Cucurbitacin B attenuates osteoarthritis development by inhibiting NLRP3 inflammasome activation and pyroptosis through activating Nrf2/HO-1 pathway.

Osteoarthritis (OA) is a complicated joint disorder characterized by inflammation that causes joint destruction. Cucurbitacin B (CuB) is a naturally occurring triterpenoid compound derived from plants in the Cucurbitaceae family. The aim of this study is to investigate the potential role and mechanisms of CuB in a mouse model of OA. This study identified the key targets and potential pathways of CuB through network pharmacology analysis. In vivo and in vitro studies confirmed the potential mechanisms of CuB in OA. Through network pharmacology, 54 potential targets for CuB in treating OA were identified. The therapeutic potential of CuB is associated with the nod-like receptor pyrin domain 3 (NLRP3) inflammasome and pyroptosis. Molecular docking results indicate a strong binding affinity of CuB to nuclear factor erythroid 2-related factor 2 (Nrf2) and p65. In vitro experiments demonstrate that CuB effectively inhibits the expression of pro-inflammatory factors induced by interleukin-1ß (IL-1ß), including cyclooxygenase-2, inducible nitric oxide synthase, IL-1ß, and IL-18. CuB inhibits the degradation of type II collagen and aggrecan in the extracellular matrix (ECM), as well as the expression of matrix metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin motifs-5. CuB protects cells by activating the Nrf2/hemeoxygenase-1 (HO-1) pathway and inhibiting nuclear factor-κB (NF-κB)/NLRP3 inflammasome-mediated pyroptosis. Moreover, in vivo experiments show that CuB can slow down cartilage degradation in an OA mouse model. CuB effectively prevents the progression of OA by inhibiting inflammation in chondrocytes and ECM degradation. This action is further mediated through the activation of the Nrf2/HO-1 pathway to inhibit NF-κB/NLRP3 inflammasome activation. Thus, CuB is a potential therapeutic agent for OA.

Microneedles loaded with cerium-manganese oxide nanoparticles for targeting macrophages in the treatment of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a prevalent inflammatory autoimmune disease characterised by persistent inflammation and joint damage with elevated levels of reactive oxygen species (ROS). Current treatment modalities for RA have significant limitations, including poor bioavailability, severe side effects, and inadequate targeting of inflamed joints. Herein, we synthesised cerium/manganese oxide nanoparticles (NPs) as efficient drug carriers with antioxidant and catalytic-like functions that can eliminate ROS to facilitate the polarization of macrophages phenotype from M1 to M2 and alleviate inflammation. Methotrexate (MTX), a first-line RA medication, was loaded into the NPs, which were further modified with bovine serum albumin (BSA) and integrated into dissolving hyaluronic acid-based microneedles (MNs) for transdermal delivery. RESULT: This innovative approach significantly enhanced drug delivery efficiency, reduced RA inflammation, and successfully modulated macrophage polarization toward an anti-inflammatory phenotype. CONCLUSION: This research not only presents a promising drug delivery strategy for RA but also contributes broadly to the field of immune disease treatment by offering an advanced approach for macrophage phenotypic reprogramming.

Asiatic acid prevents glucocorticoid-induced femoral head osteonecrosis via PI3K/AKT pathway.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) represents a predominant etiology of non-traumatic osteonecrosis, imposing substantial pain, restricting hip mobility, and diminishing overall quality of life for affected individuals. Centella asiatica (L.) Urb. (CA), an herbal remedy deeply rooted in traditional oriental medicine, has exhibited noteworthy therapeutic efficacy in addressing inflammation and facilitating wound healing. Drawing from CA's historical applications, its anti-inflammatory, anti-apoptotic, and antioxidant attributes may hold promise for managing GIONFH. Asiatic acid (AA), a primary constituent of CA, has been substantiated as a key contributor to its anti-apoptotic, antioxidant, and anti-inflammatory capabilities, showcasing a close association with orthopedic conditions. For the investigation of whether AA could alleviate GIONFH through suppressing oxidative stress, apoptosis, and to delve into its potential cellular and molecular mechanisms, the connection between AA and disease was analyzed through network pharmacology. DEX-induced apoptosis in rat osteoblasts and GIONFH in rat models, got utilized for the verification in vitro/vivo, on underlying mechanism of AA in GIONFH. Network pharmacology analysis reveals a robust correlation between AA and GIONFH in multiple target genes. AA has demonstrated the inhibition of DEX-induced osteoblast apoptosis by modulating apoptotic factors like BAX, BCL-2, Cleaved-caspase3, and cleaved-caspase9. Furthermore, it effectively diminishes the ROS overexpression and regulates oxidative stress through mitochondrial pathway. Mechanistic insights suggest that AA's therapeutic effects involve phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT) pathway activation. Additionally, AA has exhibited its potential to ameliorate GIONFH progression in rat models. Our findings revealed that AA mitigated DEX-induced osteoblast apoptosis and oxidative stress through triggering PI3K/AKT pathway. Also, AA can effectively thwart GIONFH occurrence and development in rats.

