Study The Theory Of "Mother-organ Disorder Involving Its Child-organ" Based On Nonalcoholic Steatohepatitis Inducing Cardiovascular Injury

ObjectiveNon-alcoholic steatohepatitis(NASH)is one of the most common chronic liver diseases in modern society,mounting evidence suggest that NASH is an important risk factor for clinical cardiovascular disease(CVD).The "mother-organ disorder involving its child-organ"is the theory of traditional Chinese medicine(TCM),in the theory of five elements,liver is pertain to wood,heart to fire,wood generates fire,the disorder of liver may lead to cardiac diseases.The pathogenesis of non-alcoholic fatty liver disease in TCM was considered as improper diet,the liver lose the function of convey and disperse,and liver qi stagnation lead to heart diseases,including blood stasis and palpitation.This study explored the mechanisms of CVD induced by murine NASH and provided a modern medical explanation and evidence in "mother-organ disorder involving its child-organ" between heart and liver.Methods1.Murine model of NASH was established.C57BL/6J mice were randomly assigned to 6 groups and fed with methionine choline supplemented(MCS)diet or methionine choline deficient(MCD)diet respectively for 2,4 or 6 weeks and weigh weekly.Blood was taken from the abdominal aorta after being anesthetized,the serum was used to detect AST,ALT and other indicators,a portion of the liver was fixed with paraformaldehyde for HE staining,and the remainder was stored at80℃ refrigerator for oil red O staining to confirm the severity of NASH.The heart was stored at-80℃ refrigerator for subsequent analysis.2.The study of vascular endothelial and vascular permeability function based on NASH.(1)C57BL/6J mice were randomly assigned to 2 groups and fed with MCS diet or MCD diet respectively for 4 weeks.The thoracic aorta was harvest,after being fixed,balanced,activated,and diastolic function detection in multi myograph system,the thoracic aorta was dilated by acetylcholine and sodium nitroprusside respectively to detect diastolic effect of intact endothelium and evaluate the endothelial function in mice under NASH condition.(2)C57BL/6J mice were randomly assigned to 2 groups and fed with MCS diet or MCD diet respectively,after 6 weeks,Evans blue dye was injected into the mice via tail vein,excessive isoflurane anesthesia was performed after 60min,next,the heart was perfused with 50ml PBS solution to rinse off the blood through auricle.The heart was incubated in 1ml formamide at 60℃ for 48 hours and the dye content was determined at 620nm by ultraviolet spectrophotometer to evaluate the function of cardiovascular permeability in mice.(3)The heart was obtained from mice being fed with 2,4 or 6 weeks diet,frozen sections was performed,the expression of eNOS and tight junction protein ZO-1,ZO-2 and Vecadherin in coronary artery endothelial cells was detected by immunofluorescence.3.The effects of NASH condition on NLRP3 inflammation in coronary artery endothelial cells.(1)The heart was obtained from mice being fed with 2,4 or 6 weeks diet,frozen sections was performed,the expression of NLRP3 and Caspase-1 in coronary artery endothelial cells was detected by immunofluorescence.(2)NLRP3-/-mice were randomly assigned to 2 groups and fed with MCS diet or MCD diet respectively for 2 weeks.Blood was taken from the abdominal aorta after being anesthetized,the serum was used to detect AST and ALT,and the heart was used to detect the expression of ZO-1 and ZO-2 in coronary artery endothelial cells.4.The effects of exosomes that isolated from NASH liver on mice vascular endothelium NLRP3 inflammation and permeability function.(1)Liver exosomes were isolated.Exosomes were isolated from mice liver after being fed with MCS or MCD diet for 6 weeks by ultracentrifugation,the particle size and density of exosomes was detected by Nanosight,and the physical characteristics were observed by transmission electron microscopy(TEM),furthermore,western blotting was used to detect exosomes markers,including CD63,TSG101 and HSP70.(2)Then,two kinds of exosomes were injected into naive C57BL/6J mice respectively via tail vain for 48 hours,the thoracic aorta was used for endothelial function analysis by multi myograph system,the heart was used to detect the expression of NLRP3,Caspase-1,ZO-1 and ZO-2 in coronary artery endothelial cells.