| Object:Objective to dynamically evaluate the changes of serum matrix metalloproteinase-7(MMP-7)in patients with biliary atresia(BA)and its value in diagnosis and prognosis.To preliminarily clarify the effect of MMP-7 gene polymorphism on serum MMP-7.To explore the therapeutic value of neutralizing MMP-7 on BA in animal experiment.Methods:Cholangiography was used as the gold standard for the diagnosis of BA.Serum MMP-7 was detected in BA group and non-BA cholestasis(Non-BA)group to evaluate the value of serum MMP-7 in the diagnosis of BA.It is estimated that the sensitivity of serum MMP-7 in diagnosis of BA was 87%,and the tolerance of sensitivity was 5%;The specificity was 90%,and the tolerance of specificity was 10%;Select confidence level 1α=0.95.The BA group and non-BA group used the same sample size,calculated by PASS 15 software,193 BA patients and 193 non-BA patients were included.From September 2017 to June 2020,403 patients with cholestasis(direct bilirubin level>17μmol/L)were recruited in 4 domestic centers,of which 21 cases refused to be tested for serum MMP-7,and the remaining 382 cases(136 cases from Wuhan Union Hospital,125 cases from Xi’ an Children’s Hospital,45 cases from Wuhan Children’s Hospital and 76 cases from Shenzhen Children’s Hospital)were included in this study.Exclusion criteria included refusal of MMP-7 test and knowing the specific cause of cholestasis before MMP-7 test.After the patients were enrolled,age,gender,medical history and examination results were collected,and serum MMP-7 was detected in Wuhan Union Hospital.The final diagnosis of each patient was made by a pediatric liver specialist and a pediatric surgeon.Routine follow-ups were performed 1 week,2 weeks,1 month,2 months,3 months,6 months,9 months and 12 months after hepatoportoenterostomy(HPE).Serum MMP-7 levels were detected in BA patients during each follow-up.The follow-up endpoint was death,liver transplantation or native liver survival for 1 year after HPE.Informed and written consent to participate in the study was obtained from the parents of all patients.It was approved by the ethics committee of Tongji Medical College,Huazhong University of Science and Technology(No.:2020S029),and approved by China Clinical Trial Registration Center(No.:ChiCTR1900028456).The Sanger method was used to detect the exons and promoter regions of MMP-7 in the whole blood of some children;some liver tissues were stained by immunohistochemical staining to observe the expression and distribution of MMP-7.RT-qPCR was used to detect the mRNA expression of MMP-7 gene in liver tissues.The animal model of BA was established by rotavirus(RRV).Newborn Balb/c mice were randomly divided into 4 groups:1.Normal group:25 μL medium was injected intraperitoneally into newborn Balb/c mice within 12 hours after birth as control group.2.RRV group:25 μL RRV(1 × 106 PFU)was injected intraperitoneally into newborn Balb/c mice within 12 hours after birth to establish BA model.3.MMP-7 control group:RRV was injected intraperitoneally into newborn Balb/c mice within 12 hours after birth,and 10 μL PBS was injected from 1 to 5 days after birth.4.MMP-7 intervention group:RRV was injected intraperitoneally into newborn Balb/c mice within 12 hours after birth,and MMP-7 antibody(10 μl,1 μg/μl)was injected from 1 to 5 days after birth.Blood and liver samples were collected on the 7th and 14th day after birth.In different periods,the general physiological state,incidence of clay colored stool,jaundice rate and overall survival rate of the four groups were observed.Histology was used to evaluate the degree of inflammatory infiltration in the liver of BA mice.The level of MMP-7 in serum was detected by ELISA,and the mRNA level of MMP-7 gene in liver tissues was detected by RT-qPCR.The measurement data were represented by median(interquartile range,IQR:first quartile,third quartile)or mean ± standard error,and statistical analysis was performed with SPSS 26.0,Graphpad prism 6.0,Image Pro plus software.Mann Whitney U test(nonparametric inspection)was used to test the