CN110551791A - method for detecting autophagy effect of anthocyanin-induced rat islet beta cells - Google Patents
method for detecting autophagy effect of anthocyanin-induced rat islet beta cells Download PDFInfo
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Abstract
The invention belongs to the technical field of applied cytobiology detection, and discloses a method for detecting autophagy effect of a rat islet beta cell induced by anthocyanin. The invention establishes a high-throughput screening method for the autophagy effect of the anthocyanin-induced rat islet beta cells, and provides a theoretical basis for the autophagy effect of the anthocyanin-induced rat islet beta cells to prevent diabetes.
Description
Technical Field
the invention belongs to the technical field of detection by using cell biology, and particularly relates to a method for detecting the autophagy effect of a rat islet beta cell induced by anthocyanin.
background
Currently, the closest prior art: according to the world health organization, the worldwide diabetic patients break through 6 hundred million in 2025, and the growth rate is as high as 55%. Worldwide diabetes diagnosis and treatment costs up to 825 billion dollars per year. Diabetes is mainly classified into type 1, type 2 and gestational diabetes, wherein type 2 diabetes accounts for more than 90%. Insulin is secreted in vivo in two phases, with insulin in type 2 diabetics being higher than normal or normal in one phase and delayed in the peak of two phases, primarily due to decreased islet beta cell numbers, impaired function, and insulin resistance. The reduction of the number and functional disorder of islet beta cells are closely related to factors such as glucotoxicity and lipotoxicity. Short-term hyperglycemia stimulates the synthesis and secretion of insulin and the utilization of glucose, while long-term hyperglycemia is called glucotoxicity, and not only causes the reduction of the number and the functional disorder of islet beta cells, but also enhances the insulin resistance in vivo, mainly because the islet beta cells are subjected to apoptosis and necrosis due to glycotoxin. Studies have reported that the signal transduction pathways of islet β apoptosis mainly include the extrinsic pathway, the intrinsic pathway, and the granzyme β pathway. Common features of these apoptotic signaling pathways are: various oxidative stress and stimulus signals activate specific transduction pathways, ultimately activating caspases (caspases). Activated caspases cleave intracellular substrates, thereby disrupting the cellular characteristic morphological structure and affecting the metabolic activity of the cell, resulting in apoptosis. Therefore, islet beta cell damage and dysfunction are central to the onset of type 2 diabetes, and islet beta cell apoptosis plays an important role here.
+ 2+autophagy is a process in which cells degrade damaged macromolecular substances and organelles by utilizing lysosomes, and autophagy continuously cleans up organelles such as misfolded proteins, harmful metabolites, aged mitochondria and the like, thereby maintaining the normal state of islet beta cells.
2 2the anthocyanin is mainly present in fruits, vegetables and grains, and mainly comprises six anthocyanin monomers through different acyl groups and glycosylation in the nature, the six anthocyanin monomers form six anthocyanin monomers due to the difference of methoxy groups and hydroxyl groups connected with R1 and R2 on a B ring, and the six anthocyanin monomers have different biological efficacies.
in summary, the problems of the prior art are as follows: different anthocyanin monomers have not been reported to induce autophagosome capacity of rat islet beta cells in normal and apoptosis models and the relationship between autophagy capacity and structure-activity of the rat islet beta cells.
the difficulty of solving the technical problems is as follows: anthocyanin as a natural plant polyphenol has the effects of resisting oxidation and inflammation, preventing cardiovascular diseases and the like, particularly has obvious effect on preventing and treating diabetes, and researches show that the anthocyanin mainly prevents and treats type 2 diabetes through 4 aspects of reducing oxidative stress, improving insulin resistance, promoting insulin secretion, protecting islet beta cells and the like. Autophagy is considered to be one of the self-protective mechanisms of islet beta cells and is critical for maintaining the structure, number and function of islet beta cells. At present, no technology exists for effectively detecting the autophagy effect of the anthocyanin on the rat islet beta cells, and further evaluating the effect of the anthocyanin on the prevention of diabetes according to the autophagy effect of the anthocyanin on the rat islet beta cells.
