Application of substance for inhibiting CD36 gene expression in preparation of preparation for enhancing anti-fibrosis ability of liver NK cells

Abstract

The invention relates to the technical field of biological pharmacy, in particular to application of a substance for inhibiting CD36 gene expression in preparation of a preparation for enhancing the anti-fibrosis capacity of liver NK cells. Results of the invention show that during hepatic fibrosis, CD36 mediates ferroptosis of liver NK cells to influence the anti-fibrosis ability of the NK cells. A transmission experiment proves that the CD36-NK has a better anti-fibrosis capability in vivo. The invention provides a new perspective for recovering the anti-fibrosis capability of the NK cells in the hepatic fibrosis period so as to treat fibrosis, and provides a theoretical basis for clinically applying the NK cells to treat hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to the use of a substance that inhibits CD36 gene expression in the preparation of a preparation for enhancing the anti-fibrosis ability of liver NK cells. Background Art

[0002] Chronic liver disease is one of the major diseases that seriously threaten human health. In the course of chronic liver disease, liver fibrosis is a key link that directly affects the course and prognosis of the disease. Chronic portal hypertension caused by liver fibrosis is its main clinical complication, which can lead to serious consequences such as hypersplenism, bleeding and hepatic encephalopathy in patients. If not intervened in time, liver fibrosis will further develop into cirrhosis and liver cancer, which is the main cause of death in patients.

[0003] Liver fibrosis is a pathological process that occurs after chronic liver damage due to an imbalance in repair, and is characterized by excessive deposition of extracellular matrix (ECM) and abnormal remodeling of liver structure. During liver injury, damaged hepatocytes and infiltration of immune cells activate hepatic stellate cells (HSCs) and cause them to trans-differentiate into myofibroblasts that produce collagen, which physiologically participate in tissue repair. Ultimately, under the regulation of anti-fibrotic mechanisms, myofibroblasts are inactivated or apoptotic, and scars disappear. However, under the continuous stimulation of chronic liver disease, the balance between pro-fibrotic and anti-fibrotic mechanisms is broken, resulting in continuous activation of myofibroblasts, which in turn triggers excessive production of ECM and ultimately forms liver fibrosis.

[0004] In recent years, studies have found that activated natural killer cells (NK) can selectively kill activated HSCs and play a unique anti-fibrotic role. Experiments have shown that NK cells play a unique anti-fibrotic role by directly killing activated HSCs through the secretion of IFN-γ, and induce HSCs apoptosis by expressing death receptor ligands (such as TRAIL and FASL). In addition, NK cells can also eliminate senescent HSCs, thereby promoting the alleviation of fibrosis. However, although NK cells show certain therapeutic effects in early fibrosis, their reduced activity and dysfunction limit their efficacy and clinical application as the disease progresses. Therefore, it is urgent to develop a new strategy that can enhance the anti-fibrotic ability of liver NK cells. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of a substance that inhibits CD36 gene expression in the preparation of a preparation for enhancing the anti-fibrosis ability of liver NK cells.

[0006] The present invention is achieved through the following technical solutions:

[0007] Application of a substance that inhibits CD36 gene expression in the preparation of a preparation for enhancing the anti-fibrosis ability of liver NK cells.

[0008] Preferably, the substance that inhibits CD36 gene expression comprises any one of a CD36 blocking antibody, a CD36 inhibitor and an RNA interference molecule targeting CD36.

[0009] Preferably, the CD36 blocking antibody is scavenger receptor class B type 2, purchased from Gayman, catalog number 188150-1ea.

[0010] Preferably, the preparation contains a substance that inhibits CD36 as the only active ingredient.

[0011] Preferably, the preparation further comprises a pharmaceutically acceptable excipient.

[0012] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0013] Preferably, the diluent is any one or more of lactose, microcrystalline cellulose and mannitol; the disintegrant is any one or more of cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone and sodium carboxymethyl starch.

[0014] Preferably, the preparation enhances the anti-fibrosis ability of liver NK cells by any of the following methods:

[0015] Promote NK cells to secrete IFN-γ and / or Perforin.

[0016] Inhibits ferroptosis of NK cells.

[0017] Promote the expression of ALT and AST.

[0018] Reduce the expression of fibroblast-related genes Col1a1, Acta2, and Des.

[0019] Increase the expression of NK cell activating receptor NKG2D.

[0020] Preferably, the preparation can be used to prepare a drug for treating liver fibrosis, liver cirrhosis or liver cancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides the use of a substance that inhibits CD36 gene expression in the preparation of a preparation that enhances the anti-fibrosis ability of liver NK cells. The present invention screens out the differentially expressed gene CD36 through bioinformatics analysis, and through experimental verification, it is first discovered that CD36 mediates ferroptosis of liver NK cells during liver fibrosis, affecting the anti-fibrosis ability of NK cells. The transfer experiment proves that CD36 - NK has better anti-fibrosis ability in vivo. The present invention provides a new perspective for restoring the anti-fibrosis ability of NK cells during liver fibrosis to treat fibrosis, and provides a theoretical basis for the clinical application of NK cells to treat liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a graph showing the up-regulation of the NK cell CD36 gene during liver fibrosis of the present invention; Figure 1 In the figure, A is the result of CD36 gene expression in liver tissue; B is the result of multiple tissue immunofluorescence analysis of CD36 expression in liver tissue of normal subjects and patients with liver fibrosis.

