Application of tilianin in preparation of medicine for treating sepsis myocardial injury disease

Abstract

The invention provides application of tilianin in preparation of a medicine for treating sepsis myocardial injury diseases, and belongs to the technical field of medicines. According to the application disclosed by the invention, the tilianin is found to be capable of reducing the levels of TNF-alpha, IL-6 and IL-1beta in serum of a sepsis myocardial injury (SIMI) model rat, and the inflammatory response caused by LPS (Lipopolysaccharide)-induced SIMI is relieved; tLR4 / NF-kB / NLRP3 signal channels can be inhibited, the rat sepsis myocardial damage protection effect is achieved, and more thoughts are provided for tilianin medication treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of cynaroside in preparing medicine for treating sepsis-induced myocardial injury. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection with various pathogens such as bacteria, fungi or viruses, which can cause multiple organ dysfunction in the body, and cardiac dysfunction is an important part of it. Statistics show that about half of sepsis patients are accompanied by septicemia induced myocardial injury (SIMI), the main feature of which is impaired left ventricular systolic function.

[0003] SIMI has a complex pathogenesis and rapidly progresses. Inflammatory response and oxidative stress may be important factors leading to the development of SIMI. Currently, traditional drugs (statins, positive inotropic drugs, glucocorticoids, antibiotics) have a general therapeutic effect on SIMI. Recent studies have found that in recent years, more and more natural products have been proven to have the effect of improving myocardial damage in sepsis. More and more natural products, such as anthocyanin B2, puerarin, and resveratrol, have been proven to reduce myocardial damage through various biological processes such as anti-inflammatory and antioxidant processes, and play a cardioprotective role.

[0004] Tilianin is one of the main effective monomers in the Xinjiang specialty ethnic medicine Xiangqinglan, which belongs to the flavonoids. Current studies have found that tilianin has a certain anti-inflammatory effect in the treatment of ischemia-perfusion re-injury and arteriosclerosis. However, there are no reports on whether tilianin has a protective effect on myocardial injury caused by sepsis in rats. Summary of the invention

[0005] The purpose of the present invention is to provide an application of cynarin in the preparation of a drug for treating sepsis-induced myocardial injury. The present invention finds that cynarin can significantly improve the cardiac function of rats with sepsis-induced myocardial injury, effectively improve the coordination of SIMI left ventricular wall fluctuations, improve the contractility of SIMI heart left ventricular wall, and reduce the levels of SIMI myocardial injury markers myocardial enzymes LDH and CK-MB.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of cynaroside in preparing a medicine for treating sepsis-induced myocardial injury.

[0008] The present invention provides application of cynaroside in preparing a medicine for improving the contractility of the left ventricular wall after myocardial damage caused by cardiac sepsis.

[0009] The present invention provides the use of cynaroside in preparing a medicine for reducing the levels of lactate dehydrogenase and creatine kinase isoenzymes in cardiac tissues with myocardial damage caused by sepsis.

[0010] The present invention provides the use of cynaroside in preparing a medicine for inhibiting the expression of TNF-α, IL-6 and IL-1β in cardiac tissues with sepsis-induced myocardial damage.

[0011] The present invention provides the use of cynaroside in preparing a medicine for inhibiting the TLR4 / NF-κB / NLRP3 signaling pathway in heart tissues with sepsis-induced myocardial damage.

[0012] According to the above application, the medicine comprises cynaroside and pharmaceutically acceptable excipients.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention finds that cynaroside can significantly improve the cardiac function of rats with septic myocardial injury, effectively improve the coordination of left ventricular wall fluctuations, enhance the contractility of the left ventricular wall, and reduce the levels of myocardial enzymes LDH and CK-MB, which are markers of myocardial injury in septic myocardial injury.

[0015] Cynarin can downregulate the levels of TNF-α, IL-6, and IL-1β in the serum of SIMI model rats, and alleviate the inflammatory response caused by LPS-induced SIMI; it can inhibit the TLR4 / NF-κB / NLRP3 signaling pathway, exert a protective effect on myocardial damage in rats with sepsis, and provide more ideas for the treatment of cynarin. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The M-mode images of echocardiography of SIMI rats induced by cynarin.

[0017] Figure 2 This is the result of the test of cynarin on LVEF, LVFS, LVIDs and LVIDd of SIMI rats.

[0018] Figure 3 This is the result of the detection of cynaroside on serum LDH and CK-MB in SIMI rats.

[0019] Figure 4 This is the result of the detection of cynarin on inflammatory factors TNF-ɑ, IL-6, and IL-1β in SIMI rat group.

[0020] Figure 5 This is the HE staining result of cynarin on myocardial tissue of SIMI rats.

[0021] Figure 6 This is the graph showing the effect of cynarin on the expression of p-NF-κB p65 protein in the myocardial tissue of SIMI rats.

