Immunofluorescence Staining Of Cells Research Articles

Abstract 4137339: Application of PLGA-PEG-PLGA Thermosensitive Hydrogel Loaded with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Ginsenoside Rb3 in the Treatment of Acute Myocardial Infarction

Background: Ginsenoside Rb3 (GSRb3) is a traditional Chinese medicine monomer. We aim to investigate the effects of GSRb3 on the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and the reparative effects of PLGA-PEG-PLGA thermosensitive hydrogel loaded with hiPSC-CMs and GSRb3 (NP GSRb3 @Gel+iCM) on myocardial infarction (MI). Methods: 1. Western Blot, RT-qPCR, cell immunofluorescence staining, and CCK-8 assays were employed to assess the proliferative effects of different concentrations of GSRb3 on neonatal mouse primary cardiomyocytes (NCMs) and hiPSC-CMs. 2. Transcriptomic gene sequencing technology was utilized to investigate the mRNA expression changes of hiPSC-CMs induced by GSRb3; The NP GSRb3 @Gel+iCM was injected into the left ventricular myocardium of the MI mice; the same volume injections of hiPSC-CMs (iCM group) and PLGA-PEG-PLGA thermosensitive hydrogel loaded with GSRb3 (NP GSRb3 @Gel group) served as control; Additionally, MI model group without additional treatment and sham group were established. 3. At 4 weeks post-MI, echocardiography, Sirius Red/Fast Green staining and immunofluorescence staining were utilized to evaluate the heart function, infarction size and survival rate of transplanted hiPSC-CMs. Results: GSRb3 inhibit the apoptosis of NCMs while promote proliferation of NCMs and hiPSC-CMs significantly. After 4 weeks of treatment, the engraftment rate of hiPSC-CMs in NP GSRb3 @Gel+iCM group (36.7%) was 4.8 times that of iCM group (7.7%); Compared to the MI group, NP GSRb3 @Gel group and iCM group, NP GSRb3 @Gel+iCM group showed significant improvement in cardiac function; The myocardial repair effect in NP GSRb3 @Gel+iCM group was significantly superior to NP GSRb3 @Gel group and iCM group, with a 47% reduction in infarct size compared to MI group, and further reduction in left ventricular anterior wall thickness compared to NP GSRb3 @Gel group and iCM group. Conclusion: GSRb3 promotes the proliferation of hiPSC-CMs both in vitro and in vivo. NP GSRb3 @Gel+iCM significantly increase the engraftment of hiPSC-CMs, promote angiogenesis, reduce infarction size, inhibit left ventricular remodeling and improve cardiac function.

IMMU-35. TARGETING THE AHR IMMUNOMETABOLIC HUB DURING VIROIMMUNOTHERAPY OF GLIOMAS

Abstract Glioblastoma (GBM) poses a significant therapeutic challenge in oncology. Recent clinical trials have shown the feasibility of virotherapy to treat patients with GBM. Our laboratory developed an oncolytic adenovirus termed Delta-24-RGD that has been tested in clinical trials for recurrent GBM patients with encouraging results (NCT00805376, NCT02798406). These clinical studies, together with preclinical data, showed that the efficacy of Delta-24-RGD was due to direct tumor cell oncolysis and indirect activation of anti-tumor immune response. To further leverage this immune component, our group generated Delta-24-RGDOX, an armed version of Delta-24-RGD expressing the T-cell activator OX40-L, which exhibits a superior anti-cancer effect. In this project, we aim to further boost the effectiveness of Delta-24-RGDOX by targeting factors that maintain the immunosuppressive characteristic of gliomas. Specifically, we identified the environmental sensor and transcription factor Aryl Hydrocarbon Receptor (AhR) as one of the immunosuppressive factors. We show that AhR is overexpressed in GBM surgical specimens compared to healthy brain tissue, and high AhR expression correlates with poorer survival in glioma patients. Immunofluorescence staining of glioma cells treated with Delta-24-RGDOX demonstrated that AhR translocates to the nucleus, and qPCR showed that translocation resulted in the enhanced transcription of AhR-responsive elements. Additionally, bulk RNA-seq reveals that in vivo glioma models treated with Delta-24-RGDOX exhibit activation of the AhR pathway network. This unexpected and paradoxical activation may be responsible for immune suppression mechanisms, which might hinder the effect of Delta-24-RGDOX. Based on these data, we combined two innovative strategies, the oncolytic virus Delta-24-RGDOX and AhR inhibitors. Survival analysis in immunocompetent models of gliomas treated with this combined therapy showed the greatest therapeutic benefit compared to monotherapy controls. These findings highlight the critical role of AhR activation triggered by Delta-24-RGDOX and position AhR as a potential therapeutic target for enhancing the efficacy of oncolytic adenovirus therapy in GBM.

