Study Of Effects And Mechanism Of Nordihydroguaiaretic Acid For The Treatment Of Alveolar Echinococcosis And Liver Fibrosis

BackgroundAlveolar echinococcosis(AE)is a serious zoonotic parasitosis caused by Echinococcus multilocularis larvae or metacestodes.The parasitic metacestodes mostly reside in the liver(>97%)of humans and rodents as the intermediate host.It damages the infected liver with a tumor-like invasive growth manner,such as mainly progressive liver fibrosis,or the development into cirrhosis,and it is often termed“parasitic cancer”.Albendazole(ABZ),a priority drug of choice for the clinical treatment of AE,can limit rapid growth for E.multilocularis metacestodes.However,the poor water solubility,low oral availability,and obvious side effects after its long-time use make it difficult to meet clinical goals.Therefore,safe and effective anti-echinococcal and anti-liver fibrosis drugs are crucial and urgent demand.Nordihydroguaiaretic acid(NDGA)was reported to exert antioxidant,antiviral,antibacterial,anti-tumor,anti-pulmonary fibrosis and anti-angiogenesis biological functions.Furthermore,on the molecular docking virtual screening technology and anti-echinococcosis test in our previous studies,NDGA exhibited a significant alleviation in cystic echinococcosis.However,the effect and mechanism of NDGA for the treatment of cancer-like AE and its associated liver fibrosis are still unclear.Therefore,this study should explore the effect and mechanism of NDGA against E.multilocularis metacestodes,and investigate the effects and mechanism of NDGA for the treatment of Echinococcus or not Echinococcus infection-induced liver fibrosis through the regulation of hepatic stellate cells(HSC),providing new references for drugs application to treat AE and liver fibrosis in the future.Methods1.E.multilocularis metacesodes were isolated from the laboratory infected gerbils,and after treatment with NDGA and ABZ-SO(an anthelmintic active form of ABZ,a positive control)for 7 days in vitro,the viability of E.multilocularis protoscoleces(PSC)and Em ALP content in its culture supernatant,the proliferation rate of germinal layer cells(GCs),and the viability of microcysts were detected using chemiluminescence assay and electron microscopy.Furthermore,the survival rate,the ultrastructural alterations,the expression of Notch1,Jagged1,Akt protein and m RNA in PSC after exposure to NDGA(50?mol/L)or in combination with JAG1(a Notch signaling agonist,0.5?g/m L)and DAPT(a Notch signaling inhibitor,40?mol/L)for7 days were investigated by electron microscopy,immunofluorescence,and RT-q PCR.Subsequently,on a murine model with liver AE established by in situ intrahepatic implanted infections,and after treatment with NDGA(100mg/kg·day)and ABZ(a positive control,100mg/kg·day)by oral gavage for 3 months,the weight of E.multilocularis cysts,and the expression of Notch1,Jagged1,Akt protein and m RNA in E.multilocularis metacestodes and its infected liver were measured using immunofluorescence,western blotting(WB)and real-time quantitative PCR(RT-q PCR).2.After the mice with liver AE were treated with NDGA(100 mg/kg·day)for 3months,HA,LN,CIV,PC?,?-SMA,and Col1 content in the serum were detected by immunoradiometric assay and ELISA.At the same time,the deposition of collagen fiber,the expression of?-SMA,Col1 protein and m RNA in the liver were investigated by Sirius red staining,Masson staining,WB and RT-q PCR.Furthermore,the primary HSC were isolated from the mice with AE after treated or untreated with NDGA by density gradient centrifugation method,and then were cultured for 120 hours in vitro,?-SMA and Col1 protein in the primary HSC and its culture supernatant were investigated by immunofluorescence assay and ELISA.3.After HSC,both HSC-T6 cell and LX-2 cell,were treated with NDGA(50?mol/L)or in combination with JAG1(0.5?g/m L)or DAPT(50?mol/L)for 24hours,the rates of proliferation,migration,invasion,and contraction were detected using cell proliferation assay,migration assay,transwell assay,and contraction assay,respectively.Furthermore,Col1 and?-SMA content in the culture supernatant of HSC-T6 cells were detected by ELISA,and the expression of Col1,?