Genome-wide identification and analysis of WD40 proteins reveal that NtTTG1 enhances drought tolerance in tobacco (Nicotiana tabacum).

BACKGROUND: WD40 proteins, which are highly prevalent in eukaryotes, play important roles in plant development and stress responses. However, systematic identification and exploration of WD40 proteins in tobacco have not yet been conducted. RESULTS: In this study, a total of 399 WD40 regulatory genes were identified in common tobacco (Nicotiana tabacum). Gene structure and motif analysis revealed structural and functional diversity among different clades of tobacco WD40 regulatory genes. The expansion of tobacco WD40 regulatory genes was mainly driven by segmental duplication and purifying selection. A potential regulatory network of NtWD40s suggested that NtWD40s might be regulated by miRNAs and transcription factors in various biological processes. Expression pattern analysis via transcriptome analysis and qRT-PCR revealed that many NtWD40s exhibited tissue-specific expression patterns and might be involved in various biotic and abiotic stresses. Furthermore, we have validated the critical role of NtTTG1, which was located in the nuclei of trichome cells, in enhancing the drought tolerance of tobacco plants. CONCLUSIONS: Our study provides comprehensive information to better understand the evolution of WD40 regulatory genes and their roles in different stress responses in tobacco.

Exploring the therapeutic potential of isoorientin in the treatment of osteoporosis: a study using network pharmacology and experimental validation.

BACKGROUND: Isoorientin (ISO) is a glycosylated flavonoid with antitumor, anti-inflammatory, and antioxidant properties. However, its effects on bone metabolism remain largely unknown. METHODS: In this study, we aimed to investigate the effects of ISO on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in vitro and bone loss in post-ovariectomy (OVX) rats, as well as to elucidate the underlying mechanism. First, network pharmacology analysis indicated that MAPK1 and AKT1 may be potential therapeutic targets of ISO and that ISO has potential regulatory effects on the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathways, as well as oxidative stress. ISO was added to RAW264.7 cells stimulated by RANKL, and its effects on osteoclast differentiation were evaluated using tartrate-resistant acid phosphatase (TRAP) staining, TRAP activity measurement, and F-actin ring analysis. Reactive oxygen species (ROS) production in osteoclasts was detected using a ROS assay kit. The effects of ISO on RANKL-triggered molecular cascade response were further investigated by Western blotting, quantitative real-time polymerase chain reaction, and immunofluorescence staining. In addition, the therapeutic effects of ISO were evaluated in vivo. RESULTS: ISO inhibited osteoclastogenesis in a time- and concentration-dependent manner. Mechanistically, ISO downregulated the expression of the main transcription factor for osteoclast differentiation by inhibiting MAPK and PI3K/AKT1 signaling pathways. Moreover, ISO exhibited protective effects in OVX-induced bone loss rats. This was consistent with the results derived from network pharmacology. CONCLUSION: Our findings suggest a potential therapeutic utility of ISO in the management of osteoclast-associated bone diseases, including osteoporosis.

Genome-wide identification of the N6-methyladenosine regulatory genes reveals NtFIP37B increases drought resistance of tobacco (Nicotiana tabacum L.).

BACKGROUND: N6-methyladenosine (m6A) is one of the common internal RNA modifications found in eukaryotes. The m6A modification can regulate various biological processes in organisms through the modulation of alternative splicing, alternative polyadenylation, folding, translation, localization, transport, and decay of multiple types of RNA, without altering the nucleotide sequence. The three components involved in m6A modification, namely writer, eraser, and reader, mediate the abundance of RNA m6A modification through complex collaborative actions. Currently, research on m6A regulatory genes in plants is still in its infancy. RESULTS: In this study, we identified 52 candidate m6A regulatory genes in common tobacco (Nicotiana tabacum L.). Gene structure, conserved domains, and motif analysis showed structural and functional diversity among different subgroups of tobacco m6A regulatory genes. The amplification of m6A regulatory genes were mainly driven by polyploidization and dispersed duplication, and duplicated genes evolved through purified selection. Based on the potential regulatory network and expression pattern analysis of m6A regulatory genes, a significant number of m6A regulatory genes might play important roles in growth, development, and stress response processes. Furthermore, we have confirmed the critical role of NtFIP37B, an m6A writer gene in tobacco, in enhancing drought resistance. CONCLUSIONS: This study provides useful information for better understanding the evolution of m6A regulatory genes and the role of m6A modification in tobacco stress response, and lays the foundation for further elucidating the function of m6A regulatory genes in tobacco.

