A surface molecule for inhibiting hepatocyte apoptosis and its application

Abstract

The present invention discloses a surface molecule that inhibits hepatocyte apoptosis and its application. The present invention experimentally proves for the first time that hepatocyte CD1d (cluster of differentiation 1d) has a highly effective anti-apoptotic liver protection effect, and technically uses adeno-associated virus to specifically overexpress mouse hepatocyte CD1d, or activates CD1d by injecting agonistic monoclonal antibodies into the mouse tail vein, and shows a good anti-apoptotic liver protection effect on hepatocytes in both the non-alcoholic fatty liver hepatitis model and the apoptotic acute liver damage model. The present invention can specifically enhance the anti-apoptosis and anti-damage ability of hepatocytes in advance or actively, and can be used not only for the treatment of acute and chronic liver diseases, but also for the prevention of liver damage. The mechanism of action is specific, so theoretically the toxic and side effects may also be relatively small.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a liver cell surface molecule CD1d (cluster of differentiation 1d) that can effectively inhibit liver cell apoptosis after high expression or activation by antibodies, and can be used as a drug for preventing and / or treating liver damage in acute and chronic liver diseases. Background Art

[0002] Over 1 billion people worldwide, and over 300 million in China, suffer from liver disease, a serious threat to human health. Hepatocytes are the most important cell population in the liver and the primary target of damage in various acute and chronic liver diseases. Hepatocyte apoptosis is a type of programmed cell death regulated by multiple genes. Under normal physiological conditions, hepatocytes can eliminate aging or damaged hepatocytes through a process of "self-destruction" called apoptosis, thereby maintaining liver homeostasis. However, a variety of damaging factors can also induce excessive apoptosis in hepatocytes, thereby mediating the occurrence and development of various acute and chronic liver diseases, such as poisoning, viral hepatitis, steatohepatitis, alcoholic hepatitis, drug-induced hepatitis, autoimmune diseases, cholestatic hepatitis, and ischemia-reperfusion injury. However, there is currently a lack of effective clinical interventions to improve the anti-apoptotic capacity of hepatocytes and thus control liver damage.

[0003] Cluster of differentiation 1d (CD1d), a non-classical MHC class I-like molecule with high homology to MHC class I molecules, is expressed on the surfaces of various cells. CD1d expressed on antigen-presenting cells can promote tissue inflammation by activating NKT cells through the presentation of lipid antigens. CD1d is also constitutively expressed on some tissue cells, such as hepatocytes and intestinal epithelial cells. Recent studies suggest that CD1d on tissue cells is primarily involved in maintaining the stability of the local microenvironment.

[0004] The anti-apoptotic drugs currently in clinical trials are mainly chemical inhibitors (such as emricasan, VX-166, etc.) that target key enzymes (such as Caspase family molecules) that activate or execute apoptosis when liver damage occurs. These are pathological targets that only appear when liver damage has already occurred significantly, and are therefore only suitable for treatment. Current clinical studies suggest that such treatments are ineffective and short-lived, and the toxic side effects of chemical drugs may cause some patients to suffer further liver damage, leading to worsening of the disease. The present invention, however, can preemptively or proactively enhance the anti-apoptotic ability of liver cells through the following technical solutions. Therefore, it is not only suitable for treatment, but also has certain preventive value for liver damage. In addition, due to its specific mechanism of action, the toxic side effects may theoretically be smaller. Summary of the Invention

[0005] In response to the current lack of clinical means to specifically enhance the anti-apoptosis ability of hepatocytes, the present invention provides a cell surface intervention target that can conveniently and effectively enhance the anti-apoptosis and anti-injury abilities of hepatocytes. In the future, viral vectors can be used to target and enhance the expression level of CD1d in hepatocytes, or monoclonal antibodies or small molecule drugs can be used to specifically activate CD1d on the surface of hepatocytes, thereby being applied to the prevention and treatment of liver damage in acute and chronic liver diseases in the clinic.

