Application of a marsdenia tenacissima alcohol extract in myocardial protection

Abstract

The present application relates to the technical field of biological medicine, in particular to application of aconitum brachypodum franch alcohol extract in myocardial protection, and discloses that the aconitum brachypodum franch alcohol extract mainly inhibits formation of myocardial fibrosis, reduces inflammatory reaction and reduces cell apoptosis by adjusting a TLR4 / MyD88 / NF-kappa B channel, thereby protecting ISO-induced myocardial injury of a mouse; the present application provides a theoretical basis for improving myocardial injury by the aconitum brachypodum franch, and also provides a scientific basis for development and utilization of the aconitum brachypodum franch.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of a deer herb extract in myocardial protection. Background Technology

[0002] With the increasing incidence of cardiovascular diseases, myocardial injury has gradually become one of the major diseases seriously endangering human health. Balanophora dioica, a plant belonging to the genus Balanophora in the family Balanophoraceae, is widely used as a traditional herbal medicine by the Lahu ethnic group and is currently widely applied in the food industry. Balanophora dioica is pungent and slightly sweet, and has a cooling nature. It possesses the effects of cooling the blood and stopping bleeding, clearing heat and detoxifying, and is mainly used to treat cough, hematemesis, schizophrenia, hemorrhoids, chronic hepatitis, impotence, and gastrointestinal bleeding. Although Balanophora dioica has been widely used, its molecular mechanisms in improving myocardial injury have not yet been studied in detail.

[0003] The chemical components of *Deer Antler Grass* mainly include triterpenoids, phenylpropanoids, flavonoids, steroids, and tannins. However, the specific mechanism of action of *Deer Antler Grass* on myocardial injury has not yet been fully reported.

[0004] Therefore, in-depth research on the role of *Desmodium styracifolium* ethanol extract in myocardial injury will provide important reference for the high-value-added development and utilization of *Desmodium styracifolium*. At the same time, this research also provides a scientific basis for using *Desmodium styracifolium* as a dietary intervention strategy for the prevention of myocardial diseases. Summary of the Invention

[0005] Based on the above objectives, the present invention proposes an application of *Deer Antler Grass* in any of the following:

[0006] A) The application of Deer Antler Grass in the preparation of products for myocardial protection;

[0007] B) The application of Deer Antler Grass in the preparation of products for the treatment of myocardial injury;

[0008] C) Application of Deer Antler Grass in the preparation of products for reducing oxidative stress levels;

[0009] D) The application of Deer Antler Grass in the preparation of products for inhibiting myocardial fibrosis;

[0010] E) Application of Deer Antler Grass in the preparation of products for reducing inflammatory responses caused by myocardial injury;

[0011] F) Application of Deer Antler Grass in the preparation of products for inhibiting cell apoptosis.

[0012] Preferably, the herb is an ethanol extract of the herb.

[0013] Preferably, the extraction method of the ethanol extract of *Deer Antler Grass* is as follows:

[0014] After drying and pulverizing the herb, the herb was extracted by ethanol reflux to obtain the herb ethanol extract.

[0015] Preferably, the ethanol reflux ratio is 1:(8-12).

[0016] Preferably, after drying and pulverizing *Deer Antler Grass*, it is extracted by ethanol reflux, the solvent is recovered, and then freeze-dried to obtain an extract, which is the *Deer Antler Grass* ethanol extract.

[0017] Compared with existing technologies, its advantages are as follows:

[0018] This invention discloses that the alcohol extract of Deer Antler Grass mainly inhibits the formation of myocardial fibrosis, reduces inflammatory response, and reduces cell apoptosis by regulating the TLR4 / MyD88 / NF-κB pathway, thereby protecting against ISO-induced myocardial damage in mice. This invention provides a theoretical basis for Deer Antler Grass to improve myocardial injury and also provides a scientific basis for the development and utilization of Deer Antler Grass. Attached Figure Description

[0019] Figure 1 The effects of BDEE on ISO-induced morphology of mouse heart and liver tissues were investigated. A shows the heart morphology of each group of mice; B shows the calculation of the cardiac index; C shows the myocardial tissue morphology detected by H&E and Sirius red staining (scale bar: 50 μm); D shows the BDEE hepatotoxicity detected by H&E and Masson staining. Data are expressed as mean ± SEM, n = 3; *P < 0.05 vs. control group; #P < 0.05 vs. ISO group.

