CN117045759A - Application of curcuma wenyujin source lipid nano vesicle in preparation of medicine for relieving non-alcoholic fatty liver disease - Google Patents
Application of curcuma wenyujin source lipid nano vesicle in preparation of medicine for relieving non-alcoholic fatty liver disease Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/906—Zingiberaceae (Ginger family)
- A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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Abstract
The application discloses an application of curcuma wenyujin source lipid nanovesicles in preparing a medicine for relieving non-alcoholic fatty liver disease, and belongs to the technical field of biological medicines. The nano particles are extracted from the plant curcuma aromatica through wall breaking, differential centrifugation, ultracentrifugation and sucrose density gradient centrifugation, and are characterized by transmission electron microscopy and particle size analysis, so that the nano particles are found to be of a nano vesicle-like structure. The method utilizes high-fat diet feeding to construct a non-alcoholic fatty liver disease mouse model, and further treats the high-fat fed mouse by intraperitoneal injection of curcuma wenyujin source lipid nanovesicles, and the result shows that the curcuma wenyujin source lipid nanovesicles can relieve liver lipid deposition and liver inflammation, so that a new strategy is provided for developing medicines for relieving and treating the non-alcoholic fatty liver disease.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to application of curcuma wenyujin source nano vesicles in preparation of medicines for relieving non-alcoholic fatty liver diseases.
Background
Nonalcoholic fatty liver disease (Nonalcoholic fatty liver disease, NAFLD) is the most common chronic liver disease, and the incidence is in a rapidly growing trend and younger situation. NAFLD includes a range of liver lesions, from simple liver steatosis, nonalcoholic steatohepatitis characterized by hepatocyte damage and lobular inflammation (Nonalcoholic steatohepatitis, NASH), possibly leading to cirrhosis, hepatocellular carcinoma, etc. Although drugs such as antioxidants, insulin sensitizers and cytoprotective agents are applied to NAFLD treatment in recent years, the treatment effect is poor, and no ideal preventive and relieving drugs exist in clinic at present.
In recent years, researchers find that a natural nano vesicle exists in fresh plants, and most of the nano vesicles are lipid-coated proteins, miRNAs and the like, can be used as information carriers, participate in intercellular communication, and can be taken up by most cells including macrophages to play a role. Therefore, the research on the effect of the lipid nanovesicles on relieving the non-alcoholic fatty liver disease is expected to provide a new strategy for developing new drugs for relieving the non-alcoholic fatty liver disease.
Disclosure of Invention
The application aims to provide an application of curcuma wenyujin source lipid nanovesicles in preparing a medicine for relieving non-alcoholic fatty liver disease, so as to solve the problems in the prior art.
In order to achieve the above object, the present application provides the following solutions:
one of the technical schemes of the application is as follows: application of radix Curcumae-derived lipid nanovesicles in preparing medicine for relieving non-alcoholic fatty liver disease is provided.
The second technical scheme of the application is as follows: the lipid nanovesicles derived from Curcuma wenyujin are extracted from root tuber of Curcuma wenyujin.
The third technical scheme of the application: the preparation method of the curcuma wenyujin source lipid nano vesicle is characterized by comprising the following steps:
s1, taking fresh radix curcumae tuberous root, cleaning and peeling with clear water, then washing with 1 XPBS for 3-5 times, and stirring and grinding the radix curcumae tuberous root to obtain stirred and ground radix curcumae juice;
s2, carrying out differential centrifugation on the stirred and ground curcuma wenyujin juice to obtain supernatant after differential centrifugation;
s3, centrifuging the supernatant after differential centrifugation at the temperature of 4 ℃ and the pressure of 100000-200000g for 60-120min, after the ultracentrifugation is finished, re-suspending the sediment in 1 XPBS and slightly adding the sediment to the uppermost layer of sucrose gradient solution with the concentration gradient of 60 wt%, 45 wt%, 30 wt% and 8 wt% from bottom to top, centrifuging at the temperature of 4 ℃ and the pressure of 100000-200000g for 60-120min, sucking a visible strip between the 8 wt% and 30 wt% sucrose layers after the centrifugation is finished, re-suspending the sediment in 1 XPBS, and centrifuging at the temperature of 4 ℃ and the pressure of 100000-200000g for 60-120min again, wherein the obtained sediment is the curcuma wenyujin source lipid nano vesicle.
