A method for constructing a murine model of non - alcoholic steatohepatitis by Helicobacter muridarum infection

Abstract

The present invention relates to a construction method for preparing a mouse model of non-alcoholic steatohepatitis by infecting with Helicobacter muridarum, which is characterized by abnormal serum liver function, infiltration of inflammatory cells in the liver, liver fat deposition, ballooning degeneration, and fibrosis, etc., and is a non-alcoholic steatohepatitis animal model caused by a high-fat diet and intraperitoneal injection of Helicobacter muridarum. BALB / c mice are selected and randomly divided into a control group, a high-fat diet group, a Helicobacter muridarum infection group, and a high-fat diet combined with Helicobacter muridarum infection group. The body weight of the mice is weighed once a week. After 14 and 22 weeks, blood is taken to prepare serum and liver function is detected, and at the same time the mice are sacrificed; fresh liver tissue is taken to determine the total triglyceride and total cholesterol content in the liver, and liver tissue sections are prepared for H&E, Oil Red O, and Sirius red staining to judge the effect of the NASH model. Through the present invention, it can provide a good animal model for the research and development of new drugs for NASH, and also provide a convenient animal model for studying the mechanism of intestinal microorganisms in the pathogenesis of NASH.

Description

Technical Field

[0001] The present invention relates to a construction method for preparing a mouse model of non-alcoholic steatohepatitis by infecting with Helicobacter muridarum, and specifically to the establishment of a rapid non-alcoholic steatohepatitis (NASH) animal model induced by a high-fat diet combined with intraperitoneal injection of an isolated strain of Helicobacter muridarum Hm17, that is, the establishment of a non-alcoholic steatohepatitis animal model with characteristics such as abnormal serum liver function, a large number of inflammatory cell infiltrations in the liver, ballooning degeneration, and fibrosis. Background Art

[0002] In recent years, the number of patients with non-alcoholic fatty liver disease (NAFLD) has increased sharply globally and has become one of the most common liver diseases in the world. In some regions of China, the incidence of NAFLD is as high as over 43%. NAFLD includes simple steatosis, non-alcoholic steatohepatitis (NASH), and its related cirrhosis, liver cancer, etc. Among them, about 40% of patients with simple steatosis can progress to the more severe NASH stage, which is mainly manifested as hepatocyte steatosis, inflammation, fibrosis, etc., and 15%-30% of patients will develop cirrhosis within 10 years. At present, there is still no standard treatment plan or targeted drug for NASH, and its pathogenic mechanism still needs to be further studied. Research has shown that the intestinal microbiota is highly associated with the occurrence and deterioration of NASH. Enterohepatic Helicobacter species (EHS) is a pathogenic symbiotic bacterium colonizing the lower digestive tract such as the cecum and liver of humans and animals, and is associated with various diseases such as inflammatory bowel disease, malignant hepatobiliary tumors, and NAFLD. Helicobacter muridarum ( Helicobacter mastomyrinus, H.mastomyrinus ) can cause symptoms such as focal necrotic hepatitis and cecitis in some mice and is a typical representative of EHS.

[0003] Animal models are important platforms to support the treatment of NASH and drug development. However, current animal models of NASH still have deficiencies in simulating the causes, pathological morphology, and clinical characteristics of human NASH. Clinical studies have shown that the main manifestations of NASH include abnormal liver function tests, abnormal blood lipid levels, insulin resistance, hepatic steatosis, vacuolar degeneration, and liver fibrosis. The mouse NASH model induced by a high-fat diet is the most common induction method, which can simulate some symptoms of NASH caused by human overnutrition and lack of exercise, including typical hepatic steatosis and inflammation, but has a long modeling period (more than 30 weeks) and lacks symptoms such as progressive fibrosis. Therefore, there is an urgent need to develop an NASH animal model that is more similar to the clinical symptoms of human NASH. Based on the current situation of existing technologies, the inventors of this application plan to optimize the construction of existing relevant models and establish a rapid, typical, and reliable NASH model by infecting with the intestinal microorganism Helicobacter muridarum for preclinical evaluation of drug efficacy. Summary of the Invention

[0004] The object of the present invention is to overcome the deficiencies of the prior art and provide a construction method for preparing non-alcoholic steatohepatitis in mice by infecting with Helicobacter muridarum, which uses a high-fat diet and intraperitoneal injection infection H. mastomyrinus to establish a method for establishing a rapid non-alcoholic steatohepatitis (NASH) animal model induced by the isolate Hm17. The established model has a rapid, stable, and typical NASH phenotype.

