Primary Rat Research Articles

Exploring Syndecan-4 and MLP and Their Interaction in Primary Cardiomyocytes and H9c2 Cells.

The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4-MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4-MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4-MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4-MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.

Uncovering kinesin dynamics in neurites with MINFLUX

Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study the substeps of a truncated kinesin-1 mutant in primary rat hippocampal neurons, which have so far been mainly observed on polymerized microtubules deposited onto glass coverslips. A gentle fixation protocol largely maintains the structure and surface modifications of the microtubules in the cell. By analyzing the time between the substeps, we identify the ATP-binding state of kinesin-1 and observe the associated rotation of the kinesin-1 head in neurites. We also observed kinesin-1 switching microtubules mid-walk, highlighting the potential of MINFLUX to study the details of active cellular transport.

Protective effect of dexmedetomidine against delayed bone healing caused by morphine via PI3K/Akt mediated Nrf2 antioxidant defense system.

As a class of analgesics, opioids are frequently used to treat both acute and chronic moderate to severe pain. Patients frequently receive opioid painkillers after orthopedic accidents or surgeries. Evidence suggests that opioid drug users have a 55.1% higher risk of fracture and poor bone repair than non-users of opioid drugs. The key pathogenic alterations in the incidence and progression of poor bone repair are over apoptosis and aging of osteoblasts due to the stress caused by oxidation. Dexmedetomidine (Dex) has been proven to protect against a variety of degenerative illnesses by reducing oxidative stress. However, nothing is known about how it affects bone repair. PI3K/Akt/Nrf2 pathway was detected by immunofluorescence and Western blot. SOD, CAT, JC-1, dihydroethidium and mitosox were used in the Oxidative Stress. Micro-CT, H&E and Masson's staining, immunohistochemically were performed to evaluate the therapeutic effects of DEX on calvarial defects in the morphine-induced rat model. We found that morphine-induced an imbalance in the metabolism and catabolism of primary rat Osteoblasts. However, these conditions could be inhibited by DEX treatment. In the meantime, DEX induced the expression of Nrf2-regulated antioxidant enzymes such as NQO1, HO-1, GCLm, GCLc, and TrxR1. DEX-mediated Nrf2 activation is linked to the PI3K/Akt signaling system. Furthermore, it has been established that intravenous DEX enhanced the growth of bone healing in a model of a surgically produced rat cranial lesion. This is the first description of the unique DEX mechanism acting as a Nrf2 activator against morphine-mediated oxidative harm, raising the possibility that the substance may be used to prevent bone defects.

Regulatory role of oxidative stress in retrorsine – Induced apoptosis and autophagy in primary rat hepatocytes

Pyrrolizidine alkaloids (PAs) are a group of naturally occurring alkaloids widely present in plants. PAs are highly hepatotoxic and have been documented to cause many incidents of human and animal poisoning. Retrorsine (RTS) is a pyrrolizidine alkaloid (PA) derived from the Compositae Senecio, which has been shown to cause hepatotoxicity. Human liver poisoning occurs through the consumption of RTS-contaminated food, and animals can also be poisoned by ingesting RTS-containing toxic plants. The mechanism of RTS-induced liver toxicity is not fully understood. In this study, we demonstrated that RTS-induced oxidative stress plays a pivotal role in RTS-induced liver toxicity involving apoptosis and autophagy. The results showed that RTS treatment in the cultured Primary rat hepatocytes caused cytotoxicity and release of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a time- and dose-dependent manner. Our study showed that treatment of RTS induced ROS and MDA (malondialdehyde, a lipid peroxidation marker) in the hepatocytes, and reduced antioxidant capacity (GSH content, SOD activity), suggesting RTS treatment caused oxidative stress response in the hepatocytes. Furthermore, we found that RTS induced apoptosis and autophagy in the hepatocytes, and RTS-induced apoptosis and autophagy could be alleviated by ROS scavenger N-acetylcysteine (NAC) and the MAPK pathway inhibitors suggesting ROS/MAPK signaling pathway plays a role in RTS induced apoptosis and autophagy. Collectively, this study reveals the regulatory mechanism of oxidative stress in RTS-induced apoptosis and autophagy in the hepatocytes, providing important insights of molecular mechanisms of hepatotoxicity induced by RTS and related pyrrolizidine alkaloids in liver. This mechanism provides a basis for the prevention and treatment of PA poisoning in humans and animals.

