Nanoemulsion containing Jatropha gossypiifolia leaf extract reduces dermonecrosis induced by Bothrops erythromelas venom and accelerates wound closure.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Jatropha gossypiifolia, popularly known as "pinhão-roxo", is distributed throughout Brazil, is commonly employed for topical or oral administration in treating wounds, inflammations, and snake bites. Given the significant impact of snakebites on public health and the limitations of antivenom, coupled with the diverse molecular composition of this plant species, investigating its healing and antidermonecrotic capacities is relevant. AIM OF THE STUDY: This study aimed to develop a topical nanoemulsion incorporating the hydroethanolic extract of J. gossypiifolia leaves, to evaluate its therapeutic potential, particularly in terms of its efficacy in wound healing and inhibition of dermonecrosis induced by B. erythromelas venom (BeV). MATERIAL AND METHODS: The extract of J. gossypiifolia (JgE) leaves was obtained by maceration and remaceration. The phytochemical analysis was conducted and J. gossypiifolia nanoemulsion (JgNe) was obtained, characterized and assessed for stability. The cytotoxicity was determined in normal cells (erythrocytes and 3T3) using hemolytic assay and cell viability assay using crystal violet staining. The antioxidant activity was evaluated by the reduction of ABTS and DPPH radicals. The evaluation of wound healing was conducted in vivo following treatment with JgNe, wherein the percentage of wound closure and inflammatory mediators. The skin irritation test was assessed in vivo by applying JgNe directly to the animal's skin. In vitro, the antivenom capacity was evaluated through enzymatic inhibition assays (phospholipase A2 and hyaluronidase) of BeV. Additionally, the in vivo antidermonecrotic activity of JgNe was evaluated by measuring the reduction of the dermonecrotic halo. RESULTS: The HPLC-DAD analysis identified flavonoids, specifically vitexin, luteolin derivatives and apigenin derivatives. In addition, 95.08 ± 5.46 mg of gallic acid/g of extract and 137.92 ± 0.99 mg quercetin/g extract, was quantified. JgNe maintained stability over a 4-week period. Moreover, JgE and JgNe demonstrated no cytotoxicity in human erythrocytes and murine fibroblasts at tested concentrations (32.25-250 µg/mL). Additionally, exhibited significant antioxidant activity by reducing ABTS and DPPH radicals. The treatment with JgNe did not induce skin irritation and accelerated wound healing, with significant wound closure observed from 5th day and reduction in nitrite levels, myeloperoxidase activity, and cytokine. Both JgE and JgNe demonstrated in vitro inhibition of the phospholipase and hyaluronidase enzymes of BeV. Moreover, JgNe exhibited antidermonecrotic activity by reducing the dermonecrotic halo caused by BeV after 24 h. CONCLUSIONS: JgNe and JgE exhibited no cytotoxicity at the tested concentrations. Additionally, our findings demonstrate that JgNe has the ability to accelerate wound closure and reduce dermonecrosis caused by BeV, indicating to be promising formulation for complementary therapy to antivenom treatment.

Amine functionalised graphene embedded polyvinyl alcohol (PVA) and PVA-chitosan hydrogel composites.

A novel amine-functionalized graphene oxide (AFG) doped polyvinyl alcohol (PVA)/chitosan (PVA-Ch) composite film was developed using an eco-synthesis approach, eliminating the need for halogenated compounds. The resulting AFG-doped PVA/Chitosan (PVA-Ch/AFG) polymer film exhibited promising properties for controlled delivery and biosensing applications. The investigation included assessing the swelling behaviour, dissolution percent, gel fraction, and mechanical properties of the polymer film. The swelling characteristics of PVA-Ch and PVA-Ch/AFG were found to be pH and temperature-dependent across various pH ranges (3, 5, 7, and 9). Interestingly, PVA-Ch/AFG demonstrated a stable swelling pattern at pH 5 and 7, unaffected by changes in chitosan concentration, indicating enhanced stability compared to PVA-Ch. The study also explored the use of PVA-Ch/AFG in a drug delivery system, revealing controlled release of the model antibiotic amphicillin, emphasizing its potential in medical applications. Furthermore, the eco-friendly synthesis route underscored the safety of PVA-Ch/AFG for use in food and medical applications. Biocompatibility assessments, including biodegradability studies and cytotoxicity tests on fibroblasts (3T3 cells), confirmed the safety profile of PVA-Ch/AFG. In conclusion, the study suggests that PVA-Ch/AFG holds promise for bio-sensing applications, offering a flexible and colorimetric platform capable of encapsulating, adsorbing, and desorbing biomolecules such as drugs and sensing compounds.

