CN111632059B - New application of swertiamarin - Google Patents

Abstract

The invention provides application of swertiamarin in preparing a medicament for treating, preventing, alleviating and/or relieving diseases related to heart failure. The research of the invention shows that the swertisin has the functions of inhibiting the excessive thickening of the wall of the chamber, delaying the myocardial remodeling, improving the myocardial fibrosis and improving the cardiac function; in addition, the swerosides can promote the recovery of the contraction/relaxation function of damaged cardiac muscle cells and have a protective effect on the ischemia-reperfusion injury of the heart.

Description

New application of swertiamarin

Technical Field

The invention belongs to the field of medicines, particularly relates to a new application of swertiamarin, and more particularly relates to a new application of swertiamarin in treating heart failure, promoting recovery of contraction/relaxation functions of damaged cardiac muscle cells and improving myocardial ischemia-reperfusion injury.

Background

Heart failure refers to a clinical syndrome in which the systolic function (ejection) and/or diastolic function (filling) of the ventricles are impaired due to structural and functional abnormalities of the myocardium resulting from various causes. In the Chinese cardiovascular disease report 2018 (Marli, Wu Asia phile, Chen Wei, et al, the main point of the Chinese cardiovascular disease report 2018 is introduced [ J ]. the China J.T. 2019,27(08):712 Yuan Yi 716), it is pointed out that the number of patients with heart failure is up to 450 ten thousand, and the prevalence rate increases with age. With the gradual step of aging society in China, the heart failure prevention and treatment work is an important task of medical and health work in China.

Currently, for the treatment of heart failure, western medicine mostly adopts treatment strategies of cardiotonic, diuresis, vascular dilation and renin-angiotensin system inhibition (sensitization, menwill. pathogenesis of heart failure and drug treatment progress [ J ]. clinical medicine 2015,35(05): 118-. For example, cardiac glycosides have the disadvantages of narrow safety range and susceptibility to poisoning.

Clinically, patients with coronary atherosclerotic heart disease and myocardial infarction often adopt therapy such as thrombolysis, percutaneous coronary intervention, aortic-coronary artery bypass transplantation and the like to open occluded coronary arteries, perform reperfusion intervention on ischemic and anoxic myocardial tissues and restore blood oxygen supply. However, it has been shown that Ischemia-Reperfusion often exacerbates damage to myocardial structures, and this irreversible damage is referred to as myocardial Ischemia-Reperfusion Injury (I/R). The Langendorff isolated heart local myocardial ischemia reperfusion injury model can eliminate the influence of nerve fluid regulatory factors and other organs, directly judge the influence of the medicament on the cardiac contraction function, and better simulate the occurrence and development of clinical myocardial I/R injury.

The traditional Chinese medicine is an important treasury of Chinese medicine and has the characteristics of multi-level, multi-path and small side effect. Studies have confirmed that the efficacy of treating heart failure is definite, and the mechanism research is relatively extensive, but the concept of compound holistic research (Li C, Wang J, Wang Q, et al, Qiagen granules inhibiting biologic infection and nerve stimulating metabolism [ J ] Sci Rep,2016,6:36949) is mostly adopted, and the effective components and mechanism of the stilbene-ginseng granules are not clear at present.

Disclosure of Invention

The invention aims to provide a new application of swertiamarin in treating heart failure, aiming at solving the problems in the prior art in treating heart failure.

The purpose of the invention is realized by the following technical scheme:

as one aspect of the present invention, the present invention provides the use of swertiamarin for the preparation of a medicament for the treatment, prevention, alleviation and/or alleviation of diseases associated with heart failure.

Further, the disease associated with heart failure includes cardiac hypertrophy.

Further, the diseases associated with heart failure include myocardial fibrosis.

Furthermore, the action target of the swertiamarin is ATP1A 1.

As another aspect of the invention, the invention provides the use of swertiamarin in the preparation of a medicament for improving and/or restoring the systolic-diastolic function of damaged cardiomyocytes.

As another aspect of the invention, the invention provides the use of swertiamarin in the preparation of a medicament for treating, preventing, alleviating and/or alleviating myocardial ischemia-reperfusion injury.

Further, the swertiamarin is swertiamarin bulk drug or medicinal derivatives thereof.

Further, the swertiamarin is prepared into a dosage form suitable for in vivo administration.

Further, the preparation is tablets, hard capsules, soft capsules, granules, pills, oral liquid or injection.

As another aspect of the present invention, the present invention provides a pharmaceutical composition characterized in that the composition comprises swertiamarin or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

aiming at the diseases related to heart failure with the symptoms of increased heart weight, thickened chamber wall, reduced compliance, reduced heart function and the like, the swertisin has the functions of inhibiting excessive thickening of the chamber wall, delaying myocardial remodeling, improving myocardial fibrosis and improving the heart function. In addition, the swertisin can promote the recovery of the contraction/relaxation function of damaged cardiac muscle cells and has a protective effect on the ischemia-reperfusion injury of the heart.

Drawings

Figure 1A shows the effect of swertisin on EF in excess ISO-induced heart failure mice, where, # # indicates: p <0.01 compared to blank; denotes: p <0.01 compared to model group.

Fig. 1B shows the effect of swertisin on FS in excess ISO heart failure mice, where, # # indicates: p <0.01 compared to blank; denotes: p <0.01 compared to model group.

Fig. 1C shows the effect of swertisin on LVIDd in excess ISO heart failure-causing mice, where # indicates: p <0.05 compared to blank.

FIG. 1D shows the effect of swertisin on LVIDs in excess ISO heart failure-causing mice, where, # # indicates: p <0.01 compared to blank; denotes: p <0.01 compared to model group.

Fig. 2A shows the morphology of the heart in the blank group of mice.

Fig. 2B shows the heart morphology of the model group mice.

Fig. 2C shows the heart morphology of the captopril group mice.

FIG. 2D shows the morphology of the heart of the sweroside group mice.

