| Epimedium(Epimedium davidii Franch)is one of traditional Chinese herbal medicines for replenishing.It has the function of "promoting vital energy,strengthening bones,making up waist and knees,and strengthening the heart." With the development of its pharmacological function in recent years,it has been found that icariin(ICA)is the main pharmacological active ingredient of Epimedium.A number of clinical and experimental pharmacological studies have shown that ICA has antiviral,anti-oxidant,and immune-enhancing effects.However,due to poor water solubility of flavonoids,it often causes some inconvenience in practical clinical applications.In order to improve the bioavailability of ICA,to better exert its antiviral effect,and facilitate its application in clinical practice,phosphorylation of ICA was successfully performed by a mixed phosphate method to obtain phosphorylated icariin(pICA).In the previous experiments of us,we found that the infection of Duck Hepatitis A virus(DHAV-1)can lead to severe liver injury and severe oxidative stress in the ducklings.The significant correlation shows that the inflammatory lesions caused by DHAV-1 infection in ducklings are closely related to oxidative stress injury.After the treating with ICA and pICA,the levels of inflammatory factors in ducklings were significantly decreased,and the activities of antioxidant enzymes were significantly increased.The final survival rate was significantly improved and showed a good therapeutic effect,which may be related to the anti-inflammatory and anti-oxidative stress effects of ICA and pICA.However,the above studies are limited to the in vivo experiment,and the molecular mechanism remains unclear.Therefore,in order to further explore the anti-DHAV-1 mechanism of ICA and pICA,verifying the previous guesses in in vivo experiments,we investigated the effects of ICA and pICA on DHAV-1 induced inflammatory response,oxidative stress,mitochondrial damage,apoptosis and cell proliferation in Duck embryonic hepatocytes(DEHs).The test is divided into the following six parts:Test I ICA and pICA Relieve Inflammatory Responses to DEHs Induced by DHAV-1 This study was designed to investigate the mechanism of DHAV-1-induced inflammatory response in DEHs and the alleviation of ICA and pICA.First,the levels of inflammatory cytokines IL-?,IL-6,IL-8,TNF-?,and their mRNA levels of DEHs after treatment with DHAV-1 and ICA and pICA were measured.Afterwards,we examined the genes mRNA expression of key regulatory nodes in the NF-?B pathway affected by inflammation,such as the TLR family key factors(TLR 2,TLR 3,TLR 4,TLR 5,TLR 7,Myd88)and protein levels of TLR 2,TLR 4,Myd88,and NF-?B p65.The results showed that the levels of IL-1 ?,IL-6,IL-8 and TNF-? secreted by DEHs after infection with DHAV-1 were significantly increased.The results of mRNA expression of related genes also confirmed this phenomenon.Meanwhile,TLR 2,TLR 3,TLR 4,TLR 5,TLR 7,Myd88 mRNA expression and TLR 2,TLR 4,Myd88 protein expressions were also significantly enhanced,and promoted the phosphorylation of NF-?B p65 protein.The addition of ICA and pICA significantly decreased the levels of IL-1 P,IL-6,IL-8,TNF-? and mRNA expression,reduced the genes mRNA expression of TLR 2,TLR 3,TLR 4,TLR 5,TLR 7,Myd88 and proteins expression of TLR 2,TLR 4,Myd88,while phosphorylation of NF-?B p65 protein was inhibited.This indicated that DHAV-1 could activate NF-? pathway by TLR from both Myd88-dependent and non-Myd88-dependent pathways,and eventually caused cell inflammatory response.ICA and pICA can reduce the above-mentioned indicators and play a therapeutic role by reducing the degree of inflammation of the body.Compared with ICA,pICA is more effective in relieving inflammation.Test II Study on ICA and pICA relieving the oxidative stress to DEHs induced by DHAV-1 This study was designed to investigate the Mitigation effect of ICA and pICA on DHAV-1 induced DEHs oxidative stress.Firstly,the oxidative stress related indicators(SOD,MDA,CAT,GSH,GSH-Px)of DEHs after treatment with DHAV-1 and ICA and pICA were detected.After the addition of H2O2 as the pro-oxidant(divided two ways including DHAV-1 followed by H2O2 and then H2O2 followed by DHAV-1),this reverse intervention was used to verify the antioxidant effect of ICA and pICA.Finally,the effects of ICA and pICA on the virulence of the virus were examined before and after H2O2 