CN112891361B

CN112891361B - Application of Pubescenoside C in preparation of medicine for preventing and treating myocardial ischemia-reperfusion injury

Info

Publication number: CN112891361B
Application number: CN202110106886.8A
Authority: CN
Inventors: 刘中秋; 程媛媛; 吴鹏; 周华; 廖国超; 张容容; 黄秋菊; 陈思璇
Original assignee: Guangzhou University of Traditional Chinese Medicine
Current assignee: Guangzhou University of Traditional Chinese Medicine
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2023-01-06
Anticipated expiration: 2041-01-26
Also published as: CN112891361A

Abstract

The invention provides application of pubescent holly root triterpenoid saponin compound Pubescenoside C in preparation of a medicine for preventing and treating myocardial ischemia-reperfusion injury. The triterpene saponin compound Pubescenoside C can mediate macrophage polarization from M1 type to M2 type by regulating HSP90/GSK-3 beta interaction, so that the triterpene saponin compound Pubescenoside C has the effects of improving myocardial ischemia reperfusion injury and myocardial tissue lesion and reducing myocardial injury. The ilex pubescens triterpenoid saponin compound Pubescenoside C can also be combined with other pharmaceutical excipients to prepare medicines of different dosage forms, and the prepared medicine can be used for preventing and treating myocardial ischemia reperfusion injury.

Description

Application of Pubescenoside C in preparation of medicine for preventing and treating myocardial ischemia-reperfusion injury

Technical Field

The invention belongs to the field of biological medicine. More particularly relates to an application of ilex pubescens triterpenoid saponin compound Pubescenoside C in preparing a medicament for preventing and treating myocardial ischemia-reperfusion injury.

Background

Myocardial Ischemia Reperfusion Injury (MIRI) refers to a pathological process that blood supply is recovered within a certain time after myocardial ischemia and ischemic myocardium is more seriously injured in the process of blood perfusion, and the ischemic reperfusion injury myocardial can account for 50% of the area of the whole infarcted myocardium. It is currently believed that the causes of myocardial ischemia-reperfusion injury are mainly oxygen radical (OFR) overproduction, intracellular calcium overload, endothelial cell activation, inflammatory response, etc., and the cytological basis thereof is myocardial cell apoptosis. Aiming at myocardial ischemia reperfusion injury, medicaments such as calcium ion antagonist, receptor antagonist, oxygen radical scavenger and the like are commonly used in experiments to prevent and control the occurrence of cardiovascular diseases such as arrhythmia, cardiac arrest, sudden cardiac death and the like induced by the myocardial ischemia reperfusion injury, but the clinical effect is not satisfied. The pubescent holly root is Ilex pubescens hook, which belongs to Aquifoliaceae (Aquifoliaceae) Ilex plant, is a common Chinese medicine in south China, has the medicinal part of root, has the effects of promoting blood circulation, removing obstruction in channels, relieving swelling and pain, clearing heat and detoxicating, and is widely used for treating coronary heart disease, angina, thromboangiitis obliterans and the like clinically.

Chinese patent CN 106892958A discloses a cyclocaryal double bond ursane-type triterpenoid saponin compound, its preparation method and application, the triterpenoid saponin compound is obtained by extracting radix Ilicis Pubescentis with ethanol under reflux, and has antiinflammatory activity; chinese patent CN 109045051A discloses an application of an E-ring cracked ursane type triterpenoid saponin compound, and the compound is also derived from hairy holly root and can be used for preparing medicines for preventing and treating acute myocardial infarction.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide the application of the ilex pubescens triterpenoid saponin compound Pubescenoside C in preparing a medicament for preventing and treating myocardial ischemia-reperfusion injury.

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

reflux extraction of ilex pubescens dry roots with ethanol, and separation and purification by column chromatography to obtain a triterpenoid saponin compound Pubescenoside C, wherein the structure of the compound is shown as the formula (I):

the study of the invention shows that the ilex pubescens triterpenoid saponin compound Pubescenoside C can mediate macrophage polarization from M1 to M2 through regulating the interaction of HSP90/GSK-3 beta, so that the ilex pubescens triterpenoid saponin compound Pubescenoside C has the effects of improving myocardial ischemia reperfusion injury and myocardial tissue lesion and reducing myocardial injury, and can be used for preparing the medicine for preventing and treating myocardial ischemia reperfusion injury. Therefore, the invention firstly requests to protect the application of the ilex pubescens triterpenoid saponin compound Pubescenoside C in preparing the medicine for preventing and treating myocardial ischemia-reperfusion injury.