Targeting SPP1-orchestrated neutrophil extracellular traps-dominant pre-metastatic niche reduced HCC lung metastasis.

The mechanisms by which tumor-derived factors remodel the microenvironment of target organs to facilitate cancer metastasis, especially organ-specific metastasis, remains obscure. Our previous studies have demonstrated that SPP1 plays a key role in promoting metastasis of hepatocellular carcinoma (HCC). However, the functional roles and mechanisms of tumor-derived SPP1 in shaping the pre-metastatic niche (PMN) and promoting lung-specific metastasis are unclear. Orthotopic metastasis models, experimental metastasis models, CyTOF and flow cytometry were conducted to explore the function of SPP1 in shaping neutrophil-dominant PMN and promoting HCC lung metastasis. The main source of CXCL1 in lung tissues was investigated via fluorescence activated cell sorting and immunofluorescence staining. The expression of neutrophils and neutrophil extracellular traps (NETs) markers was detected in the lung metastatic lesions of HCC patients and mouse lung specimens. The therapeutic significance was explored via in vivo DNase I and CXCR2 inhibitor assays. SPP1 promoted HCC lung colonization and metastasis by modifying pulmonary PMN in various murine models, and plasma SPP1 levels were closely associated with lung metastasis in HCC patients. Mechanistically, SPP1 binded to CD44 on lung alveolar epithelial cells to produce CXCL1, thereby attracting and forming neutrophil-abundant PMN in the lung. The recruited neutrophils were activated by SPP1 and then formed NETs-dominant PMN to trap the disseminated tumor cells and promote metastatic colonization. Moreover, early intervention of SPP1-orchestrated PMN by co-targeting the CXCL1-CXCR2 axis and NETs formation could efficiently inhibit the lung metastasis of HCC. Our study illustrates that HCC-lung host cell-neutrophil interactions play important roles in PMN formation and SPP1-induced HCC lung metastasis. Early intervention in SPP1-orchestrated PMN via CXCR2 inhibitor and DNase I is a potential therapeutic strategy to combat HCC lung metastasis.

Trifolirhizin targets PTK6 to induce autophagy and exerts antitumor effects in nasopharyngeal carcinoma.

Trifolirhizin, a natural flavonoid glycoside, has been proved to exert antitumor activities in various human malignant tumors. PTK6 was identified as a direct target of trifolirhizin based on public database SuperPred (https://prediction.charite.de/). Overexpressed PTK6 in a variety of tumors is closely associated with the malignant development of tumors. Herein, this present research was formulated to elaborate the effects of trifolirhizin on the biological behaviors of nasopharyngeal carcinoma (NPC) cells and to probe into the intrinsic mechanisms. The current study firstly elucidated the tumor-inhibiting functions of trifolirhizin in NPC malignant progression from the perspective of targeting inhibition of PTK6. In this work, CCK-8 for cell viability, EdU staining for cell proliferation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for cell apoptosis and immunofluorescence staining for LC3 expression were performed. Besides, levels of proliferation-related, apoptosis-related and autophagy-related proteins were detected by western blot analysis. Moreover, molecular docking of trifolirhizin with PTK6 was conducted to seek the compound-protein binding potential. It was demonstrated that trifolirhizin treatment inhibited the proliferation and promoted the apoptosis of NPC cells as well as strengthened autophagy in NPC cells. Furthermore, it was verified that trifolirhizin targeted PTK6 and negatively regulated PTK6 expression. The suppressive effects of trifolirhizin on the malignant behaviors of NPC cells and the enhancing effect of trifolirhizin on autophagy in NPC cells were partly abolished upon upregulation of PTK6. To conclude, findings suggested that trifolirhizin may downregulate PTK6 expression to induce autophagy and exert the antitumor activities in NPC.

