In vitro culture of muscle cells derived from myofascial trigger points.

OBJECTIVE: To examine for the in vitro existence of contractile nodules on the taut band of muscle fibers where myofascial trigger points (MTrPs) are located (using cell culture). METHODS: Sixteen male Sprague-Dawley rats (7 weeks old) were randomly divided into experimental and control groups. A blunt striking injury and eccentric exercise were applied to the gastrocnemius muscle of rats in the experimental group once a week for 8 weeks to establish an MTrP model. Subsequently, the rats were reared normally and rested for 4 weeks. After modeling, the skeletal muscles at the MTrPs (and non-MTrPs at the same anatomical position) were extracted from the two groups of rats for in vitro cell culture experiments of single muscle fibers. Potential contractile nodules in the MTrP group were exposed to different concentrations of acetylcholinesterase, whereas non-MTrP cells were exposed to acetylcholine. The morphological changes of muscle cells in each group were observed. RESULTS: By culturing MTrP cells in vitro, large contractile nodules remained in single MTrP muscle fibers, whereas some contractile nodules were twisted and deformed. After the addition of different acetylcholinesterase concentrations, no obvious morphological changes were observed in the contractile nodules in the MTrP group. After the non-MTrP cells were exposed to different acetylcholine concentrations, no significant morphological changes were observed in the single muscle fibers. CONCLUSION: MTrP cells can continue to maintain contractile morphology in vitro, but whether the recovery of such contractile nodules is related to acetylcholine remains uncertain.

A simple and robust serum-free media for the proliferation of muscle cells.

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat.

Oral administration of wild plant-derived minerals and red ginseng ameliorates insulin resistance in fish through different pathways.

Many kinds of fish are characterized by a limited efficiency to use carbohydrates. For this reason, raw fish and mixed feed containing a lot of fish meal have been used as feed for fish farming. However, continuing to use high-protein diets not only increases the cost of fish farming, but may also fuel animal protein shortages. Furthermore, carbohydrates are added to improve the texture of the feed and act as a binding agent and are usually contained at 20% in the feed. It makes sense, therefore, to find ways to make good use of carbohydrates rather than wasting them. The physiological mechanisms of glucose intolerance in fish are not yet well understood. Therefore, we investigated the glucose utilization of fish, omnivorous goldfish Carassius auratus and carnivorous rainbow trout Oncorhynchus mykiss. Furthermore, the effects of oral administration of wild plant-derived minerals and red ginseng on the glucose utilization in these fish muscle cells were investigated. As a result, we found the following. (1) An extremely high insulin resistance in fish muscle and the symptom was more pronounced in carnivorous rainbow trout. (2) Administration of wild plant-derived minerals promotes the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface of white muscle via activation of the PI3 kinase axis, whereas administration of red ginseng not only promotes GLUT4 transfer and translocation to the cell surface of white muscle via AMPK activation as well as promoting glucose uptake into muscle cells via a pathway separate from the insulin signaling system. (3) In fish, at least goldfish and rainbow trout, both PI3K/Akt and AMPK signaling cascades exist to promote glucose uptake into muscle cells, as in mammals.

[Protective effect of anthocyanins on apoptosis of gastrocnemius muscle myocytes of rats after intense exercise].

