Application of magnesium sulfate in preparation of medicine for treating acute kidney injury caused by sepsis

Abstract

The invention discloses application of magnesium sulfate in preparation of a medicine for treating acute kidney injury caused by sepsis, and belongs to the technical field of medicines. By collecting and analyzing a large number of clinical data of patients, in-vivo animal experiments, bioinformatics analysis, in-vitro cell experiments and the like, the protection effect and potential mechanism of magnesium sulfate on sepsis acute kidney injury are clarified, a theoretical basis and a scientific basis are provided for taking magnesium sulfate as a new treatment means for sepsis acute kidney injury, and the application has a wide application prospect. Wide clinical application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of magnesium sulfate in preparing a medicament for treating acute kidney injury caused by sepsis. Background Art

[0002] Sepsis-associated acute kidney injury (SA-AKI) is an acute renal dysfunction caused by sepsis. It is a common complication of sepsis patients, with high morbidity and mortality, increasing the short-term and long-term risk of death in patients. Survivors of SA-AKI may also face complications such as chronic kidney disease, end-stage renal disease, and adverse cardiovascular events. At present, the treatment of SA-AKI focuses on supportive treatment of sepsis, including the use of antibiotics, fluid resuscitation, and the application of vasoactive drugs. For renal damage that has already occurred, renal replacement therapy (RRT) is mainly used. Because the pathogenesis and pathophysiology of SA-AKI are complex and have not been fully elucidated, medical staff face challenges in treating SA-AKI and lack specific and effective treatment methods. Therefore, exploring new safe and effective strategies for the treatment of SA-AKI to improve the prognosis of patients remains an urgent problem to be solved in this field.

[0003] SA-AKI patients are often prone to hypomagnesemia (serum magnesium level less than 0.66mmol / L). In addition to the disease itself increasing the risk of hypomagnesemia, SA-AKI patients receiving RRT will also increase magnesium loss, especially in the absence of routine intravenous magnesium supplementation. In addition, the clinical manifestations of hypomagnesemia are usually lacking in specificity and are easily overlooked by medical staff. Although studies have shown that magnesium has a significant renal protective effect in multiple renal injury models such as ischemia-reperfusion-induced renal injury model, cisplatin-induced renal injury model, elderly renal injury model, and acetaminophen-induced renal injury model, its role in SA-AKI is still unclear. In addition, compared with AKI, the treatment of SA-AKI also needs to pay attention to controlling the source of infection and regulating the inflammatory response in addition to conventional treatment. For this reason, a therapeutic drug that is specific and effective for SA-AKI is urgently needed. Summary of the invention

[0004] In view of the defects of the prior art, the present invention clarifies the mechanism of action of magnesium sulfate in SA-AKI, and can be used to treat sepsis-induced acute kidney injury.

[0005] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions:

[0006] First, the present invention explores the effect of magnesium sulfate on mortality and renal function in SA-AKI patients based on the MIMIC-IV database. This part evaluates the role of magnesium sulfate in improving the prognosis of SA-AKI patients and promoting renal function recovery by collecting and analyzing clinical data of a large number of patients; and then reveals the protective effect of magnesium sulfate on SA-AKI and its potential mechanism through in vivo animal experiments, bioinformatics analysis and in vitro cell experiments. This part will use experimental animal models, combined with molecular biology techniques, to clarify the specific mechanism by which magnesium sulfate plays a therapeutic role in SA-AKI.

[0007] In one aspect, the present invention provides the use of magnesium sulfate in the preparation of a medicament for treating acute kidney injury caused by sepsis.

[0008] Preferably, the magnesium sulfate can reduce the expression of kidney injury markers NGAL and KIM1.

[0009] Preferably, the magnesium sulfate can reduce serum creatinine and urea nitrogen levels.

[0010] Preferably, the magnesium sulfate can reduce the level of serum inflammatory factors.

[0011] More preferably, the inflammatory factors are IL-6, 1L-1β and TNF-α.

[0012] Preferably, the magnesium sulfate can reduce lipopolysaccharide-induced septic acute renal injury cellular oxidative stress dysfunction.