Further, 1×pbs is added during the stirring and grinding process in step S1, and the solid-to-liquid ratio of the tuber of curcuma wenyujin to 1×pbs is 1g:2mL.
Further, the rotation speed of the stirring and grinding in the step S1 is 15000-28000rpm, the stirring and grinding is repeatedly carried out for 3-10 times, each time is 1-5min, and stirring is suspended for 1-5min in the middle of two times of stirring and grinding.
Further, the differential centrifugation in step S2 specifically includes: centrifuging at 500-2000g for 10-30min, discarding the precipitate, collecting the supernatant, centrifuging at 2000-5000g for 10-30min, discarding the precipitate, collecting the supernatant, centrifuging at 8000-15000g for 30-60min, discarding the precipitate, and collecting the supernatant after differential centrifugation.
Further, the concentration gradient in step S3 is 60 wt%, 45 wt%, 30 wt%, 8 wt% sucrose gradient solution from bottom to top, and the preparation method comprises: firstly, preparing sucrose solutions with the concentration of 8 wt%, 30 wt%, 45 wt%, 60 wt% by taking 1 XPBS or double distilled water as a solvent, and then taking a clean super-separation tube, and sequentially adding 6-8mL of each of the sucrose solutions with the concentration of 60 wt%, 45 wt%, 30 wt%, 8 wt% to prepare a gradient, so as to obtain the sucrose gradient solution.
Further, the preservation of the curcuma wenyujin source lipid nanovesicles also comprises the steps of S4: and (3) re-suspending the curcuma wenyujin source lipid nano vesicle sediment obtained in the step (S3) by using 1 XPBS to obtain a curcuma wenyujin source lipid nano vesicle suspension, and transferring the suspension into a clean enzyme-removed EP tube for preservation at-80 ℃.
The application discloses the following technical effects:
(1) The application discloses an application of curcuma wenyujin source lipid nanovesicles in preparation of a medicine for relieving non-alcoholic fatty liver disease. The curcuma wenyujin-derived lipid nanovesicles can relieve non-alcoholic fatty liver disease and infiltration of inflammatory cells in liver tissues, and can also reduce inflammatory cytokines IL-6 and IL-1 beta in serum and tissues.
(2) The application adopts the methods of wall breaking grinding, differential centrifugation and sucrose density gradient centrifugation (differential centrifugation is used for removing large particles in juice one by one, 500-2000g is used for removing large plant residues which are not fully ground, 2000-5000g is used for further removing medium-sized residues, 8000-15000g can basically remove macroscopic residues, the next step of super separation is used for obtaining nano-scale vesicle precipitation, and sucrose gradient centrifugation is used for further purifying to obtain vesicles with different densities), nano particles are extracted from plant radix curcumae, and the nano particles are characterized by a Transmission Electron Microscope (TEM) and particle size analysis, so that the nano vesicle structure is found. The method utilizes high-fat diet feeding to construct a non-alcoholic fatty liver disease mouse model, and further treats the high-fat fed mouse by intraperitoneal injection of curcuma wenyujin source lipid nanovesicles, and the result shows that the curcuma wenyujin source lipid nanovesicles can relieve liver lipid deposition and liver inflammation, so that a new strategy is provided for developing medicines for relieving and treating the non-alcoholic fatty liver disease.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a picture of the extraction process and characterization after extraction of curcuma wenyujin derived lipid nanovesicles, wherein: a is a picture of a root tuber of a raw material curcuma wenyujin; b is sucrose density gradient centrifugation; c is a TEM image of curcuma wenyujin source lipid nano vesicles; d is a particle size analysis chart of the curcuma wenyujin source lipid nano vesicle.
