Comparison of the inhibition effects of naringenin and its glycosides on LPS-induced inflammation in RAW 264.7 macrophages.

BACKGROUND: Inflammation is intricately linked to the development of various diseases, such as diabetes, cardiovascular diseases, and cancer. Flavonoids, commonly found in plants, are known for their diverse health benefits, including antioxidant and anti-inflammatory properties. These compounds are categorized into different classes based on their chemical structure. structures. However, limited research has compared the effects of flavonoid aglycones and flavonoid glycosides. This study aims to assess the anti-inflammatory effects of naringenin and its glycosides (naringin and narirutin) in RAW264.7 macrophages. METHODS AND RESULTS: RAW264.7 cells were treated with naringenin, naringin, and narirutin, followed by stimulation with lipopolysaccharide. The levels of inflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), nitric oxide (NO), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), were assessed. Additionally, the study examined nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation using western blot analysis. Among the compounds tested, narirutin exhibited the most potent anti-inflammatory effect against TNF-α, NO, and iNOS. Naringin and narirutin showed comparable inhibitory effects on IL-1ß and COX-2. Both naringin and narirutin suppressed the expression of pro-inflammatory mediators by targeting different levels of the NF-κB and MAPK pathways. Naringenin demonstrated the weakest anti-inflammatory effect, primarily inhibiting NF-κB and reducing the phosphorylation levels of p38. CONCLUSIONS: This study suggests that the presence of glycosides on naringenin and the varied binding forms of sugars in naringenin glycosides significantly influence the anti-inflammatory effects compared with naringenin in RAW 264.7 macrophages.

Tea seed saponin­reduced extract ameliorates palmitic acid­induced insulin resistance in HepG2 cells.

Tea (Camellia sinensis) seed cake is a potential resource that contains a wealth of bioactive compounds. However, the high toxicity of tea saponins in tea seed cake restricts its applications. The present study aimed to i) develop a method of extracting bioactive compounds and reducing tea saponins during the process of tea seed cake extraction and ii) investigate the anti­insulin resistance effect of tea seed saponin­reduced extract (TSSRE) in a palmitic acid (PA)­induced insulin resistance HepG2­cell model. The concentration of tea saponins in TSSRE was ~10­fold lower than that in tea seed crude extract (TSCE) after the saponin­reduction process. In addition, TSSRE cytotoxicity was significantly lower than that of TSCE in HepG2 cells. TSSRE treatment improved glucose consumption as well as glucose transporter (GLUT) 2 and GLUT4 expression levels in PA­stimulated HepG2 cells. Moreover, TSSRE enhanced the phosphorylation of the insulin receptor substrate 1/protein kinase B/forkhead box protein O1/glycogen synthase kinase 3ß and inhibited the elevated expression of phosphoenolpyruvate carboxykinase in PA­exposed HepG2 cells. The effect of TSSRE on the mediation of the insulin signaling pathway was attributed to the inhibition of PA­induced mitogen­activated protein kinase activation. The findings of the present study indicated that TSSRE ameliorates hepatic insulin resistance by ameliorating insulin signaling and inhibiting inflammation-related pathways.

Repairing TALEN-mediated double-strand break by microhomology-mediated recombination in tobacco plastids generates abundant subgenomic DNA.

Transcription activator-like effector nuclease (TALEN) technology has been widely used to edit nuclear genomes in plants but rarely for editing organellar genomes. In addition, ciprofloxacin, commonly used to cause the double-strand break of organellar DNA for studying the repair mechanism in plants, confers no organellar selectivity and site-specificity. To demonstrate the feasibility of TALEN-mediated chloroplast DNA editing and to use it for studying the repair mechanism in plastids, we developed a TALEN-mediated editing technology fused with chloroplast transit peptide (cpTALEN) to site-specifically edit the rpoB gene via Agrobacteria-mediated transformation of tobacco leaf. Transgenic plants showed various degrees of chlorotic phenotype. Repairing damaged plastid DNA resulted in point mutation, large deletion and small inversion surrounding the rpoB gene by homologous recombination and/or microhomology-mediated recombination. In an albino line, microhomology-mediated recombination via a pair of 12-bp direct repeats between rpoC2 and ycf2 genes generated the chimeric ycf2-rpoC2 subgenome, with the level about 3- to 5-fold higher for subgenomic DNA than ycf2. Additionally, the expression of chimeric ycf2-rpoC2 transcripts versus ycf2 mRNA agreed well with the level of corresponding DNA. The ycf2-rpoC2 subgenomic DNA might independently and preferentially replicate in plastids.

