A pinellia ternata exosome and application thereof in preparation of macrophage immune activator

Abstract

The application discloses raw pinellia ternate exosome and application thereof in preparation of macrophage immune activator, and belongs to the technical field of traditional Chinese medicines and their application. The raw pinellia ternate exosome is separated from raw pinellia ternate. The raw pinellia ternate is peeled, and is beaten in water to obtain a suspension. The suspension is centrifuged at a speed of 8000-12000xg for 2-3 times, and the precipitate is removed. The supernatant is subjected to ultracentrifugation, and the precipitate is collected. The precipitate is resuspended with a PBS solution, and is subjected to ultracentrifugation again, and the precipitate is collected, which is the raw pinellia ternate exosome. The raw pinellia ternate exosome acts on M0 type macrophages, and the expression of M1 type macrophage immune related genes CD86, iNOS and IL-1beta is obviously increased, and the expression of M2 type macrophage immune related genes CD206, Arg-1 and IL-10 is obviously decreased, which indicates that the raw pinellia ternate exosome has the function of stimulating macrophage immune activation, and can be applied to preparation of a macrophage immune activator, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine and its application, and particularly relates to raw pinellia exosomes and its application in preparing a macrophage immune activator. Background Art

[0002] Exosomes are membrane-bound vesicles released into the extracellular matrix after fusion of intracellular multivesicular bodies with the cell membrane. Almost all cell types can produce and release exosomes. Secreted exosomes act on other cells or tissues in the external environment, triggering various signaling pathways and significantly affecting the phenotype and function of the recipient cells. Plant-derived exosomes mediate cell signaling and communication with the external environment, participating in important biological processes such as immune response, apoptosis, and angiogenesis. For example, recent reports suggest that nanovesicles from plants such as ginseng may inhibit cancer growth and enhance the effectiveness of immunotherapy.

[0003] Macrophages are an important component of the innate immune response to maintain homeostasis in the body and play an active role in inflammation, infection or metabolic disorders. Macrophages are plastic and can differentiate into different types under the influence of different environments and cytokines. There are two common types of macrophages: (1) M1-like macrophages are typical activated macrophages that can produce a large number of pro-inflammatory factors such as TNF-α, IL-6 and IL-1β. M1-like macrophages have strong antigen presentation ability, which can promote immune activity, resist pathogen infection, phagocytose tumors, and inhibit tumor proliferation. They also express factors that can activate NK cells and T cells to kill tumors. (2) M2-like macrophages are selectively activated macrophages that can produce a large number of anti-inflammatory factors such as TGF-β, IL-4 and IL-10, as well as higher M2-like macrophage markers such as Arg-1, Ym-1, Fizz-1 and CCL18. M2-like macrophages can inhibit inflammatory responses and promote angiogenesis. The two functional antagonistic phenotypes can transform into each other under various stimuli. The effects of natural plant medicines on macrophage function are attracting increasing attention, and people hope to develop new ingredients with high efficiency and low toxicity immunomodulatory effects with anti-inflammatory, anti-cancer, and immune regulation effects.

[0004] From ancient times to the present, Pinellia ternata must be processed before use as medicine. The toxicity of raw Pinellia ternata manifests primarily as strong mucosal irritation, hepatotoxicity, and pregnancy toxicity. Improper processing or ingestion of small amounts of raw Pinellia ternata can cause severe irritation to the lips, throat, oral cavity, vocal cords, and gastrointestinal mucosa, leading to a tingling sensation, swelling of the mouth and tongue, sore throat, and even loss of voice, vomiting, and diarrhea. Excessive consumption can cause burning, swelling, and loss of voice, salivation, vomiting, general numbness, slowed breathing, cramps, and difficulty breathing. Medicinal Pinellia ternata is typically processed with alum, lime, and ginger, such as in ginger Pinellia, French Pinellia, and clear Pinellia ternata. This process is intended to both facilitate preservation and remove toxic components. However, processing can alter the effective bioactive components of Pinellia ternata. There are no reports on whether the extracellular vesicles of raw pinellia can affect the function of macrophages and regulate the body's immunity through the proteins, lipids, RNA and other components they contain, thereby playing a role in the prevention and treatment of diseases. Summary of the Invention

[0005] In response to the problems of high toxicity and low development and utilization of raw pinellia in the existing technology, the present invention provides a raw pinellia exosome and its application in the preparation of a macrophage immune activator. The prepared raw pinellia exosome has the effect of stimulating macrophage immune activation.

[0006] The present invention is achieved through the following technical solutions:

[0007] A raw pinellia exosome is isolated from the raw pinellia.