[0022] Figure 7 This is the immunofluorescence staining of cynarin on the nuclear translocation of NF-κB p65 in SIMI rats. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.

[0029] Example

[0030] 1 Materials and methods

[0031] 1.1 Materials

[0032] 1.11 Experimental Animals

[0033] Forty 8-week-old SPF male SD rats weighing 240-300 g were purchased from Xinjiang Medical University (Animal Production License No.: SCXK(Xin)2023-0002). During the feeding period, the temperature and humidity of the environment were maintained according to standard requirements, with 12-h light and dark alternation, and sufficient feed and drinking water were provided. The experimental operations followed animal ethical regulations.

[0034] This animal experiment was reviewed and approved by the Experimental Animal Ethics Committee of Xinjiang Uygur Autonomous Region Institute of Pharmacology (Ethics Approval No.: XJIMM-20240801).

[0035] 1.12 Experimental drugs and reagents

[0036] Cynarin (HPLC purity 98.13%) was prepared by Xinjiang Uygur Autonomous Region Drug Research Institute (batch number: 20170805); LPS (sigma); rat creatine kinase isoenzyme CK-MB kit, lactate dehydrogenase LDH activity detection kit (Nanjing Jiancheng); TNF-α, IL-6, IL-1β kits (Elabscience); NF-κB p65 immunofluorescence detection kit (Nanjing Jiancheng); TLR4, p-NF-κBp65, NLRP3 antibodies (abcam), MyD88 antibody (Cell Signaling Technology); β-Tubulin antibody, HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG antibody (ZSGB-BIO); ultrasensitive ECL chemiluminescent liquid (BOSTER); PVDF membrane (0.45 μm) (Millipore).

[0037] 1.13 Experimental Instruments

[0038] Small animal intelligent anesthesia machine (Beijing Zhongshidichuang Technology Development Co., Ltd.); portable color Doppler ultrasound system (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.); multifunctional microplate reader (TECAN SPARK, Switzerland); desktop high-speed refrigerated centrifuge TGL-16K (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); multifunctional imaging system (VILBER, France); upright fluorescence microscope (LEICA, Germany).

[0039] 1.2 Methods

[0040] 1.21 Model building and grouping

[0041] Male SD rats were randomly divided into 4 groups, 10 in each group: control group (Control), model group (LPS), low-dose tilianin group 7.5 mg / kg (LPS+Tilianin L), and high-dose tilianin group 15 mg / kg (LPS+Tilianin H). Adaptive feeding was performed for one week, and then tilianin was pre-administered and intragastrically administered for one week. The control group and the model group were given an equal amount of normal saline. After 7 days, the model group and the drug group were intraperitoneally injected with 10 mg / kg LPS once to establish the animal model, and the control group was intraperitoneally injected with an equal amount of normal saline.

[0042] 1.22 Assessment of left ventricular function in rats by echocardiography

[0043] 6 hours after the intraperitoneal injection, the rats were anesthetized by isoflurane inhalation, the rats' chest hair was shaved, and the rats were evaluated using a portable color Doppler ultrasound system. The instrument was cut to the long axis of the left ventricle, and the images were recorded using M-ultrasound at the chordae tendineae of the mitral valve. Four consecutive cardiac cycles were selected to measure the left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs), and the rats' left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) were calculated.

[0044] 1.23 Detection of related myocardial enzyme activities in rat serum

[0045] After the completion of the cardiac ultrasound test, the rats were anesthetized by intraperitoneal injection of pentobarbital, and then blood was collected from the abdominal aorta of the rats. After the blood collection was completed, the blood was left to stand at room temperature, and then centrifuged at 3000rpm for 10min. The supernatant was divided and packaged, one part was used for subsequent testing, and the other part was stored in a -80℃ refrigerator for standby use. The serum of the rats in the above groups was taken, and the LDH level of the samples was detected by the microplate method, and the CK-MB level was detected by the immunosuppression method in strict accordance with the operating procedures in the instructions.

[0046] 1.24 Determination of serum inflammatory factor levels

[0047] The serum of rats in the above groups was taken, and rat TNF-α, IL-6, and IL-1β detection kits were used for detection by enzyme-linked immunosorbent assay (ELISA). The absorbance was determined according to the steps, and the concentration of inflammatory factors in the serum was calculated.

[0048] 1.25HE staining to observe myocardial morphological changes

[0049] After blood was drawn from the rat abdominal aorta, the heart was carefully removed, and the fresh heart tissue sample was placed in PBS to pump out the blood. The liquid was then absorbed with absorbent paper (part of it was directly placed in a -80°C refrigerator for subsequent experiments) and fixed in 4% paraformaldehyde. The sample was taken out from the fixative within 48 hours, and was sequentially dehydrated, transparent, paraffin-embedded, and tissue sectioned (4-6 μm). Then, it was stained using the conventional HE staining method, and the myocardial morphological changes were observed under an optical microscope.