intervention.The results showed that the MDA content of DEHs increased significantly after treatment with DHAV-1,and the activities of SOD,CAT,GSH,and GSH-Px decreased significantly.ICA and pICA reduced the MDA content of DEHs,and increased SOD,CAT,GSH,and GSH-Px activity.In the two different sequential interventions,the antiviral effect of the intervention group after H2O2 addition was lower than that of the control group without H2O2 intervention.At the same time before and after the intervention,the TCID50 of DHAV-1 in the ICA and pICA groups were less than that of the VC group,and the TCID50 of DHAV-1 in each group after the H2O2 intervention was greater than that of before the intervention.The experiments in this chapter showed that ICA and pICA can play an antioxidant role in vitro by reducing the MDA content and increasing the activity of related antioxidative enzymes,The H2O2 intervention experiment approved the proof that the anti-oxidation effect of ICA and pICA in vitro was necessary for anti-DHAV-1.In addition,compared with ICA,pICA could significantly increase the activity of SOD and GSH-Px in vitro.Test III Mechanism of ICA and pICA Regulating the Mechanism of DHAV-1-induced Oxidative Stress in DEHs This study was designed to investigate the specific regulatory mechanisms of ICA and pICA in relieving DHAV-1 DEHs oxidative stress induced by DHAV-1DEHs in vitro.Firstly,the protein expression of SOD and GSH-Px in DEHs after treatment with DHAV-1 and ICA and pICA were detected.Secondly,The mRNA expression of anti-oxidative stress-related genes(Nrf2,NQO1,HO-1,GST,GCLC and GCLM)were detected.Finally,we examined the protein expression of ERK1/2,JNK,and p38 proteins in the MAPKs signaling pathway.The results showed that the abundances of SOD and GSH-Px proteins in pICA were significantly higher than those in ICA,whereas the mRNA expression of Nrf2,NQO1,HO-1,GST,GCLC,and GCLM in DEHs infected with DHAV-1 were significantly decreased,the mRNA expression of Nrf2,NQO1,HO-1,GST,GCLC and GCLM all increased after ICA and pICA were added.The genes mRNA expression of GST and GCLC genes in pICA group was were significantly higher than that in ICA group and the genes mRNA expression of GCLM gene in ICA group was significantly higher than that in pICA group.The results of MAPKs signaling pathway-associated proteins showed that phosphorylation of ERK1/2,JNK,and p38 proteins was significantly increased in DEHs after infection with DHAV-1,whereas ICA and pICA significantly decreased phosphorylation of ERK1/2,JNK,and p38 proteins,and the pICA group was also significantly expressed lower than ICA group.The experiments in this chapter showed that DHAV-1 can significantly reduce the gene mRNA expression of Nrf2 and inhibit the genes mRNA expression of downstream(NQO1,GST,HO-1,GCLC and GCLM),resulting in severe oxidative stress injury.However,ICA and pICA can play an antioxidant role by increasing the mRNA expression of upstream gene of Nrf2 and increasing the mRNA expression of downstream genes of NQO1,GST,HO-1,GCLC and GCLM.DHAV-1 can also promote the phosphorylation of ERK1/2,JNK,and p38 proteins in the MAPKs signaling pathway,while ICA and pICA can relatively suppress the phosphorylation of ERK1/2,JNK,and p38 proteins.This shows that ICA and pICA can not only improve the body antioxidant activity against the virus,may also have an antiviral effect by affecting other pathways mediated by MAPKs such as inflammation,apoptosis,cell proliferation,etc.,whereas pICA may be more resistant to oxidation than ICA.Test IV ICA and pICA Mitigates the Effect of DHAV-1 on Mitochondrial Damage in DEHs This study was aimed to investigate the mechanism of mitochondrial damage caused by DHAV-1 in DEHs and the alleviation of ICA and pICA.Firstly,the mitochondrial membrane potential,mitochondrial ROS level,and Ca2+ concentration of DEHs after treatment with DHAV-1 and ICA and pICA were detected.The enzyme activities of COX and SDH were detected,and finally the ATP content was detected.The results showed that DHAV-1 can significantly reduce the mitochondrial membrane potential,increase mitochondrial ROS levels and Ca2+ concentration,and inhibit the enzyme activities of COX and SDH,resulting in a significant decrease in ATP content.The addition of ICA and pICA can maintain the mitochondrial membrane potential stability,reduce mitochondrial ROS levels and Ca2+ concentration,while increasing the enzyme ctivities of COX and SDH,maintaining the stable production of ATP.This shows that DHAV-1 can damage mitochondrial membrane permeability through oxidative stress to change mitochondrial structure,inhibit mitochondrial respiratory chain enzyme activity,reduce ATP production,and ultimately play a pathogenic role.ICA and pICA can protect against mitochondrial damage caused by DHAV-1 with maintaining the stability of internal functions such as mitochondrial oxidative respiratory chain and tricarboxylic acid cycle.In addition,pICA is more protective of mitochondrial function than ICA.Test V Study on ICA and pICA Relieving DHAV-1-induced DEHs Apoptosis The aim of this study was to investigate the mechanism of DHAV-1 induced DEHs apoptosis and the alleviation of ICA and pICA.Flow cytometry was used to observe the apoptosis of DEHs after infection with DHAV-1 and ICA and pICA treatments.Secondly,The effect of DHAV-1,ICA,and pICA on genes mRNA expression of related apoptotic factors such as Caspase family(Caspase 3,Caspase 8,Caspase 9)and Bcl-2 family(Bcl-2,Bax)was compared.Finally,the effects of DHAV-1,ICA and pICA on the proteins expressions of Caspase 3 and Caspase 8 proteins were compared.The results showed that the apoptosis rate of DEHs after infection with DHAV-1 was significantly increased,and the gene mRNA expression of Bcl-2 was significantly decreased,and the genes mRNA expression of Bax,Caspase 3,Caspase 8,and Caspase 9 increased significantly.The Caspase 3 and Caspase 8 proteins were significantly increased.After ICA and pICA were added,the apoptosis rate of DEHs was significantly decreased,the mRNA expression of Bcl-2 was significantly increased,the genes mRNA expression of Bax,Caspase 3,Caspase 8,and Caspase 9 was significantly decreased,and the protein expression of Caspase 3 and Caspase 8 were significantly decreased.This shows that DHAV-1 can upregulate the gene mRNA expression of Bax and downregulate the gene mRNA expression of Bcl-2,and finally upregulate the apoptosis initiation factors Caspase 8,Caspase 9 and apoptotic implementation of shadow Caspase 3 to promote DEHs apoptosis.ICA and pICA can inhibit mitochondrial pore formation by increasing the Bcl-1/Bax ratio,thereby inhibiting downstream apoptosis-related factors such as Caspase 8,Caspase 9 and Caspase 3 to inhibit apoptosis.Compared with ICA,pICA can better inhibit the apoptosis induced by dhav-1.Test VI ICA and pICA Relieve DHAV-1 Inhibiting Cell Proliferation in DEHs This study was designed to investigate the mechanisms by which DHAV-1 inhibits the proliferation of DEHs cells and the alleviation of ICA and pICA.Flow cytometry was used to observe the cell cycle of DEHs infected with DHAV-1 and treated with ICA and pICA.After that,the genes mRNA expression of the related proliferation factors(CCND1 and PCNA)and the protein expression of PCNA were compared between DHAV-1,ICA and pICA.Finally,we observed their effect on protein phosphorylation of AKT and CREB.The results showed that the cell cycle G0/G1 phase of DEHs increased significantly and the S+G2+M phase decreased significantly after infection with DHAV-1,the genes mRNA expression of CCND1 and PCNA,the protein expression of PCNA were significantly decreased,and the phosphorylations of AKT and CREB proteins were inhibited.Adding ICA and pICA can reduce the ratio of G0/G1 phase and increase the ratio of S+G2/M phase,increase the genes mRNA expression of CCND1 and PCNA and protein expression of PCNA,and increase the protein phosphorylations of AKT and CREB.This shows that DHAV-1 can inhibit the phosphorylation of downstream CREB protein by inhibiting the phosphorylation of AKT,further affect the expressions of CCND1 and PCNA,reduce the ratio of S+G2/M phase cells,and eventually block the G1/G0 phase of the cell cycle.Ultimately inhibit cell proliferation.However,ICA and pICA can increase the expressions of CCND1 and PCNA by activating phosphorylations of AKT and CREB,so that DNA can be synthesized normally,which makes cell proliferation normal.Compared with ICA,pICA can better antagonize the inhibition of AKT protein phosphorylation by DHAV-1.Finally,there was a strong correlation between inflammatory injury,oxidative stress,mitochondrial damage,apoptosis,and cell proliferation through Spearman correlation analysis. 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