ASK1-mediated Endoplasmic Reticulum Stress Promotes Hepatic Fibrosis Through Exosome

Liver fibrosis is a common chronic liver disease,which can develop into liver cirrhosis and liver cancer.Factors,including alcohol stimulation,virus infection,drug poisoning,obesity may induce liver fibrosis.Hepatic stellate cells(HSCs),as the most important mesenchymal cells in the liver,play an important role in the process of liver fibrosis.Normally,HSCs are resting without proliferative activity,fibrotic characteristics,inflammatory response and so on.While,HSCs are activated response to inducement,resulting in fibrogenesis,characterized by increased proliferation viability and excessive accumulation of matrix such as α-smooth muscle actin(αSMA),type I collagen(Col-1)and type III collagen(Col-Ⅲ).Liver transplantation is the most effective treatment for patients with liver fibrosis.However,organ supply problems often limit the implementation of this treatment program.Therefore,the specific mechanism of liver fibrosis and HSC activation is of great significance for the development of new treatments.However,at present,the mechanism of activation of HSCs and the origin of hepatic fibrosis is still not very clear,and of HSCExosomes belong to a kind of extracellular vesicles secreted by cells,with diameter ranging from 50 to 150 nm.Exosomes play an important role in information transduction in liver fibrosis.Cell-to-cell communicates can be carried by exosomes via containing miRNAs or proteins..Almost all cells secret exosomes.For example,mesenchymal stem cells-derived exosomes are considered to have a potential role in reversing fibrosis.The exosomes secreted by activated HSCs mediating the glycolysis-related proteins in target cells,resulting in promotin liver fibrosis.The important role of exosomes in hepatic fibrosis has been demonstrated.However,the mechanism of exosomes releasing is not completely clear..ASK1(Apoptosis signal-regulating kinase 1)is a member of the MAPK kinase(MAP3Ks)family and it mediates the activation of c-Jun N-terminal kinase(JNK)and p38MAPK signaling pathways.ERS(endoplasmic reticulum stress),ROS(reactive oxygen species)or LPS(lipopolysaccharide)promotes cell proliferation,apoptosis and inflammation by activating JNK and p38MAPK by mediating ASK1 phosphorylation.Evidence shows that ASK1 expression is positively correlated with hepatic fibrosis stress.However,the potential relationship between ASK1 and ERS in Ang Ⅱ-activated HSCs,is still unclear and further research is needed.This study was started with an in vitro experiemnts in Ang Ⅱ-activated HSCs.This study was designed to clarify:1)The characteristics of ASK 1 expression in HSCs activated,as well as the effect on endoplasmic reticulum stress.2)The role of ASK1 pathway in the activation of hepatic stellate cells,and 3)The role of endoplasmic reticulum stress-mediated exosome in HSCs.In summary,the objective of this study is to clarify the mechanism of activated hepatic stellate cells inducing endoplasmic reticulum stress,regulating exosome release and further activating resting HSCs through ASK1 pathway.Part one:Activation of Resting Cells Mediated by Exosome of Activated Hepatic Stellate Cells.Objective:This part of experiments was to verify the characteristics of activated HSCs,and the phenomenon of activation of resting cells by activated cell-derived exosomes.Methods:1)The expression of α-SMA,Col Ⅰ and Col Ⅲ was detected by qRT-PCR,Western blot and immunofluorescence in Ang Ⅱ-activated HSCs.The cell viability was detected by CCK8 kit.2)the expression of ASK1 and endoplasmic reticulum stress-related proteins was detected by Western blot,and the production of ROS was detected by flow cytometry.3)the inflammatory response of cells was detected by ELISA.4)the exosomes of HSCs treated with Ang Ⅱ were isolated and incubated with resting cells.Meanwhile,the activated stellate cells were co-cultured with resting cells,and then the expression of α-SMA in resting cells was detected.5)the exosomes secreted by activated HSCs were blocked with Annexin V,and the expression of αSMA and cell viability was detected after incubation.Results:The expression of α-SMA was significantly up-regulated and the activity of cell viability was also significantly increased in Ang Ⅱ-treated LX-2 cells.Ang Ⅱpromoted the expression of ASK1 and endoplasmic reticulum stress-related proteins,enhanced ROS production and cell viability.The expression of α-SMA was increased in resting stellate cells co-cultured with Ang Ⅱ-treated cells or incubated with their exosomes.The exosome treated with Annexin could not activate the resting cells.Summary:1)Ang Ⅱ promoted the cell viability of HSCs and activated HSCs to develop fibrosis.2)Ang Ⅱ upregulated the expression of ASK1 and induced endoplasmic reticulum stress,ROS production and inflammatory response.3)HSCs treated with Ang Ⅱ activated resting HSCs,which