Screening of immune-enhancing Lactobacillus in mice by using a cell-line.

In this study, we found that it is possible to screen Lactobacillus strains that enhance the immune function of mice using HCT-8 cells. Lactobacillus were co-incubated with intestinal epithelial HCT-8 cells to detect and screen the strains that induced more interleukin-6 (IL-6) in the culture supernatant. Simultaneously, a mouse model of low immunity was established to administer the screened lactobacilli by gavage. After 4 weeks of continuous gavage, related cytokines in blood and immune cell indexes in organs were detected to comprehensively evaluate the feasibility of in vitro cell culture model for screening immune-enhancing strains. The content of IL-6 in the culture supernatant of HCT-8 cells induced by the three tested strains increased approximately 5, 8 and 15 fold compared with that of the control group. IL-6 content in serum of mice was significantly higher than that of the control group provided with cyclophosphamide (CTX). Lactobacillus paracasei ZLPC01 presented a higher ability to protect against the immune damage of CTX by decreasing the serum IgG level, increasing the transformation of mouse splenocytes, and the activity of NK cells. Furthermore, L. paracasei ZLPC01 increased cytokine content in serum (IL-6, IL-2, TNF-α and IFN-γ) and colon (IL-6 and TNF-α) in CTX-treated mice. Screening strains that enhance immunity via an in vitro cell-line is simple in operation, and the results are well correlated with those of animal experiments, which is feasible and effective in practice. In addition, L. paracasei ZLPC01 could have the potential to enhance the immunity of mice effectively through inducing intestinal cells to produce IL-6, TNF-α and other cytokines.

[Jiedu Huoxue Decoction improves mitochondrial damage of the prostate in rats with chronic nonbacterial prostatitis].

OBJECTIVE: To explore the possible pathogenesis of chronic nonbacterial prostatitis (CNP) in rats from the perspective of mitochondria, and the interventional effect of Jiedu Huoxue Decoction (JHD) on CNP. METHODS: Forty clean-grade SD male rats were randomly divided into 4 groups of an equal number, sham control, CNP model control, Qianliekang Tablets intervention (QLK) and JHD intervention, those in the former two groups treated intragastrically with normal saline, and those in the latter two with QLK and JHD, respectively, at 2g/kg qd for 30 successive days. Then serum and prostate tissue samples were collected from the rats for calculation of the organ coefficients, HE staining, extraction of mitochondria in the prostate tissue, measurement of the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and Na+-K+-ATPase by colorimetric assay, and observation of the ultrastructural changes of the prostatic epithelial cells under the transmission electron microscope (TEM). RESULTS: The organ coefficient of the prostate was significantly higher in the CNP model controls (ï¼»1.95 ± 0.39ï¼½%) than in the sham control (ï¼»1.50 ± 0.42ï¼½%, P < 0.05), QLK (ï¼»1.54 ± 0.32ï¼½%, P < 0.05) and JHD groups (ï¼»1.47 ± 0.53ï¼½%, P < 0.05). TEM showed significant hyperplasia of the interstitial fibrous tissue, glandular structural disorder and inflammatory cell immersion in the CNP model controls, decreased inflammatory cells and reduced hyperplasia of epithelial cells in the acinar and interstitial fibrous tissues in the QLK and JHD groups, but no significant changes in the sham controls. The CNP model controls, compared with the QLK and JHD groups, exhibited remarkably lower levels of SOD (ï¼»17.42 ± 2.91ï¼½ vs ï¼»23.47 ± 5.79ï¼½ and ï¼»22.52 ± 3.88ï¼½ U/mg prot, P < 0.05), GSH-PX (ï¼»38.35 ± 6.98ï¼½ vs ï¼»47.68 ± 10.37ï¼½ and ï¼»89.95 ± 7.65ï¼½ U/mg prot, P < 0.05 or P < 0.01), and Na+-K+-ATPase in the prostatic mitochondria (ï¼»0.98 ± 0.40ï¼½ vs ï¼»1.37 ± 0.29ï¼½ and ï¼»1.85 ± 0.32ï¼½ µmol Pi/mg prot/h, P < 0.05 or P < 0.01), but a higher level of MDA (ï¼»1.70 ± 0.22ï¼½ vs ï¼»0.54 ± 0.14ï¼½ and ï¼»0.59 ± 0.17ï¼½ nmol/mg prot, P < 0.01). Significant mitochondrial damage was observed in the prostate tissue of the CNP model controls, and markedly enhanced mitochondrial autophagy was seen in the JHD group. CONCLUSIONS: Chronic nonbacterial prostatitis induces mitochondrial dysfunction in the prostate of rats, and Jiedu Huoxue Decoction can promote the recovery of mitochondrial function, which may be related to mitochondrial autophagy.

miRNA-877-5p inhibits malignant progression of prostate cancer by directly targeting SSFA2.

