Raw pinellia ternate exosome and application thereof in preparation of macrophage immune activator

Abstract

The invention discloses a raw pinellia ternate exosome and application thereof in preparation of a macrophage immune activator, and belongs to the technical field of traditional Chinese medicines and application thereof. The method comprises the following steps: separating raw pinellia ternate exosomes from raw pinellia ternate, peeling the raw pinellia ternate, adding water, pulping to obtain a suspension, centrifuging at the speed of 8000-12000 * g for 2-3 times, and removing precipitates; carrying out ultracentrifugation on the supernate, and collecting the precipitate; and re-suspending the precipitate by using a PBS solution, continuously carrying out ultracentrifugation, and collecting the precipitate, namely the raw pinellia ternate exosome. The raw rhizoma pinelliae exosome acts on M0 type macrophages, the expressions of M1 type macrophage immune related genes CD86, iNOS and IL-1beta are obviously increased, and the expressions of M2 type macrophage immune related genes CD206, Arg-1 and IL-10 are obviously reduced, which indicates that the raw rhizoma pinelliae exosome has the effect of stimulating the immune activation of the macrophages, is applied to the preparation of the macrophage immune activator, and can be used for preparing the macrophage immune activator. Wide application prospects are realized.

Description

Technical Field

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

[0002] Exosomes are membrane vesicles released into the extracellular matrix after the fusion of intracellular multivesicular bodies with the cell membrane. Almost all types of cells can produce and release exosomes. The secreted exosomes act on other cells or tissues in the outside world, triggering various signaling pathways, thereby having a significant impact on the phenotype and function of the recipient cells. Plant-derived exosomes participate in important biological processes such as immune response, apoptosis, and angiogenesis by mediating cell signaling and communication with the outside world. For example, it has been recently reported that nanovesicles in plants such as ginseng may inhibit cancer growth and improve 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 be transformed into each other under various stimuli. The effects of natural plant drugs on macrophage function are attracting more and more 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 it can be used as medicine. The toxicity of raw Pinellia ternata mainly manifests as strong mucosal irritation toxicity, liver and kidney toxicity, and pregnancy toxicity. If it is improperly processed or taken in small amounts, it will cause strong irritation to the lips, throat, oral cavity, vocal cords, and gastrointestinal mucosa in contact, leading to a needle-like tingling sensation and swelling of the mouth and tongue, tingling in the throat or even loss of voice, vomiting, diarrhea, etc., and a large amount will cause burning and swelling of the throat, inability to speak, drooling, vomiting, numbness of the whole body, slow breathing, spasms, and difficulty breathing. Medicinal Pinellia ternata is usually processed with alum, lime, ginger, etc., such as ginger Pinellia ternata, French Pinellia ternata, and Qing Pinellia ternata. The purpose is to facilitate preservation on the one hand, and to remove its toxic components on the other hand. However, after processing, the effective biological active components of Pinellia ternata will also undergo certain changes. There has been no report on whether the extracellular vesicles based on 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 view of the problems of high toxicity and low development and utilization of raw pinellia in the prior art, 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: A raw pinellia exosome, wherein the raw pinellia exosome is separated from the raw pinellia.

[0007] Furthermore, the raw pinellia exosomes are prepared by the following method: (1) Peel the raw Pinellia tuber, add water to make a suspension, centrifuge the suspension at 8000-12000 × g for 2-3 times, and remove the precipitate; (2) After removing the precipitate from step (1), the supernatant is subjected to ultracentrifugation to collect the precipitate; (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.

[0008] Furthermore, in step (1), the mass ratio of raw pinellia to water is 1:2.

[0009] Furthermore, the centrifugation time in step (1) is 10 to 30 minutes.

[0010] Furthermore, the ultracentrifugation conditions in step (2) and step (3) are 120,000 × g, 50-90 min.

[0011] In the present invention, the raw pinellia exosomes are used in the preparation of a macrophage immune activator.

[0012] Furthermore, the raw pinellia exosomes increase the expression of immune-related genes in M1 macrophages and decrease 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.