[0005] To achieve the above object of the invention, the technical scheme adopted by the present invention is as follows: A construction method for preparing non-alcoholic steatohepatitis in mice by infecting with Helicobacter muridarum. Intraperitoneally inject 10 9 CFU / mouse of Helicobacter muridarum ( Helicobacter mastomyrinus , Hm) isolate Hm17 (Hm17 is deposited in the China Center for Type Culture Collection, the deposit date is October 17, 2023, and the deposit number is CCTCC M 20231918) to promote hepatic steatosis, inflammation, and fibrosis of hepatocytes in BALB / c mice induced by a high-fat diet, and rapidly form an NASH animal model. It includes feeding mice a high-fat diet and infecting them with Helicobacter muridarum for 14 and 22 weeks, constructing a mouse NASH model that is extremely similar to the pathological characteristics of clinical NASH, and measuring and describing its pathological histology, liver biochemical indicators, and lipid metabolism changes, determining that a rapid and reliable NASH animal model has been constructed. The steps included in establishing the model are:

[0006] 1). Select mouse strains: SPF-grade male mice (including but not limited to the BALB / c strain), 6 weeks old, weighing 20g ± 1g, raised in a barrier environment, negative pressure IVC, and adaptively raised for 1 week.

[0007] 2). Grouping and model establishment: The mice were randomly divided into a control group (CFD), a high-fat group (HFD), a Helicobacter muridarum-infected group (Hm), and a high-fat + Helicobacter muridarum-infected group (HFD + Hm), with 5 mice in each cage and 1 cage per group. The mice were fed once a day, and the remaining feed from the previous day was discarded. The drinking water was changed every 3 days, and the bedding was changed every 2 - 3 days. The bedding and drinking water (dispenser) were autoclaved, the water source was sterilized water, and the body weight was measured once a week. All operations of changing the bedding, feed, and drinking water dispenser, as well as weighing, were carried out in a biosafety cabinet.

[0008] 3). Sampling and detection: All the mice in each group were sacrificed at the same time. 12 hours before sacrifice, the mice were fasted but not water-deprived. After anesthesia, blood was collected. The whole blood was allowed to stand at 37°C for 1 hour, centrifuged at 2000g for 5 minutes to obtain serum, which was aliquoted and stored in a -80°C refrigerator for serological index detection. Detection of liver lipid content: After blood collection from the mice in each group, the abdominal cavity was opened, the inferior vena cava was cut open to drain the blood completely, and then the liver was taken, rinsed with PBS, photographed, and about 100 mg of fresh liver was taken from each. According to the weight, 10 times the mass of lysis buffer was added (1 ml of lysis buffer for 100 mg of tissue). After thorough homogenization, half was used to detect the content of triglyceride (TG) and total cholesterol (TC) in the liver, and the other half was used to measure its protein content. Finally, the TG and TC must be corrected with the protein concentration per milligram. Liver pathology detection: The liver of each mouse in each group was taken from the same liver lobe and divided into two parts. One part was fixed with paraformaldehyde and prepared into paraffin sections for H&E staining and Sirius red staining, and the other part was directly frozen in a -80°C refrigerator for frozen sections for oil red O staining.

[0009] 4). Conduct a systematic analysis of step 3) to determine that the model established by combining a high-fat diet with Helicobacter muridarum infection is a NASH model with abnormal liver function, aggravated liver fat deposition, inflammatory cell infiltration, a large number of ballooning changes, and fibrosis.

