| ObjectivesIron load refers to the reserved level of iron in the body and meets the requirement of the body's physiological function and metabolic levels.Iron deficiency and iron overload affect on people's health.With the development of chronic diseases,the detection rate of iron overload increased year by year.With increasing consumption of alcohol and red meat?or many iron supplements taken?,it had a negative impact on human's health.This study aimed to analyze the relationship between iron status and abnormal liver function in adults,and evaluate the risk of iron overload and alcohol consumption for abnormal liver function.On this basis,rat models of liver injury induced by alcohol or combined with iron overload and alcohol were established to observe the effect of Aplysin supplementation on improving liver injury and intestinal microecology in rats and its possible mechanism.It provides scientific basis for guiding people to reasonably consume iron-rich food?or iron supplements?and moderate alcohol,and effectively prevent and control liver injury.MethodsThis study included cross-sectional survey and animal experiments.Study participants and grouping in cross-sectional survey.The study used cross-sectional survey and selected 6,858 adults aged 35 to 76 who participated in the health physical examination in two hospitals of Qingdao city from January 2014 to October 2015,and collected the data of their basic information,lifestyle,physical examination,biochemical test and other information.Serum ferritin?SF?levels were analyzed at the quartile of low iron load group?Q1?,medium low iron load group?Q2?,medium high iron load group?Q3?and high iron load group?Q4?.The relationship between different ferritin levels and abnormal liver function was analyzed by regression analysis.On this basis,3,133 males were selected to analyze the the effects of iron overload and alcohol consumption on abnormal liver function.World Health Organization used serum ferritin level as the criteria for iron status,including iron deficiency<15?g/L;normal iron:15-200?g/L for men and 15-150?g/L for women;iron overload:>200?g/L for men and>150?g/L for women.Serum ferritin?SF?levels in this study were classified into the non-iron overload group??200?g/L?and the iron overload group?>200?g/L?.Combined with drinking status,the participants can be divided into non-iron overload and non-drinking group?n=1756?,iron overload and non-drinking group?n=178?,non-iron overload and dringking group?n=1033?and iron overload and drinking group?n=166?.Demography,height,weight,blood glucose,blood lipids,blood pressure,uric acid and biochemical detection information were collected.The effect of iron overload and alcohol consumption on abnormal liver function was analyzed with the additive model.Experimental animal studies with Aplysin supplementation.Rats model of alcoholic liver injury was established.Sixty male Wistar rats with a body weight of180-220 g were selected and randomly divided into 4 groups?15/group?:control group,ethanol model group,Aplysin group and Aplysin+ethanol group.In the ethanol model group,56%?v/v?ethanol was given 8 mL·(kg-1·d-1)for 2 weeks and 12 mL·(kg-1·d-1)for weeks.In the Aplysin+ethanol group,the rats were given Aplysin 150 mg·(kg-1·d-1).The normal control group and the ethanol model group were given soybean oil with 5mL·(kg-1·bw-1·d-1),and the experiment lasted for 8 weeks.After 12 h from last invervention,fecal feces of rats in each group were collected by metabolic cages,rats were anesthetized intraperitoneally with 3%pentobarbital sodium,blood samples were collected from abdominal aorta,and liver and small intestine tissues were collected.Hematolyxin and eosin?HE?staining and transmission electron microscopy?TEM?were used to observe the pathological changes of liver tissue and ultrastructural changes of liver cells in rats,and the levels of ALT,AST and ALP in serum were detected.The levels of plasma IL-1?,IL-6,IL-10,TGF-?and TNF-?were detected.The levels of plasma endotoxin,diamine oxidase?DAO?,D-lactic acid?D-LA?and fatty acid binding protein?FABP2?were measured in each group.16S rDNA high-throughput sequencing was used to detect the effects of Aplysin supplementation on intestinal microecology in rats,and analyze the changes in intestinal pathology and intestinal barrier function.On this basis,the rats model of combined ethanol and iron overload exposed and its effect on intestinal flora were established.Forty male Wistar rats with body weight of 180-220 g were selected and randomly divided into into 4 groups?10/group?:control group,ethanol+iron overload group,Aplysin group,and Aplysin+ethanol+iron overload group.The nomal diet contained 50 mg/kg of iron was fed in control group,while the diet contained1000 mg/kg of iron was fed in the ethanol and iron overload group.Meanwhile,56%?v/v?ethanol was given 8 mL·(kg-1·d-1)for 2 weeks,12 mL·(kg-1·d-1)for last 10 weeks.In the Aplysin and Aplysin+ethanol+iron overload groups,the rats were given Aplysin 150mg·(kg-1·d-1).The control group and the ethanol+iron overload group were given soybean oil with 5 mL·(kg-1·bw-1·d-1),and the experiment lasted for 12 weeks.Serum iron?SI?,ferritin?SF?,hepcidin?HEPC?levels and liver tissue iron?LIC?levels were measured in each group.The expression levels of divalent metal ion transporter 1?DMT1?and ferroportin 1?FPN1?in the duodenum of rats were analyzed.The purpose of study was to observe the pathological changes of small intestine and the ultrastructural changes of small intestinal mucosa in rats,and detect the levels of plasma endotoxin,DAO,D-LA and FABP2 in each group of rats.The amounts