| Duck virus hepatitis(DVH)caused by duck hepatitis A virus type 1(DHAV-1)is an acute and severe infectious disease that mainly harms ducklings within 3 weeks.However,no clinical treatment is available for this disease except for the yolk antibody at present.Therefore,once the ducklings were ill,they will die soon,which will cause huge economic losses to the poultry breeding industry in China.Traditional Chinese medicine(TCM)has been used for thousands of years in China,and it still provides important protection for human and animal health there.According to the theory of traditional Chinese veterinarians medicine(TCVM),the pathogenesis of DVH is believed to be caused by evil heat and wind in the liver,and the syndrome type is a high-heating wind syndrome.The treatment principle should be clearing heat and cooling blood,nourishing yin and stopping phlegm.Therefore,Hypericum japonicum.Thunb,Radix Rehmanniae Recens and Salvia plebeia R.Brown were selected as the prescription medicines.Among them,Hypericum japonicum.Thunb,is cool,sweet and slightly bitter,and has the effects of clearing away heat and dampness,dispersing phlegm and relieving pain,reducing swelling,detoxification and so on;Radix Rehmanniae Recens has the effects of clearing heat and cooling blood,nourishing yin and fluid and so on.Salvia plebeia R.is cool,bitter,has the effects of clearing away heat,detoxification,cooling blood,hemostasis,diuretic and so on.Meanwhile,for the convenience of research and quality control of the drug,we used the active ingredients in the Chinese herbal medicines to formulate the flavone-polysaccharide based prescription(Hypericum japonicum flavones-Radix Rehmanniae Recens polysaccharides-Salvia plebeia flavones,HRS).The rationality and effectiveness of the prescription was determined by comparing the anti-DHAV-1 activities of the single ingredients with the HRS prescription in vitro and in vivo.Then,the effectiveness of HRS on the treatment of DVH was confirmed by in vivo clinical studies and it was found that its therapeutic effect may be based on its hepatoprotective activity and anti-DHAV-1 proliferative activity.In order to explore its anti-DHAV-1 proliferation activity mechanism,the effect of HRS on the proliferation of DHAV-1 in duck embryonic hepatocytes(DEHs)was studied.At the same time,oxidative damage,an important mechanism of liver injury during the pathogenesis of DVH,was used as an entry point to explore the antioxidant activity of HRS in vitro and in vivo.Subsequently,based on the antioxidant signaling pathway of Nrf2/ARE,the molecular regulation mechanism of antioxidative damage of HRS was studied.The specific content is divided into the following seven parts:Test Ⅰ.Development of a traditional Chinese medicine ingredient prescription against duck type 1 viral hepatitis In order to develop a clinically effective DVH therapeutic drug,the traditional Chinese veterinary theory dialectically selected Hypericum japonicum flavones(HJF),Radix Rehmanniae Recens polysaccharides(RRRP)and Salvia plebeia flavones(SPF)to form the HRS prescription.First,the safe concentrations of HJF,RRRP,SPF and HRS on DEHs and their respective protective effects on DHAV-1 infected DEHs were examined by MTT assay.Meanwhile,the DVH model was established by artificial injecting the DHAV-1 to the ducklings,and then the infected ducklings were treated with corresponding drug at a dose of 3 mg/feather·d for 5 days by drinking water.Finally the effects of the active ingredients of the three TCM and their prescription on the course and the final mortality of DHAV-1 infected ducklings were counted.In order to further systematically evaluate the clinical therapeutic effect of HRS on DVH,the DVH model of artificially infected DHAV-1 ducklings was established again,and DVH ducklings were treated with HRS by drinking water at a dose of 3 mg/feather·d for 5 days.Fianlly,the influence of HRS on the mortality rate,visual pathological changes of liver,liver tissue microscopic pathological changes,liver function biochemical indicators and the DHAV-1 gene expression level in the blood of DHAV-1 infected ducklings,were detected.The results showed