Echinacoside promotes osteogenesis and angiogenesis and inhibits osteoclast formation.

PURPOSE: The purpose of this research is to demonstrate echinacoside promotes osteogenesis and angiogenesis and inhibits osteoclast formation. METHODS: We conducted a cell experiment in vitro to study how echinacoside affects angiogenesis, osteogenesis and osteoclast formation. We used polymerase chain reaction and Western blotting to detect the expression levels of proteins and genes related to angiogenesis, osteogenesis and osteoclast formation. We established a bone fracture model with rats to test angiogenesis, osteogenesis and osteoclast formation of echinacoside. We labelled osteogenic markers, blood vessels and osteoclastic markers in fracture sections of rats. RESULTS: The in vitro cell experiments showed echinacoside improved the osteogenic activity of mouse embryo osteoblast precursor cells and promoted the migration and tube formation of human umbilical vein endothelial cells. In addition, it inhibited differentiation of mouse leukaemia cells of monocyte macrophage. Echinacoside increased the expression of related proteins and genes and improved angiogenesis and osteogenesis while inhibiting osteoclast formation by repressing the expression of related proteins and genes. From in vivo experiments, the results of IHC and HE experiments demonstrated echinacoside significantly decreased the content of MMP-9 and improved the content of VEGF and OCN. The fluorescence immunoassay showed echinacoside promoted the activities of RUNX2 and VEGF and inhibited CTSK. Echinacoside reduced the content of TNF-α, IL-1ß and IL-6, thus demonstrating its anti-inflammatory activity. CONCLUSION: Echinacoside improved angiogenesis and osteogenesis and inhibited osteoclast formation to promote fracture healing.

Diosgenin prevents periodontitis by inhibiting inflammation and promoting osteogenic differentiation.

Diosgenin, an essential dietary steroidal sapogenin, possess multiple pharmacological activities. This study aimed to assess the effects of diosgenin on periodontitis and elucidate the mechanisms. Lipopolysaccharide (LPS)-stimulated human periodontal ligament stem cells (hPDLCs) and a Porphyromonas gingivalis (P.g) plus ligation-induced animal model were used for in vitro and in vivo studies, respectively. Inflammatory responses, nuclear factor κ-B (NF-κB) signaling and osteogenesis-related markers were measured both in LPS-stimulated hPDLSCs and in gingival tissue of periodontitis rats. Treatment with diosgenin significantly inhibited the production of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, and interleukin (IL)-6 and the activation of NF-κB pathway in LPS-stimulated hPDLSCs. Further, treatment with diosgenin enhanced the expression of osteoblast-related genes and increased the osteogenic differentiation capacity. Further, activation NF-κB pathway largely abolished the protective effects of diosgenin. Consistent with the in vitro studies, in vivo studies showed that administering diosgenin to periodontitis rats significantly lowered the levels of the TNF-α, IL-1ß, and IL-6 and the inflammatory transcription factor NF-κB in gingival tissue. In addition, osteoblast-related genes were promoted. Diosgenin attenuates periodontitis by adjusting NF-κB signaling to inhibit inflammatory effects and promoting osteogenesis, suggesting diosgenin might be developed as a therapeutic strategy for treating periodontitis in the future.

A combined metabolomics and molecular biology approach to reveal hepatic injury and underlying mechanisms after chronic l-lactate exposure in mice.

This study aimed to explore whether chronic l-lactate exposure could affect the peripheral tissues of mice and to determine the underlying pathogenesis. Herein, male C57BL/6 mice were divided into control and l-lactate groups. After l-lactate treatment for eight weeks (1 g/kg), metabolic changes in liver, kidney, muscle, and serum samples were determined by 1H nuclear magnetic resonance (1H NMR)-based metabolomics. Additionally, organ function was evaluated by serum biochemical and histopathological examinations. Reactive oxygen species (ROS) levels were measured using dihydroethidium staining; levels of signals involved in lactate metabolism and ROS-related pathways were detected using western blotting or polymerase chain reaction. Apoptosis was detected by TUNEL-fluorescence staining. Metabolomic analysis revealed that l-lactate mice showed decreased levels of glutathione (GSH), taurine, ATP, and increased glucose content, compared to control mice. Furthermore, l-lactate mice presented significantly higher serum levels of alanine aminotransferase and aspartate aminotransferase and increased glycogen content in hepatic tissues, compared to control mice. l-lactate mice also had a greater number of apoptotic nuclei in the livers than controls. Moreover, l-lactate exposure reduced mRNA and protein levels of superoxide dismutase-2 and c-glutamylcysteine ligase, elevated levels of cytochrome P450 2E1 and NADPH oxidase-2, and increased the protein expressions of LDHB, Bax/Bcl-2, cleaved caspase-3, and sirtuin-1 in hepatic tissues. Together, these results indicate that chronic l-lactate exposure increases oxidative stress and apoptosis in hepatocytes via upregulation of Bax/Bcl-2 expression and the consequent mitochondrial cytochrome-C release and caspase-3 activation, which contributes to the pathogenesis of hepatic dysfunction.

