Application of magnolol in preparation of medicine for treating kidney injury

Abstract

The invention provides application of magnolol in preparation of a medicine for treating kidney injury, and belongs to the technical field of medicines. Researches find that the compound magnolol has the effect of effectively relieving kidney injury, especially acute kidney injury, can be used for protecting renal functions, and has low toxic and side effects, and the action mechanism of the magnolol is related to iron homeostasis recovery, ferroptosis inhibition and inflammatory factor level reduction. Therefore, the compound magnolol can be used as a therapeutic agent for acute kidney injury, and has a wide clinical application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of magnolol in preparing medicines for treating kidney damage. Background Art

[0002] Acute Kidney Injury (AKI) is a common and serious clinical illness and has become a public health problem that needs to be urgently addressed worldwide. The incidence of AKI is high, with approximately 13.3 million people suffering from AKI each year and approximately 1.7 million people dying from AKI and its complications. Severe or recurrent AKI can progress to chronic kidney disease or even end-stage renal disease. Although researchers from various countries are committed to research in this direction, unfortunately, there is currently a lack of effective and specific treatments other than conservative treatment. Therefore, studying its pathogenesis and developing highly effective drugs has great clinical significance.

[0003] Recent studies have found that ferroptosis is involved in the pathological process of AKI and is one of the important causes of AKI, suggesting that targeting ferroptosis may become a new strategy for treating AKI. Although previous studies have shown that many anti-lipid peroxidation drugs can effectively prevent ferroptosis and the pathological process of all its induced diseases, there are only a few such drugs that can be truly applied in clinical practice, and more drugs for the treatment of AKI are still to be developed. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide the use of magnolol in the preparation of a drug for treating kidney damage. The present invention has found that magnolol has the effect of effectively alleviating kidney damage, especially acute kidney damage, can be used to protect kidney function, and has low toxic and side effects, and is expected to be used clinically as a drug for treating kidney damage.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a use of magnolol in the preparation of a medicine for treating kidney damage.

[0007] Magnolol is a new biologically active lignin extracted from the root and stem bark of the Chinese herbal medicine Magnolia officinalis. It has a wide range of biological activities, such as anti-tumor, hypoglycemic, and antibacterial activities. The molecular formula of Magnolol is C 18 H 18 O2, the structural formula is as follows:

[0008]

[0009] The present invention found that the compound magnolol has the effect of effectively reducing kidney damage, especially acute kidney damage, and can be used to protect kidney function with low toxic side effects. Its mechanism of action is related to restoring iron homeostasis, inhibiting ferroptosis and reducing the level of inflammatory factors. Therefore, the compound magnolol can be used as a therapeutic agent for acute kidney damage and has broad clinical application prospects.

[0010] In some embodiments of the present invention, the kidney injury is acute kidney injury.

[0011] In some embodiments of the present invention, the acute kidney injury is acute kidney injury induced by folic acid and / or ischemia-reperfusion.

[0012] When magnolol is used to treat kidney damage, the dosage is preferably 20-30 mg / kg, more preferably 25 mg / kg.

[0013] In a second aspect, the present invention provides a use of magnolol in the preparation of a drug for simultaneously reducing blood creatinine content and blood urea nitrogen content.

[0014] In a third aspect, the present invention provides a use of magnolol in the preparation of a drug for protecting renal tubular epithelial cells and / or human embryonic kidney cells.

[0015] In some embodiments of the present invention, the renal tubular epithelial cells include human renal tubular epithelial cells HK-2, and the human embryonic kidney cells include human embryonic kidney cells HEK293T.

[0016] When magnolol is used to protect renal tubular epithelial cells and / or human embryonic kidney cells in vitro (at the cell level), its concentration is preferably 5-20 μM, more preferably 10 μM.

[0017] In a fourth aspect, the present invention provides a use of magnolol in the preparation of a drug for regulating the expression level of ferroptosis-related proteins and / or the expression level of iron metabolism-related proteins.

[0018] In some embodiments of the present invention, the ferroptosis-related proteins include one or more of ACSL4, GPX4, SLC7A11 and HO-1.

[0019] In some embodiments of the present invention, the iron metabolism-related proteins include one or more of TfR1, FPN1, FTH and FTL.

[0020] When magnolol is used to regulate the expression level of ferroptosis-related proteins and / or iron metabolism-related proteins in vivo, the dosage is preferably 20-30 mg / kg, more preferably 25 mg / kg.

[0021] When magnolol is used to regulate the expression level of ferroptosis-related proteins and / or iron metabolism-related proteins in vitro (at the cellular level), its concentration is preferably 5-20 μM, more preferably 10 μM.

[0022] In a fifth aspect, the present invention provides a use of magnolol in the preparation of a drug for down-regulating the mRNA expression level of inflammation-related factors.