[0020] Figure 2 The experimental results of BDEE in reducing ISO-induced oxidative stress are shown below. A represents the effect of BDEE on CK levels, B on LDH levels, C on MDA content, D on SOD activity, E on CAT activity, and F on GSH-Px activity. Results are expressed as mean ± SEM, n = 6; ***P < 0.001, **P < 0.01, *P < 0.05 vs. control; #P < 0.001, ##P < 0.01, #P < 0.05 vs. ISO group.

[0021] Figure 3The experimental results of BDEE inhibiting ISO-induced myocardial fibrosis are shown. A represents immunohistochemical micrographs of cardiac tissue sections containing COL1A1 and α-SMA, magnification: 20×, scale bar = 50 μm; B represents quantitative image analysis of COL1A1 and α-SMA immunohistochemical staining, expressed as optical density (OD); C represents the results obtained by Western blotting. Blot analysis was performed to determine the effect of BDEE on the expression of COL1A1, COL3A1, and α-SMA in cardiac tissue; D represents the quantitative expression of COL1A1, COL3A1, and α-SMA proteins; E represents the effect of BDEE on the expression of TGF-β1, P-Smad3, and Smad3 in each group; F represents the quantitative result of normalized TGF-β1 protein expression; G represents the quantitative result of normalized P-Smad3 / Smad3 protein expression; results are expressed as mean ± SEM, n = 3; ***P < 0.001, **P < 0.01 vs control group; ###P < 0.001, ##P < 0.01, #P < 0.05 vs ISO group;

[0022] Figure 4 To demonstrate that BDEE can alleviate the inflammatory response in mice with ISO-induced myocardial injury, the following data were presented: A) Immunohistochemical micrographs of TLR4 heart sections (magnification: 20×, scale bar: 50 μm); B) Quantitative image analysis of TLR4 immunohistochemical staining, expressed as optical density (OD); C) Effect of BDEE on the expression of TLR4, MyD88, IRAK-1, TRAF-6, and NF-κB proteins in cardiac tissue determined by Western blot; D) Relative intensities of TLR4, MyD88, IRAK-1, TRAF-6, and NF-κB in each group calculated using normalized GAPDH. Values ​​are mean ± SEM, n = 3; ***P < 0.001, **P < 0.01, *P < 0.05 vs. control group; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. ISO group.

[0023] Figure 5To investigate the potential role of BDEE in cardioprotection by inhibiting ISO-induced apoptosis, the following data are presented: A: Microscopic photographs of TUNEL-stained heart sections, magnification: 20×, scale bar = 50 μm; B: Relative proportion of TUNEL-positive cells in the hearts of mice in each group; C: Effect of BDEE on Bcl-2 and Bax expression in each group; D: Quantitative results of Bax protein expression normalized to GAPDH; E: Quantitative results of Bcl-2 protein expression normalized to GAPDH; F: Quantitative results of the Bcl-2 / Bax ratio at the protein expression level; G: Effect of BDEE on Caspase-9 and Caspase-3 expression in each group; H: Quantitative results of Caspase-9 and Caspase-3 protein expression normalized to GAPDH. Results are expressed as mean ± SEM, n = 3; ***P < 0.001, **P < 0.01 vs. control group; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. ISO group. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The isoproterenol used in the following examples was purchased from Sigma (USA), the ECL chemiluminescence solution was purchased from Servicebio (Wuhan, China), the antibodies were all from Beyotime Biotechnology (Shanghai, China), and the kits were all purchased from Nanjing Jiancheng (Nanjing, China).

[0026] Example 1: Preparation of Ethanol Extract from Deer Antler Grass

[0027] Fresh whole plant of *Deer Herb* was rinsed with water, dried and pulverized. It was then extracted with 75% ethanol under reflux for one hour at a material-to-liquid ratio of 1:(8-12). The extraction was repeated twice, the solvent was recovered and the extract was freeze-dried to obtain the extract, namely *Deer Herb* ethanol extract (BDEE).