Preferably, the medicament is in the form of injection, capsule or oral tablet.

Preferably, the compound is present in the medicament in an amount of 10% to 50% by weight.

The invention also provides a medicine for preventing and treating myocardial ischemia reperfusion injury, which comprises a compound shown in the formula (I) and a pharmaceutically acceptable medicinal carrier thereof.

Preferably, the medicament is in the form of injection, capsule or oral tablet.

Preferably, the weight percentage of the compound shown in the formula (I) in the medicine is 10-50%.

Preferably, the injection is prepared by the following method: dissolving the compound shown in the formula (I) in water for injection, adjusting the pH value with sodium carbonate, sterilizing, filtering, filling and sealing, sterilizing by flowing steam at 100 ℃ for 10-20 min, and preparing 1-2 mg/mL injection for injection.

More preferably, the injection is prepared by the following method: dissolving the compound shown in the formula (I) in water for injection, adjusting pH with sodium carbonate, sterilizing, filtering, bottling, sterilizing with flowing steam at 100 deg.C for 15min, and making into 2mg/2mL injection for injection.

Preferably, the injection has a pH value of 7 to 7.5.

Preferably, the capsule is prepared by the following method: the compound shown in the formula (I) is fully mixed with microcrystalline cellulose, sodium carboxymethyl starch and sodium dodecyl sulfate according to the mass ratio of 45-55: 35-45: 2-8, dry granulation is carried out by adopting a rolling method, and then the mixture is uniformly mixed with a proper amount of magnesium stearate and filled into hollow capsules to prepare capsules with the specification of 80-100 mg/capsule for oral administration.

More preferably, the capsule is prepared by the following method: the compound shown in the formula (I) is fully mixed with microcrystalline cellulose, sodium carboxymethyl starch and sodium dodecyl sulfate according to the mass ratio of 50: 40: 5: 4, dry granulation is carried out by adopting a rolling method, and then the mixture is uniformly mixed with a proper amount of magnesium stearate and filled into empty capsules to prepare capsules with the specification of 100 mg/granule for oral administration.

Preferably, the tablet is prepared by the following method: uniformly mixing the compound shown in the formula (I) with starch, cross-linked PVP and sodium carboxymethyl starch according to the mass ratio of 45-55: 35-45: 1-5, using 75% ethanol solution containing 3% -5% of PVP as a binding agent to prepare soft materials, granulating through a sieve of 18-20 meshes, drying at 55-65 ℃ for 1h, adding a proper amount of talcum powder into the granules of 18-20 meshes, uniformly mixing, tabletting and preparing tablets with the specification of 80-100 mg/tablet for oral administration.

More preferably, the tablet is prepared by the following method: mixing the compound shown in formula (I) with starch, cross-linked PVP and sodium carboxymethyl starch at a mass ratio of 50: 40: 2: 3, preparing into soft material with 75% ethanol solution containing 5% PVP as binder, granulating with 18 mesh sieve, drying at 60 deg.C, adding appropriate amount of pulvis Talci into 1h,20 mesh granules, mixing, and tabletting to obtain 100 mg/tablet for oral administration.

The invention has the following beneficial effects:

the invention provides a new application of ilex pubescens triterpenoid saponin compound Pubescenoside C in preparing a medicine for preventing and treating myocardial ischemia-reperfusion injury. The research of the invention shows that the ilex pubescens triterpenoid saponin compound Pubescenoside C can mediate the polarization of macrophages from M1 to M2 by regulating the interaction of HSP90/GSK-3 beta, so that the ilex pubescens triterpenoid saponin compound Pubescenoside C has the effects of improving myocardial ischemia reperfusion injury and myocardial tissue lesion and reducing myocardial injury, and can be used for preparing a medicament for preventing and treating myocardial ischemia reperfusion injury. The invention is not only beneficial to the development and utilization of pubescent holly root, but also has important significance for the research and development of the medicine for treating myocardial ischemia reperfusion injury.

