Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus.

The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side effect of this widely used antimalarial drug. Here, we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and coexpress gastrin-releasing peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with the MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.

A therapeutic ERCP in an only 5.9 kg infant with obstruction jaundice using JF-260V duodenoscope.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) has found extensive use in pediatric patients; however, challenges persist in the application of therapeutic ERCP in infants. CASE PRESENTATION: This case report details the presentation of a 5.9-kilogram infant with obstructive jaundice and suspected hemolytic anemia who underwent ERCP to alleviate biliary obstruction. The infant was admitted due to clay-colored stools, jaundice, and liver injury. Ultrasound and magnetic resonance cholangiopancreatography (MRCP) revealed dilation of the common bile duct (CBD) accompanied by the presence of stones. ERCP was conducted using a JF-260V duodenoscope under general anesthesia. Successful stone extraction and biliary drainage were achieved. CONCLUSIONS: In centers with considerable expertise in ERCP and pediatric anesthesia, the use of a conventional adult duodenoscope for therapeutic ERCP in infants can be considered safe and feasible, provided careful and stringent patient selection criteria are applied. In the future, clear guidelines and standardized protocols for the indications and procedures of pediatric ERCP should be established.

Clinical validation and utility of Chinese Eppendorf Itch Questionnaire in adults with chronic pruritus symptoms.

BACKGROUND: Pruritus, or itch, is a prevalent symptom causing profound health burden in many dermatological and non-dermatological disorders. Several itch questionnaires have been created to assess itch. Particularly, Eppendorf Itch Questionnaire (EIQ) is widely accepted since it encompasses various aspects of itch, including intensity, affects, coping behavior, and motivation to scratch. METHODS: In a cross-sectional survey, we examined the validity, reliability and clinical utility of Traditional Chinese EIQ. RESULTS: We administered the consensus version to 128 adults (median: 48.5 years, interquartile range [IQR]: 39-63) with active itch for more than 6 weeks at the Outpatient Clinics of three medical centers in Taiwan. Clinical diagnoses included psoriasis (N = 82), xerosis (N = 34), or other dermatitis (N = 12). Cronbach's alpha for each EIQ scale ranged 0.82-0.98, suggesting good to excellent internal consistency and reliability. Three EIQ scales significantly correlated with visual analogue scale (VAS) for itch intensity (P ≤ 0.001 for median test), supporting for its concurrent validity. None of EIQ scale was statistically correlated with Psoriasis Area Severity Index (PASI) scores in psoriasis patients, confirming its discriminant validity. Moreover, patients of different diagnoses had distinct responses to the multi-scale EIQ index, affording it a better clinical test (area-under-the-ROC curve [AUC]: 0.76, 95% CI: 0.63-0.90) than VAS alone (AUC: 0.42, 95%CI: 0.24-0.59) in distinguishing dermatitis/eczema-related itch from psoriasis or xerosis-related itch. CONCLUSION: We demonstrated the reliability and validity of Chinese EIQ in adult patients with chronic itch at the outpatient setting. The study also revealed the diversified aspects of itch across patients with various dermatoses.

LncRNA-PAGBC acts as a microRNA sponge and promotes gallbladder tumorigenesis.

Long noncoding RNAs (lncRNAs) play roles in the development and progression of many cancers; however, the contributions of lncRNAs to human gallbladder cancer (GBC) remain largely unknown. In this study, we identify a group of differentially expressed lncRNAs in human GBC tissues, including prognosis-associated gallbladder cancer lncRNA (lncRNA-PAGBC), which we find to be an independent prognostic marker in GBC Functional analysis indicates that lncRNA-PAGBC promotes tumour growth and metastasis of GBC cells. More importantly, as a competitive endogenous RNA (ceRNA), lncRNA-PAGBC competitively binds to the tumour suppressive microRNAs miR-133b and miR-511. This competitive role of lncRNA-PAGBC is required for its ability to promote tumour growth and metastasis and to activate the AKT/mTOR pathway. Moreover, lncRNA-PAGBC interacts with polyadenylate binding protein cytoplasmic 1 (PABPC1) and is stabilized by this interaction. This work provides novel insight on the molecular pathogenesis of GBC.

