Application of sauchinone in cardiac fibrosis

Abstract

The invention discloses application of sauchinone in cardiac fibrosis, relates to the technical field of medicines, and particularly relates to application of pharmaceutically acceptable salt derivatives of sauchinone in preparation of medicines for treating and / or relieving cardiac fibrosis. The sauchinone disclosed by the invention inhibits proliferation and migration of fibroblasts induced by Ang II. The sauchinone has a potential anti-cardiac fibrosis effect. The sauchinone can reduce the area of cardiac fibrosis and down-regulate mRNA level and protein expression of fibrosis related indexes, so that the sauchinone shows a potential anti-fibrosis effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to the role of sauchinone in cardiac fibrosis. BACKGROUND

[0002] Cardiac fibrosis is a common pathophysiological remodeling process that occurs in various cardiovascular diseases, greatly affecting the structure and function of the heart, and gradually leading to the development of heart failure. Cardiac fibrosis is mainly manifested as excessive deposition of extracellular matrix, and abnormal proliferation, differentiation and migration of cardiac fibroblasts are the cause of excessive deposition of extracellular matrix in myocardium.

[0003] Sauchinone (Sau) is a lignan compound extracted from the plant Saururus chinensis. It has multiple biological activities and pharmacological effects, such as anti-inflammatory and anti-oxidative stress, anti-tumor and liver protection. Sauchinone has been studied in various fields and has shown certain therapeutic effects. For example, it can inhibit the Akt-CREB-MMP13 signaling pathway to inhibit the proliferation, migration and invasion of breast cancer cells; it can promote Th17 cells to produce IL-10 through Blimp-1 to improve intestinal inflammation; it can also prevent neurotoxicity by inhibiting the production of NO. In summary, the therapeutic effect of sauchinone in cardiac fibrosis is not clear in view of the current research status of sauchinone in the heart.

[0004] Transverse aortic constriction (TAC) is an animal surgical method that induces myocardial remodeling by increasing cardiac pressure load, and is an animal experimental model commonly used to induce myocardial hypertrophy, myocardial fibrosis and heart failure. Therefore, the present application uses transverse aortic constriction to construct a myocardial fibrosis model to evaluate the therapeutic potential of sauchinone for TAC-induced myocardial fibrosis. SUMMARY

[0005] The present application aims to provide a new use of sauchinone in the treatment of cardiac fibrosis, thereby providing a new choice for the effective treatment of cardiac fibrosis.

[0006] The sauchinone or its pharmaceutically acceptable salt derivative of the present application is used in the preparation of a medicament for treating and / or alleviating cardiac fibrosis.

[0007] Further, the sauchinone or its pharmaceutically acceptable salt derivative accounts for 0.05% to 99% of the total mass of the medicament.

[0008] Further, the sauchinone or its pharmaceutically acceptable salt derivative accounts for 1% to 5% of the total mass of the composition.

[0009] Further, the molar concentration of the tripterygium wilfordii hook f. ketone or its pharmaceutically acceptable salt derivative is 10-40 μM.

[0010] Further, the tripterygium wilfordii hook f. ketone or its pharmaceutically acceptable salt derivative is used for preparing a drug for inhibiting myocardial fibrosis induced by pressure load.

[0011] Further, the tripterygium wilfordii hook f. ketone or its pharmaceutically acceptable salt derivative is used for preparing a drug for inhibiting the up-regulation of mRNA and protein levels of Collagen I, Collagen III, alpha-SMA and CTGF in fibroblasts induced by Ang II, thereby causing proliferation and migration of cardiac fibroblasts.

[0012] Further, the treatment and / or alleviation of cardiac fibrosis is achieved by reducing the area of cardiac fibrosis, and down-regulating the mRNA level and protein expression of fibrosis-related indicators.

[0013] The present application comprises the following beneficial effects:

[0014] 1. In the fibrosis model induced by pressure load, the tripterygium wilfordii hook f. ketone (Sau) can reduce the area of cardiac fibrosis, and down-regulate the mRNA level of Collagen I, Collagen III, alpha-SMA and CTGF. It is indicated that the tripterygium wilfordii hook f. ketone (Sau) can alleviate cardiac fibrosis.