[0023] In some embodiments of the present invention, the inflammation-related factors include one or more of MCP-1, FN14, TWEAK, IL-6, CXCL16 and CD44.

[0024] When magnolol is used to down-regulate the mRNA expression level of inflammation-related factors, the dosage is preferably 20-30 mg / kg, more preferably 25 mg / kg.

[0025] In a sixth aspect, the present invention provides a drug for treating kidney damage, wherein the drug comprises magnolol.

[0026] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0027] In some embodiments of the present invention, the dosage form of the drug is injection, tablet, pill or capsule.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention found that the compound magnolol has the effect of effectively reducing kidney damage, especially acute kidney damage, and can be used to protect kidney function with low toxic side effects. Its mechanism of action is related to restoring iron homeostasis, inhibiting ferroptosis and reducing the level of inflammatory factors. Therefore, the compound magnolol can be used as a therapeutic agent for acute kidney damage and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1a It is a survival rate curve of HK-2 cells and HEK293T cells treated with different concentrations of RSL3;

[0031] Figure 1b The survival rate curves of HK-2 cells and HEK293T cells treated with different concentrations of erastin;

[0032] Figure 1c The survival rate curves of HK-2 cells and HEK293T cells treated with different concentrations of magnolol;

[0033] Figure 1d The bar graph is the survival rate of HK-2 cells co-treated with RSL3 and different concentrations of magnolol;

[0034] Figure 1e The bar graph is a survival rate bar graph of HEK293T cells co-treated with RSL3 and different concentrations of magnolol;

[0035] Figure 1f The bar graph is a survival rate bar graph of HK-2 cells co-treated with erastin and different concentrations of magnolol;

[0036] Figure 1g The bar graph is a survival rate bar graph of HEK293T cells co-treated with erastin and different concentrations of magnolol;

[0037] Figure 1h The bar graph is the LDH content in the supernatant of HK-2 cells treated with RSL3 and magnolol alone or together;

[0038] Figure 1i The bar graph shows the LDH content in the supernatant of HEK293T cells treated with RSL3 and magnolol alone or together;

[0039] Figure 2a The electrophoresis diagram of TfR1, FPN1, FTH and FTL proteins expressed in HK-2 cells treated with RSL3 and magnolol alone or together;

[0040] Figure 2b Electrophoresis of TfR1, FPN1, FTH and FTL proteins expressed in HEK293T cells treated with RSL3 and magnolol alone or together;

[0041] Figure 2c The bar graph shows the relative expression of TfR1, FPN1, FTH and FTL proteins in HK-2 cells treated with RSL3 and magnolol alone or together;

[0042] Figure 2d The bar graph shows the relative expression levels of TfR1, FPN1, FTH and FTL proteins in HEK293T cells treated with RSL3 and magnolol alone or together;

[0043] Figure 2e The fluorescence intensity graph of HK-2 cells treated with RSL3 and magnolol alone or together after calcein staining;

[0044] Figure 2f The fluorescence intensity graph of HEK293T cells treated with RSL3 and magnolol alone or together after calcein staining;

[0045] Figure 3a Electrophoresis diagram of ACSL4, SLC7A11, HO-1 and GPX4 proteins expressed in HK-2 cells treated with RSL3 and magnolol alone or together;

[0046] Figure 3b Electrophoresis diagram of ACSL4, SLC7A11, HO-1 and GPX4 proteins expressed in HEK293T cells treated with RSL3 and magnolol alone or together;

[0047] Figure 3c The bar graph shows the relative expression levels of ACSL4, SLC7A11, HO-1 and GPX4 proteins in HK-2 cells treated with RSL3 and magnolol alone or together;

[0048] Figure 3d The bar graph shows the relative expression levels of ACSL4, SLC7A11, HO-1 and GPX4 proteins in HEK293T cells treated with RSL3 and magnolol alone or together;

[0049] Figure 3e The bar graphs are for the MDA and GSH contents of HK-2 cells treated with RSL3 and magnolol alone or together;

[0050] Figure 3f The bar graphs are for MDA and GSH contents in HEK293T cells treated with RSL3 and magnolol alone or together;

[0051] Figure 4a The bar graph is a graph showing the blood creatinine content of mice with acute kidney injury model treated with or without magnolol;

[0052] Figure 4b The bar graph is a graph showing the blood urea nitrogen content of acute kidney injury model mice treated with or without magnolol.

[0053] Figure 4c HE staining of kidney tissues of mice with acute kidney injury model treated with or without magnolol;

[0054] Figure 5a The bar graph shows the relative mRNA expression of MCP-1 in kidney tissue of mice with acute kidney injury model treated with or without magnolol.

