Quercetin Ameliorates Renal Injury and Pyroptosis in Lupus Nephritis through Inhibiting IL-33/ST2 Pathway In Vitro and In Vivo.

Lupus nephritis (LN) is a common and serious symptom in patients with systemic lupus erythematosus (SLE). Tubular interstitial fibrosis is a common underlying mechanism in the development of lupus nephritis to end-stage renal failure (ESRD). Quercetin is widely proven to prevent tissue fibrosis. Therefore, the purpose of this study is to investigate the beneficial effects of quercetin on the inhibition of fibrosis and inflammation pathways in in vitro and in vivo lupus nephritis models. In the current study, MRL/lpr mice as animal models, and HK-2 human renal tubular epithelial cells were stimulated by interleukin-33 (IL-33) to mimic the cellular model of lupus nephritis. Immunohistochemical staining, immunoblotting assay, immunofluorescence staining, and quantitative real-time polymerase chain reaction assay were used. The in vivo results showed that quercetin improved the renal function and inhibited both fibrosis- and inflammation-related markers in MRL/lpr mice animal models. The in vitro results indicated that quercetin ameliorated the accumulation of fibrosis- and inflammation-related proteins in IL-33-induced HK-2 cells and improved renal cell pyroptosis via the IL33/ST2 pathway. Overall, quercetin can improve LN-related renal fibrosis and inflammation, which may offer an effective potential therapeutic strategy for lupus nephritis.

Effects of Rice-Husk Silica Liquid in Streptozotocin-Induced Diabetic Mice.

Type 2 diabetes mellitus is a complex multifactorial disease characterized by poor glucose tolerance and insulin resistance. Rice-husk silica liquid (RHSL) derived from rice husk has the ability to improve the dysfunction of pancreatic ß-cells. This study aimed to confirm the potential protective effects of RHSL in streptozotocin (STZ)-induced diabetic mice. Diabetes was induced in male C57BL/6J mice by intraperitoneal administration of STZ (200 mg/kg BW). RHSL, food-grade silica liquid (FDSL), and rosiglitazone (RSG) were administered to diabetic mice for 12 weeks after successful induction of diabetes. During the experiment, fasting blood glucose, serum insulin, and organ weights were measured. The histopathology of liver tissue was evaluated by H&E staining. Western blotting was performed to assess protein expression levels. The results showed that RHSL significantly reversed the serum insulin levels and improved oral glucose tolerance test (OGTT) results (p < 0.05). In addition, liver sections of STZ-induced diabetic mice after RHSL treatment showed neatly arranged and intact hepatocytes. Furthermore, RHSL was more effective than FDSL in increasing the expression of SIRT1 and decreasing the expression of the PPAR-γ and p-NF-κB proteins. Taken together, this study demonstrated that RHSL ameliorated STZ-induced insulin resistance and liver tissue damage in C57BL/6J mice.

Spirulina phycocyanin extract and its active components suppress epithelial-mesenchymal transition process in endometrial cancer via targeting TGF-beta1/SMAD4 signaling pathway.

Metastasis is a major challenge in aggressive endometrial cancer treatment accounting for the high recurrence risk and poor prognosis of epithelial-mesenchymal transition (EMT), regulated by the transforming growth factor beta (TGFß) signaling pathway, facilitates tumor metastasis. Spirulina phycocyanin extract (SPE) and its purified products allophycocyanin (APC) and C-phycocyanin (C-PC), derived from Spirulina platensis, can be considered a nutraceutical compound with the ability to inhibit tumor growth and metastasis. Current study aims to investigate the anti-metastatic potential of SPE, and its purified products APC, and C-PC on endometrial cancer both in vitro and in vivo. Firstly, human endometrial cancer cell lines (HEC-1A and Ishikawa) as an in vitro model. Secondly, HEC-1A cells transfected with luminescence gene were implanted into female nude mice as a xenograft model. MTT assay, transwell migration assay, immunoblotting assay, quantitative real-time polymerase chain reaction assay, and IVIS XRMS analysis techniques were used. The in vitro results showed that SPE and its purified products APC and C-PC inhibited cell migration, and altered the expression of EMT-related phenotypes by reversing the TGFß/SMADs signaling pathway. The in vivo results indicated that SPE repressed the metastasis of HEC-1A-LUC cells through modulating EMT-related markers expression. Overall, SPE and its efficient components APC and C-PC reversed the EMT through targeting the TGFß/SMADs signaling pathway, suggesting an effective therapeutic strategy for metastatic endometrial cancer.

