Application of forsythiaside A in preparation of medicine for relieving or treating diabetic cardiomyopathy

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of forsythiaside A in preparation of a medicine for relieving or treating diabetic cardiomyopathy. The forsythiaside A relieves or treats diabetic cardiomyopathy by regulating NLRP3 activation induced by endoplasmic reticulum stress. The forsythiaside A can reduce the levels of BG, GSP, TG, LDH, CK-MB, cTnI, MDA, IL-1beta, IL-6 and TNF-alpha of STZ induced diabetic mice, and meanwhile, the levels of IN and SOD are increased. The forsythiaside A can be used for reducing the expression levels of IL-1beta, IL-6, TNF-alpha, GRP78, TXNIP and NLRP3 in H9c2 cells induced by Glu. Therefore, forsythiaside A can induce NLRP3 inflammasome activation through endoplasmic reticulum stress to relieve diabetic cardiomyopathy, and a theoretical basis is provided for treating diabetic cardiomyopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the use of forsythiaside A in the preparation of a medicine for alleviating or treating diabetic cardiomyopathy. Background Art

[0002] Diabetic cardiomyopathy (DCM) is characterized by abnormal myocardial function and structure, including myocardial dysfunction, myocardial cell death, myocardial fibroblast activation and metabolic disorders. Among them, myocardial cell death and myocardial fibrosis are the core characteristics of DCM. They can induce ventricular remodeling and lead to left ventricular dysfunction. The early clinical manifestations of DCM are mainly diastolic dysfunction, mainly manifested as diastolic filling obstruction and reduced myocardial compliance. As the disease progresses, the late symptoms turn into cardiac systolic dysfunction and eventually develop into congestive heart failure. The pathogenesis of DCM is quite complex. Current studies have shown that the occurrence and development of diabetic cardiomyopathy may be slowed down by regulating the release of inflammasomes and cytokines and the expression of related genes during cell pyroptosis. There is no clear explanation at present, and there is also a lack of effective molecular intervention strategies in treatment. Therefore, it is imperative to find better drugs to treat DCM. Summary of the invention

[0003] The object of the present invention is to provide the use of forsythiaside A in preparing a medicine for alleviating or treating diabetic cardiomyopathy.

[0004] The implementation process of the present invention is as follows:

[0005] Application of forsythiaside A in the preparation of a medicine for alleviating or treating diabetic cardiomyopathy.

[0006] Furthermore, the forsythiaside A alleviates or treats diabetic cardiomyopathy by regulating endoplasmic reticulum stress-induced NLRP3 activation.

[0007] Furthermore, the forsythiaside A can reduce the levels of blood glucose and glycosylated serum protein in STZ-induced diabetic mice, while increasing the level of insulin.

[0008] Furthermore, the forsythiaside A can reduce the levels of TG, LDH, CK-MB, cTnI, and MDA in the serum of STZ-induced diabetic mice, while increasing the level of SOD.

[0009] Furthermore, the forsythiaside A can reduce the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the serum of STZ-induced diabetic mice.

[0010] Furthermore, the forsythiaside A can significantly reduce the pathological damage of myocardial tissue in STZ-induced diabetic mice, while increasing the levels of LVEF and LVFS, reducing the level of LVEDD, and improving cardiac function.

[0011] Furthermore, the forsythiaside A can reduce the expression levels of GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, Caspase-1, and IL-1β in the heart tissue of STZ mice.

[0012] Furthermore, the forsythiaside A can reduce the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in Glu-induced H9c2 cells.

[0013] Furthermore, the forsythiaside A can reduce the expression levels of GRP78, TXNIP and NLRP3 in Glu-induced H9c2 cells.

[0014] Positive effects of the present invention:

[0015] (1) The forsythiaside A described in the present invention alleviates or treats diabetic cardiomyopathy by regulating endoplasmic reticulum stress-induced nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) activation, providing a theoretical basis for alleviating or treating diabetic cardiomyopathy.

[0016] (2) The forsythiaside A described in the present invention can reduce the levels of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (P-PERK), phosphorylated eukaryotic initiation factor α (P-EIF-α), X-box binding protein 1 (XBP1), thioredoxin interacting protein (TXNIP), NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, and interleukin-1β (IL-1β) in the heart tissue of STZ mice, as well as the levels of GRP78, TXNIP and NLRP3 in glucose (Glu)-induced H9c2 cardiomyocytes.