the significance of solving the technical problems is as follows: the method combines the detection of the static autophagosome number and the dynamic autophagy flow of the anthocyanin-induced rat islet beta cell and the establishment of a high-sugar rat islet beta cell apoptosis model, and can objectively evaluate the effect of the anthocyanin-induced rat islet beta cell autophagy; a method for detecting the autophagy effect of the anthocyanin-induced rat islet beta cells in a high-throughput manner is constructed by establishing an RFP-GFP-RIN stable cell strain; a high-throughput screening method is established for detecting the autophagy effect of the anthocyanin-induced rat islet beta cells, and a theoretical basis is provided for the autophagy effect of the anthocyanin-induced rat islet beta cells to prevent diabetes.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for detecting the autophagy effect of a rat islet beta cell induced by anthocyanin, which takes an anthocyanin monomer as an experimental material, takes a rat islet beta (RIN-m5F) cell as a research object, primarily determines the conditions of a high-glucose apoptosis model through the influence of high glucose on the cell activity and the apoptosis rate, and analyzes the influence of the high-glucose apoptosis model on the intracellular Reactive Oxygen Species (ROS) level and the apoptosis protein to further verify the high-glucose apoptosis model. Secondly, detecting the autophagosome effect of the anthocyanin in the normal and high-glucose apoptosis model to induce the rat islet beta cell in a normal and high-glucose apoptosis model by a cell immunofluorescence method; establishing an RFP-GFP-RIN stable cell strain to detect the autophagy flow effect of the rat islet beta cells induced by the anthocyanin; and finally judging the autophagosome and autophagic flux effects generated by inducing rat islet beta cells by combining the anthocyanin. The method is expected to establish a high-throughput screening method for detecting the autophagy effect of the anthocyanin-induced rat islet beta cells, and provide a theoretical basis for the autophagy effect of the anthocyanin-induced rat islet beta cells to prevent diabetes.
The invention is realized in such a way that a method for detecting the autophagy effect of the rat islet beta cell induced by the anthocyanin comprises the following steps:
step one, respectively dissolving anthocyanin in dimethyl sulfoxide to prepare a stock concentration of 400mM, and storing the stock concentration at minus 20 ℃ in a dark place;
step two, culturing rat islet beta cells by using RPMI 1640 culture solution containing 10% fetal calf serum in an incubator at 37 ℃ and 5% carbon dioxide;
Step three, screening the toxic concentration range of the rat islet beta cells by the anthocyanin;
Fourthly, constructing a high-sugar rat islet beta cell apoptosis model by using a flow cytometry technology;
fifthly, verifying a high-glucose rat islet beta cell apoptosis model by a reactive oxygen species ROS level experiment;
Step six, verifying a high-sugar rat islet beta cell apoptosis model by using the expression level of the apoptosis protein;
seventhly, detecting the autophagosome effect of the rat islet beta cells induced by the anthocyanin by using a cell immunofluorescence method;
Step eight, establishing an RFP-GFP-RIN stable cell strain: constructing a lentivirus vector, packaging and preparing lentivirus, and screening a stable cell strain;
step nine, detecting the autophagy flow effect of the rat islet beta cells induced by the anthocyanin by using a cell immunofluorescence method;
And step ten, judging the autophagosome and autophagy flow effects generated by inducing the rat islet beta cells by combining the anthocyanin.
Further, the screening of the anthocyanin in the step III on the toxic concentration range of the rat islet beta cells comprises the following steps:
1) Inoculating 2X 10 4 cell suspensions into a 96-well plate, taking 200 mu L of each well, and placing the well in an incubator at 37 ℃ for 12 h;
2) after the cell monolayer grows in an adherent manner, adding six kinds of anthocyanin according to the final concentration of 25-200 mu M, and continuously culturing for 8h in a 37 ℃ culture box;
3) After the culture is finished, adding 20 mu LCCK-8 solution into each hole according to the proportion of 1:10 to continue culturing for 2 h;
4) measuring the OD value of each sample at 450nm by using a microplate reader;
5) simultaneously setting a blank group, and only adding a culture medium and a CCK-8 reagent; the negative group is not added with the drug cell hole to be detected, and each group is provided with 5 multiple holes; calculating the cell survival rate according to a formula;
cell viability (%) × 100% (treatment OD-blank OD)/(negative OD-blank OD), and cytotoxicity (%) × 100% (negative OD-treatment OD)/(negative-blank OD).
