CN111467331A - Application of 1-indanone in preparation of medicine for treating or preventing autosomal dominant hereditary polycystic kidney disease - Google Patents

Abstract

The invention provides the application of 1-indanone in preparing a medicine for treating or preventing autosomal dominant polycystic kidney disease. The present invention uses the MDCK vesicle model to prove that 1-indanone can inhibit the formation and growth of vesicles, and determines the intrarenal pharmacological activity of 1-indanone through the in vitro embryonic kidney vesicle model, which has significant effects on the development of renal vesicles. Finally, in the polycystic kidney mouse model, it was further proved that 1-indanone also has the effect of inhibiting the occurrence and development of vesicles in vivo. The above vesicle inhibition effects in vitro and in vivo are in a dose-effect relationship.

Description

Application of 1-indanone in the preparation of drugs for the treatment or prevention of autosomal dominant polycystic kidney disease application

technical field

The invention provides the application of 1-indanone in preparing a medicine for treating or preventing autosomal dominant polycystic kidney disease, and belongs to the field of biomedicine.

Background technique

Pteridium aquilinum (scientific name: Pteridium aquilinum (L.)) is a varietal of Pteridaceae (Pteridaceae) genus European fern, 1-indanone (1-indanone) is extracted from dried young bracken leaves, which has various Biological activities, including anti-inflammatory, antibacterial, antiviral, treatment of Alzheimer's disease, anti-tumor, etc. Previous studies have shown that 1-indanone derivatives can act as acetylcholinesterase inhibitors to treat Alzheimer's disease. In addition, 1-indanone can induce the apoptosis of multi-drug-resistant tumor cells (such as MCF-7, HL-60, MES-SA), and it has been shown to a variety of malignant tumors such as colon cancer, breast cancer, leukemia, etc. significant inhibitory activity. The chemical structure of 1-indanone is as follows:

Autosomal dominant polycystic kidney disease (ADPKD) is the most common single-gene inherited kidney disease, with an incidence of 1/1000 to 1/400, and is the leading cause of end-stage renal disease. The fourth cause of ESRD for short is the clinical pathological manifestation of progressive fluid-filled vesicles formation and continuous expansion in the bilateral kidneys of patients. The vesicular epithelial cells proliferate, and the vesicle fluid is continuously secreted, accompanied by interstitial fibrosis in the renal tissue, gradually compressing the renal parenchyma, and eventually leading to the loss of renal function and even renal failure. At present, there is a lack of ideal ADPKD treatment drugs that can intervene in the occurrence and growth of vesicles early and have fewer side effects. Patients can only maintain their lives through hemodialysis or transplantation, which brings a huge burden to the patient's family and society. Therefore, the development of ADPKD-specific therapeutic drugs that can significantly delay the occurrence and development of vesicles has important guiding significance for the treatment of clinical diseases.

At present, the only clinically approved drug for the treatment of ADPKD is the angiotensin type II receptor (V ₂ R) blocker tolvaptan, which exerts its inhibitory effect on vesicles by down-regulating cAMP levels in the body. The data indicated that V ₂ R antagonists only exerted an inhibitory effect on vesicles derived from collecting ducts, but had no significant effect on vesicles derived from proximal convoluted tubule cells, and tolvaptan had certain hepatotoxicity. At present, there is still a lack of ideal therapeutic drugs for ADPKD in clinical practice. According to the pathogenesis of ADPKD, screening inhibitors of vesicle formation and growth, and developing new drugs to treat ADPKD are still hot research topics in this field.

SUMMARY OF THE INVENTION

In order to solve the defects of the prior art, the present invention provides the application of 1-indanone in preparing a medicine for treating or preventing autosomal dominant polycystic kidney disease.

The invention also provides the application of the 1-indanone in the preparation of a medicine for treating or preventing the autosomal dominant polycystic kidney disease caused by the mutation of the Pkd1 gene.

The invention also provides the application of the 1-indanone in the preparation of a medicine for inhibiting the production of MDCK vesicles and embryonic kidney vesicles.

The present invention also provides the application of 1-indanone in preparing a medicine for inhibiting the generation and/or growth of renal vesicles.

The present invention also provides the application of 1-indanone in preparing a medicine for inhibiting the signal pathway of vesicle proliferation.

For the above application, the in vivo dose of PKD mice is 50-125 mg/kg/d, preferably 100 mg/kg/d; the in vitro experimental dose is 1 μM-25 μM, preferably 1 μM, 5 μM and 25 μM.