PFAS-mediated disruptions on thyroid histology

Abstract Introduction/Objective Per-and polyfluroalkyl substances (PFAS), are environmental contaminants of emerging concern due to their abundance in the environment and potential adverse health impacts. PFAS are used in waterproof clothing, makeup, carpets, upholstery, cookware, and fast-food containers. These organic pollutants have been found in the blood of 98% of Americans and have been linked to disruption in thyroid hormones. During fetal life, thyroid hormones are crucial for brain development and alterations in these hormones can induce long-term impacts on intellectual and brain impairments. T3 and T4 are known to be altered by PFAS exposure, but the mechanism by which this is done remains unclear. Methods/Case Report Normal thyroid cells were treated for 72 hours with 1000 nM solution containing either GEnX, PFOA, PFOS, or 1% DMSO control. Immunofluorescence for actin, tubulin, and DAPI was performed using a Nikon spinning disk. In vivo studies where 8-week-old mice are gavaged with 7.5 mg/kg of PFOS, PFOA, and GenX or PBS control for 8 weeks. Results (if a Case Study enter NA) We hypothesized the PFAS treatment alters thyroid cells morphology and histology. Immunofluorescence staining of normal thyroid cells treated with PFAS show a decrease in actin stress fibers compared to control cells. PFAS treated mice show a decrease in follicle size compared to control (average 1017 um2 size, p=0.0483). Conclusion This is the first study to show changes in thyroid histology after 8 weeks of PFAS exposure. As structure and function are closely linked, it is likely that this reduction in follicle size and actin reorganization contribute to alterations in thyroid function. Future studies are needed to illuminate the mechanism by which PFAS alter thyroid function in vivo. Understanding the role of PFAS-mediated thyroid dysregulation will have far reaching implications on environmental regulations and considerations for limiting PFAS in our environment.

Clinical efficacy of Fufang Yinhua Jiedu (FFYH) granules in mild COVID-19 and its anti-SARS-CoV-2 mechanism by blocking autophagy through inhibiting the AKT/mTOR signaling pathway.

Fufang Yinhua Jiedu (FFYH) granules are recommended for treating coronavirus pneumonia (COVID-19) in China. However, its anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity and clinical efficacy against COVID-19 remain to be confirmed. Our study aimed to investigate the anti-SARS-CoV-2 effect and potential mechanism of FFYH. The activity of FFYH against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was evaluated via cell pathogenic effects, immunoblotting, immunofluorescence staining, and qRT-PCR. The potential mechanism of FFYH against SARS-CoV-2 was investigated by immunoblotting. One head-to-head randomized controlled trial was designed to evaluate the clinical efficacy of FFYH in mild COVID-19. Two hundred patients were randomly recruited to receive either FFYH or LHQW (Lianhua Qingwen) granules. The in vitro results indicated that FFYH effectively inhibited SARS-CoV-2 replication by suppressing CPE and decreasing viral RNA and protein expression. A time-of-drug-addition assay confirmed that FFYH mainly targeted the binding and replication stages of the SARS-CoV-2 life cycle. Mechanistic studies revealed that blocking SARS-CoV-2-triggered autophagy may be the primary mechanism by which FFYH protects against SARS-CoV-2 infection by regulating the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Clinical results confirmed that FFYH effectively shortened the recovery time of clinical symptoms and viral nucleic acid negativity, improved abnormal hematology parameters, and controlled excessive cytokine responses in mild COVID-19 patients. Subgroup analysis revealed that FFYH improved the recovery time of clinical symptoms, improved hematological parameters, and controlled excessive cytokine storms to a greater extent in the mild COVID-19 male subgroup, abnormal hematology subgroup, and 32-42-year-old subgroup than in the corresponding LHQW subgroup (P < 0.05). No patients progressed to severe or critical cases. Our results indicate that FFYH not only has good anti-viral activity against SARS-CoV-2 but also has significant efficacy against COVID-19, indicating that FFYH may be a novel complementary option for treating COVID-19.

Cocaine amphetamine-regulated transcription peptide inhibits apoptosis in oxygen-glucose deprived neural stem cells.

