Application of salvianolic acid A in preparation of anti-vascular aging product

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of salvianolic acid A in preparation of anti-vascular aging products. The invention provides application of salvianolic acid A in preparation of products for promoting endoplasmic reticulum stress and / or resisting vascular aging. The research finds that the salvianolic acid A can effectively reduce the load of vascular senescence cells caused by high fat diet, reduce the expression of vascular senescence markers p16 and p21 and delay vascular senescence. Moreover, the danshinolic acid A can activate an endoplasmic reticulum stress PERK / eIF2alpha / CHOP signal channel, so that the apoptosis of stress senescence vascular smooth muscle cells is remarkably induced, and the number of senescence vascular smooth muscle cells after PERK is knocked out cannot be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of salvianolic acid A in the preparation of anti-vascular aging products. Background Art

[0002] Vascular aging is the pathological basis of various chronic cardiovascular diseases. At the cellular level, vascular aging is driven by the accumulation of senescent cells. Vascular smooth muscle cells are an important component of blood vessels. Under physiological conditions, vascular smooth muscle cells are statically located in the middle layer of blood vessels and are responsible for the relaxation and contraction of blood vessels. Under disease conditions, vascular smooth muscle cells with a contractile phenotype will transform into a senescent phenotype, manifested as an increase in the secretion of inflammatory factors, a decrease in the synthesis of extracellular matrix, and the secretion of MMP9 that degrades the extracellular matrix, which has an adverse effect on the occurrence and development of cardiovascular diseases.

[0003] Current anti-atherosclerotic drugs achieve the purpose of delaying vascular aging by reducing stressors and improving the stress resistance of cells. However, such drugs often ignore the impact of already senescent vascular smooth muscle cells on vascular health. Senescent vascular smooth muscle cells will have an adverse effect on blood vessels through the senescence-associated secretory phenotype, and in addition, induce secondary senescence of surrounding cells in a signal transduction manner, resulting in limited ability of anti-atherosclerotic drugs to delay vascular aging. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of salvianolic acid A in the preparation of anti-vascular aging products, aiming at the problem of the effectiveness of clinical anti-vascular aging therapies, taking endoplasmic reticulum stress as a new therapeutic target for vascular aging, and enriching drugs for delaying vascular aging and reducing the load of senescent vascular smooth muscle cells.

[0005] The present invention provides the application of salvianolic acid A in the preparation of products for promoting endoplasmic reticulum stress and / or anti-vascular aging.

[0006] Preferably, the anti-vascular aging products include products that reduce the number of senescent vascular smooth muscle cells and delay vascular aging.

[0007] Preferably, the senescent vascular smooth muscle cells include human aortic vascular smooth muscle cells.

[0008] Preferably, the anti-vascular aging products include products that promote the apoptosis of senescent vascular smooth muscle cells.

[0009] Preferably, the promotion of endoplasmic reticulum stress includes activating the endoplasmic reticulum stress signaling pathway PERK / eIF2α / CHOP.

[0010] Preferably, the vascular aging includes vascular aging caused by a high-fat diet.

[0011] Preferably, the product includes a drug.

[0012] Preferably, the concentration of salvianolic acid A in the drug is ≥20 μM.

[0013] Preferably, the concentration of salvianolic acid A in the drug is 60 - 100 μM.

[0014] The present invention also provides a drug for promoting endoplasmic reticulum stress and / or anti-vascular aging, and the active ingredient of the drug includes salvianolic acid A.

[0015] Beneficial effects:

[0016] The present invention provides the application of salvianolic acid A in the preparation of a product for promoting endoplasmic reticulum stress and / or anti-vascular aging. The research of the present invention finds that salvianolic acid A can effectively reduce the burden of vascular aging cells caused by a high-fat diet, reduce the expression of vascular aging markers p16 and p21, and delay vascular aging. And salvianolic acid A can activate the endoplasmic reticulum stress PERK / eIF2α / CHOP signaling pathway, significantly induce the apoptosis of stress-induced senescent vascular smooth muscle cells, and cannot reduce the number of senescent vascular smooth muscle cells after knocking out PERK. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0018] Figure 1 It is the Western Blot result of the detection of senescence markers in each treatment group of Example 1;

[0019] Figure 2 It is the Western Blot result of successfully knocking out PERK in Example 2;

[0020] Figure 3 It is the in-situ staining result of apoptotic cells in each treatment group of Example 2;

[0021] Figure 4 It is the Western Blot result of the detection of endoplasmic reticulum stress in each treatment group of Example 2;

[0022] Figure 5 It is the Western Blot result of the detection of senescence markers in each treatment group of Example 2;

[0023] Figure 6 It is the β-galactosidase staining result of the detection of senescence markers in each treatment group of Example 2. Detailed Embodiments

[0024] The present invention provides the application of salvianolic acid A in the preparation of a product for promoting endoplasmic reticulum stress and / or anti-vascular aging.

