Rat Muscle Cells Research Articles

Bergenin from <i>Cissus javana</i> DC. (Vitaceae) root extract enhances glucose uptake by rat L6 myotubes

Purpose: To examine the glucose uptake stimulatory activity of the root extract of Cissus javana DC. (Vitaceae) in Lδ myotubes of rat, and also to identify the extract’s active principles.Methods: The methanol extract was prepared from Cissus javana tuberous roots and evaluated for glucose uptake stimulatory effects on Lδ rat muscle cells and inhibitory activity against α-glucosidase. The chemical components were isolated using several chromatographic techniques, and their structures characterized by spectroscopic methods. Each isolate was then assayed for glucose uptake stimulatory and α-glucosidase inhibitory activities.Results: The extract (100 μg/ml) exhibited glucose uptake stimulatory effect (70.9 % enhancement) and α-glucosidase enzyme inhibitory activity (100 % inhibition). Through chromatographic separation, bergenin, stigmast-4-en-3-one and β-sitosterol were isolated and identified. Bergenin, at 100 μg/ml (0.3046 mM), increased glucose uptake by Lδ myotubes by 50.5 % without toxicity. At the same concentration, bergenin showed no inhibition on α-glucosidase enzyme, while stigmast-4-en-3-one and β-sitosterol exhibited 98.6 and 40.6 %, inhibition, respectively.Conclusion: This study is the first report on the chemical constituents, and the glucose uptake stimulatory and α-glucosidase inhibitory activities of Cissus javana DC. roots. The findings reveal the antidiabetic potential of the plant and the glucose-uptake enhancing activity of bergenin.
 Keywords: Cissus javana, α-Glucosidase, Antidiabetes, Rat skeletal muscle cells, Bergenin

The effect of endurance training on levels of LINC complex proteins in skeletal muscle fibers of STZ-induced diabetic rats

The changes of the linker of nucleoskeleton and cytoskeleton (LINC) complex have been studied in many muscular abnormality conditions; however, the effects of diabetes and physical activities on it have still remained to be defined. Therefore, the purpose of the this study was to evaluate the impacts of a six-week endurance training on the levels of SUN1 and Nesprin-1 proteins in Soleus and EDL muscles from diabetic wistar rats. A total number of 48 male Wistar rats (10 weeks, 200-250 gr) were randomly divided into healthy control (HC, N = 12), healthy trained (HT, N = 12), diabetic control (DC, N = 12), and diabetic trained (DT, N = 12) groups. Diabetes was also induced by a single intraperitoneally injection of streptozocin (45 mg/kg). The training groups ran a treadmill for five consecutive days within six weeks. The levels of the SUN1 and the Nesprin-1 proteins were further determined via ELISA method. The induction of diabetes had significantly decreased the levels of Nesprin-1 protein in the soleus and EDL muscles but it had no effects on the SUN1 in these muscles. As well, the findings revealed that six weeks of endurance training had significantly increased the levels of Nesprin-1 in DT and HT groups in the soleus as well as the EDL muscles; however, it had no impacts on the SUN1 in these muscles. The muscle fiber cross-sectional area (CSA) and myonuclei also decreased in diabetic control rats in both studied muscles. The training further augmented these parameters in both studied muscles in HT and DT groups. The present study provides new evidence that diabetes changes Nesprin-1 protein levels in skeletal muscle and endurance exercise training can modify it.

Intermedin1-53 attenuates aging-associated vascular calcification in rats by upregulating sirtuin 1.

Vascular calcification is a common phenomenon in older adults. Intermedin (IMD) is a cardiovascular bioactive peptide inhibiting vascular calcification. In this study, we aimed to investigate whether IMD1-53 attenuates aging-associated vascular calcification. Vascular calcification was induced by vitamin D3 plus nicotine (VDN) in young and old rats. The calcification in aortas was more severe in old rats treated with VDN than young control rats, and IMD expression was lower. Exogenous administration of IMD1-53 significantly inhibited the calcium deposition in aortas and the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) in VDN-treated old rats. Moreover, levels of aging-related p16, p21 and β-galactosidase were all greatly decreased by IMD1-53. These results were further confirmed in rat and human VSMCs in vitro. In addition, IMD-deficient mouse VSMCs showed senescence features coinciding with osteogenic transition as compared with wild-type mouse VSMCs. Mechanistically, IMD1-53 significantly increased the expression of the anti-aging factor sirtuin 1 (sirt1); the inhibitory effects of IMD1-53 on calcification and senescence were blocked by sirt1 knockdown. Furthermore, preincubation with inhibitors of PI3K, AMPK or PKA efficiently blunted the upregulatory effect of IMD1-53 on sirt1. Consequently, IMD1-53 could attenuate aging-associated vascular calcification by upregulating sirt1 via activating PI3K/Akt, AMPK and cAMP/PKA signaling.

