Application and method of magnolol in preparation of medicine for treating acute inflammatory diseases

Abstract

The invention relates to the field of biological medicines, in particular to application and a method of magnolol in preparation of a medicine for treating acute inflammatory diseases. The invention provides an application of magnolol in preparation of a medicine for treating acute inflammatory diseases, which is characterized in that the medicine is used for treating subjects with purine nucleotide cycle metabolism abnormality and mitochondrial energy metabolism abnormality. The invention also provides a method for evaluating the protective effect of magnolol. By callback of fumaric acid level of subjects, abnormal purine nucleotide circulation metabolic pathways and mitochondrial energy metabolic pathways of acute pancreatitis patients can be effectively improved, and individualized treatment of the acute pancreatitis patients is realized.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to an application and method of magnolol in preparing a medicine for treating acute inflammatory diseases. Background Art

[0002] Acute pancreatitis refers to a disease characterized by activation of pancreatic enzymes due to various causes, followed by local inflammation of the pancreas. In severe cases, systemic inflammatory response syndrome (SIRS) may occur, and it may be accompanied by organ dysfunction. The main symptoms of acute pancreatitis are acute onset of persistent severe upper abdominal pain, which often radiates to the back, often accompanied by abdominal distension, nausea and vomiting. The incidence of acute pancreatitis is increasing year by year.

[0003] The condition of acute pancreatitis changes rapidly, especially severe acute pancreatitis (SAP), which has many complications and a high mortality rate. Systemic inflammatory response syndrome (SIRS) may occur in the early stage, leading to circulatory, respiratory or renal failure, accompanied by problems such as intestinal dysfunction and intra-abdominal hypertension. Acute pancreatitis is often accompanied by a variety of complications, such as pancreatic necrosis, infected necrosis (IPN), and multiple organ failure. These complications are difficult to treat, especially the treatment of infected necrosis, which requires timely diagnosis and active intervention, otherwise it will significantly increase the mortality rate.

[0004] Acute pancreatitis is a complex inflammatory disease of the pancreas, often caused by a variety of reasons, including cholelithiasis, alcohol, hyperlipidemia, etc. At present, there is no completely accurate standard for the diagnosis of acute pancreatitis. Clinically, the diagnosis of acute pancreatitis is mainly assisted by observing laboratory test results such as blood and urine amylase, serum lipase, inflammatory indicators, and abdominal B-ultrasound, CT and other imaging examinations. In addition, the basic treatment principles of acute pancreatitis are fasting and drinking, gastrointestinal decompression, active and effective fluid resuscitation, enzyme inhibition, acid inhibition, and anti-infection, hormone, surgery and other treatments as needed. It is basically symptomatic supportive treatment, and there is no specific treatment method.

[0005] Despite the improved understanding of the pathogenesis of acute pancreatitis, patients are still at significant risk due to the lack of targeted drug treatments. Therefore, effective drug treatment measures are urgently needed to control the occurrence and progression of acute pancreatitis and prevent subsequent organ failure. Summary of the invention

[0006] In a first aspect, the present invention provides a use of magnolol in the preparation of a medicament for treating acute inflammatory diseases, wherein the medicament is used to treat subjects with abnormal purine nucleotide cycle metabolism and abnormal mitochondrial energy metabolism.

[0007] In some embodiments, the medicament contains magnolol as the only active ingredient.

[0008] In some embodiments, the subject has reduced fumarate levels.

[0009] In some embodiments, the acute inflammatory disease comprises acute pancreatitis.

[0010] In some embodiments, the subject has or is suspected of having acute pancreatitis.

[0011] As used herein, "subject" refers to a subject in need of disease treatment, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses and cows, but the present invention is not limited thereto.

[0012] In some embodiments, the medicament may further include a pharmaceutically acceptable carrier.

[0013] In some embodiments, the medicament is formulated for intravenous injection, subcutaneous injection, or oral administration.

