Potential Mechanisms of Guizhi Fuling Wan in Treating Endometriosis: An Analysis Based on TCMSP and DisGeNET Databases.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Fuling Wan (GFW), is a traditional Chinese herbal formula that consists of Cinnamomi Ramulus (Guizhi), Poria Cocos(Schw.) Wolf. (Fuling), Persicae Semen (Taoren), Radix Paeoniae Rubra (Chishao), and Cortex Moutan (Mudanpi). This formula has been used in traditional Chinese medicine for more than 1800 years to treat disorders caused by stagnation of circulation and qi (air). AIM OF THE STUDY: Based on pre-clinical and clinical studies, this review aimed to reveal the potential mechanisms of GFW in inhibiting endometriosis. The enhancement of therapeutic effects of western medications on endometriosis by GFW was also shown. MATERIALS AND METHODS: A bibliographic assessment of publications on "Guizhi Fuling Wan" and "endometriosis" indexed in PubMed, Science Direct, and China National Knowledge Infrastructure (CNKI) was conducted. Five pre-clinical studies and 13 clinical studies were selected for this review. Moreover, the targeted molecules of each herb were first extracted from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform followed by obtaining the endometriosis-related genes from DisGeNET. Subsequently, pathway and gene ontology analyses using David Bioinformatics Resources explored the potential mechanisms of therapeutic effects of GFW in treating endometriosis. RESULTS: Pre-clinical and clinical studies showed that GFW might inhibit the growth of endometriotic lesion through the modulation of immunity, apoptosis-regulating molecules, and angiogenesis-associated factors, while enhancing the therapeutic effects of western medications in treating endometriosis. Furthermore, pathway and gene ontology analyses demonstrated that GFW might attenuate the disease primarily by affecting AGE-RAGE signaling pathway in diabetic complications (hsa04933) as well as pathways involved in Kaposi sarcoma-associated herpesvirus infection (hsa05167), human cytomegalovirus infection (has05163), and fluid shear stress and atherosclerosis (hsa05418). These pathways were all involved in the regulation of inflammation, angiogenesis, and apoptosis and commonly affected by all herbs. CONCLUSIONS: The current review revealed that endometriosis is highly associated with aberrant inflammatory, angiogenic, and apoptotic activities. The therapeutic effects of GFW on endometriosis are likely to act through regulating these activities.

[Total saponins of Panax japonicus alleviates CCl4-induced acute liver injury in rats by regulating the PI3K/AktNF-κB signaling pathway].

OBJECTIVE: To investigate the protective effect of total saponins of Panax japonicus (TSPJ) against CCl4-induced acute liver injury (ALI) in rats and explore the underlying pharmacological mechanisms. METHODS: Male SD rat models of CCl4-induced ALI were given intraperitoneal injections of distilled water, 100 mg/kg biphenyl bisabololol, or 50, 100, and 200 mg/kg TSPJ during modeling (n=8). Liver functions (AST, ALT, TBil and ALP) of the rats were assessed and liver pathologies were observed with HE staining. Immunohistochemistry was used to detect the expressions of PI3K/Akt/NF-κB signaling pathway molecules in liver tissue; ELISA was used to determine the levels of T-SOD, GSH-Px, and MDA. Western blotting was performed to detect the expression levels of PI3K-Akt and SIRT6-NF-κB pathways in the liver tissue. RESULTS: Network pharmacological analysis indicated that the key pathways including PI3K/Akt mediated the therapeutic effect of TSPJ on ALI. In the rat models of ALI, treatments with biphenyl bisabololol and TSPJ significantly ameliorated CCl4-induced increase of serum levels AST, ALT, ALP, TBil and MDA and decrease of T-SOD and GSH-Px levels (all P < 0.01). The rat models of ALI showed significantly increased expression of p-NF-κB (P < 0.01), decreased expressions of PI3K, p-Akt and SIRT6 proteins, and elevated expression levels of p-NF-κB, TNF-α and IL-6 proteins in the liver, which were all significantly improved in the treatment groups (P < 0.05 or 0.01). CONCLUSION: TSPJ can effectively alleviate CCl4-induced ALI in rats by suppressing inflammatory responses and oxidative stress in the liver via regulating the PI3K/Akt and SIRT6/NF-κB pathways.