Extraction, Biochemical Characterization, and Health Effects of Native and Degraded Fucoidans from Sargassum crispifolium.

In the current investigation, a native crude fucoidan (Ex) was extracted from Sargassum crispifolium, pretreated by single-screw extrusion, and two degraded fucoidans, i.e., ExAh (degradation of Ex by ascorbic acid) and ExHp (degradation of Ex by hydrogen peroxide), were obtained. The extrusion pretreatment increased the extraction yield of fucoidan by approximately 1.73-fold as compared to the non-extruded sample. Among Ex, ExAh, and ExHp, their molecular weight and chemical compositions varied, but the structural features were similar. ExHp possessed the greatest antioxidant activities among the extracted fucoidans. According to the outcome, ExAh exhibited the maximum immune promoting effects via enhanced NO, TNF-α, IL-1ß, IL-6, and IL-10 secretion. Thus, both ExHp and ExAh may potentially be used as an effective antioxidant and as immunostimulant agents, which could be of great value in the development of food and nutraceutical products.

The Role of Cell Proliferation and Extracellular Matrix Accumulation Induced by Food Additive Butylated Hydroxytoluene in Uterine Leiomyoma.

Leiomyoma is the most common benign uterine tumor in reproductive-age women. Increasing numbers of studies are focusing on the effects of environmental exposure on the incidence and progression of tumors. One major step taken in the food industry is the addition of food preservatives to maintain freshness. Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant, which is widely used as an additive to develop fat-soluble characteristics, as well as in cosmetics and rubber. Previous studies also highlighted that BHT may be related to increased fibrosis capacity and carcinogenic effects. In this study, we explored the effects of the commonly used food additive BHT on leiomyoma progression, and the related mechanism. The exposure of the ELT-3 leiomyoma cell line to BHT for 48 h increased the proliferative effect. Since leiomyoma progression is related to increases in extracellular matrix (ECM) accumulation and matrix metalloproteinase (MMP), BHT could effectively increase ECM-related protein expression, as well as MMP-2 and MMP-9 protein expression. This increase in ECM, in response to BHT, may be linked to the activation of the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling pathway. Through PI3K inhibition, BHT's effect on leiomyoma progression could be partially modulated. These results suggest the harmful effect of BHT exposure on leiomyoma progression may relate to PI3K modulation. However, an in vivo study is necessary to confirm these findings.

Rice Husk Silica Liquid Protects Pancreatic ß Cells from Streptozotocin-Induced Oxidative Damage.

Type 2 diabetes mellitus is a complex multifactorial disease characterized by insulin resistance and dysfunction of pancreatic ß-cells. Rice husk silica liquid (RHSL) is derived from rice husks and has not been explored in diabetes mellitus until now. Previous studies showed that rice husk is enriched with silica, and its silica nanoparticles are higher more biocompatible. To investigate the potential protective role of RHSL on pancreatic ß cells, we utilized RIN-m5F pancreatic ß cells and explored RHSL effect after streptozotocin (STZ)-stimulation. The recovery effects of RHSL were evaluated using flow cytometry, Western blotting, and immunofluorescence analysis. Results of our study showed that RHSL reversed the cell viability, insulin secretion, reactive oxygen species (ROS) production, and the change of mitochondria membrane potential (ΔΨm) in STZ-treated RIN-m5F cells. Moreover, the expression of phospho-receptor-interacting protein 3 (p-RIP3) and cleaved-poly (ADP-ribose) polymerase (PARP), phospho-mammalian target of rapamycin (p-mTOR), and sequestosome-1 (p62/SQSTM1) were significantly decreased, while the transition of light chain (LC)3-I to LC3-II was markedly increased after RHSL treatment in STZ-induced RIN-m5F cells. Interestingly, using autophagy inhibitors such as 3-methyladenine (3-MA) and chloroquine (CQ) both showed an increase in cleaved-PARP protein level, indicating apoptosis induction. Taken together, this study demonstrated that RHSL induced autophagy and alleviated STZ-induced ROS-mediated apoptosis in RIN-m5F cells.