[0025] Figure 2 This is a result diagram showing the correlation between decreased NK cell activity and upregulation of CD36 during liver fibrosis of the present invention; Figure 2 In the figure, A is the CD36 expression graph of NK cells in the peripheral blood of normal control and cirrhotic patients in the bioinformatics analysis of the public data GSE128726; B is the CD36 expression graph of NK cells in the liver and spleen of the control group mice and CCL4-induced liver fibrosis mice detected by flow cytometry; C is the proportion statistical graph; D is the mean fluorescence intensity statistical graph; E is the proportion statistical graph; F is the mean fluorescence intensity statistical graph, n=5 / group, data are shown as mean±SD; *p<0.05; **p<0.01; ***p<0.001; NS, no statistical difference; G is the correlation analysis graph of NK cell CD36 expression and NKG2D expression; H is the correlation analysis graph of NK cell CD36 expression and CD69 expression; I is the correlation analysis graph of NK cell CD36 expression and NKG2A expression, and J is the correlation analysis graph of NK cell CD36 expression and IFN-γ expression.

[0026] Figure 3 This is a result diagram showing the increase in NK cell lipid peroxidation level during liver fibrosis of the present invention; Figure 3 In the figure, A is the serum cholesterol content; B is the cholesterol content in liver tissue; C is the free fatty acid content; D is the ROS expression of liver NK cells detected by flow cytometry, and E is the proportion statistics; F is the mean fluorescence intensity statistics; G is the immunofluorescence expression of ROS in liver NK cells; H is the lipid peroxidation of liver NK cells detected by flow cytometry; I is the PE / FITC ratio statistics, the lower the PE / FITC ratio, the higher the level of lipid peroxidation; n=3-5 / group, data are shown as mean±SD; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS, no statistical difference.

[0027] Figure 4 This is a diagram showing that blocking CD36 in the present invention can restore NK cell activity; Figure 4 In the figure, A is the lipid peroxidation level of liver NK cells purified by magnetic bead sorting; B is the IFN-γ expression level of liver NK cells detected by flow cytometry; C is the Perforin expression level of liver NK cells detected by flow cytometry; D is the PE / FITC ratio statistical graph, the lower the PE / FITC ratio, the higher the lipid peroxidation level; E is the IFN-γ expression statistical graph; F is the Perforin expression statistical graph; G is the killing efficiency of NK cells when NK and HSC were co-cultured (NK: HSCs = 10:1); n = 3-5 / group, data are shown as mean ± standard deviation (mean ± SD); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS, no statistical difference.

[0028] Figure 5 This is a graph showing the results of NK cell ferroptosis when CD36 upregulation aggravates liver fibrosis in the present invention; Figure 5 In summary, A is the result of Acsl4 RT-PCR detection of ferroptosis gene expression; B is the result of COX-2 RT-PCR detection of ferroptosis gene expression; C is the result of GPX4 RT-PCR detection of ferroptosis gene expression; D is the result of immunofluorescence detection of ferroptosis protein COX-2 expression; E is the result of immunofluorescence detection of ferroptosis protein GPX4 expression.

[0029] Figure 6 The result diagram of the invention showing that adding ferroptosis inhibitor can restore NK cell activity; Figure 6In the figure, A is the expression level of IFN-γ in liver NK cells detected by flow cytometry; B is the expression level of Perforin in liver NK cells detected by flow cytometry; C is the statistical graph of IFN-γ expression; D is the statistical graph of Perforin expression; E is the killing efficiency of NK cells when NK and HSC were co-cultured (NK: HSCs = 10:1); n = 3-5 / group, data are shown as mean ± standard deviation (mean ± SD); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS, no statistical difference.

[0030] Figure 7 This is a graph showing the results of NK cell ferroptosis when blocking CD36 to restore liver fibrosis in the present invention; Figure 7 In the figure, A shows the results of Acsl4 RT-PCR detection of ferroptosis gene expression after adding α-CD36 (10 μg / mL) to block; B shows the results of GPX4 RT-PCR detection of ferroptosis gene expression after adding α-CD36 (10 μg / mL) to block; n=5 / group, data are shown as mean±SD; ***p<0.001; ****p<0.0001; NS, no statistical difference.

[0031] Figure 8 This is a graph showing the results of the present invention's transfer of CD36-NK cells to significantly improve liver fibrosis in mice in vivo; Figure 8 Middle; A is a gross photograph of the liver; B is the liver weight to body weight ratio; C is the expression of ALT in mouse peripheral blood serum; D is the expression of ALT in mouse peripheral blood serum; E is the expression of γ-GT in mouse peripheral blood serum; F is the ratio of AST / ALT in mouse peripheral blood serum; G is the result of Col1a1 RT-PCR detection of fibrogenic gene expression; H is the result of Acta2 RT-PCR detection of fibrogenic gene expression; I is the result of Desmin RT-PCR detection of fibrogenic gene expression; J is the HE and Sirius red staining of liver tissue; K is the fibrosis pathological score (Ishak score); L is the statistical graph of Sirius red staining positive area; n=5-7 / group, data are shown as mean±SD; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS, no statistical difference.