Drawings

FIG. 1 shows Pubescenoside C ¹ H-NMR chart (400 MHz).

FIG. 2 is a drawing of Pubescenoside C ¹³ C-NMR chart (100 MHz).

FIG. 3 is a DEPT diagram of Pubescenoside C.

FIG. 4 is a graph of the effect of Pubescenoside C on myocardial infarction area and myocardial injury markers in myocardial ischemia-reperfusion-injured rats, wherein Panel A is Evans blue-TTC staining of heart sections from Pubescenoside C-treated myocardial ischemia-reperfusion-injured rats; figure B is the effect of Pubescenoside C on myocardial infarction area in myocardial ischemia reperfusion injury rats; panel C is the effect of Pubescenoside C on the electrocardiogram ST segment of myocardial ischemia reperfusion injury rats; FIG. D is the effect of Pubescenoside C on the myocardial injury marker CK-MB in rats with myocardial ischemia-reperfusion injury; panel E is the effect of Pubescenoside C on cardiac tissue morphology in myocardial ischemia reperfusion injury rats.

FIG. 5 is a graph of the effect of Pubescenoside C on macrophage M1 and M2 markers, wherein Panel A is the effect of Pubescenoside C on iNOS and COX-2 protein expression; FIG. B is a statistical chart of iNOS and COX-2 protein expression; panel C is the effect of Pubescenoside C on macrophage M1 marker mRNA levels; panel D is the effect of Pubescenoside C on macrophage M2-type marker mRNA levels; panel E is the effect of Pubescenoside C on ED 1-type macrophages in cardiac tissue from myocardial ischemia reperfusion injury rats; panel F is the effect of Pubescenoside C on ED 2-type macrophages in cardiac tissue from myocardial ischemia reperfusion injury rats.

FIG. 6 shows the effect of Pubescenoside C treated macrophages on cardiomyocytes, wherein FIG. A is a schematic representation of co-culture of macrophages RAW264.7 with H9C2 cardiomyocytes; FIG. B is a graph of the effect of Pubescenoside C treated macrophages on the survival of hypoxic reoxygenation H9C2 cardiomyocytes; panel C is the effect of Pubescenoside C treated macrophages on hypoxia reoxygenation H9C2 cardiomyocyte apoptosis.

FIG. 7 is a graph of the effect of Pubescenoside C on HSP90 and GSK-3 β, wherein Panel A is the effect of Pubescenoside C on HSP90, p-GSK-3 β and GSK-3 β protein levels; panel B is a statistical plot of HSP90, p-GSK-3 β and GSK-3 β protein levels; panel C is the interaction of Pubescenoside C with HSP90; panel D shows the interaction of Pubescenoside C with GSK-3 β; FIG. E is a three-dimensional view of molecular docking of Pubescenoside C with HSP90; FIG. F is a two-dimensional diagram of molecular docking of Pubescenoside C with HSP90; panel G is a two-dimensional map of molecular docking of Pubescenoside C with HSP 90.

FIG. 8 is a graph of the effect of Pubescenoside C on HSP90/GSK-3 β interaction, wherein Panel A is the effect of Pubescenoside C on HSP90 and GSK-3 β cell localization; panel B shows the transfection plasmids GSK3 beta and Myc-HSP90; panel C is a statistical plot of GSK3 β and Myc-HSP90 protein expression; panel D is the effect of Pubescenoside C on GSK3 β and HSP90 interaction; panel E is a statistical plot of the effect of Pubescenoside C on GSK3 β and HSP90 interaction.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1

1. Sequentially extracting radix Ilicis Pubescentis with 12, 10 and 8 times of 70% ethanol under reflux for 2 hr, mixing the ethanol extractive solutions, filtering, recovering ethanol, and concentrating under reduced pressure until no ethanol smell exists to obtain total extract 2.2kg;

2. dissolving the obtained total extract in water, passing through D-101 type macroporous resin column, sequentially eluting with 30% ethanol, 60% ethanol and 95% ethanol; collecting 60% ethanol eluate, recovering solvent under reduced pressure to obtain 566g of thick extract, passing through 200-300 mesh silica gel column, eluting with chloroform-methanol as solvent at a gradient of chloroform-methanol of 100: 1-0: 1; collecting chloroform-methanol eluate at ratio of 7:3, concentrating, passing through ODS column, eluting with methanol: and (7) water: eluting with eluent, collecting eluate, concentrating, separating with Sephadex LH-20 column, collecting methanol eluate, concentrating, and separating with preparative liquid phase (methanol: water 73: 27, collecting chromatographic peak with retention time of 52.37min, and concentrating to obtain white powdery crystal 100mg.

