A method for promoting the proliferation of anti-inflammatory macrophages and improving their anti-inflammatory effect

Abstract

A method for promoting the proliferation of anti-inflammatory macrophages and improving their anti-inflammatory effects belongs to the field of regenerative technology. In order to promote the proliferation of anti-inflammatory macrophages and enhance their anti-inflammatory effects, the present invention combines sonosensitizers and low-frequency, low-intensity ultrasound to intervene in anti-inflammatory macrophages and atherosclerosis model mice. In vitro and in vivo experiments confirm that sonosensitizers combined with low-frequency, low-intensity ultrasound can promote the proliferation of anti-inflammatory macrophages, thereby achieving the accumulation of the number of anti-inflammatory macrophages. At the same time, it can also regulate the expression levels of various pro-inflammatory / anti-inflammatory factors of anti-inflammatory macrophages, which helps to subside inflammation.

Description

Technical Field

[0001] The invention belongs to the field of regeneration technology, and specifically relates to a method for promoting the proliferation of anti-inflammatory macrophages and improving their anti-inflammatory effects. Background Art

[0002] Macrophages are widely present in the body and can maintain homeostasis and resist pathogen invasion. Macrophages can be roughly divided into classically activated pro-inflammatory types and selectively activated anti-inflammatory types. Pro-inflammatory macrophages are involved in the initiation, development and resistance of inflammatory responses, while anti-inflammatory macrophages have the function of promoting the resolution of inflammatory responses and tissue regeneration / wound healing.

[0003] Previous studies have found that a variety of cytokines can stimulate macrophage proliferation, such as: M-CSF, GM-CSF, IL-34 and IL-4. There are also patents that disclose methods that can proliferate macrophages, such as "A Soft Coral Polysaccharide and Its Preparation Method and Application" (CN202110909696.X) disclosed in 2021, which found that soft coral polysaccharides can promote the proliferation of RAW264.7 macrophages. However, the above studies and patents did not distinguish between specific subtypes of macrophages, nor did they study the extent to which substances or methods that can promote macrophage proliferation affect the proliferation ability of anti-inflammatory macrophages. Therefore, there is currently a lack of methods that can target and promote the proliferation of anti-inflammatory macrophages. Summary of the invention

[0004] In order to promote the proliferation of anti-inflammatory macrophages and enhance their anti-inflammatory effects, the present invention provides a method for promoting the proliferation of anti-inflammatory macrophages and improving their anti-inflammatory effects. The specific technical scheme is as follows:

[0005] The first object of the present invention is to provide a method for promoting the proliferation of anti-inflammatory macrophages, which is any one of the following:

[0006] Method A: Anti-inflammatory macrophages are incubated with sonosensitizers. When the sonosensitizers in the cells reach an effective concentration, low-frequency and low-intensity ultrasound is used to irradiate the anti-inflammatory macrophages.

[0007] Method B: The sonosensitizer is injected intravenously into the body, and when the sonosensitizer in the macrophages in the body's atherosclerotic plaques reaches an effective concentration, low-frequency and low-intensity ultrasound is used to locally irradiate the body's atherosclerotic plaques.

[0008] It is further defined that the sonosensitizer is an organic molecular sonosensitizer, an inorganic nanosonosensitizer or an organic-inorganic hybrid nanosonosensitizer.

[0009] In one embodiment of the present invention, the sonosensitizer is sodium porphyrin, 5-aminolevulinic acid, curcumin or rhamnosine.

[0010] In one embodiment of the present invention, the sonosensitizer is sodium porphyrin, the concentration of sodium porphyrin in method A is 0.1-0.5 μmol / L, and ultrasonic irradiation is performed 2-24 hours after treatment, and the concentration of sodium porphyrin in method B is 1-4 mg / kg, and ultrasonic irradiation is performed 4-24 hours after treatment.

[0011] In one embodiment of the present invention, the low-frequency and low-intensity ultrasound of method A has an ultrasound frequency of 0.5 MHz to 1.5 MHz and an ultrasound intensity of 0.4 to 1.0 W / cm 2 , irradiation time is 3-7min.

[0012] In one embodiment of the present invention, the low-frequency and low-intensity ultrasound of method B has an ultrasound frequency of 0.5 MHz to 1.5 MHz and an ultrasound intensity of 0.6 W / cm 2 -1.2W / cm 2 , the irradiation time is 10-20min.