Mineral matrix deposition of MC3T3-E1 pre-osteoblastic cells exposed to silicocarnotite and nagelschmidtite bioceramics: In vitro comparison to hydroxyapatite.

This work presents the effect of the silicocarnotite (SC) and nagelschmidtite (Nagel) phases on in vitro osteogenesis. The known hydroxyapatite of biological origin (BHAp) was used as a standard of osteoconductive characteristics. The evaluation was carried out in conventional and osteogenic media for comparative purposes to assess the osteogenic ability of the bioceramics. First, the effect of the material on cell viability at 24 h, 7 and 14 days of incubation was evaluated. In addition, cell morphology and attachment on dense bioceramic surfaces were observed by fluorescence microscopy. Specifically, alkaline phosphatase (ALP) activity was evaluated as an osteogenic marker of the early stages of bone cell differentiation. Mineralized extracellular matrix was observed by calcium phosphate deposits and extracellular vesicle formation. Furthermore, cell phenotype determination was confirmed by scanning electron microscope. The results provided relevant information on the cell attachment, proliferation, and osteogenic differentiation processes after 7 and 14 days of incubation. Finally, it was demonstrated that SC and Nagel phases promote cell proliferation and differentiation, while the Nagel phase exhibited a superior osteoconductive behavior and could promote MC3T3-E1 cell differentiation to a higher extent than SC and BHAp, which was reflected in a higher number of deposits in a shorter period for both conventional and osteogenic media.

Morphological Changes of 3T3 Cells under Simulated Microgravity.

BACKGROUND: Cells are sensitive to changes in gravity, especially the cytoskeletal structures that determine cell morphology. The aim of this study was to assess the effects of simulated microgravity (SMG) on 3T3 cell morphology, as demonstrated by a characterization of the morphology of cells and nuclei, alterations of microfilaments and microtubules, and changes in cycle progression. METHODS: 3T3 cells underwent induced SMG for 72 h with Gravite®, while the control group was under 1G. Fluorescent staining was applied to estimate the morphology of cells and nuclei and the cytoskeleton distribution of 3T3 cells. Cell cycle progression was assessed by using the cell cycle app of the Cytell microscope, and Western blot was conducted to determine the expression of the major structural proteins and main cell cycle regulators. RESULTS: The results show that SMG led to decreased nuclear intensity, nuclear area, and nuclear shape and increased cell diameter in 3T3 cells. The 3T3 cells in the SMG group appeared to have a flat form and diminished microvillus formation, while cells in the control group displayed an apical shape and abundant microvilli. The 3T3 cells under SMG exhibited microtubule distribution surrounding the nucleus, compared to the perinuclear accumulation in control cells. Irregular forms of the contractile ring and polar spindle were observed in 3T3 cells under SMG. The changes in cytoskeleton structure were caused by alterations in the expression of major cytoskeletal proteins, including ß-actin and α-tubulin 3. Moreover, SMG induced 3T3 cells into the arrest phase by reducing main cell cycle related genes, which also affected the formation of cytoskeleton structures such as microfilaments and microtubules. CONCLUSIONS: These results reveal that SMG generated morphological changes in 3T3 cells by remodeling the cytoskeleton structure and downregulating major structural proteins and cell cycle regulators.

