Application of Mongolian medicine rhaponticum uniflorum alcohol extract in preparation of medicine for treating acute liver injury of mice

Abstract

The invention discloses application of a Mongolian medicine rhaponticum uniflorum alcohol extract in preparation of a medicine for treating acute liver injury of mice. A blank group (Con), a model group (APAP), a rhaponticum uniflorum alcohol extract low / medium / high dose group (LLF (L / M / H)) and a positive control group (NAC) are set. Results show that the LLF can improve APAP-induced general liver morphological injury and relieve tissue pathological necrosis and inflammatory infiltration. By activating an antioxidant pathway of Nrf2 / HO-1 / NQO1, the expression of Nrf2, HO-1 and NQO1 is up-regulated, the MDA level is reduced, and the activity of SOD and GSH is improved so as to resist oxidation. Meanwhile, proinflammatory factors such as TNF-alpha and IL-6 are inhibited, and the levels of liver injury enzymes such as ALT and AST are reduced. The liver protection effect is enhanced along with the increase of the LLF dosage, and the high-dosage group is close to the NAC group. It is shown that the rhaponticum uniflorum alcohol extract can relieve APAP acute liver injury in a dose-dependent mode, the mechanism relates to oxidation resistance, inflammation resistance and liver function repairing, and a basis is provided for development of the rhaponticum uniflorum alcohol extract into a liver protection medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Mongolian medicine application and drug research and development, and particularly relates to application of Mongolian medicine alcohol extract of radix eupatorii in preparation of a drug for treating acute liver injury of mice. BACKGROUND

[0002] As an important metabolism and detoxification organ of human body and animals, the liver is easily damaged by hepatitis virus, drug toxicity, food mold, alcohol and other factors, causing acute liver injury (ALF), leading to massive necrosis or apoptosis of hepatocytes, fatty degeneration, inflammatory reaction and oxidative stress, and even liver function damage, with a high mortality rate. Therefore, effective improvement of liver damage is crucial for the prevention and treatment of liver diseases.

[0003] Drug-induced liver injury (DILI) is a global serious public health problem, and ALF induced by acetaminophen (APAP) accounts for a dominant position. Excessive APAP produces a large amount of reactive oxygen species (ROS) in the liver, causing severe oxidative damage to acute liver injury. The drug N-acetylcysteine (NAC) for treating APAP-induced liver injury has certain deficiencies and side effects.

[0004] Rhaponticum uniflorum (L.) DC. is widely distributed, and its roots and rhizomes are used as medicine in traditional Chinese medicine, which has the effects of clearing heat, etc. In Mongolian medicine, its inflorescences are used as medicine, and the Mongolian Medicine Selection records that it has the functions of clearing epidemic heat, etc. and has a long history of clinical use in Mongolian medicine. Studies have shown that alcohol extract of Rhaponticum uniflorum can protect mice from acute liver injury caused by APAP and D-galactosamine, and the alcohol extract of Rhaponticum uniflorum can resist LPS-induced inflammatory response, which is closely related to the Nrf2 signaling pathway. Given the close relationship between APAP-induced acute liver injury and Nrf2, it is of great significance to explore the protective effect of alcohol extract of Rhaponticum uniflorum on APAP-induced acute liver injury in mice. However, the existing technology lacks systematic application and mechanism research of alcohol extract of Rhaponticum uniflorum in treating APAP-induced acute liver injury in mice. SUMMARY

[0005] The present application aims to solve the above technical problems, and provides application of Mongolian medicine alcohol extract of Rhaponticum uniflorum in preparation of a drug for treating acute liver injury of mice.

[0006] To solve the above technical problems, the technical scheme provided by the present application is as follows: application of Mongolian medicine alcohol extract of Rhaponticum uniflorum in preparation of a drug for treating acute liver injury of mice, which comprises inflorescences of Rhaponticum uniflorum (L.) DC. The alcohol extract can reduce liver injury by activating the Nrf2 / HO-1 signaling pathway in the liver. The drug is continuously administered by gavage for 7 days before APAP modeling, and significantly reduces serum ALT / AST levels within 6 hours after modeling.