[0028] Example 2: Pharmacological study of the cardioprotective effect of BDEE prepared in Example 1.

[0029] I. Treatment of mice

[0030] Isoproterenol (ISO) is a β-adrenergic receptor (β-AR) agonist that can induce myocardial injury by activating β1 receptors on the myocardial cell membrane, thereby increasing heart rate, myocardial oxygen consumption, and causing ischemic-like changes in the myocardium. Isoproterenol-induced myocardial injury models are characterized by short modeling time, stability, reliability, low cost, and ease of mimicry, and are commonly used to induce and simulate ischemic myocardial injury, drug-induced myocardial injury, myocardial infarction, and other diseases.

[0031] Mice were randomly divided into 6 groups: control group, model group, low-dose BDEE group (100 mg / kg), medium-dose BDEE group (200 mg / kg), high-dose BDEE group (400 mg / kg), and BDEE drug group (400 mg / kg).

[0032] The control group received oral gavage and subcutaneous injection of 0.9% saline (10 mg / kg / d);

[0033] The model group was administered 0.9% saline by gavage and ISO (10 mg / kg / d) by subcutaneous injection to establish a mouse model of myocardial injury;

[0034] The low-dose BDEE group, medium-dose BDEE group, and high-dose BDEE group were administered BDEE (100, 200, and 400 mg / kg / d) by gavage, while ISO (10 mg / kg / d) was administered subcutaneously.

[0035] The BDEE drug group was given BDEE (400 mg / kg / day) by gavage.

[0036] The experiments in each group lasted for 21 days. On day 22, blood was collected from the eyes of the mice, and the hearts and livers were harvested. The hearts and livers were rinsed in physiological saline to remove blood, dried with filter paper, weighed, and the heart tissue was observed to calculate the cardiac index. The whole blood was then centrifuged at 3000 rpm for 10 minutes, the supernatant was carefully aspirated, and stored at -80°C for subsequent experiments.

[0037] II. Study on the effect of BDEE on improving ISO-induced myocardial injury in mice

[0038] Observe the heart tissue of mice and calculate the heart and liver indices. The formula for calculating the heart index is as follows:

[0039] HW / BW (mg / g) = Heart weight (mg) / Mouse body weight (g);

[0040] Results of mouse heart tissue Figure 1As shown in Figure A, the surface of normal mouse heart tissue is smooth, bright red, and of normal shape and size. The results show that, compared to the ISO group, the hearts of mice in the BDEE group are closer to normal in color and significantly smaller in volume.

[0041] The results of the cardiac index calculation are as follows Figure 1 As shown in Figure B, the results showed that the HW / BW ratio in the ISO group was significantly higher than that in the control group, while the HW / BW ratio in the BDEE group was significantly lower, indicating that BDEE can alleviate ISO-induced myocardial hypertrophy.

[0042] Heart tissue samples were collected and fixed with 4% buffered paraformaldehyde for 24 hours. The heart tissue was embedded in paraffin and sectioned into 4 μm sections using standard histological methods. Left ventricular sections were stained with hematoxylin-eosin (H&E) and Sirius red. Liver sections were stained with hematoxylin and eosin (HE) and Masson's stain.

[0043] The results of H&E and Sirius red staining of myocardial tissue are as follows: Figure 1 As shown in Figure C, H&E staining results revealed significant fiber bundles, myocardial structural disorder, and inflammatory cell infiltration in the ISO group mice. After BDEE treatment, the degree of myocardial structural disorder and fibrosis improved in a dose-dependent manner. Sirius red staining results showed that collagen fibers in the model group were significantly higher than those in the control group, consistent with H&E staining results. BDEE dose-dependently improved collagen fiber deposition. The results of H&E and Masson staining for detecting BDEE hepatotoxicity are as follows... Figure 1 As shown in Figure D, H&E and Masson staining of mouse liver tissue showed that hepatocytes were neatly arranged, with no inflammatory cell infiltration and no collagen fiber deposition, indicating that BDEE has no toxic effects.

