CN114748493A

CN114748493A - Application of cowherb seed flavonoid glycoside in preparation of medicine for treating sepsis

Info

Publication number: CN114748493A
Application number: CN202210536408.5A
Authority: CN
Inventors: 温家根; 马文贤; 王月悦; 黄申卓凡; 赵贵涛; 孟晓明; 吴婷妮
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2022-07-15
Anticipated expiration: 2042-05-17
Also published as: CN114748493B

Abstract

The invention relates to biological medicine, and discloses an application of cowherb seed flavonoid glycoside in preparing a medicine for treating sepsis, wherein the cowherb seed flavonoid glycoside has the structure as follows:

the invention has the beneficial effects that: the cowherb seed flavonoid glycoside is used for preparing the medicine for treating sepsis, can reduce the expression and secretion level of cell inflammatory factors, and inhibit a cell FKBP 5/NF-kB inflammatory signal channel; can also reduce the expression level of serum inflammatory factors IL-1 beta, IL-6, TNF-alpha and chemotactic factor MCP-1 of the animals infected with sepsis, improve the pathology of kidney tissues and lung tissues of the animals infected with sepsis, and inhibit NF-kB signal path tables such as P-P65, NLRP3 and C-caspase-1 and the like in the kidney tissuesThe method can reduce the levels of creatinine and urea nitrogen in the serum of kidney tissues, improve pulmonary edema, improve the survival rate of animals infected with sepsis, and has potential treatment effect on sepsis.

Description

Application of cowherb seed flavonoid glycoside in preparation of medicine for treating sepsis

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of cowherb seed flavonoid glycoside in preparing a medicine for treating sepsis.

Background

Sepsis (sepsis) is a systemic inflammatory response syndrome caused by infection and is one of the causes of death worldwide. Among patients with sepsis, critically ill patients account for a large proportion. In high income countries, 3-10 out of every 1000 people per year suffer from sepsis. Statistically, about 11% of ICU patients develop sepsis, but the therapeutic strategy for sepsis is not yet standardized. The currently used therapeutic strategies include fluid resuscitation, anti-infective therapy, glucocorticoid application, hemodialysis, and the like, but are still controversial. Despite the continuous improvement of medical conditions, the mortality rate of sepsis still reaches 25-30%, and even 40% when patients have shock, and sepsis and related diseases seriously affect the life health of people.

Sepsis is characterized by an unregulated severe systemic inflammatory response of the host to infection, accompanied by multiple organ dysfunction (e.g., lung, kidney, liver, and blood vessels). At present, a number of studies have shown that the production of inflammatory factors and inflammatory chemokines plays a key role in the progression of sepsis disease. The nuclear factor activated B cell kappa-light chain enhancement (NF-kappa B) is an important transcription factor for regulating and controlling inflammation related genes, plays an important regulation role in cascade reaction of inflammatory factors, and the activated NF-kappa B enters the nucleus to promote the transcription and synthesis of genes such as IL-6, TNF-alpha, MCP-1 and the like, so that the inflammatory injury of tissues and organs is finally caused.

FK506 binding protein 51(FKBP51, also known as FKBP5) belongs to the immunophilin FKBP family, FKBP5 is capable of binding to IKK α and plays a key role in regulating the NF- κ B signaling pathway. It has been shown that inhibition of FKBP5 can affect the development of cardiovascular disease through NF-. kappa.B signaling.

The semen Vaccariae is dried mature seed of Garcke of Vaccinium macrocephalum (Neck.) DC of Caryophyllaceae, and contains triterpene saponin, cyclic peptide, flavone, amino acid and polysaccharide as main ingredients. The cowherb seed flavonoid glycoside (Vaccarin, VA) is a natural flavonoid glycoside and is the main active ingredient of the traditional Chinese medicine cowherb seed. Research proves that VA has the functions of promoting blood circulation to restore menstrual flow, inducing lactation to reduce swelling, inducing diuresis to treat stranguria and the like, but reports of VA in the aspect of preventing and treating sepsis are not seen at present.

Disclosure of Invention

The invention aims to solve the technical problem of how to provide the application of the cowherb seed flavonoid glycoside in preparing the medicament for treating the sepsis, and the medicament has good treatment effect on the sepsis.