[0008] Furthermore, the raw pinellia exosomes are prepared by the following method:

[0009] (1) Peel the raw Pinellia tuber, add water and beat to obtain a suspension. Centrifuge the suspension at 8000-12000 × g for 2-3 times to remove the precipitate.

[0010] (2) After removing the precipitate from step (1), the supernatant was subjected to ultracentrifugation to collect the precipitate;

[0011] (3) The precipitate from step (2) was resuspended in PBS solution and subjected to ultracentrifugation to collect the precipitate, which was the raw Pinellia exosomes.

[0012] Furthermore, in step (1), the mass ratio of raw pinellia to water is 1:2.

[0013] Furthermore, the centrifugation time in step (1) is 10 to 30 minutes.

[0014] Furthermore, the ultracentrifugation conditions in step (2) and step (3) are 120,000 × g, 50-90 min.

[0015] In the present invention, the raw pinellia exosomes are used in the preparation of a macrophage immune activator.

[0016] Furthermore, the raw pinellia exosomes increase the expression of immune-related genes in M1 macrophages and reduce the expression of immune-related genes in M2 macrophages; the immune-related genes in M1 macrophages are CD86, iNOS and IL-1β; the immune-related genes in M2 macrophages are CD206, Arg-1 and IL-10.

[0017] Beneficial effects

[0018] The present invention obtains raw pinellia exosomes by isolating from raw pinellia. After the raw pinellia exosomes act on M0 macrophages for 24 hours, the expression of immune-related genes CD86, iNOS and IL-1β in M1 macrophages is significantly increased, while the expression of immune-related genes CD206, Arg-1 and IL-10 in M2 macrophages is significantly reduced, indicating that raw pinellia has the effect of stimulating macrophage immune activation and is applicable to the preparation of a macrophage immune activator. In addition, in vivo experiments show that the raw pinellia exosomes isolated from raw pinellia do not cause damage to the internal organs of mice, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an electron micrograph of the exocrine secretion of raw Pinellia ternata;

[0020] Figure 2 This is the particle size distribution diagram of the exosomes of raw Pinellia ternata;

[0021] Figure 3 Figure 2 is the expression level of CD86 in cells of the experimental group and the control group;

[0022] Figure 4 Figure 2 is the expression level of IL-1β in cells of the experimental group and the control group;

[0023] Figure 5 is the expression level of iNOS in cells of the experimental group and the control group;

[0024] Figure 6 Figure 2 is the expression level of CD206 in cells of the experimental group and the control group;

[0025] Figure 7 is the expression level of Arg-1 in cells of the experimental group and the control group;

[0026] Figure 8 The graph shows the expression of IL-10 in the cells of the experimental group and the control group.

[0027] Figure 9 HE staining of pathological sections of heart, liver, spleen, lung and kidney of mice in the experimental and control groups. DETAILED DESCRIPTION

[0028] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the implementation cases are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0029] The raw pinellia (fresh pinellia) in the present invention was purchased from Nanjing Dosfu Biotechnology Co., Ltd.; the human THP1 leukemia monocytic cell line (abbreviated as: THP1 cell) was purchased from ATCC.

[0030] Example 1

[0031] (1) Wash, dry and peel the raw Pinellia tuber. Take 100 g of the raw Pinellia tuber and add 200 g of water. Place the raw Pinellia tuber in a blender and stir for 5 times, each time for 10 seconds, with an interval of 10 seconds to obtain a suspension. Centrifuge the suspension at 10,000 g for 20 minutes, remove the precipitate, and centrifuge the supernatant at 10,000 g for 20 minutes again. Remove the precipitate and retain the supernatant for ultracentrifugation.

[0032] (2) Take the supernatant and centrifuge it at 10,000 × g for 30 minutes to remove impurities; take the supernatant and centrifuge it at 120,000 × g for 70 minutes. A precipitate can be seen on the bottom of the tube. Some samples will have color. The main component of this precipitate is exosomes.

[0033] (3) Rinse the precipitate with PBS solution, resuspend it by pipetting, and centrifuge it again at 120,000 × g for 70 minutes. Carefully discard the supernatant. The precipitate is the raw Pinellia exosomes.

[0034] (4) The exosomes of raw Pinellia ternata were reconstituted with PBS and stored at -80°C.