[0014] In some embodiments, the magnolol includes magnolol and pharmaceutically acceptable salts thereof.

[0015] In some embodiments, the dosage of the drug comprises 2.5-10 mg magnolol / kg body weight.

[0016] In certain embodiments, fumaric acid level refers to the absolute (quantitative) or relative (qualitative) value or concentration of fumaric acid in a sample. In certain embodiments, a suitable method can be used to analyze the sample collected to detect the level of fumaric acid in the sample. After the level of fumaric acid in the assay sample, the level of fumaric acid can be compared with a reference value. The reference value can be from a normal population (i.e., non-acute pancreatitis patients). If the level of fumaric acid in the experimenter is compared with the reference value, there is a reduction (i.e., not higher than, for example, the same as the reference value, substantially the same as the reference value, not statistically different from the reference value, being in a value within a predetermined range of the reference value), then the experimenter is assessed as having a reduced level of fumaric acid.

[0017] In certain embodiments, sample can comprise any biological material applicable to detecting required index, and can comprise cellular material and / or non-cellular material.In certain embodiments, sample can be separated from any suitable biological tissue or fluid.In certain embodiments, sample can be the biological material separated from experimenter.In certain embodiments, sample can be whole blood, serum or blood plasma, i.e. the fumaric acid level of whole blood, serum or blood plasma of described experimenter is reduced.

[0018] In some embodiments, the abnormal mitochondrial energy metabolism comprises an abnormality in the ATP synthesis pathway of the subject's mitochondria.

[0019] In some embodiments, the abnormal mitochondrial energy metabolism comprises an abnormality in the ATP synthesis pathway of mitochondria of pancreatic acinar cells of the subject.

[0020] In some embodiments, the subject's cellular oxygen consumption rate is reduced.

[0021] In some embodiments, the subject's pancreatic acinar cells have a reduced cellular oxygen consumption rate.

[0022] In some embodiments, the acute pancreatitis is moderate to severe acute pancreatitis or severe acute pancreatitis.

[0023] In a second aspect, the present invention also provides the use of a fumaric acid detection reagent in preparing a kit for detecting the therapeutic effect of magnolol on an acute inflammatory disease in a subject.

[0024] In some embodiments, the kit is used to detect the level of fumaric acid in a sample collected from the subject to detect whether the purine nucleotide cycle metabolic pathway and the mitochondrial energy metabolic pathway of the subject are abnormal before the administration of magnolol.

[0025] In some embodiments, when the fumarate level of the subject is reduced, the magnolol is assessed to have a therapeutic effect on the acute inflammatory disease in the subject.

[0026] In some embodiments, the reduced fumarate level is a reduced fumarate level in the subject's whole blood, serum, or plasma.

[0027] In some embodiments, the sample is a whole blood, serum or plasma sample.

[0028] In some embodiments, the acute inflammatory disease comprises acute pancreatitis.

[0029] In some embodiments, the subject has or is suspected of having acute pancreatitis.

[0030] In some embodiments, the magnolol includes magnolol and pharmaceutically acceptable salts thereof.

[0031] In some embodiments, the acute pancreatitis is moderate to severe acute pancreatitis or severe acute pancreatitis.

[0032] In a third aspect, the present invention further provides a method for evaluating the protective effect of magnolol, characterized in that the method comprises: S101 culturing the target cells to obtain a first culture; In some embodiments, the target cell may be a cell rich in mitochondria and having a large energy metabolism requirement, such as a pancreatic acinar cell, a cardiomyocyte, a hepatocyte, or a neuronal cell.

[0033] In some embodiments, the target cell is a cell model of a disease.

[0034] In some embodiments, the target cell is derived from a subject with acute pancreatitis.

[0035] In some embodiments, S101 further comprises adding a suitable amount of substance P, for example, 10 μM.