[0006] The technical solutions of the present invention are as follows:

[0007] The first object of the present invention is to provide a surface molecule for inhibiting hepatocyte apoptosis, wherein the surface molecule is a hepatocyte CD1d protein molecule.

[0008] Furthermore, the full-length amino acid sequence of the human CD1d protein molecule is shown in SEQ.ID.NO.1, and the full-length amino acid sequence of the mouse CD1d protein molecule is shown in SEQ.ID.NO.2.

[0009] In a specific embodiment, the nucleotide sequence encoding the CD1d protein is shown as SEQ ID NO.3.

[0010] A second object of the present invention is to provide the use of the aforementioned surface molecules as intervention targets in the preparation, development or screening of drugs or agents, wherein the functions of the drugs or agents include at least one of the following:

[0011] (1) Prevent and / or inhibit hepatocyte apoptosis;

[0012] (2) prevent and / or inhibit liver cell damage;

[0013] (3) Prevent and / or inhibit the activation of caspases, the execution molecules of hepatocyte apoptosis;

[0014] (4) Increase the expression of anti-apoptotic protein molecules in hepatocytes.

[0015] Furthermore, the anti-apoptotic protein molecules are Bcl-xL and Bcl-2.

[0016] The hepatocyte CD1d disclosed in the present invention is used as an intervention target. Currently, the following technical solutions can be used to enhance the anti-apoptosis ability of hepatocytes and inhibit liver damage:

[0017] (1) High expression of CD1d via hepatocyte-directed viral vectors;

[0018] (2) Specific activation of CD1d by monoclonal antibodies.

[0019] The present invention discloses an application of using hepatocyte CD1d as an intervention target, directing high expression of CD1d in hepatocytes through a viral vector, and inhibiting hepatocyte apoptosis and alleviating chronic liver damage in mice with nonalcoholic steatohepatitis (NASH) induced by HFD (high-fat diet) and MCD (methionine-choline deficient diet).

[0020] The present invention discloses the use of anti-CD1d monoclonal antibody (clone number: 19G11) in inhibiting hepatocyte apoptosis and alleviating liver damage in Fas (first apoptosis signal receptor, CD95)-induced acute liver injury in mice (simulating various acute attacks of liver injury such as clinical viral hepatitis, alcoholic hepatitis, toxic hepatitis, and drug-induced hepatitis) using hepatocyte CD1d as an intervention target.

[0021] The third object of the present invention is to provide a use of a drug or reagent for activating CD1d or increasing the expression level of CD1d in hepatocytes in the preparation of a drug for inhibiting hepatocyte apoptosis.

[0022] Furthermore, the hepatocyte apoptosis is hepatocyte apoptosis caused by non-alcoholic fatty liver disease.

[0023] The fourth object of the present invention is to provide a drug or agent for activating CD1d or increasing the expression level of CD1d in hepatocytes for use in the preparation of a drug for preventing and / or treating hepatocyte damage in liver diseases.

[0024] Furthermore, the liver injury is acute liver cell injury or chronic liver cell injury.

[0025] Furthermore, the drug or reagent of the present invention is a hepatocyte-directed CD1d1 overexpression vector or a CD1d monoclonal antibody.

[0026] Furthermore, the CD1d1 overexpression vector of the present invention is constructed based on adeno-associated virus.

[0027] The present invention observed for the first time that the expression level of CD1d on hepatocytes decreased significantly in clinical NASH patients and laboratory NASH mouse models.

[0028] After the AAV8 viral vector (containing the hepatocyte-specific promoter TBG) was used to directly overexpress CD1d in the hepatocytes of NASH mice, it was found that compared with the control group, the degree of hepatocyte apoptosis, liver inflammation, liver fibrosis, and liver cell damage in NASH mice were significantly reduced (all P < 0.05).

[0029] Mice were injected with anti-CD1d monoclonal antibodies (clone number: 19G11) in advance to activate hepatocyte CD1d, and then acute apoptotic liver injury was induced in mice by injection of Fas agonistic antibodies (simulating clinical viral hepatitis, alcoholic hepatitis, toxic hepatitis, drug-induced hepatitis and other acute liver injuries). The results showed that the degree of hepatocyte apoptosis and liver injury in mice were significantly reduced (both P < 0.05).