Fig. 3A shows HE staining results (20X) for the blank group of mice.

Fig. 3B shows HE staining results (20X) of model group mice.

FIG. 3C shows HE staining results (20X) for captopril group mice.

FIG. 3D shows HE staining results (20X) of sweroside group mice.

FIG. 4A shows the electrophoresis results of blank group, model group, captopril group and swertisin group in Western blot detection.

FIG. 4B shows the Western blot method for detecting the ATP1A1 protein expression changes of mice in the blank group, the model group, the captopril group and the swertioside group, wherein, # # represents: p <0.01 compared to blank; denotes: p <0.01 compared to model group.

Detailed Description

The present invention will be described in detail with reference to the following embodiments in order to fully understand the objects, features and effects of the invention, but the present invention is not limited thereto.

A disease associated with heart failure, or heart failure, refers to a clinical syndrome in which the systolic function (ejection) and/or diastolic function (filling) of the ventricles are impaired due to structural and functional abnormalities of the myocardium caused by various causes. Specific symptoms include: for example, increased heart weight, increased wall thickness, decreased compliance, decreased cardiac function, etc. In the present invention, the diseases associated with heart failure include cardiac hypertrophy, cardiac fibrosis, or heart failure caused by drugs (e.g., isoproterenol overdose).

Swertioside, swertiamarin, is an iridoid glycoside compound with molecular formula C₁₆H₂₂O₉Having the structure of formula (I):

current pharmacological research on current drug glycosides has focused primarily on the treatment of arthritis (Zhang R, Wang C M, Jiang H J, et al. protective Effects of Sweroside on IL-1beta-Induced Inflammation in Rapid engineering carbohydrates, prediction of NF-kappa B and mTORC1Signaling Pathway [ J ]. Infiammation, 2018), osteoporosis (Sun H, Li L, Zhang A, et al. protective Effects of Sweroside on MG-63cells and bone analytes [ J ]. Fitococcupatioia, 2013,84:174-179), diabetes (Huang X J, Li J, Mei Z Y, et al. functional sensitivity front tissue, 76. Cell et al. biochemical analysis [ J ]. 1, Cell 2016, Ak J, Biocement, et al., Cell 2016). No report on the treatment of heart failure by swertisin has been found.

The inventor also finds that the action target of the swertisin in treating the heart failure is ATP1A1(ATPase Na)⁺/K⁺Transporting neutron Alpha 1), i.e., Na⁺-K⁺-ATP(Na⁺-K⁺-ATPase, NKA) enzyme α 1 subunit. The NKA enzyme, namely adenosine triphosphate, is a membrane protein existing on a cell membrane and consists of three subunits, namely alpha subunit, beta subunit and gamma subunit, wherein the alpha subunit is the most important catalytic subunit of the three subunits, has the size of about 112kDa and contains ATP and Na⁺、K⁺And cardiac glycoside knotsA combination site. The expression of the alpha subunit has tissue specificity and species difference, the expression of the alpha 1 is the most extensive and exists in all cells, Na⁺-K⁺ATP-ase can use the hydrolysis of ATP to generate energy to make 3 Na⁺Removal of cells, 2 Ks⁺Engrafting into cells to ensure transmembrane Na⁺、K⁺The creation and maintenance of an electrochemical gradient.

In the present invention, "swertiamarin" is to be understood as including a pharmaceutically acceptable derivative thereof in addition to the iridoid glycoside compound of the above general formula (I) (swertiamarin drug substance) called swertiamarin. The swertiamarin can be obtained by market purchase. Alternatively, the swertiamarin is a main iridoid glycoside compound in honeysuckle (honeysuckle is a representative drug of the heat-clearing and detoxifying therapeutic principle in the astragalus and ginseng granules), and can be obtained by extracting and purifying the honeysuckle by conventional methods in the field, such as decoction extraction, reflux extraction, immersion extraction, ultrasonic extraction, percolation extraction, microwave extraction and the like, and by purification methods such as water extraction and alcohol precipitation, alkali solution and acid precipitation and various column chromatographic purification methods such as macroporous resin columns, silica gel columns, reversed phase columns and the like. The pharmaceutically acceptable derivative of swertiamarin refers to a derivative which retains the pharmacological activity or the main pharmacological activity of swertiamarin, for example, in the form of a pharmaceutically acceptable salt.

In the present invention, the swertiamarin may be administered alone or in any convenient pharmaceutical form. A convenient pharmaceutical form may be a dosage form suitable for in vivo administration, such as a tablet, hard capsule, soft capsule, granule, pill, oral liquid or injection. The effective amount of swertiamarin can be combined with pharmaceutically acceptable carriers or excipients by the skilled person according to the actual needs to prepare the dosage forms.

The pharmaceutically acceptable carrier or adjuvant comprises: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crospolyvinylpyrrolidone, low-substituted hydroxypropylcellulose, croscarmellose sodium, etc.; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the binder includes starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.

It is to be understood that an "effective amount" refers to a non-toxic, but sufficient amount of a drug or pharmaceutical agent to provide the desired effect, and that an "effective amount" of an ingredient or formulation unit refers to an amount of that ingredient or formulation unit which, when used in combination with other ingredients, is effective to provide the desired effect. The "effective amount" will vary from subject to subject, depending on age and general condition of the individual, the particular active agent, and the like. Thus, an exact "effective amount" may not always be possible, however, a suitable "effective amount" in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In the present invention, one reference dose administered is 80-200mg/kg/d (i.e., 120mg swertiamarin per kg subject per day); preferably, in the present invention, a reference dose of 120mg/kg/d (i.e., 120mg swertioside per kg subject per day) is administered, which may be divided into multiple divided doses or administered as a single controlled release formulation.

