| Objectives:NASH model of SD rats was established by high-fat diet,and intestinal pathological changes of NASH rats were observed and changes of intestinal permeability markers in clinical NAFLD patients were analyzed,to concentrate on the connection between non-alcoholic fatty liver disease and intestina permeability markers.Part ONE Animal experimentResearch Objects:Twenty healthy male SD rats,aged 4-5 weeks,with an average body weight of80-120 g.Research methods:（1）Twenty SD rats were fed by professionals in the Experimental Animal Center of The Clinical Medical College of Xi ’an Jiaotong University,and 7 rats were randomly selected and assigned to the control group,which were fed with ordinary diet.（2）The remaining 13 SD rats numbered 8-20 were allocated to the experimental group and fed a high-fat bite and sup.At the end of feeding every 4 weeks,2 rats were randomly selected and killed.Liver tissue was taken out for pathological section analysis,and after 12 weeks,the modeling of NASH rats was successful.（3）General information of SD rats were recorded,such as food quantity and surplus quantity,hair,excreta,body weight and activity level,etc.After the modeling was successful at the end of experiment 12,all SD rats were sacrificed after 12 hours of diet restriction.（4）After the liver and intestinal tissues of SD rats were isolated,they were fixed,dehydrated,embedded,sectioned and observed under microscope.（5）Serological indicators（including ALT,AST,GGT,TG,LDL,TC,GLU and INS）were detected by centrifugation after venous blood was collected from SD rats,and the supernatant was determined by Elisa kit instructions.（6）After intestinal tissue immunohistochemistry,the expression of intestinal permeability markers（ZO-1 and Occludin）was determined by digital scanner or imaging system of tissue section.Results:（1）Comparison of liver pathology scores: the pathological score of liver in experimental group was higher than that in control group（P<0.05）.（2）Comparison of general conditions of rats: Compared with the control group,the mental status of SD rats in the experimental group gradually deteriorated,their activity gradually decreased,their fur became dry and not smooth,their diet gradually decreased,and their stool finally appeared slight diarrhea.（3）Histopathological changes of liver: in the control group,the hepatic lobule structures were all clear,and no infiltration of fat and inflammatory cells was observed in the cytoplasm.Observation of the experimental group every 4 weeks showed that the hepatic lobule structure was gradually destroyed over time,liver cells gradually showed different degrees of steatosis,and the degree of inflammation and fibrosis in the portal area was further aggravated.At the end of the 12 th week,NASH was able to build a model.（4）Histopathological changes of intestinal tissue: Compared with the control group,the mucosa folds in the experimental group were basically intact,and slight epithelial cells were exfoliated in the mucosa layer.Lamina propria intestinal gland atrophy,accompanied by a decrease in number;The lamina propria and muscularis showed different degrees of phlogosis cell infiltration and fat vacuoles of different sizes and quantities.（5）Comparison of serological indicators:Contrasted with the benchmark group,)Serological indexes: Compared with the control group,the experimental group had higher serological indexes（ALT,GGT,LDL,TG,TC,GLU,INS,HOMA-IR）（P < 0.05）,and higher AST expression level（P > 0.05）.（6）Comparison of intestinal permeability markers: Contrasted with the benchmark group,the experimental group had lower intestinal permeability markers（ZO-1 and Occludin）（P < 0.05）.Conclusion:The markers of intestinal permeability in NASH model induced by high fat diet were reduced and Increased intestinal permeability compared with those in control group.Part Two clinical trialsResearch Objects:A total of 94 patients with fatty liver disease who visited the outpatient department and physical examination Center of The Affiliated Hospital of Yan ’an University from October 2020 to February 2022 were included in the NAFLD group based on their medical history and life history.Thirty-nine cases with normal physical examination in the physical examination center were selected as the normal control group.Research methods:（1）A total of 94 NAFLD patients diagnosed as fatty liver by abdominal Ultrasound and meeting the inclusion criteria were divided into regular liver enzyme group and unusual group according to whether their liver enzymes were unusual.Finally,a total of58 NAFLD patients were included in the regular liver enzyme group and 36 NAFLD patients in the unusual liver enzyme group,and 39 patients with normal medical physical examination at the equal time were taken as the contrast bunch.