The invention solves the technical problems through the following technical means:

the invention provides an application of cowherb seed flavonoid glycoside in preparing a medicine for treating sepsis, wherein the structure of the cowherb seed flavonoid glycoside is as follows:

the application of the cowherb seed flavonoid glycoside in preparing the medicament for treating the sepsis according to the embodiment of the invention at least has the following beneficial effects: the cowherb seed flavonoid glycoside is used for preparing the medicine for treating sepsis, and is proved to be capable of reducing the expression and secretion level of cell inflammatory factors and inhibiting a cell FKBP 5/NF-kB inflammatory signal channel; the composition can also reduce the expression levels of serum inflammatory factors IL-1 beta, IL-6, TNF-alpha and chemotactic factor MCP-1 of an animal infected with sepsis, improve the pathology of kidney tissues and lung tissues of the animal infected with sepsis, inhibit the expression of proteins related to NF-kB signal pathways such as P-P65, NLRP3 and C-caspase-1 in the kidney tissues, reduce the levels of creatinine and urea nitrogen in the blood serum of the kidney tissues, improve the lung inflammation, improve the survival rate of the animal infected with sepsis, and have potential treatment effect on sepsis.

In the present invention, sepsis also includes sepsis-related diseases such as endotoxemia (endoxemia), severe sepsis (severe septis), septic shock (septic shock), and the like. All belong to symptoms such as systemic infection caused by gram-negative bacteria and further systemic multi-organ dysfunction caused by gram-negative bacteria, and the pathogenic causes and the treatment methods of the symptoms are similar.

Preferably, the medicament for treating sepsis is a medicament capable of reducing the expression and secretion level of cell inflammatory factors.

Preferably, the inflammatory factors include IL-1 β, IL-6 and TNF- α.

Preferably, the medicament for treating sepsis is a medicament capable of inhibiting an FKBP 5/NF-kB inflammatory signaling pathway of cells.

Preferably, the NF-. kappa.B signal includes phosphorylation of the P65 subunit, NLRP3 and C-Caspase-1.

Preferably, the medicament for treating sepsis is a medicament capable of improving the survival rate of animals infected with sepsis.

Preferably, the medicament for treating sepsis is a medicament capable of reducing serum inflammatory factors of animals infected with sepsis and improving the pathology of kidney tissues and lung tissues of animals infected with sepsis.

Preferably, the medicament for treating sepsis is a medicament capable of inhibiting the expression level of inflammatory factors and chemokines in kidney tissues and lung tissues of animals infected with sepsis.

Preferably, the inflammatory factors include IL-1 beta, IL-6, TNF-alpha, and the chemokines include MCP-1.

Preferably, the medicament for treating sepsis is a medicament capable of reducing creatinine and urea nitrogen in serum of kidney tissues of animals infected with sepsis.

Preferably, the medicament for treating sepsis is a medicament capable of inhibiting the expression of NF-kB signal channels such as P-P65, NLRP3, C-caspase-1 and the like in kidney tissues of animals infected with sepsis.

Preferably, the medicament for treating sepsis is a medicament capable of improving pulmonary edema of an animal infected with sepsis.

Preferably, the medicament for treating sepsis further comprises a pharmaceutically acceptable carrier or auxiliary material. For example, oral administration preparations: tablets, capsules, pills, granules, dripping pills, oral preparations and the like; preparing a rectal administration preparation: suppositories, enemas; preparing an injection preparation: intramuscular injection preparations, intravenous injection preparations, and the like.

The invention has the advantages that:

1. the application of the cowherb seed flavonoid glycoside in preparing the medicine for treating sepsis proves that the cowherb seed flavonoid glycoside can reduce the expression and secretion level of cell inflammatory factors and inhibit a cell FKBP 5/NF-kB inflammatory signal channel.

2. The cowherb seed flavonoid glycoside is used for preparing the medicine for treating sepsis, can also reduce the expression levels of serum inflammatory factors IL-1 beta, IL-6, TNF-alpha and chemotactic factor MCP-1 of animals infected with sepsis, improves the pathology of kidney tissues and lung tissues of the animals infected with sepsis, and inhibits the expression of NF-kB signal channels such as P-P65, NLRP3, C-caspase-1 and the like in the kidney tissues.

3. The cowherb seed flavonoid glycoside is used for preparing the medicine for treating sepsis, can reduce creatinine and urea nitrogen levels in serum of kidney tissues, improves pulmonary edema, improves the survival rate of animals infected with sepsis, and has potential treatment effect on sepsis.

