Application of inosine in preparation of medicine for preventing and / or treating preeclampsia

Abstract

The invention belongs to the field of biological medicines, and provides application of inosine in preparation of a medicine for preventing and / or treating preeclampsia. According to the invention, the pathological state of preeclampsia is simulated through subcutaneous injection of a nitroso-L-arginine methyl ester solution (125 mg / kg). And from the 9th day of pregnancy, gastro-inosine is irrigated once a day (100mg / kg) until the end of pregnancy. Compared with medicines with a single treatment effect, such as an antihypertensive medicine labeolol and a spasmolysis medicine magnesium sulfate, the invention proves that oral inosine not only can remarkably improve hypertension and proteinuria caused by preeclampsia, repair placenta functions, promote intrauterine growth and development of fetuses and correct imbalance of blood vessels and anti-angiogenesis factors, but also has the effects of preventing and treating the preeclampsia. In addition, kidney and brain injuries, induced by preeclampsia, of pregnant and lying-in women can be relieved, a more comprehensive treatment effect is shown, and a novel treatment medicine is provided for preventing or treating preeclampsia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of inosine in preparing drugs for preventing and / or treating preeclampsia. Background Art

[0002] Preeclampsia (PE) is a pregnancy-specific disease. Its clinical symptoms are new-onset hypertension (systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg) and proteinuria in pregnant women after 20 weeks of gestation, accompanied by symptoms such as headache, convulsions, and visual impairment. In severe cases, it can cause damage to tissues such as the liver and kidneys, leading to multiple organ failure and even maternal and fetal death.

[0003] Current clinical treatment strategies for preeclampsia primarily include preventive and symptomatic treatment. Preventive treatment primarily involves oral low-dose aspirin, aimed at reducing the incidence of preeclampsia. Symptomatic treatment primarily involves oral antihypertensive drugs such as labetalol and nifedipine to lower blood pressure, or intravenous magnesium sulfate to relieve convulsions or spasms. However, existing treatments still have numerous adverse reactions, including gastrointestinal symptoms such as nausea, vomiting, and upper abdominal pain with oral aspirin, and headaches, nausea, and arrhythmias 24 hours after oral labetalol. These adverse reactions severely impact the maternal pregnancy. Furthermore, if medications are unable to control the condition, premature termination of pregnancy is the only way to prevent worsening of the condition and save the lives of both mother and child. However, this can increase the risk of maternal infection, fetal growth impairment, perinatal mortality, and neonatal asphyxia. Therefore, there is an urgent need to identify effective and safe therapeutic drugs.

[0004] Inosine (INO) is a nucleoside composed of adenine and ribose, and is an essential metabolite for purine biosynthesis and degradation. At the same time, inosine is a naturally occurring purine nucleoside formed by the decomposition of adenosine and is considered to be an inert adenosine metabolite. The function of inosine is mediated in a receptor-dependent or -independent manner. The receptor-mediated function of inosine is believed to be related to members of the adenosine receptor family, including G protein-coupled receptors A1, A2A, A2B and A3. Studies have shown that inosine has anti-inflammatory and immunomodulatory effects, and has potential therapeutic effects on systemic inflammation caused by PE. However, there are currently no studies or reports on the effect of inosine on PE, nor is there any evidence that it can regulate placental function or improve symptoms such as high renal damage and brain damage associated with PE. Therefore, the present invention aims to protect the important role of inosine in preventing and treating preeclampsia. Summary of the Invention

[0005] To overcome the deficiencies in the prior art, the present invention provides the use of inosine in the preparation of a drug for preventing and / or treating preeclampsia. To achieve the above objectives, the present invention employs the following technical solutions:

[0006] The first aspect of the present invention provides the use of inosine in preparing a drug for preventing and / or treating preeclampsia.

[0007] Furthermore, the drug is used to reduce hypertension caused by preeclampsia.

[0008] Furthermore, the drug is used to improve proteinuria caused by preeclampsia.

