Assessment of the Effect of Baicalin on Duck Virus Hepatitis.

BACKGROUND: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is a malignant disease in ducklings, causing economic losses in the duck industry. However, there is still no antiviral drug against DHAV-1 in the clinic. OBJECTIVE: Our aim is to investigate the anti-DHAV-1 effect of baicalin, which is a flavonoid derived from the Chinese medicinal herb huangqin (Scutellaria baicalensis Georgi). METHODS: Here, we first detected its anti-DHAV-1 ability in vitro and in vivo. At the same time, the inhibition of baicalin on DHAV-1 reproduction was determined. Finally, we tested and verified the anti-oxidative and immuno-enhancing roles of baicalin on its curative effect on DVH. RESULTS: Baicalin possessed anti-DHAV-1 effect. It improved the cytoactive of DEH which was infected by DHAV-1 as well as reduced the DHAV-1 reproduction in DEH. Under baicalin treatment, mortality of ducklings infected by DHAV-1 decreased, additionally the DHAV-1 level and liver injury in such ducklings were significantly reduced or alleviated. The in vitro mechanism study indicated baicalin inhibited DHAV-1 reproduction via interfering the viral replication and release. Furthermore, the in vivo mechanism study manifested both the anti-oxidative and immuno-enhancing abilities of baicalin, which played crucial roles in its curative effect on DVH. CONCLUSION: This study may provide a scientific basis for developing baicalin as one or a part of the anti-DHAV-1 drugs.

Inhibition mechanisms of baicalin and its phospholipid complex against DHAV-1 replication.

Duck hepatitis A virus type 1 (DHAV-1) is a serious infectious virus of ducklings. Recent study showed baicalin (BA) and baicalin phospholipid complex (BAPC) possessed anti-DHAV-1 effect. However, the antiviral mechanism is not clear. Therefore, the aim of the present work is to study influences and mechanisms of BA and BAPC on DHAV-1. The effects of BA and BAPC on DHAV-1 replication were analyzed by CCK-8 and RT-qPCR methods. And the results showed BA inhibited the replication of DHAV-1, and BAPC was more effective. Then, the influences of BA and BAPC on DHAV-1 protein translation and RNA synthesis were detected by western blot and RT-qPCR. Both BA and BAPC inhibited the protein translation, and BAPC did better. Furthermore, BAPC also inhibited the RNA synthesis. Afterwards, DHAV-1 IRES activity, DHAV-1 3D protein stability, and cellular Hsp70 expression were studied to in-depth understand the inhibition effects of BA and BAPC on DHAV-1 replication. The results indicated BA and BAPC dropped the protein translation via suppressing DHAV-1 IRES activity. Additionally, BAPC dropped the RNA synthesis via reducing the 3D protein stability and inhibiting cellular Hsp70 expression.

Phosphorylation of Icariin Can Alleviate the Oxidative Stress Caused by the Duck Hepatitis Virus A through Mitogen-Activated Protein Kinases Signaling Pathways.

The duck virus hepatitis (DVH) caused by the duck hepatitis virus A (DHAV) has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA) and its phosphorylated derivative (pICA) possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo, and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo, the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2) as an intervention method in vitro. pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs) viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2) and c-Jun N-terminal kinase (JNK), which were related to mitogen-activated protein kinases (MAPKs) signaling pathways. Ultimately, compared to ICA, pICA exhibited more antioxidant activity that could regulate oxidative stress-related indicators, and inhibited the phosphorylation of MAPKs signaling pathway.

Effects of Chrysanthemum indicum polysaccharide and its phosphate on anti-duck hepatitis a virus and alleviating hepatic injury.

