Correlation Analysis Of Degrees Of Hepatic Fibrosis And Liver Function Test In Infantile Cholestatic Liver Disease

Infantile cholestatic liver disease is a common disease during the infancystage and it has become the major cause of hospitalization for hepatic diseasein children of Chinese. Numerous and complicated reasons lead to infantilecholestatic liver disease, such as infection, abnormal development of biliarysystem, autoimmunity and genetic metabolic diseases etc. If absenceseasonable and effective treatments, cholestasis would lead to bile salts andother toxicity components accumulate both in intrahepatic cells and the wholebody, would interference developmental of children even develop intocholestasis hepatic fibrosis. Hepatic fibrosis is the common pathological andnecessary stage when the chronic hepatic disease progress to the hepaticcirrhosis. To prevent or reverse hepatic fibrosis is the future research directionin anti-hepatic fibrosis. Currently, liver biopsy has been recognized as the goldstandard for hepatic fibrosis diagnosing, but there has little acknowledgmentabout serological indexes that can reflect the degree of hepatic fibrosissensitively. Hence, this study was composed by clinical trials and animalexperiments, and set up the rat cholestasis hepatic fibrosis model by usingα-Naphthyl isothiocyanate (ANIT). We would like from the correlationanalysis of degrees of hepatic fibrosis, liver function test and pathologicalhepatic fibrosis grades both in clinic and animal studies to investigateserological indexes that can detect infantile cholestatic liver fibrosis earlier,more stable and more safety.PartⅠ: Correlation analysis of degrees of hepatic fibrosis and liverfunction test in infantile cholestatic liver disease.Objective: To study the relationship between degrees of hepatic fibrosisand liver function test in infantile cholestatic liver disease.Methods:30infants were included in the infantile cholestatic liver disease group (experimental group): infants aged within18months, visitingthe Third Hospital of Hebei Province from May2012to Dec．2013that werediagnosed as infantile cholestatic liver disease．20healthy infants wereincluded in the control group: infants aged within18months visiting the ThirdHospital of Hebei Province for normal physical examination．There is norestricting of gender. The levels of serum liver function (ALT, AST, TBIL,DBIL, IBIL, γ-GT, CHE, TBA) were detected by performance rate methodand biochemical chromometry, degrees of hepatic fibrosis (HA, PCⅢ, LN, cⅣ) in two groups were detected by radio immunoassay．Results:①The ALT, AST, TBIL, DBIL, IBIL, γ-GT, CHE, TBA inexperimental group were117.900±261.894(U/L),115.567±208.786(U/L),103.970±74.226(umol/L),55.057±41.516(umol/L),48.247±47.440(umol/L),169.300±184.600(U/L),6.639±1.936(KU/L),49.447±58.214(umol/L), respectively; in control group were11.900±1.917(U/L),29.450±3.602(U/L),7.140±1.632(umol/L),2.165±0.555(umol/L),4.975±1.212(umol/L),11.000±1.487,(U/L)9.601±1.171(KU/L),3.195±2.587(umol/L), respectively. The statistic differences were P=0.000＜0.01,P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, respectively. The ALT, AST, TBIL,DBIL, IBIL, γ-GT, TBA in experimental group were higher than the controlgroup, the CHE was lower than the control group, the statistical differencesexisted obviously.②The HA, PCⅢ, LN, cⅣ in experimental group were201.273±177.030(ng/ml),541.877±170.920(ng/ml),123.637±19.322(ng/ml),134.371±44.984(ng/ml), respectively; in control group were81.165±7.319(ng/ml),141.314±26.609(ng/ml),116.209±8.374(ng/ml),78.430±10.205(ng/ml), respectively. The statistic differences were P=0.000＜0.01, P=0.000＜0.01, P=0.452＞0.05, P=0.000＜0.01, respectively. The HA, PCⅢ, cⅣ inexperimental group were higher than control group, the statistical differencesexisted obviously.