Metformin Protects Rat Skeletal Muscle from Physical Exercise-Induced Injury.

Metformin (Met) is a drug commonly prescribed in type 2 diabetes mellitus. Its efficacy is due to the suppression of hepatic gluconeogenesis, enhancement of peripheral glucose uptake and lower glucose absorption by the intestine. Recent studies have reported Met efficacy in other clinical applications, such as age-related diseases. Despite the wide clinical use of Met, its mechanism of action on muscle and its effect on muscle performance are unclear. We investigated the effects of Met combined with training on physical performance (PP) in healthy rats receiving Met for 8 weeks while undergoing daily moderate exercise. We evaluated the following: PP through graded endurance exercise test performed before the beginning of the training protocol and 48 h before the end of the training period; blood ALT, AST, LDH and CK-MB levels in order to address muscle damage; and several blood and muscle myokines and the expression of factors believed to be involved in muscle adaptation to exercise. Our data demonstrate that Met does not improve the positive effects of exercise on performance, although it protects myocytes from exercise-induced damage. Moreover, given that Met positively affects exercise-induced muscle adaptation, our data support the idea of the therapeutic application of Met when muscle function and structure are compromised.

Metformin regulates myoblast differentiation through an AMPK-dependent mechanism.

This study aims to investigate how metformin (Met) affects muscle tissue by evaluating the drug effects on proliferating, differentiating, and differentiated C2C12 cells. Moreover, we also investigated the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) in the mechanism of action of Met. C2C12 myoblasts were cultured in growth medium with or without Met (250µM, 1mM and 10mM) for different times. Cell proliferation was evaluated by MTT assay, while cell toxicity was assessed by Trypan Blue exclusion test and Lactate Dehydrogenase release. Fluorescence Activated Cell Sorting analysis was performed to study cell cycle. Differentiating myoblasts were incubated in differentiation medium (DM) with or without 10mM Met. For experiments on myotubes, C2C12 were induced to differentiate in DM, and then treated with Met at scalar concentrations and for different times. Western blotting was performed to evaluate the expression of proteins involved in myoblast differentiation, muscle function and metabolism. In differentiating C2C12, Met inhibited cell differentiation, arrested cell cycle progression in G2/M phase and reduced the expression of cyclin-dependent kinase inhibitor 1. These effects were accompanied by activation of AMPK and modulation of the myogenic regulatory factors. Comparable results were obtained in myotubes. The use of Compound C, a specific inhibitor of AMPK, counteracted the above-mentioned Met effects. We reported that Met inhibits C2C12 differentiation probably by blocking cell-cycle progression and preventing cells permanent exit from cell-cycle. Moreover, our study provides solid evidence that most of the effects of Met on myoblasts and myotubes are mediated by AMPK.

Serum NT-pro-BNP Levels Predict Cardiovascular Events in Acromegaly Patients.

BACKGROUND: Acromegaly is associated with an increased risk of fatal and non-fatal cardiovascular (CV) events. Controlling acromegaly decreases, but does not normalize this risk. Brain natriuretic peptide (BNP) assessment is used in the general population for the diagnosis of heart failure and to predict ischemic recurrences and mortality. This is a retrospective, longitudinal, monocenter study that evaluates the role of serum N-terminal fragment of BNP (NT-pro-BNP) for predicting CV events in acromegaly patients. METHODS: Serum NT-pro-BNP levels were measured in 76 patients with acromegaly (23 males, 57.7±1.5 years), and compared with other predictors of CV events. NT-pro-BNP cut-off value discriminating the occurrence of CV events was determined by ROC analysis. CV events were recorded during a follow-up of 78.6±6.4 months. RESULTS: CV events occurred in 9.2% of patients. Mean log(NT-pro-BNP) concentration was higher in patients who experienced CV events than in those who did not (p<0.01) and in patients who died due to CV events than in those who died due to other causes (p<0.01). Based on the ROC curve, a cut-off value of 91.55 pg/mL could predict CV events (OR 19.06). Log(NT-pro-BNP) was lower in surgically treated patients by surgery (p<0.05), and in those cured by neurosurgery (p<0.02). CONCLUSIONS: High NT-pro-BNP value is an independent middle-term predictor of fatal or non-fatal CV events in patients with acromegaly. According to this parameter, surgically treated patients show lower CV risk than those managed with medical therapy, especially if the disease is cured.

