阅读说明：本技术 丁香脂素在制备防治糖尿病肾病药物中的应用 (Application of syringaresinol in preparation of medicine for preventing and treating diabetic nephropathy ) 是由 杨亮 栗广如 漆智 于 2021-08-23 设计创作，主要内容包括：本发明公开了丁香脂素在制备防治糖尿病肾病药物中的应用,属于丁香脂素应用技术领域。丁香脂素结构式为：丁香脂素的给药剂量25mg/kg,可制成注射液、片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂、喷雾剂、气雾剂等多种形式中的任意一种。本发明的有益效果是：丁香脂素在不影响糖尿病小鼠血糖的情况下能够显著改善糖尿病引起的肾损伤,为糖尿病肾病的新药研发提供了新思路,同时也拓展了丁香脂素的新用途。(The invention discloses an application of syringaresinol in preparing a medicament for preventing and treating diabetic nephropathy, and belongs to the technical field of syringaresinol application. Syringaresinol has a structural formula as follows: the syringaresinol is administered at a dose of 25mg/kg, and can be made into any one of injection, tablet, powder, granule, capsule, oral liquid, unguent, cream, spray, aerosol, etc. The invention has the beneficial effects that: syringaresinol can remarkably improve renal injury caused by diabetes under the condition of not influencing the blood sugar of diabetic mice, and provides a new medicine for developing diabetic nephropathyThe idea also expands the new application of syringaresinol.)

1. The application of syringaresinol in preparing medicine for preventing and treating diabetic nephropathy includes preparing medicine for preventing and/or treating diabetic nephropathy.

2. The syringaresinol of claim 1, having the following chemical formula:

3. the use according to claim 1, wherein said syringaresinol is administered at a dose of 25 mg/kg.

4. The use according to claim 1, wherein the product for preventing and/or treating diabetic nephropathy is a medicament or a health product.

5. The use according to claim 4, wherein the medicament or nutraceutical comprises syringaresinol and one or more pharmaceutically acceptable carriers.

6. The use according to claim 5, wherein the carrier is any one or more of a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

7. The use according to claim 4, wherein the medicament or health product is prepared into any one of injection, tablet, powder, granule, capsule, oral liquid, ointment, cream, spray and aerosol.

Technical Field

The invention relates to an application of syringaresinol in preparing a medicament for preventing and treating diabetic nephropathy, belonging to the technical field of syringaresinol application.

Background

Diabetic Nephropathy (DN) is a leading cause of End Stage Renal Disease (ESRD) and death of diabetic patients, and has a 3-12 times higher risk of death compared with simple diabetic patients, and has become a global public health problem seriously harming human health. DN is a microvascular disease caused by diabetes, characterized clinically by progressive renal function impairment, with clinical manifestations of proteinuria, edema, hypertension, reduced glomerular filtration rate, etc. Extracellular matrix (ECM) protein deposition, glomerular basement membrane thickening and loss of charge barrier, glomerular mesangial matrix expansion, depletion of foot process fusion and loss of podocytes are the major pathological features of DN. DN pathogenesis is extremely complex and is currently thought to be related to many aspects such as metabolic abnormalities, hemodynamic changes, the activation of the renin-angiotensin system (RAS), apoptosis, oxidative stress, fibrosis, and the like. Therefore, how to effectively prevent and treat DN is a great problem to be solved clinically.

Syringaresinol (Syringaresinol, SYR) is a natural polyphenol compound, and is an important active ingredient of various medicinal plants, including frankliniella occidentalis, eucommia ulmoides, sargentgloryvine stem, acanthopanax senticosus and the like. Researches find that syringaresinol has the effects of resisting aging, tumors and neuritis and protecting cardiovascular system. So far, no reports on syringaresinol in preparing medicine for preventing and treating diabetic nephropathy are available.

The invention discloses the prevention and treatment effects of syringaresinol on kidney injury caused by diabetes for the first time, suggests that syringaresinol is expected to be used for preparing medicines for preventing and treating diabetic nephropathy, and develops a new application of syringaresinol.

Disclosure of Invention

The invention aims to provide application of syringaresinol in preparing a medicament for preventing and treating diabetic nephropathy. The syringaresinol serving as a plant extract has small toxic and side effects, is used for preparing the medicine for preventing and treating diabetic nephropathy, and has a promising clinical application prospect. The invention provides a new idea for the research and development of new drugs for diabetic nephropathy and also opens up a new application of syringaresinol.

The technical scheme for solving the problems is as follows, and the syringaresinol is applied to the preparation of the medicine for preventing and treating diabetic nephropathy, and has the following chemical structural formula:

furthermore, the product for preventing and/or treating diabetic nephropathy is a medicine or a health-care product. The medicine or health care product comprises syringaresinol and one or more pharmaceutically acceptable carriers. The carrier is any one or more of a sustained release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant or a lubricant.

Furthermore, the medicine or the health care product can be prepared into any one of injection, tablets, powder, granules, capsules, oral liquid, paste, cream, spray and aerosol.