[0013] Beneficial Effects The present invention obtains raw pinellia exosomes by isolating them from raw pinellia. The raw pinellia exosomes act on M0 macrophages for 24 hours, and the expressions of immune-related genes CD86, iNOS and IL-1β of M1 macrophages are significantly increased, and the expressions of immune-related genes CD206, Arg-1 and IL-10 of M2 macrophages are significantly reduced, indicating that raw pinellia has the effect of stimulating macrophage immune activation, and is applicable to the preparation of macrophage immune activators; and in vivo experiments show that the raw pinellia exosomes isolated from raw pinellia do not damage the internal organs of mice, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an electron micrograph of the exocytosis of raw Pinellia ternata; Figure 2 This is the particle size distribution diagram of exosomes from raw Pinellia ternata; Figure 3 The expression of CD86 in cells of the experimental group and the control group; Figure 4 The expression of IL-1β in cells of the experimental group and the control group; Figure 5 The expression of iNOS in cells of the experimental group and the control group; Figure 6 The expression of CD206 in cells of the experimental group and the control group; Figure 7 It is the expression level of Arg-1 in cells of the experimental group and the control group; Figure 8 The graph shows the expression of IL-10 in the cells of the experimental group and the control group. Fig. 9 HE staining images of pathological sections of heart, liver, spleen, lung and kidney of mice in the experimental group and control group. DETAILED DESCRIPTION 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.

[0015] 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.

[0016] Example 1 (1) Wash, dry and peel the raw Pinellia tuber, take 100 g of it, add it to 200 g of water, place it in a blender and stir it for 5 times, each time for 10 seconds, with an interval of 10 seconds to obtain a suspension. The suspension is centrifuged at 10,000 g for 20 minutes, the precipitate is removed, the supernatant is centrifuged at 10,000 g for 20 minutes again, the precipitate is removed, and the supernatant is retained for ultracentrifugation; (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 layer of sediment can be seen on the bottom of the tube. Some samples will have color. The main component of this sediment is exosomes. (3) Rinse the precipitate with PBS solution, resuspend it by pipetting, and centrifuge it again at 120,000 × g for 70 min. Carefully discard the supernatant. The precipitate is the exosomes of raw Pinellia ternata. (4) The exosomes of raw Pinellia ternata were reconstituted with PBS and stored at -80°C.

[0017] Take 10 μl of the reconstituted exosome suspension from step (4) and adsorb it onto a 200-mesh carbon film grid. After absorbing the liquid, add 2% uranyl acetate dye solution for 2 minutes. Absorb the dye solution, 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 in the figure below. Figure 1 As shown, typical saucer-like exosome particles can be seen, indicating that exosomes were successfully extracted from raw Pinellia tuber; The particle size distribution of exosomes from raw Pinellia ternata is shown in the figure Figure 2 As shown, the results showed that the particle size was consistent with the size of exosomes (30-200nm), indicating that the raw pinellia exosomes were successfully prepared.

[0018] Example 2 Effects of raw pinellia exosomes on the immune function of macrophages (1) THP1 cells were inoculated into 6-well plates and cultured in 1640 medium. The cell density was adjusted to 500,000 / ml. PMA 100 nM was given and the induction time was 24 h until the cells adhered to the wall, indicating that M0 macrophages were successfully induced. (2) The cells successfully induced into M0 macrophages were divided into experimental group and control group, and treated with raw Pinellia exosomes 5uM (M0+pina 5uM) and 20uM (M0+pina 20uM) or PBS (M0) for 24 hours; (3) Trizol reagent was used to extract RNA from cells in the experimental and control groups. Specifically: 5×106 Add 1 ml Trizol to the cells, let stand for 10 min, centrifuge at 4°C 12000 rpm for 15 min; take the supernatant, add 0.2 ml (0.1 ml) chloroform, mix, let stand for 10 min, centrifuge at 4°C 12000 rpm for 15 min; take the supernatant, add 0.5 ml (0.25 ml) isopropanol, mix by inversion, let stand for 10 min, centrifuge at 4°C 12000 rpm for 10 min, discard the supernatant, wash the precipitate with 1 ml 75% ethanol (prepared with DEPC-treated water), centrifuge at 7500 rpm for 5 min, and dry; add appropriate amount of deionized water to dissolve, and store at -80°C; (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 to 25℃ 5min, 55℃ 15min, 85℃ 5sec, and cDNA was obtained; (5) Real-time quantitative PCR analysis was performed using the SYBR Green qPCR Master Mix kit. Each PCR reaction system was: 2×PCR Master Mix 5ul, cDNA 100ng, each gene primer was 0.2uM, and water was added to a total volume of 10ul; the PCR program was 95°C for 5min; PCR was performed for 40 cycles, each cycle included denaturation at 95°C for 10s, annealing at 58°C for 10s, and extension at 72°C for 10s; CD86 (M1), iNOS (M1), IL-1β (M1), CD206 (M2), Arg-1 (M2), IL-10 (M2), and β-actin (reference gene) are shown in Table 1 below.