Myeloid zinc finger 1 knockdown promotes osteoclastogenesis and bone loss in part by regulating RANKL-induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway.

The overactivation of the osteoclasts is a crucial pathological factor in the development of osteoporosis. MZF1, belonging to the scan-zinc finger family, plays a significant role in various processes associated with tumor malignant progression and acts as an essential transcription factor regulating osteoblast expression. However, the exact role of MZF1 in osteoclasts has not been determined. In this study, the purpose of our study was to elucidate the role of MZF1 in osteoclastogenesis. First, we established MZF1-deficient female mice and evaluated the femur bone phenotype by micro-computed tomography and histological staining. Our findings indicate that MZF1-/- mice exhibited a low bone mass osteoporosis phenotype. RANKL could independently induce the differentiation of RAW264.7 cells into osteoclasts, and we found that the expression level of MZF1 protein decreased gradually. Then, the CRISPR/Cas 9 gene-editing technique was used to build a RAW264.7 cell model with MZF1 knockout, and RANKL was used to independently induce MZF1-/- and wild-type cells to differentiate into mature osteoclasts. Tartrate-resistant acid phosphatase staining and F-actin fluorescence results showed that the MZF1-/- group produced more tartrate-resistant acid phosphatase-positive mature osteoclasts and larger actin rings. The expression of osteoclast-associated genes (including tartrate-resistant acid phosphatase, CTSK, c-Fos, and NFATc1) was evaluated by reverse transcription quantitative polymerase chain reaction and Western blot. The expression of key genes of osteoclast differentiation in the MZF1-/- group was significantly increased. Furthermore, we found that cell viability was increased in the early stages of RANKL-induced cell differentiation in the MZF1-/- group cells. We examined some prevalent ferroptosis markers, including malondialdehyde, glutathione, and intracellular Fe, the active form of iron in the cytoplasm during the early stages of osteoclastogenesis. The results suggest that MZF1 may be involved in osteoclast differentiation by regulating RANKL-induced ferroptosis of osteoclasts. Collectively, our findings shed light on the essential involvement of MZF1 in the regulation of osteoclastogenesis in osteoporosis and provide insights into its potential underlying mechanism.

The Potential of Natural Compounds Regulating Autophagy in the Treatment of Osteoporosis.

The maintenance of bone homeostasis is dynamically regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Abnormal differentiation of osteoclast and insufficient osteoblast production can cause bone diseases such as osteoporosis. As one of the highly conserved catabolic pathways in eukaryotic cells, autophagy plays an important role in maintaining cell homeostasis, stress injury repair, proliferation and differentiation. Numerous studies have found that autophagy activity is essential for the survival, differentiation and function of bone cells, and that regulation of autophagy can affect the metabolism of osteoblasts and osteoclasts, thus affecting bone homeostasis. Therefore, using autophagy as a theme, this review outlines the basic process of autophagy, the relationship between autophagy and osteoblasts and osteoclasts, and summarizes the latest research progress of common autophagic signaling pathways in osteoblasts and osteoclasts. The regulatory effects of protein molecules and natural compounds on the autophagy pathway of osteoblasts and osteoclasts discovered in current research are summarized and discussed. This will help to further clarify the mechanism of osteoporosis, understand the relationship between autophagy and osteoporosis, and propose new therapeutic strategies and new ideas for anti-osteoporosis.

Carbon nanosol-induced assemblage of a plant-beneficial microbiome consortium.