A Dual Strategy-In Vitro and In Silico-To Evaluate Human Antitetanus mAbs Addressing Their Potential Protective Action on TeNT Endocytosis in Primary Rat Neuronal Cells.

Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.

Network pharmacology integrated with experimental validation to elucidate the mechanisms of action of the Guizhi-Gancao Decoction in the treatment of phenylephrine-induced cardiac hypertrophy

Context The mechanisms of Traditional Chinese Medicine (TCM) Guizhi-Gancao Decoction (GGD) remain unknown. Objective This study explores the mechanisms of GGD against cardiac hypertrophy. Materials and methods Network pharmacology analysis was carried out to identify the potential targets of GGD. In vivo experiments, C57BL/6J mice were divided into Con, phenylephrine (PE, 10 mg/kg/d), 2-chloroadenosine (CADO, the stable analogue of adenosine, 2 mg/kg/d), GGD (5.4 g/kg/d) and GGD (5.4 g/kg/d) + CGS15943 (a nonselective adenosine receptor antagonist, 4 mg/kg/d). In vitro experiments, primary neonatal rat cardiomyocytes (NRCM) were divided into Con, PE (100 µM), CADO (5 µM), GGD (10−5 g/mL) and GGD (10−5 g/mL) + CGS15943 (5 µM). Ultrasound, H&E and Masson staining, hypertrophic genes expression and cell surface area were conducted to verify the GGD efficacy. Adenosine receptors (ADORs) expression were tested via real-time polymerase chain reaction (PCR), western blotting and immunofluorescence analysis. Results Network pharmacology identified ADORs among those of the core targets of GGD. In vitro experiments demonstrated that GGD attenuated PE-induced increased surface area (with an EC50 of 5.484 × 10−6 g/mL). In vivo data shown that GGD attenuated PE-induced ventricular wall thickening. In vitro and in vivo data indicated that GGD alleviated PE-induced hypertrophic gene expression (e.g., ANP, BNP and MYH7/MYH6), A1AR over-expression and A2aAR down-expression. Moreover, CADO exerts effects similar to GGD, whereas CGS15943 eliminated most effects of GGD. Discussion and conclusions Our findings suggest the mechanism by which GGD inhibits cardiac hypertrophy, highlighting regulation of ADORs as a potential therapeutic strategy for HF.

Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway

Based on the secreted frizzled-related protein 2 (SFRP2)-Wnt/β-catenin signaling pathway, this study explored the effect and mechanism of Cuiru Keli (CRKL) in the treatment of postpartum hypogalactia. A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery. Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups, including a normal group (without any modeling or medication), a model group, a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg, a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg, a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg, a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg, and a negative control (NC) group of model rats receiving normal saline. Each group contained 6 rats. Except for the normal and model groups, the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days. Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured, and HE staining was performed to identify pathological changes in the mammary tissue (MT). Six groups of rats (excluding the positive control group) were used to observe the pathological changes of eosinophils in pituitary tissue. ELISA was performed to determine the content of prolactin (PRL) in serum, immunohistochemical staining was used to determine the expression of prolactin receptor (PRLR) in MT, and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT. Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia, particularly whether it acted through the SFRP2-Wnt/β-catenin signaling pathway. The mechanism of CRKL treatment was further validated by detecting mRNA (RT-qPCR) and protein expression (Western blot) of related pathway genes. Cell experiments were conducted using primary culture rat mammary epithelial cells (RMEC) from rat MT. RMEC were divided into four groups, including a normal group (primary culture RMEC, untreated), SFRP2 overexpression group (primary cultured RMEC treated with SFRP2 overexpression vector), SFRP2 overexpression+CRKL group (receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum), and negative control group (primary culture RMEC treated with empty vector). The effect of CRKL on the expression of lactation-related genes FASN, CSN2, and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR. In this study, CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group, 6 g/kg in the medium-dose group, and 9 g/kg in the high-dose group (P<0.05 or P<0.01). Compared with the model group, CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days (P<0.05 or P<0.01), and effectively increased lactation (P<0.01), the area of mammary lobules, and the size and filling of acinar cavities. CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model, and increased the content of PRL in the serum (P<0.05 or P<0.01). CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats. In addition, it significantly promoted the expression of genes related to milk fat, milk protein, and lactose synthesis in MT (P<0.05 or P<0.01). Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia. The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2. Compared with the model group, CRKL at all doses inhibited the expression of SFRP2 gene in vivo (P<0.01) and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/β-catenin signaling pathway in MT (P<0.05 or P<0.01). Cell experiments showed that, compared to the normal group, SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN, CSN2, and GLUT1 in RMEC (P<0.01). The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells (P<0.01). After administering drug-containing serum, the expression of the lactation-related genes FASN, CSN2, and GLUT1 were up-regulated (compared with the SFRP2 overexpression group, P<0.01). CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/β-catenin signaling pathway. SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia. This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.

