CN111467331B

CN111467331B - Application of 1-indanone in preparation of medicine for treating or preventing autosomal dominant hereditary polycystic kidney disease

Info

Publication number: CN111467331B
Application number: CN202010423754.3A
Authority: CN
Inventors: 杨宝学; 周虹; 李小为
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2021-01-26
Anticipated expiration: 2040-05-19
Also published as: CN111467331A

Abstract

The invention provides application of 1-indanone in preparing a medicament for treating or preventing autosomal dominant polycystic kidney disease. The MDCK vesicle model is used for proving that the 1-indanone can inhibit the formation and growth of vesicles, the intrarenal pharmacological activity of the 1-indanone is determined by the in vitro embryo renal vesicle model, the 1-indanone has obvious inhibition effect on the development of the intrarenal vesicles, and finally, the 1-indanone also has the effect of inhibiting the generation and development of the vesicles in vivo in a polycystic kidney mouse model, and the in vitro and in vivo vesicle inhibition effects are in a dose effect relationship.

Description

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

Technical Field

The invention provides application of 1-indanone in preparation of a medicine for treating or preventing autosomal dominant hereditary polycystic kidney disease, belonging to the field of biological medicine.

Background

Fern (with the scientific name of Pteridium aquilinum (L.)) is a variant of Pteridaceae (Pteridaceae) pteris europeltis, and 1-indanone (1-indanone) is extracted from dried young leaves of Pteridophyllum vulgare, and has various biological activities, including anti-inflammatory, antibacterial, antiviral, Alzheimer's disease treatment, antitumor, etc. Previous studies have shown that 1-indanone derivatives can be used as acetylcholinesterase inhibitors to treat Alzheimer's disease. In addition, the 1-indanone can induce apoptosis of various drug-resistant tumor cells (such as MCF-7, HL-60 and MES-SA), and shows remarkable inhibitory activity on various malignant tumors such as colon cancer, breast cancer, leukemia and the like. The chemical structure of 1-indanone is as follows:

the Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common monogenic hereditary nephropathy, the incidence rate is 1/1000-1/400, the fourth cause of End-stage nephropathy (ESRD), and the clinical pathological manifestation is that the bilateral kidneys of a patient have progressive fluid-filled vesicles and are continuously amplified. The vesicle epithelial cells proliferate, and the cyst fluid is continuously secreted and accompanied with the interstitial fibrosis of the kidney tissue to gradually press the renal parenchyma, finally leading to the loss of the kidney function and the renal failure. At present, ideal ADPKD treatment medicines which can intervene vesicle generation and growth in an early stage and have few side effects are clinically lacked, and patients can only maintain life through hemodialysis or transplantation, so that huge burden is brought to families and society of the patients. Therefore, the research and development of ADPKD specific therapeutic drugs capable of obviously delaying the occurrence and development of vesicles have important guiding significance for the treatment of clinical diseases.

The only clinically approved drug currently on the market for the treatment of ADPKD is the angiotensin ii receptor (V)₂R) blocking agent tolvaptan, exerts its inhibitory effect on vesicles by downregulating cAMP levels in vivo, but clinical data indicate V₂The R antagonist only plays an inhibiting role in the collecting duct derived vesicle, but has no obvious influence on the proximal tubular cell derived vesicle, and tolvaptan has certain liver toxicity. There is still a lack of ideal therapeutic drugs for ADPKD in the clinic today. Screening for inhibitors of vesicle formation and growth based on the pathogenesis of ADPKD, and developing new drugs for the treatment of ADPKD remain the current research focus in this field.

Disclosure of Invention

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

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

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

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

The invention also provides application of the 1-indanone in preparation of medicines for inhibiting vesicle proliferation signal pathways.

In the application, the administration dosage of the in-vivo PKD mouse is 50-125 mg/kg/d, and the preferred dosage is 100 mg/kg/d; the ex vivo experimental dose is 1 μ M to 25 μ M, preferably 1 μ M, 5 μ M and 25 μ M.

The invention also provides a composition for treating and/or preventing autosomal dominant polycystic kidney disease, which comprises 1-indanone and pharmaceutically acceptable salts thereof and at least one pharmaceutically acceptable carrier, diluent, excipient or auxiliary agent.