[0032] Fig. 9 This is a graph showing the phenotype changes of NK cells after transfection of CD36-NK cells in the present invention; Fig. 9In the figure, A is the flow cytometric graph of the expression of liver NK cell activating receptor NKG2D, B is the corresponding statistical graph of the flow cytometric graph; C is the flow cytometric graph of the expression of liver NK cell activating receptor CD69; D is the corresponding statistical graph of the flow cytometric graph; E is the expression graph of liver NK cell inhibitory receptor NKG2A; F is the corresponding statistical graph of the flow cytometric graph; G is the expression graph of liver NK cell inhibitory receptor TIGIT; H is the corresponding statistical graph of the flow cytometric graph; n=5-7 / group, data are shown as mean±SD; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS, no statistical difference. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in the present invention. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] The beneficial effects of the present invention are described below through specific examples.

[0036] The CD36 blocking antibody in the present invention is scavenger receptor B type 2, the English name is Scavenger Receptor B2, purchased from Gayman, the product number is 188150-1ea.

[0037] The English abbreviations of the present invention are shown in Table 1.

[0038] Table 1

[0039]

[0040]

[0041] Example 1

[0042] 1. Experimental Materials

[0043] 1. Experimental animals

[0044] Six-week-old male C57BL / 6J mice were selected, and all experimental animals were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The mice were raised in the Experimental Animal Center of Jinzhou Medical University. The breeding environment was kept quiet and ventilated, and a light-dark cycle of 12 hours of light / 12 hours of darkness was adopted. During the experiment, the mice were free to eat standard feed and drink water. All experimental operations strictly complied with the ethical approval requirements of the Animal Research Committee of Jinzhou Medical University and followed the relevant provisions of the "Guidelines for the Care and Use of Laboratory Animals" issued by the National Health Commission.

[0045] (II) Experimental cells

[0046] HSCs mouse hepatic stellate cells were purchased from Beina Biotechnology.

[0047] 2. Experimental Methods

[0048] (I) Establishment of mouse liver fibrosis model

[0049] Ten 6-week-old C57BL / 6J male mice were randomly divided into two groups: control group (oil group) and model group (CCl4 group), with 5 mice in each group. A 99% pure CCl4 solution was dissolved in olive oil at a ratio of 1:4 to prepare a 20% CCl4 solution by volume. The model group mice were intraperitoneally injected with 20% CCl4 at a dose of 2.5 mL / kg, twice a week for 4 weeks. The control group mice were injected with an equal volume of olive oil. 48 hours after the last injection, the mice were killed and peripheral blood, spleen, liver and other tissues were collected for subsequent experiments.

[0050] (II) Detection of ALT, AST and γ-GT in mouse serum

[0051] The mouse eyeballs were removed to obtain the mouse orbital venous blood. The collected blood was left to stand at room temperature for 2 hours. Centrifuged at 3000r, 4℃, 9-speed rise and 9-speed fall for 10 minutes, and the upper serum was collected. 50μL of serum was diluted 5 times with 1×PBS, and the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyltransferase (γ-GT) were detected using an automatic biochemical analyzer.

[0052] 3. Histopathological staining

[0053] Preparation of paraffin sections: Remove the complete liver from the mouse, take the left lobe of the liver, trim it to the appropriate size with a sharp blade, and quickly put it into a 10% formalin fixative for at least 48 hours. Take out the tissue block from the 10% formalin fixative, rinse it with running water overnight, and then dehydrate the tissue. After the tissue dehydration, soak the samples in environmentally friendly dewaxing transparent liquid I for 5 minutes. After the wax immersion is completed, take out the tissue, put it in the center of the wax mold, drip wax liquid for embedding, and then cool and solidify it in a refrigerator. After the wax block is placed at 4℃ overnight until it is completely solidified, use a paraffin slicer to make 5μm thick tissue sections, spread the sections in 37℃ warm water, bake the sections at 60℃ for 1h to dry the tissue sections, and then store them in a section box at room temperature.

[0054] 2. H&E staining

[0055] Strictly follow the instructions of the hematoxylin and eosin staining kit.

[0056] 3. Sirius Red Staining

[0057] Strictly follow the instructions of the modified Sirius red staining kit.

[0058] 4. Isolation of Mouse Liver Mononuclear Cells

[0059] Remove the mouse eyeballs and bleed completely. After killing the mouse by dislocating the neck, carefully remove the mouse liver and place it in pre-cooled 1×PBS. Place the mouse liver in a clean culture dish, cut the liver into small pieces, and then grind it with a grinding rod until there is no obvious tissue block. Use a 200-mesh screen to filter into a 50mL centrifuge tube. Centrifuge at 2200r, 4℃, speed 9, speed 9, speed 9 for 10min, and discard the supernatant after centrifugation. Add 3mL of 40% percoll separation solution to each tube and resuspend, transfer to a 15mL centrifuge tube, 1260g, 24℃, speed 6, speed 2, speed 30min, and discard the supernatant after centrifugation. Add 3mL of 1×RBC Lysis Buffer to each tube, transfer to a new 15mL centrifuge tube, and lyse the red blood cells in the dark for 15min. Fill with 1×PBS and turn it upside down to stop the lysis, 2200r, 4℃, speed 9, speed 9, speed 9, speed 9, centrifuge for 10min, discard the supernatant, and the cell precipitate is liver mononuclear cells.

