Dibutyltin depressed immune functions via NF-κB, and JAK/STAT signaling pathways in zebrafish (Danio rerio).

Dibutyltin (DBT) is the degradation products of TBT, which is generally considered higher toxicity than TBT in the immune system. In order to learn more about the mechanisms of immune-toxic of DBT, we exposed zebrafish (Danio rerio) to 0, 1, 10 and 100 ng/L DBT for 8 weeks. At the end of the experiment, we determined the immune parameters and immune-related genes. The results showed that with an increase in TBT dose, lysozyme activities and IgM, C3, C4 content in intestine, skin and spleen were all significantly inhibited by the DBT exposure. Fish exposed to 10 ng/L and 100 ng/L showed significantly lower lysozyme activities and IgM, C3, C4 content than those of the control group. Zebrafish exposed to 10 ng/L and 100 ng/L DBT, the mRNA transcript levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), interferon γ2 (INFγ2), nuclear factor-κB p65 (NF-kB p65), inhibitor protein-κBα (IκBα), IκB kinases ß (IKKß), Janus family of protein tyrosine kinases (JAKs) and the signal transducers and activators of transcription proteins (STATs) all increased with the DBT levels in the intestine and spleen. Those parameters showed significantly higher values in 10 ng/L and 100 ng/L than those of fish in the control group. However, no significant difference was found in IκB kinases α (IKKα) and IκB kinase γ (IKKγ) mRNA levels in the intestine and spleen. These data imply that DBT might be via suppression on IKKß/IkBa/NF-kBp65 and JAK/STAT signaling pathways to regulate the immunity of zebrafish.

Identification and characterization of known and novel microRNAs in three tissues of Chinese giant salamander base on deep sequencing approach.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although animal miRNAs have been extensively studied in model systems, less is known in other animal with limited genome sequence data, including Chinese giant salamander (Andrias davidianus). The identification of the full repertoire of miRNAs expressed in the liver, spleen and muscle of A. davidianus would significantly increase our understanding for physiological function of A. davidianus, in this ancient and endangered urodele amphibian. In this study, three independent small RNA libraries were constructed from the liver, spleen and muscle of A. davidianus. The libraries were subjected to high-throughput sequencing by using the Illumina deep sequencing. As a result, a total of 12,831,239, 13,592,195 and 9,887,531 raw reads representing 2,240,771, 1,363,266 and 1,964,252 clean reads per library were obtained separately. Through bioinformatics analysis, we identified total of 553 known miRNAs and 44 putative novel miRNAs in our small RNA dataset from liver, spleen and muscle tissues. Five known miRNAs (gga-miR-10a-5p, pma-miR-29d-5p, aca-miR-338-3p, hsa-miR-455-3p and ssa-miR-2184-5p_R-1) and three novel miRNAs (PC-5p-891_1763, PC-5p-32538_50 and PC-3p-33645_48) showed different expression in eight different tissues as revealed by stem-loop qPCR analysis. This study characterized the miRNA of A. davidianus for the first time, which provides an opportunity for further understanding of miRNA regulation function in A. davidianus ranked as living fossils.

Effect of tributyltin on antioxidant ability and immune responses of zebrafish (Danio rerio).

Tributyltin (TBT) is a toxic compound released into aquatic ecosystems through antifouling paints. This study was designed to examine the effects of TBT on antioxidant ability and immune responses of zebrafish (Danio rerio). Three hundred sixty healthy zebrafish were randomly grouped into four groups and exposed to different doses of TBT (0, 1, 10 and 100ngL-1). At the end of 8 weeks, the fish were sampled, and antioxidant capability, immune parameters and immune-related genes were assessed. The results showed that with an increase in TBT dose, the concentration of malonaldehyde in the liver was significantly increased (p<0.05), whereas the activities of total superoxide dismutase, catalase and glutathione peroxidase were significantly decreased (p<0.05) compared to the control. The activity and expression of lysozyme and the content of immunoglobulin M were significantly decreased compared to those of the fish exposed to 0ngL-1 TBT (p<0.05). However, the expression of the HSP70, HSP90, tumor necrosis factor-α(TNF-α), interleukins (IL-1ß, IL-6), and nuclear factor-kappa B p65 (NF-κ B p65) genes were all enhanced with an increase in TBT dose. The results indicated that TBT induced oxidative stress and had immunotoxic effects on zebrafish.