[0017] (3) The forsythiaside A described in the present invention can reduce blood sugar levels and the amount of glycosylated serum protein, while increasing insulin levels.

[0018] (4) The forsythiaside A described in the present invention can reduce the levels of triglycerides, lactate dehydrogenase, creatine kinase isoenzymes, cardiac troponin, and malondialdehyde in serum, while increasing the level of superoxide dismutase.

[0019] (5) The forsythiaside A described in the present invention can reduce the levels of proinflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in STZ mice and Glu-induced H9c2 cells.

[0020] (6) The forsythiaside A described in the present invention can improve the pathological damage of cardiac tissue in STZ mice, increase the levels of LVEF and LVFS, and reduce the level of LVEDD, thereby further improving cardiac function.

[0021] (7) The forsythiaside A described in the present invention can reduce the expression levels of GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, Caspase-1, and IL-1β in the heart tissue of STZ mice.

[0022] (8) The forsythiaside A described in the present invention can reduce the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in Glu-induced H9c2 cells.

[0023] (9) Forsythiaside A described in the present invention can reduce the expression levels of GRP78, TXNIP and NLRP3 in H9c2 cells induced by Glu. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure shows the effect of FA on the levels of blood glucose, glycosylated serum protein and insulin in the serum of STZ mice. All data are expressed as mean ± standard deviation, n = 6. Compared with the control group, ## P<0.05; compared with the STZ group, ** P < 0.01;

[0025] Figure 2 The figure shows the effect of FA on the levels of serum triglyceride, lactate dehydrogenase, creatine kinase isoenzyme, cardiac troponin, malondialdehyde, and superoxide dismutase in STZ mice. All data are expressed as mean ± standard deviation, n = 6. Compared with the control group, ## P<0.05; compared with the STZ group, ** P < 0.01;

[0026] Figure 3 The figure shows the effect of FA on the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the serum of STZ mice. All data are expressed as mean ± standard deviation, n = 6. Compared with the control group, ## P<0.05; compared with the STZ group, ** P < 0.01;

[0027] Figure 4 The figure shows the effect of FA on H&E staining of cardiac tissue pathological damage in STZ mice (n=3);

[0028] Figure 5Figure 1 is the effect of FA on cardiac function in STZ mice, where A is cardiac ultrasound, BD is LVEF, LVFS and LVEDD; all data are expressed as mean ± standard deviation, n = 6; compared with the control group, ## P<0.05; compared with the STZ group, ** P < 0.01;

[0029] Figure 6 The figure shows the effect of FA on the expression levels of GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, Caspase-1, and IL-1β in the heart tissue of STZ mice, where A is protein expression and B is the quantitative value analysis of protein expression. All data are expressed as mean ± standard deviation, n = 3. Compared with the control group, ## P<0.05; compared with the STZ group, ** P < 0.01;

[0030] Figure 7 Figure 2 is the effect of FA on the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in H9c2 cells induced by Glu, where all data are expressed as mean ± standard deviation, n = 6; compared with the control group, ## P<0.05; compared with the STZ group, ** P < 0.01;

[0031] Figure 8 Figure 2 is the effect of FA on Glu-induced endoplasmic reticulum stress-related TXNIP / NLRP3 pathway in H9c2 cells. A is immunofluorescence detection of GRP78, TXNIP and NLRP3 protein expression; B is statistical analysis of GRP78, TXNIP and NLRP3 fluorescence expression. All data are expressed as mean ± standard deviation, n = 3. Compared with the control group, ## P<0.05; compared with the STZ group, ** P<0.01. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the embodiments.

[0033] 1 Reagents

[0034] Forsythiaside A (FA) was purchased from Shanghai Shiguibao Pharmaceutical Co., Ltd. Streptozotocin (STZ) was purchased from Yarebio and is widely used in experimental animals to induce diabetes models. Tunicamycin (TM, an endoplasmic reticulum stress inducer, mainly used to verify that FA exerts its efficacy by regulating endoplasmic reticulum stress) and glucose (Glu) were purchased from MCE. Related kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and all other antibodies were purchased from Cell Signaling Technology Company.

[0035] 2 Animals

[0036] Twenty-four C57BL / 6J mice (male, 6 weeks, 16–18 g) were purchased from the Animal Experiment Center of the Air Force Medical University, and the experimental protocol was approved by the Research Committee and the Animal Care and Use Committee of the Air Force Medical University.

