Composite Hydrogel Containing Collagen-Modified Polylactic Acid-Hydroxylactic Acid Copolymer Microspheres Loaded with Tetramethylpyrazine Promotes Articular Cartilage Repair.

Articular cartilage defects pose a significant challenge due to the limited self-healing ability of cartilage. However, traditional techniques face limitations including autologous chondrocyte expansion issues. This study aims to investigate the effects of the polylactic acid-glycolic acid (PLGA) and collagen-surface modified polylactic acid-glycolic acid (CPLGA) microspheres loaded with tetramethylpyrazine (TMP) on two cell types and the regeneration potential of articular cartilage. CPLGA microspheres are prepared by Steglich reaction and characterized. They evaluated the effect of TMP-loaded microspheres on HUVECs (Human Umbilical Vein Endothelial Cells) and examined the compatibility of blank microspheres with BMSCs (Bone marrow mesenchymal stromal cells) and their potential to promote cartilage differentiation. Subcutaneous implant immune tests and cartilage defect treatment are conducted to assess biocompatibility and cartilage repair potential. The results highlight the efficacy of CPLGA microspheres in promoting tissue regeneration, attributed to improved hydrophilicity and collagen-induced mitigation of degradation. Under hypoxic conditions, both CPLGA and PLGA TMP-loaded microspheres exhibit inhibitory effects on HUVEC proliferation, migration, and angiogenesis. Notably, CPLGA microspheres show enhanced compatibility with BMSCs, facilitating chondrogenic differentiation. Moreover, the CPLGA microsphere-composite hydrogel exhibits potential for cartilage repair by modulating angiogenesis and promoting BMSC differentiation.

Mechanisms of vascular invasion after cartilage injury and potential engineering cartilage treatment strategies.

Articular cartilage injury is one of the most common diseases in orthopedic clinics. Following an articular cartilage injury, an inability to resist vascular invasion can result in cartilage calcification by newly formed blood vessels. This process ultimately leads to the loss of joint function, significantly impacting the patient's quality of life. As a result, developing anti-angiogenic methods to repair damaged cartilage has become a popular research topic. Despite this, tissue engineering, as an anti-angiogenic strategy in cartilage injury repair, has not yet been adequately investigated. This exhaustive literature review mainly focused on the process and mechanism of vascular invasion in articular cartilage injury repair and summarized the major regulatory factors and signaling pathways affecting angiogenesis in the process of cartilage injury. We aimed to discuss several potential methods for engineering cartilage repair with anti-angiogenic strategies. Three anti-angiogenic tissue engineering methods were identified, including administering angiogenesis inhibitors, applying scaffolds to manage angiogenesis, and utilizing in vitro bioreactors to enhance the therapeutic properties of cultured chondrocytes. The advantages and disadvantages of each strategy were also analyzed. By exploring these anti-angiogenic tissue engineering methods, we hope to provide guidance for researchers in related fields for future research and development in cartilage repair.

CD62E- and ROS-Responsive ETS Improves Cartilage Repair by Inhibiting Endothelial Cell Activation through OPA1-Mediated Mitochondrial Homeostasis.

Background: In the environment of cartilage injury, the activation of vascular endothelial cell (VEC), marked with excessive CD62E and reactive oxygen species (ROS), can affect the formation of hyaluronic cartilage. Therefore, we developed a CD62E- and ROS-responsive drug delivery system using E-selectin binding peptide, Thioketal, and silk fibroin (ETS) to achieve targeted delivery and controlled release of Clematis triterpenoid saponins (CS) against activated VEC, and thus promote cartilage regeneration. Methods: We prepared and characterized ETS/CS and verified their CD62E- and ROS-responsive properties in vitro. We investigated the effect and underlying mechanism of ETS/CS on inhibiting VEC activation and promoting chondrogenic differentiation of bone marrow stromal cells (BMSCs). We also analyzed the effect of ETS/CS on suppressing the activated VEC-macrophage inflammatory cascade in vitro. Additionally, we constructed a rat knee cartilage defect model and administered ETS/CS combined with BMSC-containing hydrogels. We detected the cartilage differentiation, the level of VEC activation and macrophage in the new tissue, and synovial tissue. Results: ETS/CS was able to interact with VEC and inhibit VEC activation through the carried CS. Coculture experiments verified ETS/CS promoted chondrogenic differentiation of BMSCs by inhibiting the activated VEC-induced inflammatory cascade of macrophages via OPA1-mediated mitochondrial homeostasis. In the rat knee cartilage defect model, ETS/CS reduced VEC activation, migration, angiogenesis in new tissues, inhibited macrophage infiltration and inflammation, promoted chondrogenic differentiation of BMSCs in the defective areas. Conclusions: CD62E- and ROS-responsive ETS/CS promoted cartilage repair by inhibiting VEC activation and macrophage inflammation and promoting BMSC chondrogenesis. Therefore, it is a promising therapeutic strategy to promote articular cartilage repair.