[0010] The high-fat diet described above is: XTHF60 (Jiangsu Xietong Biotech Co., Ltd., referring to Research Diet D12492 in the United States), containing 60% fat + ordinary drinking water;

[0011] The Helicobacter muridarum infection described above is an intraperitoneal injection of 2×10 9 CFU / mouse;

[0012] The Helicobacter muridarum infection in combination with the high-fat diet described above is: an intraperitoneal injection of 2×10 9 CFU / mouse, and feeding with the high-fat diet XTHF60, containing 60% fat + ordinary drinking water.

[0013] The pathological and histological indicators described above include: hepatocyte steatosis, ballooning degeneration of hepatocytes, hepatic inflammatory infiltration, and liver fibrosis.

[0014] The method of the present invention is advanced and scientific. Through the present invention, a method for constructing a mouse non-alcoholic steatohepatitis (NASH) model includes constructing a mouse NASH model that is extremely similar to the clinical pathological characteristics of NASH by feeding mice a high-fat diet and intraperitoneal injection of Hm17, and measuring and describing its pathological histology (hepatocyte steatosis, vacuolar degeneration, inflammatory infiltration, liver fibrosis), liver function, etc., to determine that a rapid and stable NASH model has been constructed.

[0015] The present invention uses SPF-grade 6-week-old male BALB / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd. and raised in the animal house of the Comparative Medicine Center of Yangzhou University. The breeding conditions are barrier environment, temperature 22-24°C, and free diet) to construct a non-alcoholic steatohepatitis (NASH) model. Specifically, the method for constructing a mouse non-alcoholic steatohepatitis (NASH) model of the present invention includes:

[0016] 1) Grouping methods, including: normal feed group (CFD), high-fat feed group (HFD), Helicobacter muridarum-infected group (Hm), high-fat feed and Helicobacter muridarum-infected group (HFD+Hm);

[0017] 2) The feeding period is 14 and 22 weeks;

[0018] 3) After feeding and / or infecting for 14 weeks and 22 weeks, detect the changes in histopathology (hepatocyte fatty degeneration, ballooning degeneration, hepatic lymphocytic infiltration, and liver fibrosis) and serum liver function of the 4 groups of animals, and judge whether these indicators are consistent with the clinical lesions of NASH patients;

[0019] In the present invention, the differences in NASH-related indicators of mice in each group of CFD, Hm, HFD, and HFD+Hm were compared. The results showed that compared with the CFD group:

[0020] At 14 weeks, obvious hepatocyte fatty degeneration, vacuolar degeneration, lymphocyte infiltration, and moderate fibrosis (obvious fibrous tissue hyperplasia, showing sheet-like or band-like, but still not forming nodules) appeared in the HFD+Hm group, accompanied by increased serum AST, ALT, and ALP enzyme activities, and increased liver TC and TG levels; there were also obvious lymphocyte infiltrations in the liver of the Hm group, and increased serum ALT and ALP enzyme activities, but no typical hepatocyte fatty degeneration and vacuolar degeneration occurred, and a small amount of perisinusoidal fibrosis appeared; only mild hepatocyte fatty degeneration (confined near the central vein) occurred in the HFD group, without obvious inflammation and fibrosis, and the serum ALT enzyme activity did not increase.

[0021] At 22 weeks, severe hepatocyte vacuolar degeneration, lymphocyte infiltration, and fibrosis (a large amount of perisinusoidal fibrosis combined with periportal fibrosis and a small amount of bridging fibrosis, but the hepatic lobule structure could still be retained) occurred in the HFD + Hm group, accompanied by increased serum AST, ALT, and ALP enzyme activities, as well as elevated liver triglyceride and cholesterol levels. In the Hm group, obvious lymphocyte infiltration, severe liver fibrosis, mild hepatocyte steatosis and vacuolar degeneration also occurred in the liver, with significantly increased TC and TG contents in the liver tissue and significantly increased serum AST, ALT, and ALP enzyme activities; in the HFD group, only hepatocyte steatosis (confined near the central vein) and a small number of ballooning cells appeared, without obvious inflammation or fibrosis, and the serum ALT enzyme activity did not increase significantly.