Effects of Fibroblast Growth Factor 21 on Lactate Uptake and Usage in Mice with Diabetes-Associated Cognitive Decline.

Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exerts beneficial effects on glucose and lipid metabolic homeostasis. However, the impact of FGF21 on type 1 diabetes-associated cognitive decline (DACD) and its mechanisms of action remain unclear. In this study, we aimed to evaluate the effects of FGF21 on lactate uptake and usage in a mouse model of streptozotocin-induced DACD. Six-week-old male C57BL/6 mice were divided into the control, diabetic, and FGF21 (which received 2 mg/kg recombinant human FGF21) groups. At the end of the treatment period, learning and memory performance, nuclear magnetic resonance-based metabonomics, and expressions of various hippocampal protein were analyzed to determine the efficacy of FGF21. The results showed that compared to the control mice, the diabetic mice had reduced long-term memory performance after the hyperglycemic insult; decreased hippocampal levels of lactate dehydrogenase-B (LDH-B) activity, bioenergy metabolites, and monocarboxylate transporter 2 (MCT2); and increased lactate levels. Impaired phosphoinositide 3-kinase (PI3K) signaling was also observed in the diabetic mice. However, FGF21 treatment improved LDH-B activity, ß-nicotinamide adenine dinucleotide, and ATP levels, and increased MCT2 expression and PI3K signaling pathway, which in turn improved the learning and memory defects. These findings demonstrated that the effects of FGF21 on DACD were associated with its ability to improve LDH-B-mediated lactate usage and MCT2-dependent lactate uptake. Further, these beneficial effects of FGF21 in the hippocampus were mediated by the PI3K signaling pathways.

Improvement of polydopamine-loaded salidroside on osseointegration of titanium implants.

BACKGROUND: Microarc oxidation (MAO) on the surface of medical pure titanium can improve its histocompatibility, and loading drugs on the surface can resist excessive intimal hyperplasia. METHODS: In this study, salidroside (SAL) was loaded on the surface of porous titanium (Ti) with polydopamine (PDA) carrier. The effects of SAL on the osteogenesis and angiogenesis of Ti implants were studied by phalloidin staining, alizarin red staining, ALP staining, wound-healing assay, cell transwell assay, matrigel tube formation, and osteogenic and angiogenic genes and proteins expression detected by PCR and western blot in vitro. The bone defect model experiments in rats was established in vivo including X-ray, micro CT, hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Goldner's trichrome analysis, Safranin O-fast green staining and determination of contents of TNF-α and IL-6 in serum. RESULTS: EDS and EDS mapping showed that SAL could be loaded on the surface of the MAO coating by PDA. A drug release experiment showed that SAL loaded on the Ti coating could release slowly and stably without sudden release risk. In vitro cell experiments showed that the SAL coating could promote the proliferation, morphology, calcification and alkaline phosphate activity of MC3T3-E1 cells. At the same time, it promoted the migration and tube formation of HUVEC cells. The SAL coating promoted osteogenesis and angiogenesis by promoting the expression of genes and proteins related to. In vivo experiments, HE and IHC showed that SAL significantly promoted the expression of COL-1 and CD31. Goldner's trichrome and Safranin O-fast green staining showed that SAL coating could increase the new bone tissue around the implantation site. The SAL coating had anti-inflammatory activity by reducing the levels of TNF-α and IL-6 in vivo. CONCLUSION: Therefore, SAL could improve osteogenesis and angiogenesis in conjunction with the Ti-PDA coating.

Copper coating formed by micro-arc oxidation on pure Mg improved antibacterial activity, osteogenesis, and angiogenesis in vivo and in vitro.