Effects of dietary essential oil supplementation on growth performance, carcass yield, meat quality, and intestinal tight junctions of broilers with or without Eimeria challenge.

The effects of dietary supplementation of essential oil on growth performance, carcass yield, meat quality, serum antioxidant capacity, and intestinal tight junctions of broilers with or without Eimeria challenge were investigated. A total of 576 one-day-old male broilers were randomly separated into 8 treatments (6 replication floor-pens per treatment, 12 broilers per pen) in a 4 × 2 factorial design. The 4 diets consisted of 1) a corn and soybean meal basal diet, 2) an anticoccidial diet (60 g nicarbazin and 60 g narasin per ton of feed), 3) an oregano oil diet (500 ppm oregano oil), and 4) a clove oil diet (500 ppm clove oil). On d 10, half chicks were challenged with 1 × 104 sporulated oocysts of E. tenella, E. acervulina, and E. maxima per chick, whereas the others were inoculated with an equal amount of dilution (0.5 mL). The Eimeria challenge induced a higher fecal oocyst output on d 18, a lower duodenum Occludin expression level on d 28, a lower serum catalase level, and a higher cook loss and protein loss in thigh muscle on d 42. The anticoccidial diet lowered fecal Eimeria output and increased d 1 to 42 BW gain as compared to the control diet. The clove oil treatment enhanced duodenum ZO-1 expression level in nonchallenged birds, increased BW gain from d 1 to 14 and breast yield on d 42. The oregano oil treatment enhanced ZO-1 expression of challenged birds, reduced feed intake from 15 to 28 d, and helped broilers gain more tender meat. For those Eimeria-challenged broilers, both clove and oregano oil treatments recovered drip loss in breast muscle. Our results suggested that Eimeria challenge in broiler early age could interrupt later serum antioxidant capacity and damage meat quality. The dietary supplementation of clove or oregano essential oils could improve broiler growth performance and partially relieve the coccidial damage in gut integrity and meat quality.

Chondroitin Sulfate Enhances Proliferation and Migration via Inducing ß-Catenin and Intracellular ROS as Well as Suppressing Metalloproteinases through Akt/NF-κB Pathway Inhibition in Human Chondrocytes.

BACKGROUND: Chondroitin sulfate (CS) is found in humans' cartilage, bone, cornea, skin, and arterial wall. It consists of the foundation substance in the extracellular matrix (ECM) of connective tissue. The oral supplement form of CS is clinically used in treating osteoarthritis (OA). METHODS: Cell migration was observed by the transwell assay. The EMT, Akt/IKK/IκB pathways, TIMPs, collagen and MMPs in cell lysate were determined by Western blotting. The expression of MMP activity was determined by gelatin zymography. The production of reactive oxygen species (ROS) was determined by using a fluorescence spectrophotometer. RESULTS: In the current report, we demonstrated that CS can increase the cell proliferation and migration of chon-001 chondrocytes. Treatment with CS induced the epithelial-mesenchymal transition and increased the expression of type II collagen and TIMP-1/TIMP2 and inhibited the expressions and activities of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2). The phosphorylation of Akt, IκB kinase (IKK), IκB and p65 was decreased by CS. CS treatment resulted in ß-catenin production and XAV939, a ß-catenin inhibitor, and inhibited the cell proliferation by CS treatment. In addition, also significantly induced intracellular ROS generation. Treatment with antioxidant propyl gallate blocked cell migration induced by CS. CONCLUSION: We demonstrated that CS induced cell proliferation and migration of chondrocytes by inducing ß-catenin and enhancing ROS production. Moreover, our studies demonstrated that CS can increase the activity of chondrocytes and help patients with osteoarthritis to restore cartilage function.

Combinatory effects of current regimens and Guizhi Fuling Wan on the development of endometriosis.