Hinokitiol Exhibits Antitumor Properties through Induction of ROS-Mediated Apoptosis and p53-Driven Cell-Cycle Arrest in Endometrial Cancer Cell Lines (Ishikawa, HEC-1A, KLE).

Hinokitiol is a natural tropolone derivative that is present in the heartwood of cupressaceous plants, and has been extensively investigated for its anti-inflammatory, antioxidant, and antitumor properties in the context of various diseases. To date, the effects of hinokitiol on endometrial cancer (EC) has not been explored. The purpose of our study was to investigate the anti-proliferative effects of hinokitiol on EC cells. Cell viability was determined with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the quantification of apoptosis and reactive oxygen species (ROSs) was performed by using flow cytometry, while protein expression was measured with the Western blotting technique. Hinokitiol significantly suppressed cell proliferation through the inhibition of the expression of cell-cycle mediators, such as cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the induction of the tumor suppressor protein p53. In addition, hinokitiol increased the number of apoptotic cells and increased the protein expression of cleaved-poly-ADP-ribose polymerase (PARP) and active cleaved-caspase-3, as well as the ratio of Bcl-2-associated X protein (Bax) to B-cell lymphoma 2 (Bcl-2). Interestingly, except for KLE cells, hinokitiol induced autophagy by promoting the accumulation of the microtubule-associated protein light chain 3B (LC3B) and reducing the sequestosome-1 (p62/SQSTM1) protein level. Furthermore, hinokitiol triggered ROS production and upregulated the phosphorylation of extracellular-signal-regulated kinase (p-ERK1/2) in EC cells. These results demonstrate that hinokitiol has potential anti-proliferative and pro-apoptotic benefits in the treatment of endometrial cancer cell lines (Ishikawa, HEC-1A, and KLE).

Physicochemical and Antioxidant Properties of Gelatin and Gelatin Hydrolysates Obtained from Extrusion-Pretreated Fish (Oreochromis sp.) Scales.

Fish gelatin and its hydrolysates exhibit a variety of biological characteristics, which include antihypertensive and antioxidant properties. In this study, fish gelatins were extracted from extrusion-pretreated tilapia scales, and then subjected to analyses to determine the physicochemical properties and antioxidant activity of the extracted gelatins. Our findings indicate that TSG2 (preconditioned with 1.26% citric acid) possessed the greatest extraction yield, as well as higher antioxidant activities compared with the other extracted gelatins. Hence, TSG2 was subjected to further hydrolyzation using different proteases and ultrafiltration conditions, which yielded four gelatin hydrolysates: TSGH1, TSGH2, TSGH3, and TSGH4. The results showed that TSGH4 (Pepsin + Pancreatin and ultrafiltration < 3000 Da) had a higher yield and greater antioxidant activity in comparison with the other gelatin hydrolysates. As such, TSGH4 was subjected to further fractionation using a Superdex peptide column and two-stage reverse-phase column HPLC chromatography, yielding a subfraction TSGH4-6-2-b, which possessed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the other fractions. Further LC-ESI/MS/MS analysis of TSGH4-6-2-b suggested two novel peptides (GYDEY and EPGKSGEQGAPGEAGAP), which could have potential as naturally-occurring peptides with antioxidant properties. These promising results suggest that these antioxidant peptides could have applications in food products, nutraceuticals, and cosmetics.

