Application of TRIM65 as a target in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an application of TRIM65 as a target in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease, wherein the drug is a substance that can upregulate the expression of TRIM65. The present invention discovers for the first time the new function of TRIM65 in NAFLD, and can be used as a new target for the preparation of drugs for treating NAFLD, providing new ideas and theoretical basis for the prevention and treatment of NAFLD in clinic and the selection of molecular targets for the development of related drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of TRIM65 as a target in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common liver disease. It is a disease induced by factors other than alcohol and defined by fatty degeneration of more than 5% of liver cells. The main clinical pathological feature is excessive fat deposition in liver cells. NAFLD includes simple steatosis (SS) and non-alcoholic steatohepatitis (NASH), the latter of which can develop into cirrhosis and hepatocellular carcinoma (HCC). NAFLD has now become an important cause of chronic liver disease in developed countries such as Europe and the United States and in wealthy areas of my country. The prevalence of NAFLD in ordinary adults is 10% to 30%, of which 10% to 20% is NASH, and the incidence of cirrhosis in the latter is as high as 25% within 10 years. Due to NAFLD and its serious complications and comorbidities, it has placed a heavy burden on the healthcare systems of various countries. In the United States alone, it is estimated that the annual direct medical expenses exceed 100 billion US dollars, but there are currently no approved drug interventions for the treatment of NAFLD. Therefore, exploring new diagnostic and therapeutic targets for NAFLD will provide theoretical and experimental basis for the development of new anti-NAFLD drugs in clinical practice, which has important scientific significance and value. Summary of the invention

[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an application of TRIM65 as a target in the preparation of drugs for the prevention and treatment of NAFLD, so as to reveal the new function of TRIM65 in NAFLD and provide new ideas and theoretical basis for the selection of molecular targets for the prevention and treatment of NAFLD and related drug development in clinical practice.

[0004] The purpose of the present invention is to provide an application of TRIM65 as a target in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease (NAFLD).

[0005] TRIM65 is a member of the TRIM (tripartite motif-containing protein) protein family and has E3 ubiquitin ligase activity. Studies have found that TRIM65 plays an important regulatory role in cancer occurrence, innate immunity, and inflammatory response, but its role in NAFLD has not yet been studied. Therefore, the purpose of the present invention is to use systemic TRIM65 gene knockout mice and construct a NAFLD model by inducing a high-fat diet to explore the role of TRIM65 in NAFLD.

[0006] Furthermore, in the above technical solution, the drug is a substance that can upregulate the expression of TRIM65. Studies have shown that systemic knockout of TRIM65 in mice can significantly increase liver lipid accumulation, reduce glucose tolerance, increase insulin resistance, increase serum TG content and reduce HDL-C content, and increase the content of inflammatory factors in liver tissue and serum; we also found that TRIM65 knockout can promote liver lipid accumulation by upregulating the expression of lipid synthesis genes SREBP1c and FASN in the liver and downregulating the expression of lipid decomposition-related genes HSL, thereby aggravating the development of NAFLD. Therefore, it can be explained that TRIM65 expression has a negative regulatory effect on NAFLD, and TRIM65 can be used as a target to treat NAFLD by using drugs that can promote high expression of TRIM65.

[0007] Furthermore, in the above technical solution, the drug comprises AAV-TRIM65 (adenovirus vector overexpressing TRIM65) or TRIM65 purified protein. The drug of the present invention can be AAV-TRIM65, or TRIM65 purified protein, or a polypeptide, etc., as long as it is a TRIM65 product that can be obtained at the biological level and targets TRIM65.

[0008] Furthermore, in the above technical solution, the drug is a Chinese herbal medicine that can upregulate the expression of TRIM65 protein or mRNA. As an exogenous drug, Chinese herbal medicine has different action points. In the present invention, the Chinese herbal medicine that can upregulate the expression of TRIM65 protein or mRNA belongs to the scope of protection of the present invention.