Apoptosis has been recognized as a critical pathophysiological process during cerebral ischemia. The neuroprotective effect of CART on ischemic brain injury is determined. However, there is little research on the protective effect of CART on neural stem cells (NSCs). Primary cultured rat NSCs were utilized as the research subject. In vitro oxygen glucose deprivation (OGD) treatment was employed, and NSCs were extracted from SD pregnant rats following previous experimental protocols and identified through cell immunofluorescence staining. The appropriate concentration of CART affecting OGD NSCs was initially screened using Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) assays. EdU staining and Western blotting (WB) techniques were employed to assess the impact of the suitable CART concentration on the proliferation and apoptosis of OGD NSCs. Finally, Western blot analysis was conducted to investigate the cAMP-response element binding protein (CREB) pathway and expression levels of related proteins after KG-501 treatment in order to elucidate the mechanism underlying apoptosis and proliferation regulation in OGD NSCs. CCK-8 and LDH assays indicated that a concentration of 0.8 nM CART may be the optimal concentration for modulating the proliferation of OGD NSCs. Subsequently, cellular immunofluorescence and EdU detection experiments further confirmed the findings obtained from CCK-8 analysis. Western blot analysis of apoptosis-related protein expression also demonstrated that an appropriate concentration of CART could suppress the apoptosis of OGD NSCs. Finally, Western blotting was conducted to examine the CREB pathway and related protein expression after treatment with KG-501, revealing that an appropriate concentration of CART regulated both apoptosis and proliferation in OGD NSCs through CREB signaling. The concentration of CART at 0.8 nM may be deemed appropriate for inhibiting apoptosis and promoting proliferation in OGD NSCs in vitro. The mechanism maybe through activating the CREB pathway.

Preparation of Biomimetic Selenium-Baicalein Nanoparticles and Their Targeted Therapeutic Application in Nonsmall Cell Lung Cancer.

In this study, we prepared bionic selenium-baicalein nanoparticles (ACM-SSe-BE) for the targeted treatment of nonsmall cell lung cancer. Due to the coating of the A549 membrane, the system has homologous targeting capabilities, allowing for the preparation of target tumor cells. The borate ester bond between selenium nanoparticles (SSe) and baicalein (BE) is pH-sensitive and can break under acidic conditions in the tumor microenvironment to achieve the targeted release of BE at the tumor site. Moreover, SSe further enhances the antitumor effect of BE by increasing the production of ROS in tumor cells. Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) showed that the ACM-SSe-BE had a particle size of approximately 155 ± 2 nm. FTIR verified the successful coupling of SSe and BE. In vitro release experiments indicated that the cumulative release of ACM-SSe-BE at pH 5.5 after 24 h was 69.39 ± 1.07%, which was less than the 20% release at pH 7.4, confirming the pH-sensitive release of BE in ACM-SSe-BE. Cell uptake experiments and in vivo imaging showed that ACM-SSe-BE had good targeting ability. The results of MTT, flow cytometry, Western blot, and cell immunofluorescence staining demonstrated that ACM-SSe-BE promoted A549 cell apoptosis and inhibited cell proliferation. The in vivo antitumor results were consistent with those of the cell experiments. These results clearly suggested that ACM-SSe-BE will be a promising bionic nanosystem for the treatment of nonsmall cell lung cancer.

The regulatory effect of TiO2 nanotubes loaded with graphene oxide on macrophage polarization in an inflammatory environment

BackgroundExcessive inflammation is a major cause of implant failure. The surface morphology, hydrophilicity, and loading of biomaterials are major properties modulating anti-inflammatory macrophage activation. This paper investigates the regulatory effects of modifying the surface of Titanium dioxide nanotubes (TNTs) with graphene oxide (GO) on the polarization of mouse monocyte macrophages (RAW264.7).MethodsTNT was produced by the anodic oxidation of titanium. GO was subsequently electrodeposited on the TNT to obtain a TNT–GO composite. The samples were characterised through scanning electron microscopy (SEM), Raman spectroscopy, and X-ray diffraction. RAW264.7 cells were separately seeded onto the surface of three groups of samples: pure Ti, TNT, and TNT–GO. Under the condition of lipopolysaccharide stimulation, the influence of the sample surfaces on the gene expression profiles was investigated through RNA sequence analysis. In addition, cell spreading was observed through SEM, cell adhesion and proliferation were analysed using the CCK8 assay, and the expression of inflammation-related factors was investigated by ELISA and cellular immunofluorescence staining. The production of reactive oxygen species (ROS) in the RAW264.7 cells on the surface of the three groups was detected via immunofluorescence staining.ResultsThe CCK8 results indicated that the adhesion and proliferation of the RAW264.7 cells were reduced on the TNT and TNT–GO surfaces. ELISA results revealed significant differences in the pro-inflammatory factors tumour necrosis factor-α and interleukin-6 secretion among the three groups at 24 h (p < 0.05). The secretion of pro-inflammatory factors significantly reduced and the expression of anti-inflammatory factor IL-10 increased on the TNT and TNT–GO surfaces. The RNA sequencing, ELISA, and cell immunofluorescence staining test results suggested that the inflammatory response of M1 polarization was reduced and the M2 polarization of macrophages was induced on the TNT–GO surface, which may be attributed to the reduction in ROS production.ConclusionsUnder lipopolysaccharide stimulation, the inflammatory response of the RAW264.7 cells was reduced and the M2 polarization of macrophages was promoted on the TNT–GO surface, which may be caused by the reduced ROS production. Consequently, the designed TNT–GO material is promising for implants owing to its excellent inflammation regulation ability.