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

Has the advantages that: the MDCK vesicle model is used for proving that the 1-indanone can inhibit the formation and growth of vesicles, the intrarenal pharmacological activity of the 1-indanone is determined by the in vitro embryo renal vesicle model, the 1-indanone has obvious inhibition effect on the development of the intrarenal vesicles, and finally, the 1-indanone also has the effect of inhibiting the generation and development of the vesicles in vivo in a polycystic kidney mouse model, and the in vitro and in vivo vesicle inhibition effects are in a dose effect relationship.

The invention also shows that the 1-indanone does not influence the activity of kidney cells, which indicates that the inhibition effect of the 1-indanone on polycystic kidney is irrelevant to the cytotoxicity; the regulation effect of 1-indanone on intracellular proliferation signal pathways is one of important mechanisms for inhibiting the occurrence and development of renal vesicles.

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

Drawings

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

FIG. 2 is a growth diagram and a statistical chart of the effect of 1-indanone on MDCK vesicle inhibition; wherein, the upper graph is a growth graph of the effect of the 1-indanone on inhibiting MDCK vesicles, and the lower graph is a graph of the inhibition of the 1-indanone on the growth of the vesicles.

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

FIG. 4 is a growth chart and a statistical chart of the effect of 1-indanone on inhibition of mouse embryonic kidney vesicles; wherein, the upper graph is a growth graph of the effect of 1-indanone on inhibiting the mouse embryonic kidney vesicles, and the lower graph is a graph of the effect of 1-indanone with different doses on inhibiting the mouse embryonic kidney vesicles.

Pkd1 in FIG. 5^flox/flox(ii) a A picture of the kidney of the mouse and a statistical picture of the kidney weight index of the Ksp-Cre mouse model after administration; wherein the left picture is Pkd1^flox/flox(ii) a Mouse kidney after administration of Ksp-Cre mouse modelPhotographs of the viscera, right side, renal weight index statistical plot.

Pkd1 in FIG. 6^flox/flox(ii) a HE staining pattern of kidney tissue sections and cystic index pattern of kidney after administration of Ksp-Cre mouse model.

FIG. 7 is a schematic representation of 1-indanone promoted formation of MDCK tubule-like structures.

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

FIG. 9 is a graph showing the effect of 1-indanone on mouse renal proliferation signals.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Canine kidney cells (MDCK) in the following examples are ATCC cell bank products and are numbered CCL-34. Wherein, the MDCK vesicle and the embryo kidney vesicle experiment respectively adopt 1-indanone with three doses of 1 mu M, 5 mu M and 25 mu M.

Example 11 inhibition of vesicle growth by Indanone

Canine kidney collecting duct cells (MDCK) were cultured in vitro in three-dimensional matrigel (Purecol Collagen, incorporated Biomaterials Fremont, cat # 5409).

The culture medium 1 was prepared by adding three-dimensional matrigel, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), penicillin and streptomycin to 10 XMEM culture medium to obtain a culture medium with a concentration of three-dimensional matrigel of 2.9mg/ml, a concentration of HEPES of 10mM, a concentration of penicillin of 100U/ml and a concentration of streptomycin of 100. mu.g/ml, and had a pH of 7.4.

The culture solution 2 was prepared by adding FBS and forskolin (FSK, forskolin, Sigma Co., Ltd., Cat. No. F6886) to DMEM/F12 culture solution to obtain a culture solution having an FBS concentration of 10% and a forskolin concentration of 10. mu.M, and the DMEM/F12 culture solution was prepared by mixing DMEM medium (Invitrogen, Cat. No. 12100-046) and F12 medium (Invitrogen, Cat. No. 21700-075) in equal volumes.

The MDCK cells were mixed in 400. mu.L of the culture medium 1 in a 24-well plate, and the mixture was added to one well of the 24-well plate, wherein the number of cells per well was the same, and was 800 cells/well. Placing 24-well plate in 37 deg.C cell culture box for about 90 min, adding 1.5ml of culture solution 2 into each well after three-dimensional matrigel solidification, and placing in 37 deg.C 5% CO₂Culturing in an incubator for about 4 days to observe single-layer epithelial-coated single-vesicular vesicles under a microscope; the cells were then incubated with 1-indanone at final concentrations of 1. mu.M, 5. mu.M and 25. mu.M, respectively, in a repeat of 3 wells per dose. Replacing fresh culture solution containing 1-indanone and forskolin every 12h, tracking and recording each vesicle every two days, measuring the diameter of the vesicle to evaluate the inhibition effect of 1-indanone with different concentrations on the growth of the vesicle, observing for 8 days, counting more than 20 vesicles per hole, and drawing a vesicle growth curve.