[0044] III. Study on the effect of BDEE in reducing ISO-induced oxidative stress levels in myocardial tissue. Following the kit operation method, mouse serum was tested for CK (creatine kinase) and LDH (lactate dehydrogenase).

[0045] Following the kit instructions, mouse heart tissue was tested for MDA (malondialdehyde), SOD (superoxide dismutase), CAT (catalase), and GSH-Px (glutathione peroxidase).

[0046] When the body produces excessive reactive oxygen species (ROS) or clears them slowly, it can damage cells and tissues. The heart is one of the organs most affected by ROS. ROS can generate excessive free radicals through oxidative stress, leading to myocardial damage. Its morphological changes and energy metabolism are similar to those of human myocardial ischemia. During this process, SOD activity decreases, lipid peroxidation increases, and MDA content increases. During oxidative stress, myocardial cell rupture or increased membrane permeability causes the leakage of enzymes such as CK and LDH, leading to increased enzyme activity in the serum of mice with myocardial injury. Experimental results are as follows... Figure 2 The experimental results show that BDEE can significantly reduce leakage of CK and LDH. Figure 2 A and Figure 2 B), inhibiting the accumulation of lipid peroxidation metabolite MDA in the myocardium ( Figure 2 C), and increase the levels of SOD, CAT and GSH-Px ( Figure 2 (DF). This indicates that it can reduce ISO-induced oxidative stress, stabilize cell membranes, and protect against ISO-induced myocardial damage in mice.

[0047] IV. Research on the role of BDEE in inhibiting ISO-induced myocardial fibrosis

[0048] Fixed heart samples were embedded in paraffin and cut into 4 μm sections. After deparaffining and hydration, the heart sections were immersed in sodium citrate-sodium citrate buffer for antigen retrieval. The sections were then incubated in 3% hydrogen peroxide to quench endogenous peroxidase activity and blocked with goat serum to prevent nonspecific antibody binding. Next, the tissue samples were incubated overnight at 4°C with primary antibody, and the secondary antibody was left at room temperature for 15 minutes. The slides were then washed with Tris-buffered saline (TBS). Horseradish peroxidase (HRP)-labeled secondary antibody was added, and the samples were incubated at 37°C for 15–20 minutes, followed by diazolinone (DAB) staining and counterstaining with hematoxylin. Finally, the target protein was quantitatively analyzed.

[0049] Mouse myocardial tissue was homogenized using a tissue homogenizer. The homogenate was placed in a refrigerator for 30 min, centrifuged at 12000g, -4℃ for 10 min, and the supernatant was collected. The protein content of the sample was determined using the BCA method. Protein samples were boiled for 10 min and then separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk at room temperature for 1.5 h. It was then incubated overnight with primary antibody at 4℃. The membrane was washed 5 times with TBST. It was then incubated with anti-mouse (1:2000) and anti-rabbit (1:1000) secondary antibodies containing horseradish peroxidase. Finally, protein was detected by enhanced chemiluminescence immunoassay, and quantitative immunoblotting was performed using image analysis software.

[0050] Immunohistochemical results of COL1A1 and α-SMA as follows Figure 3 As shown in Figures AB. Compared with the control group, the expression of COL1A1 and α-SMA was increased in the model group, but decreased in a dose-dependent manner after BDEE administration. Western blot results are shown below. Figure 3 As shown in CG, compared with the control group, the protein expression of COL1A1, COL3A1, α-SMA, TGF-β1, and P-Smad3 / Smad3 in cardiomyocytes of the model group was significantly increased. Compared with the model group, the protein expression levels of COL1A1, COL3A1, α-SMA, TGF-β1, and P-Smad3 / Smad3 in the BDEE-treated group decreased in a dose-dependent manner. This suggests that BDEE may improve ISO-induced myocardial fibrosis in mice through the TGF-β1 / Smad3 pathway.

