Tail Vein Research Articles

Abstract B012: HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy

Abstract The high rate of locoregional/distant recurrence in triple-negative breast cancer (TNBC) is a major driver of the decreased survival observed in breast cancer patients. Radiation therapy is a reliable treatment modality to limit recurrence for many cancers, but TNBC patients have a higher chance for relapse despite receiving increased doses of radiation compared to patients with other breast cancer subtypes. This study investigated the differential pathways in radioresistant TNBC tumors that impact recurrence. We developed two radioresistant cell line models (468-RR and 4T1-RR) by administering a radiation dose of 50Gy over the course of 8 weeks to the human TNBC cell line, MDA-MB-468, and the mouse TNBC cell line, 4T1, respectively. Migration and invasion were assessed using the scratch-wound assay and transwell assay, respectively. We assessed in vivo metastasis by injecting the cells in the mammary fat pad, tail vein, or left ventricle. RNA-sequencing of the cell lines was done to identify global transcriptomic changes and regulatory pathways induced by radiation therapy. The mRNA stability of integrin b3 was assessed by inhibiting transcription using actinomycin D. Moreover, we implemented the newly developed InduroRT-mediated circRNA-sequencing (IMRT-seq) to identify the circular RNAs processed by HNRNPL that are present in radioresistant cells. We observed enhanced lung and bone metastasis, based on bioluminescent imaging, in both the spontaneous and vascular metastasis models for the radioresistant cells compared to the parental cells. Genset enrichment analysis identified mRNA stability and metabolism as upregulated pathways in radioresistant cells with HNRNPL, a ribonucleoprotein involved in mRNA stability, as one of the top genes. By knocking down HNRNPL, we observed decreased migration capacity of the radioresistant cells. Given the role of HNRNPL in integrin and extracellular matrix genes, which are key components associated with metastasis in cancer cells, we screened for these genes in our RNA-seq dataset and identified integrin b3 as a possible target. More specifically, knocking down HNRNPL decreased the mRNA stability of integrin b3 as assessed by actinomycin d treatment. By expressing integrin b3 in the HNRNPL-depleted cells, we were able to rescue the migration capacity of these cells. We believe a subset of HNRNPL-mediated circular RNAs are responsible for integrin b3 mRNA stability by sequestering miRNAs that target integrin b3. With IMRT-seq we are exploring the potential circRNA-miRNA interactions present in radioresistant TNBC responsible for its metastatic phenotype. The radioresistant cells derived from TNBC cell lines have an enhanced metastatic potential than the treatment-naive cell lines and our data suggest that RNA metabolism is pivotal to this phenotype. The upregulation of HNRNPL upon radiation by Nrf2 increases integrin b3 expression by stabilizing its transcripts and preventing its degradation. This novel mechanism of radioresistant metastasis provides a therapeutic approach to minimize TNBC recurrence. Citation Format: Ayush Kumar, Kensei Kishimoto, Christi Wisniewski, Shivam Goel, Arthur Mercurio. HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B012.

Microvesicle-Shuttled microRNA-130b Activates the Hepatic Inflammation by Inhibiting Glucocorticoid-Receptor-Mediated Immunosuppression in High-Fat Diet-Induced Obese Mice

Metabolism-disorder-induced liver diseases have become increasingly prevalent worldwide and are clinically linked to obesity and type 2 diabetes. In addition, a large number of previous literature studies have indicated that plasma miR-130b is a promising biomarker for the early diagnosis and treatment of obesity. However, whether miRNA-130b that was positively correlated with obesity resulted in hepatic inflammation needs to be further studied. Therefore, the study aims to determine the effect of microvesicle-shuttled miRNA-130b (miR-130b-MV) on the hepatic inflammation and its potential mechanism in high-fat diet-induced obese mice. Three-week-old C57BL/6 mice were fed a high-fat diet for eight weeks. Then, the obese mice received tail vein injections of MV-packaged scrambled control microRNA (miR-SC-MV) or miR-130b-MV every other day for 10 days. Compared with the control group, the miR-130b-MV injection significantly reduced the body weight while increasing the ratio of liver wet weight to total body weight. In addition, the miR-130b-MV injection significantly activated the hepatic inflammation by increasing the expression of proinflammatory genes, although the plasma concentrations of IL-6 and TNF-α were only slightly increased. Furthermore, the miR-130b-MV injection significantly increased the hepatic miR-130b expression while significantly suppressing the protein expression and phosphorylation of GR, a potential target of miR-130b. Moreover, the miR-130b overexpression results in a decrease in the expression of endogenous GR protein and a decrease in the activity of the luciferase reporter of GR 3′-UTR. In addition, the miR-130b-MV injection significantly upregulated NF-kB (p50) in both the cytoplasm and nucleus, showing enhanced proinflammation response. The above results demonstrated that miR-130b-MV activated the hepatic inflammation by inhibiting GR-mediated immunosuppression in high-fat diet-induced obese mice, suggesting a novel mechanism underlying the obesity-induced hepatic inflammation, and the inhibition of miR-130b may serve as a new molecular therapeutic target for the prevention and treatment of hepatic inflammation.

