hippocampal tissues was observed by Hematoxylin-Eosin (HE) staining. Western blot was used to detect the protein expression levels of AIM2, cysteinyl aspartate specific proteinase-1 (caspase-1) , apoptosis-associated speck-like protein (ASC) , interleukin-1β (IL-1β) , IL-18, ionic calcium-binding articulation molecule-1 (Iba-1) , and glial fibrillary acidic protein (GFAP) . The expression of both Iba-1 and GFAP in hippocampal tissue was assessed by immunohistochemical staining. The co-localization of AIM2 with Iba-1 or GFAP was determined by immunofluorescence double staining. Results: Compared with the control group, the escape latency of rats in the noise group was increased by 16.29 s, 17.71 s, and 20.26 s on days 3, 4, and 5, respectively. On day 6, the noise-exposed rats spent shorter time in the target quadrant and had fewer times in crossing the platform[ (7.25±2.27) s and (1.13±0.64) times] than the control group[ (15.64±3.99) s and (4.25±2.12) times] (P<0.05) . After noise exposure, hippocampal neurons of rats displayed marked nuclear hyperchromatic and pyknosis phenomenon. The noise-exposed rats had higher numbers of both microglia and astrocytes (27.00±2.65 and 43.33±5.51) in the DG area of the hippocampus relative to the control group (14.67±3.06 and 20.00±4.58) (P<0.05) . Moreover, the glial cells in the noise group had larger cell cytosol with more and thicker branches. The protein expression levels of inflammatory cytokines Cleaved-IL-1β and Cleaved-IL-18 in the hippocampus of rats in the noise group (1.55±0.19 and 1.74±0.12) were significantly higher than the control group (1.00±0.11 and 1.00±0.13) (P<0.05) . After noise exposure, the protein expression levels of AIM2, Cleaved-Caspase-1 and ASC (1.19±0.09, 1.34±0.07 and 1.14±0.01) were higher than the control group (1.00±0.07, 1.00±0.14 and 1.00±0.06) and differences between the two groups were statistically significant (P<0.05) . A significant increase in the number of cells co-localizing AIM2 with Iba-1 or GFAP in the noise group (28.67±4.04 and 40.67±5.13) compared with the control group (15.67±4.04 and 17.67±3.79) , and statistically significant differences were observed between the two groups (P<0.05) . Conclusion: Noise exposure may activate the AIM2 inflammasome in hippocampal glial cells of rats, releasing excessive inflammatory cytokines and causing neuroinflammation that damages neurons.

The combination of Butyricicoccus pullicaecorum and 3-hydroxyanthranilic acid prevents postmenopausal osteoporosis by modulating gut microbiota and Th17/Treg

BackgroundPostmenopausal osteoporosis (PMO) is a chronic condition characterized by decreased bone strength. This study aims to investigate the effects and mechanisms of the combination of Butyricicoccus pullicaecorum (Bp) and 3-hydroxyanthranilic acid (3-HAA) on PMO.MethodsThe effects of Bp and 3-HAA on PMO were evaluated in ovariectomized (OVX) rats by assessing stereological parameters, femur microstructure, and autophagy levels. The T helper (Th) 17/Regulatory T (Treg) cells of rats were detected using flow cytometric analysis. Furthermore, the impact of Bp and 3-HAA on the gut microbiota of rats was assessed using 16S rRNA gene sequencing. The correlation between the gut microbiota of rats and Th17/Treg immune factors, as well as femoral stereo parameters, was separately assessed using Spearman rank correlation analysis.ResultsBp and 3-HAA treatments protected OVX rats by promoting osteogenesis and inhibiting autophagy. Compared to the Sham group, OVX rats showed an increase in Th17 cells and a decrease in Treg cells. Bp and 3-HAA reversed these changes. Enterorhabdus and Pseudomonas were significantly enriched in OVX rats. Bp and 3-HAA regulated the gut microbiota of OVX rats, enriching pathways related to nutrient metabolism and immune function. There was a correlation between the gut microbiota and the Th17/Treg, as well as femoral stereo parameters. The concurrent administration of Bp and 3-HAA medication facilitated the enrichment of gut microbiota associated with the improvement of PMO.ConclusionThe combination therapy of Bp and 3-HAA can prevent PMO by modulating the gut microbiota and restoring Th17/Treg immune homeostasis.