-SMA,Notch/Akt signaling-related protein and m RNA in HSC-T6 cells were measured using immunofluorescence assay,WB and RT-q PCR.In addition,after HSC-T6 cells and LX-2 cells were treated with NDGA(50?mol/L)for 24 hours,the cell cycle distribution and apoptosis rate were investigated by flow cytometry.4.Three rat and mouse models with liver fibrosis were induced by intraperitoneal injection of tetrachloromethane(CCL₄),bile duct ligation(BDL)surgery,and Schistosoma japonicum infection,respectively,and then were treated with NDGA(100 mg/kg·day)by gavage for 8 weeks.HA,LN,CIV,PC?,?-SMA and Col1content in the serum were detected using immunoradiometric assay and ELISA.At the same time,the deposition of collagen fiber and the expression of?-SMA,Col1,Notch/Akt signaling-related protein and m RNA in the liver were measured by Hematoxylin-eosin(HE)staining,Sirius red staining,Masson staining,immune-histochemistry and RT-q PCR.5.In HL-7702 cells and HK-2 cells after exposure to NDGA or ABZ-SO for 48hours in vitro,the proliferation rate and cell morphology were assessed by Cell Counting Kit-8(CCK-8)method and crystal violet staining.Furthermore,after the healthy Kunming mice received intragastric administration of NDGA(100 mg/kg·day)or ABZ(100 mg/kg·day)for 8 weeks,some biochemical indicators of liver and kidney function in the serum were measured using the chemiluminescence method,and the histopathological change in the liver and kidney were assessed by HE staining.Results1.Compared with the negative control(NC)group,the NDGA(50?mol/L)group and ABZ-SO(50?mol/L)group showed a significant decrease in the survival rate of PSC(P<0.05),and a significant increase of Em ALP content in its culture supernatant(P<0.05).Among them,the survival rate of PSC in the NDGA group was lower than that in the ABZ-SO group(P<0.05),and Em ALP content in the NDGA-treated group was higher than that in the ABZ-SO group(P<0.05).Meanwhile,PSC after exposure to NDGA exhibited the disappearance of PAS positive materials,syncytial layer(SL),and microvillus,and the presentation of abundant cytoplasm vacuolization and apoptotic bodies,whilst PSC after exposure to ABZ-SO only presented a gentle reduction of PAS-positive material,shorten of microvillus and thinned syncytium layer.Compared with the NC group,the NDGA group showed a significant decrease in the proliferation rate of germinal layer cells(P<0.05),an obvious inhibition in the growth of E.multilocularis microcysts,accompanying by a significant increase of Em ALP content in its culture supernatant(P<0.05),and showed a significant decrease of Notch1,Jagged1,Akt protein and m RNA expression in PSC(P<0.05).Furthermore,compared with the NC group,the JAG1(0.5?g/m L)group showed an obvious pro-proliferation effect for PSC,with no obvious changes in Em ALP content of its culture supernatant(P>0.05);whilst the DAPT(40?mol/L)group showed a significant reduction in the survival rate of PSC(P<0.05),and a significant increase in Em ALP content in its culture supernatant(P<0.05).Compared with the JAG1group,the NDGA+JAG1 group exhibited a significant reduction in the survival rate of PSC(P<0.05),and a significant increase in Em ALP content in its culture supernatant(P<0.05).However,compared with the NDGA group,the NDGA+DAPT group showed no significant changes in the survival rate of PSC(P>0.05),the Em ALP content in its culture supernatant(P>0.05),and the expression of Notch1,Jagged1,and Akt m RNA(P>0.05).2.Compared with the uninfected group,the mice in E.multilocularis-infected group showed a significant increase in HA,LN,C?,PC?,?-SMA and Col1 content in the serum(P<0.05),in the accumulation of collagen fiber in the liver around E.multilocularis metacestodes(P<0.05),and in the expression of?-SMA,Col1,Notch1,Jagged1,Akt protein and m RNA(P<0.05).Compared with the infected group,the mice in the NDGA group and ABZ group showed a significant decrease in the wet weight of E.multilocularis cysts(P<0.05),in HA,LN,PC?and Col1content in the serum(P<0.05),in the accumulation of collagen fiber in the liver(P<0.05),and in the expression of?-SMA,Col1,Notch1,Jagged1,Akt protein and m RNA(P<0.05).In addition,compared with the primary HSC from the uninfected mice,the HSC from E.multilocularis metacestodes-infected mice showed a significant increase of?-SMA and Col1 protein expression in vitro(P<0.05).However,compared with the infected group,the HSC from the mice in the NDGA-treated group showed a significant reduction in?