A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment.

In vitro systems that mimic organ functionality have become increasingly important tools in drug development studies. Systems that measure the functional properties of skeletal muscle are beneficial to compound screening studies and also for integration into multiorgan devices. To date, no studies have investigated human skeletal muscle responses to drug treatments at the single myotube level in vitro. This report details a microscale cantilever chip-based assay system for culturing individual human myotubes. The cantilevers, along with a laser and photo-detector system, enable measurement of myotube contractions in response to broad-field electrical stimulation. This system was used to obtain baseline functional parameters for untreated human myotubes, including peak contractile force and time-to-fatigue data. The cultured myotubes were then treated with known myotoxic compounds and the resulting functional changes were compared to baseline measurements as well as known physiological responses in vivo. The collected data demonstrate the system's capacity for screening direct effects of compound action on individual human skeletal myotubes in a reliable, reproducible, and noninvasive manner. Furthermore, it has the potential to be utilized for high-content screening, disease modeling, and exercise studies of human skeletal muscle performance utilizing iPSCs derived from specific patient populations such as the muscular dystrophies.

Long-Term Electrical and Mechanical Function Monitoring of a Human-on-a-Chip System.

The goal of human-on-a-chip systems is to capture multi-organ complexity and predict the human response to compounds within physiologically relevant platforms. The generation and characterization of such systems is currently a focal point of research given the long-standing inadequacies of conventional techniques for predicting human outcome. Functional systems can measure and quantify key cellular mechanisms that correlate with the physiological status of a tissue, and can be used to evaluate therapeutic challenges utilizing many of the same endpoints used in animal experiments or clinical trials. Culturing multiple organ compartments in a platform creates a more physiologic environment (organ-organ communication). Here is reported a human 4-organ system composed of heart, liver, skeletal muscle and nervous system modules that maintains cellular viability and function over 28 days in serum-free conditions using a pumpless system. The integration of non-invasive electrical evaluation of neurons and cardiac cells and mechanical determination of cardiac and skeletal muscle contraction allows the monitoring of cellular function especially for chronic toxicity studies in vitro. The 28 day period is the minimum timeframe for animal studies to evaluate repeat dose toxicity. This technology could be a relevant alternative to animal testing by monitoring multi-organ function upon long term chemical exposure.

Investigation on vitamin D knowledge, attitude and practice of university students in Nanjing, China.

OBJECTIVE: To investigate university students' knowledge, attitudes and practice (KAP) regarding vitamin D. DESIGN: The students were requested to answer a questionnaire related to vitamin D and sun exposure. The consumption frequency of foods rich in vitamin D was assessed. Additionally, the intake of vitamin D-containing supplements was recorded. SETTING: A medical university in Nanjing, China. SUBJECTS: Five hundred and fifteen medical students were included. RESULTS: The highest rate of correct responses for the quiz was 68·0 %, while the lowest was 9·6 %. Most students lacked sun exposure because they did not want to get tanned; 82·7 % of students used some sun protection and sunscreen use was more popular in the female group. The consumption frequency of foods rich in vitamin D was low and 5·6 % of the students used vitamin D supplements. The students' knowledge on vitamin D was derived mainly from the media and health professionals. Most of the students were interested to know more about vitamin D. CONCLUSIONS: The present study suggested that medical students had little knowledge and unfavourable behaviours. They should get more health education through the media and health professionals. It is advisable to increase their consumption of foods rich in vitamin D.

Liquiritigenin Potentiates the Inhibitory Effects of Cisplatin on Invasion and Metastasis Via Downregulation MMP-2/9 and PI3 K/AKT Signaling Pathway in B16F10 Melanoma Cells and Mice Model.

Liquiritigenin (LQ) is a flavanone extracted from glycyrrhizae. Previous studies have demonstrated that LQ possesses antimigration properties in HELA and A549 cells. The present research, as an extension of our earlier ones, investigated whether LQ can enhance the antimigration and antiinvasion effect of cis-diamine dichloroplatinum (CDDP) in B16F10 melanoma cell. The data indicated that LQ (25, 50, 100, 200 µM) combined with CDDP (2 µM) significantly reduced B16F10 cell viability compared to CDDP (2 µM)-treated only. The different doses of LQ combined with CDDP significantly suppressed cell migration (21.5%, 49.6%, 75.6%) and cell invasion (26.2%, 51.4%, 69.5%) compared with CDDP-treated alone, suggesting that LQ enhance the inhibition action of CDDP on cell migration and invasion. Moreover, LQ/CDDP combination led to the downregulation of protein expression of MMP-2/9, PI3 K, p-AKT, and upregulated PTEN protein level that play an important role in tumor metastasis progression. Further study demonstrated the enhancement effect of LQ on CDDP suppressing lung metastasis in a mice model being inoculated by the B16F10 melanoma cells. In conclusion, the results suggested that LQ plays an intensive role on CDDP suppressing invasion and metastasis through regulating the PI3 K/AKT signal pathway and suppressing the protein expression of MMP-2/9.