Oroxylin A inhibits inflammatory cytokines in periodontitis via HO‑1.

Periodontal disease is a common infectious disease that can lead to the loss of teeth. Hower how to effectively suppress the inflammation with medication is unclear. The aim of the present study was to investigate the anti‑inflammatory effect of Oroxylin A in periodontitis and its potential role through heme oxygenase‑1 (HO‑1). Primary rat gingival fibroblasts (RGFs) were cultured using the tissue block method and identified by immunofluorescence. Following lipopolysaccharide (LPS) stimulation of RGFs, Oroxylin A was administered at 50, 100, 200 or 400 µg/ml. Reverse transcription‑quantitative PCR was used to assess mRNA expression of cyclooxygenase (COX)‑2, TNF‑α, RANKL and osteoprotegerin (OPG). Western blotting was used to detect protein expression levels of COX ‑2, TNF‑α, RANKL and OPG. Following HO‑1 knockdown, the same treatment was performed. The expression of COX‑2 in rat gingival tissue was observed by immunohistochemistry. One‑way analysis of variance and Student's t test were used for statistical analysis. Oroxylin A downregulated mRNA expression of COX‑2, TNF‑α, RANKL and OPG in LPS‑induced RGFs. With increase of Oroxylin A dose, the expression of HO‑1 was gradually upregulated. When HO‑1 was knocked down, Oroxylin A did not downregulate the expression of COX‑2, TNF‑α, RANKL and OPG in LPS‑induced RGFs. Immunohistochemical results showed that expression of COX‑2 was downregulated by Oroxylin A, and the expression of TNF‑α, RANKL and OPG were also downregulated. Oroxylin A decreased expression of inflammatory cytokines in LPS‑induced RGFs and had a good inhibitory effect on periodontitis in rats.

Nobiletin regulates intracellular Ca2+ levels via IP3R and ameliorates neuroinflammation in Aβ42-induced astrocytes

Astrocytes are the major glial cells in the human brain and provide crucial metabolic and trophic support to neurons. The amyloid-β peptide (Aβ) alter the morphological and functional properties of astrocytes and induce inflammation and calcium dysregulation, contributing to Alzheimer's disease (AD) pathology. Recent studies highlight the role of Toll-like receptor (TLR) 4/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in inflammation. Reactive oxygen species (ROS) generated due to Aβ, induce apoptosis in the brain cells worsening AD progression. Astrocytic cell surface receptors, such as purinergic receptors (P2Y1 and P2Y2), metabotropic glutamate receptor (mGLUR)5, α7 nicotinic acetylcholine receptor (α7nAChR), and N-methyl-d-aspartate receptors (NMDARs), have been suggested to interact with inositol trisphosphate receptor (IP3R) on the endoplasmic reticulum (ER) to induce Ca2+ movement from ER to cytoplasm, causing Ca2+ dysregulation. We found that the citrus flavonoid nobiletin (NOB) protected primary astrocytes from Aβ42-induced cytotoxicity and inhibited TLR4/NF-κB signaling in Aβ42-induced primary rat astrocytes. NOB was found to regulate Aβ42-induced ROS levels through Keap1-Nrf2 pathway. The receptors P2Y1, P2Y2, mGLUR5, α7nAChR, and NMDARs induced intracellular Ca2+ levels by activating IP3R and NOB regulated them, thereby regulating intracellular Ca2+ levels. Molecular docking analysis revealed a possible interaction between NOB and IP3R in IP3R regulation. Furthermore, RNA sequencing revealed various NOB-mediated biological signaling pathways, such as the AD-presenilin, AD-amyloid secretase, and Wnt signaling pathway, suggesting possible neuroprotective roles of NOB. To conclude, NOB is a promising therapeutic agent for AD and works by modulating AD pathology at various levels in Aβ42-induced primary rat astrocytes.