Drawings

FIG. 1 is a graph showing the effect of different concentrations of VA on the viability of RAW264.7 cells in example 1 of the present application;

FIG. 2 is a graph showing the change of inflammatory response of LPS-induced RAW264.7 cells in VA measured by Real-time PCR in example 2 of the present application;

FIG. 3 is a graph showing the change of inflammatory response of VA to LPS-induced RAW264.7 cells measured by Elisa kit in example 2 of the present application;

FIG. 4 is a graph of NF- κ B inflammatory signal pathways of RAW264.7 cells of different culture groups determined by Western blot in example 3 of the present application;

FIG. 5 is a graph showing the effect of VA on survival in mice of LPS model and CLP model in example 4 of the present application;

FIG. 6 is a graph showing the effect of VA on serum inflammatory factor levels in LPS model mice in example 5 of the present application;

FIG. 7 is a graph showing the effect of VA on serum inflammatory factor levels in CLP model mice in example 5 of the present application;

FIG. 8 is a graph showing the effect of VA on serum urea nitrogen levels in LPS model and CLP model mice in example 6 of the present application;

FIG. 9 is a graph showing the effect of VA on the serum creatinine level in mice in the LPS model and CLP model in example 6 of the present application;

FIG. 10 is a graph showing the effect of VA on the expression levels of mRNA for inflammatory factors and chemokines in kidney tissue of LPS model mice as determined by Real-time PCR in example 6 of the present application;

FIG. 11 is a graph showing the effect of VA on the expression levels of mRNA for inflammatory factors and chemokines in kidney tissue of CLP model mice as determined by Real-time PCR in example 6 of the present application;

FIG. 12 is a graph showing the results of staining mouse kidney tissues with hematoxylin eosin (H & E) staining kit according to example 7 of the present application;

FIG. 13 is a graph showing the evaluation of renal tubular injury in mice of LPS model and CLP model in example 7 of the present application;

FIG. 14 is a diagram showing the NF- κ B inflammatory signal pathway of kidney tissue of LPS model mouse determined by Western blot in example 8 of the present application;

FIG. 15 is a graph showing the NF- κ B signaling pathway of kidney tissue of CLP model mice determined by Western blot in example 8 of the present application;

FIG. 16 is a graph showing the effect of VA determination on the mRNA expression levels of inflammatory factors and chemokines in lung tissues of LPS model mice by Real-time PCR in example 9 of the present application;

FIG. 17 is a graph showing the effect of VA on the expression levels of mRNA for inflammatory factors and chemokines in lung tissue of CLP model mice as determined by Real-time PCR in example 9 of the present application;

FIG. 18 is a graph showing the results of staining mouse lung tissues with hematoxylin eosin (H & E) staining kit in example 10 of the present application;

FIG. 19 is a graph showing the lung tissue inflammation scores of mice in LPS model and CLP model according to example 10 of the present application;

FIG. 20 is a graph showing the effect of VA on the dry-to-wet weight ratio of the lung in LPS model and CLP model mice in example 11 of the present application;

FIG. 21 is a graph showing the prediction of binding patterns of VA and FKBP5 by molecular docking in example 12 of the present application;

FIG. 22 is a graph showing the results of cell heat transfer experiments conducted on the binding of VA to FKBP5 in example 12 of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Those skilled in the art who do not specify any particular technique or condition in the examples can follow the techniques or conditions described in the literature in this field or follow the product specification.

The compound used in the following examples is cowherb seed flavonoid glycoside (VA), which has the structure:

purchased from Dalian Melam Biotechnology Ltd.

Example 1: study of the Effect of VA concentration on in vitro cell viability

An in-vitro cell model is constructed by using RAW264.7 cells, the RAW264.7 cells are cultured by using culture media containing VA with different concentrations, and the survival and growth conditions of the cells are detected by adopting an MTT colorimetric method, so that the cell viability is judged. Wherein, the concentration of VA is 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400 μ M, and the detection result is shown in figure 1. As can be seen from FIG. 1, VA was not significantly toxic to RAW264.7 cells at a concentration of 0-400. mu.M.

Example 2: study of the Effect of VA on LPS (lipopolysaccharide) induced inflammatory response in vitro

The RAW264.7 cells were cultured in media of different experimental groups, which were grouped as follows: normal control group (NC), VA high dose group (VA 100. mu.M), LPS model group (LPS 1. mu.g/ml), LPS model + VA low dose group (LPS 1. mu.g/ml + VA 25. mu.M), LPS model + VA medium dose group (LPS 1. mu.g/ml + VA 50. mu.M), LPS model + VA high dose group (LPS 1. mu.g/ml + VA 100. mu.M).