[0009] Furthermore, the drug is used to improve placental function damage caused by preeclampsia.

[0010] Furthermore, the drug is used to improve fetal intrauterine growth restriction caused by preeclampsia.

[0011] Furthermore, the drug is used to improve the imbalance between angiogenic factors and anti-angiogenic factors.

[0012] Furthermore, the angiogenic factors include vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), and the anti-angiogenic factors include soluble fms-like tyrosine kinase-1 (sFlt-1).

[0013] Furthermore, the drug is used to improve renal damage induced by preeclampsia. Alleviating renal damage includes alleviating glomerular endothelial cell damage, mesangial cell proliferation, and tubulointerstitial inflammation. The drug can reduce the expression of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) in the kidney, and increase the expression of podocyte protein (podocin).

[0014] Furthermore, the drug is used to reverse brain damage induced by preeclampsia, including blood-brain barrier damage. The drug can protect the blood-brain barrier and reduce the expression of central nervous system-specific protein (S100B) and glial fibrillary acidic protein (GFAP) in the circulatory system.

[0015] A second aspect of the present invention provides a drug for preventing and / or treating preeclampsia, wherein the drug contains an effective amount of inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

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

[0017] Furthermore, the dosage form of the drug is tablets, capsules, oral liquids, granules, granules, pills, powders, suspensions, powders or injections.

[0018] Furthermore, the drug is administered orally or by injection.

[0019] The present invention has the following beneficial effects:

[0020] The present invention simulates the pathological state of preeclampsia by subcutaneous injection of nitroso-L-arginine methyl ester solution (125 mg / kg). And from the 9th day of pregnancy, inosine (100 mg / kg) is gavaged once a day until the end of pregnancy. Compared to the antihypertensive drug labetalol, antispasmodic drug magnesium sulfate and other drugs with single therapeutic effects, the present invention confirms that oral inosine can not only significantly reduce hypertension and proteinuria caused by preeclampsia, repair placental function, promote fetal intrauterine growth and development, correct the imbalance of blood vessels and anti-angiogenic factors, but also reduce maternal kidney and brain damage induced by preeclampsia, showing a more comprehensive therapeutic effect, and providing a new therapeutic drug for preventing or treating preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Comparison of blood pressure of mice in each group in Example 1 (n=6);

[0022] Figure 2 Comparison of urine protein levels in mice of each group in Example 2 (n=6);

[0023] Figure 3 Comparison of placenta weights of mice in each group in Example 3 (n=6);

[0024] Figure 4 Comparison of placental divisions of mice in each group in Example 4 (n=4);

[0025] Figure 5 Comparison of fetal weights of mice in each group in Example 5 (n=6);

[0026] Figure 6 Comparison of the levels of angiogenic and anti-angiogenic factors in the serum and placenta of mice in each group in Example 6 (n=6);

[0027] Figure 7 Comparison of renal injury indicators in mice of each group in Example 7 (n=6);

[0028] Figure 8 Comparison of brain damage indicators in the brains of mice in each group in Example 8 (n=6). DETAILED DESCRIPTION

[0029] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, but it should not be understood as limiting the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0031] 1. Preparation of experimental reagents, materials and animals.

[0032] Drug ingredients:

[0033] Inosine, chemical formula is C 10 H 12 N4O5, with a molecular weight of 268.23, is a nucleoside compound formed by the combination of hypoxanthine and ribose.

[0034] Experimental basis:

[0035] 1 Experimental animals

[0036] Male and female C57BL / 6 mice (8–10 weeks, weighing 18–20 g) were purchased from SPF (Beijing, China) Biotechnology Co., Ltd. (Beijing, China). Mice were housed under a standard light and dark cycle (12 h:12 h) with free access to food and water. After one week of acclimation, healthy male mice were mated with female mice at a ratio of 1:2 overnight. Female mice with sperm plugs were considered pregnant, and this day was defined as gestational day 0 (GD0). All animal studies were performed in accordance with protocols and guidelines approved by the Animal Ethics Committee of the Laboratory Animal Center of Southern Medical University.

