Rotundic acid improves nonalcoholic steatohepatitis in mice by regulating glycolysis and the TLR4/AP1 signaling pathway.

BACKGROUND: Steatosis and inflammation are the hallmarks of nonalcoholic steatohepatitis (NASH). Rotundic acid (RA) is among the key triterpenes of Ilicis Rotundae Cortex and has exhibited multipronged effects in terms of lowering the lipid content and alleviating inflammation. The study objective is to systematically evaluate the potential mechanisms through which RA affects the development and progression of NASH. METHODS: Transcriptomic and proteomic analyses of primary hepatocytes isolated from the control, high-fat diet-induced NASH, and RA treatment groups were performed through Gene Ontology analysis and pathway enrichment. Hub genes were identified through network analysis. Integrative analysis revealed key RA-regulated pathways, which were verified by gene and protein expression studies and cell assays. RESULTS: Hub genes were identified and enriched in the Toll-like receptor 4 (TLR4)/activator protein-1 (AP1) signaling pathway and glycolysis pathway. RA reversed glycolysis and attenuated the TLR4/AP1 pathway, thereby reducing lipid accumulation and inflammation. Additionally, lactate release in L-02 cells increased with NaAsO2-treated and significantly decreased with RA treatment, thus revealing that RA had a major impact on glycolysis. CONCLUSIONS: RA is effective in lowering the lipid content and reducing inflammation in mice with NASH by ameliorating glycolysis and TLR4/AP1 pathways, which contributes to the existing knowledge and potentially sheds light on the development of therapeutic interventions for patients with NASH.

Gestational folic acid supplement prevents vitamin D deficiency-induced depression-like behavior by reversing cortical DNA hypomethylation in adult offspring.

Depression is a common mental disorder with an increasing incidence. Several studies have demonstrated that cortical DNA hypomethylation is associated with depression-like behaviors. This study aims to investigate whether maternal vitamin D deficiency (VDD) induces depression-like behaviors and to explore the effects of folic acid supplement on VDD-induced cortical DNA hypomethylation in adult offspring. Female mice were fed with a VDD diet, beginning at 5 weeks of age and throughout pregnancy. Depression-like behaviors were evaluated, and cortical 5-methylcytosine (5mC) content was detected in adult offspring. Results showed that depression-like behaviors were observed in adult offspring of the VDD group. Cortical Ache and Oxtr mRNAs were upregulated in female offspring of the VDD group. Cortical Cpt1a and Htr1b mRNAs were increased in male offspring of the VDD group. Moreover, cortical 5mC content was reduced in offspring of VDD-fed dams. The additional experiment showed that serum folate and cortical S-adenosylmethionine (SAM) contents were decreased in the offspring of the VDD group. Folic acid supplement attenuated VDD-induced SAM depletion and reversed cortical DNA methylation. Moreover, folic acid supplement attenuated VDD-induced upregulation of depression-related genes. In addition, folic acid supplement alleviated maternal VDD-induced depression-like behaviors in adult offspring. These results suggest that maternal VDD induces depression-like behavior in adult offspring by reducing cortical DNA methylation. The gestational folic acid supplement prevents VDD-induced depression-like behavior by reversing cortical DNA hypomethylation in adult offspring.

Development of a sensitive and reliable ELISA kit of urinary haptoglobin to predict progress of diabetic kidney disease.