Lasting effects of general anesthetics on the brain in the young and elderly: "mixed picture" of neurotoxicity, neuroprotection and cognitive impairment.

General anesthetics are commonly used in major surgery. To achieve the depth of anesthesia for surgery, patients are being subjected to a variety of general anesthetics, alone or in combination. It has been long held an illusory concept that the general anesthesia is entirely reversible and that the central nervous system is returned to its pristine state once the anesthetic agent is eliminated from the active site. However, studies indicate that perturbation of the normal functioning of these targets may result in long-lasting desirable or undesirable effects. This review focuses on the impact of general anesthetic exposure to the brain and summarizes the molecular and cellular mechanisms by which general anesthetics may induce long-lasting undesirable effects when exposed at the developing stage of the brain. The vulnerability of aging brain to general anesthetics, specifically in the context of cognitive disorders and Alzheimer's disease pathogeneses are also discussed. Moreover, we will review emerging evidence regarding the neuroprotective property of xenon and anesthetic adjuvant dexmedetomidine in the immature and mature brains. In conclusion, "mixed picture" effects of general anesthetics should be well acknowledged and should be implemented into daily clinical practice for better patient outcome.

[Shenjing Guben Pills for repairing testicular spermatogenic injury induced by oxidative stress in rats].

OBJECTIVE: To investigate the antioxidative and spermatogenesis-repairing effects of Shenjing Guben Pills (SGP), a Chinese medicine for invigorating the kidney and blood circulation, on the testis, epididymis and sperm in rats with oxidative stress injury (OSI) induced by cadmium chloride. METHODS: Seventy-two male Wistar rats were equally randomized into six groups: normal control, OSI model control, Wuzi Yanzong Pills (WYP) and low-, medium- and high-dose SGP. The OSI model was made in the latter five groups by intraperitoneal injection of cadmium chloride at 1 mg/kg, and 24 hours later, the rats of the normal and model control groups treated intragastrically with 0.9% normal saline, those of the WYP group with WYP at 4.5 g/kg/d, and those of the low-, medium- and high-dose SGP groups with SGP at 2.8, 5.6 and 11.2 g/kg/d, respectively, all for 56 days. Then, all the animals were sacrificed for obtainment of the visceral indexes and histopathological changes of the testis, epididymis and seminal vesicle, measurement of sperm concentration and motility and the percentage of morphologically normal sperm (MNS) in the epididymis, and determination of the levels of glutathione perox-idase (GSH-PX), superoxide dismutase (SOD), malondial-dehyde aldehyde (MDA) and serum testosterone (T). RESULTS: Compared with the OSI model controls, the rats in the high-, medium- and low-dose SGP groups showed significantly higher visceral indexes of the testis (ï¼»0.237 ± 0.098ï¼½ vs ï¼»0.403 ± 0.090ï¼½, ï¼»0.357 ± 0.150ï¼½ and ï¼»0.348 ± 0.140ï¼½ g/100 g, P < 0.05) and seminal vesicle (ï¼»0.241 ± 0.118ï¼½ vs ï¼»0.347 ± 0.115ï¼½, ï¼»0.336 ± 0.090ï¼½ and ï¼»0.320 ± 0.065ï¼½ g/100 g, P < 0.05) and those of the high-dose SGP group in the epididymal index (ï¼»0.099 ± 0.088ï¼½ vs ï¼»0.156 ± 0.030ï¼½ g/100 g, P < 0.05). In comparison with the OSI model controls, the animals of the high-, medium- and low-dose SGP groups exhibited significant increases in sperm concentration (ï¼»10.5 ± 17.7ï¼½ vs ï¼»58.1 ± 32.2ï¼½, ï¼»36.0 ± 36.2ï¼½ and ï¼»31.9 ± 32.7ï¼½ ×106/ml, P < 0.05) and serum T (ï¼»2.56 ± 0.75ï¼½ vs ï¼»3.62 ± 0.96ï¼½, ï¼»3.48 ± 1.33ï¼½ and ï¼»3.24 ± 0.83ï¼½ nmol/L, P < 0.05 or P < 0.01), and those of the high- and medium-dose SCG groups in total sperm motility (ï¼»9.5 ± 13.0ï¼½% vs ï¼»26.5 ± 15.5ï¼½% and ï¼»18.9 ± 8.2ï¼½%, P < 0.05) and MNS (ï¼»36.2 ± 40.2ï¼½% vs ï¼»85.3 ± 23.3ï¼½% and ï¼»65.8 ± 28.1ï¼½%, P < 0.05) and the levels GSH-PX (ï¼»3.62 ± 2.22ï¼½ vs ï¼»5.70 ± 1.73ï¼½ and ï¼»5.42 ± 2.35ï¼½ U/mg prot, P < 0.05 ) and SOD (ï¼»41.3 ± 8.8ï¼½ vs ï¼»52.7 ± 14.6ï¼½ and ï¼»51.3 ± 14.7ï¼½ U/mg prot, P < 0.05). The MDA level, however, was markedly decreased in the high-, medium- and low-dose SGP groups (ï¼»0.41 ± 0.29ï¼½, ï¼»0.44 ± 0.19ï¼½ and ï¼»0.47 ± 0.20ï¼½ nmol/mg prot) as compared with that in the OSI model controls (ï¼»0.69 ± 0.28ï¼½ nmol/mg prot) (P < 0.05). Histopathological examinations manifested coagulative necrosis, calcification and disappearance of spermatogenic and Sertoli cells in the seminiferous tubules of the OSI model controls, with decreased intraluminal secretions and atrophic epithelial papillae in the seminal vesicles and non-sperm cells in the narrowed lumens of the atrophic epididymis. With the increased dose of SGP, the proportion of normal seminiferous tubules was enlarged, the epithelia of the seminal vesicle became column-shaped again, and the epididymal lumens grew lager with more sperm cells, which indicated a dose-dependent therapeutic efficacy. Medium- and high-dose SGP achieved a significantly better effect than WYP. CONCLUSIONS: Shenjing Guben Pills can antagonize oxidative stress, elevate the levels of testicular antioxidant enzymes and serum T, repair pathological injury of the testis, epididymis and seminal vesicle, and improve semen quality and spermatogenic function.