Examples

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1: study on drug effect of swertisin on treating heart failure

1 Material

1.1 animals

The SPF grade C57/BL6N mouse has a weight of 18-22 g and is provided by the Beijing Witonglihua laboratory animal technology company Limited, and the license number of the laboratory animal is SCXK (Jing) 2016-0006. The feed is bred in SPF animal laboratory of Beijing university of traditional Chinese medicine in constant temperature and humidity environment, is free to drink water and common feed, and is subjected to mold-making administration after adaptive feeding for one week.

1.2 reagents

Swertisin is provided by kyowski biotechnology limited, lot number: PS 0111123. Captopril was prepared by shanghai schubanbo pharmaceutical limited, china and america, lot number: AAQ 3404. Isoproterenol hydrochloride (ISO) is supplied by sigma-aldrich trade, ltd, lot number: 101722747. the sodium potassium atpase α 1 antibody is supplied by Abcam (shanghai) inc, cat no: ab 7671. Goat anti-mouse IgG-HRP labeled antibody is provided by beijing bayer died biotechnology, ltd, cat #: DE 0602. The RIPA lysate is provided by beijing prilley gene technology ltd, cat No.: C1053.

1.3 Instrument: canada

The ultrasonic image platform of the small animal, Pannoramic 250/MIDI digital section scanning system of 3DHIESTECH Hungary, Universal Hood II of Biorad gel imaging system of America, and Mini vertical electrophoresis transfer system of Bio-Rad of America.

1.4BATMAN-TCM on-line biological information database

http://bionet.ncpsb.org/batman-tcm/

2 method

2.1 grouping and model preparation

C57/BL6N mice were bred conventionally, and after one week of acclimatization, they were randomly divided into a blank group (8 mice), a model group (11 mice), a captopril group (9 mice), and an swertioside group (9 mice). The model group and the administration group (i.e. captopril group and swertisin group) adopt an isoproterenol hydrochloride (ISO) gradient modeling method: a mouse model of heart failure was constructed by subcutaneous injection of an excess of isoproterenol (20 mg/kg on day 1, 10mg/kg on day 2, 5mg/kg on days 3-14). The captopril group is administered with 16.5mg/kg/d of captopril, the swertioside group is administered with 120mg/kg/d of swertioside, and the administration is carried out for 14 days from the 1 st day of model building. The blank group was given daily with an equal amount of physiological saline.

3 detection index and method

3.1 cardiac function testing

Experiment ofEnd point, using Canada

And evaluating the cardiac function of the mouse by using an ultrasonic image platform of the mouse. The left ventricular end systolic diameter (LVIDs), left ventricular end diastolic diameter (LVIDd) were measured, and the Ejection Fraction (EF) and the short axis shortening (FS) were calculated. Each animal was sampled 3 times and the average of 3 consecutive cardiac cycles was recorded for subsequent analysis.

3.2 morphological and pathological examination

When the material is taken, the heart is placed in 1% PBS buffer solution to wash blood, and the whole shape of the heart is photographed and observed by using a digital camera.

Cutting heart tissue along the maximum transverse diameter of the center, immersing the bottom of the heart in 4% paraformaldehyde for fixation for 24h, performing conventional ethanol gradient dehydration, xylene transparentization, wax immersion embedding, slicing and sticking, and performing Hematoxylin-eosin staining (HE staining).

3.3 database target prediction

Openhttp://bionet.ncpsb.org/batman-tcm/Example 3 was selected, Sweroside was entered, and the pubmed CID of the compound was 161036, Score cutoff was determined to be 20, and P was used<0.05 is a statistical significance range to search the target of the swertisin for treating the heart failure disease.

3.4Western blot candidate target detection

The myocardial tissue is quickly frozen by liquid nitrogen and preserved at-80 ℃. The RIPA lysate is used for extracting total protein of the myocardial tissue, the loading amount is 30 mu g, the voltage is 80V when gel is electrophoretically concentrated, the voltage is 120V when gel is electrophoretically separated, and the electrophoresis time is 1.5 h. The electric conversion adopts a constant current of 300mA for 1.5 h. Blocking with 5% skimmed milk powder for 1h, incubating overnight at 4 deg.C (ATP1A1, 1:5000), rinsing with TBS-T buffer solution for 3 times and 10 min/time; the polyvinylidene fluoride membrane (PVDF) was taken out and added with a secondary antibody (goat anti-mouse IgG-HRP label, 1:5000), incubated at room temperature for 1h, washed with TBS-T buffer for 3 times, and then dropped with ECL luminescence solution for exposure. The grey value of each protein band was recorded and quantified using Image lab.

3.5 statistical methods

Data analysis was performed using SAS8.4 statistical software, all data being mean. + -. standard deviation

Showing that the comparison between groups is performed by analysis of variance, expressed as P<A difference of 0.05 is statistically significant.

4. Results of the experiment

4.1 mouse Heart ultrasound results

Referring to fig. 1A to 1D, EF and FS values were significantly decreased (P <0.01), left ventricular LVIDs values were significantly increased (P <0.01), and LVIDd values were increased (P <0.05) in the model group compared to the blank group; compared with a model group, the swertisin can obviously improve EF and FS values (P <0.01) of mice with excessive heart failure caused by isopropylidene, and obviously reduce the left ventricular end systolic internal diameter (LVIDs) (P <0.01) and the left ventricular end diastolic internal diameter (LVIDd), and the effect is similar to that of the positive drug captopril.

4.2 morphological and pathological examination

Referring to the morphology of mouse heart shown in fig. 2A-2D and HE staining shown in fig. 3A-3D, the blank group showed normal myocardial tissue with well-ordered arrangement of myocardial cells; the mouse myocardial cells in the model group are disorderly arranged, the myocardial cells are necrotic, lose normal structures and undergo myofiber lysis; when the myocardial cells of the drug glycoside group are arranged more orderly, the cells basically maintain the original shape, which is similar to the shape of the captopril group.

