Advantages of Using 3D Spheroid Culture Systems in Toxicological and Pharmacological Assessment for Osteogenesis Research.

Bone differentiation is crucial for skeletal development and maintenance. Its dysfunction can cause various pathological conditions such as rickets, osteoporosis, osteogenesis imperfecta, or Paget's disease. Although traditional two-dimensional cell culture systems have contributed significantly to our understanding of bone biology, they fail to replicate the intricate biotic environment of bone tissue. Three-dimensional (3D) spheroid cell cultures have gained widespread popularity for addressing bone defects. This review highlights the advantages of employing 3D culture systems to investigate bone differentiation. It highlights their capacity to mimic the complex in vivo environment and crucial cellular interactions pivotal to bone homeostasis. The exploration of 3D culture models in bone research offers enhanced physiological relevance, improved predictive capabilities, and reduced reliance on animal models, which have contributed to the advancement of safer and more effective strategies for drug development. Studies have highlighted the transformative potential of 3D culture systems for expanding our understanding of bone biology and developing targeted therapeutic interventions for bone-related disorders. This review explores how 3D culture systems have demonstrated promise in unraveling the intricate mechanisms governing bone homeostasis and responses to pharmacological agents.

Plant Alkaloid Tetrandrine Is a Nuclear Receptor 4A1 Antagonist and Inhibits Panc-1 Cell Growth In Vitro and In Vivo.

The orphan nuclear receptor 4A1 (NR4A1) is highly expressed in human pancreatic cancer cells and exerts pro-oncogenic activity. In a previous study, we demonstrated that fangchinoline (FCN), a natural inhibitor of nuclear NR4A1, induces NR4A1-dependent apoptosis in human pancreatic cancer cells. In this study, we evaluated FCN and its structural analogs (berbamine, isotetrandrine, tetrandrine, and tubocurarine) for their inhibitory effects on NR4A1 transactivity, and confirmed that tetrandrine (TTD) showed the highest inhibitory effect in pancreatic cancer cells. Moreover, in a tryptophan fluorescence quenching assay, TTD directly bound to the ligand binding domain (LBD) of NR4A1 with a KD value of 10.60 µM. Treatment with TTD decreased proliferation and induced apoptosis in Panc-1 human pancreatic cancer cells in part through the reduced expression of the Sp1-dependent anti-apoptotic gene survivin and induction of ROS-mediated endoplasmic reticulum stress, which are the well-known NR4A1-dependent proapoptotic pathways. Furthermore, at a dose of 25 mg/kg/day, TTD reduced tumor growth in an athymic nude mouse xenograft model bearing Panc-1 cells. These data show that TTD is an NR4A1 antagonist and that modulation of the NR4A1-mediated pro-survival pathways is involved in the antitumor effects of TTD.

Elecampane (Inula helenium) Root Extract and Its Major Sesquiterpene Lactone, Alantolactone, Inhibit Adipogenesis of 3T3-L1 Preadipocytes.

Recent studies have shown that Nur77 and AMPKα play an important role in regulating adipogenesis and isoalantolactone (ISO) dual-targeting AMPKα and Nur77 inhibits adipogenesis. In this study, we hypothesized that Inula helenium (elecampane) root extract (IHE), which contains two sesquiterpene lactones, alantolactone (ALA) and ISO, as major compounds, might inhibit adipogenesis. Here, we found that ALA and IHE simultaneously target AMPKα and Nur77 and inhibited adipogenic differentiation of 3T3-L1 cells, accompanied by the decreased expression of adipocyte markers. Further mechanistic studies demonstrated that IHE shares similar mechanisms of action with ISO that reduce mitotic clonal expansion during the early phase of adipogenic differentiation and decrease expression of cell cycle regulators. These results suggest that IHE inhibits adipogenesis, in part, through co-regulation of AMPKα and Nur77, and has potential as a therapeutic option for obesity and related metabolic dysfunction.

Mealworm Oil (MWO) Enhances Wound Healing Potential through the Activation of Fibroblast and Endothelial Cells.