Further, the construction of the high-glucose rat islet beta cell apoptosis model by using the flow cytometry technology in the fourth step comprises the following steps:
1) Inoculating 5 × 10 5 cell suspensions into 12-well cell plates, and culturing for 12 h;
2) Adding glucose with final concentration of 10, 20 and 30mM into each well, and culturing for 24, 36 and 48 h;
3) After the culture is finished, respectively sucking the culture medium in each hole into a centrifuge tube (1), washing each hole for 1 time by using PBS and transferring the hole into the corresponding centrifuge tube (1);
4) Then 200-300ul pancreatin without EDTA is added into each hole, 2mL of RPMI 1640 culture solution of 10% fetal bovine serum is directly added after the cells are digested for 30s and is lightly blown to make the adherent cells suspended in the culture solution, and then the adherent cells are moved into a new centrifuge tube (2);
5) Centrifuging the centrifugal tube (2) for 8min at 1000rpm, transferring the supernatant into the corresponding centrifugal tube (1), centrifuging for 8min at 3000rpm, and removing the supernatant;
6) adding 2ml of LPBS into the centrifuge tubes (1) and (2) respectively, and gently resuspending the cells by using a pipette gun;
7) centrifuging the suspended cells in the centrifugal tube (1) once again at 1000rpm multiplied by 8min respectively, transferring the supernatant into the corresponding centrifugal tube (2), centrifuging at 3000rpm multiplied by 8min, and removing the supernatant;
8) adding 300-500ul of Bindbuffer to resuspend the cells, and mixing the centrifuge tube (1) and the corresponding cell (2);
9) after being mixed gently and evenly, the mixture is sieved into a centrifuge tube through a 200-mesh cell sieve to obtain a cell suspension to be detected;
10) before using a flow cytometry instrument, adding 5uL annexin-V-FICT into 500uL of the solution each time, incubating for 10min at room temperature, and mixing uniformly;
11) then adding 5ul PI, mixing uniformly, and incubating for 5min at room temperature;
12) detecting the apoptosis condition of the rat islet beta cells by using a flow cytometry instrument with an excitation wavelength Ex being 488nm and an emission wavelength Em being 530 nm;
13) according to the apoptosis condition of the rat islet beta cells, the rat islet beta cell apoptosis model condition is determined to be 30mM and 36 h.
further, the ROS level test of the reactive oxygen free radicals in the fifth step comprises the following steps:
1) labeling intracellular ROS levels by fluorescent probe DCFH-DA;
2) preparing a cell climbing sheet according to a cell immunofluorescence method; DCFH-DA at a final concentration of 50. mu. mol/L was added to each treatment group, incubated at 37 ℃ for 50min, and then washed 3 times with PBS; the fluorescence intensity of the cells was examined using the maximum excitation wavelength and emission wavelength of 488nm and 510nm, respectively, and photographed.
further, the verification of the apoptosis model of the islet beta cells of the high-glucose rat by the expression level of the apoptosis protein in the sixth step comprises the following steps:
1) Cell culture, in which RIN-m5F cell strain is subcultured conventionally in RPMI 1640 medium containing 10% fetal calf serum at 37 deg.C under 100% saturation humidity and 5% CO 2;
2) Grouping cells, namely inoculating cells with the density of 1 multiplied by 10 6/mL into a culture plate, adding anthocyanin extract into a culture solution to induce for 8 hours after the cells grow adherent to a monolayer, and then adding H 2 O 2 to induce for 30 minutes under the condition of oxidative stress state, namely preprocessing the anthocyanin extract for 2 hours;
3) protein recovery: after the cell treatment is finished, removing the culture solution, and washing twice with precooled PBS; add 10% SDS into each well; mixing, transferring into new centrifuge tube, and incubating on ice for 20-30 min; centrifuging at 14000g for 10min, transferring the supernatant into a new centrifuge tube, performing metal bath at 100 ℃ for 10min, adding 6 × loading Buffer, fully oscillating and uniformly mixing, performing metal bath at 100 ℃ for 10min again, and storing in a refrigerator at-80 ℃ for later use;
4) protein quantification: preparing BSA standard substances and working solution with different concentrations according to the BSA kit; adding BSA standard substances with different concentrations and protein samples to be detected into a 96-well plate respectively, incubating for 30min at 37 ℃, and making 5 samples in each group in parallel; measuring the absorbance at 562 nm; calculating the protein concentration in the sample according to the standard curve of the BSA standard substance;
5) And (3) carrying out western blot detection: preparing concentrated glue and separation glue, wherein 10-12% of separation glue is selected as Caspase 3;