[0009] Furthermore, in the above technical solution, the drug also includes pharmaceutically acceptable excipients.

[0010] Furthermore, in the above technical solution, the dosage form of the drug includes one or more of tablets, granules, solutions, emulsions, and suspensions.

[0011] Furthermore, in the above technical solution, diseases related to NAFLD include one or more of cirrhosis, hepatocellular carcinoma, and other chronic liver diseases.

[0012] Advantages compared to the prior art:

[0013] The present invention uses a recognized classic high-fat diet to induce the establishment of a NAFLD mouse model, and uses gene knockout technology to target TRIM65 to knock out the expression of TRIM65 in systemic tissues to explore the possibility of TRIM65 as a target for gene therapy to treat NAFLD. The experimental results show that after TRIM65 systemic knockout mice are fed a high-fat diet, liver lipid accumulation can be significantly aggravated, glucose tolerance can be reduced, insulin resistance can be increased, serum TG content can be increased and HDL-C content can be reduced, and the content of inflammatory factors in liver tissue and serum can be increased; we also found that TRIM65 knockout can promote liver lipid accumulation by upregulating the expression of lipid synthesis genes SREBP1c and FASN in the liver and downregulating the expression of lipid decomposition-related genes HSL, ultimately exacerbating the development of NAFLD.

[0014] The present invention discovered for the first time the new function of TRIM65 in NAFLD, which can be used as a new target for the preparation of drugs for the treatment of NAFLD, and provides new ideas and theoretical basis for the prevention and treatment of NAFLD in clinical practice and the selection of molecular targets for related drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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.

[0016] Figure 1 It is a process flow chart of the present invention;

[0017] Figure 2 This is an analysis diagram of the effect of systemic TRIM65 knockout on lipid accumulation in the liver of mice induced by a high-fat diet;

[0018] Figure 3 This is an analysis diagram of the effects of systemic TRIM65 knockout on glucose tolerance and insulin resistance in mice induced by a high-fat diet;

[0019] Figure 4 This is an analysis diagram of the effect of systemic TRIM65 knockout of the present invention on the levels of TG, TC, LDL-C and HDL-C in the serum of mice induced by a high-fat diet;

[0020] Figure 5 This is an analysis diagram of the effect of systemic TRIM65 knockout on the levels of inflammatory factors in liver tissue and serum of mice induced by a high-fat diet;

[0021] Figure 6This is an analysis diagram of the effect of systemic TRIM65 knockout on the expression of genes related to lipid synthesis and degradation in the liver of mice induced by a high-fat diet. DETAILED DESCRIPTION

[0022] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0024] The purpose of the present invention is to use systemic TRIM65 gene knockout mice to induce a NAFLD model through a high-fat diet to explore the role of TRIM65 in NAFLD. The overall concept process flow chart is as follows: Figure 1 This invention will reveal the new function of TRIM65 in NAFLD and provide new ideas and theoretical basis for the prevention and treatment of NAFLD in clinic and the selection of molecular targets for the development of related drugs.

[0025] Experimental instruments and reagents:

[0026] Instruments: pipette gun (manufactured by Eppendorf); mini centrifuge (manufactured by QILINBEIER); small centrifuge, refrigerated low-temperature centrifuge, -80℃ low-temperature refrigerator, cell culture incubator, living cell workstation, living cell workstation, tissue dehydrator, tissue embedding machine, paraffin slicer, cryosectioner (manufactured by Thermo Fisher Scientific); vortex oscillator, constant temperature metal bath, shaker (manufactured by QILINBEIER); water bath (manufactured by Yongguangming Company); electronic balance, electronic analytical balance (manufactured by Mettler Toledo); multifunctional microplate reader (manufactured by Molecular Devices); cell clean bench (manufactured by AIRTECH); biochemical incubator (manufactured by Zhicheng Company); upright fluorescence microscope, inverted fluorescence microscope (manufactured by Olympus), etc.

