| Objective:Hepatic alveolar echinococcosis is a serious zoonotic parasitic disease caused by Echinococcus multilocularis infection of the liver.As a chronic infectious wasting disease,so far,there has been no objective quantitative assessment of the nutritional status of skeletal muscle in patients with HAE.In addition,the molecular mechanism of the development and occurrence of skeletal muscle atrophy caused by Echinococcus multilocularis infection in the liver is still unclear.Therefore,this study evaluated the changes of skeletal muscle mass in patients with HAE through imaging data analysis and explored the diagnostic value of skeletal muscle nutrition for postoperative complications in patients with HAE.The effects of Echinococcus multilocularis infection on skeletal muscle atrophy in the host and changes in metabolic pathways in skeletal muscle were investigated by establishing a mouse model of Echinococcus multilocularis infection in the liver.At the same time,the protoscoleces of Echinococcus multilocularis were cultured in vitro,and the conditioned medium of protoscoleces was constructed and obtained.In vitro cell experiments were conducted to investigate the effects of Echinococcus multilocularis infection on protein metabolism of C2C12 myotube cells treated with Echinococcus multilocularis protocephalal supernatant(conditioned medium)to simulate the microenvironment in skeletal muscle.On this basis,liquid chromatography mass spectrometry(LC-MS)and transcriptome RNA sequencing were used to further study the changes of signal pathways related to skeletal muscle atrophy after hepatic Echinococcus multilocularis infection,so as to provide molecular intervention targets for the reversal or remission of muscle atrophy caused by hepatic Echinococcus multilocularis infection.Materials and Methods:(1)The clinical data of patients undergoing curative liver resection for HAE at West China Hospital of Sichuan University between January 2008 and December 2018 were retrospectively analyzed.We segmented skeletal muscle and adipose tissue and measured the area of skeletal muscle tissue and adipose tissue and the degree of muscle steatosis at the L3 plane by manually tracing the preoperative plain CT images using three-dimensional visualization software.Psoas muscle index(PMI),Skeletal muscle index(SMI),the visceral to subcutaneous adipose tissue area ratio(VSR),and intramuscular adipose tissue content(IMAC)were calculated for each patient.The clinical data of healthy people undergoing routine physical examination in West China Hospital of Sichuan University during the same period were collected as the normal control group to compare the differences of above muscle nutrition indexes between the HAE group and the normal control group(healthy people).In addition,we evaluated postoperative complications in patients undergoing curative liver resection for HAE according to the Clavien-Dindo complication classification.We used clinical data from patients with HAE treated with curative liver resection in our hospital as a training set,we based on the LASSO logistic regression model to build muscle nutrition score,and then combined with the important risk factors affecting postoperative complications of HAE patients screened out by multivariate analysis,we established nomogram to predict the occurrence of postoperative complications in HAE.Harrell Concordance index(C-index)and Calibration Cure were used to evaluate and verify the discrimination and Calibration degree of the nomogram.The results were validated using clinical data from 6 other HAE centers in the hydatid epidemic area of western China.(2)Female BALB/c mice were selected as inoculated animals to establish a mouse model of hepatic Echinococcus multilocularis infection through abdominal muscular liver puncture.The growth of lesions in mice was observed regularly after surgery,and the relevant data were recorded.The changes of skeletal muscle mass in mice were evaluated by electronic gripper and rotary bar kinesiometer.The changes of body composition and back muscle mass of mice were analyzed by imaging method.At the same time,the changes of biochemical indexes and inflammatory factors in mice were analyzed.The muscle tissue of both lower limbs of mice was stripped and collected,and its corresponding weight was recorded.Then the maximum diameter and cross-sectional area of muscle fiber were