[0007] Further, the drug is administered at a dose of 100-400 mg / kg body weight by gavage, continuously for 1 week, and the efficacy is evaluated by taking samples within 6 hours after APAP modeling.

[0008] Further, the liver protection includes at least one of the following mechanisms: (a) reducing the levels of ALT, AST, ALP and LDH in serum; (b) increasing the content of GSH and the activity of SOD in liver tissue; (c) reducing the levels of MDA, TNF-α, IL-6, IL-1β and IL-18 in liver tissue; (d) inhibiting liver macrophage infiltration, manifested as a decrease in F4 / 80 positive cells; (e) up-regulating the protein expression of Nrf2, HO-1 and NQO1 in liver; Wherein, the mechanisms are verified by immunohistochemistry, Western blotting or RT-PCR detection.

[0009] Further, the preparation method of the alcohol extract includes using dried inflorescences of Echinops ritro as raw material, and reflux extraction with 50-95% ethanol solution, and the extract is concentrated and dried to obtain; and the drug is configured to prevent or reduce APAP-induced liver histopathological damage, including hepatocyte necrosis, fatty degeneration and inflammatory infiltration.

[0010] Further, the application uses a BALB / c mouse model, the mice are male and weigh 18-22 g; modeling is performed by intraperitoneal injection of 400 mg / kg APAP; the drug is used to replace or supplement N-acetylcysteine (NAC) in the treatment of APAP-induced liver injury.

[0011] Further, the drug is prepared in the form of oral preparations, including tablets, capsules, granules or oral liquids; and the efficacy evaluation is based on experimental grouping, including blank group, APAP group, LLF low (L), medium (M) and high (H) dose groups, and NAC group.

[0012] A pharmaceutical composition for treating acute liver injury in mice, comprising a therapeutically effective amount of the Echinops ritro flower alcohol extract of claim 1 as an active ingredient, and a pharmaceutically acceptable carrier; the composition is used to reduce hepatocyte necrosis and oxidative stress.

[0013] A method for evaluating the effect of alcohol extract of Echinops setiferum on acute liver injury, comprising the following steps: (a) Establishing an APAP-induced acute liver injury model in mice, using male BALB / c mice (body weight 20±2g), and grouping them into a blank group, an APAP group, an LLF dose group, and an NAC group; (b) Administering 100-400mg / kg of alcohol extract of Echinops setiferum to the model animals, and orally administering the drug for one week; (c) Taking samples at 6 hours after APAP modeling, and detecting serum ALT / AST levels, liver tissue Nrf2 / HO-1 / NQO1 protein expression, and IL-6 or TNF-α levels; (d) Analyzing the liver protection effect by pathological morphology observation, WB detection, or immunohistochemical analysis.

[0014] Compared with the prior art, the present application has the advantages that: the present application uses alcohol extract of Echinops setiferum, which is a traditional Mongolian medicine inflorescence with few side effects. By activating the Nrf2 / HO-1 signaling pathway, the present application reduces APAP-induced acute liver injury in mice through multiple pathways (regulating oxidative stress, inflammatory response, etc.), solves the shortcomings of existing NAC treatment, provides a new choice for the treatment of drug-induced liver injury, and lays a foundation for clinical application and mechanism research. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is an embodiment flowchart of the present application.

[0016] Figure 2 is a comparison chart of the actual morphology of the liver of different groups in Example 1 of the present application.

[0017] Figure 3 is an HE staining pathological morphology observation chart of different groups in Example 1 of the present application.

[0018] Figure 4 is an immunohistochemical staining result chart of Nrf2 protein in mouse liver tissue in Example 1 of the present application. Figure 5 is an immunohistochemical staining result chart of NQO1 protein in mouse liver tissue in Example 1 of the present application.

