| BackgroundHepatic cirrhosis is a pathological change caused by excessive deposition of extracellular matrix due to various acute or chronic liver injuries,and is a common outcome of the progression of various chronic liver diseases.Esophageal varices bleeding is one of the most serious complications in cirrhotic patients.Despite the use of esophageal variceal ligation in combination with non-selective β Receptor blockers recommended by the Baveno Ⅶ Portal Hypertension Consensus,the rate of EV rebleeding in patients who recovered from their first EV bleeding within 2 years still remains 37-50%.Therefore,the prediction of rebleeding after recommended treatments and the identification of high-risk patients with rebleeding are urgent issues that can help improve the prognosis of cirrhotic patients.It is well known that endoscopy and hepatic vein pressure gradient are the important methods for evaluating EV rebleeding,but cannot be used as a routine examination due to their disadvantages such as expensive and invasive.Therefore,accurate,effective and noninvasive alternative methods are helpful for predicting EV rebleeding.Radiomics can extract and screen the most valuable imaging features through high-throughput mining of medical image information,thereby providing information for disease diagnosis and treatment.In recent years,radiomics has great potential in the diagnosis and evaluation of hepatic cirrhosis,portal hypertension,and risk assessment of EV bleeding.However,whether radiomics can accurately predict EV rebleeding after treatment is unknown.Hepatocellular carcinoma(HCC)is one of the common complications of hepatic cirrhosis,and surgical resection is the first line treatment for HCC.For HCC patients with hepatic cirrhosis,most of them have impaired liver function and are prone to post-hepatectomy liver failure(PHLF).Therefore,preoperative liver function assessment and PHLF risk assessment are crucial for predicting the prognosis of HCC patients with hepatic cirrhosis.Currently,several methods have been reported to evaluate preoperative liver function,such as Child-Pugh grading,indocyanine green test.However,those methods are not accurate and have not been widely used in clinical practice.Gadobenate dimegramine(Gd-BOPTA)is a hepatobiliary specific MRI imaging contrast agent that is not only used for the detection of liver lesions,but also for the evaluation of liver function.Our previous works have shown that liver parenchyma imaging and biliary tract imaging biliary imaging using hepatobiliary specific contrast agents are critical in predicting clinical outcomes in patients with hepatic cirrhosis.However,the clinical feasibility of biliary imaging using hepatobiliary specific contrast agents for predicting PHLF in HCC patients with hepatic cirrhosis needs further study.Although the prediction of prognosis of hepatic cirrhosis and its complications are helpful for cirrhotic patients,inhibiting the progression of hepatic cirrhosis through treatment is also important for improving liver function and preventing the occurrence of complications.Although many drugs have been reported to be effective in treating hepatic cirrhosis,there is still a lack of a well-recognized drug for hepatic cirrhosis.Activation of hepatic stellate cell(HSC)is a major point during the progress of hepatic cirrhosis,with high expression of α-smooth actin(α-SMA)and secrete ECM such as collagen.Senescence of hepatocytes can induce HSC activation and improving the progress of hepatic cirrhosis,while inhibition of HSC activation and senescence of hepatocyte aging can significantly inhibit the progress of hepatic cirrhosis.Therefore,we use a multifunctional nanomaterial Fe3O4-MnO2(referred to as Fe-Mn)that can both load drugs and clear reactive oxygen species(ROS),loading insulin like growth factor I(IGF-1)and CXC chemokine receptor 4(CXCR4)antagonist AMD3100 to construct a multifunctional nanomaterial-based drug carrier nanoparticle Fe-Mn-IGF-AMD.Fe-Mn-IGF-AMD utilizes ROS in the cirrhotic microenvironment to react with MnO2,resulting in the disintegration of nanoparticle,thereby release of IGF-1 and CXCR4 into the liver,exerting its anti-cirrhotic effect.Meanwhile,the decomposition of Mn2+and Fe3O4 can provide signals for T1 and T2 imaging of magnetic resonance imaging(MRI)to diagnose hepatic cirrhosis through quantitative analysis of T1 and T2 signal values.This study includes three parts.In the first