Gremlin-1 promotes IL-1ß-stimulated chondrocyte inflammation and extracellular matrix degradation via activation of the MAPK signaling pathway.

The role and mechanism of Gremlin-1 in osteoarthritis (OA) were expected to be probed in this study. Firstly, an in vitro OA model was constructed by stimulating human chondrocyte cell line CHON-001 with IL-1ß. Next, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were utilized for assessing the effect of IL-1ß with different concentrations (5, 10, and 20 ng/mL) on the activity and Gremlin-1 messenger RNA of CHON-001 cells, respectively. Besides, the influence of knocking down/over-expressing Gremlin-1 on the inflammatory factors (IL-6, TNF-α, IL-18 and PGE2), oxidative stress-related substances (malondialdehyde [MDA]; superoxide dismutase [SOD]; lactate dehydrogenase [LDH]), extracellular matrix (ECM) degradation-related proteins, and mitogen-activated protein kinase (MAPK) pathway proteins in IL-1ß-stimulated CHON-001 cells were tested by enzyme-linked immunosorbent assay, related kits, qRT-PCR, and western blot, respectively. IL-1ß inhibited CHON-001 cell proliferation and upregulated Gremlin-1 expression in a concentration-dependent manner. Overexpression of Gremlin-1 increased the IL-6, TNF-α, IL-18, PGE2, and MDA levels, enhanced the LDH activity, and decreased the SOD activity in IL-1ß-induced CHON-001 cells; while the effect of Gremlin-1 knockdown on the above factors was in contrast with that of the overexpression. Furthermore, overexpression of Gremlin-1 upregulated protein expression of matrix metalloproteinase (MMP)-3, MMP-13, and ADAMTS4 while downregulated protein expression of collagen III, aggrecan, and SOX-9 in IL-1ß-stimulated CHON-001 cells. Besides, overexpression of Gremlin-1 increased the p-p38/p38 value while decreased the p-JNK/JNK value in L-1ß-stimulated CHON-001 cells; however, knockdown of Gremlin-1 reversed the above results. Gremlin-1 may promote IL-1ß-stimulated CHON-001 cell inflammation and ECM degradation by activating the MAPK signaling pathway.

The Effect of Endogenous PARP-1 in Different Phases of IL-1ß-Induced Chondrocyte Degeneration.

Objective: Poly (ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in DNA damage repair, gene transcription, cell growth, death and apoptosis. In our study, we aimed to explore the dynamic role of PARP-1 in chondrocyte (CH) degeneration in vitro. Methods: We used the primary CHs and treated them with interleukin-1 beta for up to 5 days. (IL-1ß) to induce degeneration. Meanwhile, we used AG-14361 (AG) to inhibit endogenous PARP-1 expression. Cell survival and collagen II expression were used to define the cell function of CHs. In addition, other metabolic indicators were measured containing the reactive oxygen species (ROS) level, 8-Hydroxy-2'-deoxyguanosine (8-OH-dG), IL-1ß, tumor necrosis factor alpha (TNF-α) and caspase 3/9 expression. Results: With IL-1ß treatment, the PARP1 expression of CHs was gradually increased from day 1 to day 5, accompanied by a reduction in cell survival and collagen II expression, and an increase in ROS, 8-OH-dG, IL-1ß, TNF-α and caspase 3/9 levels. We suppressed PARP1 expression on the first day of IL-1ß stimulation and found severe destruction of cell survival and collagen II content with a higher expression of caspase 3/9. However, when we cultured the CHs with AG from day 3 of the 5-day IL-1ß stimulation, cell survival and collagen II expression were rescued, and the ROS, 8-OH-dG, IL-1ß, TNF-α, and caspase 3/9 were downregulated. Conclusions: On day 1 of degeneration, increased PARP-1 played a protective role in CHs. However, from days 3 to 5 of degeneration, the accumulated PARP-1 presented a more destructive function in CHs.

Gastrodin Ameliorates the Inflammation, Oxidative Stress, and Extracellular Matrix Degradation of IL-1ß-Mediated Fibroblast-Like Synoviocytes via Suppressing the Gremlin-1/NF-κB Pathway.