Mealworm and mealworm oil (MWO) have been reported to affect antioxidant, anti-coagulation, anti-adipogenic and anti-inflammatory activities. However, the function of MWO in wound healing is still unclear. In this study, we found that MWO induced the migration of fibroblast cells and mRNA expressions of wound healing factors such as alpha-smooth muscle actin (α-SMA), collagen-1 (COL-1) and vascular endothelial growth factor (VEGF) in fibroblast cells. The tube formation and migration of endothelial cells were promoted through the activation of VEGF/VEGF receptor-2 (VEGFR-2)-mediated downstream signals including AKT, extracellular signal-regulated kinase (ERK) and p38 by MWO-stimulated fibroblasts for angiogenesis. Moreover, we confirmed that MWO promoted skin wound repair by collagen synthesis, re-epithelialization and angiogenesis in an in vivo excisional wound model. These results demonstrate that MWO might have potential as a therapeutic agent for the treatment of skin wounds.

Dual targeting of Nur77 and AMPKα by isoalantolactone inhibits adipogenesis in vitro and decreases body fat mass in vivo.

BACKGROUND: Suppression of adipogenesis has been considered as a potential target for the prevention and treatment of obesity and associated metabolic disorders, and the nuclear receptor 4A1 (NR4A1/Nur77) and AMPKα are known to play important roles during early and intermediate stages of adipogenesis. Therefore, we hypothesized that dual targeting Nur77 and AMPKα would show strong inhibitory effect on adipogenesis. METHODS: We screened a herbal medicine-based small molecule library to identify novel natural compounds dual targeting Nur77 and AMPKα, and the antiadipogenic effects and mechanisms of action of a "hit" compound were studied in 3T3-L1 cells. In vivo antiobesity effects of the compound were also investigated in high-fat diet (HFD)-induced obese mice. RESULTS: We identified isoalantolactone (ISO) as a new NR4A1 inactivator that also activates AMPKα in 3T3-L1 cells. ISO, as expected, inhibited adipogenic differentiation of 3T3-L1 preadipocytes, accompanied by reduced mitotic clonal expansion (MCE) which occurs in the early stage of adipogenesis and decreased expression of genes required for MCE and cell cycle markers including cyclin A, cyclin D1. Furthermore, ISO reduced body weight gain and fat mass (epididymal, subcutaneous, perirenal, and inguinal white adipose tissues) in the high-fat diet-fed C57BL/6 N mice. Serum levels of triglycerides, aspartate transaminase, and alanine transaminase and hepatic steatosis were also significantly improved in the ISO-treated group compared to the high-fat diet control group. CONCLUSIONS: These results suggest that ISO dual targeting Nur77 and AMPKα during adipogenesis represents a novel class of mechanism-based antiadipogenic agents for treatment of obesity and associated metabolic disorders, including hyperlipidemia and fatty liver.

Cold stress aggravates inflammatory responses in an LPS-induced mouse model of acute lung injury.

Although the relationship between environmental cold temperature and susceptibility to respiratory infection is generally accepted, the effect of ambient cold temperature on host reactivity in lung inflammation has not been fully studied. To examine the function of ambient cold temperature on lung inflammation, mice were exposed to 4 °C for 8 h each day for 14 days. In the lungs of mice exposed to cold stress, inflammatory cells in bronchoalveolar lavage (BAL) fluid and lung tissues were slightly increased by about twofold. However, the structures of pulmonary epithelial cells were kept within normal limits. Next, we examined the effect of cold stress on the inflammatory responses in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The infiltration of neutrophils and inflammation of lung tissue determined by histology were significantly increased by exposure to ambient cold temperature. In addition, the production of pro-inflammatory cytokines including interleukin (IL)-12, IL-17, and monokine induced by gamma interferon (MIG) was elevated by exposure to cold stress. Therefore, we suggest that cold stress is a factor that exacerbates lung inflammation including ALI. To our knowledge, this is the first report on the relationship between cold stress and severity of lung inflammation.

Outcomes of bedside peripherally inserted central catheter placement: a retrospective study at a single institution.