AIMS: Urinary haptoglobin (UHp) is a potential biomarker for predicting progress of diabetic kidney disease (DKD) to remedy the defects of currently used urinary albumin. The clinical application of UHp is however limited, owing to the extremely low level in urine. This study aims to establish an enzyme-linked immunosorbent assay (ELISA) kit specifically for detecting UHp in urine samples of patients with diabetes and DKD. MATERIALS AND METHODS: Supersensitive human haptoglobin antibodies were generated for ELISA kit development, and the sensitivity, specificity and reproducibility of the kit was evaluated. This kit was used to detect UHp in 246 healthy individuals and 83 patients with type 2 diabetes (T2D). The interference of blood haptoglobin genotypes on UHp measurement was analysed. RESULTS: The UHp ELISA kit had a standard curve ranging from 5 to 200 ng/ml. The low detection limit was 0.11 ng/ml. The coefficients of variation of intra- and interassay were 5.5% and 8.3%, respectively. The kit showed high accuracy with 100.9% mean recovery rate, and linearity R2  = 0.999. The reference range of UHp was 0-42.3 ng/g creatinine (0-Q95) in the healthy individuals. UHp level was significantly higher in T2D patients with microalbuminuria and macroalbuminuria than that in T2D without microalbuminuria (p < 0.01). The UHp concentration measured by this kit was not affected by haptoglobin genotypes. CONCLUSIONS: We have generated an ELISA kit to accurately detect UHp levels, which is potentially a reliable biomarker of DKD.

Adaptability of small brown planthopper to four rice cultivars using life table and population projection method.

In this study, we evaluated the adaptability of the small brown planthopper (SBPH), Laodelphax striatellus (Hemiptera: Delphacidae) to four rice cultivars including Shengdao13 (SD13), Shengdao14 (SD14), Shengdao15 (SD15), and Zixiangnuo (ZXN) using the age-stage, two-sex life table with a simplified method for recording egg production (i.e., every five days vs. daily). The intrinsic rate of increase (r) of the SBPH was the highest (0.1067 d-1) on cultivar SD15, which was similar to the rate on SD14 (0.1029 d-1), but was significantly higher than that occurring on ZXN (0.0897 d-1) and SD13 (0.0802 d-1). The differences of the finite rate of increase (λ) on the four rice cultivars were consistent with the r values. Population projection predicted an explosive population growth of the SBPH occurring in a relatively short time when reared on SD14 and SD15. These findings demonstrated that the SBPH can successfully survive on the four rice cultivars, although there were varying host adaptabilities.

Optimal FSH usage in revascularization of allotransplanted ovarian tissue in mice.

BACKGROUD: Ovarian transplantation is a useful method for preserving the fertility of young women with cancer who undergo radiotherapy and chemotherapy. Follicle-stimulating hormone (FSH) is use to protect transplanted ovarian tissues from ischemia injury through promoting revascularization after transplantation, but the side effect of high level FSH is ovarian overstimulation leading to substantial follicular loss. In this study, we investigated the optimal usage of FSH on revascularization in the in vitro cultured ovarian tissues before and after transplantation. RESULTS: FSH mainly exhibited an additive response in the gene and protein expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and follicle stimulating hormone receptor (FSHR) with its raised concentrations (0.15 IU/ml, 0.30 IU/ml and 0.60 IU/ml) and prolonged treatment (3 h, 6 h, 12 h, 24 h). The concentrations with 0.60 IU/ml FSH could obviously promoted the expression of VEGF, bFGF and FSHR, but under this concentration FSH could also overstimulated the ovarian tissue leading to follicular loss. With the increase of culture time, the gene and protein expression of VEGF and bFGF both were up-regulated in all of the FSH added groups, but FSHR expression decreased when culture time exceeded 12 h. So we chose 0.30 IU/ml FSH added concentration and 6 h culture time as the FSH usage condition in functional revascularization verification experiment, and found that under this condition FSH promoted 2.5 times increase of vascular density in treated group than in control group after ovarian tissues transplantation. CONCLUSION: Ovarian intervention with 0.30 IU/ml FSH for 6 h is an optimal FSH usage condition which could accelerate the revascularization in the allotransplanted ovarian tissue and can not produce ovarian overstimulation.

Identification and bioinformatics analysis of microRNAs associated with stress and immune response in serum of heat-stressed and normal Holstein cows.