Farnesoid X receptor activation is required for the anti-inflammatory and anti-oxidative stress effects of Alisol B 23-acetate in carbon tetrachloride-induced liver fibrosis in mice.

Previous studies have shown that Alisol B 23-acetate (23ABA) had potent liver-protection effects, however, its roles and potential mechanisms in carbon tetrachloride (CCl4)-induced liver fibrosis remain to be determined. The present study aimed to investigate the effects of 23ABA on CCl4-induced liver fibrosis and tried to elucidate the underlying mechanisms by focusing on regulating of farnesoid X receptor (FXR). In vivo study found that 23ABA alleviated the CCl4-induced liver injury, and showed no obvious systemic toxicity on mice. 23ABA inhibited the collagen production, decreased sera levels of hyaluronic acid (HA) and procollagen type III (PC-III), lowered mRNA expression of α-smooth muscle actin (α-SMA), fibronectin, collagen I and collagen III in livers of mice. 23ABA inhibited the mRNA expressions and the sera levels of interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α), as well as decreased the expression of cyclooxygenase 2 (COX-2) in fibrotic livers of mice. Besides, 23ABA decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased glutathione (GSH) level, enhanced activities of superoxide dismutase (SOD) and glutathione reductase (GR) as well as increased mRNA expression of nuclear factor-E2-related factor 2 (Nrf2), glutamate-cysteine ligase, catalytic subunit (GCLC) and glutamate-cysteine ligase, modifier subunit (GCLM). Further study showed that the anti-liver injury and anti-fibrotic effects of 23ABA were abrogated by FXR antagonist guggulsterone (GS) in vivo. In addition, the inhibition effects of 23ABA on liver inflammation and oxidative stress were also weakened by treatment with GS in CCl4-induced fibrotic mice livers. In conclusion, the protective effects of 23ABA against CCl4-induced liver injury and fibrosis, due to FXR-mediated regulation of liver inflammation and oxidative stress.

Methylenetetrahydrofolate reductase C677T polymorphism is associated with estimated glomerular filtration rate in hypertensive Chinese males.

BACKGROUND: Plasma level of total homocysteine (tHcy) is negatively correlated with kidney function in general population. However, the causal mechanism of this correlation is poorly understood. The purpose of this study is to investigate the association of methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism, which is a major genetic determinant of the plasma tHcy level, with estimated glomerular filtration rate (eGFR) in Chinese. METHODS: A total of 18 814 hypertensive patients (6,914 males, 11,900 females) were included in the study. RESULTS: Association between the eGFR and MTHFR C677T genotype was examined by sex-specific regression analyses. In males, TT genotype was associated with 1.37 ml/min/1.73 m(2) decrease in eGFR (p = 0.004) and with an increased risk (OR = 1.32, p = 0.008) for the lowest quintile of eGFR after adjusting for age, BMI, and blood pressures. However, such association was not observed in females (p > 0.05). This association suggests MTHFR C677T polymorphism may play a role in the regulation of eGFR in males. CONCLUSIONS: MTHFR 677 T is a risk allele for decreased kidney function in Chinese males, implicating this gene in the pathogenesis of chronic kidney disease (CKD).

Inhibitory effect of liquiritigenin on migration via downregulation proMMP-2 and PI3K/Akt signaling pathway in human lung adenocarcinoma A549 cells.