The results of the inhibition of vesicle growth by 1-indanone are shown in FIG. 2.

The upper graph is a growth chart of the effect of 1-indanone on inhibiting MDCK vesicles; the control group was a treatment group with a 1-indanone concentration of 0. Wherein the first row shows that the culture solution only containing forskolin is used for culturing on the 5 th to 12 th days, the second row shows that the culture solution containing 1 mu M of 1-indanone and forskolin is used for co-culturing on the 5 th to 12 th days, the third row shows that the culture solution containing 5 mu M of 1-indanone and forskolin is used for co-culturing on the 5 th to 12 th days, the fourth row shows that the culture solution containing 25 mu M of 1-indanone and forskolin is used for co-culturing on the 5 th to 12 th days, the fifth row shows that the culture solution containing 25 mu M of 1-indanone and forskolin is used for co-culturing on the 5 th to 9 th days, and the culture solution only containing forskolin is used for culturing on the 9 th to 12 th days.

It can be seen that 1-indanone can significantly inhibit the growth of vesicles, and the inhibition of vesicles by 1-indanone increases with increasing concentration, indicating that the inhibition of MDCK vesicle growth by 1-indanone has a dose effect.

The lower panel shows the inhibition curve of 1-indanone on vesicle growth;

the inverted triangle curve represents the culture with culture solution containing forskolin only on days 5 to 12, the black sphere curve represents the culture with culture solution containing 1 μ M of 1-indanone and forskolin on days 5 to 12, the square curve represents the culture with culture solution containing 5 μ M of 1-indanone and forskolin on days 5 to 12, and the positive triangle curve represents the culture with culture solution containing 25 μ M of 1-indanone and forskolin on days 5 to 12.

The lower right of the figure is a graph showing the reversible inhibition of vesicle growth by 1-indanone;

the black ball curve represents the culture with culture solution containing forskolin only on days 5-12, the square curve represents the culture with culture solution containing 25 μ M of 1-indanone and forskolin on days 5-9, and the culture with culture solution containing forskolin only on days 9-12.

Example 21 inhibition of growth of kidney vesicles from mouse embryos by Indanone

Mating C57BL/6 mice (Experimental animal center of department of medicine of Beijing university) with one cage in a ratio of 1: 1 for more than 6 weeks, observing whether the female mice have vaginal embolus in the morning on day 2, if so, indicating that the female mice are pregnant for 0.5 day, separating the mice without vaginal embolus into cages, closing the cages at night, and observing the mice on the second day; pregnant female mice were further fed alone for 13 days, and embryonic kidneys on day 13 were cultured on transwell plates (Corning Corp., Cat. 3401).

The mouse embryonic kidney of 13.5 days is placed in an upper chamber of a transwell, DMEM culture solution containing 8-Br-cAMP (Sigma company, product number B-5386) with the final concentration of 100 mu M is added into a lower culture hole for culture, and under the action of the 8-Br-cAMP, kidney vesicles which grow frequently and progressively are formed in kidney tissues and can be used as an in-vitro whole organ level drug screening model for evaluating the prevention and/or treatment of ADPKD by 1-indanone.

The effect of 1-indanone on inhibiting mouse embryonic kidney vesicles is shown in FIG. 4.

Wherein, the upper graph is a growth graph of the effect of inhibiting the mouse embryo kidney vesicles by the 1-indanone, the first row is that the embryo kidney is continuously cultured to the 6 th day by adding 100 mu M8-Br-cAMP, the second, third and fourth rows are that the embryo kidney is treated by adding 1 mu M, 5 mu M and 25 mu M1-indanone on the basis of adding 100 mu M8-Br-cAMP, and the embryo kidney is cultured to the 6 th day by replacing fresh corresponding culture solution every 12 h. And the fifth element is that the embryonic kidney is added with 25 mu M1-indanone for treatment on the basis of adding 100 mu M8-Br-cAMP for stimulation, the embryonic kidney is cultured to the 4 th day, the embryonic kidney is cultured in a culture solution only containing 8-Br-cAMP for 5 to 6 days, the condition of the kidney is recorded by tracking and photographing every day, and the experiment is repeated three times.