[0037] 2 Experimental reagents

[0038]

[0039]

[0040] 3. Drug preparation

[0041] 3.1 N-nitro-L-arginine methyl ester (L-NAME) solution: Weigh 125 mg of L-NAME powder and dilute to 10 mL with normal saline.

[0042] 3.2 Inosine solution: Weigh 10 mg of inosine powder and dilute to 1 mL with normal saline.

[0043] 4 Establishment and evaluation of preeclampsia model

[0044] 4.1 Establishment of preeclampsia model

[0045] Starting from GD9, pregnant mice were subcutaneously injected with 125 mg / kg of L-nitroso-L-arginine methyl ester (L-NAME) solution every day for 9 days.

[0046] 4.2 Evaluation of preeclampsia models

[0047] 4.2.1 Blood pressure

[0048] The blood pressure of mice in the preeclampsia model group was significantly higher than that of mice in the normal pregnancy group, indicating that the model was successfully established.

[0049] 4.2.2 Proteinuria

[0050] The urine protein content of mice in the preeclampsia model group was significantly higher than that in the normal pregnancy group, indicating that the model was successfully established.

[0051] 5Real-time fluorescence quantitative PCR

[0052] 5.1 RNA Reverse Transcription

[0053] Prepare a 20 μL total reaction volume according to the reverse transcription kit instructions. Add the desired RNA and 4 μL of HiScript IIQ RT SuperMix for qPCR reagent to a 200 μL PCR tube, then fill to 20 μL with ddH2O. Place the tube in a PCR amplifier and run under the following conditions: 50°C for 15 min; 85°C for 5 s; and 4°C for ∞. After the reaction is complete, centrifuge, mark the time, and store the resulting cDNA at -80°C.

[0054] 5.2 Real-time quantitative PCR (RT-qPCR)

[0055] All reverse transcription products were subjected to real-time fluorescence quantitative PCR, and the reaction system is shown in Table 1. The primer sequences of relevant genes are shown in Table 2, and a two-step PCR amplification system was used, and the method settings are shown in Table 3.

[0056] Table 1 PCR reaction system

[0057]

[0058]

[0059] Table 2 Primer sequences

[0060]

[0061] Table 3 RT-qPCR reaction procedure

[0062]

[0063] Data processing: Gapdh was used as the internal reference gene, and the relative expression of the target gene was calculated using the 2-ΔΔCt method.

[0064] 2. Example

[0065] Example 1: Inosine can significantly reduce hypertension caused by preeclampsia

[0066] Hypertension is the most critical clinical indicator of preeclampsia. To investigate the effect of inosine on the blood pressure of preeclampsia mice, the mice were randomly divided into three groups in this example: normal pregnancy group (NP, n=6), preeclampsia model group (PE, n=6), and inosine group (INO, n=6). Mice in the inosine intervention group were gavaged with inosine (100 mg / kg) from gestational day 9 (GD9), while mice in the normal pregnancy group and preeclampsia model group were gavaged with the same volume of 0.9% saline. Once a day for 9 days. The results are shown in Figure 2. Figure 1 The results showed that the blood pressure of mice in the preeclampsia model group was significantly higher than that of the normal pregnancy group and the inosine group after GD9, indicating that inosine intervention can significantly reduce hypertension (or elevated blood pressure) in preeclampsia mice. (a represents the comparison of systolic blood pressure between the NP group and the PE group on the corresponding day; aaa represents P < 0.001; b represents the comparison of systolic blood pressure between the INO group and the PE group on the corresponding day; bbb represents P < 0.001; c represents the comparison of systolic blood pressure between the NP group and the INO group on the corresponding day; cc represents P < 0.01, and ccc represents P < 0.001.)