OBJECTIVE: Endometriosis, defined as the growth of endometrial glands and stromal cells in a heterotopic location under the cyclic influence of ovarian hormones, is a common gynecological disorder manifested by chronic pelvic pain and infertility. In traditional Chinese medicine, endometriosis is characterized by stagnation of vital energy (qi) and blood stasis. Guizhi Fuling Wan (GFW) was first described in Chinese canonical medicine to treat disorders associated with stagnation of qi and blood stasis, including endometriosis. Therefore, the current study aimed to test the effects of combining GFW with western medicine on the suppression of endometriosis. MATERIALS AND METHODS: Endometriosis was generated by suturing endometrial tissue on the peritoneal wall of C57BL/6JNarl mice. The mice were subsequently treated with either GFW or current hormonal therapies or in combination for 28 days. RESULTS: Endometriosis development was inhibited by GFW, Gestrinone, Visanne, GFW + Gestrinone or GFW + medroxyprogesterone acetate (MPA). The expression of intercellular adhesion molecule 1 (ICAM-1) was inhibited by GFW, Gestrinone, MPA, Visanne, GFW + Gestrinone, GFW + MPA and GFW + Visanne. Vascular endothelial growth factor (VEGF) expression was inhibited by GFW, Gestrinone, Visanne, GFW + Gestrinone and GFW + MPA. Both ICAM-1- and VEGF-reducing effects of GFW were attenuated by western medicines. Administration of GFW, MPA, Visanne, GFW + MPA and GFW + Visanne also correspondingly reduced macrophage population in peritoneal fluid. GFW, MPA, Visanne, GFW + MPA and GFW + Visanne enhanced B-cell population in peritoneal fluid. CONCLUSION: The current study reveals the therapeutic effects of GFW on endometriosis. However, the combination of GFW and current hormonal therapies potentially impedes the efficacy of each individual agent in treating endometriosis.

Effects of Glycyrrhiza polysaccharide in diet on growth performance, serum antioxidant capacity, and biochemistry of broilers.

In the present study, we analyzed the effects of Glycyrrhiza polysaccharide (GCP) on growth performance, serum antioxidant capacity, and biochemistry of broilers. A total of 600, one-day-old AA broilers randomly divided into 5 treatment groups with 6 replicate pens of 20 birds per cage received dietary supplementation with GCP (0, 200, 500, 1,000, and 1,500 mg/kg) for 42 d. The supplementation of GCP linearly decreased (P < 0.05) feed conversion rate on day 22 to 42. Dietary supplementation with GCP reduced (P < 0.05) serum total cholesterol on day 21 and 42 and linearly improved (P < 0.05) albumin and high-density lipoprotein cholesterol. Dietary supplementation with 1,000 or 1,500 mg/kg GCP significantly increased (P < 0.05) serum total superoxide dismutase (T-SOD) activity on day 21 and 42 and reduced (P < 0.05) serum malondialdehyde content on 21 d. Dietary supplementation with 1,000 or 1,500 mg/kg GCP significantly improved (P < 0.05) interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ) expressions in liver on day 21 and 42. At the end of the experiment, we randomly selected 20 broilers from 3 treatment groups (0, 1,000, and 1,500 mg/kg), respectively, to perform an lipopolysaccharide (LPS)-induced acute stress experiment. The 60 broilers were divided into 6 treatment groups with 10 birds per cage. The experiment was designed as a 3 × 2 factorial arrangement with GCP (0, 1,000, or 1,500 mg/kg) and LPS (injection of saline or 1 mg/kg body weight) levels as treatments. When the grouping was finished, the broilers were immediately intraperitoneally injected with LPS or normal saline. Six hours after challenged, serum antioxidant and liver immunity were analyzed. The results showed that dietary GCP prevented LPS-induced reductions in T-SOD activity and increases in malonaldehyde content (P < 0.05). Also, dietary GCP supplementation mitigated the LPS-induced increase in IL-1ß and IFN-γ in the liver. Supplementation with 1,500 mg/kg GCP showed the most optimal effect in broilers. GCP has the potential to be used as feed additive in broilers.

Predictive Value of Serum Creatinine/Cystatin C in Acute Ischemic Stroke Patients under Nutritional Intervention.

