| BACGROUND Liver fibrosis(LF)is an intermediate process of various chronic liver diseases such as viral hepatitis,alcoholic liver disease,metabolic-related fatty liver disease,and autoimmune diseases.During the development of LF,enormous extracellular matrix(ECM)deposited between liver sinusoidal endothelial cells(LSECs)and hepatocyte parenchymal cells.With the structure changes,the elasticity of the liver decreases,and the liver stiffness can even reach 6 times that of the normal liver,which leading to the mechanical force around the LSECs changes significantly.Cells sense extracellular mechanical changes through mechanosensory receptors and translate this information into signaling events that trigger biological responses.In the face of the change in the mechanical environment of the liver microenvironment,the balance between hepatocytes and liver interstitial cells is disturbed,LSECs showed fenestration and capillarization.On the one hand,fenestrated LSEC activates HSCs by secreting pro-inflammatory and pro-fibrotic factors,secretes ECM and accumulates;on the other hand,the massive accumulation of ECM in turn restricts the blood flow of liver sinusoidal microvessels,at the same time,stenosis and deformation of hepatic sinusoid lumen caused by hypoxia aggravate the phenomenon of capillaryization.It summary,changes in the mechanical characteristics of the pathological microenvironment are not only the result of the development of fibrosis,but also the reason of disease progression.Understanding the mechanisms by which endothelial cells and ECM mechanics reciprocally sense and transmit mechanical cues is a major challenge in liver fibrosis research.Cells sense extracellular mechanical forces through mechanosensory receptors and convert these information into signaling events triggerring biological responses,this process called mechanotransduction.Biophysical signals are ubiquitous in the human body and are involved in the regulation of all basic life activities.At the same time,the pathological process will also be accompanied by changes in biophysical signals,providing the possibility for targeted localization of lesions.Most of the current pharmacodynamic studies in pharmacology rely on biochemical signals.The mechano-biological response mechanism of endothelial cells perception of mechanical force and how to convert this biological force into biological response is a popular research areas in currently.In the field of pharmacology research,efficacy is the ultimate goal of drug clinical application,and the regulation of physical and mechanical forces in the microenvironment can obtain different biological effects,so we can also consider ’biological force’as a multi-target ’drug’.Biomechanopharmacology embodies the combined effect of drugs and mechanical factors.On the one hand,it involves the pharmacodynamic effect of the drug itself;at the same time,the drug may also induce biological responses by adjusting the mechanotransduction to produce secondary pharmacological effects.Qizhu granule is a clinically effective prescription for anti-liver fibrosis.Preliminary studies have shown that Qizhu granule can inhibit the proliferation of hepatic stellate cells,reduce the formation of type Ⅰ,Ⅲ,and Ⅳ collagen and resist ECM deposition,at the same time,which can reduce the density of hepatic sinusoidal microvessels and prevent hepatic sinusoidal capillaryization;in addition,Qizhu granules can also help maintain the number and diameter of the LSECs fenestrae,the mechanism is related to cytoskeletal regulation.According to previous studies,the pharmacological mechanism of Qizhu granule anti-LF is related to the regulation of ECM deposition,maintenance of LSECs fenestrae and regulation of LSECs biological function,however,the relationship between its effect on regulating ECM microenvironment mechanical force and its efficacy of anti-liver sinusoidal capillary remains unclear.This study from the perspective of mechanotransduction and effect of down-regulating liver elasticity to explore the biomechanopharmacology mechanism of Qizhu Granules anti-liver sinusoidal capillary,which will reflect the holistic,multi-target,and multi-level comprehensive pharmacological effects of TCM The purpose is to provide new ideas and new methods for TCM clinical and experiment research on anti liver fibrosis.OBJECTIVE This study includes two parts:animal experiments and cell experiments.The general objective is to explore the biomechanical pharmacological mechanism of Qizhu granules to inhibit hepatic sinusoidal capillary vascularization by regulating matrix hardness and triggering mechanotransduction,which including:1.Objective of animal experiments(1)Study on the effectiveness of Qizhu Granules against liver sinusoidal capillaryization;(2)Study on the effect of Qizhu Granules on the adjustment of liver tissue stiffness;(3)Bioinformatics analysis of the relationship between Qizhu Granules’ anti-liver sinus Study on the effectiveness of Qizhu Granules against liver sinusoidal capillaryization and liver stiffness regulation;(4)Evaluation of Qizhu Granules on regulating liver transport