BATMAN-TCM predicted target point

BATMAN-TCM database predicts that the target of swertisin for treating heart failure is ATP1A1(ATPase Na)⁺/K⁺Transporting Subunit Alpha 1)。

Western blot results

Referring to fig. 4A and 4B, the model group significantly down-regulated ATP1a1 protein expression level (P <0.01) compared to the blank group; compared with the model group, the expression of ATP1A1 in the drug glycoside group is increased (P < 0.01).

The experimental results prove that the swertisin can obviously improve the heart failure caused by the excessive isoproterenol, and the action mechanism is related to the prediction target ATP1A 1. When heart failure occurs, the expression of the NKA enzyme alpha subunit is altered.

Example 2: study on promotion of myocardial contraction/relaxation function recovery of primary myocardial cells of suckling mice by swertisin

1. Material

1.1 animals

SD rat suckling mouse (1d), purchased from sbefu (beijing) biotechnology limited, laboratory animal license number: SCXK (Kyoto) 2016-.

1.2 reagents

Swertia japonica glycosides are available from Dode plus Biotech, Inc., cat # 14215-86-2.

The collagenic Type II was supplied by Gibco, USA, Cat.17101-.

Trypsin is supplied by VWR-amresco, USA, under the trade name 0458-25G.

5-brdu (5-Bromo-2' -deoxyuridine) is available from Sigma Aldrich trade, Inc., Cat B5002.

DMEM (sugar free) is supplied by Gibco, USA, Cat 11966-.

DMEM powder is supplied by Gibco, Inc. of USA, with the trade name 12100-.

D-MEM/F-12 powder was supplied by Gibco, Inc. of U.S.A., Cat. No. 12400-.

Penicilin/Streptomyces is supplied by Corning, USA, under the product number 15140122.

Fetal bovine serum is supplied by corning, usa under the cat number 35-081-cv.

The special horse serum is provided by Tianjin Kangyuan biotechnology limited, and has a cargo number of DE0475-200 ml.

The CCK8 test kit is supplied by Dongren chemical technology (Shanghai) Co., Ltd., cat # CK 04.

Flou-4, AM is available from Saimer Feishale science (China) Inc., cat # F14201.

1.3 instruments

Phase contrast microscopes are available from olympus (china) ltd under model number CXK 53.

The multifunctional microplate reader is available from the Megasolen instruments (Shanghai) Co., Ltd., model SpectraMax i3 x.

The cell hypoxia culture chamber is supplied by Qizizalmeiword Industrial products, Inc., model GC-C.

Multifunctional imaging cytoanalyzer is supplied by the Megaku instruments (Shanghai) Co., Ltd., model ImageXpress Micro XLS.

The laser scanning confocal microscope is provided by olympus (china) ltd, model FV 3000.

2. Method of producing a composite material

2.1 extraction and isolation of Primary cardiomyocytes in suckling mice

Disinfecting the suckling mouse, cutting the chest, picking up the heart and cutting into paste. Placing the mixture into a 50ml centrifuge tube, adding digestive juice (0.06% trypsin and 0.04% collagenase II are dissolved in D-hanks liquid), digesting in a 37 ℃ water bath kettle for 3-5 times (the digestion time is 10min, 25min, 30min, 20min and 15min in sequence), shaking for several minutes, adding 1-2ml special horse serum after each digestion to terminate the digestion, discarding the supernatant after the first digestion, collecting the remaining supernatant after several digestions, filtering by a 200-mesh filter screen, centrifuging at 1500rpm for 5min, re-suspending the culture medium, inoculating the culture medium into a 10cm culture dish, adding DMEM culture medium (containing 10% FBS and 5% HS), adding 5-brdu (the final concentration is 100 mu mol/L), placing the culture box, culturing for at least 1.5h, sucking out the supernatant, centrifuging at 1500rpm for 5min, re-suspending, counting cells, and paving the plate according to the experimental requirements. Changing the culture medium after 12-18h, and avoiding shaking the culture plate as much as possible during the period, otherwise, influencing the myocardial cell adherence. After 24-36h, the cardiomyocytes had spontaneous beating. And continuously culturing for 24-48 h, wherein the large-area uniform synchronous pulsation of the myocardial cells occurs. And taking the primary cardiomyocytes cultured for 3-5 days and in good growth state for subsequent experiments.

2.2 construction of hypoxia/Reoxygenation (H/R) model

Taking primary cells which are cultured for 3-5 days and have good growth state, replacing culture medium with anoxic liquid (sugar-free DMEM), placing the cells in a cell hypoxia culture chamber, and adjusting the gas proportion to be O₂(0.5％)、CO₂(5％)、N₂(94.5%), culturing for 6 hr in oxygen-poor condition, taking out, and changing culture medium to obtain oxygen-enriched liquid (normal)Cardiomyocyte medium), and then subjecting the cells to CO₂The incubator is used for 1 h.

3. Detection index and method

3.1 determining the concentration of swertisin for protecting H/R injured myocardial cells

The cardiomyocytes with good growth state in 3-5 days were used and divided into a blank group (without any treatment), a 0.01. mu. mol/L group (0.01. mu. mol/L swertiamarin incubation for 24 hours), a 0.1. mu. mol/L group (0.1. mu. mol/L swertiamarin incubation for 24 hours), a 1. mu. mol/L group (1. mu. mol/L swertiamarin incubation for 24 hours), a 10. mu. mol/L group (10. mu. mol/L swertiamarin incubation for 24 hours), and a 100. mu. mol/L group (100. mu. mol/L swertiamarin incubation for 24 hours). After treatment, the ratio of CCK8 solution: adding CCK8 solution into the culture medium according to the proportion of 1:9, placing the culture medium into an incubator for incubation for 4h, detecting the absorbance OD value by using an enzyme-labeling instrument, calculating the cell activity according to a specification formula, and determining the non-toxic concentration range of the effect of the swertisin on normal myocardial cells.