Overexpression of a multifunctional ß-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter.

The ß-glucosidase, which hydrolyzes the ß(1-4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of ß-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including ß-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2-4. We overexpressed the ß-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with ß-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of ß-glucosidase in plants could further increase. The plant-expressed ß-glucosidase had optimal activity at 80 °C and pH 5-6. In addition, the plant-expressed ß-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with ß-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial ß-glucosidase gene as a reporter in plants based on alternative ß-galactosidase activity.

Flavonoids from Camellia sinensis (L.) O. Kuntze seed ameliorates TNF-α induced insulin resistance in HepG2 cells.

The aim of this study is to discuss the non-catechin flavonoids (NCF) from Camellia sinensis (L.) O. Kuntze seed improving TNF-α impaired insulin stimulated glucose uptake and insulin signaling. Flavonoids had anti-metabolic syndrome and anti-inflammatory properties. It had widely been known for biological activity of catechins in tea, but very few research reports discussed the biological activity of non-catechin flavonoids in tea seed. We used HepG2 cell to treat with 5 µM insulin or with 5 µM insulin + 30 ng/ml TNF-α. Detecting the glucose concentration of medium, insulin decreased the glucose levels of medium meant that insulin promoted glucose uptake into cells, but TNF-α inhibited the glucose uptake effect of insulin. Furthermore, insulin increased the protein expressions of IR, IRS-1, IRS-2, PI3K-α, Akt/PKB, GLUT-2, AMPK, GCK, pyruvate kinase, and PPAR-γ. TNF-α activated p65 and MAPKs (p38, JNK1/2 and ERK1/2), iNOS and COX-2 which worsened the insulin signaling expressions of IR, IRS-1, IRS-2, PI3K-α, Akt/PKB, GLUT-2, AMPK, GCK, pyruvate kinase, and PPAR-γ. We added NCF (500, 1000, 2000 ppm) to cell with insulin and TNF-α. Not only glucose levels of medium were lowered, and the protein expressions of insulin signaling were increased, but p38, JNK1/2, iNOS and COX-2 were also reduced. NCF could ameliorate TNF-α induced insulin resistance through inhibiting p38, JNK1/2, iNOS and COX-2, and suggested that it might be used in the future to help control insulin resistance. This finding is the first report to present the discovery.

PGBR extract ameliorates TNF-α induced insulin resistance in hepatocytes.

Pre-germinated brown rice (PGBR) could ameliorate metabolic syndrome, however, not much research estimates the effect of PGBR extract on insulin resistance. The aim of this study is to examine the effects of PGBR extract in TNF-α induced insulin resistance. HepG2 cells, hepatocytes, were cultured in DMEM medium and added with 5 µM insulin or with insulin and 30 ng/ml TNF-α or with insulin, TNF-α and PGBR extract (50, 100, 300 µg/ml). The glucose levels of the medium were decreased by insulin, demonstrating insulin promoted glucose uptake into cell. However, TNF-α inhibited glucose uptake into cells treated with insulin. Moreover, insulin increased the protein expressions of AMP-activated protein kinase (AMPK), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase-α (PI3K-α), serine/threonine kinase PI3K-linked protein kinase B (Akt/PKB), glucose transporter-2 (GLUT-2), glucokinase (GCK), peroxisome proliferator activated receptor-α (PPAR-α) and PPAR-γ. TNF-α activated p65 and MAPKs (JNK1/2 and ERK1/2) which worsened the expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, glycogen synthase kinase-3 (GSK-3), PPAR-α and PPAR-γ. Once this relationship was established, we added PGBR extract to cell with insulin and TNF-α. We found glucose levels of medium were lowered and that the protein expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, GSK-3, PPAR-α, PPAR-γ and p65, JNK1/2 were also recovered. In conclusion, this study found that TNF-α inhibited insulin stimulated glucose uptake and aggravated related proteins expressions, suggesting that it might cause insulin resistance. PGBR extract was found to ameliorate this TNF-α induced insulin resistance, suggesting that it might be used in the future to help control insulin resistance.

Argon-plasma-treated chitosan: surface characterization and initial attachment of osteoblasts.

Chitosan is a biocompatible, biodegradable and non-toxic material. It is also an advanced material used for wound dressings, drug and gene delivery, and tissue engineering. In this study, we treated chitosan film with argon plasma and characterized both its physical and chemical properties. We found that argon plasma increased nano-scale roughness and introduced a carbonyl group to the chitosan surface. Meanwhile, the contact angles also decreased as the duration of the plasma treatment on chitosan was prolonged, indicating that the increased roughness and carbonyl group promote the hydrophilicity of the chitosan film surface. Moreover, the number of osteoblasts adhering to chitosan films increased and their morphology became flatter with longer plasma treatments. In conclusion, this study first demonstrates that plasma treatment on chitosan exerting surface-property change is capable of triggering the initial attachment of osteoblasts to chitosan surfaces.

