Use of compound with Anti-kidney injury activity in preparation of Anti-kidney injury drug

Abstract

Provided is the use of a compound with an anti-kidney injury activity in the preparation of an anti-kidney injury drug. 7-O-ethyl-Morroniside from Corni fructus has an anti-kidney injury effect, and such effect may be related to 7-O-ethyl-Morroniside from Corni fructus having the function of inhibiting kidney injury induced by advanced glycation end products (AGEs). Therefore, 7-O-ethyl-Morroniside from Corni fructus has the prospect of being developed into an anti-kidney injury drug.

Description

Application of a compound with anti-kidney injury activity in the preparation of anti-kidney injury drugs Technical Field

[0001] The present invention belongs to the field of medicine and relates to new uses of known compounds, and specifically relates to the use of a compound with anti-kidney injury activity in the preparation of an anti-kidney injury drug. Background Art

[0002] 7-O-ethyl-Morroniside is a natural compound isolated from Cornus officinalis. Its chemical structure is as follows:

[0003] Based on the research results on the activity of Cornus officinalis 7-ethoxymorroniside, the present invention is proposed.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide an application of a compound with anti-kidney injury activity in the preparation of an anti-kidney injury drug.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The invention relates to a medical use of Cornus officinalis 7-ethoxymorroniside in preparing a drug for preventing kidney damage.

[0008] Furthermore, the drug uses Cornus officinalis 7-ethoxymorroniside as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable carriers or excipients.

[0009] Furthermore, the carrier or auxiliary material is solid, liquid or semi-solid.

[0010] Furthermore, the dosage forms include tablets, injections, capsules, and pills. Beneficial effects:

[0011] The present invention discovered that 7-O-ethyl-Morroniside has an anti-kidney injury effect, which may be related to its ability to inhibit AGEs-induced podocyte damage. Therefore, 7-O-ethyl-Morroniside has the potential to be developed into a drug for anti-kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows the effect of 7-O-ethyl-Morroniside on AGEs-induced LDH release in renal podocytes of MPC-5 mice in a renal injury model; AGEs are advanced glycation end products, and the administration concentrations are 5, 10, and 20 μM.

[0013] Figure 2 shows the effect of 7-O-ethyl-Morroniside on AGEs-induced renal podocyte cell viability in a renal injury model; AGEs are advanced glycation end products, and the administration concentrations are 5, 10, and 20 μM. DETAILED DESCRIPTION

[0014] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0015] 1. Experimental Materials

[0016] 1. Experimental drugs and cells:

[0017] 7-O-ethyl-Morroniside was prepared in-house, with a purity of no less than 95% as determined by HPLC. Mouse kidney podocytes MPC-5 were cryopreserved in the applicant's laboratory.

[0018] 2. Experimental reagents:

[0019] Table 1 Experimental reagents and manufacturer names

[0020] 2. Experimental Methods

[0021] 1. Cell culture

[0022] (1) Preparation of culture medium: The culture medium for mouse renal podocytes MPC-5 is based on RPMI 1640. All culture media contain 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin.

[0023] (2) Cell recovery: Take the experimental cells frozen in the liquid nitrogen tank and place them in a 37°C water bath prepared in advance for rapid thawing. Then mix the cell suspension with complete culture medium in a ratio of 1:2, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, take an appropriate amount of complete culture medium to resuspend the cells, and evenly inoculate them into a 10 cm diameter culture dish.

[0024] (3) Cell passaging: After cell recovery, the medium is changed once a day, and the cells are passaged when the fusion rate reaches 90%. First, discard the original culture medium and wash the cells with 2 mL of preheated PBS buffer. Drain the PBS and add 1 mL of 0.25% trypsin cell digestion solution (containing EDTA). Ensure that all cells are covered with the digestion solution. Place the cells in a cell culture incubator and incubate for about 2 minutes until the intercellular gaps become larger and the bottom of the culture bottle becomes mottled. Then, use 2 mL of complete culture medium to terminate the digestion. Gently blow and collect the cell suspension into a 5 mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells with complete culture medium. Take an appropriate amount of cell suspension and evenly inoculate it into a new 10 cm culture dish for culture.

