Cardamonin attenuates iron overload-induced osteoblast oxidative stress through the HIF-1α/ROS pathway.

BACKGROUND: Osteoporosis(OP) is a bone disease under research. Iron overload is a significant risk factor. Iron balance is crucial for bone metabolism and biochemical processes. When there is an excess of iron in the body, it tends to produce reactive oxygen species (ROS) which can cause oxidative damage to cells. The flavonoid compound, Cardamonin (CAR), possesses potent anti-inflammatory and anti-iron overload properties that can be beneficial in mitigating the risk of OP. PURPOSE: This study investigates the potential therapeutic interventions and underlying mechanisms of CAR for treating OP in individuals with iron overload. METHODS: The model of iron-overloaded mice was established by intraperitoneally injecting iron dextran(ID) into the mice. OP severity was evaluated with micro-CT and Hematoxylin-Eosin (HE) staining in vivo. In vitro, the iron-overloaded osteoblast model was induced by ferric ammonium citrate. Cell counting kit 8 assay to evaluate cell viability, Annexin V-FITC/PI assay to detect cell apoptosis. A range of cellular markers were detected, including the variation in mitochondrial membrane potential (MMP), levels of malondialdehyde (MDA), ROS, and lipid hydroperoxide (LPO). RESULTS: CAR can reverse bone loss in iron overload-induced OP mouse models in vivo. CAR attenuates the impairment of iron overload on the activity and apoptosis of MC3T3-E1 cells as well as the accumulation of ROS and LPO activation via HIF-1α/ROS pathways. CONCLUSION: CAR downregulating HIF-1α pathways prevents inhibition of iron overload-induced osteoblasts dysfunctional by attenuating ROS accumulation, reducing oxidative stress, promotes bone formation, and alleviates OP.

Pulmonary neuroendocrine cells: crucial players in respiratory function and airway-nerve communication.

Pulmonary neuroendocrine cells (PNECs) are unique airway epithelial cells that blend neuronal and endocrine functions, acting as key sensors in the lung. They respond to environmental stimuli like allergens by releasing neuropeptides and neurotransmitters. PNECs stand out as the only lung epithelial cells innervated by neurons, suggesting a significant role in airway-nerve communication via direct neural pathways and hormone release. Pathological conditions such as asthma are linked to increased PNECs counts and elevated calcitonin gene-related peptide (CGRP) production, which may affect neuroprotection and brain function. CGRP is also associated with neurodegenerative diseases, including Parkinson's and Alzheimer's, potentially due to its influence on inflammation and cholinergic activity. Despite their low numbers, PNECs are crucial for a wide range of functions, highlighting the importance of further research. Advances in technology for producing and culturing human PNECs enable the exploration of new mechanisms and cell-specific responses to targeted therapies for PNEC-focused treatments.

Curcumenol regulates Histone H3K27me3 demethylases KDM6B affecting Succinic acid metabolism to alleviate cartilage degeneration in knee osteoarthritis.

