Effect Of ICG001 Combined With TGF-β On Liver Fibrosis In Mice

Research BackgroundHepatic fibrosis is a repair response to chronic liver injury.It is characterized by an imbalance in the synthesis and degradation of extracellular matrix（ECM）and abnormal net deposition of ECM.It is the result of a comprehensive pathological response of injury-inflammation-regeneration-repair.Repeated injury and inflammatory responses It is an important mechanism for the progress of liver fibrosis.The incidence of liver fibrosis is high,and the prognosis is poor.The consequence of not undergoing clinical intervention is the development of liver cirrhosis and hepatocellular carcinoma.The course of liver cirrhosis is longer,and its severe complications affect the patient’s quality of life.The economic pressure is heavy;Factors directly threaten the lives of patients.The lack of approved medications is a major cause of high morbidity and mortality in liver fibrosis.As the only effective solution for patients with end-stage liver disease,liver transplantation is extremely expensive;and for liver transplantation,there are insufficient liver-derived donors nationwide,with strict implementation conditions and technical difficulties,which cannot be widely promoted.In the past few decades,the research on liver fibrosis generally believed that the liver has potential regeneration ability,and liver fibrosis is reversible.Hepatic stellate cells（HSC）are the main regulators of liver fibrosis pathogenesis,TGF-βactivates HSCs in a paracrine or autocrine manner,regulating the synthesis,degradation,and abnormal accumulation of extracellular matrix such as collagen fibers during liver fibrosis.Transforming growth factor-β（TGF-β）is a superfamily of structurally related pleiotropic secreted cytokines.It consists of ligands found in various cell and tissue types.Play a ubiquitous role in a large number of basic cellular events during tissue homeostasis and disease processes.The TGF-βsuperfamily consists of 33 members,see Table 1 for details.Members of the TGF-βfamily have different time and tissue expression specificities,and play a key role in the differentiation,proliferation,and activation of many different cell types,including immune cells.Play an important role in maintenance and repair.All immune cell lineages,including B cells,T cells and dendritic cells and macrophages secrete TGF-β,negatively regulate its proliferation,differentiation and activation of other cytokines,interference with TGF-βsignaling and autoimmunity,inflammation and Cancer is closely related.TGF-βplays a key role in the progress of organ fibrosis and is the main factor that causes most chronic liver,kidney,heart,skin and other organ fibrosis.TGF-β₁ can induce organ fibrosis by activating the classical Smad pathway and non-classical Smad pathway[1],which leads to the activation of myofibroblasts,excessive production of ECM,and inhibition of ECM degradation.Inhibition of the TGF-βsignaling pathway can limit renal fibrosis in disease models.However,since TGF-βis involved in other life processes,direct targeting of TGF-βis unlikely to achieve a viable antifibrotic therapeutic approach;it has been shown in clinical trials Therapies that block TGF-β₁ have proven ineffective.In diabetic nephropathy,a phase II clinical trial of TGF-β₁ monoclonal antibody for renal fibrosis showed that there was no statistically significant difference in serum creatinine between the TGF-β₁monoclonal antibody treatment group and the placebo group;TGF-β₁ deficiency Compared with control mice,mice showed severely impaired wound repair,including re-epithelialization and reduced collagen deposition;mice lacking TGF-β₁ also exhibited severe wasting syndrome with a marked systemic inflammatory response and Tissue necrosis can lead to organ failure and death;TGF-βand its signaling pathway genes are expressed in the developing and adult heart,and targeted destruction of the TGF-β₁ and TGF-β₂ genes leads to varying degrees of heart defects,including the atrium The myocardialization of the mesenchymal cells of the septum and ventricular outflow tract fails and the valve is poorly differentiated.Recent studies have also shown that the absence of Smad5 in the TGF-β₁signaling pathway leads to abnormal cardiac development and cardiomyocyte apoptosis;the above results illustrate the limitations of using TGF-β₁ as a target for antifibrosis.Objective:1.CCL₄ was used to interfere with mice,and an animal model of mouse liver fibrosis was induced.2.The serum levels of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）in animal models of liver fibrosis were measured,and the pathological manifestations of the model were observed histologically.After the application of ICG001 and ICG001 in combination with TGF-βThe serum ALT and AST levels of mice were observed histologically after pathological changes.3.To detect the expression of Col-Ⅰ,α-SMA and FOXO1 protein and gene expression in CCL₄-induced liver fibrosis in mice by ICG001 combined with TGF-β.4.To study the role of ICG001,TGF-βand FOXO1 in the treatment of liver fibrosis.use CCL₄ to induce a mouse liver fibrosis model and explore the effects of ICG001 combined with TGF-βon the expression of Col-Ⅰandα-SMA in liver fibrosis