Regulator of G protein signalling 18 promotes osteocyte proliferation by activating the extracellular signal­regulated kinase signalling pathway.

Osteocyte function is critical for metabolism, remodelling and regeneration of bone tissue. In the present study, the roles of regulator of G protein signalling 18 (RGS18) were assessed in the regulation of osteocyte proliferation and bone formation. Target genes and signalling pathways were screened using the Gene Expression Omnibus (GEO) database and Gene Set Enrichment Analysis (GSEA). The function of RGS18 and the associated mechanisms were analysed by Cell Counting Kit 8 assay, 5­ethynyl­2'­deoxyuridine assay, flow cytometry, reverse transcription­quantitative PCR, western blotting and immunostaining. Overlap analysis of acutely injured subjects (AIS) and healthy volunteers (HVs) from the GSE93138 and GSE93215 datasets of the GEO database identified four genes: KIAA0825, ANXA3, RGS18 and LIPN. Notably, RGS18 was more highly expressed in peripheral blood samples from AIS than in those from HVs. Furthermore, RGS18 overexpression promoted MLO­Y4 and MC3T3­E1 cell viability, proliferation and S­phase arrest, but inhibited apoptosis by suppressing caspase­3/9 cleavage. Silencing RGS18 exerted the opposite effects. GSEA of GSE93138 revealed that RGS18 has the ability to regulate MAPK signalling. Treatment with the MEK1/2 inhibitor PD98059 reversed the RGS18 overexpression­induced osteocyte proliferation, and treatment with the ERK1/2 activator 12­O­tetradecanoylphorbol­13­acetate reversed the effects of RGS18 silencing on osteocyte proliferation. In conclusion, RGS18 may contribute to osteocyte proliferation and bone fracture healing via activation of ERK signalling.

Characterization of gelatin-based wound dressing biomaterials containing increasing coconut oil concentrations.

This study determined the influence and ideal ratios of various coconut oil (CO) amounts in gelatin (G) based-films as wound dressings since there are limited comparative studies to evaluate the sole effect of increasing CO on protein-based biomaterials. Homogenous films at G:CO ratio of 4:0,4:2,4:3,4:4 (w:w) corresponding to CO-0, CO-2, CO-3, CO-4, respectively, were obtained using solution casting. SEM showed CO caused rougher surfaces decreasing mechanical strength. However, no pores were observed in CO-4 due to bigger clusters of oil improving stretchability compared to CO-3; and durability since aging of CO-4 was >10% lower than CO-0 in aqueous media. FTIR showed triglycerides' band only in CO films with increasing amplitude. Moreover, amide-I of CO-2 was involved in more hydrogen bonding, therefore, CO-2 had the highest melt-like transition temperatures (Tmax) at ∼163 °C while others' were at ∼133 °C; and had more ideal mechanical properties among CO films. XTT showed that increased CO improved 3T3 cell viability as CO-0 significantly decreased viability at 10,50,75,100 µg/mL (p < 0.05), whereas CO-2 and CO-3 within 5-75 µg/mL and CO-4 within 5-100 µg/mL range increased viability ≥100% suggesting proliferation. All CO samples at 25 µg/mL stimulated 3T3 cell migration in Scratch Assay indicating wound healing. CO amounts mainly improved thermal and healing properties of gelatin-based biomaterial. CO-2 was more thermally stable and CO-4 had better influence on cell viability and wound healing than CO-0. Therefore, increased CO ratios, specifically 4:2 and 4:4, G:CO (w:w), in gelatin-based films can be ideal candidates for wound dressing materials.

Capacitance Contribution of NIH/3T3 Cells Existing on and between Electrodes of an Impedance Biosensor.

