Preliminary investigation on the effect of extracorporeal shock wave combined with traction on joint contracture based on PTEN-PI3K/AKT pathway.

To investigate the intervention effect of extracorporeal shock wave combined with manual traction on fixation-induced knee contracture and its influence on PTEN-PI3K/AKT signaling pathway. Thirty-six SD male rats were randomly divided into six groups. The left knee joints were not fixed in the control group (C group). Rats in other groups underwent brace fixation in the extended position of the left knee. After 4 weeks of bracing, it is randomly divided into five groups: Model group (M group), natural recovery group (NR group), extracorporeal shock wave treatment group (ET group), manual traction group (MT group), and extracorporeal shock wave combined with manual traction group (CT group). Joint range of motion (ROM) of left knee was carried out to assess joint function. Hematoxylin and eosin (HE) staining and Masson staining were respectively used to assess the cell number and collagen deposition expression. Immunohistochemical staining and Western blot were used to assess protein levels of phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (AKT). The combined therapy was more effective than extracorporeal shock wave therapy or manual traction alone against the joint ROM, cell number and the collagen deposition, low-expression of PTEN, and overexpression of PI3K/AKT in the anterior joint capsule of rats with knee extension contracture. Extracorporeal shock wave combined with manual traction can promote the histopathological changes of anterior joint capsule fibrosis, upregulate the protein expression of PTEN and downregulate the protein expression of PI3K/AKT in the fibrotic joint capsule in a rat joint contracture model.

Formation process of extension knee joint contracture following external immobilization in rats.

BACKGROUND: Current research lacks a model of knee extension contracture in rats. AIM: To elucidate the formation process of knee extension contracture. METHODS: We developed a rat model using an aluminum external fixator. Sixty male Sprague-Dawley rats with mature bones were divided into the control group (n = 6) and groups that had the left knee immobilized with an aluminum external fixator for 1, 2, and 3 d, and 1, 2, 3, 4, 6, and 8 wk (n = 6 in each group). The passive extension range of motion, histology, and expression of fibrosis-related proteins were compared between the control group and the immobilization groups. RESULTS: Myogenic contracture progressed very quickly during the initial 2 wk of immobilization. After 2 wk, the contracture gradually changed from myogenic to arthrogenic. The arthrogenic contracture progressed slowly during the 1st week, rapidly progressed until the 3rd week, and then showed a steady progression until the 4rd week. Histological analyses confirmed that the anterior joint capsule of the extended fixed knee became increasingly thicker over time. Correspondingly, the level of transforming growth factor beta 1 (TGF-ß1) and phosphorylated mothers against decapentaplegic homolog 2 (p-Smad2) in the anterior joint capsule also increased with the immobilization time. Over time, the cross-sectional area of muscle fibers gradually decreased, while the amount of intermuscular collagen and TGF-ß1, p-Smad2, and p-Smad3 was increased. Unexpectedly, the amount of intermuscular collagen and TGF-ß1, p-Smad2, and p-Smad3 was decreased during the late stage of immobilization (6-8 wk). The myogenic contracture was stabilized after 2 wk of immobilization, whereas the arthrogenic contracture was stabilized after 3 wk of immobilization and completely stable in 4 wk. CONCLUSION: This rat model may be a useful tool to study the etiology of joint contracture and establish therapeutic approaches.

LIPUS Alleviates Knee Joint Capsule Fibrosis in Rabbits by Regulating SOD/ROS Dynamics and Inhibiting the TGF-ß1/Smad Signaling Pathway.