Oxytocin Attenuates Sympathetic Innervation with Inhibition of Cardiac Mast Cell Degranulation in Rats after Myocardial Infarction.

Sympathetic hyperinnervation is the leading cause of fatal ventricular arrhythmia (VA) after myocardial infarction (MI). Cardiac mast cells cause arrhythmias directly through degranulation. However, the role and mechanism of mast cell degranulation in sympathetic remodeling remain unknown. We investigated the role of oxytocin (OT) in stabilizing cardiac mast cells and improving sympathetic innervation in rats. MI was induced by coronary artery ligation. Western blotting, immunofluorescence, and toluidine staining of mast cells were performed to determine the expression and location of target protein. Mast cells accumulated significantly in peri-infarcted tissues and were present in a degranulated state. They expressed OT receptor (OTR), and OT infusion reduced the number of degranulated cardiac mast cells post-MI. Sympathetic hyperinnervation was attenuated as assessed by immunofluorescence for tyrosine hydroxylase (TH). Seven days post-MI, the arrhythmia score of programmed electrical stimulation was higher in vehicle-treated rats with MI than in rats treated with OT. An in vitro study showed that OT stabilized mast cells via the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Further in vivo studies on OTR-deficient mice showed worsening mast cell degranulation and worsening sympathetic innervation. OT pretreatment inhibited cardiac mast cell degranulation post-MI and prevented sympathetic hyperinnervation, along with mast cell stabilization via the PI3K/Akt pathway. SIGNIFICANCE STATEMENT: This is the first study to elucidate the role and mechanism of oxytocin (OT) in inflammatory-sympathetic communication mediated sympathetic hyperinnervation after myocardial infarction (MI), providing new approaches to prevent fatal arrhythmias.

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

Role of ERK and NF-KB pathway in neurally mediated enhanced expression and traffcking of SGLT2 in congestive heart failure

Congestive heart failure (CHF) is characterized by the activation of neurohumoral systems concomitant with avid fluid retention. Recently we showed enhanced expression of sodium-glucose cotransporter 2 (SGLT2) in rats with CHF. However, the exact molecular mechanism linking enhanced sympathetic tone and enhanced expression and translocation of SGLT2 in CHF is not clear. The objective of this study was to investigate the role of enhanced sympathetic tone on the expression and traffcking of SGLT2 via activation of protein kinase A (PKA), extracellular signal-regulated kinases (ERK), and nuclear factor kappa B (NF-KB) in the renal cortex of CHF rats. Male Sprague Dawley rats were subjected to either the left coronary artery ligation for the CHF group or Sham surgery. Four weeks after surgery, expression of protein levels (PKA, ERK and NF-KB) were assessed by Western blotting, whereas the location of SGLT2 expression was determined by immunohistological and immunofluorescent staining in the cortical region of kidneys from Sham and CHF rats. Effect of exogenous norepinephrine (NE) (10μM for 24hrs) on expression and traffcking of SGLT2, phosphorylation of ERK and NF-KB in HK2 cells were examined in an in vitro model. Expression of SGLT2 was significantly enhanced in the renal cortex of rats with CHF versus Sham (0.83±0.06 vs. 0.58±0.02). Also, the translocation of SGLT2 was significantly enhanced towards the membrane in CHF rats (0.92±0.11 vs. 0.33±0.07). Phosphorylation of PKA (0.05±0.01 vs. 0.01±0.01), ERK (0.32±0.02 vs. 0.23±0.02) and NF-KB (0.71±0.14 vs. 0.08±0.06) were significantly enhanced in CHF compared to Sham. NE treatment significantly increased expression of SGLT2 (0.26±0.03 vs. 0.15±0.02), traffcking of SGLT2 (0.45 ± 0.09 vs. 0.15 ± 0.03) towards membrane, phosphorylation of ERK (0.38±0.05 vs. 0.14±0.02) and NF-KB (0.61±0.08 vs. 0.17±0.07) in HK2 cells. Thus, NE treatment enhanced the phosphorylation of ERK, NF-kB as well as traffcking of SGLT2. Immunohistological and immunofluorescent staining demonstrated increased expression of SGLT2 in the kidneys of rats with CHF. Similar results were observed in immunofluorescent staining of human kidney cells treated with NE. In conclusion, these findings indicate that NE activates ERK and NF-KB pathways which leads to enhanced traffcking and overexpression of SGLT2 in CHF. These results provide a molecular link between enhanced sympathetic tone, ERK and NF-KB pathway and the traffcking of SGLT2 to the luminal membrane of renal proximal tubular cells and consequent increase in sodium retention in rats with CHF. This study was funded by NIH R01-DK-129311. 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.