Expression of osteonectin/secreted protein acidic and rich in cysteine and matrix metalloproteinases in ameloblastoma.

BACKGROUND: Ameloblastoma is the most common clinically-significant epithelial odontogenic tumor, and is considered a benign but locally-aggressive tumor of the craniofacial region. Osteonectin/secreted protein acidic and rich in cysteine (SPARC) is induced in response to a number of biological processes such as tumor growth and metastasis, whereas matrix metalloproteinases (MMPs) degrade the extracellular matrix and participate in various biological processes including tumor invasion and metastasis. We hypothesize that SPARC acts with MMPs for the local invasiveness of ameloblastoma. The aim of this study was to examine the association of SPARC with MMP-1, MMP-2, and MMP-9 in ameloblastoma. METHOD: Immunohistochemical expression of SPARC, MMP-1, MMP-2, and MMP-9 as well as co-expression of SPARC and MMP-9 were examined in a cohort of 23 cases of ameloblastoma. RESULTS: SPARC, MMP-1, -2, and -9 were detected in the cytoplasm of the ameloblastic-like columnar cells and stellate-reticulum-like cells as well as in the stromal tissues of fibroblasts and endothelial cells of our cohort of ameloblastoma patients. Furthermore, co-expression of SPARC and MMP-9 were found in 23 cases of ameloblastoma. This may be the first study to demonstrate that the expression level of SPARC was statistically correlated with MMP-9 but not with MMP-1 or -2 in ameloblastoma. CONCLUSION: Our results suggest a putative association between SPARC and MMPs (especially MMP-9) in ameloblastoma to regulate tumor invasion.

Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma--in vitro and in vivo studies.

Regional lymph node and distant organ metastasis of oral squamous-cell carcinoma (OSCC) has been associated with increased production of matrix metalloproteases (MMPs), and scientific data showed that doxycycline (Dox) could down-regulate the expression of MMPs. The objective of this study was to evaluate the effect of Dox on the expression of MMPs in vitro using the SCC-15 cell line and in vivo SCC-15 xenografted nude mice. SCC-15 cells maintained under distinct culture conditions expressed high levels of pro-MMP-2 and pro-MMP-9; however, as determined by zymography and Western blot analysis, Dox significantly reduced the production of pro-MMP-2 and pro-MMP-9 after 24h of treatment in a dose-dependent manner (2.5-40 microg/ml). Dox (10 microg/ml) decreased the expression of MMP-9 mRNA but did not alter the level of MMP-2 mRNA after 24h of treatment. In addition, this drug significantly inhibited the invasive and migration activities of SCC-15 cells in vitro (>75% inhibition at 10 microg/ml). On the other hand, daily administration of Dox (3mg/mice) restrained tumor growth in SCC-15 xenografted nude mice, with an inhibition rate of 85.6%. Compared with the control group (treated with normal saline), MMP-9 mRNA levels in the fresh tumor tissue decreased upon Dox treatment (P<0.01) while MMP-2 mRNA levels were unchanged. In conclusion, reduced expression of MMP-9 at the transcriptional level and MMP-2 at the post-transcriptional level caused by Dox was found to be associated with decreased invasion of oral SCC in vitro. Moreover, Dox exerted a significant suppressive effect on tumor growth in an in vivo nude mice model. Taken together, these results, to our knowledge, may first imply that Doxycycline has an adjuvant therapeutic effect on OSCC that is associated with inhibition of MMPs expression.

A comparative study of the physical and mechanical properties of three natural corals based on the criteria for bone-tissue engineering scaffolds.

Coral has been used for bone grafts since 1970. Because coral has the advantages of good osteoconduction, biocompatibility, and biodegradation, it is also suitable for scaffolds used in bone-tissue engineering. However, the skeletons of different species of corals often vary significantly, and very few studies focus on the assessment of the permeability and mechanical properties of coral structure. In order to better understand the use of coral in bone tissue-engineering, we selected three typical models (Acropora sp., Goniopora sp., and Porites sp.) to analyze for pore size, porosity, permeability, and mechanical strength. We found Goniopora and Porites had homogenous structure and Acropora had oriented pores and irregular pore size. Acropora had the largest permeability, however, the transverse section was closed and the useful size was limited because of its habitat type. Porites had the smallest pore size and had the lowest permeability. Our data indicated that Goniopora sp. can be considered as the most promising source of scaffolds for bone-tissue engineering because of its high porosity (73%) and that its permeability and mechanics were similar to those in human cancellous bone. In conclusion, we analyzed the impact of the macroporous structure of coral on the permeability and mechanical properties that provide indicators for designing the optimal scaffold for bone-tissue engineering.

