TLR4 promotes liver inflammation by activating the JNK pathway.

OBJECTIVE: Drug-induced liver injury has become a serious public health problem that cannot be ignored. Although the mechanism of acetaminophen (APAP)-induced liver injury has been investigated for several decades, there are still many deficiencies. However, only a deeper study of its mechanism can provide more effective measures of prevention and treatment for APAP-induced liver injury. The aim of this study was to investigate whether toll-like receptor 4 (TLR4) participates in and regulates APAP-induced liver injury, which may provide a new direction for the prevention and treatment of clinical drug-induced hepatitis. MATERIALS AND METHODS: WT mice were treated with APAP (300 mg/kg) or equivalent PBS. The livers of mice were taken at 1 h, 3 h, 6 h and 12 h after treatment. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect TLR4 mRNA expression level in the liver. After TLR4 involving in APAP-induced liver injury was confirmed, we investigated the relationship between TLR4 expression and hepatic inflammation. WT and TLR4-/- mice received APAP (3000 mg/kg) intraperitoneal injection after 16 h of fasting; serum was collected after 8 h and 24 h, and serum alanine aminotransferase (ALT) and reduced glutathione (GSH) activity were measured. Rat liver tissue was observed for histological changes by hematoxylin and eosin (H&E) staining. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) assay were performed to analyze proinflammatory cytokines expression (such as TNF-α, IL-1ß, MCP-1, IL-6). After isolating mononuclear cells (MNCs) in the liver of mice, flow cytometry was used to detect cell activation level and infiltration of macrophages and neutrophils. Western blotting was used to analyze the activation of phosphorylated JNK and p38 signaling pathways in livers of WT and TLR4-/- mice. In addition, after stimulated with APAP, the silence of TLR4 in RAW264.7 cells could activate phosphorylated JNK and p38 signaling pathways. RESULTS: After APAP stimulation, WT mice exhibited more severe liver injury than TLR4-/- mice, with higher ALT levels, lower GSH levels, and more necrotic or apoptotic cells. TLR4-/- mice have lower levels of inflammatory cytokines including MCP-1 and IL-6; at the same time, the number of infiltrating macrophages and neutrophils in liver tissue of TLR4-/- mice was significantly lower than that of WT mice. The activation of JNK signaling pathway was strikingly enhanced in WT mice treated with APAP, but no significant difference was observed in the activation of JNK phosphorylation in TLR4-/- mice after the same dose of APAP stimulation. Similarly, in RAW264.7 cells, the activation of phosphorylated JNK and p38 was remarkably inhibited by TLR4-siRNA, but was activated in the control group, which was consistent in vivo. CONCLUSIONS: APAP-treated TLR4-/- mice showed milder liver injury compared to WT mice. It was confirmed that TLR4 could activate the JNK signaling pathway to induce the secretion of inflammatory factors and the infiltration of macrophages to promote APAP-induced liver injury. This finding might provide a new prevention and treatment idea for clinical drug-induced hepatitis.

[Research Progress on the Detection Method of DNA Methylation and Its Application in Forensic Science].

As an important part of epigenetic marker, DNA methylation involves in the gene regulation and attracts a wide spread attention in biological auxology, geratology and oncology fields. In forensic science, because of the relative stable, heritable, abundant, and age-related characteristics, DNA methylation is considered to be a useful complement to the classic genetic markers for age-prediction, tissue-identification, and monozygotic twins' discrimination. Various methods for DNA methylation detection have been validated based on methylation sensitive restriction endonuclease, bisulfite modification and methylation-CpG binding protein. In recent years, it is reported that the third generation sequencing method can be used to detect DNA methylation. This paper aims to make a review on the detection method of DNA methylation and its applications in forensic science.

[Genetic diversity evaluation of cultivars (G. hirsumtum L.) from the Changjiang river valley and Tellow river valley by RAPD markers].

Forty-one polymorphic decamer primers with clear and repeated band pattern, which were screened out from 300 randomly selected primers, were applied to assess genetic diversity of cultivars (G. hirsumtum L.) from the Changjiang river valley and the Yellow river valley by RAPD analysis. Eighty-four polymorphic loci were obtained from 84 cultivars from the Changjiang river valley and 96 polymorphic loci were obtained from 68 cultivars from the Yellow river valley. Jaccard's genetic similarity coefficients were calculated using the software of NTSYS-pc version 1.80, and two dendrograms were constructed by the unweighted pair group method of arithmetic average (UPGMA). Mean similarity coefficient among cultivars from the Changjiang river valley was up to 0.631. And cultivars from the Yellow river valley had an average similarity coefficient of 0.632. Compared similarity coefficient matrices and frequency distribution of pairwise similarity coefficients, a conclusion was drawn that cultivars from the two areas had nearly the same level of genetic diversity. Same base germplasm, breeding objectives, breeding methods and strategy could be attributed to similar genetic diversity between the two areas.

Genetic diversity evaluation of some Chinese elite cotton varieties with RAPD markers.

RAPDs analysis was used to assess the genetic diversity of some elite cotton varieties. 17 cotton varieties or breeding lines were screened with 200 arbitrary primers to generate 113 polymorphic bands. Cluster analysis and UPGMA showed that 17 varieties could be put into 3 clusters corresponding to genome difference. The genetic similarity among them ranged from 0.2000 to 0.8451. Three elite varieties Ejing-1, Sumian-3 and Zhongmiansuo-12 are highly similar in genetic base. 7581, an offspring from Gossypium hirsutum x G. hirsutum race Latifolium, is distinct from other upland cotton varieties. A trend in germplasm usage in the same research center was also shown in the study. To sustain progress in genetic improvement, we suggest that some good gene from other species should be considered in the upland cotton breeding procedure.

Antibody against Testudo herpesvirus is not common in Chinese soft-shelled turtles.

Seventy-six serum samples of Chinese soft-shelled turtles (Trionyx sinensis) were collected at Jiangsu Province, China. The neutralization test (NT) was performed with the sera, Testudo herpesvirus (THV) and turtle heart cells (THC). Neutralizing antibodies were detected in five of 76 samples and the titres were 1:10-1:20. Having optimized the conditions, the dot-enzyme-linked immunosorbent assay (Dot-ELISA) was developed and eight serum samples exhibited positive results. Five samples were positive by both NT and Dot-ELISA. The percentage of positive samples was only 6.6% (NT) and 11% (ELISA). It is suggested that THV infection is not a serious problem for the Chinese soft-shelled turtle culture in this region.