Inhibition of HDAC6 promotes microvascular endothelial cells to phagocytize myelin debris and reduces inflammatory response to accelerate the repair of spinal cord injury.

AIMS: To identify an effective strategy for promoting microvascular endothelial cells (MECs) to phagocytize myelin debris and reduce secretion of inflammatory factors following spinal cord injury (SCI). METHODS: We established a coculture model of myelin debris and vascular-like structures. The efficiency with which MECs phagocytize myelin debris under different conditions was examined via ELISA, flow cytometry, and immunofluorescence. Tubastatin-A was used to interfere with the coculture model. The anti-inflammatory effects of Tubastatin-A were observed by HE staining, flow cytometry, immunofluorescence, and ELISA. RESULTS: MECs phagocytized myelin debris via IgM opsonization, and phagocytosis promoted the secretion of inflammatory factors, whereas IgG-opsonized myelin debris had no effect on inflammatory factors. Application of the HDAC6 inhibitor Tubastatin-A increased the IgG levels and decreased the IgM levels by regulating the proliferation and differentiation of B cells. Tubastatin-A exerted a regulatory effect on the HDAC6-mediated autophagy-lysosome pathway, promoting MECs to phagocytize myelin debris, reducing the secretion of inflammatory factors, and accelerating the repair of SCI. CONCLUSIONS: Inhibition of HDAC6 to regulate the immune-inflammatory response and promote MECs to phagocytize myelin debris may represent a novel strategy in the treatment of SCI.

Iron induces B cell pyroptosis through Tom20-Bax-caspase-gasdermin E signaling to promote inflammation post-spinal cord injury.

BACKGROUND: Immune inflammatory responses play an important role in spinal cord injury (SCI); however, the beneficial and detrimental effects remain controversial. Many studies have described the role of neutrophils, macrophages, and T lymphocytes in immune inflammatory responses after SCI, although little is known about the role of B lymphocytes, and immunosuppression can easily occur after SCI. METHODS: A mouse model of SCI was established, and HE staining and Nissl staining were performed to observe the pathological changes. The size and morphology of the spleen were examined, and the effects of SCI on spleen function and B cell levels were detected by flow cytometry and ELISA. To explore the specific mechanism of immunosuppression after SCI, B cells from the spleens of SCI model mice were isolated using magnetic beads and analyzed by 4D label-free quantitative proteomics. The level of inflammatory cytokines and iron ions were measured, and the expression of proteins related to the Tom20 pathway was quantified by western blotting. To clarify the relationship between iron ions and B cell pyroptosis after SCI, we used FeSO4 and CCCP, which induce oxidative stress to stimulate SCI, to interfere with B cell processes. siRNA transfection to knock down Tom20 (Tom20-KD) in B cells and human B lymphocytoma cell was used to verify the key role of Tom20. To further explore the effect of iron ions on SCI, we used deferoxamine (DFO) and iron dextran (ID) to interfere with SCI processes in mice. The level of iron ions in splenic B cells and the expression of proteins related to the Tom20-Bax-caspase-gasdermin E (GSDME) pathway were analyzed. RESULTS: SCI could damage spleen function and lead to a decrease in B cell levels; SCI upregulated the expression of Tom20 protein in the mitochondria of B cells; SCI could regulate the concentration of iron ions and activate the Tom20-Bax-caspase-GSDME pathway to induce B cell pyroptosis. Iron ions aggravated CCCP-induced B cell pyroptosis and human B lymphocytoma pyroptosis by activating the Tom20-Bax-caspase-GSDME pathway. DFO could reduce inflammation and promote repair after SCI by inhibiting Tom20-Bax-caspase-GSDME-induced B cell pyroptosis. CONCLUSIONS: Iron overload activates the Tom20-Bax-caspase-GSDME pathway after SCI, induces B cell pyroptosis, promotes inflammation, and aggravates the changes caused by SCI. This may represent a novel mechanism through which the immune inflammatory response is induced after SCI and may provide a new key target for the treatment of SCI.