Management of pernio-like cutaneous manifestations in children during the outbreak of COVID-19.

During the outbreak of COVID-19 many pernio-like lesions have been increasingly reported. The aim of the study is to describe our management of these skin manifestations and to evaluate a possible correlation to SARS-CoV-2 infection. All patients underwent clinical and laboratory tests to detect a possible underlying connective disease and also to specific SARS-CoV-2 investigations such as oropharyngeal swab and IgG-IgM serology. Nine patients aged between 5 and 15 years old were evaluated. Skin lesions observed were purplish, erythematous and oedematous, in some cases painful and itchy. Six out of nine had respiratory and systemic symptoms (cough, nasal congestion, chills, fever, and asthenia) that preceded cutaneous findings of approximately 2 weeks. Concerning blood exams, three out of nine had D-dimer weakly increased, four had ANA positivity: two with a title 1:160, one with 1:320, and one with 1:5120 and a speckled pattern. The latter patient had also ENA SS-A positive and RF positivity, confirmed at a second check, so as to allow us to make a diagnosis of connective tissue disease. Four out of nine had aPL positivity (IgM). Reactants acute phase were all negative. Oropharyngeal swabs and serology tests for SARS-CoV-2 was negative (borderline in one patient for IgM). No treatment was needed. Even if we do not have enough data to prove it, we hypothesize a correlation between pernio-like lesions and SARS-CoV-2 infection for an increased number of these lesions described during the pandemic and also because such manifestations appeared when temperatures were mild and patients were at home in isolation for the lockdown. Many questions remain open about interaction host-virus.

Correct management and low rate of contagiousness of healthcare workers in a University Hospital in Southern Italy: from contact tracing to serological investigation.

The COVID-19 epidemic, which began in Wuhan in December 2019, quickly spread all over the world, leading in a few months to a high number of deaths also in healthcare workers. The purpose of the study is to a) describe the importance of a correct management of SARS-CoV-2 infections; b) report the number of positive healthcare workers after the epidemic phase and to describe their socio-characteristics data, the main methods of transmission and the symptoms; c) to report the seroconversion rate of healthcare workers  (HCWs). The study was conducted from March 9, 2020 to June 19, 2020 in three phases:1) in a first phase, we implemented the guidelines to be followed for patient care in our hospital; 2) in a second phase, we provided the epidemiological investigation/contact tracing of HCWs; 3)  we collected swabs on all healthcare workers and we also performed serological investigation. The number of healthcare workers under surveillance is of 2611 subjects and, of these, only 0.65% contracted COVID-19. In particular, 70.6% of these have been infected in the healthcare setting, 11, 8% in the family and 17.6% returning from high risk areas. Ultimately, only 0.1% of HCWs dedicated to the treatment of COVID-19 patients contracted the infection (one was asymptomatic). Only 2% of HCWS were positive for serological investigation.

Assessment of serum thyroid hormone autoantibodies in the first trimester of gestation as predictors of postpartum thyroiditis.

BACKGROUND: Measurement of serum thyroperoxidase autoantibodies (TPOAb) during gestation as a classical marker for the risk of postpartum thyroiditis (PPT) predicts PPT in 1/3 to 1/2 of women. Very few studies have measured serum thyroid hormone Ab (THAb) during gestation, and none as a possible marker for PPT. METHODS: In 412 women who were followed up from 7 to 11 weeks of gestation through 12 months after delivery, we measured THAb (T3.IgM, T3.IgG, T4.IgM, T4.IgG), thyroglobulin autoantibodies (TgAb) and TPOAb at study entry (7-11 week of gestation). RESULTS: Sixty-three women (15.3%) developed PPT, which progressed to permanent hypothyroidism (PH) in 34/63 (54%). THAb+ve were 21/412 women (5.1%), the frequency being greater in those who then developed PPT (12/63 [19.0%] vs. 9/349 [2.6%], P = 4.6 × 10-8), and in the PH subgroup (26.5% [9/34] vs. 10.3% [10/29], P = 0.12). THAb positivity occurred in 9/76 women (11.8%) who were TgAb and/or TPOAb+ve compared to 12/336 women who were TgAb and TPOAb negative (3.6%, P = 0.0031). Of these 9 THAb+ve, TgAb and/or TPOAb+ve women, all (100%) developed PPT compared to 3/11 (27.3%, P = 0.0011) THAb+ve, TgAb and/or TPOAb negative women. Of these 9 and 3 PPT women, 8 and 1 progressed to PH (88.9% and 33.3%, respectively, P = 0.12). CONCLUSIONS: Gestational positivity of THAb enhance enormously the predictivity for PPT of gestational positivity of TPOAb/TgAb. However, their low frequency (5.1%) and their sensitivity (17.5% [21/63]) go against their application in lieu of TPOAb/TgAb.