Fig. 2 is a graph showing the results of a cell uptake experiment and a cell inflammation inhibition experiment of curcuma wenyujin-derived lipid nanovesicles, wherein: a is a confocal picture of a Kupffer cell and a HepG2 cell taking the curcuma wenyujin source lipid nano vesicle for 24 hours; b is the experimental result of inhibiting inflammation of RAW264.7 cells by curcuma wenyujin-derived lipid nanovesicles, wherein P <0.05 is represented by P <0.01, and P <0.001 is represented by P.
Fig. 3 is a graph showing the results of curcuma wenyujin-derived lipid nanovesicles for alleviating non-alcoholic fatty liver disease in mice induced by high fat diet, wherein: a is a general diagram of liver tissue of a mouse; b is the detection result of the liver tissue TC and TG of the mouse, P <0.05, P <0.01, P <0.001; c is the detection result of liver tissue inflammatory factor of mice, P <0.05, P <0.01, P <0.001; d is the observation result of HE staining of liver tissue of the mice.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The turmeric root tuber used in the following examples is turmeric root tuber of a Chinese Wenzhou plant, and the picking period is 10-12 months, but experimental researches for one year show that lipid nanovesicles extracted from turmeric root tuber in each period in one year have obvious inflammation inhibition effect.
Example 1 extraction of Curcuma wenyujin derived lipid nanovesicles
(1) Fresh radix Curcumae tuberous root (shown in figure 1A) is taken, cleaned with clear water and peeled, and then washed with 1 XPBS for 3 times. 100g of the cleaned and peeled radix Curcumae tubers were then weighed into a Blendtec mixer, 200mL (solid-to-liquid ratio 1:2) of pre-chilled 1 XPBS was added, and the mixture was thoroughly ground (stirring and grinding for 1min, suspending stirring for 1min, and circulating 3 times) at 28000 rpm.
(2) The ground juice was poured into a clean 50mL EP tube and then subjected to differential centrifugation: sequentially 1000g for 10min (discard sediment, leave supernatant), 3000g for 20min (discard sediment, leave supernatant), 10000g for 40min (discard sediment, leave supernatant).
(3) The final supernatant was carefully equilibrated in an ultracentrifuge tube and ultracentrifuged at 150000g for 90min at 4 ℃. During the preparation, sucrose gradient solutions with concentration of 8 wt%, 30 wt%, 45 wt%, 60 wt% and 7mL of each sucrose solution with concentration of 60 wt%, 45 wt%, 30 wt% and 8 wt% are prepared into gradient by taking a clean super-separation tube.
(4) After completion of the 90min ultracentrifugation, the pellet was resuspended in 7mL of 1 XPBS and lightly added to the uppermost sucrose gradient and centrifuged at 150000g for 120min at 4 ℃. After centrifugation was completed, the visible band between the 8wt.% and 30wt.% sucrose layers was carefully aspirated into another clean super-tube (band shown in FIG. 1B), and 1 XPBS was added to supplement 36mL, and after accurate trimming, the remaining sucrose was removed by centrifugation at 15000g for 90min at 4℃again.
(5) And after the centrifugation is finished, the supernatant is discarded, the paper towel is used for wiping the residual liquid on the wall of the super-isolated pipe, and the sediment is obtained, namely the required curcuma wenyujin source lipid nano vesicle. The pellet was resuspended in 16mL of 1 XPBS to give a Curcuma wenyujin-derived lipid nanovesicle suspension and transferred to a clean, de-enzymed EP tube, and the protein concentration was determined to be 2.818. Mu.g/. Mu.L using the BCA assay kit and stored at-80 ℃.
Effect verification
1. Characterization of Curcuma wenyujin derived lipid nanovesicles
1. Transmission Electron Microscope (TEM)
(1) Taking out the curcuma wenyujin source lipid nano vesicle suspension in the step (5) of the example 1, taking 10 mu L of the suspension to drop on a sample-carrying copper net with the aperture of 2nm, and standing at room temperature.