[0027] Reagents Sterile mouse bedding (manufacturer: Beijing Keao); Normal SPF mouse feed (manufacturer: Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); MD12033 high-fat feed (manufacturer: Medison Biotechnology); Glucose (manufacturer: Sigma); Insulin (manufacturer: Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd.); Neutral gum (manufacturer: Biosharp); BCA protein quantification kit (manufacturer: Thermo); PMSF, SDS, TEMED, red blood cell lysis buffer, EDTA trypsin, penicillin-streptomycin mixture 100X (manufacturer: Solebao); Protease inhibitor cocktail, Phosphatese inhibitor cocktail, Western blot luminescence kit (manufacturer: GLPBIO); Methanol, xylene (manufacturer: Xilong Scientific Co., Ltd.); Protein lysis buffer, 30% polyacrylamide, APS, Tris-HCl pH6.8, Tris-HCl pH8.8, Goat anti-rabbit IgG, Goat anti-mouse IgG, paraformaldehyde, hematoxylin-eosin stain, DAPI anti-fluorescence quenching mounting medium, sterile PBS, fetal bovine serum, DMEM medium, RPMI1640 medium (manufactured by Sevier Biotechnology); NC membrane (manufactured by Millipore); antibody diluent (manufactured by Beyotime Biotechnology Co., Ltd.); Elisa kit (TNFα), Elisa kit (IL-6), Elisa kit (IL-1β) (manufactured by ELISA Biotechnology Co., Ltd.); DMSO (manufactured by Yishen Biotechnology Co., Ltd.), etc.

[0028] Experimental animals TRIM65 heterozygous mice (C57BL / 6J background) were obtained from Cyclic Acid Company.

[0029] The present invention is further described in detail below in conjunction with specific embodiments:

[0030] Example 1: Establishment of a high-fat diet-induced NAFLD model in TRIM65 knockout mice

[0031] 1. Acquisition of TRIM65 knockout mice: TRIM65 heterozygous mice (TRIM65 + / - ) mice, and mated heterozygous male and female mice to obtain WT, TRIM65 + / - 、TRIM65 - / - Mouse, in which TRIM65 - / - The mice were homozygous TRIM65 knockout mice. - / -Mice were fed a high-fat diet to establish a NAFLD model.

[0032] 2. Establishment of NAFLD model induced by high-fat diet

[0033] WT and TRIM65 cells were obtained from the same littermates at about 8 weeks of age. - / - The mice were fed a high-fat diet (Research Diets, D12492, 60% fat) for 15 weeks to obtain the wild-type high-fat diet-induced NAFLD model mice (HFD-WT) and the TRIM65 gene knockout mice high-fat diet-induced NAFLD model mice (HFD-TRIM65 - / - ).

[0034] Example 2: Effect of systemic TRIM65 knockout on high-fat diet-induced liver lipid accumulation in mice

[0035] The main pathological feature of NAFLD is excessive fat deposition in hepatocytes. Therefore, the inventors first detected the effect of TRIM65 knockout on lipid accumulation in mouse liver.

[0036] First, the liver tissues of the mice were photographed and weighed after sampling, and it was found that HFD-TRIM65 - / - The fatty liver formation in the mice of group A was more obvious than that in the HFD-WT group ( Figure 2 A), and the liver weight also increased significantly ( Figure 2 B).

[0037] Secondly, the liver tissues of mice in each group were stained with H&E and Oil Red O to further analyze the accumulation of lipids in the liver; finally, the liver function indicators AST and ALT in the serum of mice were detected. The steps of H&E staining, Oil Red O staining and liver function indicator detection are as follows:

[0038] 1. The experimental method of paraffin-embedded sections and H&E staining is as follows:

[0039] 1) Dehydration, embedding and sectioning of liver tissue

[0040] (1) The mice were killed, the liver tissue was removed, and 2 cm × 2 cm tissue blocks were cut and fixed in 4% PFA (paraformaldehyde) overnight;