BACKGROUND: Cartilage metabolism dysregulation is a crucial driver in knee osteoarthritis (KOA). Modulating the homeostasis can mitigate the cartilage degeneration in KOA. Curcumenol, derived from traditional Chinese medicine Curcuma Longa L., has demonstrated potential in enhancing chondrocyte proliferation and reducing apoptosis. However, the specific mechanism of Curcumenol in treating KOA remains unclear. This study aimed to demonstrate the molecular mechanism of Curcumenol in treating KOA based on the transcriptomics and metabolomics, and both in vivo and in vitro experimental validations. MATERIALS AND METHODS: In this study, a destabilization medial meniscus (DMM)-induced KOA mouse model was established. And the mice were intraperitoneally injected with Curcumenol at 4 and 8 mg/kg concentrations. The effects of Curcumenol on KOA cartilage and subchondral was evaluated using micro-CT, histopathology, and immunohistochemistry (IHC). In vitro, OA chondrocytes were induced with 10 µg/mL lipopolysaccharide (LPS) and treated with Curcumenol to evaluate the proliferation, apoptosis, and extracellular matrix (ECM) metabolism through CCK8 assay, flow cytometry, and chondrocyte staining. Furthermore, transcriptomics and metabolomics were utilized to identify differentially expressed genes (DEGs) and metabolites. Finally, integrating multi-omics analysis, virtual molecular docking (VMD), and molecular dynamics simulation (MDS), IHC, immunofluorescence (IF), PCR, and Western blot (WB) validation were conducted to elucidate the mechanism by which Curcumenol ameliorates KOA cartilage degeneration. RESULTS: Curcumenol ameliorated cartilage destruction and subchondral bone loss in KOA mice, promoted cartilage repair, upregulated the expression of COL2 while downregulated MMP3, and improved ECM synthesis metabolism. Additionally, Curcumenol also alleviated the damage of LPS on the proliferation activity and suppressed apoptosis, promoted ECM synthesis. Transcriptomic analysis combined with weighted gene co-expression network analysis (WGCNA) identified a significant downregulation of 19 key genes in KOA. Metabolomic profiling showed that Curcumenol downregulates the expression of d-Alanyl-d-alanine, 17a-Estradiol, Glutathione, and Succinic acid, while upregulating Sterculic acid and Azelaic acid. The integrated multi-omics analysis suggested that Curcumenol targeted KDM6B to regulate downstream protein H3K27me3 expression, which inhibited methylation at the histone H3K27, consequently reducing Succinic acid levels and improving KOA cartilage metabolism homeostasis. Finally, both in vivo and in vitro findings indicated that Curcumenol upregulated KDM6B, suppressed H3K27me3 expression, and stimulated collagen II expression and ECM synthesis, thus maintaining cartilage metabolism homeostasis and alleviating KOA cartilage degeneration. CONCLUSION: Curcumenol promotes cartilage repair and ameliorates cartilage degeneration in KOA by upregulating KDM6B expression, thereby reducing H3K27 methylation and downregulating Succinic Acid, restoring metabolic stability and ECM synthesis.

Growth of Size-Tunable Ag2O Polyhedra and Revelation of Their Bulk and Surface Lattices.

By primarily adjusting the reagent amounts, particularly the volume of AgNO3 solution introduced, Ag2O cubes with decreasing sizes from 440 to 79 nm, octahedra from 714 to 106 nm, and rhombic dodecahedra from 644 to 168 nm are synthesized. 733 nm cuboctahedra are also prepared for structural analysis. With in-house X-ray diffraction (XRD) peak calibration, shape-related peak shifts are recognizable. Synchrotron XRD measurements at 100 K reveal the presence of bulk and surface layer lattices. Bulk cell constants also deviate slightly. They show a negative thermal expansion behavior with shrinking cell constants at higher temperatures. The Ag2O crystals exhibit size- and facet-dependent optical properties. Bandgaps red-shift continuously with increasing particle sizes. Optical facet effect is also observable. Moreover, synchrotron XRD peaks of a mixture of Cu2O rhombicuboctahedra and edge- and corner-truncated cubes exposing all three crystal faces can be deconvoluted into three components with the bulk and the [111] microstrain phase as the major component. Interestingly, while the unheated Cu2O sample shows clear diffraction peak asymmetry, annealing the sample to 450 K yields nearly symmetric peaks even when returning the sample to room temperature, meaning even moderately high temperatures can permanently change the crystal lattice.

Tumor cell-released autophagosomes (TRAPs) induce PD-L1-decorated NETs that suppress T-cell function to promote breast cancer pulmonary metastasis.