[0013] The second object of the present invention is to provide a method for improving the anti-inflammatory effect of anti-inflammatory macrophages, the method being any one of the following:

[0014] Method A: Anti-inflammatory macrophages are incubated with sonosensitizers. When the sonosensitizers in the cells reach an effective concentration, low-frequency and low-intensity ultrasound is used to irradiate the anti-inflammatory macrophages.

[0015] Method B: The sonosensitizer is injected intravenously into the body, and when the sonosensitizer in the macrophages in the body's atherosclerotic plaques reaches an effective concentration, low-frequency and low-intensity ultrasound is used to locally irradiate the body's atherosclerotic plaques.

[0016] It is further defined that the sonosensitizer is an organic molecular sonosensitizer, an inorganic nanosonosensitizer or an organic-inorganic hybrid nanosonosensitizer.

[0017] In one embodiment of the present invention, the sonosensitizer is sodium porphyrin, the concentration of sodium porphyrin in method A is 0.1-0.5 μmol / L, and ultrasonic irradiation is performed 2-24 hours after treatment, and the concentration of sodium porphyrin in method B is 1-4 mg / kg, and ultrasonic irradiation is performed 4-24 hours after treatment.

[0018] In one embodiment of the present invention, the ultrasonic frequency of the low-frequency and low-intensity ultrasound is 0.5 MHz-1.5 MHz; the ultrasonic intensity of the low-frequency and low-intensity ultrasound in method A is 0.4-1.0 W / cm 2 The irradiation time is 3-7min; the ultrasound intensity of the low-frequency and low-intensity ultrasound in method B is 0.6-1.2W / cm 2 , the irradiation time is 10-20min.

[0019] The third object of the present invention is to provide anti-inflammatory macrophages obtained by the above method.

[0020] The fourth object of the present invention is to provide the use of the anti-inflammatory macrophages obtained by the above method in the preparation of anti-inflammatory drugs.

[0021] Beneficial effects of the present invention:

[0022] The present invention uses low-frequency and low-intensity ultrasound to activate sonosensitizers accumulated in anti-inflammatory macrophages, and proves through various evaluation methods (transcriptomic sequencing, cell immunofluorescence, real-time fluorescence quantitative PCR, Western Blot) that sonosensitizers combined with low-frequency and low-intensity ultrasound treatment can promote the proliferation of anti-inflammatory macrophages while improving their anti-inflammatory ability. This not only increases the number of anti-inflammatory macrophages, but also regulates the expression levels of various pro-inflammatory / anti-inflammatory factors of anti-inflammatory macrophages, improves the anti-inflammatory effect of anti-inflammatory macrophages, and helps to subside inflammation.

[0023] The present invention administers ApoE - / - Mice were fed a high-fat diet, and an animal model of atherosclerosis was established. The atherosclerosis model mice were intervened using sonosensitizers and low-frequency, low-intensity ultrasound. After sampling, immunofluorescence and immunohistochemistry were used to prove that sonosensitizers and low-frequency, low-intensity ultrasound can promote the proliferation of anti-inflammatory macrophages in atherosclerotic plaques and enhance their anti-inflammatory ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the result of identification of anti-inflammatory macrophages; Figure 1 A in the figure is a light microscopic image of anti-inflammatory macrophages. Figure 1 B is a representative image of CD163 immunofluorescence staining of anti-inflammatory macrophages, scale bar = 50 μm;

[0025] Figure 2 The figure shows the effect of porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound treatment on the expression of p53 in anti-inflammatory macrophages; Figure 2 A in the figure is the result of sequencing the transcriptome of anti-inflammatory macrophages. Figure 2 B in the figure is the result of detecting the mRNA level of p53 in anti-inflammatory macrophages in the Control group and the SDT group, * indicates P<0.05, n=3 cases / group, Figure 2 C in the figure is the protein level detection result of p53 in anti-inflammatory macrophages of Control group, Ultrasound group, DVDMS group and SDT group. Figure 2 D in Figure 2 In the statistical result graph corresponding to C in the figure, * indicates P < 0.05, n = 4 cases / group;