Currently, in sports medicine, much attention is paid to the prevention and treatment of delayed muscle soreness syndrome (DOMS), which occurs several hours or days after unusual or intense physical activity, as well as the state of athlete overtraining. One of the main pathogenetic factors in the development of this syndrome is myocyte ultrastructural damage with apoptosis activation. Therefore, using natural antioxidants in sports nutrition for the relief of this pathology is of particular relevance. The aim of the study was to study the effect of an anthocyanin-enriched diet on apoptosis of gastrocnemius muscle myocytes of rats after intense exercise. Material and methods. The experiment was carried out for 4 weeks on 4 groups of male Wistar rats (12 animals in each, initial body weight ~300 g). Animals were divided into groups of rats (groups 1 and 2), whose motor activity was limited by standard conditions for keeping animals in vivarium, and groups of physically active rats (groups 3 and 4), which received additional physical activity - treadmill training. Before the end of the experiment, the animals of groups 3 and 4 were given debilitating (until the rats refused to continue the exercise) physical activity on a treadmill. Rats of all four groups received a standard semi-synthetic diet, water ad libitum. Animals in groups 2 and 4 were additionally given blueberry and blackcurrant extract (30% anthocyanins) as part of the diet at a daily dose of 15 mg anthocyanins/kg body weight. The intensity of apoptosis of gastrocnemius muscle myocytes was studied by flow cytometry. Cells were stained with fluorochrome-conjugated annexin V and vital dye 7-aminoactinomycin. The results are presented as the percentage of intact cells and cells at different stages of apoptosis per 100 000 counted objects in each sample. Results. The enrichment of the diet of control group rats with blueberry and black currant extract did not have a significant effect on the relative content of intact cells and the studied parameters of apoptosis of gastrocnemius muscle myocytes of rats of the 2nd group. Intense physical activity in rats of the 3rd group led to a statistically significant (p<0.05) decrease in the relative content of intact (live) cells compared with this indicator in rats of other groups (85.32±1.44 vs 90.87±0.66% - in the 1st group; 90.16±0.79% - in the 2nd group; 89.01±0.81% - in the 4th group, р<0.05). After intense physical activity in rats of the 3rd group, activation of apoptosis of gastrocnemius muscle myocytes was found, as evidenced by an increase in the relative content of objects in apoptosis compared with other groups (11.61±1.45 vs 7.88±0.60% - in the 1st group, р<0.05; 8.01±0.70% - in the 2nd group, p<0.10; 7.93±0.59% - in the 4th group р<0.05). Enrichment of the diet of exercise rats with blueberry and blackcurrant extract (4th group) had a protective effect on the intensity of the apoptosis process, the studied parameters did not differ significantly from those in rats of the control and the 2nd groups. Conclusion. The results of the study indicate the activation of the process of apoptosis of gastrocnemius muscle myocytes of rats after intense physical activity. Enrichment of rats' diet with anthocyanins from blueberry and black currant extracts ensures the restoration of the studied apoptosis parameters to the level of control group rats. In the control group of rats with normal physical activity, the addition of anthocyanins to the diet does not have a significant effect on the physiological process of apoptosis of gastrocnemius muscle myocytes. In this way, an evidence base for the effectiveness of the use of biologically active substances - anthocyanins - in sports nutrition for the restoration of skeletal muscles has been obtained.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy.

While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

Dendropanax trifidus Sap-Mediated Suppression of Obese Mouse Body Weight and the Metabolic Changes Related with Estrogen Receptor Alpha and AMPK-ACC Pathways in Muscle Cells.

Dendropanax trifidus (DT) is a medicinal herb native to East Asia, which has been used extensively for its therapeutic properties in traditional medicine. In this study, we examined the effects of DT sap on the regulation of body weight and muscle metabolism in mice. Obese model db/db mice were administered daily with DT sap or vehicle control over a 6-week period. The effects of DT sap on muscle metabolism were studied in C2C12 muscle cells, where glycolytic and mitochondrial respiration rates were monitored. As AMP-activated protein kinase (AMPK) is a master regulator of metabolism and plays an important function as an energy sensor in muscle tissue, signaling pathways related with AMPK were also examined. We found that DT sap inhibited body weight increase in db/db, db/+, and +/+ mice over a 6-week period, while DT sap-treated muscle cells showed increased muscle metabolism and also increased phosphorylation of AMPK and Acetyl-CoA Carboxylase (ACC). Finally, we found that DT sap, which is enriched in estrogen in our previous study, significantly activates estrogen alpha receptor in a concentration-dependent manner, which can drive the activation of AMPK signaling and may be related to the muscle metabolism and weight changes observed here.