[0025] As an implementation manner, the CAS number of salvianolic acid A in the present invention is 96574-01-5.

[0026] As an implementation manner, the vascular aging in the present invention includes the vascular aging caused by a high-fat diet. As an implementation manner, the vascular aging caused by a high-fat diet in the present invention includes the vascular aging caused by palmitate.

[0027] As an implementation manner, the anti-vascular aging product in the present invention may include a product that reduces the number of senescent vascular smooth muscle cells and delays vascular aging. As an implementation manner, the senescent vascular smooth muscle cells in the present invention include human aortic vascular smooth muscle cells. As an implementation manner, the anti-vascular aging product in the present invention may also include a product that promotes the apoptosis of senescent vascular smooth muscle cells. As an implementation manner, the promotion of endoplasmic reticulum stress in the present invention includes activating the endoplasmic reticulum stress signaling pathway PERK / eIF2α / CHOP.

[0028] The present invention utilizes salvianolic acid A to effectively reduce the burden of vascular aging cells caused by a high-fat diet, reduce the expression of vascular aging markers p16 and p21, and delay vascular aging. And it utilizes salvianolic acid A to activate the endoplasmic reticulum stress PERK / eIF2α / CHOP signaling pathway, significantly induce the apoptosis of stress-induced senescent vascular smooth muscle cells, and cannot reduce the number of senescent vascular smooth muscle cells after knocking out PERK.

[0029] As an implementation manner, the product in the present invention includes a drug. As an implementation manner, the concentration of salvianolic acid A in the drug in the present invention is ≥20 μM; as another implementation manner, the concentration of salvianolic acid A in the drug in the present invention is 60-100 μM. In the present invention, the concentration of salvianolic acid A in the drug is based on the dosage administered to stress-induced senescent vascular smooth muscle cells in vitro.

[0030] The present invention also provides a drug for promoting endoplasmic reticulum stress and / or anti-vascular aging, and the active ingredient of the drug includes salvianolic acid A.

[0031] As an implementation manner, the concentration of salvianolic acid A in the drug in the present invention is ≥20 μM; as another implementation manner, the concentration of salvianolic acid A in the drug in the present invention is 60-100 μM. In the present invention, the concentration of salvianolic acid A in the drug is based on the dosage administered to stress-induced senescent vascular smooth muscle cells in vitro.

[0032] To further illustrate the present invention, the application of salvianolic acid A provided by the present invention in the preparation of anti-vascular aging products will be described in detail below with reference to the accompanying drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0033] Example 1

[0034] Salvianolic acid A delays vascular aging in ApoE mice on a high-fat diet - / - in mice

[0035] 1. Experimental animals: 10 male ApoE mice at 8 weeks old (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), and 5 male C57BL / 6J mice at 8 weeks old with the same genetic background (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). - / -

[0036] 2. Experimental grouping and establishment of vascular aging model

[0037] The ApoE mice - / - were randomly divided into a disease model group and a salvianolic acid A group, with 5 mice in each group. The C57BL / 6J mice served as a normal control group (WT). All experimental animals were housed in cages with 5 mice per cage, with free access to food and water, and were raised in an environment with 12 hours of light and 12 hours of darkness. The specific feeding methods were as follows:

[0038] Salvianolic acid A group: Male ApoE mice were fed with a high-fat diet (containing 0.15% cholesterol and 21% fat) for 12 weeks; among them, starting from the 9th week, 10 mg / kg of salvianolic acid A was intraperitoneally injected daily; - / -

[0039] Disease model group: Male ApoE mice were fed with a high-fat diet (containing 0.15% cholesterol and 21% fat) for 12 weeks; among them, starting from the 9th week, normal saline with the same volume as that in the salvianolic acid A group was intraperitoneally injected daily; - / -

[0040] Normal control group: C57BL / 6J mice were fed with a high-fat diet (containing 0.15% cholesterol and 21% fat) for 12 weeks; among them, starting from the 9th week, normal saline with the same volume as that in the salvianolic acid A group was intraperitoneally injected daily.