Involvement of microRNA-141-3p in 5-fluorouracil and oxaliplatin chemo-resistance in esophageal cancer cells via regulation of PTEN.

microRNAs (miRNAs) act as a major regulator of acquired chemo-resistance in various types of cancer therapeutics. This study investigated the contribution of miRNAs in influencing multiple drug resistance in esophageal squamous cell carcinoma (ESCC). The sensitivity of four ESCC cell lines (EC109, EC9706, TE-1 and KYSE-150) to 5-fluorouracil (5-FU) and oxaliplatin (OX) was determined by MTT assay. A 5-FU and OX-resistant subline, EC9706R, was established by continuous exposure to stepwise increasing concentration of 5-FU and OX. Microarray technology was used to compare the differential expression of miRNAs between resistant cells and parental cells. Chemo-sensitivity assay was performed to evaluate drug response in EC9706R cells transfected with miRNA mimic or inhibitor. The direct targets of miRNA were identified by employing pathway analysis and then confirmed with luciferase assay. Sixty ESCC tissue samples and their paired adjacent normal tissues were collected to validate the expression of identified miRNA. Mouse models were further utilized to investigate the function of miRNA on acquired chemo-resistance. MicroRNA panel results indicated that a total of 12 miRNAs were differentially expressed and miR-141-3p was highly over expressed in resistant cells. Inhibition of miR-141-3p reversed acquired chemo-resistance in EC9706R cells by stimulating apoptosis. The expression of miR-141-3p was significantly increased in ESCC tissue samples compared to their matched distant normal tissues. In addition, the elevated miR-141-3p expression was found to be associated with ESCC differentiation status and TNM stage. Moreover, Phosphatase and tensin homolog (PTEN) was identified as direct target of miR-141-3p. Western blot exhibited altered protein levels of PTEN, Akt, and PI3k with miR-141-3p inhibitor. An inverse correlation between PTEN expression and miR-141-3p expression was also observed in tissue samples. EC9706R xenograft mouse model became sensitized to 5-FU and OX treatment following miR-141-3p inhibitor transfection in vivo. Our study demonstrated that miR-141-3p contributed to an acquired chemo-resistance through PTEN modulation both in vitro and in vivo.

Cloning, characterization and mRNA expression of interleukin-6 in blunt snout bream (Megalobrama amblycephala).

In the present study, the interleukin-6 gene (IL-6) cDNA in blunt snout bream (Megalobrama amblycephala) was identified and its expression profiles under ammonia stress and bacterial challenge were investigated. The IL-6 sequence consisted of 1045 bp, including a 696 bp ORF which translated into a 232 amino acid (AA) protein. The protein contained a putative signal peptide of 24 AA in length. IL-6 expression analysis showed that the it is differentially expressed in various tissues under normal conditions and the highest IL-6 level was observed in the intestine tissue, followed by the liver, and then in the gills. Under ammonia stress, the IL-6 mRNA level both in spleens and intestine increased significantly (P < 0.05), with the maximum levels attained at 6 h, 12 h (72, 10-fold, respectively). Thereafter, they all significantly decreased (P < 0.01) and returned to the basal value within 48 h. Whereas, in livers it slightly decreased at 3 h firstly (0.5-fold), and then significantly (P < 0.05) increased with the maximum level attained 12 h (3-fold). Further expression analysis showed that the mRNA level of IL-6 in spleens, intestine and livers of blunt snout bream all increased significantly (P < 0.05), with maximum values attained at 6 h, 3 h, 6 h (10, 6, 18-fold, respectively) after Aeromonas hydrophila (A. hydrophila) injection, and then decreased to the basal value within 24 h which suggested that IL-6 was involved in the immune response to A. hydrophila. The cloning and expression analysis of the IL-6 provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions in blunt snout bream.

Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus).

MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21-22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem-loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

IDENTIFCATION AND VALIDATION OF NOVEL microRNA MOLECULE FROM THE PELODISCUS SINENSIS BY BIOINFORMATICS APPROACHES.