Protective Effects of Fucoxanthin on Hydrogen Peroxide-Induced Calcification of Heart Valve Interstitial Cells.

Cardiovascular diseases such as atherosclerosis and aortic valve sclerosis involve inflammatory reactions triggered by various stimuli, causing increased oxidative stress. This increased oxidative stress causes damage to the heart cells, with subsequent cell apoptosis or calcification. Currently, heart valve damage or heart valve diseases are treated by drugs or surgery. Natural antioxidant products are being investigated in related research, such as fucoxanthin (Fx), which is a marine carotenoid extracted from seaweed, with strong antioxidant, anti-inflammatory, and anti-tumor properties. This study aimed to explore the protective effect of Fx on heart valves under high oxidative stress, as well as the underlying mechanism of action. Rat heart valve interstitial cells under H2O2-induced oxidative stress were treated with Fx. Fx improved cell survival and reduced oxidative stress-induced DNA damage, which was assessed by cell viability analysis and staining with propidium iodide. Alizarin Red-S analysis indicated that Fx has a protective effect against calcification. Furthermore, Western blotting revealed that Fx abrogates oxidative stress-induced apoptosis via reducing the expression of apoptosis-related proteins as well as modulate Akt/ERK-related protein expression. Notably, in vivo experiments using 26 dogs treated with 60 mg/kg of Fx in combination with medical treatment for 0.5 to 2 years showed significant recovery in their echocardiographic parameters. Collectively, these in vitro and in vivo results highlight the potential of Fx to protect heart valve cells from high oxidative stress-induced damage.

Anti-Inflammatory and Anti-Hyperuricemic Effects of Chrysin on a High Fructose Corn Syrup-Induced Hyperuricemia Rat Model via the Amelioration of Urate Transporters and Inhibition of NLRP3 Inflammasome Signaling Pathway.

Hyperuricemia is the main cause of gout and involved in the occurrence of many other diseases such as hyperlipidemia and hypertension correlated with metabolic disorders. Chrysin is a flavonoid compound found naturally in honey, propolis, and mushrooms and has anti-inflammatory and antioxidant effects. However, its mechanism of action is not clear yet. This study investigated the mechanism of chrysin's anti-hyperuricemic effect in hyperuricemia-induced rats fed with high-fructose corn syrup. Orally administrated chrysin for 28 consecutive days effectively decreased uric acid by inhibiting the activity of xanthine oxidase (XO) in the liver. Moreover, chrysin markedly downregulated the protein expression of uric acid transporter 1 (URAT1) and glucose transporter type 9 (GLUT9) and upregulated the protein expression of organic anion transporter 1 (OAT1) and human ATP-binding cassette subfamily G-2 (ABCG2). In addition, chrysin showed prominent anti-oxidative and inflammatory effects as the malondialdehyde (MDA) and interleukin 1 beta (IL-1ß) concentration was reduced in both rat kidney and serum, which aligned with the inhibition of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway activation. Collectively, our results strongly suggest that chrysin exhibits potent anti-hyperuricemic and anti-inflammatory effects that may yield new adjuvant treatments for gout.

Effect of Oversulfation on the Composition, Structure, and In Vitro Anti-Lung Cancer Activity of Fucoidans Extracted from Sargassum aquifolium.

Intensive efforts have been undertaken in the fields of prevention, diagnosis, and therapy of lung cancer. Fucoidans exhibit a wide range of biological activities, which are dependent on the degree of sulfation, sulfation pattern, glycosidic branches, and molecular weight of fucoidan. The determination of oversulfation of fucoidan and its effect on anti-lung cancer activity and related signaling cascades is challenging. In this investigation, we used a previously developed fucoidan (SCA), which served as a native fucoidan, to generate two oversulfated fucoidan derivatives (SCA-S1 and SCA-S2). SCA, SCA-S1, and SCA-S2 showed differences in compositions and had the characteristic structural features of fucoidan by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) analyses. The anticancer properties of SCA, SCA-S1, and SCA-S2 against human lung carcinoma A-549 cells were analyzed in terms of cytotoxicity, cell cycle, Bcl-2 expression, mitochondrial membrane potential (MMP), expression of caspase-3, cytochrome c release, Annexin V/propidium iodide (PI) staining, DNA fragmentation, and the underlying signaling cascades. Our findings indicate that the oversulfation of fucoidan promotes apoptosis of lung cancer cells and the mechanism may involve the Akt/mTOR/S6 pathway. Further in vivo research is needed to establish the precise mechanism whereby oversulfated fucoidan mitigates the progression of lung cancer.