The experiment was divided into three groups: control group, model group, drug treatment group. The invention firstly constructs a diabetes mouse model, and then carries out syringaresinol intragastric therapy on a drug treatment group for 8 weeks continuously. Observing whether syringaresinol has the effect of treating diabetic nephropathy. Research results show that syringaresinol has no significant influence on blood sugar of diabetic mice, but the treatment of syringaresinol obviously improves the renal function of the diabetic mice (the urinary albumin excretion is reduced in 24h, and the urinary albumin/urinary creatinine ratio is reduced); syringaresinol treatment reduces renal fibrosis, oxidative stress, renal cell burn and the like caused by diabetes. In conclusion, syringaresinol has a remarkable treatment effect on diabetic nephropathy. Therefore, the syringaresinol can be used for preparing the medicine for preventing and treating diabetic nephropathy.

The administration dosage of the syringaresinol is 25mg/kg, and pharmacological experiments prove that the syringaresinol has a remarkable treatment effect on diabetic nephropathy by adopting the dosage.

The invention has the beneficial effects that: the syringaresinol serving as a plant extract has small toxic and side effects, can remarkably improve the kidney injury caused by diabetes, can be used for preparing medicines for preventing and treating diabetic nephropathy, and has a promising clinical application prospect. Provides a new idea for the research and development of new drugs for diabetic nephropathy and also expands the new application of syringaresinol.

Drawings

FIG. 1: and (5) detecting results of blood sugar, urine albumin and urine creatinine of the mouse. Wherein A is the blood sugar value of the mouse every week, B is the excretion detection result of the mouse urine albumin for 24 hours, C is the ratio of the mouse urine albumin to the urine creatinine (CON: a control group; DN: a modeling group; DN + SYR: a drug treatment group;^*P＜0.05；^**P＜0.01；^***P＜0.001)。

FIG. 2: masson staining of kidney tissue and Tianlang scarlet staining pictures (CON: control group; DN: modeling group; DN + SYR: drug treatment group).

FIG. 3: kidney tissue transmission electron microscope (CON: control group; DN: modeling group; DN + SYR: drug treatment group).

FIG. 4: and detecting the relevant indexes of the oxidative stress of the kidney tissues. Wherein A is a Westernblot image for detecting the expression levels of antioxidant proteins Nrf2 and HO-1, and B is an image for detecting the ROS concentration in kidney tissues by HDE staining (CON: a control group; DN: a modeling group; DN + SYR: a drug treatment group).

FIG. 5: detecting the relative index of the kidney tissue scorching. Wherein A is the expression level of Real-time PCR detection NLRP3, Caspase-1, GSDMD, IL-1 beta and IL-18 genes, B is the detection result of IL-1 beta ELISA in serum, and C is the detection result of IL-18ELISA in serum (CON: control group; DN: modeling group; DN + SYR: drug treatment group;^*P＜0.05；^**P＜0.01；^***P＜0.001)。

FIG. 6: graph comparing syringaresinol in treating diabetic nephropathy.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings.

The first embodiment is as follows: the detection results of blood sugar, urine albumin and urine creatinine of a mouse are shown in figure 1.

Randomly dividing 24 male C57BL/6 mice 6-8 weeks old into a control group (CON group, 8) and a molding group (16); carrying out intraperitoneal injection on STZ (STZ formulation) of the mice of the model group for 5 consecutive days according to the dose of 50 mg/kg; after 1 week of STZ injection, cutting off the tail tip of the mouse and collecting tail vein blood to detect the blood sugar level of the mouse, wherein the blood sugar is more than 11.4mM, and the blood sugar is used as a diabetic mouse which is successfully modeled; randomly dividing diabetic mice into a building block (DCM group, 8) and a drug treatment group (DCM + SYR group, 8); the mice in the drug-treated group were treated with SYR at a 25mg/kg dose for 8 weeks, the mice in the other groups were treated with the same dose of saline as a control, and the blood glucose of the mice in each group was measured every week.

After 8 weeks of syringaresinol treatment, mouse urine is collected, and mouse urine albumin and urine creatinine levels are determined according to mouse urine albumin and urine creatinine detection kit instructions.

Example two: masson staining of renal tissue

Soaking the trimmed kidney tissues in 4% paraformaldehyde, fixing at room temperature for more than 24h, performing gradient dehydration and embedding, and preparing into 6 μm tissue slices; performing dewaxing and rehydration, and dyeing with hematoxylin for 5-10 min; acid ethanol is differentiated for 5-15 s; returning blue to the bluing liquid for 3-5 min; dyeing ponceau for 5-10 min; dehydrating and transparent; sealing the neutral gum into a piece; and (4) observing and taking a picture under a 400-fold microscope, and referring to the attached figure 2.