Carbon nanosol (CNS) is a carbon-based nanomaterial that promotes plant growth; however, its functional mechanisms and effects on the microbiome are not fully understood. Here, we explored the effects of CNS on the relationship between the soil, endophytic microbiomes and plant productivity. CNS treatment increased the fresh biomass of tobacco (Nicotiana tabacum L.) plants by 27.4% ± 9.9%. Amplicon sequencing analysis showed that the CNS treatment significantly affected the composition and diversity of the microbial communities in multiple ecological niches associated with tobacco, especially the bulk soil and stem endophytic microbiome. Furthermore, the application of CNS resulted in enhanced network connectivity and stability of the microbial communities in different niches, particularly in the soil, implying a strengthening of certain microbial interactions. Certain potentially growth-promoting root endophytic bacteria were more abundant under the CNS treatment. In addition, CNS increased the abundance of some endophytic microbial functional genes known to enhance plant growth, such as those associated with nutrient metabolism and the plant hormone biosynthesis pathways. We isolated two bacterial strains (Sphingopyxis sp. and Novosphingobium sp.) that were enriched under CNS treatment, and they were confirmed to promote tobacco plant growth in vitro. These results suggested that CNS might, at least in part, promote plant growth by enriching beneficial bacteria in the microbiome.

The Therapeutic Effect of Natural Compounds on Osteoporosis through Ferroptosis.

Ferroptosis is a newly discovered non-apoptotic cell death whose key is lipid peroxidation. It has been reported that ferroptosis is involved in the occurrence and development of tumors and nervous system and musculoskeletal diseases. Cellular ferroptosis contributes to the imbalance of bone homeostasis and is involved in the development of osteoporosis; however, the detailed mechanism of which is still unclear though it may provide a new direction for anti-osteoporosis. The current drugs used in the treatment of osteoporosis, such as bisphosphonates and teriparatide, have many side effects, increasing people's search for natural compounds to treat osteoporosis. This review paper briefly summarizes the current research regarding the mechanisms of ferroptosis and natural anti-osteoporosis compounds targeting its pathway.

The Molecular Mechanisms Study of Engeletin Suppresses RANKL-Induced Osteoclastogenesis and Inhibits Ovariectomized Murine Model Bone Loss.

Objective: Osteoclastogenesis, the process of osteoclast differentiation, plays a critical role in bone homeostasis. Overexpression of osteoclastogenesis can lead to pathological conditions, such as osteoporosis and osteolysis. This study aims to investigate the role of Engelitin in the process of RAW264.7 cell differentiation into osteoclasts induced by RANKL, as well as in a mouse model of bone loss following ovariectomy. Methods: We used RANKL-stimulated RAW264.7 cells as an in vitro osteoclast differentiation model. The effects of Eng on morphological changes during osteoclast differentiation were evaluated using TRAP and F-actin staining. The effects of Eng on the molecular level of osteoclast differentiation were evaluated using Western blot and q-PCR. The level of reactive oxygen species was evaluated using the DCFH-DA staining method. We then used ovariectomized mice as a bone loss animal model. The effects of Eng on changes in bone loss in vivo were evaluated using micro-CT and histological analysis staining. Results: In the in vitro experiments, Eng exhibited dose-dependent inhibition of osteoclast formation and F-actin formation. At the molecular level, Eng dose-dependently suppressed the expression of specific RNAs (NFATc1, c-Fos, TRAP, Cathepsin K, MMP-9) involved in osteoclast differentiation, and inhibited the phosphorylation of proteins such as IκBα, P65, ERK, JNK, and P38. Additionally, Eng dose-dependently suppressed ROS levels and promoted the expression of antioxidant enzymes such as Nrf2, HO-1, and NQO1. In the in vivo experiments, Eng improved bone loss in ovariectomized mice. Conclusion: Our study found that Eng inhibited RANKL-induced osteoclast differentiation through multiple signaling pathways, including MAPKs, NF-κB, and ROS aggregation. Furthermore, Eng improved bone loss in ovariectomized mice.

CRISPR/Cas9 knockout of MTA1 enhanced RANKL-induced osteoclastogenesis in RAW264.7 cells partly via increasing ROS activities.