[0060] (V) Preparation of mouse spleen single cell suspension

[0061] Remove the mouse eyeballs and bleed completely. After killing the mouse by dislocating the neck, carefully remove the mouse spleen and place it in pre-cooled 1×PBS. Place the mouse spleen in a clean culture dish and grind it with a grinding rod until there are no obvious tissue blocks. Use a 200-mesh screen to filter into a 50mL centrifuge tube. Centrifuge at 2200r, 4℃, speed up 9 and speed down 9 for 10min, and discard the supernatant after centrifugation. Add 3mL 1×RBC Lysis Buffer to each tube, transfer to a new 15mL centrifuge tube, and lyse red blood cells for 15min in the dark. Fill with 1×PBS and turn upside down to stop lysis. Centrifuge at 2200r, 4℃, speed up 9 and speed down 9 for 10min to prepare a mouse spleen single cell suspension.

[0062] 6. Flow cytometry

[0063] 1. Surface Molecular Detection

[0064] After isolating liver mononuclear cells or spleen mononuclear cells according to the above methods (iv) and (v), calculate the number of cells. Block with rat serum at 4°C for 15 minutes, then fill up with 1×PBS to wash the cells once. Resuspend the cells with 100μL 1×PBS, add flow cytometry antibodies according to the recommended ratio in the antibody manual, and incubate at 4°C in the dark for 30 minutes. Fill up with 1×PBS, wash the cells once upside down, centrifuge at 3000r, 4°C, speed up 9 and speed down 9 for 5 minutes, resuspend the cells with 300μL 1×PBS, filter through a 300-mesh filter into a flow tube, and detect on the machine.

[0065] 2. Intracellular cytokine detection

[0066] After the liver mononuclear cells were isolated and obtained according to the above method (IV), the number of cells was calculated. 6Mononuclear cells were cultured with 1mL 1640 medium. 1μL CellActivation Cocktail stimulator was added to the medium and mixed well for 3h. Then 1μL Monensin Solution (1000×) was added to inhibit for 1h. Collect the cells in a 1.5mL EP tube, centrifuge at 4000r, 4℃ for 5min to collect the cells, and resuspend them in 100μL 1× PBS solution. Block with rat serum at 4℃ for 15min, and then wash the cells once with 1× PBS. Surface molecule markers: Add flow cytometry antibodies according to the recommended ratio in the antibody manual, and incubate at 4℃ in the dark for 30min. Wash the cells twice with 1× PBS solution (4000r, 4℃, 5min), and discard the supernatant. Resuspend the cells with 100μL of the fixation working solution in the intracellular detection kit, fix the cells at 4℃ for 30min, wash the cells once with 1× membrane-penetrating working solution (6000r, 4℃, 5min), and discard the supernatant. Resuspend in 100 μL 1× transmembrane buffer, then directly add intracellular factor detection antibody and incubate at 4°C in the dark for 1 h. Add 1× transmembrane buffer to wash cells twice (8000r, 4°C, 3min), resuspend cells in 300 μL 1× PBS, filter through a 300-mesh filter into a flow tube, and detect on the machine.

[0067] 3. Flow cytometry sorting of liver NK cells

[0068] According to the above method (IV), liver mononuclear cells were isolated and obtained, and the number of cells was calculated. The cells were blocked with rat serum at 4°C for 15 minutes, and then filled with 1×PBS to wash the cells once. The cells were resuspended in 1×PBS, and flow cytometry antibodies were added according to the recommended ratio in the antibody instruction manual. Incubate at 4°C in the dark for 30 minutes. Fill with 1×PBS, wash the cells upside down once, centrifuge at 4000r, 4°C, speed up 9 and speed down 9 for 5 minutes, resuspend the cells in 1640 culture medium, filter through a 300-mesh filter into a flow tube, and sort CD36 by flow cytometry. + NK cells and CD36 - NK cells. Take some cells and use flow cytometry to detect purity according to the method in (VI) 1 above. If the purity is greater than 90%, proceed to the subsequent experiments.

[0069] (VII) Magnetic bead separation of liver NK cells

[0070] According to the above method (IV), liver mononuclear cells were isolated and counted. Centrifuge at 300g, 4°C for 5 min and discard the supernatant. Add 40 μL Buffer / 10 7 Resuspend the cells and add 10 μL NK Cell Biotin-Antibody / 10 7 Mix the cells and incubate at 4℃ in the dark for 5 min. Add 2mL Buffer / 10 7Wash the cells once (300g, 4°C, 5min). Discard the supernatant and add 80μL Buffer / 10 7 Resuspend the cells and add 20 μL Anti-Biotin / 10 7 Mix the cells and incubate at 4℃ in the dark for 10min. Add 2mL Buffer / 10 7 Wash the cells once (300g, 4℃, 10min). Discard the supernatant, rinse the MS sorting column with 500μL Buffer, and resuspend the cells with 500μL Buffer. Add the cell suspension to the sorting column, and the cells that flow down are NK cells (negative selection), and then add 500μL Buffer to rinse the sorting column. After the sorting is completed, add 1×PBS to wash the cells once (300g, 4℃, 10min). Count the number of cells. Take some cells and use flow cytometry to detect the purity according to the above (VI) 1 method. After the purity is greater than 90%, proceed with subsequent experiments.

