Application of 5-methyltetrahydrofolic acid in preparation of medicine for treating renal fibrosis

Abstract

The invention relates to application of 5-methyltetrahydrofolic acid in preparation of a medicine for preventing or treating renal fibrosis. It is found that 5-methyltetrahydrofolic acid can inhibit expression of HK2 cells and animal kidney fibrosis injury markers COL-1, alpha-SMA and FN, reduce the cell fibrosis level, relieve mouse kidney fibrosis and improve histopathological changes in the kidney fibrosis process, and has remarkable prevention and treatment effects on chronic kidney injury.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to application of 5-methyltetrahydrofolate in treating renal fibrosis. Background Art

[0002] Chronic kidney disease (CKD) is a global public health crisis. Epidemiological studies have shown that more than 800 million people suffer from CKD, with a greater burden in middle-income and low-income countries, and the incidence of CKD in my country is significantly higher than the global average. It is estimated that by 2040, chronic kidney disease will become the fifth leading cause of life loss worldwide. The main pathological characteristics of CKD are tubular atrophy, interstitial fibrosis, and glomerular sclerosis. Among them, renal fibrosis, especially tubulointerstitial fibrosis, is the common final pathway of almost all progressive chronic kidney diseases. In CKD patients, regardless of the initial cause, renal fibrosis is the main cause of renal function loss. Renal fibrosis refers to the pathological process in which normal structures in renal tissue are replaced by fibrous tissue. Specifically, renal fibrosis is a process in which abnormal accumulation of extracellular matrix (ECM) components (such as collagen and fibronectin) in the glomeruli, tubules, and renal interstitium caused by various reasons (such as chronic inflammation, persistent hypertension, diabetes, drug toxicity, or immune response) leads to irreversible damage to renal structure and function. Among them, tubular epithelial cells are the most abundant kidney-resident cells and are the strongest predictor of disease progression in terms of renal pathology, tubulointerstitial fibrosis. When damaged, they lose their phenotype, age, and secrete pro-inflammatory and pro-fibrotic cytokines, perpetuating the damage and leading to the progression of tubulointerstitial fibrosis.

[0003] 5-Methyltetrahydrofolate (Levomefolic acid), also known as active folic acid, is a derivative of folic acid. As a member of the vitamin B9 family, 5-methyltetrahydrofolate is an essential micronutrient. Since humans cannot produce folic acid from scratch, they need to meet metabolic needs through dietary intake. Active folic acid, which usually works in a reduced form in the human body, is an essential substance in the metabolic process. Its uses include nucleotide synthesis required for cell proliferation, cysteine ​​cycle, homocysteine ​​regulation, and the supply of methyl groups required for methylation reactions (such as methylation reactions required for epigenetics). The main understanding of folic acid at present is that there is a correlation between insufficient folic acid supply and neural tube defects, but there is no research on whether 5-methyltetrahydrofolate has the effect of preventing or treating renal fibrosis.

[0004] The main clinical treatments for renal fibrosis are renal replacement therapy (RRT) and dialysis. Although they play an important role, they also have defects, such as dependence on equipment and professionals, risk of infection and complications, vascular access problems, high costs, and inability to reverse renal failure. Currently, there are almost no approved treatments specifically for renal fibrosis, so the development of a new drug to prevent or treat renal fibrosis has broad clinical application prospects. Summary of the invention

[0005] To solve the above technical problems, the present invention includes the following aspects:

[0006] A first aspect of the present invention provides use of 5-methyltetrahydrofolate in the preparation of a medicament for preventing or treating renal fibrosis.

[0007] Preferably, the drug includes a pharmaceutically acceptable carrier or excipient.

[0008] Preferably, the drug is an oral preparation, a transdermal preparation or an injectable preparation.

[0009] Preferably, the dosage form of the drug is tablet, capsule, granule, oral solution, patch, ointment, gel or injection.

[0010] The second aspect of the present invention provides the use of 5-methyltetrahydrofolate in the preparation of a medicament for preventing or treating acute kidney injury turning into chronic kidney disease.

[0011] Preferably, the drug includes a pharmaceutically acceptable carrier or excipient.

[0012] Preferably, the drug is an oral preparation, a transdermal preparation or an injectable preparation.

[0013] Preferably, the dosage form of the drug is tablet, capsule, granule, oral solution, patch, ointment, gel or injection.

[0014] The third aspect of the present invention provides the use of 5-methyltetrahydrofolate in the preparation of a drug for reducing the expression level of cell fibrosis markers COL-1, α-SMA and / or FN.

[0015] Preferably, the present invention provides the use of 5-methyltetrahydrofolate in the preparation of a drug for reducing the expression levels of cell fibrosis markers COL-1, α-SMA and FN.

[0016] Preferably, the drug includes a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, the drug is an oral preparation, a transdermal preparation or an injectable preparation.