Micro-arc oxidation (MAO) was used to improve the resistance of pure magnesium (Mg). Copper (Cu), a good antibacterial, angiogenic, and osteogenic element, was added by reaction in a Cu-containing electrolyte to improve the osteogenic and pro-angiogenic activities of Mg. The surface microstructures of the resulting MAO were evaluated by a scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) mapping. The release of Cu ions was detected by ICP-OES. The antibacterial activity of films with different concentrations of Cu ions was assessed against Staphylococcus aureus (S. aureus). The osteogenesis of films was confirmed by cell morphology and proliferation, ALP activity, alizarin red staining, and osteogenic-related gene expression in the MC3T3-E1 cell line. The angiogenesis of the films was tested in human umbilical vein endothelial cells (HUVECs) by cell migration, tube formation, and VEGF quantification in vitro, and by a chicken embryo chorioallantoic membrane (CAM) assay in vivo. The results showed that the microporous structure was shaped by MAO, and the Cu group was denser and more uniform. The Cu coating showed effective antibacterial activity against S. aureus while also enhancing osteogenesis and angiogenesis in vitro. According to the CAM assay, the Cu group showed not only biocompatibility but also a significant angiogenic response, which was consistent with in vitro studies. The findings indicate that a Cu coating on Mg-MAO enhances osteogenesis and angiogenesis.

Effect of extract from Yiyuan Yiliu Tang on human lung adenocarcinoma and human hepatoma cell lines.

OBJECTIVE: To investigate the efficacy of the extract from Yiyuan Yiliu Tang (, YYYLT) on human lung adenocarcinoma cells A549 and human hepatoma cells Bel7402. METHODS: The cancer cell lines were treated with various concentrations (0, 100, 200, 300, and 400 µg/mL) of the crude water extract of YYYLT and then cell viability, toxicity, cytokine secretion, and cell cycle/apoptosis were determined by MTT assay, LDH assay, and flow cytometry, respectively. RESULTS: The extract from YYYLT significantly suppressed the proliferation of the cancer cell lines and the release of interleukin-2 and tumor necrosis factor-α in a dose-dependent manner. The extract also promoted apoptosis, caused cell cycle arrest at G0/G1 phase, and increased the expression of caspase-3, B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X proteins. CONCLUSION: The extract from YYYLT might be a potential treatment for human lung and liver cancers.

Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity.

OBJECTIVES: This study aimed to investigate the factors affecting the quantity of DNA and RNA extractable from human formalin-fixed paraffin-embedded (FFPE) tissues stored for different lengths of time. METHODS: We randomly selected 20 FFPE specimens harvested from hysteromyoma patients with uterine fibroids during 2010, 2015, and 2017 at the Department of Pathology, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences. DNA and RNA extractions were performed using a DNA/RNA FFPE kit. DNA and RNA concentrations and their OD260/OD280 ratios were determined by a NanoDrop 2000 spectrophotometer. The human ß-globin gene and aldehyde dehydrogenase-2 (ALDH2) gene were amplified from nucleic acids using a LightCycler 480 Real-Time PCR System, and PCR amplification products were electrophoresed on 1% agarose gels. RESULTS: Specimens that were stored for longer showed more degradation and a reduced concentration of DNA and RNA after nucleic acid extraction. However, there was no significant difference in DNA or RNA purity. ß-globin and ALDH2 genes could be amplified from more than 99% of specimens. CONCLUSION: We found that FFPE tissues stored for longer had a reduced quantity of extractable DNA and RNA. However, these tissues could be used for the analysis of some small target genes.

Antiviral activity of cathelicidin 5, a peptide from Alligator sinensis, against WSSV in caridean shrimp Exopalaemon modestus.

White spot disease caused by white spot syndrome virus (WSSV) is responsible for harming shrimp aquaculture industry and results in a pandemic throughout the world. Cathelicidin 5 treatment enhanced immune parameters including antioxidant enzyme activity and immune-related genes expression in shrimp Exopalaemon modestus. Shrimp treated with cathelicidin 5 and inoculated with white spot syndrome virus (WSSV) exhibited a significantly lower mortality rate and lower viral VP28 amplification and expression than control. This study addresses the role of cathelicidin 5 in immune stimulatory and antiviral activities that could protect E. modestus from WSSV infection.