Liquiritigenin (LQ) is a flavanone extracted from Glycyrrhizae, which has multiple biological effects, such as antiinflammation and anticancer. This study is the first to investigate the effect of LQ on the migration of human lung adenocarcinoma A549 cells in vitro. First, LQ exhibited inhibitory effects on the adhesion and migration of A549 cells in the absence of cytotoxicity. Gelatin zymography and Western blot analysis showed that LQ significantly reduced the expression of promatrix metalloproteinase-2 (proMMP-2) in A549 cells in terms of both activity and protein level. Second, LQ inhibited the phosphorylation of Akt and activated the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Furthermore, the treatment of inhibitors specific for Akt (LY294002) and ERK1/2 (U0126) to A549 cells resulted in reduced activity of proMMP-2. These results suggested that the inhibition on proMMP-2 expression by LQ may be through suppression on PI3K/Akt signaling pathway, which in turn led to the inhibition of lung adenocarcinoma A549 cells migration. However, activation of ERK might not be involved in the regulation of proMMP-2. Taken together, LQ may be considered as a potential interfering agent of cancer progression.

Liquiritigenin inhibits serum-induced HIF-1α and VEGF expression via the AKT/mTOR-p70S6K signalling pathway in HeLa cells.

Liquiritigenin (LQ) is a non-toxic dietary flavonoid with chemopreventive and anticancer properties. However, the mechanism of its antiangiogenesis remains unclear. Hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumour angiogenesis and represent an attractive chemotherapeutic target. In this study, we investigated the effect of LQ on the molecular mechanism of angiogenesis. We found that LQ inhibited VEGF expression at both mRNA and protein levels. Liquiritigenin did not affect HIF-1α expression at the mRNA level, but it dramatically inhibited both serum- and mimicked hypoxic-induced HIF-1α protein accumulation in HeLa cells. Furthermore, we showed that LQ inhibited serum-induced expression of HIF-1α by reducing its stability and decreased the synthesis in a dose-dependent manner. Mechanistically, we demonstrated that LQ inhibited HIF-1α and VEGF expression involved in blocking the protein kinase B (PKB/Akt) signalling pathway, and the mechanisms correlated with dephosphorylation of the mammalian target of rapamycin (mTOR) and its effector ribosomal protein S6 kinase (p70S6K). In addition, LQ inhibited VEGF-induced formation of capillary-like structures in human umbilical vein endothelial cells (HUVEC). Taken together, our study provided valuable insights into the mechanism of antiangiogenic effect of LQ.

Liquiritigenin inhibits tumor growth and vascularization in a mouse model of HeLa cells.

Angiogenesis is one of the crucial steps in the transition of a tumor from a small, harmless cluster of mutated cells to a large, malignant growth, capable of spreading to other organs throughout the body. Vascular endothelial growth factor (VEGF) that stimulates vasculogenesis and angiogenesis is thought to be as an anti-angiogenic target for cancer therapy. Liquiritigenin (LQ), a flavanone existing in Radix glycyrrhiza, shows extensive biological activities, such as anti-inflammatory and anti-cancer properties. In our studies, liquiritigenin effectively inhibited the growth of tumors xenografted in nude mice from human cervical cancer cell line HeLa cells, and microvascular density (MVD) of the tumor exposed to liquiritigenin was reduced in a dose dependent manner, especially in the high dose group. Moreover, the expression and secretion of VEGF were down-regulated by the drug in vivo and in vitro. Therefore, liquiritigenin can be further studied on cancer and other diseases associated with VEGF up-regulation.

ALS mutations in both human skeletal muscle and motoneurons differentially affects neuromuscular junction integrity and function.

There is evidence for the involvement of human skeletal muscle (hSKM) in ALS neuromuscular junction (NMJ) dysfunction. However, the specific avenue by which the hSKM contributes to NMJ disruption is not well understood due to limited human-based studies performed to investigate the subject. Thus, hSKM and human motoneurons (hMN) generated from induced pluripotent stem cells of healthy individuals (WT) and ALS patients with two different SOD1 mutations were integrated into functional NMJ systems to investigate and compare the pathological contribution of the hSKM and hMN to ALS NMJ disruption. Morphological assessment of ALS NMJs demonstrated reduced acetylcholine receptor clustering in the post-synaptic membrane of co-cultures with ALS hSKM (hSKMSOD1-hMNWT and hSKMSOD1-hMNSOD1). Significantly reduced functional NMJ numbers, NMJ stability, contraction fidelity and increased fatigue index were observed in all ALS co-cultures compared to WT. However, these disease phenotypes were comparatively more severe in microphysiologic systems with hSKMSOD1-hMNWT or hSKMSOD1-hMNSOD1 than those with hSKMWT-hMNSOD1 co-cultures. Results from this study affirm that the inherent pathological defects in ALS hSKM, independent of motoneurons, significantly contributes to NMJ dysfunction. As such, therapeutically targeting the ALS hSKM may be just as, if not more critical than, the hMN in alleviating disease phenotypes and attenuating disease progression.