In this study, we aimed to investigate the role of miR-877-5p in the malignant phenotypes of prostate cancer (PCa) cells and its underlying mechanism. RT-qPCR analysis was performed to examine the expression of miR-877-5p and sperm-specific antigen 2 (SSFA2) in PCa tissues and cells. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-20-deoxyuridine (EdU) assay, flow cytometry, wound-healing assay, and Transwell invasion assay were performed to determine the functional roles of miR-877-5p in PCa cells. The association of miR-877-5p with SSFA2 was determined by luciferase reporter and RNA pull-down assays. In this study, we found that the expression level of miR-877-5p was decreased in PCa tissues and cells. Functionally, overexpression of miR-877-5p exerted tumor suppressor properties in PCa cells. Mechanistically, SSFA2 was identified as a target gene of miR-877-5p, while overexpression of SSFA2 could abrogate the anti-tumor effects of miR-877-5p in PCa cells. These findings demonstrated that miR-877-5p/SSFA2 axis functioned as a potential target for PCa treatment.

Oxidative stress and antioxidant defense in detoxification systems of snake venom-induced toxicity.

BACKGROUND: Snakebites remain a major life-threatening event worldwide. It is still difficult to make a positive identification of snake species by clinicians in both Western medicine and Chinese medicine. The main reason for this is a shortage of diagnostic biomarkers and lack of knowledge about pathways of venom-induced toxicity. In traditional Chinese medicine, snakebites are considered to be treated with wind, fire, and wind-fire toxin, but additional studies are required. METHODS: Cases of snakebite seen at the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine were grouped as follows: fire toxin - including four cases of bites by Agkistrodon acutus and three bites by Trimeresurus stejnegeri - and wind-fire toxin - four cases of bites by vipers and three bites by cobras. Serum protein quantification was performed using LC-MS/MS. Differential abundance proteins (DAPs) were identified from comparison of snakebites of each snake species and healthy controls. The protein interaction network was constructed using STITCH database. RESULTS: Principal component analysis and hierarchical clustering of 474 unique proteins exhibited protein expression profiles of wind-fire toxins that are distinct from that of fire toxins. Ninety-three DAPs were identified in each snakebite subgroup as compared with healthy control, of which 38 proteins were found to have significantly different expression levels and 55 proteins displayed no expression in one subgroup, by subgroup comparison. GO analysis revealed that the DAPs participated in bicarbonate/oxygen transport and hydrogen peroxide catabolic process, and affected carbon-oxygen lyase activity and heme binding. Thirty DAPs directly or indirectly acted on hydrogen peroxide in the interaction network of proteins and drug compounds. The network was clustered into four groups: lipid metabolism and transport; IGF-mediated growth; oxygen transport; and innate immunity. CONCLUSIONS: Our results show that the pathways of snake venom-induced toxicity may form a protein network of antioxidant defense by regulating oxidative stress through interaction with hydrogen peroxide.

NOL6, a new founding oncogene in human prostate cancer and targeted by miR-590-3p.

We identified a new human prostate cancer oncogene, nucleolar protein 6 (NOL6), and screened for microRNAs that interfere with its expression in prostate cancer cells. A NOL6 shRNA plasmid was constructed and packaged into lentivirus to infect PC-3 cells. The ability of cell proliferation was evaluated by cell counting and colony formation. Cell cycle progression and apoptosis of PC-3 cells were detected by flow cytometry. A retrieval database was used to screen possible target microRNAs, and the effect of target miRNA overexpression on PC-3 cells was observed. The results showed that after NOL6 gene knockdown, PC-3 cell mitosis was blocked, proliferation was decreased, and the number of apoptotic cells were increased. The microRNA, hsa-miR-590-3p, that can regulate the NOL6 gene expression was identified. Overexpression of miR-590-3p in PC-3 cells by synthetic mimics resulted in abnormal mitosis, decreased cell proliferation, and an increase in apoptosis. In summary, we identified NOL6 as a novel oncogene in the human prostate cancer PC-3 cell line. The miRNA miR-590-3p interferes with NOL6 expression making it a potential treatment for prostate cancer.

Preventive effect of Lactobacillus reuteri on melanoma.