[0013] Preferably, the drug can inhibit apoptosis of renal tissue and renal cells and reduce inflammation of renal tissue and renal cells.

[0014] Preferably, the dosage form of the drug includes tablets, granules, capsules, pills, oral solutions or injections.

[0015] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention clarifies that magnesium sulfate reduces sepsis-induced acute kidney injury through anti-inflammatory, anti-oxidative and anti-apoptotic effects by collecting and analyzing clinical data of a large number of patients, as well as in vivo animal experiments, bioinformatics analysis and in vitro cell experiments, thereby verifying the protective effect of magnesium sulfate on sepsis-induced acute kidney injury and its potential mechanism, providing a theoretical basis and scientific basis for magnesium sulfate as a new treatment for sepsis-induced acute kidney injury, and also providing a new idea for preparing sepsis-induced acute kidney injury drugs, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the 28-day survival curve of magnesium sulfate in patients with SA-AKI.

[0020] Figure 2 This is a graph showing the detection results of injury markers in mice with acute kidney injury caused by sepsis under different drug interventions. In the graph: A is NGAL; B is KIM1.

[0021] Figure 3 This is a graph showing the test results of serum creatinine and urea nitrogen in mice with acute kidney injury due to sepsis under different drug interventions. In the graph: A is creatinine; B is urea nitrogen.

[0022] Figure 4 This is a graph showing the results of serum inflammatory factor detection in mice with acute kidney injury caused by sepsis under the intervention of different drugs. In the graph: A is IL-6; B is IL-1β; C is TNF-α.

[0023] Figure 5 This is a graph showing the results of oxidative stress detection of HK-2 cells induced by LPS under the intervention of different drugs. In the graph: A is SOD; B is MDA; C is GSH.

[0024] Figure 6 The figures are the results of apoptosis detection in mice with acute kidney injury caused by sepsis under different drug interventions. In the figure: A is the expression of BCL-2, BAX and β-actin proteins detected by Western Blot; B is the relative expression of BAX / BCL-2 proteins; C is the relative expression of BCL-2 / β-actin proteins; and D is the relative expression of BAX / β-actin proteins.

[0025] Figure 7 The figures are the results of apoptosis detection in mice with acute kidney injury caused by sepsis under different drug interventions. In the figure: A is the expression of P65, Caspase-3 and GAPDH proteins detected by Western Blot; B is the relative expression of P65 / GAPDH proteins; C is the relative expression of Caspase-3 / GAPDH proteins.

[0026] Figure 8 This is a diagram of apoptosis signals in mice with acute kidney injury caused by sepsis under the intervention of different drugs. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only 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 creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0029] All animal experiments in the following examples were reviewed and approved by the Animal Care Committee of Jinan University.

[0030] Example 1

[0031] Analysis of the protective effect of magnesium sulfate on mortality and renal function in patients with SA-AKI:

[0032] This example is a retrospective cohort study based on the MIMIC-IV database. MIMIC is a retrospective database that has no impact on the clinical treatment and care of patients, and all protected privacy information has been identified and removed. According to the ethics committee policy, this study waived the requirement for individual patient informed consent.

[0033] 1. Subject inclusion criteria:

[0034] (1) Adult patients aged ≥18 years;

[0035] (2) patients who met the diagnostic criteria for SA-AKI;

[0036] (3) Patients with combined hypomagnesemia or normomagnesemia. The diagnosis of SA-AKI is based on the latest definition of the consensus report of the 28th Acute Disease Quality Initiative Working Group, that is, sepsis and AKI (acute kidney injury) coexist, and AKI occurs within 7 days of the diagnosis of sepsis. The diagnosis of sepsis is based on the Sepsis 3.0 standard, and the diagnosis of AKI is based on the KDIGO standard. Hypomagnesemia is defined as a serum magnesium level <0.66mmol / L (1.6mg / dL), and normomagnesemia is defined as a serum magnesium level between 0.66 and 1.07mmol / L (1.6 and 2.6mg / dL).