A new method to predict return of spontaneous circulation by peripheral intravenous analysis during cardiopulmonary resuscitation: a rat model pilot study

Abstract Background Enhancing venous return during cardiopulmonary resuscitation (CPR) can lead to better hemodynamics and improved outcome after cardiac arrest (CA). Peripheral Intravenous Analysis (PIVA) provides feedback on venous flow changes and may indicate an increase in venous return and cardiac output during CPR. We hypothesize PIVA can serve as an early indicator of increased venous return, preceding end-tidal CO2 (etCO2) increase, before the return of spontaneous circulation (ROSC) in a rat model of CA and CPR. Results Eight male Wistar rats were intubated and ventilated, and etCO2 was measured. Vessels were cannulated in the tail vein, femoral vein, femoral artery, and central venous and connected to pressure transducers. Ventilation was discontinued to achieve asphyxial CA. After 8 min, CPR began with ventilation, epinephrine, and automated chest compressions 200 times per minute until mean arterial pressure increased to 120 mmHg. Waveforms were recorded and analyzed. PIVA was calculated using a Fourier transformation of venous waveforms. Data are mean ± SE. Maximum PIVA values occurred in the tail vein 34.7 ± 2.9 s before ROSC, with subsequent PIVA peaks in femoral vein and centrally at 30.9 ± 5.4 and 25.1 ± 5.0 s, respectively. All PIVA peaks preceded etCO2 increase (21.5 ± 3.2 s before ROSC). Conclusion PIVA consistently detected venous pressure changes prior to changes in etCO2. This suggests that PIVA has the potential to serve as an important indicator of venous return and cardiac output during CPR, and also as a predictor of ROSC.

Effect of Different Rates of Rambutan Peel Extract on Visfatin and Cardiac Troponin I Response in Type 2 Diabetic Rats Induced with Streptozotocin

Background: The aim of this study was to investigate the effects of STZ (Streptozotocin) and Rambutan peel extract (RPE) on serum visfatin and cardiac response in experimental Type 2 diabetic rats. Methods: In this study, 64 adult male Wistar albino rats, aged between 8-10 weeks, were used. 8 groups were randomly selected, each containing 8 rats as Control (C), R100 (RPE 100 mg/kg group given with oral gavage route of administration (OGRA), R200 (RPE 200 mg/kg group given with OGRA), R300 (RPE 300 mg/kg group given with OGRA); Diabetes Control (DC): STZ 50 mg/kg i.p administered group, DR100 (D+100 mg/kg JPE), DR200 (D+200 mg/kg JPE), DR300 (D+300 mg/kg JPE). The study lasted a total of 31 days, including adaptation (7 days), induction of diabetes (3 days), and trial period (21 days). Blood samples were taken from the tail vein (vena caudalis) of all subjects on days 0 and 21 of the study. Visfatin and cTnI levels in the serum samples were measured and evaluated by ELISA method. Result: While the increase in mean serum visfatin and cTnI levels in diabetic groups was seen mostly in DC groups, the most significant decrease in mean serum visfatin level due to the addition of RPE was determined in DR100 groups (p less than 0.01).As a result, it was concluded that the administration of 100 mg/kg RPE in rats had no adverse effects. It was concluded that RPE, which has a cardioprotective effect by regulating the visfatin response that plays a role in the development of diabetes, may be useful in supporting the impaired cardiovascular system function that occurs in diabetes.

EXTH-25. OPTIMIZATION OF NEW CHEMOTHERAPEUTIC AGENTS BY INTRA-ARTERIAL INFUSION IN GLIOBLASTOMAS

Abstract Being the most aggressive malignant primary brain tumor, glioblastoma (GBM) is a difficult disease to treat. The first line treatment is well established, but most patients will relapse before or during the treatments. The overall survival is only 14,6 months while the progression-free survival is 6,9 months. In Sherbrooke, Dr Fortin prioritize the intra-arterial (IA) administration of chemotherapeutic agents (CTA) as a second line treatment. Unfortunately, certain patients will not respond or will even develop chemoresistance to the agents. Therefore, there is a strong need for bringing more CTAs onto clinical care. The maximal tolerable dosage (MTD) of every agent was first determined. Every CTAs were then administered IA by retrograde infusion in the external carotid but also intravenously (IV) by the caudal vein. We used the Fischer-F98 glioma rat model in our studies. For the efficacy study, we implanted 10 000 F98 cells into their brain. After 10 days, they received the treatment associated to their group. The animals were followed every day and were euthanized when their condition was too deteriorated. The dosages found in the MTD study for topotecan and cytarabine was 4 mg/kg and 8,75 mg/kg respectively while paclitaxel was too toxic. As for the efficacy study, the IA group of topotecan showed a promising outcome with a higher survival rate (29 days) compared to the IV one (23,5 days). As for the cytarabine, the opposite was observed (IA: 22 days, IV: 26,5 days). While topotecan and cytarabine could be given intra-arterially, only topotecan showed conclusive results. More animals will be added to this group to confirm this outcome. Cytarabine is not a good option since the IV group survived a longer time than the IA group. As for the paclitaxel, the intra-tumoral infusion will be tested to see if we can get positive results.

