Method for preparing NAFLD animal model and application of GREB1 agonist in preparation of medicine for preventing or treating NAFLD

Abstract

According to the invention, the expression of GREB1 is knocked down in PVN of CD mice, the body weight and the one-week food intake of the mice are obviously improved, multiple biological indexes in plasma are obviously increased, the liver volume is obviously increased compared with a control group, the liver weight / body weight ratio is also obviously increased, lipid droplet deposition in the liver is obviously increased, and the expression level of adipogenic differentiation related proteins is obviously increased; the above prompts that after the expression of the GREB1 is knocked down by the PVN of the CD mouse, the NAFLD animal model can be obtained. The invention also discloses that after GREB1 is over-expressed by PVN of a mouse, the weight rise, total cholesterol and low-density lipoprotein increased in serum, increased liver volume and increased lipid droplet deposition in the liver of the mouse due to NAFLD diet induction can be improved. The above all prompt that the GREB1 agonist can prevent or treat the NAFLD disease.

Description

Technical Field

[0001] The present invention relates to a method for preparing a NAFLD animal model and application of a GREB1 agonist in preparing a medicine for preventing or treating NAFLD. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic chronic syndrome that includes a series of disease processes, from simple fatty degeneration to non-alcoholic fatty liver hepatitis, followed by cirrhosis and even liver cancer. NAFLD is mainly caused by excessive accumulation of TG (triglycerides) and TC (total cholesterol) in the liver. At present, NAFLD has rapidly become the most common disease in the world, accounting for 90% of liver diseases, with an incidence of about 30%-40% in adults and even as high as 80%-90% in obese people. Patients with NAFLD will have excessive fat accumulation in their bodies, which, if left untreated, will lead to the emergence of more diseases. NAFLD has gradually threatened human health, so we are urgently seeking potential drugs to treat NAFLD.

[0003] The paraventricular nucleus (PVN) is a nucleus in the hypothalamus. Anatomically, it is adjacent to the third ventricle and has many neurons that project to the posterior pituitary. These projecting neurons secrete oxytocin and small amounts of vasopressin, and the nucleus also secretes corticotropin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH). CRH and TRH are secreted into the pituitary portal system and act on different target neurons in the anterior pituitary. The PVN regulates many different functions through these different hormones, including osmoregulation, appetite, and the body's response to stress.

[0004] Growth regulating estrogen receptor binding 1 (GREB1) is an estrogen-responsive gene and an early response gene in the estrogen receptor regulatory pathway, which plays an important role in hormone-responsive tissues and cancers. However, its role in PVN is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a NAFLD animal model.

[0006] The technical solution adopted by the present invention is:

[0007] A method for preparing a NAFLD animal model, specifically knocking down the expression of GREB1 in the animal PVN.

[0008] Preferably, the method for knocking down the expression of GREB1 is to package the shRNA targeting GREB1 into a viral vector, wherein the sequence of the shRNA targeting GREB1 is shown in any one of SEQ ID No. 1-3.

[0009] Preferably, the structure of the successfully packaged viral vector is pAAV-U6-shRNA(GREB1)-CMV-EGFP-WPRE.

[0010] Preferably, the animal is a mouse.

[0011] The present invention also discloses the use of GREB1 shRNA in preparing a NAFLD animal model, wherein the GREB1 shRNA sequence is shown in any one of SEQ ID No. 1-3.

[0012] The invention also discloses the use of the GREB1 agonist in preparing medicine for preventing or treating NAFLD.

[0013] Preferably, the NAFLD agonist is sgRNA of GREB1, and its sequence is shown in SEQ ID No.5.

[0014] Preferably, the GREB1 sgRNA is packaged into a lentiviral vector.

[0015] When GREB1 expression was knocked down in the PVN of mice on control diets (CD), the body weight and weekly food intake of mice increased significantly, and biochemical indicators such as alanine aminotransferase ALT, aspartate aminotransferase AST, triglyceride TG, total cholesterol CHO, and low-density lipoprotein LDL in plasma increased significantly. The liver volume increased significantly compared with the control group, and the liver weight / body weight ratio also increased significantly. The lipid droplet deposition in the liver increased significantly, and the expression level of adipogenic differentiation-related proteins increased significantly. All of the above indicate that an animal model of NAFLD can be obtained by knocking down GREB1 expression in the PVN of CD mice.

[0016] The present invention also discloses that after GREB1 is overexpressed in the PVN of mice, the weight gain, increased total cholesterol and low-density lipoprotein in serum, increased liver volume, and increased lipid droplet deposition in the liver induced by NAFLD diet in mice can be improved. All of the above indicate that GREB1 agonists can prevent or treat NAFLD diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A map of adenoviral vectors.

