CN115778965B - New application of ligustrum lucidum glycoside G13 in preparing anti-myocardial fibrosis medicine - Google Patents
New application of ligustrum lucidum glycoside G13 in preparing anti-myocardial fibrosis medicine Download PDFInfo
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- CN115778965B CN115778965B CN202211301805.0A CN202211301805A CN115778965B CN 115778965 B CN115778965 B CN 115778965B CN 202211301805 A CN202211301805 A CN 202211301805A CN 115778965 B CN115778965 B CN 115778965B
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a novel application of ligustrin G13 in preparing medicines for resisting myocardial fibrosis, which belongs to the technical field of medicines, namely the novel application of ligustrin G13 in preparing medicines for treating, preventing, relieving or alleviating myocardial fibrosis related diseases, wherein the myocardial fibrosis related diseases refer to diseases caused by myocardial fibrosis or diseases which can develop into myocardial fibrosis or diseases with clinical symptoms of myocardial fibrosis, including but not limited to coronary heart disease, myocardial ischemia, myocardial infarction and heart failure. The ligustrin G13 can reduce myocardial apoptosis by relieving oxidative stress injury, inhibit TGF-beta 1/Smads signal pathway and relieve ISO-induced myocardial fibrosis, play a role in resisting myocardial fibrosis, provide a new treatment strategy for preparing anti-myocardial fibrosis medicine research, and have good prospect for developing and applying the anti-myocardial fibrosis medicine.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a novel application of ligustroside G13 in preparation of an anti-myocardial fibrosis medicine.
Background
Cardiovascular disease is a well-recognized disease with a high incidence and mortality rate worldwide. Myocardial fibrosis is a core pathological change of various cardiovascular diseases (coronary heart disease, myocardial ischemia and myocardial infarction) which progress to a certain stage, and is also an important cause of heart failure, and is an important point and difficulty of the current clinical treatment. The medicines for treating myocardial fibers are few in clinic, and the existing western medicines have the problem of insufficient safety in clinical application and are accompanied with certain adverse reactions and side effects. Therefore, the anti-myocardial fibrosis medicine which is purely natural in source, good in treatment effect and low in toxic and side effects has important clinical requirements.
The molecular formula of the ligustrum lucidum glycoside G13 is C48H64O27, and the ligustrum lucidum glycoside G is a main active ingredient in Chinese medicinal ligustrum lucidum fruits and osmanthus fragrans fruits. Previous studies have found that ligustrin G13 can increase blood glucose consumption, affect cellular glucose metabolism, and improve insulin resistance in HepG2 insulin resistance cell model. Meanwhile, in osteoblast UMR-106, ligustrum japonicum G13 exerts an anti-osteoporosis effect by enhancing alkaline phosphatase (ALP) activity in serum. In addition, ligustrin G13 was found to have anti-platelet aggregation, antioxidant and anti-inflammatory activity. However, no report is available on the prevention and treatment effect of ligustrin G13 on myocardial fibrosis at present.
Isoproterenol (ISO) is a beta receptor agonist used in bronchial asthma and atrioventricular block, and can induce myocardial fibrosis. ISO-induced myocardial fibrosis is a classical animal model of myocardial fibrosis research that can mimic the pathological process of clinical myocardial fibrosis for evaluation of the effects of drugs on oxidative stress, inflammation, and apoptosis of the heart. Experimental data in the technical scheme disclosed by the invention is provided by constructing an ISO-induced mouse myocardial fibrosis model.
Disclosure of Invention
The invention aims at providing a new application of ligustrin G13 in preparing medicines for resisting myocardial fibrosis. Namely, the novel application of the ligustrum lucidum glycoside G13 in preparing medicaments for treating, preventing, relieving or alleviating myocardial fibrosis related diseases reduces myocardial cell apoptosis by alleviating oxidative stress injury, inhibits TGF-beta 1/Smads signal channels to alleviate ISO-induced myocardial fibrosis, plays a role in resisting myocardial fibrosis, and provides a novel treatment strategy for preparing anti-myocardial fibrosis medicament researches.
In order to achieve the above purpose, the technical scheme adopted is as follows:
the novel application of the ligustrum lucidum glycoside G13 in preparing medicaments for resisting myocardial fibrosis, namely in preparing medicaments for treating, preventing, relieving or alleviating myocardial fibrosis related diseases.
Wherein the myocardial fibrosis related diseases refer to diseases caused by myocardial fibrosis or diseases which can develop into myocardial fibrosis or diseases with clinical symptoms of myocardial fibrosis, including but not limited to coronary heart disease, myocardial ischemia, myocardial infarction and heart failure.