BACKGROUND AND PURPOSE: As a very common risk of adverse outcomes of the ischemic stroke patients, sarcopenia is associated with infectious complications and higher mortality. The goal of this retrospective study is to explore the predictive value of serum Cr/CysC ratio in acute ischemic stroke patients receiving nutritional intervention. METHODS: We reviewed adult patients with AIS from December 2019 to February 2020. Patients with acute kidney injury were excluded and all patients received nutritional intervention during a 3-month follow-up period. We collected baseline data at admission including creatinine and cystatin C. The primary poor outcome was major disability (modified Rankin Scale score ≥ 4) at 3 months after AIS. RESULTS: A total of 217 patients with AIS were identified for this study. Serum Cr/CysC ratio was significantly correlated with NIHSS at discharge, 1-month modified Rankin Scale score, and 3-month modified Rankin Scale score. During 3 months, 34 (15.70%) patients had a poor outcome after AIS and 11 (5.10%) patients died within 30 days. In multivariable logistic regression analyses, serum Cr/CysC ratio at admission was independently associated with 3-month poor outcomes (OR: 0.953, 95% CI: 0.921-0.986, p = .006) and 30-day mortality (OR: 0.953, 95% CI: 0.921-0.986, p = .006). CONCLUSION: As a blood biochemical indexes reflecting the muscle mass and aiding in risk stratification, Cr/CysC ratio at admission could be used as a predictor of 30-day mortality and long-term poor prognosis in AIS patients.

[Screening of drugs that selectively inhibit uveal melanoma cells with SF3B1 mutations].

OBJECTIVE: To screen compounds that can selectively inhibit uveal melanoma cells with splicing factor 3B subunit 1 (SF3B1) mutations in comparison with isogenic SF3B1 wild-type counterparts in a cell model of SF3B1 mutant allele knockout. METHODS: Principal component analysis was used to analyze transcriptome alternative splicing in TCGA cohorts of uveal melanoma with wild-type SF3B1 and SF3B1 mutations, and abnormal alternative splicing events derived from SF3B1 mutations were identified. The SF3B1 mutant allele in Mel202 cells was knocked out using CRISPR-Cas9 technology, and Sanger sequencing was used to verify the edited sequence. MTT and colony formation assays were used to assess the proliferation of Mel202 and Mut-KO cells. RT-PCR agarose electrophoresis combined with Sanger sequencing was used to determine alternative splicing events in Mel202 and Mut-KO cells. MTT assay was performed to screen the compounds that showed selective inhibitory effect against Mel202 cells with SF3B1 mutation. RESULTS: Specific knockout of SF3B1 mutant allele in Mel202 cells obviously promoted the cell proliferation and caused changes in alternative splicing of ZDHHC16 and DYNLL1 transcripts. The screening data showed that 13 compounds had selective inhibitory activity against Mel202 cells with SF3B1 mutation (Fold change≥2), and among them, tetrandrine and lapatinib showed good dose-effect curves. CONCLUSION: This study provides a cell screening model for identification of potential individualized treatment drugs for patients with uveal melanoma with SF3B1 mutation.

Chrysophanol attenuates hepatitis B virus X protein-induced hepatic stellate cell fibrosis by regulating endoplasmic reticulum stress and ferroptosis.

Hepatitis B virus X protein (HBx) and hepatic stellate cells (HSCs) are critical for liver fibrosis development. Anti-fibrosis occurs via reversion to quiescent-type HSCs or clearance of HSCs via apoptosis or ferroptosis. We aimed to elucidate the role of chrysophanol in rat HSC-T6 cells expressing HBx and investigate whether chrysophanol (isolated from Rheum palmatum rhizomes) influences cell death via ferroptosis in vitro. Analysis of lipid reactive oxygen species (ROS), Bip, CHOP, p-IRE1α, GPX4, SLC7A11, α-SMA, and CTGF showed that chrysophanol attenuated HBx-repressed cell death. Chrysophanol can impair HBx-induced activation of HSCs via endoplasmic reticulum stress (ER stress) and ferroptosis-dependent and GPX4-independent pathways.

