A Combined Strategy Based On Hepatic Sinusoidal Endothelial Cell Fenestration Regulation For The Treatment Of Liver Fibrosis

BackgroundLiver fibrosis is a common pathological process involved in many chronic diseases,including hepatitis B and nonalcoholic steatohepatitis.The main strategy for liver fibrosis treatment is to remove the causative factors like the primary disease.There is no drug approved for liver fibrosis treatment.The liver comprises hepatocyte,hepatic stellate cells（HSC）,and hepatic sinusoidal endothelial cells（LSEC）.Hepatocytes are damaged after being stimulated by oxidative stress and other external factors.The damaged cells release damage-related molecules and stimulate the activation of HSC.HSC are located in the subendothelial space of Disse,activated by injury factors and the main source of extracellular matrix in the liver.LSEC constitutes the hepatic sinusoidal capillary channels and displays a unique fenestrated phenotype,facilitating the bi-directional transfer of substrates and oxygen between the blood and HSCs.Liver sinusoid endothelial cells（LSEC）become capillarized in response to injury factors.Liver sinusoid capillarization may impair the pass of molecules in the blood,impede the appropriate oxygenation exchange and promote hepatic fibrosis progression.Activated hepatic stellate cells（HSC）are considered the major scar-producing cells in liver fibrosis.The damage-related molecules of hepatocytes are the important factors that stimulate HSC activation.Nanomedicine is a new drug form with good targeting,biocompatibility,and diverse biological functions.It can improve the therapeutic effect of small-molecule drugs with reduced toxic and side effects.The capillarized LSEC mechanically restricts the transport efficiency of therapeutic nanomedicines.Based on the above background,breaking the barrier of LSEC hence improving the transport efficacy,ameliorating liver hypoxia,and combining other strategies,is expected to show better anti-hepatic fibrosis efficacy.There are two problems caused by LSEC capillarization that decrease the transport efficacy of substance between the blood and cells in the Disse,further causing hepatic hypoxia and exacerbated cellular apoptosis.We developed two strategies to solve the problems above.In the first study,riociguat（Rio）was introduced to help maintain the fenestration of LSEC and improve the delivery efficacy of nanomedicine toward HSC.In the second part of our work,we ameliorated hepatic hypoxia with riociguat then synergistically inhibited cellular apoptosis with Sel@GBRNPs,which could scavenge ROS and inhibit the activation of ASK1.The study provides a further understanding of the liver sinusoid as a transfer barrier in liver fibrosis.Part 1 Restoration of sinusoid fenestrae followed by targeted delivery of an antifibrotic agent via nanoassembly improves treatment efficacy in liver fibrosisObjectiveThis part of the study aims to study the changes of hepatic sinusoidal porosity and permeability during the progression of chronic liver disease and develop a new strategy to improve the delivery efficacy and antifibrotic effect of nanomedicine toward HSCs.Methods1.The porosity and permeability of hepatic sinusoid in liver fibrosisThe liver specimens with clinical and experimental liver fibrosis（0 weeks,4 weeks,6 weeks,8 weeks）were collected.The samples were stained with hematoxylin and eosin,sirius red,andα-SMA immunohistochemical.The hepatic sinusoid morphology was examined with scanning electron microscopy.The permeability was examined by fluospheres（100 nm）perfusion via the portal vein.The permeability of the hepatic sinusoids was valued by the retention of fluospheres in the liver sections using a confocal fluorescence microscope.2.Effect of riociguat on LSEC porosity and permeabilityThe effect of riociguat on LSEC porosity was evaluated both in vitro and in fibrotic mice.LSEC and HSC were isolated using gradient density centrifugation.LSEC were cultured with PBS or riociguat（10μM）for 72h at 37℃.LSEC were then prepared for scanning electron microscope for porosity evaluation.At the same time,the c GMP content of LSEC was evaluated using ELISA.The effect of riociguat on liver sinusoid porosity was also evaluated in the carbon tetrachloride（CCl₄）-induced liver fibrosis model.Liver fibrotic mice（4 weeks,5 weeks,and 6 weeks）were then treated with PBS or riociguat（1 mg/kg,once a day）for two weeks.Fluospheres prefusion was also introduced to value the permeability of hepatic sinusoid.3.Peptide synthesis and characterizationTheHSC-targetingpeptideIG:VHWDFRQWWQPSDDEGK（VHWDFRQWWQPSDDEG）K（C18）and the control peptide AB:ERNECFLQHKDDNQLDK（ERNECFLQHWDDNFLD）K（C18）were synthesized by solid-phase synthesis method.HPLC and mass spectrometry were used to characterize peptide synthesis and purity.IGNP-JQ1 and ABNP-JQ1 were synthesized via the ultrasonic and self-assemble method.Transmission electron