Experimental study on direct tensile fatigue performance of basalt fiber reinforced concrete.

Conducting research on the fatigue performance of concrete materials is of great significance for the anti fatigue design of concrete structures. Currently, indirect tensile or compressive strength tests are commonly used to study the fatigue performance of basalt fiber reinforced concrete, but there is little research on its fatigue performance under direct tensile conditions. Using a fatigue testing machine and a self-developed concrete axial tensile device, direct tensile fatigue tests of basalt fiber reinforced concrete were conducted under different fiber content and stress levels. Based on fatigue test data, the entire fatigue tensile process of basalt fiber reinforced concrete was analyzed, and the effects of fiber content and stress level on the fatigue life of concrete specimens were explored. Strain fatigue life curves of concrete with different fiber content were plotted. The experimental results indicate that the failure mode of basalt fiber reinforced concrete under cyclic loading is brittle failure; with the increase of basalt fiber content, the fatigue life of concrete first increases and then decreases. When the fiber content is 0.3%, the fatigue life of basalt fiber concrete is the highest compared to the benchmark concrete. When the fiber content is the same, the fatigue life of concrete decreases with the increase of stress level. The fatigue deformation process of basalt fiber reinforced concrete can be divided into three stages: the stage of fast strain growth, the stage of uniform strain growth, and the stage of rapid strain growth.

Carnitine functions as an enhancer of NRF2 to inhibit osteoclastogenesis via regulating macrophage polarization in osteoporosis.

Osteoporosis, which manifests as reduced bone mass and deteriorated bone quality, is common in the elderly population. It is characterized by persistent elevation of macrophage-associated inflammation and active osteoclast bone resorption. Currently, the roles of intracellular metabolism in regulating these processes remain unclear. In this study, we initially performed bioinformatics analysis and observed a significant increase in the proportion of M1 macrophages in bone marrow with aging. Further metabolomics analysis demonstrated a notable reduction in the expression of carnitine metabolites in aged macrophages, while carnitine was not detected in osteoclasts. During the differentiation process, osteoclasts took up carnitine synthesized by macrophages to regulate their own activity. Mechanistically, carnitine enhanced the function of Nrf2 by inhibiting the Keap1-Nrf2 interaction, reducing the proteasome-dependent ubiquitination and degradation of Nrf2. In silico molecular ligand docking analysis of the interaction between carnitine and Keap1 showed that carnitine binds to Keap1 to stabilize Nrf2 and enhance its function. In this study, we found that the decrease in carnitine levels in aging macrophages causes overactivation of osteoclasts, ultimately leading to osteoporosis. A decrease in serum carnitine levels in patients with osteoporosis was found to have good diagnostic and predictive value. Moreover, supplementation with carnitine was shown to be effective in the treatment of osteoporosis.

Taxifolin-mediated Nrf2 activation ameliorates oxidative stress and apoptosis for the treatment of glucocorticoid-induced osteonecrosis of the femoral head.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is the main complication secondary to long-term or excessive use of glucocorticoids (GCs). Taxifolin (TAX) is a natural antioxidant with various pharmacological effects, such as antioxidative stress and antiapoptotic properties. The purpose of this study was to explore whether TAX could regulate oxidative stress and apoptosis in GIONFH by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. We conducted qRT-PCR, Western blotting, TUNEL assays, flow cytometry, and other experiments in vitro. Microcomputed tomography analysis, hematoxylin-eosin staining, and immunohistochemical staining were performed to determine the therapeutic effect of TAX in vivo. TAX mitigated the overexpression of ROS and NOX gene expression induced by DEX, effectively reducing oxidative stress. Additionally, TAX could alleviate DEX-induced osteoblast apoptosis, as evidenced by qRT-PCR, Western blotting, and other experimental techniques. Our in vivo studies further demonstrated that TAX mitigates the progression of GIONFH in rats by combating oxidative stress and apoptosis. Mechanistic exploration revealed that TAX thwarts the progression of GIONFH through the activation of the Nrf2 pathway. Overall, our research herein reports that TAX-mediated Nrf2 activation ameliorates oxidative stress and apoptosis for the treatment of GIONFH.