Glutaminase-1 (GLS1) inhibition limits metastatic progression in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is a malignant bone tumor that often develops during the period of rapid growth associated with adolescence. Despite successful primary tumor control accompanied by adjuvant chemotherapy, death from pulmonary metastases occurs in approximately 30% of patients within 5 years. As overall survival in patients remains unchanged over the last 30 years, urgent needs for novel therapeutic strategies exist. Cancer metastasis is characterized by complex molecular events which result from alterations in gene and protein expression/function. Recent studies suggest that metabolic adaptations, or "metabolic reprogramming," may similarly contribute to cancer metastasis. The goal of this study was to specifically interrogate the metabolic vulnerabilities of highly metastatic OS cell lines in a series of in vitro and in vivo experiments, in order to identify a tractable metabolically targeted therapeutic strategy for patients. METHODS: Nutrient deprivation and drug treatment experiments were performed in MG63.3, 143B, and K7M2 OS cell lines to identify the impact of glutaminase-1 (GLS1) inhibition and metformin treatment on cell proliferation. We functionally validated the impact of drug treatment with extracellular flux analysis, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry. 13C-glucose and 13C-glutamine tracing was employed to identify specific contributions of these nutrients to the global metabolic profiles generated with GLS1 inhibition and metformin treatment in vivo. RESULTS: Highly metastatic OS cell lines require glutamine for proliferation, and exposure to CB-839, in combination with metformin, induces both primary tumor growth inhibition and a distinct reduction in metastatic outgrowth in vivo. Further, combination-treated OS cells showed a reduction in cellular mitochondrial respiration, while NMR confirmed the pharmacodynamic effects of glutaminase inhibition in tumor tissues. We observed global decreases in glycolysis and tricarboxylic acid (TCA) cycle functionality, alongside an increase in fatty acid oxidation and pyrimidine catabolism. CONCLUSIONS: This data suggests combination-treated cells cannot compensate for metformin-induced electron transport chain inhibition by upregulating glutaminolysis to generate TCA cycle intermediates required for cell proliferation, translating into significant reductions in tumor growth and metastatic progression. This therapeutic approach could be considered for future clinical development for OS patients presenting with or at high risk of developing metastasis.

n-3 PUFA and caloric restriction diet alters lipidomic profiles in obese men with metabolic syndrome: a preliminary open study.

PURPOSE: For people with metabolic syndrome (MetS), altering the macronutrient composition of their diets might ameliorate metabolic abnormalities. The common method of clinical assessment only measures total lipid concentrations but ignores the individual species that contribute to these total concentrations. Thus, to predict the amelioration of MetS following caloric restriction (CR) and the intake of fish oil, we used lipidomics to investigate changes in plasma lipids and identify potential lipid metabolites. METHODS: Lipidomics was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry on plasma samples from a clinical trial conducted over 12 weeks. Subjects were randomized into two groups: CR (n = 12) and CR with fish oil (CRF, n = 9). Anthropometric and clinical parameters were measured and correlated with plasma lipidomics data. RESULTS: Compared with baseline, significant differences were observed in body weight, waist circumference, blood pressure and interleukin-6 in both groups, but triglyceride (TG) levels significantly decreased in only the CRF group (all p < 0.05). A total of 138 lipid species were identified. Levels of species containing long-chain polyunsaturated fatty acids were significantly elevated-greater than twofold-following fish oil intake, these included TG (60:9) and phosphatidylcholine (p40:6) (all q < 0.05). TG (60:9) tended to correlate negatively with body weight, body mass index, blood pressure, and HbA1c following fish oil intake. CONCLUSION: CR and fish oil can ameliorate MetS features, including anthropometric parameters, blood pressure, and blood lipid concentrations. The levels of particular lipid species such as TG-containing docosapentaenoic acid were elevated post-intervention and negatively associated with MetS features. TG (60:9) may be proposed as a lipid metabolite to predict amelioration in MetS following the intake of CR and fish oil.

A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice.