OBJECTIVE: The aim of the work described here was to investigate the efficacy and potential mechanisms of low-intensity pulsed ultrasound (LIPUS) for the treatment of arthrogenic contracture induced by immobilization in rabbits. METHODS: The left knee joint of rabbits was immobilized for 6 wk to establish the model of extending knee joint contracture. The rabbits were divided into a control group (C), a group immobilized for 6 wk (IM-6w), a group remobilized for 1 wk (RM-1w), a group subjected to LIPUS intervention for 1 wk (LIPUS-1w), a group remobilized for 2 wk (RM-2w) and a group subjected to LIPUS intervention for 2 wk (LIPUS-2w). The degrees of arthrogenic contracture and joint capsule fibrosis were assessed, as were the levels of reactive oxygen species (ROS) and the activation status of the TGF-ß1/Smad signaling pathway in the joint capsule. RESULTS: After immobilization for 6 wk, the degrees of arthrogenic contracture and joint capsule fibrosis increased. The ROS level increased, as evidenced by an increase in malondialdehyde content and a decrease in superoxide dismutase content. In addition, the TGF-ß1/Smad signaling pathway was significantly activated. The degrees of knee joint contracture increased in the first week after remobilization and decreased in the second week. Furthermore, joint capsule fibrosis continued to develop during the 2 wk of remobilization, and the ROS level increased, while the TGF-ß1/Smad signaling pathway was significantly activated. LIPUS effectively reduced the level of ROS in the joint capsule, which further inhibited activation of the TGF-ß1/Smad signaling pathway, thereby improving joint capsule fibrosis and reducing arthrogenic contracture. CONCLUSION: The high ROS levels and overactivation of the TGF-ß1/Smad signaling pathway may be reasons why immobilization induces knee joint capsule fibrosis. LIPUS can alleviate the degree of knee joint capsule fibrosis induced by immobilization by inhibiting the production of ROS and the activation of the TGF-ß1/Smad signaling pathway.

The worsening of skeletal muscle atrophy induced by immobilization at the early stage of remobilization correlates with BNIP3-dependent mitophagy.

BACKGROUND: Recent studies have shown that immobilization enhances reactive oxygen species (ROS) production and mitophagy activity in atrophic skeletal muscle. However, there are relatively few studies examining the biological changes and underlying mechanisms of skeletal muscle during remobilization. In this study, we aimed to investigate the effects of remobilization on skeletal muscle and explore the role of BNIP3-dependent mitophagy in this process. METHODS: Thirty rats were randomly divided into six groups based on immobilization and remobilization time: control (C), immobilization for two weeks (I-2w), and remobilization for one day (R-1d), three days (R-3d), seven days (R-7d), and two weeks (R-2w). At the end of the experimental period, the rectus femoris muscles were removed and weighed, and the measurements were expressed as the ratio of muscle wet weight to body weight (MWW/BW). Sirius Red staining was performed to calculate the values of cross-sectional area (CSA) of rectus femoris. Oxidative fluorescent dihydroethidium was used to evaluate the production of ROS, and the levels of superoxide dismutase (SOD) were also detected. The morphological changes of mitochondria and the formation of mitophagosomes in rectus femoris were examined and evaluated by transmission electron microscope. Immunofluorescence was employed to detect the co-localization of BNIP3 and LC3B, while Western blot analysis was performed to quantify the levels of proteins associated with mitophagy and mitochondrial biogenesis. The total ATP content of the rectus femoris was determined to assess mitochondrial function. RESULTS: Within the first three days of remobilization, the rats demonstrated decreased MWW/BW, CSA, and ATP concentration, along with increased ROS production and HIF-1α protein levels in the rectus femoris. Results also indicated that remobilization triggered BNIP3-dependent mitophagy, supported by the accumulation of mitophagosomes, the degradation of mitochondrial proteins (including HSP60 and COX IV), the elevation of BNIP3-dependent mitophagy protein markers (including BNIP3, LC3B-II/LC3B-I, and Beclin-1), and the accumulation of puncta representing co-localization of BNIP3 with LC3B. Additionally, PGC-1α, which is involved in the regulation of mitochondrial biogenesis, was upregulated within the first seven days of remobilization to counteract this adverse effect. CONCLUSION: Our findings suggested that BNIP3-denpendent mitophagy was sustained activated at the early stages of remobilization, and it might contribute to the worsening of skeletal muscle atrophy.

Metformin reduces myogenic contracture and myofibrosis induced by rat knee joint immobilization via AMPK-mediated inhibition of TGF-ß1/Smad signaling pathway.