part,we used the CT-based radiomics information of 173 cirrhotic patients to develop a radiomics model to predict EV rebleeding after treatment by establishing a "liver-spleen" joint model.At the same time,we used X-tile software to divide the population into three risk levels,making the management of EV rebleeding population more accurate.In the second part,we discussed the predictive effect of the relative enhancement ratio of the biliary system(REB)on the prediction of liver function in HCC patients with hepatic cirrhosis.We found that REB can predict the occurrence of PHLF in those patients.HCC patients with REB-2.21 have a higher incidence of PHLF than those with REB>2.21.In the third part,we used multifunctional nanomaterials Fe-Mn to encapsulate two drugs,IGF-1 and AMD3100,to form a drug-loaded particle Fe-Mn-IGF-AMD.We confirmed that Fe-Mn-IGF-AMD has a good therapeutic effect on hepatic cirrhosis.Subsequently,we confirmed that Fe-Mn-IGF-AMD has the function of inhibiting HSC activation and senescence of hepatocyte,scavenging ROS,and improving intrahepatic hypoxia.In addition,Fe-Mn-IGF-AMD can also diagnose hepatic cirrhosis through T1 and T2 imaging signals of MRI.In summary,this study provides a noninvasive and accurate imaging-based new model for predicting the prognosis of hepatic cirrhosis complications(EV rebleeding and PHLF in HCC patients with hepatic cirrhosis).At the same time,a multifunctional drug loaded nanoparticle Fe-Mn-IGF-AMD,which has the inhibitory effect on hepatic cirrhosis and can be used for diagnosis of hepatic cirrhosis has been constructed,providing a feasible method for realizing the integration of diagnosis and treatment of liver cirrhosis.Part 1 CT-based radiomics score can accurately predict esophageal variceal rebleeding in cirrhotic patientsAimEV rebleeding is one of the most serious complications of hepatic cirrhosis.Predicting EV rebleeding can help improve the prognosis of patients and reduce unnecessary endoscopy.The purpose of this study was to extract radiomics features from CT images of the liver and spleen of cirrhotic patients and construct a radiomics score(Rad-score)to predict the risk of EV rebleeding.Methods1.This study included 173 cirrhotic patients after EV bleeding treatment,and the preoperative examination information and abdominal CT data were collected.2.Among the 2264 radiomics features extracted from liver and spleen CT data,the least absolute shrinkage and selection operator(LASSO)-Cox regression was used for feature screening and development of radiomics scores(Rad-scoreLiver,Rad-scorespleen,and Rad-scoreLiver-spleen)based on liver,spleen and liver-spleen,respectively.3.Evaluating the accuracy of Rad score in predicting EV rebleeding in cirrhotic patients through concordance index(C-index),calibration curve and decision curve analysis(DCA).Kaplan-Meier survival analysis was used to evaluate the risk stratification ability of Rad-score.Results1.Rad-scoreLiver,Rad-scorespleen and Rad-scoreLiver-spleen were the independent risk factors of EV rebleeding in cirrhotic patients.2.The Rad-scoreLiver-spleen,which is composed of ten imaging features,showed good predictive ability for EV rebleeding.The C-index of the training group and the validation group were 0.853[95%confidence interval,CI),0.776-0.904]and 0.822(95%CI,0.749-0.875),respectively.3.The calibration curve and DCA showed that the EV rebleeding probability predicted by Rad-scoreLiver-Spleen was very close to the actual probability,and its reliability in clinical practice was very high.In the training and validation queue,Rad-scoreLiver-Spleen could accurately divide patients into three risk groups:high,medium and low.4.The calibration curve results showed that the Rad-scoreLiver-Spleen has high accuracy in predicting EV rebleeding,which is very close to the actual probability.The DCA indicated that Rad-scoreLiver-Spleen generates more clinical benefits than Rad-scoreLiver and Rad-scorespleen,,suggesting that Rad-scoreLiver-Spleen is more practical than Rad-scoreLiver and Rad-scorespleen in predicting EV rebleeding in cirrhotic patients.In both the training and validation cohorts,Rad-scoreLiver-spleen can accurately classify patients into low,medium,and high-risk groups for EV rebleeding,and there are statistically significant differences in the 12-month EV rebleeding rates among the groups.5.Subgroup analysis showed that the C-index of Rad-scoreLiver-Spleen