a. electrophoresis: filling the prepared gel into an electrophoresis tank, and adding 5-20 mu L of protein to be detected into each lane; the voltage is stabilized at 50-80v according to the molecular weight of Caspase3 protein, and electrophoresis is carried out for 1-2.5 h;
b. Film transfer: checking the target protein according to the molecular size, cutting off the gel block, and then putting the protein into a film loading groove according to the sandwich mode of a sandwich;
c. And (3) sealing: sealing the membrane with a sealing solution, and incubating on a shaking table for 1 h;
d. antibody incubation: diluting primary antibody with a ratio of 1:1000, incubating overnight in a refrigerator at 4 deg.C, removing primary antibody, and cleaning membrane with TPBST for 5-10min three times;
e. antibody incubation: diluting the secondary antibody with a ratio of 1:5000, incubating for 1h in a shaking table, absorbing the secondary antibody, and washing the membrane with PBST for three times, each time for 5-10 min;
f. adding chemiluminescence developing solution, developing with an Image Quant LAS 4000mini gel imager, taking pictures, and storing; each band was quantitatively analyzed using Image J analysis software.
further, the step seven of detecting the autophagosome effect of the rat islet beta cells induced by the anthocyanin by using a cellular immunofluorescence method comprises the following steps:
1) Under normal conditions and in a high-sugar rat islet beta cell apoptosis model, respectively inoculating 1 × 105 cell suspensions into 12-hole culture plates paved with slides to culture for 12 h;
2) adding 50 mu M anthocyanin solution into each hole, and culturing for 8 h;
3) Absorbing the culture solution by using a liquid transfer gun, and washing for 2-3 times by using PBS; adding 4% paraformaldehyde, fixing at room temperature for 10min, removing paraformaldehyde, and washing with PBS for 3 times; removing PBS, adding 10% goat serum to obtain sealing solution, and sealing at room temperature for 30 min;
4) Adding 0.1% saponin into 10% confining liquid prepared from goat serum to obtain diluent, mixing the diluent with one antibody LC3, MBL, PM036 according to the ratio of 1:1000, coating the climbing sheet on an anti-dilution solution, and incubating for 1h at 37 ℃ in a dark place;
5) washing the climbing sheet with sealing liquid for three times;
6) Incubating the second antibody Southern Biotech,4050-05, with the same antibody, and applying the slide to the blocking solution three times;
7) dropping a proper amount of the sealing tablet on a glass slide, covering the sealing tablet with the front face of the climbing tablet downwards, and drying in a dark place;
8) The number of fluorescent point-like aggregation is observed by a laser confocal microscope, namely the number of autophagosomes, and the point-like concentration degree can preliminarily evaluate the autophagosomal capacity of the rat islet beta cells; after autophagy occurs, LC3-I which is dispersed in cytoplasm is subjected to ubiquitination processing modification and is combined with phosphatidylethanolamine PE on the surface of an autophagosome membrane to form LC 3-II; the number of the point-shaped aggregates is observed by adopting laser confocal focusing through a cell immunofluorescence method, namely the number of autophagosomes, and the point-shaped concentration degree can preliminarily evaluate the cell autophagy;
9) And (6) taking a picture.
Further, the establishment of RFP-GFP-RIN stable cell strain in the step eight comprises the following steps:
1) amplifying an RFP-GFP-LC3 fragment from a PCMV-RFP-GFP-LC3 plasmid by a PCR technology; the amplification product is transformed by 10g/L agar, and recombinant plasmids are screened out to obtain RFP-GFP-LC3-GV374 plasmids; confirming the construction of the lentiviral vector through sequencing comparison;
2) culturing 293FT cells to 90%, digesting with pancreatin, subculturing in a cell plate at the density of 1 × 10 6/mL for 24h, performing operation according to the instruction of a Lipo3000 transfection kit after the logarithmic phase of cell growth, collecting cell supernatant after culturing for 24h, performing 3000r/min, centrifuging for 10min, and taking the supernatant;
3) adding the collected lentivirus particles into RIN-m5F cells for culturing for 48 h; during the period, new culture medium is observed and replaced, and after the culture is finished, puromycin with the volume ratio of 6 mug/mL is added for screening infected RIN-m5F cells; after culturing for 7 days, screening out cells with red and green fluorescence under a fluorescence microscope; selecting positive clone cells, transferring the positive clone cells to a new culture dish, and continuing culturing to obtain the RFP-GFP-LC3 islet beta stable cell strain.