[0050] 1.26 Immunofluorescence staining of cardiac tissue First, the paraffin sections of the heart were dewaxed and hydrated routinely, and immunostaining blocking solution was added and blocked at room temperature for 1 hour; then the immunostaining blocking solution was removed, and NF-κB p65 mouse monoclonal antibody was added and incubated at 4°C overnight; the monoclonal antibody was removed overnight, and the sections were washed 3 times with washing solution for 5 minutes each; then anti-mouse Cy3 was added and incubated at room temperature for 1 hour; anti-mouse Cy3 was removed, and the sections were washed 2 times with washing solution for 5 minutes each; nuclear staining solution (DAPI) was added and stained at room temperature for about 5 minutes; nuclear staining solution was removed, and the sections were washed 3 times with washing solution for 3 minutes each. After the last wash, the washing solution was completely removed, and an appropriate amount of anti-fluorescence quenching sealing solution was added, and the sections were observed under a fluorescence microscope after the coverslip was sealed. NF-κBp65 was stained with red fluorescence, and DAPI staining of the cell nucleus was stained with blue fluorescence.

[0051] 1.27 Western blot detection of the expression of cardiac tissue-related proteins

[0052] The obtained heart tissue samples were taken out of the -80℃ refrigerator, and the myocardial tissue of appropriate weight was cut and cut into small pieces. The tissue and lysate were added to the lysate at a ratio of 10mg:100μL, and the tissue was ground and crushed using a handheld tissue homogenizer, and then further fully lysed using an ultrasonic cell crusher, placed on ice for 20min, and then centrifuged at 12000rpm for 15min, and the supernatant was aspirated as the tissue protein extract. The concentration was determined by the BCA protein quantification method, 5× protein buffer was added, 100℃ metal bath was used for 5min, 30-50μg sample was quantified, and the protein bands were transferred to PVDF membranes by SDS-PAGE gel electrophoresis, blocked with 5% skim milk powder for 2h, incubated with primary antibody at 4℃ overnight, and washed with 1×TBST the next day, and then incubated with HRP-labeled secondary antibody at room temperature on a shaker for 2h, and finally washed and placed in a multifunctional imaging system for recording. Image J software was used to determine the gray value of each band, normalized with the internal reference as the baseline, and the relative expression level of the target protein was analyzed.

[0053] 1.28 Statistical analysis

[0054] GraphPad Prism 10.1.2 was used for statistical analysis and plotting, and one-way ANOVA was used for comparative analysis. The difference was considered significant when p < 0.05.

[0055] 2 Results

[0056] 2.1 Effects of cynaroside on cardiac ultrasound parameters in SIMI rats

[0057] In order to study whether cynaroside can improve the cardiac structure and function of rats with LPS-induced SIMI, the present invention used a color Doppler ultrasound system to perform ultrasound electrocardiogram detection, and the results showed that ( Figure 1 ). Compared with the control group, the coordination of left ventricular wall fluctuations in the model group decreased, the fluctuation amplitude decreased, the ejection fraction (LVEF) and shortening fraction (LVFS) were significantly decreased (p < 0.01), the left ventricular end-systolic internal diameter (LVIDs) was significantly increased (p < 0.01), and the left ventricular end-diastolic internal diameter (LVIDd) did not change significantly, indicating that the contractile capacity of the left ventricular wall in the model group was reduced; compared with the model group, the LVEF and LVFS of the high-dose cynarin group were significantly improved (p < 0.01), the LVEF of the low-dose cynarin group was also improved (p < 0.05), and the LVIDs of the high-dose cynarin group was significantly reduced (p < 0.05), indicating that cynarin may improve myocardial injury in rats caused by sepsis by improving the contractile capacity of the left ventricular wall of the heart. See Figure 2 .

[0058] 2.2 Effects of cynaroside on myocardial enzyme activities in SIMI rats

[0059] Lactate dehydrogenase and creatine kinase isoenzymes are common myocardial enzymes that are characteristic of myocardial injury. Figure 3 ), compared with the control group, the levels of LDH and CK-MB in the serum of the model group rats were significantly increased (p<0.01); compared with the model group, the levels of LDH and CK-MB in the serum of the low-dose and high-dose cynarin groups were significantly reduced, and the differences were statistically significant (p<0.01). This suggests that cynarin can alleviate myocardial damage in SIMI rats.