Production of Plant-Based, Film-Type Scaffolds Using Alginate and Corn Starch for the Culture of Bovine Myoblasts.

Natural scaffolds have been the cornerstone of tissue engineering for decades, providing ideal environments for cell growth within extracellular matrices. Previous studies have favored animal-derived materials, including collagen, gelatin, and laminin, owing to their superior effects in promoting cell attachment, proliferation, and differentiation compared to non-animal scaffolds, and used immortalized cell lines. However, for cultured meat production, non-animal-derived scaffolds with edible cells are preferred. Our study represents the first research to describe plant-derived, film-type scaffolds to overcome limitations associated with previously reported thick, gel-type scaffolds completely devoid of animal-derived materials. This approach has been employed to address the difficulties of fostering bovine muscle cell survival, migration, and differentiation in three-dimensional co-cultures. Primary bovine myoblasts from Bos Taurus Coreanae were harvested and seeded on alginate (Algi) or corn-derived alginate (AlgiC) scaffolds. Scaffold functionalities, including biocompatibility and the promotion of cell proliferation and differentiation, were evaluated using cell viability assays, immunofluorescence staining, and reverse transcription-quantitative polymerase chain reaction. Our results reveal a statistically significant 71.7% decrease in production time using film-type scaffolds relative to that for gel-type scaffolds, which can be maintained for up to 7 days. Film-type scaffolds enhanced initial cell attachment owing to their flatness and thinness relative to gel-type scaffolds. Algi and AlgiC film-type scaffolds both demonstrated low cytotoxicity over seven days of cell culture. Our findings indicated that PAX7 expression increased 16.5-fold in alginate scaffolds and 22.8-fold in AlgiC from day 1 to day 3. Moreover, at the differentiation stage on day 7, MHC expression was elevated 41.8-fold (Algi) and 32.7-fold (AlgiC), providing initial confirmation of the differentiation potential of bovine muscle cells. These findings suggest that both Algi and AlgiC film scaffolds are advantageous for cultured meat production.

Glutenin-chitosan 3D porous scaffolds with tunable stiffness and systematized microstructure for cultured meat model

A glutenin (G)-chitosan (CS) complex (G-CS) was cross-linked by water annealing with aim to prepare structured 3D porous cultured meat scaffolds (CMS) here. The CMS has pore diameters ranging from 18 to 67 μm and compressive moduli from 16.09 to 60.35 kPa, along with the mixing ratio of G/CS. SEM showed the porous organized structure of CMS. FTIR and CD showed the increscent content of α-helix and β-sheet of G and strengthened hydrogen-bondings among G-CS molecules, which strengthened the stiffness of G-CS. Raman spectra exhibited an increase of G concentration resulted in higher crosslinking of disulfide-bonds in G-CS, which aggrandized the bridging effect of G-CS and maintained its three-dimensional network. Cell viability assay and immuno-fluorescence staining showed that G-CS effectively facilitated the growth and myogenic differentiation of porcine skeletal muscle satellite cells (PSCs). CLSM displayed that cells first occupied the angular space of hexagon and then ring-growth circle of PSCs were orderly formed on G-CS. The texture and color of CMS which loaded proliferated PSCs were fresh-meat like. These results showed that physical cross-linked G-CS scaffolds are the biocompatible and stable adaptable extracellular matrix with appropriate architectural cues and natural micro-environment for structured CM models.

CPEB1 induces autophagy and promotes apoptosis in ovarian granulosa cells of polycystic ovary syndrome.