Wherein, the ligustrum lucidum glycoside G13 plays a role in resisting myocardial fibrosis by reducing oxidative stress injury, reducing myocardial apoptosis and inhibiting TGF-beta 1/Smads signaling pathway to reduce ISO-induced myocardial fibrosis.
Wherein the ligustrin G13 reduces myocardial apoptosis by relieving oxidative stress injury, and inhibits TGF-beta 1/Smads signaling pathway to relieve ISO-induced myocardial fibrosis, and the effective concentration for resisting myocardial fibrosis is 20-80mg/kg/d.
Wherein the ligustrum lucidum glycoside G13 has a structural formula of (1):
wherein the ligustrin G13 is extracted from glossy privet fruits and osmanthus fruits, but is not limited to glossy privet fruits and osmanthus fruits, wherein the extraction process from the osmanthus fruits comprises the following steps: degreasing dried osmanthus fragrans fruits with petroleum ether, performing ultrasonic extraction for 1h by adopting 55% ethanol according to a feed-liquid ratio of 1:10, recovering the extracting solution by using a rotary evaporator at 40 ℃, freeze-drying to obtain an osmanthus fragrans fruit extract, dissolving the extract with water, and preparing a mother solution with a concentration of 5 mg/mL; enriching the ligustrum lucidum glycoside G13 by using macroporous resin with the model of XAD-16, loading the sample at the flow rate of 1mL/min, and stopping loading the sample when detecting the concentration of the ligustrum lucidum glycoside G13 in the resin effluent to 5% of the concentration in the mother liquor by using HPLC; after the adsorption reaches equilibrium, eluting impurities with 30% ethanol, detecting by HPLC until the occurrence of ligustroside G13 in the eluent begins, eluting with 40% ethanol in a large amount, collecting 40% ethanol eluent, recovering by rotary evaporator at 40deg.C, and lyophilizing to obtain ligustroside G13 concentrate; dissolving the ligustrum glycoside G13 concentrate with methanol, filtering with 0.22 μm microporous membrane, purifying and preparing ligustrum glycoside G13 with 0.1% phosphoric acid water (A) -acetonitrile (B) as mobile phase, and performing gradient elution under conditions of 0-10min and 10-30% B with YMC-Pack ODS-A C chromatographic column (20×250mm,5 μm) at flow rate of 8mL/min and detection wavelength of 210 nm; 10-25min,30%; collecting the ligustroside G13 flowing out from the chromatographic column according to the retention time of the ligustroside G13 by adopting a manual collection mode, recovering the ligustroside G13 by using a rotary evaporator at 40 ℃, and freeze-drying to obtain the ligustroside G13.
Wherein, when the ligustrum lucidum glycoside G13 is used as a medicament for treating, preventing, relieving or alleviating myocardial fibrosis, the ligustrum lucidum glycoside G13 can be directly used or used in the form of a pharmaceutical composition, and the pharmaceutical composition contains 0.1-99% of the ligustrum lucidum glycoside G13 and the balance of a medicinal carrier or excipient.
A medicament for treating, preventing, alleviating or alleviating myocardial fibrosis, comprising ligustroside G13 and a pharmaceutically acceptable carrier.
Wherein the pharmaceutically acceptable carrier is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants, including fillers, diluents, emulsifiers, disintegrants, binders and drug-carrying carriers without toxic or side effects.
Wherein the dosage form of the medicine is as follows: tablets, injections, suspensions, emulsions, solutions, syrups, tablets, capsules, granules, electuaries, sprays and aerosols.
Compared with the prior art, the ligustrum lucidum glycoside G13 has never been reported to have anti-myocardial fibrosis activity and is used for treating myocardial fibrosis related diseases. The invention discloses application of ligustrin G13 in preparing medicines for treating, preventing, relieving or alleviating myocardial fibrosis, and experimental data show that the exact improvement and treatment effect of ligustrin G13 on myocardial fibrosis can obviously improve ISO-induced myocardial fibrosis. Animal experiments show that the natural iridoid glycoside component ligustrin G13 can reduce myocardial apoptosis by relieving oxidative stress injury, inhibit TGF-beta 1/Smads signal pathway to relieve ISO-induced myocardial fibrosis, and play an anti-myocardial fibrosis role.