Hedyotis diffusa Willd inhibits inflammation and oxidative stress to protect against chronic prostatitis via the NRF2/ARE signaling pathway.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a common and serious disease with unclear pathogenesis and recurrent symptoms. Hedyotis diffusa Willd (HDW) has been recognized for its potential in managing various chronic inflammatory diseases. This research aimed to interrogate the mechanism of HDW in treating CP/CPPS. Complete Freund Adjuvant (CFA) and LPS were utilized to establish the rat and cell models of CP/CPPS. Results showed that HDW decreased levels of inflammation-related factors in CP rat prostate tissue and LPS-elicited RWPE-1 cell injury model. Moreover, HDW administration impaired oxidative stress in the prostate and RWPE-1 cells. In addition, HDW treatment activated the NRF2/ARE signaling in rat prostate tissue and cell models. Interestingly, NRF2/ARE pathway inhibitor ML385 reversed the inhibition effects of cell apoptosis, inflammation, and oxidative stress triggered by HDW. In summary, HDW alleviated inflammation and oxidative stress by activating NRF2/ARE signaling in CP/CPPS rat model and human prostate epithelial cell injury model.

Noninvasive focused ultrasound-mediated delivery of rAAV9-EGFP vectors for neuronal targeting in rats

ObjectiveTo evaluate the synergistic potential of Focused Ultrasound (FUS) in conjunction with microbubbles (MB) and recombinant adeno-associated virus serotype 9 (rAAV9) vectors for targeted gene delivery to neuronal cells in rats, optimizing gene expression conditions and assessing any adverse effects. MethodsThe parameters for permeability enhancement of the rat's blood-brain barrier (BBB) were established using FUS+MB, with MRI scans and Evans Blue (EB) dye assisting in the evaluation. Rats underwent FUS-mediated transfection using rAAV9-Syn-EGFP vectors produced via a triple-transfection in HEK293T cells. Following this, the uptake and expression of GFP in targeted brain regions were evaluated using confocal fluorescence microscopy at various time intervals. Inflammatory responses post-FUS treatment were tracked by observing levels of GFAP, a marker for astrocytic activation, and TNF-α, a pro-inflammatory cytokine. Motor behavior effects post-intervention were gauged using the Rotarod test across multiple groups over a span of four weeks. ResultsFUS+MB affected BBB permeability, with optimal results at 4 W for 200 s showing 85 % permeability and evident Gd-DTPA leakage. Settings beyond these resulted in tissue damage. Control groups exhibited a basal GFP expression of 2 % ± 0.5 %, whereas FUS+MB with rAAV-EGFP injections substantially increased GFP expression to about 67 % ± 6 % in targeted neurons. This GFP expression peaked at three weeks post-treatment and remained evident six months later. Following FUS treatment, both GFAP and TNF-α levels underwent fluctuations before eventually nearing their baseline values. The Rotarod test revealed no significant behavioral differences post-treatments among the groups. ConclusionsCombining FUS+MB with rAAV offers an innovative approach to enhance therapeutic delivery to the central nervous system (CNS) by transiently adjusting BBB permeability.

Cytotoxicity of novel hybrid composite materials for making bone fracture plates

Bone fracture plates are usually made from steel or titanium, which are much stiffer than cortical bone. This may cause bone ‘stress shielding’ (i.e. bone resorption leading to plate loosening) and delayed fracture healing (i.e. fracture motion is less than needed to stimulate callus formation at the fracture). Thus, the authors previously designed, fabricated, and mechanically tested novel ‘hybrid’ composites made from inorganic and organic materials as potential bone fracture plates that are more flexible to reduce these negative effects. This is the first study to measure the cytotoxicity of these composites via the survival of rat cells. Cubes of carbon fiber/flax fiber/epoxy and glass fiber/flax fiber/epoxy had better cell survival vs. Kevlar fiber/flax fiber/epoxy (57% and 58% vs. 50%). Layers and powders made of carbon fiber/epoxy and glass fiber/epoxy had higher cell survival than Kevlar fiber/epoxy (96%–100% and 100% vs. 39%–90%). The presence of flax fibers usually decreased cell survival. Thus, carbon and glass fiber composites (with or without flax fibers), but not Kevlar fiber composites (with or without flax fibers), may potentially be used for bone fracture plates.