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that have an important regulatory function in animal growth and developmental processes. However, the differential expression of miRNA and the role of these miRNAs in heat-stressed Holstein cows are still unknown. In this study, the profile of differentially expressed miRNAs and the target genes analysis in the serum of heat-stressed and normal Holstein cows were investigated by a Solexa deep-sequencing approach and bioinformatics. The data identified 52 differentially expressed miRNAs in 486 known miRNAs which were changed significantly between heat-stressed and normal Holstein cows (fold change >2, P < 0.001). Target genes analysis showed that at least 7 miRNAs (miR-19a, miR-19b, miR-146a, miR-30a-5p, miR-345-3p, miR-199a-3p, and miR-1246) were involved in the response to stress, oxidative stress, development of the immune system, and immune response among the identified 52 differentially expressed miRNAs. Five miRNAs (miR-27b, miR-181a, miR-181b, miR-26a, and miR-146b) were involved in stress and immune responses and the expression of five miRNAs was striking (P < 0.001). In addition, RT-qPCR and deep-sequencing methods showed that 8 miRNAs among the 12 selected miRNAs (miR-19a, miR-19b, miR-27b, miR-30a-5p, miR-181a, miR-181b, miR-345-3p, and miR-1246) were highly expressed in the serum of heat-stressed Holstein cows. GO and KEGG pathway analysis showed that these differentially expressed miRNAs were involved in a pathway that may differentially regulate the expression of stress response and immune response genes. Our study provides an overview of miRNAs expression profile and the interaction between miRNAs and their target genes, which will lead to further understanding of the important roles of miRNAs in heat-stressed Holstein cows.

Expression of FGF4 mRNA is mediated by mating behaviours in mice.

In mammals, breeding is preceded by species-specific mating behaviours. In this study, we investigated whether parthenogenetic embryo quality could be improved by mating behaviours in mice. To investigate this hypothesis, female mice were mated with vasectomized Kunming white male mice after superovulation. Oocytes were collected and counted at 16 h after superovulation. The oocytes were then artificially activated by medium containing 10 mM strontium chloride and 5 µg/ml cytochalasin B. Blastocysts were obtained by cultivating activated oocytes in vitro. Expression levels of reprogramming transcription factors (i.e. Oct4, Sox2, Klf4 and c-Myc) in oocytes, apoptosis-related genes (i.e. Bax, Bcl2 and c-Myc) in cumulus cells and pluripotency-related transcription factors (i.e. Oct4, Nanog and FGF4) in blastocysts were analysed in samples collected from mated and unmated mice. Additionally, developmental competence of parthenogenetic embryos was used to assess following fibroblast growth factor 4 (FGF4) treatment. The results showed that the formation rate of blastocysts in unmated mice was significantly higher than that in mated mice (p < 0.05). Embryo development was primarily blocked at the eight-cell stage in mated mice; however, the blastocyst formation rate did not differ significantly between groups after the addition of 25 ng/ml FGF4 to the medium at the four-cell stage (p > 0.05). Moreover, the expression of the reprogramming factor Sox2 was significantly different in oocytes collected from mated versus unmated mice. Taken together, our results demonstrated that mating behaviours influenced embryonic development in vitro by decreasing FGF4 expression.

Preoperative Cyfra21-1 and SCC-Ag serum titers predict survival in patients with stage II esophageal squamous cell carcinoma.