Drawings
FIG. 1 shows the chemical structure of ligustrum lucidum glycoside G13;
FIG. 2 is the effect of ligustrin G13 on cardiac morphology, cardiac index and cardiac function in mice;
FIG. 3 is the effect of ligustrum lucidum glycoside G13 on oxidative stress markers;
FIG. 4 is a graph showing that ligustrin G13 induces the expression of type I collagen, type III collagen and alpha-SMA in myocardial tissue of mice;
FIG. 5 is the effect of ligustrin G13 on heart histopathology and hepatotoxicity;
FIG. 6 is the effect of ligustrin G13 on apoptosis in mice with myocardial fibrosis;
FIG. 7 is the effect of ligustrum lucidum glycoside G13 on TGF-beta 1/Smads signaling pathway.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
Example 1
The novel application of the ligustrum lucidum glycoside G13 in preparing medicaments for resisting myocardial fibrosis, namely in preparing medicaments for treating, preventing, relieving or alleviating myocardial fibrosis related diseases.
Wherein the myocardial fibrosis related diseases refer to diseases caused by myocardial fibrosis or diseases which can develop into myocardial fibrosis or diseases with clinical symptoms of myocardial fibrosis, including but not limited to coronary heart disease, myocardial ischemia, myocardial infarction and heart failure.
Wherein, the ligustrum lucidum glycoside G13 plays a role in resisting myocardial fibrosis by reducing oxidative stress injury, reducing myocardial apoptosis and inhibiting TGF-beta 1/Smads signaling pathway to reduce ISO-induced myocardial fibrosis.
Wherein the ligustrin G13 reduces myocardial apoptosis by alleviating oxidative stress injury, and inhibits TGF-beta 1/Smads signaling pathway to alleviate ISO-induced myocardial fibrosis, and the effective concentration for resisting myocardial fibrosis is 20mg/kg/d.
Wherein the ligustrum lucidum glycoside G13 has a structural formula of (1):
wherein the ligustrin G13 is extracted from glossy privet fruits and osmanthus fruits, but is not limited to glossy privet fruits and osmanthus fruits, wherein the extraction process from the osmanthus fruits comprises the following steps: degreasing dried osmanthus fragrans fruits with petroleum ether, performing ultrasonic extraction for 1h by adopting 55% ethanol according to a feed-liquid ratio of 1:10, recovering the extracting solution by using a rotary evaporator at 40 ℃, freeze-drying to obtain an osmanthus fragrans fruit extract, dissolving the extract with water, and preparing a mother solution with a concentration of 5 mg/mL; enriching the ligustrum lucidum glycoside G13 by using macroporous resin with the model of XAD-16, loading the sample at the flow rate of 1mL/min, and stopping loading the sample when detecting the concentration of the ligustrum lucidum glycoside G13 in the resin effluent to 5% of the concentration in the mother liquor by using HPLC; after the adsorption reaches equilibrium, eluting impurities with 30% ethanol, detecting by HPLC until the occurrence of ligustroside G13 in the eluent begins, eluting with 40% ethanol in a large amount, collecting 40% ethanol eluent, recovering by rotary evaporator at 40deg.C, and lyophilizing to obtain ligustroside G13 concentrate; dissolving the ligustrum glycoside G13 concentrate with methanol, filtering with 0.22 μm microporous membrane, purifying and preparing ligustrum glycoside G13 with 0.1% phosphoric acid water (A) -acetonitrile (B) as mobile phase, and performing gradient elution under conditions of 0-10min and 10-30% B with YMC-Pack ODS-A C chromatographic column (20×250mm,5 μm) at flow rate of 8mL/min and detection wavelength of 210 nm; 10-25min,30%; collecting the ligustroside G13 flowing out from the chromatographic column according to the retention time of the ligustroside G13 by adopting a manual collection mode, recovering the ligustroside G13 by using a rotary evaporator at 40 ℃, and freeze-drying to obtain the ligustroside G13.
Wherein, when the ligustrum lucidum glycoside G13 is used as a medicament for treating, preventing, relieving or alleviating myocardial fibrosis, the ligustrum lucidum glycoside G13 can be directly used or used in the form of a pharmaceutical composition, and the pharmaceutical composition contains 0.1-99% of the ligustrum lucidum glycoside G13 and the balance of a medicinal carrier or excipient.
A medicament for treating, preventing, alleviating or alleviating myocardial fibrosis, comprising ligustroside G13 and a pharmaceutically acceptable carrier.
Wherein the pharmaceutically acceptable carrier is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants, including fillers, diluents, emulsifiers, disintegrants, binders and drug-carrying carriers without toxic or side effects.
Wherein the dosage form of the medicine is as follows: a tablet.