Erythropoietin hyporesponsiveness in non-alcoholic fatty liver disease.

Treatment with erythropoietin (EPO) can correct anaemia in chronic kidney disease (CKD) patients; however, up to 10% exhibit resistance or hyporesponsiveness to EPO. Non-alcoholic fatty liver disease (NAFLD), prevalent liver disease in CKD patients, may limit EPO response because of thrombopoietin deficiency, iron homeostasis disorder and inflammation. Therefore, we hypothesized NAFLD is a risk factor for EPO responsiveness. To test our hypothesis, we evaluated the effect of EPO in healthy rats and rats with NAFLD induced by a high-fat, high-carbohydrate (HFHC) diet. After 12 weeks on the HFHC diet, NAFLD rats showed lower erythroid response to EPO treatment than healthy rats. We, then, determined that the primary cause of EPO hyporesponsiveness could be iron deficiency associated with inflammation, which reduces erythroid cell production. Specifically, the concentrations of hepcidin, ferritin, transferrin and white blood cells in NAFLD rats were 12.8-, 16.4-, 2.51- and 1.40-fold higher than those in healthy rats, respectively. However, erythroid cell types in the bone marrow of NAFLD rats were significantly reduced. In conclusion, our data suggest that NAFLD could be a risk factor for EPO responsiveness, which is attributed to functional iron deficiency associated with inflammation.

Caveolin-1 scaffolding domain-derived peptide enhances erectile function by regulating oxidative stress, mitochondrial dysfunction, and apoptosis of corpus cavernosum smooth muscle cells in rats with cavernous nerve injury

AimIncreased corpus cavernosum smooth muscle cells (CCSMCs) apoptosis in the penis due to cavernous nerve injury (CNI) is a crucial contributor to erectile dysfunction (ED). Caveolin-1 scaffolding domain (CSD)-derived peptide has been found to exert potential antiapoptotic properties. However, whether CSD peptide can alleviate CCSMCs apoptosis and ED in CNI rats remains unknown. The study aimed to determine whether CSD peptide can improve bilateral CNI-induced ED (BCNI-ED) by enhancing the antiapoptotic processes of CCSMCs. Main methodsFifteen 10-week-old male Sprague-Dawley (SD) rats were randomly classified into three groups: sham surgery (Sham) group and BCNI groups that underwent saline or CSD peptide treatment respectively. At 3 weeks postoperatively, erectile function was assessed and the penis tissue was histologically examined. Furthermore, an in vitro model of CCSMCs apoptosis was established using transforming growth factor-beta 1 (TGF-β1) to investigate the mechanism of CSD peptide in treating BCNI-ED. Key findingsIn BCNI rats, CSD peptide significantly prevented ED and decreased oxidative stress, the Bax/Bcl-2 ratio, and the levels of caspase3. TGF-β1-treated CCSMCs exhibited severe oxidative stress, mitochondrial dysfunction, and apoptosis. However, CSD peptide partially reversed these alterations. SignificanceExogenous CSD peptide could improve BCNI-ED by inhibiting oxidative stress, the Bax/Bcl-2 ratio, and caspase3 expression in penile tissue. The underlying mechanism might involve the regulatory effects of CSD peptide on oxidative stress, mitochondrial dysfunction, and apoptosis of CCSMCs following CNI. This study highlights CSD peptide as an effective therapy for post-radical prostatectomy ED (pRP-ED).

Mesenteric Lymphatic B Cells Migrate to the Intestine and Aggravate DSS-Induced Colitis via the CXCR5-CXCL13 Axis.