that the maximum safe concentrations of HJF,RRRP,SPF and HRS on DEHs were 12.5 μg/mL,1250 μg/mL,2000 μg/mL and 2500 μg/mL,and HJF,RRRP,SPF and HRS all had protective effects on DHAV-1 infected DEHs,and their maximum hepatoprotective rates were 37.0%,9.9%,14.0%,and 64.3%,respectively,and all the active ingredients of the three TCM and their prescription can effectively shorten the course of DVH caused by DHAV-1 and the final mortality rates of HJF,RRRP,SPF and HRS groups were:57.1%,62.9%,60.0%and 42.9%.It indicated that after the combination of these three active ingredients,HRS prescription showed a superior effect on the protection of hepatocytes and on the therapeutic effect on duck virus hepatitis compared with the single active ingredients in vitro and in vivo.A more systematic in vivo test,found that HRS treatment can significantly reduce the high mortality of ducklings with DVH,while the degree of pathological damage of liver tissue was significantly reduced,the area of inflammatory cell infiltration and necrosis of liver tissue was reduced,plasma ALT and LDH levels were significantly decreased,and the expression of DHAV-1 virus gene in duckling blood was significantly reduced.It indicated that the therapeutic effect of HRS on DVH was exact,which may be based on its hepatoprotective activity and anti-DHAV-1 proliferative activity.Test Ⅱ.Effect of HRS on the proliferation of DHAV-1 on DEHs This study was designed to investigate the influence of HRS on the adsorption,replication and release of DHAV-1 on DEHs.The effects of HRS on DHAV-1 adsorption on DEHs were detected by qRT-PCR with two different drug adding manners:add drug after virus inoculation and inoculate virus after adding drug.Meanwhile,the effect of HRS on DHAV-1 replication and release of DHAV-1 on DEHs were also detected with qRT-PCR method.The results showed that,in the adsorption phase,the DHAV-1 gene expression level in the HRS group was at the same level as that in the VC group when the drug was added after virus addition and the expression level of DHAV-1 gene in the high concentration HRS group was significantly lower than that in the VC group when the virus was added after drug addition;The expression levels of DHAV-1 gene in HRS group at high,medium and low concentrations were significantly lower than that in the VC group in virus replication phase;There was no significant difference in DHAV-1 gene expression level between the HRS group and the VC group during the virus release.It indicated that the effect of HRS against DHAV-1 infection on DEHs was mainly by inhibiting the replication of the virus,and the inhibitory effect of the drug on the virus adsorption could only be exerted under the high concentration and with the drug adding manner of adding the virus after drug addition.Test Ⅲ.Study on the antioxidant activity of HRS in vitro This study was designed to investigate the free radical scavenging activity of HRS and its effects on oxidative damage to DEHs caused by DHAV-1 infection.Firstly,the abilities of HRS to scavenge ABTS free radical,DPPH free radical and hydrogen peroxide in vitro was examined.Then,the effects of HRS on the superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPX)activities and(malonodialdehyde)MDA content in DHAV-1 infected DEHs were investigated.Meanwhile,the influence of HRS on ROS content in DHAV-1 infected DEHs were detected by flow cytometry and immunofluorescence,respectively.It showed that HRS had good ABTS,DPPH free radicals and hydrogen peroxide scavenging ability in a certain concentration range,and could significantly enhance the SOD,CAT and GPX,and could significantly decrease intracellular MDA and ROS levels in DEHs infected with DHAV-1.It demonstrated that HRS had good antioxidant activity in vitro and could significantly alleviate the oxidative damage of DEHs caused by DHAV-1 infection.Test Ⅳ,Protective effect of HRS on oxidative damage in ducklings induced by DHAV-1 infection The aim of this study was to investigate whether HRS can effectively alleviate oxidative damage in ducklings caused by DHAV-1 infection.Firstly,The DVH model was established by artificial inoculation of