Application of targeted intervention of ANGPTL8 mRNA in the preparation of drugs for the treatment of hyperhomocysteinemia

Abstract

The present invention relates to the application of targeted intervention of ANGPTL8 mRNA in the preparation of drugs for treating hyperhomocysteinemia, and belongs to the field of biomedical technology. The present invention first discovered that targeted inhibition of ANGPTL8 gene expression level can effectively reduce the Hcy level in mouse serum, increase the expression level of CBS protein, and increase the activity of CBS protein, thereby achieving the effect of treating hHcy, and is applied to the research and development and preparation of drugs for treating hyperhomocysteinemia. In addition, shRNA that interferes with ANGPTL8 gene expression by knocking down the expression level of ANGPTL8 gene mRNA is designed, which successfully inhibits lipid deposition and inflammatory response of glomerular vascular endothelial cells caused by hyperhomocysteinemia, and restores the normal skeleton structure of endothelial cells. Therefore, the ANGPTL8 gene can be used as a therapeutic target to prepare drugs for treating renal damage caused by hyperhomocysteinemia.

Description

Technical Field

[0001] The present invention relates to application of targeted intervention of ANGPTL8 mRNA in preparing a drug for treating hyperhomocysteinemia, and belongs to the technical field of biomedicine. Background Art

[0002] Homocysteine ​​(Hcy) is an intermediate product of methionine metabolism in the body and a non-essential amino acid containing a sulfhydryl group. Abnormal methionine metabolism will lead to the accumulation of Hcy, an intermediate metabolite, in plasma. When the Hcy level in plasma rises above 10μM, it is defined as hyperhomocysteinemia (hHcy).

[0003] Hyperhomocysteinemia (hHcy) is a rare disease characterized by elevated plasma homocysteine ​​(Hcy) levels, and it has been shown that hHcy significantly increases the risk of coronary heart disease, peripheral vascular disease, chronic kidney disease, and cerebrovascular disease. As an important key enzyme for hHcy genetic deficiency, the main function of cystathionine β-synthetase (CBS) is to convert homocysteine ​​into other amino acids and reduce the concentration of homocysteine ​​in the blood. Reduced activity of CBS will lead to hyperhomocysteinemia. Therefore, in clinical applications, CBS is widely used for the detection of homocysteine ​​in the blood. Given that pathogenic risk factors such as hHcys require a long period of sustained action to lead to the occurrence of related diseases, there is still a sufficient time window for intervention treatment after the diagnosis of hyperHcyemia; however, there is currently no specific drug for hHcys, and existing intervention treatment methods are limited to supplementing food ingredients such as folic acid, vitamin B6, and B12, which are not effective for hHcys patients whose diet is not deficient in the above vitamins; therefore, the development of new anti-hHcys drugs targeting the disease state has important clinical value.

[0004] RNA interference (RNAi) technology is a commonly used gene silencing experimental tool. It specifically inhibits the expression of target genes by using specific RNA molecules to mediate the degradation of target genes or hinder their translation process. At present, this technology has important and extensive applications in gene function research, biological trait research and gene therapy. Since the success or failure of this technology mainly depends on the precise sequence pairing between small molecule RNA (including siRNA and shRNA) and the target gene, it is particularly important to select a highly efficient and specific shRNA to improve the interference efficiency of this technology. At the same time, the application of this technology to specifically silence highly pathogenic genes related to hHcys is of great significance for clarifying the new pathogenic mechanism of hHcys and finding its potential therapeutic targets. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of targeted intervention of ANGPTL8 mRNA in the preparation of a drug for treating hyperhomocysteinemia.

[0006] The technical solution of the present invention is as follows:

[0007] Application of ANGPTL8 mRNA as a target in the preparation of drugs for the treatment of hyperhomocysteinemia.

[0008] Preferably according to the present invention, the NCBI accession number of the ANGPTL8 mRNA is NM_018687.7.

[0009] Application of shRNA that specifically interferes with ANGPTL8 gene expression in the preparation of drugs for treating hyperhomocysteinemia.

[0010] Preferably according to the present invention, the nucleotide sequence of the shRNA that specifically interferes with the expression of the ANGPTL8 gene is as shown in SEQ ID NO.1.

[0011] Use of shRNA that specifically interferes with ANGPTL8 gene expression in the preparation of drugs for preventing / treating secondary diseases caused by hyperhomocysteinemia.

[0012] Preferably according to the present invention, the secondary diseases are diseases caused by hyperhomocysteinemia, including chronic kidney disease, cardiovascular and cerebrovascular diseases, Alzheimer's disease, stroke, venous thrombosis, neonatal defects and habitual abortion.