The pathogenesis of inflammatory bowel disease (IBD) is still unknown. Mesenteric lymphatics (MLs), which are closely related to the intestine in both anatomy and physiology, have been suggested to be involved in IBD. In the present study, we aim to investigate the effects of ML immune cells on IBD and explore the potential associated mechanisms. Acute colitis was induced in rats using dextran sulfate sodium salt (DSS). Mesenteric lymphangiogenesis, ML stenosis, and dilation were observed, with an increased proportion of MLB cells in DSS-induced colitis rats. The adoptive transfer of B cells isolated from ML (MLB) was employed to investigate their effects on colitis. MLB cells derived from DSS-induced colitis rats exhibited a higher propensity to migrate to the intestine. The proportion of colonic T cells was altered, along with the aggravated colitis induced by the adoptive transfer of MLB cells derived from DSS-induced colitis rats. RNA sequencing revealed increased Cxcr5 expression in MLB cells from colitis rats, while real-time PCR indicated an upregulation of its ligand Cxcl13 in the colon of colitis rats. These findings suggest that MLB cells may migrate to the intestine and aggravate colitis. In summary, colonic T cells respond to MLB cells from colitis rats, and MLB cells aggravate DSS-induced colitis via the CXCR5-CXCL13 axis.

Network Pharmacology Combined with Animal Models to Investigate the Mechanism of ChangPu YuJin Tang in the Treatment of Tourette Syndrome.

ChangPu YuJin Tang (CPYJT) is a Chinese herbal formula that has been shown to be an effective therapeutic strategy for pediatric patients with Tourette Syndrome (TS). Using an integrated strategy of network pharmacology and animal model, the aim of this study was to investigate the mechanism of CPYJT in the treatment of TS. Compound libraries of CPYJT were established using databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The TCMSP database and Swiss Target Prediction database were used to predict the targets. The above results were constructed into a CPYJT-Drug-Component-Target network. Moreover, TS targets were predicted using GeneCards and other databases. The targets corresponding to the potential ingredients in CPYJT and the targets corresponding to TS were taken as the intersections to construct the CPYJT-TS network. The target network was analysed by PPI using the string database. GO and KEGG enrichment analyses were performed on the target network. The whole process was performed using Cytoscape 3.7.2 to make visual network diagrams of the results. CPYJT was characterised by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS). Transmission Electron Microscopy (TEM) was used to observe the structural changes of CPYJT on the neuronal cells of the IDPN model rats. RT-PCR and Western Blot were used to analyse the changes in the mRNA and protein expression levels of BDNF, TrkB, PI3K, and AKT in the cortex, striatum, and thalamus brain regions after CPYJT administration in IDPN model rats. Network pharmacology and UHPLC-MS studies revealed that CPYJT acted on the TS through multiple neurotransmitters and the BDNF/TrkB and PI3K/AKT signalling pathways. CPYJT ameliorated neurocellular structural damage in the cortex, striatum, and thalamus of TS model rats. Additionally, CPYJT up-regulated the levels of BDNF, TrkB, PI3k, and AKT in the cortex, striatum, and thalamus of TS model rats. It was found that CPYJT protected neuronal cells from structural damage in multiple brain regions and affected the expression levels of BDNF, TrkB, PI3K, and Akt in the cortex, striatum, and thalamus during TS treatment.

The mitochondrial tRNA-derived fragment, mt-tRF-LeuTAA, couples mitochondrial metabolism to insulin secretion

ObjectiveThe contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. MethodsWe used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. ResultsOur study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic β-cells. ConclusionsOur findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.

Spinal TNF-α receptor expression in a rodent model of respiratory motor neuron death