Regulatory effects of Prohibitin 1 on proliferation and apoptosis of pulmonary arterial smooth muscle cells in monocrotaline-induced PAH rats

BackgroundPulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by unbalanced proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Prohibitin 1 (PHB1) is known for its significant anti-proliferative activity. However, the role of PHB1 in PASMCs and PAH have not been elucidated. MethodsMonocrotaline (MCT 60 mg/kg) was used to build a PAH model in SD rats. Right ventricular systolic pressure (RVSP) and right ventricle (RV) hypertrophy were measured. Morphology of pulmonary vessels was observed by Hematoxylin-Eosin (HE) staining. Expression of PHB1 in pulmonary arteries and PASMCs was determinated by immunoblot and immunofluorescence. Cell proliferation was detected by CCK8 and EDU when PASMCs were stimulated by PDGF-BB (20 ng/mL). Furthermore, siRNA for PHB1 and Akt inhibitor were conducted to investigate the mechanism behind the role of PHB1 and AKT signaling pathway in PASMCs proliferation and apoptosis. ResultsThe protein expression of PHB1 in PAH rats lung tissue was significantly up-regulated accompanied by elevated RVSP and enhanced RV hypertrophy. Immunohistochemistry showed that PHB1 was mainly localized in the pulmonary vascular smooth muscle layer. PDGF-BB significantly up-regulated the expression of PHB1 in rat primary PASMCs in a time- and dose-dependent manner. After PHB1 knock down, PASMCs proliferation was significantly suppressed while apoptosis was significantly recovered. Meanwhile the level of proliferating cell nuclear antigen (PCNA) and P-Akt were significantly down-regulated. Perifosine (Akt inhibitor) also significantly inhibit proliferation of PASMCs. ConclusionPHB1 contributes to pulmonary vascular remodeling by accelerating proliferation of PASMCs which involves AKT phosphorylation.

MiR-137 and its target T-type CaV 3.1 channel modulate dedifferentiation and proliferation of cerebrovascular smooth muscle cells in simulated microgravity rats by regulating calcineurin/NFAT pathway.

ObjectivesPostflight orthostatic intolerance has been regarded as a major adverse effect after microgravity exposure, in which cerebrovascular adaptation plays a critical role. Our previous finding suggested that dedifferentiation of vascular smooth muscle cells (VSMCs) might be one of the key contributors to cerebrovascular adaptation under simulated microgravity. This study was aimed to confirm this concept and elucidate the underlying mechanisms.Materials and MethodsSprague Dawley rats were subjected to 28‐day hindlimb‐unloading to simulate microgravity exposure. VSMC dedifferentiation was evaluated by ultrastructural analysis and contractile/synthetic maker detection. The role of T‐type CaV3.1 channel was revealed by assessing its blocking effects. MiR‐137 was identified as the upstream of CaV3.1 channel by luciferase assay and investigated by gain/loss‐of‐function approaches. Calcineurin/nuclear factor of activated T lymphocytes (NFAT) pathway, the downstream of CaV3.1 channel, was investigated by detecting calcineurin activity and NFAT nuclear translocation.ResultsSimulated microgravity induced the dedifferentiation and proliferation in rat cerebral VSMCs. T‐type CaV3.1 channel promoted the dedifferentiation and proliferation of VSMC. MiR‐137 and calcineurin/NFATc3 pathway were the upstream and downstream signalling of T‐type CaV3.1 channel in modulating the dedifferentiation and proliferation of VSMCs, respectively.ConclusionsThe present work demonstrated that miR‐137 and its target T‐type CaV3.1 channel modulate the dedifferentiation and proliferation of rat cerebral VSMCs under simulated microgravity by regulating calcineurin/NFATc3 pathway.

