Application of isorhamnetin in preparation of anti-myocardial fibrosis drugs

Abstract

The invention discloses an application of isorhamnetin in preparation of an anti-myocardial fibrosis medicine, and belongs to the technical field of medicines. The invention proposes that the isorhamnetin can effectively reduce myocardial tissue collagen deposition for the first time, and the medical application of the isorhamnetin is expanded. The isorhamnetin is used as the anti-myocardial fibrosis medicine, can effectively relieve the increase of mRNA expression levels of alpha-SMA, COL-1, COL-3, p53, PAI-1, IL-1beta and IL-6, and has the function of retarding myocardial fibrosis. The isorhamnetin is used as the active component of the anti-myocardial fibrosis medicine, the safety is high, the side effect is small, a novel anti-myocardial fibrosis treatment strategy is provided, and the application blank of the isorhamnetin in the field is filled.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of isorhamnetin in the preparation of anti-myocardial fibrosis drugs. Background Technology

[0002] Myocardial fibrosis is a common pathological feature of many cardiovascular diseases. Due to excessive activation of cardiac fibroblasts, excessive deposition of extracellular matrix occurs, ultimately damaging the structure and function of the heart, leading to increased cardiac stiffness and decreased contractile function. While current clinical drugs for treating myocardial fibrosis can relieve symptoms, they have adverse reactions and toxic side effects. Myocardial fibrosis is a core pathological change in many cardiovascular diseases (such as hypertensive heart disease, myocardial infarction, and diabetic cardiomyopathy). Currently, there are no specific anti-fibrotic drugs available clinically; treatment focuses on controlling the primary disease (such as ACEIs / ARBs), with limited efficacy.

[0003] Isorhamnetin is a plant flavonoid found in ginkgo leaves and sea buckthorn fruit. As an active ingredient in traditional Chinese medicine, it possesses the characteristics of high efficacy and low toxicity. Current research has found that isorhamnetin has anti-inflammatory, anti-tumor, and antioxidant effects.

[0004] No studies have yet demonstrated the role of isorhamnetin in the prevention of myocardial fibrosis. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide the application of isorhamnetin in the preparation of anti-myocardial fibrosis drugs, thus expanding the pharmaceutical applications of isorhamnetin. The isorhamnetin has the following structural formula and its chemical name is 3,5,7-trihydroxy-4'-methoxyflavone.

[0006] .

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the use of isorhamnetin in the preparation of anti-myocardial fibrosis drugs.

[0008] Based on the above technical solution, the anti-myocardial fibrosis drug uses isorhamnetin as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0009] Based on the above technical solution, the excipient is further defined as a solid excipient, a liquid excipient, or a semi-solid excipient.

[0010] Based on the above technical solution, the dosage form is further defined as tablets, capsules, or injections.

[0011] Based on the above technical solution, the effective content of isorhamnetin is further 5-10 mg / kg.

[0012] Secondly, the present invention provides an anti-myocardial fibrosis pharmaceutical composition, wherein the active ingredient of the anti-myocardial fibrosis pharmaceutical composition includes isorhamnetin.

[0013] Based on the above technical solution, the sole active ingredient of the anti-myocardial fibrosis drug composition is isorhamnetin.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to propose that isorhamnetin can effectively reduce collagen deposition in myocardial tissue, thus expanding the pharmaceutical applications of isorhamnetin.

[0015] 2. This invention is the first to propose that isorhamnetin, as an anti-myocardial fibrosis drug, can effectively alleviate the increase in mRNA expression levels of α-SMA, COL-1, COL-3, p53, and PAI-1, and has the function of slowing down myocardial fibrosis.

[0016] 3. This invention is the first to propose that isorhamnetin, as an anti-myocardial fibrosis drug, can effectively alleviate the increase in mRNA expression levels of inflammatory factors IL-1β and IL-6.

