Lidocaine inhibits migration of tenocytes by downregulating focal adhesion kinase and paxillin phosphorylation.

Lidocaine is the most frequently applied local infiltration anesthetic agent for treating tendinopathies. However, studies have discovered lidocaine to negatively affect tendon healing. In the current study, the molecular mechanisms and effects of lidocaine on tenocyte migration were evaluated. We treated tenocytes intrinsic to the Achilles tendons of Sprague-Dawley rats with lidocaine. The migration ability of cells was analyzed using electric cell-substrate impedance sensing (ECIS) and scratch wound assay. We then used a microscope to evaluate the cell spread. We assessed filamentous actin (F-actin) cytoskeleton formation through immunofluorescence staining. In addition, we used Western blot analysis to analyze the expression of phospho-focal adhesion kinase (FAK), FAK, phospho-paxillin, paxillin, and F-actin. We discovered that lidocaine had an inhibitory effect on the migration of tenocytes in the scratch wound assay and on the ECIS chip. Lidocaine treatment suppressed cell spreading and changed the cell morphology and F-actin distribution. Lidocaine reduced F-actin formation in the tenocyte during cell spreading; furthermore, it inhibited phospho-FAK, F-actin, and phospho-paxillin expression in the tenocytes. Our study revealed that lidocaine inhibits the spread and migration of tenocytes. The molecular mechanism potentially underlying this effect is downregulation of F-actin, phospho-FAK, and phospho-paxillin expression when cells are treated with lidocaine.

Lidocaine Inhibited Tendon Cell Proliferation and Extracellular Matrix Production by Down Regulation of Cyclin A, CDK2, Type I and Type III Collagen Expression.

Lidocaine injection is a common treatment for tendon injuries. However, the evidence suggests that lidocaine is toxic to tendon cells. This study investigated the effects of lidocaine on cultured tendon cells, focusing on the molecular mechanisms underlying cell proliferation and extracellular matrix (ECM) production. Tendon cells cultured from rat Achilles tendons were treated with 0.5, 1.0, or 1.5 mg/mL lidocaine for 24 h. Cell proliferation was evaluated by Cell Counting Kit 8 (CCK-8) assay and bromodeoxyuridine (BrdU) assay. Cell apoptosis was assessed by Annexin V and propidium iodide (PI) stain. Cell cycle progression and cell mitosis were assessed through flow cytometry and immunofluorescence staining, respectively. The expression of cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2), p21, p27, p53, matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), type I collagen, and type III collagen were examined through Western blotting, and the enzymatic activity of MMP-9 was determined through gelatin zymography. Lidocaine reduced cell proliferation and reduced G1/S transition and cell mitosis. Lidocaine did not have a significant negative effect on cell apoptosis. Lidocaine significantly inhibited cyclin A and CDK2 expression but promoted p21, p27, and p53 expression. Furthermore, the expression of MMP-2 and MMP-9 increased, whereas that of type I and type III collagen decreased. Lidocaine also increased the enzymatic activity of MMP-9. Our findings support the premise that lidocaine inhibits tendon cell proliferation by changing the expression of cell-cycle-related proteins and reduces ECM production by altering levels of MMPs and collagens.

Simvastatin Downregulates Cofilin and Stathmin to Inhibit Skeletal Muscle Cells Migration.

Statins are the most effective therapeutic agents for reducing cholesterol synthesis. Given their widespread use, many adverse effects from statins have been reported; of these, musculoskeletal complications occurred in 15% of patients after receiving statins for 6 months, and simvastatin was the most commonly administered statin among these cases. This study investigated the negative effects of simvastatin on skeletal muscle cells. We performed RNA sequencing analysis to determine gene expression in simvastatin-treated cells. Cell proliferation and migration were examined through cell cycle analysis and the transwell filter migration assay, respectively. Cytoskeleton rearrangement was examined through F-actin and tubulin staining. Western blot analysis was performed to determine the expression of cell cycle-regulated and cytoskeleton-related proteins. Transfection of small interfering RNAs (siRNAs) was performed to validate the role of cofilin and stathmin in the simvastatin-mediated inhibition of cell migration. The results revealed that simvastatin inhibited the proliferation and migration of skeletal muscle cells and affected the rearrangement of F-actin and tubulin. Simvastatin reduced the expression of cofilin and stathmin. The knockdown of both cofilin and stathmin by specific siRNA synergistically impaired cell migration. In conclusion, our results indicated that simvastatin inhibited skeletal muscle cell migration by reducing the expressions of cofilin and stathmin.