[0019] Figure 6 is an immunohistochemical staining result chart of F4 / 80 protein in mouse liver tissue in Example 1 of the present application.

[0020] Figure 7 is an immunohistochemical staining result chart of HO-1 protein in mouse liver tissue in Example 1 of the present application.

[0021] Figure 8is a histogram of ALP (alkaline phosphatase) level detection in serum and tissue homogenate of different groups in Example 1 of the present application.

[0022] Figure 9 is a histogram of ALT (alanine aminotransferase) level detection in tissue homogenate and serum of different groups in Example 1 of the present application.

[0023] Figure 10 is a histogram of AST (aspartate aminotransferase) level detection in tissue homogenate and serum of different groups in Example 1 of the present application.

[0024] Figure 11 is a histogram of GSH (glutathione) level detection in tissue homogenate and serum of different groups in Example 1 of the present application.

[0025] Figure 12 is a histogram of LDH (lactate dehydrogenase) level detection in tissue homogenate and serum of different groups in Example 1 of the present application.

[0026] Figure 13 is a histogram of MDA (malondialdehyde) level detection in tissue homogenate and serum of different groups in Example 1 of the present application.

[0027] Figure 14 is a histogram of SOD (superoxide dismutase) level detection in tissue homogenate and serum of different groups in Example 1 of the present application.

[0028] Figure 15 is a histogram of TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6) level detection in serum of different groups in Example 1 of the present application.

[0029] Figure 16 is a histogram of IL-1β (interleukin-1β) and IL-18 (interleukin-18) level detection in serum of different groups in Example 1 of the present application. DETAILED DESCRIPTION

[0030] (I) Material Preparation 1. Experimental animals: 72 male BALB / c mice were selected, with a body weight in the range of 18-22 g (actual experiment used mice with a body weight of 20±2 g), purchased from a supplier with experimental animal production qualification, and raised in an environment meeting animal ethics requirements. After adapting to the environment for several days, the experiment was carried out.

[0031] 2. Reagents and drugs: The dried inflorescences of Rhaponticum uniflorum (L.) DC. were used as the raw material for the preparation of the alcohol extract of LLF. APAP (acetaminophen) and NAC (N-acetylcysteine) were purchased from a regular chemical reagent company. Ethanol (50-95% for the extraction of the alcohol extract of LLF), ELISA detection kits (for the detection of ALT, AST, ALP, LDH, GSH, MDA, TNF-a, IL-6, IL-1b, IL-18, and other indicators in serum), immunohistochemical reagents (such as specific antibodies F4 / 80, Nrf2, HO-1, NQO1, and staining reagents), WB detection reagents (protein extraction reagents, electrophoresis reagents, membrane transfer reagents, and antibodies), and RT-PCR reagents (RNA extraction reagents, reverse transcription reagents, and PCR amplification reagents) were purchased from a well-known biological reagent company to ensure the quality of the reagents.

[0032] 3. Instruments and equipment: Reflux extraction device (for the extraction of the alcohol extract of LLF, to realize the ethanol reflux extraction process), microplate reader (for ELISA detection, to read the absorbance value for the quantification of indicators), tissue sectioning machine, microscope (for pathological morphological observation and immunohistochemical result observation), protein electrophoresis instrument, membrane transfer instrument, chemiluminescence imaging system (for WB detection, to realize protein electrophoresis separation, membrane transfer, and result imaging analysis), PCR amplifier (for RT-PCR detection, to amplify gene fragments), and the like. The instruments were calibrated to ensure the accuracy of the experimental data.