NIMG-32. ORAL ADMINISTRATION OF DEUTERATED CHOLINE AS A NEW APPROACH TO PROVIDE HIGH TUMOR-TO-BRAIN IMAGE CONTRAST IN DEUTERIUM METABOLIC IMAGING (DMI)

Abstract BACKGROUND Metabolic imaging using FDG-PET is a key component of disease management in many cancers outside of the brain, but in brain tumors it often shows low tumor-to-brain contrast because of high background signal from normal brain. We explore generating cancer-specific metabolic image contrast using Deuterium Metabolic Imaging (DMI), which is a novel 2H magnetic resonance spectroscopic imaging-based technique. DMI was applied after oral administration of deuterated choline, an essential nutrient needed for membrane synthesis whose uptake is often increased in tumors. METHODS Total choline (tCho) DMI was performed in an orthotopic model of glioblastoma, F344 rats (n=11) implanted with RG2 cells, on an 11.7 tesla MRI scanner, with isotropic spatial resolution of 2.5 mm. tCho-DMI data were collected for 36 min during intravenous infusion (IV, n=6) or after 3 days of oral administration (PO, n=5) of [2H9]-choline chloride dissolved in sterile water. At a dose of 285 mg/kg body weight, bolus-continuous IV infusion via the tail vein required 1.57 ml for a 250-gram rat. PO administration via gavage on 3 consecutive days used the max daily dose recommended for human adults, 50 mg/kg. Results - In the PO group, average tumor tCho concentration was 0.78±0.14 mM and 0.20±0.05 mM in contralateral normal brain. In the IV group, the tCho concentration was 0.88±0.18 mM in the tumor, and 0.18±0.03 mM in normal brain (PO vs. IV: p=0.52). Tumor-to-brain contrast was 5.9±1.2 in PO-administered animals, and 7.1±3.3 for animals receiving IV 2H-choline, (p=0.54). CONCLUSIONS - In a preclinical GBM model oral administration of [2H9]-choline resulted in comparably high tumor-to-brain image contrast on tCho-DMI-based maps as during IV infusion. Work is ongoing to identify the different choline metabolites that contribute to the tCho signal. Because oral administration of choline is very safe, this approach could facilitate the translation of tCho-DMI to human subjects.

EXTH-80. A NOVEL BISAMINOQUINOLINE DERIVATIVE NANOPARTICLE (BAQ13-NP) FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Aminoquinolines, such as hydroxycholoquine, are known to inhibit autophagy and slow cancer cell growth, improving survival in preclinical models of glioblastoma. Unfortunately, they failed to demonstrate benefit in clinical trials due to a lack of sufficient potency and BBB penetration. We have developed a novel bisaminoquinoline derivative packaged in a nanoparticle (BAQ13-NP) with improved potency and intratumoral accumulation after systemic administration, confirmed by distribution and toxicity studies in animals. In this study, we evaluated the impact of BAQ13-NP on tumor cell autophagy and survival using patient-derived glioma cell lines and murine models. GBM patient-derived cell lines (n=4) and murine glioma cells (GL261, CT-2A) were treated with BAQ13-NP (1-4 µM) in culture, demonstrating growth restriction to less than 50% of control at 5-7 days (p<0.01) by MTT assay. Cells treated with BAQ13-NP also demonstrated reduced autophagy with significant increases in accumulation of LC3B and p62 (p<0.01) and reduced IL-6 dependent STAT3 phosphorylation in tumor cells, T cells, and myeloid cells (p<0.01). In addition, treatment of tumor cells with BAQ13-NP reduced IL-6 production over 50% (p<0.01). Mice with orthotopic GL261 or CT2A tumors were treated with BAQ13-NP by tail vein injection (25 mg/kg q2 days) starting 7 days post implantation. Significantly increased survival was observed in BAQ13-NP treated GL261 (HR 0.75, p=0.030) and CT2A (HR 0.78, p=0.033) bearing mice compared to vehicle control treated animals. Reduced tumor size at specific intervals was also observed with BAQ13-NP treatment, corelating with improved survival. BAQ13-NP can inhibit autophagy and slow glioma tumor growth, improving survival in preclinical models. Given the excellent distribution and low toxicity of this novel agent when delivered intravenously, we believe it has great potential to improve outcomes for GBM patients. Our drug has recently received IND approval and will soon be tested in a phase I clinical trial.