③Correlation analysis of degrees of hepatic fibrosis and liver function test. HA and AST, γ-GT were positively related, r=0.780, P=0.000＜0.01，r=0.599, P=0.000＜0.01, respectively. HA and ALT, CHE, TBA were negativelyrelated, r=-0.627, P=0.000＜0.01，r=-0.427, P=0.042＜0.05，r=-0.463,P=0.035＜0.05, respectively. cⅣ and γ-GT was positively related, r=0.466,P=0.025＜0.05. cⅣ and CHE was negatively related, r=-0.520, P=0.011＜0.05.Conclusion：①ALT, AST, TBIL, DBIL, IBIL, γ-GT, CHE, TBA of liverfunction test, HA, PCⅢ, cⅣ of degrees of hepatic fibrosis had statisticaldifference between infantile cholestatic liver disease group and healthy infantsgroup.②Serum level of HA, cⅣ, γ-GT and CHE were sensitive indicators ofinfantile cholestatic liver fibrosis.PartⅡ: Correlation analysis of degrees of hepatic fibrosis, liver functiontest and pathological hepatic fibrosis grade of neonatal cholestastic rat.Object: To study the relationship between degrees of hepatic fibrosis,liver function test and pathological hepatic fibrosis grade of neonatalcholestastic rat.Methods:38healthy SD rats aged3weeks, weighing50-70g, obtainedfrom Experimental Animal Center of Hebei Province. After24h feedingadaption rats were randomly assigned to3groups as follows:20were inexperimental group(model group): intragastric administration with1%ANIT75mg/kg;10were in control group: intragastric administration with equalvolume corn oil at the same time with experimental group;8were in blankcontrol group: synchronous feeding. The groups were divided no related withgender. Before rats were intragastric administrated, they were fasted for12h.48h after intragastic administration, blood samples from all groups werecollected by decapitation for liver function and degrees of hepatic fibrosisanalysis. The levels of serum liver function (ALT, AST, TBIL, DBIL, IBIL,γ-GT, CHE, TBA) were detected by performance rate method and biochemicalchromometry, degrees of hepatic fibrosis (HA, PCⅢ, LN, cⅣ) were detectedby radio immunoassay. And then liver tissues were removed into10%neutralformalin solution. After embedding, sectioning and dehydration, did the hematoxylin-eosin, Masson, Sirius red-saturated picric acid staining.Results:①The ALT, AST, TBIL, DBIL, IBIL, γ-GT, CHE, TBA inexperimental group were633.167±248.799(U/L),2080.167±704.386(U/L),144.811±24.901(umol/L),132.789±23.431(umol/L),12.022±5.597,(umol/L)19.944±10.367(U/L),0.256±0.041(KU/L),675.656±84.736(umol/L); in control group were48.875±14.035(U/L),349.500±114.409(U/L),3.275±0.870(umol/L),2.850±0.907(umol/L),0.425±0.292(umol/L),0.750±1.035(U/L),0.256±0.038(KU/L),198.013±120.474(umol/L); in blank control group were36.143±11.335(U/L),320.714±148.833(U/L),2.429±0.665(umol/L),1.927±0.687(umol/L),0.500±0.332(umol/L),0.143±1.574(U/L),0.267±0.026(KU/L),113.029±87.370(umol/L), respectively. The statistic differences between experimentalgroup and control group were P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.141＞0.05,P=0.000＜0.01, respectively. The statistic differences between experimentalgroup and blank control group were P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.000＜0.01, P=0.687＞0.05,P=0.000＜0.01, respectively. The statistic differences between control groupand blank control group were P=0.078＞0.05, P=0.679＞0.05, P=0.094＞0.05,P=0.072＞0.05, P=0.649＞0.05, P=0.463＞0.05, P=0.053＞0.05, P=0.147＞0.05, respectively. The ALT, AST, TBIL, DBIL, IBIL, γ-GT, TBA inexperimental group were higher than control group, the statistical differencesexisted obviously.