The lower panel shows the inhibition effect of different doses of 1-indanone on mouse embryonic kidney vesicles on the left side. The right side of the lower graph shows that the 1-indanone has reversibility on the inhibition effect of the embryo kidney vacuole.

The results show that the 1-indanone obviously inhibits the development of kidney vesicles, and the inhibition effect of the 1-indanone on the embryo kidney vesicles is in a dose-dependent relationship. And when the drug is removed on days 5-6, the vesicle can grow again.

Example 3 in vivo experiments

The mice used were obtained as follows: pkd1 will be mixed^flox/floxMating the mouse and Ksp-Cre mouse to obtain a first filial generation Pkd1^+/-(ii) a Ksp-Cre mouse, Pkd1^+/-(ii) a Mating male mouse and female mouse of Ksp-Cre mouse to obtain wild type mouse Pkd1^+/+(ii) a Ksp-Cre and Pkd1^flox/flox(ii) a Ksp-Cre mice (kPKD mice). Wherein, Pkd1^flox/floxThe genetic background of both mice and Ksp-Cre mice is C57BL/6 mice, both described in the literature (Wang W, Li F, Sun Y, et al, Aquaporin-1 retards fresh cell level in multicyclic reagent by inhibition of Wnt signaling. FASEB J.2015; 29(4): 1551. sub.1563.). Pkd1^flox/floxThe mouse is obtained by knocking out Pkd1 gene in a whole kidney specificity manner under the background of a C57BL/6 mouse, so that ADPKD which develops rapidly and progressively occurs after the mouse is born, the mouse can survive for about 7-10 days after the mouse is born, and the gene identification is carried out on the first day after the mouse is born, so that the genotype of the mouse is determined. Pkd1^flox/floxThe mouse corresponded wild type C57BL/6 mouse was assigned Pkd1^+/+A mouse.

Wild type mice (Pkd 1)^+/+(ii) a Ksp-Cre) and PKD mice (Pkd 1)^flox/flox(ii) a Ksp-Cre) were randomly divided into two groups, a blank control group (CTRL) (empty solvent group, i.e., physiological injection group)Saline) and administration groups (IND) (mice are given 1-indanone at a daily dose of 100mg per kg body weight) with no less than 5 mice per group. Each mouse was administered by subcutaneous injection using an insulin syringe (each injection amount was 30. mu.l) every 24 hours from day 1 after birth, a blank control group (CTRL) was administered by injection of 30. mu.l of 1-indanone solution every mouse, and an administration group (IND) was administered by injection of 30. mu.l of 1-indanone solution every mouse (the 1-indanone solution is a solution obtained by dissolving commercial 1-indanone powder in physiological saline), and administration was continued until day 5 after birth. Weigh, sacrifice, and remove tissue.

There was no significant difference between the groups as seen by the kidney size and body weight of the mice (FIG. 5, left panel Pkd1 flox/flox; Ksp-Cre mouse model post-dose photographs of the mice's kidneys, right side statistical body weight). From the kidney size, at postnatal day 5, significant polycystic kidneys occurred in knockout PKD mice, and the kidney volume became significantly smaller after 1-indanone treatment. While 1-indanone had no significant effect on normal kidney size.

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

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 kidney, and administration of 1-indanone significantly reduced the mouse kidney and improved the kidney tissue structure (fig. 6).

Example 41-Indanone promotes the formation of MDCK tubule-like structures.

In an MDCK cell tubule generation experiment, MDCK cells are planted in three-dimensional matrigel, 3T3 conditioned medium (3T3 fibroblasts are cultured in complete medium for 2-3 days, the obtained culture solution is 3T3 conditioned medium which contains hepatocyte growth factors and has the effect of promoting cell differentiation) is directly added for culture, fresh culture solution is replaced every 24 hours, and the MDCK cells are cultured to 12 days, wherein the MDCK cell colonies gradually form tubule-like structures because the hepatocyte growth factors can promote the differentiation of endothelial cells. 1-indanone with different final concentrations (the concentrations are 1 mu M, 5 mu M and 25 mu M respectively) is added into the 3T3 conditioned medium at the same time, fresh 1-indanone-containing medium is replaced every 24h, at least 3 wells in each group are replaced, at least more than 10 cell colonies are tracked in each well, tracking photographing is carried out every 2 days, and the number of small tubes formed on each cell colony and the length of the longest small tube on each colony are counted on day 12, so that the influence of the 1-indanone with different concentrations on MDCK cell differentiation is researched.