significance of the data.Chi square test or Fisher test were used to test the rate of the two groups,and P<0.05 was considered to be statistically significant.Results:1.1.Dynamic changes of serum MMP-7 and its significance:The serum level of MMP-7 in BA group(67.26 ng/ml,IQR:48.02,100.60)was significantly higher than that in Non-BA group(12.52 ng/ml,IQR:8.12,18.71)(P<0.0001).The cut-off value of serum MMP-7 in the diagnosis of BA was 35.76 ng/ml(sensitivity 97.40%,specificity 93.68%,AUC 0.9723).The positive predictive value,negative predictive value and diagnostic efficiency of serum MMP-7 level>35.76 ng/ml for BA were 97.31%,93.94%and 95.61%,respectively.Age group analysis showed that the level of MMP-7 in BA patients with 61-90 days and more than 90 days was significantly higher than that in BA patients with<28 days.There was no significant difference in serum MMP-7 level between BA patients of<28 days and BA patients of 29-60 days.However,the level of MMP-7 did not change with age in Non-BA.1.2.The dynamic change of serum MMP-7 after HPE was that the serum MMP-7 level decreased first one week after HPE,then increased.Until the fourth week,the serum MMP-7 in the native liver survival group(NLS)began to decrease gradually,while the serum MMP-7 in liver transplantation group(LT)was higher and higher.ROC curve analysis:three months after HPE,the critical value of serum MMP-7 for predicting liver transplantation was 56.68 ng/ml(sensitivity 95.74%,specificity 86.79%,AUC 0.9422).1.3.The mRNA level of MMP-7 in BA group was 4.38 ±0.58 times higher than that in Non-BA group.1.4.The results of Spearman correlation analysis of serum MMP-7 levels were as follows:Serum MMP-7 levels were positively correlated with fibrosis stage,inflammatory grade,total bilirubin(TBIL),direct bilirubin(DBIL),alanine aminotransferase(ALT),aspartate aminotransferase(AST),gamma glutamyl transpeptidase platelet(γ-GT).The results of multiple linear regression model of serum MMP-7 level showed that inflammation grade,γ-GT and fibrosis stage were important factors affecting serum MMP-7 level.2.In order to evaluate the cause of low serum level of MMP-7 in a small number of patients in this study,we sequenced the exon and promoter regions of MMP-7.The heterozygous mutation rate of c.410G/A in exon 3(rs17884789)of BA patients with low serum MMP-7 level(<35.76 ng/ml)was significantly higher than that of BA patients with high serum MMP-7 level(>35.76 ng/ml)(P=0.010).3.In animal model,on the 7th day after birth,serum MMP-7 in RRV group was higher than that in normal group(2.24±0.40 ng/ml vs 1.01±0.13 ng/ml,P=0.0186).Serum MMP-7 in MMP-7 intervention group was lower than that in MMP-7 control group(1.23±0.12 ng/ml vs 2.21±0.28 ng/ml,P=0.0118).On the 14th day,serum MMP-7 in RRV group was higher than that in normal group(3.76±0.85 ng/ml vs 1.39±0.27 ng/ml,P=0.0284).Serum MMP-7 in MMP-7 intervention group was lower than that in MMP-7 control group(1.59±0.17 ng/ml vs 3.71±0.68 ng/ml,P=0.0161).Through animal intervention experiment,we found that compared with MMP-7 control group,the incidence of jaundice and clay colored stool decreased significantly,and the degree of inflammation in liver tissue decreased significantly.The time of death of mice was delayed,and the overall survival rate increased significantly.Conclusions:1.Multi center prospective study found that serum MMP-7 level could be used for the early diagnosis of BA.The important factors influencing serum MMP-7 level were inflammation grade,γ-GT and fibrosis stage.2.The liver transplantation probability of serum MMP-7>55.68 ng/ml was significantly higher than that of children with serum MMP-7 level<55.68 ng/ml at 3 months after HPE.3.The mutation of c.410 G/A(rs17884789)in exon 3 caused MMP-7 to be membrane type(preferentially located in cell membrane)and low expression in serum.4.Neutralizing MMP-7 could alleviate bile duct injury,reduce jaundice rate and prolong survival rate of mice,providing a new target for the treatment of BA in the future. |
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