Dexmedetomidine attenuates spinal cord ischemia-reperfusion injury through both anti-inflammation and anti-apoptosis mechanisms in rabbits.

BACKGROUND: Dexmedetomidine (Dex) can improve neuronal viability and protect the spinal cord from ischemia-reperfusion (I/R) injury, but the underlying mechanisms are not fully understood. This study investigated the effects of dexmedetomidine on the toll-like receptor 4 (TLR4)-mediated nuclear factor κB (NF-κB) inflammatory system and caspase-3 dependent apoptosis induced by spinal cord ischemia-reperfusion injury. METHODS: Twenty-four rabbits were divided into three groups: I/R, Dex (10 µg/kg/h prior to ischemia until reperfusion), and Sham. Abdominal aortic occlusion was carried out for 30 min in the I/R and Dex groups. Hindlimb motor function was assessed using the Tarlov scoring system for gait evaluation. Motor neuron survival and apoptosis in the ventral grey matter were assessed by haematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling staining. The expression and localisation of ionised calcium-binding adaptor molecule 1, TLR4, NF-κB and caspase-3 were assessed by immunoreactivity analysis. The levels of interleukin 1ß and tumour necrosis factor α were assessed using enzyme-linked immunosorbent assays. RESULTS: Perioperative treatment with dexmedetomidine was associated with a significant preservation of locomotor function following spinal cord ischemia-reperfusion injury with increased neuronal survival in the spinal cord compared to control. In addition, dexmedetomidine suppressed microglial activation, inhibited the TLR4-mediated NF-κB signalling pathway, and inhibited the caspase-3 dependent apoptosis. CONCLUSIONS: Dexmedetomidine confers neuroprotection against spinal cord ischemia-reperfusion injury through suppression of spinal cord inflammation and neuronal apoptosis. A reduction in microglial activation and inhibition of both the TLR4-mediated NF-κB signalling pathway and caspase-3 dependent apoptosis are implicated.

Fecal bacteria from treatment-naive Crohn's disease patients can skew helper T cell responses.

