Effects of miR-9 and tetramethylpyrazine on activation of hepatic stellate cells.

Micro-RNAs (miRNAs) are involved in regulation of the incidence and development of several hepatic diseases. Thus manipulating miRNAs may be a promising therapeutic strategy against these entities. In this study hepatic stellate cells (HSCs) were transfected with hsa-miR-9 or anti-hsa-miR-9, treated with tetramethylpyrazine (TMP), or subjected to treatment with TMP and hsa-miR-9 transfection (combined treatment group). Then, real-time polymerase chain reaction (PCR) was performed to measure mRNA levels of hsa-miR-9. Expression of hsa-miR-9 was highest in the combination treatment group compared with other groups, and significantly higher than TMP-treated and hsa-miR-9-transfected groups (both p<0.05). The anti-hsa-miR-9-transfected group expressed the lowest mRNA level of hsa-miR-9 with marked decrease versus control (p<0.05). Downstream factors that may be affected by miR-9 such as leptin, α-smooth muscle actin (SMA), and collagen I, as well as phosphorylation levels of Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) were investigated at the protein level. All these factors were regulated contrariwise to expression trends of hsa-miR-9, showing the lowest level in the combination treatment group and highest level in anti-hsa-miR-9-transfected group. These results suggest that both transfection of hsa-miR-9 and TMP can lead to upregulated endogenous expression of hsa-miR-9, inhibit activation of JAK1/STAT3 signal pathway induced by leptin, and lead to reduction of α-SMA and collagen I-thus impeding activation of HSC.

[Effect of echinacoside on replication and antigen expression of hepatitis B virus].

To verify the effect of echinacoside on replication and antigen expression of hepatitis B virus (HBV) by using HBV-transfected HepG2. 2. 15 cells as the in vitro model. The ELISA method was used to determine HBeAg and HBsAg levels in cellular supernatants. The effect of echinacoside on HBV replication was studied by using HBV transgenic mice as the in vivo model. First of all, the HBV DNA level in hepatic tissues was quantified with PCR method. Meanwhile, the serum transaminase levels and hepatic pathological changes were also evaluated. Subsequently, HBV transgenic mice were divided into five groups: the control group, the lamivudine group (50 mg · kg(-1)) and echinacoside high, medium and low dose group (50, 25 and 12.5 mg · kg(-1)). The mice were orally administered with drugs once per day for 30 days. At the 31st day, the mice serum was separated to measure HBsAg, HBeAg and HBV DNA. Additionally, the liver HBV DNA level and histopathological change were detected. The results indicated that echinacoside at 50 and 100 mg · L(-1) suppressed significantly HBsAg and HBeAg expressions on the sixth day, with the maximum inhibition ratios of 42.68% and 46.29%; And echinacoside at 100 mg · L(-1) also showed an inhibitory effect on HBV DNA. Besides, echinacoside at 50 mg · kg(-1) inhibited significantly HBsAg and HBeAg expressions of HBV transgenic mice, with the inhibition ratios of 42.82% and 29.12%, and reduced markedly the serum HBV DNA level in HBV transgenic mice. In conclusion, the study suggested that echinacoside has a strong effect against HBV replication and antigen expression.

Aqueous synthesis and fluorescence-imaging application of CdTe/ZnSe core/shell quantum dots with high stability and low cytotoxicity.

CdTe/ZnSe core/shell quantum dots were directly synthesized in an aqueous condition by heating a mixed solution of ZnCI2, NaHSe and CdTe QDs in the presence of mercaptosuccinic acid as a stabilizer. By controlling the size and composition, the CdTe/ZnSe QDs with emission wavelength ranging from 540 to 630 nm, high quantum yield (44%) and narrow full width at half maximum (FWHM) could be obtained. Characterization results with HRTEM, XRD and EDX have shown that the synthesized CdTe/ZnSe QDs have good monodispersity and a nice crystal structure, and exhibited better stability and less cytotoxicity as compared with CdTe QDs. Furthermore, luminescent QD-IgG bioprobes were produced to detect the breast cancer marker Her2 on the surface of fixed MCF-7 cancer cells for their optical imaging.

Hepatitis E virus chimeric DNA vaccine elicits immunologic response in mice.

AIM: To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice. METHODS: The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 microg. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected. RESULTS: After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 (3.1+/-0.49) was higher than that in the control group (0.787+/-0.12, P<0.01). None in the control group had a detectable level of anti-HEV IgG. CONCLUSION: DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.

Dynamic correlation between induction of the expression of heme oxygenase-1 and hepatitis B viral replication.