MicroRNAs (miRNAs) are a group of endogenous, short non-coding RNAs with the length of about 22 nt, which mediate gene expression at the post-transcriptional level through mRNA degradation or translational repression. Among them, some are highly evolutionally conserved in the animal kingdom; this provides a powerful strategy for identifying miRNAs in new species. The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important freshwater aquaculture reptilian species in China, but related miRNAs have not been identified up to now. In the present study, a total of 10 Pelodiscus sinensis miRNAs were identified according to Pelodiscus sinensis ESTs and GSSs information in NCBI database by bioinformatics approaches. The RT-PCR-based assays were performed and found that 10 Pelodiscus sinensis miRNAs were expressed. Using these miRNAs, 22 target genes were identified. These genes encode 22 proteins involved in metabolism, signal transduction, transcriptional regulation, and development. These miRNAs and their targets will serve as useful resources for their, functional analyses in miRNA-regulated processes in Pelodiscus sinensis breeding and genetic research.

Association between genetic polymorphisms in AURKA (rs2273535 and rs1047972) and breast cancer risk: a meta-analysis involving 37,221 subjects.

BACKGROUND: Published data on the association between AURKA polymorphisms and breast cancer (BC) risk are inconclusive. This meta-analysis was performed to derive a more precise estimation on the relationship between AURKA polymorphisms (rs2273535 and rs1047972) and BC risk. METHODS: PubMed, Web of Knowledge and Embase were searched for relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to estimate the strength of associations. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were performed for allele contrast genetic model, homozygous genetic model, heterozygote genetic model, dominant model, and recessive model, respectively. RESULTS: A total of 13 studies (16,349 BC patients and 20,872 case-free controls) were involved in this meta-analysis. Meta-analysis showed that there was significant association between rs2273535 and BC risk in three genetic models in the overall population (A vs. T: OR = 1.08, 95% CI = 1.01-1.15, P = 0.02; AA vs. TT: OR = 1.36, 95% CI = 1.06-1.73, P < 0.00001; AA vs. TT + TA: OR = 1.15, 95% CI = 1.01-1.31, P = 0.04). In the subgroup analysis by ethnicity, the effects remained in Asians (allele contrast genetic model: OR = 1.12, 95% CI = 1.00-1.26, P = 0.04 and homozygote comparison: OR = 1.22, 95% CI = 1.06-1.41, P = 0.007). However, no genetic models reached statistical association in Cauasians. Rs1047972 polymorphism was associated with BC risk in the overall population based on homozygote comparison (AA vs. GG: OR = 0.81, 95% CI = 0.66-0.99, P = 0.04). When stratified by ethnicity, rs1047972 polymorphism had a decreased association with BC risk in Caucasians based on allele contrast genetic model, homozygote comparison, the dominant model and the recessive model. However, there was no association in any genetic model in Asians. CONCLUSIONS: This meta-analysis suggests that AURKA rs2273535 polymorphism has an increased risk with BC, especially in Asians. However, rs1047972 polymorphism has a decreased BC risk in Caucasians. Further large scale multicenter epidemiological studies are warranted to confirm this finding.

Functional promoter -765 G > C variant in COX-2 gene is associated with the susceptibility of breast cancer in Chinese Han women.

BACKGROUND: Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandins synthesis which exists in two isoforms, COX-1 and COX-2. Over-expression of COX-2 was considered to increase the proliferation and enhance the invasiveness of breast cancer cells. It was suggested that genetic variations in COX-2 could influence its expression. Herein, the present study was aimed to investigate the associations between two mostly studied functional polymorphisms (-765 G > C and 8473 C > T) in COX-2 and breast cancer risk in Chinese Han women. METHODS: In the hospital-based case-control study, 465 breast cancer patients and 799 cancer-free controls were genotyped for the COX-2 -765 G > C and 8473 C > T polymorphisms using TaqMan assay. We estimated odds ratios (ORs) and 95% confidence intervals (95% CIs) using the logistic regression. RESULTS: Compared with the wild genotype of -765 G > C, we found a statistically significant increased risk of breast cancer associated with the variant genotypes [GC/CC vs. GG: OR = 1.56, 95% CI = 1.11-2.21]. In the stratified analysis, the increased risk was more predominant among the subgroups of younger subjects (OR = 1.61, 95% CI = 1.00-2.61). Furthermore, the variant genotypes were associated with large tumor size (OR = 3.01, 95% CI = 1.47-6.12). No significant association was observed for the 8473 C > T polymorphism. CONCLUSIONS: Our results suggest that the functional -765 G > C polymorphism in the promoter of COX-2 may influence the susceptibility and progression of breast cancer in the Chinese Han population.

Associations between C1772T polymorphism in hypoxia-inducible factor-1α gene and breast cancer: a meta-analysis.