Many studies have demonstrated that the inflamed mucosa of Crohn's disease (CD) patients presented a disturbed gut commensal community, and the shift in microbial composition and species variety is associated with disease severity. To establish a link between changes in the intestinal bacterial composition and the alteration of inflammation, we obtained fecal bacteria from CD patients and non-CD controls. The bacteria were then used to stimulate the peripheral blood mononuclear cells (PBMCs) from one non-CD individual. We found that the frequency of IFN-γ- and IL-17-expressing CD4 T cells was significantly higher after stimulation with CD bacteria than with non-CD bacteria, while the frequency of IL-4- and IL-10-expressing CD4 T cells was significantly decreased after stimulation with CD bacteria. A similar trend was observed in the level of cytokine expression and transcription expression. However, this difference was not clear-cut, as overlapping regions were observed between the two groups. With longer stimulation using CD bacteria, the skewing toward Th1/Th17 responses were further increased. This increase depended on the presence of monocytes/macrophages. Interestingly, we also found that B cells presented an inhibitory effect in CD bacteria-mediated skewing toward Th1/Th17 cells and promoted IL-10 secretion in CD bacteria-stimulated PBMCs. Together, our results demonstrated that CD bacteria could promote Th1/Th17 inflammation in a host factor-independent fashion.

Correction: Xiang, S., et al. Schisandrin B Induces Apoptosis and Cell Cycle Arrest of Gallbladder Cancer Cells. Molecules 2014, 19, 13235-13250.

We would like to change the Affiliation addresses on Page 13235 of paper [1], [...].

Total mesopancreas excision for pancreatic head cancer: analysis of 120 cases.

OBJECTIVE: To evaluate the feasibility and safety of total mesopancreas excision (TMpE) in the treatment of pancreatic head cancer. METHODS: The clinical and pathological data of 120 patients with pancreatic head cancer who had undergone TMpE in our center from May 2010 to January 2014 were retrospectively analyzed. RESULTS: The mean operative time was (275.0±50.2) min and the average intra-operative blood loss was (390.0±160.5) mL. Post-operative complications were reported in 45 patients, while no peri-operative death was noted. The specimen margins were measured in three dimensions, and 86 patients (71.6%) achieved R0 resection. CONCLUSIONS: TMpE is safe and feasible for pancreatic head cancer and is particularly helpful to increase the R0 resection rate.

SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway.

BACKGROUND: Gallbladder cancer (GBC) is a leading cause of cancer-related death worldwide, and its prognosis remains poor, with 5-year survival of approximately 5%. In this study, we analyzed the involvement of a novel proteoglycan, Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), in the tumor progression and prognosis of human GBC. METHODS: SPOCK1 expression levels were measured in fresh samples and stored specimens of GBC and adjacent nontumor tissues. The effect of SPOCK1 on cell growth, DNA replication, migration and invasion were explored by Cell Counting Kit-8, colony formation, EdU retention assay, wound healing, and transwell migration assays, flow cytometric analysis, western blotting, and in vivo tumorigenesis and metastasis in nude mice. RESULTS: SPOCK1 mRNA and protein levels were increased in human GBC tissues compared with those in nontumor tissues. Immunohistochemical analysis indicated that SPOCK1 levels were increased in tumors that became metastatic, compared with those that did not, which was significantly associated with histological differentiation and patients with shorter overall survival periods. Knockdown of SPOCK1 expression by lentivirus-mediated shRNA transduction resulted in significant inhibition of GBC cell growth, colony formation, DNA replication, and invasion in vitro. The knockdown cells also formed smaller xenografted tumors than control GBC cells in nude mice. Overexpression of SPOCK1 had the opposite effects. In addition, SPOCK1 promoted cancer cell migration and epithelial-mesenchymal transition by regulating the expression of relevant genes. We found that activation of the PI3K/Akt pathway was involved in the oncogenic functions of SPOCK1 in GBC. CONCLUSIONS: SPOCK1 activates PI3K/Akt signaling to block apoptosis and promote proliferation and metastasis by GBC cells in vitro and in vivo. Levels of SPOCK1 increase with the progression of human GBC. SPOCK1 acts as an oncogene and may be a prognostic factor or therapeutic target for patients with GBC.

Magnolol inhibits growth of gallbladder cancer cells through the p53 pathway.