Construction and optimization of a coculture system of mouse brain microvascular endothelial cells and myelin debris.

Microvascular endothelial cells are a newly discovered cell type involved in the phagocytosis of myelin debris, which play a key role in the repair of spinal cord injuries. Several methods for the preparation of myelin debris and parameters for constructing a coculture system of microvascular endothelial cells and myelin debris are available, but no systematic studies have yet been conducted, which hinders further exploration of the mechanisms of demyelinating disease repair. Herein, we aimed to develop a standardized method for this process. Myelin debris of different sizes was obtained from the brains of C57BL/6 mice by stripping the brains under aseptic conditions, multiple grinding, gradient centrifugation, etc. Transmission electron microscopy and nanoparticle size analysis were used to characterize myelin debris. Microvascular endothelial cells were cultured on a matrix gel, and myelin debris of different sizes (fluorescently labeled using CFSE) was placed in coculture after forming a vascular-like structure. Subsequently, myelin debris of different concentrations was cocultured in the vascular-like structure, and phagocytosis of myelin debris by microvascular endothelial cells was detected using immunofluorescence staining and flow cytometry. We found that myelin debris could be successfuly obtained from the mouse brain with secondary grinding and other steps and cocultured with microvascular endothelial cells at a concentration of 2 mg/mL, which promoted the phagocytosis of microvascular endothelial cells. In conclusion, we provide a reference for the protocol of a coculture system of microvascular endothelial cells and myelin debris.

Wen-Shen-Tong-Luo-Zhi-Tong Decoction regulates bone-fat balance in osteoporosis by adipocyte-derived exosomes.

CONTEXT: Wen-Shen-Tong-Luo-Zhi-Tong (WSTLZT) Decoction is a Chinese prescription with antiosteoporosis effects, especially in patients with abnormal lipid metabolism. OBJECTIVE: To explore the effect and mechanism of WSTLZT on osteoporosis (OP) through adipocyte-derived exosomes. MATERIALS AND METHODS: Adipocyte-derived exosomes with or without WSTLZT treated were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blotting (WB). Co-culture experiments for bone marrow mesenchymal stem cells (BMSCs) and exosomes were performed to examine the uptake and effect of exosome in osteogenesis and adipogenic differentiation of BMSC. MicroRNA profiles, luciferase and IP were used for exploring specific mechanisms of exosome on BMSC. In vivo, 80 Balb/c mice were randomly divided into four groups: Sham, Ovx, Exo (30 µg exosomes), Exo-WSTLZT (30 µg WSTLZT-exosomes), tail vein injection every week. After 12 weeks, the bone microstructure and marrow fat distribution were analysed by micro-CT. RESULTS: ALP, Alizarin red and Oil red staining showed that WSTLZT-induced exosomes from adipocyte can regulate osteoblastic and adipogenic differentiation of BMSC. MicroRNA profiles observed that WSTLZT treatment resulted in 87 differentially expressed miRNAs (p < 0.05). MiR-122-5p with the greatest difference was screened by q-PCR (p < 0.01). The target relationship between miR-122-5p and SPRY2 was tested by luciferase and IP. MiR-122-5p negatively regulated SPRY2 and elevated the activity of MAPK signalling pathway, thereby regulating the osteoblastic and adipogenic differentiation of BMSC. In vivo, exosomes can not only improve bone microarchitecture but also significantly reduce accumulation of bone marrow adipose. CONCLUSIONS: WSTLZT can exert anti-OP effect through SPRY2 via the MAKP signalling by miR-122-5p carried by adipocyte-derived exosomes.