Example 2
The novel application of the ligustrum lucidum glycoside G13 in preparing medicaments for resisting myocardial fibrosis, namely in preparing medicaments for treating, preventing, relieving or alleviating myocardial fibrosis related diseases.
Wherein the myocardial fibrosis related diseases refer to diseases caused by myocardial fibrosis or diseases which can develop into myocardial fibrosis or diseases with clinical symptoms of myocardial fibrosis, including but not limited to coronary heart disease, myocardial ischemia, myocardial infarction and heart failure.
Wherein, the ligustrum lucidum glycoside G13 plays a role in resisting myocardial fibrosis by reducing oxidative stress injury, reducing myocardial apoptosis and inhibiting TGF-beta 1/Smads signaling pathway to reduce ISO-induced myocardial fibrosis.
Wherein the ligustrin G13 reduces myocardial apoptosis by alleviating oxidative stress injury, and inhibits TGF-beta 1/Smads signaling pathway to alleviate ISO-induced myocardial fibrosis, and the effective concentration for resisting myocardial fibrosis is 80mg/kg/d.
Wherein the ligustrum lucidum glycoside G13 has a structural formula of (1):
wherein the ligustrin G13 is extracted from glossy privet fruits and osmanthus fruits, but is not limited to glossy privet fruits and osmanthus fruits, wherein the extraction process from the osmanthus fruits comprises the following steps: degreasing dried osmanthus fragrans fruits with petroleum ether, performing ultrasonic extraction for 1h by adopting 55% ethanol according to a feed-liquid ratio of 1:10, recovering the extracting solution by using a rotary evaporator at 40 ℃, freeze-drying to obtain an osmanthus fragrans fruit extract, dissolving the extract with water, and preparing a mother solution with a concentration of 5 mg/mL; enriching the ligustrum lucidum glycoside G13 by using macroporous resin with the model of XAD-16, loading the sample at the flow rate of 1mL/min, and stopping loading the sample when detecting the concentration of the ligustrum lucidum glycoside G13 in the resin effluent to 5% of the concentration in the mother liquor by using HPLC; after the adsorption reaches equilibrium, eluting impurities with 30% ethanol, detecting by HPLC until the occurrence of ligustroside G13 in the eluent begins, eluting with 40% ethanol in a large amount, collecting 40% ethanol eluent, recovering by rotary evaporator at 40deg.C, and lyophilizing to obtain ligustroside G13 concentrate; dissolving the ligustrum glycoside G13 concentrate with methanol, filtering with 0.22 μm microporous membrane, purifying and preparing ligustrum glycoside G13 with 0.1% phosphoric acid water (A) -acetonitrile (B) as mobile phase, and performing gradient elution under conditions of 0-10min and 10-30% B with YMC-Pack ODS-A C chromatographic column (20×250mm,5 μm) at flow rate of 8mL/min and detection wavelength of 210 nm; 10-25min,30%; collecting the ligustroside G13 flowing out from the chromatographic column according to the retention time of the ligustroside G13 by adopting a manual collection mode, recovering the ligustroside G13 by using a rotary evaporator at 40 ℃, and freeze-drying to obtain the ligustroside G13.
Wherein, when the ligustrum lucidum glycoside G13 is used as a medicament for treating, preventing, relieving or alleviating myocardial fibrosis, the ligustrum lucidum glycoside G13 can be directly used or used in the form of a pharmaceutical composition, and the pharmaceutical composition contains 0.1-99% of the ligustrum lucidum glycoside G13 and the balance of a medicinal carrier or excipient.
A medicament for treating, preventing, alleviating or alleviating myocardial fibrosis, comprising ligustroside G13 and a pharmaceutically acceptable carrier.
Wherein the pharmaceutically acceptable carrier is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants, including fillers, diluents, emulsifiers, disintegrants, binders and drug-carrying carriers without toxic or side effects.
Wherein the dosage form of the medicine is as follows: tablets, injections, suspensions, emulsions, solutions, syrups, capsules, granules, sprays and aerosols.
Example 3
The novel application of the ligustrum lucidum glycoside G13 in preparing medicaments for resisting myocardial fibrosis, namely in preparing medicaments for treating, preventing, relieving or alleviating myocardial fibrosis related diseases.
Wherein the myocardial fibrosis related diseases refer to diseases caused by myocardial fibrosis or diseases which can develop into myocardial fibrosis or diseases with clinical symptoms of myocardial fibrosis, including but not limited to coronary heart disease, myocardial ischemia, myocardial infarction and heart failure.