BACKGROUND: A meta-analysis was performed to estimate the association between HIF-1α polymorphism (C1772T) and breast cancer risk. MATERIAL AND METHODS: The relevant published literature was retrieved from PubMed, Web of Knowledge, and Embase. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of the associations. RESULTS: Six case-control studies, including 2043 cases and 2146 controls were identified. Meta-analysis showed that there was no marked association between C1772T polymorphism and breast cancer risk in the overall population in the dominant model. The subgroup analysis showed an increased breast cancer risk in Asians based on homozygote comparison and the recessive model. There were no associations between C1772T polymorphism with clinicopathological parameters and habits. CONCLUSIONS: The present meta-analysis suggests that HIF-1α C1772T polymorphism is a risk factor for susceptibility to breast cancer in Asians.

PiRNAs biogenesis and its functions.

piRNAs (piwi-interacting RNA) are a novel class of non-coding small single-stranded RNAs with the length of 26-33 nt. The piRNAs play important biological role through the specific interaction with the piwi proteins of the Argonaute family. piRNA function in embryonic development, maintenance of germline DNA integrity, silencing of transposon transcription, suppressionof translation, formation of heterochromatin, and epigenetic regulation of sex determination. This review summarizes recent research and progress on biogenesis and function of piRNA in eukaryotic species.

[Sequences analysis of jlFABP2 and the correlation between polymorphisms and body weight gain in Cyprinus carpio var. jian].

Two replicate intestine fatty acid binding protein genes (jlFABP2a and jlFABP2b) were cloned from Cyprinus carpio var. jian using PCR. Both ORFs were 399 bp in length sharing 92.2% similarity with each other, and 88.0% and 90.5% with their counterpart in zebrafish, respectively. The gene structure of jlFABP2s was same as other FABPs, which contained four exons and three introns. Sequences and lengths of introns between 2a and 2b. were obviously different Phylogenetic tree displayed that two jlFABP2s corresponded to one zebrafish FABP2 which matches the fact that the chromosome number of common carp was twice of zebrafish. Real time-PCR showed that jlFABP2 genes mainly expressed in intestine and the expression level was very significantly higher than other tissues such as brain, liver, muscle, kidney, and gonad (P<0.01). The expression level of jlFABP2a was significantly (male, P<0.05) or very significantly (females, P<0.01) higher than 2b in intestine; and 2b was expressed slightly higher than 2a in other tissues. It seemed that 2a expressed specifically in intestine, while 2b expressed ubiquitously. Twelve and four SNP loci were found at jlFABP2a and 2b introns through comparison sequences from 8 individuals, respectively. Genotypes of I1-A15G, I1-A99G, I2-C487T, and I3-A27T on jlFABP2a were detected using PCR-RFLP in selection population of C. carpio var. jian. The SNP genotypes and individual weight gain correlation indicated that four SNPs were significantly (P<0.05 or P<0.01) associated with adult weight gain. Diplotype analysis displayed that individuals with genotype AGGGCCXX or AGGGXXAT grew faster than other individuals by 15%. The individuals with these two genotypes only occupied 9% in total selection populations, indicating the presence of large selection space. The 4 SNPs detected in this experiment can be used in C. carpio var. Jian growth selection breeding plan.

Clinical characteristics, susceptibility profiles, and treatment of nocardiosis: a multicenter retrospective study in 2015-2021.

OBJECTIVES: This study aimed to investigate the microbiological characteristics, antimicrobial resistance profiles, antibiotic choice, and outcomes of Nocardia infection in various centers over a 7-year period (from 2015 to 2021). METHODS: We retrospectively analyzed the medical records of all hospitalized patients diagnosed with Nocardia between 2015 and 2021. The isolates were identified to the species level through the sequencing of 16S ribosomal RNA or secA1 or ropB genes. The susceptibility profiles were determined using the broth microdilution method. RESULTS: Of the 130 nocardiosis cases, 99 (76.2%) were established as pulmonary infection, of which the most common underlying disease was chronic lung disease (40.4%, 40/99), including bronchiectasis, chronic obstructive pulmonary disease, and chronic bronchitis. Among 130 isolates, 12 species were identified, with the most common species being Nocardia cyriacigeorgica (37.7%) and Nocardia farcinica (20.8%). All Nocardia strains were susceptible to linezolid and amikacin, and the susceptibility rate of trimethoprim-sulfamethoxazole (TMP-SMX) was 97.7%. Of the 130 patients, 86 (66.2%) received TMP-SMX monotherapy or multidrug regimen. Furthermore, 92.3% patients who were treated achieved clinical improvement. CONCLUSION: TMP-SMX was the treatment of choice for nocardiosis, and other combination drugs with TMP-SMX therapy yielded even better results.