BACKGROUND: Lung metastasis is the primary cause of breast cancer-related mortality. Neutrophil extracellular traps (NETs) are involved in the progression of breast cancer. However, the mechanism of NET formation is not fully understood. This study posits that tumor cell-released autophagosomes (TRAPs) play a crucial role in this process. METHODS: TRAPs were isolated from breast cancer cell lines to analyze their impact on NET formation in both human and mouse neutrophils. The study used both in vitro and in vivo models, including Toll-like receptor 4 (TLR4-/-) mice and engineered breast cancer cell lines. Immunofluorescence, ELISA, Western blotting, RNA sequencing, and flow cytometry were employed to dissect the signaling pathways leading to NET production and to explore their immunosuppressive effects, particularly focusing on the impact of NETs on T-cell function. The therapeutic potential of targeting TRAP-induced NETs and their immunosuppressive functions was evaluated using DNase I and αPD-L1 antibodies. Clinical relevance was assessed by correlating circulating levels of TRAPs and NETs with lung metastasis in patients with breast cancer. RESULTS: This study showed that TRAPs induced the formation of NETs in both human and mouse neutrophils by using the high mobility group box 1 and activating the TLR4-Myd88-ERK/p38 signaling axis. More importantly, PD-L1 carried by TRAP-induced NETs inhibited T-cell function in vitro and in vivo, thereby contributing to the formation of lung premetastatic niche (PMN) immunosuppression. In contrast, Becn1 KD-4T1 breast tumors with decreased circulating TRAPs in vivo reduced the formation of NETs, which in turn attenuated the immunosuppressive effects in PMN and resulted in a reduction of breast cancer pulmonary metastasis in murine models. Moreover, treatment with αPD-L1 in combination with DNase I that degraded NETs restored T-cell function and significantly reduced tumor metastasis. TRAP levels in the peripheral blood positively correlated with NET levels and lung metastasis in patients with breast cancer. CONCLUSIONS: Our results demonstrate a novel role of TRAPs in the formation of PD-L1-decorated NETs, which may provide a new strategy for early detection and treatment of pulmonary metastasis in patients with breast cancer.

An herbal formulation "Shugan Xiaozhi decoction" ameliorates methionine/choline deficiency-induced nonalcoholic steatohepatitis through regulating inflammation and apoptosis-related pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Shugan Xiaozhi (SGXZ) decoction is a traditional Chinese medicine used for treating nonalcoholic steatohepatitis (NASH). It has been used clinically for over 20 years and proved to be effective; however, the molecular mechanism underlying the effects of SGXZ decoction remains unclear. AIM OF THE STUDY: We analyzed the chemical components, core targets, and molecular mechanisms of SGXZ decoction to improve NASH through network pharmacology and in vivo experiments. MATERIALS AND METHODS: The chemical components, core targets, and related signaling pathways of SGXZ decoction intervention in NASH were predicted using network pharmacology. Molecular docking was performed to verify chemical components and their core targets. The results were validated in the NASH model treated with SGXZ decoction. Mouse liver function was assessed by measuring ALT and AST levels. TC and TG levels were determined to evaluate lipid metabolism, and lipid deposition was assessed via oil red O staining. Mouse liver damage was determined via microscopy following hematoxylin and eosin staining. Liver fibrosis was assessed via Masson staining. Western blot (WB) and immunohistochemical (IHC) analyses were performed to detect inflammation and the expression of apoptosis-related proteins, including IL-1ß, IL-6, IL-18, TNF-α, MCP1, p53, FAS, Caspase-8, Caspase-3, Caspase-9, Bax, Bid, Cytochrome c, Bcl-2, and Bcl-XL. In addition, WB and IHC were used to assess protein expression associated with the TLR4/MyD88/NF-κB pathway. RESULTS: Quercetin, luteolin, kaempferol, naringenin, and nobiletin in SGXZ decoction were effective chemical components in improving NASH, and TNF-α, IL-6, and IL-1ß were the major core targets. Molecular docking indicated that these chemical components and major core targets might interact. KEGG pathway analysis showed that the pathways affected by SGXZ decoction, primarily including apoptosis and TLR4/NF-κB signaling pathways, interfere with NASH. In vivo experiments indicated that SGXZ decoction considerably ameliorated liver damage, fibrosis, and lipid metabolism disorder in MCD-induced NASH mouse models. In addition, WB and IHC verified the underlying molecular mechanisms of SGXZ decoction as predicted via network pharmacology. SGXZ decoction inhibited the activation of apoptosis-related pathways in MCD-induced NASH mice. Moreover, SGXZ decoction suppressed the activation of TLR4/MyD88/NF-κB pathway in MCD-induced NASH mice. CONCLUSION: SGXZ decoction can treat NASH through multiple targets and pathways. These findings provide new insights into the effective treatment of NASH using SGXZ decoction.

Enhancing the efficacy of vaccinia-based oncolytic virotherapy by inhibiting CXCR2-mediated MDSC trafficking.