Example 2

The white amorphous powder obtained in example 1, liebermann-Burchard, reacted positively and finally showed a purple-red color, indicating that it is a triterpenoid. Mass spectrum ESI-ME m/z:933[ 2 ], [ M + Na ]] ⁺ Is combined with ¹³ C NMR and ¹ h NMR showed the formula C ₄₇ H ₇₄ O ₁₇ And unsaturation Ω =11.

¹ In the H NMR spectrum (FIG. 1), the low field delta _H 5.62 (1H, br s) shows a monoene hydrogen signal; delta _H 6.32 (1H, d, J = 8.0Hz), 5.36 (1H, d, J = 7.6Hz) and 4.85 (1H, d, J = 7.6Hz) are 2 terminal hydrogen proton signals of glucose and xylose, and the high field delta is _H 0.90 (3h, s), 1.02 (3h, d, j = 6.8hz), 1.10 (6h, s), 1.13 (3h, s), 1.29 (3h, s) and 1.82 (3h, s) are seven methyl signals, suggesting that the compound may be an ursane-type triterpenoid saponin.

¹³ The C NMR (fig. 2) and DEPT spectra (fig. 3) showed that the compound had a total of 47 carbon signals. In the low field region delta _C 175.3 for 1 ester carbonyl carbon signal, delta _C 127.5, 139.1 and delta _C 135.2, 136.1 are 2 alkene carbon signals, and 1 continuous oxygen methine carbon signal delta _C 89.1。δ _C 67.0, 71.9, 78.3, 83.6, 106.1 are group 1 xylose carbon signals, δ _C 62.9, 71.6, 77.6, 78.3, 78.6, 106.4 and delta _C 62.7, 71.3, 74.6, 79.3, 79.5 and 96.1 are glucose carbon signals of 2 groups, which indicates the triterpenoid saponin compounds.

The above data are consistent with the Pubescenoside C reported in the literature (Wang J R, zhou H, jiang Z H, et al, two new ternary saponin from the anti-inflammatory saponin fraction of Ilex Pubescens root. Chem. Biologics.2008, 5:

example 3

The effect of the compound on reducing myocardial ischemia-reperfusion injury is verified by researching the influence of Pubescenoside C on myocardial ischemia-reperfusion injury rat myocardial infarction and myocardial injury markers by adopting an SD rat myocardial ischemia-reperfusion model.

1. Animal administration group

Adult male Sprague-Dawley (SD) rats (WM: 250-280 g) were used for in vivo experiments. Each cage was housed with 4 rats and subjected to 12: 12h light-dark cycles in a SPF house at 22 ± 2 ℃ allowing the animals to freely obtain standard rodent chow and distilled water.

Rats were grouped: group 1: SHAM group, saline (i.p.); group 2: MI/R group, saline (i.p.); group 3: MI/R + Pubescenoside C (10 mg/kg, i.p.); group 4: MI/R + Pubescenoside C (30 mg/kg, i.p.). Male SD rats were subjected to left coronary ligation surgery by dissolving Pubescenoside C in absolute ethanol and diluting in physiological saline, and intraperitoneally injecting Pubescenoside C or physiological saline 10 or 30mg/kg 15min before and 24h and 48h after reperfusion.

2. Myocardial ischemia reperfusion model

Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (100. Mu.l/100 g) and kept breathing with a ventilator. The rat was supine, skin and pectoralis muscles of its left chest were cut, the intercostal space between the third and fourth ribs on the left side of the rat chest was then cut, the rat heart was exposed after opening the pericardium, and the left anterior descending coronary artery (LAD) was ligated using 7-0 sutures, a short plastic tube was placed before the ligation was complete, the anterior wall of the Left Ventricle (LV) was whitened to confirm successful ligation, while the thoracic cavity was closed rapidly and closed with 4-0 sutures. After ligation for 15 minutes (i.e., before reperfusion for 15 minutes), the rats were injected with Pubescenoside C (10 or 30 mg/ml) or normal saline in the abdominal cavity, 30 minutes after ischemia, the thoracic cavity was opened again, the plastic tube was removed and the thoracic muscle and skin were closed, and the operation was terminated.