High frequency of empty sella, with gender differences, in the early neuroradiology evaluation of patients with traumatic brain injury. A prospective study.

One-hundred four persons aged ≥ 18 years (62 males and 42 females) who were admitted for traumatic brain injury (TBI) underwent brain computed tomography (CT) scan and assay of serum cortisol, insulin-like growth factor 1 (IGF-1), thyrotropin (TSH) and free thyroxine (FT4). The main purpose was to assess any gender difference and the rate of empty sella (ES). Women were more likely to have empty sella (19/42 [45.2%] vs 19/62 [30.6%], P = 0.15, OR = 1.9), which was more frequently total ES or TES (16/19 [84.2%] vs 3/19 [15.8%], P = 0.0025, OR = 11.6). Neuroradiology was normal in the remaining 65 patients. Patients with TES were approximately 20-30 years older than both patients with partial ES (PES) and normal sella, but only the comparison with normal sella was significant (P = 0.001 all patients, P = 0.005 males). Presumed deficiency of IGF-1, cortisol or TSH occurred in 33 persons (31.7%; 20 Males [32.2%], 13 Females [30.9%]), 14 (13.5%; 10 M [16.2%], 4F [9.5%]) or 8 (7.7%; 1 M [1.7%], 7F [16.7%]), with only TSH deficiency having significant intergender difference (P = 0.007). The highest or lowest rates of IGF-1 deficiency occurred in men with PES (41.7%) or men with TES (14.3%), of cortisol deficiency in men with PES (33.3%) or women with PES (zero), and TSH deficiency in women with TES (18.7%) or both men and women with PES (zero) and men with normal sella (zero). Within ES, males with no deficiency were older compared to males with at least one hormone deficiency (75.7 ±â€¯17.4 vs 55.6 ±â€¯18.9, P = 0.022); in turn, the former males were also older compared with normal sella males having no hormone deficiency (54.1 ±â€¯25.2, P = 0.023). In conclusion, ES is detectable in almost 40% of persons who undergo CT within 24 h from TBI. A number of intergender differences concerning ES and the hormones evaluated are apparent.

Circulating thyrotropin is upregulated by estradiol.

After encountering two women with serum thyrotropin (TSH) levels greater in periovulatory phase than in other days of the menstrual cycle, we hypothesized that TSH levels could be sensitive to changes in circulating estrogens in women. The objective of this study was to evaluate whether serum TSH increases after an induced acute increase of serum estradiol, and compare serum TSH increase with that of prolactin (PRL) which is a classic estradiol-upregulated pituitary hormone. In this retrospective study, we resorted to stored frozen sera from 55 women who had undergone the GnRH agonist (buserelin)-acute stimulation test of ovarian steroidogenesis. This test, that is preceded by dexamethasone administration to suppress adrenal steroidogenesis, had been performed to show an increased buserelin-stimulated response of 17-hydroxyprogesterone, a response that is frequent in polycystic ovary syndrome. Fifty-five women had enough serum volume at pertinent times (first observation early in the follicular phase and all times of the test) to permit assay of serum estradiol, TSH and PRL. Before dexamethasone administration, estradiol averaged 26.4 ±â€¯15.5 pg/ml (reference range 23-139, follicular phase), TSH 1.78 ±â€¯0.86 mU/L (reference range 0.3-4.2) and PRL 409.4 ±â€¯356 mU/L (reference range 70.8-556) (mean ±â€¯SD). Serum estradiol, TSH and PRL averaged 47.2 ±â€¯27 pg/ml, 0.77 ±â€¯0.48 mU/L and 246.4 ±â€¯206.8 mU/L just prior to the buserelin injection, but they peaked at 253.4 ±â€¯113.5 pg/ml (nv 83-495, midcycle), 3.30 ±â€¯1.65 mU/L and 540.3 ±â€¯695.2 mU/L after injection. The responses to buserelin of estradiol, TSH and PRL were of wide magnitude. There was a significant correlation between TSH peak and serum estradiol peak, betweeen AUC0-24 h-TSH and AUC0-24 h-estradiol, or between PRL peak and estradiol peak and AUC0-24 h -PRL and AUC0-24 h-estradiol in only a subgroup of women. Therefore, women with estradiol-dependent increase in serum TSH do exist. Reference bands of serum TSH dependent on the phases of the menstrual cycle should be available.