[0055] Figure 5b The bar graph shows the relative mRNA expression of FN14 in the kidney tissue of mice with acute kidney injury who were given or not given magnolol;

[0056] Figure 5c The bar graph shows the relative mRNA expression of TWEAK in the kidney tissue of mice with acute kidney injury who were given or not given magnolol;

[0057] Figure 5d The bar graph shows the relative mRNA expression of IL-6 in kidney tissue of mice with acute kidney injury who were given or not given magnolol;

[0058] Figure 5e The bar graph shows the relative mRNA expression of CXCL16 in kidney tissue of mice with acute kidney injury who were given or not given magnolol;

[0059] Figure 5f The bar graph shows the relative mRNA expression of CD44 in kidney tissue of mice with acute kidney injury who were given or not given magnolol;

[0060] Figure 5g The electrophoresis diagrams of TfR1, FPN1, FTH and FTL proteins in kidney tissues of mice with acute kidney injury model treated with or without magnolol;

[0061] Figure 5h The bar graph shows the relative expression of TfR1, FPN1, FTH and FTL proteins in the kidney tissue of mice with acute kidney injury who were given or not given magnolol;

[0062] Figure 5i The electrophoresis diagrams of ACSL4, SLC7A11, HO-1 and GPX4 proteins in kidney tissues of mice with acute kidney injury model treated with or without magnolol;

[0063] Figure 5j The bar graph shows the relative expression of ACSL4, SLC7A11, HO-1 and GPX4 proteins in kidney tissues of mice with acute kidney injury who were given or not given magnolol;

[0064] Figure 5k The bar graph is a graph showing the MDA content in kidney tissue of mice with acute kidney injury model treated with or without magnolol;

[0065] Figure 5l The bar graph shows the GSH content in kidney tissue of mice with acute kidney injury model treated with or without magnolol. DETAILED DESCRIPTION

[0066] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. It should be understood by those skilled in the art that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0067] Unless otherwise specified, the significance symbols of P values ​​in the examples and drawings of the present invention are defined as follows: *P<0.05, **P<0.01, ***P<0.001.

[0068] Unless otherwise specified, the symbol “-” in the drawings of the present invention represents that the substance is not added, and the symbol “+” represents that the substance is added.

[0069] Example 1

[0070] Magnolia officinalis has a protective effect on human renal tubular epithelial cell (HK-2) injury and human embryonic kidney cell (HEK293T) injury induced by RSL3 (glutathione peroxidase 4 inhibitor, ferroptosis inducer) and erastin (erastin, ferroptosis inducer) in vitro.

[0071] 1. Experimental methods

[0072] 1.1 Cytotoxicity assay

[0073] Human renal tubular epithelial cells (HK-2) and human embryonic kidney cells (HEK293T) were inoculated in 96-well plates at a density of about 6000 cells / well and cultured for 24 hours. Each cell type was divided into a control group (without drug), an RSL3 group (0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, and 0.5 μM RSL3 were added to each well, respectively), an erastin group (2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM erastin were added to each well, respectively), and a magnolol group (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 100 μM magnolol were added to each well, respectively). Complete culture medium (DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 100 U / mL penicillin and 100 U / mL streptomycin) was added and cultured for another 24 h. At the same time, a blank group (without cells and without drug) was set up. After the culture is completed, the old culture medium is removed, and the complete culture medium and CCK-8 reagent (cell counting reagent) are mixed at a volume ratio of 1:10. After thorough mixing, they are added to the 96-well plate and incubated for 1 hour. The OD value of each well is measured at 450nm using an ELISA reader and the results are recorded. Figure 1a , Figure 1b , Figure 1c shown.

[0074] Cell survival rate = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0075] 1.2 Effects of magnolol on cell death induced by ferroptosis inducers

[0076] Human renal tubular epithelial cells (HK-2) and human embryonic kidney cells (HEK293T) were inoculated in 96-well plates at a density of about 6000 cells / well and cultured for 24 hours. Each cell was divided into a control group (no drug), an RSL3 group (0.1 μM RSL3 was added to each well), an RSL3+magnolia phenol group (0.1 μM RSL3 and 5 μM, 10 μM, 20 μM, 40 μM magnolia phenol were added to each well), an erastin group (10 μM erastin was added to each well), and an erastin+magnolia phenol group (10 μM erastin and 5 μM, 10 μM, 20 μM, 40 μM magnolia phenol were added to each well), and complete culture medium (DMEM high glucose medium containing 10% fetal bovine serum (FBS) and 100 U / mL penicillin and 100 U / mL streptomycin) was added, and culture was continued for 24 hours. At the same time, a blank group (no cells and no drugs) was set up. After the culture is completed, the cell survival rate is measured according to the test method in 1.1. The results are as follows Figure 1d , Figure 1e , Figure 1f , Figure 1g shown.

