Establishment Of Liver Cirrhosis Animal Model In BALB/c Mice And Its Application In Treatment Of Cirrhosis With IL-33

Liver cirrhosis and its related complications feature have extremely high mortality and poor prognosis,which makes cirrhosis one of the main causes of death in patients with liver diseases.Currently,no effective treatment method are available except liver transplantation.However,the number of liver donors is limited and the surgery is costly.Therefore,it is of great clinical significance to develop effective therapy method for liver cirrhosis.As a classic animal model of liver cirrhosis,the bile-duct-ligation(BDL)model of mice plays an important role in the research of the related mechanisms underlying liver cirrhosis.The level of Interleukin-33(IL-33),a member of the IL-1 family,was reported with an increase in many inflammatory processes in vivo,meanwhile,IL-33 can stimulate the activation of hepatic stellate cells(HSCs)to be transformed into myofibroblasts,thus aggravating fibrosis.RNA interference(RNAi)technique is a highly specific knockdown technology that inhibits the expression of targeted genes.In the process,given that the efficiency and durability,short hairpin RNA(shRNA)is often applied to the functional investigation of target genes.In order to explore a new treatment method for liver cirrhosis,adenovirus vectors which express shRNA-I133 in liver of BALB/c mice for 1133 gene silence was used in this study,and the effect of Il-33 silence on the BDL liver cirrhosis model was performed.First,the BDL model for liver cirrhosis was established by double ligation of the common bile duct in mice with minimally invasive abdominal midline surgery,and the following experiments were conducted:(1)samples were randomly collected at 7d,14d,21d,and 28d after surgery for the detection of liver index-related enzyme levels in serum;(2)liver tissue sections were prepared,and liver inflammation and fibrosis were observed by H&E and Sirius red staining;(3)immunohistochemical staining was carried out on liver tissue sections for the detection of α-SMA and Collagen Ⅰ expression and distribution;and(4)total RNA was extracted from the liver tissues,and the levels of cytokines were detected by qPCR.The results showed that the body weight of mice in the BDL group decreased continuously after the operation,and obvious symptoms presented with jaundice;the serum enzyme level in the BDL group was significantly higher than that of the sham surgery group(P<0.01);the liver cirrhosis-related cytokines like Il-6,Tnf-α,Tgf-β and Il-33 in the BDL group sharply increased during the observation period,and the level was significantly higher than that of the sham surgery group(P<0.01);in addition,pathology findings revealed that liver cirrhosis occurred 21d after the BDL operation.Then,the recombinant adenovirus expressing the specific shRNA targeting Il-33(rAd-sh-Il33)and control adenovirus(Ad-control)were injected into BDL mice and sham-treated mice via tail veins,and the samples were collected 28d after the operation.During this period,the body weight change and death of mice were recorded,and the following tests were conducted:(1)the total liver protein was extracted,and Il-33 gene silencing was evaluated by WB;(2)paraffin-embedded liver tissue sections were prepared to be stained with H&E,Sirius red,and immunohistochemistry,and liver inflammation and fibrosis were observed before and after Il-33 gene silencing for comparison;(3)the total RNA was extracted from liver tissues and submitted to qPCR assay for cytokines quantitation.Compared with the Ad-control-injected mice,the 28-day survival rate of Il-33 gene-silenced mice increased by 35%,and the serum enzyme activity and liver cytokine transcription levels significantly decreased.Furthermore,the microscopic examination on pathological sections showed that liver inflammation and liver fibrosis were alleviated.The finding of this study indicated that the inhibition of Il-33 expression can significantly alleviate liver injury,inflammation,and fibrosis in the BDL model mice,and the prolongation of survival was also observed-Therefore,inhibiting the expression of Il-33 in the liver may be a potential treatment option for liver cirrhosis.