In this study, an impedance biosensor capable of real-time monitoring of the growth and drug reactions using NIH/3T3 cells was fabricated through a semiconductor process. With the fabricated impedance biosensor, the cell growth and drug reaction states are monitored in real-time, showing the validness of the developed biosensor. By using the developed impedance biosensor, we have investigated the capacitance contribution of NIH/3T3 cells existing on electrodes and between electrodes. To compare the capacitance value contributions of the cells on and between electrodes, wide- and narrow-gap electrode patterns are manufactured with 3.7 and 0.3 mm electrode gap spacings, respectively. From the detailed analysis, the capacitance contributions of NIH/3T3 cells existing on electrodes are estimated around less than 20 percent compared to the cells existing between electrodes. In other words, a minimized electrode area with maximized electrode spacing is the promising impedance biosensor design guide for accurate cell capacitance measurements.

Phospholipase D1 activity is crucial for cytosolic phospholipase A2 -dependent prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells.

In bone, prostaglandin E2 (PGE2) is highly osteogenic and formed by osteoblasts, a key modulatory event in the regulation of bone cell activity. MC3T3-E1 cells are widely used as an in vitro model of osteoblast function. It is still not clear which pathways contribute to the release of AA in these cells. In this study we have focussed on the contribution of phospholipase D (PLD) enzymes to osteoblastic PGE2 formation after stimulation with endothelin-1 (ET-1). Using specific inhibitors of PLD1 and PLD2 we could show that PGE2 formation was strictly dependent on PLD1 but not PLD2 activity and cytosolic phospholipase A2 (cPLA2) was activated by triggering through PLD1. We have identified diacyl glycerol (DAG) as a possible effector molecule which may serve as a triggering signal for PKC activation and subsequent cPLA2 phosphorylation.

Selenomethionine against titanium particle-induced osteolysis by regulating the ROS-dependent NLRP3 inflammasome activation via the ß-catenin signaling pathway.

Wear debris-induced osteolysis, especially titanium (Ti) particles-induced osteolysis, is the most common cause of arthroplasty failure with no effective therapy. Previous studies have suggested that inflammation and impaired osteogenesis are associated with Ti particles -induced osteolysis. Selenium (Se) is an essential trace element in the human body, which forms selenomethionine (Se-Met) in nature, and selenoproteins has strong anti-inflammatory and antioxidant stress effects. In this study, the effects of Se-Met on Ti particles-induced osteolysis were observed and the potential mechanism was explored. We found that exogenous Se-Met relieved osteolysis induced by Ti particles in two animal models and MC3T3-E1 cells. We found that the addition of Se-Met effectively inhibited Ti particle-induced inflammation by regulating reactive oxygen species-dependent (ROS-dependent) NOD-like receptor protein 3 (NLRP3) inflammasome activation. These therapeutic effects were abrogated in MC3T3-E1 cells that had received a ß-catenin antagonist, suggesting that Se-Met alleviates inflammatory osteolysis via the ß-catenin signaling pathway. Collectively, these findings indicated that Se-Met may serve as a potential therapeutic agent for treating Ti particle-induced osteolysis.

Senescent adipocytes as potential effectors of muscle cells dysfunction: An in vitro model.

Recently, there has been a growing body of evidence showing a negative effect of the white adipose tissue (WAT) dysfunction on the skeletal muscle function and quality. However, little is known about the effects of senescent adipocytes on muscle cells. Therefore, to explore potential mechanisms involved in age-related loss of muscle mass and function, we performed an in vitro experiment using conditioned medium obtained from cultures of mature and aged 3 T3-L1 adipocytes, as well as from cultures of dysfunctional adipocytes exposed to oxidative stress or high insulin doses, to treat C2C12 myocytes. The results from morphological measures indicated a significant decrease in diameter and fusion index of myotubes after treatment with medium of aged or stressed adipocytes. Aged and stressed adipocytes presented different morphological characteristics as well as a different gene expression profile of proinflammatory cytokines and ROS production. In myocytes treated with different adipocytes' conditioned media, we demonstrated a significant reduction of gene expression of myogenic differentiation markers as well as a significant increase of genes involved in atrophy. Finally, a significant reduction in protein synthesis as well as a significant increase of myostatin was found in muscle cells treated with medium of aged or stressed adipocytes compared to controls. In conclusion, these preliminary results suggest that aged adipocytes could influence negatively trophism, function and regenerative capacity of myocytes by a paracrine network of signaling.