in hepatitis B virus cohort was higher than that in non-HBV cohort.ConclusionThe Rad-scoreLiver-Spleen extracted from CT images of liver and spleen can accurately predict the risk of EV rebleeding and classify patients according to the risk of EV rebleeding.This score provides a new non-invasive predictive tool for assessing the risk of EV rebleeding.Part 2 The value of gadobenate dimeglumine-enhanced biliary imaging from the hepatobiliary phase for predicting post-hepatectomy liver failure in HCC patients with hepatic cirrhosisAimGd-BOPTA-enhanced hepatobiliary imaging plays an important role in evaluating the clinical outcome of cirrhotic patients.This study aims to explore the value of Gd-BOPTA-enhanced hepatobiliary imaging in predicting the prognosis of HCC patients with hepatic cirrhosis after resection.Methods1.This study collected 221 HCC patients with hepatic cirrhosis who underwent Gd-BOPTA enhanced MRI scanning before hepatectomy in two medical centers.2.The relative enhancement ratio of the biliary system(REB)and the liver to muscle ratio(LMR)were measured in the hepatobiliary phase of MRI imaging.The capacity of REB and LMR predicting preoperative liver function was analyzed by ROC curve.3.Logistic regression was used to analyze the potential risk factors of PHLF.The capacity of REB and LMR predicting PHLF and serious complications was analyzed by ROC curve.Results1.REB showed significantly better diagnostic performance than the LMR in differentiating Child-Pugh A from Child-Pugh B,indicating that the capacity of REB for preoperative liver function is higher than that of LMR.2.REB was an independent risk factor for PHLF(odds ratio[OR]=0.127[0.047-0.348],p<0.001).Although LMR tended to be associated with PHLF(p=0.063),LMR is not an independent risk factor in multivariate analysis(OR=0.624[0.023-16.709],p=0.079).3.The area under the receiver operating characteristic curve(ROC)of REB and LMR for predicting PHLF was 0.87 and 0.60 respectively.The most appropriate cut-off value for REB was 2.21.The incidence of PHLF in HCC patients with hepatic cirrhosis with REB≤2.21 was higher than that in patients with REB>2.21(60.0%vs 8.5%,p<0.001).4.The area under the ROC curve predicted by REB and LMR for predicting major complications was 0.80 and 0.59,respectively.ConclusionGd-BOPTA-enhanced hepatobiliary imaging can accurately predict the PHLF and major complications in HCC patients with hepatic cirrhosis,providing a reliable tool for predicting the prognosis of HCC patients with hepatic cirrhosis.Part 3 The therapeutic effect for hepatic cirrhosis of drug-loaded nanoparticles based on multifunctional nanomaterialsAimHepatic cirrhosis is a complex pathophysiological process,and its mechanism such as HSC activation,senescence of hepatocyte,ROS accumulation,and intrahepatic hypoxia can promote the progression of hepatic cirrhosis.Although many drugs have been reported to be effective in treating liver cirrhosis,there is still a lack of a recognized treatment for hepatic cirrhosis.The purpose of this study is to develop a novel drug-loaded nanoparticle Fe-Mn-IGF-AMD using a multifunctional nano material Fe-Mn,IGF-1 and AMD3100,and to verify its anti-cirrhosis effect and mechanism.Methods1.Fe3O4-related nanoparticles(NP)were detected and characterized by methods such as transmission electron microscope(TEM),X-ray diffraction spectrogram(XRD),Fourier transform infrared spectroscopy(FTIR)and vibrating sample magnetometer.2.To detect the toxicity of Fe-Mn-IGF-AMD on cells,the CCK-8 method was used to evaluate the effects of different iron concentrations of Fe-Mn,Fe-Mn-IGF,Fe-Mn-AMD,and Fe-Mn-IGF-AMD on the survival rate of LX-2,HepG2,and RAW264.7 after co-culturing for 24 and 48 hours.The HE staining was used to evaluate the effects of Fe-Mn,Fe-Mn-IGF,Fe-Mn-AMD,and Fe-Mn-IGF-AMD on the structure of heart,spleen,lung,and kidney tissues.3.To evaluate the therapeutic effect of Fe-Mn-IGF-AMD on hepatic cirrhosis,Sirius red staining,Masson’s staining and measurement of hepatic hydroxyproline content were used to assess the collagen content in the liver tissues of each group.The serum AST and ALT levels were measured to evaluate liver function damage.4.In order to detect the effect of Fe-Mn-IGF-AMD on HSC activation,Prussian blue staining was used to investigate the distribution of NPs in liver and TGF-β1 activated LX-2.Immunohistochemistry and Western blot were used to detect the expression