MiR-146b-5p enriched bioinspired exosomes derived from fucoidan-directed induction mesenchymal stem cells protect chondrocytes in osteoarthritis by targeting TRAF6.

Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive cartilage degradation and inflammation. In recent years, mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) have attracted widespread attention for their potential role in modulating OA pathology. However, the unpredictable therapeutic effects of exosomes have been a significant barrier to their extensive clinical application. In this study, we investigated whether fucoidan-pretreated MSC-derived exosomes (F-MSCs-Exo) could better protect chondrocytes in osteoarthritic joints and elucidate its underlying mechanisms. In order to evaluate the role of F-MSCs-Exo in osteoarthritis, both in vitro and in vivo studies were conducted. MiRNA sequencing was employed to analyze MSCs-Exo and F-MSCs-Exo, enabling the identification of differentially expressed genes and the exploration of the underlying mechanisms behind the protective effects of F-MSCs-Exo in osteoarthritis. Compared to MSCs-Exo, F-MSCs-Exo demonstrated superior effectiveness in inhibiting inflammatory responses and extracellular matrix degradation in rat chondrocytes. Moreover, F-MSCs-Exo exhibited enhanced activation of autophagy in chondrocytes. MiRNA sequencing of both MSCs-Exo and F-MSCs-Exo revealed that miR-146b-5p emerged as a promising candidate mediator for the chondroprotective function of F-MSCs-Exo, with TRAF6 identified as its downstream target. In conclusion, our research results demonstrate that miR-146b-5p encapsulated in F-MSCs-Exo effectively inhibits TRAF6 activation, thereby suppressing inflammatory responses and extracellular matrix degradation, while promoting chondrocyte autophagy for the protection of osteoarthritic cartilage cells. Consequently, the development of a therapeutic approach combining fucoidan with MSC-derived exosomes provides a promising strategy for the clinical treatment of osteoarthritis.

Erythropoietin suppresses osteoblast apoptosis and ameliorates steroid-induced necrosis of the femoral head in rats by inhibition of STAT1-caspase 3 signaling pathway.

BACKGROUND: Steroid-induced avascular necrosis of the femoral head (SANFH) is characterized by osteoblast apoptosis, leading to a loss of bone structure and impaired hip joint function. It has been demonstrated that erythropoietin (EPO) performs a number of biological roles. OBJECTIVE: We examined the effects of EPO on SANFH and its regulation of the STAT1-caspase 3 signaling pathway. METHOD: In vitro, osteoblasts were treated with dexamethasone (Dex) or EPO. We identified the cytotoxicity of EPO by CCK-8, the protein expression of P-STAT1, cleaved-caspase9, cleaved-caspase3, Bcl-2, BAX, and cytochrome c by Western blotting, and evaluated the apoptosis of osteoblasts by flow cytometry. In vivo, we analyzed the protective effect of EPO against SANFH by hematoxylin and eosin (H&E), Immunohistochemical staining, and Micro-computed tomography (CT). RESULTS: In vitro, EPO had no apparent toxic effect on osteoblasts. In Dex-stimulated cells, EPO therapy lowered the protein expression of BAX, cytochrome c, p-STAT1, cleaved-caspase9, and cleaved-caspase3 while increasing the expression of Bcl-2. EPO can alleviate the apoptosis induced by Dex. In vivo, EPO can lower the percentage of empty bone lacunae in SANFH rats. CONCLUSION: The present study shows that EPO conferred beneficial effects in rats with SANFH by inhibiting STAT1-caspase 3 signaling, suggesting that EPO may be developed as a treatment for SANFH.

