Application of magnetic field device in promoting rapid recovery of liver function after mouse liver part resection

Abstract

The invention discloses application of a magnetic field device in promoting rapid recovery of liver functions after mouse liver part resection, and belongs to the technical field of magnetic biology. Specifically, the invention provides any one of the following applications of the magnetic field device: 1, an application of the magnetic field device in construction of a mouse model for rapid liver function recovery after partial liver resection and the like; the magnet device is a cuboid permanent magnet plate formed by fixedly embedding a plurality of cuboid neodymium iron boron with upward or downward magnetic field directions on a base and splicing the cuboid neodymium iron boron, and the average magnetic field intensity of the position 16 mm above the magnetic field device is 0.1 T. The method has the beneficial effects that a moderate-intensity steady-state magnetic field experimental device is combined with a 2 / 3 liver partial resection model, scientific experiments are carried out on the animal level and the molecular level, and it is proved for the first time that a downward steady-state magnetic field can promote cell proliferation in the liver after liver resection; an in-vitro physical auxiliary mode is provided for postoperative recovery of liver resection, and the method has high research value and application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of magnetic biology, and in particular to an application of a magnetic field device in promoting rapid recovery of liver function after partial liver resection in mice. Background Art

[0002] The liver is an organ with strong regenerative ability in the body. This ability ensures the operation of most organisms and their dependence on liver function, and is also a guarantee for the maintenance of various metabolic functions of the body. Although liver cells are quiescent under normal circumstances, when liver tissue is damaged, liver cells will re-enter the cell cycle and divide until the original liver weight is restored. This function also ensures that partial hepatectomy (PHx) or partial liver transplantation (PLTx) can be performed safely. Even if nearly 2 / 3 of the liver is removed by surgery, the remaining liver can quickly return to its original size. This surgery can remove localized liver lesions, including liver tumors, liver trauma, liver abscesses, etc., by removing liver segments, lobes and hemilivers, thereby retaining normal liver tissue sufficient to maintain function. Therefore, liver regeneration is a very important topic for scientists and clinicians. 2 / 3PHx is the most commonly used model for studying liver regeneration. In the mouse animal model, after 2 / 3PHx, hepatocytes rapidly enter the cell cycle from the resting phase, cell proliferation reaches its peak 36 to 48 hours after liver resection, and the liver can recover to its original weight in 7 to 10 days. In humans, DNA synthesis of liver tumors reaches its peak 7 to 10 days after resection, and it fully recovers in about 3 months. In contrast, failure of liver regeneration can lead to liver failure, infection, and even death.

[0003] As a non-invasive physical therapy method, magnetic fields have the characteristics of high penetration and high safety, and can cause a variety of biological effects. Studies have shown that steady-state magnetic fields can relieve pain and maintain bone health. At the same time, steady-state magnetic fields also have the potential to be used in regenerative medicine, including liver regeneration. Studies have found that steady-state magnetic fields can affect DNA synthesis and liver repair after liver damage. The patent with publication number CN113519459A discloses the use of a magnetic field generating device for regulating the level of reactive oxygen species (ROS) in hepatocytes in a mouse model of alcoholic liver disease. The invention states that a downward magnetic field can inhibit oxidative stress and improve liver damage in drinking mice, but does not explain the effect of a downward steady-state magnetic field on liver regeneration and recovery of liver function after PHx. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to provide a magnetic field device and apply it to a PHx model to reduce liver damage and improve the recovery of liver function.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] A first aspect of the present invention provides an application of a magnetic field device in any of the following aspects:

[0007] (1) Use for non-diagnostic or therapeutic purposes to increase the levels of neutrophils, hepatocyte growth factor (HGF), red blood cells (RBC), hemoglobin (HGB) and / or hematocrit (HCT);

[0008] (2) Application for non-diagnostic or therapeutic purposes in promoting liver regeneration and recovery of liver function after PHx;

[0009] The magnet device is composed of a plurality of rectangular NdFeB magnets with magnetic fields directed upward or downward, which are fixedly embedded on a base and spliced ​​to form a rectangular permanent magnet plate. The average magnetic field intensity at 16 mm above the magnetic field device is 0.1T.

[0010] Preferably, in (1), the weight of the resected liver is ≤ 2 / 3 of the original weight of the liver.

[0011] Preferably, in (1), the method for constructing the mouse model is: after subjecting the mice to 2 / 3PHx, they are exposed to a magnetic field device, and then the recovery of the mouse liver is detected by experiments.

[0012] Preferably, the position 16 mm above the magnetic field device corresponds to the position of the mouse liver.