analyzed and measured by HE staining of gastrocnemius tissue.The expression levels of ubiquitin ligase Atrogin-1 and Mu RF1 in the gastrocnemius tissues of mice were detected by q RT-PCR and Western blot.Metabonomics analysis was used to elucidate changes in biochemical metabolites and metabolic pathways in the gastrocnemius tissues of mice after hepatic Echinococcus multilocularis infection.The protoscoles with Echinococcus multilocular from gerbils were cultured in vitro and the supernatant of the medium(conditioned medium)was obtained.C2C12 myoblast cells were induced to differentiate into myotube cells and identified.In vitro experiment,Echinococcus multilocular protoscoles supernatant(conditioned medium)was used to treat C2C12 myotube cells and simulate the environment of muscle fibers in vivo under Echinococcus multilocular infection to detect the effect of Em on protein metabolism of myotube cells(3)Transcriptome sequencing was performed on the gastrocnemius tissues of mice infected with Echinococcus multilocular in liver.The Differentially expressed genes were analyzed,and their functions were annotated.Combined with the results of skeletal muscle metabolomics analysis and transcriptome sequencing,the effects of Echinococcus multilocular infection on the Akt-Fox O3a-Atrogin-1/Mu RF1 pathway in skeletal muscle were investigated in animal and cell experiments from the protein level.Results:(1)Through the statistical analysis of imaging data,it was found that the PMI value(6.21 cm~2/m~2 vs.6.74 cm~2/m~2,P=0.013)and SMI value(42.76 cm~2/m~2 vs.44.91cm~2/m~2,P=0.014)of HAE patients was lower than that of healthy people.The IMAC value(-0.544 vs.-0.575,P=0.029)of HAE patients was higher than that of healthy people.However,there was no significant difference in VSR value(0.353 VS.0.349,P=0.50)between HAE patients and healthy people.Five muscle nutrition indexes related to postoperative complications were selected by Lasso Logistic regression and muscle nutrition score was established,which was 2.091+1.109×VSR+0.797×IMAC-0.016× age-0.374×PMI-0.019×SMI.Multivariate Logistic regression analysis showed that the level of γ-GGT and the surface area of hepatectomy were independent risk factors for complications after curative liver resection in patients with HAE.Based on muscular nutrition score and the result of multivariate logistic regression analysis,the nomogram was established for predicting complications after curative liver resection in HAE patients.The model has good predictive ability,and the C-index of the nomogram in the training set and external validation set was 0.836(95% CI,0.776-0.896)and0.904(95% CI,0.811-0.996),respectively.(2)In this study,we successfully established a BALB/c mouse model of hepatic Echinococcus multilocular infection by liver injection through the abdominal muscular layer.The tibialis anterior,gastrocnemius mass and gastrocnemius muscle index of BALB/c mice in HAE group were significantly lower than those in the other two groups,while the spleen weight was increased(P<0.05).Compared with the other two groups,the grasping power and relative grasping power of HAE group were significantly decreased(P<0.05).The retention time of rotating rod of mice in HAE group was significantly shortened(P<0.05),which reflected the decrease of muscle mass in HAE group.DEXA analysis showed that the percentage of lean body mass in hepatic Echinococcus multilocularis infected mice decreased compared with Sham and Control groups.Based on the analysis of the back skeletal muscle mass by the mouse magnetic resonance imaging system,we found that in the L4 plane,the area of the lumbar dorsal muscle in the HAE group was significantly smaller than that in the Control group and the Sham group.In addition,we found that the lumbar dorsal muscle area was highly positively correlated with the lesion free body weight and gastrocnemius muscle mass.Serum biochemical results showed that Compared with the other two groups,serum ALT and AST levels of mice in HAE group were increased,and serum ALB was significantly decreased.Serum ELISA results showed that the levels of IFN-γ,TNF-α and IL-6 in HAE group were significantly higher than those in Control group and Sham group.The HE staining of gastrocnemius tissue showed that the maximum diameter and cross-sectional area of skeletal muscle fibers were decreased in HAE group(P<0.05).q RT-PCR and Western