[0026] Figure 3 The figure shows the effect of porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound on the proliferation of anti-inflammatory macrophages; Figure 3 A in the figure shows the effect of porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound on the proportion of cells expressing proliferating nuclear antigen Ki67 in anti-inflammatory macrophages. The yellow arrows indicate double-positive cells. Scale bar = 50 μm. Figure 3 B in Figure 3 Statistical results of A in Figure 1, n = 3 cases / group, ** indicates P < 0.01;

[0027] Figure 4 This is a graph showing the effects of porphyrin sonosensitizers combined with low-frequency, low-intensity ultrasound on the levels of IL-10, TNF-α, and IL-6 proteins and mRNA in anti-inflammatory macrophages; Figure 4 A in the figure is the result of the detection of IL-10, TNF-α and IL-6 protein expression levels of anti-inflammatory macrophages in the Control group, Ultrasound group, DVDMS group and SDT group. Figure 4 B in Figure 4 In the statistical result graph corresponding to A, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Figure 4 C in the figure is the result of detecting the mRNA expression levels of IL-10, TNF-α and IL-6 in anti-inflammatory macrophages in the Control group and SDT group, * indicates P < 0.05, ** indicates P < 0.01, n = 3-5 cases / group;

[0028] Figure 5 The figure shows the effect of porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound on the proportion of cells expressing proliferating nuclear antigen Ki67 in anti-inflammatory macrophages in atherosclerotic plaques; Figure 5 The A in the expression is ApoE - / - Representative images of CD163 and Ki67 immunofluorescence double staining of atherosclerotic plaques in mouse aortic root. Yellow arrows indicate double-positive cells. Scale bar = 20 μm. Figure 5 B in the figure is a statistical diagram of the proportion of Ki67 and CD163 double-positive cells to total CD163-positive cells in atherosclerotic plaques, n = 6 cases / group, **** represents P < 0.0001;

[0029] Figure 6 The figure shows the effect of porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound on the proportion of cells expressing the cell cycle protein CyclinD1 in anti-inflammatory macrophages in atherosclerotic plaques; Figure 6 The A in the expression is ApoE - / -Representative images of CD163 and CyclinD1 immunofluorescence double staining of atherosclerotic plaques in mouse aortic root. Yellow arrows indicate double-positive cells. Scale bar = 20 μm. Figure 6 B in the figure is a statistical diagram of the proportion of CyclinD1 and CD163 double-positive cells to the total CD163-positive cells in atherosclerotic plaques, n = 6 cases / group, ** represents P < 0.01;

[0030] Figure 7 Porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound for ApoE - / - The results of the effect of inflammation on atherosclerotic plaques in mice; Figure 7 A in the figure is a representative image of immunohistochemical staining of TNF-α, IL-6 and IL-10 in atherosclerotic plaques of aortic root of ApoE- / - mice in the Control group and SDT group, scale bar = 50 μm, Figure 7 B in Figure 7 Statistical results of Figure A, * represents P < 0.05, ** represents P < 0.01, n = 6 cases / group. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0032] The experimental animals used in the present invention are 6-week-old male ApoE - / - Mice (weighing 18-20 g) and 6-week-old male C57BL / 6 mice (weighing 18-20 g) were purchased from Qingzilan Technology Co., Ltd.

[0033] The present invention is based on the nucleotide sequences of mouse p53, Il10, Tnf, Il6 and Gapdh published on NCBI, and involves primers of the above genes, which are synthesized by Invitrogen Biotechnology Co., Ltd. The specific information of the primers of the above genes is shown in Table 1.

[0034] Table 1 Primer information

[0035]

[0036] Example 1: Application of sonosensitizer combined with low-frequency and low-intensity ultrasound in promoting proliferation of anti-inflammatory macrophages and enhancing their anti-inflammatory effects

[0037] (1) Establishment and identification of anti-inflammatory macrophage model:

[0038] Bone marrow cells were extracted from C57BL / 6 mice and 2×10 6 The cells were cultured in a 35 mm cell culture dish at a concentration of 100 μg / mL, and 10 ng / mL of GM-CSF was added to differentiate them into bone marrow-derived macrophages. Then, 20 ng / mL of IL-4 was added to stimulate for 12 to 24 hours to polarize the bone marrow-derived macrophages into anti-inflammatory macrophages.