[0037] 3STZ mouse model

[0038] After 1 week of adaptive feeding, the mice were randomly divided into a control group, a STZ group (intraperitoneal injection, 55 mg / kg), a STZ (intraperitoneal injection, 55 mg / kg) + FA (intraperitoneal injection, 40 mg / kg), and a STZ (intraperitoneal injection, 55 mg / kg) + FA (intraperitoneal injection, 40 mg / kg) + TM (intraperitoneal injection, 5 mg / kg) group. STZ was dissolved in 0.1 mol / L citric acid buffer solution (pH = 4.2) and injected once a day for 5 consecutive days to establish the model. The mice in the control group were injected with an equal volume of citric acid buffer solution. FA and TM were injected intraperitoneally once a day for 5 consecutive days.

[0039] 4Glu-induced H9c2 cell model

[0040] H9c2 cells were divided into control group, glucose (Glu, 5.5mmol / L), glucose (Glu, 5.5mmol / L) + FA (10μM), glucose (Glu, 5.5mmol / L) + FA (10μM) + TM (1μM) group. After Glu was co-incubated with cells for 24h, FA was co-incubated with MT for 24h, and the control group was incubated with an equal amount of PBS.

[0041] 5. Blood glucose (BG), glycosylated serum protein (GSP), insulin (IN) testing

[0042] Experimental procedure: On day 0 and day 5, mice were fasted for 12 hours, tail blood was collected, and blood glucose was measured using a blood glucose meter. Mice were fasted and allowed to drink water for 12 hours. Blood was collected from the eyeballs and centrifuged to obtain serum. The content of glycosylated serum protein was determined using the method described in the kit instructions. The serum obtained by centrifugation was used to determine the insulin level in mouse serum according to the kit instructions. The double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) method was used.

[0043] Experimental results: see Figure 1 Compared with the control group, the levels of BG and GSP in the STZ group were significantly increased; compared with the STZ group, the levels of BG and GSP in the STZ+FA group were significantly decreased; after the administration of retinal stress inducer (TM), the levels of BG and GSP in the STZ+FA+TM group increased, and were equivalent to the STZ group, with no statistical difference. Compared with the control group, the serum IN content in the STZ group was significantly decreased; compared with the STZ group, the serum IN content in the STZ+FA group was significantly increased, and after the administration of retinal stress inducer (TM), the IN level in the STZ+FA+TM group decreased, and was equivalent to the STZ group, with no statistical difference, indicating that FA exerts its efficacy by regulating endoplasmic reticulum stress. When TM induces endoplasmic reticulum stress, FA loses its efficacy.

[0044] 6. Detection of serum triglyceride (TG), lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), cardiac troponin I (cTnI), malondialdehyde (MDA), and superoxide dismutase (SOD) levels in STZ mice

[0045] Experimental process: Detect serum TG, LDH, CK-MB, cTnI, MDA, and SOD levels according to the kit instructions.

[0046] Experimental results: see Figure 2 Compared with the control group, the levels of TG, LDH, CK-MB, cTnI, and MDA in the STZ group were significantly increased, while the level of SOD was significantly decreased. Compared with the STZ group, the levels of TG, LDH, CK-MB, cTnI, and MDA in the STZ+FA group were significantly decreased, while the level of SOD in serum was significantly increased; after the administration of ER stress inducer (TM), the levels of TG, LDH, CK-MB, cTnI, and MDA in the STZ+FA+TM group were increased, while the level of SOD was decreased, and it was equivalent to the STZ group, with no statistical difference, indicating that FA exerts its efficacy by regulating ER stress. When TM induces ER stress, FA loses its efficacy.

[0047] 7 Detection of pro-inflammatory factors IL-1β, IL-6, and TNF-α levels in the serum of STZ mice

[0048] Experimental process: Detect the levels of pro-inflammatory factors in serum according to the kit instructions.

[0049] Experimental results: see Figure 3 Compared with the control group, the levels of serum interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the STZ group were significantly increased. Compared with the STZ group, the levels of IL-1β, IL-6 and TNF-α in the STZ+FA group were significantly reduced; after the administration of ER stress inducer (TM), the levels of L-1β, IL-6 and TNF-α in the STZ+FA+TM group increased and were comparable to those in the STZ group, with no statistical difference, indicating that FA exerts its efficacy by regulating ER stress. When TM induces ER stress, FA loses its efficacy.