[0060] 2.3 Effects of cynaroside on inflammatory factors TNF-ɑ, IL-6, and IL-1β in SIMI rats

[0061] In view of the important influence of inflammation on myocardial injury in SIMI rats, the present invention detected the levels of inflammatory factors in the serum of SIMI rats. The results of ELISA showed that ( Figure 4), compared with the blank group, the concentrations of inflammatory factors TNF-ɑ, IL-6, and IL-1β in the serum of the model group were significantly increased (p<0.01); compared with the model group, both the low-dose and high-dose cynaroside groups could reduce the concentrations of TNF-ɑ, IL-6, and IL-1β in the serum of rats, and the differences were statistically significant (p<0.05, p<0.01). The results showed that cynaroside can alleviate LPS-induced myocardial inflammatory response.

[0062] 2.4 Observation of myocardial morphology in each group of rats under an optical microscope

[0063] In order to further observe the effect of cynarin on the pathological damage of myocardial tissue in SIMI rats, HE staining was performed. The results of HE staining of heart tissue showed that ( Figure 5 ), the myocardial fiber gaps in the control group were dense, the structure was complete, the arrangement was neat, the coloring was uniform, and no abnormalities were found; compared with the model group, the myocardial cell arrangement in the cynaroside-treated group tended to be normal, the cell gaps were reduced, the myocardial fiber ruptures were reduced, the inflammatory cell infiltration was alleviated, and the myocardial injury showed an improving trend.

[0064] 2.5 Effect of cynaroside on NF-κB expression in myocardial tissue of SIMI rats

[0065] In order to explore the effect of cynaroside on the activation of NF-κB in the heart of SIMI rats, the present invention detected the expression of p-NF-κB p65 protein in rat myocardial tissue by Western blot experiment, and used tissue immunofluorescence to observe the nuclear entry of NF-κB p65. The Western blot test results showed that ( Figure 6 ), compared with the control group, the expression of p-NF-κBp65 protein in myocardial tissue of the model group increased (p < 0.01); compared with the model group, the cynarin-treated group significantly downregulated the expression of p-NF-κB p65 protein. The results of tissue immunofluorescence showed that compared with the control group, the nuclear entry of NF-κB p65 in the heart of the model group rats was more obvious; compared with the model group, the expression of NF-κB p65 in the nucleus of the heart tissue of the cynarin-treated group was reduced (see Figure 7 ).

[0066] 3.0 Conclusion

[0067] Sepsis can cause damage to multiple organs throughout the body, and the heart is one of the organs that is often affected. Studies have shown that SIMI may be caused by excessive cardiac inflammation, oxidative stress, myocardial cell death, or myocardial energy metabolism disorders and mitochondrial dysfunction. Therefore, it is of great clinical significance to explore the pathogenesis of SIMI and discover possible treatment pathways.

[0068] Cisin can significantly improve the cardiac function of rats with septic myocardial injury, effectively improve the coordination of left ventricular wall fluctuations, improve the contractility of the left ventricular wall, and reduce the levels of common myocardial injury markers, myocardial enzymes LDH and CK-MB, suggesting that cisin may have a positive therapeutic effect on SIMI. A large number of studies have shown that inflammatory stress permeates the entire stage of the pathological development of sepsis. After sepsis is formed, immune cells are activated and secrete a large amount of proinflammatory factors TNF-α, IL-6, and IL-1β to resist over-activated immune cells, so reducing the release of inflammatory factors is also an important part. The results of the present invention found that cisin can downregulate the levels of TNF-α, IL-6, and IL-1β in the serum of SIMI model rats, and reduce the inflammatory response caused by LPS-induced SIMI, indicating that cisin has anti-inflammatory activity.

[0069] In summary, cynarin can improve the contractility of the left ventricular wall of the heart of SIMI rats, inhibit the activity of related myocardial enzymes in serum, reduce the expression of inflammatory factors TNF-α, IL-6, and IL-1β, inhibit the TLR4 / NF-κB / NLRP3 signaling pathway, and play a protective role in myocardial injury in rats with sepsis, providing more ideas for the treatment of cynarin.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of cynaroside in the preparation of drugs for the treatment of myocardial damage caused by sepsis.

2. The use of cynaroside in the preparation of drugs for improving the contractile ability of the left ventricular wall after myocardial injury caused by cardiac sepsis.

3. The application of cynaroside in the preparation of drugs for reducing the levels of lactate dehydrogenase and creatine kinase isoenzymes in cardiac tissue with sepsis-induced myocardial damage.

4. The application of cynaroside in the preparation of drugs for inhibiting the expression of TNF-α, IL-6, and IL-1β in cardiac tissues with sepsis-induced myocardial damage.

5. Application of cynaroside in the preparation of drugs for inhibiting the TLR4 / NF-κB / NLRP3 signaling pathway in cardiac tissue with sepsis-induced myocardial injury.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine comprises cynaroside and pharmaceutically acceptable excipients.

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