Experimental study on the axial tensile properties of polypropylene fiber reinforced concrete.

In order to study the axial tensile properties of polypropylene fiber reinforced concrete, an axial tensile test device for concrete is developed in this paper. The device is composed of three parts: rigid frame, spherical hinge and puller, and specimen fabrication part. The test device can accurately measure the tensile strength and peak tensile strain of concrete, and perfectly solves the eccentricity problem of concrete specimens under tension. It can measure the post peak segment tensile strain, such that the whole process tensile stress-strain curve can be obtained. The axial tensile test of polypropylene fiber concrete was carried out using the above test device, and the results show that the tensile strength of concrete can be clearly improved by adding polypropylene fiber, which makes the tensile failure of concrete show certain plastic characteristics. The test results show that with the increase in fiber content, the tensile strength of concrete increases first and then decreases. The effects of polypropylene fiber content and curing age on the tensile properties of concrete were studied and the optimum polypropylene fiber content was determined. When the fiber content is 0.9 kg/m3, the tensile strength of concrete reaches the maximum value. The splitting tensile test of concrete under the same condition was carried out simultaneously. The damage phenomenon and test results of the axial tensile test and splitting tensile test of concrete were compared and analyzed, and the applicability of the new developed device in the concrete axial tensile test was verified.

Extracts of Oldenlandia diffusa protects chondrocytes via inhibiting apoptosis and associated inflammatory response in osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteoarthritis (OA) is a type of joint disorder that is marked by the gradual breakdown of cartilage and persistent inflammation of the synovial membrane, and is a leading cause of disability among elderly people worldwide. Oldenlandia diffusa (OD) is a member of the Rubiaceae family, and various researches have revealed that it possesses antioxidant, anti-inflammatory, and anti-tumor properties. Extracts of Oldenlandia diffusa is commonly used in traditional oriental medicine to treat various illnesses, including inflammation and cancer. AIM OF THE STUDY: This study is aimed at investigating the anti-inflammatory and anti-apoptosis effects of OD and its potential mechanisms on IL-1ß-induced mouse chondrocytes, as well as its characteristics in a mouse osteoarthritis model. MATERIALS AND METHODS: In this study, the key targets and potential pathways of OD were determined through network pharmacology analysis and molecular docking. The potential mechanism of OD in osteoarthritis was verified by in vitro and in vivo studies. RESULTS: The results of network pharmacology showed that Bax, Bcl2, CASP3, and JUN are key candidate targets of OD for the treatment of osteoarthritis. There is a strong correlation between apoptosis and both OA and OD. Additionally, molecular docking results show that ß-sitosterol in OD can strongly bind with CASP3 and PTGS2. In vitro experiments showed that OD pretreatment inhibited the expression of pro-inflammatory factors induced by IL-1ß, such as COX2, iNOS, IL-6, TNF-α, and PGE2. Furthermore, OD reversed IL-1ß-mediated degradation of collagen II and aggrecan within the extracellular matrix (ECM). The protective effect of OD can be attributed to its inhibition of the MAPK pathway and inhibition of chondrocyte apoptosis. Additionally, it was found that OD can alleviate cartilage degradation in a mouse model of knee osteoarthritis. CONCLUSION: Our study showed that ß-sitosterol, one of the active components of OD, could alleviate the inflammation and cartilage degeneration of OA by inhibiting chondrocyte apoptosis and MAPK pathway.

Gas-to-ionic liquid partition: QSPR modeling and mechanistic interpretation.

The present work was devoted to explore the quantitative structure-property relationships for gas-to-ionic liquid partition coefficients (log KILA ). A series of linear models were first established for the representative dataset (IL01). The optimal model was a four-parameter equation (1Ed) consisting of two electrostatic potential-based descriptors ( Σ V s , i n d - ${{\rm { \Sigma }}{V}_{s,ind}^{-}}$ and Vs,max ), one 2D matrix-based descriptor (J_D/Dt) and dipole moment (µ). All of the four descriptors introduced in the model can find the corresponding parameters, directly or indirectly, from Abraham's linear solvation energy relationship (LSER) or its theoretical alternatives, which endows the model good interpretability. Gaussian process was utilized to build the nonlinear model. Systematical validations, including 5-fold cross-validation for the training set, the validation for test set, as well as a more rigorous Monte Carlo cross-validation were performed to verify the reliability of the constructed models. Applicability domain of the model was evaluated, and the Williams plot revealed that the model can be used to predict the log KILA values of structurally diverse solutes. The other 13 datasets were also processed in the same way, and all of the linear models with expressions similar to equation 1Ed were obtained. These models, whether linear of nonlinear, represent satisfactory statistical results, which confirms the universality of the method adopted in this study in QSPR modeling of gas-to-IL partition.