Mechanical Stretch Promotes Macrophage Polarization and Inflammation via the RhoA-ROCK/NF-κB Pathway.

Macrophages play an essential role in the pathogenesis of most inflammatory diseases. Recent studies have shown that mechanical load can influence macrophage function, leading to excessive and uncontrolled inflammation and even systemic damage, including cardiovascular disease and knee osteoarthritis. However, the molecular mechanism remains unclear. In this study, murine RAW264.7 cells were treated with mechanical stretch (MS) using the Flexcell-5000T Tension System. The expression of inflammatory factors and cytokine release were measured by RT-qPCR, ELISA, and Western blotting. The protein expression of NF-κB p65, Iκb-α, p-Iκb-α, RhoA, ROCK1, and ROCK2 was also detected by Western blotting. Then, Flow cytometry was used to detect the proportion of macrophage subsets. Meanwhile, Y-27632 dihydrochloride, a ROCK inhibitor, was added to knockdown ROCK signal transduction in cells. Our results demonstrated that MS upregulated mRNA expression and increased the secretion levels of proinflammatory factors iNOS, IL-1ß, TNF-α, and IL-6. Additionally, MS significantly increased the proportion of CD11b+CD86+ and CD11b+CD206+ subsets in RAW264.7 macrophages. Furthermore, the protein expression of RhoA, ROCK1, ROCK2, NF-κB p65, and IκB-α increased in MS-treated RAW264.7 cells, as well as the IL-6 and iNOS. In contrast, ROCK inhibitor significantly blocked the activation of RhoA-ROCK and NF-κB pathway, decreased the protein expression of IL-6 and iNOS, reduced the proportion of CD11b+CD86+ cells subpopulation, and increased the proportion of CD11b+CD206+ cell subpopulation after MS. These data indicate that mechanical stretch can regulate the RAW264.7 macrophage polarization and enhance inflammatory responses in vitro, which may contribute to activation the RhoA-ROCK/NF-κB pathway.

Quercetin Attenuates Osteoporosis in Orchiectomy Mice by Regulating Glucose and Lipid Metabolism via the GPRC6A/AMPK/mTOR Signaling Pathway.

Quercetin, a flavonoid found in natural medicines, has shown a role in disease prevention and health promotion. Moreover, because of its recently identified contribution in regulating bone homeostasis, quercetin may be considered a promising agent for improving bone health. This study aimed to elucidate the role of quercetin in androgen deprivation therapy-induced osteoporosis in mice. C57BL/6 mice were subjected to orchiectomy, followed by quercetin treatment (75 and 150 mg/kg/d) for 8 weeks. Bone microstructure was then assessed by micro-computed tomography, and a three-point bending test was used to evaluate the biomechanical parameters. Hematoxylin and eosin (H&E) staining was used to examine the shape of the distal femur, gastrocnemius muscle, and liver. The balance motion ability in mice was evaluated by gait analysis, and changes in the gastrocnemius muscle were observed via Oil red O and Masson's staining. ELISA and biochemical analyses were used to assess markers of the bone, glucose, and lipid metabolism. Western blotting analyses of glucose and lipid metabolism-related protein expression was performed, and expression of the GPCR6A/AMPK/mTOR signaling pathway-related proteins was also assessed. After 8 weeks of quercetin intervention, quercetin-treated mice showed increased bone mass, bone strength, and improved bone microstructure. Additionally, gait analysis, including stride length and frequency, were significantly increased, whereas a reduction of the stride length and gait symmetry was observed. H&E staining of the gastrocnemius muscle showed that the cross-sectional area of the myofibers had increased significantly, suggesting that quercetin improves balance, motion ability, and muscle mass. Bone metabolism improvement was defined by a reduction of serum levels of insulin, triglycerides, total cholesterol, and low-density lipoprotein, whereas levels of insulin-like growth factor-1 and high-density lipoprotein were increased after quercetin treatment. Expression of proteins involved in glucose uptake was increased, whereas that of proteins involved in lipid production was decreased. Moreover, the GPRC6A and the phospho-AMPK/AMPK expression ratio was elevated in the liver and tibia tissues. In contrast, the phospho-mTOR/mTOR ratio was reduced in the quercetin group. Our findings indicate that quercetin can reduce the osteoporosis induced by testosterone deficiency, and its beneficial effects might be associated with the regulation of glucose metabolism and inhibition of lipid metabolism via the GPCR6A/AMPK/mTOR signaling pathway.

[Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair].

Objective: To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods: CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups ( n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results: The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 µm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes ( P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers ( P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B ( P<0.05), and the level of TIMP-1 in group C was significantly higher ( P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B ( P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B ( P<0.05). Conclusion: CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.

A new method for primary culture of microglia in rats with spinal cord injury.

At present, the primary culture method of microglia is complicated, and the culture of spinal cord microglia is rare, so we will explore to establish a new and efficient primary culture method of microglia in rats with spinal cord injury (SCI). The SCI model of SD rats was established by modified A11en's method, and the model of SCI was performed on 1 d, 3 d, 7 d and 14 d respectively. Then the injured spinal cord was removed, mechanically separated and filtered. The morphology of microglia was observed the next day and its purity was identified by CD11b and Iba1 immunofluorescence labeling. According to the above results, the morphological changes of microglia after 3 d of SCI were observed at 1 d, 2 d and 4 d. The results showed that the purity of microglia was 98%. The number of microglia after 3 d of SCI was the most. After SCI, the migration ability of microglia was enhanced, the number of microglia in the injured area increased, and the number was the highest at 3 d, then gradually decreased. In addition, the microglia after SCI would gradually change from active state to resting state with the passage of time. Therefore, we can use a simple and efficient mechanical separation method to extract primary microglia, which provides the basis for the study of microglia.

Cartilage Repair Using Clematis Triterpenoid Saponin Delivery Microcarrier, Cultured in a Microgravity Bioreactor Prior to Application in Rabbit Model.

Cartilage tissue engineering provides a promising method for the repair of articular cartilage defects, requiring appropriate biological scaffolds and necessary growth factors to enhance the efficiency of cartilage regeneration. Here, a silk fibroin (SF) microcarrier and a clematis triterpenoid saponin delivery SF (CTS-SF) microcarrier were prepared by the high-voltage electrostatic differentiation and lyophilization method, and chondrocytes were carried under the simulated microgravity condition by a rotating cell culture system. SF and CTS-SF microspheres were relatively uniform in size and had a porous structure with good swelling and cytocompatibility. Further, CTS-SF microcarriers could sustainably release CTSs in the monitored 10 days. Compared with the monolayer culture, chondrocytes under the microgravity condition maintained a better chondrogenic phenotype and showed better proliferation ability after culture on microcarriers. Moreover, the sustained release of CTS from CTS-SF microcarriers upregulated transforming growth factor-ß, Smad2, and Smad3 signals, contributing to promote chondrogenesis. Hence, the biophysical effects of microgravity and bioactivities of CTS-ST were used for chondrocyte expansion and phenotype maintenance in vitro. With prolonged expansion, SF- and CTS-SF-based microcarrier-cell composites were directly implanted in vivo to repair rabbit articular defects. Gross evaluations, histopathological examinations, and biochemical analysis indicated that SF- and CTS-SF-based composites exhibited cartilage-like tissue repair compared with the nontreated group. Further, CTS-SF-based composites displayed superior hyaline cartilage-like repair that integrated with the surrounding cartilage better and higher cartilage extracellular matrix content. In conclusion, these results provide an alternative preparation method for drug-delivered SF microcarrier and a culture method for maintaining the chondrogenic phenotype of seed cells based on the microgravity environment. CTS showed its bioactive function, and the application of CTS-SF microcarriers can help repair and regenerate cartilage defects.

Current status and development trend of miRNAs in osteoporosis-related research: A bibliometric analysis.

