Effects of probiotics on intestinal flora, inflammation and degree of liver cirrhosis in rats with liver cirrhosis by regulating Wnt/ß-catenin signaling pathway.

We aimed to explore the effects of probiotics on intestinal flora, inflammation and degree of liver cirrhosis in rats with liver cirrhosis, and to verify the Wnt/ß-catenin signaling pathway that regulates this process. A total of 30 SD rats were randomly divided into 3 groups, namely, control group (n=10), model group (n=10) and probiotic group (n=10). Rats in the model group were used to construct liver cirrhosis models using carbon tetrachloride (CCL4) method, and those in the probiotic group were administered with probiotic preparations by gavage for 8 weeks. Then the feces of rats in each group were taken to detect the composition of intestinal flora, and changes in the content of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1) and interferon-gamma (IFN-γ), in peripheral blood collected were examined by enzyme-linked immunosorbent assay (ELISA). Next, changes in the degree of liver cirrhosis were analyzed by hematoxylin and eosin (H&E) staining, and the expression levels of the Wnt/ß-catenin signaling pathway-related molecules, including ß-catenin, glycogen synthase kinase (GSK)-3ß and Frizzled-2, in liver tissues in each group were detected via polymerase chain reaction (PCR) and Western blotting (WB). Compared with rats in the control group, those in the model group had a disordered structure of hepatic lobule and hyperplasia of a large number of fibrous tissues. In contrast to those in the model group, the liver lobule structure was greatly improved, the edema cells were obviously reduced, and the hyperplasia of collagen fibers was remarkably alleviated in the probiotic group. Moreover, the degree of liver cirrhosis in the probiotic group was significantly reduced compared with that in the model group. Moreover, the rats in the model group exhibited a higher Bifidobacterium level in the intestinal tract, while those in the probiotic group displayed higher levels of microorganisms in the intestinal tract, such as Lachnospiraceae, Ruminococcaceae, Actinbacteria, Slackia and Pasteurellaceae. In comparison with that in the control group, the level of salt-tolerant Lactobacillus in the intestinal tract of rats in the model group was significantly decreased, while that in the probiotic group was partially increased (P=0.023). Meanwhile, some intestinal flora of rats in the control group, model group and probiotic group were closely correlated. Specifically, highly positive correlations were found between Bacteroidetes and Paraeggerthella (r=0.423, P=0.034) and between Firmicutes and Lactobacillus (r=0.318, P=0.027), but strongly negative associations were detected between Firmicutes and Paraeggerthella (r=-0.691, p=0.004) and between Paraeggerthella and Lactobacillus (r=-0.384, P=0.047). In addition, the levels of inflammatory cytokines TNF-α IL-6, MCP-1 and IFN-γ in the plasma of rats in the model group were markedly higher than those in the control group (P<0.05), whereas such levels in the probiotic group were decreased compared with those in the model group (P<0.05). PCR results revealed that the expression levels of ß-catenin and Frizzled-2 in the model group were higher than those in the control group, whereas they were lower in the probiotic group than those in the model group (P<0.05). Furthermore, the model group had a decreased level of GSK-3ß in comparison with the control group, but the probiotic group had a higher level of GSK-3ß than the model group (P<0.05). WB results were consistent with PCR results. Probiotics can affect intestinal flora, inflammation and degree of liver cirrhosis in rats with liver cirrhosis, and its mechanism may be related to the Wnt/ß-catenin signaling pathway.

MiR-146a relieves kidney injury in mice with systemic lupus erythematosus through regulating NF-κB pathway.