Oncolytic virotherapy is an innovative approach for cancer treatment. However, recruitment of myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment (TME) after oncolysis-mediated local inflammation leads to tumor resistance to the therapy. Using the murine malignant mesothelioma model, we demonstrated that the in situ vaccinia virotherapy recruited primarily polymorphonuclear MDSCs (PMN-MDSCs) into the TME, where they exhibited strong suppression of cytotoxic T lymphocytes in a reactive oxygen species-dependent way. Single-cell RNA sequencing analysis confirmed the suppressive profile of PMN-MDSCs at the transcriptomic level and identified CXCR2 as a therapeutic target expressed on PMN-MDSCs. Abrogating PMN-MDSC trafficking by CXCR2-specific small molecule inhibitor during the vaccinia virotherapy exhibited enhanced antitumor efficacy in 3 syngeneic cancer models, through increasing CD8+/MDSC ratios in the TME, activating cytotoxic T lymphocytes, and skewing suppressive TME into an antitumor environment. Our results warrant clinical development of CXCR2 inhibitor in combination with oncolytic virotherapy.

Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Mitochondrial One-Carbon Metabolism is Required for TGF-ß-Induced Glycine Synthesis and Collagen Protein Production.

A hallmark of Idiopathic Pulmonary Fibrosis is the TGF-ß-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by lung fibroblasts requires de novo synthesis of glycine, the most abundant amino acid in collagen protein. TGF-ß upregulates the expression of the enzymes of the de novo serine/glycine synthesis pathway in lung fibroblasts through mTORC1 and ATF4-dependent transcriptional programs. SHMT2, the final enzyme of the de novo serine/glycine synthesis pathway, transfers a one-carbon unit from serine to tetrahydrofolate (THF), producing glycine and 5,10-methylene-THF (meTHF). meTHF is converted back to THF in the mitochondrial one-carbon (1C) pathway through the sequential actions of MTHFD2 (which converts meTHF to 10-formyl-THF), and either MTHFD1L, which produces formate, or ALDH1L2, which produces CO2. It is unknown how the mitochondrial 1C pathway contributes to glycine biosynthesis or collagen protein production in fibroblasts, or fibrosis in vivo. Here, we demonstrate that TGF-ß induces the expression of MTHFD2, MTHFD1L, and ALDH1L2 in human lung fibroblasts. MTHFD2 expression was required for TGF-ß-induced cellular glycine accumulation and collagen protein production. Combined knockdown of both MTHFD1L and ALDH1L2 also inhibited glycine accumulation and collagen protein production downstream of TGF-ß; however knockdown of either protein alone had no inhibitory effect, suggesting that lung fibroblasts can utilize either enzyme to regenerate THF. Pharmacologic inhibition of MTHFD2 recapitulated the effects of MTHFD2 knockdown in lung fibroblasts and ameliorated fibrotic responses after intratracheal bleomycin instillation in vivo. Our results provide insight into the metabolic requirements of lung fibroblasts and provide support for continued development of MTHFD2 inhibitors for the treatment of IPF and other fibrotic diseases.

A classical herbal formula alleviates high-fat diet induced nonalcoholic steatohepatitis (NASH) via targeting mitophagy to rehabilitate dysfunctional mitochondria, validated by UPLC-HRMS identification combined with in vivo experiment.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) has caused a significant burden on public health care systems, the economy and society. However, there has still been no officially approved pharmacotherapy for NASH. It has been suggested that oxidative stress and mitochondrial dysfunction play vital roles in NASH pathological progression. Shugan Xiaozhi (SG) formula, as a kind of classical herbal formula, was shown to attenuate NASH. PURPOSE: This study aimed to explore the potential mechanisms of SG formula treating NASH. STUDY DESIGN AND METHODS: Ultra-high-performance liquid chromatography-high resolution mass spectrometry combined with bioinformatics analysis was applied to explore the therapeutic targets and main components of SG formula. Moreover, in vivo NASH model was utilized to confirmed the therapeutic effects of SG formula. Molecular docking analysis and further validation experiments were conducted to verify the results of bioinformatics analysis. RESULTS: The in vivo experiments confirmed SG formula significantly attenuated hepatic pathological progression and relieved oxidative stress in high-fat diet (HFD) induced - NASH model. Ultra-high-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) combined with bioinformatics analysis expounded the components of SG formula and revealed the mitochondrial regulation mechanism of SG formula treating NASH. Further in vivo experiments validated that SG formula could alleviate oxidative stress by rehabilitating the structure and function of mitochondria, which was strongly related to regulating mitophagy. CONCLUSION: In summary, this study demonstrated that SG formula, which could attenuate NASH by regulating mitochondria and might be a potential pharmacotherapy for NASH.