Example three: sirius red staining of kidney tissue

Soaking the trimmed kidney tissues in 4% paraformaldehyde, fixing at room temperature for more than 24h, performing gradient dehydration and embedding, and preparing into 6 μm tissue slices; after dewaxing and rehydration, dyeing with sirius red staining solution for 1 h; staining cell nucleus with hematoxylin for 8-10 min; dehydrating and transparency in a conventional way; sealing neutral gum into a sheet; and (4) observing and taking a picture under a 400-fold microscope, and referring to the attached figure 2.

Example four: transmission electron microscope observation of podocyte by kidney tissue

After 8 weeks of syringaresinol treatment, mice killed by cervical dislocation were immediately taken out of the kidney, 2mm × 2mm tissue blocks were taken from the renal cortex and soaked in a stationary liquid, and then were subjected to embedding, slicing, staining and photographing by a scientific compass company, see fig. 3.

Example five: westernblot for detecting expression of kidney tissue protein

Weighing 15-20mg of kidney tissue, putting the kidney tissue into a 2ml centrifuge tube, adding lysis solution, putting clean magnetic beads, and grinding for 2min at 50 Hz; inserting the centrifugal tube into ice, vortexing and shaking for 1 time every 7min, and repeating for 3 times; centrifuging at 12000rpm at 4 deg.C for 30 min; sucking the supernatant to obtain total protein; detecting protein concentration using a BCA kit; electrophoresis was performed on a 10% SDS-PAGE gel; transferring the protein to a PDF membrane; sealing 5% skimmed milk for 2 h; adding Nrf2 and HO-1 antibody, and incubating overnight at 4 ℃; washing with TBST for 3 times, adding a second antibody labeled with horseradish peroxidase, and incubating at room temperature for 1 h; the luminescence detection was performed after 3 TBST washes. The detection result is shown in the A diagram of the attached FIG. 4.

Example six: DHE staining of renal tissue

Sequentially immersing the taken fresh heart tissue into 10%, 20% and 30% of sucrose solution for gradient dehydration; adding OCT embedded tissue; slicing at a thickness of 6 μm; adding DHE working solution, and incubating at 37 deg.C for 40 min; washing with PBS for 3 times, and adding an anti-fluorescence quencher to seal; observing under a 100-fold microscope, and shooting. See fig. 4B.

Example seven: real-time PCR detection of kidney tissue gene expression

Weighing 15-20mg of kidney tissue, putting into a 2ml enzyme-free tube, adding 1ml of Trizol lysate and magnetic beads, and grinding for 2min at 50 Hz; standing the tissue lysate for 10min at room temperature; adding 200 μ l chloroform, shaking, mixing, standing at room temperature for 15 min; centrifuging at 12000g at 4 deg.C for 30 min; transferring the supernatant to a new 1.5ml enzyme-free tube, adding isopropanol with the same volume as the sucked supernatant, reversing, mixing, and standing at room temperature for 10 min; centrifuging at 12000g at 4 deg.C for 10min, and removing supernatant; adding l mL of 75% ethanol, centrifuging at 4 ℃ 8000g for 5min, pouring off the supernatant, and obtaining white precipitate at the bottom as RNA; detecting the concentration of RNA; inverting RNA into cDNA using a reverse transcription kit; designing primer sequences of NLRP3, Caspae-1, GSDMD, IL-1 beta and IL-18 by Premier 5 software; gene expression levels were detected using the Real-time PCR kit. The detection result is shown in the A diagram of the attached FIG. 5.

Example eight: IL-1 beta and LDH detection in serum

After 8 weeks of syringaresinol treatment, collecting the eyeball blood into a centrifuge tube, and standing for 3 hours at room temperature; centrifuge at 5000rpm for 15 min. The upper layer of light yellow transparent liquid is serum. IL-1 β and LDH concentrations in mouse serum were measured using IL-1 β and LDH detection kits. The detection results are shown in B and C of FIG. 5.

All data are presented as mean ± SEM and statistically analyzed using SPSS 20 software. The difference between the two groups was analyzed using the t-test. Differences between groups were analyzed using one-way ANOVA, and P <0.05 was considered statistically significant. Charts such as histograms were generated using GraphPad Prism 5 software.

Through the specific implementation of the invention, the result shows that syringaresinol has no significant influence on the blood sugar of a diabetic mouse, but the syringaresinol treatment significantly improves the renal function of the diabetic mouse (the urine albumin excretion is reduced in 24h, and the urine albumin/urine creatinine ratio is reduced); syringaresinol treatment reduces renal fibrosis, oxidative stress, renal cell burn and the like caused by diabetes. The comparison results are shown in FIG. 6. Therefore, the syringaresinol has obvious treatment effect on the diabetic nephropathy, and can be used for preparing the medicine for preventing and treating the diabetic nephropathy. Specifically, the product for preventing and/or treating diabetic nephropathy is a medicine or a health-care product. The medicine or health care product comprises syringaresinol and one or more pharmaceutically acceptable carriers. The carrier is any one or more of a sustained release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant or a lubricant. The medicine or health care product can be prepared into any one of various forms such as injection, tablets, powder, granules, capsules, oral liquid, ointment, cream, spray, aerosol and the like.