[0025] (4) Cell freezing: The freezing solution of the above cells is prepared according to the volume ratio of blank culture medium: FBS: DMSO = 7:2:1. After multiple passages, when the fusion rate reaches 90%, the original culture medium is discarded and the cells are washed with 2 mL of preheated PBS buffer. The PBS is removed and 1 mL of 0.25% trypsin cell digestion solution (containing EDTA) is added to ensure that all cells are covered with the digestion solution. The cells are placed in a cell culture incubator and digested until the gaps between most cells become larger and the bottom of the culture bottle becomes mottled. The digestion can be terminated with 2 mL of complete culture medium. The cells are gently blown and the cell suspension is collected into a 5 mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, the supernatant is discarded, and the cells are resuspended with the cell freezing solution prepared in advance. The cell suspension is divided into cryopreservation tubes and then placed in a programmed cell freezing box. It is placed at -80°C for 24 hours and finally transferred to a liquid nitrogen tank for long-term storage.

[0026] 2. Grouping, Modeling, and Drug Administration

[0027] Accurately weigh an appropriate amount of 7-O-ethyl-Morroniside and dissolve it in DMSO to prepare a 10 mM stock solution. Aliquot and store at -20°C. Use AGE-induced MPC-5 cells to establish a kidney injury model. Based on this, dilute the solution with the corresponding complete culture medium to obtain three concentrations: 5, 10, and 20 μM. The original culture medium is withdrawn, and the prepared drug solution is added to each well as a replacement medium. A control group containing 0.1% DMSO medium and a blank control group containing no cells are set up. The drug exposure time is 24 hours.

[0028] 3. LDH release level detection

[0029] (1) After the drug treatment ends, place the 96-well plate in a centrifuge and centrifuge at 400g for 5 minutes. According to the grouping, take 120 μL of the supernatant from each well and place it in a new 96-well plate for later use.

[0030] (2) Preparation of LDH working solution First, dilute the 10× INT solution in the kit 10 times as needed to prepare 1× INT solution; then prepare the LDH working solution in the ratio of lactic acid solution: 1× INT solution: enzyme solution = 1:1:1.

[0031] (3) LDH detection in samples: Take the prepared samples, add 60 μL / well of the freshly prepared LDH working solution, mix thoroughly, place on a shaker, incubate at 40 r / min, 25°C in the dark for 30 min, and use a microplate reader to detect the absorbance at 490 nm. The absorbance value of each sample group is recorded as A, the absorbance value of the corresponding maximum enzyme activity release group is recorded as AM, and the absorbance value of the blank control group is recorded as AB. The LDH release rate (%) = (A-AB) / (AM-AB)×100.

[0032] 4. CCK8 cell viability assay

[0033] (1) Obtain the MPC-5 cells to be tested and inoculate them into a 96-well plate.

[0034] (2) Add an appropriate amount of culture medium and test substances (such as drugs, compounds, etc.) to each well and perform treatments at different concentrations.

[0035] (3) Place the plate in an incubator and culture under appropriate temperature and CO2 conditions.

[0036] (4) After a certain period of incubation, CCK-8 reagent was added to each well and incubation was continued for a period of time.

[0037] (5) Detect the absorbance, generally using an enzyme-linked microplate reader or a multifunctional enzyme-linked microplate reader, and read the OD value at a wavelength of 450 nm.

[0038] (6) Calculate cell viability based on OD values.

[0039] 5. Statistical analysis

[0040] All experimental results were analyzed using GraphPad Prism 5.0 software and presented as mean ± standard deviation (mean ± SD). Comparisons of means between two groups were performed using the t-test, and comparisons of means between three or more groups were performed using the one-way ANOVA test. P < 0.05 indicated statistical significance.

[0041] 3. Experimental Results

[0042] As shown in Figure 1, the LDH release rate of MPC-5 cells in the model group was significantly higher than that in the control group, indicating successful model establishment. Compared with the model group, the 7-O-ethyl-Morroniside-treated group exhibited a concentration-dependent inhibition of LDH release in the corresponding injury model cells. As shown in Figure 2, the cell survival rate of MPC-5 cells in the model group was significantly lower than that in the control group, indicating successful model establishment. Compared with the model group, the 7-O-ethyl-Morroniside-treated group increased the survival rate of the corresponding injury model cells.

[0043] These results suggest that 7-O-ethyl-Morroniside has anti-kidney injury effects, which may be related to its ability to inhibit AGE-induced podocyte proliferation. Therefore, 7-O-ethyl-Morroniside has the potential to be developed as a drug against kidney injury.

[0044] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. The medical use of Cornus officinalis 7-ethoxymorroniside for preparing drugs for preventing kidney damage.

2. The medical use according to claim 1, wherein the drug uses 7-ethoxymorroniside from Cornus officinalis as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier or excipient.

3. The medical use according to claim 2, wherein the carrier or excipient is solid, liquid or semisolid.

4. The medical use according to claim 2, wherein the dosage form comprises tablets, injections, capsules, and pills.

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