After 24h of culture, collecting the supernatant of RAW264.7 cells, extracting RNA in the supernatant, and measuring the expression levels of mRNA of inflammatory factors such as IL-1 beta, IL-6, TNF-alpha and the like in the supernatant by a Real-time fluorescent quantitative PCR method (Real-time PCR), wherein the measurement results are shown in figure 2 (figures 2A, 2B and 2C respectively correspond to IL-1 beta, IL-6 and TNF-alpha). As can be seen from FIG. 2, VA significantly reduced the expression level of inflammatory factors mRNA such as IL-1 beta, IL-6, TNF-alpha induced in vitro by LPS.

The secretion of inflammatory factors in the supernatant of RAW264.7 cells cultured in a normal control group (NC), a VA high dose group (VA 100. mu.M), an LPS model group (LPS 1. mu.g/ml), and an LPS model + VA high dose group (LPS 1. mu.g/ml + VA 100. mu.M) was measured using an Elisa kit, and the measurement results are shown in FIG. 3 (FIGS. 3A, 3B, and 3C correspond to IL-1. beta., IL-6, and TNF-. alpha., respectively). As can be seen from FIG. 3, VA significantly inhibited secretion of inflammatory factors such as IL-1. beta., IL-6, TNF-. alpha.in the cell supernatant induced by LPS.

Example 3: study of the Effect of VA on LPS-induced in vitro cell inflammatory response Signal pathway NF-kB Signal

NF-kB inflammatory signal pathways such as P-P65, P65, NLRP3, C-Caspase-1, beta-actin and the like of RAW264.7 cells cultured in a normal control group (NC), a VA high dose group (VA 100 mu M), a LPS model group (LPS 1 mu g/ml) and a LPS model + VA high dose group (LPS 1 mu g/ml + VA 100 mu M) are measured by using a Western blot method, P-P65/P65, NLRP3 and C-Caspase-1 are used as abscissa and relative protein expression level is used as ordinate, so that the expression levels of NF-kB inflammatory signal pathway proteins of different test groups are obtained, and the measurement result is shown in figure 4 and shows that VA remarkably inhibits the expression of NF-kB inflammatory signal pathway proteins of cells induced by LPS.

Example 4: study of the effects of VA on survival of LPS model and CLP model (cecal ligation and puncture) mice

Constructing an LPS model: mice were randomly divided into Saline (Saline), LPS + VA groups, 10 per group. Wherein, mice of the salt group, LPS group and LPS + VA group are respectively injected with normal Saline, 7mg/kg LPS and 7mg/kg LPS +25mg/kg VA.

Constructing a CLP model: mice were randomly divided into Sham, CLP + VA groups, 10 per group. Among them, Sham operation was performed only in Sham group mice, cecal ligation and puncture operation was performed in CLP group mice, and cecal ligation and puncture operation was performed in CLP + VA group mice and medication was administered with VA of 25 mg/kg.

Survival of LPS model and CLP model mice was observed every 12 hours until 96 hours, and the results are shown in fig. 5 (fig. 5A, 5B correspond to LPS model and CLP model, respectively). As can be seen from FIG. 5A, LPS and CLP caused sepsis infection in mice, the survival rate of LPS group mice was less than 50% at 48 hours, while the survival rate of LPS + VA group mice stabilized at about 90% at 48 hours. As can be seen from fig. 5B, the survival rate of CLP group mice at 72 hours was 0%, while the survival rate of CLP + VA group mice at 72 hours stabilized at about 50%. The VA is shown to remarkably improve the survival rate of mice in an LPS model and a CLP model.

Example 5: study of the Effect of VA on serum inflammatory factor levels in LPS model and CLP model mice

Constructing an LPS model: mice were randomly divided into Saline (Saline), VA, LPS + VA groups, 8 per group. Wherein, mice of the salt group, the VA group, the LPS group and the LPS + VA group are respectively injected with normal Saline, 25mg/kg of VA, 7mg/kg of LPS and 7mg/kg of LPS +25mg/kg of VA.