[0030] In addition, the present invention uses a saturated fatty acid (palmitic acid, PA) (simulating the lipotoxicity in fatty liver hepatitis) to induce apoptosis of hepatocytes (AML12 mouse hepatocyte cell line) in vitro, and uses anti-CD1d (clone number: 19G11) to activate hepatocyte CD1d in vitro. The results show that activating CD1d can significantly increase the expression of hepatocyte anti-apoptotic proteins (Bcl-xL, Bcl-2) and significantly reduce the degree of hepatocyte apoptosis.

[0031] Based on the above, the hepatocyte surface molecule CD1d is used as an intervention target. After high expression or activation, it can enhance the anti-apoptosis ability of hepatocytes, and thus can be used to prevent or alleviate liver damage caused by excessive hepatocyte apoptosis in acute and chronic liver diseases.

[0032] Therefore, the present invention can effectively address a series of clinical problems: chemical drug inhibitors currently in clinical trials (such as emricasan and VX-166) primarily target key enzymes that execute or regulate apoptosis, pathological targets that only appear after significant liver damage has already occurred. Therefore, they are only suitable for treatment, and current clinical evidence suggests that the treatment effect is poor and short-lived, and some patients even experience worsening liver damage (possibly due to drug toxicity). The present invention, through the above technical solution, can preemptively or actively enhance the anti-apoptotic ability of hepatocytes. Therefore, it can be used not only for treatment but also for prevention. Moreover, due to its specific mechanism of action, the toxicity and side effects are theoretically also likely to be minimal.

[0033] The present invention can significantly enhance the anti-apoptosis ability of hepatocytes by targeting and increasing the expression level of CD1d in hepatocytes through viral vectors or specifically activating CD1d through monoclonal antibodies, thereby preventing or alleviating liver damage caused by excessive apoptosis of hepatocytes.

[0034] The present invention formulates an intervention strategy based on the changing characteristics of CD1d of hepatocytes in clinical patients with liver disease, which is not only conducive to exploring the causes and mechanisms of liver disease, but also can specifically improve the anti-apoptosis and anti-damage ability of hepatocytes in patients with liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 CD1d expression in liver cells of NASH patients and mice:

[0036] A. Immunohistochemistry technique was used to detect the expression level of CD1d in liver cells of NASH patients;

[0037] B Western blot detection of CD1d expression level in hepatocytes of NASH mice (HFD induced);

[0038] C Western blot was used to detect the expression level of CD1d in hepatocytes of NASH mice (MCD induced).

[0039] Figure 2 Hepatocyte CD1d reduces hepatocyte apoptosis, liver inflammation, and liver damage in NASH mice (HFD-induced), including:

[0040] A. Experimental flow chart: NASH was induced in mice by HFD, and CD1d was highly expressed in hepatocytes by viral vectors;

[0041] B RT-PCR detection of high expression of CD1d in mouse hepatocytes;

[0042] C Western blot detection of high expression of CD1d in mouse hepatocytes;

[0043] D. Chemical spectrophotometry was used to detect the apoptosis level of mouse liver cells;

[0044] E tissue TUNEL method was used to detect the apoptosis level of mouse liver cells;

[0045] F RT-PCR was used to detect the expression levels of inflammatory factors (IL-1β, IL-6, TNF-α, and CCL2) in mouse liver;

[0046] G RT-PCR was used to detect the expression levels of liver fibrosis-related genes (α-SMA, Col-I, Col-III) in mice;

[0047] H Western blot was used to detect the expression levels of fibrosis-related proteins (α-SMA, Col-I, and Col-III) in mouse liver;

[0048] I biochemical analyzer was used to detect the ALT content in the serum of mice, an indicator of liver damage.