[0067] Example 2: Inosine can significantly improve proteinuria caused by preeclampsia

[0068] Proteinuria is one of the key clinical indicators of preeclampsia. In this example, subcutaneous injection of nitroso-L-arginine methyl ester (L-NAME) solution was used to simulate the disease state of preeclampsia during its natural onset, and the effect of inosine on urine protein in preeclampsia mice was investigated. Figure 2 The results showed that the urine protein content of mice in the preeclampsia model group was significantly higher than that in the normal pregnancy group, and the urine protein level of mice in the model group was significantly downregulated after inosine intervention, which indicates that inosine can significantly reduce the proteinuria level of mice in the preeclampsia model group.

[0069] Example 3: Inosine can significantly increase the placenta weight of preeclamptic mice

[0070] The placenta is rich in blood vessels and nutrients, which provide the necessary substances for the growth and development of the fetus. By comparing the weight of the placenta of each group of mice, the results are as follows Figure 3 The results showed that compared with the mice in the normal pregnancy group, the placenta weight of the mice in the preeclampsia model group was significantly reduced, and the intervention of inosine significantly reversed the decrease in placenta weight, indicating that inosine is beneficial to the growth and development of the placenta in preeclampsia mice.

[0071] Example 4: Inosine can improve placental dysfunction

[0072] The placenta is mainly divided into three parts: the decidual region, the connecting region and the labyrinth. The placental labyrinth is mainly involved in the synthesis of hormones and cytokines (such as angiogenic factors and anti-angiogenic factors), maintaining normal placental function and fetal growth. The placental connecting region is mainly responsible for the exchange of oxygen, nutrients and metabolic waste. By comparing the placental divisions of each group of mice, the results are as follows Figure 4 As shown in Figure 2, the ratio of the labyrinth to the junctional area in the placentas of mice in the PE group was significantly higher than that in the NP group, suggesting pathological damage to the placental structure of PE mice. Administration of inosine reversed this phenomenon, indicating that inosine can repair pathological damage to the placental tissue of PE mice and improve placental dysfunction.

[0073] Example 5: Inosine can improve intrauterine growth restriction in mice with preeclampsia

[0074] Fetal growth restriction is one of the main complications of preeclampsia. Figure 5 The results showed that compared with the mice in the normal pregnancy group, the fetal weight of the mice in the preeclampsia model group was significantly reduced, and the intervention of inosine could significantly reverse the reduction in fetal weight, indicating that inosine can promote the growth and development of the fetus in PE mice in utero.

[0075] Example 6: Inosine can improve the imbalance between vascular and anti-angiogenic factors

[0076] During the development of preeclampsia, the abnormal function of the vascular system causes an imbalance between vascular and anti-angiogenic factors, which affects the remodeling of uterine spiral arteries. By comparing the vascular and anti-angiogenic factors in the serum and placenta of each group of mice, Figure 6 As shown, compared with the normal pregnancy group, the serum and placenta levels of soluble fms-like tyrosine kinase-1 (sFlt-1, an anti-angiogenic factor) in the preeclampsia model mice were significantly increased, while the levels of angiogenic factor (VEGF) and placental growth factor (PIGF) were significantly decreased. Furthermore, the PE disease marker sFlt-1 / PIGF was significantly increased. These phenomena were reversed by inosine intervention, indicating that inosine can improve the imbalance between vascular and anti-angiogenic factors.

