Preliminary Study Of CD4~+CD25~+Foxp3~+Treg In Alcoholic Liver Disease Mice

Alcoholic liver disease (ALD) is the result of long-term excessive alcoholdrinking. Generally, it begins with fatty liver and then turns into alcoholichepatitis (AH), alcoholic hepatic fibrosis (AHF) or alcoholic cirrhosis (AC) ifwithout taking any medication. There is high morbidity of ALD in westerncountry, while it keeps on increasing in our country recently. ALD has becomeanother major cause of liver damage. There is no specific treatment becausethe pathogenesis is still unclear so far. So it is the current focus to investigatethe mechanisms of ALD via a good alcoholic liver disease model.Undoubtedly, the research will benefit to clinical treatment.The liver is not only a metabolic organ, but also an immune organ. Theimmune system has innate immunity and adaptive subdivisions which cankeep the balance between self defence and autoimmune response accurately.CD4~+CD25~+Foxp3~+regulatory T cell (Treg) is different from the subsets ofTh1, Th2and Th17. It is critical to induce and maintain antigen specific Tcells tolerance. The abnormalities of CD4~+CD25~+Foxp3~+Treg both in numberand function are related to various autoimmune diseases and a variety of liverdiseases. But research on the relations between CD4~+CD25~+Foxp3~+Treg andALD is really rare so far. The study aims at the role ofCD4~+CD25~+Foxp3~+Treg in ALD which may provide theoretical basis forimmunotherapy.Objective:1To duplicate the model of chronic and acute ALD.2Toinvestigate the role of CD4~+CD25~+Foxp3~+Treg in ALD.Methods:(1)â‘ Chronic group,40C57BL/6mice were fed adequateLieber-DeCarli Control diet for5days, then divided into four groups randomly,the Control+CCL4(number=10), the Control+olive oil (number=10), theAlcohol+CCL4(number=10) and the Alcohol+olive oil (number=10). The Alcohol+CCL4and the Alcohol+olive oil mice were fed Lieber-DeCarlialcohol diet during the first8weeks and the others were pair-fedLieber-DeCarli control diet. Lieber-DeCarli alcohol diet must be addedalcohol gradually from1%to5%. The Control+CCL4and the Alcohol+CCL4mice were given an intraperitoneal injection of CCL4(2.5%4mL/kg, twice aweek) dissolved in olive oil at the ninth week, while the others received anintraperitoneal injection of olive oil (2.5%4mL/kg, twice a week). Micecontinued drinking the same diets. All were killed at the tenth week;â‘¡Acute group,20C57BL/6mice were divided into two groups randomly, theControl (number=10), the Alcohol (number=10). The Control mice were fedLieber-DeCarli Control diet for10days followed by single gavage of maltose(5g/kg). The Alcohol mice were fed Lieber-DeCarli diet for10days followedby single gavage of ethanol (5g/kg). All were killed10hours after gavage.(2)The general conditions of the mice were noted, such as hair gloss, appetite,behavior and so on. The body weight was measured weekly.(3) The changesof liver morphology or histopathology，liver weight, the Hepatosomaticindices, alanine aminotransferase (ALT), aspartate aminotransferase (AST)and Triglyceride (TG) were detected in each group.(4) The lymphocytes inthe liver and spleen from each group were isolated andCD4~+CD25~+Foxp3~+Treg were identified by fluorescence activated cell sorting.(5) The expressions of Foxp3mRNA, IL-ï¼'mRNA, TGF-β mRNA wereobserved by Real-time fluorescent quantitation PCR.Results:(1)â‘ Chronic group：there was no morphological change inthe livers of the Control+CCL4and Control+olive oil group. On the otherhand the livers in the Alcohol+CCL4and the Alcohol+olive oil group grewmore swelling, heavier and turned yellow; the change was the most significantin the Alcohol+CCL4group. Furthermore, the Hepatosomatic indices of theAlcohol+CCL4and Alcohol+olive oil group increased significantly comparedwith the other two groups. It reached6.33±0.47in the group ofAlcohol+CCL4, compared with Control+CCL4and Control+olive oil group,P<0.05. There showed no significance (P>0.05) between Alcohol+CCL4and Alcohol+olive oil group;â‘¡Acute group: the livers of the Alcohol groupgrew swelling, heavier, turned little yellow and the Hepatosomatic indicesincreased significantly compared with the control.