An Innovative Model of ISS-Based Multiple Fractures and Gastrointestinal Dysfunction Related to c-Kit Protein Expression on Interstitial Cells of Cajal.

OBJECTIVE: Gastrointestinal dysfunction seriously affects the prognosis and quality of life of patients with multiple fractures. However, experimental evidence of this relationship is lacking. Here we describe a newly developed mouse model of postoperative gastrointestinal dysfunction after multiple fractures. METHODS: Trauma severity was assessed using the injury severity score (ISS). Based on the ISS, a multiple fracture model was established in mice as follows: limb fractures with pelvic fractures and multiple rib fractures; limb fractures with multiple rib fractures; closed fracture of both forelegs with pelvic fracture and rib fractures; closed limb fractures; limb fracture with pelvic fracture; spinal fractures; hind leg fractures with pelvic fractures; pelvic fracture with multiple rib fractures; closed fracture of both fore legs with pelvic fracture; and closed fracture of both fore legs with multiple rib fractures. In each model group, gastrointestinal motility was assayed and the histopathology of the small intestine was examined. Western blot and immunohistochemical analyses of jejunal tissue were performed to detect c-kit protein expression, the level of which was compared with that of a control group. The results of ANOVA are expressed as mean ± standard deviation. RESULTS: In mice with multiple fractures, food intake was greatly reduced, consistent with histopathological evidence of an injured intestinal epithelium. The jejunal tissue of mice in groups a, c, f, and h was characterized by extensively necrotic and exfoliated intestinal mucosal epithelium and inflammatory cell infiltration in the lamina propria. In the gastrointestinal function assay, gastrointestinal motility was significantly reduced in groups a, b, c, f, and g; these group also had a higher ISS (p < 0.01). The expression of c-kit protein in groups with gastrointestinal dysfunction was significantly up-regulated (p < 0.001) compared with the control group. The close correlation between c-kit expression and the ISS indicated an influence of trauma severity on gastrointestinal motility. CONCLUSION: Gastrointestinal dysfunction after multiple fractures was successfully reproduced in a mouse model. In these mice, c-kit expression correlated with gastrointestinal tissue dysfunction and might serve as a therapeutic target.

Urine Metabolomics Profiling of Lumbar Disc Herniation and its Traditional Chinese Medicine Subtypes in Patients Through Gas Chromatography Coupled With Mass Spectrometry.

Lumbar disc herniation (LDH) possesses complex pathogenesis, which has not been well elucidated yet. To date, specific or early diagnosis of LDH remains unavailable, resulting in missed opportunity for effective treatment. According to Traditional Chinese medicine (TCM) theory, LDH can be divided into two subtypes (reality syndrome and deficiency syndrome). The purpose of this study was to analyze the metabolic disorders of LDH and its TCM subtypes and screen out potential biomarkers for LDH diagnosis. Gas chromatography coupled with mass spectrometry (GC-MS) was applied to test the urine samples from 66 participants (30 healthy volunteers, 18 LDH patients with deficiency syndrome and 18 patients with reality syndrome). PCA analysis showed a distinct separation tendency between the healthy subjects and LDH patients but no obvious separation between the different syndromes (reality syndrome and deficiency syndrome) of LDH patients. As a result, 23 metabolites were identified significantly altered in the LDH patients, as compared with the healthy subjects. The altered metabolites belong to amino acid metabolism, nucleic acid metabolism, carbohydrate metabolism, and vitamin metabolism, which are related to osteoporosis and inflammation. Our results indicate metabolic disorders of LDH and thereby propose a group of metabolic biomarkers for potential application in early diagnosis of LDH in clinic, which provide a reasonable explanation for the pathogenesis of LDH.

Anti-Proliferative, Pro-Apoptotic, Anti-Migrative and Tumor-Inhibitory Effects and Pleiotropic Mechanism of Theaflavin on B16F10 Melanoma Cells.