microscopy,dynamic light scattering,and HPLC were introduced to characterize the size,charge,and encapsulation efficiency of nanomedicines.The targeting property of IGNP-Cy5.5 and ABNP-Cy5.5 towards HSCs was evaluated using flow cytometry and confocal fluorescence microscope.The effect of IGNP-JQ1 on HSC activation was verified by western blot,immunofluorescence,and sirius red staining.The distribution of IGNP-Cy5.5 and ABNP-Cy5.5 was evaluated using in vivo imaging system and flow cytometry.4.Anti-fibrotic effect of Rioguat combined with IGNP-JQ1The anti-fibrotic effect of IGNP-JQ1 was valued in CCl₄ induced liver fibrosis mice.The mice were injected with PBS,Rio+JQ1,IGNP-JQ1,Rio+ABNP-JQ1,Rio+IGNP-JQ1,respectively.The degree of fibrosis was evaluated by liver sections with HE,sirius red,immunochemistry and western blot.Serum liver functions were also tested using the fully automatic analyzer.Results1.The relationship between hepatic fibrosis and hepatic sinusoidal porosity and permeabilityThe hepatic sinusoidal porosity gradually decreases in clinically collected and experimental fibrotic liver samples.The porosity of normal mice liver was 13.20%,and the porosity of mice treated with 8 weeks tetrachloride carbon decreased to 3.59%.The fluorescence of liver sections in mice treated with 4 weeks and 8 weeks of CCl₄decreased to 24%and 15%of normal mice,respectively.These data suggested that the porosity and permeability gradually decreased with liver fibrosis development.2.Effects of Riociguat on porosity and permeability of hepatic sinusoidsRiociguat contributes to the maintenance of hepatic sinusoidal porosity in vitro and in vivo.The results of LSEC cultured in vitro showed that the porosity of LSEC increased from 0.24%in the PBS group to 4.18%in the riociguat group.c GMP in LSEC increased from 132.86 fmol/mg protein in the PBS group to 344.66 fmol/mg protein in the riociguat group.Riociguat increased LSEC intracellular c GMP levels and helped maintain LSEC porosity.In the liver of fibrotic mice,riociguat helped maintain hepatic sinusoidal fenestration（porosity 9.83%in the 6-week Riociguat group vs.4.87%in the6-week PBS group）and increased hepatic sinusoids permeability.3.Characterization of nanomedicineHPLC and mass spectrometry results verified the synthesis of peptide IG and AB.The critical micelle concentration of peptide IG was 2.167μM.ABNP-JQ1 and IGNP-JQ1 were synthesized based on peptide self-assembly.Transmission electron microscopy results showed that ABNP-JQ1 and IGNP-JQ1 has a uniform size of about 100 nm.The encapsulation efficiency of peptide IG and AB on JQ1 was about 70%when the mass ratio of peptide to JQ1 was 1:1.Results of flow and confocal experiments suggest that IGNP-FITC could target HSC.Western blot results showed that INGP-JQ1 inhibited the expressions of CDK4,Collagen I,andα-SMA,indicating that IGNP-JQ1 inhibited the proliferation of HSC cells and reduced the viability of HSC.The distribution of IGNP-Cy5.5 in the fibrotic liver was stronger（1.72 times）than that of ABNP-Cy5.5.Riociguat further increased the distribution of IGNP-Cy5.5 in HSC cells.4.Anti-fibrosis effect of Riociguat combined with IGNP-JQ1The liver stiffness of mice was 19.33 k Pa after 6 weeks of CCl₄ gavage treatment,and the liver stiffness of the IGNP-JQ1 treatment group was 17.06 k Pa.In riociguat combined with the IGNP-JQ1 group,the liver stiffness decreased to 15.47 k Pa.The positive sirius red staining area was reduced from 5.2%of the PBS group to 1.53%in the combined group.Western blot and immunofluorescence results showed that the activation of HSC was inhibited.Riociguat treatment increased the anti-fibrotic effect of IGNP-JQ1.ConclusionDuring the development of chronic liver disease and liver fibrosis,the porosity and permeability of liver sinusoids gradually decreased.In the combined treatment group,riociguat maintained a relatively normal hepatic sinusoidal fenestration and porosity,breaking the hepatic sinusoidal endothelium’s transport barrier for nanomedicines.Riociguat increased the permeability of hepatic sinusoid and delivery efficacy of IGNP-JQ1 to HSC.The study emphasized the important role of LSEC as a transport barrier and developed a new strategy for liver fibrosis treatment.Part2 Hepatic hypoxia combined with and apoptosis inhibition nanomedicine for liver fibrosis treatmentObjectiveThe purpose of this part of the study was to investigate the effect of riociguat on hepatic hypoxia and the synergically anti-fibrotic and anti-apoptotic effect combined with Sel@GBNPs nanomedicine in fibrosis treatment in CCl₄-induced mouse liver fibrosis model.Methods1.Hepatic hypoxia and inflammation modulationThe liver fibrosis mice model was induced by 4 weeks CCl₄ gavage.After the model was successfully established,PBS or Riociguat（1 mg/kg,daily）were injected intraperitoneally,respectively.The livers were prepared for PCR,western blot,and flow