[0022] Therefore, the present invention determines that a high-fat diet combined with intraperitoneal injection of Helicobacter muridarum into suckling mice for 14 and 22 weeks can induce typical pathophysiological changes of NASH in BALB / c mice, which can be used as a rapid and stable mouse NASH model for evaluating NASH therapeutic drugs.

[0023] In summary, the present invention specifically features an animal model of non-alcoholic steatohepatitis caused by a high-fat diet and intraperitoneal injection of Helicobacter muridarum, characterized by abnormal serum liver function, hepatic inflammatory cell infiltration, hepatic fat deposition, ballooning, and fibrosis. The present invention selects BALB / c mice, which are randomly divided into a control group, a high-fat diet group, a Helicobacter muridarum infection group, and a high-fat diet combined with Helicobacter muridarum infection group. After 1 week of adaptive feeding of BALB / c mice, the high-fat group is fed a high-fat diet with 60% fat, the Helicobacter muridarum infection group is intraperitoneally injected with 10 9 CFU / mouse of Helicobacter muridarum and fed a normal diet, the high-fat diet + Helicobacter muridarum infection group is intraperitoneally injected with Helicobacter muridarum and fed a high-fat diet with 60% fat, and the control group is fed a normal diet. During this period, the body weight of the mice is weighed once a week. After 14 and 22 weeks, blood is taken to prepare serum and liver function is detected, and at the same time, the mice are sacrificed; fresh liver tissue is taken to measure the total triglyceride and total cholesterol contents of the liver, and liver tissue sections are prepared for H&E, Oil Red O, and Sirius red staining to determine the effect of the NASH model.

[0024] The present invention constructs a mouse NASH model by the method of intraperitoneal injection of Helicobacter muridarum + high-fat diet feeding, which not only shortens the time for high-fat diet-induced NASH phenotype in mice, provides a good animal model for the research and development of new NASH drugs, but also provides a convenient animal model for studying the mechanism of intestinal microbiota in the pathogenesis of NASH. Description of the Drawings

[0025] Figure 1 It is a line graph of the body weight changes of each group;

[0026] Among them, CFD: normal feed group; Hm: intraperitoneal injection and infection with Hm17 group; HFD: high-fat feed group; HFD + Hm: high-fat feed and intraperitoneal injection and infection with Hm17 group;

[0027] *, ** respectively represent P <0.05 and P <0.01);

[0028] The body weights of Hm at weeks 2, 12 - 14, and 16 - 21 were significantly lower than those of the CFD group; while the body weight of the HFD + Hm group was only lighter than that of the HFD group at weeks 2 and 3, and was similar to that of the HFD group at other times.

[0029] Figure 2 Results of the morphology and color during liver autopsy when sampling mice in each group;

[0030] Among them, CFD: normal feed group; Hm: intraperitoneal injection and infection with Hm17 group; HFD: high-fat feed group; HFD + Hm: high-fat feed and intraperitoneal injection and infection with Hm17 group;

[0031] From the appearance and color of the liver during autopsy, a small number of white spots could be seen on the liver surface of the Hm group at 14w, and the liver color of the HFD + Hm group was yellowish and there were obvious white spots on the surface; at 22w, the liver color of the Hm group was yellowish and obvious white spots appeared on the surface, and the liver color of the HFD + Hm group was yellowish and showed a rough "granular feeling" on the surface.