Inflammatory damage in ovarian granulosa cells (GCs) is a key mechanism in polycystic ovary syndrome (PCOS), cytoplasmic polyadenylation element binding protein-1 (CPEB1) is important in inflammatory regulation, however, its role in PCOS is unclear. We aim to research the mechanism of CPEB1 in ovarian GCs in PCOS using dehydroepiandrosterone (DHEA)-induced PCOS rat models and testosterone-incubated GC models. The pathophysiology in PCOS rats was analyzed. Quantitative-realtime-PCR, TUNEL, immunohistochemistry, and Western blot were applied for quantification. Additionally, cell counting kit-8, flow cytometry, immunofluorescence, Western blot, and Monodansylcadaverine staining were performed. We found that PCOS rat models exhibited a disrupted estrus cycle, elevated serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), increased LH/FSH ratio, and heightened ovarian index. Furthermore, reduced corpus luteum and increased follicular cysts were observed in ovarian tissue. In ovarian tissue, autophagy and apoptosis were activated and CPEB1 was overexpressed. In vitro, CPEB1 overexpression inhibited cell viability and sirtuin-1 (SIRT1), activated tumor necrosis factor-α, and interleukin-6 levels, as well as apoptosis and autophagy; however, CPEB1 knockdown had the opposite effect. In conclusion, overexpression of CPEB1 activated autophagy and apoptosis of ovarian GCs in PCOS.

Frizzled class receptor 5 contributes to ovarian cancer chemoresistance through aldehyde dehydrogenase 1A1

BackgroundChemoresistance is associated with tumor relapse and unfavorable prognosis. Multiple mechanisms underlying chemoresistance have been elucidated, including stemness and DNA damage repair. Here, the involvement of the WNT receptor, FZD5, in ovarian cancer (OC) chemoresistance was investigated.MethodsOC cells were analyzed using in vitro techniques including cell transfection, western blot, immunofluorescence and phalloidin staining, CCK8 assay, colony formation, flowcytometry, real-time PCR, and tumorisphere formation. Pearson correlation analysis of the expression levels of relevant genes was conducted using data from the CCLE database. Further, the behavior of OC cells in vivo was assessed by generation of a mouse xenograft model.ResultsFunctional studies in OC cells showed that FZD5 contributes to epithelial phenotype maintenance, growth, stemness, HR repair, and chemoresistance. Mechanistically, FZD5 modulates the expression of ALDH1A1, a functional marker for cancer stem-like cells, in a β-catenin-dependent manner. ALDH1A1 activates Akt signaling, further upregulating RAD51 and BRCA1, to promote HR repair.ConclusionsTaken together, these findings demonstrate that the FZD5-ALDH1A1-Akt pathway is responsible for OC cell survival, and targeting this pathway can sensitize OC cells to DNA damage-based therapy.

Abstract 171: Chemotherapeutic agent, cisplatin, influences the proportion of TREM2 (+) M2 macrophages in the TME of LUAD

Abstract Background: Triggering receptor expressed on myeloid cells (TREM2) serves as an immune signaling hub that senses and interacts with a wide range of ligands arising from damaged tissues in pathologic conditions. Despite TREM2’s diverse role in casting immune suppressive environment, the effects of chemotherapy on tumor microenvironment (TME), TREM2 and their interactions are not well established. In this study, we aimed to investigate the characteristics of TREM2 (+) Mφ within the lung TME (Tu), focusing on the impact of chemotherapy on the immune dynamics of lung adenocarcinoma (LUAD). Method: Changes in the number and distribution of Mφ subclusters in normal appearing lung tissue (NL) and Tu were examined using open-source single cell RNA sequencing (scRNA-seq) datasets and immunofluorescent staining of lung cancer-induced Kras-G12D mouse tissue. Clinical measures affecting the expression of TREM2 were explored using the TCGA-LUAD and -LUSC datasets, and the effects of anticancer drugs and the apoptotic cells on Mφ were observed by differentiating THP1 cells into M0, M1, and M2. Results: scRNA-seq analysis on LUAD datasets showed that Mφ were the second most abundant cell population that constitutes the lung TME, and the proportion of Mφ in the Tu decreases compared to NL. Among the Mφ subclusters in Tu, a subcluster which specifically increased in Tu shows enrichment of apoptotic cell clearance gene sets, including TREM2. In lung cancer induced Kras-G12D mouse, compared to NL the Tu has an increase in M2 proportion, showing the characteristics of interstitial Mφ in addition with overexpression of TREM2 and enrichment of monocyte chemotaxis gene set enrichment. Among TCGA-LUAD, the high TMB group has low TREM2 expression, and the proportion of TREM2 (+) M2 in the Tu decreases in cisplatin-treated Kras-G12D mouse Tu, suggesting the expression of TREM2 in M2 is suppressed by either cisplatin treatment itself or active immunity in Tu. Additionally, we are going to observe the effect on TREM2 expression by adding cisplatin treated A549-GFP cells or cisplatin alone, to the THP-1 cells that were differentiated into M0, M1, or M2 state. Conclusion: In addition to changes in the lung immune environment due to the tumor, anticancer treatment changes in the characteristics of M2 in TME, in terms of decrease in TREM2 (+) Mφ fraction. Considering that TREM2 plays a central inhibitory role in the tumor immune environment, the effect of anticancer chemotherapeutic agents should be considered in the development TREM2 targeting agents. Citation Format: Yoon Jin Cha, Min Kyung Park, Yoon Soo Chang. Chemotherapeutic agent, cisplatin, influences the proportion of TREM2 (+) M2 macrophages in the TME of LUAD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 171.