[0036] S102 detecting the fumaric acid level and the cellular oxygen consumption rate in the first culture to obtain a first fumaric acid level and a first oxygen consumption rate; S103 culturing the target cells with magnolol at the first concentration to obtain a second culture; In some embodiments, the first concentration comprises 6.25 - 25 μM.

[0037] In some embodiments, the target cell mass may be 1×10 6 – 10 8 indivual.

[0038] S104 detecting the fumaric acid level and the cellular oxygen consumption rate in the second culture to obtain a second fumaric acid level and a second oxygen consumption rate; S105: evaluating whether purine nucleotide metabolism in the target cells is restored or improved according to the first fumaric acid level and the second fumaric acid level; if so, proceeding to S106; if not, evaluating that magnolol is difficult to provide a protective effect on the target cells; In some embodiments, if the second fumarate level is increased compared to the first fumarate level, then purine nucleotide metabolism in the target cell is assessed to be restored or enhanced.

[0039] S106, evaluating whether mitochondrial energy metabolism in the target cell is restored or improved according to the first oxygen consumption rate and the second oxygen consumption rate; if so, evaluating that magnolol can provide a protective effect on the target cell; if not, evaluating that magnolol is difficult to provide a protective effect on the target cell; In some embodiments, mitochondrial energy metabolism in the target cell is assessed to be restored or enhanced if the second oxygen consumption rate is increased compared to the first oxygen consumption rate.

[0040] The protective effect includes providing a protective effect on the mitochondrial function of the target cells.

[0041] Magnolol is a natural organic compound (molecular formula C18H1802, molecular weight 266.34, CAS registration number 528-43-8), which can be extracted from the root of the plant Magnolia officinalis. It is relatively cheap and easy to obtain. The present invention finds that magnolol can effectively improve mitochondrial function by regulating the purine nucleotide cycle metabolic pathway and mitochondrial energy metabolic pathway in cells. Therefore, magnolol can be used as a more accessible mitochondrial function regulating reagent for application scenarios such as scientific research (such as research related to mitochondrial function). However, magnolol does not have a protective effect on every type of cell. In view of this, the present invention provides a method for evaluating the protective effect of magnolol. On the one hand, the method provided by the present invention can evaluate whether magnolol has a protective effect on target cells (such as providing a protective effect on the mitochondrial function of target cells) by judging the changes in the level of fumaric acid (and the changes in the cell oxygen consumption rate) caused by abnormal purine nucleotide metabolism. On the other hand, the method provided by the present invention can also be used in combination with other drugs (such as substance P) to assist in evaluating related molecular mechanisms, saving time while also saving experimental costs such as pathway analysis.

[0042] In a fourth aspect, the present invention also provides the use of magnolol in regulating the purine nucleotide cycle metabolic pathway and / or mitochondrial energy metabolic pathway of cells in vitro.

[0043] In some embodiments, the magnolol regulates the purine nucleotide cycle metabolic pathway and / or the mitochondrial energy metabolic pathway of the cell by regulating the fumarate level of the cell.

[0044] In some embodiments, the cell comprises a pancreatic acinar cell, a cardiomyocyte, a hepatocyte, or a neuronal cell.

[0045] In some embodiments, the cell is a cellular model of a disease.

[0046] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: Acute pancreatitis is a complex inflammatory disease of the pancreas, often caused by a variety of reasons, with rapid changes in the condition, many complications and high mortality. Although there are many treatment options for acute pancreatitis, they are basically symptomatic supportive treatments. In other words, the current difficulty in treating acute pancreatitis lies in the difficulty in achieving individualized treatment for patients with acute pancreatitis.