A Mutation in γ-Adducin Impairs Autoregulation of Renal Blood Flow and Promotes the Development of Kidney Disease.

The genes and mechanisms involved in the association between diabetes or hypertension and CKD risk are unclear. Previous studies have implicated a role for γ-adducin (ADD3), a cytoskeletal protein encoded by Add3. We investigated renal vascular function in vitro and in vivo and the susceptibility to CKD in rats with wild-type or mutated Add3 and in genetically modified rats with overexpression or knockout of ADD3. We also studied glomeruli and primary renal vascular smooth muscle cells isolated from these rats. This study identified a K572Q mutation in ADD3 in fawn-hooded hypertensive (FHH) rats-a mutation previously reported in Milan normotensive (MNS) rats that also develop kidney disease. Using molecular dynamic simulations, we found that this mutation destabilizes a critical ADD3-ACTIN binding site. A reduction of ADD3 expression in membrane fractions prepared from the kidney and renal vascular smooth muscle cells of FHH rats was associated with the disruption of the F-actin cytoskeleton. Compared with renal vascular smooth muscle cells from Add3 transgenic rats, those from FHH rats had elevated membrane expression of BKα and BK channel current. FHH and Add3 knockout rats exhibited impairments in the myogenic response of afferent arterioles and in renal blood flow autoregulation, which were rescued in Add3 transgenic rats. We confirmed these findings in a genetic complementation study that involved crossing FHH and MNS rats that share the ADD3 mutation. Add3 transgenic rats showed attenuation of proteinuria, glomerular injury, and kidney fibrosis with aging and mineralocorticoid-induced hypertension. This is the first report that a mutation in ADD3 that alters ACTIN binding causes renal vascular dysfunction and promotes the susceptibility to kidney disease.

Effects of Xiangsha Liujunzi decoction drug serum on gastric antrum smooth muscle cells from rats with functional dyspepsia by regulating gastrointestinal hormones

Abstract Objective To observe the effect and mechanism of Xiangsha Liujunzi decoction (XSLJZD) drug serum on gastric antrum smooth muscle cells (SMCs) in rats with functional dyspepsia (FD). Methods Gastric antrum SMCs from rats with FD were isolated, cultured, and then divided into six groups as follows: control, model, domperidone, low-dose XSLJZD (LXSLJZD), medium-dose XSLJZD (MXSLJZD), and high-dose XSLJZD (HXSLJZD). Each group was administered the corresponding drug serum for intervention. Drug serum intervention conditions and proliferative activity of SMCs were tested by cholecystokinin octapeptide. Ghrelin, gastrin, somatostatin, and substance P (SP) levels were measured by ELISA. Somatostatin and SP mRNA expression was measured by real-time PCR. Results A concentration of 10% drug serum for 24 h was decided to be the best intervention condition for later study. The mean optical density value in the model group was lower than that in the control group (P = .001). Optical density values in the domperidone and HXSLJZD groups were higher than those in the model group (P = .025, P = .032, respectively). Gastrin, SP, and ghrelin levels in the model group were lower (P = .007, P = .037, P = .005, respectively), but somatostatin levels were higher, compared with those in the control group (P = .031). Gastrin, SP, and ghrelin levels in the domperidone, MXSLJZD, and HXSLJZD groups were higher than those in the model group (all P Conclusions XSLJZD and domperidone drug serum effectively promote proliferative activity of gastric antrum SMCs in an FD model. The mechanism of this activity may be regulated by gastrointestinal hormones.