[0051] V. Study on the effect of BDEE in alleviating the inflammatory response of ISO-induced myocardial injury in mice

[0052] TLR4 is an important target protein in the inflammatory pathway. The immunohistochemical assay for TLR4 is the same as described in section four above. The immunohistochemical results of TLR4 show the following... Figure 4 As shown in Figures A and B, TLR4 expression was significantly increased in the ISO group compared to the control group, while TLR4 expression decreased in a dose-dependent manner after BDEE administration. These results indicate that TLR4 plays a role in BDEE-mediated amelioration of the inflammatory response in ISO-induced myocardial injury mice. Western blot analysis further validated the roles of TLR4 and other TLR4-related target proteins in reducing inflammation. Figure 4 Results C and D showed that, compared with the control group, the expression of TLR4, MyD88, IRAK-1, TRAF-6, and NF-κB proteins was increased in the ISO group. However, these expression levels decreased to varying degrees after BDEE administration. These results indicate that BDEE can alleviate the inflammatory response by regulating the expression of TLR4, MyD88, IRAK-1, TRAF-6, and NF-κB. Therefore, we conclude that BDEE can alleviate ISO-induced myocardial inflammation and improve myocardial injury in mice by regulating the TLR4 / MyD88 / NF-κB pathway.

[0053] VI. Research on BDEE's Cardioprotective Effect Through Inhibition of ISO-Induced Apoptosis

[0054] After euthanizing mice, heart tissue was removed, fixed in 4% paraformaldehyde for 24 hours, and embedded in paraffin. After dewaxing and rehydration, proteinase K working solution was added, and the tissue was incubated at 37°C for 22 minutes. The tissue was washed three times with PBS (pH 7.4) in a Rocker apparatus for 5 minutes each time. After the sections dried, permeabilization working solution was added, and the tissue was incubated at 37°C for 20 minutes, followed by washing with PBS. After the sections were slightly dry, buffer solution was added to the tissue, and the tissue was incubated at room temperature for 10 minutes. Appropriate amounts of TDT enzyme, dUTP, and buffer were mixed in a fixed ratio, and the mixture was used to cover the tissue. The tissue was incubated at 37°C for 2 hours, followed by washing with PBS. The liquid was removed, and the tissue was covered with a cover slide using an anti-fading mounting agent. Microscopic examination and image collection were performed using a fluorescence microscope. The Western blot procedure was the same as described in section four above.

[0055] TUNEL staining results are as follows Figure 5 As shown in A and B. Compared with the control group, the apoptosis rate of cardiomyocytes in the model group was significantly increased. Compared with the model group, the apoptosis rate was decreased after BDEE administration. Western blot results showed ( Figure 5 Compared with the control group, the model group showed decreased Bcl-2 protein expression and significantly increased expression of Bax, Caspase-9, and Caspase-3 proteins. In the BDEE-treated group, Bcl-2 expression increased in a dose-dependent manner, while the expression levels of Bax, Caspase-9, and Caspase-3 decreased in a dose-dependent manner. This indicates that BDEE inhibits ISO-induced apoptosis by regulating Bcl-2, Bax, Caspase-9, and Caspase-3, thereby improving myocardial injury.

[0056] In summary, ISO can promote the proliferation of cardiac fibroblasts and collagen formation, mediated by Ang II. After binding to its corresponding receptor, Ang II not only promotes cardiac fibroblast proliferation but also stimulates fibroblasts to secrete TGF-β1, promoting collagen production and inducing cardiac interstitial fibrosis. The TGF-β1 / Smads signaling pathway plays an important regulatory role in tissue fibrosis. Currently, many experiments have confirmed that inhibiting the TGF-β1 / Smad3 signaling pathway can suppress myocardial fibrosis, making TGF-β1 / Smad3 one of the main targets for anti-fibrotic therapy. In this invention, compared with the ISO model group, the expression of TGF-β1 / Smad3 in the myocardium of mice in all dose groups was reduced, indicating that TGF-β1 / Smad3 expression is reduced by regulating the TGF-β1 / Smad3 pathway.