[0033] (II) Preparation of the alcohol extract of LLF The dried inflorescences of Rhaponticum uniflorum (L.) DC. were crushed into coarse powder. An appropriate amount of the coarse powder was weighed and added to 50-95% ethanol solution (the solid-liquid ratio can be adjusted according to the actual situation, generally 1:10-1:20). The mixture was placed in a reflux extraction device and heated for 1-3 hours (the extraction time was determined based on the pre-experiment to ensure the sufficient extraction of effective components). The extraction was repeated 2-3 times. The extraction solutions were combined and concentrated by a vacuum concentration device (such as a rotary evaporator) to remove ethanol and obtain a concentrated solution. The concentrated solution was dried under vacuum (or freeze-dried, depending on the stability of the components) to obtain the alcohol extract of LLF, which was ready for use.

[0034] (III) Experimental grouping and drug administration 1. Grouping: 72 mice were randomly divided into 6 groups, namely, the control group (Control), the APAP group (400 mg / kg), the LLF (alcohol extract of LLF) low-dose group (100 mg / kg), the LLF medium-dose group (200 mg / kg), the LLF high-dose group (400 mg / kg), and the NAC group (as a positive control), with 12 mice in each group.

[0035] 2. Administration: LLF mice in each dose group were administered with the corresponding dose of LLF flower alcohol extract, which was prepared into appropriate concentration with normal saline, and administered by gavage once a day for one week. The blank group and the APAP group were administered with the same volume of normal saline by gavage. The NAC group was administered with appropriate concentration of NAC by gavage, and the administration period was the same as that of the LLF group. One hour after the last gavage of LLF (or normal saline, NAC), the mice in the APAP group, the LLF group in each dose, and the NAC group were injected intraperitoneally with APAP (400 mg / kg) to establish the model, except for the blank group.

[0036] (Four) Sample collection and processing Six hours after modeling, the mice were anesthetized by appropriate anesthesia (such as ether inhalation anesthesia), and the eyeball was taken for blood collection, and the serum was separated for subsequent serum index detection. The liver tissue was removed, and a part was used for pathological morphological observation (after fixation, embedding, sectioning, etc.), and a part was used for preparation of tissue homogenate (for detection of related indexes in tissue), immunohistochemical detection, protein extraction and Western blotting detection, RNA extraction and RT-PCR detection, etc., and the operation was standardized according to the sample processing requirements of each detection method.

[0037] (Five) Implementation of detection method 1. Liver tissue pathological detection: The treated liver tissue sections were stained with hematoxylin-eosin (HE), and the pathological morphological changes such as hepatocyte necrosis, fatty degeneration and inflammatory infiltration were observed under an optical microscope. Semi-quantitative or qualitative analysis was performed according to the degree of pathological change to evaluate the improvement effect of the drug on liver tissue pathological injury.

[0038] 2. Serum and tissue homogenate index detection: According to the ELISA kit instruction, the levels of ALT, AST, ALP and LDH in serum were detected, and the levels of GSH, MDA, SOD, TNF-α, IL-6, IL-1β and IL-18 in liver tissue homogenate were detected. The operation steps were strictly followed, the standard and blank controls were set, and the data were read by enzyme-labeled instrument. The contents of each index were calculated, and the regulation effect of the drug on these indexes was analyzed.

[0039] 3. Immunohistochemical detection: The liver tissue sections were prepared and stained by immunohistochemistry. The steps of dewaxing, antigen repair, blocking, primary antibody (F4 / 80, Nrf2, HO-1, NQO1 specific antibody) incubation, secondary antibody incubation, color development (DAB color development), and re-staining were carried out in turn. The positive expression (such as the number of positive cells, staining intensity, etc.) was observed under a microscope, and semi-quantitative analysis was performed by image analysis software to judge the influence of the drug on macrophage infiltration and related protein expression.

[0040] 4. Liver protein extraction and Western blotting detection: Extract liver tissue protein, after measuring protein concentration, perform SDS-PAGE electrophoresis (separate proteins according to different protein molecular weights), transfer membrane (transfer proteins to nitrocellulose membrane or PVDF membrane), after blocking, add primary antibody (specific antibody for Nrf2, HO-1, and other signal pathway related proteins) and incubate, incubate with secondary antibody, finally perform chemiluminescence color development, use chemiluminescence imaging system to obtain images, analyze target protein expression level, and explore the regulatory effect of drugs on Nrf2 / HO-1 signal pathway related protein expression.