OBJECTIVE: To explore the effect of micro ribonucleic acid (miRNA)-146a on kidney injury in mice with systemic lupus erythematosus (SLE), and to investigate its possible mechanism. MATERIALS AND METHODS: A total of 45 female MRL/lpr mice were randomly divided into control group, miR-146a mimic group and miR-146a inhibitor group. Urine protein level was measured every 2 weeks. Meanwhile, the levels of serum anti-dsdeoxyribonucleic acid (anti-dsDNA), anti-ssDNA, antinuclear antibody (ANA) and anti-chromatin were measured using enzyme-linked immunosorbent assay (ELISA). At 2 weeks after drug treatment, the effects of miR-146a mimic and inhibitor on kidney tissues of MRL/lpr mice were detected and analyzed by gene chip and gene set enrichment analysis, respectively. The mice were executed at the age of 24 weeks, and the blood samples were collected. Subsequently, the level of blood urea nitrogen (BUN) was measured using the BUN analyzer. After that, kidney tissues were taken, and the effect of drug treatment on the morphology of kidney tissues was detected via hematoxylin-eosin (HE) staining. Moreover, the effects of drug treatment on the mRNA levels of inflammatory factors and the nuclear factor-κB (NF-κB) signaling pathway in kidney tissues were detected via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. RESULTS: MiR-146a mimic significantly reduced urine protein in a time-dependent manner, which also significantly reduced BUN level at 24 weeks. The results of HE staining showed that both glomerular injury and renal vascular injury in miR-146a mimic group were significantly alleviated. In miR-146a mimic group, serum autoantibodies of anti-dsDNA, anti-ssDNA, anti-chromatin and ANA decreased significantly. However, the survival time of mice was significantly prolonged. High-throughput gene expression chip technique elucidated that in miR-146a mimic group, the expression of positive regulatory gene of NF-κB showed a decreasing trend. However, the expression of negative regulatory gene of NF-κB showed an increasing trend. MiR-146a mimic remarkably down-regulated the expression levels of RELA, IRAK1, interleukin-1B (IL1B) and IL-10 in kidney tissues. Furthermore, the results of Western blotting showed that miR-146a mimic inhibited both the classical and non-classical NF-κB signaling pathways. CONCLUSIONS: MiR-146a reduces SLE-induced kidney injury in MRL/lpr mice through regulating classical and non-classical NF-κB signaling pathways.

Autoantibody detection to tumor-associated antigens of P53, IMP1, P16, cyclin B1, P62, C-myc, Survivn, and Koc for the screening of high-risk subjects and early detection of esophageal squamous cell carcinoma.

The aim of this study was to evaluate the diagnostic values by detecting sera autoantibodies to eight tumor-associated antigens (TAAs) of P53, IMP1, P16, cyclin B1, P62, C-myc, Survivn and Koc full-length recombinant proteins for the screening of high-risk subjects and early detection of esophageal squamous cell carcinoma (ESCC). Enzyme-linked immunosorbent assay was used to detect autoantibodies against the eight selected TAAs in 567 sera samples from four groups, including 200 individuals with normal esophageal epithelia (NOR), 214 patients with esophageal basal cell hyperplasia (BCH), 65 patients with esophageal dysplasia (DYS), and 88 patients with ESCC. In addition, the expression of the eight antigens in esophageal tissues was analyzed by immunohistochemistry. Statistically significant distribution differences were identified among the four groups for each of the individual autoantibodies to six TAAs (P53, IMP1, P16, cyclin B1, P62, and C-myc); the detection rates of antoantibodies were positively correlated with the progression of ESCC. When autoantibody assay successively accumulated to six TAAs (P53, IMP1, P16, cyclin B1, P62, and C-myc), a stepwise increased detection frequency of autoantibodies was found in the four sera groups (6% in NOR, 18% in BCH, 38% in DYS, and 64% in ESCC, respectively), the risks to BHC, DYS, and ESCC steadily increased about 3-, 9-, and 27-folds. The sensitivity and the specificity for autoantibodies against the six TAAs in diagnosing ESCC reached up to 64% and 94%, respectively. The area under the receiver operating characteristic curve for the six anti-TAA autoantibodies was 0.78 (95% confidence interval 0.74-0.83). No more increasing in sensitivity was found with the addition of new anti-TAA autoantibodies. A combination detection of autoantibodies to TAAs might distinguish ESCC patients from normal individuals and the patients with esophageal precancerous lesions.

The fate of Cu, Zn, Pb and Cd during the pyrolysis of sewage sludge at different temperatures.