blot showed that the expression levels of ubiquitin ligase Atrogin-1 and Mur F1 in muscle tissues were higher than those in Control and Sham groups.Metabolomics analysis of skeletal muscle showed obvious metabolic disorder in skeletal muscle after hepatic Echinococcus multilocularis infection.150 metabolites were significantly altered,of which 111 were significantly increased in the gastrocnemius of mice in the HAE group,and 39 metabolites were significantly decreased in the gastrocnemius of mice in the HAE group,among which the changes of amino acid metabolites were the most obvious.In vitro culture system of Echinococcus multilocularis protoscoleces was successfully established,and the myoblast cells were differentiated into myotube cells.Western blot and immunofluorescence results showed that treatment of C2C12 cells with Echinococcus multilocularis protoscolex conditioned medium resulted in atrophy of myotubes,decreased expression of MHC and Myogenin,and increased apoptosis of myotube cells.(3)The transcriptome sequencing analysis of gastrocnemius showed that there were 1063 differentially expressed genes in muscle tissue of HAE group and sham group,among which 605 genes were up-regulated and 458 genes were downregulated in the HAE group.The expressions of Atrogin-1 and Mu RF1 in HAE group were significantly higher than those in Sham group,and the expression of Fox O3 a in the upstream targets of Atrogin-1 and Mu RF1 was also significantly up-regulated.GO functional annotation and enrichment analysis showed that the cell components were mainly concentrated in membrane,membrane components,cytoplasm,etc.The biological processes mainly involved in cell adhesion,signal transduction and so on;The molecular function mainly focuses on protein binding,metal ion binding,molecular function,etc.KEGG pathway enrichment and GSEA enrichment analysis showed that the atrophy of skeletal muscle caused by Echinococcus multilocularis infection might be related to the inhibition of PI3K-Akt pathway.Western blot demonstrated that the phosphorylation of Akt in skeletal muscle decreased,the pFox O3a/Fox O3 a ratio decreased significantly,and the expression of Mu RF1 and Atrogin-1 increased after hepatic Echinococcus multilocularis infection.In cell experiment,the PI3k/Akt pathway was also changed after C2C12 myotube cells were treated with Echinococcus multilocularis protoscolex conditioned medium,and the change trend was the same as that of tissue Western blot.Akt agonist could reverse the decreased phosphorylation of Akt and Fox O3 a and the increased expression of Atrogin-1 and Mu RF1 induced by Echinococcus multilocularis protoscolex conditioned medium and alleviate the atrophy of myotube cells induced by Echinococcus multilocularis protoscolex conditioned medium.Conclusion:In this study,we confirmed the presence of skeletal muscle depletion in HAE patients using imaging data.At the same time,we established muscle nutrition score in HAE patients,which proved for the first time that muscle nutrition score is an important factor affecting the occurrence of postoperative complications in HAE patients.Our prediction model for predicting postoperative complications in HAE patients based on muscular nutrition score has demonstrated good predictive ability and calibration in both the training set and the validation set.In addition,muscular nutrition score can improve the prediction efficiency of the prediction model.We successfully established a mouse model of hepatic Echinococcus multilocularis infection by liver injection through the abdominal muscular layer.In the animal model of hepatic Echinococcus multilocularis infection,the skeletal muscle mass of mice decreased significantly.Hepatic Echinococcus multilocularis infection causes complex metabolic disorders in skeletal muscle tissue,which is mainly related to increased skeletal muscle protein catabolism,resulting in the large release of amino acids in muscle tissue.After Echinococcus multilocularis infection,the phosphorylation levels of Akt and Fox O3 a in skeletal muscle were down regulated,and the ubiquitination ligases Atrogin1 and Mu RF1 were increased.Echinococcus multilocularis infection can inhibit the Akt signaling pathway in skeletal muscle and activate the skeletal muscle protein degradation pathway,leading to skeletal muscle atrophy. |
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