Response to Electrostimulation Is Impaired in Muscle Cells from Patients with Chronic Obstructive Pulmonary Disease.

Among the comorbidities associated with chronic obstructive pulmonary disease (COPD), skeletal muscle weakness and atrophy are known to affect patient survival rate. In addition to muscle deconditioning, various systemic and intrinsic factors have been implicated in COPD muscle dysfunction but an impaired COPD muscle adaptation to contraction has never been extensively studied. We submitted cultured myotubes from nine healthy subjects and nine patients with COPD to an endurance-type protocol of electrical pulse stimulation (EPS). EPS induced a decrease in the diameter, covered surface and expression of MHC1 in COPD myotubes. Although the expression of protein degradation markers was not affected, expression of the protein synthesis marker mTOR was not induced in COPD compared to healthy myotubes after EPS. The expression of the differentiation markers p16INK4a and p21 was impaired, while expression of Myf5 and MyoD tended to be affected in COPD muscle cells in response to EPS. The expression of mitochondrial biogenesis markers PGC1α and MFN2 was affected and expression of TFAM and COX1 tended to be reduced in COPD compared to healthy myotubes upon EPS. Lipid peroxidation was increased and the expression of the antioxidant enzymes SOD2 and GPx4 was affected in COPD compared to healthy myotubes in response to EPS. Thus, we provide evidence of an impaired response of COPD muscle cells to contraction, which might be involved in the muscle weakness observed in patients with COPD.

Effect of Ashwagandha Withanolides on Muscle Cell Differentiation.

Withania somnifera (Ashwagandha) is used in Indian traditional medicine, Ayurveda, and is believed to have a variety of health-promoting effects. The molecular mechanisms and pathways underlying these effects have not yet been sufficiently explored. In this study, we investigated the effect of Ashwagandha extracts and their major withanolides (withaferin A and withanone) on muscle cell differentiation using C2C12 myoblasts. We found that withaferin A and withanone and Ashwagandha extracts possessing different ratios of these active ingredients have different effects on the differentiation of C2C12. Withanone and withanone-rich extracts caused stronger differentiation of myoblasts to myotubes, deaggregation of heat- and metal-stress-induced aggregated proteins, and activation of hypoxia and autophagy pathways. Of note, the Parkinson's disease model of Drosophila that possess a neuromuscular disorder showed improvement in their flight and climbing activity, suggesting the potential of Ashwagandha withanolides for the management of muscle repair and activity.

Evaluation of therapeutic effect of Premna herbacea in diabetic rat and isoverbascoside against insulin resistance in L6 muscle cells through bioenergetics and stimulation of JNK and AKT/mTOR signaling cascade.

BACKGROUND: Premna herbacea Roxb., a perennial herb is well documented for its therapeutic uses among the traditional health care-givers of Assam, India. Scientific validation on the traditional use of the medicinal plant using modern technology may promote further research in health care. PURPOSE: This study evaluates the therapeutic potential of methanolic extract of P. herbacea (MEPH) against type 2 diabetes mellitus (T2DM) and its phytochemical(s) in ameliorating insulin resistance (IR), thereby endorsing the plant bioactives as effective anti-hyperglycemic agents. METHODS: The anti-diabetic potential of the plant extract was explored both in L6 muscle cells and high fructose high fat diet (HF-HFD) fed male Sprague Dawley (SD) rats. Bioactivity guided fractionation and isolation procedure yielded Verbascoside and Isoverbascoside (ISOVER) as bioactive and major phytochemicals in P. herbacea. The bioenergetics profile of bioactive ISOVER and its anti-hyperglycemic potential was validated in vitro by XFe24 analyzer, glucose uptake assay and intracellular ROS generation by flourometer, FACS and confocal microscopy. The potential of ISOVER was also checked by screening various protein markers via immunoblotting. RESULTS: MEPH enhanced glucose uptake in FFA-induced insulin resistant (IR) L6 muscle cells and decreased elevated blood glucose levels in HF-HFD fed rats. Isoverbascoside (ISOVER) was identified as most bioactive phytochemical for the first time from the plant in the Premna genus. ISOVER activated the protein kinase B/AMP-activated protein kinase signaling cascades and enhanced glucose uptake in IR-L6 muscle cells. ISOVER decreased the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) and increased that of mammalian target of rapamycin (mTOR), thereby attenuating IR. However, molecular docking revealed that ISOVER increases insulin sensitivity by targeting the JNK1 kinase as a competitive inhibitor rather than mTOR. These findings were further supported by the bioenergetics profile of ISOVER. CONCLUSION: This study for the first time depicts the functional properties of ISOVER, derived from Premna herbacea, in ameliorating IR. The phytochemical significantly altered IR with enhanced glucose uptake and inhibition of ROS through JNK-AKT/mTOR signaling which may pave the way for further research in T2DM therapeutics.