[0013] Preferably according to the present invention, the nucleotide sequence of the shRNA that specifically interferes with the expression of the ANGPTL8 gene is as shown in SEQ ID NO.1.

[0014] A drug for treating hyperhomocysteinemia is a nucleic acid molecule, carbohydrate, lipid, small molecule chemical drug, antibody drug, peptide, protein or virus that can specifically interfere with the expression of ANGPTL8 gene.

[0015] Preferably according to the present invention, the drug for treating hyperhomocysteinemia is a shRNA that specifically interferes with the expression of the ANGPTL8 gene, and the nucleotide sequence is shown in SEQ ID NO.1.

[0016] Beneficial effects:

[0017] 1. The present invention first discovered that targeted inhibition of ANGPTL8 gene expression levels can effectively reduce the Hcy level in mouse serum, increase the expression level of CBS protein, and increase the activity of CBS protein, thereby achieving the effect of treating hHcy. Therefore, the ANGPTL8 gene can be used as a drug target for the development and preparation of drugs for treating hyperhomocysteinemia. In addition, the present invention designs shRNA that interferes with ANGPTL8 gene expression by knocking down the expression level of ANGPTL8 gene mRNA, and successfully reverses the reduction of CBS expression and activity in hyperhomocysteinemia. Therefore, the ANGPTL8 gene can be used as a therapeutic target to prepare drugs for treating hyperhomocysteinemia.

[0018] 2. Using the ANGPTL8 gene provided by the present invention as a target, interfering with and inhibiting the expression level of the ANGPTL8 gene can also effectively inhibit the lipid deposition and inflammatory response of glomerular vascular endothelial cells caused by hyperhomocysteinemia, and restore the normal skeleton structure of vascular endothelial cells. In addition, the present invention designs shRNA that interferes with the expression of the ANGPTL8 gene by knocking down the mRNA expression level of the ANGPTL8 gene, successfully inhibiting the lipid deposition and inflammatory response of glomerular vascular endothelial cells caused by hyperhomocysteinemia, and restoring the normal skeleton structure of endothelial cells. Therefore, the ANGPTL8 gene can be used as a therapeutic target to prepare a drug for treating renal damage caused by hyperhomocysteinemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Reverse transcription-real-time fluorescence quantitative PCR and western blot analysis were used to detect the expression changes of CBS in the liver of each group of mice;

[0020] In the figure, A is the result of reverse transcription-real-time fluorescence quantitative PCR detection, and B is the result of protein immunoblotting analysis.

[0021] Figure 2 The enzyme activity of CBS in the liver of each group of mice was detected using an enzyme activity detection kit.

[0022] Figure 3 To transfect sh-ANGPTL8 into mouse hepatoma cells (Hepa1c1c-7), the mRNA level of ANGPTL8 was detected by reverse transcription-real-time fluorescence quantitative PCR, and the Student's t test was used to clarify the statistical significance of the differences among the groups.

[0023] Figure 4 To detect the expression changes of CBS in Hepa1c1c-7 cells cultured in vitro after silencing ANGPTL8 by reverse transcription-real-time fluorescence quantitative PCR and western blot analysis;

[0024] In the figure, A is the result of reverse transcription-real-time fluorescence quantitative PCR detection, and B is the result of protein immunoblotting analysis.

[0025] Figure 5 The enzyme activity detection kit was used to detect the enzyme activity of CBS in each group of Hepa1c1c-7 cells cultured in vitro.

[0026] Figure 6 To use dual luciferase assay to screen the downstream signaling pathways and key molecules that ANGPTL8 regulates CBS in hepatocytes.

[0027] Figure 7 To verify the downstream signaling pathways and key molecules of ANGPTL8 regulating CBS in hepatocytes by reverse transcription-real-time fluorescence quantitative PCR and western blot analysis;

[0028] In the figure, A is the result of reverse transcription-real-time fluorescence quantitative PCR detection, and B is the result of protein immunoblotting analysis.

[0029] Figure 8 Western blotting was used to analyze the effect of ANGPTL8 stimulation on the phosphorylation level of protein kinase B (AKT) in Hepa1c1c-7 cells.

[0030] Fig. 9 To transfect sh-ANGPTL8 into human glomerular endothelial cells (HGECs), the mRNA level of ANGPTL8 was detected by reverse transcription-real-time fluorescence quantitative PCR, and the Student's t test was used to clarify the statistical significance of the differences among the groups.

[0031] Fig.10 To transfect sh-ANGPTL8 into HGEC cells, western blot analysis was used to detect the changes of endothelial nitric oxide synthase (eNOS) and occludin in HGEC cells.

[0032] Fig.11 To transfect sh-ANGPTL8 into HGEC cells, reverse transcription-real-time fluorescence quantitative PCR and western blotting were used to detect the expression differences of intercellular adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in the cells.