The Comparison and Interpretation of Machine-Learning Models in Post-Stroke Functional Outcome Prediction.

Prediction of post-stroke functional outcomes is crucial for allocating medical resources. In this study, a total of 577 patients were enrolled in the Post-Acute Care-Cerebrovascular Disease (PAC-CVD) program, and 77 predictors were collected at admission. The outcome was whether a patient could achieve a Barthel Index (BI) score of >60 upon discharge. Eight machine-learning (ML) methods were applied, and their results were integrated by stacking method. The area under the curve (AUC) of the eight ML models ranged from 0.83 to 0.887, with random forest, stacking, logistic regression, and support vector machine demonstrating superior performance. The feature importance analysis indicated that the initial Berg Balance Test (BBS-I), initial BI (BI-I), and initial Concise Chinese Aphasia Test (CCAT-I) were the top three predictors of BI scores at discharge. The partial dependence plot (PDP) and individual conditional expectation (ICE) plot indicated that the predictors' ability to predict outcomes was the most pronounced within a specific value range (e.g., BBS-I < 40 and BI-I < 60). BI at discharge could be predicted by information collected at admission with the aid of various ML models, and the PDP and ICE plots indicated that the predictors could predict outcomes at a certain value range.

Use of Platelet-Rich Plasma Plus Suramin, an Antifibrotic Agent, to Improve Muscle Healing After Injuries.

BACKGROUND: The increasing use of platelet-rich plasma (PRP) to treat muscle injuries raises concerns because transforming growth factor-beta (TGF-ß) in PRP may promote fibrosis in the injured muscle and thus impair muscle regeneration. PURPOSE: To investigate whether suramin (a TGF-ß inhibitor) can reduce muscle fibrosis to improve healing of the injured muscle after PRP treatment and identify the underlying molecular mechanism. STUDY DESIGN: Controlled laboratory study. METHODS: Myoblasts isolated from the gastrocnemius muscle of Sprague Dawley rats were treated with PRP or PRP plus suramin. MTT assays were performed to evaluate cell viability. The expression of fibrosis-associated proteins (such as type I collagen and fibronectin), Smad2, and phosphorylated Smad2 was determined using Western blot analysis and immunofluorescent staining. An anti-TGF-ß antibody was employed to verify the role of TGF-ß in fibronectin expression. Gastrocnemius muscles were injured through a partial transverse incision and then treated using PRP or PRP plus suramin. Hematoxylin and eosin staining was conducted to evaluate the healing process 7 days after the injury. Immunofluorescent staining was performed to evaluate fibronectin expression. Muscle contractile properties-fast-twitch and tetanic strength-were evaluated through electric stimulation. RESULTS: PRP plus 25 µg/mL of suramin promoted myoblast proliferation. PRP induced fibronectin expression in myoblasts, but suramin reduced this upregulation. The anti-TGF-ß antibody also reduced the upregulation of fibronectin expression in the presence of PRP. The upregulation of phosphorylated Smad2 by PRP was reduced by either the anti-TGF-ß antibody or suramin. In the animal study, no significant difference was discovered in muscle healing between the PRP versus PRP plus suramin groups. However, the PRP plus suramin group had reduced fibronectin expression at the injury site. Fast-twitch strength and tetanic strength were significantly higher in the injured muscle treated using PRP or PRP plus suramin. CONCLUSION: Simultaneous PRP and suramin use reduced fibrosis in the injured muscle and promoted healing without negatively affecting the muscle's contractile properties. The underlying molecular mechanism may be associated with the phosphorylated Smad2 pathway. CLINICAL RELEVANCE: Simultaneous PRP and suramin use may reduce muscle fibrosis without compromising muscle contractile properties and thus improve muscle healing.

Functional Recovery Patterns of Hemorrhagic and Ischemic Stroke Patients Under Post-Acute Care Rehabilitation Program.