[0032] Figure 3 Detection of serum AST, ALT, and ALP contents in each group at 14w and 22w;

[0033] Among them, CFD: normal feed group; Hm: intraperitoneal injection and infection with Hm17 group; HFD: high-fat feed group; HFD + Hm: high-fat feed and intraperitoneal injection and infection with Hm17 group;

[0034] 3A: Detection result of serum AST enzyme activity; 3B: Detection result of serum ALT content; 3C: Serum AST / ALT result; 3D: Detection result of serum ALP content;

[0035] At 14 weeks, AST was significantly increased only in the HFD+Hm group, while ALT and ALP were significantly increased in both the Hm and HFD+Hm groups. The ratio of AST / ALT was significantly decreased in both the Hm and HFD+Hm groups, indicating severe liver cell damage. At 22 weeks, the trends of change in AST, ALT, and ALP in the HFD+Hm group were similar to those at 14 weeks, but the increases in AST, ALT, and ALP in the Hm group were significant. The ratio of AST / ALT was significantly decreased in both the Hm and HFD+Hm groups, significantly lower than those in the CFD group and the HFD group. There was no difference between the treatment groups ( P >0.05), so the letters are the same. If there is a difference ( P <0.05), the letters are different.

[0036] Figure 4 Detection of the contents of total cholesterol (TC) and total triglyceride (TG) in the livers of each group;

[0037] Among them, CFD: normal diet group; Hm: group infected with Hm17 by intraperitoneal injection; HFD: high-fat diet group; HFD+Hm: high-fat diet and group infected with Hm17 by intraperitoneal injection;

[0038] 4A: Detection results of TC content, 4B: Detection results of TG content; At 14 weeks, both TC and TG in the HFD+Hm group were significantly higher than those in other treatment groups. At 22 weeks, the contents of TC and TG in the HFD group, Hm group, and HFD+Hm group were all significantly higher than those in the CFD group, but the contents of TC and TG in the HFD group, Hm group, and HFD+Hm group were similar. There was no difference between the treatment groups ( P >0.05), so the letters are the same. If there is a difference ( P <0.05), the letters are different.

[0039] Figure 5 H&E staining of the livers of each group. The upper row is 14 weeks and the lower row is 22 weeks. a-d and e-h are the CFD, Hm, HFD, and HFD+Hm groups respectively;

[0040] The results showed that at 14 weeks, more intra-lobular inflammation was visible in the Hm group. At 14 weeks, there was mild fatty change in the HFD group, concentrated around the central vein. In the HFD+Hm group, obvious vacuolar degeneration of the liver and a large number of ballooning cells were observed. At 22 weeks, not only intra-lobular inflammation but also mild vacuolar degeneration and a small number of ballooning cells appeared in the Hm group. The range of vacuolar degeneration in the HFD group was enlarged, but there was still no inflammation and ballooning. The vacuolar degeneration in the HFD+Hm group decreased, but large-area portal area inflammation and granulomas appeared. The scale bar is 100 microns (CFD: normal diet group, Hm: group infected with Hm17 by intraperitoneal injection, HFD: high-fat diet group, HFD+Hm: high-fat diet and group infected with Hm17 by intraperitoneal injection).

[0041] Figure 6 Sirius red staining was performed on the livers of each group. The upper row was for 14 weeks and the lower row was for 22 weeks. a-d and e-h were the CFD, Hm, HFD, and HFD+Hm groups respectively.

[0042] The results showed that compared with the CFD group, at 14 weeks, there was a small amount of perisinusoidal fibrosis in the liver of the Hm group, no obvious fibrosis in the HFD group, and severe fibrosis in the HFD+Hm group, with a large amount of perisinusoidal fibrosis combined with periportal fibrosis and a small amount of bridging fibrosis. At 22 weeks, obvious perisinusoidal fibrosis combined with periportal fibrosis was visible in the Hm-infected group, no fibrosis was still found in the HFD group, and the fibrosis in the HFD+Hm group was still characterized by a large amount of perisinusoidal fibrosis combined with periportal fibrosis and a small amount of bridging fibrosis. The scale bar was 100 μm; (CFD: normal diet group, Hm: group infected with Hm17 by intraperitoneal injection, HFD: high-fat diet group, HFD+Hm: high-fat diet group and infected with Hm17 by intraperitoneal injection).

[0043] Figure 7 Statistics of NASH and fibrosis in the livers of each group.