Cells with good growth state of 3-5 days are taken and randomly divided into 4 groups, a blank group, a model group (H/R damage), a 0.1 mu mol/L group (cells are incubated with a culture medium containing 0.1 mu mol/L swertisin 24 hours in advance and then treated with H/R, and the H/R process is also treated with 1 mu mol/L swertisin), a1 mu mol/L group (cells are incubated with a culture medium containing 1 mu mol/L swertisin 24 hours in advance and then treated with H/R, and the H/R process is also treated with 1 mu mol/L swertisin), and a 10 mu mol/L group (cells are incubated with a culture medium containing 10 mu mol/L swertisin 24 hours in advance and then treated with H/R, and the H/R process is also treated with 10 mu mol/L swertisin). After treatment, the ratio of CCK8 solution: adding CCK8 solution into the culture medium according to the proportion of 1:9, placing the culture medium into an incubator for incubation for 4H, detecting the absorbance OD value by using an enzyme-labeling instrument, calculating the cell activity according to a specification formula, and analyzing the influence of the current drug glycoside on the myocardial cell activity after H/R injury.

3.2 Effect of swertisin on contractile function of H/R primary cardiomyocytes

Cells with good growth state of 3-5 days are taken and randomly divided into 4 groups, a blank group, a model group (H/R damage), a1 mu mol/L group (cells are incubated with a culture medium containing 1 mu mol/L swertisin 24 hours in advance and then treated with H/R, H/R process is also treated with 1 mu mol/L swertisin), and a 10 mu mol/L group (cells are incubated with a culture medium containing 10 mu mol/L swertisin 24 hours in advance and then treated with H/R, H/R process is also treated with 10 mu mol/L swertisin). After processing, confocal microscopy was used to record 20s bright field under 20x lens with exposure time of 40ms, at least 3 different wells per group were designed, and at least 3 fields of view were recorded per well cell. Data analysis was performed using Image J Muscle Motion Software (see Sala L, van Meer B J, Tertolole L, et al. MUSCLEOTION: A Versatile Open Software Tool to quantity Cardiocyte and Cardiac Muscle control In Vitro and In Vivo [ J ] Circ Res,2018,122(3): e5-e16), which is a multifunctional Open plug developed by Luca Sala et al, is an extended function of ImageJ Software, and by processing dynamically recorded data In a specific required format, the contractile function of cardiomyocytes, neurons, etc. of animals or humans can be accurately measured.

3.3 Swingoside on transient Effect of H/R injury on Primary cardiomyocyte calcium

3-5 days of cells with good growth state are taken, and the grouping is the same as 3.2. After the cells are treated, the culture medium is sucked out, PBS is used for washing for 3 times, Fluo-4 and AM fluorescent dye are added, incubation is carried out for 30min, then PBS is used for washing away the dye, and calcium-free desktop solution is added. After 1Hz electric field stimulation was given to each plate of cells, the changes in intracellular calcium fluorescence were recorded using a multifunctional imaging cytoanalyzer. More than 3 wells were designed per set, with 4 fields recorded for each well, exposure time 40ms, and 20s recorded. 10 cells were selected for analysis per field. The change in fluorescence intensity Δ F was analyzed using Image J, the amplitude and intensity of calcium transients are expressed as the net change in standard fluorescence intensity Δ F — F-F0, with F0 referring to the background fluorescence intensity at rest. The mean peak height of calcium transients over the detection time was analyzed by PeakCaller software (see Artimovich E, Jackson R K, Kilander M, et al, PeakCaller: an automated graphical interface for the quantification of intracellular calcium associated by high-content screening [ J ]. BMC Neurosci,2017,18(1):72), and statistical analysis was performed. PeakCaller is a script of MATLAB software developed by Elena Artimovich et al. Can be used for high-throughput detection of calcium transient in cells.

3.4 statistical analysis

Data analysis was performed using SAS8.4 statistical software, all data being mean. + -. standard deviation

It is shown that the group comparisons were statistically significant using one-way analysis of variance (Oneway-ANOVA) with P <0.05 as the difference.

4 results

4.1 concentration of swertisin effective in protecting H/R injured myocardial cells

The results are shown in tables 1 and 2.

TABLE 1 nontoxic concentration range of swertisin acting on normal myocardial cells

TABLE 2 Effect of swertisin on myocardial cell viability after H/R modeling

Grouping	N	Cell viability (%)
			Blank group	3	1.00±0.05
Model set	4	0.46±0.10###
			0.1μmol/L	5	0.50±0.08
1μmol/L	5	0.64±0.09**
			10μmol/L	5	0.66±0.05**
100μmol/L	5	0.62±0.08**

# P <0.001 compared to blank group, P <0.01 compared to model group, # P <0.001

As shown in Table 1, when the concentration of the swertisin is in the range of 0.01 mu mol/L to 100 mu mol/L, the pretreatment for 24h has no influence on the activity of the myocardial cells, which indicates that the swertisin has no toxic effect on normal myocardial cells. The results in Table 2 show that compared with the blank group, the myocardial cell activity of the model group is obviously reduced after the myocardial cells are treated by H/R (P is less than 0.001); compared with the model group, the cell viability of each swertiamarin administration group (1. mu. mol/L, 10. mu. mol/L, 100. mu. mol/L) is obviously improved (P < 0.01). The results show that the survival rate of H/R myocardial cells can be remarkably improved by the swertisin (1 mu mol/L, 10 mu mol/L and 100 mu mol/L).

4.2 swertisin can promote the recovery of the contractile function of H/R damaged cardiac muscle cells

The results are shown in Table 3.

TABLE 3 Effect of swertisin on H/R-damaged cardiomyocyte contractile function

, # P <0.01, # P <0.001 compared to the blank group, and # P <0.05, # P <0.001 compared to the model group

As shown in Table 3, after H/R injury to cardiomyocytes, heart rate decreased (P <0.001), amplitude of contraction decreased (P <0.001), systolic time course increased (P <0.001), peak time course increased (P <0.01), diastolic time course increased (P < 0.001); similarly, after H/R injury, 1 mu mol/L or 10 mu mol/L swertin is used for pretreatment, so that relevant indexes of myocardial cell contraction can be obviously improved, the heart rate of the H/R myocardial cell is increased (P <0.001), the contraction amplitude is increased (P <0.001), the contraction time course is reduced (P <0.001), the peak reaching time course is reduced (P <0.05), the diastole time course is reduced (P <0.001), and the recovery of the myocardial cell contraction/diastole function is promoted.