[0077] Example 7: Inosine can improve renal damage induced by preeclampsia

[0078] The systemic endothelial dysfunction characteristic of preeclampsia manifests in the kidneys as glomerular endothelial hyperplasia and proteinuria. The systemic inflammatory response and oxidative stress triggered by preeclampsia further exacerbate renal pathological damage, triggering a series of pathological changes, including glomerular endothelial cell damage, mesangial cell proliferation, and tubulointerstitial inflammation. These changes can ultimately lead to acute kidney injury and even chronic kidney disease, seriously compromising the long-term health of pregnant women. KIM-1 is highly expressed on the surface of renal tubular epithelial cells and released into the urine during tubular injury, serving as an early, sensitive marker of tubular injury. Its elevation precedes changes in traditional indicators such as serum creatinine, providing a timely indicator of the extent of tubular damage. Neutrophil glomerulonephritis-associated leukemia (NGAL), primarily produced by neutrophils, is also synthesized and released in large quantities by renal tubular epithelial cells and glomerular mesangial cells in response to renal ischemia and toxic insults. Its concentrations in serum and urine are significantly elevated, making it crucial for the early diagnosis and assessment of acute kidney injury. Podocin is a protein specific to podocytes and plays an important role in maintaining the normal structure and function of podocytes. When podocytes are damaged, the expression of podocin will decrease or become abnormal. By comparing the renal injury indicators of mice in each group, the results are as follows Figure 7 The results showed that compared with the preeclampsia model group, the expression of KIM-1 and NGAL in pregnant mice with preeclampsia was significantly downregulated, and the expression of Podocin was significantly upregulated after inosine intervention, indicating that inosine can improve renal damage induced by preeclampsia.

[0079] Example 8: Inosine can reverse brain damage induced by preeclampsia

[0080] Brain complications caused by preeclampsia (such as eclampsia, cerebral edema and cerebral hemorrhage) are the main causes of maternal mortality. In most cases, the onset of eclampsia precedes hypertension and there are usually no premonitory symptoms. Central nervous system-specific protein (S100B) and glial fibrillary acidic protein (GFAP) are currently being evaluated as biomarkers of brain damage. By comparing the brain damage indicators in the brains of mice in each group, the results are as follows Figure 8 As shown in the results, compared with the preeclampsia model group, the expression of S100b and Gfap in the brains of pregnant mice with preeclampsia was significantly reduced after inosine intervention, indicating that inosine can reverse the brain damage induced by preeclampsia.

[0081] The above examples further illustrate that oral administration of inosine can significantly alleviate hypertension and proteinuria caused by preeclampsia, repair pathological tissue structure and functional changes of the placenta, improve fetal intrauterine growth and development, correct the imbalance between vascular and anti-angiogenic factors, and reverse kidney and brain damage induced by preeclampsia, proving that inosine has a good therapeutic effect on preeclampsia.

[0082] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of inosine in the preparation of drugs for preventing and / or treating preeclampsia.

2. The use according to claim 1, characterized in that The drug is used to reduce high blood pressure caused by preeclampsia.

3. The use according to claim 1, characterized in that The medicine is used for improving proteinuria caused by preeclampsia.

4. The use according to claim 1, characterized in that The drug is used to improve placental function damage caused by preeclampsia.

5. The use according to claim 1, characterized in that The drug is used to improve intrauterine growth restriction of the fetus caused by preeclampsia.

6. The use according to claim 1, characterized in that The drug is used to improve renal damage induced by preeclampsia.

7. The use according to claim 6, wherein the renal injury includes tubular epithelial cell damage, glomerular endothelial cell damage, mesangial cell proliferation and tubulointerstitial inflammation, and the drug can reduce the expression of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) in the kidney, and increase the expression of podocyte protein (podocin).

8. The use according to claim 1, characterized in that The drug is used to reverse brain damage induced by preeclampsia.

9. The use according to claim 8, characterized in that The brain damage includes blood-brain barrier damage, and the drug can reduce the expression of central nervous system-specific protein (S100B) and glial fibrillary acidic protein (GFAP) in the brain.

10. A drug for preventing and / or treating preeclampsia, characterized in that: The medicine contains an effective amount of inosine or a pharmaceutically acceptable salt thereof as a main active ingredient; The drug also includes a pharmaceutically acceptable carrier or excipient; The dosage form of the drug is tablet, capsule, oral solution, granule, granule, pill, powder, suspension, powder or injection; The drug is administered orally or by injection.

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