INTRODUCTION: The occurrence of osteoporosis (OP) has drawn considerable attention from scholars around the world due to the significant impacts thereof on the social economy and the quality of human life. OP research has been rapidly expanding since the inclusion of microRNAs (miRNAs) as critical regulators of gene-expression. However, despite the ability to evaluate miRNA gene therapy in OP being enhanced, there has been a scarcity of updated citation analyses that reflect such developments. In the present study, through bibliometric analysis, the global research activity and trends in regard to the relationship between OP and miRNAs were reviewed. METHODS: Publications related to miRNA and OP from 2000 to 2021 were retrieved via Web of Science (WoS). The data included publication years, countries, journals, institutions, authors and keywords, and were sorted and summarized by bibliometrics, before being visually analyzed through VOS Viewer. RESULTS: In the past five years, 599 articles have been published, with said studies accounting for 79.11% of all relevant documents, indicating the increased interest in the present research topic. The country with the highest contribution rate was China, and the publication rate of Journal of Bone and Mineral Research was the highest, followed by Bone. The institutions with the highest contribution rate were Nanjing Medical University. The most frequently occurring keywords were clustered into five groups. The research area of the first group described that circulating miRNA would be a potential biomarker for postmenopausal osteoporosis (PMOP). The remaining four groups involved the influences of miRNAs and exosomes on the osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs), and the interactions of lncRNA and miRNA with OP. CONCLUSIONS: The results of the present study will expand the research on miRNAs and OP. The research direction with the highest frequency was the miRNAs acting on osteoblasts and osteoclasts. The influence of miRNAs carried by exosomes on the differentiation of MSCs might become an effective method for OP cell-free treatment.

Icariin promotes the repair of PC12 cells by inhibiting endoplasmic reticulum stress.

BACKGROUND: Endoplasmic reticulum stress (ERS) is one of the main mechanisms of spinal cord injury (SCI) pathology and can affect the physiological state of neurons. Icariin (ICA), the main pharmacological component of Epimedium, can relieve the symptoms of patients with SCI and has obvious protective effects on neurons through ERS. METHODS: PC12 cells were induced to differentiate into neurons by nerve growth factor and identified by flow cytometry. Cell proliferation was detected by CCK8 method, cell viability was detected by SRB assay, apoptosis was detected by flow cytometry and microstructure of ER was observed by transmission electron microscope. Western blot was used to detect the protein expression of CHOP and Grp78, and qPCR was used to detect the mRNA expression of CHOP and Grp78. RESULTS: The results of CCK8, SRB and flow cytometry showed that ICA could relieve ERS and reduce apoptosis of PC12 cells. The results of transmission microscope showed that ICA could reduce apoptosis of PC12 cells caused by ERS. The results of Western blot and q-PCR showed that ICA could inhibit ERS by down-regulating the expression of CHOP and Grp78. CONCLUSIONS: ICA can inhibit ERS and promote the repair of PC12 cells by down-regulating the expression of CHOP and Grp78. ICA has the potential to promote the recovery of spinal cord injury.

Jisuikang Promotes the Repair of Spinal Cord Injury in Rats by Regulating NgR/RhoA/ROCK Signal Pathway.

Jisuikang (JSK) is an herbal formula composed of many kinds of traditional Chinese medicine, which has been proved to be effective in promoting the rehabilitation of patients with spinal cord injury (SCI) after more than ten years of clinical application. However, the mechanisms of JSK promoting nerve regeneration are yet to be clarified. The aim of this study was to investigate the effects of JSK protecting neurons, specifically the regulation of NgR/RhoA/ROCK signal pathway. The motor function of rats was evaluated by the BBB score and inclined plate test, Golgi staining and transmission electron microscope were used to observe the microstructure of nerve tissue, and fluorescence double-labeling method was used to detect neuronal apoptosis. In this study, we found that JSK could improve the motor function of rats with SCI, protect the microstructure (mitochondria, endoplasmic reticulum, and dendritic spine) of neurons, and reduce the apoptosis rate of neurons in rats with SCI. In addition, JSK could inhibit the expression of Nogo receptor (NgR) in neurons and the NgR/RhoA/ROCK signal pathway in rats with SCI. These results indicated JSK could improve the motor function of rats with SCI by inhibiting the NgR/RhoA/ROCK signal pathway, which suggests the potential applicability of JSK as a nerve regeneration agent.