[0039] An anti-inflammatory macrophage model was established in vitro using the above-mentioned operation method. To verify whether the modeling was successful, we observed the morphology of the obtained anti-inflammatory macrophages under a light microscope and used a cell immunofluorescence method to detect whether the obtained anti-inflammatory macrophages expressed the specific surface antigen CD163 of anti-inflammatory macrophages.

[0040] The steps for detecting the surface antigen CD163 of anti-inflammatory macrophages by cell immunofluorescence are as follows: ① After washing the anti-inflammatory macrophages with PBS buffer for 3 times, fix them with 4% paraformaldehyde for 10 minutes. ② Wash the cells with PBS buffer again for 2 times, and permeabilize the membrane with 0.1% Triton for 2 minutes. ③ Wash the cells with PBS buffer twice, and block them with instant goat serum blocking solution at room temperature for 30 minutes. ④ Incubate the cells with the primary antibody working solution prepared with 1% BSA at 4°C overnight. ⑤ The next day, wash the cells with PBS buffer for 3 times, and incubate the cells with the fluorescent secondary antibody working solution at room temperature in the dark for 90 minutes. ⑥ After washing the cells with PBS buffer, stain with DAPI for 10 minutes. ⑦ Add anti-fluorescence quencher to the cell slide, invert it on a slide, collect images with a fluorescence microscope, and analyze them with Image-Pro Plus software.

[0041] The results of light microscopy observation are shown in Figure 1 A in Figure 1 shows that the anti-inflammatory macrophages are round or spindle-shaped, which is consistent with the morphological characteristics of anti-inflammatory macrophages. Representative images of CD163 immunofluorescence staining of anti-inflammatory macrophages are shown in Figure 1 B in the figure shows that the obtained anti-inflammatory macrophages are able to express the specific surface antigen CD163 of anti-inflammatory macrophages, that is, the anti-inflammatory macrophage model is successfully established.

[0042] (2) Method for treating anti-inflammatory macrophages by combining sonosensitizer with low-frequency and low-intensity ultrasound

[0043] The present invention uses a porphyrin sonosensitizer (0.5 μmol / L sodium porphyrin) to incubate anti-inflammatory macrophages in a cell packing box at 37°C in the dark for 6 hours, and then uses low-frequency and low-intensity ultrasound (ultrasound frequency of 1.0 MHz, ultrasound intensity of 0.5 W / cm 2) Anti-inflammatory macrophages incubated with sonosensitizer were irradiated for 5 minutes to activate the sonosensitizer accumulated in the cells, and the effect was detected 1 day after intervention.

[0044] (3) Sonosensitizer combined with low-frequency, low-intensity ultrasound reduced the expression of p53 in anti-inflammatory macrophages

[0045] The anti-inflammatory macrophages were divided into a control group (Control group) and a porphyrin sonosensitizer-coupled low-frequency, low-intensity ultrasound group (SDT group) (the specific treatment method is as above), and the RNA of the cells in the Control group and the SDT group was extracted for transcriptomic sequencing.

[0046] Transcriptomic sequencing results Figure 2 As shown in A in the figure, the figure shows that the method of combining porphyrin sonosensitizers and low-frequency and low-intensity ultrasound has a significant regulatory effect on the p53 signaling pathway of anti-inflammatory macrophages.

[0047] Subsequently, the present invention conducted real-time fluorescence quantitative PCR verification on the control group (Control group), porphyrin sonosensitizer-treated group and low-frequency low-intensity ultrasound group (SDT group), and the operation steps were as follows: ① The cell culture dish was taken out and placed on ice, the cell supernatant was discarded, and the cell RNA was extracted by Trizol method; ② The RNA concentration of each sample was measured in turn, and the RNA was reverse transcribed into cDNA using TOYOBO reverse transcription kit; ③ The p53 primer used for the first time was centrifuged at 12000rpm for 2-3 minutes, and DEPC water was added to prepare it into 1 00pmol / μl storage solution, diluted 10 times when used; ④ Add the following to the reaction wells for amplification: 10μl of SYBRGreen, 6μl of DEPC water, 1μl of upstream and downstream primers and 2μl of cDNA for PCR amplification reaction. The reaction procedure is pre-denaturation at 95℃ for 1 minute, 40 cycles; 95℃ for 15 seconds; 60℃ for 15 seconds; 72℃ for 45 seconds; melting curve is 95℃ for 15 seconds; 60℃ for 60 seconds; 95℃ for 30 seconds; 60℃ for 15 seconds; ④ After the PCR is completed, collect data.