Constructing a CLP model: mice were randomly divided into Sham, VA, CLP + VA groups, 8 per group. Among them, Sham operation was performed only in Sham group mice, Sham operation was performed in VA group mice and medication was given 25mg/kg of VA, cecal ligation and puncture operation was performed in CLP group mice, and medication was performed in CLP + VA group mice and medication was given 25mg/kg of VA.

After the mice were subjected to the above-described operation for 24 hours, the mice were anesthetized, and blood was collected to obtain serum. Serum inflammatory factor levels of mice in LPS model and CLP model were measured using Elisa kit, respectively, and the measurement results in LPS model are shown in FIG. 6 (FIGS. 6A, 6B, and 6C correspond to IL-1. beta., IL-6, and TNF-. alpha., respectively), and the measurement results in CLP model are shown in FIG. 7 (FIGS. 7A, 7B, and 7C correspond to IL-1. beta., IL-6, and TNF-. alpha., respectively). It can be seen from the figure that VA significantly reduced serum inflammatory factor levels in LPS model and CLP model mice.

Example 6: study of the Effect of VA on renal function Damage in LPS and CLP models

Example 5 after 24 hours from the establishment of an LPS model and a CLP model in mice, the mice were anesthetized and kidney tissues were collected.

The method comprises the following steps of (1) measuring the urea nitrogen level (BUN) in serum of kidney tissues of mice in an LPS model and a CLP model, wherein the specific operation process of the measurement is as follows: adding the components with corresponding contents into the test tubes of each test group according to the table 1, uniformly mixing, placing in a boiling water bath for 15min, and immediately cooling with tap water; the OD value of each tube was measured by zeroing double distilled water at a wavelength of 520nm and an optical path of 1 cm.

TABLE 1 test tubes of the test groups were charged with the components and their contents

[ note ] this method comprises the following steps: the 10mmol/L standard substance is 10mmol/L urea nitrogen, and the conversion is 280.1 mg/L.

The measurement results of the urea nitrogen level (BUN) in the serum of the kidney tissue of the LPS model and CLP model mice are shown in fig. 8 (fig. 8A and 8B correspond to the LPS model and CLP model, respectively). As can be seen from fig. 8, VA significantly reduced the urea nitrogen levels in the serum of the kidney tissue of mice in the LPS model and CLP model.

Measuring creatinine level (CRE) in kidney tissue blood serum of mice in LPS model and CLP model, wherein the specific operation process of measurement is as follows: adding samples with corresponding contents, standard substances (with the concentration of 442 mu mol/L), double distilled water and an enzyme solution A into test tubes of each test group according to the table 2, incubating for 5min at 37 ℃, and measuring the absorbance A1 at the wavelength of 546 nm; after the measurement, the same amount of the enzyme solution B was added to the above tubes, and the tubes were incubated at 37 ℃ for 5min and then the absorbance A2 was measured at a wavelength of 546 nm.

The formula for the creatinine content is as follows:

creatinine content (. mu.mol/L) (assay A2-K × assay A1) — (blank A2-K × blank A1) ]/[ (standard A2-K × standard A1) — (blank A2-K × blank A1) ], standard concentration (442. mu. mol/L)

Wherein the dilution factor K (amount of sample addition + volume of enzyme solution a)/(amount of sample addition + volume of enzyme solution a + volume of enzyme solution B) is 186/246.

TABLE 2 test tubes of each test group were charged with the components and their contents

The measurement results of creatinine levels (CRE) in kidney tissue of LPS model and CLP model mice are shown in fig. 9 (fig. 9A and 9B correspond to LPS model and CLP model, respectively). As can be seen from fig. 9, VA significantly reduced creatinine levels in the serum of kidney tissue in LPS model and CLP model mice.

And thirdly, the mRNA expression levels of inflammatory factors IL-1 beta, IL-6, TNF-alpha and chemotactic factor MCP-1 in the kidney tissues are measured by a Real-time fluorescence quantitative PCR method (Real-time PCR). The measurement results of the LPS model are shown in FIG. 10 (FIGS. 10A, 10B, 10C, and 10D correspond to IL-1. beta., IL-6, TNF-. alpha., and MCP-1, respectively); the measurement results of the CLP model are shown in FIG. 11 (FIGS. 11A, 11B, 11C, and 11D correspond to IL-1. beta., IL-6, TNF-. alpha., and MCP-1, respectively).