Magnolol, the major active compound found in Magnolia officinalis has a wide range of clinical applications due to its anti-inflammation and anti-oxidation effects. This study investigated the effects of magnolol on the growth of human gallbladder carcinoma (GBC) cell lines. The results indicated that magnolol could significantly inhibit the growth of GBC cell lines in a dose- and time-dependent manner. Magnolol also blocked cell cycle progression at G0 /G1 phase and induced mitochondrial-related apoptosis by upregulating p53 and p21 protein levels and by downregulating cyclin D1, CDC25A, and Cdk2 protein levels. When cells were pretreated with a p53 inhibitor (pifithrin-a), followed by magnolol treatment, pifithrin-a blocked magnolol-induced apoptosis and G0 /G1 arrest. In vivo, magnolol suppressed tumor growth and activated the same mechanisms as were activated in vitro. In conclusion, our study is the first to report that magnolol has an inhibitory effect on the growth of GBC cells and that this compound may have potential as a novel therapeutic agent for the treatment of GBC.

CLIC1 overexpression is associated with poor prognosis in gallbladder cancer.

The chloride intracellular channel 1 (CLIC1) gene family is a recently identified class of Cl channel proteins. Although CLIC1 involvement is well established in some cancers such as gastric cancer and colon cancer, its expression pattern in gallbladder cancer (GBC) remains unknown. The aim of our study was to investigate the expression of CLIC1 in relation to progression and prognosis of GBC. Eight fresh gallbladder cancers paired with adjacent non-tumour tissues were quantified using real-time PCR and Western blot. Tissue samples from resected gallbladder cancer (n = 75) and cholelithiasis (n = 75) were evaluated for CLIC1 expression by immunohistochemical staining. Their expression was correlated with different clinicopathological parameters. CLIC1 expression was significantly higher (62.7 %) in gallbladder cancer than in cholelithiasis (21.3 %, p < 0.001). CLIC1 levels were associated with the histological grade, TNM stage and perineural invasion (p < 0.05), but not with patient age, sex, lymph node metastasis or gallbladder stones (p > 0.05). Univariate Kaplan-Meier analysis showed that a positive CLIC1 expression was associated with a decreased overall survival (p < 0.001). Multivariate Cox regression analysis showed that CLIC1 expression and histological grade were independent risk factors for overall survival. Therefore, the expression of CLIC1 is closely related to the progression of GBC and may be used as an effective marker for predicting the prognosis of this disease.

Evaluation of two inflammation-based prognostic scores in patients with resectable gallbladder carcinoma.

BACKGROUND: Survival after surgery for gallbladder cancer is generally poor. A number of inflammation-based prognostic scores have been established to help predict survival after surgery for several types of cancer. The objective of this study was to analyze and compare the utility of two inflammation-based prognostic scores, the Glasgow prognostic score (GPS) and the neutrophil-to-lymphocyte ratio (NLR), for predicting survival in patients with gallbladder cancer after surgery with curative intent. METHODS: We retrospectively reviewed the medical records of 85 patients with histologically confirmed, resectable gallbladder carcinoma (GBC), who were to receive curative surgery in our department. Univariate and multivariate analyses were performed to evaluate the relationship between the variables to overall survival (OS). RESULTS: A significant difference was detected in OS in patients with low and high GPS and NLR scores. Univariate analyses using clinicopathological characteristics revealed that tumor differentiation; tumor invasion; lymph node metastasis; tumor, node, metastasis classification system stage; positive margin status; combined common bile duct resection; serum levels of C-reactive protein, albumin, carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen, and CA125; white blood cell count; and GPS and NLR were all associated with OS. Among these characteristics, multivariate analysis demonstrated that a high GPS was independently associated with poorer OS, together with tumor invasion, lymph node metastasis, and positive margin status. CONCLUSIONS: GPS is superior to NLR with respect to its prognostic value for patients with GBC after surgery with curative intent. GPS is not only associated with tumor progression but is also an independent marker of poor prognosis.

Bufalin induces cell cycle arrest and apoptosis in gallbladder carcinoma cells.