BACKGROUND: Synovial inflammation plays a crucial role in osteoarthritis (OA). Gastrodin (GAS), an active ingredient derived from the Gastrodia elata Blume rhizome, possesses antioxidant and anti-inflammatory pharmacological effects. This research aimed to evaluate the function and molecular mechanism of GAS on human fibroblast-like synoviocytes of osteoarthritis (HFLS-OA) induced by interleukin (IL)-1ß. METHODS: The impact of GAS on the viability of IL-1ß-treated HFLS-OA cells was assessed using the cell counting kit-8 (CCK-8). Quantitative real-time reverse transcription PCR (qRT-PCR) was employed to detect changes in IL-8, IL-6, monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and Gremlin-1 mRNA expression in each group. Corresponding kits were utilized to measure the catalase (CAT) and superoxide dismutase (SOD) activities, as well as the nitric oxide (NO) level. Western blot analysis was conducted to examine the expression of extracellular matrix degradation-associated proteins and nuclear factor kappa-B (NF-κB) pathway-correlated proteins in each group. RESULTS: GAS significantly promoted the proliferation of IL-1ß-induced HFLS-OA cells and concurrently down-regulated Gremlin-1 mRNA expression (p < 0.05). Through the down-regulation of Gremlin-1 expression, GAS exhibited the following effects: decreased IL-8, IL-6, and TNF-α mRNA expression, as well as NO levels (p < 0.05); increased SOD and CAT activities (p < 0.05); down-regulated matrix metallopeptidase 13 (MMP-13) and MMP-1 protein expression levels (p < 0.01); and up-regulated collagen II protein expression level (p < 0.01) in IL-1ß-treated HFLS-OA cells. Additionally, GAS decreased phospho-inhibitory kappa B (p-IκB)/IκB, phospho-inhibitory kappa B kinase (p-IKK)/IKK, and p-p65/p65 ratios in IL-1ß-induced HFLS-OA cells by inhibiting Gremlin-1 expression (p < 0.01). CONCLUSION: GAS demonstrates a positive impact on inflammation, oxidative stress, and extracellular matrix degradation in IL-1ß-mediated HFLS-OA cells. This effect is achieved by suppressing Gremlin-1 expression and reducing NF-κB pathway activity.

Acupuncture for abdominal wall endometriosis: A case report.

INTRODUCTION: Endometriosis refers to a series of symptoms caused by the presence of endometrial-like tissue outside the uterine cavity. In extrapelvic endometriosis, abdominal wall endometriosis (AWE) is very common. Acupuncture therapy has been widely used as an alternative therapy to treat multiple diseases, such as sequelae of stroke, pain, and facial paralysis. To our knowledge, case reports of acupuncture for the treatment of AWE has not been reported. We report a case of acupuncture in the treatment of abdominal endometriosis. RATIONALE: AWE could result in symptoms including pelvic pain, dysmenorrhea, and infertility. Acupuncture might be effective in the treatment of the disease. PATIENT CONCERNS: A 38-year-old woman complained of the aggregation of pain in a mass, which is located in her abdominal wall. DIAGNOSES: The patient was diagnosed with AWE, surgical history (excision of deep abdominal wall mass, repair of abdominal wall defect with patch). According to traditional Chinese medicine theory, traditional Chinese medicine diagnosis is Zhengjia (qi stagnation and blood stasis pattern). INTERVENTIONS: Combined with the theory of disentanglement, we use acupuncture, cupping, and needle therapy to promote qi circulation, activate blood circulation, relieve pain, and dissipate masses. OUTCOMES: After treatment, abdominal ultrasound showed that the mass gradually decreased. CONCLUSION: Acupuncture can effectively relieve the pain caused by abdominal endometriosis and reduce the size of abdominal endometriosis masses.

TAF15 regulates the BRD4/GREM1 axis and activates the gremlin-1-NF-κB pathway to promote OA progression.

Background: Anterior cruciate ligament (ACL) injury is recognized as a risk factor for osteoarthritis (OA) progression. Herein, the function of TAF15 in ACL injury-induced OA was investigated. Methods: OA cell model and OA mouse model were established by interleukin-1 beta (IL-1ß) stimulation and ACL transection administration, respectively. The pathological changes were analyzed by histopathology. The mRNA and protein expressions were assessed using qRT-PCR, Western blot and IHC. Chondrocyte viability and apoptosis were examined by CCK8 assay and TUNEL staining, respectively. The interactions between TAF15, BRD4 and GREM1 were analyzed by RIP or ChIP assay. Results: TAF15 expression was markedly elevated in OA, and its knockdown suppressed IL-1ß-induced chondrocyte apoptosis and ECM degradation in vivo and cartilage pathological changes in vitro. TAF15 promoted BRD4 mRNA stability, and TAF15 silencing's repression on chondrocyte apoptosis and ECM degradation induced by IL-1ß was abrogated following BRD4 overexpression. BRD4 promoted GREM1 expression by directly binding with GREM1. In addition, the GREM1/NF-κB pathway functioned as the downstream pathway of BRD4 in promoting OA progression. Conclusion: TAF15 upregulation facilitated chondrocyte apoptosis and ECM degradation during OA development by acting on the BRD4/GREM1/NF-κB axis, which provided a theoretical basis for the development of novel therapies for OA.