To explore new effective anti-duck hepatitis A virus drugs, Chrysanthemum indicum polysaccharide (CIPS) was phosphorylation modified using STMP-STPP method, and phosphorylated Chrysanthemum indicum polysaccharide (pCIPS) was obtained. Characteristic absorption peaks were observed in pCIPS using IR spectrum, suggested that CIPS was successfully modified. In addition, field emission scanning electron micro-scope (FE-SEM) was used to observe the polysaccharides' surface features. In vitro, we found that the survival rate of DHAV-infected hepatocytes increased after the two drugs treatment, indicated that the two drugs possess good anti-DHAV activity. The results of real-time PCR showed that pCIPS inhibited the virus gene replication more effectively than CIPS. Reed-Muench assay was used to observe the changes of the virulence, and the expression level of IFN-ß was observed to verify the changes of virulence. In vivo experiment, the blood virus content reduced after CIPS and pCIPS treatment. To evaluate the ducklings' hepatic injury, the serum ALT, AST, TP and ALB levels were detected. Results showed that both CIPS and pCIPS could alleviate the hepatic injury of ducklings infected DHAV, especially for pCIPS. All the results above mentioned demonstrated that the anti-DHAV activity of CIPS was enhanced after phosphorylation modification.

A flavone-polysaccharide based prescription attenuates the mitochondrial dysfunction induced by duck hepatitis A virus type 1.

The principal target organ of duck hepatitis A virus type 1 (DHAV-1) is duckling liver, which is an energy-intensive organ and plays important roles in body's energy metabolism and conversion. As the "power house" of the hepatocytes, mitochondria provide more than 90% of the energy. However, mitochondria are much vulnerable to the oxidative stress for their rich in polyunsaturated fatty acids. Although previous researches have demonstrated that DHAV-1 could induce the oxidative stress in the serum of the infected ducklings, no related study on the mitochondria during the pathological process of DVH has been reported by far. To address this issue, we examined the HE stained tissue pathological slices, detected the hepatic SOD, CAT and GPX activities and MDA contents and analyzed the ATP content, mitochondrial ultrastructure and the mitochondrial SOD, GPX activities and MDA content in the liver tissues. The results showed that the hepatic redox status was significantly disturbed so that causing the mitochondrial dysfunction, ATP depletion and mitochondrial oxidative stress during the process of the DHAV-1 infection, and a prescription formulated with Hypericum japonicum flavone, Radix Rehmanniae Recens polysaccharide and Salvia plebeia flavone (HRS), which had been demonstrated with good anti-oxidative activity in serum, could effectively alleviate the hepatic injury and the oxidative stress in liver tissue induced by DHAV-1 thus alleviating the mitochondrial injury and oxidative stress. In a word, this research discovers the oxidative stress induced mitochondrial dysfunction and oxidative stress during the DVH pathological process and demonstrates HRS exerts good anti-oxidative activity in liver tissue to protect mitochondria against reactive oxygen species (ROS).

Anti-DHAV-1 reproduction and immuno-regulatory effects of a flavonoid prescription on duck virus hepatitis.

CONTEXT: The flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) has a curative effect on duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1). However, the mechanism of this curative effect is not understood. OBJECTIVE: This study investigates the mechanism of the curative effect of BLIN on DVH caused by DHAV-1. We analyzed the anti-DHAV-1 reproduction mechanism and immuno-regulatory effect of BLIN. MATERIALS AND METHODS: The anti-DHAV-1 reproduction effects of BLIN at 20, 10, 5 and 2.5 µg/mL in vitro, as well as the influence of BLIN at 20 µg/mL on DHAV-1 adsorption, replication and release were tested using the qRT-PCR method. The promotion abilities of BLIN at 20, 10, 5 and 2.5 µg/mL on T- and B-lymphocyte proliferation were investigated by the MTT method. IL-2 and IFN-γ levels and total anti-DHAV-1 antibody secretion after treatment with DHAV-1 for 4, 8 and 54 h were determined by ELISA. RESULTS: BLIN showed a dose-dependent DHAV-1 reproduction inhibitory effect. The inhibitory effect was highest at 20 µg/mL, where DHAV-1 adsorption and release were significantly lower. Meanwhile, BLIN at 5 µg/mL significantly increased T and B lymphocyte proliferation. BLIN stimulated total anti-DHAV-1 antibody secretion in ducklings at the dosage of 4 mg per duckling, but did not stimulate IL-2 and IFN-γ secretion significantly. CONCLUSIONS: BLIN inhibits DHAV-1 reproduction by suppressing its adsorption and release. Additionally, BLIN promoted the duckling antiviral response.

Phosphorylated Codonopsis pilosula polysaccharide could inhibit the virulence of duck hepatitis A virus compared with Codonopsis pilosula polysaccharide.