Bufalin, a major digoxin-like immunoreactive component of the Chinese medicine Chan Su, has been shown to exert a potential for anticancer activity against various human cancer cell lines in vitro. However, no detailed studies have so far been reported on its action on human gallbladder carcinoma cells. In this study, bufalin remarkably inhibited growth in human gallbladder cancer cells by decreasing cell proliferation, inducing cell cycle arrest and apoptosis in a dose-dependent manner. Bufalin also disrupted the mitochondrial membrane potential (ΔΨm) and regulated the expression of cell cycle and apoptosis regulatory molecules. Activation of caspase-9 and the subsequent activation of caspase-3 indicated that bufalin may be inducing mitochondria apoptosis pathways. Intraperitoneal injection of bufalin for 3 weeks significantly inhibited the growth of gallbladder carcinoma (GBC-SD) xenografts in athymic nude mice. Taken together, the results indicate that bufalin may be a potential agent for the treatment of gallbladder cancer.

Ursolic acid induces cell cycle arrest and apoptosis of gallbladder carcinoma cells.

BACKGROUND: Ursolic acid (UA), a plant extract used in traditional Chinese medicine, exhibits potential anticancer effects in various human cancer cell lines in vitro. In the present study, we evaluated the anti-tumoral properties of UA against gallbladder carcinoma and investigated the potential mechanisms responsible for its effects on proliferation, cell cycle arrest and apoptosis in vitro. METHODS: The anti-tumor activity of UA against GBC-SD and SGC-996 cells was assessed using MTT and colony formation assays. An annexin V/PI double-staining assay was used to detect cell apoptosis. Cell cycle changes were detected using flow cytometry. Rhodamine 123 staining was used to assess the mitochondrial membrane potential (ΔΨm) and validate UA's ability to induce apoptosis in both cell lines. The effectiveness of UA in gallbladder cancer was further verified in vivo by establishing a xenograft GBC model in nude mice. Finally, the expression levels of cell cycle- and apoptosis-related proteins were analyzed by western blotting. RESULTS: Our results suggest that UA can significantly inhibit the growth of gallbladder cancer cells. MTT and colony formation assays indicated dose-dependent decreases in cell proliferation. S-phase arrest was observed in both cell lines after treatment with UA. Annexin V/PI staining suggested that UA induced both early and late phases of apoptosis. UA also decreased ΔΨm and altered the expression of molecules regulating the cell cycle and apoptosis. In vivo study showed intraperitoneally injection of UA can significantly inhibited the growth of xenograft tumor in nude mice and the inhibition efficiency is dose related. Activation of caspase-3,-9 and PARP indicated that mitochondrial pathways may be involved in UA-induced apoptosis. CONCLUSIONS: Taken together, these results suggest that UA exhibits significant anti-tumor effects by suppressing cell proliferation, promoting apoptosis and inducing 7cell cycle arrest both in vitro and in vivo. It may be a potential agent for treating gallbladder cancer.

Oridonin induces apoptosis and cell cycle arrest of gallbladder cancer cells via the mitochondrial pathway.

BACKGROUND: Gallbladder cancer is the most frequent malignancy of the bile duct with high aggressive and extremely poor prognosis. The main objective of the paper was to investigate the inhibitory effects of oridonin, a diterpenoid isolated from Rabdosia rubescens, on gallbladder cancer both in vitro and in vivo and to explore the mechanisms underlying oridonin-induced apoptosis and cell cycle arrest. METHODS: The anti-tumor activity of oridonin on SGC996 and NOZ cells was assessed by the MTT and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/PI double-staining and Hoechst 33342 staining assays. Loss of mitochondrial membrane potential was observed by Rhodamine 123 staining. The in vivo efficacy of oridonin was evaluated using a NOZ xenograft model in athymic nude mice. The expression of cell cycle- and apoptosis-related proteins in vitro and in vivo was analyzed by western blot analysis. Activation of caspases (caspase-3, -8 and -9) was measured by caspases activity assay. RESULTS: Oridonin induced potent growth inhibition, S-phase arrest, apoptosis, and colony-forming inhibition in SGC996 and NOZ cells in a dose-dependent manner. Intraperitoneal injection of oridonin (5, 10, or 15 mg/kg) for 3 weeks significantly inhibited the growth of NOZ xenografts in athymic nude mice. We demonstrated that oridonin regulated cell cycle-related proteins in response to S-phase arrest by western blot analysis. In contrast, we observed inhibition of NF-κB nuclear translocation and an increase Bax/Bcl-2 ratio accompanied by activated caspase-3, caspase-9 and PARP-1 cleavage after treatment with oridonin, which indicate that the mitochondrial pathway is involved in oridonin-mediated apoptosis. CONCLUSIONS: Oridonin possesses potent anti-gallbladder cancer activities that correlate with regulation of the mitochondrial pathway, which is critical for apoptosis and S-phase arrest. Therefore, oridonin has potential as a novel anti-tumor therapy for the treatment of gallbladder cancer.