Lactate regulates respiratory efficiency and mitochondrial dynamics in primary rat podocytes

Podocytes are crucial for regulating glomerular permeability. They have foot processes that are integral to the renal filtration barrier. Understanding their energy metabolism could shed light on the pathogenesis of filtration barrier injury. Lactate has been increasingly recognized as more than a waste product and has emerged as a significant metabolic fuel and reserve. The recent identification of lactate transporters in podocytes, the expression of which is modulated by glucose levels and lactate, highlights lactate's relevance. The present study investigated the impact of lactate on podocyte respiratory efficiency and mitochondrial dynamics. We confirmed lactate oxidation in podocytes, suggesting its role in cellular energy production. Under conditions of glucose deprivation or lactate supplementation, a significant shift was seen toward oxidative phosphorylation, reflected by an increase in the oxygen consumption rate/extracellular acidification rate ratio. Notably, lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) isoforms, which are involved in lactate conversion to pyruvate, were detected in podocytes for the first time. The presence of lactate led to higher intracellular pyruvate levels, greater LDH activity, and higher LDHB expression. Furthermore, lactate exposure increased mitochondrial DNA-to-nuclear DNA ratios and resulted in upregulation of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor coactivator-1α and transcription factor A mitochondrial, regardless of glucose availability. Changes in mitochondrial size and shape were observed in lactate-exposed podocytes. These findings suggest that lactate is a pivotal energy source for podocytes, especially during energy fluctuations. Understanding lactate's role in podocyte metabolism could offer insights into renal function and pathologies that involve podocyte injury.

Retraction: Comparison of the effects of EGF, FGF-b, and NGF on the proliferation, migration, and reprogramming of primary rat Müller cells.

[This retracts the article DOI: 10.3389/fncel.2024.1338129.].

The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity.

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.

Identification of cardioprotective substances in Panax ginseng/P. notoginseng based on mitochondrial morphological characteristics and UPLC-Triple-TOF-MS

Panax ginseng is reputed to be capable of replenishing healthy Qi and bolstering physical strength, and P. notoginseng can resolve blood stasis and alleviate pain. P. ginseng and P. notoginseng are frequently employed to treat ischemic heart diseases caused by blockages in the heart vessels. Mitochondrial dysfunction often coexists with abnormal mitochondrial morphology, and mitochondrial plasticity and dynamics play key roles in cardiovascular diseases. In this study, primary neonatal rat cardiomyocytes were exposed to 4 hours of hypoxia(H) followed by 2 hours of reoxygenation(R). MitoTracker Deep Red and Hoechst 33342 were used to label mitochondria and nuclei, respectively. Fluorescence images were then acquired using ImageXpress Micro Confocal. Automated image processing and parameter extraction/calculation were carried out using ImagePro Plus. Subsequently, representative parameters were selected as indicators to assess alterations in mitochondrial morphology and function. The active compounds of P. ginseng and P. notoginseng were screened out and identified based on the UPLC-Triple-TOF-MS results and mitochondrial morphometric parameters. The findings demonstrated that RS-2, RS-4, SQ-1, and SQ-4 significantly increased the values of three key morphometric parameters, including mitochondrial length, branching, and area, which might contribute to rescuing morphological features of myocardial cells damaged by H/R injury. Among the active components of the two medicinal herbs, 20(R)-ginsenoside Rg_3, ginsenoside Re, and gypenoside ⅩⅦ exhibited the strongest protective effects on mitochondria in cardiomyocytes. Specifically, 20(R)-ginsenoside Rg_3 might upregulate expression of optic atrophy 1(OPA1) and mitofusin 2(MFN2), and ginsenoside Re and gypenoside ⅩⅦ might selectively upregulate OPA1 expression. Collectively, they promoted mitochondrial membrane fusion and mitigated mitochondrial damage, thereby exerting protective effects on cardiomyocytes. This study provides experimental support for the discovery of novel therapeutic agents for myocardial ischemia-reperfusion injury from P. ginseng and P. notoginseng and offers a novel approach for large-scale screening of bioactive compounds with cardioprotective effects from traditional Chinese medicines.