The present invention also provides a composition for treating and/or preventing autosomal dominant polycystic kidney disease, the composition comprising 1-indanone and a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

Preferably, in the application, the pharmaceutically acceptable carrier includes a solid carrier or a liquid carrier.

Beneficial effects: The present invention uses the MDCK vesicle model to prove that 1-indanone can inhibit the formation and growth of vesicles, and determines the intrarenal pharmacological activity of 1-indanone through the in vitro embryonic kidney vesicle model, and has a positive effect on the effect of 1-indanone on intrarenal vesicles. The development has a significant inhibitory effect. Finally, it was further proved in the polycystic kidney mouse model that 1-indanone also has the effect of inhibiting the occurrence and development of vesicles in vivo. The above vesicle inhibitory effects in vitro and in vivo are in a dose-effect relationship. .

The invention also shows that 1-indanone does not affect the viability of kidney cells, indicating that the inhibitory effect of 1-indanone on polycystic kidney has nothing to do with its cytotoxicity; the regulating effect of 1-indanone on intracellular proliferation signaling pathway is its inhibition of kidney One of the important mechanisms of vesicle occurrence and development.

The above results show that: 1-indanone can be used for the treatment of autosomal dominant polycystic kidney disease.

Description of drawings

Figure 1 is a schematic diagram of MDCK cell colonies and vesicles.

Figure 2 is a growth chart and a statistical chart of the inhibitory effect of 1-indanone on MDCK vesicles; the upper figure is the growth chart of the inhibitory effect of 1-indanone on MDCK vesicles, and the lower figure is the inhibition of vesicle growth by 1-indanone Graph.

Figure 3 is a schematic diagram of a mouse embryonic kidney vesicle model.

Figure 4 is the growth chart and statistical chart of the effect of 1-indanone on the inhibition of mouse embryonic kidney vesicles; the upper figure is the growth chart of the inhibition of mouse embryonic kidney vesicles by 1-indanone, and the lower figure is the growth chart of different doses of 1 - Graph of the inhibitory effect of indanone on the growth of mouse embryonic kidney vesicles.

Fig. 5 is Pkd1 flox ^/ flox; Ksp-Cre mouse model after administration of mouse kidney photo and renal weight index statistics chart; Wherein, the left picture is Pkd1 flox ^/ flox; Ksp-Cre mouse model after administration of mouse kidney In the photo, the right side is a graph of renal BMI.

Figure 6 is a graph of HE staining of kidney tissue sections and a graph of the cystic index of the kidney after administration of the Pkd1 flox ^/ flox; Ksp-Cre mouse model.

Figure 7 is a schematic diagram showing that 1-indanone promotes the formation of MDCK tubule-like structures.

Figure 8 is a schematic diagram showing the effect of 1-indanone on the viability of MDCK vesicle cells.

Figure 9 is a schematic diagram showing the effect of 1-indanone on the proliferation signal of mouse kidney.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The canine kidney cells (Madin-Darby canine kidney cells, MDCK) in the following examples are products of the ATCC cell bank, numbered CCL-34. The MDCK vesicles and embryonic kidney vesicle experiments used three doses of 1-indanone at 1 μM, 5 μM and 25 μM, respectively.

Example 1 Inhibition of vesicle growth by 1-indanone

Madin-Darby canine kidney cells (MDCK) were cultured in vitro in three-dimensional Matrigel (Purecol Collagen, Innamed Biomaterials Fremont, Cat. No. 5409).

Culture medium 1 is the three-dimensional matrigel obtained by adding three-dimensional matrigel, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), penicillin and streptomycin to 10×MEM medium. The concentration is 2.9 mg/ml, and the concentration of HEPES is 10 mM, 100 U/ml penicillin concentration, 100 μg/ml streptomycin concentration, pH 7.4.

Culture medium 2 is a culture medium with FBS concentration of 10% and forskolin concentration of 10 μM obtained by adding FBS and forskolin (FSK, Forskolin, Sigma, Cat. No. F6886) to DMEM/F12 culture medium. DMEM/F12 culture medium It is a liquid obtained by mixing equal volumes of DMEM medium (Invitrogen, USA, catalog number 12100-046) and F12 medium (Invitrogen, USA, catalog number 21700-075).