4.3 Effect of swertisin on calcium transients in H/R injured cardiomyocytes

The results are shown in Table 4.

TABLE 4 transient effects of swertiamarin on H/R-damaged cardiomyocyte calcium

# P <0.01 compared to blank group and # P <0.05 compared to model group.

As shown in Table 4, the fluorescence intensity of calcium transient peak of the cardiomyocytes decreased (P <0.01) after H/R injury, and the calcium transient peak was restored to the level of blank group when the group was pretreated with the drug glycoside (1. mu. mol/L, 10. mu. mol/L) compared to the H/R group.

The experimental results show that the glucoside can improve the contraction/relaxation dysfunction of primary myocardial cells of suckling mice caused by H/R injury, has the effects of positive inotropic and positive inotropic, and can improve the contraction/relaxation movement of the damaged myocardial cells. The swertisin can increase calcium transient amplitude of myocardial cells damaged by H/R, and is beneficial to recovery of calcium transient, excitation-contraction coupling function of myocardial cells, and contraction/relaxation function of myocardial cells.

Example 3: protective effect of swertisin on ischemia reperfusion injury of isolated heart

1. Material

1.1 animals

About 250g of SD rat purchased from Schbefu (Beijing) Biotechnology Ltd, laboratory animal license number: SCXK (Kyoto) 2016-. Feeding in SPF animal laboratory of Beijing university of traditional Chinese medicine, keeping constant temperature and humidity, alternating day and night for 12h, freely drinking water, and using common feed

1.2 reagents

Swertia japonica glycosides are available from Dode plus Biotech, Inc., cat # 14215-86-2.

Digoxin is available from MedChemex (MCE) Inc., USA under the designation HY-B1049.

Pentobarbital sodium is supplied by sigma aldrich trade, ltd, with a cat number P3761-25G.

Heparin sodium was supplied by Beijing Byeldi Biotechnology Inc., under the trade designation DE0290-1 g.

The 2% TTC staining solution is supplied by Beijing Sorleibao technologies, Inc., cat # G3005.

Other reagents were analytically pure.

The preparation method of the K-H solution comprises the following steps: weigh NaCL6.9g, NaHCO₃2.1g、KCL0.35g、KH₂PO₄0.163g、MgSO₄0.144g of the extract is dissolved in 1L of sterilized deionized water, and 1.998g of glucose and 0.139g of anhydrous calcium chloride are added before use.

1.3 instruments

Langendorff perfusion apparatus is supplied by Olcott, Shanghai, and is model ALC-HP.

The PowerLab physiological recorder is available from Eddy instruments International trade (Shanghai) Inc., model PowerLab 16/35.

The suture needle with thread (nylon thread) is provided by Shanghai medical suture needle works Co., Ltd., type 5-0 ligature.

2. Method of producing a composite material

2.1 Langendoroff Ex vivo Heart perfusion procedure

SD rats are weighed and injected with heparin sodium (1X 10) intraperitoneally half an hour in advance³U/kg) and 1% sodium pentobarbital (5 ml/k)g) The patients are fully anesthetized. Fixed on a rat plate. Preparing skin, opening chest near diaphragm, tearing diaphragm and thymus, exposing aortic arch position, slightly lifting aorta and innominate artery junction, cutting off aorta, lifting off aortic broken end, and placing the cut heart in 4 deg.C precooled K-H solution. The wall of the aorta is clamped, the perfusion needle is inserted into the aorta, and the ligature is fixed. Start K-H perfusion (take 95% O half an hour in advance for K-H liquid)₂、5％CO₂Mixed gas oxygenation at a certain ratio). The right auricle is breached to facilitate perfusate outflow. Slightly cutting off left auricle, breaking hole of mitral valve, sending deflated self-made saccule into left ventricle, connecting pressure measuring saccule with multi-guide physiological recorder via pressure sensing device, slowly injecting deionized water into saccule, adjusting saccule liquid amount, namely saccule size, keeping diastolic pressure of left ventricle at 5-10mmHg, and recording data. Perfusate perfused into the heart should be maintained at 37 + -0.5 deg.C.

2.2 construction of isolated Heart local I/R model

After heart perfusion, the heart is balanced for 20min and is brought into an isolated heart for experiment according to the following standards (1. the diastolic pressure of the left ventricle is within the range of 5-10 mmHg; 2. the development pressure of the left ventricle is more than or equal to 60 mmHg; 3. the arrhythmia, 4. the heart rate is more than 220 times per minute). The 5-0 needle suture is carefully placed approximately 2mm below the left atrial appendage, with the needle inserted perpendicularly and obliquely upward (towards the pulmonary artery cone). The loose rubber band is used for cushioning and knotting. A significant change in left ventricular pressure was observed after ligation. After 30min of ligation, the rubber band was carefully released to allow the heart to resume perfusion. And re-irrigating for 75 min.

2.3 Experimental groups and procedures

After the isolated heart is subjected to equilibrium perfusion for 20min, the isolated heart for experiment is screened according to the inclusion standard. The blank group (6) is equilibrium perfusion for 125 min; model group (6): after 20min of balanced perfusion, ligating the left anterior descending branch of the coronary artery, stopping perfusion for 30min, loosening the ligature, and recovering perfusion for 75 min; swertioside group (5): after 20min of equilibrium perfusion, ligating the left anterior descending branch of the coronary artery, stopping perfusion for 30min, loosening the ligature, and restoring perfusion for 75min by using K-H liquid containing 10 mu mol/L swertiamarin; digoxin group (6): after 20min of equilibrium perfusion, the left anterior descending branch of the coronary artery is ligated, the perfusion is stopped for 30min, then the ligation thread is loosened, and perfusion is resumed for 75min by K-H liquid containing 1 mu mol/L digoxin.