Hepatitis B X protein upregulates decoy receptor 3 expression via the PI3K/NF-κB pathway.

Chronic hepatitis B (CHB) is associated with the development of hepatocellular carcinoma (HCC). Decoy receptor 3 (DcR3) is a tumor necrosis factor receptor that promotes tumor cell survival by inhibiting apoptosis and interfering with immune surveillance. Previous studies showed that DcR3 was overexpressed in HCC cells and that short hairpin RNA (shDcR3) sensitizes TRAIL-resistant HCC cells. However, the expression of DcR3 during hepatitis B virus (HBV) infection has not been investigated. Here, we demonstrated that DcR3 was overexpressed in CHB patients and that DcR3 upregulation was positively correlated with the HBV DNA load and liver injury (determined by histological activity index, serum alanine aminotransferase level, and aspartate aminotransferase level). We found that hepatitis B virus X protein (HBx) upregulated DcR3 expression in a dose-dependent manner, but this increase was blocked by NF-κB inhibitors. HBx also induced the activation of NF-κB, and the NF-κB subunits p65 and p50 upregulated DcR3 by directly binding to the DcR3 promoters. Inhibition of PI3K significantly downregulated DcR3 and inhibited the binding of NF-κB to the DcR3 promoters. Our results demonstrate that the HBx induced DcR3 expression via the PI3K/NF-κB pathway; this process may contribute to the development of HBV-mediated HCC.

HOXB9 inhibits proliferation in gastric carcinoma cells via suppression of phosphorylated-Akt and NF-κB-dependent Snail expression.

BACKGROUND: HOXB9 is a homeobox transcription factor which plays an important role in carcinoma development. This protein has been shown to inhibit cancer cell proliferation. However, the mechanisms that underpin HOXB9-mediated inhibition of cellular proliferation remain to be elucidated. METHODS: In this study, two gastric cancer cell lines, SGC7901 and MKN45, were transfected with plasmids pLVX-HOXB9 and shHOXB9. These transfections resulted in the over-expression of the HOXB9 gene in the SGC7901/HOXB9 cells and knockdown of the HOXB9 gene in the MKN45/shHOXB9 cells. RESULTS: Over-expression of the HOXB9 gene in the SGC7901/HOXB9 cells caused an increase in the apoptotic rate and a concomitant reduction in metastatic ability compared with the knocked-down MKN45/shHOXB9 cells. Moreover, a reduction in the expression of the phosphorylated-Akt protein was observed in the SGC7901/HOXB9 cells, while an increase in expression of the same protein was observed in the MKN45/shHOXB9 cells. We also observed that HOXB9 mediated a reduction in both NF-κB and N-cadherin and Snail protein expression. Conversely, HOXB9 caused an increase in the expression of E-cadherin. CONCLUSIONS: In summary, this study reports that HOXB9 can suppress both phosphorylated-Akt expression and NF-κB activity. The latter phenomenon affects Snail protein expression and the inhibition of gastric carcinoma proliferation.

Treatment of colon cancer in a patient with systemic lupus erythematosus: a case report.

BACKGROUND: Gastrointestinal symptoms occur in approximately 50% of patients with systemic lupus erythematous with low specificity. Although it is well established that colon cancer is one of the many gastrointestinal manifestations associated with systemic lupus erythematous, the diagnosis and treatment remains complex due to adrenal insufficiency symptoms. CASE PRESENTATION: A 43-year-old Chinese woman with a five-year history of systemic lupus erythematous was diagnosed with colon cancer based on imaging test. A radical bowel resection was performed successfully. To avoid serious complications during surgery, prednisone was replaced with methylprednisolone therefore avoiding adrenal insufficiency. The patient was subsequently treated with mFOLFOX6 chemotherapy and recovered well. CONCLUSION: There is a lack of reports for treatment of colon cancer in patients with systemic lupus erythematous. This report provides an effective way to diagnose colon cancer in patients with systemic lupus erythematous and illustrates a successful therapy strategy for this complex medical condition.

Total saponins from Albizia julibrissin inhibit vascular endothelial growth factor-mediated angiogenesis in vitro and in vivo.