[0047] The present invention finds that magnolol can effectively treat patients with acute pancreatitis with abnormal purine nucleotide cycle metabolism and abnormal mitochondrial energy metabolism. Although the purine nucleotide cycle metabolism pathway and the mitochondrial energy metabolism pathway are two relatively independent pathways, the present invention finds that the change in the level of fumaric acid can serve as a "bridge" between the above two pathways, that is, the purine nucleotide cycle metabolism pathway and the mitochondrial energy metabolism pathway of the subject can be evaluated by whether the subject's fumaric acid level (especially the fumaric acid level of whole blood, serum or plasma that is easy to collect) is reduced. Whether it is abnormal. In addition, acute pancreatitis can also be distinguished from diseases with similar symptoms by whether the subject's fumaric acid level is reduced and the degree of reduction, thereby achieving precise treatment of acute pancreatitis. The experimental verification of the present invention shows that magnolol can effectively improve the abnormal purine nucleotide cycle metabolism pathway and mitochondrial energy metabolism pathway of patients with acute pancreatitis by adjusting the fumaric acid level of the subject (for example, in acinar cells), thereby achieving individualized treatment of patients with acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0049] Figure 1 The results of fumaric acid levels in pancreatic acinar cells of different groups are shown in the graph; Figure 2 The results of the cellular oxygen consumption rate of pancreatic acinar cells in different groups are shown in FIG. Figure 3 The results of ATP content in pancreatic acinar cells of different groups are shown in Fig. Figure 4 The transmission electron microscopy results of mitochondria in different groups of pancreatic acinar cells; Figure 5 The results of metabolic enzymes related to purine nucleotide cycle in different groups of pancreatic acinar cells are shown in FIG. Figure 6 The figure is the result of fumaric acid level in peripheral blood of different clinical samples; Figure 7The results show that magnolol has an improving effect on the local and systemic symptoms of the acute pancreatitis mouse model induced by caerulein combined with substance P (A shows the plasma amylase level, plasma lipase level, pancreatic myeloid peroxidase level and pulmonary myeloid peroxidase level of mice in different groups; B shows the total pathological score, edema, inflammation and necrosis score of mice in different groups); Figure 8 For the pancreas Tacr1 The results of gene expression of key enzymes of purine nucleotide metabolism in mice after gene-specific knockout; Fig. 9 This figure shows the results that magnolia officinalis has no effect on improving local and systemic symptoms in the animal model of acute pancreatitis with normal fumaric acid levels (A shows the plasma amylase level, plasma lipase level, pancreatic myeloid peroxidase level, pulmonary myeloid peroxidase level and trypsin level of mice in different groups; B shows the representative H&E staining results of mice in different groups, as well as the total pathological score, edema, inflammation and necrosis scores). DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0051] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0052] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0053] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0054] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0055] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0056] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0057] Embodiment 1 Freshly isolated pancreatic acinar cells (approximately 3 × 10 7 The pancreatic acinar cells were homogenized in the experimental buffer and centrifuged at 13000 g for 10 min to eliminate insoluble substances. Test samples and fumaric acid standards were prepared in the experimental buffer at 50 μL / well on a 96-well plate. 100 μL of reaction mixture, fumaric acid solution / fumaric acid standard, and test sample were filled into each well. The reaction was kept away from light and incubated at 37°C for 60 min. The absorbance of the microplate reader was detected at 450 nm.

[0058] like Figure 1As shown, compared with the control group, the fumaric acid level in the pancreatic acinar cells of the model group was reduced to a certain extent, while the addition of magnolol could significantly increase the fumaric acid level in the pancreatic acinar cells of the model group, even higher than that of the control group.

[0059] Embodiment 2 The night before the test, the Seahorse xfe24 sensor was hydrated and incubated overnight at 37°C. On the first day of the experiment, 100 μL of 1× polysaccharide was added to the cell plate, incubated at 37°C for 30 minutes, and then washed with PBS.