[0041] 3. After the feeding in step 2 was completed, the aorta was taken, and the expression levels of the vascular aging markers p16 and p21 were detected by Western Blot method. The results were as Figure 1 shown. Among them, HFD is the high-fat diet, and SAA is salvianolic acid A. It can be seen from Figure 1 the results that the vascular aging markers p16 and p21 in the salvianolic acid A group were significantly lower than those in the disease model group. Salvianolic acid A can effectively reduce the vascular aging cell load in high-fat diet mice and delay vascular aging.

[0042] Example 2

[0043] Salvianolic acid A promotes apoptosis of senescent vascular smooth muscle cells by activating endoplasmic reticulum stress ​​​

[0044] 1. Experimental materials: Human aortic vascular smooth muscle cells.

[0045] 2. Normal culture medium: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0046] 3. Experimental method: Human aortic vascular smooth muscle cells were randomly divided into 9 treatment groups and were treated under the conditions of 37°C and 5% CO2 as follows:

[0047] Blank control group (PBS-BSA-PBS): After treating the cells with PBS solution containing 10% BSA for 48 hours, they were cultured with normal culture medium for 48 hours, and then PBS with the same volume as the low-dose salvianolic acid A group was added for intervention for 24 hours;

[0048] Model control group (PA-BSA-PBS): After treating the cells with PBS solution containing 200 μM palmitate and 10% BSA for 48 hours, they were cultured with normal culture medium for 48 hours, and then PBS with the same volume as the low-dose salvianolic acid A group was added for intervention for 24 hours;

[0049] Low-dose salvianolic acid A group (PA-BSA-20 μM): After treating the cells with PBS solution containing 200 μM palmitate and 10% BSA for 48 hours, they were cultured with normal culture medium for 48 hours, and then 20 μM salvianolic acid A was added for intervention for 24 hours;

[0050] Medium-dose salvianolic acid A group (PA-BSA-60 μM): After treating the cells with PBS solution containing 200 μM palmitate and 10% BSA for 48 hours, they were cultured with normal culture medium for 48 hours, and then 60 μM salvianolic acid A was added for intervention for 24 hours;

[0051] High-dose salvianolic acid A group (PA-BSA-100 μM): After treating the cells with PBS solution containing 200 μM palmitate and 10% BSA for 48 hours, they were cultured with normal culture medium for 48 hours, and then 100 μM salvianolic acid A was added for intervention for 24 hours;

[0052] Transfection control group 1 (PBS-BSA-siNC): After transfecting cells with siNC for 24 hours, the cells were treated with PBS solution containing 10% BSA for 48 hours, then cultured in normal medium for 48 hours, and intervened with PBS of the same volume as the low-dose salvianolic acid A group for 24 hours; among them, the nucleotide sequence of the sense strand of siNC is: 5'-UUCUCCGAACGUGUCACGUYY-3' (SEQ ID NO:1), and the nucleotide sequence of the antisense strand is: 5'-ACGUGACACGUUCGGAGAAYY-3' (SEQ ID NO:2), purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.; among them, Y in SEQ ID NO:1 and SEQ ID NO:2 is base T respectively;

[0053] Transfection control group 2 (PA-BSA-siNC): After transfecting cells with siNC for 24 hours, the cells were treated with PBS solution containing 200 μM palmitate and 10% BSA for 48 hours, and then intervened with PBS of the same volume as the low-dose salvianolic acid A group for 24 hours;

[0054] siNC + salvianolic acid A group (PA-BSA-siNC + 100 μM): After transfecting cells with siNC for 24 hours, the cells were treated with PBS solution containing 200 μM palmitate and 10% BSA for 48 hours, then cultured in normal medium for 48 hours, and intervened with 100 μM salvianolic acid A for 24 hours;

[0055] siPERK + salvianolic acid A group (PA-BSA-siPERK + 100 μM): After transfecting cells with siPERK for 24 hours, the transfected cells were treated with PBS solution containing 200 μM palmitate and 10% BSA for 48 hours, then cultured in normal medium for 48 hours, and intervened with 100 μM salvianolic acid A for 24 hours; among them, the nucleotide sequence of the sense strand of siPERK is: 5'-GGAACGACCUGAAGCUAUAYY-3' (SEQ ID NO:3), and the nucleotide sequence of the antisense strand is: 5'-UAUAGCUUCAGGUCGUUCCYY-3' (SEQ ID NO:4), purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.; among them, Y in SEQ ID NO:3 and SEQ ID NO:4 is base T respectively.