[0044] Among them, 8A and 8B were the NASH and fibrosis scores at 14 weeks, and 8C and 8D were the NASH and fibrosis scores at 22 weeks. Through the observation and statistics of the pathological sections of the livers of all mice in each group, it was found that at 14 weeks, 3 mice in the HFD+Hm group reached the NASH standard (5 points); at 22 weeks, 4 mice in the HFD+Hm group reached the NASH standard (5 points). For the analysis of the degree of fibrosis, in the Hm group, there were 4 cases of F1 and 1 case of F2 fibrosis at 14 weeks, and 3 cases of F2 and 2 cases of F3 fibrosis at 22 weeks; in the HFD+Hm group, there were 3 cases of F2, 1 case of F3, and 1 case of F1 fibrosis at 14 weeks, and 3 cases of F3 and 2 cases of F2 fibrosis at 22 weeks; (CFD: normal diet group, Hm: group infected with Hm17 by intraperitoneal injection, HFD: high-fat diet group, HFD+Hm: high-fat diet group and infected with Hm17 by intraperitoneal injection).

[0045] Figure 8 Oil red staining of the livers of each group.

[0046] Among them, a-d and e-h are the CFD, Hm, HFD, and HFD+Hm groups of 14w and 22w respectively; the results show that compared with the control group, at 14w, there are more small lipid droplets deposited in the liver of the Hm group, extensive small lipid droplets are deposited in the HFD group, and more large lipid droplets appear around the central vein. The lipid droplet deposition in hepatocytes of the HFD+Hm group is the most, and the lipid droplets are larger; at 22w, compared with the control group, the lipid droplet deposition in the HFD group is obvious and more large lipid droplets appear around the central vein. The lipid droplet deposition in hepatocytes of the Hm group has increased significantly compared with 14w, while the lipid droplets in the HFD+Hm group have decreased compared with 14w; the enlarged area in the lower left corner is the area within the blue dotted line box, magnified 2 times, and the scale bar is 100 microns (CFD: normal diet group, Hm: group infected with Hm17 by intraperitoneal injection, HFD: high-fat diet group, HFD+Hm: high-fat diet and group infected with Hm17 by intraperitoneal injection). Detailed implementation mode

[0047] The present invention will be clearly and completely described below in conjunction with the accompanying drawings and the description of the drawings. Example

[0048] Animals and diet: SPF-grade 6-week-old male BALB / c mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. and raised in the SPF animal house of the Comparative Medicine Center of Yangzhou University. The breeding conditions were barrier-level, the temperature was 22-24°C, and they had free access to food and water. After 1 week of adaptive breeding, the mice were randomly divided into 4 groups (n = 10 in each group): (1) normal pellet diet (CFD); (2) normal block diet + intraperitoneal injection of Hm17 2×10 9 CFU / mouse (Hm); (3) high-fat / high-calorie diet (Jiangsu Xietong Biological product number: XTHF60, referring to Research Diet D12492 in the United States); (4) high-fat / high-calorie diet (Jiangsu Xietong Biological product number: XTHF60, referring to Research Diet D12492 in the United States) + intraperitoneal injection of Helicobacter muridarum isolate Hm17 2×10 9 CFU / mouse (HFD+Hm). The drinking water was changed twice a week, and the body weight and food consumption of the mice were monitored; the animal experiment was approved by the Animal Ethics Committee of Yangzhou University; after 14 weeks or 22 weeks of breeding, the mice were sacrificed, and the liver and serum were collected and stored at -80°C for histopathological, biochemical, and molecular biological analyses.

[0049] Serological analysis: Blood was taken from the orbital cavity and centrifuged (4°C, 2000g, 10 minutes) to separate the serum. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured using a veterinary automatic biochemical analyzer (BS-240VET).

[0050] Liver biochemical analysis: After fresh liver was homogenized by an automatic homogenizer, the levels of triglyceride (TG) and total cholesterol (TC) in the mouse liver were measured using the TG and TC assay kits from Solarbio.