[0041] (2) Soak in PBS for 5 min, wash off 4% PFA, and place in an automatic tissue dehydrator;

[0042] (3) Set up the program: 70% ethanol 50 min; 85% ethanol 50 min; 95% ethanol 30 min; 95% ethanol 30 min; 100% ethanol 20 min; 100% ethanol 20 min; ½ ethanol-xylene 10 min; xylene 3 min; xylene 3 min; ½ xylene-paraffin 30 min; paraffin 90 min; paraffin 90 min;

[0043] (4) Embed the tissue block in an automatic paraffin embedding machine and store it at 4°C after the paraffin cools down;

[0044] (5) Liver tissue was sliced ​​(4 μm) and stored in a -20°C refrigerator.

[0045] 2) Hematoxylin-eosin (H&E) staining:

[0046] (1) Take the paraffin sections out of the -20℃ freezer;

[0047] (2) Soak in xylene for 15 min, twice;

[0048] (3) Soak in 100% ethanol for 3 min, twice;

[0049] (4) Soak in 95% ethanol for 3 min, once;

[0050] (5) Soak in 75% ethanol for 2 min, once;

[0051] (6) Soak in 50% ethanol for 2 min, once;

[0052] (7) Rinse with tap water for 2 minutes;

[0053] (8) Hematoxylin staining takes 1-10 min. The staining process can be observed under a microscope to determine the staining time.

[0054] (9) Rinse with tap water for 10 min;

[0055] (10) Eosin staining takes 1-2 min. The staining time can be determined by observing under a microscope during the staining process.

[0056] (11) Rinse with tap water for 1 min;

[0057] (12) Soak in 100% ethanol for 3 min, twice;

[0058] (13) Soak in xylene for 3 min, twice;

[0059] (14) Use neutral gum to seal the slides, and do not allow bubbles to form;

[0060] (15) Take photos under a microscope.

[0061] 2. The experimental method of Oil Red O staining is as follows:

[0062] 1) Frozen sections

[0063] (1) The mice were killed, the liver tissue was removed, 2 cm × 2 cm tissue blocks were cut, and fixed in 4% PFA overnight;

[0064] (2) Soak in PBS for 5 min, wash off 4% PFA, and place in 15% sucrose solution. The tissue floats on the solution. After the tissue sinks to the bottom, transfer to 30% sucrose solution at 4°C overnight.

[0065] (3) After the liver tissue sinks to the bottom, take out the tissue, dry it with filter paper, embed it with OCT, put it in a -80℃ refrigerator and freeze it for 5 min, press the embedding box, take out the embedded tissue, and store it in a -80℃ refrigerator;

[0066] (4) Precool the cryostat (-25°C) and use the cryostat to slice the liver tissue (5 μm). Place the slices in a slice box and store them in a -40°C refrigerator until use.

[0067] 2) Oil Red O staining:

[0068] (1) Take the frozen sections out of the refrigerator and place them at room temperature for 30 min;

[0069] (2) Soak in PBS for 10 min to wash away the OCT embedding medium;

[0070] (3) Soak the slide in Oil Red O working solution and stain at room temperature for 30-60 min;

[0071] (4) Soak in 50% isopropanol, 30% isopropanol, and 10% isopropanol for 5 s for differentiation;

[0072] (5) Seal the sections with glycerol gelatin;

[0073] (6) Take photos under a microscope, such as Figure 2 As shown in C.

[0074] 3. Detection of liver function indicators ALT and AST

[0075] The mice were anesthetized with isoflurane, the beards were trimmed (to prevent blood from getting on the beards and causing hemolysis), the eyeballs were removed, and 750 μL of whole blood was obtained. The blood was left to stand at room temperature for 1-2 hours, centrifuged at 2000 g for 30 minutes, and the upper layer of serum was collected and placed in an EP tube and stored at -80°C. The blood was sent to Sevier Company, and the indicators including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by a biochemical analyzer and the data were analyzed. The results are as follows: Figure 2 As shown in D.

