Orcinol glucoside targeted p38 as an agonist to promote osteogenesis and protect glucocorticoid-induced osteoporosis.

BACKGROUND: Glucocorticoids (GC)-induced osteoporosis (GIOP) is the most common cause of secondary osteoporosis, which leads to an increased risk of fracture in patients. The inhibition of the osteoblast effect is one of the main pathological characteristics of GIOP, but without effective drugs on treatment. PURPOSE: The aim of this study was to investigate the potential effects of orcinol glucoside (OG) on osteoblast cells and GIOP mice, as well as the mechanism of the underlying molecular target protein of OG both in vitro osteoblast cell and in vivo GIOP mice model. METHODS: GIOP mice were used to determine the effect of OG on bone density and bone formation. Then, a cellular thermal shift assay coupled with mass spectrometry (CETSA-MS) method was used to identify the target of OG. Surface plasmon resonance (SPR), enzyme activity assay, molecular docking, and molecular dynamics were used to detect the affinity, activity, and binding site between OG and its target, respectively. Finally, the anti-osteoporosis effect of OG through the target signal pathway was investigated in vitro osteoblast cell and in vivo GIOP mice model. RESULTS: OG treatment increased bone mineral density (BMD) in GIOP mice and effectively promoted osteoblast proliferation, osteogenic differentiation, and mineralization in vitro. The CETSA-MS result showed that the target of OG acting on the osteoblast is the p38 protein. SPR, molecular docking assay and enzyme activity assay showed that OG could direct bind to the p38 protein and is a p38 agonist. The cellular study found that OG could promote p38 phosphorylation and upregulate the proteins expression of its downstream osteogenic (Runx2, Osx, Collagen Ⅰ, Dlx5). Meanwhile, it could also inhibit the nuclear transport of GR by increasing the phosphorylation site at GR226 in osteoblast cell. In vivo GIOP mice experiment further confirmed that OG could prevent bone loss in the GIOP mice model through promoting p38 activity as well as its downstream proteins expression and activity. CONCLUSIONS: This study has established that OG could promote osteoblast activity and revise the bone loss in GIOP mice by direct binding to the p38 protein and is a p38 agonist to improve its downstream signaling, which has great potential in GIOP treatment for targeting p38. This is the first report to identify OG anti-osteoporosis targets using a label-free strategy (CETSA-MS).

Cardiac glycoside neriifolin exerts anti-cancer activity in prostate cancer cells by attenuating DNA damage repair through endoplasmic reticulum stress.

Prostate cancer (PCa) is one of the most common cancers in men. Patients with recurrent disease initially respond to androgen-deprivation therapy, but the tumor eventually progresses into castration-resistant PCa. Thus, new therapeutic approaches for PCa resistance to current treatments are urgently needed. Here, we report that cardiac glycoside neriifolin suppresses the malignancy of cancer cells via increasing DNA damage and apoptosis through activation of endoplasmic reticulum stress (ERS) in prostate cancers. We found that cardiac glycoside neriifolin markedly inhibited the cell growth and induced apoptosis in prostate cancer cells. Transcriptome sequence analysis revealed that neriifolin significantly induced DNA damage and double strand breaks (DSBs), validated with attenuation expression of genes in DSBs repair and increasing phosphorylated histone H2AX (γ-H2AX) foci formation, a quantitative marker of DSBs. Moreover, we found that neriifolin also activated ERS, evidenced by upregulation and activation of ERS related proteins, including eukaryotic initiation factor 2α (eIF2α), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and C/EBP homologous protein (CHOP) as well as downregulation of CCAATenhancerbinding protein alpha (C/EBP-α), a transcriptional factor that forms heterodimers with CHOP. In addition, neriifolin treatment dramatically inhibited the by tumor growth, which were reversed by CHOP loss or overexpression of C/EBP-α in nude mice. Mechanistically, neriifolin suppressed the tumor growth by increasing DNA damage and apoptosis through CHOP-C/EBP-α signaling axis of ERS in prostate cancers. Taken together, these results suggest that cardiac glycoside neriifolin may be a potential tumor-specific chemotherapeutic agent in prostate cancer treatment.

Effect of regional block technique on postoperative high-grade complications according to Clavien-Dindo classification in elderly patients with thoracic and abdominal cancer: a retrospective propensity score matching analysis.