Combination of curcumin and catalase protects against chondrocyte injury and knee osteoarthritis progression by suppressing oxidative stress.

Knee Osteoarthritis (KOA) is an age-related progressive degenerative joint disease, which is featured with pain, joint deformity, and disability. Accumulating evidence indicated oxidative stress plays a crucial role in the occurrence and development of KOA. Curcumin is a polyphenolic compound with significant antioxidant activity among various diseases while catalase (CAT) is an enzyme degrading hydrogen peroxide in treating oxidative diseases. We previously showed that the expression of CAT was low in cartilage. However, the combination of curcumin and CAT in KOA is still elusive. In this study, we demonstrated that the combination of curcumin and CAT has the potential to inhibit the IL1ß-induced chondrocyte apoptosis without cytotoxicity in vitro. Mechanistically, we found that the synergistic application curcumin and CAT not only promotes curcumin's regulation of the NRF2/HO-1 signaling pathway to enhance antioxidant enzyme expression to remove superoxide radicals, but also CAT can further remove downstream hydrogen peroxide which enhances the ability to scavenge reactive oxygen species (ROS). In vivo, studies revealed that combination of curcumin and catalase could better inhibit oxidative stress-induced chondrocyte injury by promoting the expression of ROS scavenging enzymes. In sum, the combination of curcumin and catalase can be used to treat KOA. Thus, combination of curcumin and catalase may act as a novel therapeutic agent to manage KOA and our research gives a rationale for their combined use in the therapeutic of KOA.

Synthesis of metakaolin-based geopolymer foamed materials using municipal solid waste incineration fly ash as a foaming agent.

The existence of metallic aluminum in municipal solid waste incineration fly ash (MSWIFA) makes it challenging to recycle MSWIFA into cement materials because expansion occurs in the resultant matrices. Geopolymer-foamed materials (GFMs) are gaining attention in the field of porous materials due to their high-temperature stability, low thermal conductivity and low CO2 emission. This work aimed to utilize MSWIFA as a foaming agent to synthesize GFMs. The physical properties, pore structure, compressive strength and thermal conductivity were analyzed to assess different GFMs which were synthesized with various MSWIFA and stabilizing agent dosages. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis were conducted to characterize the phase transformation of the GFMs. Results showed that when MSWIFA content was increased from 20 to 50%, the porosity of GFMs increased from 63.5 to 73.7%, and bulk density decreased from 890 to 690 kg/m3. The addition of stabilizing agent could trap the foam, refine the cell size, and homogenize the cell size range. With the stabilizing agent increase from 0 to 4%, the porosity increased from 69.9 to 76.8%, and the bulk density decreased from 800 to 620 kg/m3. The thermal conductivity decreased with increasing MSWIFA from 20 to 50%, and stabilizing agent dosage from 0 to 4%. Compared with the collected data from references, a higher compressive strength can be obtained at the same level of thermal conductivity for GFMs synthesized with MSWIFA as a foaming agent. Additionally, the foaming effect of MSWIFA results from the H2 release. The addition of MSWIFA changed both the crystal phase and gel composition, whereas the stabilizing agent dosage had little impact on the phase composition.

Experimental Revelation of Surface and Bulk Lattices in Faceted Cu2 O Crystals.

Semiconductor crystals have generally shown facet-dependent electrical, photocatalytic, and optical properties. These phenomena have been proposed to result from the presence of a surface layer with bond-level deviations. To provide experimental evidence of this structural feature, synchrotron X-ray sources are used to obtain X-ray diffraction (XRD) patterns of polyhedral cuprous oxide crystals. Cu2 O rhombic dodecahedra display two distinct cell constants from peak splitting. Peak disappearance during slow Cu2 O reduction to Cu with ammonia borane differentiates bulk and surface layer lattices. Cubes and octahedra also show two peak components, while diffraction peaks of cuboctahedra are comprised of three components. Temperature-varying lattice changes in the bulk and surface regions also show shape dependence. From transmission electron microscopy (TEM) images, slight plane spacing deviations in surface and inner crystal regions are measured. Image processing provides visualization of the surface layer with depths of about 1.5-4 nm giving dashed lattice points instead of dots from atomic position deviations. Close TEM examination reveals considerable variation in lattice spot size and shape for different particle morphologies, explaining why facet-dependent properties are emerged. Raman spectrum reflects the large bulk and surface lattice difference in rhombic dodecahedra. Surface lattice difference can change the particle bandgap.

Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and (ii) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity.

Short-term cultivation limiting soil aggregate stability and macronutrient accumulation associated with glomalin-related soil protein in Eucalyptus urophylla × Eucalyptus grandis plantations.

The relationship between glomalin-related soil protein (GRSP) and soil aggregation has been a hot topic of research for its close link to soil stability and quality. However, the short-term cultivation of Eucalyptus poses serious threats to soil stability and nutrient stocks, and the effects of GRSP on soil aggregate stability and macronutrient accumulation remain unclear. The aim is to clarify the potential mechanisms affecting soil aggregate stability and macronutrient accumulation in short-term Eucalyptus plantations. Five Eucalyptus urophylla × Eucalyptus grandis plantations with different cultivation periods (1-5 years) in this study were investigated, and a native evergreen broadleaf forest (0 year) was selected as control. The mean weight diameter index increased in the first 3 years and then significantly decreased during 5 years cultivation of Eucalyptus. Soil organic carbon (SOC) and total nitrogen also decreased after planting Eucalyptus for 3 years, but variation in total phosphorus was not obvious. The relative abundance of Glomeraceae and Claroideoglomeraceae decreased in the 5-year-old Eucalyptus plantations and was positively correlated with GRSP content. In pathway modeling, nutrient-acquisition enzyme activities positively affected GRSP and macronutrient content. Total GRSP (T-GRSP) had higher total effects than easily extractable GRSP on soil aggregate stability, and positively correlated with SOC in macroaggregates. Both T-GRSP and SOC had positive and direct effects on soil aggregate stability. Variance partitioning analysis further explained the contribution of GRSP and SOC to aggregate stability, particularly in >2 and 2-0.25 mm macroaggregates. Our results suggested that GRSP was directly associated with SOC content and soil aggregate stability, and was a potential key factor affecting soil aggregate stability in Eucalyptus plantations. Improving T-GRSP and SOC are efficient approaches for preventing the gradual deterioration of soil aggregate stability. Short-term cultivation should be carefully used in Eucalyptus plantations, and a new cultivation period is needed.

Quercetin protects against iron overload-induced osteoporosis through activating the Nrf2/HO-1 pathway.

AIMS: Eucommia is the tree bark of Eucommia japonica, family Eucommiaceae. In traditional Chinese medicine, Eucommia is often used to treat osteoporosis. Quercetin (QUE), a major flavonoid extract of Eucommia japonica, has been reported to have anti-osteoporosis effects. However, there are no studies reporting the mechanism of QUE in the treatment of iron overload-induced osteoporosis. This study set out to investigate the therapeutic effects of QUE against iron overload-induced bone loss and its potential molecular mechanisms. MATERIALS AND METHODS: In vitro, MC3T3-E1 cells were used to study the effects of QUE on osteogenic differentiation, anti-apoptosis and anti-oxidative stress damage in an iron overload environment (FAC 200 µM). In vivo, we constructed an iron overload mouse model by injecting iron dextrose intraperitoneally and assessed the osteoprotective effects of QUE by Micro-CT and histological analysis. KEY FINDINGS: In vitro, we found that QUE increased the ALP activity of MC3T3-E1 cells in iron overload environment, promoted the formation of bone mineralized nodules and upregulated the expression of Runx2 and Osterix. In addition, QUE was able to reduce FAC-induced apoptosis and ROS production, down-regulated the expression of Caspase3 and Bax, and up-regulated the expression of Bcl-2. In further studies, we found that QUE activated the Nrf2/HO-1 signaling pathway and attenuated FAC-induced oxidative stress damage. The results of the in vivo study showed that QUE was able to reduce iron deposition induced by iron dextrose and attenuate bone loss. SIGNIFICANCE: Our results suggested that QUE protects against iron overload-induced osteoporosis by activating the Nrf2/HO-1 signaling pathway.

