Construction Of Orosomucoid 2 Knockout Mice And Its Effect On Steatohepatitis

Fatty liver hemorrhagic syndrome(FLHS)is a serious nutritional metabolic disease in poultry.Lipid deposition in liver of diseased poultry causes liver inflammation and liver fibrosis.FLHS is one of the main causes of non-infectious death of caged laying hens,which has brought huge economic losses to poultry industry.Orosomucoid 2(ORM2)is an acute phase protein mainly produced by liver.ORM2 has functions such as regulating immunity and acting as a disease marker.Previous studies have shown that ORM2 can ameliorate LPS-induced liver injury by competitively binding CCR5 to CCL4.At present,there are few studies on ORM2,and there is no related research on constructing Orm2 knockout mice to explore its function and its role in steatohepatitis.In this study,we constructed Orm2 knockout mice to provide an exclusive animal model for studying ORM2-related functions.Then,the effect of Orm2 knockout on the body was explored by phenotype analysis of Orm2 knockout mice.Finally,we further explored the effect of ORM2 on liver inflammation and fibrosis in steatohepatitis.The main results of this study are as follows:Experiment 1: Genotypic identification,reproductive establishment and phenotypic observation of Orm2 knockout mice.After obtaining F1 generation mice from Saiye Biological Company,DNA was extracted from the tail tip of F1 generation mice and then amplified by PCR.Finally,heterozygous positive Orm2 knockout mice were identified by agarose gel electrophoresis and gene sequencing.Heterozygous Orm2 positive knockout mice were mated,and F2 generation mice were obtained 21 days later.The DNA was extracted from the tails of F2 mice at 3 weeks,and the genotype of F2 mice were identified by PCR amplification and agarose gel electrophoresis.A total of 37 mice were born in the F2 generation,including 8wild-type mice,accounting for 21%,and 18 heterozygous mice,accounting for 49%,and 11 homozygous mice,accounting for 30%.The genotyping of F2 generation mice basically conformed to Mendelian inheritance law.The appearance hair and body weight of F2 generation wild-type,homozygous and heterozygous Orm2 knockout mice were observed and no significant difference was found.Experiment 2: Orm2 knockout induced liver injury in mice.On the basis of experiment 1,the effect of Orm2 knockout on mice was investigated.F2 generation mice were bred to 8 weeks of age.Eight wild-type mice,eight heterozygous Orm2 knockout mice and eight homozygous Orm2 knockout mice(half male and half male)were taken for autopsy sampling.Firstly,the levels of alanine aminotransferase and aspartate aminotransferase in serum of mice were detected,and then the appearance of mice liver,spleen and lung were observed.The liver,spleen and lung of mice were weighed and the organ index was calculated.Finally,liver and spleen of mice were stained with HE to observe histological morphology and pathology.The results showed that there were no significant changes in liver appearance,tissue morphology and organ index in Orm2 knockout mice compared with wild-type mice aged 8 weeks.There were no significant changes in lung tissue appearance and organ index(P > 0.05).Orm2 knockout significantly increased alanine aminotransferase and aspartate aminotransferase levels in mice(P < 0.05),the index of spleen organ was significantly increased(P < 0.05)and the spleen was abnormally widened and enlarged externally.Experiment 3: Orm2 knockout exacerbates dietary methionine choline deficiency induced hepatitis and liver fibrosisOn the basis of experiment 2,the effect of Orm2 knockout on liver inflammation and liver fibrosis induced by MCD diet was investigated.Sixteen 8-week-old male wild-type C57BL/6N mice and eight male Orm2 knockout mice were randomly divided into 3 groups(n=8): 8 wild-type mice were fed normal diet(Control group);8 wild-type mice were fed MCD diet for 4 weeks(MCD group);8 Orm2 homozygous knockout mice were fed the MCD diet for 4 weeks(KO-MCD group).The weight curves of mice were drawn during the experiment.At the age of 12 weeks,anatomical samples were taken to observe the appearance of each tissue and calculate the organ index.Liver and spleen were stained with HE to observe the morphology of tissue.Serum alanine aminotransferase and aspartate aminotransferase levels were detected.Finally,the contents of total cholesterol and triglyceride in liver,m RNA expression level of inflammatory factors and liver fibrosis level were detected.The results showed that compared with Control group,the body weight of MCD group was significantly decreased(P < 0.05),the color of the liver became yellow,the hepatocyte ballooning and steatosis occurred,and the contents of total cholesterol and triglyceride in the liver were significantly increased(P < 0.05).The m RNA levels of chemokines Cxcl10,Ccl2,and Cd36 were significantly increased(P < 0.05),and the protein level of liver fibrosis marker α-SMA was significantly increased(P < 0.05).The steatohepatitis model was successfully constructed through the MCD diet.Compared with the mice in the MCD group,the liver of the KO-MCD group was more yellow in color,the hepatocyte ballooning and steatosis were more severe,and the liver total cholesterol and triglyceride contents were further increased(P < 0.05).The m RNA expression levels of Ccl2,Il1β,Cxcl10 and Cd36 were further increased(P < 0.05),and the protein levels of liver fibrosis markers α-SMA and Collagen1 were significantly increased(P < 0.05).In conclusion,the present study found that homozygous Orm2 knockout mice were not lethal and did not affect the long-term survival of mice by breeding Orm2 knockout mice,illustrating the effects of Orm2 knockout on mice,and providing an animal model for studying the related functions of Orm2.It was also found that Orm2 knockout intensified the lipid deposition and inflammatory response in the liver of mice with steatohepatitis,and accelerated the process of liver fibrosis,providing a new idea for the treatment of steatohepatitis.