[0015] 2. In the in vitro fibrosis model induced by Ang II, the tripterygium wilfordii hook f. ketone (Sau) inhibits the up-regulation of mRNA levels of Collagen-I, Collagen-III, alpha-SMA and CTGF induced by Ang II, and the protein levels of Collagen-I and Collagen-III, and 20 μM of Sau shows a relatively significant potential anti-fibrosis effect.

[0016] 3. The tripterygium wilfordii hook f. ketone (Sau) inhibits the proliferation and migration of fibroblasts induced by Ang II.

[0017] 4. The tripterygium wilfordii hook f. ketone (Sau) has a potential anti-cardiac fibrosis effect.

[0018] 5. The present application provides the effect of tripterygium wilfordii hook f. ketone (Sau) in treating myocardial fibrosis. The tripterygium wilfordii hook f. ketone can reduce the area of cardiac fibrosis, and down-regulate the mRNA level and protein expression of fibrosis-related indicators, thereby showing a potential anti-fibrosis effect. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a flow chart of TAC model mouse animal experiment;

[0020] Figure 2Figure for the effect of Sau on the heart size and fibrosis of TAC model mice;

[0021] Figure 3 Figure for the effect of Sau on the fibrosis related mRNA index of the heart tissue of TAC model mice;

[0022] Figure 4 Figure for the effect of Sau on the fibrosis related mRNA index of the primary fibroblast of mouse heart;

[0023] Figure 5 Figure for the effect of Sau on the fibrosis related protein index of the primary fibroblast of mouse heart;

[0024] Figure 6 Figure for the effect of Sau on the proliferation and migration of the primary fibroblast of mouse heart;

[0025] Figure legend: C57BL / 6: sham group; TAC group; TAC + Sau group; Sau group; primary fibroblast of heart: Control group; AngII group; AngII + Sau group, Sau group. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear and apparent, the spirit of the present application will be described in detail below, and any person skilled in the art can make changes and modifications to the technology taught by the present application content after understanding the embodiments of the present application, without departing from the spirit and scope of the present application content.

[0027] The schematic embodiments of the present application and their descriptions are used to explain the present application, but not as a limitation of the present application.

[0028] The following provides Examples 1 to 6 to illustrate the effect of Sau (Sau) on the heart size, the area of heart fibrosis, and the mRNA level of fibrosis related index.

[0029] Example 1

[0030] Construction of the animal model of heart fibrosis

[0031] Experimental method: 8-week-old C57BL / 6 mice weighing 21-23 g were selected. The mice were randomly grouped, modeled and dosed. In this experiment, a TAC operation was performed to construct a cardiac fibrosis model using a rubber ring with an inner diameter of 0.45 mm. The treatment of the rubber ring with an inner diameter of 0.45 mm: the rubber ring was cut open with a scalpel, and an 8-0 surgical suture was inserted vertically into the rubber ring. Before use, the rubber ring was immersed in 100°C water for 5 minutes to remove impurities, and then soaked in 75% ethanol for use. The mice were anesthetized with anesthetic (ready-to-use tribromoethanol solution) at a dose of 0.125 mL / 10 g. The fur on the neck and chest of the mouse was removed with depilatory cream, and the mouse was fixed on a mouse plate. The neck skin was cut, the fat and muscle were bluntly dissected, the trachea of the mouse was exposed, the tracheal intubation was performed, and after the intubation needle entered the trachea, the intubation needle was fixed on the mouse plate with tape. Continue to cut the second rib along the cut skin, expose the transverse aortic arch, clamp the surgical suture of one side of the rubber ring to cover the transverse aorta from below, and tighten the surgical suture on both sides of the rubber ring. After suturing the ribs and skin, the skin surface was disinfected with iodophor. The mouse was injected with 0.1 mL of normal saline intraperitoneally for rehydration. After the mouse turned over on its own, the tracheal intubation was removed.