further, the step nine of detecting the autophagy flow effect of the rat islet beta cells induced by the anthocyanin by using a cellular immunofluorescence method comprises the following steps:
1) in a high-glucose rat islet beta cell apoptosis model, a suspension containing 1 × 10 5/mL RFP/GFP-LC3 stable cell line was seeded into a culture plate that had been plated on a slide;
2) adding 50 mu M anthocyanin and 20 mu M chloroquine into each well, culturing for 2h, and adding 30mM glucose for culturing for 36 h;
3) after the reaction is finished, removing the culture medium, and washing the climbing film for 2-3 times by PBS; adding 4% paraformaldehyde, fixing at room temperature for 10min, removing paraformaldehyde, and washing with PBS for 3 times;
4) dropping a proper amount of the sealing tablet on a glass slide, covering the sealing tablet with the front face of the climbing tablet downwards, and drying in a dark place;
5) and (3) observing the ratio of red fluorescence to green fluorescence by adopting a laser confocal microscope observation microscope to judge autophagy flow and taking a picture.
the invention also aims to provide application of the method for detecting the autophagy effect of the anthocyanin on the rat islet beta cells in screening the autophagy effect of the anthocyanin on the rat islet beta cells.
the invention also aims to provide an application of the anthocyanin for preventing diabetes by inducing autophagy of rat islet beta cells.
in summary, the advantages and positive effects of the invention are:
the invention researches and analyzes the effect of the anthocyanin on inducing the rat islet beta cell autophagosome in normal and high-glucose cell apoptosis models, and the main conclusion is as follows:
(1) Establishes a rat islet beta cell hyperglycosis model. By adopting a CCK-8 method and a flow cytometry to analyze the survival rate and the apoptosis rate of glucose with different concentrations on rat islet beta cells, the optimal condition that 30mM glucose is used for treating the rat islet beta cells for 36h as a high-glucose cell apoptosis model is determined; and the high-glucose cell apoptosis model is further verified by two evaluation indexes of the apoptosis protein caspase3 and the reactive oxygen species ROS, and the expression levels of the caspase3 and the ROS are found to be increased with a control group, which shows that the rat islet beta cell high-glucose apoptosis model is successfully constructed.
(2) the toxicity of different anthocyanins on rat islet beta cells was established. After treating rat islet beta cells with 0-200 mu M of six anthocyanin monomers under normal conditions by using the method of CCK-8, cytotoxicity is found at a concentration of 200 mu M of the six anthocyanidins, the concentration of Dp, Pn, Pg, Cy and Pt for treating the rat islet beta cells is 25-100 mu M, and the concentration of Mv is lower than 100 mu M.
(3) The capacity of different anthocyanidins to induce rat islet beta cell autophagosomes under normal conditions is clarified. By the method of cellular immunofluorescence, it was found that four anthocyanins of Pt, Pn, Pg and Mv at 25 μ M did not induce rat islet β autophagosome, while two anthocyanins of Dp and Cy at 25 μ M induced rat islet β autophagosome. The five anthocyanidins of Dp, Cy, Pt, Pn and Pg of 50-100 mu M induce the beta-cell autophagosome of the rat islet, and the five anthocyanidins are increased in a dose gradient, and 25-80 mu M Mv do not induce the beta-cell autophagosome of the rat islet.
(4) The influence of the anthocyanin on the rat islet beta cell autophagosome in a high-carbohydrate apoptosis model is determined. It was determined by the method of cellular immunofluorescence that 0-30mM glucose does not itself induce autophagosomes in rat islet beta cells. The five anthocyanins of Dp, Cy, Pt, Pn and Pg with the concentration of 50 mu M are found to be capable of inducing rat islet beta cell autophagosome in a high-glucose apoptosis model through a cytoimmunofluorescence and western blotting method, wherein the Dp has the strongest capacity of inducing the rat islet beta cell autophagosome, and the Mv does not induce the rat islet beta cell autophagosome in the high-glucose apoptosis model.
drawings
fig. 1 is a flowchart of a method for detecting the autophagy effect of rat islet beta cells induced by anthocyanin according to an embodiment of the invention.