[0071] (VIII) Cell Immunofluorescence

[0072] Mouse liver NK cells were isolated by magnetic beads according to the method (VII) above. The cells were resuspended in 1× PBS and the concentration was adjusted to 3×10 4 Cells / 150μL 1×PBS. Add 150μL of cell suspension to each well of the smear cup and use a cell smear machine to smear the cells onto the anti-smear glass slide (1700r, 5min, middle, 3×10 per well). 4 cells). Fixation: Fix with 4% paraformaldehyde for 30 min. Wash 3 times with 1×PBS, 5 min each time. Permeabilization: Permeabilize with 0.5% Triton X-100 for 10 min. Wash 3 times with 1×PBS, 5 min each time. Blocking: Block with 5% BSA for 30 min. Wash 3 times with 1×PBS. Place the slides in a humidified box to protect from light and keep moist, and incubate with primary antibody at 37℃ for 1 h (GPX4 and COX-2 are diluted 1:500 with 5% BSA). Wash 3 times with 1×PBS, 5 min each time. Place the slides in a humidified box to protect from light and keep moist, and incubate with secondary antibody at room temperature for 1 h. Wash 3 times with 1×PBS, 5 min each time. Place the slides in a humidified box to protect from light and keep moist, and add DAPI to stain the nucleus at room temperature for 5 min. Wash 3 times with 1×PBS, 5 min each time.

[0073] Add fluorescence anti-fading sealing agent to seal the slices and examine them under a confocal fluorescence microscope.

[0074] (IX) Co-culture of NK cells and HSC in vitro

[0075] The present invention sets up OIL group, OIL+α-CD36 or Ferrostatin-1 group, CCL4 group, CCL4+α-CD36 or Ferrostatin-1 group. 5×10 3 HSCs were cultured to adhere to the wall and grow. Mouse liver NK cells were magnetically sorted according to the method (VII) above. HSCs were cultured in serum-free medium for 6 hours to activate HSCs. At the same time, NK cells in the OIL+α-CD36 or Ferrostatin-1 group and CCL4+α-CD36 or Ferrostatin-1 group were cultured in 1640 complete medium with 10μg / mL α-CD36 or 10μM Ferrostatin-1 for 6 hours. HSC serum-free culture was discarded, and the corresponding NK cells were added at a 10:1 effector-target ratio and cultured for 12 hours.

[0076] (X) NK cell toxicity experiment

[0077] Strictly follow the lactate dehydrogenase cytotoxicity detection kit.

[0078] (XI) Lipid peroxidation detection

[0079] Isolate each group of NK cells according to method (VII) or method (IX). Resuspend the cell pellet with 1× lipid peroxidation probe and incubate in a cell culture incubator at 37°C and 5% CO2 for 30 minutes. Wash the cells once with 1× PBS (3000r, 4°C, 5min). Resuspend the cells with 100μL 1× PBS, add flow cytometry antibodies and incubate at 4°C in the dark for 30min. Fill the 1× PBS, wash the cells once upside down, centrifuge at 3000r, 4°C, speed up 9 and speed down 9 for 5min, resuspend the cells with 300μL 1× PBS, filter through a 300-mesh filter into a flow tube, and detect on the machine. Analyze the PE / FITC ratio using FlowJo software. The lower the PE / FITC ratio, the higher the degree of lipid peroxidation.

[0080] (XII) Free fatty acid detection

[0081] Strictly follow the free fatty acid (NEFA / FFA) colorimetric test kit.

[0082] (XIII) Adoptive transfer of NK cells

[0083] Fifteen 6-week-old C57BL / 6J male mice were randomly divided into PBS group, CD36 + 、CD36 - There were 5 mice in each group. All mice were used to establish the mouse liver fibrosis model according to method (I). Between the 3rd, 4th, 5th, 6th, 7th, and 8th injections, the mice were injected with tail veins with high-purity CD36 isolated by method (VI)3.+ NK cells and CD36 - NK cells, 1×10 per mouse per adoptive transfer 5 The PBS group was injected with the same dose of 1×PBS. Two days after the eighth injection, the mice were killed, and the serum, liver and spleen were harvested for subsequent related experiments.

[0084] (XIV) RNA extraction, RNA microextraction, reverse transcription and qPCR

[0085] 1. RNA extraction: Take out the tissue frozen at -80℃, place it in a mortar treated with high pressure, add a small amount of liquid nitrogen to the mortar, and grind the tissue into powder with a grinding rod, adding liquid nitrogen continuously. Use an enzyme-free pipette to draw 1mL of Trizol solution into the mortar, continue to grind the tissue until it is homogenized, and use an enzyme-free pipette to transfer it to a 1.5mL enzyme-free EP tube. Add 200μL of chloroform to each EP tube, shake vigorously for 15s, put it in an ice box and let it stand for 3min, 12000g, 4℃, centrifuge for 15min. After centrifugation, put it in an ice box and let it stand for 10 minutes, and draw 200μL of supernatant into a new enzyme-free 1.5mL EP tube. Add 500μL of isopropanol to each tube and mix it by blowing, let it stand in an ice box for 10min, and then centrifuge it at 12000g, 4℃ for 10min. After centrifugation, remove the supernatant, add 1 mL of pre-cooled 75% ethanol to each tube, gently pipette to mix, centrifuge at 7500g, 4°C for 5 min, discard the supernatant, and repeat the wash. After removing the supernatant, use a pipette to absorb the remaining ethanol. After the ethanol evaporates, add 10 μL of sterile enzyme-free water to dissolve the RNA, and take 1 μL of it. Use the machine to test the concentration and purity of the RNA. The OD value of the RNA is 0. 260 / 280 A ratio between 1.8 and 2.0 indicates that the RNA is of high purity and reverse transcription can be continued.