[0018] Figure 2 This is the plasmid map of the empty lentiviral vector.

[0019] Figure 3 Plasmid map of the lentiviral vector for transcriptional activation of GREB1.

[0020] Figure 4 The results of immunofluorescence staining of brain slices from CD mice and NAFLD mice.

[0021] Figure 5 Statistical results of body weight and food intake after GREB1 knockdown in PVN of CD mice and NAFLD mice.

[0022] Figure 6 Statistical results of plasma biochemical indexes after GREB1 knockdown in PVN of CD mice and NAFLD mice.

[0023] Figure 7 Liver weight and liver Oil Red O staining results after GREB1 knockdown in PVN of CD mice and NAFLD mice.

[0024] Figure 8 These are the results of protein immunoblotting and real-time fluorescence quantitative PCR analysis of liver lipid metabolism indicators after GREB1 knockdown in PVN of CD mice and NAFLD mice.

[0025] Fig. 9 Results of plasma biochemistry, body weight, liver weight, and liver Oil Red O staining after PVN overexpression of GREB1 in NAFLD mice. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0027] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0028] Unless otherwise specified, the materials, reagents or instruments used in the following examples can be obtained from conventional commercial channels.

[0029] Example 1 Construction of an adeno-associated virus for knocking down GREB1 expression.

[0030] The interference targets selected by the adeno-associated virus for knocking down GREB1 expression are shown in Table 1 below. The interference vector selected is pscAAV-hSyn-EGFP-tWPA, and its map is shown in Figure 1As shown, the constructed adeno-associated virus structure is pAAV-U6-shRNA(GREB1)-CMV-EGFP-WPRE.

[0031] Table 1 Target sequences

[0032]

[0033] Example 2 Construction of a lentivirus that transcribes and activates GREB1

[0034] The conventional method was used to construct a lentivirus that activates GREB1 transcription. The viral vector structure before construction was LV-hSyn-SV40 NLS-dSaCas9-HA-2XSV40 NLS-VPR-nEF1α-Puro-T2A-EGFP-U6

[0035] -sasgRNA(BbsⅠ)-WPRE, such as Figure 2 The constructed viral vector structure is LV-hSyn-SV40NLS-dSaCas9-HA-2XSV40 NLS-VPR-nEF1α-Puro-T2A-EGFP-U6-sgRNA4(Greb1)

[0036] -WPRE, such as Figure 3 The sequence of sgRNA4 (Greb1) is shown in SEQ ID No.5.

[0037] Example 3 Immunofluorescence staining of GREB1 in the PVN of CD mice and NAFLD mice

[0038] The brain tissues of CD mice and NAFLD mice were fixed and then subjected to immunofluorescence staining. Figure 4 As shown, the results of immunofluorescence staining of brain slices showed that compared with the normal diet (CD) group, the expression of breast cancer estrogen-regulated protein (GREB1) in the paraventricular nucleus (PVN) of mice was reduced after they were given a NAFLD (induced non-alcoholic fatty liver disease) diet.

[0039] Example 4 Effects of knocking down GREB1 expression on CD mice and NAFLD mice

