Application of ganximatianone A in the preparation of drugs for treating myocardial fibrosis

Abstract

The present invention discloses the use of radix strychnos nucifera A in the preparation of a drug for treating myocardial fibrosis, and requests protection for the use of radix strychnos nucifera A in the preparation of an anti-myocardial fibrosis drug. The present invention finds that radix strychnos nucifera A has an anti-myocardial fibrosis effect and has the application prospect of being developed into an anti-myocardial fibrosis drug. The prior art has not disclosed this effect of radix strychnos nucifera A, nor does it have any technical inspiration, and has outstanding substantive characteristics and significant progress.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to a new use of a known compound, and specifically relates to the use of scutellaria baicalensis ketone A in preparing a medicine for treating myocardial fibrosis. Background Art

[0002] With the acceleration of the aging process and the increase in the frequency of high-calorie diets, the incidence of cardiovascular diseases has been increasing year by year. Due to its extremely high disability and mortality rates, it seriously threatens people's lives and health. Therefore, clarifying the pathogenesis of cardiovascular diseases, exploring new treatment strategies and developing effective preventive and therapeutic drugs is a major health issue that needs to be addressed urgently.

[0003] Myocardial fibrosis is a pathological phenomenon in which pathogenic factors act on the heart for a long time, myocardial fibroblasts are activated and transformed into myofibroblasts, which then causes excessive deposition of extracellular matrix in myocardial tissue, resulting in severe scarring, tissue stiffness and impaired function of the heart. Myocardial fibrosis can cause abnormalities in myocardial contraction, conduction function and metabolism, and play an important role in ventricular remodeling. It can also cause heart diseases such as heart failure and various arrhythmias, greatly increasing the risk of sudden cardiac death and cardiac arrest. In developed countries, due to the lack of effective means to treat fibrosis, about 45% of deaths are attributed to fibrotic diseases (reference: Bian Xiaohong et al., Exploring the prevention and treatment of myocardial fibrosis with traditional Chinese medicine based on the TGF-β1 signaling pathway, Hebei Medicine, 2023). Therefore, how to effectively prevent and treat myocardial fibrosis has become one of the research hotspots.

[0004] Salvia serrata A is a natural product that has been isolated from Salvia serrata a long time ago. However, there are very few reports on the activity of Salvia serrata A, and there are no reports related to its treatment of myocardial fibrosis.

[0005] Based on the findings of the study on the activity of new ketone A of Gansu rat tail, the present invention is specially proposed. Summary of the invention

[0006] The purpose of the present invention is to provide the use of ganximab ketone A in preparing medicine for treating myocardial fibrosis.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] Application of ganximab ketone A in the preparation of drugs for treating myocardial fibrosis.

[0009] Preferably, the drug uses ganseritinone A as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0010] More preferably, the auxiliary material is a solid, liquid or semi-solid auxiliary material.

[0011] More preferably, the dosage forms include tablets, capsules, and injections.

[0012] Beneficial effects:

[0013] The present invention finds that ganxiongsai rat tail ketone A has an anti-myocardial fibrosis effect and has the application prospect of being developed into an anti-myocardial fibrosis drug. The prior art has not disclosed such an effect of ganxiongsai rat tail ketone A, nor does it have any technical inspiration. It has outstanding substantive characteristics and significant progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the proliferation activity of NIH-3T3 cells in each group.

[0015] Figure 2 The expression levels of fibrosis marker proteins (α-SMA, Collagen Ⅰ and Collagen Ⅲ) in primary cardiac fibroblasts of rats in each group. DETAILED DESCRIPTION

[0016] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0017] 1. Experimental Materials

[0018] Instruments: biological safety cabinet (Thermo, HCB-900V), cell culture incubator (Thermo, Thermo 317), high-speed refrigerated centrifuge (Eppendorf, 5430R), Milli Q water purifier (Merck, Milli-Q), electronic balance (Thermo, SQP), multifunctional microplate reader (Berthold, LB942), high-speed refrigerated centrifuge (Eppendorf, 5340R), metal bath (Eppendorf, Thermo Top), Western-blot electrophoresis and transfer system (Bio-Rad, 1658033), ice machine (Skochman, AF100), Tianneng gel imaging system (Tiannian, Tanono-5200).