[0086] 2. RNA micro-extraction: Separate and obtain NK cells from each group according to method (VII) and collect them into the same enzyme-free EP tube. Add 1mL Trizol to each tube to lyse the cells. Add 200μL chloroform to each EP tube, shake vigorously for 15s, put it in an ice box and let it stand for 3min, and centrifuge it at 12000g, 4℃ for 15min. After centrifugation, put it in an ice box and let it stand for 10 minutes, then pipette 200μL supernatant into a new enzyme-free 1.5mL EP tube, add 40μL sodium acetate 3M to each EP tube, mix well, then add 15mg / mL 2μL glycogen and mix well. Add 500μL isopropanol to each tube, gently blow and shake, let it stand in the ice box for 10min, and then put it in a -80℃ refrigerator to settle overnight. Take out the -80℃ sample, put it on ice to thaw naturally, and then centrifuge it at 12000g, 4℃ for 10min. After centrifugation, the supernatant was removed. The bottom of the tube was a blue RNA precipitate. 1 mL of pre-cooled 75% ethanol was added to each tube. After gently blowing and mixing, the tube was centrifuged at 7500 g, 4 ° C, for 5 min. The supernatant was discarded and the washing was repeated once. After the supernatant was removed, the residual ethanol was sucked up with a pipette tip. After the ethanol evaporated, 10 μL of sterile enzyme-free water was added to dissolve the RNA. 1 μL was taken and the concentration and purity of the RNA were tested on the machine. The OD value of the RNA was 0. 260 / 280 A ratio between 1.8 and 2.0 indicates that the RNA purity is high and reverse transcription can continue.

[0087] 3. Reverse transcription:

[0088] (1) Reaction system: Add 4 μL Prime Script Buffer, 1 μL Oligo dTPrimer, 1 μL Prime Script RT Enzyme Mix I, 1 μg of the above RNA, and sterile enzyme-free water to a 0.2 mL 8-tube strip to make up to a total volume of 20 μL.

[0089] (2) Reaction conditions: Place the above eight tubes into the gene amplification instrument at 37°C for 15 min, 85°C for 5 s, and 4°C for infinity.

[0090] (3) The obtained cDNA was stored in a -80°C refrigerator for subsequent qPCR experiments.

[0091] 4. Fluorescence quantitative PCR

[0092] Using gapdh as the internal reference gene, the gene expression levels of ferroptosis-related genes Gpx4, Cox-2, and Acsl4 in NK cells of each group were detected. Using gapdh as the internal reference gene, the gene expression levels of liver fibrosis-related genes Acta2, Col1a1, and Desmin in liver tissue cells were detected. The operation was performed according to the ABIQuant Studio 3 quantitative PCR instrument and SYBR Green instructions. The primer sequences are shown in Table 2.

[0093] Table 2 Mouse qPCR primer sequences

[0094]

[0095]

[0096] 15. Bioinformatics data analysis

[0097] R software was used to perform bioinformatics analysis on 15 data sets numbered GSE222576 and 23 data sets numbered GSE128726 in the Gene Expression Omnibus database. Gene ontology enrichment analysis was performed on the significantly differentially expressed genes screened out. Gene set enrichment analysis was performed to comprehensively evaluate the enrichment of differentially expressed genes in specific biological functions or pathways.

[0098] 3. Experimental Results

[0099] 1. The number of NK cells decreases and their killing ability weakens in liver fibrosis

[0100] The liver fibrosis model was successfully established by intraperitoneal injection of 20% CCl4 at a concentration of 2.5 mL / kg per mouse twice a week for 4 weeks. This model is different from other animal models of liver fibrosis, and the pathological changes are obvious and reversible. After the liver fibrosis model was successfully established, serum-related biochemical indicators were detected. The results showed that the levels of ALT, AST and γ-GT in the mice in the CCl4-induced group were significantly increased, and the AST / ALT ratio was significantly decreased, indicating severe liver damage. H&E staining and Sirius red staining showed that compared with the control group, obvious collagen aggregation appeared in the portal vein and central vein regions of the liver of the mice in the CCl4-induced group, and the degree of liver fibrosis was significantly aggravated. The proportion and number of NK cells in the liver and spleen during liver fibrosis were statistically analyzed by flow cytometry. The results showed that compared with the control group, the proportion and number of NK cells in the liver and spleen of the mice in the CCl4-induced group were significantly decreased. Primary NK cells from mouse livers were isolated and co-cultured with activated HSCs cells to detect the killing ability of NK cells. The results showed that compared with the control group, the killing ability of NK cells in the liver of mice induced by CCl4 was significantly reduced against HSCs. The above results indicate that the number of NK cells decreases and their killing ability weakens during liver fibrosis.