Take a 24-well plate, mix MDCK cells in 400 μL of culture medium 1, and add them to one well of the 24-well plate, with the same number of cells per well, 800 cells/well. Place the 24-well plate in a 37°C cell incubator for about 90 minutes. After the three-dimensional matrigel solidifies, add 1.5ml of culture medium 2 to each well, and place it in a 5% _CO2 incubator at 37°C for 4 days. The monolayer epithelial-coated unicellular vesicles can be observed under the microscope; then 1-indanone with a final concentration of 1 μM, 5 μM and 25 μM was added to the cell culture wells to continue the culture, and each dose was repeated 3 times. hole. The culture medium containing 1-indanone and forskolin was replaced every 12 hours, and the vesicles were recorded and photographed every two days, and the diameter of the vesicles was measured to evaluate the inhibitory effect of different concentrations of 1-indanone on the growth of vesicles. A total of 8 observations were made. At least 20 vesicles were counted in each well, and the growth curve of vesicles was made.

Figure 2 shows the inhibitory effect of 1-indanone on vesicle growth.

The upper picture is the growth diagram of the inhibition of MDCK vesicles by 1-indanone; the control group is the treatment group with 1-indanone concentration of 0. Among them, the first row indicates that the medium containing only forskolin was incubated on days 5 to 12, the second row indicates that the medium containing 1 μM 1-indanone and forskolin was co-cultured on days 5 to 12, and the third row indicates that the 5-12 days were co-cultured with the medium containing 5 μM 1-indanone and forskolin, the fourth row indicated that the 5-12 days were co-cultured with the medium containing 25 μM 1-indanone and forskolin, and the fifth row indicated the first On days 5 to 9, the cells were co-cultured with a medium containing 25 μM of 1-indanone and forskolin, and on days 9 to 12, they were incubated with a medium containing only forskolin.

It can be seen that 1-indanone can significantly inhibit the growth of vesicles, and the inhibitory effect of 1-indanone on vesicles increases with the increase of the concentration, indicating that the inhibitory effect of 1-indanone on the growth of MDCK vesicles has a dose effect .

The left side of the figure below shows the inhibition curve of 1-indanone on vesicle growth;

Inverted triangle curves represent days 5 to 12 cultured with forskolin only, black ball curves represent days 5 to 12 cultured with 1-indanone and forskolin at 1 μM, square curves represent days 5 to 12 The cells were incubated with the medium containing 5 μM 1-indanone and forskolin, and the positive triangle curve represented the incubation with the medium containing 25 μM 1-indanone and forskolin on days 5 to 12.

The right side of the figure below is a graph showing the reversibility of the inhibitory effect of 1-indanone on vesicle growth;

The black ball curve represents day 5-12 incubation with medium containing only forskolin, the square curve represents day 5-9 incubation with medium containing 25 μM 1-indanone and forskolin, and day 9-12 incubation with medium containing only forskolin culture broth.

Example 2 Inhibition of 1-indanone on the growth of mouse embryonic kidney vesicles

C57BL/6 mice over 6 weeks old (Experimental Animal Center, Peking University Medical Department) were mated in co-sex cages at a ratio of 1:1, and on the morning of the second day, the female mice were observed for vaginal plugs. The mice had been pregnant for 0.5 days, and the mice without vaginal plugs were first divided into cages, then closed at night, and then observed on the second day; the pregnant female mice were continued to be fed alone for 13 days, and the embryonic kidneys were taken on the 13th day using transwell plates (Corning Company). , Cat. No. 3401) culture.

The above-mentioned 13.5-day-old mouse embryonic kidney was placed in the upper chamber of the transwell, and the DMEM medium containing 8-Br-cAMP (Sigma company, product number B-5386) at a final concentration of 100 μM was added to the lower culture well for cultivation. Under the action of 8-Br-cAMP, multiple and progressive renal vesicles will be formed in renal tissue, which can be used as an in vitro whole organ-level screening drug model for evaluating the prevention and/or treatment of ADPKD with 1-indanone.

Figure 4 shows the results of 1-indanone inhibiting mouse embryonic kidney vesicles.

Among them, the above picture shows the growth of 1-indanone inhibiting the growth of mouse embryonic kidney vesicles. The first row of embryonic kidney was cultured with 100 μM 8-Br-cAMP until day 6, and the second, third, and fourth rows of embryos Kidneys were stimulated by adding 100 μM 8-Br-cAMP, and then treated with 1 μM, 5 μM, and 25 μM 1-indanone, and cultured until the sixth day. The fifth row of embryonic kidneys was stimulated by adding 100 μM 8-Br-cAMP, and then treated with 25 μM 1-indanone, and cultured until the 4th day. The kidneys were cultured in liquid, followed and photographed every day to record the condition of the kidneys, and the experiment was repeated three times.

The left side of the figure below shows the inhibitory effect of different doses of 1-indanone on mouse embryonic kidney vesicles. The right side of the figure below shows that the inhibitory effect of 1-indanone on embryonic kidney vesicle growth is reversible.