(2) The liquid on the side of the filter screen is sucked by filter paper, 2% phosphotungstic acid solution is added dropwise, and the filter screen is subjected to negative dyeing for 2min at room temperature.
(3) The negative dye solution is sucked by filter paper, 10 mu L of double distilled water is dripped for 3 times, and then the solution is naturally dried at room temperature, and then the solution can be observed by a transmission electron microscope, a TEM image of the solution is shown in figure 1C, and the result shows that the extract has a vesicle-like structure.
2. Particle size analysis
The particle size analysis and loading results are shown in figure 1D, and the results show that the average particle size of the extract is 211.2+/-3.1 nm and is nano-scale.
2. Kupffer cell/HepG 2 cell uptake assay
PKH26 fluorescent dye labeled Curcuma wenyujin-derived lipid nanovesicles
(1) Taking out the curcuma wenyujin source lipid nano vesicle suspension preserved at the temperature of minus 80 ℃ in the step (5) of the example 1, and melting on ice.
(2) Under dark conditions, PKH26 fluorescent dye was removed, 1. Mu.L to 200. Mu.L PCR tube was taken, and 99. Mu.L dilution was added to dilute 100-fold.
(3) The diluted PKH26 dye is added into 1mL of curcuma wenyujin source lipid nano microvesicle suspension, vigorously vortexed for 1min and incubated for 20min in a dark place.
(4) The 1mL suspension was transferred to a super-isolation tube, and fully resuspended to 40mL with 1 XPBS, and after stringent trimming, centrifuged at 150000g for 90min at 4 ℃.
(5) The supernatant was discarded and the curcuma wenyujin-derived lipid nanovesicles were resuspended in 1mL of 1 XPBS buffer.
(6) Filtering the resuspended Curcuma wenyujin Y.H.Chen et C.Ling source lipid nano vesicle with a 0.22 μm filter membrane to obtain fluorescence labeled Curcuma wenyujin Y.H.Chen et C.Ling source lipid nano vesicle suspension, the BCA assay kit was stored at-80℃in the absence of light at a protein concentration of 0.269. Mu.g/. Mu.L.
Method for preparing PKH26 fluorescent dye marked Curcuma wenyujin source lipid nano vesicle treated Kupffer cells and HepG2 cells and climbing slices and performing laser confocal shooting
(1) Taking a 24-hole plate, putting a round cell climbing plate into each hole, inoculating 8×10 4 Individual Kupffer cells or 5X 10 4 HepG2 cells were returned to the incubator for further culture.
(2) After culturing the cells for 12 hours, 30 μg of fluorescence-labeled curcuma wenyujin-derived lipid nanovesicle suspension was added to each well, and the culture was continued for 24 hours.
(3) Subsequently, samples were collected, 500. Mu.L of 1 XPBS buffer was added to the corresponding wells, and the wells were gently shaken several times, PBS was discarded, and washing was repeated three times.
(4) 300. Mu.L of 4% paraformaldehyde was added thereto, and the mixture was fixed at room temperature for 1 hour.
(5) The immobilized slide was removed, 4% paraformaldehyde was discarded, 500. Mu.L of 1 XPBS buffer was added, the mixture was gently shaken several times, PBS was discarded, and washing was repeated three times.
(6) Taking a clean glass slide, dropwise adding a drop of anti-fluorescence quenching agent, clamping the climbing piece by using tweezers, carefully sucking water by using edge contact filter paper, reversely buckling the glass slide on the anti-fluorescence quenching agent, dropwise adding neutral resin on the periphery of the edge, standing and solidifying at room temperature in a dark place, and storing in a dark place after the glass slide is stabilized.
(7) The laser confocal observation is carried out, the photographed image is shown in fig. 2A, and the result shows that both Kupffer cells and HepG2 cells can ingest the curcuma wenyujin source lipid nanovesicles after 24h of incubation with the curcuma wenyujin source lipid nanovesicles.
