Application of extracorporeal shockwave therapy (ESWT) to the subchondral bone of medial tibia condyle has shown chondroprotective effects of the knee with decreased cartilage degradation and improved subchondral bone remodeling in the osteoarthritis (OA) of rat knee. Recently, transplantation of ex vivo preparations of mesenchymal stem cells (MSCs) to animal or human joints with OA seems to induce therapeutically effective repair because of paracrine responses from host cells including progenitor cells residing within the synovium. This study compared ESWT, Wharton's jelly mesenchymal stem cells (WJMSCs) and combination of ESWT and WJMSCs therapies for early OA of the rat knee. The results showed ESWT, WJMSCs and combination of therapies significantly improved early OA knee based on analysis of pathological findings, micro-CT and immunohistochemistry (IHC) stain. The combined therapy group increased the bone volume (61.755 ± 1.537), and trabecular thickness (0.215 ± 0.014; P < 0.01) as well as reduced synovitis (1.8 ± 0.37) more than ESWT or WJMSCs individually. However, there were no significant difference in combined ESWT and WJMSCS as shown in the expressions of IGF-1 and TGF-β1 and reduction of the TUNEL activity on OA knee. Furthermore, WJMSCs treatment significantly increased the expression of the type II collagen (22.62 ± 0.84; P < 0.001) when compared with ESWT (6.97 ± 0.54) and ESWT combined with WJMSCs (8.87 ± 0.31) in OA knee. In mechanistic factors analysis, the synergistic effect was observed by ESWT combined with WJMSCs in the expression of RUNX-2, SOX-9 and Collagen Xα1 on OA knee. Our results provided the innovative information of ESWT, and WJMSCs in the treatment of early osteoarthritis of the knee in rats.
Epithelial–mesenchymal transition (EMT) is associated with malignant tumors. In a previous study, we found that KLHL23 is a tumor suppressor gene that inhibits EMT and cancer dissemination. However, the correlation between its expression and cancer progression in urothelial carcinoma (UC) remains unknown. This study showed that the deficiency of KLHL23 in the invasive leading cancer cells is important for improving cell migration in UC. Currently, little is known about the underlying mechanisms of KLHL23-mediated cytoskeleton remodeling in the metastatic leading cells of tumors. Our findings showed that silencing of KLHL23 promotes cell migration in UC by regulating the translocation of focal adhesion proteins. Lack of KLHL23 causes abnormal formation of lamellipodia and increases the EMT phenotype and migration. Wound healing assay revealed that KLHL23 potentiates the actin bundles and intracellular focal adhesion protein formation in the invasive leading cells. Knockdown of KLHL23 abolishes the formation of actin stress fibers and translocalizes vinculin to the perimembrane, which enhances the mobility of cancer cells. To elucidate the mechanism, we found that during migration, KLHL23 appears in the leading cells in large numbers and binds to the actin stress fibers. A large amount of vinculin accumulated at both ends of the KLHL23/actin fibers, indicating an increase in cell anchorage. Thus, KLHL23 might play a critical role in enhancing actin fibers and promoting focal adhesion complex formation in the invasive leading cells. Analysis of the overall survival revealed that low KLHL23 is associated with poor survival in patients with bladder UC, indicating its clinical significance. We hypothesize that KLHL23 is involved in the formation of actin stress fibers and focal adhesion complexes in the invasive leading cells and may be associated with EMT progression and prognosis in UC patients.
Toona sinensis (TS) is a medicinal herb possessing anti-apoptotic, anti-oxidant, and anti-inflammatory properties and is used to treat diabetes, cancer, and inflammatory diseases. In traditional Chinese medicine theory, TS clears dampness and heat, strengthens the stomach function, and regulates vital energy flow. TS is also used as an astringent and a pesticide. In this study, we aimed to evaluate how TS influences autophagy and cytokines during the inflammatory process in RAW 264.7 macrophages. The treatment groups were pre-supplemented with TS leaf extract; rapamycin was used to enhance autophagy and lipopolysaccharide (LPS) was used to induce inflammation. The expression of autophagy-related proteins was analyzed by western blotting. The survival rate of, and chemokine expression and oxidative stress in the cells were also assessed. TS leaf extract inhibited mammalian target of rapamycin (mTOR) phosphorylation at site S2448 in the macrophages. At relatively higher concentrations (50 and 75 μg/mL), TS elevated the expression of light chain 3 II (LC3-II), which further modulated autophagy. Pre-supplementation with TS leaf extract elevated the total glutathione (GSH) level and GSH/oxidized GSH (GSSG) ratio, but it decreased the GSSG, total nitric oxide, nitrate, nitrite, malondialdehyde, and superoxide anion levels. TS reversed the effects of LPS-induced cytokines, including interleukin (IL)-6 and IL-10. TS did not induce significant toxicity at the studied concentrations. In conclusion, TS leaf extract may modulate autophagy during inflammation. Furthermore, it may prevent cell damage via anti-inflammation and anti-oxidation. Thus, this study supports the ethnomedical use of TS in the prevention of inflammation-related diseases.