EXTH-14. IRREVERSIBLE TARGETING OF THE C-MYC/TOPOISOMERASE I AXIS IN GLIOBLASTOMA: DUAL INHIBITION USING A NOVEL INDENOISOQUINOLINE INHIBITOR

Abstract Glioblastoma–the most common primary CNS malignancy–is notorious for its cellular and molecular complexity and uniform fatality. Overexpression of the c-MYC and TOPOISOMERASE 1 (TOP1) oncogenes is implicated in more than 50% of human cancers. They are involved in the pathogenesis of glioblastomas with overexpression correlating with decreased overall median survival in glioblastoma patients. LMP744 is a non-camptothecin indenoisoquinoline small molecule inhibitor designed to target the C-MYC/TOP1 axis. The compound is inherently stable, has high brain penetrance, and irreversibly binds to its target providing several advantages over the camptothecin class of drugs. Human glioblastoma cells treated with LMP744 show a dose-dependent decrease in viability with LD50 ranging between 50 and 100 nM compared to 100-200µM following treatment with temozolomide in the same cell lines. Treatment results in a pro-apoptotic effect in exposed cells as well as S-phase arrest. Protein arrays and confirmatory western blots demonstrate increased CHK2 and decreased p53 and GSK-a/ß in treated cells. Orthotopic xenotransplantation via stereotactic injection of human glioblastoma cells (7×105 cells/4 µL) in nude mice showed exquisite sensitivity to LMP744 without adverse events. Administration of 20 mg/kg of LMP744 via tail vein injection resulted in total eradication of tumor signal on bioluminescent imaging. These preliminary findings suggest that LMP744–and perhaps the class of non-camptothecin indenoisoquinolones–is a promising novel candidate for glioblastoma pharmacotherapy.

Knockdown of hepatic mitochondrial calcium uniporter mitigates MASH and fibrosis in mice

BackgroundMitochondrial calcium uniporter (MCU) plays pleiotropic roles in cellular physiology and pathology that contributes to a variety of diseases, but the role and potential mechanism of MCU in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH) remain poorly understood.Methods and resultsHere, hepatic knockdown of MCU in C57BL/6J mice was achieved by tail vein injection of AAV8-mediated the CRISPR/Cas9. Mice were fed a Choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) for 8 weeks to induce MASH and fibrosis. We find that expression of MCU enhanced in MASH livers of humans and mice. MCU knockdown robustly limits lipid droplet accumulation, steatosis, inflammation, and hepatocyte apoptotic death during MASH development both in vivo in mice and in vitro in cellular models. MCU-deficient mice strikingly mitigate MASH-related fibrosis. Moreover, the protective effects of MCU knockdown against MASH progression are accompanied by a reduced level of mitochondrial calcium, limiting hepatic oxidative stress, and attenuating mitochondrial dysfunction. Mechanically, RNA sequencing analysis and protein immunoblotting indicate that knockdown MCU inhibited the Hippo/YAP pathway activation and restored the AMP-activated protein kinase (AMPK) activity during MASH development both in vitro and in vivo.ConclusionsMCU is up-regulated in MASH livers in humans and mice; and hepatic MCU knockdown protects against diet-induced MASH and fibrosis in mice. Thus, targeting MCU may represent a novel therapeutic strategy for MASH and fibrosis.Graphical

The effect of methylphenidate on the reproductive function of female rats

AbstractAimResearch on the effects of methylphenidate on female fertility is limited. This study evaluated the effects of methylphenidate on reproductive function, oxidants, antioxidants, proinflammatory cytokines, prolactin, and cortisol in female rats.MethodsForty‐eight albino Wistar female rats were divided into four groups consisting of 12 rats, which were given pure water orally once daily for 7 days (HG‐1), 10 mg/kg methylphenidate orally once daily for 7 days (MP‐1), pure water orally once daily for 30 days (HG‐2), and 10 mg/kg methylphenidate orally once daily for 30 days (MP‐2). At the end of the treatment periods, tail vein blood was collected from six rats per group for prolactin and cortisol determination. Subsequently, euthanasia was performed and the ovaries were removed. Ovaries were analyzed for malondialdehyde (MDA), total glutathione (tGSH), superoxide dismutase (SOD), catalase (CAT), interleukin‐1 beta (IL‐1β), and tumor necrosis factor‐alpha (TNF‐α), and immunohistochemically. For breeding, the remaining six rats were mated with male rats for 1 month. Rats that failed to give birth were classed as infertile.ResultsA comparison of MP‐1 and MP‐2 groups to healthy controls revealed an elevation in MDA and corticosterone levels, and a decline in tGSH, SOD, and CAT levels (p < 0.001). Methylphenidate did not affect prolactin, IL‐1β, and TNF‐α levels (p > 0.05). MP‐1 and MP‐2 exhibited immunopositivity for 8‐hydroxy‐2'‐deoxyguanosine (8‐OHDG). MP‐2 rats developed 66.7% infertility while MP‐1, HG‐1, and HG‐2 rats did not.ConclusionIn ovaries, methylphenidate caused oxidative stress, but did not induce inflammation. Long‐term use of methylphenidate caused increased cortisol levels and infertility.