[0060] Freshly isolated pancreatic acinar cells were divided into five groups: control group, model group (final concentration of substance P was 10 μM), and different concentrations of drug administration groups (final concentrations of magnolol were 6.25 μM, 12.5 μM, and 25 μM, respectively). 400 μL of culture medium and 100 μL of cell solution were added to each group. Before starting the experiment, the hydration plate was calibrated. After calibration, the hydration plate was replaced with a cell plate for testing. 56 μL of 1.5 μM oligo was pipetted into well A in each well. 62 μL of 0.5 μM FCCP was pipetted into well B in each well. 69 μL of 0.5 μM Rot was pipetted into well C in each well. The OCR (oxygen consumption rate of living cells) was then automatically calculated by a Seahorse XF-24 analyzer (Seahorse Bioscience, CA, USA).

[0061] like Figure 2 As shown, compared with the control group, the oxygen consumption rate of pancreatic acinar cells in the model group was reduced to a certain extent, while the addition of magnolol (12.5 μM magnolol and 25 μM magnolol) could significantly increase the oxygen consumption rate of pancreatic acinar cells in the model group.

[0062] Embodiment 3 Freshly isolated pancreatic acinar cells were divided into three groups: control group, model group (final concentration of substance P was 10 μM), and drug group (final concentration of magnolol was 25 μM). After stimulation, the cells were lysed with lysis buffer on ice to release ATP, and then the OD value was read on an ELISA reader and the ATP level was calculated. Finally, the amount of cell protein in each group was measured for data standardization.

[0063] like Figure 3 As shown, compared with the control group, the ATP content of pancreatic acinar cells in the model group decreased to a certain extent, while the addition of magnolol could significantly increase the ATP content in pancreatic acinar cells in the model group. The above results indicate that the mitochondrial function of pancreatic acinar cells in the model group is damaged (specifically manifested as a slowdown in the rate of oxidative phosphorylation), and the addition of magnolol can protect the mitochondrial function of pancreatic acinar cells in the model group.

[0064] Embodiment 4 Freshly isolated pancreatic acinar cells were divided into three groups: control group, model group (final concentration of substance P was 10μM), and drug group (final concentration of magnolol was 25μM). They were fixed with 2.5% glutaraldehyde (pH = 7.4) at 4°C for 4h. They were fixed with 1% osmium tetroxide, dehydrated with acetone, and then infiltrated with dehydrating agent and Epon-812 embedding agent at a ratio of 3:1. After embedding, the samples were made into ultrathin sections, stained with uranyl acetate for 10-15 min, and stained with lead citrate for 1-2 min. Transmission electron microscopy was used for observation and photography.

[0065] like Figure 4 As shown, the results of transmission electron microscopy showed that the degree of mitochondrial damage in the model group was higher, and the addition of magnolol could reduce the degree of mitochondrial damage in the pancreatic acinar cells of the model group.

[0066] Embodiment 5 Freshly isolated pancreatic acinar cells were divided into three groups: control group, model group (final concentration of substance P was 10 μM) and drug group (final concentration of magnolol was 25 μM). Incubate at room temperature for 30 minutes, centrifuge and add 200 μl RIPA to lyse, use BCA kit to determine protein concentration, add SDS-PAGE loading buffer and boil immediately. Samples were cooled on ice for 2 minutes before Western blot analysis. Equal amounts of protein samples were separated on SDS-7.5% PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (10 μl per sample, 250 mA, 1.5 h), blocked with 5% dry skim milk for 1 h, and incubated with adenylo succinate synthetase (ADSS) (1:1000), adenylo succinate lyase (ADSL) (1:1000), adenosine monophosphate deaminase (AMPD) (1:1000), and internal control protein (vinculin) (1:1000) at 4°C overnight. Goat anti-mouse IgG-HRP secondary antibody (1:10000) or goat anti-rabbit IgG-HRP secondary antibody (1:40 000) was incubated at room temperature for 1 h.

[0067] like Figure 5 As shown, the protein expression of three metabolic enzymes ADSS, ADSL and AMPD in the purine nucleotide cycle of pancreatic acinar cells in the model group was reduced, while magnolol could restore their expression.

