The role of ZEB1 in mediating the protective effects of metformin on skeletal muscle atrophy.

Metformin is an important antidiabetic drug that has the potential to reduce skeletal muscle atrophy and promote the differentiation of muscle cells. However, the exact molecular mechanism underlying these functions remains unclear. Previous studies revealed that the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), which participates in tumor progression, inhibits muscle atrophy. Therefore, we hypothesized that the protective effect of metformin might be related to ZEB1. We investigated the positive effect of metformin on IL-1ß-induced skeletal muscle atrophy by regulating ZEB1 in vitro and in vivo. Compared with the normal cell differentiation group, the metformin-treated group presented increased myotube diameters and reduced expression levels of atrophy-marker proteins. Moreover, muscle cell differentiation was hindered, when we artificially interfered with ZEB1 expression in mouse skeletal myoblast (C2C12) cells via ZEB1-specific small interfering RNA (si-ZEB1). In response to inflammatory stimulation, metformin treatment increased the expression levels of ZEB1 and three differentiation proteins, MHC, MyoD, and myogenin, whereas si-ZEB1 partially counteracted these effects. Moreover, marked atrophy was induced in a mouse model via the administration of lipopolysaccharide (LPS) to the skeletal muscles of the lower limbs. Over a 4-week period of intragastric administration, metformin treatment ameliorated muscle atrophy and increased the expression levels of ZEB1. Metformin treatment partially alleviated muscle atrophy and stimulated differentiation. Overall, our findings may provide a better understanding of the mechanism underlying the effects of metformin treatment on skeletal muscle atrophy and suggest the potential of metformin as a therapeutic drug.

Metformin Ameliorates Postoperative Cognitive Dysfunction through Regulation of the AMPK/SIRT1 Pathway.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a common postoperative complication in elderly patients. The purpose of this study was to investigate the mechanism through which metformin improves postoperative cognitive function. METHODS: In the in vivo experiment, 18-month-old Sprague-Dawley (SD) rats were randomly divided into four groups (n = 12 in each group): the control, metformin, operation, and operation plus metformin groups. The animals were pretreated with metformin by gavage once daily for two weeks. The Morris water maze (MWM) was used to measure cognitive ability. In the in vitro experiment, BV2 cells were divided into five groups: the control, metformin, lipopolysaccharide (LPS), LPS plus metformin, and LPS plus metformin plus compound C groups. We stimulated microglia with LPS (500 ng/mL). Immunofluorescence and Western blotting were used to assess ROS (reactive oxygen species) levels, autophagy-associated protein levels and adenosine monophosphate-activated protein kinase (AMPK)/regulator factor 2-related enzyme 1 (SIRT1) signaling pathway activity in the rat cortex and microglial cells. RESULTS: In the MWM test, the metformin-pretreated rats spent a higher proportion of time in the target quadrant. Immunofluorescence showed that the fluorescence intensity of LC3 in the cortex was increased in rats pretreated with metformin. Western blotting indicated that metformin upregulated the expression of autophagy-related and AMPK/SIRT1 signaling pathway-related proteins in the cortex after surgery. By activating the AMPK/SIRT1 signaling pathway in vitro, metformin reduced microglial activation and oxidative stress and promoted autophagy. CONCLUSIONS: Through the AMPK/SIRT1 pathway, metformin can boost autophagy and reduce oxidative stress in cortical microglia in older rats, in turn improving postoperative cognitive function.

Near-infrared light-triggered polypyrrole promotes C2C12 cell differentiation and inhibits TNF-α induced myotube atrophy.

Treatment of skeletal muscle atrophy and strengthening the muscles remain a challenge in modern medicine. Studies have shown that photobiomodulation can inhibit skeletal muscle atrophy and aid in functional recovery. Near-infrared radiation (NIR) therapy has emerged as a complementary therapy for the treatment of skeletal muscle atrophy, but its underlying mechanism remains unclear. Polypyrrole (PPy) is an organic polymer with strong near-infrared absorption, which can generate heat from absorbed NIR. In this study, MHC immunofluorescence staining was performed on C2C12 myoblasts to investigate the differentiation of C2C12 cells after NIR-triggered PPy exposure. As TNF-α-induced C2C12 myotubes were used as a model of muscular atrophy. Giemsa staining was used to determine the myotube diameter. Western blot analysis was performed to examine the proteins involved in the differentiation and atrophy of muscle cells, as well as in the Akt/P70S6K signaling pathway. PPy triggered by NIR promoted the differentiation of C2C12 cells, inhibited C2C12 myotube atrophy caused by TNF-α, and downregulated the expression levels of Atrogin-1 and MuRF 1 protein. In addition, we determined that Akt/P70S6K signaling pathway activity plays a crucial role in the therapeutic effect of NIR-triggered polypyrrole, which was further confirmed by the administration of the Akt inhibitor GDC0068. The optimal conditions for these effects were a PPy concentration of 0.125 mg/ml and NIR exposure for 80 s. We show that the photothermal effect of PPy triggered by near-infrared light can increase the beneficial effects of NIR, promote the differentiation of C2C12 cells, and improve C2C12 myotube atrophy, laying a foundation for its future clinical use.