The results showed that 1-indanone significantly inhibited the development of renal vesicles, and its inhibitory effect on embryonic renal vesicles was dose-dependent. And when the drug was removed on days 5-6, the vesicles resumed growth.

Example 3 In vivo experiment

The mice used were obtained as follows: Pkd1 flox ^/ flox mice and Ksp-Cre mice were bred to obtain offspring Pkd1 ^+/- ; Ksp-Cre mice, Pkd1 ^+/- ; Ksp-Cre mice male mice Mating with female mice to obtain wild-type mice Pkd1 ^+/+ ; Ksp-Cre and Pkd1 flox ^/ flox; Ksp-Cre mice (kPKD mice). Among them, the genetic backgrounds of Pkd1 flox ^/ flox mice and Ksp-Cre mice are both C57BL/6 mice, which are both recorded in the literature (Wang W, Li F, Sun Y, et al. Aquaporin-1retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling. FASEB J. 2015;29(4):1551-1563.). Pkd1 flox ^/ flox mice are mice obtained by knocking out the Pkd1 gene in the whole kidney under the background of C57BL/6 mice, so that the mice develop ADPKD rapidly and progressively after birth. After surviving for about 7 to 10 days, gene identification was performed on the first day after the mouse was born to determine the genotype of the mouse. Wild-type C57BL/6 mice corresponding to Pkd1 flox ^/ flox mice were denoted as Pkd1 ^+/+ mice.

Wild-type mice (Pkd1 ^+/+ ; Ksp-Cre) and PKD mice (Pkd1 flox ^/ flox; Ksp-Cre) were randomly divided into two groups, blank control group (CTRL) (empty solvent group, that is, injected physiological saline) and administration group (IND) (mice were administered 1-indanone at a dose of 100 mg per kilogram of body weight per day), and each group had no less than 5 mice. Each mouse was administered subcutaneously using an insulin syringe every 24 hours starting from the 1st day after birth (30 μl per injection), and each mouse in the control group (CTRL) was injected with 30 μl of 1- Indanone solution, each mouse in the administration group (IND) was injected with 30 μl of 1-indanone solution each time (1-indanone solution was a solution obtained by dissolving commercial 1-indanone powder in physiological saline), and the mice were always given 1-indanone solution. The medicine lasts until the 5th day after birth. Weigh, kill, and take tissue.

Judging from the size and body weight of the kidneys of mice (Figure 5, the left picture is Pkd1 flox/flox; the photos of mouse kidneys after administration of Ksp-Cre mouse model, the right side is the weight chart), there is no significant difference between the groups difference. In terms of kidney size, on the 5th day after birth, polycystic kidney disease occurred in knockout PKD mice, and the kidney volume was significantly reduced after 1-indanone treatment. However, 1-indanone had no significant effect on normal kidney size.

There was no significant difference in body weight between groups of mice, but in PKD mice, administration of 1-indanone significantly reduced the renal weight index (bilateral kidney weight/body weight) in polycystic kidney mice (right panel of Figure 5). .

The results of H&E staining of mouse kidney sections showed that in PKD mice, there were a large number of vesicles in the mouse kidneys, and when 1-indanone was administered, the mouse kidneys were significantly smaller and the kidney tissue structure was improved (Figure 6).

Example 4 1-Indanone promotes the formation of MDCK tubule-like structures.

In the MDCK cell tubule formation experiment, MDCK cells were seeded in three-dimensional Matrigel, and 3T3 conditioned medium was directly added (3T3 fibroblasts were cultured in complete medium for 2 to 3 days, and the resulting medium was 3T3 conditioned medium, It contains hepatocyte growth factor, which has the effect of promoting cell differentiation), and the culture medium is replaced every 24h until the 12th day. Since hepatocyte growth factor can promote the differentiation of endothelial cells, MDCK cell colonies will gradually form tubules. sample structure. Different final concentrations of 1-indanone (concentrations of 1 μM, 5 μM, 25 μM) were added to the 3T3 conditioned medium at the same time, and the culture medium containing 1-indanone was replaced every 24h. At least 10 cell colonies were tracked in the well, and the tracking photos were taken every 2 days. On the 12th day, the number of tubules formed on each cell colony and the length of the longest tubule on each colony were counted to study different concentrations of 1- The effect of indanone on MDCK cell differentiation.