[0035] Take 10 μl of the re-dissolved exosome suspension in step (4) and adsorb it onto a 200-mesh carbon film grid. After absorbing the liquid, add 2% uranyl acetate stain for 2 minutes. After absorbing the stain, bake the grid and observe and photograph under an electron microscope at 80 kV. The electron microscope image of the exosomes of raw Pinellia ternata is shown below. Figure 1 As shown, typical saucer-like exosome particles can be seen, indicating that exosomes were successfully extracted from raw Pinellia ternata;

[0036] The particle size distribution of exosomes from raw Pinellia ternata is shown in the figure below. Figure 2 As shown, the results showed that the particle size was consistent with the size of exosomes (30-200nm), indicating that the exosomes of raw Pinellia were successfully prepared.

[0037] Example 2

[0038] Effects of raw Pinellia ternata exosomes on macrophage immune function

[0039] (1) THP1 cells were seeded into 6-well plates and cultured in 1640 medium. The cell density was adjusted to 500,000 / ml. PMA 100 nM was added and the induction time was 24 h until the cells adhered to the wall, indicating that M0 macrophages were successfully induced.

[0040] (2) The cells successfully induced into M0 macrophages were divided into experimental and control groups, and treated with 5uM (M0+pina 5uM) and 20uM (M0+pina 20uM) raw Pinellia exosomes or PBS (M0) for 24 hours.

[0041] (3) Trizol reagent was used to extract RNA from the experimental and control groups, specifically: 5×10 6 Add 1 ml of Trizol to the cells, let them stand for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes. Remove the supernatant, add 0.2 ml (0.1 ml) of chloroform, mix thoroughly, let them stand for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes. Remove the supernatant, add 0.5 ml (0.25 ml) of isopropanol, mix thoroughly by inversion, let them stand for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 10 minutes. Discard the supernatant, wash the pellet with 1 ml of 75% ethanol (prepared with DEPC-treated water), centrifuge at 7,500 rpm for 5 minutes, and air dry. Dissolve the pellet in an appropriate amount of deionized water and store at -80°C.

[0042] (4) Using the SweScript RT cDNA first-strand synthesis kit, the reverse transcription system was prepared according to the following components: 5×Fast RT Buffer 4ul, RT Enzyme Mix 1ul, Random Hexamer Primer (100 μM) 1ul, Total RNA 2ug, add H2O to a total volume of 20ul, gently mix and centrifuge; the reverse transcription program was set at 25℃ for 5min, 55℃ for 15min, and 85℃ for 5sec to obtain cDNA;

[0043] (5) Real-time quantitative PCR analysis was performed using the SYBR Green qPCR Master Mix kit. Each PCR reaction system consisted of 5 μl of 2× PCR Master Mix, 100 ng of cDNA, 0.2 μM of each gene primer, and water added to a total volume of 10 μl. The PCR program was 95°C for 5 min. PCR was performed for 40 cycles, each cycle consisting of denaturation at 95°C for 10 s, annealing at 58°C for 10 s, and extension at 72°C for 10 s.

[0044] CD86 (M1), iNOS (M1), IL-1β (M1), CD206 (M2), Arg-1 (M2), IL-10 (M2), β-actin (internal reference gene) are shown in Table 1 as follows.

[0045] Table 1

[0046]

[0047] The expression amounts of CD86, IL-1β, iNOS, CD206, Arg-1 and IL-10 in the cells of the experimental groups (M0+pina 5uM, M0+pina 20uM) and the control group (M0) after 24h of PBS and raw pinellia ternate exosome treatment are shown in Table 1 as follows. Figures 3 to 8 As can be seen from the figure, after 24h of raw pinellia ternate exosome treatment, the expression of M1 type macrophage immune related genes CD86, iNOS and IL-1β is significantly increased, and the expression of M2 type macrophage immune related genes CD206, Arg-1 and IL-10 is significantly reduced; it is proved that raw pinellia ternate exosome has the effect of stimulating macrophage immune activation.

[0048] Example 3

[0049] Effect of raw pinellia ternate exosome on mouse heart, liver, spleen, lung and kidney

[0050] (1) Drug treatment: 30 C57BL / 6J mice, half male and half female, 16-22g, were randomly divided into 3 groups: normal control group: given the same volume of distilled water; low dose group: raw pinellia ternate exosome 0.25g / kg; high dose group: raw pinellia ternate exosome 1g / kg, the dosage is 20ml / kg;

[0051] (2) Sampling and fixation: the mice were continuously given intragastrically for 15 days, after the last administration, the mice were killed and dissected quickly, the heart, liver, spleen, lung and kidney were taken out and soaked in 10% paraformaldehyde solution overnight, washed with PBS for 3 times, then the tissue samples were put into a vacuum pump filled with PBS, the power of the vacuum pump was turned to the maximum, and the residual gas in the tissue samples was dried as much as possible;