[Effect of electroacupuncture preconditioning on myocyte apoptosis in myocardial ischemia-reperfusion injury rats based on FXR/SHP pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on left-cardiac function, contents of serum TNF-α and IL-6 and expression of myocardial farnesoid X receptor(FXR), small heterodimer partner (SHP), apoptosis inducing factor (AIF) and heat shock proteins 70 (HSP70) genes in myocardial ischemia-reperfusion injury (MIRI) rats, so as to explore its mechanisms underlying improvement of ischemic myocardial injury. METHODS: Forty male Wistar rats were randomly divided into normal control, sham operation, MIRI model and EA pretreatment groups, with 10 rats in each group. Rats of the sham operation group received exposure of the thorax and heart. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery (LAD). EA (2 Hz/100 Hz and 1 mA) was applied to bilateral "Neiguan" (PC6), "Zusanli" (ST36) and "Guanyuan" (CV4) for 20 min, once a day for 7 days. The left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP)and maximal rates of rise and fall of left ventricular pressure (±dp/dtmax) were detected, the contents of serum TNF-α and IL-6 were detected by using enzyme-linked immunosorbent assay (ELISA), and the expression of FXR, SHP, AIF and HSP70 apoptotic genes in the myocardial tissue were measured by fluorescent quantitative RT-PCR. RESULTS: Compared with the normal control group, the LVEDP, contents of serum TNF-α and IL-6, and the expression levels of myocardial FXR, SHP, AIF and HSP70 mRNAs were significantly increased (P<0.05), while LVSP and ±dp/dtmax levels were obviously decreased in the model group (P<0.05). In comparison with the model group, MIRI-induced increases of LVEDP, TNF-α and IL-6 contents, and FXR, SHP and AIF mRNA expression and decreases of ±dp/dtmax and LVSP levels were reversed(P<0.05), except HSP70 mRNA expression with significantly increased (P<0.05) in the EA pretreatment group. CONCLUSION: EA pretreatment can protect the left ventricular function of the ischemic heart in MIRI rats, which may be related to its effects in reliving peripheral inflammation and regulating the expression levels of apoptosis-related factors FXR, SHP, AIF and HSP70 in the myocardium.