BACKGROUND: Tailored rehabilitation programs for stroke patients cannot be made without knowledge of their recovery potential. The aim of this study is to characterize the functional recovery patterns of ischemic stroke (IS) and intracerebral hemorrhage (ICH) patients under post-acute care stroke rehabilitation. METHODS: This retrospective study analyzed the data of patients enrolled in the Post-Acute Care-Cerebrovascular Disease (PAC-CVD) rehabilitation program, which provides an individualized 1- to 3-hour intensive physical, occupational, and speech and language therapy for post-acute stroke patients in Taoyuan Chang Gung Memorial hospital in Taiwan. Our primary endpoint measure was Barthel Index (BI), and secondary endpoint measures included other 12 functional measures. RESULTS: A total of 489 patients were included for analysis. Patients with stroke history had less BI improvement than those who suffered their first-ever stroke. In first-ever stroke patients who had completed 6 to 12 weeks of PAC-CVD program, subcortical ICH patients had greater BI, quality of life, sensation, and balance improvements, and had greater late-phase recovery than their IS counterparts. In IS patients, those with age >75 had less BI improvement; those with National Institute of Health Stroke Scale (NIHSS) score 1-5 had greater Motor Activity Log quality of use (MAL-quality) improvement than those with NIHSS score >5; those with Mini-Mental State Examination (MMSE) score ≥24 had greater BI and instrumental activities of daily living (IADL) improvement. Using the general linear model, previous stroke (ß: -6.148, p=0.01) and subcortical ICH (ß: 5.04, p=0.03) were factors associated with BI improvement. CONCLUSION: Subcortical ICH patients have greater functional improvement and greater late-phase recovery than their IS counterparts following PAC rehabilitation. More studies are needed to validate our findings and unravel the underlying mechanisms of stroke recovery to optimize the treatment strategy following a stroke.

Borneol and Luteolin from Chrysanthemum morifolium Regulate Ubiquitin Signal Degradation.

Targeting the two degradation systems, ubiquitin proteasome pathway and ubiquitin signal autophagy lysosome system, plays an important function in cancer prevention. Borneol is called an "upper guiding drug". Luteolin has demonstrated anticancer activity. The fact that borneol regulates luteolin can be sufficient to serve as an alternative strategy. Borneol activates luteolin to inhibit E1 and 20S activity (IC50 = 118.8 ± 15.7 µM) and perturb the 26S proteasome structure in vitro. Borneol regulates luteolin to inhibit 26S activity (IC50 = 157 ± 19 µM), induces apoptosis (LC50 = 134 ± 4 µM), and causes pre-G1 and G0/G1 arrest in HepG2 cells. Borneol regulates luteolin to induce ubiquitin signal autophagic degradation, resulting in induction of E1, reduction of USP47, and accumulation of p62 in HepG2 reporter cells. Interestingly, luteolin decreased Ub conjugates, while borneol increased the accumulation of Ub conjugates in HepG2 reporter cells. E1, p62, and ubiquitin levels were downregulated in borneol-treated HepG2 reporter cells at 24 h. These observations suggest a potential autophagic inhibitor of borneol that may guide luteolin in the ubiquitin proteasome pathway and the ubiquitin signal autophagic degradation.

Low-level laser irradiation stimulates tenocyte proliferation in association with increased NO synthesis and upregulation of PCNA and cyclins.

Low-level laser therapy is commonly used to treat tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. There are few evidence to elucidate that low-level laser promote tenocyte proliferation. This study was designed to determine the effect of laser on tenocyte proliferation. Furthermore, the association of this effect with secretion of nitric oxide (NO) and the expressions of proliferating cell nuclear antigen (PCNA) and cyclins D1, E, A, and B1 was investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm). Tenocyte proliferation was evaluated by MTT assay and immunocytochemistry with Ki-67 stain. NO in the conditioned medium was measured by ELISA. Western blot analysis was used to evaluate the protein expressions of PCNA and cyclins D1, E, A, and B1. The results revealed that tenocytes proliferation was enhanced dose dependently by laser. NO secretion was increased after laser treatment. PCNA and cyclins E, A, and B1 were upregulated by laser. In conclusion, low-level laser irradiation stimulates tenocyte proliferation in a process that is mediated by upregulation of NO, PCNA, and cyclins E, A, and B1.

The effect of aging on migration, proliferation, and collagen expression of tenocytes in response to ciprofloxacin.

Quinolone-induced tendinopathy or tendon rupture tends to be age-related. However, the synergistic effects of quinolone and aging on tenocytes remained to be explored. Tenocytes intrinsic to rat Achilles tendon from two age groups (young: 2 months; and near senescent (old): 24 months) were treated with ciprofloxacin. Tenocyte migration and proliferation were assessed by transwell filter migration assay and MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. Messenger RNA and protein expressions of types I and III collagen were determined by reverse transcription-polymerase chain reaction (RT/PCR) and Western blot analysis, respectively. Transwell filter migration assay revealed that ciprofloxacin inhibited tenocytes migration, which became more significant in old tenocytes (p < 0.05). The results of MTT assay revealed that tenocytes proliferation decreased after ciprofloxacin treatment (p < 0.05), which also became more significant in old tenocytes. The results of RT-PCR and Western blot analysis revealed that mRNA and protein expressions of type I collagen remained unchanged in either young or old tenocytes with ciprofloxacin treatment, whereas the expressions of type III collagen were down-regulated by ciprofloxacin, which was more significant in old tenocytes. In conclusion, aging potentiated the ciprofloxacin-mediated inhibition of migration, proliferation, and expression of type III collagen of tenocytes.