Formononetin improves the inflammatory response and bone destruction in knee joint lesions by regulating the NF-kB and MAPK signaling pathways.

Formononetin (FMN) is a phytoestrogen that belongs to the isoflavone family. It has antioxidant and anti-inflammatory effects, as well as, many other biological activities. Existing evidence has aroused interest in its ability to protect against osteoarthritis (OA) and promote bone remodeling. To date, research on this topic has not been thorough and many issues remain controversial. Therefore, the purpose of our study was to explore the protective effect of FMN against knee injury and clarify the possible molecular mechanisms. We found that FMN inhibited osteoclast formation induced by receptor activator of NF-κB ligand (RANKL). Inhibition of the phosphorylation and nuclear translocation of p65 in the NF-κB signaling pathway plays a role in this effect. Similarly, during the inflammatory response of primary knee cartilage cells activated by IL-1ß, FMN inhibited the NF-κB signaling pathway and the phosphorylation of the ERK and JNK proteins in the MAPK signaling pathway to suppress the inflammatory response. In addition, in vivo experiments showed that both low- and high-dose FMN had a clear protective effect against knee injury in the DMM (destabilization of the medial meniscus) model, and the therapeutic effect of high-dose FMN was stronger. In conclusion, these studies provide evidence of the protective effect of FMN against knee injury.

Overexpression of miR-148a-3p inhibits extracellular matrix degradation and alleviates IL-1ß-induced intervertebral disc degeneration.

Objectives: Recently, studies on microRNAs (miRNAs) and their targets and related genes have provided new therapeutic opportunities for controlling intervertebral disc degeneration (IDD). We aimed to investigate the effects of miR-148a-3p overexpression on IDD progression. Materials and Methods: This study used microRNA microarrays to analyze key regulators of IDD. Q-PCR was used to verify the IL-1ß-induced down-regulation of miR-148a-3p expression both in nucleus pulposus (NP) tissues of IDD patients and in degenerated NP cells (NPCs) of rats. Rat NPC micromass cultures and ex vivo intervertebral disc (IVD) culture models were established, and histological staining was performed to verify the effect of miR-148a-3p on the general morphology and proteoglycan and collagen contents of IVDs. In addition, q-PCR and western blotting analyses were performed to examine the expression of ECM molecules and matrix-degrading enzymes. A luciferase reporter assay was used to confirm the target genes of miR-148a-3p. Results: Our data revealed that miR-148a-3p was down-regulated in IDD. Overexpression of miR-148a-3p had no effect on ACAN or COL2A1 gene expression but decreased MMP3, MMP13, and ADAMTS5 gene expression. The matrix deposited by miR-148a-3p-overexpressing rat NPCs contained high levels of proteoglycans and collagen. The ex vivo experiments verified that agomiR-148a-3p alleviated the NPC matrix degradation induced by IL-1ß. A luciferase reporter assay confirmed that miR-148a-3p directly targeted ADAMTS5 and MMP13. Conclusion: We proved that miR-148a-3p can attenuate ECM loss and protect NP function by inhibiting matrix-degrading enzymes.

Stevioside targets the NF-κB and MAPK pathways for inhibiting inflammation and apoptosis of chondrocytes and ameliorates osteoarthritis in vivo.