Metastasis-associated protein 1 (MTA1), belonging to metastasis-associated proteins (MTA) family, which are integral parts of nucleosome remodelling and histone deacetylation (NuRD) complexes. However, the effect of MTA1 on osteoclastogenesis is unknown. Currently, the regulation of MTA1 in osteoclastogenesis was reported for the first time. MTA1 knockout cells (KO) were established by CRISPR/Cas9 genome editing. RAW264.7 cells with WT and KO group were stimulated independently by RANKL to differentiate into mature osteoclasts. Further, western blotting and quantitative qRT-PCR were used to explore the effect of MTA1 on the expression of osteoclast-associated genes (including CTSK, MMP9, c-Fos and NFATc1) during osteoclastogenesis. Moreover, the effects of MTA1 on the expression of reactive oxygen species (ROS) in osteoclastogenesis was determined by 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Nuclear translocation of Nrf2 was assessed by immunofluorescence staining and western blotting. Our results indicated that the MTA1 deletion group could differentiate into osteoclasts with larger volume and more TRAP positive. In addition, compared with WT group, KO group cells generated more actin rings. Mechanistically, the loss of MTA1 increased the expression of osteoclast-specific markers, including c-Fos, NFATc1, CTSK and MMP-9. Furthermore, the results of qRT-PCR and western blotting showed that MTA1 deficiency reduced basal Nrf2 expression and inhibited Nrf2-mediated expression of related antioxidant enzymes. Immunofluorescence staining demonstrated that MTA1 deficiency inhibited Nrf2 nuclear translocation. Taken together, the above increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclast formation.

Predictive Factors for Bone Cement Displacement following Percutaneous Vertebral Augmentation in Kümmell's Disease.

Objective: To investigate the independent influencing factors of bone cement displacement following percutaneous vertebral augmentation (PVA) in patients with stage I and stage II Kümmell's disease. Methods: We retrospectively reviewed the records of 824 patients with stage Ⅰ and stage Ⅱ Kümmell's disease treated with percutaneous vertebroplasty (PVP) or percutaneous vertebroplasty (PKP) from January 2016 to June 2022. Patients were divided into the postoperative bone cement displacement group (n = 150) and the bone cement non-displacement group (n = 674) according to the radiographic inspection results. The following data were collected: age, gender, body mass index (BMI), underlying disease, bone mineral density (BMD), involved vertebral segment, Kümmell's disease staging, anterior height, local Cobb angle, the integrity of anterior vertebral cortex, the integrity of endplate in surgical vertebrae, surgical method, surgical approach, the volume of cement, distribution of cement, the viscosity of cement, cement leakage, and postoperative anti-osteoporosis treatment. Binary logistic regression analysis was performed to determine the independent influencing factors of bone cement displacement. The discrimination ability was evaluated using the area under the curve (AUC) of the receiver operating characteristic (ROC). Results: The results of logistic regression analysis revealed that thoracolumbar junction (odds ratio (OR) = 3.23, 95% confidence interval (CI) 2.12−4.50, p = 0.011), Kümmell's disease staging (OR = 2.23, 95% CI 1.81−3.41, p < 0.001), anterior cortex defect (OR = 5.34, 95% CI 3.53−7.21, p < 0.001), vertebral endplates defect (OR = 0.54, 95% CI 0.35−0.71, p < 0.001), cement distribution (OR = 2.86, 95% CI 2.03−3.52, p = 0.002), cement leakage (OR = 4.59, 95% CI 3.85−5.72, p < 0.001), restoration of local Cobb angle (OR = 3.17, 95% CI 2.40−5.73, p = 0.024), and postoperative anti-osteoporosis treatment (OR = 0.48, 95% CI 0.18−0.72, p = 0.025) were independently associated with the bone cement displacement. The results of the ROC curve analysis showed that the AUC was 0.816 (95% CI 0.747−0.885), the sensitivity was 0.717, and the specificity was 0.793. Conclusion: Thoracolumbar fracture, stage Ⅱ Kümmell's disease, anterior cortex defect, uneven cement distribution, cement leakage, and high restoration of the local Cobb angle were risk factors for cement displacement after PVA in Kümmell's disease, while vertebral endplates defect and postoperative anti-osteoporosis treatment are protective factors.

Hypoxia-inducible factor 1α enhances RANKL-induced osteoclast differentiation by upregulating the MAPK pathway.

