Epigenetic modification of miR-217 promotes intervertebral disc degeneration by targeting the FBXO21-ERK signalling pathway.

BACKGROUND: Numerous potential therapeutic alternatives for intervertebral disc degeneration (IDD) have been investigated, the most promising of which are based on biological variables such as microRNAs (miRNAs). Therefore, we verified the hypothesis that miRNAs modulate IDD by affecting the FBXO21-ERK signalling pathway. METHODS: Microarray and quantitative real-time polymerase chain reaction (RT-qPCR) tests were used to examine the expression profiles of miRNAs in nucleus pulposus (NP) cells between patients with IDD and controls. Western blotting and luciferase reporter assays were used to identify the miRNA targets. RESULTS: Microarray and RT-qPCR assays confirmed that the expression level of miR-217 was significantly decreased in degenerative NP cells. CpG islands were predicted in the miR-217 promoter region. The IDD group had considerably higher methylation than the control group. Gain- and loss-of-function experiments revealed that miR-217 mimics inhibited apoptosis and extracellular matrix (ECM) breakdown in NP cells. Bioinformatic analyses and luciferase assays were used to determine the connection between miR-217 and FBXO21. In vitro tests revealed that miR-217 mimics inhibited the expression of FBXO21, pERK, MMP13, and ADAMTS5 proteins, successfully protecting the ECM from degradation. Additionally, in vivo investigation using the IDD mouse model demonstrated that the miR-217 agonist may sufficiently promote NP cell proliferation, decrease apoptosis, promote ECM synthesis, and suppress the expression of matrix-degrading enzymes in NP cells. CONCLUSIONS: Overexpression of miR-217 inhibits IDD via FBXO21/ERK regulation. TRIAL REGISTRATION: This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the human research ethics committee of Wuhan University Renmin Hospital (Approval No. RMHREC-D-2020-391), and written informed consent was obtained from each participant.

Innate/Inflammatory Bioregulation of Surfactant Protein D Alleviates Rat Osteoarthritis by Inhibiting Toll-Like Receptor 4 Signaling.

Osteoarthritis (OA) is a deteriorating disease of cartilage tissues mainly characterized as low-grade inflammation of the joint. Innate immune molecule surfactant protein D (SP-D) is a member of collectin family of collagenous Ca2+-dependent defense lectins and plays a vital role in the inflammatory and innate immune responses. The present study investigated the SP-D-mediated innate/inflammatory bioregulation in OA and explored the underlying molecular mechanism. Transcriptome analysis revealed that SP-D regulated genes were strongly enriched in the inflammatory response, immune response, cellular response to lipopolysaccharide (LPS), PI3K-Akt signaling, Toll-like receptor (TLR) signaling, and extracellular matrix (ECM)-receptor interaction pathways. Knockdown of the SP-D gene by the recombinant adeno-associated virus promoted the macrophage specific markers of CD68, F4/80 and TLR4 in the articular cartilage in vivo. SP-D alleviated the infiltration of synovial macrophages and neutrophils, and inhibited TLR4, TNF-α and the phosphorylation of PI3K, Akt and NF-κB p65 in cartilage. SP-D suppressed cartilage degeneration, inflammatory and immune responses in the rat OA model, whilst TAK-242 strengthened this improvement. In in vitro conditions, SP-D pre-treatment inhibited LPS-induced overproduction of inflammation-correlated cytokines such as IL-1ß and TNF-α, and suppressed the overexpression of TLR4, MD-2 and NLRP3. SP-D prevented the LPS-induced degradation of ECM by down-regulating MMP-13 and up-regulating collagen II. Blocking of TLR4 by TAK-242 further enhanced these manifestations. We also demonstrated that SP-D binds to the TLR4/MD-2 complex to suppress TLR4-mediated PI3K/Akt and NF-κB signaling activation in chondrocytes. Taken together, these findings indicate that SP-D has chondroprotective properties dependent on TLR4-mediated PI3K/Akt and NF-κB signaling and that SP-D has an optimal bioregulatory effect on the inflammatory and innate responses in OA.

Effect and Repair Mechanism of Nano Ag Sponge Dressing Combined with Gelatin-Bletilla Striata Gum/Salvia Miltiorrhiza on Refractory Orthopedic Wounds.

OBJECTIVE: To explore the effect and mechanism of the sponge dressing on the healing of refractory orthopedic wound, and the gelatin-Bletilla striata gum/Salvia miltiorrhiza nano Ag (GBS-Ag) sponge dressing was prepared. METHODS: GBS-Ag sponge dressing was prepared by the freeze-drying method. Twenty male SD rats were randomly divided into the control group (Ctrl group) and GBS-Ag group, with 10 rats in each group, and the rats in the two groups were established a model of back wound infection. The Ctrl group was treated with gauze, while the GBS-Ag group was treated with GBS-Ag sponge dressing. Wound healing rate, blood immune indexes, Ag content in each organ, morphological changes of wound, and expression of transforming growth factor-ß1 (TGF-ß1) in wound transformation were detected in the two groups of rats. RESULTS: The mechanical properties of GBS-Ag sponge dressing were all in line with the standard, and it had good killing effect on the conventional strain after being incubated for 24 hours. Compared with the Ctrl group, the healing rate and lymphocyte percentage in the GBS-Ag group were significantly increased on day 4 and day 10 (P < 0.05), while the total number of white blood cells and the percentage of neutrophils were significantly decreased (P < 0.05). Compared with Ctrl group, the Ag content in liver, spleen, and kidney of rats in the GBS-Ag group was significantly increased (P < 0.05). The histological results showed that the Ctrl group lacked collagen fibers in the dermis, and the angiogenesis was not rich, accompanied by a large number of inflammatory cell infiltration. The epidermal repair of rats in the GBS-Ag group was complete and partially keratinized, the dermis was rich in collagen fibers, with elastic fibers and new blood vessels, inflammatory cells were rare, and new hair follicles and thick-walled blood vessels were also observed. The expression of TGF-ß1 protein in the wounds of rats in the GBS-Ag group was higher than that of the Ctrl group. CONCLUSION: GBS-Ag sponge dressing had multiple effects of sterilization and promoting wound healing, and its mechanism may be related to promoting the TGF-ß1 protein expression.