As can be seen from FIGS. 10 and 11, the mRNA expression levels of inflammatory factors IL-1 beta, IL-6, TNF-alpha and chemokine MCP-1 in the kidney tissues of mice in the CLP group and LPS group are remarkably increased, so that the mice are infected with sepsis; the inflammatory factors and chemokines of mice in CLP + VA group and LPS + VA group are remarkably reduced, which shows that VA can remarkably inhibit the expression of the inflammatory factors and chemokines, so that renal function injury of mice infected with sepsis is improved, and the levels of creatinine and urea nitrogen in serum of renal tissues of the mice are reduced.

Example 7: study of the Effect of VA on the renal histopathology of LPS model and CLP model mice

Example 5 after 24 hours from the establishment of an LPS model and a CLP model in mice, the mice were anesthetized and kidney tissues were collected. Staining mouse kidney tissue with hematoxylin eosin (H & E) staining kit, wherein the staining result is shown in FIG. 12 (FIGS. 12A and 12B correspond to LPS model and CLP model, respectively); the renal tubular injury of LPS model and CLP model mice was observed, and the results are shown in fig. 13 (fig. 13A and 13B correspond to LPS model and CLP model, respectively). From fig. 12 and fig. 13, it can be seen that the renal tubular injury levels of the mice in the CLP group and the LPS group are significantly increased, while the renal tubular injury levels of the mice in the CLP + VA group and the LPS + VA group are significantly reduced, which indicates that VA has a significant improvement effect on renal histopathology of the sepsis mice.

Example 8: study of the Effect of VA on the histopathology of the kidney of mice in LPS and CLP models

Example 5 after 24 hours from the establishment of an LPS model and a CLP model in mice, the mice were anesthetized and kidney tissues were collected. The NF-kB inflammatory signal pathways such as P-P65, P65, NLRP3, C-Caspase-1, beta-actin and the like of kidney tissues of mice of an LPS model and a CLP model are respectively measured by adopting a Western blot method, the protein expression levels of the NF-kB inflammatory signal pathways of different test groups are obtained by taking P-P65/P65, NLRP3 and C-Caspase-1 as abscissa and taking the relative protein expression level as ordinate, and the measurement results are shown in figures 14 and 15. From FIGS. 14 and 15, VA can inhibit the expression of NF-kB inflammatory signaling pathway proteins such as P-P65, NLRP3 and C-caspase-1 in kidney tissues of mice in LPS model and CLP model.

Example 9: study of the Effect of VA on the expression levels of inflammatory factor and chemokine mRNA in Lung tissues of LPS model and CLP model mice

Example 5 after 24 hours from the establishment of an LPS model, a CLP model, in mice, the mice were anesthetized and lung tissues were collected. The mRNA expression levels of inflammatory factors IL-1 beta, IL-6, TNF-alpha and chemokine MCP-1 in lung tissues are measured by a Real-time fluorescent quantitative PCR method (Real-time PCR). The measurement results of the LPS model are shown in FIG. 16 (FIGS. 16A, 16B, 16C, and 16D correspond to IL-1. beta., IL-6, TNF-. alpha., and MCP-1, respectively); the measurement results of the CLP model are shown in FIG. 17 (FIGS. 17A, 17B, 17C, and 17D correspond to IL-1. beta., IL-6, TNF-. alpha., and MCP-1, respectively).

As can be seen from FIGS. 16 and 17, VA can significantly inhibit the mRNA expression levels of inflammatory factors IL-1 beta, IL-6, TNF-alpha and chemokine MCP-1 in lung tissues of mice in LPS model and CLP model.

Example 10: study of the Effect of VA on the Lung tissue pathology of LPS model and CLP model mice

Example 5 after 24 hours from the establishment of an LPS model, a CLP model, in mice, the mice were anesthetized and lung tissues were collected. Staining mouse lung tissue with hematoxylin eosin (H & E) staining kit, with staining results as shown in FIG. 18 (FIGS. 18A and 18B correspond to LPS model and CLP model, respectively); lung histopathology was observed in LPS model and CLP model mice, and the results are shown in fig. 19 (fig. 19A and 19B correspond to LPS model and CLP model, respectively). As can be seen from fig. 18 and 19, the lung injury of the mice in the CLP group and the LPS group is significantly increased, while the lung injury of the mice in the CLP + VA group and the LPS + VA group is significantly reduced, which indicates that VA has a significant improvement effect on the lung tissue pathology of the sepsis mice.