[0050] 8 Changes in cardiac pathology and cardiac function

[0051] Experimental procedure: The mice were anesthetized with 1.5% isoflurane and placed in a supine position for hair removal. The VEVO2100 ultrasound imaging system was used to obtain echocardiographic images and evaluate the cardiac function of the mice. The mouse hearts were fixed with paraformaldehyde solution, sliced ​​using hematoxylin and eosin (HE) staining, and the pathological changes of the heart tissue were observed under an optical microscope.

[0052] Experimental results: see Figure 4 The control group was normal myocardial cells, with neatly arranged myocardial fibers and normal structure, and no inflammatory cell infiltration. Compared with the control group, the myocardial tissue of mice in the STZ group was significantly damaged, with loose and disordered arrangement of myocardial cells, broken myocardial fibers, and inflammatory cell infiltration in the myocardial interstitium. Compared with the STZ group, the pathological damage of myocardial tissue in mice in the STZ+FA group was significantly alleviated, and the infiltration of inflammatory cells was significantly reduced; after the administration of ER stress inducer (TM), the infiltration of inflammatory cells in the myocardium of mice in the STZ+FA+TM group increased significantly, the arrangement of cells was obviously disordered, and obvious pathological damage of tissue appeared, indicating that FA exerts its efficacy by regulating endoplasmic reticulum stress. When TM induces endoplasmic reticulum stress, FA loses its efficacy.

[0053] See Figure 5 The results of cardiac function showed that compared with the control group, the left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) in the STZ group were significantly reduced, and the left ventricular end-diastolic diameter (LVEDD) was significantly increased. Compared with the STZ group, the LVEF and LVFS levels in the STZ+FA group were significantly increased, and the LVEDD level was significantly decreased; after the administration of reticular stress inducer (TM), the LVEF and LVFS levels in the STZ+FA+TM group increased, while the LVEDD level was significantly decreased, and was equivalent to the STZ group, with no statistical difference, indicating that FA exerts its efficacy by regulating endoplasmic reticulum stress. When TM induces endoplasmic reticulum stress, FA loses its efficacy.

[0054] 9. Western Blot

[0055] Experimental process: Myocardial total protein was extracted from myocardial tissue, and the protein concentration was determined by the BCA method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins of different relative molecular weights. After electrophoresis, the membrane was transferred. Seal with 5% skim milk for 1 hour at room temperature and recover. The test strip was incubated with the corresponding antibody and other primary antibodies in a 4°C refrigerator overnight. The strips were incubated with the secondary antibody at room temperature for 1 hour. Finally, ECL chemiluminescent substrate was added, and protein expression was detected with a chemiluminescent detector to obtain imaging.

[0056] Experimental results: see Figure 6 A and Figure 6 B, Compared with the control group, the levels of GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, Caspase-1, and IL-1β in the STZ group were significantly increased ( Figure 6 A), gray value analysis showed statistically significant differences ( Figure 6 B). Compared with the STZ group, the levels of GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, Caspase-1, and IL-1β in the STZ+FA group were significantly decreased ( Figure 6 A), gray value analysis showed statistically significant differences ( Figure 6 B); after administration of TM, the levels of GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, and Caspase-1 in the STZ+FA+TM group increased and were comparable to those in the STZ group ( Figure 6 A), no statistical difference ( Figure 6 B), indicating that FA exerts its pharmacological effect by regulating ER stress. When TM induces ER stress, FA loses its pharmacological effect.

[0057] Effects of 10FA on Glu-induced cytokine expression in H9c2 cells

[0058] Experimental procedure: The levels of pro-inflammatory factors in the cell supernatant were detected according to the kit instructions.

[0059] Experimental results: see Figure 7Compared with the control group, the levels of serum interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the Glu group were significantly increased. Compared with the Glu group, the levels of IL-1β, IL-6 and TNF-α in the Glu+FA group were significantly decreased; after the administration of ER stress inducer (TM), the levels of L-1β, IL-6 and TNF-α in the Glu+FA+TM group increased and were comparable to those in the Glu group, with no statistical difference, indicating that FA exerts its efficacy by regulating ER stress. When TM induces ER stress, FA loses its efficacy.