Isoliquiritigenin Reverses Epithelial-Mesenchymal Transition Through Modulation of the TGF-ß/Smad Signaling Pathway in Endometrial Cancer.

Endometrial cancer is a common gynecological cancer with a poor prognosis, mostly attributed to tumor metastasis. Epithelial-mesenchymal transition (EMT) can be mediated via transforming growth factor beta (TGF-ß) signaling pathway, facilitating the ability of cancer cell invasion and migration. Isoliquiritigenin (ISL) is a flavonoid derived from licorice with reported antineoplastic activities. This study aims to investigate the anti-metastatic potential of ISL on endometrial cancer both in vitro and in vivo. First, human endometrial cancer cell lines (HEC-1A, Ishikawa, and RL95-2) were treated with ISL and then subjected to functional assays such as migration assay as well as molecular analyses including immunoblotting, immunofluorescence and RT-qPCR. In addition, HEC-1A-LUC cells were implanted into female nude mice and treated with ISL by intraperitoneal injection for four weeks. Results showed that ISL inhibited cell migration and reversed the effect of TGF-ß on the expression of E-cadherin, N-cadherin, vimentin, α-SMA, p-Smad3, and TWIST1/2 In vitro. Interestingly, In vivo study revealed that ISL reduced peritoneal dissemination and serum level of TGF-ß1, as well as decreased the expression levels of N-cadherin, p-Smad2/3, TWIST1/2, while increased E-cadherin. Overall, ISL reverses the EMT through targeting the TGF-ß/Smad signaling pathway and features a potential therapeutic treatment for metastatic endometrial cancer.

Salvia sclarea L. Essential Oil Extract and Its Antioxidative Phytochemical Sclareol Inhibit Oxytocin-Induced Uterine Hypercontraction Dysmenorrhea Model by Inhibiting the Ca2+-MLCK-MLC20 Signaling Cascade: An Ex Vivo and In Vivo Study.

Salvia sclarea essential oil is used as an aromatic therapy for dysmenorrhea. Sclareol-one of the natural products isolated from S. sclarea-displays anti-inflammatory and antioxidant activities; however, researchers have not yet evaluated the mechanism related to the pain-relieving effect of sclareol. In the present study, we aimed to investigate the potential effect of sclareol in ex vivo and in vivo dysmenorrhea models, as well as its possible mechanism. In the ex vivo study of uterine tissue from Sprague Dawley (SD) rats, the uterine contraction amplitude was observed and recorded. In the in vivo study, we measured the uterine contraction pressure of SD rats and performed writhing tests on mice. The uterine tissues from the writhing test subjects were collected and analyzed by Western blot. The results demonstrated that sclareol inhibited prostaglandin (PG) F2α-, oxytocin-, acetylcholine-, carbachol-, KCl-, and Bay K 8644-induced uterine contraction and possessed an analgesic effect in the writhing test. Sclareol affects the Ca2+ level and regulates oxytocin receptor (OTR), myosin light chain kinase (MLCK), extracellular signal-regulated kinase, p-p38, cyclooxygenase-2 (COX-2), and phospho-myosin light chain 20 (p-MLC20) protein expression. Integrating these results, we suggest that sclareol is a potential alternative supplement for dysmenorrhea.

Impacts of Endocrine Disruptor di-n-Butyl Phthalate Ester on Microalga Chlorella vulgaris Verified by Approaches of Proteomics and Gene Ontology.