Dried stem bark from Albizia julibrissin (AJ) is a highly valued Traditional Chinese Medicine, which has been shown to suppress tumor growth and angiogenesis. Total saponins from AJ (TSAJ) are one of the most bioactive components of AJ extract. The present study evaluated the anti­tumor and anti­angiogenic effects of TSAJ in vitro and in vivo. The anti­angiogenic activity of TSAJ was investigated by measuring the effects on vascular endothelial growth factor (VEGF)­induced proliferation, migration and tube formation of Ea.hy926 endothelial cells in vitro. The expression levels of proteins associated with VEGF­induced angiogenesis were determined by western blotting. Furthermore, in vivo Matrigel™ plug and H22 hepatoma tumor models were used to verify the anti­angiogenic effects of TSAJ. The present study demonstrated that TSAJ significantly inhibited VEGF­mediated endothelial cell proliferation, migration and tube formation of Ea.hy926 cells in vitro. The anti­angiogenic effects of TSAJ were modulated by suppression of phosphorylated­(p­) focal adhesion kinase, p­Akt, and p­extracellular signal­regulated kinase in the VEGF/VEGF receptor 2 (R2) signaling pathway. Furthermore, oral administration of TSAJ significantly inhibited tumor growth and tumor­induced angiogenesis, as well as the formation of functional vessels, in the Matrigel™ plug model. These results suggest that TSAJ may be a potential anti­angiogenic agent that targets the VEGF/VEGFR2 signaling pathway, and inhibits tumor­induced angiogenesis.

[A primary study of evolution of hepatitis B virus based on motif discovery].

Hepatitis B is a serious infectious disease worldwide, and hepatitis B virus (HBV) is the direct cause of this disease. In recent years, as an essential part of its evolutionary process, HBV mutation has been extensively studied domestically and globally. However, the study on the conserved sequences in HBV sequences is still in its infancy. In this study, we applied multiple EM for motif elicitation (MEME) algorithm to discover HBV motif and proposed a new metric, conservative index (CI), to carry out phylogenetic analysis based on HBV sequences. Then, the constructed phylogenetic tree was subjected to reliability assessment. The results demonstrated that the new metric CI combined with the MEME algorithm can effectively help to discover motifs in HBV sequences and construct a phylogenetic tree based on them and to analyze the evolutionary relationship between HBV sequences; in addition, the possible ancestral sequences of samples may be obtained by conservative analysis. The proposed method is valuable for the exploratory study on large HBV sequence data sets.

[Analysis of B/C recombinant structure of C gene in a newly found HBV subtype].

Hepatitis B is one of the most serious global threats to human health. Phylogenetic analysis of hepatitis B virus (HBV) can reveal the evolutionary relationship between HBV sequences and thus provide a basis for the prediction and treatment of hepatitis B and other aspects. In this study, we performed sequence analyses on the HBV sequences of five clinical HBV samples and the HBV sequences retrieved from the GenBank, EMBL, and DDBJ to construct a phylogenetic tree and analyze sequence structures. The experimental results revealed that the C gene of one cloned sequence had a recombinant structure of HBV B/ C subtype. Moreover, the phylogenetic results proved the existence of a newly found subtype HBV/B6 in Xishuangbanna of Yunnan Province, China. The experimental conclusion represents certain value for phylogenetic studies of HBV in Yunnan ethnic minority groups.

Lipid droplets may lay a spacial foundation for vasculogenic mimicry formation in hepatocellular carcinoma.

Vasculogenic mimicry is a highly patterned vascular channel distinguished from the endothelium-dependent blood vessel. Vasculogenic mimicry is lined by highly aggressive tumor cells, and is associated with tumor grade, invasion and metastasis, and poor clinical prognosis. Much attention has been focused on the signaling pathways and the tumor microenvironment needed for vasculogenic mimicry formation, however, the studies on the spacial foundation for vasculogenic mimicry formation are limited. There are many lipid droplets in hepatocellular carcinoma due to steatosis, while increased numbers of lipid droplets also have been reported in many other neoplastic processes. The role of lipid droplets in tumor is still unclear. Based on the similar structural and morphological characteristics between vasculogenic mimicry and lipid droplet, we speculate that the lipid droplets may lay a spacial foundation for vasculogenic mimicry formation by a way of "space placeholder" in HCC. Experimental data and limited clinical literatures support the hypothesis to a certain degree. This hypothesis may provide a new idea for the study of vasculogenic mimicry and also provide a new direction for the functional study of lipid droplets in tumor.