Novel Peptide-Based 68Ga-Labeled Radiotracer for Preclinical Studies of TIM3 Expression.

T-cell immunoglobulin and mucin domain-3 (TIM3) is an immune checkpoint that plays a negative regulatory role in the immune response. TIM3-targeted drugs inhibit this negative regulation, thereby modulating the level of immune response activation. In the previous investigation, several peptides targeting TIM3 were identified through screening from a phage peptide library. In this research, three peptides were selected to construct the radioactive molecular probes according to the characteristic that targeting TIM3 drugs would lead to the increase of interferon-γ (IFN-γ) secretion. Molecular docking was performed to assess the binding properties of the selected peptides with the TIM3 protein. To further enhance the targeting properties, one of the peptides with a higher-affinity peptide was structurally modified. Then, 68Ga was used to construct the peptide probe 68Ga-DOTA-peptide by labeling the six peptides with 68Ga riboprobes, and the binding affinity and specificity were assessed using TIM3 overexpressing cell line A549TIM3 and the parental A549 cells. In addition, in Micro-PET/CT imaging, transfected model mice were dynamically imaged for 30 min after injection of 3.7-7.4 MBq 68Ga-DOTA-peptides via the tail vein. Meanwhile, the same dose of molecular probes was injected in the MC38 model (colorectal cancer in mice) and the CCRCC (clear cell renal cell carcinoma) xenografted model, followed by static scans at 15, 30, and 60 min postinjection. Finally, immunohistochemical (IHC) staining was performed to assess TIM3 expression in the dissected tumor tissues. The molecular docking results showed that the binding energy of P26 to TIM3 protein was -6.5 kcal/mol, which was lower than that of P24 to TIM3 protein, -3.6 kcal/mol, indicating that the affinity of P26 peptide to TIM3 protein was higher than that of P24 and P20 peptide. After structural modification of the P26 peptide, P26NH2, r-NH2, and P26X2 were obtained, and the above peptides were successfully constructed into six targeting TIM3 peptide probes by 68Ga labeling. Cellular uptake experiments demonstrated that 68Ga-DOTA-P26, 68Ga-DOTA-P26NH2, and 68Ga-DOTA-r-NH2 showed significantly higher uptake in A549TIM3 cells than in A549 cells and could be blocked by the unlabeled peptide. Micro-PET imaging experiments showed that the uptake of each probe in the A549TIM3 model tumor tissue was significantly higher than that in the A549 model tumor tissue, and a comparison of the tumor-to-cardiac uptake ratios of each group showed that the 68Ga-DOTA-P26 had a better tumor-to-cardiac uptake ratio in the A549TIM3 model than several other molecular probes, and in the MC38 model, similar results were obtained, with the difference that the 68Ga-DOTA-P26NH2 had the highest tumor-to-cardiac uptake ratio in the CCRCC model. Finally, validation by IHC showed that A549TIM3, MC38, and CCRCC tumor tissues had varying degrees of TIM3 expression. Upon comparison of ex vivo and in vivo studies, one of them, the 68Ga-DOTA-P26 probe, demonstrated significant target specificity for TIM3. These results suggest that studying peptide probes targeting TIM3 will promote the process of TIM3-targeted drug research and is expected to guide the application of TIM3 immune checkpoint drugs in immunotherapy.

Bone marrow mesenchymal stem cells overexpressing stromal cell- derived factor 1 aid in bone formation in osteoporotic mice