BACKGROUND: The purpose of this study is to evaluate the predictive significance of preoperative serum level of cytokeratin 19 fragments (Cyfra21-1) and squamous cell carcinoma antigen (SCC-Ag) after complete resection in patients with stage II esophageal squamous cell carcinoma (ESCC). METHODS: Between 1995 and 2006, a total of 379 patients in stage II ESCC who underwent complete resection were consecutively recruited. Statistical analyses were applied to test the associations between preoperative serum titers of Cyfra21-1 and SCC-Ag, clinicopathological factors and prognoses. RESULTS: Preoperative high and normal serum level of Cyfra21-1 and SCC-Ag were found in 47.8%, 52.2% and 72.8%, 27.2%, respectively. The 1-, 3-, 5-year overall survival rate for the entire cohort of patients was 95%, 78%, and 56%, respectively. Median overall survival (OS) was 45.3 months longer in patients with low preoperative serum level of Cyfra21-1 (91.9 months) than those with high preoperative serum level of Cyfra21-1 (46.6 months) (P < 0.001). Median OS among patients with SCC-Ag-low level was also longer than those with SCC-Ag-high level (89.7 vs. 63.7 months, P < 0.001), especially for those with stage IIB (P < 0.001). After multivariate analysis, along with pTNM stage, preoperative serum level of Cyfra21-1 and SCC-Ag were independently and significantly predictive factors (P < 0.001, P < 0.001). Furthermore, the five-year survival rate in double-low subset, either-low subset and double-high subset was 100%, 83% and 27%, respectively (P < 0.001). CONCLUSIONS: The preoperative serum level of Cyfra21-1 and SCC-Ag are independently significant predictors which negatively affected the survivals of patients with stage II ESCC.

[Analysis of genetic diversity and biological functional site of HIV-1 gp120 from a patient with AIDS dementia complex].

To explore the relationship between the genetic diversity and biological functional site of human immunodeficiency virus HIV-1 gp120 and the pathogenesis of AIDS dementia complex (ADC), the full length sequences of gp120 gene was amplified with PCR from genomic DNA which was extracted from lymphoid and different brain department (periaortic lymphoid, temporal gray/white matter junction, periventricular tissue, choroids plexus, occipital white matter and occipital gray/white matter junction.) of a patient who died of ADC. PCR products were cloned into the pGEM-T vector and positive clones were sequenced. The analysis of neighbor-joining tree, N-glycosylation sites, values of ds/dn, and loop were then all performed. The samples were all identified as HIV-1 B and genetic variation existed in HIV-1 gp120 isolated from different tissues. Compared with standard HIV-1B gp120, biological functional sites of HIV-1 gp120 isolated from the patient changed to some extent. In addition, there were differences in some biological functional sites of HIV-1 gp120 between lymphoid and brain. Therefore, genetic diversity and alterations of some biological functional sites of HIV-1 gp120 might be associated with the pathogenesis of ADC.

Study on the relationship between G1057D variants of IRS2 gene and obese T2DM in Chinese Han subjects.

OBJECTIVE: To investigate the relationship between the G1057D variants of insulin receptor substrate-2(IRS2) gene and type 2 diabetes mellitus (T2DM) in subjects. METHODS: Four hundred and thirty-nine Chinese Han subjects, including 218 patients with T2DM and 221 normal controls, were selected from the Hans in the Liaoning area, and each group was divided into two subgroups according to body mass index. The G1057D variants of IRS2 were detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and their relationships with T2DM were analyzed. RESULTS: (1) The frequency of G1057D variant was 29% in all subjects. The frequency of DD genotype was significantly lower in non-obese DM group than in non-obese control group. The Logistic regression analysis showed that the odds ratio of DD genotype was 0.265. The frequency of DD genotype was significantly higher in obese DM group than in obese control group. The Logistic regression analysis showed that the odds ratio of DD genotype was 3.991. (2) In the non-obese control group, the FPG and 2hCP of DD genotypes were lower than those of GG genotypes (P< 0.05, P< 0.01), the HOMA-B of DD genotypes was higher than that of GG genotype (P< 0.01). In the non-obese DM group, the waistline/hip ratio (WHR) of DD genotypes was higher than that of GG genotypes(P< 0.01). In the obese DM group, the WHR, HOMA-IR, 2hPG, 2hINS and 2hCP levels of DD genotypes were higher than those of GG genotypes, while the level of HOMA-B of DD genotypes was lower than that of GG genotypes. In the obese control group, the WHR, HOMA-IR, 2hPG, 2hINS and 2hCP levels of DD genotype were higher than those of GG genotype, and the HOMA-B level of DD genotype was lower than that of GG genotypes (P< 0.05). CONCLUSION: The relationships between G1057D variants of IRS2 and T2DM are mediated by obesity.