Wherein, the ligustrum lucidum glycoside G13 plays a role in resisting myocardial fibrosis by reducing oxidative stress injury, reducing myocardial apoptosis and inhibiting TGF-beta 1/Smads signaling pathway to reduce ISO-induced myocardial fibrosis.
Wherein the effective concentration of the ligustrum lucidum glycoside G13 for exerting the anti-myocardial fibrosis effect is 40mg/kg/d by reducing oxidative stress injury, reducing myocardial apoptosis and inhibiting TGF-beta 1/Smads signal pathway to reduce ISO-induced myocardial fibrosis.
Wherein the ligustrum lucidum glycoside G13 has a structural formula of (1):
wherein the ligustrin G13 is extracted from glossy privet fruits and osmanthus fruits, but is not limited to glossy privet fruits and osmanthus fruits, wherein the extraction process from the osmanthus fruits comprises the following steps: degreasing dried osmanthus fragrans fruits with petroleum ether, performing ultrasonic extraction for 1h by adopting 55% ethanol according to a feed-liquid ratio of 1:10, recovering the extracting solution by using a rotary evaporator at 40 ℃, freeze-drying to obtain an osmanthus fragrans fruit extract, dissolving the extract with water, and preparing a mother solution with a concentration of 5 mg/mL; enriching the ligustrum lucidum glycoside G13 by using macroporous resin with the model of XAD-16, loading the sample at the flow rate of 1mL/min, and stopping loading the sample when detecting the concentration of the ligustrum lucidum glycoside G13 in the resin effluent to 5% of the concentration in the mother liquor by using HPLC; after the adsorption reaches equilibrium, eluting impurities with 30% ethanol, detecting by HPLC until the occurrence of ligustroside G13 in the eluent begins, eluting with 40% ethanol in a large amount, collecting 40% ethanol eluent, recovering by rotary evaporator at 40deg.C, and lyophilizing to obtain ligustroside G13 concentrate; dissolving the ligustrum glycoside G13 concentrate with methanol, filtering with 0.22 μm microporous membrane, purifying and preparing ligustrum glycoside G13 with 0.1% phosphoric acid water (A) -acetonitrile (B) as mobile phase, and performing gradient elution under conditions of 0-10min and 10-30% B with YMC-Pack ODS-A C chromatographic column (20×250mm,5 μm) at flow rate of 8mL/min and detection wavelength of 210 nm; 10-25min,30%; collecting the ligustroside G13 flowing out from the chromatographic column according to the retention time of the ligustroside G13 by adopting a manual collection mode, recovering the ligustroside G13 by using a rotary evaporator at 40 ℃, and freeze-drying to obtain the ligustroside G13.
Wherein, when the ligustrum lucidum glycoside G13 is used as a medicament for treating, preventing, relieving or alleviating myocardial fibrosis, the ligustrum lucidum glycoside G13 can be directly used or used in the form of a pharmaceutical composition, and the pharmaceutical composition contains 0.1-99% of the ligustrum lucidum glycoside G13 and the balance of a medicinal carrier or excipient.
A medicament for treating, preventing, alleviating or alleviating myocardial fibrosis, comprising ligustroside G13 and a pharmaceutically acceptable carrier.
Wherein the pharmaceutically acceptable carrier is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants, including fillers, diluents, emulsifiers, disintegrants, binders and drug-carrying carriers without toxic or side effects.
Wherein the dosage form of the medicine is as follows: tablets, injections, suspensions, emulsions, solutions, syrups, tablets, capsules, granules, electuaries, sprays and aerosols.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any simple modification, material transformation, equivalent changes and modification made to the above embodiment according to the technical matter of the present invention still fall within the scope of the technical scheme of the present invention.
In order to further verify the novel application of the ligustrin G13 (hereinafter referred to as G13) in preparing medicaments for treating, preventing, relieving or alleviating myocardial fibrosis related diseases, namely, reducing myocardial apoptosis by alleviating oxidative stress injury, inhibiting TGF-beta 1/Smads signal channels to alleviate ISO-induced myocardial fibrosis, playing an anti-myocardial fibrosis role, animal experiments are designed as follows.
