The Role And Mechanism Of Matrix Protein CCN1 In The Pathogenesis Of Autoimmune Hepatitis

【Objective】The aim of this research was to analyze the clinical characteristics of patients with autoimmune liver disease（ALD）,explore the diagnostic value of CCN1 in ALD patients,and provide help for the early diagnosis of ALD patients;and investigate the underlying mechanism of CCN1 involving in the pathogenesis of autoimmune hepatitis（AIH）by regulating IL-6 which provides a reliable experimental and theoretical basis for elucidating the pathogenesis of autoimmune hepatitis.【Methods】1.Analyze the clinical characteristics of ALD patients and explore the diagnostic value of CCN1 in ALD patientsCollect clinical information of patients diagnosed with autoimmune liver disease in the First Affiliated Hospital of Fujian Medical University from July 2009 to July2019.The diagnosis was re-evaluated in all cases according to the latest diagnostic criteria developed jointly with the Chinese Medical Association Hepatology Branch,Chinese Medical Association Gastroenterology Branch and Chinese Medical Association Infection.Finally,107 patients with ALD were diagnosed and further subclassified based on different disease types,including 59 cases of autoimmune hepatitis,26 cases of primary biliary cholangitis and 22 cases of autoimmune hepatitis-primary biliary cholangitis overlap syndrome.Collect general information such as age and gender,blood routine,biochemical and immunological laboratory test results,including antinuclear antibodies（ANA）,anti-mitochondrial antibodies（AMA）and anti Anti-smooth muscle antibody（SMA）which was tested by indirect immunofluorescence method,and the corresponding clinical data of 118 healthy people with matching age and sex were selected as controls.Enzyme-linked immunosorbent assay（ELISA）was used to detect the level of CCN1 in the sera of ALD patients,and the serum of healthy people with matching age and sex during the same period was selected as a control.ROC curve was used to analyze the diagnostic value of CCN1 in ALD.Statistical analysis was performed using SPSS 22.0 software.The measurement data with or without the normal distribution were expressed as"mean±standard deviation"or"median（interquartile range）".Means of groups of four samples were compared using the One-way ANOVA test or the Kruskal-Wallis H test while the means of samples between two groups using the t-test or the Mann-Whitney U test depending on whether the measurement data followed the normal distribution.The comparison of the positive rate between the two groups was using theX²test and the Fisher exact probability method.Difference was considered statistically significant when P<0.05.2.Investigation of the mechanism of CCN1 involving in the pathogenesis of autoimmune hepatitis by regulating IL-6The CCN1^flox/floxhomozygous mice expressing Cre recombinase introduced by Shanghai Jiaotong University School of Medicine were propagated and the genotype of each offspring was determined.The four to six weeks-old CCN1^flox/floxCre⁺mice were selected and tamoxifen was administered via intraperitoneal injection（1?mg/mouse/day）for 5 consecutive days to construct CCN1-konckout mice.Next,six to eight weeks-old C57BL/6J mice（NC-CCN1）and CCN1-knockout（KO-CCN1）mice were injected with Con A（20 mg/kg）via tail vein injection to induce the autoimmune hepatitis mouse model.Eight hours later,blood samples were obtained by removing the eyeball and mice were killed by cervical dislocation to collect liver samples.Then,a part of liver tissues were washed with saline,fixed with 4%paraformaldehyde solution,embedded in paraffin,sectioned,stained with hematoxylin and eosin（HE staining）and CCN1 immunohistochemical staining was also performed.The remaining part of liver tissues and serum samples were stored frozen at-80?