3. Curcuma wenyujin Y.H.Chen et C.Ling-derived lipid nano-microvesicles combined with LPS to stimulate RAW264.7 cells
1. Cell seeding and LPS stimulation
(1) A24-well plate was used to inoculate 1.0X10 each 5 RAW264.7 cells were cultured in 500. Mu.L of complete medium, and the medium was returned to the incubator.
(2) After cell culture for 12h, 30. Mu.g of Curcuma wenyujin-derived lipid nanovesicle suspension (prepared in step (5) of example 1) filtered through a 0.22 μm membrane was added to each well, and the culture was continued in an incubator for 24h.
(3) After 24h of treatment of the curcuma wenyujin source lipid nanovesicles, preparing an LPS working solution, taking out LPS storage solution (10 mg/mL) at-30 ℃, taking 0.5 mu L of the storage solution, mixing with 500 mu L of complete culture medium (1:1000 dilution), obtaining LPS working solution with the concentration of 10 mu g/mL, discarding the original culture medium in a culture plate hole, adding 500 mu L of complete culture medium containing 10 mu g/mL of LPS into each hole, and putting back into the culture box for continuous culture for 6h.
(4) The cells and culture supernatant were collected for subsequent detection.
qPCR detection of cytokines IL-6, IL-1. Beta. And TNF-alpha
(1) mu.L of Trizol reagent was added to each well to thoroughly scrape the cells in the well into 1.5mL of the enzyme-removed EP tube.
(2) Precipitation on ice for 5min was thoroughly broken, 60 μl of chloroform was added and immediately vortexed for 15s for thoroughly mixing.
(3) The mixture was again precipitated on ice for 5min, followed by centrifugation at 14000rpm at 4℃for 15min.
(4) The isopropanol was pre-chilled 4℃in advance and a corresponding number of 1.5mL of the enzyme-depleted EP tubes were prepared and labeled.
(5) 120. Mu.L of supernatant was transferred to the labeled de-enzyme EP tube described above using a pipette.
(6) Adding equal volume of isopropanol to precipitate RNA, mixing the mixture upside down for tens of times, and precipitating the mixture on ice for 10min.
(7) Centrifuge at 14000rpm for 10min at 4℃and carefully discard the supernatant.
(8) The precipitate was washed with 75wt.% ethanol (DEPC water formulation), centrifuged at 14000rpm at 4℃for 5min, washed twice repeatedly, the supernatant carefully discarded, isolated at 1min and dried at 42 ℃.
(9) RNA was dissolved by adding 20. Mu.L of DEPC water.
(10) Reverse transcription and qPCR procedure reference the Nanjinopran kit (NO: R223-01) procedure is as follows:
i, reverse transcription
(1) Removal of genomic DNA
TABLE 1 reagents for removal of genomic DNA
After gently stirring and mixing by a pipette, the PCR tube was placed into a PCR instrument and the program was run at 42℃for 2min.
(2) Preparation of reverse transcription reaction System
TABLE 2 reverse transcription reaction system
The evenly mixed solution is lightly blown by a pipetting gun, and then the PCR tube is centrifuged for 10s at 2000rpm for the next step in a PCR instrument.
(3) Reverse transcription reaction
TABLE 3 reverse transcription reaction conditions
After the PCR procedure was completed, the cDNA samples were stored in a-20℃refrigerator.
II, real-time fluorescent quantitative PCR (qRT-PCR)
The procedure for reference to the Nanjinopran kit (NO: Q712-02) is as follows:
TABLE 4 primer sequences
(1) Preparing a reaction system
TABLE 5 reaction system
Labeling was performed on a 96-well plate, and 20. Mu.L/well of the above mixture was sequentially added. After balancing the 96-well plate in a centrifuge, the reaction solution was collected by brief centrifugation.