1. In vivo experiments
Mice were randomly divided into 6 groups: the control group, the model group, the G13 low dose group (20 mg/kg), the G13 medium dose group (40 mg/kg), the G13 high dose group (80 mg/kg) and the G13 drug group (80 mg/kg), the control group is given by intragastric administration and subcutaneous injection of 0.9% physiological saline (10 mg/kg/d), the model group is given by intragastric administration and subcutaneous injection of 0.9% physiological saline ISO (10 mg/kg/d), the G13 low dose group, the G13 medium dose group and the G13 high dose group are respectively given by intragastric administration and the G13 (20, 40, 80 mg/kg/d) are given by subcutaneous injection and the G13 drug group is given by intragastric administration only (80 mg/kg/d). Each set of experiments lasted 21 days. After 21 days, each group of mice was weighed, electrocardiographic examination was performed, blood was collected from the eyes, and hearts and livers were collected after sacrifice. The heart and liver were freed from blood traces in physiological saline, dried with filter paper, weighed, and heart index calculated. The results of the CK (creatine kinase) and LDH (lactate dehydrogenase) assays on mouse serum using the kit procedure are shown in figure 2 for the effects of G13 on mouse heart morphology, heart index and heart function. (wherein A is the morphology of the heart of each group of mice; B is the heart index of each group of mice; C and D are changes in serum CK and LDH levels of mice; E is the effect of G13 on ISO-induced electrocardiography of mice); the effect of G13 on oxidative stress markers is shown in FIG. 3. ( Wherein a-D is the change in oxidative stress markers in mouse heart tissue (n=6). E is immunohistochemical staining to detect expression of Nrf2 (scale bar: 50 μm). F is Western blotting to detect Nrf2, HO-1 and NQO-1 protein expression, and GAPDH is used as a control. G-I is the gray value analysis of Nrf2, HO-1, NQO-1 (n=3). )
As can be seen from fig. 2, the heart color of the G13 mice was close to normal, and the volume was also significantly reduced. The CWI of the ISO group was significantly higher than the control group, while the CWI of the G13 group was significantly reduced, indicating that G13 could alleviate ISO-induced myocardial hypertrophy. Meanwhile, the myocardial enzyme level of the ISO group is obviously increased, and the myocardial enzyme activity is obviously reduced after G13 is added, so that the G13 has a protective effect on ISO-induced myocardial injury. The contrast group has normal electrocardiogram, while the ISO group has low T wave, reduced QRS wave amplitude and increased S-T segment, which indicates the occurrence of myocardial ischemia symptoms. After administration of G13, T-wave was elevated and S-T segment was significantly restored, indicating that G13 could ameliorate ISO-induced myocardial ischemia.
As can be seen from fig. 3, the expression of key proteins associated with oxidative stress was detected using immunohistochemical staining and western blotting, and Nrf2 was expressed significantly lower in the ISO group than in the normal group, and G13 recovered its expression in a dose-dependent manner. On the other hand, the expression of Nrf2, HO-1 and NQO-1 is down-regulated under the stimulation of ISO, and after G13 treatment, the expression levels of Nrf2, HO-1 and NQO-1 are up-regulated, so that the G13 can be proved to be capable of relieving oxidative stress injury caused by myocardial fibrosis.
2. Immunohistochemical staining and western blotting
The extent of fibrosis was assessed by measuring the expression of type I collagen and alpha-SMA in myocardial tissue using immunohistochemical staining and western blotting. The specific operation is as follows: fixed heart samples were embedded in paraffin and cut into 4 μm sections. Heart sections were deparaffinized and hydrated and then immersed in sodium citrate-sodium citrate buffer for antigen retrieval, then sections were incubated in 3% hydrogen peroxide to quench endogenous peroxidase activity and blocked with goat serum to prevent non-specific antibody binding. Subsequently, the tissue samples were incubated with primary antibody overnight at 4 ℃, 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, incubated at 37 ℃ for 15-20 min, then developed with Diaminobenzidine (DAB) and counterstained with hematoxylin, and finally the target protein was quantitatively analyzed. The results of G13 induction of the expression of type I collagen, type III collagen and α -SMA in mouse heart tissue are shown in FIG. 4 (wherein A is immunohistochemical staining analysis (scale: 50 μm) of collagen I and α -SMA in each group of mouse heart tissue; B-C is quantitative analysis of positive expression of collagen I and α -SMA; D is gray value analysis of Western blotting to detect protein expression of collagen I, collagen III and α -SMA; E is type I collagen, type III collagen and α -SMA (n=3); the effect of G13 on heart histopathology and hepatotoxicity is shown in FIG. 5 (A is HE and sirius red staining for detection of myocardial tissue (scale: 50 μm). B is a quantitative analysis of collagen fibers in heart tissue. C is H & E and Masson staining for G13 hepatotoxicity).
As can be seen from fig. 4, the expression of the collagen I and α -SMA in the ISO group was strongly positive, and the expression of both was reduced after the G13 treatment, indicating that the G13 treatment inhibited the expression of the collagen I and α -SMA, indicating that the myocardial fibrosis model was successfully established.