3. Infarct size analysis

Left ventricular function was assessed 72 hours after MI/R by electrocardiogram and echocardiogram. The rats were euthanized and venous blood was collected, the thoracic cavity was opened, the left anterior descending artery was ligated again, the aorta was perfused with 2% Evans blue dye, the heart was removed and washed with PBS, the heart was frozen in liquid nitrogen for 2 minutes, and its cross-section was cut into six pieces. Heart sections were placed in 2,3, 5-triphenyltetrazolium chloride (TTC) and incubated in a dark room at 37 ℃ for 15 minutes, then fixed with 4% paraformaldehyde at 4 ℃ overnight. After the treatment, blue is normal myocardium, pink is ischemic myocardium, and grey white is necrotic myocardium. The percent of area of the myocardium at risk, AAR, LV, left ventricle area (AAR/LV), the percent of area of the myocardium at risk, IR, infarcted myocardium, infarcted area (IR/AAR), left ventricle area (IR/LV) were calculated by Image J software.

4. Histopathological examination

After the animals were euthanized, hearts were collected and fixed in paraformaldehyde, processed into sections (5 μm) after paraffin embedding, and stained with hematoxylin-eosin (H & E), and then the slides were imaged under an optical microscope.

5. Immunofluorescence staining

Heart tissues were embedded in paraffin and processed into 5 μm thick sections according to SABC (Rabbit, igG)

POD kit for the determination of macrophage polarization. After ethanol gradient rehydration, 3% H was used at room temperature ₂ O ₂ Tissue sections are treated for 5-10 minutes to inactivate endogenous sourcesSex factor, and soaking the paraffin sections in 0.01M sodium citrate buffer (pH 6.0) placed in a microwave oven for antigen retrieval. The sections were incubated dropwise with 5% BSA blocking buffer for 20min at room temperature and overnight at 4 ℃ with the primary antibody ED1 or ED 2. After washing off the primary antibody, the reaction was carried out at 20-37 ℃ for 30 minutes using Bio-sheet anti-rabbit IgG, followed by carrying out the SABC-POD reaction at 20-37 ℃ for 30 minutes. Color development was performed using Diaminobenzidine (DAB) at room temperature, slight elution of hematoxylin, and finally sections were dehydrated, mounted and imaged on a microscope.

6. Western blot analysis

Approximately 250mg of rat left ventricle was placed in a centrifuge tube, and 500. Mu.l of 1 XPPA buffer was added and ground with a grinder. The supernatant was extracted by centrifugation at 12000rpm for 15 minutes. After the samples were prepared, the samples were subjected to separation of different protein molecular weights by SDS-PAGE at concentrations of 10% or 15% at a constant voltage of 100V and placed in a Transfer Buffer (25mM Tris,192mM glycine, 10% isopropanol) to electrotransfer the proteins onto a polyvinylidene fluoride (PVDF) membrane at a constant current of 300 mA. After blocking the membranes with 5% BSA diluted with PBS-Tween20 (0.1%) (PBST) for 1 hour, the membranes were incubated overnight at 4 ℃ in specific antibodies (HSP 90, GSK-3. Beta., p-GSK-3. Beta., GAPDH) (1/1000 in 2% BAS). The membrane was washed three times with PBS, incubated for 1 hour with a secondary goat anti-rabbit or goat anti-mouse IgG antibody (1/3000 in 2% BAS), and the amount of protein expressed was detected with a chemiluminescent solution.