[0019] Animals, cells, and reagents: SD rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., mouse embryonic fibroblasts (NIH-3T3) were purchased from the Chinese Academy of Sciences, Cell Counting Kit-8 cell proliferation toxicity detection kit (Tongren Chemical, CK04), dimethyl sulfoxide (DMSO, GENERAL-REAGENT, 67-68-5), newborn bovine serum (Newborn bovine calf serum, 10099141, Australian), DMEM medium (Keygen Biotechnology, KGM12800-500), TGF-β1 (Proteintech, AF-100-21C), trypsin (Gibco, Thermo Scientific, 25200072), neoprzewaquinone A (Neoprzewaquinone A) was purchased from Chengdu Desite Biotechnology Co., Ltd. with a purity of >98%, and the positive drug CB839 (studies have shown that CB839 can inhibit the activation of fibroblasts by inhibiting glutamine degrading enzyme 1 (GLS1)) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0020] 2. Experimental Methods

[0021] 1. Neoprzewaquinone A inhibits fibroblast proliferation

[0022] The present invention adopts the classic TGF-β1 induction method to construct a cell fibrosis model.

[0023] The principle of TGF-β1 induction method is: TGF-β is one of the important fibrogenic cytokines in the body, and is involved in regulating the entire process of fibroblast proliferation, differentiation, migration and extracellular matrix production. Its level reflects the severity of cell fibrosis to a certain extent; TGF-β1 is the main subtype of the TGF-β family, which can promote the expression of type I and type III collagen fibers in fibroblasts by activating downstream Smads protein signal transduction molecules, thereby promoting the activation process of fibroblasts.

[0024] 2. Mouse embryonic fibroblast culture

[0025] NIH-3T3 cells were cultured in DMEM containing 10% NCBS in a cell culture incubator at 37°C and 5% CO2 for 2 generations before subsequent experiments.

[0026] 3. Culture of primary rat cardiac fibroblasts

[0027] After euthanasia of 1-3 day old suckling rats by decapitation, the hearts were quickly removed and perfused with ice-cold PBS, and then the hearts were minced into 1 mm pieces with scissors. 3All the small pieces were digested with 1 mg / mL type II collagenase in a water bath at 37°C for 5 min×6 times, all the enzyme solutions were terminated by DMEM medium containing 10% FBS, and the supernatant was discarded after 2000g×5min. 500μL of red blood cell lysis solution was added to each cell to remove red blood cells, and the upper red blood cell lysis solution was discarded after 2000g×3min again. The lower layer of cells was resuspended with 10% FBS DMEM medium and purified through a 70μm cell strainer. The obtained cell suspension was pre-spread in a 100mm culture dish with 10% FBS DMEM medium. After 90min of cell adhesion, the upper layer of culture medium containing cardiomyocytes was discarded, and then the lower layer of culture medium was replaced with 3% FBS DMEM medium to culture myocardial fibroblasts, and cells of generations 1-3 were used for subsequent pharmacodynamic evaluation.

[0028] 4. CCK-8 method to determine cell proliferation activity

[0029] NIH-3T3 cells with good growth status were taken to prepare cell suspension (cell concentration 5×10 4 / mL) were inoculated in a 96-well plate, 200μL per well, divided into a control group (Control), a model group (TGF-β1) and a drug-treated group (NeoprzewaquinoneA), with 10 replicates in each group. After 24h, the control group was replaced with fresh culture medium, the model group was replaced with a fresh culture medium containing 10ng / mL TGF-β1, and the drug-treated group was replaced with a fresh culture medium containing 10ng / mL TGF-β1 and 1μM NeoprzewaquinoneA. After being placed in a cell culture incubator for 24h, 20μL CCK-8 solution was added to each well, and the cells were incubated in the cell culture incubator for 1 hour, and the absorbance was detected at 450nm by a multifunctional microplate reader. The cell proliferation activity of the model group and the drug-treated group was calculated according to the absorbance value of 450nm in each group.