The results are shown in Figure 7. The upper figure shows that MDCK cells formed branch structures under the stimulation of 3T3 conditioned medium; and administration of different concentrations of 1-indanone significantly promoted the formation and extension of branch structures on MDCK cells. The statistical results of the number and length of tubules showed that IND dose-dependently promoted the formation of tubule structures (the left side of the lower figure) and the lengthening (the right side of the lower figure) on MDCK cells, with statistical differences.

Example 5 Cytotoxicity test

Cytotoxicity of 1-indanone determined by CCK-8 assay

The log-phase MDCK cell suspension was seeded in a 96-well culture plate, each well containing 1 × 10 ⁴ cells, and each well was given 100 μl DMEM medium containing 10% fetal bovine serum (FBS, Gibco Fisher Scientific, The Netherlands). (Invitrogen, USA, catalog No. 12100-046), placed in a 5% CO ₂ incubator at 37° C. for 24 hours. FBS was removed and serum starved for 24 hours. Then, 1-indanone solution was added to the cell culture wells (dosing wells), and the volume added to each well was the same. One concentration per well was incubated for 24 hours. Remove the supernatant, add DMEM medium containing 10% CCK-8 reagent, and continue to culture for 1 hour in a 5% CO ₂ incubator at 37°C. The microplate reader detects the OD value of each well (detection wavelength 450nm), and sets to zero. Wells (containing equal amounts of medium, CCK-8, and DMSO, without 1-indanone) and control wells (containing equal amounts of cells, medium, CCK-8, and DMSO, without 1-indanone, i.e., 1 - Dosing wells with 0 μM indanone), at least 5 replicate wells were set for each group. The cell viability was calculated according to the following formula, cell viability=(dosing well-zeroing well)]/(control well-zeroing well)×100%. The experiment was repeated three times.

Figure 8 shows the results of the cytotoxicity test of 1-indanone by CCK-8 on MDCK cells. The results show that there is no significant difference between the administration group (IND) and the control group (0 μM) at different concentrations. The concentration of 1- Indanone did not inhibit the cell viability of MDCK cells, and had no toxic effect on MDCK cells, indicating that the effect of 1-indanone on vesicle growth was not related to its cytotoxicity.

Example 5 Western blot experiment

Experimental methods: The kidneys of kPkd1(-/-) and kPkd1(+/+) mice were taken, and the effect of 1-indanone on the expression level of renal proliferation signal was studied by Western blot.

Western blot method: The kidney tissue was treated with RIPA lysate, the protein was collected, and the protein was quantified by BCA method. The protein amount of the sample was adjusted for SDS-PAGE, and the electrophoretically separated proteins were transferred to PVDF membrane. Wash the membrane with PBST for 5min×3. The PVDF membrane was blocked with 5% nonfat dry milk (dissolved in PBST) for 1 h at room temperature. Anti-proliferative and fibrotic signaling molecules were then added and incubated overnight at 4°C. PBST was washed 3 times, the corresponding secondary antibody was added, incubated for 1 h, and rinsed 3 times. The PVDF membrane was developed with the luminescent reagent ECL, the images were collected with Bio-Rad gel imaging gel, and the images were analyzed in grayscale with Quantity one. The above experiments were repeated 3 to 5 times.

Experimental results: Figure 9 is a schematic diagram of the effect of 1-indanone on the proliferation signal of mouse kidney. The results show that: 1-indanone can significantly inhibit the expression level of kidney proliferation signal molecules, including ERK1/2, S6, PCNA and so on.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (8)

1. Application of 1-indanone in preparing a medicine for treating or preventing autosomal dominant polycystic kidney disease. 2. Application of 1-indanone in the preparation of a medicine for treating or preventing autosomal dominant polycystic kidney disease caused by Pkd1 gene mutation. 3. The application of 1-indanone in the preparation of drugs for inhibiting the production of MDCK vesicles and embryonic kidney vesicles. 4. The application of 1-indanone in the preparation of a drug for inhibiting the generation and/or growth of renal vesicles. 5. The application of 1-indanone in the preparation of a drug for inhibiting vesicle proliferation signaling pathway. 6. The application according to any one of claims 1 to 5, wherein the in vivo PKD mouse dosage is 50-125 mg/kg/d, and the preferred dosage is 100 mg/kg/d; the in vitro experimental dosage 1 μM-25 μM, preferably 1 μM, 5 μM and 25 μM. 7. A composition for treating and/or preventing autosomal dominant polycystic kidney disease, comprising 1-indanone and a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, Diluent, excipient or adjuvant. 8. The application of claim 7, wherein the pharmaceutically acceptable carrier is a solid carrier or a liquid carrier.

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