[0101] 2. NK cell activity decreases in liver fibrosis

[0102] The expression levels of activating receptors NKG2D and CD69, inhibitory receptors NKG2A and TIGIT on the surface of NK cells in the liver and spleen of mice were analyzed by flow cytometry. The results showed that compared with the control group, the expression of activating receptors NKG2D and CD69 of NK cells in the liver of mice in the CCl4-induced group was significantly reduced, and the expression of inhibitory receptors NKG2A and TIGIT was significantly increased. At this time, the liver NK cells were in a state of functional inhibition. Similar to the liver results, the expression of activating receptor NKG2D of NK cells in the spleen of mice in the CCl4-induced group was significantly reduced, and the expression of inhibitory receptor TIGIT was significantly increased, indicating that the spleen NK cells were also in a state of functional inhibition at this time. The expression of regulatory cytokines IFN-γ, TNF-α and cytotoxic factors Perforin and Granzyme B secreted by mouse liver NK cells was further analyzed by flow cytometry. The results showed that compared with the control group, the expression of regulatory cytokines and cytotoxic cytokines of NK cells in the liver of mice in the CCl4-induced group was significantly reduced. The above results show that the activity of NK cells decreases during liver fibrosis.

[0103] 3. Decreased NK cell activity in liver fibrosis is associated with upregulation of CD36

[0104] The differential expression of genes in the liver tissues of mice with liver fibrosis and mice in the control group in the public database (GSE222576) was analyzed. The results showed that the expression of genes related to lipid decomposition and uptake, Acnat2, Scd2, Lpl, and CD36, in the liver tissues of mice in the liver fibrosis group was significantly increased (LogFC>2). KEGG enrichment analysis was used to identify significantly enriched KEGG pathways and GO analysis was used to identify significantly enriched GO pathways in biological processes. The results showed that the genes in the liver tissues of mice in the liver fibrosis group were significantly enriched in pathways related to lipid uptake and decomposition. GSEA was used to analyze the gene sets of pathways related to lipid uptake and decomposition with gene differences P<0.05 and NES>1 in the liver tissues of mice with liver fibrosis and mice in the control group, and they were intersected with the genes with LogFC>2 differences between the control group and the liver fibrosis group to screen out the only gene CD36. Further analysis found that the expression of the CD36 gene was significantly increased in the liver tissues of animal models of liver fibrosis, such as Figure 1 As shown in A. The expression of CD36 in liver tissues of cirrhotic patients and normal subjects was analyzed by tissue multiple immunofluorescence, and the bioinformatics analysis results were verified, as shown in Figure 1 As shown in B.

[0105] The present invention focuses on the expression of CD36 in NK cells and analyzes the CD36 gene expression in peripheral blood NK cells of patients with liver cirrhosis and peripheral blood NK cells of the control group in the public database (GSE128726). Figure 2As shown in A in the figure, NK cells also express higher levels of CD36 in cirrhosis. Based on the above results, the present invention detected the expression of CD36 in liver NK cells and spleen NK cells of mice with liver fibrosis induced by CCl4 by flow cytometry. Figure 2 As shown in Figures B to F, the liver and spleen NK cells of liver fibrosis mice have higher expression of CD36 compared with the control group. The correlation analysis between the expression of NK cell CD36 and the expression of its activating receptors, inhibitory receptors and secreted cytokines was analyzed. Figure 2 As shown in G to J, the expression of CD36 in liver NK cells was negatively correlated with the expression of NK cell activating receptors NKG2D and CD69, positively correlated with the expression of NK cell inhibitory receptor NKG2A, and negatively correlated with the expression of NK cell regulatory cytokine IFN-γ, indicating that the decrease in NK cell activity may be closely related to the upregulation of CD36.

[0106] 4. Lipid peroxidation of NK cells during liver fibrosis

[0107] First, the cholesterol content in serum and liver tissue was tested. Figure 3 As shown in A and B in Figure 1, compared with the control group, the cholesterol content in the serum and liver tissue of the mice in the CCl4-induced group was significantly increased. The content of free fatty acids in the liver tissue was detected, and the results were as follows Figure 3 As shown in Figure C, the free fatty acid content in the serum of mice in the CCl4-induced group was also significantly increased. The expression of ROS in NK cells was detected by flow cytometry and immunofluorescence. Figure 3 As shown in D to G in Figure 4, the expression of ROS in the liver NK cells of mice in the CCl4-induced group was significantly increased. The lipid peroxidation levels of the liver NK cells in the two groups were detected. Figure 3 As shown in H and I, the lipid peroxidation level of NK cells in the liver of mice in the CCl4-induced group was significantly increased. The lower the PE / FITC ratio, the higher the lipid peroxidation level.

[0108] 5. Blocking CD36 can restore NK cell activity in liver fibrosis

[0109] The liver NK cells of the two groups of mice were isolated, and some of them were cultured in vitro with CD36 blocking antibodies for 18 hours, and then the lipid peroxidation levels of NK cells in each group were detected. Figure 4 As shown in A, the lipid peroxidation level of NK cells in the liver fibrosis period can be effectively restored after treatment with CD36 blocking antibodies. This indicates that the lipid peroxidation of liver NK cells during liver fibrosis is mediated by CD36. After culturing liver lymphocytes in vitro with CD36 blocking antibodies for 18 hours, the ability of NK cells in each group to secrete IFN-γ and Perforin was detected. The results are shown in Figure 4As shown in Figures B and C, compared with the liver NK cells of mice in the simple CCl4-induced group, the NK cells cultured in vitro with CD36 blocking antibodies can effectively restore the ability to secrete IFN-γ and Perforin, effectively restore the killing ability of cells to HSCs, and restore cell activity. Figure 4 As shown in D~G.