[0077] 1.3. LDH (lactate dehydrogenase) content detection

[0078] HK-2 cells in logarithmic growth phase were cultured at 1×10 4 -1×10 5 HEK293T cells were expressed at 1×10 / dish 4 -1×10 5 / dish was inoculated in a 10-cm culture dish. They were divided into a normal group (cont, without drug addition), an RSL3 group (added with 0.1μM RSL3), an RSL3+magnolia officinalis group (added with 0.1μM RSL3 and 10μM magnolia officinalis), and a magnolia officinalis group (added with 10μM magnolia officinalis). After 24 hours of treatment, the cells were collected and 500μL of 1×PBS was added to form a homogenate. Take 200μL of the homogenate, and according to the operating procedures of the LDH kit (purchased from Wanlei Biotechnology Co., Ltd.), add each reagent in turn, measure the absorbance at 450nm and calculate the LDH content. The results are as follows Figure 1h , Figure 1i Shown

[0079] 2. Experimental results

[0080] The survival rates of cells treated with different concentrations of RSL3, erastin, and magnolol were as follows: Figure 1a , Figure 1b , Figure 1cAs shown in the figure, after treating kidney cells HK-2 and HEK293T with different concentrations of classic ferroptosis inducers RSL3 and erastin for 24 hours, the survival rates of HK-2 and HEK293T cells decreased in a concentration-dependent manner ( Figure 1a , Figure 1b ), magnolol alone had no significant toxic side effects on the two cell lines ( Figure 1c ).

[0081] The survival rates of HK-2 and HEK293T cells co-treated with ferroptosis inducers (RSL3, erastin) and different concentrations of magnolol were shown in Figure 2. Figure 1d , Figure 1e , Figure 1f , Figure 1g As shown. As can be seen from the figure, compared with HK-2 and HEK293T cells treated with ferroptosis inducers (RSL3, erastin) alone, the relative survival rates of HK-2 and HEK293T cells were significantly restored after co-treatment with ferroptosis inducers (RSL3, erastin) and magnolol. The above experimental results show that magnolol has a good protective effect on HK-2 and HEK293T cells stimulated by ferroptosis inducers.

[0082] The results of LDH content in the supernatant of HK-2 and HEK293T cells treated with RSL3 and magnolol alone or together were as follows: Figure 1h , Figure 1i As shown in the figure, compared with HK-2 and HEK293T cells treated with RSL3 alone, the LDH content in the supernatant of HK-2 and HEK293T cells was significantly reduced after co-treatment with RSL3 and magnolol, indicating that magnolol can inhibit cell death caused by ferroptosis inducers, further proving that magnolol has a good protective effect on HK-2 and HEK293T cells stimulated by RSL3.

[0083] Experimental Example 2

[0084] Magnolol inhibits cellular ferroptosis by improving the accumulation of iron ions in vitro.

[0085] 1. Experimental methods

[0086] 1.1. Western Blot Experiment

[0087] HK-2 cells in logarithmic growth phase were cultured at 1×10 4 -1×10 5 HEK293T cells were expressed at 1×10 / dish 4 -1×10 5 / dish was inoculated in a 10 cm culture dish. They were divided into a normal group (cont, no drug), an RSL3 group (with 0.1 μM RSL3), an RSL3+magnolia phenol group (with 0.1 μM RSL3 and 10 μM magnolia phenol), and a magnolia phenol group (with 10 μM magnolia phenol). After 24 hours of treatment, the cells were digested with trypsin and collected in a 2 mL EP tube. The precipitate was retained by centrifugation at 4 ° C, the supernatant was discarded, and the prepared RIPA buffer strong cell lysis solution was added. The cells were fully lysed in an ice bath for 30 minutes (the oscillator was shaken and mixed once every 10 minutes). Centrifuged at 4 ° C, 12000 rpm for 20 minutes, and the supernatant (protein solution) was aspirated and placed in a new EP tube. BCA protein quantitative reagent was added according to the proportion, and the concentration of each protein sample was determined by Nanodrop (micro-ultraviolet spectrophotometer). According to the cell characteristics, the corresponding protein (TfR1, FPN1, FTH, FTL) samples were prepared and placed in a 97 ° C metal bath for 10 minutes. Protein separation was performed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) electrophoresis.

[0088] According to the needs, select different concentrations of SDS polyacrylamide gel (SDS-PAGE) for protein separation (80V, 30min, then adjust to 120V, about 2h, and stop electrophoresis when bromophenol blue runs to the bottom of the gel). The electrophoresis diagram is as follows: Figure 2a , Figure 2b As shown. The protein on the gel was then transferred to a PVDF membrane (300 mA, transfer for 90 minutes). Block with a 5% skimmed milk powder solution prepared with TBST (Tris-Borate-Sodium Tween-20 buffer) for 1 hour, and then rinse the PVDF membrane with TBST. Cut the membrane according to the amount of protein and incubate with the corresponding primary antibody, decolorize and incubate overnight on a shaker at 4°C. The next day, recover the primary antibody and use 1×TBST to wash the non-specific binding antibody (3×10 min). Incubate the secondary antibody and shake at room temperature for 1 hour. Wash the membrane with 1×TBST (4×10 min) to enhance the signal-to-noise ratio. The membrane was developed using a chemiluminescence imager, and the images were analyzed for protein quantification using Imagej software. The experiment was repeated 3 times for each group. The protein expression levels of TfR1, FPN1, FTH and FTL of the two cells were obtained respectively, and the results are shown in Figure 2c , Figure 2d shown.