As shown in fig. 4A and 4B, the myocardial infarction area of rats in the MI/R group was significantly larger than that of the Sham group, and the myocardial infarction area of rats in the MI/R + pubescoside C (15 and 30 mg/kg) groups was reduced compared to the MI/R group, and in particular, the infarct area/risk zone area of heart after MI/R treatment of rats was significantly reduced after pubescoside C (30 mg/kg, i.p.) [ n =6, ^*** P<0.001]. Electrocardiographic (FIG. 4C) results showed a significant reduction in ST segment in the MI/R + IP-8 (30 mg/kg) group rats compared to the MI/R group rats [ n =12-18, ^# P<0.05](ii) a On the other hand, as shown in fig. 4D, IP-8 (30 mg/kg) significantly decreased the value of CK-MB [ n =12-18, ^# P<0.05]. At the same time, H&E staining (FIG. 4E) also showed rat heartsHistopathological changes of left ventricular myocardium, neutrophilic infiltration, myocardial cell nucleus condensation and myocardial necrosis of left ventricular myocardial tissues of rats in MI/R group were observed compared with those in SHAM group, while the concentration of left ventricular myocardial cell nucleus in rats in MI/R + Pubescenoside C (30 mg/ml) group was significantly reduced compared with those in MI/R group.

The results show that the Pubescenoside C can improve the myocardial tissue pathological changes of rats with myocardial ischemia-reperfusion injury and reduce myocardial injury.

Example 4

The compound is proved to be capable of inhibiting apoptosis and damage of myocardial cells on a rat myocardial cell H9c2 and macrophage RAW264.7 model.

1. Cell culture and processing

Rat cardiomyocyte H9C2 and macrophage RAW264.7 were cultured in DMEM containing 10% Fetal Bovine Serum (FBS). Lipopolysaccharide (LPS) was dissolved in PBS at a concentration of 0.1mg/mL, and interferon-. Gamma. (IFN-. Gamma.) was dissolved in PBS at a concentration of 1. Mu.g/mL. In the biological assay, RAW264.7 was pretreated with IP-8 (10 or 30. Mu.M) diluted in DMEM containing 3% FBS, and after 1 hour, LPS was added to the medium at 0.1. Mu.g/mL and IFN-. Gamma.was added at 10ng/mL for 23 hours, and the samples were collected.

2. Cell co-culture

RAW264.7 cells were treated with the drug, LPS, IFN-. Gamma.for 24 hours, and then RAW264.7 cells were collected. Inoculating H9c2 cells in Transwell lower chamber overnight, changing to sugar-free and serum-free DMEM medium, placing in an anoxia device for 5H, changing to normal DMEM medium after anoxia, placing treated RAW264.7 cells in upper chamber, and reducing CO content to 5% ₂ The culture was continued for 19 hours in the incubator to form co-culture.

3. Apoptosis detection method

After cell pretreatment, the sample is processed according to the method of the Annexin V-FITC apoptosis detection kit. The medium in the cells was pipetted into a suitable centrifuge tube, the cells were washed in PBS and trypsinized at room temperature, the cells were added with the collected medium to stop the digestion, the cells were gently pipetted and transferred into the centrifuge tube, 1000g was centrifuged for 5 minutes and discardedThe cells were washed, gently resuspended in PBS, and centrifuged at 1000g for 5 minutes before counting. Take 1X 10 ⁶ The cells were centrifuged at 1000g for 5 minutes, the supernatant was discarded, 500. Mu.l Binding Buffer was added to gently resuspend the cells, 5. Mu.l Annexin v-FITC was added to gently mix, 5. Mu.l Propidium Iodide (PI) dye was added to gently mix, incubation was performed at room temperature in the dark for 10 minutes, and then placed in an ice bath with aluminum foil, and finally, the cell samples were subjected to apoptosis test using a flow cytometer to determine the effect of drug treatment on macrophages and cardiomyocytes.

4. Real-time quantitative RT-PCR (qRT-PCR) analysis

After pretreatment of the cells, total RNA was extracted using TRNzol Universal Reagent (DP 424) and PrimeScript was used ^TM RT kit (RR 037A) was used for reverse transcription to obtain cDNA. Using SYBR Premix Ex Taq ^TM II PCR kit (DRR 081A) real-time quantitative analysis was performed on the mRNA levels of IL-6, TNF-alpha, CXCL-10, IL-1 beta, iNOS, IL-10, PPAR-gamma, ARG-1, fizz-1, ym-1 and GAPDH. Finally, the target mRNA level was normalized to the geometric mean of GAPDH mRNA levels for computational analysis.