[0013] Preferably, the exposure time to the magnetic field device is 12 to 48 hours. Preferably, the exposure time to the magnetic field device is 48 hours.

[0014] Preferably, the size of the rectangular NdFeB is length×width×height=60mm×50mm×30mm; the size of the rectangular permanent magnet plate is length×width×height=250mm×160mm×45mm.

[0015] Preferably, the magnetic field direction upwards or downwards represents the north pole and south pole of the magnet to which the animal is exposed, respectively.

[0016] Preferably, the magnetic field is directed downward.

[0017] The second aspect of the present invention provides a method for promoting liver regeneration and liver function recovery in mice after PHx for non-diagnostic or therapeutic purposes, comprising the following steps: exposing the mice to the above-mentioned magnetic field device.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention combines a medium-intensity steady-state magnetic field experimental device with a 2 / 3PHx model, conducts scientific experiments at the animal level and the molecular level, and proves for the first time that a steady-state magnetic field in a downward direction can promote cell proliferation in the liver after PHx. For example, a steady-state magnetic field in a downward direction can increase the HGF level in the liver by an average of 28.4% compared with the sham exposure group after 48 hours of magnetization treatment; and can increase the cell proliferation level by an average of about 25.8% compared with the sham exposure group after 48 hours of magnetization treatment. In addition, the present invention compares a steady-state magnetic field in a downward direction with a steady-state magnetic field in an upward direction, proving that a steady-state magnetic field in a downward direction has a better effect. For example, a steady-state magnetic field in a downward direction can increase the HGF level in the liver by an average of 17.3% compared with a steady-state magnetic field in an upward direction after 48 hours of magnetization treatment; and can increase the cell proliferation level by an average of about 33.9%. This shows that a steady-state magnetic field in a downward direction can provide an in vitro physical auxiliary method for postoperative recovery of PHx, which has high research value and application prospects.

[0020] 2. The downward steady-state magnetic field provided by the medium-intensity steady-state magnetic field experimental device of the present invention can promote the recovery of liver function after PHx. Compared with the sham exposure group, the magnetic treatment for 24 hours after PHx can reduce the level of alanine aminotransferase (ALT) in the liver, which helps to improve liver function after PHx.

[0021] 3. The downward steady-state magnetic field provided by the medium-intensity steady-state magnetic field experimental device of the present invention can promote the hematopoietic function of mice after PHx. When treated with the magnetic field for 24 hours after PHx, the levels of RGB, HGB and HCT can be increased compared with the sham exposure group and the upward magnetic field treatment group. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The schematic diagram of the magnetic field device, magnetization method and magnetic scanning distribution diagram of the present invention, wherein A is the schematic diagram of the magnetic field device and the magnetization method of mice after surgery, and B is the magnetic field distribution diagram 16 mm above the magnetic field device;

[0023] Figure 2 The construction method of the 2 / 3PHx model of the present invention and the postoperative magnetic treatment time, wherein a: left lateral lobe, b, c: middle lobe, d: caudate lobe, e: right upper lobe, f: right lower lobe;

[0024] Figure 3 The liver weight and liver organ index of mice after the postoperative magnetic treatment scheme of the present invention is completed, wherein A is the liver weight of the mice, and B is the liver organ index of the mice;

[0025] Figure 4The serum biochemical test results of mice after the magnetic treatment scheme of the present invention are completed, wherein A is the serum ALT test result, and B is the serum aspartate aminotransferase (AST) test result;

[0026] Figure 5 The blood routine test results of mice treated with magnetic field for 24 hours after PHx of the present invention; RBC, HGB, HCT, MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin content), MCHC (mean corpuscular hemoglobin concentration), Gran (neutrophil number), WBC (white blood cells), PLT (platelets);

[0027] Figure 6 The H&E stained slices of the liver of mice after the magnetic treatment scheme of the present invention and the results of the HGF detection in the serum of mice, wherein A is the H&E stained slices of the liver of mice at different treatment times after surgery, the black arrows in the figure indicate cells in the mitotic phase, and B is the results of the HGF detection in the serum of mice;

[0028] Figure 7 The Ki67 staining results and positive area statistical analysis results of the liver of mice after the magnetic addition scheme of the present invention are completed, wherein Figure A is the liver Ki67 staining section result, and Figure B is the positive area statistics and analysis result. The statistical method is to randomly intercept three slices of each slice, analyze each slice and obtain the positive area percentage as a scattered point in the bar graph;

[0029] Figure 8 Figure 1 is a F4 / 80 stained slice image of the liver of mice after the magnetic treatment of the present invention and the statistical results of the positive area of ​​the F4 / 80 stained slice results, wherein A is a F4 / 80 stained slice image of the liver, and B is a statistical result of the positive area of ​​the F4 / 80 stained slice results. The statistical method is the same as Figure 7 B.