Lactobacillus is one of the bacteria strains which has been widely used in human life, has valuable probiotic properties. In the present study, L. reuteri FLRE5K1 was isolated from healthy mouse kidney and tested for its anti-melanoma activity in cell assays and animal models. The results showed that L. reuteri FLRE5K1 could significantly reduce the incidence of melanoma from 70 % to 30 % and significantly prolonged the survival of tumor-bearing mice. Despite of its significant effect on melanoma prevention, the tumor mass remained uneliminated. The alanine aminotransferase (ALT) level remained normal in tumor-bearing mice with intragastric administration of L. reuteri FLRE5K1, indicating that L. reuteri FLRE5K1 could prevent melanoma cells from damaging the liver. Cell assays showed that melanoma cells were inhibited by serum from mice with intragastric administration of L. reuteri FLRE5K1, but not by the bacteria itself. Further examination revealed that the levels of TNF-α and INF-γ in this serum were significantly increased. In summary, it was speculated that L. reuteri FLRE5K1 could stimulate the body to produce anti-cancer cytokines, inhibit the migration and colonization of melanoma cell line B16-F10, so as to prevent the onset of melanoma and prolong the survival of mice. The experiment was a useful complement to functional studies of endogenous probiotics and provided theoretical data support for the development of new microbioecologics.

Oxidative stress and antioxidant defense in detoxification systems of snake venom-induced toxicity

Snakebites remain a major life-threatening event worldwide. It is still difficult to make a positive identification of snake species by clinicians in both Western medicine and Chinese medicine. The main reason for this is a shortage of diagnostic biomarkers and lack of knowledge about pathways of venom-induced toxicity. In traditional Chinese medicine, snakebites are considered to be treated with wind, fire, and wind-fire toxin, but additional studies are required. Methods: Cases of snakebite seen at the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine were grouped as follows: fire toxin - including four cases of bites by Agkistrodon acutus and three bites by Trimeresurus stejnegeri - and wind-fire toxin - four cases of bites by vipers and three bites by cobras. Serum protein quantification was performed using LC-MS/MS. Differential abundance proteins (DAPs) were identified from comparison of snakebites of each snake species and healthy controls. The protein interaction network was constructed using STITCH database. Results: Principal component analysis and hierarchical clustering of 474 unique proteins exhibited protein expression profiles of wind-fire toxins that are distinct from that of fire toxins. Ninety-three DAPs were identified in each snakebite subgroup as compared with healthy control, of which 38 proteins were found to have significantly different expression levels and 55 proteins displayed no expression in one subgroup, by subgroup comparison. GO analysis revealed that the DAPs participated in bicarbonate/oxygen transport and hydrogen peroxide catabolic process, and affected carbon-oxygen lyase activity and heme binding. Thirty DAPs directly or indirectly acted on hydrogen peroxide in the interaction network of proteins and drug compounds. The network was clustered into four groups: lipid metabolism and transport; IGF-mediated growth; oxygen transport; and innate immunity. Conclusions: Our results show that the pathways of snake venom-induced toxicity may form a protein network of antioxidant defense by regulating oxidative stress through interaction with hydrogen peroxide.(AU)

Inhibition potential of UDP-glucuronosyltransferases (Ugts) 1A isoforms by the analogue of resveratrol, bakuchiol.

Bakuchiol is a promising anti-tumor candidate with resveratrol-like structure. The present study aims to evaluate the inhibition potential of bakuchiol towards UDP-glucuronosyltransferases (UGT) 1A isoforms. An in vitro incubation system using 4-methylumbelliferone (4-MU) glucuronidation was used to evaluate the inhibition capability of bakuchiol towards UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9 and 1A10. The glucuronidation of trifluoperazine (TFP) was employed as the probe reaction to determine bakuchiol's inhibition towards UGT1A4. At 1 microM and 10 microM of bakuchiol, no or weak inhibition was observed for all the tested UGT1A isoforms. At 100 microM of bakuchiol, the activity of UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9 and 1A10 was inhibited by -46.2%, 74.7%, 17.8%, 98.7%, 70.4%, 99.2%, 75.8%, and 93.3%, respectively. Further inhibition kinetic behaviour was determined for UGT1A6, 1A8, and 1A10. Both Dixon plot and Lineweaver-Burk plot showed the noncompetitive inhibition of bakuchiol towards all these three UGT isoforms. The inhibition kinetic parameters (Ki) were calculated to be 5.3, 1.8, and 92.6 microM for UGT1A6, 1A8, and 1A10, respectively. In combination with the in vivo exposure of bakuchiol, the high possibility of in vivo inhibition of UGT1A6 and 1A8 was predicted. However, relatively low possibility of in vivo inhibition towards UGT1A10 was predicted due to lower in vivo concentration of bakuchiol than its inhibition parameter (Ki). All these information will be helpful for the R&D of bakuchiol as a promising anti-tumor drug.