induced by CCL₄.Methods:1.Use CCL₄ to establish an animal model of mouse liver fibrosis:C57BL/6J male mice were randomly divided into a control group and a model group.The control group was intraperitoneally injected with 0.1 mL of olive oil and the experimental group was intraperitoneally injected with 20%CCL₄（the solute was olive Oil）,twice a week,until the end.Treatment was performed every 2 weeks,and 3 mice were randomly selected from the experimental group and the model group to be sacrificed.Blood was collected from the eyeballs,and liver tissue was collected.The general condition was observed under a stereomicroscope,and sections of the liver were fixed.2.Treatment of mouse CCL₄-induced liver fibrosis model:48 C57BL/6J male mice were randomly divided into control group,model group,ICG001 group and ICG001combined with TGF-βgroup（IT group）;model group,ICG001 Group and IT group received intraperitoneal injection of 20%CCL ₄0.1 mL twice a week for 6 weeks;the control group received intraperitoneal injection of 0.2 mL physiological saline twice a week for 6 weeks;starting from the 5th week of modeling,ICG001 group and IT The group was intraperitoneally injected with ICG001（5mg/kg）once a day for two weeks;the IT group was intraperitoneally injected with TGF-β（5mg/kg）once every 2 days for a total of two weeks.At the end of the 6th week,the contents of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）in the serum of the mice in each group were detected;the liver tissues of each group of mice were collected,and the general conditions were observed under a stereomicroscope.The sections were fixed and the remainder was stored at-80℃for further experiments.3.Extract the liver tissue protein of each group of mice,extract the protein of each group of mice,and detect the protein expression ofα-SMA,Col-Ⅰ,FOXO1 by Western Blot.4.RNA was extracted from liver tissue of each group of mice,and the expression of IL-6,IL-10,TNF-α,α-SMA,Col-Ⅰ,FOXO1 was detected by q-PCR;5.According to the type of data,first use single factor analysis of variance to test whether each sample comes from the same population,and then use t test（Fisher’s exact probability method）to analyze the differences between groups.Results:1.The first part:the serological indexes AST and ALT in the model group far exceed the control group;the second part:the serological indexes AST and ALT in the model group far exceed the control group（ALT:t=18.36,P<0.01;AST:t=8.83,P<0.01）;Compared with the model group,the serological index in the ICG001 group decreased（ALT:t=8.12,P<0.01;AST:t=5.59,P<0.01）,and the reduction in the IT group was more significant（ALT:t=11.75,P<0.01;AST:t=7.76,P<0.01）;2.Histological examination showed that the inflammatory cells in the model group gathered around the portal vein and the central vein,and a large amount of inflammatory cell infiltration was also seen in the parenchymal cells.In the ICG001 group and the IT group,the inflammatory cells in the parenchymal cells were reduced,and the IT group was improved.The degree is more obvious than the ICG001 group;the staining of the reticular fibers shows that the reticular fibers in the model group are thick and have protrusions and are irregularly distributed;compared with the model group,the fibers in the ICG001 group are thinner and the protrusions are smaller than in the model group.The degree of improvement was more obvious than the ICG001 group.3.Western Blot results:Compared with the control group,α-SMA and Col-Ⅰproteins were significantly increased in the model group（Col-Ⅰ:t=7.22,P<0.0;α-SMA:t=17.72,P<0.011）,ICG001 group decreased（Col-Ⅰ:t=8.08,P<0.01;α-SMA:t=8.42,P<0.01）,the degree of decline in the IT group was more significant Col-Ⅰ:t=5.87,P<0.01;α-SMA:t=4.22,P<0.05);there was no statistical difference in FOXO1 between the model group and the control group（t=1.44,P=0.22>0.05）,and there was no statistical difference between the ICG001 group and the model group（t=1.48,P=0.212>0.05）,the IT group was higher than the ICG group（t=3.51,P<0.05.）4.q-PCR results showed that the inflammatory factors IL-6,TNF-α,α-SMA,and Col-Ⅰwere significantly improved in the model group（IL-6:t=10.61,P<0.01;TNF-α:t=89.83,P<0.01;α-SMA:t=22.92,P<0.01;Col-Ⅰ:t=13.73,P<0.01）,the ICG001 group was lower than the model group（IL-6:t=9.91,P<0.01;TNF-α:t=58.93,P<0.01;α-SMA:t=16.57,P<0.01;Col-Ⅰ:t=6.22,P<0.01）,the IT group was significantly lower than the ICG001group（IL-6:t=5.39,P<0.01;TNF-α:t=4.54,P<0.05;α-SMA:t=4.58,P<0.05;Col-Ⅰ:t=3.36,P<0.05）;Anti-inflammatory factor IL-10 in the model Group（t=6.48,P<0.01）,the ICG001 group was higher than the model group（t=3.67,P<0.05）,and the IT group increased more significantly（t=3.35,P<0.05）;FOXO1 was It increased in the model group（t=10.06,P<0.01）.There was no statistical difference between the ICG001 group and the model group（t=2.51,P=0.066>0.05）.The IT group increased（t=4.99,P<0.01）.).Conclusion:1.An animal model of mouse liver fibrosis was successfully established using CCL₄.After comparison,the 5th week was selected as the node for drug intervention.2.Compared with ICG001 alone,ICG001 combined with TGF-βhas a better effect on liver fibrosis caused by CCL₄,which may be related to the change of FOXO1 caused by ICG001’s action on the TGF-βpathway.