Identification of housekeeping genes suitable for gene expression analysis in Jian carp (Cyprinus carpio var. jian).

Jian carp (Cyprinus carpio var. jian) is an important economic fish species cultured in China. In this report, we performed a systematic analysis to identify an appropriate housekeeping (HK) gene for the study of gene expression in Jian carp. For this purpose, partial DNA sequences of four potential candidate genes (elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate (GAPDH), beta-actin (ACTB), and 18S ribosomal RNA (18S rRNA) were isolated, and their expression levels were studied using RNA extracted from nine tissues (forebrain, hypothalamus, liver, fore-intestine, hind-intestine, ovary, muscle, heart, kidney) in juvenile and adult Jian carp. Gene expression levels were quantified by quantitative real time RT-PCR (qRT-PCR), and expression stability was evaluated by comparing the coefficients of variation (CV) of the Ct values. The results showed that EF-1α was the most suitable HK gene in all tissues of juvenile and adult Jian carp. However, at distinct juvenile and adult developmental stages, there was not a single optimal gene for normalization of expression levels in all tissues. EF-1α was the most stable gene only in forebrain, hypothalamus, liver, heart, and kidney. These results provide data that can be expected to aid gene expression analysis in Jian carp research, but underline the importance of identifying the optimal HK gene for each new experimental paradigm.

Effects of Zinc Adaptation on Histological Morphology, Antioxidant Responses, and Expression of Immune-Related Genes of Grass Carp (Ctenopharyngodon idella).

The study was conducted to evaluate the effect of zinc adaptation on histological morphology and antioxidant and immune responses of grass carp(Ctenopharyngodon idella). A total of 180 young grass carp (20.0 ± 2.0 g) was equally distributed into 9 groups, and triplicate groups were subjected to 0 µg/L Zn2+ (control group), 200 µg/L Zn2+, and 300 µg/L Zn2+ solution for 42 days, respectively. The results indicated that the liver and gill have obvious pathological changes after long-term adaptation to zinc except the intestine; the zinc adaptation can positively influence intestinal morphology. The activities of GPX (glutathione peroxidase activity), SOD (superoxide dismutase), and CAT (Catalase) were significantly increased in zinc treatment groups (P < 0.05). The genes expression levels of CuZnSOD (copper zinc superoxide dismutase), CAT, Hsp70 (heat shock protein-70), IL-1b (interleukin-1-b), and TGF-ß1 (transforming growth factor-ß1) were upregulated in the gill and intestine of grass carp following waterborne adaptation to zinc solution for 42 days (P < 0.05). In conclusion, zinc adaptation has different effects on organs of grass carp and may reduce the inflammatory response of the body's gills and intestines by improving the body's antioxidant and anti-stress defense capabilities.

Effect of Spirulina and Ferrous Fumarate on Intestinal Morphology and the Diversity of Gut Microbiota of Yellow River Carp.

The study aimed to investigate the effect of diets supplemented with Spirulina and the mixture of Spirulina and ferrous fumarate on intestinal morphology and the diversity of gut microbiota of Yellow River carp. The results showed that the Spirulina and the mixture of Spirulina and ferrous fumarate could promote the development of intestinal villi, increase the thickness of intestinal muscular layer. Additionally, high-throughput sequencing of 16S rRNA gene revealed that the Spirulina and the mixture of Spirulina and ferrous fumarate could alter the composition, diversity, and richness of intestinal microbial communities. The relative abundances of the predominant phyla Firmicutes and Verrucomicrobia showed significant changes at the phylum level after fed with Spirulina and ferrous fumarate. At the genus level, the predominant genera with marked differences in abundances were Flavobacterium, Aeromonas, and Brevinema. In conclusion, this study indicated the Spirulina and the ferrous fumarate could alter the intestinal microbiota structure and could also cause positive impacts on the health of Yellow River carp. This study provides the valuable information for elucidating the mechanisms of Spirulina and ferrous fumarate in aquatic animals in the future.

Association of ABO Blood Groups and Risk of Gastric Cancer.