As can be seen from fig. 5, after H & E staining, the ISO group of mice showed significant fiber bundles, myocardial structural disorders and inflammatory cell infiltration. After G13 treatment, myocardial structural disorders and fibrosis levels improved in a dose-dependent manner. Sirius red staining results showed that model group collagen fibers were significantly higher than control group, while G13 dose-dependent improved collagen fiber deposition. In addition, H & E and Masson staining of mouse liver tissue showed neat alignment of hepatocytes, no inflammatory cell infiltration, no collagen fiber deposition, demonstrating no toxic effects of G13.
3. Assessment of extent of myocardial apoptosis following myocardial fibrosis using TUNEL staining
TUNEL staining was used to assess the extent of cardiomyocyte apoptosis following myocardial fibrosis. The specific operation is as follows: after the mice were sacrificed, heart tissue was removed, 4% paraformaldehyde was fixed for 24 hours, and paraffin embedded. Dewaxing, rehydration, adding proteinase K working solution, incubating at 37℃for 22 min, washing 3 times with PBS (pH 7.4) in a Rocker apparatus for 5min each. After the sections were dried, a permeabilization working solution was added for 20 minutes at 37℃and then washed with PBS. After the sections were slightly dried, buffer was added to the tissue in the sheet and the buffer incubated at room temperature for 10 minutes. Appropriate amounts of TDT enzyme, dUTP and buffer were mixed in fixed proportions, covered tissue, incubated in a 37 ℃ wet box for 2 hours, and washed with PBS. DAPI dye solution was added and incubated for 10 minutes at room temperature, washed with PBS, the liquid removed, and then coverslips with fade-resistant mounting agent. Microscopy and image collection were performed by fluorescence microscopy. The effect of G13 on apoptosis in mice with myocardial fibrosis is shown in FIG. 6 (wherein A is TUNEL fluorescent staining (scale: 20 μm) of heart tissue sections, apoptotic cells (green) and nuclei (blue). B is a gray-scale analysis of Western blotting for detection of Bcl-2, bax and Caspase-3 protein expression, GAPDH as control. C-F is Bcl-2, bax and Caspase-3 (n=3)).
Fig. 6 shows that the number of myocardial apoptosis increases significantly under ISO stimulation, while the number of myocardial apoptosis decreases in a dose-dependent manner under G13, indicating that myocardial fibrosis is involved in apoptosis. Meanwhile, bcl-2 is found to be significantly down-regulated under ISO stimulation, bax and Caspase-3 are significantly up-regulated as pro-apoptotic molecules, and after G13 is added for treatment, the ratio of Bcl-2/Bax is increased, so that G13 can be proved to reduce myocardial fibrosis and inhibit apoptosis.
4. Study of the Effect of glossy privet glycoside G13 on TGF-beta 1/Smads Signal pathway by Wsterin blot technique
The influence of the ligustrum lucidum glycoside G13 on the classical TGF-beta 1/Smads signal path of the tissue fibrosis is studied by adopting Wsterin blot and other technologies. The specific operation is as follows: the myocardial tissue of the mice was ground with a tissue grinder, the homogenate was placed in a refrigerator for 30min, centrifuged at-12000 g for 10min at-4℃and the supernatant was collected, and the protein content of the samples was determined by BCA method. The protein samples were boiled for 5 minutes and then separated by SDS polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes. 5% skim milk was soaked for 1.5 hours at room temperature. Incubate with primary antibody overnight at 4 ℃. Membranes were washed three times with TBS/0.1% Tween 20. Anti-mouse (1:2000) and anti-rabbit antibodies (1:1000) contain horseradish peroxidase. Finally, the protein was detected by enhanced chemiluminescence and immunoblots were quantified using image analysis software. The effect of G13 on the TGF-beta 1/Smads signaling pathway is shown in FIG. 7 (where A is the result of G13 affecting the TGF-beta 1/Smads signaling pathway. B is the P-Smad2 protein gray value analysis, smad2 is the control. C is the P-Smad3 protein gray value analysis, smad3 is the control. D-E is the gray value analysis of Smad7 and Smad 4).
As can be seen from fig. 7, the expression levels of TGF- β1, p-Smad2, p-Smad3 and Smad4 were significantly up-regulated in the ISO group, and the expression level of Smad7 was significantly down-regulated in the G13-administered group, compared to the control group, and the above expression was reversed, indicating that G13 has a cardioprotective effect and that myocardial fibrosis was inhibited by modulating TGF- β1/Smad signaling pathway.