may be related to the uptake of exosomes by resting cells.Part two:ASK1 Mediates Endoplasmic Reticulum Stress and Involves in the Activation of Hepatic Stellate CellsObjective:This study was aiming to explore the role of ASK1 in the in vitro model of hepatic fibrosis induced by Ang Ⅱ and verify the mechanism of endoplasmic reticulum stress and exosome in the transformation of HSCs from resting state to activated state.Methods:Based on the in vitro model induced by Ang Ⅱ,1)the cells were treated with ASK1 inhibitor or ASK1 siRNA.Afterwards,the expression of α-SMA,Col I and Col Ⅲ were detected by qRT-PCR,Western blot and immunofluorescence.The cell viability was detected by CCK8 kit.The expression of ASK1 and endoplasmic reticulum stress-related proteins was detected by Western blot.The production of ROS in LX-2 was detected by flow cytometry.The inflammatory factors were detected by ELISA.The exosomes of HSCs in each group were isolated and incubated with resting cells,and HSCs were co-cultured with resting cells meanwhile.Afterwards,the expression of α-SMA in resting cells was detected.2)ASK1 inhibitor was used as treatment group,and endoplasmic reticulum stress inducer(tunicamycin)and exosome release inhibitor(GW-4869)were used to treat cells for the same detection as in 1).Results:1)The expression of α-SMA was significantly down-regulated in LX-2 after treatment with GS-4997 or ASK1 siRNA.Cell viability was significantly decreased,the endoplasmic reticulum stress-related proteins,ROS production and inflammatory response were significantly weakened.2)the inhibitory effect of GS-4997 on endoplasmic reticulum stress,ROS production and inflammatory reaction of HSCs was reversed after LX-2 treated with tunicamycin.While the activation of resting cells by corresponding exosome was also enhanced.Further exosome release did not affect the endoplasmic reticulum stress,ROS production and inflammatory response of HSCs,but the activation of resting cells under the action of exosome was significantly weaker than that of tunicamycin treatment group.Summary:1)ASK1 synergized activation of Ang Ⅱ inHSCs,and promoted in endoplasmic reticulum stress,ROS production and inflammatory response.2)the exosome of activated HSCs mediates the activation of resting cells.3)endoplasmic reticulum stress pathway is a key link in ASK1-mediated stellate cell activation.Part three:ASK1/endoplasmic Reticulum Stress Mediates Eosome Release and Activation of Resting Hepatic Stellate Cells.Objective:This study was to verify the role of ASK 1-mediated endoplasmic reticulum stress in hepatic fibrosis in hepatic fibrosis rat model,and to explore the activation effect of serum exosome on resting cells in vitro.Methods:1)the animal model of hepatic fibrosis was made by intraperitoneal injection of carbon tetrachloride oil solution.ASK1 inhibitor,GS-4997,was used as a drug in model rats.Western blot was used to detect the expression of ASK1,a-SMA and endoplasmic reticulum stress-related proteins in liver.H&E staining and Masson staining were used to detect liver pathology and fibrosis.Immunohistochemical staining was used to detect the expression of ASK1,and serum ALT and SOD were detected.2)the serum exosomes of animals in each group were isolated,and the resting cells were incubated to detect the activation of the cells.Results:1)the results showed that the expression of ASK1 in the liver of the model group was up-regulated,the level of ALT was up-regulated,and the level of SOD was decreased.Inflammatory cell infiltrated in the liver of the model group,accompanied by hyperfibrosis.The expression of α-SMA,Col I and Col Ⅲ in the tissue increased.Expressionof endoplasmic reticulum stress-related proteins wasincreased in the model group.After GS-4997 treatment,the above phenomena were reversed.2)the particle size of serum exosome of each group was about 100 nm,and CD63 and TSG101 proteins were expressed.After co-cultured with exosomes,the expression of a-SMA was down-regulated in resting LX-2 cells.Summary:1)ASK1 inhibitor inhibited the progression of hepatic fibrosis and weaken the stress signal of endoplasmic reticulum in rats.2)the serum exosome of the animal model of hepatic fibrosis regulated the activation of resting HSCs in vitro.Conclusion1)HSCs showed the characteristics of fibroblasts after Ang Ⅱ treatment.In addition,the expression of ASK 1 and endoplasmic reticulum stress were promoted by Ang Ⅱ.2)the exosome secreted by HSCs treated with Ang Ⅱ can induce the activation of resting cells,which depends on the endoplasmic reticulum stress mediated by ASK1,and this activation can be weakened immediately after the exosome release pathway is blocked.3)GS-4997,an inhibitor of ASK1,inhibited hepatic fibrosis and has a potential therapeutic effect on the progression of hepatic fibrosis,which may be achieved through the release of exosomes and the activation of HSCs.