of Bifidobacterium,Escherichia coli,Lactobacillus,Enterococcus,Clostridium tender and Bacteroides fragilis of rats in each group were measured by real-time quantitative PCR.ResultsThe epidemiological cross-sectional study showed that increased serum ferritin levels in adults may increase the risk of abnormal liver function.After adjusting for confounding factors,the results from multivariate logistic regression indicated that the risk of abnormal liver function in the high ferritin group was 2.96 times higher than that in the low ferritin group?95%CI:1.74-5.02??P<0.05?.The risk for men and women was 1.88 times?95%CI:1.20-2.96?and 4.18 times?95%CI:1.74-10.05?,respectively?P<0.05?.The rate of abnormal liver function in the iron overload group?12.5%?was significantly higher than that in the iron deficiency group?1.0%?and the iron normal group?3.1%??P<0.05?.Further analysis of the effect of iron overload and alcohol consumption on abnormal liver function in men was conducted through multiple logistic regression analysis.After adjusting for confounding factors,the results showed that the risk of abnormal liver function in iron overload and drinking group was 5.12 times higher than that in the normal group?95%CI:3.06-8.57??P<0.05?.The results of interaction analysis showed that the combined effect of iron overload and drinking was 1.36 times than the combined effects when the two factors were alone,and the risk of abnormal liver function caused by the interaction between the two factors was 1.10.Experimental studies showed the protective effects of Aplysin supplementation on alcoholic liver injury caused by alcohol.Compared with the control group,the ethanol model group had liver injury.After Aplysin supplementation,the hepatic lobular structure was normal,the arrangement of hepatic cords was gradually restored,and the organizational structure tended to be normal.The nuclear membrane of liver cells in rats was intact,the number of lipid drops was reduced,and the mitochondrial structure was intact.The levels of ALT,AST and ALP in serum in the ethanol model group was significantly increased in contrast with the control group?P<0.05?,and Aplysin could effectively inhibit these changes.TNF-?and TGF-?increased by 1.9 and 1.2 times,respectively,while IL-10 decreased by 59.7%?P<0.05?.The levels of plasma TGF-?and TNF-?decreased by 37.0%and 34.7%,respectively,compared with the ethanol model group,while the level of IL-10 increased by 2.0 times?P<0.05?.Transmission electron microscopy?TEM?showed that rats in long-term alcohol exposed,intestinal villi were significantly damaged,a large number of mucosal epithelial cells were fallen off,and the intestinal mucosa structure of rats gradually returned to normal after Aplysin supplementation.Compared with the control group,the levels of plasma endotoxin,D-LA,DAO and FABP2 in the ethanol group were increased.These results showed a decreasing trend after Aplysin supplementation.In the ethanol model group,the abundance of Bacteroides and Lactobacillus were significantly reduced,and Clostridium IV,Paraprevotella and Anaerostipes were significantly increased,but the abundance of Prevotella was significantly increased with the Aplysin supplementation.Aplysin supplementation significantly improved intestinal mucosal injury and intestinal flora disorder caused by alcohol and iron overload.Intestinal villus and glands were damaged significantly and many mucosal epithelial were fallen off in the ethanol and iron overload?model?group compared with the control group after 12 weeks.However,Aplysin supplementation improved the pathological injuries.Transmission electron microscopy?TEM?revealed that sparse small intestine microvilli and incompleted tight junctions were apparent by co-treatment of ethanol plus iron.These changes were significantly improved in the Aplysin supplementation group.The levels of SI,SF,LIC and HEPC in the model group were significantly higher than those in the control group,and the expression levels of DMT1 and FPN1 in small intestine were significantly upregulated?P<0.05?.Compared with the model group,the levels of SI,SF,LIC and HEPC showed a decreasing trend and the expression levels of DMT1 and FPN1showed a downregulating trend after the Aplysin supplementation?P<0.05?.Compared with the control group,the levels of plasma endotoxin,D-LA,DAO and FABP2 in the model group were increased.However,after the supplementation,these levels showed a decreasing trend.The analysis of intestinal flora showed that compared with the control group,the amount of Escherichia coli and Bacteroides fragils increased significantly?P<0.05?,and the amount of Bifidobacterium,Lactobacillus and Clostridium tender decreased significantly?P<0.05?.After the Aplysin supplementation,the amount of Escherichia coli and Bacteroides fragils in rat was significantly reduced?P<0.05?,and the amount of Bifidobacterium,Clostridium tender and Lactobacillus increased significantly?P<0.05?.Conclusions1.Serum ferritin levels in this population?35-76 years?can increase the risk of abnormal liver function.2.The risk of abnormal liver function in men with iron overload?SF>200?g/L?and drinking was 5.12 times higher than with no history of alcohol consumption and with normal ferritin levels.3.This study established alcohol,combined alcohol and iron overload animal models,and observed Aplysin supplementation can effectively improve liver and intestinal mucosa injury caused by alcohol exposure,combined alcohol and iron overload exposure,and restore the intestinal barrier function and intestinal flora disorder. |
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