BackgroundOsteoporosis is characterized by low systemic bone mineral content and destruction of bone microarchitecture. Promoting bone regeneration and reversing its loss by infusion of exogenous bone marrow mesenchymal stem cells (BMSCs) is a potentially effective treatment for osteoporosis. However, their limited migration to target organs reduces the therapeutic effect of the cells. Stromal cell-derived factor 1 (SDF1) is a chemokine that induces targeted cell migration through the SDF1/CXCR4 (C-X-C chemokine receptor 4) axis and can induce migration of exogenous mesenchymal stem cells to sites of high SDF1 concentration. There are no studies on BMSCs overexpressing SDF1 (SDF1-BMSCs) in osteoporotic mice in vivo. We aimed to investigate if the increased SDF1 concentration facilitated cell migration to the bone.MethodsWe used lentivirus to construct BMSCs overexpressing SDF1 or knocking down CXCR4. We verified the proliferation ability of the cells in vitro using Cell Counting Kit-8 (CCK8) and 5-Bromodeoxyuridinc (BrdU), the migration ability of the cells using Transwell, and the osteogenic and lipogenic ability of the cells using osteogenic and lipogenic induction solutions. In in vivo experiments, we induced osteoporosis in 72 female mice by ovariectomy and injected different groups of cells via the tail vein. Femoral tissue samples were collected for a fixed time, and the osteogenic and homing abilities of the cells were verified by MicroCT and tissue section staining.ResultsWe successfully demonstrated that high expression of SDF1 promoted cell proliferation and migration in vitro, without affecting their cell differentiation ability. In an ovariectomized mouse model, SDF1-BMSCs were more likely to be home to the femur than the BMSCs, had a better pro-osteogenic ability, and had higher expression of Wnt-1. Blocking the SDF1/CXCR4 axis reduced the homing of exogenous mesenchymal stem cells (MSCs) to the femur and their osteogenic capacity.ConclusionsSDF1-BMSCs can further promote bone formation by increasing the number of cells homing to the femur in osteoporotic mice. Our study shows that stem cells can promote their proliferation and home to the femur via the SDF1/CXCR4 axis and further help bone formation via Wnt-1 signaling.

Targeting GSDME-mediated macrophage polarization for enhanced antitumor immunity in hepatocellular carcinoma.

Despite the notable efficacy of anti-PD1 therapy in the management of hepatocellular carcinoma (HCC) patients, resistance in most individuals necessitates additional investigation. For this study, we collected tumor tissues from nine HCC patients receiving anti-PD1 monotherapy and conducted RNA sequencing. These findings revealed significant upregulation of GSDME, which is predominantly expressed by tumor-associated macrophages (TAMs), in anti-PD1-resistant patients. Furthermore, patients with elevated levels of GSDME+ macrophages in HCC tissues presented a poorer prognosis. The analysis of single-cell sequencing data and flow cytometry revealed that the suppression of GSDME expression in nontumor cells resulted in a decrease in the proportion of M2-like macrophages within the tumor microenvironment (TIME) of HCC while concurrently augmenting the cytotoxicity of CD8 + T cells. The non-N-terminal fragment of GSDME within macrophages combines with PDPK1, thereby activating the PI3K-AKT pathway and facilitating M2-like polarization. The small-molecule Eliprodil inhibited the increase in PDPK1 phosphorylation mediated by GSDME site 1. The combination of Eliprodil and anti-PD1 was effective in the treatment of both spontaneous HCC in c-Myc + /+;Alb-Cre + /+ mice and in a hydrodynamic tail vein injection model, which provides a promising strategy for novel combined immunotherapy.

Ethanolamine and Vinyl–Ether Moieties in Brain Phospholipids Modulate Behavior in Rats

Plasmalogens are brain-enriched phospholipids with a vinyl–ether bond at the sn-1 position between the glycerol backbone and the alkyl chain. Previous studies have suggested that plasmalogens modulate locomotor activity, anxiety-like behavior, and cognitive functions in rodents; however, the specific moieties contributing to behavioral regulation are unknown. In this study, we examined the behavioral modulation induced by specific phospholipid moieties. To confirm the permeability of phospholipids in injected liposomes, we measured the fluorescence intensity following intravenous injection of liposomes containing ATTO 740-labeled dioleoylphosphatidylethanolamine. Then, we compared the behavioral effects following injection of liposomes composed of egg phosphatidylcholine (PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (PE 18:0/22:6), PC 18:0/22:6, 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (PE P-18:0/22:6), or PC P-18:0/22:6, into the tail vein of male rats. The time spent in the central region of the open field was significantly reduced after injection of PE 18:0/22:6, harboring an ester bond at sn-1 compared to controls. Furthermore, the discrimination ratio in the novel object recognition test was significantly higher in PC 18:0/22:6 compared to PE 18:0/22:6, suggesting that the substitution of ethanolamine with choline can enhance recognition memory. We demonstrate that the structures of the sn-1 bond and the hydrophilic moiety in the phospholipids can modulate exploratory behaviors and recognition memory in rodents.

Intravenous Regeneration-associated Cell Transplantation Enhances Tissue Recovery in Mice with Acute Ischemic Stroke.