2.4TTC staining

After the perfusion is finished, taking down the heart, placing the heart in physiological saline for rinsing for several times, placing the heart in a heart cutting groove, cutting the heart into 5 slices with the thickness of about 2mm, placing the heart slices in 2% TTC solution, reacting for 15min in a 37-DEG thermostat in a dark place, taking out the heart slices, placing the heart slices in 4% paraformaldehyde, soaking the heart slices overnight, and taking a picture by using a Nikon single lens reflex camera (D7200). Images were analyzed using Image J software to calculate infarct/heart area values.

2.5 hemodynamic indices

After the isolated heart starts perfusion, a PowerLab multi-lead physiological recorder is used, biological signals such as pressure and the like are recorded through a sensor, then a LabChart data analysis system is used for analyzing ventricular pressure signals, and Left ventricular end-to-septum pressure (LVEDP), Left ventricular systolic pressure (LVESP), Left Ventricular Developed Pressure (LVDP) and Left ventricular maximum rate of rise/fall of the Left ventricular pressure in isovolumetric contraction period (Maximal rise/fall of Left ventricular pressure, +/-dp/dt) are calculated_max) And isohemodynamic parameters.

2.6 statistical analysis

Data analysis was performed using SAS8.4 statistical software, all data being mean. + -. standard deviation: (

) It is shown that the group comparisons were statistically significant using One way-ANOVA with P <0.05 as the difference.

3 results

3.1 Effect of swertisin on infarct size of isolated Heart I/R injured myocardial tissue

The results are shown in Table 5.

TABLE 5 influence of swertiamarin on infarct area of local I/R injured myocardial tissue of isolated heart

# P <0.01 compared to blank group, and # P <0.001 compared to model group

As shown in table 5, the myocardial infarction area of the model group significantly increased to 44.33 ± 0.08% (P <0.001) by local I/R injury compared to the blank group; after 10 mu mol/L swertiamarin re-perfusion, the myocardial infarction area is obviously reduced (P is less than 0.001); after 1 mu mol/L digoxin repeated irrigation, the myocardial infarction area also has a tendency to decrease (P is less than 0.001).

3.2 Effect of swertisin on isolated Heart regional I/R injury LVEDP

The results are shown in Table 6.

TABLE 6 Effect of swertisin on isolated Heart regional I/R injury LVEDP

# P <0.001 compared to blank group and # P <0.001 compared to model group

As shown in Table 6, when the isolated heart perfusion was in equilibrium for 20min, the LVEDP mean values of the blank group (6.97 + -1.04 mmHg), the model group (8.70 + -1.82 mmHg), the swertide group (8.74 + -1.18 mmHg), and the digoxin group (8.29 + -1.30 mmHg) were all 5-10mmHg, and were in accordance with the inclusion criteria.

When ligation is carried out for 30min, compared with a blank group, the LVEDP value of the model group is obviously increased (P is less than 0.001), and after ligation, compared with the model group, the LVEDP group (P is more than 0.05) and the digoxin group (P is more than 0.05) are not obviously different, which indicates that the weight consistency of each group of models is higher.

Within 75min after re-irrigation, the trend of the LVEDP value of the blank group is stable, and the LVEDP value of the model group is in an ascending trend. The swertia herb glycosides and digoxin group also show an ascending trend. When the mixture is irrigated again for 75min, the LVEDP of the model group is obviously increased compared with that of the blank group. Compared with the model group, the swertiamarin group and the digoxin group have a descending trend but have no statistical difference.

3.3 Effect of swertisin on isolated Heart local I/R injury LVESP

The results are shown in Table 7.

TABLE 7 Effect of swertisin on isolated cardiac local I/R injury LVESP

# P <0.05, # P <0.001 compared to the blank group, and # P <0.01, # P <0.001 compared to the model group

As shown in Table 7, the values of LVESP were at the same level in the blank group (81.54. + -. 8.33mmHg), model group (87.87. + -. 7.07mmHg), swertide group (88.94. + -. 6.09mmHg), digoxin group (81.49. + -. 11.63mmHg) and four groups at 20min of equilibration of isolated heart perfusion. Compared with the blank group, the LVESP of the model group (P >0.05), the swertisin group (P >0.05) and the digoxin group (P >0.05) has no significant difference.

After 30min of ligation, the value of model group (P <0.05) was significantly reduced compared to the blank group, and there was no significant difference in LVESP between the drug glycoside group (P >0.05) and digoxin group (P >0.05) compared to the model group.

Within 75min after re-irrigation, the trend of the LVESP value of the blank group is stable, the LVESP of the model group is remarkably reduced, and the LVESP is reduced from 73.03 +/-2.46 mmHg when ligation is carried out for 30min to 58.73 +/-2.71 mmHg when re-irrigation is carried out for 75min, which indicates that the injury is the injury caused by I/R. The contents of the drug glycosides and digoxin are also decreased. When the culture medium is irrigated again for 75min, the LVESP of the model group is obviously reduced compared with that of the blank group (P < 0.001). Compared with the model group, the drug glycoside group (P <0.001) and digoxin group (P <0.01) are obviously increased.

3.4 Effect of swertisin on isolated Heart I/R injury LVDP values

The results are shown in Table 8.

TABLE 8 Effect of swertisin on isolated Heart regional I/R injury LVDP

# P <0.001 compared to blank group, P <0.01 compared to model group, # P <0.001

As shown in Table 8, LVDP values of the blank group (74.57 + -8.14 mmHg), the model group (79.17 + -7.29 mmHg), the swertide group (80.19 + -6.51 mmHg), and the digoxin group (73.20 + -10.98 mmHg) were at the same level at 20min of equilibration of the isolated heart perfusion. Compared with the blank group, the LVEP of the model group (P >0.05), the swertisin group (P >0.05) and the digoxin group (P >0.05) has no significant difference.