The effect of GelMA/alginate interpenetrating polymeric network hydrogel on the performance of porous zirconia matrix for bone regeneration applications.

Bone tissue is a natural composite, exhibiting complicated structures and unique mechanical/biological properties. With an attempt of mimicking the bone tissue, a novel inorganic-organic composite scaffolds (ZrO2-GM/SA) was designed and prepared via the vacuum infiltration method and the single/double cross-linking strategy by blending GelMA/alginate (GelMA/SA) interpenetrating polymeric network (IPN) into the porous zirconia (ZrO2) scaffold. The structure, morphology, compressive strength, surface/interface properties, and biocompatibility of the ZrO2-GM/SA composite scaffolds were characterized to evaluate the performance of the composite scaffolds. Results showed that compared to ZrO2 bare scaffolds with well-defined open pores, the composite scaffolds prepared by double cross-linking of GelMA hydrogel and sodium alginate (SA) presented a continuous, tunable and honeycomb-like microstructure. Meanwhile, GelMA/SA showed favorable and controllable water-uptake capacity, swelling property and degradability. After the introduction of IPN components, the mechanical strength of composite scaffolds was further improved. The compressive modulus of composite scaffolds was significantly higher than the bare ZrO2 scaffolds. In addition, ZrO2-GM/SA composite scaffolds had highly biocompatibility and displayed a potent proliferation and osteogenesis of MC3T3-E1 pre-osteoblasts compared to bare ZrO2 scaffolds and ZrO2-GelMA composite scaffolds. At the same time, ZrO2-10GM/1SA composite scaffold regenerated significantly greater bone than other groups in vivo. This study demonstrated that the proposed ZrO2-GM/SA composite scaffolds had great research and application potential in bone tissue engineering.

Protective effects of Cu/Zn-SOD and Mn-SOD on UVC radiation-induced damage in NIH/3T3 cells and murine skin.

Superoxide dismutase (SOD) is an antioxidant enzyme with multiple metal cofactors that can specifically clear reactive oxygen species (ROS), which plays an important role in a variety of ultraviolet-induced lesions. Therefore, SOD has the anti-ultraviolet radiation effect. The objective of this study was to compare the differences in the anti-ultraviolet radiation effect of SOD with distinct metal cofactors: Cu/Zn-SOD and Mn-SOD. SOD was first purified using hydrophobic interaction chromatography and ion-exchange chromatography. Second, the Methylthiazolyldiphenyl-tetrazolium bromide method and cell senescence kits were used to study the protective effect of SOD on ultraviolet-induced cell damage. Finally, the protective effect of SOD on ultraviolet -induced skin damage was histopathologically evaluated, and the expression levels of malondialdehyde (MDA) and matrix metalloproteinases (MMPs) in tissues were detected. The results showed that Cu/Zn-SOD was superior to Mn-SOD in promoting cell proliferation, alleviating cell damage, protecting skin structure, and regulating the expression levels of MDA and MMPs, and it has no side effects. In conclusion, Cu/Zn-SOD had a better anti-ultraviolet radiation effect than Mn-SOD, and it can be used in anti-aging and anti-ultraviolet skin-care products.

UPLC-ESI-Q-TOF-MSE-based metabolomics analysis of Acer mono sap and evaluation of osteogenic activity in mouse osteoblast cells.