[0017] 4. This invention is the first to propose the use of isorhamnetin as the active ingredient in an anti-myocardial fibrosis drug, which has high safety and few side effects, providing a new strategy for the treatment of myocardial fibrosis and filling the gap in the application of isorhamnetin in this field. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0019] Figure 1 MASSON collagen staining images of myocardial sections from mice in different experimental groups: A is the CON group; B is the ISO group; C is the isorhamnetin (10 mg / kg ISOR) single-drug group; D is the ISO+ISOR group; E is the ISO+PRO group. Figure 2 Bar charts showing the mRNA expression levels of myocardial fibrosis-related genes α-SMA, COL-1, and COL-3 in mice from different experimental groups: Figure A shows the mRNA expression level of α-SMA; Figure B shows the mRNA expression level of COL-1; Figure C shows the expression levels of COL-1 and COL-3. Figure 3 Bar charts showing the mRNA expression results of p53 and PAI-1, genes related to myocardial fibrosis, in mice from different experimental groups: Figure A shows the mRNA expression level of p53; Figure B shows the mRNA expression level of PAI-1. Figure 4The bar chart shows the mRNA expression levels of IL-1β and IL-6, genes related to myocardial fibrosis, in mice from different experimental groups: Figure A shows the mRNA expression level of IL-1β; Figure B shows the mRNA expression level of IL-6.

[0020] Note: * indicates p < 0.05 compared to the con group; ** indicates p < 0.005 compared to the con group; *** indicates p < 0.0005 compared to the con group; **** indicates p < 0.0001 compared to the con group; NS indicates p > 0.05 compared to the con group, with no significant difference between the two groups; # indicates p < 0.05 compared to the iso group; ## indicates p < 0.005 compared to the iso group; ### indicates p < 0.0005 compared to the iso group; #### indicates p < 0.0001 compared to the iso group; ns indicates p > 0.05 compared to the iso group, with no significant difference between the two groups. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0022] This invention has discovered that isorhamnetin can effectively inhibit the occurrence and development of myocardial fibrosis, thus revealing a new application of isorhamnetin in the field of anti-myocardial fibrosis. Developing new uses for isorhamnetin in this field has significant clinical implications. In the following examples, myocardial fibrosis was used as the research object to investigate the mitigating effect of isorhamnetin on the reduction of collagen deposition in myocardial tissue, and the mRNA expression levels of α-SMA, COL-1, COL-3, p53, PAI-1, and inflammatory factors IL-1β and IL-6 caused by myocardial fibrosis. Isorhamnetin is commercially available.

[0023] Example 1 To construct the experimental model, 40 male 8-week-old C57BL / 6 mice were randomly divided into 5 groups: (1) Experimental control group (CON): Free access to water and food; (2) Myocardial fibrosis model group (ISO): Myocardial fibrosis was induced by daily intraperitoneal injection of ISO (10 mg / kg day) for 21 days; (3) Control group receiving only one dose (ISOR): ISOR was administered by gavage daily (10 mg / kg day) for 21 days; (4) Drug treatment group A (ISO + 5 mg / kg.day ISOR): Mice were administered ISOR (5 mg / kg.day) by gavage. The specific procedure was as follows: ISO (10 mg / kg.day) was injected intraperitoneally daily for 7 days. After 7 days, ISOR (5 mg / kg.day) was administered by gavage daily, followed by an intraperitoneal injection of ISO (10 mg / kg.day) 2 hours later, for 14 days. (5) Drug treatment group B (ISO + 10 mg / kg.day ISOR): Mice were administered ISOR (10 mg / kg.day) by gavage. The specific procedure was as follows: ISO (10 mg / kg.day) was injected intraperitoneally daily for 7 days. After 7 days, ISOR (10 mg / kg.day) was administered by gavage daily, followed by an intraperitoneal injection of ISO (10 mg / kg.day) 2 hours later, for 14 days. (6) Drug treatment group C (ISO + 20 mg / kg / day ISOR): Mice were administered ISOR (20 mg / kg / day) by gavage. The specific procedure was as follows: ISO (10 mg / kg / day) was injected intraperitoneally daily for 7 days. After 7 days, ISOR (20 mg / kg / day) was administered by gavage daily, followed by an intraperitoneal injection of ISO (10 mg / kg / day) 2 hours later, for 14 days. (7) Positive control group (ISO+PRO): The specific procedure was as follows: ISO (10 mg / kg.day) was injected intraperitoneally daily for 7 days. After 7 days, ISO (10 mg / kg.day) was injected intraperitoneally daily, followed by PRO (40 mg / kg.day) 2 hours later, for 14 days. The mice in each group were sacrificed 14 days after the culture was completed.

[0024] ISO refers to isoproterenol, chemically named 4-[(2-isopropylamino-1-hydroxy)ethyl]-1,2-benzenediol.

[0025] ISOR refers to isorhamnetin.

[0026] Propranolol is abbreviated as PRO and its chemical name is 1-isopropylamino-3-(1-naphthoxy)-2-propanol.