To screen effective anti-duck hepatitis A virus (DHAV) drugs, we applied STMP-STPP method to prepare phosphorylated Codonopsis pilosula polysaccharide (pCPPS), the phosphorylation-modified product of Codonopsis pilosula polysaccharide (CPPS). The IR spectrum and field emission scanning electron microscope (FE-SEM) were subsequently used to analyze the structure of pCPPS. Several tests were conducted to compare the anti-DHAV activities of CPPS and pCPPS. The MTT method was used to compare the effect of the drugs on DHAV-infected duck embryonic hepatocytes (DEHs), and the Reed-Muench assay was employed to observe changes in the virulence of DHAV. We also applied real-time PCR to examine the relationship between virus replication and the expression of IFN-ß. The results indicated that CPPS could not inhibit the replication of DHAV. In contrast, pCPPS increased the virus TCID50, inhibited viral replication and, accordingly, increased the survival rate of DEHs infected with DHAV. Because DHAV induced the expression of IFN-ß, and the IFN-ß expression level was positively associated with the number of DHAV, the reduction of IFN-ß expression levels after pCPPS treatment demonstrated a decrease in the number of virus particles. These results indicated that pCPPS, which reduces the number of DHAV, was more effective than CPPS in anti-DHAV activity.

Polysaccharides from Traditional Chinese Medicines: Extraction, Purification, Modification, and Biological Activity.

Traditional Chinese Medicine (TCM) has been used to treat diseases in China for thousands of years. TCM compositions are complex, using as their various sources plants, animals, fungi, and minerals. Polysaccharides are one of the active and important ingredients of TCMs. Polysaccharides from TCMs exhibit a wide range of biological activities in terms of immunity- modifying, antiviral, anti-inflammatory, anti-oxidative, and anti-tumor properties. With their widespread biological activities, polysaccharides consistently attract scientist's interests, and the studies often concentrate on the extraction, purification, and biological activity of TCM polysaccharides. Currently, numerous studies have shown that the modification of polysaccharides can heighten or change the biological activities, which is a new angle of polysaccharide research. This review highlights the current knowledge of TCM polysaccharides, including their extraction, purification, modification, and biological activity, which will hopefully provide profound insights facilitating further research and development.

Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes.

Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection, and the virus particles with size of about 50 nm including more than 20 nm of vacuum drying gold were observed on the infected cells surface. What's more, the replication lasted around 13 h after the early protein synthesis for about 5h, and the release of DHAV-1 was in steady state after 32 h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies.

Assessment of a Flavone-Polysaccharide Based Prescription for Treating Duck Virus Hepatitis.

Because polysaccharide and flavone ingredients display good antiviral activity, we developed a flavone/polysaccharide-containing prescription that would be effective against duck viral hepatitis (DVH) and investigated its hepatoprotective effects. Flavones were derived from Hypericum japonicum (HJF) (entire herb of Hypericum japonicum Thunb) and Salvia plebeia (SPF) (entire herb of Salvia plebeia R. Br.), and polysaccharides were derived from Radix Rehmanniae Recens (RRRP) (dried root of Rehmannia glutinosa Libosch). This prescription combination was based on the theory of syndrome differentiation and treatment in traditional Chinese veterinary medicine. In vitro and in vivo experiments were conducted using the three single ingredients compared to the combined HRS prescription to determine their anti-duck hepatitis A viral (anti-DHAV) activity. The results showed that all experimental conditions displayed anti-DHAV activity, but the HRS prescription presented the best effect. To further investigate the hepatoprotective effect of the HRS prescription on DHAV-induced hepatic injury, we tested the mortality rate, the hepatic pathological severity score, plasma biochemical indexes of hepatic function, blood DHAV gene expression levels and peroxidation damage evaluation indexes and then analyzed correlations among these indexes. The results demonstrated that the HRS prescription significantly decreased the mortality rate, reduced the severity of hepatic injury, decreased the hepatic pathological severity score, depressed blood DHAV gene expression levels, and returned the indexes of hepatic function and peroxidation almost to a normal level. These results indicate that the HRS prescription confers an outstanding hepatoprotective effect, and we expect that it will be developed into a new candidate anti-DHAV drug.