Investigation of phytochemicals and bioactive molecules is tremendously vital for the applications of new plant resources in chemistry, food, and medicine. In this study, the chemical profiling of sap of Acer mono (SAM), a Korean syrup known for its anti-osteoporosis effect, was performed using UPLC-ESI-Q-TOF-MSE analysis. A total of 23 compounds were identified based on the mass and fragmentation characteristics and most of the compounds have significant biomedical applications. The in vitro antioxidant assessment of SAM indicated excellent activity by scavenging DPPH and ABTS-free radicals and were found to be 23.35 mg mL-1 and 29.33 mg mL-1, respectively, as IC50 concentrations. As well, the in vitro proliferation effect of the SAM was assessed against mouse MC3T3-E1 cells, and the results showed that the SAM enhanced the proliferation of the cells, and 12.5 mg mL-1 and 25 mg mL-1 of SAM were selected for osteogenic differentiation. The morphological analysis clearly evidenced the SAM enhanced the osteogenic activity in MC3T3-E1 cells by the increased deposition of extracellular calcium and nodule formation. Moreover, the qRT-PCR analysis confirmed the increased expression of osteoblast marker gene expression including ALP, osteocalcin, osteopontin, collagen1α1, Runx2, and osterix in SAM-treated MC3T3-E1 cells. Together, these results suggest that SAM possesses osteogenic effects and can be used for bone regeneration and bone loss-associated diseases such as osteoporosis.

Delivery of the reduced form of vitamin K2(20) to NIH/3T3 cells partially protects against rotenone induced cell death.

Mitochondria generate energy through the action of the electron transport chain (ETC) and ATP synthase. Mitochondrial malfunction can lead to various disorders, including neurodegenerative diseases. Several reports have shown that menaquinone-4 (MK-4, vitamin K2(20)), a safe drug for osteoporosis, may improve mitochondrial function. Here, we hypothesized that the efficient delivery of menahydroquinone-4 (MKH), an active form of MK-4, could exert a supporting effect. We verified the effects of MKH delivery on mitochondrial dysfunction by using MK-4 and MKH ester derivatives in NIH/3T3 mouse fibroblast cells treated with mitochondrial inhibitors. MK-4 and MKH derivatives suppressed cell death, the decline in mitochondrial membrane potential (MMP), excessive reactive oxygen species (ROS) production, and a decrease in intrinsic coenzyme Q9 (CoQ9) induced by rotenone (ROT, complex I inhibitor). MK-4 and MKH derivatives delivered MKH to NIH/3T3 cells, acting as an effective MKH prodrug, proving that the delivered MKH may reflect the mitigation effects on ROT-induced mitochondrial dysfunction. MKH prodrugs are also effective against 3-nitropropionic acid (3-NP, complex II inhibitor) and carbonyl cyanide-m-chlorophenylhydrazone (CCCP, uncoupler)-induced cell death. In conclusion, MKH delivery may mitigate mitochondrial dysfunction by maintaining MMP, ROS, and CoQ9, indicating that MKH prodrugs may be good candidates for treating mitochondrial disorders.

Activation of Focal Adhesion Kinase Restores Simulated Microgravity-Induced Inhibition of Osteoblast Differentiation via Wnt/Β-Catenin Pathway.

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/ß-catenin pathway. However, the mechanism by which SMG alters the Wnt/ß-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/ß-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/ß-catenin and Wnt/ß-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/ß-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients.

PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment.

Chemokines are highly expressed in tumor microenvironment and play a critical role in all aspects of tumorigenesis, including the recruitment of tumor-promoting immune cells, activation of cancer-associated fibroblasts, angiogenesis, metastasis, and growth. Poly (ADP-ribose) polymerase (PARP) is a multi-target transcription regulator with high levels of poly(ADP-ribose) (pADPr) being reported in a variety of cancers. Furthermore, poly (ADP-ribose) glycohydrolase (PARG), an enzyme that degrades pADPr, has been reported to be downregulated in tumor tissues with abnormally high levels of pADPr. In conjunction to this, we have recently reported that the reduction of pADPr, by either pharmacological inhibition of PARP or PARG's overexpression, disrupts renal carcinoma cell malignancy in vitro. Here, we use 3 T3 mouse embryonic fibroblasts, a universal model for malignant transformation, to follow the effect of PARG upregulation on cells' tumorigenicity in vivo. We found that the overexpression of PARG in mouse allografts produces significantly smaller tumors with a delay in tumor onset. As downregulation of PARG has also been implicated in promoting the activation of pro-inflammatory genes, we also followed the gene expression profile of PARG-overexpressing 3 T3 cells using RNA-seq approach and observed that chemokine transcripts are significantly reduced in those cells. Our data suggest that the upregulation of PARG may be potentially useful for the tumor growth inhibition in cancer treatment and as anti-inflammatory intervention.