Background: Postoperative complications have an influence on postoperative rehabilitation, length of hospital stay and hospitalization expenses in elderly patients, especially those with higher Clavien-Dindo (C-D) classification. Patients with cancers often experience more serious postoperative complications after surgery. Different anesthesia methods can affect the postoperative outcomes of cancer patients. Regional block techniques have been recommended in guidelines for enhanced recovery after surgery. However, the relationship between regional blocks and high-grade postoperative complications remains unclear, thus, the study explored the relationship between regional block techniques and high-grade postoperative complications graded by C-D classification in elderly patients with thoracic and abdominal cancer. Method: Retrospective enrollment of eligible elderly patients admitted to Peking University People's Hospital between January 2018 and March 2022 was conducted. Propensity score matching (PSM) and univariate and multivariate regression analyses were used to analyze the potential benefits of regional blocks for elderly patients in real world practice. Results: A total of 2769 patients were enrolled in this study, including 568 who underwent colorectal resection, 2201 who underwent video-assisted thoracoscopic pneumonectomy. Among them, 2033 patients received regional block, while 736 patients did not. Statistical analysis indicated that regional blocks could reduce the incidence of postoperative complications of C-D classification Grade II or higher, with an Odds ratio (OR) of 0.742, 95% Confidence interval (CI) (0.552 to 0.996) (P = 0.047). Conclusion: Regional block is associated with a reduction in the occurrence of postoperative complications graded by C-D classification in elderly patients with thoracic and abdominal cancer. The application of regional blocks can lower the risk of high-risk complications and mortality.

Animal Models of Rotator Cuff Injury and Repair: A Systematic Review.

There are a large number of animal studies on rotator cuff injury and repair, but a lack of detailed research and evaluation on the animal models. This systematic review aims to provide a framework for animal studies and repair patches for rotator cuff injury. Four hundred nine animal studies were included, of which the most common animal model of rotator cuff injury is rat (53.56%), the most common site of rotator cuff injury is the supraspinatus tendon (62.10%), and the most common injury type (degree) is acute tear (full thickness) (48.41%). The most common research purpose is to evaluate the repair effect of the patch (24.94%), followed by the observation of pathophysiological changes after rotator cuff injury (20.87%). Among the five types of repair patch materials including nondegradable and degradable synthetic materials, autologous and allogeneic tissues, and naturally derived biomaterial, the last one is the mostly used (52.74%). For different animal models, the rodent models (rat and mouse) are the most commonly used and probably the most suitable species for preliminary studies of rotator cuff injury; the rabbit, canine, sheep, and goat models are more suitable for biomechanical performance testing, rehabilitation training, and validation of surgical methods; and the nonhuman primate models (monkey and baboon) are the closest to human, but it is more difficult to carry out the animal studies on them because of ethical issues, high feeding cost, and management difficulties.

Osteoblastic protein kinase D1 contributes to the prostate cancer cells dormancy via GAS6-circadian clock signaling.

Disseminated prostate cancer (PCa) is known to have a strong propensity for bone marrow. These disseminated tumor cells (DTCs) can survive in bone marrow for years without obvious proliferation, while maintaining the ability to develop into metastatic lesions. However, how DTCs kept dormant and recur is still uncertain. Here, we focus on the role of osteoblastic protein kinase D1 (PKD1) in PCa (PC-3 and DU145) dormancy using co-culture experiments. Using flow cytometry, western blotting, and immunofluorescence, we observed that in co-cultures osteoblasts could induce a dormant state in PCa cells, which is manifested by a fewer cell divisions, a decrease Ki-67-positive populations and a lower ERK/p38 ratio. In contrast, silencing of PKD1 gene in osteoblasts impedes co-cultured prostate cancer cell's dormancy ability. Mechanismly, protein kinase D1 (PKD1) in osteoblasts induces PCa dormancy via activating CREB1, which promoting the expression and secretion of growth arrest specific 6 (GAS6). Furthermore, GAS6-induced dormancy signaling significantly increased the expression of core circadian clock molecules in PCa cells, and a negative correlation of circadian clock proteins (BMAL1, CLOCK and DEC2) with recurrence-free survival is observed in metastatic prostate cancer patients. Interestingly, the expression of cell cycle factors (p21, p27, CDK1 and PCNA) which regulated by circadian clock also upregulated in response to GAS6 stimulation. Taken together, we provide evidence that osteoblastic PKD1/CREB1/GAS6 signaling regulates cellular dormancy of PCa cells, and highlights the importance of circadian clock in PCa cells dormancy.

Application of Artificial Intelligence in Radiotherapy of Nasopharyngeal Carcinoma with Magnetic Resonance Imaging.

