Effect Of Xanthohumol On Hepatic Alveolar Echinococcosis In Mice

Objective:1.Establish an in vitro culture model of endothelial progenitor cells(EPCs)to explore the effects of xanthohumol(XN)on the proliferation,migration,tube formation,apoptosis and related protein expression of endothelial progenitor cells.The role and possible mechanism of angiogenesis.2.Establish an in vitro culture system for alveolar echinococcus protocercaria,explore whether xanthohumol has pharmacological activity against alveolar echinococcus protocercaria in vitro,and further explore the possibility of xanthohumol against alveolar protocercaria Mechanisms.3.Establish a mouse liver alveolar hydatid disease model,verify the effect of xanthohumol on the growth of hepatic alveolar hydatid in vivo,and hope to clarify the mechanism of this effect may be through indirect or direct host angiogenesis Anti-foamed hydatid to play a role.4.Detect the expression of P65,p-P65,IκBa,p-IκBa,VEGF and CD34 in human hepatic alveolar hydatid tissue.Methods:1.Isolation,cultivation and identification of EPCs derived from mouse bone marrow.Experimental grouping: 5,10,20μmol/L xanthohumol group,with the same dimethyl sulfoxide(DMSO,DMSO<0.1%)solution as the control group,respectively 6,24,48 h after treatment,into a tube test To detect the effect of xanthohumol on the formation of endothelial tubules in EPCs;CCK-8 method to detect the effect of xanthohumol on the proliferation of EPCs;scratch test and Transwell test to detect the effect of xanthohumol on the migration of EPCs;flow cytometry,TUNEL apoptosis The effect of xanthohumol on the apoptosis of EPCs was detected by staining method;the expression of AKT,p-AKT,P65,p-P65,IκBa,p-IκBa,IKKβ,VEGF,VEGFR2,Bcl-2 and Bax protein in EPCs was detected by Western blot method.2.Extraction and identification of alveolar hydatid protocercaria.Experimental groups: blank control group,different concentrations of xanthohumol(10,20,40,80 μmol/L)group and solvent(DMSO)control group.Eosin staining method was used to observe the activity of protocercaria,and calculate the activity rate;after xanthohumol acted on alveolar hydatid protocercaria for 3 days,Western-blot detected caspase-3,Bcl-2/Bax and caspase in protocercaria-9 protein expression;Mitochondrial Membrane Potential(JC-1)is used to detect the changes in mitochondrial membrane potential in protocercaria.3.Establish a mouse liver alveolar hydatid disease model.After 30 days,they were randomly divided into 3 groups,namely the model control group,the solvent group,and the xanthohumol treatment group,with10 animals in each group,and a blank control group.Xanthohumol group was given intraperitoneal injection of xanthohumol(10mg/Kg.wk)30 days after modeling,while the solvent group was given intraperitoneal injection of the same xanthohumol solution(DMSO).After 60 days of treatment,the mice were dissected,blood,liver,and hepatic alveolar hydatid tissues were collected.ELISA kits were used to detect serum VEGF.Immunohistochemistry and Western blot were used to detect NF-κBP65 and p-P65 in mouse hepatic alveolar cysts.,VEGF and CD34 expression.4.Immunohistochemical method was used to detect the expression of NF-κBP65,VEGF and CD34 in hepatic alveolar hydatid tissue;Western blot was used to detect NF-κBP65,p-P65,IκBa and p-IκBa in hepatic alveolar hydatid tissue And VEGF protein expression;Vascular endothelial growth factor A(VEGFA)detection kit detects the content of VEGF in serum.Results:1.Xanthohumol can significantly inhibit the proliferation of EPCs.The IC50 of EPCs growth at 24 and48 h is(22.19 ± 0.98)μmol/L and(11.51 ± 1.25)μmol/L;compared with the control group,5,10 and 20 μmol The migration ability of EPCs in the Xanthohumol group decreased with the increase of concentration(P <0.05);the apoptosis rate of Xanthohumol increased significantly(P<0.05);AKT,p-AKT,P-P65,p-IκBa,Bcl-2,VEGF protein expression decreased,and Bax protein expression increased(P <0.05).2.With the increase of drug concentration,the activity of alveolar hydatid protocercaria gradually decreases;the expression of Bcl-2 decreases,the expression of Bax,caspase-3 and caspase-9 increases with the increase of xanthohumol concentration,and Compared with the blank control group or the solvent control group,the difference was statistically significant(P <0.05).The fluorescence intensity of the mitochondrial membrane potential in the protocercaria of Echinococcus alveolar increased with the increase of the drug concentration.3.The wet weight of the cysts in the xanthohumol group was lower than that of the model group and the solvent group(all P <0.05),and the cyst inhibition rate of the xanthohumol group was higher than that of the solvent group and the model group(all P <0.05);the xanthohumol group The concentration of VEGF in the serum of mice was lower than that of the model group and the solvent group(both P <0.05),and higher than that of the blank control group;NF-κBP65,MVD-CD34,VEGF and Bcl-2 were weakly positive in the xanthohumol group.The expression of NF-κBP65,p-P65,CD34 and VEGF protein in the xanthohumol group was lower than that in the model group and the solvent group(all P <0.05).4.The serum VEGF concentration in the case group was higher than that in the control group(P <0.05);NF-κBP65,MVD-CD34,VEGF were strongly positive in the case group,and individual positive markers were occasionally found in the control group;NF-κBP65 in the case group The protein expressions of,p-P65,p-IκBa and VEGF were higher than those of the control group(P <0.05).Conclusion:1.Xanthohumol can inhibit the proliferation,migration and tube formation of EPCs,and promote their apoptosis,and play a direct role in inhibiting angiogenesis.The mechanism may be related to the inhibition of the NF-κB signaling pathway.2.Xanthohumol has the effect of killing protocercaria in vitro,and its mechanism may be related to the induction of cell apoptosis of alveolar echinococcal protocercaria through the mitochondrial pathway.3.Xanthohumol can inhibit the growth of hepatic alveolar echinococcosis,which may be related to direct anti-hepatic alveolar echinococcosis or indirectly through NF-κB signaling pathway against host angiogenesis to inhibit the growth of hepatic alveolar echinococcosis.4.The NF-κB pathway in human hepatic alveolar hydatid tissue is positively correlated with angiogenesis.