°C for later analysis.Serum ALT and AST concentrations in mice were determined by enzyme colorimetry on the Siemens ADVIA 2400 biochemical analyzer.The expression level of CCN1 m RNA and protein were measured using RT-PCR,Western blot and immunohistochemistry.The IL-6 m RNA in liver tissue and blood samples was confirmed by RT-PCR.CCN1 si RNA 794 were transfected into the normal human hepatic cell line LO2,the IL-6 m RNA and protein expression were respectively determined by RT-PCR or Western blot and chemiluminiscent assay was used to measure IL-6 in cell-culture supernatant.【Results】1.Clinical features of ALD patients and CCN1 levels in serumThere was no significant difference in age and gender among AIH,PBC,AIH-PBC OS and HC（F=0.367,P=0.777;X²=5.553,P=0.125）.Compared with the three groups of patients with PBC,AIH-PBC OS and HC,the laboratory indicators of AIH patients were significant differences in hemoglobin content[127（18.5）g/L,119（25.75）g/L,119.5（20.5）g/L,134（13.75）g/L,X²=51.67,P<0.001],γ-glutamyl transferase[133（134.5）U/L,302（401）U/L,317.5（411.75）U/L,16（9）U/L,X²=151.74,P<0.001],lactate dehydrogenase[231（65）U/L,191（69.5）U/L,200.5（41）U/L,185（37）U/L,X²=34.446,P<0.001],aspartate aminotransferase[194（298.5）U/L,86（74.75）U/L,92.5（119.75）U/L,20（5）U/L,X²=161.311,P<0.001],low-density lipoprotein cholesterol[2.1（1.01）mmol/L,3.065（1.2675）mmol/L,2.815（1.8275）mmol/L,3.04（0.755）mmol/L,X²=40.377,P<0.001],total cholesterol[4.22（0.95）mmol/L,5.21（3.065）mmol/L,5.385（2.235）mmol/L,4.61（0.74）mmol/L,X²=35.306,P<0.001],alkaline phosphatase[128（70.5）U/L,224.5（238.75）U/L,160.5（169.75）U/L,67（21）U/L,X²=127.992,P<0.001].Of 54 AIH patients,33（61.1%）were positive for antinuclear antibodies（ANA）,13 patients（24.1%）were positive for anti-smooth muscle antibodies（SMA）,and 10 patients（18.5%）were negative for autoantibodies;Of 19 PBC patients,15（78.9%）were positive for anti-mitochondrial antibody（AMA）,14 patients（73.7%）were positive for ANA and3 patients（12.5%）were AMA-and AMA-M2-negative;Of 16 AIH-PBC OS patients,10（62.5%）were positive for ANA,9 patient（56.3%）were positive for AMA and 6patient（37.5%）were positive for AMA-M2.The level of CCN1 in the serum of AIH patients was significantly higher than that of healthy people,P value<0.05.ROC curve analysis showed that the areas under the AIH,PBC and OS curves were 0.8755,0.7657 and 0.9097,respectively.2.CCN1 participated in the pathogenesis of autoimmune hepatitis by regulating IL-6The CCN1 knockout mice were successfully established.Compared with the control group,the CCN1 m RNA and protein expression in the liver tissue of KO-CCN1 mice significantly decreased.After the mouse model of Con A-induced autoimmune hepatitis were established,the liver injury of KO-CCN1 and NC-CCN1mice was compared.The results demonstrated that the levels of AST and ALT,the biomarker for liver damage,and inflammatory marker IL-6 were decreased in KO-CCN1 mice compared to NC-CCN1 mice.Pathology reports also showed that the liver damage in NC-CCN1 mice was relatively moderate.These suggested that CCN1may play a critical role in mediating liver cell damage in AIH.CCN1 si RNA 794 had the best effect on interfering with LO2 cells.After transfection,compared with the control group,the levels of CCN1 m RNA and CCN1 protein in the cells decreased significantly,and the levels of IL-6 m RNA and IL-6 in the cell culture supernatant were also significantly decreased.【Conclusions】1.ALD patients have different laboratory test results;serum CCN1 is elevated in AIH,which is expected to become a new laboratory diagnostic indicator of AIH.2.This study successfully constructed CCN1 conditional knockout mice.3.CCN1 may act as a pro-inflammatory factor by promoting the expression of IL-6 to involve in the pathogenesis of AIHand this research would provide a theoretical and experimental basis for elucidating the pathogenesis of autoimmune hepatitis.