[0068] The above results indicate that the purine nucleotide cycle metabolism of pancreatic acinar cells in the model group is abnormal, and magnolol can improve mitochondrial damage by improving the purine nucleotide metabolism-mitochondrial mechanism.

[0069] Embodiment 6 Based on the above results, this example detected the fumaric acid levels in peripheral blood of 147 clinical samples. The results are as follows: Figure 6 As shown. The results showed that the peripheral blood fumaric acid levels of most healthy people (HV) were high, while the peripheral blood fumaric acid levels of some patients with acute pancreatitis were reduced. Overall, compared with normal subjects, patients with moderate to severe acute pancreatitis (MSAP) and severe acute pancreatitis (SAP) had lower fumaric acid levels. The above results show that the experiments carried out on pancreatic acinar cells in the above embodiments are representative, and some patients with acute pancreatitis have abnormal purine nucleotide cycle metabolism and abnormal mitochondrial energy metabolism. Therefore, for patients with acute pancreatitis with reduced fumaric acid levels, magnolol can regulate their abnormal purine nucleotide metabolism and mitochondrial energy metabolism by adjusting their fumaric acid levels, which is particularly suitable for individualized treatment of patients with moderate to severe acute pancreatitis and severe acute pancreatitis.

[0070] Embodiment 7 Model establishment: Male C57BL / 6 mice were randomly divided into groups and intraperitoneally injected with 50 μg / kg caerulein for 7 consecutive times, with an interval of 1 h between each injection.

[0071] Animal grouping and treatment: Male C57BL / 6 mice were randomly divided into five groups: normal control group (N=6), acute pancreatitis group (N=7), and three dose groups of magnolol compounds: low, medium, and high (2.5, 5, and 10 mg / kg, N=7, respectively).

[0072] Normal control group: intraperitoneal injection of saline solution with the same dose as cerulein solution was performed 7 times in succession, with an interval of 1 hour between each injection. The first injection of cerulein was recorded as 0 h, and the same amount of solvent as the test drug was injected at 4, 5 and 6 h.

[0073] Acute pancreatitis group: cerulein was injected intraperitoneally for 7 times, with an interval of 1 hour between each injection. The first injection of cerulein was recorded as 0 h, and substance P (449.2 μg / kg) and the same amount of solvent as the test drug were injected at 4, 5 and 6 h.

[0074] Low-dose drug group: intraperitoneal injection of caerulein, 7 times in succession, 1 hour interval between each injection. The first injection of caerulein was marked as 0 h, and substance P (449.2 μg / kg) and drug (2.5 mg / kg) were injected at 4, 5 and 6 h.

[0075] Medium-dose drug group: intraperitoneal injection of caerulein, 7 times in succession, 1 hour interval between each injection. The first injection of caerulein was marked as 0 h, and substance P (449.2 μg / kg) and drug (5 mg / kg) were injected at 4, 5 and 6 h.

[0076] High-dose drug group: intraperitoneal injection of caerulein, 7 times in succession, 1 hour interval between each injection. The first injection of caerulein was marked as 0 hour, and substance P (449.2μg / kg) and drug (10 mg / kg) were injected at 4, 5 and 6 hours.

[0077] Sample collection and testing: 12 hours after modeling: (1) Blood was collected under anesthesia and centrifuged at 1500 g for 15 min. The supernatant was collected and frozen at -80 °C for the determination of pancreatic amylase and pancreatic lipase. (2) The pancreatic tissue was divided into two parts. The head was placed in an embedding box and dynamically immersed in a 10% neutral formalin solution prepared in PBS. After embedding, HE staining and related research were performed; the other part of the pancreatic tissue was frozen in a -80 ℃ refrigerator for measuring myeloperoxidase activity; (3) The lung tissue was divided into two parts. One part was placed in an embedding box and placed in a 10% neutral formalin solution prepared with PBS. After embedding, it was stained with HE. The other part of the lung tissue was frozen in a -80 ℃ refrigerator for measuring myeloperoxidase activity.