[0040] Adeno-associated virus knocking down GREB1 and control sequence virus were stereotactically injected into the bilateral paraventricular nucleus (PVN) of the CD group mice and the NAFLD group mice of the same age at 16 weeks old, respectively. The virus titer was 1.0*10 12 vg / mL, 100 nL was injected on each side, and the body weight and food intake were monitored for 6 consecutive weeks after virus injection. It was found that after GREB1 knockdown in PVN in the CD group, the body weight ( Figure 5 b) and food intake ( Figure 5 a) were significantly increased compared with the CD control group, but in the NAFLD group, the above changes were not significant ( Figure 5 a, b). At the same time, the biochemical indexes were tested by sampling the medial canthus venous blood of mice at 1, 3, 5, and 7 weeks after virus injection. It was found that after knocking down GREB1 in PVN, the plasma alanine aminotransferase (ALT) in the CD group was significantly decreased. Figure 6 a), aspartate aminotransferase AST ( Figure 6 b), triglyceride TG ( Figure 6 c), total cholesterol CHO ( Figure 6 d), low-density lipoprotein LDL ( Figure 6 f) levels were significantly higher than those in the CD control group, while high-density lipoprotein HDL ( Figure 6 e) levels were not significantly different, while in the NAFLD group, knockdown of GREB1 expression in PVN had little effect on the above plasma biochemicals ( Figure 6 af). At the 7th week after virus injection, i.e. the end point of the experiment, the livers of mice were harvested, photographed and weighed, and sections were prepared and stained. It was found that after PVN knockdown of GREB1 in the CD group, the liver volume increased significantly compared with the control group ( Figure 7 a), and the liver weight / body weight ratio (LW / BW) also increased significantly ( Figure 7 b), while in the NAFLD group, this trend was not significant. The results of Oil Red O staining of liver tissue showed that in the CD group, lipid droplet deposition in the liver increased significantly after GREB1 knockdown in PVN ( Figure 7 c), the statistical results also show this point ( Figure 7 d). At the same time, compared with the CD group, the liver lipid deposition in the NAFLD group was more severe ( Figure 7 c), but knocking down GREB1 expression in the PVN of NAFLD mice did not affect the level of hepatic lipid droplet deposition ( Figure 7 d). Western blotting experiments on liver tissues showed that after knocking down GREB1 in PVN in the CD group, the expression levels of adipogenic differentiation-related proteins PPARa, PPARy, and CPT1B were significantly increased ( Figure 8 a), while cholesterol metabolism related indicators such as ABCG5, ABCA1, and LXRa showed no significant differences ( Figure 8 a). At the same time, compared with the two CD groups, the expression levels of adipogenic differentiation-related proteins in the two NAFLD groups were higher ( Figure 8 a), but knocking down GREB1 expression in the PVN of NAFLD mice did not affect the levels of hepatic adipogenic differentiation-related proteins ( Figure 8 a). At the same time, the results of real-time fluorescence quantitative PCR for liver tissue showed that the adipogenic differentiation-related index, PPARa ( Figure 8 b), PPARy( Figure 8 c),FASN( Figure 8 d),CEBPa( Figure 8 e) showed a trend consistent with the protein level ( Figure 8 a).

[0041] The above results show that knocking down the expression of GREB1 in the PVN of CD mice can quickly produce a mouse model of NAFLD mice.

[0042] Example 5 Effect of overexpression of GREB1 on NAFLD mice

[0043] The GREB1 transcriptional activation lentivirus was stereotactically injected into the bilateral paraventricular nucleus (PVN) of the NAFLD group mice at 16 weeks of age, and the control virus was injected into the CD group mice of the same age at the same time. The virus titer was 1.0*10 12 vg / mL, 100 nL was injected on each side. After virus injection, the weight of mice was continuously monitored for 6 weeks. At the same time, the medial canthus venous blood of mice was collected for biochemical indicators 1-6 weeks after virus injection. The liver of mice was taken for photographing and weighing at the 6th week after virus injection, which was the end point of the experiment. It was found that after PVN overexpression of GREB1, the weight of NAFLD group was significantly reduced ( Fig. 9 a), total cholesterol CHO ( Fig. 9 b) and low-density lipoprotein (LDL) Fig. 9 c) levels were also significantly reduced. At the same time, after overexpression of GREB1 in the PVN of the NAFLD group, the liver volume was significantly reduced compared with the control group ( Fig. 9 d), and the liver weight / body weight ratio (LW / BW) was also significantly reduced ( Fig. 9 e) Oil red O staining of liver tissue showed that lipid droplet deposition in the liver of the NAFLD group was significantly reduced after GREB1 was overexpressed in the PVN ( Fig. 9 f), the statistical results also show this point ( Fig. 9 g). The above results show that overexpression of GREB1 in the PVN of NAFLD mice can improve plasma lipid biochemistry and liver lipid deposition in NAFLD mice.

[0044] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a NAFLD animal model, characterized in that: The expression of GREB1 was knocked down in the PVN of animals.

2. The method according to claim 1, characterized in that: The method for knocking down the expression of GREB1 is to package the shRNA targeting GREB1 into a viral vector, wherein the sequence of the shRNA targeting GREB1 is shown in any one of SEQ ID No. 1-3.

3. The method according to claim 2, characterized in that: The structure of the successfully packaged viral vector was pAAV-U6-shRNA(GREB1)-CMV-EGFP-WPRE.

4. The method according to any one of claims 1 to 3, characterized in that: The animal is a mouse.

5. Use of GREB1 shRNA in preparing a NAFLD animal model, wherein the GREB1 shRNA sequence is shown in any one of SEQ ID No. 1-3.

6. Use of GREB1 agonists in the preparation of drugs for preventing or treating NAFLD.

7. The use according to claim 6, characterized in that: The NAFLD agonist is sgRNA of GREB1, and its sequence is shown in SEQ ID No.

5.

8. The use according to claim 7, characterized in that: The GREB1 sgRNA was packaged into a lentiviral vector.

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