Structural insights into broadly neutralizing antibodies elicited by hybrid immunity against SARS-CoV-2.

ABSTRACTIncreasing spread by SARS-CoV-2 Omicron variants challenges existing vaccines and broadly reactive neutralizing antibodies (bNAbs) against COVID-19. Here we determine the diversity, potency, breadth and structural insights of bNAbs derived from memory B cells of BNT162b2-vaccinee after homogeneous Omicron BA.1 breakthrough infection. The infection activates diverse memory B cell clonotypes for generating potent class I/II and III bNAbs with new epitopes mapped to the receptor-binding domain (RBD). The top eight bNAbs neutralize wildtype and BA.1 potently but display divergent IgH/IgL sequences and neuralization profiles against other variants of concern (VOCs). Two of them (P2D9 and P3E6) belonging to class III NAbs display comparable potency against BA.4/BA.5, although structural analysis reveals distinct modes of action. P3E6 neutralizes all variants tested through a unique bivalent interaction with two RBDs. Our findings provide new insights into hybrid immunity on BNT162b2-induced diverse memory B cells in response to Omicron breakthrough infection for generating diverse bNAbs with distinct structural basis.

Metformin suppresses Oxidative Stress induced by High Glucose via Activation of the Nrf2/HO-1 Signaling Pathway in Type 2 Diabetic Osteoporosis.

BACKGROUND: Metformin (MET) is widely used as a first-line hypoglycemic agent for the treatment of type 2 diabetes mellitus (T2DM) and was also confirmed to have a therapeutic effect on type 2 diabetic osteoporosis (T2DOP). However, the potential mechanisms of MET in the treatment of T2DOP are unclear. OBJECTIVE: To clarify the effect of MET in T2DOP and to explore the potential mechanism of MET in the treatment of T2DOP. METHODS: In vitro, we used MC3T3-E1 cells to study the effects of MET on osteogenic differentiation and anti-oxidative stress injury in a high glucose (Glucose 25 mM) environment. In vivo, we directly used db/db mice as a T2DOP model and assessed the osteoprotective effects of MET by Micro CT and histological analysis. RESULTS: In vitro, we found that MET increased ALP activity in MC3T3-E1 cells in a high-glucose environment, promoted the formation of bone mineralized nodules, and upregulated the expression of the osteogenesis-related transcription factors RUNX2, Osterix, and COL1A1-related genes. In addition, MET was able to reduce high glucose-induced reactive oxygen species (ROS) production. In studies on the underlying mechanisms, we found that MET activated the Nrf2/HO-1 signaling pathway and alleviated high-glucose-induced oxidative stress injury. In vivo results showed that MET reduced bone loss and bone microarchitecture destruction in db/db mice. CONCLUSION: Our results suggest that MET can activate the Nrf2/HO-1 signaling pathway to regulate the inhibition of osteogenic differentiation induced by high glucose thereby protecting T2DOP.

Shape-Tunable BaTiO3 Crystals Presenting Facet-Dependent Optical and Piezoelectric Properties.

BaTiO3 octahedra, edge-, and corner-truncated cubes, and cubes with four tunable sizes from 132 to 438 nm are synthesized by a solvothermal growth approach. Acetic acid treatment can cleanly remove BaCO3 impurity. Rietveld refinement of X-ray diffraction patterns and Raman spectra help to confirm the particles have a tetragonal crystal structure. The crystals also exhibit size- and facet-dependent bandgap shifts. BaTiO3 octahedra show larger piezoelectric, ferroelectric, and pyroelectric effects than truncated cubes and cubes. The measured dielectric constant differences should be associated with their various facet-dependent behaviors. Piezoelectric nanogenerators fabricated from BaTiO3 octahedra consistently show the best performance than those containing truncated cubes and cubes. In particular, a nanogenerator with 30 wt.%-incorporated octahedra displays an open-circuit voltage of 23 V and short-circuit current of 324 nA. The device performance is also highly stable. The maximum output power reaches 3.9 µW at 60 MΩ. The fabricated nanogenerator can provide sufficient electricity to power light-emitting diodes. This work further demonstrates that various physical properties of semiconductor crystals show surface dependence.