and metabolism in mice with liver fibrosis.2.Objective of cell experiments(1)Construction of 2D hydrogel substrates with different hardness and extraction of primary mice LSECs;(2)The effect of different substrate stiffness on capillaryization of LSECs;(3)Exploration of the mechanotransduction mechanism of matrix stiffness regulating LSECs fenestrae.METHODS Experimental methods1.Animal experiments The liver fibrosis mouse model was established by intraperitoneal injection of CCl4,and a blank group,a model group,a positive drug/silibinin group,a low-dose group of Qizhu granules,a medium-dose group of Qizhu granules,and a high-dose group of Qizhu granules were set.The main purpose is to study the effectiveness of Qizhu Granules in anti-liver sinus capillary and regulating liver tissue stiffness,and make preliminary bioinformatics prediction and analysis on the relationship between regulating liver stiffness and anti-liver sinus capillary.(1)The first part of animal experiment is to explore the anti-liver sinus capillaryization effect of Qizhu granules:The environmental scanning electron microscope(ESEM)to directly observe the fenestrae on the surface of LSECs.Western blotting(WB)semi-quantitative detection of the LSECs’fenestration regulatory proteins eNOS and caveolin-1.The expression of CD31,a marker of sinus capillaryization,was semi-quantitatively detected by immunofluorescence;the expressions of CD31 and VEGFA in liver tissue were determined semi-quantitatively by Western blotting(WB).(2)The second part of animal experiment is to study the regulating effect of Qizhu granules on liver tissue stiffness:firstly,the stiffness of liver tissue was directly mechanically tested,which the liver stiffness is detected by liver ultrasound in vivo;then,Young’s modulus of liver tissue sections is detected by atomic force microscopy(AFM),and liver tissue elastic is tested by Tensile Strength Tester equipments.Then the extracellular matrix(ECM)of liver tissue was detected to indirectly reflect the stiffness of liver tissue,the method used HE and Sirius red staining of liver tissue sections,and immunofluorescence staining of liver tissue sections for MMP-2 and Col.-Ⅰ.Analysis of serological matrix components(including type Ⅲ procollagen(PCⅢ),type Ⅳ collagen(Ⅳ-C),laminin(LN),hyaluronic acid(HA)).(3)The third part of animal experiment is to predict and analyze the biomolecular mechanism of the relationship between Qizhu Granules anti-liver sinus capillary and regulation effect on liver stiffness.ceRNA gene chip is used to perform the differential gene analysis of mRNA,miRNA,circRNA and lncRNA between control group.model group and Qizhu Granules treatment group,and the ceRNA regulatory network was constructed to analyze and predict the relationship between the effect of Qizhu Granules on anti-liver sinus capillaryization and the effect on regulating liver stiffness.(4)The fourth part of the animal experiment evaluated the function of Qizhu granules in regulating the transport and metabolism of liver fibrosis:The near-infrared photoacoustic real-time imaging technology of small animals was used to monitor the ability of indocyanine green(ICG)transport and metabolism of liver fibrosis mice.2.Cell experiments:In order to further explore the effect of extracellular matrix stiffness changes on liver sinusoidal capillaryization,hydrogel 2D medium substrates with different gradient hardness were constructed in experiments,and primary mice liver sinusoidal endothelial cells(LSECs)were extracted and cultured for observation.(1)The first part of cell experiment is constructed 2D hydrogel substrates with different hardnesses and extracted primary mouse LSECs for culture:Polyacrylamide hydrogels(PAM)were used to simulate the stiffness of healthy liver tissue,advanced liver fibrosis tissue and liver fibrosis/cirrhosis tissue,respectively.The bottom of a glass dish was set as a control group.The primary LSECs were extracted from C57BL/6J mice,which were consistent with animal experiments,and were optimized and perfected based on the extraction of LSECs from early-stage rats in the research group.Mouse primary LSECs were obtained by adherent separation by density gradient centrifugation.(2)The second part of cell experiment is to observe the effect of different substrate stiffness on the capillaryization of LSECs:The surface apertures of LSECs were directly observed by ESEM.and the the expression of LSECs skeleton and CD31 were semi-quantitatively detected by immunofluorescence.(3)The third part of cell experiment conducted a preliminary exploration of the mechanotransduction mechanism of substrate stiffness regulating LSECs fenestrae:DAF-FM DA probe solution was used to label LSECs,and Ca2+carrier A23187 was used as an inducer to stimulate LSECs releasing NO,and the NO release of LSECs was monitored in real time under a confocal laser microscope.RESULTS 1.Results of animal experiments(1)The research results of Qizhu granules on anti-liver sinus capillary:①ESEM detection results showed that,the number and diameter of the LSECs fenestrae in treatment groups remained normal;while that in the model group,the liver sinus surface became smooth by a decreased LSECs fenestrae,which showing a typical liver capillarization pathological manifestations;② WB protein detection analysis showed that Qizhu granules had a regulatory effect on the fenestration regulatory proteins eNOS and caveolin-1 of LSECs.The expression of eNOS was up-regulated,and the expression of caveolin-1 was down-regulated.