Early-senescent bone marrow mesenchymal stem cells promote C2C12 cell myogenic differentiation by preventing the nuclear translocation of FOXO3.

AIMS: Mouse bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells with self-renewal and differentiation abilities. Since the effects of senescent BMSCs on C2C12 cells are not fully clear, the present study aimed to elucidate these effects. MAIN METHODS: Senescence-associated ß-galactosidase staining and western blotting were performed to confirm the senescence of BMSCs. Immunofluorescence and western blotting were used to assess myoblast differentiation in each group. The role of the AKT/P70 signaling pathway and forkhead box O3 (FOXO3) nuclear translocation was explored by western blotting. BMSC-derived exosomes were injected into the tibialis anterior of mice, and RT-qPCR was used to assess the role of exosomes in promoting muscle differentiation. KEY FINDINGS: Conditioned medium (CM) from early-senescent BMSCs promoted myogenic differentiation in vitro, which was detected as enhanced expression of myosin heavy chain (MHC), myogenin (MYOG), and myogenic differentiation 1 (MyoD). The AKT signaling pathway was found to be regulated by CM, which inhibited FOXO3 nuclear translocation. RT-qPCR analysis results showed that MHC, MyoD, and MYOG mRNA expression increased in the tibialis anterior of mice after exosome injection. SIGNIFICANCE: The present study demonstrated that early-senescent BMSCs accelerated C2C12 cell myogenic differentiation, and the transcription factor, FOXO3, was the target of senescent cells. Collectively, our results suggest that the AKT/P70 signaling pathway mediates the effect of BMSCs on neighboring cells.

MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation.

BACKGROUND: Sarcopenia is a common skeletal disease related to myogenic disorders and muscle atrophy. Current clinical management has limited effectiveness. We sought to investigate the role of miR-1290 in myoblast differentiation and muscle atrophy. METHODS: By transfecting miR-1290 into C2C12 cells, we investigated whether miR-1290 regulates myogenesis and myotube atrophy via AKT/P70 signaling pathway. MHC staining was performed to assess myoblast differentiation. Differentiation-related MHC, Myod, and Myog protein levels, and atrophy-related MuRF1 and atrogin-1 were explored by western blot. An LPS-induced muscle atrophy rat model was developed. RT-PCR was conducted to analyze miR-1290 serum levels in muscle atrophy patients and normal controls (NCs). RESULTS: The miR-1290 transfection increased MHC-positive cells and MHC, Myod, and Myog protein levels in the miR-1290 transfection group, demonstrating that miR-1290 promoted C2C12 myoblast differentiation. Myotube diameter in the miR-1290 transfection group was higher than in the TNF-α-induced model group. Western blot analysis showed decreased MuRF1 and atrogin-1 levels in the miR-1290 transfection group compared with the model group, demonstrating that miR-1290 protected against myoblast cellular atrophy. Luciferase assay and western blot analysis showed that miR-1290 regulation was likely caused by AKT/p70/FOXO3 phosphorylation activation. In the LPS-induced muscle atrophy rat model, miR-1290 mimics ameliorated gastrocnemius muscle loss and increased muscle fiber cross-sectional area. Clinically, miR-1290 serum level was significantly decreased in muscle atrophy patients. CONCLUSIONS: We found that miR-1290 enhances myoblast differentiation and inhibits myotube atrophy through Akt/p70/FoxO3 signaling in vitro and in vivo. In addition, miR-1290 may be a potential therapeutic target for sarcopenia treatment.

A Randomized Controlled Pilot Study Comparing the Efficacy of Pulsed Radiofrequency Combined With Exercise Versus Exercise Alone in Pain Relief and Functional Improvement for Chronic Knee Osteoarthritis.