FIG. 2 is a schematic diagram of the method of the present invention using CCK-8 to study the effect of different concentrations of glucose on the viability of rat islet beta cells (RIN-m 5F).
FIG. 3 is a schematic diagram illustrating the effect of analyzing the effect of glucose treatment on the apoptosis rate of rat islet beta cells at different concentrations for 36h by using flow cytometry.
FIG. 4 is a schematic diagram of the effect of high sugar on the expression level of apoptotic protein in rat islet beta cells studied by Western blotting method according to the present invention.
FIG. 5 is a graph demonstrating the effect of high sugar on the level of Reactive Oxygen Species (ROS) expression in rat islet beta cells, as provided by the examples of the present invention.
FIG. 6 is a graph showing the effect of different concentrations of anthocyanins on rat islet beta cell autophagosomes under normal conditions, as provided by the examples of the present invention.
FIG. 7 is a schematic diagram showing the effect of glucose at different concentrations on the induction of autophagosome numbers of islet beta cells in rats by the method of cellular immunofluorescence provided in the example of the present invention.
FIG. 8 is a schematic diagram of how Dp, Cy, Pt, Pn, Pg and Mv provided in the examples of the present invention induce rat islet beta cell autophagosome in a high-glucose apoptosis model.
FIG. 9 is a schematic diagram showing the expression level of marker protein LC3 for detecting autophagy by a Western blotting method in a high-carbohydrate apoptosis model.
Detailed Description
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
aiming at the problems in the prior art, the invention provides a method for detecting the autophagy effect of the anthocyanin on rat islet beta cells, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for detecting the autophagy effect of anthocyanin on rat islet beta cells provided by the embodiment of the invention comprises the following steps:
S101: preparing an anthocyanin solution: dissolving Paeoniflorin, pelargonidin, malvidin, cyanidin, delphinidin and petunidin in dimethyl sulfoxide respectively, configuring to 400mM stock concentration, and storing at-20 deg.C in dark.
s102: rat islet beta cell culture: rat islet beta cells (RIN-m5F) were established as subjects and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, BI, 150772) at 37 ℃ in a 5% carbon dioxide incubator.
s103: screening of the cytotoxic concentration range of rat islet beta with anthocyanin.
S104: constructing a high-sugar rat islet beta cell apoptosis model by using a flow cytometry technology.
s105: ROS level experiment of active oxygen free radical verifies the islet beta cell apoptosis model of high-sugar rats.
s106: the expression level of the apoptosis protein verifies the islet beta cell apoptosis model of the high-sugar rat.
S107: detecting the autophagosome effect of the rat islet beta cells induced by the anthocyanin by using a cell immunofluorescence method.
s108: establishing an RFP-GFP-RIN stable cell strain: comprises the steps of lentivirus vector construction, lentivirus packaging preparation and stable cell strain screening.
s109: detecting the autophagy effect of the rat islet beta cells induced by the anthocyanin by using a cell immunofluorescence method.
S110: the effect of the anthocyanin on inducing autophagosome and autophagy flow generated by rat islet beta cells is combined for judgment.
The technical effects of the present invention will be further described below with reference to experiments.
1. Experimental materials and methods
1.1 materials and reagents
six anthocyanins, including peonidin, pelargonidin, malvidin, cyanidin, delphinidin and petunidin, were purchased from chromomadex corporation, usa, respectively. The materials and reagents required for the experiments of the present invention are shown in table 1.
TABLE 1 materials and reagents required for the experiment
1.2 instruments and devices
the instruments and equipment required for the experiments of the present invention are shown in table 2.
TABLE 2 instruments and Equipment required for the experiment
1.3 Experimental methods
1.3.1 technical route
The method comprises the steps of preparing an anthocyanin solution, culturing rat islet beta cells, screening the toxic concentration range of the rat islet beta cells by the anthocyanin, constructing a high-glucose rat islet beta cell apoptosis model by a flow cytometry, verifying the high-glucose rat islet beta cell apoptosis model by an active oxygen radical ROS level experiment, verifying the high-glucose rat islet beta cell apoptosis model by an expression level of an apoptosis protein, detecting the autophagy effect of the rat islet beta cells induced by the anthocyanin by a cell immunofluorescence method, establishing an RFP-GFP-RIN stable cell strain, detecting the autophagy effect of the rat islet beta cells induced by the anthocyanin by the cell immunofluorescence method, and judging the autophagy effect and the autophagy effect of the rat islet beta cells induced by the anthocyanin.