BACKGROUND: Bedside insertion of peripherally inserted central catheters (PICCs) has higher rates of malposition than fluoroscopic-guided PICC placement. This study evaluated the success rate of bedside PICC placement, variations in tip location, and risk factors for malposition. METHODS: This retrospective study included patients who underwent bedside PICC placement from January 2013 to September 2014 in a single institution. The procedure was conducted under ultrasound guidance or by a blind method. After PICC placement, tip location was determined by chest X-ray. RESULTS: The overall venous access success rate with bedside PICC placement was 98.1% (1,302/ 1,327). There was no significant difference in the venous access success rate between ultrasound-guided placement (868/880, 98.6%) and a blind approach placement (434/447, 97.1%). Optimal tip position was achieved on the first attempt in 1,192 cases (91.6%). Repositioning was attempted in 65 patients; 60 PICCs were repositioned at the bedside, two PICCs were repositioned under fluoroscopic guidance, and three PICCs moved to the desired position without intervention. Final optimal tip position after repositioning was achieved in 1,229 (94.4%). In logistic regression analysis, five factors associated with tip malposition included female sex (Exp(B), 1.687; 95% confidence interval [CI], 1.180 to 2.412; P=0.004), older age (Exp(B), 1.026; 95% CI, 1.012 to 1.039; P<0.001), cancer (Exp(B), 0.650; 95% CI, 0.455 to 0.929; P=0.018), lung disease (Exp(B), 2.416; 95% CI, 1.592 to 3.666; P<0.001), and previous catheter insertions (Exp(B), 1.262; 95% CI, 1.126 to 1.414; P<0.001). CONCLUSIONS: Bedside PICC placement without fluoroscopy is effective and safe in central venous catheters. Potential risk factors associated with catheter tip malposition include older age, female sex, cancer, pulmonary disease, and previous central vein catheterizations.

Comparative study of liver injury induced by high-fat methionine- and choline-deficient diet in ICR mice originating from three different sources.

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. It is characterized by the accumulation of lipids without alcohol intake and often progresses to non-alcoholic steatohepatitis (NASH), liver fibrosis, and end-stage liver diseases such as cirrhosis or cancer. Although animal models have greatly contributed to the understanding of NAFLD, studies on the disease progression in humans are still limited. In this study, we used the recently reported high-fat L-methionine-defined and choline-deficient (HFMCD) diet to rapidly induce NASH and compared the responses to HFMCD in ICR mice from three different countries: Korea (supplied by the National Institute of Food and Drug Safety Evaluation), USA, and Japan during 6 weeks. Feeding HFMCD did not cause significant differences in weight gain in comparison with mice fed control diet. Relative weight of the liver increased gradually, while the relative weight of the kidneys remained unchanged. The parameters of liver injury (serum activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase) increased rapidly from 1 week and remained elevated for as long as 6 weeks. Histopathological analysis showed that the accumulation of hepatic lipids induced by HFMCD was prominent at 1 week after diet supplementation and increased further at 6 weeks. Inflammatory markers were significantly increased in a time-dependent manner by HFMCD. The mRNA levels of TNF-α and IL-6 were elevated approximately 15-fold relative to control diet and that of IL-1ß was increased more than 20-folds at 6 week after the onset of HFMCD intake. In addition, mRNA expression of fibrosis markers such as α-SMA, TGFß1, and Col1a1 were also significantly increased at 6 week. In summary, the responses of Korl:ICR mice by intake of HFMCD diet were similar to those of ICR mice from other sources, which suggests that Korl:ICR mice is also a useful resource to study the pathogenesis of diet-induced NAFLD.

Comparison of toxic responses to acetaminophen challenge in ICR mice originating from different sources.

Acetaminophen (APAP) is the most common antipyretic analgesic worldwide. However, APAP overdose causes severe liver injury, especially centrilobular necrosis, in humans and experimental animals. At therapeutic dosage, APAP is mainly metabolized by sulfation and glucuronidation, and partly by cytochrome P450-mediated oxidation. However, APAP overdose results in production of excess reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by cytochromes P450; NAPQI overwhelms the level of glutathione (GSH), which could otherwise detoxify it. NAPQI binds covalently to proteins, leading to cell death. A number of studies aimed at the prevention and treatment of APAP-induced toxicity are underway. Rats are more resistant than mice to APAP hepatotoxicity, and thus mouse models are mainly used. In the present study, we compared the toxic responses induced by APAP overdose in the liver of ICR mice obtained from three different sources and evaluated the usability of the Korl:ICR stock established by the National Institute of Food and Drug Safety Evaluation in Korea. Administration of APAP (300 mg/kg) by intraperitoneal injection into male ICR mice enhanced CYP2E1 protein expression and depleted hepatic GSH level 2 h after treatment accompanied with significantly increased level of hepatic malondialdehyde, a product of lipid peroxidation. Regardless of the source of the mice, hepatotoxicity, as evidenced by activity of serum alanine aminotransferase, increased from 8 h and peaked at 24 h after APAP treatment. In summary, hepatotoxicity was induced after the onset of oxidative stress by overdose of APAP, and the response was the same over time among mice of different origins.