Ultrastructural Characteristics of Slow and fast twitch Muscle Fibers of Rats after a Forced Swimming Cycle against a Background of Introduction of Green Tea Extract and Ammonium Salts

Finding and studying the mechanisms of action of substances of natural origin that would reduce fatigue and restore the performance of skeletal muscles under extreme physical loads are very important tasks of sports physiology and medicine. A comparison of the effects of decaffeinated green tea extract (GTE) and ammonium chloride (AChl), which we previously tested separately and in combination with GTE in the forced swimming model, allowed it to be established that has a greater stimulative effect than does GTE. In the present work, on the basis of electron microscopy data, morphometric studies of m. soleus (SOL) and m. extensor digitorum longus (EDL) muscle fibers of rats were conducted after a forced swimming cycle after the effect of GTE and low doses of ammonium salts (chloride (AChl) or carbonate (ACar)). In GTE group rats, the adaptation to physical load mainly occurs due to an increase in the average area of mitochondria in SOL muscles. In AChl group rats, the adaptation is caused by an increase in the average area of mitochondria in EDL muscles, while, at the same time, the average area of transversely oriented (in relation to the cut plain) T-tubules (TTs) in these muscles decreases along with an increase in the portion of longitudinally oriented TTs, indicating muscle fiber breaks. The combined action of AChl and GTE provides an additive functional effect due to an increase in the average area and volumetric density of mitochondria in EDL muscles and average area of TTs in SOL and EDL muscles, as well as their amount in EDL muscles. In ACar group rats, the functional effect is caused by an increase in the average area of TTs in EDL muscles and their amount in SOL muscles. At the same time, the portion of longitudinal TTs in EDL muscles (and therefore muscle fiber breaks) increases. In the group of combined ACar and GTE effect, changes in mitochondrial apparatus were detected in slow twitch, but especially in fast twitch muscles, as well as an increase in the area of TTs in SOL muscles. Analysis of ultrastructural characteristics indicates that the efficiency of preparations is mainly caused by adaptive changes of TTs and fast twitch muscle mitochondria, but it increases with the involvement of slow twitch muscle fibers in structural and functional adaptation to extreme physical load.

Age-Related Changes of Procollagen Alpha Polypeptide in Vascular Remodeling in Rat Vascular Smooth Muscle Cell

The current study revealed that increased synthesis and secretion of collagen types I and III play major roles in arterial wall remodeling, aneurysm formation, and atherosclerotic cap stability. The aim is to investigate the age-related changes of the procollagen alpha polypeptide gene mRNA and protein expression in the vascular smooth muscle cells (VSMCs) in rats, as well as the possible underlying mechanisms. We tested in vitro culture of VSMC from the thoracoabdominal aorta in neonate and 9-month-old healthy male Wistar rats; procollagen alpha polypeptide mRNA and procollagen alpha polypeptide protein expression were detected, using RT-PCR, VG staining, Western blot and ELISA methods. Semi-quantitative analysis displayed that, in the real-time reverse transcription polymerase chain reaction (RT-PCR), the type I collagen α polypeptide chain mRNA increased in the adult group, but not significantly (P = 0.05). Further, there was no significant difference between the two groups of type III collagen α polypeptide chain mRNA (P > 0.05). Both the type I and type III procollagen alpha polypeptide protein expression were increased significantly in the older group as compared with the young group (P < 0.05). This phenomenon mainly lies in the fact that the regulatory pathway on age-related changes of procollagen alpha polypeptides may be one of the molecular mechanisms in vascular remodeling during aging.

冷热型哮喘大鼠平滑肌内钙通道特性与差异

冷热型哮喘大鼠平滑肌内钙通道特性与差异

Decreased inward rectifier and voltage-gated K+ currents of the right septal coronary artery smooth muscle cells in pulmonary arterial hypertensive rats.

In vascular smooth muscle, K+ channels, such as voltage-gated K+ channels (Kv), inward-rectifier K+ channels (Kir), and big-conductance Ca2+-activated K+ channels (BKCa), establish a hyperpolarized membrane potential and counterbalance the depolarizing vasoactive stimuli. Additionally, Kir mediates endothelium-dependent hyperpolarization and the active hyperemia response in various vessels, including the coronary artery. Pulmonary arterial hypertension (PAH) induces right ventricular hypertrophy (RVH), thereby elevating the risk of ischemia and right heart failure. Here, using the whole-cell patch-clamp technique, we compared Kv and Kir current densities (IKv and IKir) in the left (LCSMCs), right (RCSMCs), and septal branches of coronary smooth muscle cells (SCSMCs) from control and monocrotaline (MCT)-induced PAH rats exhibiting RVH. In control rats, (1) IKv was larger in RCSMCs than that in SCSMCs and LCSMCs, (2) IKv inactivation occurred at more negative voltages in SCSMCs than those in RCSMCs and LCSMCs, (3) IKir was smaller in SCSMCs than that in RCSMCs and LCSMCs, and (4) IBKCa did not differ between branches. Moreover, in PAH rats, IKir and IKv decreased in SCSMCs, but not in RCSMCs or LCSMCs, and IBKCa did not change in any of the branches. These results demonstrated that SCSMC-specific decreases in IKv and IKir occur in an MCT-induced PAH model, thereby offering insights into the potential pathophysiological implications of coronary blood flow regulation in right heart disease. Furthermore, the relatively smaller IKir in SCSMCs suggested a less effective vasodilatory response in the septal region to the moderate increase in extracellular K+ concentration under increased activity of the myocardium.