Liquiritigenin induces mitochondria-mediated apoptosis via cytochrome c release and caspases activation in HeLa Cells.

It has been demonstrated that many flavonoids possess a potent and broad spectrum of antitumor activity. Liquiritigenin is a flavanone extracted from Glycyrrhizae. This study investigated the effects of liquiritigenin on cell viability and apoptosis induction in human cervical carcinoma (HeLa) cells. The results show that liquiritigenin significantly suppressed cell proliferation in a dose- and time-dependent manner in HeLa cells. In addition, liquiritigenin promoted apoptosis in HeLa cells, evidenced by apoptotic morphological changes and Annexin-V binding. The apoptosis induction with liquiritigenin is associated with the up-regulation of p53 and Bax, along with down-regulation of Bcl-2 and survivin. Finally, examination of the mitochondrial pathway of apoptosis revealed that cytochrome c is released from mitochondria to cytosol, associated with the activation of caspase-9 and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP). Overall, the results indicate that liquiritigenin induces apoptosis in part via the mitochondrial pathway, which is associated with p53 up-regulation, release of cytochrome c and elevated activity of caspase-9 and -3 in HeLa cells.

Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Inhibition of hepatoma 22 tumor by Liquiritigenin.

The purpose of this study was to evaluate the in vivo antitumor effects of liquiritigenin (LQ) on H(22) Hepatocarcinoma. After mice were administrated liquiritigenin (10, 20 and 40 mg/kg) intragastrically for 15 days, tumor volume, indices of thymus and spleen, MDA level in serum, optical microscopy, electron microscopy were determined. We have found that LQ had inhibitory effects on transplanted tumors and that the middle dose of LQ was more effective than the others. All LQ groups could increase thymus weight but it had no obvious effect on the spleen. MDA content decreased with LQ treatment but there wasn't a significant difference. In the group treated with LQ, we observed that the nuclei changed markedly and had ultrastructural morphological changes to apoptosis. The study supports that LQ significantly inhibits the growth of H(22) in vivo, and might be a promising antihepatoma agent.

A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing.

Loss of the neuromuscular junction (NMJ) is an early and critical hallmark in all forms of ALS. The study design was to develop a functional NMJ disease model by integrating motoneurons (MNs) differentiated from multiple ALS-patients' induced pluripotent stem cells (iPSCs) and primary human muscle into a chambered system. NMJ functionality was tested by recording myotube contractions while stimulating MNs by field electrodes and a set of clinically relevant parameters were defined to characterize the NMJ function. Three ALS lines were analyzed, 2 with SOD1 mutations and 1 with a FUS mutation. The ALS-MNs reproduced pathological phenotypes, including increased axonal varicosities, reduced axonal branching and elongation and increased excitability. These MNs formed functional NMJs with wild type muscle, but with significant deficits in NMJ quantity, fidelity and fatigue index. Furthermore, treatment with the Deana protocol was found to correct the NMJ deficits in all the ALS mutant lines tested. Quantitative analysis also revealed the variations inherent in each mutant lines. This functional NMJ system provides a platform for the study of both fALS and sALS and has the capability of being adapted into subtype-specific or patient-specific models for ALS etiological investigation and patient stratification for drug testing.

The protection of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium-induced LLC-PK(1) cells.

Selenium, an essential trace element, showed the significant protective effects against liver and kidney damage induced by some heavy metals. However, the mechanism how selenium suppresses cadmium (Cd)-induced cytotoxicity remains unclear. In this study, we investigated the protective mechanism of selenium on Cd-induced apoptosis in LLC-PK(1) cells via reactive oxygen species (ROS) and mitochondria linked signal pathway. Studies of PI and Annexin V dual staining analysis demonstrated that 20 microM Cd-induced apoptosis as early as 18 h. A concomitant by the generation of ROS, the loss of mitochondrial membrane potential, cytochrome c (cyt c) release, activation of caspase-9, -3 and regulation of Bcl-2 and Bax were observed. N-acetylcysteine (NAC, 500 microM), a free radical scavenger, was used to determine the involvement of ROS in Cd-induced apoptosis. During the process, selenium played the same role as NAC. The anti-apoptosis exerted by selenium involved the blocking of Cd-induced ROS generation, the inhibition of Cd-induced mitochondrial membrane potential collapse, the prevention of cyt c release, subsequent inhibition of caspase activation and the changed level of Bcl-2 and Bax. Taken together, we concluded that Cd-induced apoptosis was mediated by oxidative stress and selenium produced a significant protection against Cd-induced apoptosis in LLC-PK(1) via ameliorating the mitochondrial dysfunction.