[0030] The Shapiro-Wilk test was used for statistical analysis to determine whether the data conformed to normal distribution. If the data conformed to normal distribution, the two-tailed Student's t test was used for inter-group comparison, otherwise the Mann-Whitney U test was used for analysis. The control group was compared with the sham exposure group, and the groups were compared with each other at the same magnetic field treatment time. The statistical significance of the difference in the figure is the result of the comparison analysis between the two groups with the line, where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0031] 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 in combination with 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 creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0033] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0034] Embodiment 1:

[0035] A medium-intensity steady-state magnetic field experimental device (such as Figure 1 A), which is mainly composed of 12 permanent magnets (length × width × height = 60mm × 50mm × 30mm) spliced ​​together to form a rectangular magnetic plate (length × width × height = 250mm × 160mm × 45mm), which is fixed to the base by rivets. These magnetic plates can generate a relatively uniform steady-state magnetic field on each horizontal plane (the average magnetic field intensity is 0.1T at 16mm above the magnet). We use the direction upward and the direction downward to represent the north pole and south pole of the magnet to which the animal is exposed, respectively. In order to minimize experimental differences, mice in the sham exposure group were placed in the same non-magnetic flat-plate device. In order to measure the magnetic field distribution at the location of the mouse liver, a surface magnetic field distribution meter was used to detect the distribution of the magnetic field 16mm above the magnet (as shown in Figure 1). Figure 1 B).

[0036] Animal model construction

[0037] This example uses 12-week-old male C57BL / 6J mice, in which the mice are divided into a control group (5 mice), a sham exposure group (4-5 mice), an upward direction group (4-5 mice), and a downward direction group (4-5 mice), a total of 46 mice, and the mice are raised in a 12-hour light-dark cycle, an indoor temperature of 20-26°C, a relative humidity of 40-70%, and a feeding room with free access to water and food. The PHx model is constructed by anesthetizing 12-week-old mice with isoflurane, cutting the abdominal hair and disinfecting the skin, and then opening the abdominal cavity to expose the liver. While the mice are under isoflurane anesthesia, the left lateral lobe and middle lobe of the mouse liver are ligated and removed in turn (accounting for about 70% of the total liver weight). After the liver lobe is removed, the peritoneum and cortex of the mouse are sutured separately with sterile sutures and disinfected with iodine. Each PHx modeling experimental mouse is continuously exposed to a steady-state magnetic field for 24 hours / day. In addition, the control group consisted of healthy mice, which did not undergo surgery or magnetic field treatment. The mice were sacrificed and samples were collected at time 0 from the beginning of the experiment.

[0038] Euthanasia and biological material collection

[0039] At the end of the experimental period, mice were euthanized by cervical dislocation. Blood was collected after weighing the mice, and blood routine analysis was performed on whole blood and serum obtained by centrifugation was performed on blood biochemistry analysis. After rapid laparotomy, the liver was carefully isolated and weighed. Part of the liver tissue was fixed in 4% paraformaldehyde for histological analysis.

[0040] Serum ALT, AST, HGF detection

[0041] Whole blood was collected in EP tubes and centrifuged at 4°C and 3000 rpm for 15 minutes to separate serum. The levels of ALT and AST in serum were detected by the Lysine method, and the level of HGF was detected by enzyme-linked immunosorbent assay (ELISA). Then, the absorbance (OD value) was measured using an enzyme-labeled instrument to calculate the sample concentration.

[0042] Routine blood analysis

[0043] Before the mice were sacrificed, whole blood was collected and placed in an EP tube containing an anticoagulant containing EDTA, and then routine blood tests were performed using a fully automatic animal blood analyzer.

[0044] Immunohistochemical staining

[0045] The liver tissue was fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and cut into 5-10 μm slices. Before staining, the slices were dewaxed and stained with hematoxylin and eosin or anti-Ki67 antibody or anti-F4 / 80 antibody. After staining, the slices were rinsed and sealed, and then the slices were observed under a microscope and the results were scanned.