[0049] Figure 3 Hepatocyte CD1d reduces hepatocyte apoptosis, liver inflammation and damage in NASH mice (MCD-induced), including:

[0050] A. Experimental flow chart: NASH in mice was induced by MCD, and CD1d was highly expressed in hepatocytes via viral vectors;

[0051] B RT-PCR detection of high expression of CD1d in mouse hepatocytes;

[0052] C Western blot detection of high expression of CD1d in mouse hepatocytes;

[0053] D. Chemical spectrophotometry was used to detect the apoptosis level of mouse liver cells;

[0054] E tissue TUNEL method was used to detect the apoptosis level of mouse liver cells;

[0055] F RT-PCR was used to detect the expression levels of inflammatory factors (IL-1β, IL-6, TNF-α, and CCL2) in mouse liver;

[0056] G RT-PCR was used to detect the expression levels of liver fibrosis-related genes (α-SMA, Col-I, and Col-III) in mice;

[0057] H. Sirius red staining of liver tissue sections was used to detect the degree of liver fibrosis in mice;

[0058] I biochemical analyzer was used to detect the ALT content in the serum of mice, an indicator of liver damage.

[0059] Figure 4 Activation of hepatocyte CD1d reduces hepatocyte apoptosis and liver damage in mice with acute apoptotic liver disease, including:

[0060] A. Gross appearance of the liver and H&E staining to observe liver necrosis;

[0061] B. TUNEL assay to detect apoptosis of mouse liver cells;

[0062] C biochemical analyzer was used to detect the levels of ALT and AST, indicators of liver damage, in mouse serum.

[0063] Figure 5 In vitro activation of hepatocyte CD1d enhances its anti-apoptotic ability, including:

[0064] A. Immunoprecipitation technique combined with Western blot was used to detect the expression level of CD1d downstream molecule p-Tyrosine;

[0065] B. Flow cytometry was used to detect the level of hepatocyte apoptosis (Annexin + cell);

[0066] Flow cytometry was used to detect the apoptosis level of hepatocytes (TUNEL + cell);

[0067] D Western blot was used to detect the expression of hepatocyte apoptosis execution molecules (cleaved-caspase 3, cleaved-caspase 7) and anti-apoptotic molecules (Bcl-xL, Bcl-2).

[0068] Figure 6 Destination vector map of HFD-induced mouse NASH model.

[0069] Figure 7 Map of the target vector for the MCD-induced NASH model in mice. DETAILED DESCRIPTION

[0070] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0071] Example 1: CD1d expression in liver cells of NASH patients and mice.

[0072] (1) NASH patient sample collection and testing: 54 clinically obese patients underwent routine liver biopsy during bariatric surgery to assess liver pathology. Based on the NAFLD (nonalcoholic fatty liver disease) activity score (NAS), 8 normal livers (without fat deposition and inflammation) and 32 NASH liver tissue samples were selected. All the above samples were obtained with the informed consent of the patients and the approval of the ethics committee. Liver tissue samples were sectioned and immunohistochemically tested for CD1d expression on hepatocytes.

[0073] (2) Modeling and detection of NASH in mice: Mice (C57BL / 6 strain) were fed an HFD (high-fat diet) or an MCD (methionine-choline deficient diet) to induce NASH (nonalcoholic steatohepatitis). Control mice were simultaneously fed a normal chow diet. After 24 weeks (HFD) or 8 weeks (MCD) of feeding, the mice were anesthetized and primary hepatocytes were isolated by perfusion. Cell proteins were further extracted, and the expression level of CD1d in hepatocytes was detected by Western blot.

[0074] The results are as attached Figure 1 As shown, NASH patients ( Figure 1 A) HFD-induced NASH mice ( Figure 1 B) MCD-induced NASH mice ( Figure 1 C) The expression level of CD1d protein in hepatocytes was significantly decreased.

[0075] Example 2: Hepatocyte CD1d reduces hepatocyte apoptosis, liver inflammation and liver damage in NASH mice.