Mechanism of AMPK-mediated apoptosis of rat gastric smooth muscle cells under high glucose condition.

To observe changes in AMP-activated protein kinase (AMPK) activity and phosphorylation changes in AMPK signaling pathway in gastric smooth muscle cells of rats with diabetic gastroparesis (DGP), investigate the effect of AMPK on apoptosis and explore the underlying mechanism. After establishing rat model of DGP, rats were divided into normal control (NC) and DGP groups. The phosphorylation changes in AMPK pathway were detected by AMPK Signaling Phospho-Antibody Array, and the apoptosis-related proteins were determined. Rat gastric smooth muscle cells were cultured in vitro under different glucose conditions, and divided into normal and high glucose groups. The AMPK activity and intracellular Ca2+ changes in cells were observed. After AMPK silencing, cells were divided into high glucose-24h, high glucose-48h and high glucose-48h+siRNA groups. Changes in expression of apoptosis-related proteins were observed. AMPK activity and apoptosis rates were both increased in gastric smooth muscle tissues in DGP rats (P<0.05, P<0.001, respectively). A total of 14 apoptosis-related differentially phosphorylated proteins were identified. Under high-glucose condition, AMPK activity and intracellular Ca2+ concentrations in rat gastric smooth muscle cells were increased (both P<0.05). After AMPK silencing, p53 expression was decreased, Akt and p70 S6 ribosomal protein kinase (p70S6K) activities were were increased, Bcl-2 expression was increased, CaMKII activity was decreased in the high glucose-48h group. Under high-glucose condition, activated AMPK can directly or indirectly promote cells apoptosis by regulating the expression and activity of p53, Akt, p70S6K, Protein kinase A (PKA), Phospholipidol C (PLC)-β3, CaMKII, CaMKIV and eukaryotic translation initiation factor 4E binding protein1 (4E-BP1) in rat gastric smooth muscle cells.

Acid mine drainage in an Indian high-sulfur coal mining area: Cytotoxicity assay and remediation study

Opencast mining causes significant environmental concern due to acid mine drainage (AMD) caused by the oxidation of pyrites and other sulfur-bearing minerals. The present study intends to determine the seasonal variability of AMD in the affected area of the Ledo opencast mining, the cytotoxicity of the AMD, and the AMD remediation process. The physicochemical properties of the collected samples were analyzed by using laboratory-based methods and sophisticated instrumental tools. The cytotoxicity study of AMD water was performed by using different cell lines such as normal rat muscle and human carcinoma cells. The study demonstrates that the mine water samples have high conductivity (1.30–2.49 ms cm−1) with high total dissolved solids (1068−1339 ppm) which can change the ionic composition of water. The concentration level of trace elements are also found to be higher than the permissible limit during monsoon season. A simple laboratory-based remediation process of AMD has been carried out in the current study by using size segregated pulverized limestone and the process reveals the decrease in elemental concentrations of AMD water. This study will be useful to develop a remediation technique to minimize the concentration levels of hazardous elements and ions in the AMD water.