Osteoarthritis (OA) is a joint disease that is characterized by articular cartilage degeneration and destruction. Stevioside (SVS) is a diterpenoid glycoside extracted from Stevia rebaudiana Bertoni with some specific effects against inflammatory and apoptotic, whereas it is still unclear what function SVS has in osteoarthritis. This study focuses on the anti-inflammatory and anti-apoptosis functions of SVS on chondrocytes induced by interleukin (IL)-1beta, and the role of SVS in an osteoarthritis model for mice. We can detect the production of inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2) using real-time quantitative polymerase chain reaction (RT-qPCR), the Griess reaction, and enzyme linked immunosorbent assay (ELISA). On the basis of Western blot, we have observed the protein expressions of cartilage matrix metabolism, inflammatory factors, and apoptosis of chondrocytes. Simultaneously, the pharmacological effects of SVS in mice were evaluated by hematoxylin and eosin (HE), toluidine blue, Safranin O, and immunohistochemical staining. The results show that SVS slows extracellular matrix degradation and chondrocyte apoptosis. In addition, SVS mediates its cellular effect by inhibiting the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways. Meanwhile, molecular docking studies revealed that SVS has excellent binding capabilities to p65, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). The study suggests that SVS can be developed as a potential osteoarthritis treatment.

Plantamajoside suppresses the activation of NF-κB and MAPK and ameliorates the development of osteoarthritis.

Osteoarthritis (OA) is a common degenerative bone and joint disorder characterized by progressive cartilage degeneration and secondary synovial inflammation. It is a common chronic joint disorder that affects people of all ages (especially the old). Plantamajoside is a phenylpropanoside derived from plantain. It has a variety of biological properties, including antioxidant, anti-malignant cell proliferation, and anti-inflammatory properties. In this study, the latent mechanism of plantamajoside was explored by slowing the in-vivo and in-vitro progression of osteoarthritis. The results revealed that plantamajoside pre-conditioning inhibited IL-1ß induced pro-inflammatory factors like COX-2, iNOS, IL-6, and TNF-α. Moreover, plantamajoside also reversed the IL-1 ß mediated type II collagen and aggrecan degradation within the extracellular matrix (ECM). The protective effects of plantamajoside have been attributed to the inhibition of both MAPK and NF-κB pathways. Furthermore, our in-vivo research found that plantamajoside could slow the progression of OA in mice. Finally, all findings point to plantamajoside as a potential anti-OA therapeutic candidate.

XIAP and PHB1 Regulate Anoikis through Competitive Binding to TRAF6.

Anoikis resistance is a prerequisite for circulating tumor cells to survive. However, the mechanism underlying anoikis resistance is poorly understood. In the current study, the effect of TNF receptor-associated factor 6 (TRAF6)-induced NF-kB activation on anoikis susceptibility in tumor cells was evaluated. Differential TRAF6-binding proteins in anoikis-sensitive versus anoikis-resistant tumor cells were screened by LC/MS-MS analysis. The effects of TRAF6-binding proteins on the stability of TRAF6, the activation of NF-kB signaling and anoikis susceptibility in tumor cells were detected. We found that the loss of TRAF6 expression is an important molecular event linked to anoikis. X-linked inhibitor of apoptosis protein (XIAP), an E3 ligase, can bind, ubiquitinate, and degrade TRAF6 and may lead to inactivation of NF-κB signaling and anoikis sensitivity. High expression of prohibitin 1 (PHB1) competes with XIAP for binding to TRAF6 and confers anoikis resistance to tumor cells. PHB1 and TRAF6 knockdown eliminated tumor cells from the circulation in vivo. Significant correlations between elevated PHB1 and TRAF6 expression and distant metastasis were observed in patients with oral cancer. Collectively, we elucidated a novel mechanism governing anoikis. Our data also indicated that TRAF6 and PHB1 are potential therapeutic targets for tumor cells disseminating in the circulation. IMPLICATIONS: Our data implicate that PHB1 competes with XIAP for binding to TRAF6 and confers anoikis resistance to tumor cells.

MiR-148a deletion protects from bone loss in physiological and estrogen-deficient mice by targeting NRP1.

Bone metabolic homeostasis is largely dependent on the dynamic balance between osteoblasts and osteoclasts. MicroRNAs (miRNAs) play critical roles in regulating bone metabolism. In this study, we explored the role of a new miRNA (miR-148a) in osteoporosis. We compared the bone phenotype between miR-148a knockout (KO) mice and the wild-type (WT) littermates. We found miR-148a KO mice exhibited an increased bone mass phenotype and decreased osteoclastogenesis compared to the WT group. In vitro, miR-148a overexpression promoted osteoclastogenesis and bone resorption function. Mechanistically, NRP1 was identified as a novel direct target of miR-148a, and NRP1 silencing reversed the effect of miR-148a knockout. In OVX and calvarial osteolysis models, miR-148a KO protects mice against excessive bone resorption, while miR-148a agomiR/AAV-shNRP1 accelerates pathologic bone loss. Finally, the miR-148a level was found to be positively correlated with ß-CTX in postmenopausal osteoporosis (PMOP) serum specimens. In summary, our findings revealed that miR-148a genetic deletion ameliorates bone loss under physiological and pathological conditions by targeting NRP1. In osteoclast-related bone metabolic diseases such as PMOP, miR-148a may be an attractive therapeutic target in the future.