[0089] 1.2 Flow cytometry

[0090] HK-2 cells in logarithmic growth phase were cultured at 1×10 4 -1×10 5 HEK293T cells were expressed at 1×10 / dish 4 -1×10 5 / dish was inoculated in a 10-cm culture dish. They were divided into a normal group (cont, without drug addition), an RSL3 group (added with 0.1μM RSL3), an RSL3+magnolia officinalis group (added with 0.1μM RSL3 and 10μM magnolia officinalis), and a magnolia officinalis group (added with 10μM magnolia officinalis). After 24 hours of treatment, the cells were digested with trypsin, collected in a 15mL centrifuge tube, and the supernatant was removed by centrifugation. Resuspend with 1mL of 1×PBS and transfer to a 1.5mL EP tube. Add calcein probe with a final concentration of 1μM to the EP tube and incubate in a 37°C constant temperature incubator away from light for 30 minutes. After the end, wash and resuspend with 1mL of 1×PBS, and use a flow cytometer to detect fluorescence changes. The results are as follows Figure 2e , Figure 2f shown.

[0091] 2. Experimental results

[0092] The electrophoretic images of TfR1, FPN1, FTH and FTL proteins expressed in HK-2 cells and HEK293T cells treated with RSL3 and magnolol (MA) alone or together (with β-actin as internal reference) are shown in Figure 2 Figure 2a , Figure 2b The relative expression bar graphs of TfR1, FPN1, FTH and FTL proteins in HK-2 cells and HEK293T cells treated with RSL3 and magnolol (MA) alone or together are shown in Figure 2c , Figure 2d As shown. As can be seen from the figure, compared with the normal group (Control, no drug), the expression of TfR1 decreased after treatment with RSL3 (0.1μM), while the combined treatment of RSL3 and magnolol (10μM) inhibited this phenomenon. Compared with the normal group (Control, no drug), FPN1, FTH and FTL showed an upward trend after induction by RSL3 (0.1μM), and the addition of magnolol (10μM) could inhibit the changes in the expression of these three proteins.

[0093] Calcein is a fluorescent dye that can enter living cells and will be quickly quenched after binding to intracellular iron. Therefore, the fluorescence intensity can reflect the changes in intracellular iron. The fluorescence intensity graphs of HK-2 cells and HEK293T cells treated with RSL3 and magnolol (MA) alone or together after calcein staining are shown in Figure 2. Figure 2e , Figure 2fAs shown. As can be seen from the figure, after RSL3 (0.1μM) treatment, the intracellular fluorescence decreased, indicating that the content of free iron pool (LIP) in the cell increased. The addition of magnolol (10μM) can significantly inhibit the increase of intracellular LIP caused by RSL3. The above experimental results show that magnolol can alleviate cell ferroptosis by inhibiting iron accumulation.

[0094] Experimental Example 3

[0095] Magnolol inhibits cellular ferroptosis in vitro by improving iron ion and lipid peroxidation.

[0096] 1.1、Western Blot

[0097] HK-2 cells in logarithmic growth phase were cultured at 1×10 4 -1×10 5 HEK293T cells were expressed at 1×10 / dish 4 -1×10 5 / dish was inoculated in a 10 cm culture dish. They were divided into a normal group (cont, no drug), an RSL3 group (with 0.1 μM RSL3), an RSL3+magnolia phenol group (with 0.1 μM RSL3 and 10 μM magnolia phenol), and a magnolia phenol group (with 10 μM magnolia phenol). After 24 hours of treatment, the cells were digested with trypsin and collected in a 2 mL EP tube. The precipitate was retained by centrifugation at 4 ° C, the supernatant was discarded, and the prepared RIPA buffer strong cell lysis solution was added. The cells were fully lysed in an ice bath for 30 minutes (the oscillator was shaken and mixed once every 10 minutes). Centrifuged at 4 ° C, 12000 rpm for 20 minutes, and the supernatant (protein solution) was aspirated and placed in a new EP tube. BCA protein quantification reagent was added according to the proportion, and the concentration of each protein sample was determined by Nanodrop. According to the characteristics of the cells, the corresponding protein (ACSL4, SLC7A11, HO-1, GPX4) samples were prepared and placed in a 97 ° C metal bath for 10 minutes.