Clinical and prognostic significance of preoperative plasma hyperfibrinogenemia in gallbladder cancer patients following surgical resection: a retrospective and in vitro study.

BACKGROUND: Coagulation and fibrinolysis activation is frequently observed in cancer patients, and the tumors in these cases are thought to be associated with a higher risk of invasion, metastasis, and worse long-term outcome. The objective of this study was to elucidate the prognostic significance of blood coagulation tests and various clinicopathological characteristics in patients with gallbladder cancer (GBC) after surgical resection. METHODS: We retrospectively reviewed the medical records of 115 patients with histologically confirmed GBC who underwent surgical resection in our department. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), international normalized ratio (INR), fibrinogen levels, and platelet counts were measured pretreatment at the time of diagnosis. The predictive value of fibrinogen levels for tumor staging was evaluated using a receiver operating characteristic (ROC) curve analysis. Correlations between the preoperative hyperfibrinogenemia and clinicopathological characteristics were analyzed, and univariate and multivariate survival analyses were performed to identify the factors associated with overall survival (OS). Cancer cell migration and invasion in vitro were examined to investigate the function of fibrinogen in GBC cell migration. RESULTS: The plasma levels for all coagulation tests, with the exception of INR, were significantly different between the GBC patients and control patients (p < 0.001). Hyperfibrinogenemia (>402 mg/dL) was associated with poorly differentiated tumors, advanced tumor invasion, lymphatic metastasis, and advanced tumor stage (p < 0.001), and had a statistically significant adverse effect on survival (p = 0.001). In the multivariate analysis, hyperfibrinogenemia (p = 0.031) was independently associated with worse OS, tumor stage (p = 0.016), margin status (p < 0.001), and lymphatic metastasis (p = 0.035). Moreover, cell migration and invasion in vitro were significantly enhanced by fibrinogen. CONCLUSIONS: Preoperative plasma fibrinogen levels was associated with tumor progression and may be an independent marker of poor prognosis in GBC patients. Furthermore, fibrinogen may contribute to cell migration by inducing epithelial-mesenchymal transition.

Effects of oxymatrine on the apoptosis and proliferation of gallbladder cancer cells.

Gallbladder carcinoma is the most common malignancy of the biliary tract and is associated with a very poor outcome. The aim of the present study was to investigate the effects of oxymatrine (OM) on gallbladder cancer cells and the possible mechanism of its effects. The effects of OM on the proliferation of gallbladder cancer cells (GBC-SD and SGC-996) were investigated using cell counting kit-8 and colony formation assays. Annexin V/propidium iodide double staining was performed to investigate whether OM could induce apoptosis in gallbladder cancer cells. The mitochondrial membrane potential (ΔΨm) and expression of apoptosis-associated proteins were evaluated to identify a mechanism for the effects of OM. In addition, the RNA expression of relevant genes was measured by qRT-PCR using the SYBR Green method. Finally, a subcutaneous implantation model was used to verify the effects of OM on tumor growth in vivo. We found that OM inhibited the proliferation of gallbladder cancer cells. In addition, Annexin V/propidium iodide double staining showed that OM induced apoptosis after 48 h and the ΔΨm decreased in a dose-dependent manner after OM treatment. Moreover, the activation of caspase-3 and Bax and downregulation of Bcl-2 and nuclear factor κB were observed in OM-treated cells. Finally, OM potently inhibited in-vivo tumor growth following subcutaneous inoculation of SGC-996 cells in nude mice. In conclusion, OM treatment reduced proliferation and induced apoptosis in gallbladder cancer cells, which suggests that this drug may serve as a novel candidate for adjuvant treatment in patients with gallbladder cancer.