[Shikonin induces cell death by inhibiting glycolysis in human testicular cancer I-10 and seminoma TCAM-2 cells].

OBJECTIVE: To investigate the pattern of shikonin-induced cell death in testicular cancer cell I-10 and seminoma TCAM-2 cells and explore the possible mechanism in light of mitochondrial function and glycolysis. METHODS: I-10 cells treated with 0, 1.2, 1.4 and 1.6 µmol/L shikonin and TCAM-2 cells treated with 0, 0.5, 1 and 1.5 µmol/L shikonin were examined for mitochondrial membrane potential and production of reactive oxygen species (ROS) using JC-1 kit and ROS kit, respectively. The levels of intracellular lactic acid in the cells were detected using a lactic acid kit. The inhibitory effect of shikonin on the proliferation of the cells was assessed with MTT assay. The death patterns of the cells were observed by transmission electron microscopy, and annexin V-FITC/PI double staining was used to detect cell apoptosis. Western blotting was used to detect the relative expression levels of the apoptotic proteins Bax, Bcl-2, and cleaved caspase-3, the autophagy- related protein LC3B and glycolysis- related proteins PKM2, GLUT1 and HK2. RESULTS: MTT assay showed that shikonin significantly inhibited the proliferation of I-10 and TCAM-2 cells in a time- and dose-dependent manner (P < 0.05). The IC50 values of shikonin in I-10 cells at 24, 48, and 72 h were 1.8, 1.36 and 1.16 µmol/L, as compared with 2.37, 0.8 and 0.41 µmol/L in TCAM-2 cells, respectively. Shikonin treatment significantly reduced mitochondrial membrane potential, increased ROS levels and lower the level of lactic acid in both I-10 and TCAM-2 cells (P < 0.05). Transmission electron microscopy and annexin V-FITC/PI double staining demonstrated that shikonin induced apoptosis and excessive autophagy in I-10 and TCAM-2 cells (P < 0.05). In both I-10 and TCAM cells, shikonin treatment significantly down- regulated the expressions of Bax, Bcl-2, cleaved caspase-3, PKM2, GLUT1 and HK2, and up-regulated the expression of autophagy-related protein LC3B (P < 0.05). CONCLUSIONS: Shikonin can inhibit the proliferation, induce apoptosis and increase autophagy in both I-10 and TCAM-2 cells probably by affecting energy metabolism of the cells.

Characterization of the cellulolytic bacteria communities along the gastrointestinal tract of Chinese Mongolian sheep by using PCR-DGGE and real-time PCR analysis.

A balanced gastrointestinal microbial ecosystem is crucial for the health and growth of animals. In the gastrointestinal tract (GIT) of ruminants, cellulolytic bacteria aid in the digestion and absorption of nutrients. Rumen contents and feces in ruminants are often used to assess gastrointestinal microbial communities; however, these sites do not guarantee to represent the diversity of microbes found in the entire GIT. In this study, we investigated the microbiota along the GIT of five Chinese Mongolian sheep using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR analysis. Results indicated that microbiota were more abundant in the stomach and large intestine than in the small intestine. DGGE and real-time PCR revealed the predominance of Firmicutes and Bacteroidetes in the GIT. Meanwhile, Ruminococcus flavefaciens and Clostridium cluster IV showed significant difference in their abundance along the GIT (P < 0.05). Fibrobacter succinogenes was the most dominant species, followed by Ruminococcus albus and R. flavefaciens. The ileum harbored a larger number of cellulolytic bacteria, particularly-Clostridium cluster IV, than reported previously. In addition, comparisons between microbiota in the rumen and rectum indicated similar number of total bacteria, Firmicutes, Bacteroidetes, F. succinogenes, Butyrivibrio fibrisolvens, Clostridium cluster IV, and Clostridium cluster XIVa, whereas the number of R. albus and R. flavefaciens was higher in the rumen. This study investigated the composition and quantification of GIT microbial community in Chinese Mongolian sheep, and revealed for the first time the cellulolytic bacterial community in these sheep.

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro.

BACKGROUND: For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes. RESULTS: We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells. CONCLUSION: In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

Viable intestinal passage of a canine jejunal commensal strain Lactobacillus acidophilus LAB20 in dogs.

The strain Lactobacillus acidophilus LAB20 with immunomodulatory properties was previously found dominant in the jejunal chyme of four dogs, and the novel surface layer protein of LAB20 suggested its competitive colonization in canine gut. To evaluate the persistence and survival of LAB20 in healthy dogs, LAB20 was fed to five healthy pet dogs for 3 days, at a dosage of 10(8) CFU daily as fermented milk supplement. The fecal samples, from 1 day prior to feeding, three continuous feeding days, and on day 5, 7, 14, and 21, were collected for strain-specific detection of LAB20 using real-time PCR. We found that LAB20 count was significantly increased in dog fecal samples at the second feeding day, but rapidly decreased after feeding ceased. The fecal samples from prior to feeding, during feeding, and post-cessation days were plated onto mLBS7 agar, from where LAB20 was recovered and distinguishable from other fecal lactobacilli based on its colony morphotype. Using strain-specific PCR detection, the colonies were further verified as LAB20 indicating that LAB20 can survive through the passage of the canine intestine. This study suggested that canine-derived strain LAB20 maintained at high numbers during feeding, viably transited through the dog gut, and could be identified based on its colony morphotype.

Dominance of Lactobacillus acidophilus in the facultative jejunal Lactobacillus microbiota of fistulated beagles.

Lactobacilli were isolated from jejunal chyme from five fistulated beagles. Cultivable lactobacilli varied from 10(4) to 10(8) CFU/ml. Seventy-four isolates were identified by partial 16S rRNA gene sequencing and differentiated by repetitive element PCR (Rep-PCR), Lactobacillus acidophilus was dominant, and nearly 80% of 54 isolates shared the same DNA fingerprint pattern.