[0051] Liver histopathological examination: Fresh mouse livers were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. The liver sections were stained with hematoxylin and eosin (H&E), and the NAFLD activity NAS score of the H&E sections was semi-quantitatively scored by professional pathologists based on histological features. The score was divided into three categories: hepatocyte steatosis 0-3 points, inflammation 0-3 points, ballooning degeneration 0-2 points, with a total score of 8 points. (1) Hepatocyte steatosis: 0 points (<5%); 1 point (5% - 33%); 2 points (34% - 66%); 3 points (>66%). (2) Lobular inflammation (counting necrosis foci under 20× magnification): 0 points, none; 1 point (<2); 2 points (2 - 4); 3 points (>4). (3) Hepatocyte ballooning degeneration: 0 points, none; 1 point, rare; 2 points, common. The scores of the above three items, namely hepatocyte steatosis, hepatocyte ballooning degeneration, and lobular inflammation, were added together to obtain the NAS score. If the NAS of the liver specimen was ≥5, the sample was defined as "NASH", 3-4 points were suspected of NASH, and if NAS was less than 3, it was indicated as "non-NASH". Sirius red staining was used to evaluate the degree of liver fibrosis, which was defined from F0 to F4 as follows: F0 (no fibrosis); F1 (perisinusoidal or periportal fibrosis); F2 (perisinusoidal fibrosis combined with periportal fibrosis); F3 (formation of bridging fibrosis); F4 (cirrhosis); The liver was embedded in OTC and cut into 8-μm-thick frozen sections, fixed in 4% neutral paraformaldehyde, and stained with oil red to show the fat droplet deposition in hepatocytes.

[0052] All data were expressed as mean ± SEM. Statistical analysis was performed using SPSS 25.0 software. Analysis of variance was used to compare component differences, and multiple comparisons between groups were completed by LSD test. P <0.05 was considered to have significant statistical significance. P <0.01 was considered to have extremely significant statistical significance.

[0053] The results showed that in the high-fat diet + Hm17-infected group (HFD + Hm), obvious hepatic lipid deposition, fatty degeneration of hepatocytes, vacuolar degeneration, lymphocyte infiltration, and moderate fibrosis (a large amount of perisinusoidal fibrosis combined with periportal fibrosis and a small amount of bridging fibrosis, but no nodules were formed yet) occurred at 14 weeks. At the same time, there were increases in the activities of serum AST, ALT, and ALP enzymes, as well as increases in the levels of total hepatic triglyceride and total hepatic cholesterol. The Hm17-infected group (Hm) caused obvious lymphocyte infiltration and mild hepatic fibrosis, with increases in the activities of serum ALT and ALP enzymes, but fatty degeneration and vacuolar degeneration of hepatocytes were not obvious; the high-fat diet only showed mild fatty degeneration of hepatocytes (confined near the central vein), without obvious inflammation or fibrosis, and there were no increases in the activities of serum AST, ALT, and ALP enzymes. At 22 weeks, the high-fat diet + Hm17-infected group (HFD + Hm) showed more severe inflammation and fibrosis within the hepatic lobules (obvious hepatic sinusoidal fibrosis and bridging fibrosis occurred), along with increases in the activities of serum AST, ALT, and ALP enzymes, as well as increases in the levels of total hepatic triglyceride and total cholesterol. The Hm17-infected group (Hm) showed obvious inflammation within the hepatic lobules, moderate hepatic fibrosis, mild fatty degeneration and vacuolar degeneration of hepatocytes, and more obvious increases in the activities of serum AST, ALT, and ALP enzymes; the high-fat diet still only showed fatty degeneration of hepatocytes (confined near the central vein) and a small number of balloon-like cells, without obvious inflammation or fibrosis, and there were no obvious increases in the activities of serum AST, ALT, and ALP enzymes. Therefore, the present invention determined that the high-fat diet + infection with the Helicobacter muridarum isolate Hm17 for 14 and 22 weeks could induce NASH pathophysiological changes similar to those of clinical NASH patients in mice, and could be used as a rapid and stable mouse NASH model for studying the pathogenesis of NASH promoted by gut microbiota and evaluating NASH therapeutic drugs.