Deoxypodophyllotoxin in Anthriscus sylvestris alleviates fat accumulation in the liver via AMP-activated protein kinase, impeding SREBP-1c signal.

Deoxypodophyllotoxin (DPT) is a naturally occurring flavolignan in Anthriscus sylvestris known as cow parsley or wild chervil, and has been reported to have inhibitory effects against several pathological processes including cancer, inflammation and infection. Here, we report the effects of DPT in the fatty liver induced by high fat diet in vivo as well as its regulatory mechanism related with the transcription factor for lipogenic genes such as sterol regulatory element binding protein-1c (SREBP-1c) in vitro. C57BL/6 mice were fed high fat diet for 10 weeks and also orally administrated with DPT for additional 4 weeks. 5 and 10 mg/kg of DPT decreased lipid accumulation in the liver induced by high fat diet, as indicated by histological parameters such as Oil Red O staining and hematoxylin & eosin as well as the contents of hepatic triglyceride and cholesterol. In hepatocytes, DPT inhibited the liver X receptor α-mediated SREBP-1c induction and expression of the lipogenic genes, including fatty acid synthase, acetyl-CoA carboxylase and stearoyl-CoA desaturase-1. Moreover, DPT induced AMP-activated protein kinase (AMPK) activation, which has been known to inhibit the expression of SREBP-1c in hepatocyte. Also this compound restored the dysregulation of AMPK and SREBP-1c induced by high fat diet in mice. In conclusion, we demonstrated that DPT significantly inhibited fatty liver by adjusting lipid metabolism coordinated with AMPK activation and SREBP-1c inhibition.

Comparision of doxorubicin-induced cardiotoxicity in the ICR mice of different sources.

Doxorubicin is a widely used chemotherapeutic agents and is now part of standard therapeutic regimens for a variety of cancers (eg, hematopoietic malignancies and advanced solid tumors of the breast, ovary, thyroid, and bone). However, a potentially lethal and dose-dependent cardiotoxicity that appears within a short time after treatment limits the usage of doxorubicin in cancer patients. Although the mechanism of doxorubicin-induced cardiotoxicity is not completely understood, it is thought that free radical-induced oxidative stress and excessive production of reactive oxygen species are primary drivers of its toxicity. In this study, we compared the doxorubicin-induced cardiotoxicity of ICR mice obtained from three different sources and evaluated the utility of Korl:ICR stock established by the Korean FDA. Because doxorubicin-induced cardiotoxicity is thought to involve the excessive generation of ROS followed by oxidative stress, we determined the representative tissue index of oxidation, lipid peroxidation, and antioxidant, glutathione (GSH), as well as the parameters of heart injury. Doxorubicin treatment successfully induced cardiotoxicity as evidenced by histological examination and serum parameters (eg, levels of LDH and CK activities) in ICR mice. It was accompanied by increased lipid peroxidation and a decrease in both cysteine and GSH, further supporting previous reports that oxidative stress is a potential mechanism of doxorubicin-induced cardiotoxicity. Of interest, we did not observe a significant difference in doxorubicin-induced cardiotoxicity among mice of different origins. Collectively, our results suggest that Korl:ICR strain may be useful in the research of doxorubicin-induced cardiotoxicity.

Erratum: Comparison of doxorubicin-induced cardiotoxicity in the ICR mice of different sources.

[This corrects the article on p. 165 in vol. 33, PMID: 28747983.].

Comparative study of fatty liver induced by methionine and choline-deficiency in C57BL/6N mice originating from three different sources.