[0056] 4. After the treatment of each treatment group in step 3 was completed, Western Blot was used to detect protein kinase R-like endoplasmic reticulum kinase (PERK) in the cells of the blank control group, siNC + salvianolic acid A group and siPERK + salvianolic acid A group. The results are as Figure 2As shown in the figure; among them, Control, Stress, and siPERK represent the blank control group, siNC + salvianolic acid A group, and siPERK + salvianolic acid A group, respectively. According to Figure 2 It can be seen that the knockdown of protein kinase R-like endoplasmic reticulum kinase in the siPERK + salvianolic acid A group was successful.

[0057] 5. After the treatment of each treatment group in step 3 was completed, in situ staining of apoptotic cells was performed, and the results were as Figure 3 shown. Among them, the red fluorescence is live cells with mitochondrial membrane potential maintained, labeled with the mitochondrial membrane potential-dependent red fluorescent probe Mito Tracker Red CMXRo; the green fluorescence is cells that have undergone apoptosis and necrosis, labeled with the apoptotic cell green fluorescent probe AnnexinV-FITC; the blue fluorescence is the cell nucleus labeled with the blue fluorescence Hoechst 33341. According to Figure 3 It can be seen that 100 μM salvianolic acid A significantly induced the apoptosis of stress-induced senescent vascular smooth muscle cells, and knocking down PERK partially rescued the selective clearance of stress-induced senescent vascular smooth muscle cells induced by salvianolic acid A, suggesting that the effect of salvianolic acid A depends on the activation of endoplasmic reticulum stress.

[0058] 5. After the treatment of each treatment group in step 3 was completed, endoplasmic reticulum stress was detected. Specifically: Western Blot was used to detect the expression of the endoplasmic reticulum stress signaling pathway PERK / eIF2α / CHOP, and the results were as Figure 4 shown. Among them, PA represents palmitate, SAA represents salvianolic acid A, L represents low-dose salvianolic acid A (20 μM), M represents medium-dose salvianolic acid A (60 μM), and H represents high-dose salvianolic acid A (100 μM). According to Figure 4 It can be seen that 100 μM salvianolic acid A can activate the endoplasmic reticulum stress PERK / eIF2α / CHOP signaling pathway.

[0059] 6. After the treatment of each treatment group in step 3 was completed, senescence markers were detected. Specifically: Western Blot was used to detect the expression of the senescent cell markers p16 and p21; β-galactosidase staining was used. If β-galactosidase-positive cells showed blue staining, they were generally considered senescent cells. The results were as Figure 5 and Figure 6 shown. Among them, Figure 5 PA represents palmitate, SAA represents salvianolic acid A, L represents low-dose salvianolic acid A (20 μM), M represents medium-dose salvianolic acid A (60 μM), and H represents high-dose salvianolic acid A (100 μM). According to Figure 5 and Figure 6 It can be seen that salvianolic acid A cannot reduce the number of senescent vascular smooth muscle cells after knocking out PERK.

[0060] It can be seen from the above content that salvianolic acid A can activate endoplasmic reticulum stress in senescent vascular smooth muscle cells, promote apoptosis of senescent vascular smooth muscle cells, reduce the load of senescent vascular smooth muscle cells, and effectively delay vascular aging.

[0061] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of salvianolic acid A in preparing a product for promoting endoplasmic reticulum stress and / or anti-vascular aging.

2. The application according to claim 1, wherein The anti-vascular aging product includes a product for reducing the number of senescent vascular smooth muscle cells and delaying vascular aging.

3. The application according to claim 2, characterized in that, The senescent vascular smooth muscle cells include human aortic vascular smooth muscle cells.

4. The application according to claim 1, wherein The anti-vascular aging product includes a product for promoting apoptosis of senescent vascular smooth muscle cells.

5. The application according to claim 1, characterized in that, The promotion of endoplasmic reticulum stress includes activating the endoplasmic reticulum stress signaling pathway PERK / eIF2α / CHOP.

6. The application according to claim 1, characterized in that The vascular aging includes vascular aging caused by a high-fat diet.

7. The application according to any one of claims 1 to 6, characterized in that, The product includes a drug.

8. The application according to claim 7, characterized in that, The concentration of salvianolic acid A in the drug is ≥20 μM.

9. The application according to claim 8, wherein The concentration of salvianolic acid A in the drug is 60 - 100 μM.

10. A drug for promoting endoplasmic reticulum stress and / or anti-vascular aging, characterized in that, The active ingredient of the drug includes salvianolic acid A.

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