Previously, we reported that transplantation of regeneration-associated cells (RACs) via the ipsilateral external carotid artery reduced stroke volume in mice with permanent occlusion of the middle cerebral artery (MCA). However, intracarotid arterial transplantation is invasive and requires skill, and severe complications may occur, such as thromboembolism, infection, and decreased cerebral blood flow. This study aimed to investigate the efficacy of intravenous injection of RACs in reducing stroke volume and increasing anti-inflammatory and angiogenic factors in mice with focal cerebral ischemia. Mice with occluded MCAs received intravenous injections of phosphate-buffered saline (PBS) (control), low-dose RACs, or high-dose RACs. The proximal part of the left MCA was occluded to induce permanent focal ischemia. After 3 days, we administered PBS or low-dose (1 × 104 /50 µL) or high-dose RACs (1 × 105 /50 µL) through the tail vein and assessed the infarct volume on day 7. High-dose RACs significantly decreased infarct volume compared to PBS, whereas low-dose RACs showed no effect. The number of interleukin-10 (IL-10)-positive and vascular endothelial growth factor (VEGF)-positive cells in the peri-infarct area on day 7 was significantly higher in mice treated with low-dose and high-dose RACs than in the PBS control group. Intravenous injection of RACs can reduce ischemic stroke volume; however, a higher dose of RACs is required than the dose used in intraarterial transplantation. By assessing IL-10 and VEGF expression, the study sheds light on the underlying mechanisms of RAC therapy, revealing its potential anti-inflammatory and angiogenic properties in the treatment of cerebral ischemia.

Sex and Age-Dependent Effects of miR-15a/16-1 Antagomir on Ischemic Stroke Outcomes

Ischemic stroke is a leading cause of disability and mortality worldwide. Recently, increasing evidence implicates microRNAs (miRs) in the pathophysiology of ischemic stroke. Studies have shown that miR-15a/16-1 is abnormally expressed in brains after ischemic stroke, and its upregulation may increase ischemic damage. Given that sex and age are significant modifiers of stroke outcomes, here we investigated whether inhibiting miR-15a/16-1 with antagomirs mitigates cerebral ischemia/reperfusion (I/R) injury in a sex- and age-dependent manner. Young (3 months) and aged (18 months) male and female C57/BL mice underwent 1-h middle cerebral artery occlusion and 3–7 days reperfusion (tMCAO). We administered miR-15a/16-1 antagomir (30 pmol/g) or control antagomir (NC, 30 pmol/g) via tail vein 2 h post-MCAO. Neurobehavioral testing and infarct volume assessment were performed on days 3 and 7. Compared to controls, antagomir treatment significantly improved neurobehavioral outcomes and reduced infarct volume in tMCAO mice at day 7, with the effects being more pronounced in young mice. Notably, young female mice exhibited superior survival and sensorimotor function compared to young male mice. These results were also replicated in a permanent MCAO (pMCAO) mice model. This suggests miR-15a/16-1 antagomir and estradiol may synergistically regulate genes involved in neurovascular cell death, inflammation, and oxidative stress, with sex and age-dependent expression of miR-15a/16-1 and its targets likely underlying the observed variations. Overall, our findings identify miR-15a/16-1 antagomir as a promising therapeutic for ischemic stroke and suggest that sex and age should be considered when developing miR-based therapeutic strategies.

Overexpression of SULT1E1 alleviates salt-processed Psoraleae Fructus-induced cholestatic liver damage

ObjectiveSalt-processed Psoraleae fructus (SPF) is widely used as a phytoestrogen-like agent in the treatment of osteoporosis. However, due to improper clinical use or misuse, resulting in liver damage. In this study, network pharmacology was employed to analyze the mechanism of cholestatic liver damage. An adeno-associated virus overexpressing SULT1E1 (rAAV8-SULT1E1) was constructed and the hepatotoxicity of SPF, psoralen, and isopsoralen was determined. MethodsBy utilizing three databases inclding TCMSP, TCMID, and BATMAN- TCM, the targets of the three databases were summarized, and a total of 45 psoralen compounds were included. Network pharmacology analysis was then performed. The adenoviral vectors were injected into the tail vein of C57BL6 mice to elucidate the role of SULT1E1 in SPF-induced cholestasis-mediated hepatotoxicity in vivo. SPF (10 g/kg), psoralen, and isopsoralen (50 mg/kg each) were intragastrically administered to mice for 30 d. B-ultrasound and samples were collected and examined for follow-up experiments. ResultsA total of 854 targets were predicted for 45 active components, with 151 cholestasis-mediated hepatotoxicity-related disease targets obtained for SPF. A total of 126 pathways were enriched based on KEGG pathway analysis, with the “estrogen signaling pathway” identified as one of the top 20 pathways. In terms of pathological hepatic changes, treated mice had visually swollen hepatocytes, dilated bile ducts, and elevated serum biochemical markers, which were more prominent in mice treated with isopsoralen than in those treated with other compounds. Notably, the overexpression of SULT1E1 could reverse liver damage in each treatment group. B-ultrasound was used to observe the size of the gallbladder in vivo. The size of the gallbladder was found to significantly increase on day 30 after treatment in the SPF-, psoralen-, and isopsoralen-treated groups, especially the SPF group. Compared with the expression levels in the negative control group (rAAV8-empty+con), the expression levels of FXR, Mrp2, Bsep, SULT1E1, SULT2A1, Ntcp, and Nrf2 decreased, whereas those of CYP7a1 and IL-6 increased in the SPF-, psoralen-, and isopsoralen-treated groups. ConclusionThe overexpression of SULT1E1 could alleviate the decreased or increased expression of indicators, indicating that SULT1E1 is an important target gene for SPF-induced liver damage. The severity of liver damage was significantly lower in the rAAV8-SULT1E1 groups than in the rAAV8-empty groups.