[0030] 5. Western blot to determine the expression level of fibrosis-related proteins

[0031] Primary cardiac fibroblasts of rats in good growth condition were used to prepare cell suspension (cell concentration 2×10 5 / mL) were inoculated in 6-well plates, 2mL per well, and divided into control group (Control), model group (TGF-β1) and drug-treated group, with 5 replicates in each group. After 24 hours of culture, the control group was replaced with fresh culture medium, the model group was replaced with fresh culture medium containing 10ng / mL TGF-β1, and the drug-treated group was replaced with fresh culture medium containing 10ng / mL TGF-β1 and different drugs (1μM NeoprzewaquinoneA, 2μM NeoprzewaquinoneA or 2.5μM CB839). After culturing for 24 hours in a cell culture incubator, the culture medium was discarded, and the cells were washed three times with PBS. 120 μL of Western and IP cell lysis buffer containing 1×PMSF was added to each well. After being fully lysed on ice for 10 minutes, the cells were scraped off the bottom of the culture dish with a clean cell scraper and transferred to a pre-prepared 1.5 mL EP tube. The cells were then centrifuged at 4°C and 12,000 g for 15 minutes. The supernatant was gently removed and transferred to a clean centrifuge tube. 3× loading buffer was added to make the final loading buffer concentration 1×. The cells were boiled in a 100°C metal bath for 10 minutes until the proteins were denatured. The samples were then collected and stored in a -20°C refrigerator for testing. The expression levels of α-SMA, CollagenⅠ and CollagenⅢ proteins were detected by Western blot.

[0032] 6. Statistical analysis

[0033] All experiments were designed with randomization, and all data were expressed as mean ± standard deviation. The confidence standard for distinguishing significant differences was set at p < 0.05, ### represents p < 0.0001 compared with the normal group, ## represents p < 0.001 compared with the normal group, *** represents p < 0.0001 compared with the TGF-β1 group, ** represents p < 0.001 compared with the TGF-β1 group, * represents p < 0.01 compared with the TGF-β1 group, NS represents no significant difference compared with the TGF-β1 group, and the p value was evaluated based on one-way ANOVA with Tukey's test.

[0034] 3. Experimental Results

[0035] 1. Neoprzewaquinone A inhibits fibroblast proliferation

[0036] Based on the results of fibroblast (NIH-3T3) proliferation assessment ( Figure 1) showed that compared with the control group, the proliferation activity of fibroblasts stimulated by TGF-β1 was significantly enhanced, indicating that the model was successfully established; compared with the model group, the proliferation activity of cells in the drug-treated group was significantly reduced, indicating that Neoprzewaquinone A effectively inhibited the proliferation of NIH-3T3 cells. Enhanced fibroblast proliferation activity is a core cell effect in the pathological process of fibrosis. Therefore, the above research results show that Neoprzewaquinone A has anti-fibrotic activity.

[0037] 2. Expression levels of fibrosis marker proteins

[0038] The characteristic proteins α-SMA, Collagen Ⅰ and Collagen Ⅲ secreted after activation of cardiac fibroblasts were used as evaluation indicators to investigate the efficacy of ganximab tail new ketone A in inhibiting the activation of cardiac fibroblasts. The results of the study based on Western-blot are as follows Figure 2 As shown in the figure, compared with the control group, the expression levels of α-SMA, CollagenⅠ and CollagenⅢ proteins in cardiac fibroblasts were significantly upregulated after TGF-β1 stimulation, indicating that the cardiac fibroblast activation model was successfully established; compared with the model group, after cardiac fibroblasts were treated with NeoprzewaquinoneA (1μM), NeoprzewaquinoneA (2μM) or CB839 (2.5μM), the expression levels of α-SMA, CollagenⅠ and CollagenⅢ in the cells were significantly downregulated, indicating that NeoprzewaquinoneA can play an anti-cardiac fibroblast activation effect similar to the positive drug CB839, and shows obvious dose dependence.

[0039] The above experiments prove that Gansu rat tail ketone A has an anti-myocardial fibrosis effect and has the application prospect of being developed into an anti-myocardial fibrosis drug. The prior art has not disclosed this effect of Gansu rat tail ketone A, nor does it have any technical inspiration. It has outstanding substantive characteristics and significant progress.

[0040] The purpose of the above-mentioned 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 the specific embodiments.

Claims

1. Application of ganximab tail ketone A in the preparation of drugs for the treatment of myocardial fibrosis.

2. The use according to claim 1, wherein the drug uses ganximab tail new ketone A as the active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

3. The use according to claim 2, wherein the auxiliary material is a solid, liquid or semi-solid auxiliary material.

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

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