PURPOSE: Theaflavin (TF) is a primary pigment of tea, exhibiting anti-proliferative, pro-apoptotic and anti-metastatic activities on cancer cell lines. However, it is unknown whether TF is effective in treating melanoma cells. METHODS: To determine the effects of TF on melanoma cells, we conducted in vitro assays of cell viability, DAPI staining, wound healing, transwell, and flow cytometry as well as in vivo experiments on B16F10-bearing mouse model. Real-time PCR (qPCR) and Western blot (WB) were conducted to explore the molecular actions of TF. RESULTS: The cell viability assay showed that TF exerted inhibitory effect on B16F10 cells in a dose-dependent manner from 40 to 400 µg/mL, with IC50 values ranging from 223.8±7.1 to 103.7±7.0 µg/mL. Moreover, TF induced early and late apoptosis and inhibited migration/invasion of B16F10 cells in a dose-dependent manner, indicating its pro-apoptotic and anti-migrative effects. In vivo, TF significantly inhibited B16F10 tumor size in mice model from 40 to 120 mg/kg, which exerted higher effect than that of cisplatin. The molecular data showed that TF significantly up-regulated the mRNA expressions of pro-apoptotic genes (Bax, Casp3, Casp8, c-fos, c-Jun, and c-Myc), up-regulated the protein expressions of apoptosis-related p53 and JNK signaling molecules (ASK1, phosphorylated Chk1/2, cleaved caspase 3, phosphorylated JNK, c-JUN, cleaved PARP, and phosphorylated p53), and down-regulated the protein expressions of proliferation-related MEK/ERK and PI3K/AKT signaling molecules (phosphorylated MEK1/2, phosphorylated ERK1/2, phosphorylated PI3K, and phosphorylated AKT) as well as the expressions of MMP2 and MMP9. CONCLUSION: It can be concluded that TB exhibited anti-proliferative, pro-apoptotic, anti-migrative, and tumor-inhibitory effects on melanoma cells through pleiotropic actions on the above pathways. This study provides new evidence of anti-melanoma efficacy and mechanism of TF, contributing to the development of TF-derived natural products for melanoma therapy.

Theaflavin Induces Apoptosis of A375 Human Melanoma Cells and Inhibits Tumor Growth in Xenograft Zebrafishes Through P53- and JNK-Related Mechanism.

Theaflavin (TF) is a major active pigment and polyphenol of tea, possessing anti-cancer activities. However, little is known about its activity and mechanism on melanoma cells. To fill this gap, we conducted in vitro experiments (cell viability assay, morphology observation, DAPI staining, and flow cytometry) and in vivo experiment by using a xenograft model of larval zebrafishes. Real-time PCR (qPCR) and Western blot (WB) analyses were conducted to explore the mechanism of TF. The in vitro data showed that TF exerted significant anti-proliferative and pro-apoptotic effects on A375 cells in a concentration-dependent manner. In vivo, TF significantly inhibited A375 tumor growth in larval zebrafishes at 0.67 and 2.0 µg/ml (1.3 to 3.9 µM). qPCR and WB data showed that TF significantly activated the P53 pathway-related proteins (ATM, CHK1/2, P53, and CASP8/3) and the JNK pathway-related proteins (ASK1, JNK, and C-JUN) through phosphorylation and cleavage, followed by activation of pro-apoptotic molecules (PARP, BAX, BIM, PUMA, and P53). In sum, TF possessed cytotoxic pro-apoptotic and tumor-inhibitory effects on A375 cells through activations of P53 and JNK pathways. This is the first report on TF regarding its effects and mechanism on A375 cells, making it a promising candidate of natural products for clinical treatment of melanoma.

Genetic diversity and population history of golden monkeys (Rhinopithecus roxellana).

Golden monkey (Rhinopithecus roxellana), namely the snub-nosed monkey, is a well-known endangered primate, which distributes only in the central part of mainland China. As an effort to understand the current genetic status as well as population history of this species, we collected a sample of 32 individuals from four different regions, which cover the major habitat of this species. Forty-four allozyme loci were surveyed in our study by allozyme electrophoresis, none of which was found to be polymorphic. The void of polymorphism compared with that of other nonhuman primates is surprising particularly considering that the current population size is many times larger than that of some other endangered species. Since many independent loci are surveyed in this study, the most plausible explanation for our observation is that the population has experienced a recent bottleneck. We used a coalescent approach to explore various scenarios of population bottleneck and concluded that the most recent bottleneck could have happened within the last 15,000 years. Moreover, the proposed simulation approach could be useful to researchers who need to analyze the non- or low-polymorphism data.