[0048] The results of real-time fluorescence quantitative PCR are shown in Figure 2 B, the figure shows that porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound treatment downregulated the mRNA level of p53, and the difference was significant (P<0.05).

[0049] Furthermore, the present invention divides the anti-inflammatory macrophages into a control group (Control group), a low-frequency and low-intensity ultrasound group (Ultrasound group), a porphyrin sonosensitizer group (DVDMS group) and a porphyrin sonosensitizer combined with a low-frequency and low-intensity ultrasound group (SDT group). The specific operating parameters of the Ultrasound group and the DVDMS group refer to the conditions in the above step (2). The changes in the expression level of p53 protein in each group of cells were detected by Western blotting. The specific steps were as follows: ① Place the cells taken out from the packing box on an ice plate, collect the cell supernatant, wash the cells with PBS buffer precooled at 4°C, add 70μl~80μl of cell lysis buffer containing protease inhibitors and phosphatase inhibitors to the culture dish according to the cell density for lysis, use the BCA protein concentration determination kit to detect the protein concentration, add 1 / 4 protein sample volume of 5× protein loading buffer and heat it in a 100°C metal bath for 10 minutes to obtain the cell protein sample; ② Prepare PAGE gel according to the molecular weight of the target protein, calculate the protein loading volume according to the protein sample concentration, perform electrophoresis for 1~2 hours, and stop electrophoresis when the blue band approaches the lower edge of the separation gel; ③ When transferring to the membrane, prepare 0.45μ according to the molecular weight of the target protein. m or 0.2μm pore size PVDF membrane, and activate it with methanol, assemble the PVDF membrane, gel and filter paper in the transfer folder and put it into the transfer tank for transfer, the transfer current is 300 mA, and the transfer time is set according to the molecular weight of the target protein; ④ After the transfer, the PVDF membrane is placed in 5% milk blocking solution. After blocking at room temperature for 2 hours, the PVDF membrane can be placed in the primary antibody working solution and incubated overnight at 4℃. The next day, the PVDF membrane is washed 3 times with PBST buffer, and then the PVDF membrane is placed in the HRP-labeled secondary antibody configured with PBST and incubated at room temperature for 2 hours. The PVDF membrane is washed 3 times with PBST again, each time for 10 minutes; ⑤ Then the PVDF membrane is spread flat on the ChemiDocTMMP imaging system, and the ultra-sensitive ECL color developing solution is evenly added for color development, and Image Lab or Image J software is used for analysis.

[0050] The results are as follows Figure 2 C and Figure 2 As shown in D, the p53 protein in anti-inflammatory macrophages was significantly reduced after the intervention of porphyrin sonosensitizers and low-frequency and low-intensity ultrasound (P<0.05). Since the activation of p53 transcription factor can lead to cell cycle arrest and inhibit cell cycle progression, the combined action of porphyrin sonosensitizers and low-frequency and low-intensity ultrasound may promote the proliferation of anti-inflammatory macrophages.

[0051] (4) Sonosensitizer combined with low-frequency and low-intensity ultrasound promotes the proliferation of anti-inflammatory macrophages

[0052] The present invention divides anti-inflammatory macrophages into a control group (Control group) and a porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound group (SDT group), and uses immunofluorescence double staining to detect anti-inflammatory macrophage markers CD163 and cell proliferation nuclear antigen Ki67 to observe the proliferation level of anti-inflammatory macrophages. Figure 3 As shown in the results, after the intervention of porphyrin sonosensitizers and low-frequency and low-intensity ultrasound on anti-inflammatory macrophages, the proportion of proliferating cells increased significantly (P<0.01).

[0053] (5) Sonosensitizer combined with low-frequency, low-intensity ultrasound enhances the anti-inflammatory effect of anti-inflammatory macrophages

[0054] In order to observe the effect of porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound on the anti-inflammatory effect of anti-inflammatory macrophages, the present invention uses Western blot and real-time fluorescence quantitative PCR technology to detect the protein and mRNA expression levels of anti-inflammatory factor IL-10 and proinflammatory factors TNF-α and IL-6 in different groups of anti-inflammatory macrophages. As shown in the figure, after porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound intervention in anti-inflammatory macrophages, the protein expression level of IL-10 increased (P<0.01), and the expression levels of TNF-α and IL-6 were significantly reduced (P<0.001, P<0.05), and similar results were obtained at the mRNA level.