Background: Hypoxia (low-oxygen tension) and excessive osteoclast activation are common conditions in many bone loss diseases, such as osteoporosis, rheumatoid arthritis (RA), and pathologic fractures. Hypoxia-inducible factor 1 alpha (HIF1α) regulates cellular responses to hypoxic conditions. However, it is not yet known how HIF1α directly affects osteoclast differentiation and activation. This study sought to. explore the effects of HIF1α on osteoclast differentiation and it's molecular mechanisms. Methods: L-mimosine, a prolyl hydroxylase (PHDs) domain inhibitor, was used to stabilize HIF1α in normoxia. In the presence of receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL), RAW264.7 cells were cultured and stimulated by treatment with L-mimosine at several doses to maintain various levels of intracellular HIF1α. The multi-nucleated cells were assessed by a tartrate-resistant acid phosphatase (TRAP) and F-actin ring staining assays. The osteoclast-specific genes, such as Cathepsin K, ß3-Integrin, TRAP, c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and matrix metallo-proteinase 9 (MMP9), were analyzed by real time-polymerase chain reaction (RT-PCR). The expression of relevant proteins was analyzed by Western blot. Results: L-mimosine increased the content of intracellular HIF1α in a dose-dependent manner, which in turn promoted RANKL-induced osteoclast formation and relevant protein expression by upregulating the mitogen-activated protein kinase (MAPK) pathways. Conclusions: Our findings suggest that HIF1α directly increases the osteoclast differentiation of RANKL-mediated RAW264.7 cells in vitro by upregulating the MAPK pathways.

Hydrogel-based delivery system applied in the local anti-osteoporotic bone defects.

Osteoporosis is an age-related systemic skeletal disease leading to bone mass loss and microarchitectural deterioration. It affects a large number of patients, thereby economically burdening healthcare systems worldwide. The low bioavailability and complications, associated with systemic drug consumption, limit the efficacy of anti-osteoporosis drugs currently available. Thus, a combination of therapies, including local treatment and systemic intervention, may be more beneficial over a singular pharmacological treatment. Hydrogels are attractive materials as fillers for bone injuries with irregular shapes and as carriers for local therapeutic treatments. They exhibit low cytotoxicity, excellent biocompatibility, and biodegradability, and some with excellent mechanical and swelling properties, and a controlled degradation rate. This review reports the advantages of hydrogels for adjuvants loading, including nature-based, synthetic, and composite hydrogels. In addition, we discuss functional adjuvants loaded with hydrogels, primarily focusing on drugs and cells that inhibit osteoclast and promote osteoblast. Selecting appropriate hydrogels and adjuvants is the key to successful treatment. We hope this review serves as a reference for subsequent research and clinical application of hydrogel-based delivery systems in osteoporosis therapy.

Receptor activator of nuclear factor-κB ligand-mediated osteoclastogenesis signaling pathway and related therapeutic natural compounds.

Osteoclast is a hematopoietic precursor cell derived from the mononuclear macrophage cell line, which is the only cell with bone resorption function. Its abnormal activation can cause serious osteolysis related diseases such as rheumatoid arthritis, Paget's disease and osteoporosis. In recent years, the adverse effects caused by anabolic anti-osteolytic drugs have increased the interest of researchers in the potential therapeutic and preventive effects of natural plant derivatives and natural compounds against osteolytic diseases caused by osteoclasts. Natural plant derivatives and natural compounds have become major research hotspots for the treatment of osteolysis-related diseases due to their good safety profile and ability to improve bone. This paper provides an overview of recent advances in the molecular mechanisms of RANKL and downstream signaling pathways in osteoclast differentiation, and briefly outlines potential natural compounds with antiosteoclast activity and molecular mechanisms.

Urolithin B suppresses osteoclastogenesis via inhibiting RANKL-induced signalling pathways and attenuating ROS activities.