[0098] According to the needs, select different concentrations of SDS polyacrylamide gel (SDS-PAGE) for protein separation (80V, 30min, then adjust to 120V, about 2h, and stop electrophoresis when bromophenol blue runs to the bottom of the gel). The electrophoresis diagram is as follows: Figure 3a , Figure 3bAs shown. The protein on the gel was then transferred to a PVDF membrane (300 mA, transfer for 90 minutes). Block with a 5% skimmed milk powder solution prepared with TBST for 1 hour, and then rinse the PVDF membrane with TBST. Cut the membrane according to the amount of protein and incubate with the corresponding primary antibody, and incubate overnight on a 4°C decolorizing shaker. The next day, the primary antibody was recovered, and non-specific binding antibodies were washed with 1×TBST (3×10 min). Incubate with secondary antibody and shake at room temperature for 1 hour. Wash the membrane with 1×TBST (4×10 min) to enhance the signal-to-noise ratio. The membrane was developed using a chemiluminescence imager, and the images were analyzed for protein quantification using Imagej software. The experiment was repeated 3 times for each group. The protein expression levels of ACSL4, SLC7A11, HO-1 and GPX4 of the two cells were obtained, and the results are shown in Figure 3c , Figure 3d shown.

[0099] 1.2. MDA (malondialdehyde) and GSH (cofactor glutathione) detection

[0100] HK-2 cells in logarithmic growth phase were cultured at 1×10 4 -1×10 5 HEK293T cells were expressed at 1×10 / dish 4 -1×10 5 / dish was inoculated in a 10-cm culture dish. They were divided into a normal group (Control, without drug addition), an RSL3 group (added with 0.1μM RSL3), an RSL3+magnolia officinalis group (added with 0.1μM RSL3 and 10μM magnolia officinalis), and a magnolia officinalis group (added with 10μM magnolia officinalis). After 24 hours of treatment, the cells were digested with trypsin and collected into a 2mL EP tube. The precipitate was retained by centrifugation at 4°C, and 1mL of 1×PBS was added to form a homogenate. According to the relevant steps of the MDA kit (Wanlei Biotechnology Co., Ltd.) and the GHS kit (Solabao Technology Co., Ltd.), the supernatant was taken to a 96-well plate, and the absorbance values ​​were measured at 532nm (MDA) and 405nm (GHS), respectively, and the contents of MDA and GSH were calculated. The results are shown in the following table. Figure 3e , Figure 3f shown.

[0101] 2. Experimental results

[0102] In order to further explore the molecular inhibition of magnolol on ferroptosis, the present invention detected the expression changes of biomarkers ACSL4, SLC7A11, HO-1 and GPX4 when ferroptosis occurred. ACSL4 induces ferroptosis by accumulating oxidized cellular phospholipids; GPX4 and HO-1 are important antioxidant defense enzymes in the body and are also important inhibitors of ferroptosis; SLC7A11 is an important component of systemXc- (cystine / glutamate antiporter) and is involved in lipid peroxidation and ferroptosis.

[0103] The electrophoretic diagrams of ACSL4, SLC7A11, HO-1 and GPX4 proteins expressed in HK-2 cells and HEK293T cells treated with RSL3 and magnolol (MA) alone or together (with β-actin as internal reference) are shown in Figure 2 Figure 3a , Figure 3b The relative expression bar graphs of ACSL4, SLC7A11, HO-1 and GPX4 proteins in HK-2 cells and HEK293T cells treated with RSL3 and magnolol (MA) alone or together are shown in Figure 2. Figure 3c , Figure 3d As shown. As can be seen from the figure, compared with the normal group (Control, no drug), after RSL3 treatment of HK-2 and HEK293T cells, ACSL4 and HO-1 expression was upregulated, and GPX4 and SLC7A11 expression was significantly downregulated. After combined treatment with magnolol, the expression levels of these proteins were restored. These results indicate that magnolol can restore the changes in the expression of target proteins related to cell ferroptosis induced by RSL3.

[0104] The MDA and GSH contents of HK-2 cells and HEK293T cells treated with RSL3 and magnolol (MA) alone or together were as shown in Figure 3e , Figure 3f As shown. The change in MDA content directly reflects the degree of lipid peroxidation in the body. As can be seen from the figure, compared with the normal group (cont, no drug), after treatment with RSL3, the MDA level in the cells was significantly upregulated, indicating that the degree of lipid peroxidation increased, and this phenomenon was inhibited after the addition of magnolol. The activity of GPX4 depends on GSH, and GSH can also reflect the body's own degree of oxidation. After treatment with RSL3, the GSH level was significantly downregulated, indicating that the body's degree of oxidation increased, and this downregulation phenomenon was inhibited after the addition of magnolol. These results show that magnolol can significantly inhibit the increase in intracellular lipid peroxidation levels caused by RSL3. At the same time, magnolol also limits the depletion of GSH and restores the oxidative balance in cells.

[0105] Example 4

[0106] Magnolia officinalis inhibits folic acid-induced renal tissue damage and renal dysfunction.