[0033] Fig.12 To transfect sh-ANGPTL8 into HGEC cells, reverse transcription-real-time fluorescence quantitative PCR was used to detect the expression difference of phospholipid synthase in HGEC cells. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0035] Mouse hepatoma cells (Hepa1c1c-7) are available from ATCC.

[0036] Human glomerular endothelial cells (HGEC) are available from ATCC.

[0037] The ANGPTL8 gene knockout mouse (ANGPTL8 flox) was constructed by entrusting Shanghai Model Organisms Research Co., Ltd. according to the existing gene knockout method.

[0038] The plasmid expressing CBS Luc (PGL3-basic-CBS) was commissioned to be constructed and synthesized by Boshang Biotech (Jinan, China).

[0039] ANGPTL8 protein is commercially available from Wuxi OriGene Biotechnology Co., Ltd. (OriGene China).

[0040] Example 1: Construction of a mouse model of hyperhomocysteinemia

[0041] A mouse model of hyperhomocysteinemia was constructed using ANGPTL8 knockout mice, and the differences in homocysteine ​​levels in the mouse blood circulation were detected.

[0042] The specific implementation process is as follows:

[0043] (1) Prepare 24 6-week-old male C57 / BL6J normal mice and 24 6-week-old ANGPTL8 knockout mice;

[0044] (2) In the first week, both groups of mice were given a normal diet:

[0045] (3) In the second week, half of the mice in each group were given a FR diet (0 g folic acid / kg), and the other half continued to be given a diet containing sufficient folic acid (0.2 g folic acid / kg). A total of four groups of mice were obtained, namely the normal folic acid sufficient group (FS), the normal folic acid-free group (FR), the knockout mouse folic acid sufficient group (ANGPTL8 fl / fl +FS) and knockout mice without folate (ANGPTL8 fl / fl +FR), where the normal folic acid-free group of mice (FR) was a mouse model of hyperhomocysteinemia;

[0046] (4) After the above four groups of mice were fed for 10 weeks, the blood of each group of mice was collected using a test tube that did not contain pyrogens and endotoxins. The blood was then centrifuged at 3000 rpm for 10 minutes to quickly and carefully separate the serum and red blood cells.

[0047] (5) The serum homocysteine ​​level was detected using a mouse homocysteine ​​ELISA kit (Senbega, SBJ-M0905). The specific results are shown in Table 1.

[0048]

[0049] As shown in Table 1, compared with mice fed with sufficient folic acid (normal folic acid sufficient group), the concentration of homocysteine ​​in the blood circulation of normal mice fed with folic acid-free diet (normal folic acid-free group) increased significantly, indicating that the mouse model of hyperhomocysteinemia was successfully established. At the same time, compared with normal mice fed with folic acid-free diet (normal folic acid-free group), the level of homocysteine ​​(Hcy) in the serum of knockout mice fed with folic acid-free diet (knockout mice folic acid-free group) decreased significantly, which fully demonstrated that inhibiting the expression of ANGPTL8 can effectively reduce the level of homocysteine ​​(Hcy) in the serum of mice.

[0050] Example 2: Effect of ANGPTL8 knockout on CBS mRNA expression

[0051] A mouse model of hyperhomocysteinemia was constructed according to the method described in Example 1, and reverse transcription-real-time fluorescence quantitative PCR was used to detect the expression changes of cystathionine β-synthetase (CBS) mRNA, a key enzyme of hyperhomocysteinemia (hHcy) genetic deficiency, in the liver of mice.

[0052] The specific implementation process is as follows:

[0053] (1) The normal folate sufficient group (FS), normal folate deficient group (FR), knockout mouse folate sufficient group (ANGPTL8 fl / fl +FS) and knockout mice without folate (ANGPTL8 fl / fl Liver tissues were harvested from +FR) mice;

[0054] (2) Liver tissue was ground and lysed using TRAzol RNA extraction and lysis buffer (Dongsheng Biotechnology, R1021 / R1022);

[0055] (3) Collect the lysed liver tissue and isolate total RNA from cells using an RNA rapid extraction kit (ES Science);

[0056] (4) The integrity of the extracted RNA was tested by agarose gel electrophoresis, and the concentration of the extracted RNA was measured using the Tuohe Tnano-800;

[0057] (5) Using the RNA described in step (4) as a template, reverse transcription was performed using the Revertra Ace qPCR RT Kit (TOYOBO FSQ-101) to synthesize cDNA products;

[0058] (6) Real-time fluorescence quantitative PCR was performed using Ultra SYBR Mixture (CWBIO), the data were normalized to the expression of cellular actin, and analyzed using the 2-ΔΔCt method. The specific results are as follows: Figure 1 As shown in A.