[0018] Preferably, the dosage form of the drug is tablets, capsules, granules, oral liquid, patches, ointments, gels, intravenous injections or intramuscular injections.

[0019] The technical effects produced by the present invention are:

[0020] The present invention unexpectedly discovered that 5-methyltetrahydrofolate can inhibit the expression of COL-1, α-SMA and FN, the fibrosis markers of HK2 cells and damaged animals' kidneys, reduce the level of cellular fibrosis, alleviate renal fibrosis in mice, improve the pathological changes of renal tissues in chronic kidney disease, and has a significant preventive and therapeutic effect on chronic kidney damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the dosing regimen for establishing a mouse model of acute to chronic renal injury and renal fibrosis using bilateral renal artery ischemia-reperfusion in Experimental Example 1 of the present invention;

[0022] Figure 2 The results of Western blot and Real-time PCR detection of fibrosis indexes in each test group in Test Example 1 of the present invention;

[0023] Figure 3 The results of immunofluorescence (IF) detection of the expression changes of COL-1 and α-SMA in each test group in Test Example 1 of the present invention are as follows;

[0024] Figure 4 The figure is a staining result of kidney tissue of mice in each test group using Masson staining kit in Test Example 1 of the present invention;

[0025] Figure 5 The results of Western blot and Real-time PCR detection of fibrosis indexes in HK2 cells of each test group in Test Example 2 of the present invention;

[0026] Figure 6 These are the results of immunofluorescence (IF) detection of changes in α-SMA expression in each test group in Test Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The 5-methyltetrahydrofolate used in the test examples of the present invention was purchased from MedChemExpress, USA (HY-14781), and its chemical structural formula is The English abbreviation is LA (Levomefolic acid).

[0028] Experimental Example 1: Effect of 5-methyltetrahydrofolate on IRI-induced renal fibrosis in mice

[0029] The ischemia-reperfusion injury model of both kidneys in mice was used for renal fibrosis modeling. 6-8 week old C57BL / 6 mice were adaptively raised for 7 days and anesthetized by intraperitoneal injection of 5% chloral hydrate (0.1ml / 10g). A 0.8-1.0cm incision was made on both sides of the back of the mouse. After finding the renal pedicle, the renal pedicles on both sides were quickly blocked with non-damaging micro-arterial clamps. The kidneys turned from red to purple-black, indicating successful clamping, and the clamping lasted for 40min. The back skin of the mouse was closed in two layers using standard 6-0 sutures. During the entire operation, the body temperature was maintained between 35℃-37.5℃, the vital signs of the mice were closely observed, and water and feed were supplemented. Blood and kidney samples of the mice were collected 14 days after modeling for further study.

[0030] The mice were divided into control group (NC group), simple drug administration group (10 mg / kg LA, HD group), model group (IRI group), 5-methyltetrahydrofolate low-dose group (IRI+2.5 mg / kg LA, IRI+LD group), medium-dose group (IRI+5 mg / kg LA, IRI+MD group) and high-dose group (IRI+10 mg / kg LA, IRI+HD group), with 6 mice in each group. The mice in each drug administration group were gavaged with 5-methyltetrahydrofolate once a day before IRI and on the 2nd, 4th, 6th, 8th, 10th, 12th and 14th day after IRI, and the kidney samples of the mice were collected on the 14th day (dosage schedule see Figure 1 ).

[0031] Western blot: extract tissue protein and perform immunoblotting experiment. Figure 2 As shown in A (each test group shows the test results of two samples), the level of α-SMA in renal tissue was significantly increased in the IRI-induced renal fibrosis model, while the expression of α-SMA was significantly inhibited after 5-methyltetrahydrofolate treatment, suggesting that 5-methyltetrahydrofolate has a good effect in inhibiting renal fibrosis.

[0032] Real-time PCR: Extract tissue RNA, reverse transcribe, and amplify. Real-time PCR results are as follows: Figure 2 As shown in B, the levels of COL-1, α-SMA and FN in renal tissue were significantly increased in the IRI-induced renal fibrosis model, while the expression of COL-1, α-SMA and FN was significantly inhibited after 5-methyltetrahydrofolate treatment, suggesting that 5-methyltetrahydrofolate has a good effect in inhibiting renal fibrosis.

[0033] Immunofluorescence: Dewax the tissue sections, fix them, seal the slides with a histochemical pen, and block with 10% BSA (PBS). Then add COL-1 and α-SMA primary antibodies and incubate overnight at 4°C. After returning to room temperature the next day, wash three times with PBS, incubate with secondary antibodies for 1.5 hours, wash three times with PBS, incubate with DAPI for 10 minutes, wash three times with PBS, and seal with anti-fluorescence quencher. The results of the immunofluorescence experiment are as follows: Figure 3 As shown in A and 3B, in the IRI-induced renal fibrosis model, the expression levels of COL-1 and α-SMA in the renal tissue were significantly increased, while the expression of COL-1 and α-SMA was significantly inhibited after 5-methyltetrahydrofolate treatment, which verified that 5-methyltetrahydrofolate has a good effect in inhibiting renal fibrosis.