Carboxymethylcellulose with phenolic hydroxyl microcapsules enclosinggene-modified BMSCs for controlled BMP-2 release in vitro.

OBJECTIVES: The present study aimed to develop microparticles of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) via Co-flow microfluidics technology and encapsulated gene-modified rat bone mesenchymal stem cells (BMSCs) for the detection of the growth factor release was controlled by Tet-on system. Meanwhile, we investigated the effect of the CMC-Ph microcapsules and Lentiviral transduction on osteogenesis of BMP2-BMSCs. METHODS: The middle size of CMC-Ph microcapsules was prepared by optimized co-flow microfluidics through ejecting fluid CMC-Ph suspension (mixed with HRP) into co-flowing liquid paraffin which blends H2O2 at priority. The Lentivirus-encoding hBMP-2 and Tet-On system were constructed and amplified by RT-PCR, then encapsulated in the microcapsules. The cellular viability of CMC-Ph microparticles was assessed by Live/dead staining and metabolic activity was estimated by colorimetric assay kit. In addition, BMP-2 secretion and kinetic studies were determined by ELISA, alkaline phosphatase (ALP) activity was evaluated using ALP assay kit, and ALP staining as well as mineral calcium deposition was detected by alizarin red S staining. KEY FINDINGS: The diameter of CMC-Ph microparticles was controlled between 100 and 150 µm by altering the flow speed of liquid paraffin and then encapsulated bone morphogenetic protein 2 (BMP-2) gene modified BMSCs transduced by a lentiviral vector. Moreover, the mitochondrial activity of the encapsulated cells was maintained at least 24 d and BMP-2 protein secretion into the supernatant sustained for 35 d without significant loss of efficiency under the induction of the doxycycline. Furthermore, mineral deposition staining and ALP activity detection showed that encapsulated lentiviral-BMP2 transduced BMSCs possess more osteogenic differentiation potential than normal cells. CONCLUSIONS: Co-flow microfluidics and phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) provide a promising strategy for cell-enclosed microcapsules in combination with BMP-2 gene and Tet-on system modified BMSCs and then controlled BMP-2 protein released effectively as well as promoted the osteogenic differentiation of BMSCs.

Parathyroid Hormone-Induced Bone Marrow Mesenchymal Stem Cell Chondrogenic Differentiation and its Repair of Articular Cartilage Injury in Rabbits.

BACKGROUND We explored the effect of parathyroid hormone (PTH)-induced bone marrow stem cells (BMSCs) complexed with fibrin glue (FG) in the repair of articular cartilage injury in rabbits. MATERIAL AND METHODS Forty-eight rabbits randomized into four groups were subjected to articular surgery (cartilage loss). The PTH and non-PTH intervention groups included transplantation with PTH/BMSC/FG xenogeneic and BMSC/FG xenogeneic complexes, respectively, into the injured area. The injured group contained no transplant while the control group comprised rabbits without any articular injury. Samples were monitored for cartilage repair up to three months post-surgery. Immunohistochemistry as well as real-time fluorescent quantitative PCR and Western blot were used to analyze the expression of type II collagen and aggrecan in the repaired tissue. RESULTS At 12 weeks post-surgery, the loss of articular cartilage in the PTH group was fully repaired by hyaline tissue. Typical cartilage lacunae and intact subchondral bone were found. The boundary separating the surrounding normal cartilage tissue disappeared. The gross and International Cartilage Repair Society (ICRS) histological ranking of the repaired tissue was significantly higher in the PTH intervention group than in the non-PTH intervention and injury groups (p<0.05) without any significant difference compared to the control group (p>0.05). Type II collagen and aggrecan stained positive and the average optical density, relative mRNA expression and protein-integrated optical density in the PTH group were higher than in non-PTH and injured groups (p<0.05) but not significantly different from the control group (p>0.05). CONCLUSIONS PTH/BMSC/FG xenogeneic complexes effectively repaired the loss of cartilage in rabbit knee injury.