PURPOSE: The two structural components contributing to joint contracture formation are myogenic and arthrogenic contracture, and myofibrosis is an important part of myogenic contracture. Myofibrosis is a response to long-time immobilization and is described as a condition with excessive deposition of endomysial and perimysial connective tissue components in skeletal muscle. The purpose of this study was to confirm whether metformin can attenuate the formation of myogenic contracture and myofibrosis through the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK) and inhabitation of subsequent transforming growth factor beta (TGF-ß) 1/Smad signaling pathway. MATERIALS AND METHODS: An immobilized rat model was used to determine whether metformin could inhibit myogenic contracture and myofibrosis. The contents of myogenic contracture of knee joint was calculated by measuring instrument of range of motion (ROM), and myofibrosis of rectus femoris were determined by ultrasound shear wave elastography and Masson staining. Protein expression of AMPK and subsequent TGF-ß1/Smad signaling pathway were determined by western blot. Subsequently, Compound C, a specific AMPK inhibitor, was used to further clarify the role of the AMPK-mediated inhibition of TGF-ß1/Smad signaling pathway. RESULTS: We revealed that the levels of myogenic contracture and myofibrosis were gradually increased during immobilization, and overexpression of TGF-ß1-induced formation of myofibrosis by activating Smad2/3 phosphorylation. Activation of AMPK by metformin suppressed overexpression of TGF-ß1 and TGF-ß1-induced Smad2/3 phosphorylation, further reducing myogenic contracture and myofibrosis during immobilization. In contrast, inhibition of AMPK by Compound C partially counteracted the inhibitory effect of TGF-ß1/Smad signaling pathway by metformin. CONCLUSION: Notably, we first illustrated the therapeutic effect of metformin through AMPK-mediated inhibition of TGF-ß1/Smad signaling pathway in myofibrosis, which may provide a new therapeutic strategy for myogenic contracture.

Effects of lentivirus-mediated CYP17A1 gene silencing on the biological activity of glioma.

Gliomas are the most common malignant primary brain tumors with poor prognosis. We attempted to explore the role of CYP17A1 in glioma progression. We demonstrated that the expression of CYP17A1 was significantly higher in the glioma tissues than the normal brain tissues, especially in malignant glioma. Moreover, the expression of CYP17A1 gene was positively correlative with glioma pathological grades. In vitro, CYP17A1 gene silence inhibited the proliferation and invasion of glioma cells and promoted the apoptosis in glioma cells. Also, the subcutaneously transplanted tumour in BALB/C-nu showed that CYP17A1 gene silence inhibited glioma growth. These results reveal that CYP17A1 plays a major role in the progress of glioma.

G-protein-coupled receptor kinase-5 promotes glioblastoma progression by targeting the nuclear factor kappa B pathway.

G-protein-coupled receptor kinase-5 (GRK5) plays essential roles in multiple celluar events. However, its role in the development and progression of glioma is poorly understood. In this research, we found that GRK5 is significantly upregulated in human gliomas. For the first time, a close relationship was noted between GRK5 expression and blood vessel development in human glioma. Specifically co-expression of GRK5 and the tumor stem cell marker CD133 was observed in the cytoplasm of high grade glioma cells. The depletion of GRK5 suppressed the proliferation, migration and invasion in glioma cells, and promoted apoptosis. We next discovered that GRK5 knockdown inhibits the nuclear factor kappa B (NF-κB) pathway, thus resulting in downregulation of key downstream secretory products CCL2, IL-6 and IL-8 in glioma cell conditioned medium (CM). In addition, treatment of cells with the NF-κB stimulator PMA reversed this effect and increased the GRK5 level. Our results demonstrate an oncogenic role for GRK5 and reveal an activation of the GRK5-NF-κB pathway during the malignant progression of glioma.

The long non-coding RNA CRNDE acts as a ceRNA and promotes glioma malignancy by preventing miR-136-5p-mediated downregulation of Bcl-2 and Wnt2.

The colorectal neoplasia differentially expressed (CRNDE) gene encodes a long non-coding RNA (lncRNA) that is the most unregulated among 129 lncRNAs differentially expressed in gliomas. In this study, we confirmed high CRNDE expression in clinical glioma specimens and observed through experiments in human glioma cell lines a novel molecular mechanism by which CRNDE may contribute to glioma pathogenesis. By inducing or silencing CRNDE expression, we detected a positive correlation between CRNDE levels and the proliferative, migratory, and invasive capacities of glioma cells, which were concomitant with a decreased apoptosis rate. Our experiments also suggest that these effects are mediated by downregulation of miR-136-5p, which correlated with the glioma WHO grade. Based on predicted CRNDE/miR-136-5p/mRNA interactions, both the mRNA and protein expression analyses suggested that miR-136-5p-mediated repression of Bcl-2 and Wnt2 underlies the pro-tumoral actions of CRNDE. We therefore propose that CRNDE functions as a competing endogenous RNA (ceRNA) that binds to and negatively regulates miR-136-5p, thereby protecting Bcl-2 and Wnt2 from miR-136-5p-mediated inhibition in glioma.