-SMA and Col1 protein expression(P<0.05).3.Compared with the NC group,HSC-T6 cells and LX-2 cells in the JAG1 group showed a significant increase in the rates of proliferation,migration,invasion,and contraction(P<0.05),and in the expression of?-SMA,Col1,Notch1,Jagged1,Hes1,Akt,p-Akt protein and m RNA in HSC-T6 cells(P<0.05).However,compared with the NC group,the two types of cells in the NDGA group or DAPT group exhibited a significant decrease in the rates of proliferation,migration,invasion,and contraction(P<0.05),and in the expression of?-SMA,Col1,Notch1,Jagged1,Hes1,Akt,p-Akt protein and m RNA in HSC-T6 cells(P<0.05).Compared with the JAG1 group,the two types of cells in the NDGA+JAG1 group showed a significant decrease in the rates of proliferation,migration,invasion,and contraction(P<0.05),and in the expression of?-SMA,Col1,Notch1,Jagged1,Hes1,Akt,p-Akt protein and m RNA in HSC-T6 cells(P<0.05).Compared with the NDGA group,the two types of cells in the NDGA+DAPT group showed a significant decrease in the rates of migration,invasion,and contraction(P<0.05),and a significant decrease in?-SMA and Col1protein expression in HSC-T6 cells(P<0.05),but no obvious changes in the expression of otch1,Jagged1,Hes1,Akt,p-Akt protein and m RNA in HSC-T6 cells(P>0.05).In addition,compared with the NC group,the two types of cells in the NDGA group revealed a significant increase in the percentage of cells arrested in the G2/M phase(P<0.05),and in the apoptotic rates(P<0.05).4.Compared with the Oil,Sham or Control group,the rats and/or mice in the CCL₄-induced group,BDL-induced group and S.japonicum-infected group showed a significant increase in?-SMA and Col1 content in the serum(P<0.05),in the accumulation of collagen fiber(P<0.05),and in the expression of?-SMA,Col1,Sm22,Desmin,Notch1,Notch3,Jagged1,Hes1,Akt protein and/or m RNA in the liver(P<0.05).However,compared with the CCL₄-induced group,BDL-induced group and S.japonicum-infected group,the NDGA group showed a significant reduction or reduce in?-SMA and Col1 content in the serum(P<0.05),in the accumulation of collagen fiber in the liver(P<0.05),and in the expression of?-SMA,Col1,Sm22,Desmin,Notch1,Jagged1,Hes1,Akt protein and/or m RNA in the liver(P<0.05).5.Compared with the NC group,the survival rates of HL-7702 cells and HK-2cells after exposure to the different concentrations of NDGA or ABZ-SO(10,20,50,100,200,and 400?mol/L)in vitro showed an obvious dose-dependent decrease.Among them,at the drug concentrations of 20?mol/L and more,the survival rates of the two types of cells in ABZ-SO group were lower than that in the NDGA group(P<0.05).In addition,compared with the normal control group,the mice in the NDGA group showed a significant increase in ALT content in the serum(P<0.05),and no apparent pathological alterations in the liver and kidney;meanwhile,the ABZ group showed a significant increase in ALT and IBIL content in the serum(P<0.05),and obvious pathological alterations in the liver,including dilated intercellular space,liver cells swelling and hydropic degeneration,and no obvious pathological alterations in the kidney.Conclusions1.The activation of Notch1/Akt signaling pathway could boost the invasive proliferation of E.multilocular metacestodes.NDGA could inhibit the growth and proliferation of E.multilocular metacestodes through inhibiting Notch1/Akt signaling pathway,accompanying with its insecticidal effect superior to ABZ-SO,providing new references for exploring drug-targets and chemotherapeutic drugs application for AE.2.Notch1/Akt signaling pathway is a common regulatory mechanism in E.multilocularis-infected,chemical injury-induced(CCL₄),mechanical injury-induced(BDL)and S.japonicum-infected liver fibrosis.NDGA can inhibit activation and proliferation of HSC to relieve these types of liver fibrosis by inhibiting Notch1/Akt signaling pathway,providing a new idea for exploring anti-fibrosis drug-targets and clinical drugs.3.Cell toxicity of NDGA was lower than that of ABZ-SO in normal human liver cells and kidney cells in vitro,and the sub-acute toxicity of NDGA was lower than that of ABZ in the liver of the healthy mice,providing more references of NDGA for the clinical treatment of AE and liver fibrosis.