Platelet-rich plasma and skeletal muscle healing: a molecular analysis of the early phases of the regeneration process in an experimental animal model.

Platelet-rich plasma (PRP) has received increasing interest in applied medicine, being widely used in clinical practice with the aim of stimulating tissue healing. Despite the reported clinical success, there is still a lack of knowledge when considering the biological mechanisms at the base of the activity of PRP during the process of muscle healing. The aim of the present study was to verify whether the local delivery of PRP modulates specific molecular events involved in the early stages of the muscle regeneration process. The right flexor sublimis muscle of anesthetized Wistar rats was mechanically injured and either treated with PRP or received no treatment. At day 2 and 5 after surgery, the animals were sacrificed and the muscle samples evaluated at molecular levels. PRP treatment increased significantly the mRNA level of the pro-inflammatory cytokines IL-1ß, and TGF-ß1. This phenomenon induced an increased expression at mRNA and/or protein levels of several myogenic regulatory factors such as MyoD1, Myf5 and Pax7, as well as the muscular isoform of insulin-like growth factor1 (IGF-1Eb). No effect was detected with respect to VEGF-A expression. In addition, PRP application modulated the expression of miR-133a together with its known target serum response factor (SRF); increased the phosphorylation of αB-cristallin, with a significant improvement in several apoptotic parameters (NF-κB-p65 and caspase 3), indexes of augmented cell survival. The results of the present study indicates that the effect of PRP in skeletal muscle injury repair is due both to the modulation of the molecular mediators of the inflammatory and myogenic pathways, and to the control of secondary pathways such as those regulated by myomiRNAs and heat shock proteins, which contribute to proper and effective tissue regeneration.

Experimental model for the study of the effects of platelet-rich plasma on the early phases of muscle healing.

BACKGROUND: There is abundant evidence suggesting that growth factors may play a key role in the healing process, especially in the early stages of inflammation. Despite the reported clinical successes with the use of growth factors there is still a lack of knowledge on the biological mechanism underlying the activity of platelet-rich plasma during the process of muscle healing. The aim of this study was to analyse the early effects of platelet- rich plasma in an easily reproducible animal model. MATERIALS AND METHODS: Wistar male adult rats (n=102) were used in this study. The muscle lesion was created with a scalpel in the flexor sublimis muscles. Platelet-rich plasma was applied immediately after surgery. Treated, untreated and contralateral muscles were analysed by morphological evaluation and western blot assay. RESULTS: Leucocyte infiltration was significantly greater in muscles treated with platelet-rich plasma than in both untreated and contralateral muscles. The latter showed greater leucocyte infiltration when compared to the untreated muscles. Platelet-rich plasma treatment also modified the cellular composition of the leucocyte infiltration leading to increased expression of CD3, CD8, CD19 and CD68 and to decreased CD4 antigen expression in both platelet-rich plasma treated and contralateral muscles. Blood vessel density and blood vessel diameters were not statistically significantly different between the three groups analysed. DISCUSSION: The results of this study showed that treatment with platelet-rich plasma magnified the physiological early inflammatory response following a muscle injury, modifying the pattern of cellular recruitment. Local platelet-rich plasma treatment may exert a direct or, more plausibly, indirect systemic effect on healing processes, at least in the earliest inflammatory phase.