OBJECTIVES: To compare the long-term efficacy between pulsed radiofrequency (PRF) combined with passive stretching (PRF-PS) exercise and PS exercise alone in reducing pain and improving quadriceps muscle strength and knee function. METHODS: Sixty-two participants were randomly assigned with a 1:1 allocation to the PRF-PS exercise group or the PS exercise group. Level of pain, muscle strength, and knee function were assessed from baseline to the first, third, and sixth months after treatment using the VAS, peak torque (PT), and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), respectively. RESULTS: There were no significant differences at baseline between the 2 groups. Compared to exercise alone, participants achieved superior efficacy with PRF-PS in pain relief, improvement of muscle strength, and knee function. Moreover, the improvement of all variables was maintained for a longer period of time in the PRF-PS group. The reduction in participants' VAS pain intensity scores was superior for PRF-PS vs. PS with overall estimation (adjusted mean difference: -1.85 cm; 95% confidence interval [CI] -2.25, -1.45 cm; P = 0.000). The increase in participants' PT scores was superior for PRF-PS vs. PS with overall estimation (adjusted mean difference: 15.53 N. m; 95% CI 7.07, 23.98 N. m; P = 0.000; and 12.62 N. m; 95% CI 0.96, 24.28 N. m; P = 0.000 for PT 60 degrees/s and PT 180 degrees/s, respectively). The reduction in participants' WOMAC scores was superior for PRF-PS vs. PS with overall estimation (adjusted mean difference: -16.43; 95% CI -22.22, -10.64; P = 0.000). DISCUSSION: The improvement in pain relief and knee function might be associated with restoration of muscle strength after PRF-PS exercise by overcoming muscle inhibition.

Effects of Trigger Point Dry Needling on Neuromuscular Performance and Pain of Individuals Affected by Patellofemoral Pain: A Randomized Controlled Trial.

PURPOSE: To investigate the effects of trigger point dry needling (TrP-DN) on exercise-induced patellofemoral pain syndrome (PFPS). PATIENTS AND METHODS: In this randomized, single-blind, parallel-group trial, 50 patients with PFPS were randomly allocated to the following two groups: the TrP-DN group (n = 25) and the Sham needling group (n = 25). Patients in both groups were asked to perform a stretching exercise of the quadriceps daily after needling. The needling group received a single session of TrP-DN to trigger points (TrPs) in the vastus medialis oblique (VMO), vastus lateralis (VL), and rectus femoris muscles (once a week for 6 weeks), and the Sham group received placebo needling. Visual analogue scale (VAS) for pain intensity and Kujala questionnaire for the functional status were assessed before treatment, 3 and 6 weeks after treatment, and at the 3-month follow-up. The ratio of the myoelectric amplitude of the vastus medialis oblique and vastus lateralis muscles (VMO/VL) was assessed before treatment and 6 weeks after treatment. RESULTS: There was no significant difference in the general data between the two groups. The VAS scores and Kujala scores in the TrP-DN group were significantly improved and increased at the 3-week treatment visit, 6-week treatment visit, and 3-month follow-up compared to the scores before treatment; and the scores in the Sham group were only significantly improved at the 3-week treatment visit, and 6-week treatment visit. VAS scores in the TrP-DN group were significantly lower and Kujala scores were significantly higher at the 6-week treatment visit and the 3-month follow-up compared to those in the Sham group. The VMO/VL ratio in the TrP-DN group was significantly increased at the 6-week treatment visit compared to that before treatment. CONCLUSION: TrP-DN at the quadriceps combined with stretch can reduce the pain, and improves the clinical symptoms and function, the VMO/VL ratio, and the coordination of VMO and VL in patients with PFPS.

Preoperative Serum Metabolites Are Associated With Postoperative Delirium in Elderly Hip-Fracture Patients.

BACKGROUND: Hypotheses on the development of postoperative delirium (PD) include "neuroinflammatory," "neuronal aging," "oxidative stress," "neurotransmitter deficiency," and "neuroendocrine." Here, we employed metabolomics to determine the serum metabolites in the baseline associated with an increased risk of PD. METHODS: Two hundred and nine elderly hip-fracture patients who had undergone hemiarthroplasty and had completed our assessments were selected. Fasting venous blood was collected at 7:00 on the morning of surgery and a serum sample bank was created for analysis. On the first 3 postoperative days, the patients were assessed twice daily using the Confusion Assessment Method - Chinese Revision. Ultimately, 43 patients were diagnosed with PD, who comprised the PD group. Meanwhile, 43 matched non-PD patients were selected based on age, sex, and body mass index. Serum samples from the two groups were analyzed by gas chromatography-time-of-flight mass spectrometry and Acquity ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. RESULTS: The demographic characteristics of the groups were matched. Four metabolites associated with an increased risk of PD were identified, including S-methylcysteine, linolenic acid, eicosapentaenoic acid, and linoleic acid. CONCLUSIONS: Multiple metabolic pathways in the PD group altered before surgery, including deficiency of ω3 and ω6 fatty acids, energy metabolism and oxidative stress with interactions between hypoxia and mitochondrial dysfunction, in addition to glutamate-glutamine cycle dysfunction. These metabolic abnormalities could possibly increase the fragility of the brain and then contribute to PD.

