[Effect of Vitamin D3 to Platelet Activation Mediated by Tumor Cell Culture Medium].

OBJECTIVE: To investigate the effect of vitamin D3 to platelet activation by tumor cell culture medium. METHODS: The peripheral blood platelets of BALB/c mice were isolated. The platelets were activated in 4T1 culture fluid for 24 h. The platelets were divided into 7 groups: control group, activation group, 1 nmol/L vitamin D3 group, 10 nmol/L vitamin D3 group, 50 nmol/L vitamin D3 group, 100 nmol/L vitamin D3 group, and positive drug (0.1 µmol/L eptifibatide) group. CCK-8 assay was used to detect the platelet proliferation at 24, 48 and 72 h. Flow cytometry was used to detect the expression of CD61 and CD62p and receptor for advanced glycation end products (RAGE) at 24, 48 and 72 h. ELISA was used to detect the level of platelet-endothelial cell adhesion molecule-1 (PECAM-1) at 24, 48 and 72 h. RESULTS: The CD41+/CD61+ and CD41+/CD62p+ ratio, PECAM-1 content and RAGE expression of platelets in activated group were all significantly increased as compared with those in control group (P<0.05). Compared with the activated group, the platelet proliferation, proportion of CD41+/CD61+ and CD41+/CD62p+, content of PECAM-1 and RAGE expression in vitamin D3 groups were all decreased (P<0.05). CONCLUSION: Vitamin D3 shows antiplatelet effect and can inhibit platelet proliferation and activation.

[Detection of HHV-8 Virus in 450 Free Blood Donons in Wuhan and Its Genotyping Based on ORFK1 Gene].

OBJECTIVE: To investigate the infection of HHV-8 virus in free blood donors in Wuhan. METHODS: Whole blood samples were collected from 450 free blood donors in Wuhan, and the genomic DNA extraction and serum collection were performed respectively. And then, the positive rate of HHV-8 was detected by PCR and ELISA, the positive detection rates were compared between them. Finally, HHV-8 ORFK1 gene was cloned by PCR in the positive samples, and the HHV-8 ORFK1 gene th was genetyped through sequencing analysis, homology comparison and phylogenetic tree construction. RESULTS: 25 and 23 cases of positive samples were detected by PCR and ELISA, their positive rate were 5.56% and 5.11% respectively, and without statistically significant difference using χ2 test analysis (P > 0.05). Based on the results of ORFK1 gene cloning and sequence analysis in 23 positive samples, 15 samples C subtype of had HHV-8 ORFK1 gene, and 8 cases had A subtype had HHV-8 ORFK1 gene. CONCLUSION: There is a certain percentage of HHV-8 infection in the free blood donors in Wuhan. It is suggested to increase the HHV-8 virus screening for free blood donors, so as to ensure the quality of blood.

Inducible CYP4F12 enhances Hepatitis C virus infection via association with viral nonstructural protein 5B.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) functions as an RNA-dependent RNA polymerase in the HCV replication complex derived from the endoplasmic reticulum in hepatic cells. In this study, NS5B was used as bait in a yeast two-hybrid assay to screen a human liver cDNA library. We confirmed that CYP4F12, a member of the cytochrome P450 superfamily, interacted with NS5B. Furthermore, overexpression of CYP4F12 facilitated HCV replication. In contrast, knockdown of CYP4F12 by specific shRNA decreased HCV replication and viral protein expression. Moreover, our results demonstrated that HCV infection increased the binding of the transcription factor SREBP1 to the CYP4F12 promoter and activated the promoter activity, which indicated that HCV infection increased the expression of CYP4F12 through the SREBP1 pathway. Our results showed that HCV infection induced expression of CYP4F12 protein, which bound to the HCV replication complex to facilitate viral replication.

Soluble interleukin-6 receptor is elevated during influenza A virus infection and mediates the IL-6 and IL-32 inflammatory cytokine burst.

Influenza A virus (IAV) infection is a major worldwide public health problem. However, the factors involved in mediating the inflammatory response to this infection and their relationships remain poorly understood. Here, we show that IAV infection stimulates the expression of the soluble IL-6 receptor (sIL-6R), a multifunctional protein involved in IL-6 signaling. Interestingly, sIL-6R expression upregulated the levels of its own ligand, IL-6 and those of the pro-inflammatory cytokine IL-32. shRNA-mediated knockdown of sIL-6R suppressed IL-6 and IL-32, indicating that this regulation is dependent on sIL-6R during IAV infection. Furthermore, our results demonstrate that IL-32 participates in a negative feedback loop that inhibits sIL-6R while upregulating IL-6 expression during IAV infection. Therefore, we show that sIL-6R is a critical cellular factor involved in the acute inflammatory response to viral infection.

HCV NS4B induces apoptosis through the mitochondrial death pathway.

The hepatitis C virus (HCV) NS4B protein is known to induce the formation of a membranous web that is thought to be the site of viral RNA replication. However, the exact functions of NS4B remain poorly characterized. In this study, we found that NS4B induced apoptosis in 293T cells and Huh7 cells, as confirmed by Hoechst staining, DNA fragmentation, and annexin V/PI assays. Furthermore, protein immunoblot analysis demonstrated that NS4B triggered the cleavage of caspase 3, caspase 7, and poly(ADP-ribose) polymerase (PARP). Further studies revealed that NS4B induced the activation of caspase 9, the reduction of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. However, NS4B expression did not trigger XBP1 mRNA splicing and increase the expression of binding immunoglobulin protein (BiP, or GRP78) and C/EBP homologous protein (CHOP), which serves as the indicators of ER stress. Taken together, our results suggest that HCV NS4B induces apoptosis through the mitochondrial death pathway.