OBJECTIVE: We previously reported that genetic ablation of (Fibroblast Growth Factors Receptors) FGFR1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice, which suggests that FGFR1 is a potential targeting molecule for osteoarthritis (OA). Here, we identified R1-P1, an inhibitory peptide for FGFR1 and investigated its effect on the pathogenesis of OA in mice induced by destabilization of medial meniscus (DMM). DESIGN: Binding ability between R1-P1 and FGFR1 protein was evaluated by enzyme-linked immuno sorbent assay (ELISA) and molecular docking. Alterations in cartilage were evaluated histologically. The expression levels of molecules associated with articular cartilage homeostasis and FGFR1 signaling were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry (IHC). The chondrocyte apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. RESULTS: R1-P1 had highly binding affinities to human FGFR1 protein, and efficiently inhibited extracellular signal-regulated kinase (ERK)1/2 pathway in mouse primary chondrocytes. In addition, R1-P1 attenuated the IL-1ß induced significant loss of proteoglycan in full-thickness cartilage tissue from human femur head. Moreover, this peptide can significantly restore the IL-1ß mediated loss of proteoglycan and type II collagen (Col II) and attenuate the expression of matrix metalloproteinase-13 (MMP13) in mouse primary chondrocytes. Finally, intra-articular injection of R1-P1 remarkably attenuated the loss of proteoglycan and the destruction of articular cartilage and decreased the expressions of extracellular matrix (ECM) degrading enzymes and apoptosis in articular chondrocytes of mice underwent DMM surgery. CONCLUSIONS: R1-P1, a novel inhibitory peptide for FGFR1, attenuates the degeneration of articular cartilage in adult mice, which is a potential leading molecule for the treatment of OA.

Navel Orange Peel Essential Oil To Control Food Spoilage Molds in Potato Slices.

The aim of this study was to investigate the efficacy of navel orange, Citrus sinensis (L.) Osbeck, peel essential oil (NOPEO) for inhibiting spoilage fungi in potato slices. Sixteen different components accounting for 99.79% of the headspace components of NOPEO were identified by gas chromatography-mass spectrometry. d-Limonene was the major component of NOPEO. Antifungal activity of NOPEO was tested in vitro and in vivo against four foodborne fungi. A MIC of NOPEO against the four fungal species was 9.40 µL/mLair. NOPEO provided about 74, 74, 73, and 69% protection against Aspergillus niger, Mucor wutungkiao, Penicillium funiculosum, and Rhizopus oryzae at 2.00 µL/mLair concentration, respectively. NOPEO has been demonstrated to significantly improve the microbiological quality of potato slices.

Effects of extruded flaxseed on layer performance, nutrient retention and yolk fatty acid composition.

1. This study was conducted to determine the effects of feeding an extruded flaxseed (EF) on layer performance, apparent total tract nutrient retention (ATTR) and egg yolk fatty acid concentrations. 2. Seventy-two White Leghorn laying hens (58-week-old; three per cage) were randomly allotted to one of four dietary treatments: 0%, 7.5%, 15.0% and 22.5% of EF-supplemented diets for 8 weeks. 3. Supplementation with EF had no effect on feed intake, egg production, feed conversion ratio and egg weight. Egg components (yolk, albumen and shell percentages) were similar among treatments, except that shell percentage was greater for layers fed 22.5% EF than those fed 7.5% and 15% EF. The ATTR of dry matter and organic matter were highest for 0% and 7.5% EF, intermediate for 15% EF and lowest for 22.5% EF. Similar reductions on ATTR of crude protein and nitrogen-corrected apparent metabolisable energy were observed for layers fed 22.5% EF relative to those fed 0% or 7.5% EF. 4. Feeding EF at 7.5%, 15.0% and 22.5% of the diet markedly increased (by 92%, 198% and 271%, respectively) egg yolk concentrations of n-3 polyunsaturated fatty acid (PUFA) and reduced saturated fatty acid and n-6 PUFA concentrations. 5. It was concluded that omega-3 labelled eggs (300 mg/60 g of egg) may be produced with low (7.5% of diet) levels of dietary EF without compromising egg production parameters. However, feeding moderate to high levels of EF (i.e. 15% and 22.5% EF) may reduce total tract nutrient and energy utilisation.

A comparative scanning electron microscopy evaluation of smear layer removal with chitosan and MTAD.