Claims

1. A construction method for preparing non-alcoholic steatohepatitis in mice by Helicobacter muridarum infection, characterized in that, Intraperitoneal injection of 2×10 9 CFU / mouse of Helicobacter muridarum ( Helicobacter mastomyrinus , Hm) isolate Hm17 promotes hepatocyte steatosis, inflammation and fibrosis induced by high-fat diet in BALB / c mice, and rapidly forms a NASH animal model. It includes feeding mice a high-fat diet and infecting them with Helicobacter muridarum for 14 and 22 weeks to construct a mouse NASH model that is extremely similar to the pathological characteristics of clinical NASH, and measuring and describing its pathological histology, liver biochemical indexes, and lipid metabolism changes, and determining that a rapid and reliable NASH animal model has been constructed; Among them, Hm17 is deposited in the China Center for Type Culture Collection. The deposit date is October 17, 2023, and the deposit number is CCTCC M 20231918.

2. The construction method according to claim 1, wherein The steps included are as follows: 1). Select mouse species: SPF-grade male mice, 6 weeks old, weighing 20 g ± 1 g, raised in a barrier environment, negative pressure IVC, and adaptively raised for 1 week; 2). Grouping and modeling: Randomly divide the mice into a control group CFD, a high-fat group HFD, a Helicobacter mastomyrinus-infected group Hm, and a high-fat + Helicobacter mastomyrinus-infected group HFD + Hm, with 5 mice in each cage and 1 cage per group; Feed once a day, discard the remaining feed from the previous day, change the drinking water every 3 days, and change the bedding every 2 - 3 days. The bedding and water dispenser are autoclaved. The water source is sterilized water. Weigh the body weight once a week. All operations of changing the bedding, feed, and water dispenser and weighing are carried out in a biosafety cabinet; 3). Sampling and detection: All mice in each group are sacrificed at the same time. Fast for 12 hours before sacrifice without restricting water intake. Anesthetize and take blood. Let the whole blood stand at 37°C for 1 hour, centrifuge at 2000 g for 5 min to obtain serum, aliquot and store in a -80°C refrigerator for serological index detection; Detection of liver lipid content: After blood collection from mice in each group, open the abdominal cavity, cut the inferior vena cava to fully drain the blood, take the liver, wash it with PBS, take pictures, take about 100 mg of fresh liver from each, and add 10 times the mass of lysis buffer according to the weight, 100 mg of tissue corresponding to 1 ml of lysis buffer. After sufficient homogenization, half is used to detect the content of triglyceride TG and total cholesterol TC in the liver, and the other half is used to measure its protein content respectively. Finally, TG and TC must be corrected with the protein concentration per milligram; Liver pathology detection: Take the same liver lobe from each mouse in each group and divide it into two parts. One part is fixed with paraformaldehyde and prepared into paraffin sections for H&E staining and Sirius red staining, and the other part is directly frozen in a -80°C refrigerator for frozen sections for oil red O staining; 4). Conduct a systematic analysis of step 3) to determine that the model established by combining a high-fat diet with Helicobacter mastomyrinus infection is a NASH model with abnormal liver function, aggravated liver fat deposition, inflammatory cell infiltration, a large number of ballooning changes, and fibrosis.

3. The construction method according to claim 2, wherein In step 1), the SPF-grade male mice include but are not limited to the BALB / c strain.

4. The construction method according to claim 2, characterized in that The high-fat diet described is: XTHF60, containing 60% fat + ordinary drinking water; The Helicobacter muridarum infection described above is achieved by intraperitoneal injection of 2×10 9 CFU per mouse; The Helicobacter muridarum infection combined with a high-fat diet is as follows: Inject 2×10 9 CFU per mouse intraperitoneally with the Hm17 isolate, and feed the high-fat diet XTHF60, which contains 60% fat + ordinary drinking water.

5. The construction method according to claim 2, characterized in that The pathological histological indicators described include: hepatocyte steatosis, hepatocyte ballooning degeneration, liver inflammatory infiltration, and liver fibrosis.

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