[0059] Depend on Figure 1 A shows that the expression level of CBS mRNA in the liver of mice in the normal folic acid group (FR) was significantly lower than that in the normal folic acid sufficient group (FS). fl / fl Compared with the normal folic acid-free group (FR), the expression level of CBS mRNA in the liver of mice in the folic acid-free group (+FR) was significantly increased, which fully demonstrated that inhibiting the expression of ANGPTL8 can effectively increase the expression level of CBS mRNA, thereby achieving the effect of treating hyperhomocysteinemia (hHcy).

[0060] Example 3: Effect of ANGPTL8 knockout on cystathionine β-synthetase (CBS) protein expression

[0061] A mouse model of hyperhomocysteinemia was constructed according to the method described in Example 1, and reverse transcription-real-time fluorescence quantitative PCR was used to detect the protein expression changes of CBS, a key enzyme of hyperhomocysteinemia (hHcy) genetic deficiency, in the liver of mice.

[0062] The specific implementation process is as follows:

[0063] (1) The normal folate sufficient group (FS), normal folate deficient group (FR), knockout mouse folate sufficient group (ANGPTL8 fl / fl +FS) and knockout mice without folate (ANGPTL8 fl / fl Liver tissues were harvested from +FR) mice;

[0064] (2) Liver tissue was lysed with RIPA lysis buffer (Aladdin, R301899-100ml) on ice for 15 min;

[0065] (3) Collect the lysed liver tissue and measure the protein concentration using the BCA protein concentration detection kit (Shangbao Bio, 34001);

[0066] (4) Separate the proteins using 10% SDS-PAGE and transfer the proteins to PVDF membrane;

[0067] (5) After labeling with CBS antibodies (MCE, YA2148), the target protein bands were observed by chemiluminescence. The specific results are as follows: Figure 1 As shown in B.

[0068] Depend on Figure 1 B shows that the protein expression level of cystathionine β-synthetase (CBS) in the liver of mice was significantly reduced compared with the normal folate sufficient group (FS). fl / fl Compared with the normal folic acid-free group (FR), the protein expression level of cystathionine β-synthetase (CBS) in the liver of mice in the folic acid-free group (FR) was significantly increased, which fully demonstrated that inhibiting the expression of ANGPTL8 can effectively increase the expression level of CBS protein, thereby achieving the effect of treating hyperhomocysteinemia (hHcy).

[0069] Example 4: Effect of ANGPTL8 knockout on cystathionine β-synthetase (CBS) activity

[0070] A mouse model of hyperhomocysteinemia was constructed according to the method described in Example 1, and reverse transcription-real-time fluorescence quantitative PCR was used to detect the protein expression changes of cystathionine β-synthetase (CBS), a key enzyme of hHcy genetic deficiency, in the liver of mice.

[0071] The specific implementation process is as follows:

[0072] (1) The normal folate sufficient group (FS), normal folate deficient group (FR), knockout mouse folate sufficient group (ANGPTL8 fl / fl +FS) and knockout mice without folate (ANGPTL8 fl / fl 500 mg of liver tissue was removed from +FR) mice and weighed;

[0073] (2) Use the cell CBS activity analysis kit-colorimetric method (GENMED, GMS50546.1) to detect the enzyme activity of CBS. For specific methods, refer to the instructions of the kit. The specific results are as follows: Figure 2 shown.

[0074] Depend on Figure 2 It can be seen that the activity of CBS in the liver of mice in the normal folic acid-deficient group (FR) was significantly reduced compared with the normal folic acid-sufficient group (FS). fl / fl Compared with the normal folic acid-free group (FR), the activity of CBS in the liver of mice fed folate+FR was significantly increased, which fully demonstrated that inhibiting the expression of ANGPTL8 can effectively increase the activity of CBS protein, thereby achieving the effect of treating hyperhomocysteinemia (hHcy).

[0075] Example 5. Effect of sh-ANGPTL8 that specifically interferes with ANGPTL8 gene expression on ANGPTL8 mRNA expression level in mouse hepatocellular carcinoma cells (Hepa1c1c-7)

[0076] Based on the nucleotide sequence of ANGPTL8 mRNA, sh-ANGPTL8 which can specifically knock down the expression level of ANGPTL8 mRNA was designed. The same doses of sh-ANGPTL8 and sh-NC were transfected into Hepa1c1c-7 cells, respectively, and the ANGPTL8 mRNA level was detected by reverse transcription-real-time fluorescence quantitative PCR.

[0077] Among them, the nucleotide sequence of sh-ANGPTL8 is: 5'-GCCTACCGAGAATTTGAGGTC-3' (SEQ ID NO.1), and sh-NC is an existing public sequence, which was artificially synthesized by Shanghai GenePharma Company, China according to the sequence information.