The aim of the present study was to compare the efficacy of chitosan and MTAD for the smear layer removal from the root canal through a scanning electron microscope (SEM). Thirty teeth were randomly divided into three groups according to the final irrigants: 0.2% chitosan, MTAD, saline (control group). After the mechanical preparation, the samples were irrigated with saline (control group), 0.2% chitosan and MTDA respectively. Then, the samples were split and the smear layer at the apical, middle, and coronal thirds of each root canal was imaged using SEM. The statistical analysis was performed using the Kruskal-Wallis test and the Mann-Whitney U test (α = 5%). The difference between chitosan and MTDA was statistically significant in the apical region (p < 0.05), no significant difference was obtained in the coronal and middle regions in these two experiment groups (p > 0.05). The control group exhibited the lowest efficacy in smear layer removal in all regions. Thus, from the result of the present study, we may conclude that chitosan was more effective in smear layer removal than MTAD especially in the apical third. CONTEXT: Irrigation, which serves a variety of purposes including antibacterial action, tissue dissolution, cleaning and chelating, plays a centric role in the final success of root canal treatment. Thus, more and more attention has been put on the improvement and development of various irrigation techniques or systems. AIMS: The aim of the present study was to compare the efficacy of chitosan and MTAD for the smear layer removal from the root canal through scanning electron microscopy (SEM). SETTINGS AND DESIGN: Thirty single-canal premolars were instrumented with rotary-files and then, randomly assigned to test groups which were irrigated with chitosan and MTDA, and control group was treated with saline. Thereafter, the efficacy of smear layer removal was evaluated by SEM. MATERIALS AND METHODS: Thirty teeth were randomly divided into three groups according to the final irrigants: 0.2% chitosan, MTAD, saline (control group). After the mechanical preparation, the samples were irrigated with saline (control group), 0.2% chitosan and MTDA respectively. Then, the samples were split and the smear layer at the apical, middle, and coronal thirds of each root canal was imaged using SEM. STATISTICAL ANALYSIS USED: Kruskal-Walli test and Mann-Whitney U test Results: The difference between chitosan and MTDA was statistically significant in the apical regions (p < 0.05), no significant difference was obtained in the coronal and middle regions in these two experiment groups (p > 0.05). The control group exhibited the lowest efficacy in smear layer removal in all regions. CONCLUSION: Thus, from the result of present study, we may conclude that chitosan was more effective in smear layer removal than MTAD, especially in the apical third.

Celastrol, an active constituent of the TCM plant Tripterygium wilfordii Hook.f., inhibits prostate cancer bone metastasis.

BACKGROUND: Treatment failure of prostate cancer (PCa) is often due to bone metastasis. Celastrol, an active constituent of Tripterygium wilfordii roots, has shown anti-tumor effects in previous studies in accordance with its indication in traditional Chinese medicine. METHODS: Using a PC-3 cell model, in vitro assays were performed to evaluate the effects of celastrol on proliferation, migration (wound healing assay), tissues invasion (Transwell-Matrigel penetration assay) and vascular endothelial growth factor (VEGF) secretion (enzyme-linked immunosorbent assay). An intra-tibia injection mouse model was used to assess the effect of celastrol on PCa bone metastasis in vivo. RESULTS: Pretreatment with celastrol significantly reduced proliferation of PC-3 cells in a dose-dependent manner and cell migration was much slower than in controls. Significantly fewer cells penetrated the gel-membrane after celastrol administration and their skeletal invasive ability was significantly reduced in a dose-dependent manner. Correspondingly, a significant, dose-dependent decrease in VEGF secretion was observed. In the in vivo mouse model, pretreatment with celastrol (8 µmol l-1) inhibited the tumorigenicity of PC-3 cells so that almost no bone invasion occurred as compared with control injections. Histological examinations using hematoxylin and eosin staining showed that tibiae injected with celastrol pretreated PC-3 cells retained their natural bone structure. CONCLUSIONS: Celastrol may have preventive potential against PCa bone metastasis.

Saikosaponin a Induces Apoptosis through Mitochondria-Dependent Pathway in Hepatic Stellate Cells.

Saikosaponin a (SSa) is one of the main active components of Bupleurum falcatum. It is commonly used to treat liver injury and fibrosis in traditional Chinese medicine. Our previous study showed that SSa induces apoptosis and inhibits the proliferation of rat hepatic stellate cell (HSC) line HSC-T6. The aim of the present study was to elucidate the mechanism of SSa-mediated apoptosis. Rat HSC cell line HSC-T6 and human HSC cell line LX-2 were used in this study. SSa triggered cell death mainly by apoptosis, as indicated by the typical morphological changes, sub-G1 phase of cell cycle increase, and activation of the caspase-9/caspase-3 cascade. In addition, SSa-induced apoptosis was partially inhibited by the caspase-3 inhibitor Z-DEVD-FMK, suggesting an involvement of caspase-3 dependent and independent pathways. Moreover, SSa upregulated pro-apoptotic proteins [BAK, Bcl-2-associated death promoter (BAD), and p53 upregulated modulator of apoptosis (PUMA)] and downregulated anti-apoptotic proteins (Bcl-2). In the mitochondria, SSa triggered the translocation of BAX and BAK from the cytosol to the outer membrane, resulting in a reduction of mitochondrial functions and membrane potential and subsequent release of apoptotic factors. Therefore, this study demonstrates that SSa induces apoptosis through the intrinsic mitochondrial-dependent pathway in HSCs.

Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells.

BACKGROUND: Saikosaponin d (SSd) is one of the main active triterpene saponins in Bupleurum falcatum. It has a steroid-like structure, and is reported to have pharmacological activities, including liver protection in rat, cell cycle arrest and apoptosis induction in several cancer cell lines. However, the biological functions and molecular mechanisms of mammalian cells under SSd treatment are still unclear. METHODS: The cytotoxicity and apoptosis of hepatic stellate cells (HSCs) upon SSd treatment were discovered by MTT assay, colony formation assay and flow cytometry. The collage I/III, caspase activity and apoptotic related genes were examined by quantitative PCR, Western blotting, immunofluorescence and ELISA. The mitochondrial functions were monitored by flow cytometry, MitoTracker staining, ATP production and XF24 bioenergetic assay. RESULTS: This study found that SSd triggers cell death via an apoptosis path. An example of this path might be typical apoptotic morphology, increased sub-G1 phase cell population, inhibition of cell proliferation and activation of caspase-3 and caspase-9. However, the apoptotic effects induced by SSd are partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK, suggesting that SSd may trigger both HSC-T6 and LX-2 cell apoptosis through caspase-3-dependent and independent pathways. We also found that SSd can trigger BAX and BAK translocation from the cytosol to the mitochondria, resulting in mitochondrial function inhibition, membrane potential disruption. Finally, SSd also increases the release of apoptotic factors. CONCLUSIONS: The overall analytical data indicate that SSd-elicited cell death may occur through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian HSCs, and thus can delay the formation of liver fibrosis by reducing the level of HSCs.

No significant effects of Poly(I:C) on human umbilical cord-derived mesenchymal stem cells in the treatment of B6.MRL-Fas(lpr) mice.

OBJECTIVE: Our study aimed to compare the curative effect and immunoregulation between MSCs activated by Poly(I:C) for 24hours and unactivated MSCs on lupus mice. MATERIALS AND METHODS: MSCs were pretreated by Poly(I:C) at 50µg/mL for 24h. B6.MRL-Fas(lpr) mice were divided into UC-MSC treated group, FLS treated group, Poly(I:C) preconditioned MSC treated group (P-MSC) and untreated group randomly. All treated mice were infused with 1×10(6) MSCs or FLSs at the 24th week and were sacrificed 4 weeks later. The spleen weight, serum immunoglobulin G (IgG) levels, serum anti-double stranded DNA (anti-dsDNA) antibody levels, immune cell subsets, renal lesions and IgG deposition in the kidney were evaluated. The effects of two kinds of MSCs on the proliferation and apoptosis of CD4+ T cells were detected by flow cytometry. The TLR3 expression at protein level in MSCs was assessed with and without Poly(I:C) treatment. The expression of immunoregulatory factors were detected by qRT-PCR in different dose and duration of Poly(I:C). RESULT: Poly(I:C) preconditioned MSCs had similar therapeutic effects in lupus mice compared with untreated MSCs in vivo. Furthermore, Poly(I:C) treated MSCs and untreated MSCs had comparable inhibitory effects on proliferation of T cells, and Poly(I:C) could enhance the expression of TLR3 at protein and mRNA level. Poly(I:C) could partly alter the mRNA levels of immunoregulatory factors, such as hepatocyte growth factor, transforming growth factor ß1, vascular endothelial growth factor, but did not have significant changes in cyclooxygenase 2, interleukin 6, tumor necrosis factor α, indoleamine 2,3-dioxygenase, interferon γ and chemokine (C-C motif) ligand 2. CONCLUSION: Our study did not find that Poly(I:C) treatment could enhance the therapeutic effect of MSCs in lupus mice in vivo.