[0055] Example 2: Sonosensitizer combined with low-frequency and low-intensity ultrasound promotes the proliferation of anti-inflammatory macrophages in tissues and enhances the anti-inflammatory effect

[0056] (1) Modeling and grouping of atherosclerosis mice

[0057] The present invention administers ApoE - / - Mice were fed a high-fat diet (0.15% cholesterol + 22% fat) for 24 weeks and ApoE was successfully produced - / - Mouse atherosclerosis model. The atherosclerosis model mice were randomly divided into a control group (Control group) and a porphyrin sonosensitizer combined with low-frequency low-intensity ultrasound group (SDT group). The intervention process of the SDT group was as follows: ApoE - / - Mice were injected with a porphyrin sonosensitizer (4 mg / kg sodium porphyrin) via the tail vein. After 4 hours of exposure to light, the heart was irradiated with low-frequency, low-intensity ultrasound for 15 minutes. In animal studies, the ultrasound frequency was 1.0 MHz and the ultrasound intensity was 0.8 W / cm 2 . SDT group ApoE - / - The mice were sampled 1 week after the intervention. The control group was not intervened and samples were collected at the same time as the SDT group.

[0058] (2) Sonosensitizer combined with low-frequency, low-intensity ultrasound promotes the proliferation of anti-inflammatory macrophages in atherosclerotic plaques

[0059] The present invention observes the changes in the proliferation level of anti-inflammatory macrophages in atherosclerotic plaques by immunofluorescence double staining of CD163 and Ki67. The specific operation steps are as follows: ① After taking out the frozen section from the -80°C refrigerator, rewarm it at room temperature for 10-15 minutes, wash it once with PBST, and the washing time is 5 minutes; ② drip 0.1% Triton on the tissue section for permeabilization for 10 minutes, and after the permeabilization, wash it once with PBST, and the washing time is 5 minutes; ③ After wiping the section dry, drip the ready-to-use goat serum blocking solution on the tissue to make it uniform. ④ After the blocking, prepare the primary antibody working solution with 1% BSA solution, place the slices in a humidified box, put them in a 4°C refrigerator, and incubate with the primary antibody for 24 hours in the dark; ⑤ After the incubation, wash with PBST 3 times, 5 minutes each time, and incubate with the secondary antibody for 90 minutes at room temperature in the dark; ⑥ After washing with PBST 3 times, add DAPI to the slices and stain the nucleus for 10 minutes; ⑦ After washing with PBST 3 times, add anti-fluorescence quencher to prevent quenching, and cover the slices with coverslips; ⑧ Collect images with a fluorescence microscope and analyze them using Image-Pro Plus software.

[0060] The results are as follows Figure 5 As shown in the results, after the intervention of atherosclerotic plaques with porphyrin sonosensitizers combined with low-frequency and low-intensity ultrasound, the proportion of proliferating anti-inflammatory macrophages increased significantly (P<0.0001).

[0061] At the same time, the present invention performs double fluorescence staining of the anti-inflammatory macrophages in the atherosclerotic plaques with the marker CD163 and the cell cycle protein CyclinD1 to observe the changes in the cell cycle level of the anti-inflammatory macrophages in the atherosclerotic plaques. Figure 6 As shown in the results, after porphyrin sonosensitizer combined with low-frequency and low-intensity ultrasound intervention in atherosclerotic plaques, the proportion of double-positive cells increased significantly (P<0.01).

[0062] (3) Sonosensitizer combined with low-frequency, low-intensity ultrasound reduces inflammation in atherosclerotic plaques