[0032] Example 2

[0033] Experimental method: The experimental subjects constructed in Example 1 were divided into sham group, TAC group, TAC+Sau group, and Sau group, a total of 4 groups, 5 mice in each group. One week after the construction of the cardiac fibrosis model, i.e. on the eighth day, the TAC+Sau group and the Sau group were given tripterygium wilfordii honnigmann ex hand-masson (Sau) intraperitoneally (20 mg / kg / day / mouse), and the intraperitoneal injection lasted for three weeks. The samples were taken on the 29th day. (See Figure 1 ) The mouse heart was sectioned and stained, and the changes in fibrosis area were observed by Masson staining. Masson staining: according to the instructions, the section was routinely deparaffinized with water, stained with prepared Weigert iron hematoxylin staining solution for 5-10 min, differentiated with acid ethanol differentiation solution, washed with water, blued with Masson blue solution, washed with water, washed with distilled water for 1 min, stained with Lichun red staining solution for 5-10 min, during the above operation process, weak acid working solution was prepared according to the ratio of distilled water: weak acid solution = 2:1, washed with weak acid working solution for 1 min, washed with phosphomolybdic acid solution for 1-2 min, washed with prepared weak acid working solution for 1 min, directly put into aniline blue staining solution for 1-2 min, washed with prepared weak acid working solution for 1 min, quickly dehydrated with 95% ethanol, dehydrated with anhydrous ethanol for 3 times, each time for 5-10 s, transparent with xylene for 3 times, each time for 1-2 min, and sealed with neutral resin.

[0034] Example 3

[0035] Experimental method: At the end of Example 1 and Example 2, the heart RNA was extracted, and the transcription level of fibrosis-related factors was observed using RT-qPCR experiment. The heart was placed in a 1.5 mL EP tube, a little Trizol was added, and the tissue was ground with a grinding rod, and then Trizol was added to make the volume of the EP tube 1 mL. 200 μl of chloroform was added to each tube, and the vortex machine was shaken three times at 13500 rpm for 15 min. The supernatant was aspirated into a clean centrifuge tube, and an equal volume of isopropanol was added, and it was placed on ice for half an hour, and then centrifuged at 13500 rpm for 15 min. The supernatant was discarded, 0.5 mL of 75% alcohol was added to wash the precipitate, and then centrifuged at 10600 rpm for 6 min to discard the supernatant. The water was wiped off with a water-absorbing paper, and then 15 μL of DEPC water was added to the EP tube after it was dried, and then mixed evenly. The RNA content was detected using a Nano Drop spectrophotometer, and the RNA was reversely transcribed into cDNA using a HiScript III All-in-one RT SuperMix Perfect for qPCR kit. The sample-primer-ChamQ Universal SYBR qPCR Master Mix reaction system was prepared, the target gene was amplified using a QPCR instrument, and the relative quantitative analysis was carried out by the ΔΔCt value method.

[0036] In summary, Examples 1 to 3, the following experimental results can be obtained (see Figure 2 ): In the TAC-induced fibrosis model, Sau can reduce the area of cardiac fibrosis, and down-regulate the transcription levels of Collagen III, Collagen I, α-SMA and CTGF. It is suggested that Sau can alleviate cardiac fibrosis.

[0037] Example 4

[0038] Construction of fibrosis cell model

[0039] Experimental method: The experimental object constructed in Example 1 is divided into a control group; an AngII group, an AngII+Sau (10 μM) group, an AngII+Sau (20 μM) group, an AngII+Sau (40 μM) group, a total of 4 groups, 5 samples in each group. To observe the effect of different concentrations of three white grass ketones (Sau) on the AngII-induced fibrosis model. Cells were treated with (1 μM) AngII and different concentrations of Sau, and AngII was used for 48 hours. Extract cell RNA, and use RT-qPCR to observe the transcription level of fibrosis-related factors in fibroblasts. After the cells are scraped with appropriate Trizol, they are placed in a 1.5 mL EP tube, 100 μL chloroform is added to each 0.5 mL Trizol, and vortexed for three times, 13500 rpm, 15 min centrifugation. The supernatant is aspirated into a clean centrifuge tube, and an equal volume of isopropanol is added, and it is placed on ice for half an hour, 13500 rpm, 15 min centrifugation. Discard the supernatant, add 500 μL of 75% alcohol to wash the precipitate, 10600 rpm, 6 min centrifugation. Discard the supernatant, wipe off the water with a water-absorbing paper, and after the EP tube is dried, add 15 μL of DEPC water and mix well. Use NanoDrop spectrophotometer to detect the content of RNA, and use HiScript Ⅲ All-in-one RT SuperMix Perfect for qPCR reagent kit to reverse transcribe RNA into cDNA. Prepare the sample-primer-ChamQ Universal SYBR qPCR MasterMix reaction system, use QPCR instrument to amplify the target gene, and use the ΔΔCt value method for relative quantitative analysis.