[0052] (3) Dehydration and clearing: Place the above tissue specimens in gradient ethanol of different concentrations in the order of 70% (2 min) → 75% (2 min) → 80% (2 min) → 85% (2 min) → 90% (5 min) → 95% (5 min) → 95% (5 min) → 100% (10 min) → 100% (10 min); use alcohol from low to high concentration as dehydrating agent to gradually remove water from the tissue block, and then place the tissue block in xylene, a clearing agent that is soluble in both alcohol and paraffin, to clear it, and replace the alcohol in the tissue block with xylene;

[0053] (4) Wax embedding: Place the transparent tissue block in the melted paraffin and put it in a wax melting box to keep it warm. After the paraffin is completely immersed in the tissue block, embed it: first prepare a container (such as a folded small paper box), pour in the melted paraffin, quickly pick up the tissue block soaked in paraffin and put it in it, and cool it to solidify into a block.

[0054] (5) Sectioning and mounting: Use a fully automatic paraffin embedding machine to embed the dehydrated tissue specimens in paraffin. The embedded tissue sections are fixed on the fully automatic microtome. First, trim the paraffin tissue block and then slice it. The thickness of the slice is 4 μm. Use a brush to put the cut specimen slices into 37°C water, then adhere them to a poly-lysine-coated slide and mark them. All slices are placed in a 60°C oven overnight.

[0055] (6) Dewaxing: Place the slides in xylene → xylene → 100% ethanol → 100% ethanol → 95% ethanol → 90% ethanol → 80% ethanol → 70% ethanol for dewaxing, and rinse in clean water after dewaxing;

[0056] (7) HE staining: Hematoxylin (H) is a basic dye that can stain the cell nucleus and ribosomes in the cell into blue-purple. The structures stained by basic dyes are basophilic. Eosin (E) is an acidic dye that can stain the cytoplasm into red or light red. The structures stained by acidic dyes are eosinophilic. The sections that have been immersed in distilled water are placed in a hematoxylin aqueous solution for several minutes, rinsed with running water for 1 hour, and then placed in distilled water for a while. Dehydrated in 70% and 90% alcohol for 10 minutes each, and then stained in alcohol eosin staining solution for 2-3 minutes.

[0057] (8) Apply gum to the stained sections and seal with a coverslip.

[0058] After 15 days of oral administration of raw Pinellia exosomes to mice, the heart, liver, spleen, lung, and kidney of the experimental and control groups were harvested for pathological staining. Figure 9 As shown by Figure 9It can be seen that there were no obvious abnormalities in the heart, liver, spleen, lungs, and kidneys before and after the action of Pinellia exosomes, indicating that Pinellia exosomes did not cause damage to the organs of mice.

Claims

1. A use of raw pinellia exosomes in the preparation of an M1 macrophage immune activator, characterized in that: The raw pinellia exosomes are isolated from raw pinellia, and the M1 macrophage immune activator is a drug for inhibiting tumors.

2. The use of the raw pinellia exosomes in the preparation of an M1 macrophage immune activator according to claim 1, characterized in that: Raw Pinellia exosomes were prepared by the following method: (1) Peel the raw Pinellia tuber, add water and beat to obtain a suspension, centrifuge the suspension at 8000-12000 × g for 2-3 times to remove the precipitate; (2) After removing the precipitate from step (1), the supernatant was subjected to ultracentrifugation to collect the precipitate; (3) The precipitate from step (2) was resuspended with PBS solution, and ultracentrifuged continuously to collect the precipitate, which was the raw Pinellia exosomes.

3. The use of the raw pinellia exosomes in the preparation of an M1 macrophage immune activator according to claim 2, characterized in that: The mass ratio of raw pinellia to water in step (1) is 1:

2.

4. The use of the raw pinellia exosomes in the preparation of an M1 macrophage immune activator according to claim 2, characterized in that: The centrifugation time in step (1) is 10 to 30 minutes.

5. The use of the raw pinellia exosomes in the preparation of an M1 macrophage immune activator according to claim 2, characterized in that: The ultracentrifugation conditions in step (2) and step (3) are 120,000×g, 50 to 90 min.

6. The use of raw pinellia exosomes in the preparation of an M1 macrophage immune activator according to claim 1, characterized in that: The raw pinellia exosomes increase the expression of M1 macrophage immunity-related genes; the M1 macrophage immunity-related genes are CD86, iNOS and IL-1β.

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