The results are shown in fig. 7, which shows that MDCK cells form branched structures under the stimulation of 3T3 conditioned medium; and significantly promotes the formation and elongation of branched structures on MDCK cells after administration of different concentrations of 1-indanone. Statistics on tubule number and length show that IND dose-dependent promotes formation (left panel) and elongation (right panel) of tubule structures on MDCK cells with statistical differences.

Example 5 cytotoxicity assay

Cytotoxicity of 1-Indenones by CCK-8 method

Logarithmic phase MDCK cell suspension was seeded in 96-well plates containing 1X 10 cells per well⁴Each well was dosed with 100. mu.l of DMEM medium (Invitrogen, USA, catalog No. 12100-046) containing 10% fetal bovine serum (FBS, Gibco Fisher Scientific, Netherlands) at 37 ℃ in 5% CO₂The culture was carried out in an incubator for 24 hours. FBS was removed and serum starved for 24 hours. Then, 1-indanone solution was added to the cell culture wells (administration wells) in the same volume per well, and the concentrations of 1-indanone were 0. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, and 200. mu.M, respectively. One concentration per well, incubated for 24 hours. Removing supernatant, adding DMEM containing 10% CCK-8 reagent, 5% CO at 37 deg.C₂And (3) continuously culturing for 1 hour in the incubator, detecting the OD value (the detection wavelength is 450nm) of each hole by using an enzyme-labeling instrument, setting a zero setting hole (containing the same amount of culture medium, CCK-8 and DMSO and not containing 1-indanone) and a control hole (containing the same amount of cells, culture medium, CCK-8 and DMSO and not containing 1-indanone, namely a 0 mu M administration hole of 1-indanone), and setting at least 5 multiple holes in each group. Cell viability was calculated according to the following formula (administration well-zero well)]V (control wells-zero wells). times.100%. The experiment was repeated 3 times.

FIG. 8 is a chart showing the results of cytotoxicity test of MDCK cells with CCK-8 being 1-indanone, and shows that: no obvious difference exists between the administration groups (IND) with different concentrations and the control group (0 mu M), the 1-indanone with the concentration of 200 mu M does not inhibit the cell viability of MDCK cells, and has no toxic effect on the MDCK cells, which indicates that the effect of the 1-indanone on inhibiting the vesicle growth is irrelevant to the cytotoxicity.

Example 5 Western blot experiment

The experimental method comprises the following steps: the influence of 1-indanone on the expression level of a kidney proliferation signal is researched by a Western blot method by taking the kidney of a kPkd1(-/-), a kPkd1(+/+) mouse respectively.

Western blot method: renal tissue was treated with RIPA lysate, protein was recovered, and protein quantification was performed by BCA method. SDS-PAGE was performed by adjusting the amount of protein in the sample, and the electrophoretically separated protein was transferred to a PVDF membrane. PBST membrane washing 5min 3. PVDF membrane was blocked with 5% skimmed milk powder (PBST dissolved) for 1h at room temperature. Then, a signal molecule antibody such as antiproliferation and fibrosis was added thereto, and the mixture was incubated at 4 ℃ overnight. PBST was eluted 3 times, and corresponding secondary antibody was added, incubated for 1h, and rinsed 3 times. And (3) developing the PVDF membrane by using a luminescent reagent ECL, collecting an image by using a Bio-Rad gel imaging gel, and performing gray level analysis on the image by using Quantity one. The experiments are repeated for 3-5 times.

The experimental results are as follows: FIG. 9 is a graph showing the effect of 1-indanone on mouse kidney proliferation signals, and the results show that: the 1-indanone can obviously inhibit the expression level of kidney proliferation signal molecules, including ERK1/2, S6, PCNA and the like.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1.1-indenone in the preparation of a medicament for treating or preventing autosomal dominant polycystic kidney disease.

2.1-Indanone for the preparation of a medicament for the treatment or prevention ofPkd1The application of the medicine for treating autosomal dominant polycystic kidney disease caused by gene mutation.

3. Use according to any one of claims 1 to 2, characterized in that: the administration dosage of the in vivo PKD mouse is 50-125 mg/kg/d; the dose for the in vitro experiment is 1-25 muM.