[0078] The results are as follows Figure 7 ((A) Plasma amylase level; plasma lipase level; pancreatic myeloid peroxidase level; pulmonary myeloid peroxidase level; (B) total pathological score, edema, inflammation and necrosis score) showed that the pancreatic amylase and lipase of mice in the acute pancreatitis group were significantly increased, while the three concentrations of magnolol compounds at low, medium and high concentrations could significantly reduce these two indicators. Myeloperoxidase level is related to the content of neutrophils in tissues, from Figure 7 A It can be seen that the three concentrations of magnolol, low, medium and high, can significantly inhibit the content of myeloperoxidase. The pancreas of the acute pancreatitis model induced by caerulein combined with substance P in mice showed severe edema, inflammatory infiltration and necrosis, and different concentrations of magnolol compounds can alleviate various pathological scores to varying degrees. In summary, magnolol can alleviate the local and systemic symptoms of acute pancreatitis in mice by adjusting the level of fumaric acid.

[0079] Embodiment 8 Model establishment: Male C57BL / 6 mice were operated under sterile conditions. Isoflurane inhalation anesthesia was used, and the laparotomy was performed at a width of two fingers below the xiphoid process. After the liver was seen, the incision was started downwards, and a cotton swab soaked in saline was inserted into the abdominal cavity. The duodenum was found on the back of the liver. After it was turned out, it was placed on a sterile gauze. The pancreatic bile duct and duodenal papilla were found. A hole was made in the duodenum with a 1 mL syringe needle aligned with the papilla. A 24G indwelling needle hose was inserted into the pancreatic bile duct along the papilla. The common bile duct below the liver was closed with an arterial vascular clamp, and the injection was performed. Tacr1Knockout virus (AP_ Tacr1 -KO, 1.2×10 12 IU / ml), and then continue to close the common bile duct, slowly remove the hose, remove the vascular artery clamp, return the duodenum, double-layer suture the abdominal cavity, and feed for 28 days to obtain pancreatic specific Tacr1 Knockout mice.

[0080] Grouping and drug administration: After 28 days of feeding, 12 male Tacr1 The knockout mice were randomly divided into two groups (N=6): a control group and a drug-treated group (10 mg / kg).

[0081] Normal control group: intraperitoneal injection of cerulein, 7 times in succession, each injection 1 hour apart. The first injection of cerulein was recorded as 0 h, and the same amount of solvent as the test drug was injected at 4, 5 and 6 h.

[0082] Drug group: intraperitoneal injection of caerulein, 7 times in succession, 1 hour interval between each injection. The first injection of caerulein was recorded as 0h, and magnolol (10mg / kg) was injected at 4, 5 and 6 hours.

[0083] Sample collection and testing 12 hours after modeling: (1) Blood was collected under anesthesia and centrifuged at 1500 g for 15 min. The supernatant was collected and frozen at -80 °C for the determination of pancreatic amylase and pancreatic lipase. (2) The pancreatic tissue was divided into three parts. One part was placed in an embedding box and placed in a 10% neutral formalin solution prepared with PBS. After embedding, HE staining and related research were performed. The second part was added with RNA lysis solution, and then tissue RNA was extracted and reverse transcribed into cDNA for PCR detection. Adss , Adsl and Ampd The expression levels of three genes; the third part of the pancreatic tissue was taken and frozen in a -80 ℃ refrigerator and divided into two parts, which were used to measure trypsin activity and myeloperoxidase activity respectively; (3) The lung tissue was divided into two parts. One part was placed in an embedding box and placed in a 10% neutral formalin solution prepared with PBS. After embedding, it was stained with HE. The other part of the lung tissue was frozen in a -80 ℃ refrigerator for measuring myeloperoxidase activity.