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kills respiratory motor neurons, and mimics aspects of neurodegenerative diseases including breathing deficits. In this model, phrenic long-term facilitation (pLTF) induced by acute intermittent hypoxia is enhanced following 7 days (d) of CTB-SAP vs. control rats, whereas pLTF in 28d CTB-SAP treated rats is comparable to pLTF in controls suggesting that the underlying mechanisms of plasticity at both time points differ. Inflammation is a hallmark of neurodegenerative diseases and is known to prevent pLTF. Furthermore, ketoprofen (NSAID) delivery attenuates pLTF in 7d CTB-SAP-treated rats, but enhances it in 28d CTB-SAP-treated rats; this suggests that inflammation contributes to pLTF in 7d CTB-SAP-treated rats, but undermines pLTF in 28d CTB-SAP-treated rats. In addition, cervical spinal inflammatory marker gene expression ( e.g., TNF-a) is increased in CTB-SAP rats, and pilot data suggest that soluble TNF receptor 1 (sTNFR1) inhibition differentially affects pLTF (Nichols and Smith , ibid). Thus, the goal of the current study is to investigate TNFR1 as a pharmacological target that can modulate inflammation to maximize pLTF and breathing in CTB-SAP rats. We hypothesized that C4 TNFR1 expression would be increased on phrenic motor neurons and glial cells of CTB-SAP rats vs. controls. To address this, we studied TNFR1 expression on phrenic motor neurons and glial cells using immunohistochemistry, confocal microscopy, and ImageJ analysis in male Sprague Dawley rats 7 and 28 days after intrapleural injection of: CTB-SAP or un-conjugated CTB and SAP (control); n=8/group. Our preliminary data (n=4/group) suggest that phrenic TNFR1 expression is increased in 7d CTB-SAP rats vs. controls (p&lt;0.05), but is unaffected in 28d CTB-SAP rats vs. controls (p&gt;0.05). Further data analysis is underway for phrenic as well as glial TNFR1 expression in CTB-SAP rats vs. controls. If C4 TNFR1 expression is increased, this would suggest that TNFR1 is a viable target for therapeutic intervention in CTB-SAP rats, and potentially in patients with breathing deficits due to respiratory motor neuron death. NSF Computational Neuroscience grant, MU CVM COR grant, and Spinal Cord Injury &amp; Disease Research Program grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protective effects of Dioscin against sepsis-induced cardiomyopathy via regulation of toll-like receptor 4/MyD88/p65 signal pathway.

Dioscin has many pharmacological effects; however, its role in sepsis-induced cardiomyopathy (SIC) is unknown. Accordingly, we concentrate on elucidating the mechanism of Dioscin in SIC rat model. The SIC rat and H9c2 cell models were established by lipopolysaccharide (LPS) induction. The heart rate (HR), left ventricle ejection fraction (LVEF), mean arterial blood pressure (MAP), and heart weight index (HWI) of rats were evaluated. The myocardial tissue was observed by hematoxylin and eosin staining. 4-Hydroxy-2-nonenal (4-HNE) level in myocardial tissue was detected by immunohistochemistry. Superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) activities in serum samples of rats and H9c2 cells were determined by colorimetric assay. Bax, B-cell lymphoma-2 (Bcl-2), toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), phosphorylated-p65 (p-p65), and p65 levels in myocardial tissues of rats and treated H9c2 cells were measured by quantitative real-time PCR and Western blot. Viability and reactive oxygen species (ROS) accumulation of treated H9c2 cells were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and dihydroethidium staining assays. Dioscin decreased HR and HWI, increased LVEF and MAP, alleviated the myocardial tissue damage, and reduced 4-HNE level in SIC rats. Dioscin reversed LPS-induced reduction on SOD, CAT, GSH, and Bcl-2 levels, and increment on Bax and TLR4 levels in rats and H9c2 cells. Overexpressed TLR4 attenuated the effects of Dioscin on promoting viability, as well as dwindling TLR4, ROS and MyD88 levels, and p-p65/p65 value in LPS-induced H9c2 cells. Protective effects of Dioscin against LPS-induced SIC are achieved via regulation of TLR4/MyD88/p65 signal pathway.

Protective effects of oyster polypeptide on cyclophosphamide-induced immunosuppressed rats.