[0027] Example 2 To investigate the mitigating effect of isorhamnetin on collagen deposition in myocardial tissue caused by myocardial fibrosis, Masson staining was performed on myocardial tissue from mice sacrificed in Example 1. The specific procedures were as follows: Tissue samples were taken from the apex of the heart of each group of mice, fixed in 10% formalin, and embedded in paraffin the next day for sectioning.

[0028] (1) Dewaxing and hydration: Xylene I 20 min, Xylene II 20 min, Anhydrous ethanol I 5 min, Anhydrous ethanol II 5 min, 75% ethanol 5 min, rinse with tap water for a few seconds; (2) Staining: Soak sections in Masson A solution overnight and wash with tap water; immerse sections in a mixture of equal parts Masson B and Masson C solutions for 1 min, wash with tap water, differentiate with 1% hydrochloric acid alcohol, and wash with tap water; immerse sections in Masson D solution for 6 min and rinse with tap water; immerse sections in Masson E solution for 1 min, drain slightly, and directly immerse in Masson F solution for 20-30 seconds; differentiate sections by rinsing with 1% glacial acetic acid and dehydrate twice with anhydrous ethanol; (3) Mounting: Place the slide in anhydrous ethanol for 5 minutes, then in xylene for 5 minutes to make it clear; then add two drops of neutral resin to mount the slide.

[0029] MASSON collagen staining images of myocardial sections from different experimental groups of mice are shown below. Figure 1 As shown, compared with the CON group, the MASSON collagen staining images of myocardial sections from the ISO group mice showed a large amount of collagen deposition. Figure 1 (A, 1B) In contrast, the MASSON collagen staining images of myocardial tissue sections from mice treated with ISOR 15 mg / kg / day showed a significant reduction in myocardial collagen deposition, which was not significantly different from the experimental results after treatment with the positive drug propranolol PRO. Figure 1 C,1D).

[0030] The results showed that isorhamnetin effectively slowed down collagen deposition in myocardial tissue caused by myocardial fibrosis.

[0031] Example 3 The effect of isorhamnetin on mitigating the mRNA expression levels of α-SMA, COL-1, COL-3, p53, and PAI-1 induced by myocardial fibrosis was investigated. Real-time quantitative qRT-PCR was used to detect the mRNA expression levels of COL-1, COL-3, α-SMA, p53, PAI-1, and GAPDH in the myocardial tissue of mice sacrificed in Example 1. The specific operational steps were as follows: (1) Primer design: Follow the principles of primer design; (2) RNA extraction: Small pieces of mouse heart tissue, about the size of a grain of rice, were cut and placed in sterile, enzyme-free 1.5 mL centrifuge tubes. The tissue was minced using ophthalmic scissors, and the tubes were placed on ice. 400 μL of RNAisoPlus was added to each tube, and the tissue was homogenized by sonication. Another 400 μL of RNAisoPlus was added, and the mixture was incubated at room temperature for 5 min. The tubes were then centrifuged at 13000 g for 15 min at 4°C. The top layer of clear liquid was transferred to a new sterile, enzyme-free centrifuge tube using a pipette. 500 μL of isopropanol was added to each tube, and the mixture was inverted to mix. The tubes were incubated at room temperature for 20 min, then centrifuged at 13000 g for 15 min at 4°C. The supernatant was discarded (do not discard the bottom precipitate). Add 1 mL of 75% ethanol prepared with DEPC-treated water to each centrifuge tube, invert the tube, wash thoroughly, and centrifuge at 13000 rpm for 15 min at 4°C. Discard the supernatant, retain the precipitate, and air dry it upside down on clean paper. Add 10-50 μL of DEPC-treated water according to the amount of precipitate and dissolve it completely. Finally, determine the RNA concentration and purity using a micro spectrophotometer (ensuring the A260 / 280 value is between 1.8 and 2.0).

[0032] (3) Reverse transcription: 1. Genomic DNA removal reaction: Thaw the reagents on ice, prepare according to Table 1, add the reagents sequentially to 0.2 mL centrifuge tubes, centrifuge at low speed for about 20 s, place the centrifuge tubes in a PCR instrument, incubate at 42℃ for 2 min, and cool at 4℃. 2. Reverse transcription reaction: Prepare the premix according to Table 2, add the premix to the above centrifuge tubes, centrifuge at low speed for about 20 s, place in a PCR instrument, incubate at 37℃ for 15 min, incubate at 85℃ for 5 s, and cool at 4℃ for storage.

[0033] Table 1 shows the reaction configurations for removing genomic DNA: .

[0034] Table 2 shows the transcriptome response configurations: .