[Determination of inorganic elements in rat serum, and vegetable and fruit ferment liquid by ICP-MS].

In the present paper, the contents of thirteen inorganic elements in rat serum, and vegetable and fruit ferment liquid (VFFL) were measured by ICP-MS in order to study the anti-tumor effect of VFFL. Serum or VFFL was digested in nitric and perchloric acids at room temperature and then heated until dryness. The residue was dissolved with 1% (phi) nitric acid prior to ICP-MS analysis. The element contents were quantitated by using 45Sc, 103Rh and 187Re as the internal standards, respectively, according to the rule of close mass number. Certificate references bovine serum (GBW(E)090006) and tea (GBW070605) were employed to validate the proposed method, and the analysis results of most elements in two certificate references were in agreement with their reference values. The intra-day and inter-day precisions of the method in terms of relative standard deviation (RSD) were mainly below 10% and below 15%, respectively. The spiked recoveries for most of studied elements were 80%-110% in rat serum and 90%-120% in VFFL. This method was rapid, highly sensitive, and especially suitable to being applied to small quantity of biological samples with greatly different elements contents. Therefore, we measured the content of thirteen elements in the sera of rats, where in were induced liver cancer by revulsant, and the rate were fed with different dosage of VFFL in intragastric infusion at the same time. It was preliminarily found that the concentrations of some elements in sera of different experiment groups of rats were significantly different, implying the potential anti-tumor effects of VFFL.

Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics.

There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle's contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs.

Cytoprotective effects of selenium on cadmium-induced LLC-PK1 cells apoptosis by activating JNK pathway.

Extensive studies have indicated that the apoptosis pathway appears to be associated with intracellular reactive oxygen species (ROS) production in cadmium-induced nephrotoxicity, however, the precise cellular mechanism remains unclear. The purpose of this study was to determine the relationships between the activation of phosphorylated c-jun N-terminal kinase (JNK) and cadmium-induced apoptosis, and assess the possible cytoprotective mechanism of selenium. Our study clearly revealed cadmium treatment caused apoptosis in LLC-PK1 cells, which was partially suppressed by pretreatment with selenium, an antioxidant nutrient. Further studies found the phosphorylation of JNK kinase increased with exposure to cadmium for 3 h, even remained elevated at 9 h in the time course study, and the activation of phosphorylated JNK was detected in a dose-dependent manner. In addition, a concomitant time-dependent increase in caspase-3 activities was observed by cadmium treatment. During the process, selenium played the same role as N-acetyl-L-cysteine (NAC), a free radical scavenger. Pretreatment of cells with selenium partially suppressed of the phosphorylation of JNK, coupled with caspase-3 activation involved in cadmium-induced apoptosis. In conclusion, our studies provided a molecular linkage between the phosphorylation of JNK and cadmium-induced LLC-PK1 cells apoptosis, and demonstrated selenium also contributed a potentially protection to prevent cadmium-cytotoxicity.

Evaluation of Holistic Treatment for ALS Reveals Possible Mechanism and Therapeutic Potential.

There has been a tremendous amount of research into the causes of Amyotrophic Lateral Sclerosis (ALS), but yet very few treatment options beyond amelioration of symptoms. A holistic approach has shown anecdotal evidence of slowing disease progression and this treatment, known as the Deanna protocol (DP), postulates that ALS is a metabolic disease caused by glutamate that induces toxicity. In this study, glutamate exposure to human motoneurons was investigated and found not to significantly affect cell viability or electrophysiological properties. However, varicosities were observed in axons suggestive of transport impairment that was dose dependent for glutamate exposure. Surprisingly, a subset of the components of the DP eliminated these varicosities. To verify this finding a human SOD1 patient-derived iPSC line was examined and significant numbers of varicosities were present without glutamate treatment, compared to the iPSC control, indicating the possibility of a common mechanism despite different origins for the varicosities. Importantly, the DP ameliorated these varicosities by over 70% in the patient derived cells as well. These results are consistent with much of the literature on ALS and give hope for treatment not only for arresting disease progression using compounds considered safe but also the potential for restoration of function.