[0060] Effect of 11FA on Glu-induced endoplasmic reticulum stress-related TXNIP / NLRP3 inflammasome pathway in H9c2 cells

[0061] Experimental process: After cell inoculation, cells were collected and fixed, and then permeabilized and blocked at room temperature for 30 minutes. The blocking solution was removed, and the primary antibody was added and incubated overnight at 4°C. The fluorescent secondary antibody was added and incubated at room temperature for 1 hour, and then the DAPI solution was added and incubated for 10 minutes. The sections were sealed with an anti-fluorescence quencher and observed under a fluorescence microscope.

[0062] Experimental results: see Figure 8 A and Figure 8 B, Compared with the control group, the expression of GRP78, TXNIP and NLRP3 in the Glu group was significantly increased ( Figure 8 A), the fluorescence positive rate was statistically significant ( Figure 8 B). Compared with the Glu group, the expression levels of GRP78, TXNIP and NLRP3 in the Glu+FA group were significantly decreased (Figure A), and the fluorescence positive rate was statistically significant ( Figure 8 B); after administration of TM, the expression levels of GRP78, TXNIP and NLRP3 in the Glu+FA+TM group increased ( Figure 8 A), and was comparable to the Glu group, with no statistical difference ( Figure 8 B), indicating that FA exerts its pharmacological effect by regulating ER stress. When TM induces ER stress, FA loses its pharmacological effect.

[0063] The present invention has found through research that FA alleviates DCM by regulating NLRP3 inflammasome activation induced by endoplasmic reticulum stress. Activation of NLRP3 inflammasome can promote the maturation and secretion of proinflammatory cytokines IL-1β, IL-6, and TNF-α. Therefore, NLRP3 inflammasome activation plays a vital role in regulating inflammatory factors. It is composed of intracellular innate immune receptor NLRP3, junction protein ASC, and protease caspase-1. Studies have shown that endoplasmic reticulum stress can affect the activation of inflammasomes, and TXNIP plays an important role in the maturation of IL-1β mediated by endoplasmic reticulum stress. Overactivated IRE1α increases the stability of TXNIP mRNA by reducing the level of microRNA-17 (miR-17), which usually leads to increased translation of TXNIP and increased expression of TXNIP protein, which subsequently leads to further activation of NLRP3 inflammasome and secretion of caspase-1 and IL-1β. In this study, FA reduced GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, and Caspase-1 in the hearts of STZ mice; and inhibited the levels of GRP78, TXNIP, and NLRP in Glu-induced H9c2 cells. Therefore, FA can induce NLRP3 inflammasome activation through endoplasmic reticulum stress to alleviate diabetic cardiomyopathy, providing a theoretical basis for the treatment of diabetic cardiomyopathy.

[0064] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and the specific implementation of the present invention cannot be limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. Use of forsythiaside A in the preparation of a drug for alleviating or treating diabetic cardiomyopathy.

2. The application according to claim 1, characterized in that: The forsythiaside A alleviates or treats diabetic cardiomyopathy by regulating endoplasmic reticulum stress-induced NLRP3 activation.

3. The application according to claim 1, characterized in that: The forsythiaside A can reduce the levels of blood sugar and glycosylated serum protein in STZ-induced diabetic mice, and increase the level of insulin.

4. The application according to claim 1, characterized in that: The forsythiaside A can reduce the levels of TG, LDH, CK-MB, cTnI and MDA in the serum of STZ-induced diabetic mice, and increase the level of SOD.

5. The application according to claim 1, characterized in that: The forsythiaside A can reduce the levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α in the serum of STZ-induced diabetic mice.

6. The use according to claim 1, characterized in that: The forsythiaside A can significantly reduce the pathological damage of myocardial tissue in STZ-induced diabetic mice, while increasing the levels of LVEF and LVFS, reducing the level of LVEDD, and improving cardiac function.

7. The use according to claim 1, characterized in that: The forsythiaside A can reduce the expression levels of GRP78, P-PERK, P-EIF-α, XBP1, TXNIP, NLRP3, ASC, Caspase-1, and IL-1β in the heart tissue of STZ mice.

8. The use according to claim 1, characterized in that: The forsythiaside A can reduce the levels of proinflammatory factors IL-1β, IL-6 and TNF-α in Glu-induced H9c2 cells.

9. The use according to claim 1, characterized in that: The forsythiaside A can reduce the expression levels of GRP78, TXNIP and NLRP3 in Glu-induced H9c2 cells.

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