[0035] (4) Real-Time PCR Reaction: Thaw the required reagents on ice, prepare the premix according to Table 3, add cDNA and premix to a 96-well plate, seal the plate, and centrifuge until no air bubbles remain in the wells. Set the amplification reaction program: 95℃ pre-denaturation for 30s, 95℃ reaction for 5s, 60℃ reaction for 30s, for 39 cycles. GAPDH was used as the internal control. Each sample was tested in triplicate. The reaction was repeated 2 times. ﹣∆∆CtThe relative expression level of the target gene was calculated using a method. Table 4 shows the primer sequences used in this experiment.

[0036] Table 3 shows the configuration of Real-Time PCR amplification reactions: .

[0037] Table 4 shows the gene primer sequences:

[0038] The mRNA expression levels of the myocardial fibrosis-related gene α-SMA in different experimental groups of mice are as follows: Figure 2 As shown, compared with the control group, the mRNA levels of α-SMA, COL-1, and COL-3 in the myocardial tissue of mice in the ISO group were significantly increased, and ISOR 10 mg / kg / day had a significant alleviating effect on the ISO-induced increase in α-SMA, COL-1, and COL-3 mRNA levels. Figure 2 The results for A-2C were slightly better than those for the positive control drug propranolol PRO. Furthermore, the efficacy was superior to the ISOR 5 mg / kg / day group. The ISOR 20 mg / kg / day group increased the mRNA levels of α-SMA, COL-1, and COL-3 in mouse cardiomyocytes compared to the ISO group, suggesting potential drug toxicity at this dosage. The results indicate that ISOR can alleviate ISO-induced myocardial fibrosis in mice, with 10 mg / kg / day being the optimal dose.

[0039] The results showed that isorhamnetin effectively slowed down the expression levels of α-SMA, COL-1, and COL-3 mRNA caused by myocardial fibrosis, and the optimal dose was 10 mg / kg / day.

[0040] The mRNA expression levels of p53 and PAI-1, genes related to myocardial fibrosis, in mice from different experimental groups are as follows: Figure 3 As shown, compared with the control group, the mRNA levels of p53 and PAI-1 in the myocardial tissue of mice in the ISO group were significantly increased, and ISOR 10 mg / kg / day had a significant alleviating effect on the ISO-induced increase in p53 and PAI-1 mRNA levels. Figure 3 The results (A, 3B) showed no significant difference from those after treatment with the positive control drug propranolol PRO. Furthermore, the effect on PAI-1 mRNA expression was superior to the ISOR 5 mg / kg / day experimental group. The ISOR 20 mg / kg / day experimental group increased the levels of p53 and PAI-1 mRNA in mouse cardiomyocytes compared to the ISOR experimental group, suggesting potential drug toxicity at this dosage. These results indicate that ISOR may regulate p53 expression through PAI-1, and that 10 mg / kg / day is the optimal dose.

[0041] The results showed that isorhamnetin effectively reduced the expression levels of p53 and PAI-1 mRNA caused by myocardial fibrosis, and the optimal dose was 10 mg / kg / day.

[0042] The mRNA expression levels of IL-1β and IL-6, genes related to myocardial fibrosis, in mice from different experimental groups are as follows: Figure 4 As shown, compared with the control group, the levels of IL-1β and IL-6 mRNA in the myocardial tissue of mice in the ISO group were significantly increased, and ISOR 10 mg / kg / day had a significant alleviating effect on the increase in IL-1β and IL-6 mRNA levels induced by ISO. Figure 4 The results (A, 4B) showed no significant difference from those of the positive drug propranolol PRO treatment.

[0043] The results showed that ISOR could inhibit the increase of inflammatory factors caused by myocardial fibrosis.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of isorhamnetin in the preparation of anti-myocardial fibrosis drugs.

2. The application according to claim 1, characterized in that, The anti-myocardial fibrosis drug uses isorhamnetin as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The excipients are solid excipients, liquid excipients, or semi-solid excipients.

4. The application according to claim 2, characterized in that, The dosage form is tablet, capsule, or injection.

5. The application according to any one of claims 1 to 4, characterized in that, The effective content of isorhamnetin is 5-10 mg / kg.

6. A pharmaceutical composition for treating myocardial fibrosis, characterized in that, The active ingredient in the anti-myocardial fibrosis drug composition includes isorhamnetin.

7. The anti-myocardial fibrosis pharmaceutical composition according to claim 6, characterized in that, The sole active ingredient in the anti-myocardial fibrosis drug composition is isorhamnetin.

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