(2) PCR reaction (in CFX96 Real-Time PCR System instrumentation)
TABLE 6PCR reaction procedure
The relative expression level of the genes after the reaction was according to 2 -△△Ct ( △ Ct value = Ct Purpose(s) -Ct Internal reference ， △△ Ct value = △ Ct Experimental group － △ Ct Control group ) Calculating to obtain relative expression level of gene, and detecting mRNA expression level of inflammatory factor in cell by using Gapdh gene as control gene (result is shown in figure 2B), wherein RT-qPCR result shows that Curcuma wenyujin source lipid nanovesicle +The relative expression of the mRNA of inflammatory factors IL-6, IL-1 beta and TNF-alpha in RAW264.7 cells of LPS group is lower than that of the LPS-stimulated group alone, and has obvious difference (P < 0.05). The curcuma wenyujin source lipid nanovesicles can inhibit cell inflammation.
4. Feeding process and intraperitoneal injection operation of high-fat mice
1.20 mice of comparable body weight, 8 week old, were randomly divided into 4 groups, wherein the high-fat group was fed with 45% kcal fat diet, the control group was fed with 10% kcal fat diet, and the total was fed for three months, during which time the mice of the different groups were intraperitoneally injected with a lipid nanovesicle heavy suspension of Curcuma wenyujin origin/1 XPBS.
2. Intraperitoneal injection procedure
(1) The suspension of the lipid nanovesicles derived from Curcuma wenyujin Y.H. Chen et C.Ling and 1 XPBS were prepared in advance, and the suspension was filtered through a 0.22 μm filter, and 150. Mu.g of the suspension of the lipid nanovesicles derived from Curcuma wenyujin Y.H.Chen et C.Ling or an equal volume of 1 XPBS was injected into each mouse.
(2) The method comprises the steps of grabbing a mouse by the left hand, holding a 1mL syringe on the right with the abdomen facing upwards, determining the position of the central line of the abdomen of the mouse, avoiding important organs on the left side or the right side of the central line, slowly injecting the abdominal cavity with the inclined surface of the needle of the syringe facing upwards, then placing the mouse back into a cage, and observing the state of the mouse.
5. Picking up mouse liver
The method comprises the following specific steps:
(1) Firstly, the mice are anesthetized, the weights of the mice are weighed, the anesthetic dosage of the mice is calculated, and the 1% sodium pentobarbital dosage of the anesthetic is 8 mu L/g. The method comprises the steps of grabbing the mouse, exposing the abdomen of the mouse, puncturing along the upper position of the root of the thigh, performing anesthetic intraperitoneal injection, and putting the mouse back into the cage after the injection is finished, so as to wait for the mouse to be thoroughly comatose.
(2) Dissecting the mice, cutting abdominal cavity and thoracic cavity, fully exposing each organ, observing changes of liver color, morphology and the like, and photographing and recording, as shown in fig. 3A, the liver surface of the mice fed by the control feed is smooth, the color is dark red, the liver of the high-fat group mice is enlarged in volume, the color is light red, the surface is visible as tiny particles, however, compared with the high-fat+wenyujin lipid nanovesicle group and the high-fat+1xPBS group, the liver morphology changes are relieved to a certain extent.
(3) The heart blood of the mice is firstly extracted by a 1mL syringe for subsequent detection, and then the liver is extracted for subsequent detection.
6. Mouse liver tissue TC, TG detection
1. Accurately weighing 20mg of mouse liver tissue in a 1.5mL EP tube according to the weight (g): volume (mL) =1: 9, and adding 9 times of absolute ethyl alcohol by volume.
2. Mechanically homogenizing in ice-water bath, and fully grinding. And then centrifuging at 2500rpm for 10min, and taking the supernatant to be measured.
3. The detection kit is purchased from Nanjing's institute of biological engineering (goods No. A111-1-1, A110-1-1). As shown in fig. 3B, it can be seen that the TG level of liver tissue of mice treated with the nano-vesicle of the lipid derived from wenyujin by intraperitoneal injection was reduced compared to that of the pure high-fat group, and the TG level was significantly different (P < 0.05). It is demonstrated that curcuma wenyujin-derived lipid nanovesicles can alleviate lipid deposition in the liver of mice caused by high-fat diet.