[0076] (1) The HFD-induced NASH model in mice demonstrated that:

[0077] Mice were fed an HFD diet and injected with adeno-associated virus serotype 8 (AAV8)-constructed CD1d1 overexpression vector (containing the hepatocyte-specific promoter TBG: pAAV-TBG-CD1d1, abbreviated as AAV-m-CD1d1) via the tail vein at the 10th and 17th weeks, respectively, to overexpress CD1d specifically in hepatocytes. The control group mice were injected with the empty virus vector AAV-Ctrl. Each mouse was injected with 10 11 Virus particles ( Figure 2 A) The following tests were performed at week 24.

[0078] The CD1d1 overexpression vector constructed by adeno-associated virus type 8 in this experiment was constructed by Hanbio Biotech Co., Ltd. (Shanghai). The map of the target vector AAV-m-CD1d1 is shown in Figure 2. Figure 6 The specific construction plan is briefly described as follows:

[0079] 1) Synthesize the full-length sequence template of the target gene: SEQ ID NO.3;

[0080] 2) Design primers for PCR of the target gene template and recover the PCR product by agarose gel electrophoresis;

[0081] The primer sequences are as follows:

[0082] F1: 5'-GCCACCATGCGGTACCTACCAT-3', (SEQ ID NO. 4);

[0083] R1: 5'-CCGGATGTCTTGATAAGCGCT-3', (SEQ ID NO. 5).

[0084] 3) The PCR product of the target gene and the basic vector containing the TBG promoter (pAAV-ZsGreen, purchased from Hanbio, Shanghai) were double-digested with restriction enzymes (EcoRI and BamHI), and the digestion products (linearized vector and target gene) were recovered by agarose gel electrophoresis.

[0085] 4) Ligate the linearized vector and target gene fragment using the T4 ligation method;

[0086] 5) The ligation product was transformed into DH5α competent cells for selective culture, and single clones were selected for PCR and sequencing to screen and confirm the successful construction of the plasmid (named: pAAV-TBG-CD1d1, abbreviated as AAV-m-CD1d1). The bacterial culture was further amplified and the plasmid was extracted and purified;

[0087] 6) Virus packaging using a three-plasmid adeno-associated virus system: Three plasmid vectors (pAAV-TBG-CD1d1 carrying the target gene, pAAV-RC adeno-associated virus packaging vector plasmid, and pAAV-Helper adeno-associated virus packaging vector plasmid; the latter two were purchased from Hanbio Biotech Co., Ltd., Shanghai) were extracted with high purity and endotoxin-free, and then purified using Hanbio's Lipofiter. TM The three plasmids were co-transfected into 293T cells using a transfection reagent; cell pellets were collected 72 hours after transfection; high-titer adeno-associated virus storage fluid was obtained using column purification, and virus quality was tested.

[0088] (2) MCD-induced NASH model in mice demonstrated:

[0089] Mice were injected with adeno-associated virus serotype 8 (AAV8)-constructed CD1d1 overexpression vector (pAAV-CMV-CD1d1, abbreviated as AAV003-m-CD1d1) via the tail vein, and the control group mice were injected with the virus empty vector AAV-Ctrl. Each mouse was injected with 10 11 One week after the injection, the two groups of mice were fed MCD diet for 8 weeks to induce NASH ( Figure 3 A), and then perform the following test.

[0090] The CD1d1 overexpression vector constructed by adeno-associated virus type 8 in this experiment was constructed by Hanbio Biotech Co., Ltd. (Shanghai). The map of the target vector AAV003-m-CD1d1 is as follows Figure 7 The specific construction plan is briefly described as follows:

[0091] 1) Synthesize the full-length sequence of the target gene: SEQ ID NO. 3; and add restriction enzyme sites (BamHI, SpeI) at both ends of the sequence;

[0092] 2) The above-synthesized sequence and the basic vector containing the CMV promoter (pAAV-MCS; purchased from Hanbio, Shanghai) were double-digested with restriction enzymes (BamHI and SpeI), and the digestion products (linearized vector and target gene) were recovered by agarose gel electrophoresis.