Di-n-butyl phthalate (DBP) is an extensively used plasticizer. Most investigations on DBP have been concentrated on its environmental distribution and toxicity to humans. However, information on the effects of plasticizers on algal species is scarce. This study verified the impacts of endocrine disruptor di-n-butyl phthalate ester on microalga Chlorella vulgaris by approaches of proteomics and gene ontology. The algal acute biotoxicity results showed that the 24h-EC50 of DBP for C. vulgaris was 4.95 mg L-1, which caused a decrease in the chlorophyll a content and an increase in the DBP concentration of C. vulgaris. Proteomic analysis led to the identification of 1257 C. vulgaris proteins. Sixty-one more proteins showed increased expression, compared to proteins with decreased expression. This result illustrates that exposure to DBP generally enhances protein expression in C. vulgaris. GO annotation showed that both acetolactate synthase (ALS) and GDP-L-fucose synthase 2 (GER2) decreased more than 1.5-fold after exposure to DBP. These effects could inhibit both the valine biosynthetic process and the nucleotide-sugar metabolic process in C. vulgaris. The results of this study demonstrate that DBP could inhibit growth and cause significant changes to the biosynthesis-relevant proteins in C. vulgaris.

Degradation of Sargassum crassifolium Fucoidan by Ascorbic Acid and Hydrogen Peroxide, and Compositional, Structural, and In Vitro Anti-Lung Cancer Analyses of the Degradation Products.

Fucoidans possess multiple biological functions including anti-cancer activity. Moreover, low-molecular-weight fucoidans are reported to possess more bioactivities than native fucoidans. In the present study, a native fucoidan (SC) was extracted from Sargassum crassifolium pretreated by single-screw extrusion, and three degraded fucoidans, namely, SCA (degradation of SC by ascorbic acid), SCH (degradation of SC by hydrogen peroxide), and SCAH (degradation of SC by ascorbic acid + hydrogen peroxide), were produced. The extrusion pretreatment can increase the extraction yield of fucoidan by approximately 4.2-fold as compared to the non-extruded sample. Among SC, SCA, SCH, and SCAH, the chemical compositions varied but structural features were similar. SC, SCA, SCH, and SCAH showed apoptotic effects on human lung carcinoma A-549 cells, as illustrated by loss of mitochondrial membrane potential (MMP), decreased B-cell leukemia-2 (Bcl-2) expression, increased cytochrome c release, increased active caspase-9 and -3, and increased late apoptosis of A-549 cells. In general, SCA was found to exhibit high cytotoxicity to A-549 cells and a strong ability to suppress Bcl-2 expression. SCA also showed high efficacy to induce cytochrome c release, activate caspase-9 and -3, and promote late apoptosis of A-549 cells. Therefore, our data suggest that SCA could have an adjuvant therapeutic potential in the treatment of lung cancer. Additionally, we explored that the Akt/mammalian target of rapamycin (mTOR) signaling pathway is involved in SC-, SCA-, SCH-, and SCAH-induced apoptosis of A-549 cells.

Dietary Compound Isoliquiritigenin, an Antioxidant from Licorice, Suppresses Triple-Negative Breast Tumor Growth via Apoptotic Death Program Activation in Cell and Xenograft Animal Models.

Patients with triple-negative breast cancer have few therapeutic strategy options. In this study, we investigated the effect of isoliquiritigenin (ISL) on the proliferation of triple-negative breast cancer cells. We found that treatment with ISL inhibited triple-negative breast cancer cell line (MDA-MB-231) cell growth and increased cytotoxicity. ISL reduced cell cycle progression through the reduction of cyclin D1 protein expression and increased the sub-G1 phase population. The ISL-induced apoptotic cell population was observed by flow cytometry analysis. The expression of Bcl-2 protein was reduced by ISL treatment, whereas the Bax protein level increased; subsequently, the downstream signaling molecules caspase-3 and poly ADP-ribose polymerase (PARP) were activated. Moreover, ISL reduced the expression of total and phosphorylated mammalian target of rapamycin (mTOR), ULK1, and cathepsin B, whereas the expression of autophagic-associated proteins p62, Beclin1, and LC3 was increased. The decreased cathepsin B cause the p62 accumulation to induce caspase-8 mediated apoptosis. In vivo studies further showed that preventive treatment with ISL could inhibit breast cancer growth and induce apoptotic and autophagic-mediated apoptosis cell death. Taken together, ISL exerts an effect on the inhibition of triple-negative MDA-MB-231 breast cancer cell growth through autophagy-mediated apoptosis. Therefore, future studies of ISL as a supplement or alternative therapeutic agent for clinical trials against breast cancer are warranted.