A hybrid platform featuring nanomagnetic ligand fishing for discovering COX-2 selective inhibitors from aerial part of Saussurea laniceps Hand.-Mazz.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea laniceps Hand.-Mazz. (Compositae) is a representative "snow lotus" herb well known in Chinese folk medicine to treat inflammation-related diseases such as arthritis. S. laniceps (SL) shows anti-inflammatory and analgesic potencies and contains various constituents potentially with cyclooxygenase-2 (COX-2) selective inhibition. The herb is a valuable source of natural alternatives to synthetic COX-2 selective nonsteroidal anti-inflammatory drugs, a common medication for rheumatoid arthritis (RA) and osteoarthritis (OA) reported with serious cardiovascular side effects. AIM OF THE STUDY: Based on an innovative drug screening platform, this study aimed to discover safe, effective COX-2 selective inhibitors from SL. MATERIALS AND METHODS: An enzyme-anchored nanomagnetic fishing assay was developed to separate COX-2 ligands from SL. Cell and animal models of cardiomyocytes, lipopolysaccharide-stimulated macrophages, rat adjuvant-induced arthritis, and anterior cruciate ligament transection-induced OA rats, were adopted to screen the single/combined ligands regarding toxicity and bioactivity levels. Molecular docking was employed to unravel binding mechanisms of the ligands towards COX-1 and COX-2. RESULTS: Four COX-2 selective compounds were separated from SL using optimized COX-2-functionalized magnetic nanoparticles. All the four ligands were proved with evidently lower cardiotoxicity both in vitro and in vivo than celecoxib, a known COX-2 selective inhibitor. Two ligands, scopoletin and syringin, exhibited potent anti-arthritic activities in rat models of RA and OA by alleviating clinical statuses, immune responses, and joint pathological features; their optimum combination ratio was discovered with stronger remedial effects on rat OA than single administrations. The COX-1/2 binding modes of the two phytochemicals contributed to explain their cardiac safety and therapeutic performances. CONCLUSIONS: The screened chemicals are promising to be developed as COX-2 selective inhibitors as part of treating RA and OA. The hybrid strategy for discovering therapeutic agents from SL is shown here to be efficient; it should be equally valuable for finding other active chemicals in other natural sources.

Berberine improves intralipid-induced insulin resistance in murine.

Insulin resistance (IR) is a major metabolic risk factor even before the onset of hyperglycemia. Recently, berberine (BBR) is found to improve hyperglycemia and IR. In this study, we investigated whether BBR could improve IR independent of hyperglycemia. Acute insulin-resistant state was induced in rats by systemic infusion of intralipid (6.6%). BBR was administered via different delivery routes before or after the beginning of a 2-h euglycemic-hyperinsulinemic clamp. At the end of experiment, rats were sacrificed, gastrocnemius muscle was collected for detecting mitochondrial swelling, phosphorylation of Akt and AMPK, as well as the mitochondrial permeability regulator cyclophilin D (CypD) protein expression. We showed that BBR administration markedly ameliorated intralipid-induced IR without affecting blood glucose, which was accompanied by alleviated mitochondrial swelling in skeletal muscle. We used human skeletal muscle cells (HSMCs), AML12 hepatocytes, human umbilical vein endothelial cells, and CypD knockout mice to investigate metabolic and molecular alternations. In either HSMCs or AML12 hepatocytes, BBR (5 µM) abolished palmitate acid (PA)-induced increase of CypD protein levels. In CypD-deficient mice, intralipid-induced IR was greatly attenuated and the beneficial effect of BBR was diminished. Furthermore, we demonstrated that the inhibitory effect of BBR on intralipid-induced IR was mainly mediated by skeletal muscle, but not by intestine, liver, or microvasculature; BBR administration suppressed intralipid-induced upregulation of CypD expression in skeletal muscle. These results suggest that BBR alleviates intralipid-induced IR, which is related to the inhibition of CypD protein expression in skeletal muscle.

Cell type-selective secretome profiling in vivo.

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

The cardiovascular protective effect and mechanism of calycosin and its derivatives.