The bidirectional effects of APPswe on the osteogenic differentiation of MSCs in bone homeostasis by regulating Notch signaling

Amyloid precursor protein (APP), especially Swedish mutant APP (APPswe), is recognized as a significant pathogenic protein in Alzheimer's disease, but limited research has been conducted on the correlation between APPswe and the osteogenic differentiation of mesenchymal stem cells (MSCs). The effects of APPswe and its intracellular and extracellular segments on the osteogenic differentiation of bone morphogenetic protein 2 (BMP2)-induced MSCs were analyzed in this study. Our analysis of an existing database revealed that APP was positively correlated with the osteogenic differentiation of MSCs but negatively correlated with their proliferation and migration. Furthermore, APPswe promoted BMP2-induced osteogenic differentiation of MSCs, while APPswe-C (APPswe without an intracellular segment) had the opposite effect; thus, the intracellular domain of APPswe may be a key factor in promoting the osteogenic differentiation of MSCs. Additionally, both APPswe and APPswe-C inhibited the proliferation and migration of MSCs. Furthermore, the intracellular domain of APPswe inhibited the activity of the Notch pathway by regulating the expression of the Notch intracellular domain to promote the osteogenic differentiation of MSCs. Finally, APPswe-treated primary rat bone marrow MSCs exhibited the most favorable bone repair effect when a GelMA hydrogel loaded with BMP2 was used for in vivo experiments, while APPswe-C had the opposite effect. These findings demonstrate that APPswe promotes the osteogenic differentiation of MSCs by regulating the Notch pathway, but its extracellular segment blocks the self-renewal, proliferation, and migration of MSCs, ultimately leading to a gradual decrease in the storage capacity of MSCs and affecting long-term bone formation.

Abstract 1157: Inhibition Of P90rsk Ameliorates Pdgf-bb-induced Vascular Smooth Muscle Cell Phenotypic Change And Neointimal Hyperplasia

Platelet-derived growth factor type BB (PDGF-BB) has been reported to induce vascular smooth muscle cell (VSMC) proliferation and migration, which are major events that are highly linked to vascular diseases such as atherosclerosis and restenosis. Although p90RSK has been shown to regulate multiple cellular processes including cell growth, proliferation, survival and motility through various downstream substrates in different cell types, the role of p90RSK on PDGF-BB-induced VSMC proliferation and migration, and vascular neointima formation remains unknown. In this study, we determine whether inhibition of p90RSK protects from PDGF-BB-induced cellular phenotypic change and investigate the molecular mechanisms underlying the effect of p90RSK inhibition. In cultured primary rat VSMCs, we found that pretreatment with FMK or BI-D1870, specific inhibitors of p90RSK, suppressed PDGF-BB-induced VSMC phenotypic alteration, ECM accumulation, proliferation, and migration. In addition, FMK and BI-D1870 repressed cell cycle regulatory genes cyclin D1 and Cdk4 induced by PDGF-BB, whereas inhibition of p90RSK augmented cyclin-dependent kinase inhibitor p27 downregulated by PDGF-BB in VSMCs, suggesting that p90RSK induces VSMC proliferation via cell cycle regulation of G0/G1 phase. Notably, using the mouse model of partial carotid ligation model, FMK was found to attenuate the neointimal hyperplasia of carotid arteries. These results suggest that p90RSK impedes PDGF-BB induced VSMC phenotypic switching, proliferation, migration, and neointima formation.

Effects of MicroRNA-24 on Myocardial Fibrosis in Rats After Myocardial Infarction by Targeting Wnt Family Member 4/Dvl-1/β-Catenin Signaling Pathway