RUNX2 Regulates Osteoblast Differentiation via the BMP4 Signaling Pathway.

RUNX2 is a master osteogenic transcription factor, and mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Studies have revealed that RUNX2 is not only a downstream target of the bone morphogenetic protein (BMP) pathway but can also regulate the expression of BMPs. However, the underlying mechanism of the regulation of BMPs by RUNX2 remains unknown. In this project, we diagnosed a CCD patient with a 7.86-Mb heterozygous deletion on chromosome 6 containing all exons of RUNX2 by multiplex ligation-dependent probe amplification (MLPA) and whole-genome sequencing (WGS). Bone marrow mesenchymal stem cells (BMSCs) were further extracted from patient alveolar bone fragments (CCD-BMSCs), an excellent natural model to explore the possible mechanism. The osteogenic differentiation ability of CCD-BMSCs was severely affected by RUNX2 heterozygous deletion. Also, BMP4 decreased most in BMP ligands, and CHRDL1, a BMP antagonist, was abnormally elevated in CCD-BMSCs. Furthermore, BMP4 treatment essentially rescued the osteogenic capacity of CCD-BMSCs, and RUNX2 overexpression reversed the abnormal expression of BMP4 and CHRDL1. Notably, we constructed CRISPR/Cas9 Runx2+/m MC3T3-E1 cells, which simulated a variant in CCD-BMSCs, to exclude the interference of other gene deletions and the heterogeneity of the genetic background of primary cells, and verified all findings from the CCD-BMSCs. Moreover, the luciferase reporter experiment showed that RUNX2 could inhibit the transcription of CHRDL1. Through immunofluorescence, the inhibitory effect of CHRDL1 on BMP4/Smad signaling was confirmed in MC3T3-E1 cells. These results revealed that RUNX2 regulated the BMP4 pathway by inhibiting CHRDL1 transcription. We collectively identified a novel RUNX2/CHRDL1/BMP4 axis to regulate osteogenic differentiation and noted that BMP4 might be a valuable therapeutic option for treating bone diseases.

Investigating the Anti-Inflammatory Effects of RCI001 for Treating Ocular Surface Diseases: Insight Into the Mechanism of Action.

The ocular surface is continuously exposed to various environmental factors, and innate and adaptive immunity play crucial roles in ocular surface diseases (OSDs). Previously, we have reported that the topical application of RCI001 affords excellent anti-inflammatory and antioxidant effects in dry eye disease and ocular chemical burn models. In this study, we examined the inhibitory effects of RCI001 on the Rac1 and NLRP3 inflammasomes in vitro and in vivo. Following RCI001 application to RAW264.7 and Swiss 3T3 cells, we measured Rac1 activity using a glutathione-S-transferase (GST) pull-down assay and G-protein activation assay kit. In addition, we quantified the expression of inflammatory cytokines (interleukin [IL]-1ß, IL-6, and tumor necrosis factor [TNF]-α) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells using ELISA and real-time PCR. In the mouse ocular alkali burn model, RCI001 was administered via eye drops (10 mg/mL, twice daily) for 5 days, and 1% prednisolone acetate (PDE) ophthalmic suspension was used as a positive control. Corneal epithelial integrity (on days 0-5) and histological examinations were performed, and transcript and protein levels of Rac1, NLRP3, caspase-1, and IL-1ß were quantified using real-time PCR and western blotting in corneal tissues collected on days 3 and 5. We observed that RCI001 dose-dependently inhibited Rac1 activity and various inflammatory cytokines in LPS-stimulated murine macrophages. Furthermore, RCI001 restored corneal epithelial integrity more rapidly than corticosteroid treatment in chemically injured corneas. Compared to the saline group, activation of Rac1 and the NLRP3 inflammasome/IL-1ß axis was suppressed in the RCI001 group, especially during the early phase of the ocular alkali burn model. Topical RCI001 suppressed the expression of activated Rac1 and inflammatory cytokines in vitro and rapidly restored the injured cornea by inhibiting activation of Rac1 and the NLRP inflammasome/IL-1ß axis in vivo. Accordingly, RCI001 could be a promising therapeutic agent for treating OSDs.