Comparison of mini-open, anteroinferior psoas approach and mini-open, direct lateral transpsoas approach for lumbar burst fractures: A retrospective cohort study.

Objective: We evaluated the effect of a novel modified OLIF technique (anteroinferior psoas approach, AIPA) for anterior decompression reconstruction in lumbar burst fractures, and compared the clinical, radiological outcomes and approach-related complications with the mini-open, lateral transpsoas approach (LTPA). Methods: From March 2016 to November 2019, 68 patients with lumbar burst fractures underwent one-stage monosegmental posterior/anterior surgery from L1-L4 segments. 35 patients included in AIPA and 33 patients in LTPA group underwent anterior decompression reconstruction. The clinical, radiological and functional evaluation outcomes were recorded during the 16-60 months follow-up period. Results: At the latest follow up, neurological state of one or more ASIA grades were achieved in AIPA (90.9%) and LTPA group (94.9%). No significant differences were noted between the two groups regarding preoperative and postoperative Cobbs angle. The surgery time (192.29 vs. 230.47 min, P = 0.02) in AIPA group was better compared with LTPA. The AIPA showed better improvement on Oswestry Disability Index (43.4% vs. 60.8%, P < 0.05) and Mental Component Score (49.0% vs. 43.7%, P < 0.05) one month after surgery, but no difference at the latest follow-up. 10 patients (9 in LTPA and 1 in AIPA) experienced temporary motor deficits in hip flexor and groin or thigh numbness, which disappeared six months after surgery. Conclusions: Compared with lateral transpsoas approach, anterior decompression reconstruction via mini-open, anteroinferior psoas approach was a safe and less invasive approach, with fewer approach-related complications in the treatment for unstable lumbar burst fractures.

N-cadherin protects oral cancer cells from NK cell killing in the circulation by inducing NK cell functional exhaustion via the KLRG1 receptor.

BACKGROUND: Circulating tumor cells (CTCs) can survive in the circulation and return to primary tumors through a self-seeding process. However, the mechanisms underlying CTCs escape from natural killer (NK) cell-mediated immune surveillance remain unclear. METHOD: Self-seeded tumor cells were isolated and characterized using a modified contralateral seeding model. A comparison of transcriptional profiles was performed between the parental cells and self-seeded cells. The molecular mechanism of self-seeded tumor cells escaping from NK cell was demonstrated through in vitro experiments and verified in a CTC-mimicking in vivo model. Then, the expression level of key protein mediating CTCs immune escape was detected in 24 paired primary and recurrent tumor samples of patients with oral cancer by the immunohistochemical method. RESULT: Self-seeded cells displayed resistance to NK cell-mediated lysis and a higher tumor seeding ability than their parental cells. Elevated expression levels of the CDH2 gene and its protein product, N-cadherin were found in self-seeded cells. NK cells secreted cytokines, and fluid shear stress facilitated N-cadherin release by promoting A disintegrin and metalloprotease 10 (ADAM10) translation or converting the precursor ADAM10 to the mature form. Soluble N-cadherin triggered NK cell functional exhaustion by interacting with the killer cell lectin-like receptor subfamily G member 1 (KLRG1) receptor and therefore protected tumor cells from NK cell killing in the circulation. In vivo experimental results showed that overexpression of N-cadherin promoted tumor self-seeding and facilitated the survival of CTCs. Compared with primary tumors, N-cadherin expression was significantly increased in matched recurrent tumor tissues. CONCLUSION: Together, our findings illustrate an unknown mechanism by which CTCs evaded NK cell-mediated immune surveillance, and indicate that targeting N-cadherin is an effective strategy to prevent CTCs from homing to primary tumor.

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.

BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.