Production of inflammatory cytokines, cortisol, and Aß1-40 in elderly oral cancer patients with postoperative delirium.

AIM: Pathophysiological disorders after surgery might be related to postoperative delirium (POD). This study was designed to elucidate the pathogenesis of POD in elderly oral cancer patients by determining the perioperative kinetics of inflammatory cytokines, cortisol, and amyloid ß1-40 (Aß1-40). METHODS: A total of 257 elderly oral cancer patients who underwent tumor resection surgery were selected. Venous blood was collected prior to surgery (T0), at the end of surgery (T1), and at 12 hours after surgery (T2). During the first three postoperative days, patients were examined using the confusion assessment method twice a day (8 am and 8 pm). Mini-Mental State Examination scores were recorded at T0 and on postoperative days 1, 3, and 7. Ultimately, 56 patients suffering from POD made up the POD group, and 56 patients randomly selected from a cohort of patients without POD were allocated to the no POD (NPOD) group. Subsequently, interleukin-6, C-reactive protein, procalcitonin, cortisol, and Aß1-40 in plasma from the two groups were measured. RESULTS: The two groups displayed comparable basic characteristics. There were no differences in all tested biomarkers between the two groups at T0. However, after surgery, the biomarker levels displayed distinct patterns between the two groups. The peak levels of all biomarkers were higher in the POD group than in the NPOD group. Conversely, the Mini-Mental State Examination scores after surgery were lower in the POD group than in the no POD group. CONCLUSION: The boost of inflammatory cytokines, cortisol, and Aß1-40 after surgery might be involved in POD onset among elderly oral cancer patients. POD was accompanied by progressive cognitive deficiency.

Prevalence and risk factors of postoperative delirium in elderly hip fracture patients.

OBJECTIVE: To explore the risk factors associated with postoperative delirium (PD) in elderly patients following total hip arthroplasty (THA) for hip fracture. METHODS: This prospective study enrolled elderly patients (≥ 65 years) with hip fractures who underwent THA under general anaesthesia, and who had a complete set of postoperative observations. Detailed medical history and perioperative characteristics were recorded. During the postoperative period, patients were assessed twice daily for PD using the Confusion Assessment Method. RESULTS: A total of 572 patients were eligible for inclusion in the study. Of these, 120 patients (21.0%) were diagnosed with PD and 452 patients (79.0%) did not experience PD. Multivariate stepwise logistic regression analyses showed that older age, a history of stroke, lower albumin, higher blood glucose, higher total bilirubin, higher C-reactive protein, longer surgery duration and higher volume of red blood cell transfusions were independent risk factors for PD. CONCLUSIONS: Correcting the modifiable risk factors might help prevent PD. Strategies might include nutritional support, tight blood glucose control, improvement of liver function, preoperative infection control and minimizing surgical injury or blood loss.

Impact of multicomponent, nonpharmacologic interventions on perioperative cortisol and melatonin levels and postoperative delirium in elderly oral cancer patients.

OBJECTIVE: To investigate the impact of multicomponent, nonpharmacologic interventions (MNI) on perioperative cortisol and melatonin levels, as well as postoperative delirium (PD), in elderly oral cancer patients. METHODS: A total of 160 elderly oral cancer patients who underwent tumor resection surgery and completed our investigation were included in this study. The cancer patients were randomly divided into 2 groups: Group U or Group I. During the perioperative period, Group U received usual care, while Group I received MNI, which is based on usual care and aims to decrease the risk of PD. MNI focused on general geriatric approaches and supportive nursing care. On the day before surgery and the first three postoperative days, nocturnal (20:00-8:00) urine samples were collected. The melatonin sulfate and cortisol levels in the urine samples were determined. Moreover, the RASS (Richmond Agitation Sedation Scale), CAM-ICU (Confusion Assessment Method for the Intensive Care Unit) and QoR40 (40-item quality of recovery score) scores were dynamically monitored. RESULTS: There were no significant differences in the general characteristics between the 2 groups. After surgery, the melatonin sulfate levels in the nocturnal urine of Group I were higher than those in Group U. The cortisol concentrations were lower in Group I compared to those in Group U. Group I achieved better postoperative RASS and QoR40 scores than Group U. Compared to Group U, Group I also experienced less PD (incidence and duration). CONCLUSIONS: MNI ameliorated some postoperative disturbances regarding sleep and stress, decreased the incidence of PD and improved recovery quality.