[0107] 1. Experimental methods

[0108] 1.1. Detection of serum creatinine and blood urea nitrogen levels

[0109] In order to adapt the mice to the environment, the experiment was started after the mice were left to rest for one week. The mice were divided into NaHCO3 group, FA (folic acid) group, FA + magnolol group, and magnolol group.

[0110] Among them, mice in the FA+magnolia nobile group were intraperitoneally injected with 250 mg / kg of folic acid (FA) to induce acute renal injury. Folic acid was dissolved in 0.3 mol / L NaHCO3 aqueous solution, and magnolia nobile (25 mg / kg) was gavaged once 6 hours after the injection of folic acid, and then gavage was performed every 12 hours;

[0111] Mice in the NaHCO3 group were intraperitoneally injected with the same volume of NaHCO3 aqueous solution as folic acid solution as a negative control and then fed normally;

[0112] Mice in the FA group were induced with acute renal injury using the same method as mice in the FA + magnolol group and then maintained normally;

[0113] The mice in the magnolol group were gavaged with magnolol (25 mg / kg) at the same time point as those in the FA+magnoliaol group without FA treatment.

[0114] All mice were anesthetized and killed 48 h after folic acid injection, and two kidney samples were collected for subsequent experiments.

[0115] The levels of creatinine and urea nitrogen in the serum of the animal model were detected according to the instructions of the blood creatinine and urea nitrogen kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0116] Creatinine is measured as follows:

[0117]

[0118] Creatinine content (μmol / L) = [(Assay A2-K × Assay A1) - (Blank A2-K × Blank A1)] / [(Standard A2-K × Standard A1) - (Blank A2-K × Blank A1)] × standard concentration.

[0119] Note: Dilution factor K = (sample volume + enzyme solution A volume) / (sample volume + enzyme solution A volume + enzyme solution B volume) = 186 / 246. The concentration of the standard is 442 μmol / L. The blank sample is double distilled water.

[0120] The urea nitrogen test method is as follows:

[0121]

[0122] Urea nitrogen content (mmol / L) = (measured OD value - blank OD value) / (standard OD value - blank OD value) × standard concentration × sample dilution factor before testing. The standard concentration is 10mmol / L, and the blank sample is double distilled water.

[0123] The test results of serum creatinine and blood urea nitrogen content are as follows Figure 4a , Figure 4b shown.

[0124] 1.2. HE staining shows kidney tissue morphology

[0125] After sampling, rinse with 1×PBS and immerse in fixative for fixation. Rinse with running water before staining, immerse in ethanol for dehydration, and immerse in xylene solution until transparent. Embed in paraffin, place the tissue slices in a 65℃ oven for 4h, then stain with hematoxylin and eosin staining solution, seal with neutral gum, and observe under a microscope. The results are as follows Figure 4c shown.

[0126] 2. Experimental results

[0127] from Figure 4a and Figure 4b It can be seen that compared with the negative control group (NaHCO3), the levels of serum creatinine and blood urea nitrogen in the folic acid-induced model group (FA) were significantly increased, and renal function deteriorated; while magnolol administration effectively reduced the serum creatinine level in the model group (see Figure 4a ) and blood urea nitrogen levels (see Figure 4b HE staining results showed that compared with the FA group, the FA+magnolia phenol group mice had significantly alleviated renal tubular epithelial cell edema, severe tubular dilatation, and obvious tubular cavitation after oral administration of magnolia phenol, indicating that magnolia phenol can effectively treat acute kidney injury ( Figure 4c ). The above experimental results further confirmed that magnolol has a protective effect on the kidneys in acute kidney injury.

[0128] Example 5

[0129] Effects of magnolol on inflammatory response, iron metabolism, and ferroptosis proteins in a folic acid-induced acute kidney injury model.

[0130] 1. Experimental methods

[0131] In this example, the mouse kidney samples obtained in Example 4 were used for experiments.

[0132] 1.1 Real-time PCR (RT-PCR)

[0133] During the sampling process, the mouse kidneys were frozen in liquid nitrogen and then added with 1 mL of sterile 1×PBS. The kidneys were pre-cooled and then crushed with a crusher. 50 μL of homogenate was added with 500 μL of RNA-easy reagent. The procedure in the instruction manual was followed. After inverting and drying, an appropriate amount of RNase-free ddH2O was added to dissolve the RNA precipitate at the bottom of the EP tube. Reverse transcription and amplification were performed. The experiment was repeated 3 times for each group. The mRNA expression levels of inflammatory factors MCP1, TWEAK, IL-6, CD44, FN14, and CXCL16 in the kidney tissues of each group of mice were obtained. The results are shown in Figure 5a to Figure 5f shown.