Hypoglycemic activity of Phaseolus vulgaris (L.) aqueous extract in type 1 diabetic rats

Abstract The aim of the present study was to evaluate the hypoglycemic activity of the aqueous extract from the fruit walls of Phaseolus vulgaris pods and to examine the potential mechanism underlying the improvement of the glycemic level. In the course of the study, diabetes mellitus was induced in rats with a single intraperitoneal injection of streptozotocin (45 mg·kg−1 b.w.). Diabetic and control rats were then orally administered with a single-dose or repeated-dose (28 day) of P. vulgaris extract (200 mg·kg−1). Results show that the extract was found to possess significant hypoglycemic activity, and the study of glucose utilization by isolated rat hemidiaphragm suggests that the aqueous extract may enhance the peripheral utilization of glucose. The subsequent experiments have revealed that the P. vulgaris extract could increase glucose transporter 4 (GLUT-4) content in skeletal muscle cells of control and diabetic rats. Our data also indicate that the P. vulgaris extract did not affect the content of the insulin receptor, but significantly reduced the total tyrosine kinase activity in skeletal muscle cells of both experimental groups of rats. The present results clearly indicated that P. vulgaris extract may be beneficial for reducing hyperglycemia through its potency in regulation of glucose utilization via GLUT-4, but the current mechanism remains to be unidentified.

Downregulation of miR-542-3p promotes osteogenic transition of vascular smooth muscle cells in the aging rat by targeting BMP7

BackgroundAging is believed to have a close association with cardiovascular diseases, resulting in various pathological alterations in blood vessels, including vascular cell phenotypic shifts. In aging vessels, the microRNA(miRNA)-mediated mechanism regulating the vascular smooth muscle cell (VSMC) phenotype remains unclarified. MiRNA microarray was used to compare the expressions of miRNAs in VSMCs from old rats (oVSMCs) and young rats (yVSMCs). Quantitative reverse transcription real-time PCR (qRT-PCR) and small RNA transfection were used to explore the miR-542-3p expression in oVSMCs and yVSMCs in vitro. Calcification induction of yVSMCs was conducted by the treatment of β-glycerophosphate (β-GP). Alizarin red staining was used to detect calcium deposition. Western blot and qRT-PCR were used to investigate the expression of the smooth muscle markers, smooth muscle 22α (SM22α) and calponin, and the osteogenic markers, osteopontin (OPN), and runt-related transcription factor 2 (Runx2). Lentivirus was used to overexpress miR-542-3p and bone morphogenetic protein 7 (BMP7) in yVMSCs. Luciferase reporter assay was conducted to identify the target of miR-542-3p.ResultsCompared with yVSMCs, 28 downregulated and 34 upregulated miRNAs were identified in oVSMCs. It was confirmed by qRT-PCR that oVSMC expressed four times lower miR-542-3p than yVSMCs. Overexpressing miR-542-3p in yVSMCs suppressed the osteogenic differentiation induced by β-GP. Moreover, miR-542-3p targets BMP7 and overexpressing BMP7 in miR-542-3p–expressing yVSMCs reverses miR-542-3p’s inhibition of osteogenic differentiation.ConclusionsmiR-542-3p regulates osteogenic differentiation of VSMCs through targeting BMP7, suggesting that the downregulation of miR-542-3p in oVSMCs plays a crucial role in osteogenic transition in the aging rat.

Exogenous hydrogen sulfide improves erectile dysfunction by inhibiting apoptosis of corpus cavernosum smooth muscle cells in rats with cavernous nerve injury