Oyster polypeptide (OP) is a mixture of oligopeptides extracted from oysters through enzyme lysis, separation, and purification. It is associated with immunomodulatory effects, but the underlying mechanisms are not known. This study therefore combined proton nuclear magnetic resonance (1H-NMR) urinary metabolomics and 16S rRNA gene sequencing of the gut microbiome to determine the immunoprotective mechanisms of OP in rats subjected to cyclophosphamide-induced immunosuppression. Oyster polypeptide restored the body weight and the structure of spleen and thymus in rats with cyclophosphamide-induced immunosuppression. It upregulated the levels of white blood cells (WBCs), hemoglobin (HGB), platelets (PLT), red blood cells (RBCs), immunoglobulin G (IgG), immunoglobulin M (IgM), cytokines such as interleukin‑6 (IL-6) and tumor necrosis factor-α (TNF-α), and increased the numbers of CD3+ and CD4+ T cells in the immunosuppressed rats. The 1H-NMR metabolomics results showed that OP significantly reversed the levels of ten metabolites in urine, including 2-oxoglutarate, citrate, dimethylamine, taurine, N-phenylacetylglycine, alanine, betaine, creatinine, uracil, and benzoate. The 16S rRNA gene sequencing results showed that OP restored the gut microbiome homeostasis by increasing the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria. Finally, a combination of metabolomics and microbiomics found that the metabolism of taurine and hypotaurine, and the metabolism of alanine, aspartate, and glutamate were disturbed, but these metabolic pathways were restored by OP. This study demonstrated that OP had immunoprotective effects in rats with cyclophosphamide-induced immunosuppression by restoring key metabolic pathways and the gut microbiome homeostasis. Our findings provide a framework for further research into the immunoregulatory mechanisms of OP and its potential use in drugs and nutritional supplements. © 2024 Society of Chemical Industry.

AGE-RELATED CHARACTERISTICS OF GLUTATHIONE-DEPENDENT ENZYMES FUNCTIONING IN RATS UNDER CONDITIONS OF TOXIC INJURY BY ACETAMINOPHEN

Aim. The study is dedicated to evaluating the enzymatic activities of the glutathione system in liver cells of rats from different age groups under conditions of toxic injury caused by acetaminophen. In the experiments, rats of two age categories were used: young (138–150 days) and mature age (348–360 days). Mehods. Acute toxic injury from acetaminophen was induced by administering it per os at a dose of 1250 mg/kg of animal body weight during the last two days of the experiment. Results. Under conditions of simulated acute toxic injury by the medicinal xenobiotic acetaminophen in the livers of young and mature rats, a decrease was observed in glutathione-S-transferase, Se-dependent glutathione peroxidase, and glutathione reductase activities. A significant decrease in non-Se-glutathione peroxidase in liver cells is observed only in mature animals that were administered toxic doses of acetaminophen. The influence of the age component can be considered as one of the critically important factors in suppressing the functional activity of the glutathione-dependent enzymatic system, manifested by decreased activity of glutathione-S-transferase, non-Se-glutathione peroxidase, Se-dependent glutathione peroxidase and glutathione reductase under conditions of toxic injury caused by the medicinal xenobiotic acetaminophen. A more pronounced decrease in glutathione-dependent enzymes under conditions of acetaminophen intoxication is observed in animals aged 360 days. Cunclusion. The consequence of the established changes could be the disruption of acetaminophen biotransformation in Phase II involving the glutathione system, which can be considered as one of the risk factors for the development of drug-induced hepatotoxicity in different age groups.

Bioactive metabolites from endophytes of African club moss- Selaginella kraussiana (Kunze) A. Braun exhibit broad-spectrum pharmaceutical utilities