The value of automatic organ-at-risk outlining software for radiotherapy is based on artificial intelligence technology in clinical applications. The accuracy of automatic segmentation of organs at risk (OARs) in radiotherapy for nasopharyngeal carcinoma was investigated. In the automatic segmentation model which is proposed in this paper, after CT scans and manual segmentation by physicians, CT images of 147 nasopharyngeal cancer patients and their corresponding outlined OARs structures were selected and grouped into a training set (115 cases), a validation set (12 cases), and a test set (20 cases) by complete randomization. Adaptive histogram equalization is used to preprocess the CT images. End-to-end training is utilized to improve modeling efficiency and an improved network based on 3D Unet (AUnet) is implemented to introduce organ size as prior knowledge into the convolutional kernel size design to enable the network to adaptively extract features from organs of different sizes, thus improving the performance of the model. The DSC (Dice Similarity Coefficient) coefficients and Hausdorff (HD) distances of automatic and manual segmentation are compared to verify the effectiveness of the AUnet network. The mean DSC and HD of the test set were 0.86 ± 0.02 and 4.0 ± 2.0 mm, respectively. Except for optic nerve and optic cross, there was no statistical difference between AUnet and manual segmentation results (P > 0.05). With the introduction of the adaptive mechanism, AUnet can achieve automatic segmentation of the endangered organs of nasopharyngeal carcinoma based on CT images more accurately, which can substantially improve the efficiency and consistency of segmentation of doctors in clinical applications.

Evaluation of patches for rotator cuff repair: A systematic review and meta-analysis based on animal studies.

Based on the published animal studies, we systematically evaluated the outcomes of various materials for rotator cuff repair in animal models and the potentials of their clinical translation. 74 animal studies were finally included, of which naturally derived biomaterials were applied the most widely (50.0%), rats were the most commonly used animal model (47.0%), and autologous tissue demonstrated the best outcomes in all animal models. The biomechanical properties of naturally derived biomaterials (maximum failure load: WMD 18.68 [95%CI 7.71-29.66]; P = 0.001, and stiffness: WMD 1.30 [95%CI 0.01-2.60]; P = 0.048) was statistically significant in the rabbit model. The rabbit model showed better outcomes even though the injury was severer compared with the rat model.

A bioceramic scaffold composed of strontium-doped three-dimensional hydroxyapatite whiskers for enhanced bone regeneration in osteoporotic defects.

Reconstruction of osteoporotic bone defects is a clinical problem that continues to inspire the design of new materials. Methods: In this work, bioceramics composed of strontium (Sr)-doped hydroxyapatite (HA) whiskers or pure HA whiskers were successfully fabricated by hydrothermal treatment and respectively named SrWCP and WCP. Both bioceramics had similar three-dimensional (3D) porous structures and mechanical strengths, but the SrWCP bioceramic was capable of releasing Sr under physiological conditions. In an osteoporotic rat metaphyseal femoral bone defect model, both bioceramic scaffolds were implanted, and another group that received WCP plus strontium ranelate drug administration (Sr-Ran+WCP) was studied for comparison. Results: At week 1 post-implantation, osteogenesis coupled blood vessels were found to be more common in the SrWCP and Sr-Ran+WCP groups, with substantial vascular-like structures. After 12 weeks of implantation, comparable to the Sr-Ran+WCP group, the SrWCP group showed induction of more new bone formation within the defect as well as at the implant-bone gap region than that of the WCP group. Both the SrWCP and Sr-Ran+WCP groups yielded a beneficial effect on the surrounding trabecular bone microstructure to resist osteoporosis-induced progressive bone loss. While an abnormally high blood Sr ion concentration was found in the Sr-Ran+WCP group, SrWCP showed little adverse effect. Conclusion: Our results collectively suggest that the SrWCP bioceramic can be a safe bone substitute for the treatment of osteoporotic bone defects, as it promotes local bone regeneration and implant osseointegration to a level that strontium ranelate can achieve.

Application of hydroxyapatite nanoparticles in tumor-associated bone segmental defect.

Hydroxyapatite (HA) has been widely applied in bone repair because of its superior biocompatibility. Recently, a proliferation-suppressive effect of HA nanoparticles (n-HA) against various cancer cells was reported. This study was aimed at assessing the translational value of n-HA both as a bone-regenerating material and as an antitumor agent. Inhibition of tumor growth, prevention of metastasis, and enhancement of the survival rate of tumor-bearing rabbits treated with n-HA were demonstrated. Activated mitochondrial-dependent apoptosis in vivo was confirmed, and we observed that a stimulated immune response was involved in the n-HA-induced antitumor effect. A porous titanium scaffold loaded with n-HA was fabricated and implanted into a critical-sized segmental bone defect in a rabbit tumor model. The n-HA-releasing scaffold not only showed a prominent effect in suppressing tumor growth and osteolytic lesion but also promoted bone regeneration. These findings provide a rationale for using n-HA in tumor-associated bone segmental defects.

The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway.