5. Western blot analysis

RAW26.47 or H9c2 cells were washed once with 1x PBS and protein was extracted with 1x RIPA buffer containing PMSF and a protein phosphatase inhibitor. After the samples were prepared, the samples were separated for different protein molecular weights by 10% or 15% SDS-PAGE at 100V constant voltage and placed in a Transfer Buffer (25mM Tris,192mM glycine, 10% isopropanol) to electrotransfer the proteins onto polyvinylidene fluoride (PVDF) membranes at 300mA constant current. PVDF membranes were blocked with 5% BSA diluted with PBS-Tween20 (0.1%) (PBST) for 1 hour, and the membranes were incubated overnight at 4 ℃ in specific antibodies (HSP 90, GSK-3. Beta., p-GSK-3. Beta., GAPDH) (1/1000 in 2% BAS). The membrane was washed three times with PBS, incubated for 1 hour with secondary goat anti-rabbit or goat anti-mouse IgG antibodies (1/3000 in 2% BAS), and the amount of protein expressed was detected with chemiluminescence solution.

As shown in FIGS. 5A and 5B, pubescenoside C selectively inhibited the expression of iNOS and COX-2, suggesting that Pubescenoside C may affect macrophage polarization. The macrophage M1 and M2 type markers were detected by qRT-PCR, and the results (FIGS. 5C and 5D) showed that Pubescenoside C could significantly inhibit M1 type-related markers such as IL-6, CXCL10, IL-1 β, iNOS, TNF α; meanwhile, IP-8 can improve the expression of M2 type related markers. To further confirm the macrophage modulating effect of Pubescenoside C, we examined macrophage expression in heart tissue. As shown in FIGS. 5E and 5F, pubescenoside C administration significantly reduced left ventricular M1-type macrophages by IHC staining; in contrast, pubescenoside C significantly increased left ventricular M2 macrophages after administration.

As shown in FIG. 6A, we simulated the damage of ischemia reperfusion to myocardial cells by using the OGD/R model, cultured macrophages treated with drugs and LPS + IFN-gamma with H9c2 cells in tranwell, and then tested the cell viability and apoptosis, as shown in FIG. 6B, the cell viability of the OGD/R + LPS + IFN-gamma group was significantly reduced compared to the control group. In contrast, pubescenoside C dose-dependently significantly enhanced cell viability compared to the OGD/R + LPS + IFN-. Gamma.group. On the other hand, we analyzed apoptosis of H9c2 cells by flow cytometry. As shown in FIG. 6C, apoptosis was most severe in the OGD/R + LPS + IFN-y group, whereas apoptosis was reduced in the OGD/R + LPS + IFN-y + IP-8 (10. Mu.M) group compared to the OGD/R + LPS + IFN-y group, especially the number of apoptotic cells was significantly reduced in the OGD/R + LPS + IFN-y + IP-8 (30. Mu.M) group, and these results indicate that Pubescenoside C can inhibit cardiomyocyte apoptosis and damage by regulating macrophage polarization.

As shown in the attached figures 7A-C, pubescenoside C can recover the reduction of GSK3 beta ser9 site phosphorylation level caused by LPS + IFN-gamma, and up-regulate HSP90 expression, which shows that Pubescenoside C can obviously inhibit GSK3 beta activity to regulate macrophage polarization from M1 to M2. In order to explore the mechanism of pubescoside C in regulating GSK3 beta activity, we used SPR experiments to detect the binding of pubescoside C to HSP90 and GSK3 beta, respectively. As shown in FIG. 7D, SPR experiments showed that Pubescenoside C bound HSP90 but not GSK-3 β, as shown by molecular docking (FIGS. 7E and 7F). Pubescenoside C can form hydrogen bonds with amino acid residues HIS210, SER50, PHE213, LYS242 and ASP170 on HSP90 proteins, while forming pi bonds with TRP 168. To demonstrate whether Pubescenoside C affects HSP90 interaction with GSK-3 β, we verified by infocal and Co-IP, first we Co-localized HSP90 and GSK-3 β using immunofluorescence. As shown in figure 8A, blue light represents cell nucleus, green light represents HSP90, red light represents GSK-3 beta, and after IP-8 (30 mu M) is administered, the fluorescence intensity of the cell is obviously higher than that of a normal cell, and simultaneously, the HSP90 (Myc) and GSK-3 beta plasmids are transfected into the cell, as shown in figures 8B and 8C, pubescenoside C can obviously increase the expression of HSP90 and GSK-3 beta compared with the cell without overexpression, which indicates the successful overexpression. Further using the CO-IP approach, as shown in FIGS. 8D and 8E, the results indicate that Pubescenoside C significantly promotes the binding of HSP90 and GSK-3 β.