[0078] The specific implementation process is as follows:

[0079] (1) Hepa1c1c-7 cells were selected and inoculated and cultured in a 6-well plate one day before transfection;

[0080] (2) When the degree of confluence reached 70%, the liposome transfection reagent Lipofectamine™ 2000 Transfection Reagent (Invitrogen™, 11668019) was used to transfect the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC into Hepa1c1c-7 cells, respectively, and they were recorded as sh-ANGPTL8 group and Control group;

[0081] (3) After 48 hours of transfection, cells from each group were collected and the level of ANGPTL8 mRNA was detected by reverse transcription-real-time fluorescence quantitative PCR. The specific method is shown in Example 3. The specific results are as follows: Figure 3 shown.

[0082] Depend on Figure 3 It can be seen that transfection of sh-ANGPTL8 can effectively reduce the level of ANGPTL8 mRNA in Hepa1c1c-7 cells and interfere with the expression of ANGPTL8 gene.

[0083] Example 6: Effect of silencing ANGPTL8 on CBS mRNA expression

[0084] The same doses of sh-ANGPTL8 and sh-NC were first transfected into Hepa1c1c-7 cells, respectively. Then, 100 μM Hcy was used to stimulate two groups of Hepa1c1c-7 cells by co-incubation. Finally, reverse transcription-real-time fluorescence quantitative PCR was used to detect the mRNA level of CBS in the cells.

[0085] The specific implementation process is as follows:

[0086] (1) Normal Hepa1c1c-7 cells were divided into two groups and transfected with the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC, respectively. Then, one group of transfected cells was selected from each of the two groups of cells and incubated with 100 μM Hcy for 24 h. A total of 4 groups of cells were obtained, which were recorded as Control group, Hcy group, sh-ANGPTL8 group and sh-ANGPTL8+Hcy group. The specific transfection method is shown in Example 5.

[0087] (2) After culturing for 48 hours, the cells in each group were collected and the ANGPTL8 mRNA level in each group of cells was detected by reverse transcription-real-time fluorescence quantitative PCR. The specific method is shown in Example 2. The specific results are as follows: Figure 4 As shown in A.

[0088] Depend on Figure 4 A shows that transfection of sh-ANGPTL8 can effectively reverse the reduced CBS level in Hepa1c1c-7 cells due to Hcys stimulation, thereby achieving the effect of treating hyperhomocysteinemia (hHcy).

[0089] Example 7 Effect of sh-ANGPTL8 that specifically interferes with ANGPTL8 gene expression on the expression level of CBS protein in mouse hepatoma cells (Hepa1c1c-7 cells)

[0090] The same doses of sh-ANGPTL8 and sh-NC were first transfected into Hepa1c1c-7 cells, respectively. Then, 100 μM Hcy was used to stimulate two groups of Hepa1c1c-7 cells by co-incubation. Finally, protein immunoblotting was used to detect the changes in the expression level of CBS in Hepa1c1c-7 cells.

[0091] The specific implementation process is as follows:

[0092] (1) Normal Hepa1c1c-7 cells were divided into two groups and transfected with the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC, respectively. Then, one group of transfected cells was selected from each of the two groups of cells and incubated with 100 μM Hcy for 24 h. A total of 4 groups of cells were obtained, which were recorded as Control group, Hcy group, sh-ANGPTL8 group and sh-ANGPTL8+Hcy group. The specific transfection method is shown in Example 5.

[0093] (2) After culturing for 48 hours, the cells in each group were collected and the CBS (MCE, YA2148) protein level in each group of cells was detected by western blotting. The specific method was the same as that in Example 3. The specific results are shown in FIG. Figure 4 As shown in B.

[0094] Depend on Figure 4 B shows that transfection of sh-ANGPTL8 can effectively reverse the CBS level in Hepa1c1c-7 cells that was reduced by Hcys stimulation. This fully demonstrates that inhibiting the expression of ANGPTL8 can increase the CBS expression level in Hepa1c1c-7 cells and treat hyperhomocysteinemia (hHcy).

[0095] Example 8: Effect of ANGPTL8 knockout on cystathionine β-synthetase (CBS) activity

[0096] The same doses of sh-ANGPTL8 and sh-NC were first transfected into Hepa1c1c-7 cells, respectively. Then, 100 μM Hcy was used to stimulate two groups of Hepa1c1c-7 cells by co-incubation. Finally, protein immunoblotting was used to detect the changes in the expression level of CBS in Hepa1c1c-7 cells.

[0097] The specific implementation process is as follows:

[0098] (1) Normal Hepa1c1c-7 cells were divided into two groups and transfected with the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC, respectively. Then, one group of transfected cells was selected from each of the two groups of cells and incubated with 100 μM Hcy for 24 h. A total of 4 groups of cells were obtained, which were recorded as Control group, Hcy group, sh-ANGPTL8 group and sh-ANGPTL8+Hcy group. The specific transfection method is shown in Example 5.