(3)To verify the key role of NLRP3 gene,two kinds of exosomes were injected into NLRP3-/-mice respectively via tail vain for 48 hours,the thoracic aorta was used for endothelial function analysis by multi myograph system,the heart was used to detect the expression of ZO-1 and ZO-2 in coronary artery endothelial cells.(4)The in vitro study of exosomes isolated from NASH liver on mice vascular endothelial function.To verify the effect of exosomes in vivo,the endothelial cells were treated with exosomes in a concentration of 120μg/ml for 24 hours,after that,NLRP3 mRNA level was detected by RT-PCR,the expression of NLRP3 and Caspase-1 was detected by immunofluorescence,moreover,the expression of IL-1β,downstream protein of NLRP3,was determined by western blotting.The leakage of FITC-dextran and transepithelial electrical resistance(TEER)was detected in monolayer endothelial cells,and ZO-1 expression was determined by immunofluorescence.5.The sequencing analysis of exosomes and potential target research.To further verify the small molecule cargos in exosomes that induced NLRP3 inflammasome activation,miRNAs in exosomes were determined by RNA sequencing.After analysis,we hypothesized AhR as the target gene and selected the target miRNAs.The miRNAs level in exosomes was detected by RT-PCR and with the highest expression was selected for further study,subsequently,the expression of miRNA after incubation with exosomes in endothelial cells was detected.To confirm whether miRNA would lead to NLRP3 inflammasome activation and destroy tight junction proteins of endothelial cells,endothelial cells were transfected with mimic,the expression of AhR,NLRP3,Caspase-1,ZO-1 and ZO-2 was determined by western blotting.Results1.The mice developed steatohepatitis after fed with MCD diet for 2,4 or 6 weeks,and getting worse over time.In mice fed with MCD diet,AST and ALT value was significantly increased,HE staining and oil red O staining showed significant fat accumulation and fat vacuoles.2.Moreover,vascular endothelial dysfunction was found in NASH mice by impaired endothelium-dependent diastolic and increased the leakage of Evans blue,besides,the expression of eNOS,ZO-1,ZO-2,VE-cadherin in coronary artery endothelial cells was decreased.3.In NASH mice,the expression of NLRP3 and Caspase-1 in coronary artery endothelial cells was increased,no matter in 2,4 or 6 weeks,which was represent the NLRP3 inflammasome assembled.Serum AST,ALT value was significantly increased in NLRP3-/mice,the expression of ZO-1 and ZO-2 in coronary artery endothelial cells was unchanged.4.(1)Exosomes were isolated from liver successfully.After being injected with exosomes isolated from NASH liver,in line with NASH mice,the mice displayed endothelial-dependent diastolic dysfunction,the expression of NLRP3 and Caspase-1 in coronary artery endothelial cells was increased while decreased ZO-1 and ZO-2 expressions.The damage effect of exosomes was attenuate after knockout NLRP3 gene,in NLRP3-/-mice,exosomes from NASH liver neither impaired endothelial diastolic function or reduced the expression of ZO-1 and ZO-2.(2)In vitro study showed that there were an increase effects in NLRP3 mRNA level,NLRP3,Caspase-1 and IL-1β expressions after being treated with exosomes from NASH liver,in addition,permeable FITC-dextran and TEER value was increased,while ZO-1 expression was significantly down-regulated,which indicated permeability dysfunction in endothelial cells.5.AhR targeted miRNAs were obtained by RNA sequencing via profiling,and the miRNAs expressions in exosomes detected by RT-PCR were consistent with the RNA sequencing results,and novel-miRNA-126 level was the highest.In addition,the expression of novel-miRNA-126 was increased after NASH liver exosomes treatment in contrast with control group,overexpression of novel-miRNA-126 lead to suppress AhR level and increase NLRP3,Caspase-1 expressions while reduced the expressions of ZO-1,ZO-2.ConclusionThe murine NASH induced by MCD diet was associated with cardiovascular endothelial dysfunction,we identified the exosomes cargo novel-miRNA-126,which was increased in NASH liver exosomes,as a regulator of NLRP3 inflammasome activation by inhibiting the AhR expression,leading to increase the vascular endothelial permeability eventually.This study enriched the theory of "mother-organ disorder involving its child-organ" between the liver and heart within modern medicine.