Application of dihydrotanshinone I in preparation of medicine for treating cerebral cavernous malformation

Abstract

The invention aims to give full play to the advantages of traditional Chinese medicines for treating cavernous blood vessel deformity, and particularly relates to a traditional Chinese medicine monomer dihydrotanshinone I for treating cavernous blood vessel deformity, which has a good curative effect. The chemical name of the traditional Chinese medicine monomer is (1R)-1, 6-dimethyl-1, 2-dihydronaphtho [1, 2-g] [1] benzofuran-10, 11-dione, and the molecular formula of the traditional Chinese medicine monomer is C18H14O3. The invention further discloses a preparation method of the traditional Chinese medicine monomer. The research finds that dihydrotanshinone I has a good effect of resisting cavernous vessel deformity in vivo, can inhibit mRNA levels of KLF2 and KLF4 of hCMEC / D3-CCM1KD cells in vitro, and can be used for preparing the medicine for resisting cavernous vessel deformity.

Description

Technical Field

[0001] The invention relates to dihydrotanshinone I, a natural product derived from a traditional Chinese medicine salvia miltiorrhiza, and in particular to application of dihydrotanshinone I in preparing a drug for resisting cerebral cavernous malformation, belonging to the field of traditional Chinese medicine pharmacy. Background Art

[0002] Cerebral cavernous malformations (CCMs) are a common cerebrovascular disease with an incidence of about 0.5%. Clinical manifestations of CCMs patients include intracranial hemorrhage, stroke, epilepsy and even sudden death. Pathological features include lumen dilation, thinning of the wall, destruction of endothelial cell connections, and lack of mature vascular wall components such as smooth muscle cells. Currently, CCMs disease relies on surgical treatment and lacks effective drug treatment.

[0003] The mitogen-activated protein kinase kinase kinase kinase 3 (MEKK3)-KLF transcription factor 2 / 4 (KLF2 / 4) signaling cascade in endothelial cells provides a direct driving force for the progression of CCMs. Endothelial cell-specific knockout mice with cerebral cavernous malformation 1 gene (Ccm1) ECKO ) will show obvious symptoms of CCMs disease damage, leading to a significant upregulation of MEKK3 and its downstream target genes KLF2 / 4. Endothelial-specific double knockout of Ccm genes and Klf2 / 4 genes can significantly reduce CCMs damage. Therefore, it is crucial to find drugs that effectively inhibit MEKK3-KLF2 / 4, which has become a hot topic of concern for scholars at home and abroad.

[0004] Dihydrotanshionone I (DHT) is extracted from the traditional Chinese medicine Salvia miltiorrhiza and is an important active ingredient of tanshinones. A large number of studies have shown that DHT has anti-tumor and cardiovascular protective effects. Our cell and animal experiments show that DHT has a good effect in improving CCMs diseases, and there are no reports on the use of DHT to fight CCMs diseases. Summary of the invention

[0005] The purpose of the present invention is to provide the use of dihydrotanshinone I in the preparation of anti-cerebral cavernous malformation drugs.

[0006] The anti-cerebral cavernous malformation drug involved in the present invention is an important tanshinone active ingredient extracted from the traditional Chinese medicine Danshen, and its chemical name is:

[0007] (1R)-1,6-dimethyl-1,2-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione, molecular formula C18 H 14 O3, its structural formula is as follows:

[0008]

[0009] The dihydrotanshinone I is used in the preparation of a drug for treating cerebral cavernous malformations. The dihydrotanshinone I is prepared with pharmaceutically acceptable excipients into one of an oral preparation and an injection.

[0010] The dihydrotanshinone I is used in the preparation of a drug for treating cerebral cavernous malformations, and the oral preparation is one of a tablet, a hard capsule, a soft capsule, and a granule.

[0011] The technical solution of the present invention is to use Ccm1 ECKO Mouse model was used to study the pharmacodynamic effects of DHT in mice as a whole; and the hCMEC / D3-CCM1KD cell model was constructed using a human brain microvascular endothelial cell line (hCMEC / D3) to investigate the effect of DHT on the mRNA levels of KLF2 and KLF4 in hCMEC / D3-CCM1KD cells at the molecular level, revealing the molecular mechanism of DHT in treating cerebral cavernous malformations, and providing important experimental basis for the preparation of drugs against cerebral cavernous malformations.

[0012] The experimental results of the present invention show that DHT has a good effect on improving cerebral cavernous malformations in vivo, whether in inhibiting collagen deposition or relieving endothelial structure. In vitro cell experiments show that DHT significantly reduces the mRNA levels of KLF2 and KLF4. The dihydrotanshinone I is a new compound that can treat cerebral cavernous malformations and can be used to prepare anti-cerebral cavernous malformation drugs.