[0057] ISO can induce myocardial injury by triggering an inflammatory response. In this invention, the levels of TLR4, MyD88, and NF-κB proteins were elevated in a myocardial injury model mouse. TLRs are an important family of pattern recognition receptors that can induce innate immune responses. TLR4 is a type of TLR. As an important receptor for inflammatory responses, it is a crucial member of the immune defense system and a bridge between immune responses and chronic inflammation. TLR4 activates the MyD88 pathway and ultimately activates NF-κB, a key transcription factor that activates inflammatory responses. TLR4 activates NF-κB through MyD88, IRAK, and TRAF6. NF-κB p65 dissociates and translocates to the nucleus, activating and entering the cell nucleus. Anti-inflammatory effects are one of the main functions of BDEE. After administration of BDEE to myocardial injury mice, H&E staining showed a significant reduction in myocardial necrosis and inflammatory infiltration, and Sirius red staining showed a reduction in myocardial collagen deposition. This suggests that BDEE can play a protective role in myocardial injury by inhibiting myocardial inflammation, protecting cardiomyocytes, reducing myocardial necrosis and inflammatory responses, and reducing myocardial collagen deposition. Based on the results of this invention and numerous earlier studies, it can be seen that BDEE may exert a protective effect against myocardial injury by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway to suppress myocardial inflammation.

[0058] In recent years, reports have indicated that cardiomyocyte apoptosis plays a crucial role in the development and progression of cardiovascular diseases such as atherosclerosis and myocardial ischemia-reperfusion injury. Excessive cardiomyocyte apoptosis is a significant factor leading to cardiac dysfunction and structural changes, resulting in myocardial injury. Therefore, inhibiting cardiomyocyte apoptosis can reduce myocardial damage. TUNEL staining is a classic method for assessing apoptosis. In this invention, TUNEL staining showed that BDEE significantly improved ISO-induced cardiomyocyte apoptosis. Bcl-2 family proteins play an important role in the regulation of apoptosis. Bcl-2 can inhibit the occurrence of apoptosis, while Bax can promote the production of apoptosis. The two work synergistically to control apoptosis by controlling their levels. Studies have found that Bcl-2 can regulate mitochondrial membrane permeability, triggering caspase cascade reactions, leading to cardiomyocyte apoptosis. Precise regulation of the cardiomyocyte apoptosis pathway is inseparable from the caspase family. Caspase 9 belongs to the initiator type of cysteine ​​protease, whose main function is to initiate apoptosis and regulate the activity of effector cysteine ​​proteases. Caspase 3 belongs to the effector type of cysteine ​​protease, whose main function is to act as the executor and terminator of apoptosis. Its activation can promote the formation of apoptotic bodies, inducing cardiomyocyte apoptosis. Based on the results of this invention, it was found that BDEE can improve myocardial injury by inhibiting cardiomyocyte apoptosis and regulating Bcl-2, Bax, Caspase-9, and Caspase-3.

[0059] In summary, this invention demonstrates that BDEE primarily inhibits the formation of myocardial fibrosis, reduces inflammatory responses, and decreases cell apoptosis by regulating the TLR4 / MyD88 / NF-κB pathway, thereby protecting against ISO-induced myocardial damage in mice. This invention provides a theoretical basis for *Deer Antler Grass* to improve myocardial injury and also provides a scientific basis for the development and utilization of *Deer Antler Grass*.

[0060] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An application of *Deer Antler Grass* in any of the following: A) The application of Deer Antler Grass in the preparation of drugs for inhibiting myocardial fibrosis; B) The application of Deer Antler Grass in the preparation of drugs for reducing inflammatory responses caused by myocardial injury; in, The deer herb is an ethanol extract of the deer herb. The extraction method of the ethanol extract of Deer Antler Grass is as follows: after drying and pulverizing the Deer Antler Grass, it is subjected to 75% ethanol reflux extraction to obtain the ethanol extract of Deer Antler Grass; the material-to-liquid ratio of the ethanol reflux is 1:(8-12); after drying and pulverizing the Deer Antler Grass, it is subjected to ethanol reflux extraction, the solvent is recovered, and then it is freeze-dried to obtain an extract, which is the ethanol extract of Deer Antler Grass.

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