A novel control scheme for inducing angiostatin-human IgG fusion protein production using recombinant CHO cells in a oscillating bioreactor.

In this study, a novel control scheme for inducing protein production using a recombinant CHO cell line in a BelloCell bioreactor was developed. This control scheme was applied in a simple regular semi-batch process. Production of angiostatin-human IgG fusion protein in a suspension recombinant CHO cell culture and a protein-free medium was used for this study. The bottom holding time (BH) was the sole operating variable to control the exposure time of the cells immobilized on the carriers to the air and allow the nutrient remained on the liquid film of the carriers to be consumed to a threshold level so that the cells can be arrested and promoted for protein production. In the cell cultures with various BH (1.5-90 min), final cell densities of 1.6-4.0 x 10(9) have been obtained in 20 days while total angiostatin-human IgG production of 228-388 mg have been harvested. In general, low BH will minimize the nutrient limitation and favor the cell growth, while high BH will restrict the nutrient and promote the production in this type of non-growth associated production systems. It was found that specific production rate was generally inversely proportional to the specific growth rate. In this case of study, BH of 30 and 60 min were found to be about 72% better than BH of 1.5 min and 35% better than BH of 9 and 90 min in term of the total angiostatin-human IgG production. In comparison to a conventional spinner flask study, a 3.8-fold increase of the total angiostatin-human IgG production was realized in a 35-day culture. This study illustrated that a simple method of using BH in a semi-batch process can effectively control the apparent nutrient concentration to the cells, and thus regulate the cell growth and protein production in a novel oscillating bioreactor.

Bone tissue engineering evaluation based on rat calvaria stromal cells cultured on modified PLGA scaffolds.

Using natural materials to coat the scaffolds used for tissue-engineered bone-repair techniques is expected to increase osteoblast adhesion to the scaffold and to express normal physiological function. To test this hypothesis, we therefore modified poly(DL-lactic-co-glycolic acid) (PLGA) substrate by coating it with natural biomaterial solutions of collagen, chitosan, or N-succinyl-chitosan, and then used these three combinations as scaffolds to evaluate their effects on osteoblast attachment, proliferation, and differentiation. The results demonstrated that the pore size of scaffolds ranging from 125-500 microm did not affect the osteoblast phenotype; however, the surface modification of the scaffolds coated with these natural biomaterials did. Collagen increased cell attachment and proliferation, but chitosan and N-succinyl-chitosan decreased them. Chitosan and N-succinyl-chitosan increased differentiation, but collagen decreased it. These results provide us a new strategy for modifying microenvironments to increase osteoblast adhesion, proliferation, and differentiation on PLGA scaffolds, a strategy that might be useful for tissue regeneration.

Stereoselective esterase from Pseudomonas putida IFO12996 reveals alpha/beta hydrolase folds for D-beta-acetylthioisobutyric acid synthesis.

Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-beta-acetylthioisobutyrate (dl-MATI) to produce d-beta-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67 degrees C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the alpha/beta hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket approximately 12 A deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.

Preparation of macroporous biodegradable PLGA scaffolds for cell attachment with the use of mixed salts as porogen additives.

In the present study, a mixture of ammonium-bicarbonate (NH(4)HCO(3)) and sodium-chloride (NaCl) particles was used as a porogen additive to fabricate highly macroporous biodegradable poly(lactic-co-glycolic acid) (PLGA) scaffolds. A two-step salt-leaching process was performed after the sample had become semisolidified. Compared to the standard solvent-casting/particulate-leaching (SC/PL) technique, the processing time of this approach was significantly shorter: Instead of several days, only half a day was required. In addition, the polymer/salts/solvent mixture can be easily handled and molded into scaffolds of any specific shape-for example, thin sheet, cylindrical, or bone-shaped-for special applications in tissue engineering. Our results demonstrate that these scaffolds have a highly interconnected open-pore structure as well as greater mechanical properties than those made using the standard SC/PL technique. Primary rat osteoblasts seeded into the scaffolds exhibited good seeding efficiency. The method presented here is a promising approach for fabricating scaffolds for tissue engineering applications.