[0041] 5. RT-PCR detection: Extract liver tissue RNA, after detecting RNA quality and concentration, perform reverse transcription to synthesize cDNA, use cDNA as template for PCR amplification, amplify Nrf2, HO-1, NQO1 and other related gene fragments, detect amplification products by agarose gel electrophoresis or real-time fluorescent quantitative PCR, and analyze related gene transcription level to explore drug mechanism from gene level.

[0042] (Six) Conclusion Multi-dimensional result verification of drug hepatoprotective effect 1. Liver gross morphology Figure 2 ) APAP group: liver swelling, dark color, irregular surface, showing typical acute liver injury morphology; LLF group: with increasing dose, liver swelling is reduced, color is gradually reddish, surface is smoother, and high-dose group (LLF(H)) is close to normal; NAC group: liver injury is improved, and the LLF group verifies that the drug can repair the gross morphology of liver damage.

[0043] 2. Histopathological morphology Figure 3 ) APAP group: hepatocyte necrosis and obvious inflammatory infiltration (red arrow mark damage area); LLF group: hepatocyte necrosis and inflammation degree decrease with increasing dose, and high-dose group liver lobule structure is basically clear; Conclusion: Eupatorium lindleyanum flower alcohol extract can repair liver histopathological damage and reduce cell necrosis and inflammation.

[0044] 3. Antioxidant pathway protein expression Figures 4-7 ) APAP group: Nrf2, HO-1, and NQO1 positive staining is weak, indicating that the oxidative stress pathway is inhibited; LLF group: with increasing dose, positive staining is enhanced (brown particles increase), indicating that the drug activates the Nrf2 / HO-1 pathway and up-regulates the expression of antioxidant proteins; Mechanism support: through the activation of "Nrf2→HO-1 / NQO1" antioxidant pathway, enhance the antioxidant capacity of the liver.

[0045] 4. Inflammation and oxidative indicators (MDA, SOD, GSH) Figures 8-16 ) Oxidative damage: MDA in APAP group increased, SOD / GSH decreased; LLF group reversed the regulation, with the increase of dose, MDA decreased, SOD / GSH increased; Inflammatory factors: TNF-α, IL-6 and other pro-inflammatory factors in APAP group soared; pro-inflammatory factors in LLF group decreased with the increase of dose, and the anti-inflammatory effect was significant; Liver function enzymes: ALT, AST, ALP and LDH in APAP group increased (liver damage markers); enzyme levels in LLF group decreased with the increase of dose, suggesting that the drug repaired hepatocytes and improved liver function Echinops flower alcohol extract can dose-dependently improve APAP-induced acute liver injury in mice, the mechanism includes: repair the gross morphology and histopathological structure of the liver; activate Nrf2 / HO-1 / NQO1 antioxidant pathway, reduce oxidative damage; inhibit the release of pro-inflammatory factors, relieve liver inflammation; reduce liver damage enzyme levels, restore hepatocyte function.

[0046] This example verifies the hepatoprotective effect and dose-effect relationship of Echinops flower alcohol extract through "gross morphology→tissue→molecule→biochemistry" multidimensional verification, and provides a complete evidence chain for its development as a liver injury treatment drug.

[0047] The above describes the present application and its embodiments, which is not restrictive, and the shown in the drawings is only one of the embodiments of the present application, and the actual structure is not limited thereto. In summary, if a person skilled in the art is inspired thereby, without departing from the purpose of the present application, without creative design, similar structure and embodiments of the technical solution can be designed, which shall belong to the protection scope of the present application.