In this study, we investigated the impact of miR-24 on myocardial fibrosis severity in rats following myocardial infarction (MI) and explored its underlying mechanisms. We established an MI-induced myocardial fibrosis rat model and assessed cardiac function via echocardiography. We employed Western blotting and RT-qPCR to examine the effects of agomiR-24 on key fibrotic markers, including COL1A1, COL3A1, and α-SMA. Microarray analysis, pathway enrichment, and proteinprotein interaction network analysis revealed the signaling pathways and genes influenced by agomiR-24. Primary rat cardiac fibroblasts (CFs) were isolated, and miR-24’s direct target was confirmed using luciferase reporter assays. We modulated miR-24 expression in CFs and assessed cell proliferation and invasion through CCK-8 and Transwell assays, respectively. Furthermore, we investigated the impact of miR-24 on the Wnt4/Dvl-1/β-catenin signaling pathway by Western blotting. Finally, we examined mRNA expression levels of key genes (Cyclin D1, p27, p21, MMP-3, and MMP-9) through RT-qPCR. Our findings demonstrated that agomiR-24 improved cardiac function and reduced fibrotic marker expression in rat myocardial tissues. MiR-24 inhibited CF proliferation and invasion, potentially by targeting Wnt4/Dvl-1/β- catenin signaling. It also regulated mRNA expression of genes associated with cell proliferation and matrix remodeling. Overall, our study suggests that miR-24 may attenuate myocardial fibrosis in post-MI rats by suppressing the Wnt4/Dvl- 1/β-catenin pathway.

Dicliptera chinensis-derived polysaccharide enhanced the growth activity of submandibular gland cells in vitro after radiotherapy

ObjectiveRadiotherapy for head and neck can damage the salivary gland cells, which can easily result in xerostomia. No effective treatment for radiation-induced salivary gland dysfunction currently exists. Thus, we aimed to study the protective effect of Dicliptera chinensis polysaccharides (DCP) on the prevention of submandibular gland (SMG) cell damage caused by radiotherapy in Sprague-Dawley rats. DesignMechanical enzyme digestion was used to extract primary rat SMG cells. A radiation injury model was established by treating these cells with a dose of 8 Gy, followed by intervention using different DCP concentrations. The cell counting kit 8 assay was used to determine the inhibition rate of SMG cells in each group. The rates of apoptosis and cell cycle progression were detected using flow cytometry. Expression of the Mre11/Rad50/Nbs1 complex (MRN) was detected using western blotting. ResultsDCP increased the proliferation of SMG cells after irradiation, and cell growth activity positively correlated with polysaccharide concentration. Flow cytometry analysis of SMG cell apoptosis revealed that DCP markedly reduced the total apoptosis rate after irradiation, especially the early apoptosis rate. Cell cycle results suggested that DCP reduced the number of cells in the S and G2 phases after irradiation and alleviated the S and G2 blocks. Western blot results indicated that the expression of Mre11, Rad50, and Nbs1 decreased in the radiation-injured group, whereas their expression increased after DCP treatment. ConclusionsDCP can protect the rat SMG cells after radiation and be used as a protective agent against salivary gland cell damage caused by radiotherapy.

Application of Digital Holographic Imaging to Monitor Real-Time Cardiomyocyte Hypertrophy Dynamics in Response to Norepinephrine Stimulation.

Cardiomyocyte hypertrophy, characterized by an increase in cell size, is associated with various cardiovascular diseases driven by factors including hypertension, myocardial infarction, and valve dysfunction. In vitro primary cardiomyocyte culture models have yielded numerous insights into the intrinsic and extrinsic mechanisms driving hypertrophic growth. However, due to limitations in current approaches, the dynamics of cardiomyocyte hypertrophic responses remain poorly characterized. In this study, we evaluate the application of the Holomonitor M4 digital holographic imaging microscope to track dynamic changes in cardiomyocyte surface area and volume in response to norepinephrine treatment, a model hypertrophic stimulus. The Holomonitor M4 permits non-invasive, label-free imaging of three-dimensional changes in cell morphology with minimal phototoxicity, thus enabling long-term imaging studies. Untreated and norepinephrine-stimulated primary neonatal rat cardiomyocytes were live-imaged on the Holomonitor M4, which was followed by image segmentation and single-cell tracking using the HOLOMONITOR App Suite software version 4.0.1.546. The 24 h treatment of cultured cardiomyocytes with norepinephrine increased cardiomyocyte spreading and optical volume as expected, validating the reliability of the approach. Single-cell tracking of both cardiomyocyte surface area and three-dimensional optical volume revealed dynamic increases in these parameters throughout the 24 h imaging period, demonstrating the potential of this technology to explore cardiomyocyte hypertrophic responses with greater temporal resolution; however, technological limitations were also observed and should be considered in the experimental design and interpretation of results. Overall, leveraging the unique advantages of the Holomonitor M4 digital holographic imaging system has the potential to empower future work towards understanding the molecular and cellular mechanisms underlying cardiomyocyte hypertrophy with enhanced temporal clarity.

Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis.

Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.