DHAV-1 in ducklings.DHAV-1 infected ducklings were treated with HRS by drinking water.The effects of HRS treatment on SOD,CAT and GPX activities and MDA content,liver tissue ATP content,mitochondrial ultrastructural changes,mitochondrial SOD and GPX activity and MDA content in DHAV-1 infected ducklings were studied.The results showed that HRS could significantly increase the activities of SOD,CAT and GPX,and significantly reduce the content of MDA in plasma and in liver tissues of infected ducklings.Meanwhile,HRS could also significantly increase the ATP content in liver tissue and increase SOD and GPX activities in liver tissue mitochondria,and reduce MDA content in liver tissue mitochondria,and alleviate mitochondrial ultrastructural damage in liver tissue.It indicated that DHAV-1 infection caused severe oxidative damage in the body,liver tissue and mitochondria,disrupted mitochondrial function and destroyed the internal structure of mitochondria,and HRS could enhance the antioxidant enzyme activities and reduce the production of MDA in plasma,liver tissue and mitochondria by its antioxidant activity to reduce oxidative damage in the body,liver tissue and mitochondria,thereby playing a role in liver protection against injury.Test Ⅴ.Verification the effect of HRS on the DHAV-1 induced oxidative damage by intervention test This study aimed to further validate that the therapeutic effect of HRS on DVH is dependent on its antioxidant activity.Firstly,the DVH model was established by artificially inoculate the DHAV-1 to ducklings,Then,the infected ducklings were treated with HRS.Meanwhile,the intramuscular dose of 80 mg/kg hinokitiol was used as an pro-oxidant intervention agent in the treatment of HRS in vivo.The activities of SOD,CAT,GPX and MDA content in liver tissues of ducklings,as well as liver lesion scores and mortality rate were determined in each group.The results showed that the activities of SOD,CAT and GPX in the liver tissues of the ducklings in the HRS group were significantly decreased and the MDA content and the liver lesion scores and the mortality rate were significantly increased after the intervention of hinokitiol.It indicated that the pro-oxidant intervention agent hinokitiol significantly reduced the antioxidant effect of HRS during DVH treatment,which significantly affected its therapeutic effect.Therefore,the therapeutic effect of HRS on DVH is directly related to its antioxidant effect.Test Ⅵ.Exploration of the regulatory mechanism of HRS against oxidative damage induced by DHAV-1 infection based on Nrf2/ARE signaling pathway The aim of this study was to investigate the molecular regulatory mechanisms of HRS against oxidative damage caused by DHAV-1 infection.The effect of HRS on the mRNA expression levels of SOD-1,CAT,GPX-1 and Nrf2 in DHAV-1 infected DEHs was detected by qRT-PCR in vitro,and Western Blot method was used to detect the effect of HRS on the expression of SOD-1,CAT,GPX-1,total Nrf2 protein and nuclear Nrf2 protein in DHAV-1 infected DEHs.Subsequently,Nrf2 specific inhibitor ML385 was used to pretreat DHAV-1 infected DEHs to intervene Nrf2/ARE signaling pathway.qRT-PCR was used to detect the effect of HRS on the mRNA expression levels of SOD-1,CAT,GPX-1 and Nrf2 in DHAV-1 infected DEHs after ML385 pretreatment,and Western Blot method was used to detect the effect of HRS on the expression of SOD-1,CAT,GPX-1,total Nrf2 protein and nuclear Nrf2 protein in DHAV-1 infected DEHs after ML385 pretreatment.The results showed that DHAV-1 infection significantly reduced the mRNA and protein expression levels of SOD-1,CAT,GPX-1 and Nrf2 genes in DEHs.Meanwhile,the mRNA and protein expression levels of the above-mentioned indicators in infected DEHs treated with HRS were significantly up-regulated,and ML385 intervention significantly reduced the up-regulation of the mRNA and protein expression levels of SOD-1,CAT,GPX-1 and Nrf2 genes in infected DEHs caused by HRS treatment.These results indicated that the molecular mechanism of HRS against oxidative damage induced by DHAV-1 infection was based on Nrf2/ARE signaling pathway. |
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