Example 11: study of the Effect of VA on pulmonary edema in LPS model and CLP model mice

Example 5 after 24 hours from the establishment of LPS model and CLP model in mice, the mice were anesthetized and lung tissue was collected. The results of measuring the dry-wet weight ratio of the lungs of the LPS model and CLP model mice are shown in fig. 20 (fig. 20A and 20B correspond to the LPS model and CLP model, respectively). As can be seen from FIG. 20, the dry-wet weight ratio of the lung of the mice in the CLP + VA group and the LPS + VA group is lower than that of the mice in the CLP group and the LPS group, which indicates that VA has obvious improvement effect on pulmonary edema of the sepsis mice.

Example 12: investigation of the binding Pattern of VA and FKBP5

The results of Molecular Docking of the active region of FKBP5 with VA using Molecular Docking service (Molecular Docking) method and predicting its possible binding pattern are shown in fig. 21. The specific operation is that FKBP5 and ligand eutectic ID is 7AWF as a template, and a binding mode of VA and FKBP5 is established through a CDOCKER module of molecular simulation software Discovery Studio 2018. As shown in fig. 21, the hydroxyl group of VA forms hydrogen bond interactions with multiple amino acid residues of FKBP5 (Tyr57, Asp68, Ser70, VAl86 and Lys88), and at the same time, the five-carbon sugar moiety linked to the flavone parent nucleus phenyl also occupies the hydrophobic region of the protein (formed by Ile122, Gly59 and Phe 130), so that VA firmly occupies the active site of FKBP 5.

The VA-treated FKBP5 was subjected to a cell heat transfer experiment (CETSA) at temperatures of 42, 47, 52, 57 and 62 ℃, and the results are shown in fig. 22. As can be seen from fig. 22, the VA-treated FKBP5 has significantly improved stability at temperatures of 52, 57 and 62 ℃. It is shown that VA may be directly combined with FKBP5 protein to exert anti-inflammatory protection effect.

In conclusion, experiments prove that VA can play an important anti-inflammatory role in sepsis diseases and has an obvious protective effect on tissue and organ damage caused by sepsis. Therefore, the medicine is expected to become a key medicine for preventing and treating the sepsis.

The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. An application of cowherb seed flavonoid glycoside in preparing a medicament for treating sepsis is characterized in that: the structure of the cowherb seed flavonoid glycoside is shown as follows:

2. the use of vaccaria flavonoid glycoside according to claim 1 for the preparation of a medicament for the treatment of sepsis, characterized in that: the medicament for treating sepsis is a medicament capable of reducing the expression and secretion level of cell inflammatory factors.

3. The use of vaccaria flavonoid glycoside according to claim 1 for the preparation of a medicament for the treatment of sepsis, characterized in that: the medicament for treating sepsis is a medicament capable of inhibiting an FKBP 5/NF-kB inflammatory signal pathway of cells.

4. The use of the cowherb seed flavonoid glycoside according to claim 1 in the preparation of a medicament for the treatment of sepsis, characterized in that: the medicament for treating sepsis is a medicament capable of improving the survival rate of animals infected with sepsis.

5. The use of vaccaria flavonoid glycoside according to claim 4, characterized by: the medicament for treating sepsis is a medicament capable of reducing serum inflammatory factors of animals infected with sepsis and improving the pathology of kidney tissues and lung tissues of the animals infected with sepsis.

6. The use of vaccaria flavonoid glycoside according to claim 5, characterized by: the medicament for treating sepsis is a medicament capable of inhibiting the expression level of inflammatory factors and chemokines in kidney tissues and lung tissues of animals infected with sepsis.

7. The use of vaccaria flavonoid glycoside according to claim 6, characterized by: the inflammatory factors include IL-1 beta, IL-6, TNF-alpha, and the chemokines include MCP-1.

8. The use of vaccaria flavonoid glycoside according to claim 5, characterized by: the medicament for treating sepsis is a medicament capable of reducing creatinine and urea nitrogen in serum of kidney tissues of animals infected with sepsis.

9. The use of vaccaria flavonoid glycoside according to claim 5, characterized by: the medicament for treating sepsis is a medicament capable of improving pulmonary edema of an animal infected with sepsis.

10. Use of a cowherb seed flavonoid glycoside according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of sepsis, characterized in that: the medicament for treating sepsis further comprises a pharmaceutically acceptable carrier or auxiliary material.