Taken together, the analysis shows that ligustrin G13 can reduce myocardial apoptosis by reducing oxidative stress injury, and inhibit TGF-beta 1/Smads signaling pathway to reduce ISO-induced myocardial fibrosis. Wherein, the ligustrum lucidum glycoside G13 has the best anti-myocardial fibrosis effect under the administration condition of high dose (80 mg/kg/d), has no toxicity to liver and has very good prospect in developing and preparing anti-myocardial fibrosis medicines.
Claims (6)
1. The application of the ligustrum lucidum glycoside G13 as the only medicinal component in preparing the anti-myocardial fibrosis medicament is characterized in that: the structural formula of the compound is shown in the formula (1) in the specification, and the compound is applied to the preparation of medicines for treating, preventing, relieving or alleviating myocardial fibrosis related diseases:
2. the use of ligustrum lucidum glycoside G13 as the only active ingredient in the preparation of an anti-myocardial fibrosis drug according to claim 1, characterized in that: the myocardial fibrosis related diseases refer to diseases caused by myocardial fibrosis or diseases which can be developed into myocardial fibrosis or diseases with clinical symptoms of myocardial fibrosis, including but not limited to coronary heart disease, myocardial ischemia, myocardial infarction and heart failure.
3. The use of ligustrum lucidum glycoside G13 as the only active ingredient in the preparation of an anti-myocardial fibrosis drug according to claim 1, characterized in that: the ligustrum lucidum glycoside G13 has the effects of reducing myocardial cell apoptosis by relieving oxidative stress injury, inhibiting TGF-beta 1/Smads signal channels, relieving ISO-induced myocardial fibrosis and playing an anti-myocardial fibrosis role.
4. The use of ligustrum lucidum glycoside G13 as the only active ingredient in the preparation of an anti-myocardial fibrosis drug according to claim 1, characterized in that: the ligustrum lucidum glycoside G13 reduces myocardial cell apoptosis by reducing oxidative stress injury, inhibits TGF-beta 1/Smads signal channels to reduce ISO-induced myocardial fibrosis, and has an effective concentration of 20-80mg/kg/d for playing an anti-myocardial fibrosis role.
5. The use of ligustrum lucidum glycoside G13 as the only active ingredient in the preparation of an anti-myocardial fibrosis drug according to claim 1, characterized in that: the process for extracting osmanthus fragrans fruits comprises the following steps: degreasing dried osmanthus fragrans fruits with petroleum ether, performing ultrasonic extraction with 55% ethanol according to a feed-liquid ratio of 1:10 to obtain 1h, recovering the extracting solution at 40 ℃ with a rotary evaporator, freeze-drying to obtain an osmanthus fragrans fruit extract, dissolving the extract with water, and preparing a mother solution with a concentration of 5 mg/mL; enriching the ligustrum lucidum glycoside G13 by using macroporous resin with the model of XAD-16, loading the sample at the flow rate of 1mL/min, and stopping loading the sample when detecting the concentration of the ligustrum lucidum glycoside G13 in the resin effluent to 5% of the concentration in the mother liquor by using HPLC; after the adsorption reaches equilibrium, eluting impurities with 30% ethanol, detecting by HPLC until the occurrence of ligustroside G13 in the eluent begins, eluting with 40% ethanol in a large amount, collecting 40% ethanol eluent, recovering by rotary evaporator at 40deg.C, and lyophilizing to obtain ligustroside G13 concentrate; dissolving the ligustrum glycoside G13 concentrate with methanol, passing through a 0.22 μm microporous filter membrane, purifying and preparing ligustrum glycoside G13 by adopting a preparation liquid phase, taking 0.1% phosphoric acid water A-acetonitrile B as a mobile phase, selecting a YMC-Pack ODS-A C chromatographic column as a chromatographic column, and performing gradient elution under the conditions of 0-10min and 10-30% B, wherein the flow rate is 8mL/min, the detection wavelength is 210 nm; 10-25min,30%; collecting the ligustroside G13 flowing out from the chromatographic column according to the retention time of the ligustroside G13 by adopting a manual collection mode, recovering the ligustroside G13 by using a rotary evaporator at 40 ℃, and freeze-drying to obtain the ligustroside G13.
6. The use of ligustroside G13 as a sole active ingredient in the manufacture of a medicament for the treatment of myocardial fibrosis according to claim 1, wherein the ligustroside G13 is used directly or in the form of a pharmaceutical composition comprising 0.1 to 99% of ligustroside G13, the remainder being a pharmaceutically acceptable carrier or excipient.
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