Claims

1. The use of Mongolian medicine Radix Erodii Herba alcohol extract in the preparation of a drug for treating acute liver injury in mice, characterized in that: The plantain flower is a dry inflorescence of Rhaponticum uniflorum (L.) DC. of the Compositae family; The alcohol extract reduces liver injury by activating the liver Nrf2 / HO-1 signaling pathway; The drug is continuously administered by gavage for 7 days before APAP modeling, and significantly reduces serum ALT / AST levels within 6 hours after modeling.

2. The use of the Mongolian medicine Erodii Herba alcohol extract of claim 1 in the preparation of a drug for treating acute liver injury in mice, characterized in that: The drug is administered at a dose of 100-400 mg / kg of body weight, administered by gavage, administered continuously for 1 week, and sampled within 6 hours after APAP modeling to evaluate efficacy.

3. The use of the alcohol extract of the Mongolian medicinal plant Erodium stephanianum Willd. according to any one of claims 1 or 2 for the preparation of a medicament for the treatment of acute liver injury in mice, characterized in that: The liver protection includes at least one of the following mechanisms: (a) reducing the levels of ALT, AST, ALP, and LDH in serum; (b) increasing the content of GSH and the activity of SOD in liver tissue; (c) reducing the levels of MDA, TNF-α, IL-6, IL-1β, and IL-18 in liver tissue; (d) inhibiting liver macrophage infiltration, manifested as a decrease in F4 / 80 positive cells; (e) up-regulating the protein expression of Nrf2, HO-1, and NQO1 in the liver; The mechanisms are verified by immunohistochemistry, Western blotting, or RT-PCR detection.

4. The use of the alcohol extract of the Mongolian medicine Erodium stephanianum Willd. in the preparation of a drug for treating acute liver injury in mice according to claim 1, characterized in that: The preparation method of the alcohol extract includes using 50-95% ethanol solution to reflux extract the dry inflorescence of Rhaponticum uniflorum (L.) DC. as the raw material, and obtaining the extract after concentration and drying; and the drug is configured to prevent or reduce APAP-induced liver histopathological injury, including hepatocyte necrosis, fatty degeneration, and inflammatory infiltration.

5. The use of the Mongolian medicine Erodii Herba alcohol extract of claim 1 in the preparation of a medicament for treating acute liver injury in mice, characterized in that: The application uses a BALB / c mouse model, the mice are male and weigh 18-22 g; modeling is performed by intraperitoneal injection of 400 mg / kg APAP; and the drug is used to replace or supplement N-acetylcysteine (NAC) in the clinical application of treating APAP liver injury.

6. The use of the alcohol extract of the Mongolian medicine Erodium stephanianum Willd. in the preparation of a drug for treating acute liver injury in mice according to claim 2, characterized in that: The drug is prepared in the form of oral preparations, including tablets, capsules, granules, or oral liquids; and the efficacy evaluation is based on experimental grouping, including a blank group, an APAP group, LLF low (L), medium (M), and high (H) dose groups, and an NAC group.

7. A pharmaceutical composition for treating acute liver injury in mice, characterized by: The composition contains a therapeutically effective amount of the plantain flower alcohol extract of claim 1 as an active ingredient, and a pharmaceutically acceptable carrier; and is used to reduce hepatocyte necrosis and oxidative stress.

8. A method for evaluating the effect of a liquorice flower alcohol extract on acute liver injury, characterized in that: The method includes the following steps: (a) establishing an APAP-induced acute liver injury model in mice, using male BALB / c mice (20±2 g in weight), and grouping the mice into a blank group, an APAP group, LLF dose groups, and an NAC group; (b) administering 100-400 mg / kg of the plantain flower alcohol extract to the model animals by gavage for continuous one week; (c) sampling within 6 hours after APAP modeling, detecting serum ALT / AST levels, liver tissue Nrf2 / HO-1 / NQO1 protein expression, and IL-6 or TNF-α levels; (d) analyzing the liver protection effect by pathological morphology observation, WB detection, or immunohistochemical analysis.

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