③Immunofluorescence and WB protein detection analysis showed that Qizhu granules could down-regulate the pro-capillarization factor VEGFA and the capillarization marker factor CD31.Based on the above experimental results,it is proved that Qizhu granule has anti-liver sinusoidal capillarization effect,and its mechanism may be related to regulating the expression of eNOS and caveolin-1 to regulate the shape of LSECs fenestration,and reduce the expression of VEGFA.(2)The results of Qizhu Granules on regulating the stiffness of liver tissue:①The results of liver ultrasound showed that compared with the control group,the liver of the model group was enhanced and rougher,and the gray value of the liver parenchyma area increased.And Qizhu granules treatment groups compared with the model group,the echo of the liver parenchyma and the gray value was significantly reduced,indicating that Qizhu granules could significantly reduce the liver stiffness.At the same time,the treatment group showed a tendency to reduce the inner diameter of the portal vein.② The AFM results of frozen liver sections showed that:the Young’s modulus of liver tissue in the model group was significantly higher than control group,however the Young’s modulus in treatment groups were significantly lower than that in model group.Which indicating that Qizhu granules can reduce the stiffness of liver fibrosis tissue.③The results of the tensile test curve of fresh liver tissue showed that the Qizhu Granule treatment group significantly improved the elasticity of liver fibrosis tissue,which was significantly different from the model group.The results showed that Qizhu Granules increased the elasticity of liver tissue and reduced tissue stiffness,which was consistent with the AFM test results.④The liver fibrosis model mice was rough in texture,obvious on the surface,dull in color and less glossy;The results of HE and Sirius red staining of liver tissue sections showed that:HE staining showed disorder of liver cords,necrosis of hepatocytes,pyknosis,and pseudopyknosis,and leaflet formation;Sirius red staining showed a large number of specific red-stained collagen fibers,forming pseudolobules.On the other hand,the Qizhu granule treatment groups significantly improved liver sinus disorder,hepatocyte necrosis and inflammation,and significantly reduced collagen fiber deposition in mice with liver fibrosis.⑤ IMatrix metalloproteinase-2(MMP-2)and collagen type Ⅰ(Col-Ⅰ)in liver tissues were fluorescently labeled and detected by immunofluorescence detection technology.The results showed that treatment groups significantly increased MMP-2 expresses and reduces the expression of Col-Ⅰ,which indicates that Qizhu granules couold directly degrading ECM;⑥ Serological test results showed that Qizhu granule significantly reduced the extracellular matrix components PCⅢ,Ⅳ-C and LN in liver fibrosis,the composition of HA.(3)ceRNA gene chip is used to perform the differential gene analysis of mRNA,miRNA,circRNA and lncRNA between control group,model group and Qizhu Granules treatment group,and the ceRNA regulatory network was constructed to analyze and predict the relationship between the effect of Qizhu Granules on anti-liver sinus capillaryization and the effect on regulating liver stiffness.The results of ceRNA chip detection showed that,a large number of differential genes between model and treatment groups are related to ECM.GO enrichment results proved that the corresponding cellular components(CC),molecular functions(MF)and biological processes(BP)of Qizhu granules treatment group had significant correlations with ECM components(such as type Ⅳ collagen,fibrin,etc.)and ECM production and degradation biological processes.At the same time,KEGG resultst was also enriched with TGF-β signaling pathway,PI3K-Akt signaling pathway,ECM-receptor interaction pathway,which are all related to the ECM biological processes.The ceRNA regulatory network of miRNA-mRNA and lncRNA-miRNA-mRNA showed,the screened housekeeping target genes involve miR199a,miR199b,miR34a,miR223 and miR7018,miR7118,etc.Among them,the miR199 gene cluster has been widely reported to be related to angiogenesis and extracellular matrix The generation and degradation of fibrosis are closely related to the TGF-β signaling pathway.It can be speculated that there may be a regulatory relationship between Qizhu granule’s anti-liver sinus capillary vascularization and regulating liver tissue stiffness,and miR199 and TGF-β signaling pathways may play the key role in this progress.