Osteoporosis (OP) has severely affected human health, which is characterized by abnormal differentiation of osteoclasts. Urolithin B (UB), as a potential natural drug, has been reported to exhibit numerous biological activities including antioxidant and anti-inflammatory but its effects on OP, especially on RANKL-stimulated osteoclast formation and activation, are still understood. In our study, we have demonstrated for the first time that UB inhibits RANKL-induced osteoclast differentiation and explored its potential mechanisms of action. The RAW264.7 cells were cultured and induced with RANKL followed by UB treatment. Then, the effects of UB on mature osteoclast differentiation were evaluated by counting tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and F-actin ring analysis. Moreover, the effects of UB on RANKL-induced reactive oxygen species (ROS) were measured by 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Further, we explored the potential mechanisms of these downregulation effects by performing Western blotting and quantitative RT-PCR examination. We found that UB represses osteoclastogenesis, F-actin belts formation, osteoclast-specific gene expressions and ROS activity in a time- and concentration-dependent manner. Mechanistically, UB attenuates intracellular ROS levels by upregulation of Nrf2 and other ROS scavenging enzymes activation. Furthermore, UB also inhibited RANKL-induced NF-κB, MAPK and Akt signalling pathway and suppressed expression of c-Fos and nuclear factor of activated T cells 1 (NFATc1), which is the master transcription factor of osteoclast differentiation. Taken together, our findings confirm that UB is a polyphenolic compound that can be a potential therapeutic treatment for osteoclast-related bone diseases such as osteoporosis.

Tanshinone Ameliorates Glucocorticoid-Induced Bone Loss via Activation of AKT1 Signaling Pathway.

Purpose: Osteoporosis, a common disorder especially prevalent in the postmenopausal women and the elderly, is becoming a worldwide public health problem. Osteoporosis can cause severe joint pain, fragility fractures, and other symptoms, which can seriously impair the daily lives of affected patients. Currently, no gold-standard drug is available that can completely cure osteoporosis. Tanshinone is a traditional Chinese medicine, which can exhibit multiple biological activities. It might also display a protective effect on osteoporosis. However, the molecular mechanism through which tanshinone can improve osteoporosis remain unclear. The objective of our study is to explore the underlying mechanism behind the protective actions of tanshinone. Methods: The common KEGG pathways of tanshinone-targeted genes and osteoporosis were analyzed by using bioinformatics analysis. The bioinformatics analysis results were further validated both by in vitro and in vivo experiments. Results: 21 common KEGG pathways were identified between osteoporosis and tanshinone-targeted genes. It was further found that tanshinone could induce expression of AKT1, promote the proliferation of MSCs, and ultimately suppress their apoptosis. Conclusion: Taken together, our findings indicate that tanshinone can alleviate osteoporosis, its effect was potentially mediated through modulating AKT1 expression. Thus, tanshinone could serve as a promising treatment option for osteoporosis.

Relationship between degenerative scoliosis and lower extremity mechanical parameters based on EOS imaging system.

OBJECTIVE: This study aimed to assess the correlation between coronal imbalance and lower-limb physiological parameters in degenerative scoliosis using the biplanar whole body imaging system (EOS). MATERIALS AND METHODS: A total of 101 successive EOS images were selected between January 2018 and December 2021. Of the selected images, 63 patients were in the degenerative scoliosis group (DSG) and 38 patients were in the control group (CG). Two independent observers performed measurements of the parameters and compared the two groups. RESULTS: Among parameters examined, significant inter-group differences were found for coronal pelvic tilt angle (CPT), bilateral femoral length difference (ΔFL), and bilateral total lower limb length (ΔTL) difference. Additionally, the knee and ankle joints had more severe degeneration on the main curved side in patients with degenerative scoliosis. In the left curved group, 18 (42.86%) and 24 (57.1%) patients had more severe degeneration in the left knee and left ankle, respectively. In the right lateral bending group, 13 (61.9%) and 14 (66.7%) patients had more severe degeneration in the right knee and right ankle, respectively. Statistical differences were found in the degree of degeneration in both knee and ankle joints bilaterally. CONCLUSION: This study showed that biomechanical parameters of the lower limbs are affected in cases of degenerative scoliosis with altered coronal balance. The lower limb on the main curve side became shorter compared to its counterpart, and joint degeneration of the knee and ankle joints became more severe.