Dietary Glycotoxins, Advanced Glycation End Products, Inhibit Cell Proliferation and Progesterone Secretion in Ovarian Granulosa Cells and Mimic PCOS-like Symptoms.

Women with polycystic ovary syndrome (PCOS) have been reported to have an elevated serum advanced glycation end product (AGE) level. However, the effect of AGEs on the pathophysiological ovarian granulosa cells of PCOS is still unclear. In this study, five indented BSA-derived AGE products were used to evaluate their effect on the function of human granulosa cells. We found that the proliferation of both primary human ovarian granulosa (hGC) cells and human granulosa-like tumor (KGN) cells were inhibited by treatment with these five AGE products. The progesterone secretion level was also reduced in both hGC and KGN cells by treatment with these AGE products through downregulation of LH receptor/cAMP regulatory activity. The granulosa cell layer and serum progesterone level were reduced in rats by treatment with MG-BSA; moreover, an increased number of follicle cysts and an irregular estrous cycle were observed. MG-BSA treatment had a similar effect on the phenotypes of the DHEA-induced PCOS model. Additionally, the insulin resistance and hepatic lesions seen in the DHEA-induced PCOS model were observed in the MG-BSA treatment group. Taken together, we found that AGEs exert a toxic effect on ovarian granulosa cells, ovarian morphology, and the estrous cycle that mimics the DHEA-induced PCOS phenotypes.

Natural Antioxidant Resveratrol Suppresses Uterine Fibroid Cell Growth and Extracellular Matrix Formation In Vitro and In Vivo.

Resveratrol (RSV) is a polyphenolic phytoalexin found in peanuts, grapes, and other plants. Uterine fibroids (UF) are benign growths that are enriched in extracellular matrix (ECM) proteins. In this study, we aimed to investigate the effects of RSV on UF using in vivo and in vitro approaches. In mouse xenograft models, tumors were implanted through the subcutaneous injection of Eker rat-derived uterine leiomyoma cells transfected with luciferase (ELT-3-LUC) in five-week-old female nude (Foxn1nu) mice. When the tumors reached a size of 50-100 mm3, the mice were randomly assigned to intraperitoneal treatment with RSV (10 mg·kg-1) or vehicle control (dimethyl sulfoxide). Tumor tissues were assayed using an immunohistochemistry analysis. We also used primary human leiomyoma cells as in vitro models. Cell viability was determined using the sodium bicarbonate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The protein expression was assayed using Western blot analysis. The messenger ribonucleic acid (mRNA) expression was assayed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Cell apoptosis was assayed using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) and Hoechst 33342 staining. RSV significantly suppressed tumor growth in vivo and decreased the proportion of cells showing expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA). In addition, RSV decreased the protein expression of PCNA, fibronectin, and upregulated the ratio of Bax (Bcl-2-associated X) and Bcl-2 (B-cell lymphoma/leukemia 2) in vivo. Furthermore, RSV reduced leiomyoma cell viability, and decreased the mRNA levels of fibronectin and the protein expression of collagen type 1 (COL1A1) and α-SMA (ECM protein marker), as well as reducing the levels of ß-catenin protein. RSV induced apoptosis and cell cycle arrest at sub-G1 phase. Our findings indicated the inhibitory effects of RSV on the ELT-3-LUC xenograft model and indicated that RSV reduced ECM-related protein expression in primary human leiomyoma cells, demonstrating its potential as an anti-fibrotic therapy for UF.