[0034] Masson staining: The mouse kidney tissue was stained with a Masson staining kit. The results of Masson staining showed that the kidney damage of mice in the IRI group was obvious, while the degree of kidney damage of mice in the 5-methyltetrahydrofolate treatment group was reduced (see Figure 4 ), indicating that 5-methyltetrahydrofolate has a significant improvement effect on IRI-induced renal tissue pathology in mice.

[0035] Experimental Example 2: Effect of 5-methyltetrahydrofolate on TGF-β-induced cell fibrosis in vitro

[0036] HK2 cells in the logarithmic growth phase were inoculated in 6-well plates and divided into a normal group (NC), a model group (TGF-β), a 5-methyltetrahydrofolate simple administration group (5 μM), and a model + 5-methyltetrahydrofolate group (TGF-β + LA 5 μM). The experiment was repeated 3 times for each group. The cell inoculation density was approximately 1.0×10 5 / ml, incubate for 24 hours, starve with serum-free medium for 12 hours, then stimulate and add drugs, and continue to culture for 36 hours.

[0037] Western Blot: Extract tissue proteins and perform immunoblotting experiments. Western Blot results are as follows Figure 5 As shown in A, in the TGF-β-induced HK2 fibrosis model, the expression levels of COL-1 and α-SMA proteins were significantly increased, while the expression of COL-1 and α-SMA was significantly inhibited after 5-methyltetrahydrofolate treatment. This indicates that 5-methyltetrahydrofolate can significantly reduce the level of cell fibrosis in the TGF-β-induced in vitro fibrosis model.

[0038] Real-time PCR: Extract tissue RNA, reverse transcribe, and amplify. Real-time PCR results are as follows: Figure 5As shown in B, the expression levels of COL-1 and FN were significantly increased in the TGF-β-induced HK2 fibrosis model, while the expression of COL-1 and FN was significantly inhibited after 5-methyltetrahydrofolate treatment, indicating that 5-methyltetrahydrofolate can significantly reduce the level of cell fibrosis in the TGF-β-induced in vitro fibrosis model.

[0039] Immunofluorescence: Wash three times with PBS, fix with polymethanol for 15 minutes, seal the slide with a histochemical pen, block with 10% BSA (prepared in PBS), add α-SMA primary antibody and incubate overnight at 4°C. After returning to room temperature the next day, wash three times with PBS, incubate with secondary antibody for 1.5 hours, wash three times with PBS, incubate with DAPI for 10 minutes, wash three times with PBS, and seal with anti-fluorescence quencher. The results of immunofluorescence experiments are shown in Figure 2. Figure 6 As shown, the expression level of α-SMA was significantly increased in the TGF-β-induced HK2 fibrosis model, while the expression of α-SMA was significantly inhibited after 5-methyltetrahydrofolate treatment, verifying that 5-methyltetrahydrofolate has a good function of alleviating fibrosis in vitro.

[0040] The above experimental results show that 5-methyltetrahydrofolate can inhibit the expression of COL-1, α-SMA and FN, the fibrosis markers in HK2 cells and injured animals' kidneys, reduce the level of cellular fibrosis, alleviate renal fibrosis in mice, improve the pathological changes of renal tissue in chronic kidney disease, and has a significant preventive and therapeutic effect on chronic kidney damage.

[0041] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

Use of 1,5-methyltetrahydrofolate in preparing medicine for preventing or treating renal fibrosis.

2. The use according to claim 1, characterized in that: The medicine includes a pharmaceutically acceptable carrier or excipient.

3. The use according to claim 1, characterized in that: The medicine is an oral administration preparation, a transdermal administration preparation or an injection preparation.

4. The use according to claim 1, characterized in that: The dosage form of the drug is tablet, capsule, granule, oral solution, patch, ointment, gel or injection. Application of 5.5-methyltetrahydrofolate in the preparation of drugs for preventing or treating acute kidney injury turning into chronic kidney disease.

6. The use according to claim 5, characterized in that: The medicine includes a pharmaceutically acceptable carrier or excipient.

7. The use according to claim 5, characterized in that: The medicine is an oral administration preparation, a transdermal administration preparation or an injection preparation.

8. The use according to claim 5, characterized in that: The dosage form of the drug is tablet, capsule, granule, oral solution, patch, ointment, gel or injection. Use of 9.5-methyltetrahydrofolate in the preparation of a drug for reducing the expression level of cell fibrosis markers COL-1, α-SMA and / or FN. Application of 10.5-methyltetrahydrofolate in the preparation of drugs for reducing the expression levels of animal fibrosis markers COL-1, α-SMA and FN.

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