Decreased proliferation of aging tenocytes is associated with down-regulation of cellular senescence-inhibited gene and up-regulation of p27.

Symptomatic tendinopathy tends to be age-related. However, the molecular mechanisms of ageing and its effects on tenocyte proliferation and cell cycle progression are unknown. We examined tenocytes from Achilles tendons in rats from three age groups (young, 2 months; middle-aged, 12 months, and near senescence, 24 months). Tenocyte proliferation was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Senescence-associated ß-galactosidase (SA ß-gal) staining was performed in all groups of tenocytes. mRNA and protein expression of cellular senescence-inhibited gene (CSIG) and p27 was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results of MTT assay revealed that tenocyte proliferation decreased with age (p < 0.05). Cell cycle progression was arrested at G0/G1 phase in senescent tenocytes. More senescent tenocytes expressed SA ß-gal than young tenocytes did. By RT-PCR and Western blot analysis, the gene and protein expression of CSIG was found to be down-regulated, whereas that of p27 was up-regulated with age. In conclusion, the proliferation of tenocytes declines with age and is associated with the down-regulation of CSIG and up-regulation of p27.

Ciprofloxacin up-regulates tendon cells to express matrix metalloproteinase-2 with degradation of type I collagen.

Ciprofloxacin-induced tendinopathy and tendon rupture have been previously described, principally affecting the Achilles tendon. This study was designed to investigate the effect of ciprofloxacin on expressions of matrix metalloproteinases (MMP)-2 and -9, tissue inhibitors of metalloproteinase (TIMP)-1 and -2 as well as type I collagen in tendon cells. Tendon cells intrinsic to rat Achilles tendon were treated with ciprofloxacin and then underwent MTT (tetrazolium) assay. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis were used, respectively, to evaluate the gene and protein expressions of type I collagen, and MMP-2. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and -9. Reverse zymography was used to evaluate TIMP-1 and -2. Immunohistochemical staining for MMP-2 in ciprofloxacin-treated tendon explants was performed. Collagen degradation was evaluated by incubation of conditioned medium with collagen. The results revealed that ciprofloxacin up-regulated the expression of MMP-2 in tendon cells at the mRNA and protein levels. Immunohistochemistry also confirmed the increased expressions of MMP-2 in ciprofloxacin-treated tendon explants. The enzymatic activity of MMP-2 was up-regulated whereas that of MMP-9, TIMP-1 or TIMP-2 was unchanged. The amount of secreted type I collagen in the conditioned medium decreased and type I collagen was degraded after ciprofloxacin treatment. In conclusion, ciprofloxacin up-regulates the expressions of MMP-2 in tendon cells and thus degraded type I collagen. These findings suggest a possible mechanism of ciprofloxacin-associated tendinopathy.

Ibuprofen upregulates expressions of matrix metalloproteinase-1, -8, -9, and -13 without affecting expressions of types I and III collagen in tendon cells.

Nonsteroidal antiinflammatory drugs are widely used to treat sports-related tendon injuries or tendinopathy. This study was designed to investigate the effect of ibuprofen on expressions of types I and III collagen, as well as collagen-degrading enzymes including matrix metalloproteinase (MMP)-1, -2, -8, -9, and -13. Rat Achilles tendon cells were treated with ibuprofen and then underwent MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Reverse transcription-polymerase chain reaction was used to evaluate mRNA expressions of types I and III collagen, MMP-1, -2, -8, -9, and -13. Protein expressions of types I and III collagen, MMP-1, -8, and -13 were determined by Western blot analysis. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and MMP-9. The results revealed that ibuprofen upregulated expressions of MMP-1, -8, -9, and -13, both at mRNA and protein levels. There was no effect of ibuprofen on mRNA and protein expressions of types I and III collagen. Gelatin zymography revealed that the enzymatic activity of MMP-9 was upregulated after ibuprofen treatment. In conclusion, ibuprofen upregulates the expressions of collagenases including MMP-1, -8, -9, and -13 without affecting the expressions of types I and III collagen. These findings suggest a molecular mechanism potentially accounting for the inhibition of tendon healing by ibuprofen.