PURPOSE: Evidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer. However, the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown. The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells. METHODS: The cytotoxicity and IC50 of five small molecule compounds (oleandrin, neriifolin, strophanthidin, gitoxigenin, and convallatoxin) in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays, respectively. Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide, followed by flow cytometry. Intracellular Ca2+ was determined using Fluo-3 AM,glutathione (GSH) levels were measured using a GSH detection kit,and the activity of caspase-3, -9 was measured using a peptide substrate. BAX, pro-caspase-3, -9, cytochrome C and BCL-2 expression were determined by Western blotting. RESULTS: Oleandrin significantly decreased cell viabilities in SW480, HCT116 and RKO cells. The IC50 for SW480 cells was 0.02 µM, whereas for NCM460 cells 0.56 µM. More interestingly, the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells. Oleandrin downregulated protein expression of pro-caspase-3, -9, but enhanced caspase-3, -9 activities. These effects were accompanied by upregulation of protein expression of cytochrome C and BAX, and downregulation of BCL-2 protein expression in a concentration-dependent manner. Furthermore, oleandrin increased intracellular Ca2+ concentration, but decreased GSH concentration in the cells. CONCLUSIONS: The present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway. Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.

A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development.

The protein kinase D family of serine/threonine kinases, particularly PKD1, has been implicated in the regulation of a complex array of fundamental biological processes. However, its function and mechanism underlying PKD1-mediated the bone development and osteoblast differentiation are not fully understood. Here we demonstrate that loss of PKD1 function led to impaired bone development and osteoblast differentiation through STAT3 and p38 MAPK signaling using in vitro and in vivo bone-specific conditional PKD1-knockout (PKD1-KO) mice models. These mice developed markedly craniofacial dysplasia, scapula dysplasia, long bone length shortage and body weight decrease compared with wild-type littermates. Moreover, deletion of PKD1 in vivo reduced trabecular development and activity of osteoblast development, confirmed by Micro-CT and histological staining as well as expression of osteoblastic marker (OPN, Runx2 and OSX). Mechanistically, loss of PKD1 mediated the downregulation of osteoblast markers and impaired osteoblast differentiation through STAT3 and p38 MAPK signaling pathways. Taken together, these results demonstrated that PKD1 contributes to the osteoblast differentiation and bone development via elevation of osteoblast markers through activation of STAT3 and p38 MAPK signaling pathways.

The US/China workshop: Regulation, standards, and innovation IV, organized by the Chinese Society for Biomaterials (CSBM) and the US Society for Biomaterials (SFB) in Minneapolis 2017.

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Rictor Regulates Spermatogenesis by Controlling Sertoli Cell Cytoskeletal Organization and Cell Polarity in the Mouse Testis.

Maintenance of cell polarity is essential for Sertoli cell and blood-testis barrier (BTB) function and spermatogenesis; however, the signaling mechanisms that regulate the integrity of the cytoskeleton and polarity of Sertoli cells are not fully understood. Here, we demonstrate that rapamycin-insensitive component of target of rapamycin (TOR) (Rictor), a core component of mechanistic TOR complex 2 (mTORC2), was expressed in the seminiferous epithelium during testicular development, and was down-regulated in a cadmium chloride-induced BTB damage model. We then conditionally deleted the Rictor gene in Sertoli cells and mutant mice exhibited azoospermia and were sterile as early as 3 months old. Further study revealed that Rictor may regulate actin organization via both mTORC2-dependent and mTORC2-independent mechanisms, in which the small GTPase, ras-related C3 botulinum toxin substrate 1, and phosphorylation of the actin filament regulatory protein, Paxillin, are involved, respectively. Loss of Rictor in Sertoli cells perturbed actin dynamics and caused microtubule disarrangement, both of which accumulatively disrupted Sertoli cell polarity and BTB integrity, accompanied by testicular developmental defects, spermiogenic arrest and excessive germ cell loss in mutant mice. Together, these findings establish the importance of Rictor/mTORC2 signaling in Sertoli cell function and spermatogenesis through the maintenance of Sertoli cell cytoskeletal dynamics, BTB integrity, and cell polarity.

Rictor/mTORC2 pathway in oocytes regulates folliculogenesis, and its inactivation causes premature ovarian failure.

Molecular basis of ovarian folliculogenesis and etiopathogenesis of premature ovarian failure (POF), a common cause of infertility in women, are not fully understood. Mechanistic target of rapamycin complex 2 (mTORC2) is emerging as a central regulator of cell metabolism, proliferation, and survival. However, its role in folliculogenesis and POF has not been reported. Here, we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide (VCD)-induced POF mouse model. Notably, mice with oocyte-specific ablation of Rictor, a key component of mTORC2, demonstrated POF phenotypes, including massive follicular death, excessive loss of functional ovarian follicles, abnormal gonadal hormone secretion, and consequently, secondary subfertility in conditional knock-out (cKO) mice. Furthermore, reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins, Bad, Bax, and cleaved poly ADP-ribose polymerase (PARP), were observed in cKO mice, replicating the signaling alterations in 4-VCD-treated ovaries. These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis. Interestingly, loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice, suggesting that maternal Rictor is dispensable for preimplantation embryonic development. Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis, follicle survival, and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POF.