[0076] The results showed that TRIM65 knockout significantly increased lipid accumulation in the mouse liver ( Figure 2 C), and also leads to partial damage to liver function ( Figure 2 D), indicating that TRIM65 knockout can promote the formation of NAFLD.

[0077] Example 3: Effects of systemic TRIM65 knockout on glucose tolerance and insulin resistance in mice induced by a high-fat diet

[0078] Insulin resistance is closely related to the development of hepatic fatty degeneration. Therefore, the inventors collected tail vein blood from membrane-forming mice before the end of the experiment and performed glucose tolerance (GTT) and insulin tolerance (ITT) tests using a blood glucose meter and related test strips.

[0079] 1. Glucose tolerance test (GTT)

[0080] The mice were fasted for 16 h and intraperitoneally injected with D-glucose (1.5 g / kg). Blood samples were collected from the tail vein at different time points (0, 30, 60, 90, and 120 min) after glucose administration, and blood glucose levels were measured using a blood glucose meter.

[0081] Testing steps:

[0082] (1) Prepare glucose before use. Weigh 1 g of D-glucose, dissolve it in 5 mL, and filter it with a filter.

[0083] (2) Prepare 1 mL syringe, pipette, scissors, alcohol cotton pads, blood glucose meter, and blood glucose test strips;

[0084] (3) Weigh the mice, record their weight, and calculate the amount of glucose to be injected into each mouse based on their weight;

[0085] (4) Measure the fasting blood glucose value before injecting glucose, that is, the blood glucose value at 0 min;

[0086] (5) After the fasting blood glucose value is measured, glucose is injected, and then the blood glucose value at each time point is measured and recorded based on the time of the first injection of the mouse.

[0087] 2. Insulin tolerance (ITT) test:

[0088] The mice were fasted for 4 h and injected intraperitoneally with insulin (0.5 / 0.75 U / kg). Blood samples were collected from the tail vein at different time points (0, 30, 60, 90, and 120 min) after insulin administration, and blood glucose levels were measured using a blood glucose meter.

[0089] Testing steps:

[0090] (1) Dilute insulin: the working solution is 0.1 U / mL (take 25 μL insulin and add 9.975 mL sterile PBS);

[0091] (2) Prepare 1 mL syringe, pipette, scissors, alcohol cotton pads, blood glucose meter, and blood glucose test strips;

[0092] (3) Weigh the mice, record their weight, and calculate the amount of insulin required for each mouse based on their weight;

[0093] (4) Measure fasting blood glucose before injecting insulin, i.e., blood glucose value at 0 min;

[0094] (5) After the fasting blood glucose value is measured, insulin is injected, and then the blood glucose value at each time point is measured and recorded based on the time of the first injection of the mouse.

[0095] The test results are statistically shown as follows Figure 3 As shown in Figure 2, the results showed that TRIM65 knockout significantly reduced glucose tolerance ( Figure 3 A, B), and also reduce insulin sensitivity leading to insulin resistance ( Figure 3 C, D), indicating that TRIM65 knockout can promote the occurrence of NAFLD by increasing insulin resistance.

[0096] Example 4: Effect of systemic TRIM65 knockout on the levels of TG, TC, LDL-C and HDL-C in the serum of mice induced by a high-fat diet

[0097] Hyperlipidemia is also one of the causes of NAFLD. Therefore, the inventors further detected the levels of TG, TC, LDL-C and HDL-C in mouse serum.

[0098] The blood lipid analysis method is as follows: anesthetize the mouse with isoflurane, cut the beard (to prevent blood from getting on the beard and causing hemolysis), remove the eyeball, and obtain 750 μL of whole blood. Let it stand at room temperature for 1-2 hours, centrifuge at 2000 g for 30 minutes, take the upper serum into an EP tube, and store it at -80°C. Send it to Sevier Company, and use a biochemical analyzer to detect indicators including triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) and analyze the data. The results are as follows Figure 4 shown.