Role of Glutamine Synthetase on Vascular Permeability in Gliomas.

This study aimed to investigate the effect and underlying mechanism of inhibiting glutamine synthetase (GS) on the vascular permeability of gliomas. C6 glioma rat models were randomly divided into control and L-methionine sulfoximine (MSO) treatment groups. MSO was intraperitoneally injected once every other day for a total of three injections in the MSO group. We assessed the effect of MSO on tumor vascular permeability by tail vein injection of Evans blue dye. GS activity, glutamate (Glu) concentration, glutamine (Gln) concentration, and arginine concentration in tumor tissues were measured using the corresponding kits. qPCR experiments were then conducted to examine the effect of glutamate concentration on N-methyl-D-aspartate (NMDA) receptor expression. Finally, the nitric oxide synthase (NOS) assay kit and the nitric oxide (NO) assay kit were employed to detect NOS activity and NO concentration changes, respectively. Increased glioma tumor vascular permeability was observed after intraperitoneal injection of MSO; MSO acted as an inhibitor of GS, leading to a decrease in GS activity; increased glutamate levels caused activation of NMDA receptors and further activation of NOS; additionally, elevated NO levels were detected in association with an increase in arginine and NOS. Inhibiting GS results in increased vascular permeability in gliomas, which is associated with elevated NO levels and the vasodilatory effects of NO.

Anti-Thrombotic Effect of Protoparaxotriol Saponins From Panax notoginseng Using Zebrafish Model.

Panax notoginseng has the effect of stimulating circulation to end stasis. Our study was designed to evaluate the anti-thrombotic effect of protoparaxotriol saponins (PTS) from P. notoginseng and the involved mechanisms. A thrombosis model was constructed, and the anti-thrombotic activity of PTS was determined by erythrocyte staining, heart rate, and blood flow velocity. In addition, quantitative real-time polymerase chain reaction was used to identify changes in the expression of genes related to coagulation, inflammation, and apoptosis. PTS alleviated arachidonic acid-induced caudal vein thrombosis, restored blood flow, and increased the area of cardiac erythrocyte staining, heart rate, and blood flow velocity. It reduced the ponatinib-induced cerebral thrombus area and decreased the intensity of erythrocyte staining. The quantitative polymerase chain reaction data showed that the anti-thrombotic effect of PTS was mediated by suppression of genes related to coagulation, inflammation, and apoptosis and also involved inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathways.

(Pro)renin receptor aggravates myocardial pyroptosis in diabetic cardiomyopathy through AMPK-NLRP3 pathway

IntroductionAs one of the most common complications of diabetes, diabetic cardiomyopathy (DCM) is the main cause of heart failure in patients with diabetes. However, the lack of effective treatments for DCM remains a clinical challenge. (Pro) renin receptor (PRR) is a member of renin angiotensin aldosterone system (RAAS). Here, we aim to determine whether PRR is involved in myocardial pyroptosis in diabetic cardiomyopathy.MethodsWe established diabetic rats model by intraperitoneal injection of streptozotocin (STZ). PRR overexpression adenovirus or PRR knockdown adenovirus was injected into the tail vein. Western blot, histopathology and immunohistochemistry staining, ELISA and Echocardiography were used to detect cardiac function changes and myocardial injury levels of DCM rats. Primary cardiomyocytes were stimulated with high glucose and PRR overexpression or PRR knockdown was achieved by adenovirus transfection, we also used the inhibitor of AMPK to decrease the activity of AMPK. Western blot, Real-time PCR, Immunofluorescence and ELISA were used to detect the level of PRR and pyroptosis in cardiomyocyte.ResultsWe found that high glucose increased the expression of PRR in heart. After overexpression of PRR, the expression of the pyroptosis related proteins such as Caspase-1, IL-1β, IL-18, and NLRP3 was significantly increased, the phosphorylation level of AMPK was significantly decreased, and the fibrosis level was significantly increased, thus aggravating the cardiac function injury of DCM. On the contrary, PRR knockdown can alleviate the level of myocardial pyroptosis in DCM and improve cardiac function. The related mechanism was that PRR could inhibit AMPK phosphorylation and promote the activation of NLRP3 inflammasome.DiscussionPRR aggravated pyroptosis of cardiomyocyte, increased the dysfunction of cardiomyocyte, and may be related to the decrease of AMPK phosphorylation and the overactivation of NLRP3. This may provide new ideas and targets for the treatment of DCM.