Heme oxygenase­1 (HO­1) possesses significant potential as a drug target for hepatitis B, which may be transferable to patient therapy. The aim of the present study was to clarify the dynamic correlation between the hepatitis B virus (HBV) and HO­1. The levels of HBV replication and expression of HO­1 were investigated in HepG2.2.15 hepatoma cells following exposure to 5­50 µM hemin for 1­6 days. The mRNA expression levels of HO­1 were then detected using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). HBV replication levels were determined by enzyme­immunoassay and a PCR­fluorescence quantitation assay. The results of the present study demonstrated that the mRNA expression levels of HO­1 increased in a dose­dependent manner in the HepG2.2.15 cells, following exposure to 5­50 µM hemin. The mRNA expression levels of HO­1 reached a peak following exposure of the cells to hemin for three days, subsequently the expression of HO­1 decreased. Following exposure to hemin at an optimal concentration of 50 µM for 1­6 days, the levels of the hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the cells were significantly reduced. This marked reduction in the expression of HBsAg and HBeAg reached its peak on the first day, following which the inhibition weakened as the duration of exposure increased. In addition, the inhibition of HBV DNA replication increased with the a longer duration of exposure. Furthermore, HBV DNA levels were significantly decreased following exposure to hemin for 3­6 days. In conclusion, the present study demonstrated that induced expression of HO­1 interfered with HBV replication in a dose and time­dependent manner, implying that a reduction of the HBV viral load may contribute to upregulation in the expression of HO­1.

Hepatoprotective and antiviral properties of isochlorogenic acid A from Laggera alata against hepatitis B virus infection.

ETHNOPHARMACOLOGICAL RELEVANCE: The aim of this study was to determine the anti-hepatitis B effect of isochlorogenic acid A isolated from Laggera alata (Asteraceae), a traditional Chinese herbal medicine. MATERIALS AND METHODS: The anti-hepatitis B activity of isochlorogenic acid A was evaluated by the D-galactosamine (D-GalN)-induced HL-7702 hepatocyte damage model and the HBV-transfected HepG2.2.15 cells. RESULTS: Isochlorogenic acid A significantly improved HL-7702 hepatocyte viability and markedly inhibited the productions of HBsAg and HBeAg. The inhibitory rates of isochlorogenic acid A on the HBsAg and HBeAg expressions were 86.9% and 72.9%, respectively. In addition, isochlorogenic acid A declined markedly the content of hepatitis B virus covalently closed circular DNA (HBV cccDNA) and induced significantly the heme oxygenase-1 (HO-1) expression in HepG2.2.15 cells. CONCLUSIONS: Isochlorogenic acid A was verified to possess the potent anti-hepatitis B activity. The anti-HBV target of isochlorogenic acid A is probably associated with blocking the translation step of the HBV replication. Overexpression of HO-1 may contribute to the anti-HBV activity of isochlorogenic acid A by reducing the stability of the HBV core protein and thus blocking the refill of nuclear HBV cccDNA. Additionally, the hepatoprotective effect of isochlorogenic acid A could be achieved by its antioxidative property and induction of HO-1.

Identification of two hsp90 genes from the marine crab, Portunus trituberculatus and their specific expression profiles under different environmental conditions.

Hsp90, a molecular chaperone, was generally thought to be a unique cytoplasmic form in invertebrates, playing important roles in multiple cellular stress responses. Now, two cytoplasmic Hsp90 cDNAs (ptHSP90-1 and ptHSP90-2 genes) were isolated from an invertebrate - crab Portunus trituberculatus. Main features, sequence identities and phylogenetic analysis with other species were described. Expression profiles in tissues and under stressful conditions were analyzed using semi-quantitative RT-PCR method. ptHSP90-1 and ptHSP90-2 were constitutively expressed with higher transcript levels in ovary and muscle, respectively. A cold treatment rapidly activated both ptHSP90s transcription in hepatopancreas and gill, but caused the ptHSP90-2 mRNA decrease in muscle and ovary. Under heat treatment ptHSP90-1 mRNA was accumulated in hepatopancreas and muscle (but down-regulated in ovary), while ptHSP90-2's transcription tendency in each tissue was the same as that in cold shock. Moreover, the transcriptional levels of both ptHSP90 genes under Cu(2+) stress were evaluated. This crab exposed to the low and high salinity exhibited either lower expression levels of both ptHSP90s or no changes in four tissues except the up-regulation of ptHSP90-2 transcription in hepatopancreas. These results suggested there were at least two Hsp90s in P. trituberculatus, which played differing roles in physiological and stressful conditions.