Suplementación con hierro en la actividad física / Iron supplementation in sport activity

Mientras que la recomendación de prestar una especial atención para mantener un consumo adecuado de hierro siempre es una estrategia dietética aconsejable, el empleo de suplementos de hierro debería ser una opción cautelosa basada en una cuidadosa evaluación hematológica, principalmente por los posibles peligros para la salud que provienen de un tratamiento injustificado. Respecto a la suplementación en el deporte, actualmente no existe ninguna directriz estandarizada para la administración de hierro y la decisión sobre administrar o no hierro oral en deportistas se basa principalmente en recomendaciones empíricas o en la esperanza de mejorar el rendimiento. Estudios recientes han permitido definir un nivel de ferritina, independiente del sexo, en el que una suplementación oral de hierro es útil y ventajosa y por encima de la cual esta práctica no sería aconsejable por no ser útil y ser potencialmente peligrosa para la salud del deportista. De hecho, se ha clarificado científicamente que algunas situaciones que se caracterizan por una transferencia reducida de hierro de los enterocitos duodenales al plasma aumenta la concentración intracelular de hierro. En estas condiciones, la suplementación oral de hierro no sólo sería innecesaria, sino también potencialmente contraproducente debido a los posibles efectos secundarios en el tracto gastro-intestinal derivados del aumento de la concentración intracelular de hierro como malestar, náuseas, vómitos, diarrea, o estreñimiento. Tales efectos secundarios adquieren aún más importancia en los deportistas debido a las consecuencias negativas sobre la capacidad de entrenamiento y de rendimiento que pueden producir. Un abordaje científico y un posible estudio de nuevos parámetros podrían guiar a los clínicos hacia la definición de la verdadera necesidad de suplementación con hierro. Este abordaje reduciría las posibles contraindicaciones y efectos secundarios relacionados con una inapropiada suplementación de hierro. De hecho, una suplementación incorrecta podría afectar negativamente el rendimiento, antes que mejorarlo (AU)

While the recommendation to pay particular attention in maintaining an adequate consumption iron would be always advisable in a dietary strategy, the use of iron supplements should be a cautious choice based on a careful hematological evaluation, mainly because of the possible health risks deriving from an unjustified treatment. With particular regard to the sport related supplementation, currently no standardized guidelines for iron administration are available and the decision if administrating or not oral iron in athletes is mainly based on empiric recommendations or on performance enhancing hopes. Recent studies have permitted to define a specific and sex-independent ferritin cut-off below which an oral iron supplementation is useful and advantageous and above which this practice would not be advisable because useless and potentially hazardous for the athlete’s health. In fact, it is becoming scientifically clear that conditions characterized by a reduced transfer of dietary iron from duodenal enterocytes to plasma rises intracellular iron concentration. An oral iron supplementation in these conditions would be not only unnecessary but also potentially disadvantageous because of the likely side effects deriving from the increased intracellular iron concentration in the gastro-intestinal tract including discomfort, nausea, vomiting, diarrhea, or constipation. Such side effects gain even more importance in athletes because of the negative consequences on training capacity and performances that they imply. A scientifically based approach and a possible study of new parameters should always guide clinicians to define the real necessity of iron supplementation. This approach may reduce possible contraindications and side effects linked to improper iron supplementation. In fact, an incorrect supplementation may negatively affect the performance, rather than improve it (AU)

Circulating beta-endorphin, adrenocorticotrophic hormone and cortisol levels of stallions before and after short road transport: stress effect of different distances.

BACKGROUND: Since transport evokes physiological adjustments that include endocrine responses, the objective of this study was to examine the responses of circulating beta-endorphin, adrenocorticotrophic hormone (ACTH) and cortisol levels to transport stress in stallions. METHODS: Forty-two healthy Thoroughbred and crossbred stallions were studied before and after road transport over distances of 100, 200 and 300 km. Blood samples were collected from the jugular vein: first in a single box immediately before loading (pre-samples), then immediately after transport and unloading on arrival at the breeding stations (post-samples). RESULTS: An increase in circulating beta-endorphin levels after transport of 100 km (P < 0.01), compared to basal values was observed. Circulating ACTH levels showed significant increases after transport of 100 km (P < 0.001) and 200 km (P < 0.001). Circulating cortisol levels showed significant increases after road transport over distances of 100, 200 and 300 km (P < 0.001). An effect of transport on beta-endorphin, ACTH and cortisol variations was therefore evident for the different distances studied. No significant differences (P > 0.05) between horses of different ages and different breeds were observed for beta-endorphin, ACTH and cortisol levels. CONCLUSION: The results obtained for short term transportation of stallions showed a very strong reaction of the adrenocortical system. The lack of response of beta-endorphin after transport of 200-300 km and of ACTH after transport of 300 km seems to suggest a soothing effect of negative feedback of ACTH and cortisol levels.