Down-regulation of insulin-like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway.

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down-regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT-PCR and Western blot analyses were performed to examine the levels of apoptosis-related factors, including Bax, caspase-3 and Bcl2. The silencing of IGFBP5 up-regulated the levels of Bax and caspase-3 and down-regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.

The effects of chitosan oligosaccharides on OPG and RANKL expression in a rat osteoarthritis model.

PURPOSE:: To investigate the effect of chitosan oligosaccharides (COS) against osteoarthritis (OA) and preliminarily discuss the osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) and RANK expression in a rat OA model. METHODS:: Thirty-six 6-week-old Male SD rats were randomly divided into three groups: sham-operated group(CON), OA-induction group(OA), COS intervention group(n=12/group). At 4 weeks after the operation, COS (50 ul) intervention weekily for consecutive 5 weeks. The OA and CON groups received an injection of 50 ul physiological saline. At death, 11 weeks following surgery, cartilage was harvested and total RNA and protein were extracted. Both the morphological changes of the cartilage were observed and harvested the total RNA and protein. Meanwhile, the expression of OPG, RANKL and RANK in cartilage were determined. RESULTS:: The expression of OPG and RANKL were both enhanced in the cartilage of the OA model. Compared with the OA group, COS treatment improved the cartilage damage (both extent and grade). Furthermore, the COS group showed highly OPG and lower RANKL. Simultaneously, COS treatment upregulated the ratio of OPG/RANKL and downregulated the RANKL/RANK. CONCLUSION:: Chitosan oligosaccharides may be used as a unique biological agent to prevent and treat osteoarthritis, and this effect is associated with modulation of the expression of osteoprotegerin and receptor activator of NF-κB ligand.

The effects of chitosan oligosaccharides on OPG and RANKL expression in a rat osteoarthritis model

Abstract Purpose: To investigate the effect of chitosan oligosaccharides (COS) against osteoarthritis (OA) and preliminarily discuss the osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) and RANK expression in a rat OA model. Methods: Thirty-six 6-week-old Male SD rats were randomly divided into three groups: sham-operated group(CON), OA-induction group(OA), COS intervention group(n=12/group). At 4 weeks after the operation, COS (50 ul) intervention weekily for consecutive 5 weeks. The OA and CON groups received an injection of 50 ul physiological saline. At death, 11 weeks following surgery, cartilage was harvested and total RNA and protein were extracted. Both the morphological changes of the cartilage were observed and harvested the total RNA and protein. Meanwhile, the expression of OPG, RANKL and RANK in cartilage were determined. Results: The expression of OPG and RANKL were both enhanced in the cartilage of the OA model. Compared with the OA group, COS treatment improved the cartilage damage (both extent and grade). Furthermore, the COS group showed highly OPG and lower RANKL. Simultaneously, COS treatment upregulated the ratio of OPG/RANKL and downregulated the RANKL/RANK. Conclusion: Chitosan oligosaccharides may be used as a unique biological agent to prevent and treat osteoarthritis, and this effect is associated with modulation of the expression of osteoprotegerin and receptor activator of NF-κB ligand.

Surgical factors contributing to nonunion in femoral shaft fracture following intramedullary nailing.

PURPOSE: To explore the possible surgical factors related with nonunion in femoral shaft fracture following intramedullary nailing. METHODS: We retrospectively analyzed totally 425 patients with femoral shaft fracture in level I urban trauma center, including 254 males and 171 females, with an average age of 37.6 (ranging from 21 to 56) years old. The inclusion criteria included: (1) traumatically closed fracture of femoral shaft, with pre- operative films showing non-comminuted fracture, such as transverse fracture, oblique fracture or spiral fracture; (2) closed reduction and fixation with interlocking intramedullary nail at 3-7 days after trauma; (3) complete follow-up data available. The relationship between the following factors (fracture site, reduction degree, direction of nail insertion and nail size) and nonunion was studied. RESULTS: The incidence of femoral nonunion was 2.8% in patients with closed simple fracture undergoing interlocking intrameduallary nailing, including 11 cases of hypertrophic nonunion. Nonunion was related significantly to distal fracture, unsatisfactory reduction and unreamed nail (p < 0.05). There was no significant difference between antegrade nail and retrograde nail (p > 0.05). CONCLUSIONS: Nonunion in femoral shaft facture following interlocking intramedullary nailing is related to fracture site, fracture reduction and nail diameter. The choice of reamed nails or unreamed nails depends on the fracture site and reduction degree.