To explore the effects of exogenous hydrogen sulfide (H2S) on apoptosis of corpus cavernosum smooth muscle cells (CCSMCs) and erectile dysfunction (ED) in rats with bilateral cavernous nerve injury (BCNI). Twentyfour male SD rats were randomly divided into 3 groups (n=8):sham operation group, bilateral cavernous nerve injury group (BCNI group) and H2S intervention group (BCNI+NaHS group). In BCNI and BCNI+NaHS groups, BCNI was induced by clamp injury of the bilateral cavernous nerves, and the rats were subjected to daily intraperitoneal injection of normal saline and 100 μmol/kg NaHS solution for 4 weeks, respectively. After the treatment, the intracavernous pressure (ICP) and mean arterial pressure (MAP), ) of the rats were measured. Western blotting was used to detect the expressions of cystathionine β synthetase (CBS), cystathionine γ lyase (CSE), α-SMA, collagen-I, caspase-3, Bax and Bcl-2 in the penile cavernous tissue, and the expressions of CBS and CSE were also detected immunohistochemically. The ratio of cavernous smooth muscle to collagen was detected using Masson's Trichrome staining. The apoptosis level of CCSMC was detected by TUNEL + α-SMA immunofluorescence double staining. After 4 weeks of treatment, the rats in BCNI+NaHS group showed a significantly higher ICP/MAP ratio than those in BCNI group (P < 0.05). The results of Masson's Trichrome staining showed that the ratio of cavernous smooth muscle/collagen was significantly higher in BCNI + NaHS group than in BCNI group (P < 0.05). Western blotting showed a significantly higher expression of α-SMA protein but a lower expression of collagen-I protein in BCNI + NaHS group than in BCNI group (P < 0.05). TUNEL+α-SMA immunofluorescence double staining revealed a significantly lower number of apoptotic CCSMCs in BCNI+NaHS group than in BCNI group (P < 0.05). Compared with those in BCNI group, the rats in BCNI+NaHS group had significantly decreased expressions of caspase-3 and Bax proteins (P < 0.05) with significantly enhanced Bcl-2 protein expression and an increased Bcl-2/Bax ratio (P < 0.05). The expressions of CBS and CSE were significantly lower in BCNI group than in the other two groups (P < 0.05). Exogenous H2S enhance the expression of the classic apoptotic protein Bcl-2 and reduces apoptosis of CCSMC to improve the erectile function in rats with BCNI.

Effects of S-glutathionylation on the passive force-length relationship in skeletal muscle fibres of rats and humans.

This study investigated the effect of S-glutathionylation on passive force in skeletal muscle fibres, to determine whether activity-related redox reactions could modulate the passive force properties of muscle. Mechanically-skinned fibres were freshly obtained from human and rat muscle, setting sarcomere length (SL) by laser diffraction. Larger stretches were required to produce passive force in human fibres compared to rat fibres, but there were no fibre-type differences in either species. When fibres were exposed to glutathione disulfide (GSSG; 20mM, 15min) whilst stretched (at a SL where passive force reached ~ 20% of maximal Ca2+-activated force, denoted as SL20% max), passive force was subsequently decreased at all SLs in both type I and type II fibres of rat and human (e.g., passive force at SL20% max decreased by 12 to 25%). This decrease was fully reversed by subsequent reducing treatment with dithiothreitol (DTT; 10mM for 10min). If freshly skinned fibres were initially treated with DTT, there was an increase in passive force in type II fibres (by 10 ± 3% and 9 ± 2% in rat and human fibres, respectively), but not in type I fibres. These results indicate that (i) S-glutathionylation, presumably in titin, causes a decrease in passive force in skeletal muscle fibres, but the reduction is relatively smaller than that reported in cardiac muscle, (ii) in rested muscle in vivo, there appears to be some level of reversible oxidative modification, probably involving S-glutathionylation of titin, in type II fibres, but not in type I fibres.

Wnt/β-catenin signaling pathway regulates asthma airway remodeling by influencing the expression of c-Myc and cyclin D1 via the p38 MAPK-dependent pathway.

Airway remodeling is the main characteristic of asthma; however, the mechanisms underlying this pathophysiological change have not been fully elucidated. Previous studies have indicated that the Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling pathway are involved in the development of airway remodeling during asthma. Therefore, the present study established an airway remodeling rat model, after which β-catenin, cyclin D1 and c-Myc protein expressions were analyzed via western blotting in the lung tissue and airway smooth muscle cells (ASMCs) of rats. The mRNA expression of the aforementioned proteins were evaluated via reverse transcription-quantitative PCR. β-catenin, cyclin D1 and c-Myc are core transcription factors and target genes of the Wnt/β-catenin and MAPK signaling pathways. Furthermore, β-catenin, c-Myc and cyclin D1 protein expression were determined following blocking of the p38 MAPK signaling pathway in vitro. The results demonstrated that higher expressions of β-catenin, cyclin D1 and c-Myc were detected in lung tissues and ASMCs in the asthma group compared with the control. Blocking the p38 MAPK signaling pathway with a specific inhibitor SB203580 also downregulated the expressions of β-catenin, cyclin D1 and c-Myc in vitro. Taken together, these results indicated that the Wnt/β-catenin signaling pathway may regulate the process of airway remodeling via the p38 MAPK-dependent pathway.