7. Mouse liver tissue inflammatory factor IL-6, IL-1 beta detection (qPCR)
1.20 mg of mouse liver tissue was cut into 1.5mL of a deenzyme EP tube, 300. Mu. LTrilzol was added, and the mixture was thoroughly ground at 4 ℃. And then allowed to stand on ice for 5min.
2. 60 μl of chloroform was added to each tube and vortexed immediately for 15s to mix thoroughly.
3. The mixture was again precipitated on ice for 5min, followed by centrifugation at 14000rpm at 4℃for 15min.
4. The isopropanol was pre-chilled 4℃in advance and a corresponding number of 1.5mL of the enzyme-depleted EP tubes were prepared and labeled.
5. mu.L of the supernatant was pipetted with a pipette and transferred into the labeled de-enzymed EP tube described above.
6. Adding equal volume of isopropanol to precipitate RNA, mixing the mixture upside down, and precipitating on ice for 10min.
Centrifuge at 14000rpm at 7.4℃for 10min, carefully discard the supernatant.
8. The precipitate was washed with 75wt.% ethanol (DEPC water formulation), centrifuged at 14000rpm at 4℃for 5min, washed twice repeatedly, the supernatant carefully discarded, isolated at 1min and dried at 42 ℃.
9. RNA was dissolved by adding 20. Mu.L of DEPC water.
10. Reverse transcription and qPCR steps reference was made to the Nanjinopran kit (NO: R223-01) as follows:
i, reverse transcription
(1) Removal of genomic DNA
TABLE 7 reagents for removal of genomic DNA
After gently stirring and mixing with a pipette, the EP tube was placed into a PCR instrument and the program was run at 42℃for 2min.
(2) Preparation of reverse transcription reaction System
TABLE 8 reverse transcription reaction system
The mixed solution was gently beaten by a pipette, and the EP tube was centrifuged briefly at 2000rpm for 10s and placed in a PCR instrument for the next step.
(3) Reverse transcription reaction
TABLE 9 reverse transcription reaction conditions
After the PCR procedure was completed, the cDNA samples were stored in a-20℃refrigerator.
II, real-time fluorescent quantitative PCR (qRT-PCR)
The procedure for reference to the Nanjinopran kit (NO: Q712-02) is as follows:
TABLE 10 primer sequences
(1) Preparing a reaction system
TABLE 11 reaction system
Labeling was performed on a 96-well plate, and 20. Mu.L/well of the above mixture was sequentially added. After balancing the 96-well plate in a centrifuge, the reaction solution was collected by brief centrifugation.
(2) PCR reactions (run on CFX96 Real-Time PCR System instruments)
TABLE 12 PCR reaction procedure
The relative expression level of the genes after the reaction is according to The relative expression level of the genes is calculated, the Gapdh gene is used as a control gene to detect the mRNA expression level of inflammatory factors in the liver tissues of the mice (the result is shown in figure 3C), and the RT-qPCR result shows that the relative expression level of the inflammatory factors IL-6, IL-1 beta and TNF-alpha in the liver tissues of the high-fat mice treated by intraperitoneal injection of the curcuma wenyujin-derived lipid nanovesicles is lower than that of the single high-fat group, and the IL-6 level has a significant difference (P < 0.05). The lipid nanovesicles derived from curcuma wenyujin are capable of relieving liver inflammation of mice caused by high-fat diet.
8. HE staining of mouse liver tissue
1. Immediately cutting liver tissue of the middle part of the liver lobule after the liver tissue of the mouse is removed, immersing the liver tissue in 4% paraformaldehyde fixing solution, and preserving at room temperature.