At 30min of ligation, the model group (P <0.001) was significantly reduced compared to the blank group. Compared with the model group, the LVDP of the digoxin group (P >0.05) has no obvious difference when the medicinal glycoside group (P >0.05) is used.

Within 75min after re-irrigation, the trend of LVEDP value of the blank group is stable. The model group LVDP decreased significantly. The trend of the swertia herb glycoside group and the digoxin group is stable. When the test solution is irrigated again for 75min, the LVDP value of the model group is obviously reduced (P is less than 0.001) compared with that of the blank group. Compared with the model group, the LVDP values of the swertisin group (P <0.001) and the digoxin group (P <0.01) are obviously increased.

3.5 swertisin left + dp/dt to local I/R injury of isolated heart_maxInfluence of (2)

The results are shown in Table 9.

TABLE 9 swertisin + dp/dt for local I/R injury in isolated heart_maxInfluence of (2)

Compared to the blank group, # P <0.05, # P < 0.001; p <0.05 compared to model group

As shown in Table 9, when the isolated heart perfusion was in equilibrium for 20min, the blank group (2446.04 + -251.68 mmHg/s), the model group (2881.26 + -687.05 mmHg/s), and the swertiamarin group(2486.61 + -302.46 mmHg/s), digoxin group (2643.61 + -298.32 mmHg/s), and fours group + dp/dt_maxThe values are at an approximate level. Model set (P) compared to blank set>0.05), swertisin group (P)>0.05), digoxin group (P)>0.05)+dp/dt_maxThere was no significant difference.

When the ligation is carried out for 30min, the model group has a tendency to decrease compared with the blank group, but has no statistical significance. Compared with the model group, swertia pseudochinensis glucoside group (P)>0.05), digoxin group (P)>0.05)+dp/dt_maxThere was no significant difference.

Within 75min after re-irrigation, blank group + dp/dt_maxThe numerical trend is smooth. The model group is obviously reduced, and when the glucoside group and the digoxin group are in a reduction trend, the model group is more stable than the model group. When the mixture is irrigated again for 75min, compared with a blank group, the model group is + dp/dt_maxDecrease significantly (P)<0.01). Compared with the model group, swertia pseudochinensis glucoside group (P)<0.05) has obvious rise, digoxin group has rising trend, and the statistical significance is not achieved.

3.6 swertisin has effect on local I/R injury of isolated heart left-dp/dt_maxInfluence of (2)

The results are shown in Table 10.

TABLE 10 swertisin on isolated heart local I/R injury-dp/dt_maxInfluence of (2)

Compared to the blank group, # P <0.05, # P < 0.001; p <0.05, P <0.05 compared to model group

As shown in Table 10, when the isolated heart perfusion was balanced for 20min, the blank group (-1867.03 + -180.59 mmHg/s), the model group (-2050.67 + -178.17 mmHg/s), the swertide group (-1923.80 + -190.37 mmHg/s), the digoxin group (-1978.70 + -130.82 mmHg/s), and the four groups + dp/dt_maxThe values are at the same level. Model set (P) compared to blank set>0.05), swertisin group (P)>0.05), digoxinGroup (P)>0.05)+dp/dt_maxThere was no significant difference.

When the ligation is carried out for 30min, the model group has a tendency to decrease compared with the blank group, but has no statistical significance. Compared with the model group, swertia pseudochinensis glucoside group (P)>0.05), digoxin group (P)>0.05)-dp/dt_maxThere was no significant difference.

Within 75min after re-irrigation, blank group-dp/dt_maxThe numerical trend is relatively smooth. When the mixture is irrigated again for 75min, compared with a blank group, the model group is-dp/dt_maxIncrease significantly (P)<0.01). Compared with the model group, swertia pseudochinensis glucoside group (P)<0.01), digoxin group (P)<0.01) had a significantly downward trend.

The experiment adopts a local I/R model of a rat isolated heart, focuses on the influence of the swertisin on hemodynamic indexes such as systolic pressure, development pressure and the like of the left ventricle after I/R injury, and aims to confirm that the action of the swertisin on myocardial protection is focused on the systolic function of the isolated heart organ. The experimental result shows that the swertiamarin can obviously reduce the myocardial infarction area caused by the I/R injury of the isolated heart and reduce the risk of myocardial injury; in addition, swertisin can increase LVDP, LVESP, ± dp/dt of I/R injured heart_maxAnd the myocardial contraction function is obviously enhanced.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other substitutions, modifications, combinations, changes, simplifications, etc., which are made without departing from the spirit and principle of the present invention, should be regarded as equivalent substitutions, and are included in the scope of the present invention.

Claims (8)

1. Use of swertiamarin as sole active ingredient for the manufacture of a medicament for the treatment, prevention, alleviation and/or alleviation of heart failure.

2. The use as claimed in claim 1, wherein the use of swertiamarin as the sole active ingredient in the manufacture of a medicament for ameliorating the symptoms of myocardial hypertrophy.

3. The use as claimed in claim 1, wherein the use of swertiamarin as the sole active ingredient in the manufacture of a medicament for improving the symptoms of myocardial fibrosis.

4. The use according to any one of claims 1 to 3, wherein the target of action of sweroside is ATP1A 1.

5. Use of swertiamarin as sole active ingredient for the manufacture of a medicament for improving and/or restoring the systolic and diastolic function of damaged cardiomyocytes.

6. The application of swertiamarin as the only active ingredient in preparing the medicament for treating, preventing, relieving and/or relieving myocardial ischemia-reperfusion injury.

7. The use according to any one of claims 1-3, 5, and 6, wherein the sweroside is formulated into a dosage form suitable for in vivo administration.

8. The use according to claim 7, wherein the dosage form is a tablet, a hard capsule, a soft capsule, a granule, a pill, an oral liquid or an injection.

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