[0013] The Chinese medicine monomer dihydrotanshinone I of the present invention can be used as an active ingredient and can be prepared with its pharmaceutically acceptable carrier and / or excipient into any dosage form of anti-CCMs drug suitable for humans or animals, such as oral pharmaceutical preparations, injections, tablets, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The effect of DHT on the number of cerebral cavernous malformation lesions in mice;

[0015] Figure 2 The effect of DHT on collagen deposition and vascular structure in cerebral cavernous malformations in mice;

[0016] Figure 3 This is the effect of DHT on the expression of KLF2 and KLF4 mRNA in hCMEC / D3-CCM1KD cells. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, but these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0018] The present invention proposes a potential anti-cerebral cavernous malformation drug for the treatment of cerebral cavernous malformations, which is further described in detail below in conjunction with the specific embodiments of the present invention:

[0019] Example 1: Take 10 g of dihydrotanshinone I (Shanghai Yuanye Biotechnology Co., Ltd., B20357), add starch, granulate and prepare tablets.

[0020] Example 2: Take 10 g of dihydrotanshinone I (Shanghai Yuanye Biotechnology Co., Ltd., B20357), add starch, granulate, and prepare granules.

[0021] Example 3: Take 10 g of dihydrotanshinone I (Shanghai Yuanye Biotechnology Co., Ltd., B20357), add dextrin, and fill the capsules to prepare capsules.

[0022] Example 4: Take 10 g of dihydrotanshinone I (Shanghai Yuanye Biotechnology Co., Ltd., B20357), add DMSO, TWEEN80 and physiological saline (8%:4%:88%) to prepare an injection.

[0023] Example 5: Study on the pharmacological effect of DHT on cerebral cavernous malformation in mice

[0024] 1. Animals

[0025] Cerebral cavernous malformation 1, referred to as CCM1. SPF level Ccm1 fl / fl 10 mice, Ccm1 ECKO Twenty mice were donated by Peter Choi of Centennial Research Institute, Australia.

[0026] 2. Drugs and main reagents

[0027] Dihydrotanshinone I (Shanghai Yuanye Biotechnology Co., Ltd., B20357), tamoxifen (Sigma-Aldrich, H7904), 4% paraformaldehyde (Biosharp, batch number 70071800), hematoxylin and eosin (HE) staining kit (Beyotime, C0105S), and Sirius red staining solution (Solebol, G1473).

[0028] 3. Main instruments

[0029] Stereo microscope (Leica), pathology imager (PerkinElmer).

[0030] 4. Animal Grouping

[0031] 10 Ccm1 fl / fl Mice were used as normal controls, and 20 Ccm1 ECKO The mice were randomly divided into a model group (n=10) and a dihydrotanshinone I treatment group (10 mg / kg; n=10).

[0032] 5. Animal Model Establishment

[0033] The mouse model of cerebral cavernous malformation was established by intragastric injection of tamoxifen. The specific steps are as follows: On the first day of birth (D1) of the young mice, 50 μL of tamoxifen (0.5 mg / mL) was injected intragastricly. The brain tissue of the mice was collected on D19, and then the pharmacodynamics was evaluated.

[0034] 6. Treatment Methods

[0035] Drug treatment group: The young mice were intraperitoneally administered DHT 10 mg / kg starting on the 11th day of birth (D11), and then once every 2 days, namely D13, D15, and D17; the normal control group and the model group were intraperitoneally injected with the same volume of drug solvent for a total of 4 times.

[0036] 7.Indicator detection

[0037] Stereoscopic microscopy was used to observe the overall lesions of brain tissue; HE staining was used to observe the number and area of ​​CCMs disease lesions; Sirius red staining was used to examine the collagen deposition status at the CCMs disease lesions; and scanning electron microscopy was used to detect the vascular structure at the CCMs disease lesions.

[0038] 8. Statistical methods

[0039] All data were expressed as Mean±SD. GraphPad Prism 9 software was used to analyze the differences among the groups. P<0.05 indicated a significant difference.

[0040] 9. Results and Conclusions

[0041] The results showed that DHT (10 mg / kg) can improve cerebral cavernous malformations in mice, specifically by reducing large lesions (>2000 μm 2 ) and small lesions (<2000 μm 2 ) (see Figure 1 ), while reducing collagen deposition in CCMs disease lesions and improving vascular structure (see Figure 2 ).

[0042] Conclusion: DHT can play an anti-cerebral cavernous malformation role.

