Study On The Mutual Regulation Of CD73/A2AR In Alcoholic Liver Fibrosis

Background: Ecto-5’-exonucleotidase(CD73)/adenosine A2 A receptor(A2AR)plays an extremely important role in many diseases,but we know little about its role in liver diseases.As a transmembrane hydrolase,CD73 can hydrolyze extracellular adenosine monophosphate(AMP)into adenosine,and the adenosine produced can activate four adenosine receptors: A1,A2 A,A2B and A3,in many diseases Play an important role.The important hydrolase CD73 that hydrolyzes AMP to adenosine and the adenosine A2 A receptor that binds to adenosine as common targets for immunotherapy research have been very popular in recent years.However,there are few literature reports about CD73/A2 AR co-acting on alcoholic liver fibrosis and the mutual regulation relationship.Therefore,we investigated whether CD73/A2 AR has a mutual regulatory effect in alcoholic liver fibrosis and whether it can be used as a therapeutic target.Objective:(1)To investigate the role of CD73/A2 AR in alcoholic liver fibrosis;(2)To investigate the relationship between CD73/A2 AR in alcoholic liver fibrosis.Methods: Because it is difficult to simulate alcoholic liver fibrosis in mice for a short period of time using alcohol feed alone,through reference,the mice were given liver fibrosis by feeding them alcoholic feed and intraperitoneal injection of carbon tetrachloride(CCl4).The specific experimental model was established as follows:C57BL/6J mice were fed Lieber-De Carli liquid feed containing 4% ethanol every day for 8 weeks.In the last four weeks,the mice were injected intraperitoneally with a 5% carbon tetrachloride(CCl4)olive oil solution(2m L/kg),twice a week.The specific grouping of the administration group is as follows,divided into control group(Pair-fed,n=12),alcohol plus carbon tetrachloride group(Et OH-fed,n=12),CD73 specific inhibitor group(APCP)low and medium High three dose group.The doses were(1mg/kg,=12),(3mg/kg,n=12),(9mg/kg,n=12)and the positive control drug,the colchicine group(0.2mg/ kg,n=12).In 5 to 8 weeks,mice in the administration group were intraperitoneally injected with corresponding doses of APCP and colchicine.The control group and the alcohol plus carbon tetrachloride group were injected with the same volume of normal saline.In the early morning of the 9th week,the mice were first anesthetized and then sacrificed,and then the mice’s serum and liver were taken.In vitro,200μM ethanol was used to stimulate rat hepatic stellate cells(HSCs-T6)for 48 h to establish an alcoholic liver fibrosis model.Culture hepatic stellate cells in a carbon dioxide incubator.The second component is the normal group,200μM acetaldehyde group,200μM acetaldehyde and 1m M AMP co-culture group,100μM APCP and 200μM acetaldehyde co-culture group,1m M AMP,100μM APCP and 200μM acetaldehyde co-culture group,and finally use si RNA interference Silence CD73.The third component is the normal group,200 μM acetaldehyde group,200 μM acetaldehyde and 30 μM adenosine co-culture group,1.0 μM A2 AR antagonist ZM241385 and 200 μM acetaldehyde,30 μM adenosine and 1.0 μM ZM241385 and 200 μM acetaldehyde co-culture group,;Then use 200 μM acetaldehyde and the 1.0 μM A2 AR agonist CGS 21680 co-culture group;the fourth component is the normal group,200 μM acetaldehyde group,200 μM acetaldehyde and 100 μM APCP co-culture group,Co-culture group with 1.0μM A2 AR antagonist ZM241385 and 200μM acetaldehyde,Co-culture group of 200 μM acetaldehyde,100μM APCP and1.0μM ZM241385.The detection of ALT and AST is carried out by using a microplate reader colorimetric method,and the content of HA and LN in the serum is determined by using an ELISA kit;HE staining method to evaluate liver damage;Masson and Sirius red to evaluate the degree of liver fibrosis;α-SMA immunohistochemistry was used to evaluate the expression of α-SMA in the liver.In the in vitro experiment,CCK8 kit was used to detect the cell viability of ZM241385 and CGS 21680 at different concentrations;The protein expression in vivo and in vitro was detected by Western blot;The AMP concentration determination kit was used to detect the content of AMP in the cell supernatant.Results: In the mouse experiment,compared with the control group,the serum fibrosis indicators and pathological results of the ethanol plus CCl4 group had obvious changes.Immunofluorescence showed that the expression of CD73 and A2 AR proteins in the liver was significantly increased.When using CD73 specific inhibitors,the serum and pathological indicators of the ethanol plus CCl4 group were significantly reduced.In cell experiments,we found that inhibiting CD73 and A2 AR can reduce the expression of fibrotic protein,but it will increase the content of AMP.At the same time,we found that inhibiting the expression of CD73 will also down-regulate the expression of A2 AR,and inhibiting the expression of A2 AR will also reduce the expression of CD73.Interestingly,activating the expression of A2 AR also activates the expression of CD73,which shows that there is an association between CD73/A2 AR.Conclusion:(1)Inhibition of CD73/A2 AR can reduce alcoholic liver fibrosis;(2)CD73/A2 AR has a mutual regulation relationship in alcoholic liver fibrosis;(3)Inhibition of CD73 expression can reduce the expression of A2 AR,and the same inhibition of A2 AR expression will also make CD73 expression decreased.