Cardiovascular disease is the main cause of mortality and morbidity in the world, especially in developing countries. Drug therapy is one of the main ways to treat cardiovascular diseases. Among them, great progress has been made in the treatment of cardiovascular diseases with traditional Chinese medicine. In terms of experimental research, the mechanism of traditional Chinese medicine in the treatment of cardiovascular diseases has been thoroughly discussed in vitro and in vivo. In terms of clinical treatment, traditional Chinese medicine with flavonoids, saponins and alkaloids as the main effective components has a definite effect on the treatment of cardiovascular diseases such as arrhythmia, myocardial ischemia, angina pectoris and myocardial infarction, with high safety and good application prospects. With the further research on the effective ingredients, mechanism and adverse reactions of traditional Chinese medicine, it will be beneficial to the effectiveness of traditional Chinese medicine, reduce side effects and promote the modernization of traditional Chinese medicine. Calycosin and its derivatives, the main bioactive flavonoids in Astragalus membranaceus have multiple biological effects, such as antioxidant, pro-angiogenesis, anti-tumour, and anti-inflammatory effects. Based on the above biological effects, calycosin has been shown to have good potential for cardiovascular protection. The potent antioxidant effect of calycosin may play an important role in the cardiovascular protective potential. For injured cardiac myocytes, calycosin and its derivatives can alleviate the cell damage mainly marked by the release of myocardial enzymes and reduce the death level of cardiac myocytes mainly characterized by apoptosis through various mechanisms. For vascular endothelial cells, calycosin also has multiple effects and multiple mechanisms, such as promoting vascular endothelial cell proliferation, exerting vasodilating effect and directly affecting the synthesis function of endothelial cells. The present review will address the bioactivity of calycosin in cardiovascular diseases such as protective effects on cardiac myocytes and vascular endothelial cells and elucidate main mechanism of calycosin and its derivatives to exert the above biological effects.

Peripheral EphrinB1/EphB1 signalling attenuates muscle hyperalgesia in MPS patients and a rat model of taut band-associated persistent muscle pain.

BACKGROUND: Myofascial pain syndrome (MPS) is an important clinical condition that is characterized by chronic muscle pain and a myofascial trigger point (MTrP) located in a taut band (TB). Previous studies showed that EphrinB1 was involved in the regulation of pathological pain via EphB1 signalling, but whether EphrinB1-EphB1 plays a role in MTrP is not clear. METHODS: The present study analysed the levels of p-EphB1/p-EphB2/p-EphB3 in biopsies of MTrPs in the trapezius muscle of 11 MPS patients and seven healthy controls using a protein microarray kit. EphrinB1-Fc was injected intramuscularly to detect EphrinB1s/EphB1s signalling in peripheral sensitization. We applied a blunt strike to the left gastrocnemius muscles (GM) and eccentric exercise for 8 weeks with 4 weeks of recovery to analyse the function of EphrinB1/EphB1 in the muscle pain model. RESULTS: P-EphB1, p-EphB2, and p-EphB3 expression was highly increased in human muscles with MTrPs compared to healthy muscle. EphB1 (r = 0.723, n = 11, P < 0.05), EphB2 (r = 0.610, n = 11, P < 0.05), and EphB3 levels (r = 0.670, n = 11, P < 0.05) in the MPS group were significantly correlated with the numerical rating scale (NRS) in the MTrPs. Intramuscular injection of EphrinB1-Fc produces hyperalgesia, which can be partially prevented by pre-treatment with EphB1-Fc. The p-EphB1 contents in MTrPs of MPS animals were significantly higher than that among control animals (P < 0.01). Intramuscular administration of the EphB1 inhibitor EphB1-Fr significantly suppressed mechanical hyperalgesia. CONCLUSIONS: The present study showed that the increased expression of p-EphB1/p-EphB2/p-EphB3 was related to MTrPs in patients with MPS. This report is the first study to examine the function of EphrinB1-EphB1 signalling in primary muscle afferent neurons in MPS patients and a rat animal model. This pathway may be one of the most important and promising targets for MPS.

Inhibition of Peripheral ERK Signaling Ameliorates Persistent Muscle Pain Around Trigger Points in Rats.