[0037] 2. Exclusion criteria include:

[0038] (1) Patients aged <18 years;

[0039] (2) patients with a length of ICU stay of <24 hours or >30 days;

[0040] (3) patients with hypermagnesemia (serum magnesium level >1.07 mmol / L [2.6 mg / dL]) before SA-AKI or lack of serum magnesium level data;

[0041] (4) Patients with chronic kidney disease stage 5 or receiving long-term dialysis treatment.

[0042] The exposure factor was intravenous magnesium sulfate during ICU stay, with no dose restrictions. Patients were divided into a magnesium sulfate group and a no magnesium sulfate group according to whether they were treated with magnesium sulfate. Information on magnesium sulfate use was extracted from the drug prescription form of the database. The study outcomes were 28-day mortality and MAKE30. MAKE30 is an assessment at 30 days to see whether the following endpoints were achieved:

[0043] 1. Serum creatinine value ≥ 2.0 times the baseline creatinine value at 30 days;

[0044] 2. Whether RRT was performed within 30 days;

[0045] 3. Whether the patient died at 30 days.

[0046] This example included 12,238 SA-AKI patients, including 10,411 in the magnesium sulfate group and 1,827 in the non-magnesium sulfate group. The 28-day mortality rates of patients in the magnesium sulfate group and the non-magnesium sulfate group were 20.09% (2,092 / 10,411) and 34.59% (632 / 1,827), respectively. Figure 1 The KM curve in the figure shows that the 28-day survival curve of the magnesium sulfate group in the original cohort is always above the non-magnesium sulfate group. Through the Log-rank test, we found that there was a significant statistical difference in the survival curves of the two groups of patients (P<0.05), which indicates that the 28-day survival probability of patients in the magnesium sulfate group is significantly higher than that in the non-magnesium sulfate group.

[0047] In addition, the results of univariate analysis and multivariate analysis showed that magnesium sulfate was associated with lower 28-day mortality in patients with SA-AKI (univariate analysis HR 0.51, 95% CI 0.47-0.56, P < 0.001; multivariate analysis HR 0.57, 95% CI 0.52-0.63, P < 0.001). The MAKE30 of patients in the magnesium sulfate group and the non-magnesium sulfate group were 29.84% (3107 / 10411) and 40.61% (742 / 1827), respectively. The results of univariate analysis and multivariate analysis showed that magnesium sulfate was associated with lower MAKE30 in patients with SA-AKI (univariate analysis OR 0.62, 95% CI 0.56-0.69, P = 0.007; multivariate analysis OR 0.72, 95% CI 0.63-0.81, P < 0.001). 。

[0048] Example 2

[0049] 1. Animal Experiments

[0050] 1. The sepsis-induced acute kidney injury model was established by intraperitoneal injection of LPS 20 mg / kg.

[0051] 2. Grouping

[0052] Male C57BL / 6J mice weighing 18-22g and aged 42-56 days were selected (n=40). After the mice adapted for one week without abnormality, they were weighed and numbered. The mice were randomly divided into 5 groups: (1) blank control group; (2) modeling group: LPS (lipopolysaccharide) 20mg / kg; (3) low-dose magnesium sulfate group: magnesium sulfate 50mg / kg was given after modeling; (4) medium-dose magnesium sulfate group: magnesium sulfate 100mg / kg was given after modeling; (5) high-dose magnesium sulfate group: magnesium sulfate 150mg / kg was given after modeling.

[0053] 3. Detection indicators

[0054] (1) Mice were anesthetized with 5% isoflurane. After anesthesia, blood was collected and cardiac perfusion was performed with normal saline until the kidneys turned white. The kidneys were collected for subsequent analysis. After the blood sample was allowed to stand at room temperature for 30 minutes, it was centrifuged (3500 rpm, 4°C, 10 minutes), and the serum was transferred to an EP tube and stored at -80°C. The expression of blood creatinine, urea nitrogen, NGAL and KIM1 was then detected using a kit according to the instructions.

[0055] (2) The levels of mouse serum inflammatory factors IL-6, 1L-1β, and TNF-α were measured according to the instructions of the ELISA kit (Biyuntian, China).