[0043] Example 6: Observation of the effect of DHT on the mRNA levels of KLF transcription factor 2 (KLF2) and KLF transcription factor 4 (KLF4) in human brain microvascular endothelial cells (hCMEC / D3-CCM1KD)

[0044] 1. Main cells used in the experiment

[0045] Human brain microvascular endothelial cells: hCMEC / D3

[0046] 2. Experimental drugs and main reagents

[0047] Dihydrotanshinone I (Shanghai Yuanye Biotechnology Co., Ltd., B20357), DMEM (Keygen Biotechnology, KGL1206-500), fetal bovine serum (Novozyme, F101-01), Trypsin (BIOFROX, 1004GR100), PBS (self-made in the laboratory), reverse transcription kit (Shanghai Yishen Biotechnology Co., Ltd., 11141ES60), amplification kit (Shanghai Yishen Biotechnology Co., Ltd., product number 11201ES60), Lentivirus (GFP-Puro) / U6-CCM1-i and Lentivirus (GFP-Puro) / U6-NC (Nanjing Koris Biotechnology Co., Ltd.).

[0048] 3. Main instruments

[0049] Inverted optical microscope (ZEISS), clean bench (ESCO BIOTECH), CO2 incubator (ThermoScientific), fully automatic high-pressure sterilizer (ZEALWAY), ultra-low temperature refrigerator (Haier), water purifier (Nanjing Hanlong Laboratory Equipment Co., Ltd.), refrigerator (Haier), electronic balance (Sartorius Co., Ltd., Germany), real-time fluorescence quantitative PCR instrument (Bio-Rad), Nanodrop One (ThermoFisher).

[0050] 4. Experimental Methods

[0051] First, hCMEC / D3 cells were transfected according to the instructions for lentiviral transfection, and hCMEC / D3-NC cells and hCMEC / D3-CCM1KD cells were successfully constructed. Subsequently, hCMEC / D3-CCM1KD cells that were in logarithmic growth and in good condition were taken, digested with trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the culture medium was added and blown evenly, and the cell concentration was adjusted to inoculate in 2 small dishes, 1×10 6cells / dish, when the cell density is 60%, DHT is added to make the final concentrations of 0 and 10 μM respectively. At the same time, hCMEC / D3-NC cells are used as the control group and cultured for 24 hours. Discard the culture medium, wash the cells 3 times with pre-cooled PBS, completely remove the culture medium, add 300 μl of Trizol to each small dish, blow repeatedly for 1 minute, and transfer to a 1.5 ml ep tube. Add 60 μl of chloroform to each 300 μl Trizol, cover the sample tube cap tightly, shake it vigorously by hand for 15 seconds and place it at room temperature (stand) for 3 minutes. Centrifuge at 4°C, 12000rpm for 15 minutes. Transfer the upper water layer to a new 1.5 ml centrifuge tube, add 150 μl of isopropanol to mix (turn back and forth several times), stand at room temperature for 10 minutes, centrifuge at 4°C, 12000rpm for 10 minutes, and RNA is attached to the bottom of the tube. Discard the supernatant, add 300 μl 75% ethanol, flick the tube wall, centrifuge at 4°C, 7400 rpm for 5 min. Pour off the supernatant, place in a fume hood for 10-30 min, add 20 μl DEPC water after the ethanol evaporates, and perform reverse transcription and amplification experiments according to the instructions in the kit.

[0052] 5. Results and Conclusions

[0053] The results showed that compared with hCMEC / D3-NC cells, the mRNA level of CCM1 in the hCMEC / D3-CCM1KD group was significantly decreased, indicating that hCMEC / D3-CCM1KD cells were successfully constructed. At the same time, in hCMEC / D3-CCM1KD cells, the mRNA levels of KLF2 and KLF4 were significantly increased. Compared with the hCMEC / D3-CCM1KD group, 10μM DHT could significantly reduce the mRNA levels of KLF2 and KLF4 (see Figure 3 ).

[0054] Conclusion: DHT plays an anti-cerebral cavernous malformation role by reducing the expression of KLF2 and KLF4.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of dihydrotanshinone I in the preparation of drugs for treating cerebral cavernous malformations, It is characterized in that The chemical name of dihydrotanshinone I is (1R)-1,6-dimethyl-1,2-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione, molecular formula C 18 H 14 O 3 , the structural formula is as follows: 。 2. The use of dihydrotanshinone I according to claim 1 in the preparation of a drug for treating cerebral cavernous malformations, It is characterized in that Dihydrotanshinone I and pharmaceutically acceptable excipients are prepared into an oral preparation or an injection.

3. The use of dihydrotanshinone I according to claim 2 in the preparation of a drug for treating cerebral cavernous malformations, It is characterized in that The oral preparation is one of tablets, hard capsules, soft capsules and granules.

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