In the present study, a sequential extraction procedure, recommended by the Community Bureau of Reference (BCR), was used for the fractionation of the heavy metals Cu, Zn, Pb and Cd in sewage sludge and its residues produced after pyrolysis at different temperatures from 250 to 700 degrees C. The results show that, in the sludge sample, the sum of the percentages of the reducible and oxidizable fractions for all metals except Cu was very high (65.4% for Cd, 85.7% for Pb, 78.7% for Zn), whereas the sum of the percentages of the oxidizable and residual fractions for Cu was very high (88.8%). The same result could be attained in the residues. Statistical analysis shows that at low temperatures the variation in pyrolysis temperature did not effectively contribute to the distribution of metal speciation in the residues. Meanwhile a modified Toxicity Characteristic Leaching Procedure (TCLP) was employed to determine the leachability of these four metals. The result indicates that the TCLP concentration of Cu, Zn, Pb and Cd dropped sharply after the temperature reached 350 degrees C, 550 degrees C, 500 degrees C and 400 degrees C respectively, which means pyrolysis can enhance the stability of these four metals when the temperature is high enough.

[Role of afferent renal nerves in 2K2C Goldblatt hypertension].

In order to study the role of afferent renal nerves in 2K2C Goldblatt hypertension, the renal afferent nerves were selectively lesioned by bilateral T9-L2 spinal dorsal rhizotomy before clipping (internal diameter, 0.3 mm) of birenal arteries. Systolic blood pressure of the rat was measured by tail-cuff method. Concentrations of catecholamines, Ang II, and aldosterone were determined respectively by HPLC-EC and RIA, and vascular structural changes were measured by blood vessel micro-image analysis system coupled with a computer. The results showed that the concentrations of NE and E in medulla oblongata, adrenal gland and plasma, and of plasma Ang II and aldosterone as well as body weight of the rat were all significantly increased. The heart coefficient (heart wt/body wt), the media thickness, and the media thickness/lumen diameter in superior mesenteric arteries were also increased in 2K2C hypertensive rats (clipping, 6 wk) as compared with those in control rats. Bilateral rhizotomy delayed development of 2K2C hypertension and prevented above-mentioned vascular structural changes, the NE and E concentrations of medulla oblongata, adrenal gland and plasma were all decreased, hypothalamic NE and E were increased, and plasma Ang II level was not significantly changed. These results suggest that afferent renal nerves may play a partial role in the development of hypertension in 2K2C rats by activating sympathetic nervous system as a result of affecting metabolic activities of brain catecholaminergic neurons, and that high-plasma Ang II and aldosterone as well as heart hypertrophy and proliferation of vascular smooth muscle cells may also participate in the pathogenesis of hypertension in 2K2C rats.

[A simplified method for preparation of DOCA-salt hypertension model in rats by subcutaneous implantation of DOCA silastic tube].

To study a simplified method for preparation of DOCA-Salt hypertension model in rat. 45 male SD rats (140 +/- 9 g) were used in this study. A silastic tube (length 25 mm; external diameter 4 mm; internal diameter 2.5 mm; 10-14 about 300 microns micropores on the wall) filled 100 mg DOCA in was implanted subcutaneously at the belly of the rat and followed by uninephrectomy. After operation the rat only received 1% salt solution in stead of drinking water. Hypertension developed within 3 weeks. At the 8th week after operation, systolic blood pressure (SBP) of rats was elevated to 23.3 +/- 0.37 kPa. If DOCA (10 mg/rat, weekly) was given subcutaneously, however, from control level of 16 +/- 0.16 kPa hypertension did not occur until the 5th week after injection. At the 13th week, SBP was elevated to 23.3 +/- 0.66 kPa. Difference of regression coefficients (1.295, 0.692) of SBP curves between two experimental groups was very significant (P < 0.001). Compared with subcutaneous injection, there are two advantages for method of subcutaneous implantation: (1) The rate of SBP elevation is faster and amplitude higher. (2) This method is simple and well reproducible.