of α-SMA.Immunofluorescence were used to detect the expression of CXCR4 in each group.Immunofluorescence is used to detect the expression of hepatocyte growth factor(HGF)in each group,as well as the colocalization relationship between HGF and α-SMA.5.In order to detect the effect of Fe-Mn-IGF-AMD on the senescence of hepatocyte,SA-β-Gal staining was used to detect the proportion of senescent cells in each group.Double immunofluorescence staining was used to detect the colocalization relationship among p53 with albumin,F4/80,and α-SMA.Western blot was used to detect the expression of senescent markers p21 and p16 in H2O2-treated HepG2 in each group.qRT-PCR was used to detect the expression of IL-1β、IL-6 and MMP-9 in that cell.6.In order to detect the effect of Fe-Mn-IGF-AMD on ROS and hypoxia in liver,immunofluorescence was used to detect the level of ROS and pimonidazole in each group.Immunohistochemistry was used to detect the expression of 4-HNE and cellular immunofluorescence was used to detect the ability of scavenge ROS.7.In order to detect whether Fe-Mn-IGF-AMD can be used to diagnose hepatic cirrhosis,MRI was used to detect the relaxivity of Fe-Mn.After 12 hours of injection of each NPs into normal and cirrhotic mice,signal values in both groups were detected on T1 and T2 image.Results1.The synthesized NPs have good stability,crystallinity,density and superparamagnetism.2.There was no significant difference in the toxicity of HepG2 and RAW264.7 cells treated with Fe-Mn,Fe-Mn-IGF,Fe-Mn-AMD,and Fe-Mn-IGF-AMD at Fe concentrations of 1,2,5,and 10 μg/mL.In addition,treatment with Fe-Mn-IGF-AMD at a Fe concentration of 2 μg/mL did not affect apoptosis of LX-2,HepG2,and RAW264.7 cells.Fe-Mn,Fe-Mn-IGF,Fe-Mn-AMD,and Fe-Mn-IGF-AMD have no significant effects on the structure of heart,spleen,lung,and kidney tissues.3.The Sirius red staining positive area,Masson staining positive area,liver hydroxyproline content,AST and ALT content in the Fe-Mn-IGF-AMD group were significantly lower than those of other NPs and untreated group.4.The Prussian blue staining results showed that the activated HSC increased the uptake of Fe-Mn-IGF,Fe-Mn-AMD,and Fe-Mn-IGF-AMD.Fe-Mn-IGF-AMD can be more effectively suppressed the expression of α-SMA and CXCR4 than other NPs.Fe-Mn-IGF-AMD can significantly increase the HGF content in liver by promoting HGF secretion by HSCs.5.The number of p53 positive hepatocytes and HSCs in the cirrhosis group was significantly higher than that in the control group.All NPs had no effect on p53 positive HSCs and macrophages.Fe-Mn-IGF-AMD could significantly reduce the number of p53 positive hepatocytes.In addition,Fe-Mn-IGF-AMD could significantly reduce the protein expression of p21 and p16,and the mRNA expression of IL-1β、IL-6 and MMP-9 in H2O2-treated HepG2.6.The expression of ROS,4-HNE,and MDA in cirrhosis group was higher than that in the control group,while the production of ROS,4-HNE,and MDA was significantly reduced in each NPs treatment group.Among all the groups,the content of ROS,4-HNE,and MDA in the Fe-Mn-IGF-AMD group was the lowest.Compared with normal group,the hypoxic staining area in cirrhosis group was significantly increased,and the intrahepatic hypoxic area in each NPs treatment group have different degrees of improvement.All NPs exhibited good ROS scavenging ability in HepG2 H2O2-treated HepG2.Among all the groups,Fe-Mn-IGF and Fe-Mn IGF-AMD having the strongest scavenging ability for ROS.7.Compared to the normal group of mice injected with Fe-Mn-IGF-AMD,the relative signal intensity of T1 in the liver of cirrhotic mice injected with Fe-Mn-IGF-AMD was significantly increased,while the relative signal intensity of T2 was significantly decreased.No significant differences were observed in the relative signal intensities of the normal and cirrhosis groups injected with Fe-Mn,Fe-Mn-IGF,and Fe-Mn-AMD.ConclusionThe drug-loaded nanoparitcle Fe-Mn-IGF-AMD based on multi-functional nanomaterials achieves the purpose of inhibiting hepatic cirrhosis through the inhibition of HSC activation and senescence of hypatocyte,ROS scavenging in microenvironment and improving liver tissue hypoxia.In addition,the Fe-Mn-IGF-AMD can be used as an effective diagnostic tool for hepatic cirrhosis through MRI T1 and T2 imaging,providing a new strategy for the integration of diagnosis and treatment of hepatic cirrhosis. |
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