[0110] 6. CD36 upregulation aggravates NK cell ferroptosis

[0111] The expression of ferroptosis-related genes in NK cells during liver fibrosis was detected. RT-PCR experiments showed that compared with the liver NK cells in the control group, the expression of ferroptosis-related genes Acsl4 and COX-2 in the liver NK cells of the CCl4-induced group was significantly increased, and the expression of the gene GPX4 that inhibits ferroptosis was significantly decreased. Figure 5 As shown in A to C in Figure 1. Immunofluorescence experiments also yielded the same results, such as Figure 5 As shown in D and E in Figure 1, it indicates that during liver fibrosis, liver NK cells underwent significant ferroptosis. The isolated lymphocytes were treated with ferroptosis inhibitors in vitro for 18 hours, and the ability of NK cells in the four groups to secrete IFN-γ and Perforin was detected. The results showed that compared with the liver NK cells of the CCl4-induced group, the NK cells in the liver fibrosis period treated with ferroptosis inhibitors in vitro could effectively restore the ability to secrete IFN-γ and Perforin, as shown in Figure 1. Figure 6 As shown in A and B in Figure 1, the killing ability of HSCs was effectively restored and the cell activity was restored. Figure 6 As shown in C to E.

[0112] Liver NK cells were isolated and cultured in vitro with CD36 blocking antibody for 18 h. RNA of NK cells in each group was extracted, and RT-PCR was used to detect the expression of ferroptosis-related genes in NK cells in each group. Figure 7 As shown in A and B, compared with the mouse liver NK cells in the simple CCl4-induced group, the expression of the ferroptosis-related gene Acsl4 in NK cells during the liver fibrosis period after in vitro culture with CD36 blocking antibodies was significantly reduced, and the expression of the gene GPX4 that inhibits ferroptosis was significantly increased. Blocking CD36 can effectively inhibit the ferroptosis of liver NK cells during liver fibrosis.

[0113] 7. CD36 - NK cells significantly improve liver fibrosis in mice

[0114] During the CCl4 modeling, wild-type mouse CD36 + NK cells and CD36 -NK cells were injected back into the CCl4 model mice via tail vein injection once a week for 3 weeks, with 1×10 NK cells injected into each mouse each time. 5 cells, observed CD36 + NK cells and CD36 - NK cells have the ability to restore liver fibrosis in vivo. Liver macroscopic observation showed that the transfer of CD36 + NK cells and CD36 - There was no difference in NK cells between the two groups. - The ratio of liver weight to body weight of NK cells in mice was significantly increased, suggesting that CD36 - NK cells improve liver damage, such as Figure 8 As shown in A and B in Figure 1, the expression levels of ALT, AST, and γ-GT in serum, which are biochemical indicators of liver injury, were detected. - Compared with mice transfected with CD36 + NK cell-derived mice restored the high expression of ALT and AST, and surface transfection of CD36 - NK cells can alleviate liver damage, such as Figure 8 The expression of fibroblast-related genes Col1a1, Acta2, and Des in the liver tissues of each group was detected by RT-PCR, and it was found that compared with the liver tissues of the control group, the CD36 - The liver fibrosis-related genes in mice with NK cells were significantly reduced, while the CD36 + There was no significant change in the expression of fibroblast-related genes in the liver tissue of mice with NK cells, such as Figure 8 HE staining and Sirius red staining were used to determine the degree of liver fibrosis in each group, and the same results as those obtained in the RT-PCR experiment were obtained. - NK cells can significantly improve CCL4-induced liver fibrosis in mice, while the transfer of CD36 + NK cells have no obvious effect, such as Figure 8 As shown in K to L in Figure 1, the expression of activating receptors NKG2D and CD69, and inhibitory receptors NKG2A and TIGIT on the surface of liver NK cells of each group of mice was detected by flow cytometry. - The expression of NKG2D, a NK cell activating receptor, was significantly increased in the liver of mice with NK cells. Fig. 9 shown.

[0115] In summary, the results of the present invention indicate that CD36 mediates ferroptosis of liver NK cells during liver fibrosis, affecting the anti-fibrosis ability of NK cells. -NK has a higher anti-fibrosis ability in the body. The present invention provides a new perspective for restoring the anti-fibrosis ability of NK cells during liver fibrosis to treat fibrosis, and provides a theoretical basis for the clinical application of NK cells to treat liver fibrosis.

[0116] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0117] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. For those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of substances that inhibit CD36 gene expression in the preparation of preparations for enhancing the anti-fibrosis ability of liver NK cells.

2. The use according to claim 1, characterized in that: The substance for inhibiting CD36 gene expression comprises any one of CD36 blocking antibodies, CD36 inhibitors and RNA interference molecules targeting CD36.

3. The use according to claim 2, characterized in that: The CD36 blocking antibody is scavenger receptor B type 2.

4. The use according to claim 1, characterized in that: The preparation has a substance that inhibits CD36 as the only active ingredient.

5. The use according to claim 1, characterized in that: The preparation also includes pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

7. The use according to claim 1, characterized in that: The preparation enhances the anti-fibrosis ability of liver NK cells by any of the following methods: Promote NK cells to secrete IFN-γ and / or Perforin; Inhibit NK cell ferroptosis; Promote the expression of ALT and AST; Reduced the expression of fibroblast-related genes Col1a1, Acta2, and Des; Increase the expression of NK cell activating receptor NKG2D.

8. The use according to claim 1, characterized in that: The preparation is used for preparing medicine for treating liver fibrosis, liver cirrhosis or liver cancer.

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