[0099] (2) After 48 hours of continuous culture, the cells in each group were collected and the CBS enzyme activity of each group of cells was detected using the cell CBS activity analysis kit-colorimetric method (GENMED, GMS50546.1). The specific method is referred to the kit manual. The specific results are as follows: Figure 5 shown.

[0100] Depend on Figure 5 It can be seen that transfection of sh-ANGPTL8 can effectively reverse the reduced CBS activity in Hepa1c1c-7 cells due to Hcys stimulation. This fully demonstrates that inhibiting the expression of ANGPTL8 can effectively increase the activity of CBS protein, thereby achieving the effect of treating hyperhomocysteinemia (hHcy).

[0101] Example 9: The dual luciferase assay was used to screen the downstream signaling pathways and key molecules of ANGPTL8 regulating CBS in hepatocytes.

[0102] Hepa1c1c-7 cells were transfected with a plasmid expressing CBS Luc (PGL3-basic-CBS), and then the cells were treated with ANGPTL8 protein and the pathway inhibitor to be screened, respectively. The expression level of the target gene was detected using the dual luciferase system to screen and verify the downstream signaling pathways and key molecules of CBS in hepatocytes regulated by ANGPTL8.

[0103] The specific implementation process is as follows:

[0104] (1) The promoter sequence of CBS was amplified from mouse genomic DNA, and then cloned into the PGL3-basic vector using the KpnI and MluI restriction sites to obtain the plasmid PGL3-basic-CBS;

[0105] (2) According to the instructions of the kit, 100 ng of plasmid PGL3-basic-CBS was transfected into Hepa1c1c-7 cells using Lipofectamine 3000 (Invitrogen, CA, USA);

[0106] (3) 36 hours after transfection, the transfected cells were incubated with ANGPTL8 protein, horse serum and the signaling pathway inhibitor to be screened for 2 hours, respectively, to obtain five groups of treated cells: Vehicle-1, Vehicle-2, U0126, SB203580 and Wortmannin; Vehicle-1 was treated with 50% horse serum, and Vehicle-2 was treated with 40 nM ANGPTL8 protein;

[0107] The signal pathway inhibitors to be screened and the co-incubation concentrations are 10 μM SB203580 (P38 MAPK inhibitor; Selleck, 152121-47-6), 10 μM Wortmannin (AKT inhibitor; Selleck, 19545-26-7) and 10 μM U0126 (ERK1 / 2 inhibitor; Selleck, 1173097-76-1);

[0108] (4) The relative luciferase activity of the five groups of cells after treatment was measured using the dual luciferase system (Promega, Madison, WI, USA). The specific results are as follows: Figure 6 shown.

[0109] Depend on Figure 6 It can be seen that wortmannin (AKT inhibitor) eliminated the interaction between ANGPTL8 and CBS, indicating that AKT may mediate the effect of ANGPTL8 on CBS and may achieve the effect of treating hHcy through the ANGPTL8-AKT-CBS pathway.

[0110] Example 10: Verification of downstream signaling pathways and key molecules of ANGPTL8 regulating CBS in hepatocytes by reverse transcription-real-time fluorescence quantitative PCR and western blot analysis

[0111] Hepa1c1c-7 cells were treated with ANGPTL8 protein alone or in combination with various signaling pathway inhibitors. The expression of CBS was analyzed by reverse transcription-real-time fluorescence quantitative PCR and protein immunoblotting analysis to verify the downstream signaling pathways and key molecules of CBS regulated by ANGPTL8 in hepatocytes.

[0112] The specific implementation process is as follows:

[0113] (1) Hepa1c1c-7 cells were selected and incubated with ANGPTL8 protein, horse serum and the signal pathway inhibitor to be screened for 2 hours. The specific ANGPTL8 signal pathway inhibitor and the co-incubation concentration were the same as those in Example 5.

[0114] (2) Reverse transcription-real-time fluorescence quantitative PCR was used to detect the CBS mRNA level of each group of cells. The specific detection method was the same as that in Example 2;

[0115] (3) Western blotting was used to detect the CBS protein level. The specific detection method was the same as in Example 3. The specific results are as follows: Figure 7 shown.

[0116] Depend on Figure 7 It can be seen that wortmannin (AKT inhibitor) eliminated ANGPTL8-induced CBS transcription and translation, while other pathway inhibitors could not do this. These data further indicate that AKT may be involved in mediating the effect of ANGPTL8 on the expression of CBS, that is, ANGPTL8 affects CBS expression, translation and protein activity through the AKT pathway, further confirming that the effect of treating hyperhomocysteinemia (hHcy) can be achieved through the ANGPTL8-AKT-CBS pathway.