[0040] Through Example 4, the present application finds that Sau inhibits the up-regulation of the mRNA levels of Collagen III, Collagen I, α-SMA and CTGF induced by AngII, and 20 μM of Sau shows a relatively significant potential anti-fibrosis effect. Therefore, the Sau concentration used in the subsequent experiments is 20 μM (see Figure 3 and 4 ).

[0041] Example 5

[0042] Experimental method: On the basis of Example 4, the experimental grouping is changed to control group, Ang II group, Ang II + Sau group, Sau group, a total of 4 groups, 5 samples in each group. Extract fibroblast protein, perform Western Blot experiment, and observe the protein expression of Collagen III and Collagen I. Add 20 μL protein lysate in each well of the six-well plate, scrape with a cell scraper, and aspirate the cell liquid into a 1.5 mL EP tube. Ultrasonic three times, 13500 rpm, 15 min, and aspirate the supernatant. Measure the protein concentration with a BCA kit on an enzyme marker. After measuring the concentration, calculate according to the formula, prepare the protein sample with Loading and Ripa, and perform Western Blot experiment at 100℃ for 5 min.

[0043] It is found through the example that Sau inhibits the up-regulation of Collagen III and Collagen I protein expression induced by Ang II (see Figure 5 ).

[0044] Example 6

[0045] Experimental method: On the basis of Example 5, the experimental grouping is changed to control group, Ang II group, Ang II + Sau group, Sau group, a total of 4 groups, 5 samples in each group. Scratch experiment is performed on fibroblasts to observe the effect of tripterygium wilfordii hook on the migration of mouse heart primary fibroblasts. Remove the culture medium, wash with PBS for 2-3 times, use 200 μL gun head to vertically scratch along the cover of the six-well plate, take a photo under a microscope, and mark as 0 hour. Take a photo after 24 hours of Ang II action, and take a photo after 48 hours of Ang II action. Perform migration analysis with ImageJ.

[0046] It is found through the example that Sau inhibits the proliferation and migration of fibroblasts induced by Ang II (see Figure 6 ).

[0047] In combination of all examples, Sau provided by the application has a potential anti-cardiac fibrosis effect.

Claims

1. The use of pharmaceutically acceptable salt derivatives of salicylic acid in the preparation of drugs for the treatment and / or relief of cardiac fibrosis.

2. The application according to claim 1, characterized in that, The salicylic acid or its pharmaceutically acceptable salt derivatives comprise 0.05% to 99% of the total mass of the drug.

3. The application according to claim 1, characterized in that, The salicylic acid or its pharmaceutically acceptable salt derivatives comprise 1% to 5% of the total composition by mass.

4. The application according to claim 1, characterized in that, The molar concentration of the salicylic acid or its pharmaceutically acceptable salt derivative is 10 μM to 40 μM.

5. The application according to any one of claims 1 to 4, characterized in that, The salicylic acid ketone or its pharmaceutically acceptable salt derivatives are used to prepare a treatment that inhibits stress-induced myocardial fibrosis in mice.

6. The application according to claim 5, characterized in that, The aforementioned salicylic acid or its pharmaceutically acceptable salt derivatives are used to prepare a drug that inhibits the upregulation of mRNA and protein levels of Collagen I, Collagen III, α-SMA, and CTGF in Ang II-induced fibroblasts, leading to the proliferation and migration of cardiac fibroblasts.

7. The application according to claim 1, characterized in that, The treatment and / or relief of cardiac fibrosis is achieved by reducing the area of ​​cardiac fibrosis and downregulating the mRNA levels and protein expression of fibrosis-related indicators.

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