[0084] from Figure 8 It can be seen that mice injected Tacr 1. After gene knockout virus and caerulein modeling, three key enzymes of the purine nucleotide cycle Adss , Adsl and AmpdThe gene expression of AP_ Tacr1 -KO mice had normal purine nucleotide metabolism.

[0085] from Fig. 9 ((A) Plasma amylase level; plasma lipase level; pancreatic myeloid peroxidase level; pulmonary myeloid peroxidase level; trypsin activity level; (B) Representative H&E staining images of the control group and drug group and the total pathological score, edema, inflammation and necrosis score) It can be seen that mice injected with Tacr After the 1 gene knockout virus and caerulein modeling, there was no statistical difference in the various indicators between the magnolol-treated and non-drug-treated groups. Therefore, magnolol compounds have no effect on improving the local and systemic symptoms of acute pancreatitis animal models with normal purine nucleotide metabolism (that is, normal fumaric acid levels), which indicates that magnolol mainly plays a protective role against acute pancreatitis with abnormal fumaric acid levels caused by abnormal purine nucleotide metabolism.

[0086] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Use of magnolol in preparing a medicament for treating acute inflammatory diseases, characterized in that: The drug is used to treat subjects with abnormal purine nucleotide cycle metabolism and abnormal mitochondrial energy metabolism, and the drug contains magnolol as the only active ingredient; the fumaric acid level of the subject is reduced; and the acute inflammatory disease includes acute pancreatitis.

2. The use according to claim 1, characterized in that The abnormal mitochondrial energy metabolism includes an abnormal ATP synthesis pathway in the mitochondria of the subject.

3. The use according to claim 1, characterized in that The subject's cellular oxygen consumption rate is reduced.

4. The use according to claim 1, characterized in that The acute pancreatitis is moderate to severe acute pancreatitis or severe acute pancreatitis.

5. Use of a fumaric acid detection reagent in the preparation of a kit for detecting the therapeutic effect of magnolol on acute inflammatory diseases of a subject, characterized in that: The acute inflammatory disease includes acute pancreatitis, and the kit is used to detect the fumaric acid level of the sample collected from the subject, so as to detect whether the purine nucleotide cycle metabolic pathway and the mitochondrial energy metabolic pathway of the subject are abnormal before the administration of magnolol; When the fumaric acid level of the subject is reduced, the magnolol is assessed to have a therapeutic effect on the acute inflammatory disease of the subject.

6. The use according to claim 5, characterized in that The sample is a whole blood, serum or plasma sample.

7. A method for evaluating the protective effect of magnolol, characterized in that: The method comprises: S101 culturing the target cells to obtain a first culture; S102 detecting the fumaric acid level and the cellular oxygen consumption rate in the first culture to obtain a first fumaric acid level and a first oxygen consumption rate; S103 culturing the target cells with magnolol at the first concentration to obtain a second culture; S104 detecting the fumaric acid level and the cellular oxygen consumption rate in the second culture to obtain a second fumaric acid level and a second oxygen consumption rate; S105: evaluating whether purine nucleotide metabolism in the target cells is restored or improved according to the first fumaric acid level and the second fumaric acid level; if so, proceeding to S106; if not, evaluating that magnolol is difficult to provide a protective effect on the target cells; S106, evaluating whether mitochondrial energy metabolism in the target cell is restored or improved according to the first oxygen consumption rate and the second oxygen consumption rate; if so, evaluating that magnolol can provide a protective effect on the target cell; if not, evaluating that magnolol is difficult to provide a protective effect on the target cell; The protective effect includes providing a protective effect on the mitochondrial function of the target cells.

8. The method according to claim 7, characterized in that The target cells include pancreatic acinar cells, cardiomyocytes, hepatocytes or neuronal cells.

9. The method according to claim 7, characterized in that The target cells are cell models of diseases.

10. The method according to claim 7, characterized in that S101 also includes adding a suitable amount of P substance.

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