OBJECTIVE: This study sought to investigate the relationship between ABO blood groups and the risk of gastric cancer as well as clinical pathological parameters and prognosis. METHODS: Gastric cancer patient data were collected from January 1995 to January 2012 at Jilin Cancer Hospital, and the blood group information of the blood donors at Jilin City Blood Center was recorded. The relationships between ABO blood group and both clinicopathological parameters and the risk of gastric cancer were analyzed retrospectively. The impact of ABO blood type on the 5-year survival rate of patients with gastric cancer was evaluated through outpatient and telephone interviews. RESULTS: (1) Compared with the healthy population, the frequency distribution of gastric cancer patients with the A blood group was significantly increased (χ2 = 4.708, P = 0.000), whereas the frequency distribution of gastric cancer patients with the AB blood group was significantly decreased (χ2 = 9.630, P = 0.002). However, there was no significant difference in the distributions of the B blood group and O blood group (P > 0.05). (2) The risk of gastric cancer in people with the A blood group was higher, whereas the risk of gastric cancer in people with the AB blood group was lower. There was no significant difference in the risk of gastric cancer between type B and type O patients (P > 0.05). (3) The ABO blood group was not related to pathological factors, including the size of the gastric tumor or the T stage or N stage of the disease (P > 0.05). (4) Univariate analysis results showed that the degree of differentiation, tumor size, T stage, lymph node metastasis, and type O blood were factors affecting the 5-year survival rate of gastric cancer patients (P < 0.05). Multivariate analysis results showed that tumor size, T stage, lymph node metastasis, and O blood group were independent prognostic factors. The 5-year survival rate for gastric cancer was significantly better in patients with type O blood (hazard ratio = 0.97, 95% confidence interval = 1.67-3.92). CONCLUSION: (1) The risk of gastric cancer was higher in patients with the A blood group and lower in those with the AB blood group. (2) The ABO blood group showed no significant effect on the clinicopathological parameters of gastric cancer. (3) The O blood group may be a prognostic factor for gastric cancer patients.

[Inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats].

OBJECTIVE: To investigate the inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats. METHODS: Hepatocarcinoma model rats were induced by diethylnitrosamine (DEN). Sixty SD male rats were randomly divided into 4 groups: normal control group, hepatocarcinoma model group, ESB of high dose group and ESB of low dose group. All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma-glutamyltransferase (gamma-GT) and alpha-L-fucosidase (AFU) in serum were measured by biochemical examinations; Hematoxy and eosin (HE) methods were used to examine the changes of liver pathology. RESULTS: The levels of ALT, AST, TBIL, ALP, gamma-GT, AFU in hepatocarcinoma model group and ESB groups were higher than that of control group (P < 0.05). ESB could relieve hepatic injures. The levels of liver function indexes in ESB groups were lower than that of model group. Histological examination demonstrated that the number of liver cancer nodus in ESB groups were lower than that of model group. Furthermore, ESB could attenuate the grade of cancer cell differentiation. CONCLUSION: ESB could inhibit experimental hepatocarcinoma and relieve hepatic injures in rats.

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3.

AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

[Anti-proliferative and apoptosis-inducing activity of Scutellaria barbate containing serum on mouse's hepatoma H22 cells].

OBJECTIVE: To investigate the effects of Scutellaria barbate extract (ESB) on suppressing proliferation and inducing apoptosis of mouse hepatoma H22 cells. METHODS: H22 cells cultured in vitro were divided into 5 groups: blank control group, ESB in high, medium, low dose groups and 5-Fu group. H22 cells were cultured in media with serum containing different concentrations of ESB and blank serum. The proliferation of H22 cells was determined by microculture tetrazolium (MTT) assay. Fluorescence microscopy was utilized to observe the apoptosis of H22 cells by staining with Hoechst 33258. The cell cycle and apoptosis were analyzed by flow cytometry (FCM). RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observerd in a dose and time dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48 hours. Among the various phases of cell cycle, the percentage of cells in S phase decreased significantly, while the percentage of cells in G1 phase increased. Drug-containing serum showed positive effect on cell apoptosis. The apoptosis rate of blank control group, ESB in low, medium, high dose groups and 5-Fu group were 0.51%, 1.07%, 3.15%, 7.83%, 11.26%, respectively. CONCLUSION: ESB containing serum can inhibit proliferation and induce apoptosis of H22 cells in vitro.