Background: Nonunion of long bone fractures is a significant complication following surgical fixation, with an incidence ranging from 5 to 10%. Surgical intervention is the standard treatment for nonunions, but it may come with potential complications. Nonoperative approaches, such as Extracorporeal Shockwave Therapy (ESWT), have been advocated as alternatives. Methods: In the retrospective study conducted between January 2004 and January 2018, 91 patients who underwent ESWT for tibia or femur nonunions were included. Nonunion was defined based on radiographic criteria and clinical symptoms. The nonunion morphology was categorized as hypertrophic, oligotrophic, or atrophic. ESWT was administered using the OssaTron device in a single treatment session. Bony union was defined as the presence of a bridging callus over the fracture site with more than three-fourths of the circumference in both planes within the 12-month postoperative period. Results: The study included 91 patients, with an overall union rate of 62.6%. A higher healing rate was observed in trophic nonunion(69.9%) than in atrophic nonunion(33.3%). Multivariate analysis identified the number of surgeries, maximum fracture gap, and atrophic nonunion as independent factors influencing the risk of fracture nonunion after ESWT. The receiver operating characteristic curves were generated for these factors, providing more than one surgical intervention, and fracture gap greater than 3.94 mm as negative predictors of ESWT for long bone nonunions. Conclusion: The study’s primary findings suggest that ESWT is effective in achieving bony union for nonunions in long bones(62.6%). Despite the overall positive results, the study highlights that atrophic nonunions, larger fracture gaps of more than 3.94 mm, and multiple surgeries are associated with poorer outcomes.
Abstract Background VP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell. Results An arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity. Conclusions VP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.
Adulteration by Bacopa monnieri (BM) in Portulaca oleracea (PO) plants frequently occurs; it decreases the efficacy of traditional Chinese medicine (TCM) and leads to fraud in the herbal marketplace. In this study, a diagnostic PCR assay was established for the rapid authentication of PO and BM in the herbal market. The sequence divergences in internal transcribed spacer 2 (ITS2) between PO and its adulterant species were used to design diagnostic PCR primers. The specific designed primer sets were evaluated and show that the diagnostic PCR assay can be used to verify the authenticity of PO and BM. The detection limits of the primer set for PO and BM identification were 10 pg and 1 pg, respectively. The reactivity of diagnostic PCR was 0.1% PO genomic DNA and 0.01% BM genomic DNA in the test sample during DNA amplification. In addition, multiplex PCR (mPCR) for PO and BM identification was also established. The samples were more susceptible to the effect of steaming in authentication by singleplex PCR and mPCR than boiling and drying treatment. Furthermore, commercial samples from the market were used to demonstrate the applicability of the developed diagnostic PCR for PO authentication and diagnose BM adulteration, and the investigation found that approximately 72.2% (13/18) of PO plants in the herbal market were adulterated. In conclusion, the diagnostic PCR assay was successfully developed and its specificity, sensitivity and reactivity for PO and BM authentication were proven. These developed PCR-based molecular methods can be applied as an identification tool for PO authenticity and can be practically applied for inspection of BM adulteration in the herbal market in the future.
Despite the widespread use of nerve-sparing prostatectomy techniques, the incidence of post-operative erectile dysfunction (ED) remains high. Early intracavernous (IC) injection of platelet-rich plasma (PRP) after nerve crushing improves erectile function (EF) in rats by promoting cavernous nerve (CN) regeneration and preventing structural changes in the corpus cavernosum. However, the neuroprotective effects of the in situ application of PRP glue in rats after CN-sparing prostatectomy (CNSP) remain unclear.This study aimed to investigate the effects of PRP glue treatment on EF and CN preservation in rats after CNSP.After prostatectomy, male Sprague-Dawley rats were treated with PRP glue, IC PRP injection, or their combination. The intracavernous pressure (ICP), mean arterial pressure (MAP), and CN preservation status in the rats were evaluated after 4 weeks. Results were corroborated using histology, immunofluorescence, and transmission electron microscopy.The PRP glue-treated rats showed 100% CN preservation and significantly higher ICP responses (the ratio of maximum ICP to MAP (0.79 ± 0.09)) than the CNSP rats (the ratio of maximum ICP to MAP (0.33 ± 0.04)). PRP glue also significantly increased neurofilament-1 expression, indicating its positive effect on the CNs. Furthermore, this treatment significantly increased the expression of α-smooth muscle actin. Electron micrographs revealed that PRP glue preserved the myelinated axons and prevented atrophy of the corporal smooth muscle by maintaining the adherens junctions.These results indicate that PRP glue is a potential solution for EF preservation by neuroprotection in patients with prostate cancer who are likely to undergo nerve-sparing radical prostatectomy.
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