Moxifloxacin suppresses airway inflammation and modulates expression of caveolin-1 and flotillin-1 in airway smooth muscle cells of asthmatic rats.

Moxifloxacin (MXF) possesses anti-inflammatory properties on asthmatic airway smooth muscle cells (ASMCs) beyond their antimicrobial effects, but the mechanisms are still unknown. This study was to investigate effects of MXF on expression of caveolin-1 (Cav-1) and flotillin-1 (FLOT1) in ASMCs in asthmatic rats. ASMCs were collected from the airway and cultured in vitro. Cells from normal rats were treated with normal saline (Group N); cells from asthmatic rats were incubated with normal saline (Group A) or MXF (20 mg/L) (Group M); Cav-1 expression was up-regulated by transferring Cav-1 expressing lentivirus (Group L) and FLOT1 expression down-regulated by using siRNA in cells from asthmatic rats (Group S). The expressions of Cav-1, FLOT1 and p65 NF-κB were measured by Western blotting and quantificational real-time polymerase chain reaction (qRT-PCR), and interleukin-8 (IL-8) and eotaxin contents were measured by enzyme-linked immunosorbent assay (ELISA). Compared with normal control, Cav-1 expression significantly decreased in asthmatic groups (P<0.01); MXF up-regulated Cav-1 expression in asthmatic groups (P<0.01). However, compared with normal control, the expression of FLOT1 and p65 NF-κB dramatically increased in asthmatic groups (P<0.01); MXF down-regulated the expression of FLOT1 and p65 NF-κB in asthmatic groups (P<0.01); meanwhile, the expressions of FLOT1 and p65 NF-κB decreased after up-regulation of Cav-1 expression in asthmatic groups (P=0.01). Compared with asthmatic groups, the IL-8 and eotaxin contents significantly decreased in MXF Groups, Cav-1 up-regulation asthmatic groups and FLOT1 down-regulation asthmatic groups (P<0.01). MXF can modulate the airway inflammation, upregulate Cav-1 expression, downregulate the expression of FLOT1 and p65 NF-κB in asthmatic rat ASMCs, which may be related to the anti-inflammatory effects of MXF in asthmatic ASMCs.

The comparative effects of high dose atorvastatin and proprotein convertase subtilisin/kexin type 9 inhibitor on the mitochondria of oxidative muscle fibers in obese-insulin resistant female rats

The present study aimed to compare the effects of high dose atorvastatin and a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor on the mitochondrial function in oxidative muscle fibers in obese female rats. Female Wistar rats were fed with either a normal diet (ND: n = 12) or a high-fat diet (HFD: n = 36) for a total of 15 weeks. At week 13, ND-fed rats received a vehicle, and HFD-fed rats were divided to three groups to receive either a vehicle, 40 mg/kg/day of atorvastatin, or 4 mg/kg/day of PCSK9 inhibitor (SBC-115076) for 3 weeks. Soleus muscles were investigated to assess mitochondrial ROS, membrane potential, swelling, mitochondrial-related protein expression, and level of malondialdehyde (MDA). The results showed that HFD-fed rats with vehicle developed obese-insulin resistance and dyslipidemia. Both atorvastatin and PCSK9 inhibitor reduced obesity and dyslipidemia, as well as improved insulin sensitivity in HFD-fed rats. However, the efficacy of PCSK9 inhibitor to increase weight loss and reduce dyslipidemia in HFD-fed rats was greater than those of atorvastatin. An increase in MDA level, ratio of p-Drp1ser616/total Drp1 protein, CPT1 protein, mitochondrial ROS, and membrane depolarization in the soleus muscle were observed in HFD-fed rats with vehicle. PCSK9 inhibitor enabled the restoration of all these parameters to normal levels. However, atorvastatin facilitated restoration of some parameters, including MDA level, p-Drp1ser616/total Drp1 ratio, and CPT1 protein expression. These findings suggest that PCSK9 inhibitor is superior to atorvastatin in instigating weight loss, cholesterol reduction, and attenuation of mitochondrial oxidative stress in oxidative muscle fibers of obese female rats.