[0063] The present invention performs immunohistochemical staining of pro-inflammatory factors TNF-α, IL-6 and anti-inflammatory factor IL-10 on atherosclerotic plaques, and the specific steps are as follows: ① After taking out the frozen section from a -80°C refrigerator, rewarm it at room temperature for 10-15 minutes, wash it once with PBST, and the washing time is 5 minutes; ② soak the tissue section in a 3% hydrogen peroxide solution for blocking for 15 minutes, and after the blocking is completed, wash it once with PBST, and the washing time is 5 minutes; ③ After wiping the section dry, drop the ready-to-use goat serum blocking solution on the tissue to evenly and completely cover the tissue, and block it for 30 minutes; ④ After the blocking is completed, prepare a primary antibody working solution with a 1% BSA solution, place the section in a wet box, put it in a 4°C refrigerator, and incubate the primary antibody for 24 hours; ⑤ After the incubation is completed, wash it 3 times with PBST , 5 minutes each time, and incubate with secondary antibody for 90 minutes at room temperature; ⑥ Prepare DAB working solution for DAB color development, determine the color development time under a microscope, and terminate with tap water; ⑦ Drop hematoxylin stain on the tissue to make it evenly and completely cover the tissue, stain the cell nucleus for 30 seconds, and terminate with tap water; ⑧ Before dehydration, rinse the slices once with deionized water, 75% ethanol once, 80% ethanol once, 85% ethanol once, 90% ethanol once, 95% ethanol twice, and 100% ethanol twice, dehydrate for 5 minutes each time; ⑨ After dehydration, immerse the slices in xylene twice, each time for five minutes to make them transparent; ⑩ Drop neutral resin on the tissue for sealing treatment, then put the slices in a 60℃ oven for 24-48 hours to solidify them, collect images with an optical microscope, and analyze them using Image-Pro Plus software.

[0064] The results are as follows Figure 7 As shown in the results, the combination of porphyrin sonosensitizers and low-frequency, low-intensity ultrasound reduced the proinflammatory factors TNF-α and IL-6 in atherosclerotic plaques (P<0.05, P<0.05), and increased the anti-inflammatory factor IL-10 (P<0.01), indicating that the combination of porphyrin sonosensitizers and low-frequency, low-intensity ultrasound can reduce the inflammation of atherosclerotic plaques.

[0065] The incubation concentration of sodium porphyrin selected by the inventors in the cell experiment is 0.1-0.5 μmol / L, the incubation time is 2-24 hours, the ultrasound frequency of low-frequency and low-intensity ultrasound is 0.5 MHz-1.5 MHz, and the ultrasound intensity is 0.4 W / cm 2 -1.0W / cm 2 The irradiation time is 3-7min. The incubation concentration of sodium porphyrin selected in the animal experiment is 1-4mg / kg, the incubation time is 4-24h, the ultrasound frequency of low-frequency and low-intensity ultrasound is 0.5MHz-1.5MHz, and the ultrasound intensity is 0.6W / cm 2 -1.2W / cm 2The irradiation time is 10-20min. Through cell experiments and animal experiments, the inventors found that the above treatment conditions can achieve the effects recorded in the above embodiments, and through experiments, it was found that when 5-aminolevulinic acid, curcumin or rhein was used as the sonosensitizer, similar conclusions to those in the above embodiments could be obtained.

[0066] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for promoting the proliferation of anti-inflammatory macrophages in vitro, characterized in that: The method comprises the following steps: incubating anti-inflammatory macrophages with sodium chrysoporphyrin, and when sodium chrysoporphyrin in the cells reaches an effective concentration, irradiating the anti-inflammatory macrophages with low-frequency and low-intensity ultrasound; the ultrasound frequency of the low-frequency and low-intensity ultrasound is 0.5 MHz-1.5 MHz, and the ultrasound intensity is 0.4 W / cm 2 -1.0 W / cm 2 , the irradiation time is 3-7 min.

2. The method according to claim 1, characterized in that The concentration of sodium porphyrin is 0.1-0.5 μmol / L, and ultrasonic irradiation is performed 2-24 hours after treatment.

3. A method for improving the anti-inflammatory effect of anti-inflammatory macrophages in vitro, characterized in that: The method comprises the following steps: incubating anti-inflammatory macrophages with sodium chrysoporphyrin, and when sodium chrysoporphyrin in the cells reaches an effective concentration, irradiating the anti-inflammatory macrophages with low-frequency and low-intensity ultrasound; the ultrasound frequency of the low-frequency and low-intensity ultrasound is 0.5 MHz-1.5 MHz, and the ultrasound intensity is 0.4-1.0 W / cm 2 , the irradiation time is 3-7 min.

4. The method according to claim 3, characterized in that The concentration of sodium porphyrin is 0.1-0.5 μmol / L, and ultrasonic irradiation is performed 2-24 hours after treatment.

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