(4)Evaluation of Qizhu Granules on liver fibrosis transport and metabolism.The results show that:① The near-infrared photoacoustic real-time imaging technology of small animals was used to monitor the ability of ICG transport and metabolism of liver fibrosis mice.Results showed that the peak ICG absorption value of the model group was about 300s,which was significantly lower than that of the normal group 90s,besides,the ICG metabolism rate of the model group was significantly lower than that of the normal group.At the same time,the peak ICG absorption value in the low-dose Qizhu granule group and the high-dose Qizhu granule group was about 200s,which was significantly higher than model group,but the positive drug silibinin group showed longer absorption time than that in the normal group;and the metabolic rate of the low-dose and high-dose groups of Qizhu granules was significantly faster than that of the model group;the peak time of ICG absorption in the middle-dose group of Qizhu granules was about 300s,but it was still higher than that of the model group,and the ICG absorption and metabolism curves were almost the same as those of the model group.Based on the above results,it can be seen that drug treatment groups improved the ability of the liver to absorb and metabolize ICG.② Although biochemical indicators such as serum molecular markers such as transaminase are not specific for the diagnosis of liver fibrosis,they can reflect the degree of liver cell damage and liver cell function to a certain extent.The detection results of serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)showed that Qizhu Granules significantly reduced serum AST and ALT levels in mice with liver fibrosis,suggesting that Qizhu Granules could improve liver function.2.Results of vitro experiments:(1)Construction of 2D hydrogel substrates with different hardnesses and extraction of primary mouse LSECs:① Polyacrylamide hydrogels(PAM)were used to simulate the stiffness of healthy liver tissue,advanced liver fibrosis tissue and liver fibrosis/cirrhosis tissue,respectively.Which stiffness parameters were set according to the results of the Young’s modulus test of the liver tissue of each group by AFM in the animal experiment,and the hydrogel was characterized by the Tensile Strength Tester equipments,results show that the stiffness gradient was successfully established;②The primary LSECs of C57BL/6J mice were successful extracted:the immunofluorescence results show that the primary endothelial cells presented specific CD 14 molecules.And electron microscope scanning windows were used to identify the extracted primary endothelial cells,the results showed that the cell surface presented typical LSECs fenestrae phenotype.Those results proved that the extracted endothelial cells are exactly LSECs.(2)The effect of different substrate hardness on the capillary vascularization of LSECs:① The light microscope observation of the growth morphology of basal cells with different hardness showed that:the LSECs cultured on the hydrogel soft substrate(0.7Kpa)were looked round or oval,with less interconnection between cells,few pseudopodia,and more apertures on the cell membrane surface;compared with 0.7Kpa,the LSECs cultured on the hydrogel medium substrate(4Kpa)showed more spreading area and the skeleton state increased,pseudopodia formed,the intercellular connections increased,and the number fenestrae were decreased;LSECs cultured on a hard substrate(10Kpa)showed similar growth state with cultured on the cover glass(Cov),which the pseudopodia increased and grew,the cells formed tight junctions with each other,and the cytoskeletal fibers thickened significantly.② The fluorescence detection results showed that with the increase of basal stiffness,the expression of CD31 increased,and the cytoskeletal fibers were thickened and clearly visible,indicating that the vascularization of LSECs changed with the increase of matrix stiffness,and the change of matrix stiffness play an important role on had an impact on LSECs phenotype changes.(3)Preliminary exploration of the mechanotransduction mechanism of matrix stiffness regulating the fenestration of LSECs shows that:The results of real-time monitoring of NO fluorescent probes showed that the Ca2+carrier A23187 successfully induced NO release from soft substrate(0.7Kpa)LSECs,and the average fluorescence intensity change rate was 1.65%,while the medium stiffness(4Kpa)and high stiffness(10Kpa)and cover glass(Cov)were-3.3%,-4.05%,-4.83%,respectively,that is.calcium carrier A23187 did not induce NO release on these substrate stiffness,at the same time,with the prolongation of detection time,the NO fluorescence signal gradually weakened.The results indicated that LSECs with medium and above hardness substrates lost the ability to release NO.CONCLUSION Qizhu granules showed dual effects on anti-liver sinus capillarization and regulating liver tissue stiffness;at the same time,the regulation of liver stiffness by Qizhu granules can further exert biomechanopharmacology effect on anti-liver sinus capillarization based on mechanotransduction,and the mechanism may be related to the regulation of cytoskeleton and fenestration structure by the NO-dependent pathway in LSECs triggered by Ca2+signal stimulated by matrix stiffness. |
PDF Full Text Request