The purpose of this study was to investigate whether the ERK signaling pathway was involved in ameliorating chronic myofascial hyperalgesia from contused gastrocnemius muscle in rats. We established an animal model associated with myofascial pain syndrome and described the mechanism of muscle pain in an animal model. Changes in the mechanical pain threshold were observed 0.5, 1, 2, 3, 4, 5, 8, 12, 18, and 24 h after ERK inhibitor injection around myofascial trigger points (MTrPs) of the gastrocnemius muscle in rats. Morphological changes in gastrocnemius muscle cells were observed by hematoxylin and eosin (H&E) staining. ERK signaling pathway activation was detected through immunohistochemistry and Western blotting. The main morphological characteristics of injured muscle fibers around MTrPs include gathered circular or elliptical shapes of different sizes in the cross-section and continuous inflated and tapering fibers in the longitudinal section. After intramuscular injection of U0126 (ERK inhibitor), the mechanical pain threshold significantly increased. The reduction in mechanical hyperalgesia was accompanied by reduced ERK protein phosphorylation, myosin light chain kinase (MLCK) protein, p-MLC protein expression, and the cross-sectional area of skeletal muscle cells around MTrPs. An ERK inhibitor contributed to the attenuation of mechanical hyperalgesia in the rat myofascial pain model, and the increase in pain threshold may be related to MLCK downregulation and other related contraction-associated proteins by ERK.

Active Compound of Pharbitis Semen (Pharbitis nil Seeds) Suppressed KRAS-Driven Colorectal Cancer and Restored Muscle Cell Function during Cancer Progression.

Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of Pharbitis nil, also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether Pharbitis nil seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of Pharbitis nil (Pharbitis semen) were separated by chromatography and the active compound of Pharbitis semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.

Abscisic acid enriched fig extract promotes insulin sensitivity by decreasing systemic inflammation and activating LANCL2 in skeletal muscle.

Abscisic acid is a phytohormone found in fruits and vegetables and is endogenously produced in mammals. In humans and mice, lanthionine synthetase C-like 2 (LANCL2) has been characterized as the natural receptor for ABA. Herein, we characterize the efficacy of a fig fruit extract of ABA in promoting glycemic control. This ABA-enriched extract, at 0.125 µg ABA/kg body weight, improves glucose tolerance, insulin sensitivity and fasting blood glucose in diet-induced obesity (DIO) and db/db mouse models. In addition to decreasing systemic inflammation and providing glycemic control without increasing insulin, ABA extract modulates the metabolic activity of muscle. ABA increases expression of important glycogen synthase, glucose, fatty acid and mitochondrial metabolism genes and increases direct measures of fatty acid oxidation, glucose oxidation and metabolic flexibility in soleus muscle cells from ABA-treated mice with DIO. Glycolytic and mitochondrial ATP production were increased in ABA-treated human myotubes. Further, ABA synergized with insulin to dramatically increase the rate of glycogen synthesis. The loss of LANCL2 in skeletal muscle abrogated the effect of ABA extract in the DIO model and increased fasting blood glucose levels. This data further supports the clinical development of ABA in the treatment of pre-diabetes, type 2 diabetes and metabolic syndrome.

Photobiomodulation reduces cell death and cytokine production in C2C12 cells exposed to Bothrops venoms.

Snakebites caused by the genus Bothrops are often associated with severe and complex local manifestations such as edema, pain, hemorrhage, and myonecrosis. Conventional treatment minimizes the systemic effects of venom; however, their local action is not neutralized. The purpose of this study was to evaluate the effect of photobiomodulation (PBM) on C2C12 muscle cells exposed to B. jararaca, B. jararacussu, and B. moojeni venoms on events involved in cell death and the release of inflammatory mediators. Cells were exposed to venoms and immediately irradiated with low-level laser (LLL) application in continuous wave at the wavelength of 660 nm, energy density of 4.4 J/cm2, power of 10 mW, area of 0.045 cm2, and time of 20 s. Cell integrity was analyzed by phase contrast microscope and cell death was performed by flow cytometry. In addition, interleukin IL1-ß, IL-6, and IL-10 levels were measured in the supernatant. Our results showed that the application of PBM increases cell viability and decreases cell death by apoptosis and necrosis. Moreover, the release of pro-inflammatory interleukins was also reduced. The data reported here indicate that PBM resulted in cytoprotection on myoblast C2C12 cells after venom exposure. This protection involves the modulation of cell death mechanism and decreased pro-inflammatory cytokine release.