Expression And Protection Mechanism Of IGF-1/IGFBP-3 In Mouse Ulcerative Colitis Model

Objectives: Recent studies have found that insulin-like growth factor-1(IGF-1)can promote cell differentiation and proliferation and wound repair,but its overexpression can promote fibrosis and cell malignant transformation.Binding of IGF-1 to the receptor binding regulatory protein insulin-like growth factor binding protein 3(IGFBP-3)can avoid excessive local dissociation of IGF-1 and prevent negative effects.Moreover,IGF-1 is involved in epithelialto mesenchymal transition(EMT),which is associated with inflammatory response,wound repair,invasion and metastasis of tumor cells,etc.However,there are few studies discussing the expression and role of IGF-1 and EMT in the development and tissue repair of ulcerative colitis(UC).This study intended to detect the changes of IGF-1 and IGFBP-3 expression characteristics at different stages in colon tissues of UC mouse models and the transformation of EMT in the progression of UC,to evaluate its influence on the occurrence,progression and outcome of UC,and to explore its mechanism.Methods: 120 SPF grade male BALB/c mice aged 6-8 weeks were selected and randomly divided into control group,model group and treatment group.The model group and the treatment group were treated with 4% dextran sulfate sodium(DSS)free drink to establish an acute UC model for 7 days,while the control group drank distilled water;The control group was given an equal volume of sterile normal saline by gavage.The body weight of the mice in each group was monitored,and the disease activity index(DAI)was evaluated.At 0d,4d,7d,8d,11 d and 14 d,5 mice were randomly selected from each group and killed,the whole colon was taken,the length of the colon was measured,and part of the colon tissue was HE stained,and the pathological characteristics were observed under the microscope.Store in a refrigerator at 80°C for testing.The labeled streptavidin-biotin(LSAB)method was used for immunohistochemical detection,and the detected target proteins included IGF-1,IGFBP-3,transforming growth factor-β(TGF-β)β)1,E-cadherin(E-cadherin),α-smooth muscle actin(α-smooth muscle actin,α-SMA),cytokeratin-19(cytokeratin-19,CK-19),using H The staining intensity was evaluated by using the real-time fluorescence quantitative PCR method(q RT-PCR method)to detect the relative expression of IGF-1 m RNA,IGFBP-3 m RNA,TGF-β1 m RNA,E-cadherin m RNA,α-SMA m RNA and CK-19 m RNA in colon tissue;Western The relative expressions of IGF-1 protein,IGFBP-3 protein,TGF-β1 protein,E-cadherin protein,α-SMA protein and CK-19 protein were detected by blot experiment.SPSS25.0 software was used for data analysis.Results:(1)The body weight of mice in the model group and the treatment group on d3～d6 showed a downward trend,and the DAI gradually increased,which was different from control group(P<0.05).There was no difference between the model group and the treatment group(P>0.05).(2)The colon length of mice in d4 and d7 model group and treatment group was significantly shorter than that in control group(P<0.05),and the colon length of mice in d14 treatment group was longer than that in d7 group(P<0.05).(3)Pathological examination showed that the colonic mucosa of mice in the d7 and d8 model groups and the treatment group showed typical UC manifestations,indicating that the modeling was successful.(4)The results of real-time fluorescence quantitative PCR showed that the relative expressions of IGF-1 m RNA and IGFBP-3 m RNA in the colon tissue of the model group and the treatment group were significantly decreased on d4 and d7,with the lowest on d7(P<0.05),rebounded on d8,d11 and d14,and the treatment group was significantly higher than the model group(P<0.05).Immunohistochemistry and Western blot trais showed that the IGF-1and IGFBP-3 expression in the treatment group decreased gradually on d4,d7,and d8,the lowest on d7/d8,and rebounded on d11 and d14,of which the treatment group was significantly higher(P<0.05).(5)The results of real-time fluorescence quantitative PCR showed that the epithelial-mesenchymal transition(EMT)inducer TGF-β1 m RNA and the mesenchymal marker α-SMA m RNA were significantly increased in the model group and the treatment group on d4 and d7(P<0.05),the highest on d7,and gradually decreased on d8,d11 and d14,of which the treatment group was significantly lower(P<0.05);the relative expression of epithelial markers E-cadherin and CK19 m RNA decreased significantly on d4 and d7,and the lowest on d7;It gradually increased with d14,and the treatment group was higher than the model group(P<0.05).Immunohistochemistry and Western blot trials showed that the TGF-β1 and α-SMA expression on d4,d7,and d8 in the model group and treatment group were significantly increased(P<0.05),the highest on d7/d8,and gradually decreased on d11 and d14.The expression of E-cadherin protein and CK19 protein in d4,d7,d8 decreased significantly,d7/d8 was the lowest,and d11 and d14 gradually recovered,and the treatment group was higher than the model group(P<0.05).).(6)The correlation analysis showed that the immunohistochemical H values ??of IGF-1 and IGFBP-3 in colon tissue of mice in d7 treatment group and model group were negatively correlated with TGF-β1 and α-SMA H values,and were negatively correlated with E-cadherin,CK-19 was positively correlated(P<0.05).IGF-1 immunohistochemical H values ??in colon tissue of d14 mice were negatively correlated with TGF-β1 and α-SMA,and positively correlated with E-cadherin(P<0.05);IGFBP-3immunohistochemical H values The value was negatively correlated withα-SMA(P<0.05).Conclusions: IGF-1 and IGFBP-3 can play a protective effect in the occurrence and progression of UC,which can reduce the damage of colonic mucosa and promote the repair of damage.The specific mechanism may be related to the inhibition of EMT.IGF-1 and IGFBP-3 are expected to be the targets for clinical diagnosis and treatment of UC.