[0099] from Figure 4 The results showed that compared with the HFD-WT group, HFD-TRIM65 - / - Serum TG levels in group A mice Figure 4 A) content increased significantly, HDL-C ( Figure 4 C) content decreased significantly, although TC ( Figure 4 B) and LDL-C ( Figure 4 D) content also increased, but it was not statistically significant.

[0100] Example 5: Effect of systemic TRIM65 knockout on the levels of inflammatory factors in the liver and serum of mice induced by a high-fat diet

[0101] During the development of NAFLD, the accumulation of harmful lipids in liver cells leads to chronic inflammation, which in turn promotes the development of NAFLD. Therefore, the inventors further tested the changes in the levels of inflammatory factors IL-β, IL-6, and TNFα in the liver and serum of mice. The results of the analysis are as follows: Figure 5 shown.

[0102] from Figure 5 The results show that TRIM65 knockout significantly increased liver ( Figure 5 A) and increased the expression of inflammatory factors IL-β and IL-6 mRNA in serum ( Figure 5 B) The secretion of inflammatory factors IL-β and IL-6 in the liver, indicating that TRIM65 knockout significantly increased liver tissue and systemic inflammation.

[0103] Example 6: Effects of systemic TRIM65 knockout on the expression of genes related to lipid synthesis and catabolism in the liver of mice induced by a high-fat diet

[0104] When lipid synthesis increases or lipid decomposition decreases, lipid accumulation will occur. Since TRIM65 knockout can promote liver lipid accumulation, the inventors further detected the expression changes of genes related to lipid synthesis and decomposition in the liver after TRIM65 knockout.

[0105] from Figure 6 The results showed that compared with the HFD-WT group, HFD-TRIM65 - / - The expression of lipid synthesis genes SREBP1c and FASN in the liver of mice in group A was upregulated ( Figure 6 A, while the lipid degradation-related gene HSL ( Figure 6B) Decreased expression, indicating that TRIM65 knockout promotes hepatic lipid accumulation by upregulating the expression of lipid synthesis genes and inhibiting the expression of lipid degradation genes in the liver, thereby exacerbating the development of NAFLD.

[0106] In summary, after the TRIM65 systemic knockout mice were fed a high-fat diet to establish a NAFLD model, the present invention can significantly aggravate liver lipid accumulation, reduce glucose tolerance, increase insulin resistance, increase serum TG content and reduce HDL-C content, and increase the content of inflammatory factors in liver tissue and serum; we also found that TRIM65 knockout can promote liver lipid accumulation by upregulating the expression of lipid synthesis genes SREBP1c and FASN in the liver and downregulating the expression of lipid decomposition-related gene HSL. It shows that the loss of TRIM65 expression will cause NAFLD. Therefore, by using TRIM65 as a target and preparing a drug that can promote the high expression of TRIM65, it can provide new ideas and theoretical basis for the prevention and treatment of NAFLD in clinical practice.

[0107] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of TRIM65 as a target in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease, characterized in that: The TRIM65 can directly inhibit liver lipid accumulation by downregulating the expression of lipid synthesis genes SREBP1c and FASN in the liver and upregulating the expression of lipid decomposition-related gene HSL.

2. The use according to claim 1, characterized in that: The drug is a substance that can upregulate the expression of TRIM65.

3. The use according to claim 2, characterized in that: The drug comprises AAV-TRIM65 or TRIM65 purified protein.

4. The use according to claim 2, characterized in that: The drug is a Chinese herbal medicine that can upregulate the expression of TRIM65 protein or mRNA.

5. The use according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 2, characterized in that: The dosage form of the drug includes one or more of tablets, granules, solutions, emulsions, and suspensions.

7. The use according to any one of claims 1 to 6, characterized in that: Diseases associated with nonalcoholic fatty liver disease include cirrhosis, hepatocellular carcinoma, and one or more other chronic liver diseases.

Images