Non-alcoholic fatty liver disease (NAFLD) is believed to be the most prevalent liver disease worldwide and a major cause of chronic liver injury. It is characterized by lipid accumulation in the absence of significant alcohol consumption and frequently progresses to steatohepatitis, liver fibrosis, and hepatocellular carcinoma. Although many studies have been conducted to better understand NAFLD since it was first recognized, there are still many gaps in knowledge of etiology, prognosis, prevention and treatment. Methionine-choline deficient (MCD) diet, a well-established experimental model of NAFLD in rodents, rapidly and efficiently produces the clinical pathologies including macrovesicular steatosis and leads to disease progression. In this study, we measured the response to MCD diet in C57BL/6N mice obtained from three different sources; Korea NIFDS, USA, and Japan. We evaluated changes in body weight, food consumption, and relative weights of tissues such as liver, kidney, gonadal white adipose tissue, inguinal white adipose tissue, and brown adipose tissue. These basic parameters of mice with an MCD diet were not significantly different among the sources of mice tested. After 3 weeks on an MCD diet, histopathological analyses showed that the MCD diet induced clear fat vacuoles involving most area of the acinus in the liver of all mice. It was accompanied by increased serum activities of alanine aminotransferase and aspartate aminotransferase, and decreased levels of serum triglyceride and cholesterol. In conclusion, the response of C57BL6N mice originating from different sources to the MCD diet showed no significant differences as measured by physiological, biochemical, and histopathological parameters.

Erratum: Comparative study of fatty liver induced by methionine and choline-deficiency in C57BL/6N mice originating from three different sources.

[This corrects the article on p. 157 in vol. 33, PMID: 28747982.].

Liqustri lucidi Fructus inhibits hepatic injury and functions as an antioxidant by activation of AMP-activated protein kinase in vivo and in vitro.

Medicinal herbs are used to treat or prevent various diseases, and function to regulate protective mechanisms as nutraceuticals. Fructus Ligustri lucidi is the fruit of Ligustrum lucidum and has been used for its tonic effects on the liver. This study was designed to examine the effects of Fructus Ligustri lucidi water extract (FLL) against severe oxidative stress and mitochondrial impairment in vivo and in vitro and to elucidate its cellular mechanisms of action. Treatment of HepG2 cells with arachidonic acid (AA) + iron successfully induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of ROS, decreses in mitochondrial membrane potential (Δψm), and altered expression of apoptosis-related proteins, such as procaspase-3 and Bcl-xL. FLL treatment significantly blocked these pathological changes and the mitochondrial dysfunction caused by AA + iron, which were similar with the effect of aminoimidazole-carboxamide-ß-d-ribofuranoside (AICAR). Moreover, FLL induced the activation of AMP-activated protein kinase (AMPK), which was mediated by its upstream kinase LKB1. Inhibition or activation of AMPK revealed the role of AMPK in cellular protection conferred by FLL in LKB1-deficient cells. In mice, oral administration of 100 mg/kg FLL activated AMPK in the liver, and protected against oxidative stress and liver injury induced by CCl4 injection. Among the components of FLL, chlorogenic acid was found to be responsible for the protection of hepatocytes against AA + iron-induced cellular damage. Overall, our results confirmed that FLL has the ability to protect hepatocytes against oxidative injury through regulation of the AMPK signaling pathway.

Barley Sprouts Extract Attenuates Alcoholic Fatty Liver Injury in Mice by Reducing Inflammatory Response.

It has been reported that barley leaves possess beneficial properties such as antioxidant, hypolipidemic, antidepressant, and antidiabetic. Interestingly, barley sprouts contain a high content of saponarin, which showed both anti-inflammatory and antioxidant activities. In this study, we evaluated the effect of barley sprouts on alcohol-induced liver injury mediated by inflammation and oxidative stress. Raw barley sprouts were extracted, and quantitative and qualitative analyses of its components were performed. The mice were fed a liquid alcohol diet with or without barley sprouts for four weeks. Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were used to study the effect of barley sprouts on inflammation. Alcohol intake for four weeks caused liver injury, evidenced by an increase in serum alanine aminotransferase and aspartate aminotransferase activities and tumor necrosis factor (TNF)-α levels. The accumulation of lipid in the liver was also significantly induced, whereas the glutathione (GSH) level was reduced. Moreover, the inflammation-related gene expression was dramatically increased. All these alcohol-induced changes were effectively prevented by barley sprouts treatment. In particular, pretreatment with barley sprouts significantly blocked inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression in LPS-stimulated RAW 264.7. This study suggests that the protective effect of barley sprouts against alcohol-induced liver injury is potentially attributable to its inhibition of the inflammatory response induced by alcohol.