Fucoidan Inhibits the Proliferation of Leiomyoma Cells and Decreases Extracellular Matrix-Associated Protein Expression.

BACKGROUND/AIMS: Uterine leiomyomas (ULs) are benign uterine tumors, and the most notable pathophysiologic feature of ULs is excessive accumulation of extracellular matrix (ECM). Fucoidan is a polysaccharide extracted from brown seaweeds that has a wide range of pharmacological properties, including anti-fibrotic effects. We aimed to study the effect of fucoidan on the growth of ULs activated by transforming growth factor beta (TGFß). METHODS: We used ELT-3 (Eker rat leiomyoma tumor-derived cells) and HUtSMC (human uterine smooth muscle cells) as in vitro models. Cell viability was determined by the MTT assay. Cell colony formation was stained using crystal violet. The side population, cell cycle and apoptosis were analyzed using flow cytometry. Protein expression was assayed by western blot analysis. We also conducted in vivo experiments to confirm the inhibitory effects of fucoidan in nude mouse xenograft models. Tumor tissues were assayed by immunohistochemistry analysis. RESULTS: In our study, fucoidan caused a 50% growth inhibition using a dose of 0.5 mg/ml and decreased the stem cell activity after 48 h. In addition, fucoidan induced sub-G1 cell cycle arrest and apoptosis. Fucoidan down-regulated fibronectin, vimentin, α-SMA and the COL1A1 protein levels in TGFß3-induced ELT-3 cells. In the cellular mechanism, fucoidan abrogated TGFß3-induced levels of p-Smad2 and p-ERK1/2, as well as ß-catenin translocation into the nucleus. Furthermore, fucoidan suppressed xenograft tumor growth in vivo. CONCLUSION: Fucoidan displays anti-proliferation and anti-fibrotic effects and exerts protective effects against ULs development.

Characterization and Antioxidant and Angiotensin I-Converting Enzyme (ACE)-Inhibitory Activities of Gelatin Hydrolysates Prepared from Extrusion-Pretreated Milkfish (Chanos chanos) Scale.

Fish gelatin hydrolysates have been shown to possess various biological activities due to their unique Gly-Pro-Y and Gly-X-Hyp sequences. In the current study, fish gelatin was extracted from non-extruded milkfish scale (FSG1) or extrusion-pretreated milkfish scale (FSG2); extracted gelatins were hydrolyzed with different combinations of Flavourzyme and Alcalase to give four different hydrolysates, namely: FSGH1 (FSG1 hydrolyzed with Flavourzyme), FSGH2 (FSG1 hydrolyzed with Alcalase + Flavourzyme), FSGH3 (FSG2 hydrolyzed with Flavourzyme), and FSGH4 (FSG2 hydrolyzed with Alcalase + Flavourzyme). The extrusion-pretreatment process enhanced the extraction yield of gelatin from fish scale. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) analyses showed the extracts FSG1 and FSG2 possessed characteristics of gelatin. Moreover, the physicochemical characteristics of FSGH1⁻FSGH4 were examined by analyses of their degree of hydrolysis, amino acid composition, UV spectrum, FTIR spectrum, molecular weight, and RP-HPLC profile. Additional biological functional analyses showed that all of the studied gelatin hydrolysates FSGH1⁻FSGH4 possessed antioxidant activity dose-dependently as revealed by DPPH scavenging, ABTS scavenging, and reducing power analyses. In addition, FSGH2 and FSGH4 showed higher angiotensin-I-converting enzyme (ACE)-inhibitory activity as compared to FSGH1 and FSGH3. Taken together, FSGH2 and FSGH4 showed high antioxidant activity and potent anti-ACE activity. Due to the potential antioxidant and antihypertensive properties of FSGH2 and FSGH4, further research is needed to explore their possible use as natural supplementary raw materials in food and nutraceutical products.