②The HA, PCⅢ, LN, cⅣ in experimental group, were2004.179±479.859(ng/ml),45.520±2.155(ng/ml)，48.512±6.505(ng/ml)，30.275±10.823(ng/ml); in control group were1030.073±541.191(ng/ml)，46.627±2.633(ng/ml)，34.413±10.812(ng/ml)，20.079±2.700(ng/ml); in blankcontrol group were1018.951±555.132(ng/ml),36.200±9.647(ng/ml),27.571±6.246(ng/ml),18.328±5.593(ng/ml), respectively. The statisticdifferences between experimental group and control group were P=0.000＜0.01, P=0.304＞0.05, P=0.001＜0.01, P=0.000＜0.01, respectively. The statistic differences between experimental group and blank control group wereP=0.000＜0.01, P=0.096＞0.05, P=0.003＜0.01, P=0.000＜0.01, respectively.The statistic differences between control group and blank control group wereP=0.972＞0.05, P=0.086＞0.05, P=0.476＞0.05, P=0.151＞0.05, respectively.The HA, LN, cⅣ in experimental group were higher than control group, thestatistical differences existed obviously.③Degree of hepatic fibrosis in pathology. The pathological hepaticfibrosis grade in experimental group, control group, blank control group were2.050±0.621,0.440±0.527,0.130±0.354, respectively. The statisticdifferences existed between experimental group and control group,experimental group and blank control group obviously(P=0.000＜0.01,P=0.000＜0.01).④Hepatic tissues pathological staining. Hematoxylin-eosin and ironstaining: The hepatic lobules in control group and blank control group wereholonomic and clear, hepatic cells arranged in funicular form. There wasalmost no proliferation of fiber tissue and infiltration of inflammatory cell inportal area. The connective tissue did not proliferate in Central vein and portalarea obviously. While in experimental group, the hepatic lobules weredamaged or disappear,25%-75%hepatic tissues were fatty degeneration,edema necrosis and apoptosis. Cholestasis can be found in hepatocytes andbile ducts. Only a few normal hepatocytes survived. The fibrosis generatedobviously in the majority of portal area and necrosis area, accompanied withsevere infiltration of neutrophilic granulocytes and eosinophilic granulocytes.Fibrous septum divided necrosis hepatic lobules into pseudolobules. Massonstaining: Compared with the control group and blank control group, aroundblood vessels and bile ducts there were large number of green collagen fibersdeposition in the hepatic tissue of experimental group. Sirius red-saturatedpicric acid staining: Compared with the control group and blank control group,numerous red collagen fibers were found in hepatic lobules of experimentalgroup. Through observed by polarizing microscope, large thick yellow and redfibers which had a strong refractivity and few slender green fibers which had weak birefringence were found. Thick yellow and red fibers distributed aroundor star shaped around the central vein, clearance of hepatic sinus and portalarea, some stretched into the lobule and formed interlobular separated. Fewslender green fibers mainly distributed in portal area, small blood vessels,central vein, and also can be seen discontinuous distribution around clearanceof hepatic sinus.⑤Correlation analysis of degrees of hepatic fibrosis and liver functiontest. HA and ALT, γ-GT were positively related, r=0.474, P=0.047＜0.05，r=0.531, P=0.023＜0.05, respectively. PCⅢand TBIL, DBIL, γ-GT, TBAwere positively related, r=0.645, P=0.004＜0.01, r=0.653, P=0.003＜0.01, r=0.535, P=0.022＜0.05, r=0.616, P=0.006＜0.01, respectively. cⅣ andγ-GT was positively related, r=0.495, P=0.037＜0.05.⑥Correlation analysis of liver function test and pathological hepaticfibrosis grade. Pathological hepatic fibrosis grade and γ-GT was positivelyrelated, r=0.617, P=0.006＜0.01.⑦Correlation analysis of degrees of hepatic fibrosis and pathologicalhepatic fibrosis grade. Pathological hepatic fibrosis grade and HA waspositively related, r=0.833, P=0.000＜0.01.Conclusion：①After48h neonatal cholestatic rats which were intragasticadministrated by1%ANIT75mg/kg were induced into cholestasis hepaticfibrosis models, and the pathological characteristics conform to human hepaticfibrosis.②Pathological hepatic fibrosis grade and HA, γ-GT were positivelyrelated in animal study, further confirmed serum level of HA and γ-GT weresensitive indicators of infantile cholestatic liver fibrosis in pathology.