[0093] 4) Ligate the linearized vector and target gene fragment using the T4 ligation method;

[0094] 5) Transform the ligation product into DH5α competent cells for selective culture, and select single clones for PCR and sequencing to screen and confirm the successful construction of the plasmid. Further bacterial liquid amplification and plasmid extraction and purification are performed.

[0095] 6) Virus packaging was performed using a three-plasmid adeno-associated virus system (the experimental process is as follows): three plasmid vectors (the vector plasmid carrying the target gene pAAV-CMV-CD1d1, the pAAV-RC adeno-associated virus packaging vector plasmid, and the pHelper adeno-associated virus packaging vector plasmid; the latter two were purchased from Hanbio Biotech Co., Ltd., Shanghai) were extracted with high purity and endotoxin-free, and then the cells were purified using Hanbio's Lipofiter. TM The three plasmids were co-transfected into 293T cells using a transfection reagent; cell pellets were collected 72 hours after transfection; high-titer adeno-associated virus storage fluid was obtained using column purification, and virus quality was tested.

[0096] Testing steps:

[0097] (1) Liver tissue was fixed with 4% paraformaldehyde, then embedded in paraffin and sliced. Hepatocyte apoptosis was detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling).

[0098] (2) An appropriate amount of liver was collected and total RNA was extracted, which was then reverse transcribed into cDNA. RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was used to detect the mRNA expression levels of inflammatory factor genes (IL-1β, IL-6, TNF-α, CCL2) and fibrosis-related genes (α-SMA, Col-I, Col-III) in the liver.

[0099] (3) Take appropriate amount of liver tissue and extract total protein: detect the activity of Caspase-3 (apoptosis execution molecule) in the liver using Caspase-3 activity assay kit (company: Solebo, product number BC3830-50T); detect the expression levels of liver fibrosis-related proteins α-SMA (α-smooth muscle actin), collagen types I (type I collagen), and collagen types III (type III collagen) by Western blot.

[0100] (4) Liver tissue sections were stained with Sirius red to detect the degree of liver fibrosis.

[0101] (5) Separate the mouse serum and detect the liver damage indicator ALT (alanine aminotransferase) content using an automatic biochemical analyzer.

[0102] Experimental results in HFD-induced NASH model in mice (respectively Figure 2 B-2I): AAV-CD1d significantly increased the mRNA expression level of CD1d in mouse hepatocytes ( Figure 2 B) and protein levels ( Figure 2 C). AAV-CD1d significantly inhibited the activity of Caspase 3, an apoptosis execution molecule in the liver ( Figure 3 D) Significantly reduced TUNEL expression in the liver + The number of hepatocytes ( Figure 3 E), thus proving that AAV-CD1d can reduce hepatocyte apoptosis in NASH mice. AAV-CD1d significantly reduced the mRNA expression levels of inflammatory factors (IL-1β, IL-6, TNF-α, CCL-2) in the liver of NASH mice ( Figure 2 F), thus proving that AAV-CD1d can reduce liver inflammation in NASH mice. AAV-CD1d significantly reduced the mRNA expression levels of α-smooth muscle actin, type I collagen, and type III collagen in the liver ( Figure 2 G) and protein expression levels ( Figure 2 H), thus proving that AAV-CD1d can alleviate liver fibrosis in NASH mice. AAV-CD1d significantly reduced the ALT content in the serum of NASH mice ( Figure 2 I), which demonstrated that AAV-CD1d can alleviate the degree of hepatocyte damage in NASH mice.