cytometry analysis.The expression of hepatic hypoxia-inducible protein HIF-1α,cleaved Caspase 3 was evaluated by western blot and PCR.The m RNA level of inflammatory cytokines was analyzed using PCR.2.Synthesis and characterization of Sel@GBRNPsGalactose-modified PEG-bilirubin（Gal-PEG-BR）was synthesized by ECD/NHS reaction,and Selonsertib-encapsulated nanomedicine Sel@GBRNPs was synthesized by thin-film hydration method.The encapsulation efficiency of Sel was evaluated by HPLC.To verify the targeting of GBRNPs to liver parenchymal cells,the uptake of Cy5.5@GBRNPs by AML12 cells was verified by flow cytometry and confocal laser in vitro.The distribution of Cy5.5@GBRNPs was evaluated in the liver fibrosis mice model.3.Protective effects of Sel@GBRNPs on apoptosis of AML12The protective effect of Sel@GBRNPs in AML12 cells towards injury induced by H₂O₂ was evaluated by CCK-8,transmission electron microscopy,scanning electron microscopy,and flow cytometry.The influence of Sel@GBRNPs on ASK1 activation was evaluated by western blot.4.Anti-hepatic fibrosis effectThe liver fibrosis mice model was established by CCl₄ gavage for 4 weeks.After the model was successfully established,the mice were injected with PBS,Rio+Sel,Rio+GBRNPs,Sel@GBNPs,Rio+Sel@GBNPs.The liver was sectioned and stained with HE,Sirius red,α-SMA,and p-ASK1 immunohistochemical.The liver was also stained with phosphorylated p-ASK1 and TUNEL to evaluate liver apoptosis.Serum inflammatory factor levels were measured by ELISA.Results1.Riociguat ameloriate liver hypoxiaResults showed that HIF-1αm RNA and protein level in the fibrotic liver was 2.69times and 2.39 times higher than that of olive oil control liver,respectively.The hepatic HIF-1αm RNA decreased by 52%in riociguat compared to PBS treated fibrotic mice.Flow cytometry results showed that the CD11b+Ly6c+inflammatory mononuclear cells were 9.9%in the PBS group,while the inflammatory mononuclear cells decreased to 2.2%in the riociguat group.Inflammatory factors,including IL-1β,IL-6,and TNF-αm RNA were also decreased by riociguat treatment.The above results suggested that riociguat ameliorated liver hypoxia partially alleviated liver cell apoptosis and inflammatory liver status.2.Characterization of Sel@GBRNPsTransmission electron microscopy results showed that Sel@GBRNPs were uniform nanoparticles with a size of 100 nm.Results of flow cytometry and laser confocal showed that AML12 cells had a stronger uptake of Cy5.5@GBRNPs compared to the Cy5.5 group.In fibrotic mice,Cy5.5@GBRNPs were more distributed in liver parenchymal cells than free Cy5.5,suggesting that galactose modification increased the distribution of nanoparticles to liver parenchymal cells.3.Apoptotic protective effect of Sel@GBRNPs on AML12 cellsH₂O₂（400μM）induced a 40%decrease in viability in AML12.Pretreated with Sel@GBNRPs,the viability of AML12 cells increased to 90%,suggesting the protective effect of Sel@GBRNPs on H2O2-induced injury.Cell morphology results suggested that Sel@GBNRPs reduced the shrinkage of AML12 cells and release of apoptotic bodies.Sel@GBRNPs pretreatment decreased the apoptotic ratio of AML12cells from 32%in the H₂O₂ group to 18%,inhibited ASK1 activation,and decreased intracellular ROS levels in AML12 cells.The conditioned medium of AML12 cells pretreated with Sel@GBRNPs showed reduced stimulation towards LSEC and HSCs.The above results suggested that Sel@GBRNPs protected AML12 from H₂O₂ induced apoptosis.4.Hepatic hypoxia and apoptosis modulation attenuate liver fibrosis progressionIn the CCl₄-induced liver fibrosis mice model,Sel@GBRNPs treatment decreased sirius red staining area from 4.5%in the model group to 2.0%,Rio combined Sel@GBRNPs further decreased the sirius area to 1.1%.The positive staining area ofα-SMA and p-ASK1 was 7.7%and 1.1%,respectively.In the Sel@GBRNPs treatment group,α-SMA and p-ASK1 positive areas decreased to 5.7%and 0.8%,respectively.Rio combined Sel@GBRNPs further lowered theα-SMA and p-ASK1 positive area to4.0%and 0.5%.The results of TUNEL staining showed that the proportion of apoptotic cells was the lowest in the combined treatment of Rio+Sel@GBRNPs.The serum TNF-αand IL-6 in liver fibrosis mice were 168.5 and 44.71 pg/ml,respectively.These results suggest that the pre-regulation of hepatic hypoxia by riociguat,combined with reactive oxygen species scavenging and ASK1 inhibition strategy with Sel@GBRNPs,synergically reduced liver cell apoptosis and slowed down the progression of liver fibrosis.ConclusionIn the liver fibrosis mice model,riociguat ameliorated hepatic hypoxia and removed the pathophysiological factors that could induce hepatocyte apoptosis.Hypoxia modulation with riociguat combined with Sel@GBRNPs,a nanomedicine with anti-apoptotic and ROS scavenging properties,reduced the apoptosis of liver cells and slowed down liver fibrosis progression,providing a new combined strategy for liver fibrosis treatment.