[0117] Example 11: Verification of ANGPTL8 triggering phosphorylation of AKT protein

[0118] Western blotting was used to analyze the phosphorylation level of AKT (p-AKT) in Hepa1c1c-7 cells stimulated with ANGPTL8 for the indicated time periods.

[0119] The specific implementation process is as follows:

[0120] (1) Hepa1c1c-7 cells were selected and stimulated with 40 nM ANGPTL8 protein for 5 min, 15 min, and 30 min respectively by co-incubation. At the same time, PBS buffer with the same amount and time was used as a negative control;

[0121] (2) Collect cell samples and use Western blotting to detect the phosphorylation level of AKT. The specific method is the same as Example 3. The specific results are as follows: Figure 8 shown.

[0122] Depend on Figure 8 It can be seen that ANGPTL8 can trigger the phosphorylation of AKT protein, that is, ANGPTL8 affects CBS expression, translation and protein activity by triggering the phosphorylation of AKT protein, and can achieve the effect of treating hyperhomocysteinemia (hHcy) through the ANGPTL8-AKT-CBS pathway.

[0123] Example 12 Effect of sh-ANGPTL8 that specifically interferes with ANGPTL8 gene expression on ANGPTL8 mRNA expression levels in glomerular endothelial cells (HGECs)

[0124] Based on the nucleotide sequence of ANGPTL8 mRNA, sh-ANGPTL8 which can specifically knock down the expression level of ANGPTL8 mRNA was designed. The same doses of sh-ANGPTL8 and sh-NC were transfected into HGEC cells, respectively, and the ANGPTL8 mRNA level was detected by reverse transcription-real-time fluorescence quantitative PCR.

[0125] Among them, the nucleotide sequence of sh-ANGPTL8 is: 5'-GCCTACCGAGAATTTGAGGTC-3' (SEQ ID NO.1), and sh-NC is an existing published sequence.

[0126] The specific implementation process is as follows:

[0127] (1) Select glomerular endothelial cells (HGECs), seed and culture the cells in 6-well plates one day before transfection;

[0128] (2) When the degree of confluence reached 70%, the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC were transfected into HGEC cells using Lipofectamine™ 2000 Transfection Reagent (Invitrogen™, 11668019), respectively, and recorded as sh-ANGPTL8 group and Control group;

[0129] (3) After 48 hours of transfection, cells from each group were collected and the level of ANGPTL8 mRNA was detected by reverse transcription-real-time fluorescence quantitative PCR. The specific method was as shown in Example 2. The specific results are as follows: Fig. 9 shown.

[0130] Depend on Fig. 9 It can be seen that transfection of sh-ANGPTL8 can effectively reduce the level of ANGPTL8 mRNA in HGEC cells and interfere with the expression of ANGPTL8 gene.

[0131] Example 13 Effects of sh-ANGPTL8 that specifically interferes with ANGPTL8 gene expression on the expression levels of eNOS and Occludin in glomerular endothelial cells (HGECs)

[0132] The same doses of sh-ANGPTL8 and sh-NC were transfected into HGEC cells, respectively. Then, 100 μM Hcy was used to stimulate two groups of HGEC cells by co-incubation. Finally, western blotting was used to detect the changes in the expression levels of endothelial nitric oxide synthase (eNOS) and occludin in HGEC cells.

[0133] The specific implementation process is as follows:

[0134] (1) Normal HGEC cells were divided into two groups and transfected with the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC, respectively. Then, one group of transfected cells was selected from each of the two groups of cells and co-incubated with 100 μM Hcy for 24 h. A total of 4 groups of cells were obtained, which were recorded as Control group, Hcy group, sh-ANGPTL8 group and sh-ANGPTL8+Hcy group. The specific transfection method is shown in Example 12.

[0135] (2) After culturing for 48 hours, the cells in each group were collected and the levels of eNOS (antibody: Abcam, ab199956) and Occludin (antibody: Abcam, ab222691) proteins in each group were detected by western blotting. The specific method was the same as that in Example 3. The specific results are shown in Table 3. Fig.10 shown.

[0136] Depend on Fig.10 It can be seen that transfection of sh-ANGPTL8 can effectively reverse the reduced eNOS and Occludin levels in the HGEC cell supernatant due to Hcys stimulation and maintain the normal structure and function of glomerular endothelial cells. This fully demonstrates that inhibiting the expression of ANGPTL8 can reduce the Hcy level in HGEC cells, treat hyperhomocysteinemia (hHcy), and thus affect the function of glomerular endothelial cells.