[0019] Table 1 After 24 h of treatment with PBS and raw pinellia exosomes, the expression levels of CD86, IL-1β, iNOS, CD206, Arg-1, and IL-10 in the experimental group (M0+pina 5uM, M0+pina 20uM) and the control group (M0) were as follows: Figures 3 to 8As shown in the figure, it can be seen that after 24 hours of the action of Pinellia exosomes, the expression of M1 macrophage immune-related genes CD86, iNOS and IL-1β was significantly increased, and the expression of M2 macrophage immune-related genes CD206, Arg-1 and IL-10 was significantly decreased; this indicates that Pinellia exosomes have the effect of stimulating macrophage immune activation.

[0020] Example 3 Effects of raw Pinellia exosomes on the heart, liver, spleen, lungs, kidneys and other organs of mice (1) Drug treatment: 30 C57BL / 6J mice, half male and half female, weighing 16-22 g, were randomly divided into 3 groups: normal control group: given an equal volume of distilled water; low-dose group: 0.25 g / kg of Pinellia exosomes; high-dose group: 1 g / kg of Pinellia exosomes, the dosage for both groups was 20 ml / kg; (2) Sampling and fixation: Mice were given drugs by gavage for 15 consecutive days. After the last drug administration, the mice were killed and the main organs, including heart, liver, spleen, lung, and kidney, were removed and immersed in 10% paraformaldehyde solution overnight. The tissue specimens were washed three times with PBS, and then placed in a vacuum pump filled with PBS. The power was turned on and the vacuum pump was adjusted to the maximum power to absorb as much residual gas as possible from the tissue specimens. (3) Dehydration and transparency: Place the above tissue specimens in gradient ethanol of different concentrations in the following order: 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 the water in the tissue block, and then place the tissue block in xylene, a transparent agent that is soluble in both alcohol and paraffin, to make it transparent, and use xylene to replace the alcohol in the tissue block; (4) Wax embedding: Place the transparent tissue block in the melted paraffin and put it in a wax melting box to keep 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 the melted paraffin, quickly clamp the tissue block soaked in paraffin and put it in the container, and cool it to solidify into a block. (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. (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; (7) HE staining: Hematoxylin (H) is an alkaline dye that can dye the cell nucleus and ribosomes in the cell into blue-purple. The structures stained by the alkaline dye are basophilic. Eosin (Eosin, E) is an acidic dye that can dye the cytoplasm into red or light red. The structures stained by the acidic dye are eosinophilic. The sections that have been immersed in distilled water are placed in a hematoxylin aqueous solution for staining for several minutes. After rinsing with running water for 1 hour, they are placed in distilled water for a while, and then dehydrated in 70% and 90% alcohol for 10 minutes each. They are then placed in alcohol eosin staining solution for staining for 2-3 minutes. (8) Apply gum to the stained sections and seal with a coverslip. After mice were given raw Pinellia exosomes by gavage for 15 days, the heart, liver, spleen, lung, and kidney of the experimental group and the control group were respectively removed for pathological staining. The results are as follows: Fig. 9 As shown by Fig. 9 It 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 raw pinellia exosome, characterized in that: The raw pinellia exosomes are isolated from raw pinellia.

2. The raw pinellia exosomes according to claim 1, characterized in that Raw Pinellia exosomes were prepared by the following method: (1) Peel the raw Pinellia tuber, add water to make a suspension, centrifuge the suspension at 8000-12000 × g for 2-3 times, and remove the precipitate; (2) After removing the precipitate from step (1), the supernatant is subjected to ultracentrifugation to collect the precipitate; (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.

3. The raw pinellia exosomes according to claim 2, characterized in that In step (1), the mass ratio of raw pinellia to water is 1:

2.

4. The raw pinellia exosomes according to claim 2, characterized in that The centrifugation time in step (1) is 10 to 30 minutes.

5. The raw pinellia exosomes according to claim 2, characterized in that The ultracentrifugation conditions in step (2) and step (3) are 120,000 × g, 50-90 min.

6. Use of the raw pinellia exosomes according to any one of claims 1 to 5 in the preparation of a macrophage immune activator.

7. The use according to claim 6, characterized in that: The raw pinellia exosomes increase the expression of immune-related genes in M1 macrophages and decrease the expression of immune-related genes in M2 macrophages; The M1 macrophage immunity-related genes are CD86, iNOS and IL-1β; The M2 macrophage immunity-related genes are CD206, Arg-1 and IL-10.

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