（2）The physical examination reports of the included subjects were collected at the physical examination center and the outpatient department,and the general basic information of the subjects was extracted.Age,Sex,weight,BMI,CM,SBP and DBP were included.Clinical hematological indicators of the study subjects were collected,Including ALT,AST,ALP,GGT,TB,TC,TG,HDL-C,LDL-C and GLu.All hematological indexes were more than 8-12 h fasting.Peripheral venous blood was collected at 8-9am and analyzed by automatic biochemical analyzer in the clinical laboratory of our hospital.（3）Human whole blood samples collected from 133 subjects in Clinical laboratory were stored for 2h at room temperature or overnight in a 4℃constant temperature environment.After centrifugation for 25 minutes,the supernatant was taken and stored in the-80℃ constant temperature refrigerator of our hospital.Finally,the expression of intestinal permeability markers（ZO-1 and Occludin）was measured by Elisa.（4）The changes of general baseline data,liver function enzymology,blood lipid,blood glucose and intestinal permeability markers among all groups were analyzed,and the correlation analysis was conducted.Results:（1）Comparison of general baseline data: A sum of 133 subjects were included.There were measurably huge contrasts in sex,age,body weight,BMI,SBP and DBP among the three groups（P<0.05）,however no genuinely huge contrasts in stature among the three groups（P>0.05）.BMI and DBP in normal and abnormal liver enzyme groups were taller than those in contrast bunch,and those in abnormal liver enzyme group were taller than those in ordinary group（P<0.05）.（2）The relationship of liver enzyme showed that AST,ALP and GGT in normal liver enzyme tranches were exceed than those in contrast bunch（P>0.05）,and those unusual liver enzyme bunch were higher than those in normal liver enzyme bunch and contrast bunch bunch（P<0.05）.ALT in normal and abnormal liver enzyme groups was higher than that in control group,abnormal liver enzyme group and normal group（P<0.05）.TB in normal liver enzyme group and unusual liver enzyme group was higher than that in control bunch（P<0.05）,but normal liver enzyme group and abnormal liver enzyme group（P>0.05）.（3）Looking at blood lipid series,TG and TC in ordinary and abnormal liver enzyme groups were higher than those in control bunch（P<0.05）,ordinary and unusual liver enzyme groups（P>0.05）.The HDL-C unusual liver enzyme tranches was inferior than that of the ordinary liver enzyme tranches and the contrast tranches（P<0.05）,the ordinary liver enzyme groups and the control group（P>0.05）.The LDL-C in the ordinary liver enzyme groups was higher than that in control bunch（P<0.05）,between unusual liver enzyme group and control bunch,regular liver enzyme group and unusual groups（P >0.05）.（4）Fasting blood glucose,GLU in abnormal liver enzyme group was higher than that in control group（P<0.05）,between regular liver enzyme group and unusual group,regular liver enzyme group and control bunch（P >0.05）.（5）Intestinal permeability markers were compared,ZO-1 and Occludin in the normal and unusual liver enzyme groups were lower than those in the contrast group,and those in the unusual liver enzyme groups were lower than those in the ordinary group（P<0.05）.（6）Spearman correlation analysis of influencing factors and intestinal permeability markers（ZO-1 and Occludin）:（1）ZO-1 was negatively correlated with BMI,AST,ALT,GGT and TB（P<0.05）.Among them,ZO-1 had the highest correlation with ALT（r=-0.402,P<0.001）.Hdl-c was positively correlated with ZO-1.（2）Occludin was negatively correlated with BMI,AST,ALT,GGT and GLU（P<0.05）.Occludin had the highest correlation with GGT（r=-0.402,P<0.001）.Hdl-c was positively correlated with Occludin.（7）Multiple linear regression analysis showed that TB was contrarily associated with ZO-1 after controlling jumbling factors（β =-1.174,95%CI=（-2.03,-0.32）,P<0.01）.GGT and negatively correlated with Occludin（beta = 0.472,95% CI =（0.88,0.06）,P < 0.05）.（8）Multivariate logistic regression analysis of NAFLD disease occurrence showed that:BMI and TC are risk factors for NAFLD,and ZO-1（P=0.018,OR=0.954）and Occludin（P=0.001,OR=0.982）can reduce the risk of NAFLD.（9）Multivariate logistic regression analysis of NAFLD disease progression showed that:BMI was still a risk factor for progression of NAFLD disease,and ZO-1（P=0.001,OR=0.956）and Occludin（P=0.003,OR=0.990）still reduced the risk of progression of NAFLD disease.Conclusion:The increase of intestinal permeability may be related to the occurrence and development of nonalcoholic fatty liver disease. |
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