In Situ Regulation And Mechanisms Of 3D Liver Matrix Stiffness On Activation And Reversion Of Hepatic Stellate Cells

Liver fibrosis is a major cause of morbidity and mortality worldwide.The activation of hepatic stellate cells（HSCs）is an important event in the occurrence of liver fibrosis.Activated HSCs transdifferentiate into myofibroblasts（MFs）to secrete excessive extracellular matrix（ECM）,the accumulation and remodeling of ECM result in the increase of liver matrix stiffness in the aggravation of liver fibrosis.Three-dimensional（3D）matrix stiffness has been considered as an important biophysical cue contributing to the regulation of HSCs proliferation,activation,reversion,and apoptosis.At present,it is difficult for two-dimensional（2D）or quasi-three-dimensional（quasi-3D）platforms constructed in vitro to simulate the structure,composition,and stiffness of the real 3D mechanical microenvironment of HSCs in vivo.To better understand the mechanism of HSCs activation and reversion and explore new mechanical target for the treatment of liver fibrosis,in this study,we constructed decellularized liver matrix scaffolds with different stiffness by decellularizing the rat livers at different stages of fibrosis.The effects of 3D matrix stiffness on the adhesion,proliferation,activation,reversion,and apoptosis of HSCs cultured in these developed scaffolds are studied,and a preliminary exploration of the internal mechanism is conducted.（1）Rat livers were induced to be fibrotic by artificially intraperitoneal injection of carbon tetrachloride（CCl₄）,followed by histological evaluation.Different degrees of fibrotic livers were decellularized by the method of gradient concentration perfusion processing.Decellularization,structural,and mechanical characterization of scaffolds were performed.①Rats had different degrees of liver fibrosis after injected with 40%CCl₄for 2 weeks,4 weeks,6 weeks,and 8 weeks,respectively.②Decellularized rat livers with different degrees of fibrosis had different mechanical properties,which gradually increased with the degree of liver fibrosis.（2）The resting HSCs were seeded into liver scaffolds with different matrix stiffness,and recellularization cytocompatibility of scaffolds was evaluated with hematoxylin-eosin（H-E）staining and Live/dead staining.The relationship between collagen and HSCs distribution in ECM was detected.①HSCs adhered and spread inside the scaffold with good vitality.3D decellularized liver scaffolds with different matrix stiffness had good cell compatibility.②Increased secretion of collagen I was a major factor leading to the increase of 3D liver matrix stiffness,and HSCs tended to migrate and aggregate to the thicker collagen bundles.（3）The proliferation ability of HSCs in scaffolds with different matrix stiffness was evaluated by EDU（5-ethynyl-2’-deoxyuridine）staining.The staining results preliminarily indicated that the increase of 3D matrix stiffness could enhance the proliferation ability of HSCs.（4）The resting and activated HSCs were seeded into liver scaffolds with different matrix stiffness,respectively.To study the effect of different 3D matrix stiffness on the activation and reversion of HSCs,the specific skeleton proteinα-smooth muscle actin（α-SMA）expressed by resting/activated HSCs in each group of scaffolds was detected.The effect of matrix stiffness on HSCs apoptosis was also investigated.The results showed that high matrix stiffness significantly promoted the activation of HSCs,while low matrix stiffness could reverse the activated HSCs to a certain extent,and some of activated HSCs underwent apoptosis.（5）Through immunofluorescence and immunohistochemistry staining,how the integrinβ1-YAP signaling pathway involved in the regulation of matrix stiffness on the activation and reversion of HSCs was studied.The results suggested that the activation of HSCs was accompanied by the activation of the cell membrane mechanoreceptor integrinβ1 and the nucleation of the mechanical sensitive protein Yes-associated protein（YAP）.The reversion process of HSCs was accompanied by the inactivation of integrinβ1 and YAP.The integrinβ1-YAP mechanical signaling pathway played a key regulatory role in the activation and reversion of HSCs induced by 3D matrix stiffness.In summary,3D matrix stiffness participated in the regulation of HSCs activation and reversion,suggesting the theoretical feasibility of inducing the reversion of activated HSCs to a resting state or undergo apoptosis by regulating the mechanical state of the liver matrix.The further activation of HSCs may be inhibited and even the further development of liver fibrosis may be prevented by targeting YAP or blocking the integrinβ1-YAP mechanical signaling pathway.And it can reduce the matrix stiffness by crushing and destroying the structure of collagenⅠbundle to provide a new entry point for reversing liver fibrosis.