2. HE staining of mouse liver tissue was delegated to wuhansai wilt medical test company. The pathological conditions of liver tissues of various groups of mice are observed by HE staining (shown in figure 3D), and the results show that the number of lipid droplets and the aggregation condition of inflammatory cells in liver tissues of high-fat mice treated by intraperitoneal injection of the curcuma wenyujin source lipid nanovesicles are obviously reduced, which indicates that the curcuma wenyujin source lipid nanovesicles can relieve non-alcoholic fatty liver diseases of the mice caused by high-fat diet.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Claims (8)
1. Application of radix Curcumae-derived lipid nanovesicles in preparing medicine for relieving non-alcoholic fatty liver disease is provided.
2. A curcuma wenyujin derived lipid nanovesicle as claimed in claim 1, wherein extracted from the root tuber of curcuma wenyujin.
3. A method for preparing the curcuma wenyujin-derived lipid nanovesicles as claimed in claim 2, comprising the steps of:
s1, taking fresh radix curcumae tuberous root, cleaning and peeling with clear water, then washing with 1 XPBS, and stirring and grinding the radix curcumae tuberous root to obtain stirred and ground radix curcumae juice;
s2, carrying out differential centrifugation on the stirred and ground curcuma wenyujin juice to obtain supernatant after differential centrifugation;
s3, centrifuging the supernatant after differential centrifugation at the temperature of 4 ℃ and the pressure of 100000-200000g for 60-120min, after the ultracentrifugation is finished, re-suspending the sediment in 1 XPBS and slightly adding the sediment to the uppermost layer of sucrose gradient solution with the concentration gradient of 60 wt%, 45 wt%, 30 wt% and 8 wt% from bottom to top, centrifuging at the temperature of 4 ℃ and the pressure of 100000-200000g for 60-120min, sucking a visible strip between the 8 wt% and 30 wt% sucrose layers after the centrifugation is finished, re-suspending the sediment in 1 XPBS, and centrifuging at the temperature of 4 ℃ and the pressure of 100000-200000g for 60-120min again, wherein the obtained sediment is the curcuma wenyujin source lipid nano vesicle.
4. The preparation method according to claim 3, wherein 1 XPBS is added during the stirring and grinding process in the step S1, and the solid-to-liquid ratio of the radix curcumae tuber to the 1 XPBS is 1 g/2 mL.
5. The method according to claim 3, wherein the stirring and grinding in step S1 is performed at a rotation speed of 15000-28000rpm, the stirring and grinding is repeated 3-10 times for 1-5min each time, and stirring is suspended for 1-5min in the middle of the two times of stirring and grinding.
6. The method according to claim 3, wherein the differential centrifugation in step S2 is specifically performed by: centrifuging at 500-2000g for 10-30min, discarding the precipitate, collecting the supernatant, centrifuging at 2000-5000g for 10-30min, discarding the precipitate, collecting the supernatant, centrifuging at 8000-15000g for 30-60min, discarding the precipitate, and collecting the supernatant after differential centrifugation.
7. A method according to claim 3, wherein the concentration gradient in step S3 is 60 wt%, 45 wt%, 30 wt%, 8 wt% sucrose gradient solution in the order from bottom to top is prepared by: firstly, preparing sucrose solutions with the concentration of 8 wt%, 30 wt%, 45 wt%, and 60 wt% respectively by taking 1 XPBS or double distilled water as a solvent, and then taking a clean super-separation tube, sequentially adding 6-8mL of each of the sucrose solutions with the concentration of 60 wt%, 45 wt%, 30 wt%, and 8 wt% to prepare a gradient, so as to obtain the sucrose gradient solution.
8. The method according to claim 3, further comprising the step of storing the lipid nanovesicles derived from the curcuma wenyujin: and (3) re-suspending the curcuma wenyujin source lipid nano vesicle sediment obtained in the step (S3) by using 1 XPBS to obtain a curcuma wenyujin source lipid nano vesicle suspension, and transferring the suspension into a clean enzyme-removed EP tube for preservation at-80 ℃.
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