[Research progress of different mechanical stimulation regulating chondrocytes metabolism].

As a kind of mechanical effector cells, chondrocytes can produce a variety of physical and chemical signals under the stimulation of multiaxial load in vivo, which affect their own growth, development and apoptosis. Therefore, simulating the mechanical environment in vivo has become a research hotspot in the culture of chondrocytes in vitro. Although a large number of reports have fully proved that different mechanical stimulation can regulate the metabolism of chondrocytes, the loading scheme has not been agreed. Starting from different mechanical forms, this review will explore the differences in the regulation of chondrocyte metabolism by different mechanical stimuli, so as to find an advantage scheme to promote the growth and proliferation of chondrocytes and to develop a more stable, effective and reliable experimental strategy.

Osthole-loaded N-octyl-O-sulfonyl chitosan micelles (NSC-OST) inhibits RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss in rats.

Osthole (OST), a derivative of Fructus Cnidii, has been proved to have potential anti-osteoporosis effects in our recent studies. However, its pharmacological effects are limited in the human body because of poor solubility and bioavailability. Under the guidance of the classical theory of Chinese medicine, Osthole-loaded N-octyl-O-sulfonyl chitosan micelles (NSC-OST), which has not previously been reported in the literature, was synthesized in order to overcome the defects and obtain better efficacy. In this study, we found that NSC-OST inhibited on the formation and resorption activity of osteoclasts through using a bone marrow macrophage (BMM)-derived osteoclast culture system in vitro, rather than affecting the viability of cells. We also found that NSC-OST inhibited osteoclast formation, hydroxyapatite resorption and RANKL-induced osteoclast marker protein expression. In terms of mechanism, NSC-OST suppressed the NFATc1 transcriptional activity and the activation of NF-κB signalling pathway. In vivo, ovariectomized (OVX) rat models were established for further research. We found that NSC-OST can attenuate bone loss in OVX rats through inhibiting osteoclastogenesis. Consistent with our hypothesis, NSC-OST is more effective than OST in parts of the results. Taken together, our findings suggest that NSC-OST can suppress RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss in rats and could be considered a safe and more effective anti-osteoporosis drug than OST.

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osthole inhibits osteoclasts formation and bone resorption by regulating NF-κB signaling and NFATc1 activations stimulated by RANKL.

Chinese herbal medicine Fructus Cnidii has an outstanding effect on chronic lumbar pain and impotence, also has been used against osteoporosis with high frequency. Yet, the mechanisms of osthole, a derivative of Fructus Cnidii, on osteoclasts remains barely known. In this study, it was found out that osthole (10-6 mol/L, 10-5 mol/L) had the influence of inhibiting osteoclast formation and bone resorptive activities induced by receptor activator of nuclear factor κB ligand (RANKL), rather than affecting the viability of osteoclast-like cells. Furthermore, osthole could also inhibit the messenger RNA expressions of c-Src, tartrate-resistant acid phosphatase, ß3-Integrin, matrix metallopeptidase 9, and cathepsin K. The results of the mechanistic study indicated that osthole regulated the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and nuclear factor-κB (NF-κB) activations following the RANKL stimulation. These findings suggested that the inhibitory effects of osthole were associated with restraining the activations of NFATc1 and NF-κB induced by RANKL. Thus osthole can be used as a potential treatment for abnormal bone-resorption related diseases.

[Research progress on signaling molecules involved in articular cartilage repair].

After the articular cartilage injury, the metabolic level is increased during the progressive degeneration, the chondrocytes secrete a variety of inflammatory factors, and the original cell phenotype is gradually changed. For a long time, a large number of researchers have done a lot of researches to promote anabolism of chondrocytes and to maintain the stability of chondrocyte phenotype. There are many molecular signaling pathways involved in the process of promoting cartilage repair. This review focuses on the key signaling molecules in articular cartilage repair, such as transforming growth factor-beta and bone morphogenetic protein, and reveals their roles in the process of cartilage injury and repair, so that researchers in related fields can understand the molecular mechanism of cartilage injury and repair widely and deeply. Based on this, they may find promising targets and biological methods for the treatment of cartilage injury.