The search for potent natural compounds from plant and microbial sources is increasing logarithmically to combat various types of health issues of which multidrug-resistant microbes and problems related to oxidative stress are the prime ones. Novel secondary metabolites from endophytes have always been potent alternatives to contemporary antimicrobials and antioxidants. The present study shows that two potent endophytic fungal isolates Ascochyta medicaginicola SK16 and Phomopsis sp. SK5 synthesises non-proteinaceous, thermostable secondary metabolites with broad-spectrum antimicrobial, antioxidative, and cytotoxic activity. Bioassay-guided fractionation revealed that the Ethyl acetate (EA) extract of the cell-free culture extract (CFCE) of SK5 and SK16 constitutes ten and eleven secondary metabolites of which 2,4, -di tert butyl phenol, aziridine- 1,2-amino ethyl, actinobolin, bactobolin, azulene, menthyl acetate, phenol, p-[2-(methylamino)ethyl], and imidazole components were significant. SK16 exhibited strong antimicrobial activity against seventeen pathogens including Methicillin-resistant Staphylococcus aureus (MRSA), and C. albicans with a minimum microbicidal concentration of 100- 400 µg mL−1. These metabolites disrupt the microbial cell membranes causing lethal leakage of biomacromolecules- nucleic acid, protein, and potassium ions. Enzymes involved in the EMP pathway, TCA cycle, and gluconeogenesis were also blocked. Both the biofilm formation and transition of Candida cells from the yeast-to-hyphal form were inhibited. SK5 metabolites are potent radical scavengers with an IC50 of 34.25 ± 0.87 – 195.21 ± 5.73 µg mL−1 against DPPH, ABTS, FRAP, and H2O2 free radical generators and significantly increased antioxidant parameters in treated peritoneal macrophage cells of male Wistar rats. Also, SK5 metabolites were cytotoxic against breast (T47D), pancreatic (MIA Paca-2), lung (A549) and colon (HCT-116) cancer cells. The present findings suggest that metabolites from endophytes of African club moss hold potent pharmaceutical utilities.

Effect of VBIT-4 on the functional activity of isolated mitochondria and cell viability

VBIT-4 is a new inhibitor of the oligomerization of VDAC proteins of the outer mitochondrial membrane preventing the development of oxidative stress, mitochondrial dysfunction, and cell death in various pathologies. However, as a VDAC inhibitor, VBIT-4 may itself cause mitochondrial dysfunction in healthy cells. The article examines the effect of VBIT-4 on the functional activity of rat liver mitochondria and cell cultures. We have demonstrated that high concentrations of VBIT-4 (15–30 μM) suppressed mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. VBIT-4 induced depolarization of organelles fueled by substrates of complex I but not complex II of the respiratory chain. VBIT-4 has been found to inhibit the activity of complexes I, III, and IV of the respiratory chain. Molecular docking demonstrated that VBIT-4 interacts with the rotenone-binding site in complex I with similar affinity. 15–30 μM VBIT-4 caused an increase in H2O2 production in mitochondria, decreased the Ca2+ retention capacity, but increased the time of Ca2+-dependent mitochondrial swelling. We have found that the incubation of breast adenocarcinoma (MCF-7) with 30 μM VBIT-4 for 48 h led to the decrease of the mitochondrial membrane potential, an increase in ROS production and death of MCF-7 cells. The mechanism of action of VBIT-4 on mitochondria and cells is discussed.

Huperzine A ameliorates neurological deficits after spontaneous subarachnoid hemorrhage through endothelial cell pyroptosis inhibition.

Spontaneous subarachnoid hemorrhage (SAH) is a kind of hemorrhagic stroke which causes neurological deficits in survivors. Huperzine A has a neuroprotective effect, but its role in SAH is unclear. Therefore, we explore the effect of Huperzine A on neurological deficits induced by SAH and the related mechanism. In this study, Evans blue assay, TUNEL staining, immunofluorescence, western blot analysis, and ELISA are conducted. We find that Huperzine A can improve neurological deficits and inhibit the apoptosis of nerve cells in SAH rats. Huperzine A treatment can improve the upregulation of brain water content, damage of blood-brain barrier, fibrinogen and matrix metalloprotein 9 expressions and the downregulation of ZO-1 and occludin expressions induced by SAH. Huperzine A inhibit the expressions of proteins involved in pyroptosis in endothelial cells in SAH rats. The increase in MDA content and decrease in SOD activity in SAH rats can be partly reversed by Huperzine A. The ROS inducer H 2O 2 can induce pyroptosis and inhibit the expressions of ZO-1 and occludin in endothelial cells, which can be blocked by Huperzine A. In addition, the increase in the entry of p65 into the nucleus in endothelial cells can be partly reversed by Huperzine A. Huperzine A may delay the damage of blood-brain barrier in SAH rats by inhibiting oxidative stress-mediated pyroptosis and tight junction protein expression downregulation through the NF-κB pathway. Overall, Huperzine A may have clinical value for treating SAH.