[0046] Result analysis:

[0047] After the magnetic treatment in Example 1 of the present invention, the serum ALT and AST test results of mice (such as Figure 4 ) It can be seen that compared with the control group, the ALT and AST levels in the sham magnetic exposure group increased significantly after PHx, and basically returned to normal levels in 48 hours. ALT exists in the cytoplasm of hepatocytes, and AST exists in the mitochondria and cytoplasm of hepatocytes. When hepatocytes are slightly damaged, ALT enters the blood first, and when hepatocytes are severely damaged or even cause mitochondrial damage, AST will also enter the blood. Combined with the results, it can be seen that PHx causes severe liver damage, but as time goes by, the liver damage is gradually repaired and has basically returned to normal levels in 48 hours. The downward steady-state magnetic field can promote the recovery of liver damage in mice after PHx during the recovery process compared with the sham magnetic exposure group.

[0048] The results of routine blood tests after PHx and magnetic treatment for 24 hours in Example 1 of the present invention showed that the downward steady-state magnetic field could increase the levels of RBC, HGB and HCT compared with the sham exposure group and the upward group, promote the hematopoietic function of mice after PHx, and help the regeneration of the liver. In addition, the downward steady-state magnetic field could increase the level of neutrophils in the blood of mice after PHx (such as Figure 5 ), and neutrophils have been shown to secrete HGF to accelerate hepatocyte proliferation.

[0049] The results of H&E staining after the magnetic treatment in Example 1 of the present invention showed that compared with the control group, the liver of mice in the sham magnetic exposure group had cell proliferation after PHx (e.g. Figure 6 A). In addition, the results of serum HGF detection after the magnetic treatment showed that compared with the control group, the HGF level in the sham magnetic exposure group increased significantly after PHx, and the downward steady-state magnetic field could promote the production of HGF after PHx compared with the sham magnetic exposure group (e.g. Figure 6 B), thereby accelerating the proliferation of hepatocytes, while an upward steady-state magnetic field has no such effect.

[0050] The analysis of the Ki67 section staining results and positive area results after the magnetic treatment in Example 1 of the present invention showed that compared with the control group, the liver Ki67 level increased after PHx. Ki67 is a protein related to cell division and proliferation in the nucleus and is expressed during the cell mitosis phase. Combined with the experimental results, it can be obtained that at 24 hours and 48 hours of magnetic treatment, the downward steady-state magnetic field can promote liver cell proliferation compared with the sham exposure group and the upward group (such as Figure 7 ), which helps the liver recover.

[0051] The F4 / 80 staining section diagram and positive area analysis after magnetic treatment in Example 1 of the present invention showed that the F4 / 80 level in the liver increased after treatment with a downward steady-state magnetic field. F4 / 80 in the liver of mice is mainly expressed on macrophages. Therefore, the downward steady-state magnetic field can increase the number of macrophages in the liver (such as Figure 8 ). Liver macrophages have been shown to respond rapidly after PHx, secreting auxiliary mitogens, activating downstream signaling pathways, and initiating liver cell proliferation.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A magnetic field device for use in any of the following: (1) Application for non-diagnostic or therapeutic purposes to increase the levels of neutrophils, HGF, RBC, HGB and / or HCT; (2) Application for non-diagnostic or therapeutic purposes in promoting liver regeneration and recovery of liver function after PHx; The magnet device is composed of a plurality of rectangular NdFeB magnets with magnetic fields directed upward or downward, which are fixedly embedded on a base and spliced ​​to form a rectangular permanent magnet plate. The average magnetic field intensity at 16 mm above the magnetic field device is 0.1T.

2. The use according to claim 1, characterized in that: (1) The weight of the liver removed was ≤2 / 3 of the original liver weight.

3. The use according to claim 1, characterized in that: (1) The mouse model was constructed by subjecting the mice to 2 / 3 PHx, exposing them to a magnetic field device, and then conducting experiments to detect the recovery of the mouse liver.

4. The use according to claim 1, characterized in that: The position 16 mm above the magnetic field device corresponds to the position of the mouse liver.

5. The use according to claim 1, characterized in that: The exposure time to the magnetic field device is 12 to 48 hours.

6. The use according to claim 1, characterized in that: The exposure time to the magnetic field device is 48 hours.

7. The use according to claim 1, characterized in that: The size of the rectangular NdFeB is length×width×height=60mm×50mm×30mm; the size of the rectangular permanent magnet plate is length×width×height=250mm×160mm×45mm.

8. The use according to claim 1, characterized in that: The magnetic field direction upward or downward indicates the north and south pole of the magnet to which the animal is exposed, respectively.

9. The use according to claim 1, characterized in that: The magnetic field is directed downward.

10. A method for promoting liver regeneration and liver function recovery in mice after PHx for non-diagnostic or therapeutic purposes, characterized in that: The method comprises the following steps: exposing mice to the magnetic field device according to claim 1.

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