Orai1 downregulation causes proliferation reduction and cell cycle arrest via inactivation of the Ras-NF-κB signaling pathway in osteoblasts.

BACKGROUND: The purpose of this study was to determine the role of Orai1 in the regulation of the proliferation and cell cycle of osteoblasts. METHODS: The expression of Orai1 was inhibited by Orai1 small interfering RNA (siRNA) in MC3T3-E1 cells. Following Orai1 downregulation, cell proliferation and cell cycle were examined. Furthermore, the expression of cyclin D1, cyclin E, CDK4, and CDK6 was analyzed. The activity of the Ras-NF-κB signaling pathway was investigated to identify the role of Orai1 in the regulation of osteoblast proliferation. RESULTS: Orai1 was successfully downregulated in MC3T3-E1 cells by the Orai1 siRNA transfection (p < 0.05). We found that MC3T3-E1 cell proliferation was decreased, and the cell cycle was arrested by Orai1 downregulation (p < 0.05). Additionally, the expression of cyclin D1 was decreased by Orai1 downregulation (p < 0.05), as was the activity of the Ras-NF-κB signaling pathway (p < 0.05). Orai1 siRNA did not further reduce cell proliferation, the proportion of cells in the S phase, and cyclin D1 expression after chemical blockage of the Ras signaling pathway in MC3T3-E1 cells (p > 0.05). CONCLUSIONS: The results reveal that Orai1 downregulation may reduce cyclin D1 expression by inactivating the Ras-NF-κB signaling pathway thus blocking osteoblast proliferation and cell cycle.

The Activation of GPR27 Increases Cytosolic L-Lactate in 3T3 Embryonic Cells and Astrocytes.

G-protein-coupled receptors (GPCRs) represent a family with over 800 members in humans, and one-third of these are targets for approved drugs. A large number of GPCRs have unknown physiologic roles. Here, we investigated GPR27, an orphan GPCR belonging to the family of super conserved receptor expressed in the brain, with unknown functions. Cytosolic levels of L-lactate ([lactate]i), the end product of aerobic glycolysis, were measured with the Laconic fluorescence resonance energy transfer nanosensor. In single 3T3 wild-type (WT) embryonic cells, the application of 8535 (1 µM), a surrogate agonist known to activate GPR27, resulted in an increase in [lactate]i. Similarly, an increase was recorded in primary rat astrocytes, a type of neuroglial cell abundant in the brain, which contain glycogen and express enzymes of aerobic glycolysis. In CRISPR-Cas9 GPR27 knocked out 3T3 cells, the 8535-induced increase in [lactate]i was reduced compared with WT controls. Transfection of the GPR27-carrying plasmid into the 3T3KOGPR27 cells rescued the 8535-induced increase in [lactate]i. These results indicate that stimulation of GPR27 enhances aerobic glycolysis and L-lactate production in 3T3 cells and astrocytes. Interestingly, in the absence of GPR27 in 3T3 cells, resting [lactate]i was increased in comparison with controls, further supporting the view that GPR27 regulates L-lactate homeostasis.