(2)â‘ Chronic group: thehistopathology displayed normal in the Control+CCL4and Control+olive oilgroup. On the contrary, there was more inflammation, fatty and vacuolardegeneration in the Alcohol+CCL4and Alcohol+olive oil group. Picrosiriusred staining showed Alcohol+CCL4induced liver fibrosis;â‘¡Acute group:there was mild fatty and vacuolar degeneration in the Alcohol group.(3)â‘ Chronic group: the serum levels of ALT, AST, TG in the different groupsshowed:34.76±3.90U/L,111.57±5.90U/L,0.59±0.06mmol/L in theControl+CCL4group,31.90±1.93U/L,99.61±4.88U/L,0.54±0.04mmol/L in the Control+olive oil group,124.82±9.94U/L,205.18±14.86U/L,0.88±0.06mmol/L in the Alcohol+CCL4group,195.11±3.64U/L,201.12±6.81U/L,0.76±0.06mmol/L in the Alcohol+olive oil group. ALT,AST and TG increased significantly in the Alcohol+CCL4and Alcohol+oliveoil group, compared with the Control+CCL4and Control+olive oil group,P<0.05; but there was no significance between the Alcohol+CCL4andAlcohol+olive oil group, P>0.05;â‘¡Acute group: ALT, AST, TG in theAlcohol group increased significantly compared with the Control group (95.58±10.52U/L,220.40±13.41U/L,0.86±0.05mmol/L vs31.14±2.26U/L,112.23±10.23U/L,0.52±0.06mmol/L, P<0.05).(4) The percentage ofCD4~+CD25~+Foxp3~+Treg in livers showed the Control+CCL46.45±1.17, theControl+olive oil4.78±0.84, the Alcohol+CCL412.35±0.82and theAlcohol+olive oil10.21±1.61. Compared the Alcohol+CCL4and theAlcohol+olive oil with the Control+CCL4and the Control+olive oil, thedifference was significant, P<0.05; but there showed no significance betweenthe Alcohol+CCL4and Alcohol+olive oil group or the Control+CCL4andControl+olive oil group, P>0.05. There showed no significance in spleens,P>0.05.(5)â‘ Chronic group: The expressions of Foxp3mRNA in theAlcohol+CCL4and the Alcohol+olive oil group (3.17±0.77,2.91±0.28)were significantly higher than that in the Control+CCL4and the Control+olive oil group (1.25±0.16,0.95±0.07)． The differences between theControl+olive oil and the Control+CCL4or the Alcohol+olive oil and theAlcohol+CCL4were no significance. The expressions of TGF-β mRNAdisplayed the Control+CCL4(1.81±0.57), the Control+olive oil (1.49±0.23),the Alcohol+CCL4(2.99±0.30), the Alcohol+olive oil (1.78±0.15). TheAlcohol+CCL4was significantly higher than the other three groups, P<0.05.The difference between the Control+CCL4and the Control+olive oil was nosignificance, P>0.05. The expressions of IL-2mRNA displayed theControl+CCL4(1.00±0.11)， the Control+olive oil (0.70±0.25)， theAlcohol+CCL4(2.61±0.70), Alcohol+olive oil (2.21±0.26). Alcohol+CCL4was significantly higher than the others, P<0.05; Compared the Alcohol+oliveoil with the Control+olive oil, the difference was significant; but the differencebetween the Control+CCL4and the Control+olive oil was no significance,P>0.05;â‘¡Acute group: the expressions of Foxp3mRNA was nosignificance between the Control and the Alcohol (4.93±0.32vs5.07±0.17,P>0.05). The expressions of TGF-β mRNA in the Alcohol group wassignificantly higher than that in the Control group (7.80±0.66vs2.83±1.24,P<0.05). The expressions of IL-2mRNA was no significance between theControl and the Alcohol (0.10±0.00vs0.08±0.21, P>0.05).Conclusions:1We have duplicated the model of chronic and acute ALD.2Both the percentage of CD4~+CD25~+Foxp3~+Treg and Foxp3mRNAintrahepatic increased dramatically in the mice of Alcohol+CCL4and Alcoholgroup compared with the control in the chronic group. We speculatedCD4~+CD25~+Foxp3~+Treg may be one of the mechanisms in chronic ALD. Thechange in acute ALD was not significant which need further investigate fromaspect of cytology.