[0137] Example 14 Effect of sh-ANGPTL8 that specifically interferes with ANGPTL8 gene expression on the expression levels of intercellular adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in glomerular endothelial cells (HGECs)

[0138] The same doses of sh-ANGPTL8 and sh-NC were transfected into HGEC cells, respectively. Then, two groups of HGEC cells were stimulated with 100 μM Hcy. Finally, reverse transcription-real-time fluorescence quantitative PCR and protein immunoblotting were used to detect the expression differences of intercellular adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in the cells.

[0139] The specific implementation process is as follows:

[0140] (1) Normal HGEC cells were divided into two groups and transfected with the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC, respectively. Then, one group of transfected cells was selected from each of the two groups of cells and co-incubated with 100 μM Hcy for 24 h. A total of 4 groups of cells were obtained, which were recorded as Control group, Hcy group, sh-ANGPTL8 group and sh-ANGPTL8+Hcy group. The specific transfection method is shown in Example 12.

[0141] (2) After culturing for 48 hours, the cells in each group were collected and the ICAM1 and VCAM1 mRNA levels in each group of cells were detected by reverse transcription-real-time fluorescence quantitative PCR. The specific method is as shown in Example 2.

[0142] (3) Western blotting was used to detect the protein levels of ICAM1 (antibody: MCE, YA352) and VCAM1 (antibody: Abcam, ab215380) in each group of cells. The specific method is shown in Example 3. The specific results are as follows: Fig.11 shown.

[0143] Depend on Fig.11 It can be seen that transfection of sh-ANGPTL8 can effectively reverse the elevated ICAM1 and VCAM1 levels in HGEC cells caused by Hcys stimulation and inhibit the development of glomerular endothelial cell inflammation.

[0144] Example 15 Effect of sh-ANGPTL8 that specifically interferes with ANGPTL8 gene expression on the expression levels of phospholipid synthases (ETNK2, LPCAT3, PEMT) in glomerular endothelial cells (HGECs)

[0145] The same doses of sh-ANGPTL8 and sh-NC were transfected into HGEC cells, respectively. Then, two groups of HGEC cells were stimulated with 100 μM Hcy. Finally, reverse transcription-real-time fluorescence quantitative PCR was used to detect the mRNA levels of cellular phospholipid synthases (ETNK2, LPCAT3, and PEMT) in the cells.

[0146] The specific implementation process is as follows:

[0147] (1) Normal HGEC cells were divided into two groups and transfected with the same dose (10 μM, 3 μl) of sh-ANGPTL8 and sh-NC, respectively. Then, one group of transfected cells was selected from each of the two groups of cells and co-incubated with 100 μM Hcy for 24 h. A total of 4 groups of cells were obtained, which were recorded as Control group, Hcy group, sh-ANGPTL8 group and sh-ANGPTL8+Hcy group. The specific transfection method is shown in Example 12.

[0148] (2) After culturing for 48 hours, the cells in each group were collected and the mRNA levels of ETNK2, LPCAT3 and PEMT in each group were detected by reverse transcription-real-time fluorescence quantitative PCR. The specific method is shown in Example 2. The specific results are as follows: Fig.12 shown.

[0149] Depend on Fig.12 It can be seen that transfection of sh-ANGPTL8 can effectively reverse the elevated levels of phospholipid synthases (ETNK2, LPCAT3, PEMT) in HGEC cells due to Hcys stimulation and inhibit the accumulation of phospholipids in glomerular endothelial cells. Combined with the results of Examples 13 and 14, it can be seen that ANGPTL8 mRNA can be used not only as a target for the preparation of drugs for the treatment of hyperhomocysteinemia, but also for the preparation of drugs for the prevention / treatment of chronic kidney disease, a secondary disease caused by hyperhomocysteinemia.

Claims

1. Use of shRNA that specifically interferes with ANGPTL8 gene expression in the preparation of a drug for treating hyperhomocysteinemia, characterized in that: The nucleotide sequence of the shRNA that specifically interferes with the expression of the ANGPTL8 gene is shown in SEQ ID NO.1; the NCBI accession number of the ANGPTL8 mRNA is NM_018687.

7.

2. Use of shRNA that specifically interferes with ANGPTL8 gene expression in the preparation of a drug for treating chronic kidney disease caused by hyperhomocysteinemia, characterized in that: The nucleotide sequence of the shRNA that specifically interferes with the expression of the ANGPTL8 gene is shown in SEQ ID NO.1; the NCBI accession number of the ANGPTL8 mRNA is NM_018687.

7.

3. A drug for treating hyperhomocysteinemia, characterized in that: The drug for treating hyperhomocysteinemia is a shRNA that specifically interferes with the expression of the ANGPTL8 gene, and the nucleotide sequence is shown in SEQ ID NO.1.

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