摘要Background and objective:Noninvasive non-alcoholic steatohepatitis(NASH)assessment is a clinical challenge to the management of non-alcoholic fatty liver disease.We aim to develop diagnostic models based on sequential ultrasound molecular imaging(USMI)for the noninvasive identification of NASH in mouse models.Methods:Animal experiments were approved by the Animal Ethics Committee of South China Agricul-tural University.Forty-nine C57BL/6 mice were divided into normal control,non-alcoholic fatty liver,NASH,and hepatitis groups.Sequential USMI was implemented using CD36-targeted microbubbles(MBs-CD36)and intercellular adhesion molecule-1(ICAM-1)-targeted microbubbles(MBs-ICAM-1)to visualize hepatic steatosis and inflammation.The targeting signal of USMI was quantified as the normalized intensity difference(N1D)with the destruction-replenishment method.Correlation analysis was conducted between the NID-MBs-CD36 and pathological steatosis score and between the NID-MBs-ICAM-1 and pathological inflammation score.Finally,diagnostic models combining NID-MBs-CD36 with NID-MBs-ICAM-1 were established for NASH diagnosis.Results:MBs-CD36 and MBs-ICAM-1 were successfully prepared and used for sequential USMI in all mice.NID-MBs-CD36 values increased with the progression of steatosis,while NID-MBs-ICAM-1 values increased in parallel with the progression of inflammation.A strong positive correlation was identified between N1D-MBs-CD36 and pathological steatosis grade(rs=0.9078,P＜0.0001)and between NID-MBs-ICAM-1 and pathological inflammation grade(rs=0.9071,P＜0.0001).Among various sequential USMI-based diagnostic models,the serial testing model showed high diagnostic performance in detecting NASH,with 95％sensitivity,97％specificity,95％positive predictive values,97％negative pre-dictive values,and 96％accuracy.Conclusions:Sequential USMI using MBs-CD36 and MBs-ICAM-1 allows noninvasive grading of hepatic steatosis and inflammation.Sequential USMI-based diagnostic models hold great potential in the noninvasive identification of NASH.