Novel tubulin polymerization inhibitors based on the hybridization of coumarin and indole ring: Design, synthesis and bioactivities evaluation

In order to find the potent tubulin polymerization inhibitors as anticancer drugs, a series of novel coumarin-indole derivatives were synthesized, and the bioactivity of these synthetic compounds was performed to elucidate the mechanism of anticancer activity. In vitro cell experiments indicated that among these compounds, 7a exhibited excellent inhibitory activities on MCF-7, A549 and HepG2 cells, having IC50 values ranging from 1.79 to 40.55 nanomolar, which was better than the positives cisplatin and colchicine. Tubulin polymerization inhibition assay found 7a could inhibit the polymerization of tubulin. Meanwhile, immunofluorescence staining of A549 cells showed that 7a targeted the tubulin to exert its anticancer activity. Additionally, compound 7a displayed significant inhibition of the wound-healing activity of A549 cells. The outcomes of flow cytometry demonstrated 7a could significantly halt the progression of the cell cycle during the G2/M phase and induce the apoptosis of A549 cells by reducing the mitochondrial membrane potential and increasing the level of ROS. Finally, molecular docking further demonstrated 7a could interact with tubulin.Collectively, the promising biological activity of 7a makes it valuable as further development of antitumor drugs.

Abstract 21: Similarity in the Changes of Brain Myeloid Transcriptome Signature is Shared Between Ischemic Stroke and Chronic Inflammation Caused by Diabetes Mellitus

Introduction: Ischemic stroke (IS) is a pressing global health concern. Diabetes mellitus not only heightens the risk of IS but also aggravates its outcomes. There is established evidence that neutrophil infiltration is involved in inflammation after stroke through various mechanisms, such as activating neutrophil extracellular traps. We aimed to examine the factors that contribute to the deterioration of IS in diabetic mice focusing on mechanisms underlying immune cell infiltration by single cell RNA sequencing (scRNAseq) of brain before and after stroke. Methods: Experimental stroke was induced using the distal middle cerebral artery occlusion (dMCAO) model. db/db mice were used to model type 2 diabetes and obesity, while db/+ mice served as euglycemic controls. Mononuclear cells from ipsilateral mouse brains were isolated 3 days post-dMCAO or sham surgery with the Percoll gradient following enzymatic digestion. Transcriptomic analysis was performed using scRNAseq from db/+ sham, db/db sham, db/+ stroke, and db/db stroke. Fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) staining were conducted to validate the changes detected in the transcriptome results. Results: Prior to the onset of stroke, db/db mice exhibited changes in gene expression in endothelial cells and myeloid cells including macrophages, monocytes, and microglia, mirroring patterns of changes after stroke. Among the macrophage subpopulation in particular, db/db sham and db/+ stroke mice shared an overlap of 638/901 (70.8%) differentially expressed genes compared to db/+ sham. Gene enrichment analysis indicated predominant upregulation of genes linked to leukocyte migration and neutrophil chemotaxis. FACS revealed a heightened neutrophil presence in the brain parenchyma of diabetic mice before stroke, and further elevated after stroke compared to control mice. Specifically, the increased expression of anaphylatoxin receptor C3aR and complement C3 shown by IF corroborated with the transcriptomic signature, supporting a role of complement activation and myeloid cell transmigration. Conclusions: Our findings provide valuable insights into the mechanisms whereby diabetes potentially escalates neutrophil infiltration post-stroke.