[0103] Experimental results in MCD-induced NASH mouse model (respectively Figure 3 B-3I): AAV-CD1d significantly increased the mRNA expression level of CD1d in mouse hepatocytes ( Figure 3 B) and CD1d protein levels ( Figure 3 C). AAV-CD1d significantly inhibited the activity of Caspase 3, an apoptosis execution molecule in the liver ( Figure 3 D) Significantly reduced TUNEL expression in the liver + Hepatocytes ( Figure 3E), which proved that AAV-CD1d reduced hepatocyte apoptosis in NASH mice. AAV-CD1d significantly reduced the mRNA expression levels of inflammatory factors (IL-1β, IL-6, TNF-α, CCL-2) in the liver of NASH mice ( Figure 3 F), thus proving that AAV-CD1d alleviated liver inflammation in NASH mice. AAV-CD1d significantly reduced the mRNA expression levels of α-smooth muscle actin, type I collagen, and type III collagen in the liver ( Figure 3 G) and liver tissue collagen fiber content ( Figure 3 H), which proved that AAV-CD1d can reduce the degree of liver fibrosis in NASH mice. AAV-CD1d significantly reduced the ALT content in the serum of NASH mice ( Figure 3 I), which demonstrated that AAV-CD1d alleviated the degree of hepatocyte damage.

[0104] Example 3: Activating hepatocyte CD1d reduces hepatocyte apoptosis and liver damage in mice with acute apoptotic liver disease.

[0105] Mice were first injected with CD1d monoclonal antibody anti-CD1d (clone 19G11) via the tail vein. The next day, anti-Fas monoclonal antibody (clone Jo2) was injected intraperitoneally to induce acute apoptotic liver disease. Five hours later, samples were collected and analyzed as follows.

[0106] (1) The abdominal cavity of the mouse was dissected and the gross appearance of the liver was observed. The liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Liver necrosis was observed by H&E staining (hematoxylin-eosin staining).

[0107] (2) Liver tissue sections: Hepatocyte apoptosis was detected by TUNEL method.

[0108] (3) The serum of mice was separated and the levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase), liver injury indicators, were detected using an automatic biochemical analyzer.

[0109] The results are as attached Figure 4 As shown, anti-CD1d can significantly reduce liver necrosis in mice ( Figure 4 A), hepatocyte apoptosis ( Figure 4 B) and degree of liver damage ( Figure 4 C).

[0110] Example 4: In vitro activation of hepatocyte CD1d enhances its anti-apoptosis ability.

[0111] Mouse hepatocyte AML-12 cells were cultured with the CD1d monoclonal antibody anti-CD1d (clone 19G11; working concentration: 10 μg / ml) and incubated at 37°C for 1 hour. After washing with PBS (phosphate-buffered saline), goat anti-rabbit IgG (working concentration: 10 μg / ml) was added as a crosslinker for anti-CD1d and incubated at 37°C for 1 hour. The cells were then washed with PBS and incubated in serum-free medium at 37°C for 24 hours. Finally, the cells were stimulated with PA (working concentration: 0.5 mM) for 16 hours to induce apoptosis. Cell samples were collected for analysis.

[0112] (1) Cell proteins were extracted and immunoprecipitated with protein G-Sepharose for CD1d. The expression level of p-Tyrosine, a downstream molecule of the CD1d signaling pathway, was then detected by Western blot.

[0113] (2) Collect cells and detect Annexin by flow cytometry + Hepatocytes and TUNEL + Hepatocytes (reflecting apoptosis).

[0114] (3) After extracting cell proteins, the level of anti-apoptotic molecule p-Tyrosine and the expression of apoptosis execution molecules (cleaved-caspase 3, cleaved-caspase 7) and anti-apoptotic molecules (Bcl-xL, Bcl-2) were detected by Western blot.

[0115] The results are as follows Figure 5 , anti-CD1d cross-linking of hepatocytes can activate CD1d signaling ( Figure 5 A), inhibiting hepatocyte apoptosis ( Figure 5 B- Figure 5 C) inhibit the expression of hepatocyte apoptosis execution molecules ( Figure 5 D) Inducing the expression of anti-apoptotic molecules in hepatocytes ( Figure 5 D).

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a drug or reagent that activates CD1d in the preparation of a drug for preventing liver cell damage in liver disease, wherein the liver damage is acute liver cell damage, and the drug or reagent is a CD1d monoclonal antibody; The clone number of the CD1d monoclonal antibody is 19G11.

2. The use according to claim 1, characterized in that The acute liver cell injury is acute liver injury caused by viral hepatitis, alcoholic hepatitis, toxic hepatitis, and drug-induced hepatitis.

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