In conclusion, pubescenoside C can mediate macrophage polarization from M1 to M2 by regulating HSP90/GSK-3 beta interaction, thereby reducing myocardial ischemia-reperfusion injury.

EXAMPLE 5 preparation of pharmaceutical injection

1000mg of the compound obtained by the method of example 1 is taken, 1000ml of water for injection is added, the pH value is adjusted to 7-7.5 by sodium carbonate, the mixture is stirred to be dissolved, sterilized, filtered, filled and sealed, and the mixture is sterilized by flowing steam at 100 ℃ for 15 minutes to prepare 2mg/2ml injection for injection.

EXAMPLE 6 preparation of pharmaceutical capsules

5000mg of the compound obtained by the method in example 1 is fully mixed with auxiliary materials such as 4000mg of microcrystalline cellulose, 500mg of sodium carboxymethyl starch, 400mg of sodium dodecyl sulfate and the like, dry granulation is carried out by adopting a rolling method, and then the mixture is uniformly mixed with a proper amount of magnesium stearate and filled into 3# hollow capsules to prepare capsules with the specification of 100 mg/capsule for oral administration.

EXAMPLE 7 preparation of pharmaceutical tablets

5000mg of the compound obtained by the method described in example 1 was uniformly mixed with 4000mg of starch, 200mg of crosslinked PVP and 300mg of sodium carboxymethyl starch, and using 75% ethanol solution of 5% PVP as a binder, soft mass was prepared, granulated with a 18-mesh sieve, dried at 60 ℃ and then granulated for 1h, after 20-mesh granulation, an appropriate amount of talc was added, and mixed, and tableted to prepare tablets of 100 mg/tablet for oral administration.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims

1. The application of the ilex pubescens triterpenoid saponin compound Pubescenoside C in preparing the medicine for preventing and treating myocardial ischemia-reperfusion injury is characterized in that the chemical structure of the compound is shown as the formula (I):

2. the use of claim 1, wherein the medicament is in the form of an injection, a capsule or an oral tablet.

3. The use of claim 1, wherein the compound is present in the medicament in an amount of from 10% to 50% by weight.

4. The use of claim 2, wherein the injection is prepared by the following method: dissolving the compound shown in the formula (I) in water for injection, adjusting the pH value with sodium carbonate, sterilizing, filtering, filling and sealing, sterilizing by flowing steam at 100 ℃ for 10-20 min, and preparing each injection with the concentration of 1-2 mg/mL for injection.

5. The use according to claim 4, wherein the injection has a pH of 7 to 7.5.

6. The use according to claim 2, wherein the capsule is prepared by a method comprising: the compound shown in the formula (I) is fully mixed with microcrystalline cellulose, sodium carboxymethyl starch and sodium dodecyl sulfate according to the mass ratio of 45-55: 35-45: 2-8, dry granulation is carried out by adopting a rolling method, and then the mixture is uniformly mixed with a proper amount of magnesium stearate and filled into hollow capsules to prepare capsules with the specification of 80-100 mg/capsule for oral administration.

7. The use according to claim 2, wherein the tablet is prepared by a process comprising: uniformly mixing the compound shown in the formula (I) with starch, cross-linked PVP and sodium carboxymethyl starch according to the mass ratio of 45-55: 35-45: 1-5, using 75% ethanol solution containing 3% -5% PVP as a binding agent to prepare a soft material, granulating through a sieve of 18-20 meshes, drying at 55-65 ℃ for 1h, then adding a proper amount of talcum powder after granulating through the sieve of 18-20 meshes, uniformly mixing, tabletting, and preparing tablets with the specification of 80-100 mg/tablet for oral administration.