[0134] 1.2. Western Blot method

[0135] Weigh the kidney tissue, add protein lysis buffer at a mass volume ratio of 14 μL / mg, grind thoroughly, transfer to EP tube, and lyse for 30 min on a shaker at 4°C. The whole process is performed on ice. For the subsequent steps, refer to the Western Blot method for cell experiments. The electrophoresis images of TfR1, FPN1, FTH and FTL in the kidney tissues of each group of mice were obtained ( Figure 5g ) and relative expression bar graph ( Figure 5h ), electrophoresis diagram of ACSL4, SLC7A11, HO-1 and GPX4 ( Figure 5i ) and relative expression bar graph ( Figure 5j ).

[0136] 1.3. MDA and GSH detection

[0137] The collected mouse kidney tissue was added to 1 ml PBS for ultrasonic disruption, and 500 μL of kidney homogenate was taken. According to the relevant steps of the MDA and GSH kits, the supernatant was taken to a 96-well plate, and the absorbance values ​​were measured at 532 nm (MDA) and 405 nm (GHS), respectively, and the contents of MDA and GSH were calculated. The results are as follows: Figure 5k , Figure 5l shown.

[0138] 2. Experimental results

[0139] The results of RT-PCR experiments showed that compared with the negative control group, after FA treatment, the mRNA levels of inflammatory factors monocyte chemoattractant protein-1 (MCP-1), fibroblast growth factor-inducing-14 (FN14), human tumor necrosis factor-like weak apoptosis inducer (TWEAK), interleukin-6 (IL-6), CXC chemokine ligand 16 (CXCL16), and cluster of differentiation 44 (CD44) in the mouse kidney tissue were significantly increased ( Figure 5a to Figure 5f After combined treatment with magnolol, the expression levels of six inflammatory factors in the kidney tissue of mice were significantly restored ( Figure 5a to Figure 5f ). This indicates that magnolol can inhibit the expression of inflammatory factors in the kidney.

[0140] Iron metabolism proteins in the body strictly regulate the homeostasis of iron ions. Therefore, the present invention detects the changes in iron metabolism-related proteins. The treatment of magnolol significantly restored the decrease in transferrin TfR1 and FPN1 expression caused by FA treatment ( Figure 5h The expression levels of FTH and FTL were significantly upregulated in FA-induced AKI mice, and their expression was restored to varying degrees after magnolol treatment ( Figure 5h). These results indicate that FA treatment disrupted the iron homeostasis of mouse kidneys, while magnolol treatment could significantly restore the iron homeostasis in the kidneys. This may be one of the mechanisms of magnolol in protecting against acute kidney injury.

[0141] Given that ferroptosis plays an important role in the AKI process, the present invention then explored the effect of magnolol on ferroptosis in FA-induced renal injury by detecting the expression levels of genes and proteins related to ferroptosis. The results showed that after FA treatment, the mRNA and protein expression levels of ferroptosis targets GPX4 and SLC7A11 were significantly downregulated ( Figure 5j ), ACSL4 and HO-1 levels were significantly upregulated ( Figure 5j ), and their protein levels were significantly restored after magnolol treatment ( Figure 5j ). After FA treatment, the MDA content in the mouse kidneys increased and the GSH content decreased. After treatment with magnolol, the expression levels of these two indicators were effectively restored, indicating that magnolol can reduce the degree of lipid peroxidation in the kidneys ( Figure 5k , Figure 5l ).

[0142] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of magnolol in the preparation of medicines for treating kidney damage.

2. The use according to claim 1, characterized in that: The kidney injury is acute kidney injury.

3. The use according to claim 2, characterized in that: The acute kidney injury is acute kidney injury induced by folic acid and / or ischemia-reperfusion.

4. The application of magnolol in the preparation of a drug for simultaneously reducing blood creatinine content and blood urea nitrogen content.

5. The use of magnolol in the preparation of drugs for protecting renal tubular epithelial cells and / or human embryonic kidney cells.

6. The use according to claim 5, characterized in that: The renal tubular epithelial cells include human renal tubular epithelial cells HK-2, and the human embryonic kidney cells include human embryonic kidney cells HEK293T.

7. Use of magnolol in the preparation of drugs for regulating the expression level of ferroptosis-related proteins and / or the expression level of iron metabolism-related proteins; The ferroptosis-related proteins include one or more of ACSL4, GPX4, SLC7A11 and HO-1; The iron metabolism-related proteins include one or more of TfR1, FPN1, FTH and FTL.

8. Use of magnolol in the preparation of drugs for down-regulating the mRNA expression level of inflammation-related factors; The inflammation-related factors include one or more of MCP-1, FN14, TWEAK, IL-6, CXCL16 and CD44.

9. A drug for treating kidney damage, characterized in that: The drug includes magnolol.

10. The drug according to claim 9, characterized in that The drug also includes pharmaceutically acceptable excipients; And / or, the drug is in the form of injection, tablet, pill or capsule.

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