[0056] (3) After collecting the mouse kidney, remove the capsule and renal pedicle, cut the kidney tissue of equal weight and place it in an EP tube. Add the prepared protein lysis buffer (RIPA = 100:1), add 20 ml of lysis buffer for every 1 mg of kidney tissue. Add 3 grinding magnetic beads (1 large and 2 small) to each EP tube and grind them thoroughly in a tissue grinder. Transfer the ground solution to a new EP tube and centrifuge at 12000 rpm for 20 minutes at 4°C. After centrifugation, discard the precipitate and retain the supernatant. Use BCA protein detection reagent to detect the protein concentration in the supernatant, and add protein loading buffer at a volume ratio of 4:1 and vortex to mix. Place the mixture in a 100°C water bath and heat for 15 minutes to denature the protein. After detection, the sample can be analyzed immediately or stored at -80°C. Western Blot was used to detect the expression levels of BCL-2, BAX, β-actin, P65, Caspase-3 and GAPDH proteins.

[0057] (4) Detect cell apoptosis according to the instructions of the TUNEL kit.

[0058] 2. Cell Experiment

[0059] 1. Refer to the animal experiment grouping, and randomly divide into 5 groups: ① blank control group; ② modeling group: LPS exposure; ③ low-dose magnesium sulfate group: LPS + magnesium sulfate 1μM; ④ medium-dose magnesium sulfate group: LPS + magnesium sulfate 2μM; ⑤ high-dose magnesium sulfate group: LPS + magnesium sulfate 4μM. First, treat human renal tubular epithelial cell line (HK-2) cells with high-glucose medium containing 10μg / mL LPS for 24h to establish a sepsis injury model. After the model is successfully established, normal medication is given according to the grouping.

[0060] 2. Testing indicators:

[0061] The superoxide dismutase (SOD) kit (Biyuntian, China), the malondialdehyde (MDA) kit (Biyuntian, China), and the glutathione peroxidase (GSH-Px / GPX) kit (Biyuntian, China) were used to detect the SOD activity, MDA activity, and GSH activity in mouse serum, respectively.

[0062] In summary, Figure 2 , 3 It can be seen that magnesium sulfate can reduce the degree of renal injury in mice with acute kidney injury caused by sepsis, including reducing the expression of renal injury markers NGAL and KIM1, and reducing the levels of creatinine and urea nitrogen.

[0063] Depend on Figure 4 It can be seen that magnesium sulfate significantly reduced the inflammatory response of mice with acute kidney injury caused by sepsis, including reducing the expression of IL-6, IL-1β, and TNF-α, and the protective effect became more significant as the concentration of magnesium sulfate increased.

[0064] Depend on Figure 5 It can be seen that magnesium sulfate can reduce the oxidative stress of HK-2 cells induced by LPS, including reducing MDA and GSH and increasing the expression of SOD.

[0065] Depend on Figure 6-8 It can be seen that magnesium sulfate can inhibit the apoptotic signals of mice with acute kidney injury caused by sepsis, including reducing the expression of pro-apoptotic BAX, increasing the expression of anti-apoptotic BCL2, and reducing the expression of P65 and Caspase-3, thereby reducing cell apoptosis.

[0066] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Application of magnesium sulfate in the preparation of drugs for the treatment of acute kidney injury caused by sepsis.

2. The use according to claim 1, characterized in that: The magnesium sulfate can reduce the expression of kidney injury markers NGAL and KIM1.

3. The use according to claim 1, characterized in that: The magnesium sulfate can reduce the levels of creatinine and urea nitrogen in serum.

4. The use according to claim 1, characterized in that: The magnesium sulfate can reduce the level of serum inflammatory factors.

5. The use according to claim 1, characterized in that: The magnesium sulfate can alleviate the oxidative stress dysfunction of cells in septic acute renal injury induced by lipopolysaccharide.

6. The use according to claim 1, characterized in that: The drug can inhibit apoptosis of renal tissue and renal cells and reduce inflammation of renal tissue and renal cells.

7. The use according to claim 1, characterized in that: The dosage form of the drug includes tablets, granules, capsules, pills, oral liquid or injection.

8. The use according to claim 1, characterized in that: The medicine also includes a pharmaceutically acceptable carrier or excipient.

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