1.3.2 Experimental methods
(1) Preparation of anthocyanin solutions
The six anthocyanidins are respectively dissolved in dimethyl sulfoxide, and are prepared into a stock concentration of 400mM, and are stored at minus 20 ℃ in a dark place.
(2) Culture of rat islet beta cells
Rat islet beta cells (RIN-m5F) were established as subjects and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, BI, 150772) at 37 ℃, 5% carbon dioxide (CO 2) in an incubator.
(3) Screening of anthocyanin for rat islet beta cytotoxic concentration range
Inoculating 2 × 10 4 cell suspension into 96-well plate, taking 200 μ L of each well, and culturing in 37 deg.C incubator for 12 h;
Secondly, after the cells grow in a monolayer adherent manner, adding six kinds of anthocyanin according to the final concentration of 25-200 mu M, and continuously culturing for 8 hours in an incubator at 37 ℃;
Thirdly, after the culture is finished, adding 20 mu L () CCK-8 solution into each hole according to the proportion of 1:10 to continue the culture for 2 h;
measuring the OD value of each sample at 450nm by using an enzyme-labeling instrument;
and fifthly, simultaneously setting a blank group (only adding culture medium and CCK-8 reagent) and a negative group (no cell hole of the drug to be detected), and setting 5 multiple holes in each group.
and according to the formula: cell viability (%) was calculated as (treatment OD-blank OD)/(negative OD-blank OD) × 100%. Cytotoxicity (%) - (negative group OD-treated group OD)/(negative group-blank group) × 100%
(4) Flow cytometry technology for constructing high-sugar rat islet beta cell apoptosis model
Inoculating 5 × 105 cell suspensions into 12-hole cell plates, and culturing for 12 h;
adding glucose with final concentration of 10, 20 and 30mM into each hole, and culturing for 24, 36 and 48 hours respectively;
after the culture is finished, respectively sucking the culture medium in each hole into a centrifuge tube (1), washing each hole for 1 time by PBS and transferring the hole into the corresponding centrifuge tube (1);
fourthly, 200-300ul pancreatin without EDTA is added into each hole, 2mL of RPMI 1640 culture solution of 10% fetal bovine serum is directly added after 30s of cell digestion, and the cell is lightly blown and beaten, so that adherent cells are suspended in the culture solution and then are moved into a new centrifuge tube (2);
Fifthly, centrifuging the centrifuge tube (2) for 8min at 1000rpm, transferring the supernatant into the corresponding centrifuge tube (1), centrifuging for 8min at 3000rpm, and removing the supernatant;
Sixthly, adding 2mL of PBS into the centrifuge tubes (1) and (2) respectively, and gently suspending the cells by using a pipette gun;
Seventhly, respectively centrifuging the suspended cells in the centrifugal tube (1) once again at 1000rpm multiplied by 8min, transferring the supernatant into a corresponding centrifugal tube (2), centrifuging at 3000rpm multiplied by 8min, and removing the supernatant;
adding 300 plus 500ul of Bindbuffer to resuspend the cells, and mixing the centrifuge tube (1) and the corresponding cells (2);
Ninthly, after gentle and uniform mixing, screening the cell with 200 meshes into a centrifuge tube to obtain cell suspension to be detected;
Before the application of flow cytometry, 5uL Annexin-V-FICT is added into 500uL of the filtrate, incubated at room temperature for 10min, and mixed uniformly;
then adding 5ul PI, mixing uniformly, and incubating for 5min at room temperature;
detecting the apoptosis condition of the rat islet beta cells by using a flow cytometry instrument with an excitation wavelength Ex being 488nm and an emission wavelength Em being 530 nm;
According to the apoptosis condition of the rat islet beta cells, the rat islet beta cell apoptosis model condition is determined to be 30mM and 36 h.
(5) The reactive oxygen radical ROS level test comprises the following steps:
1) labeling intracellular ROS levels by fluorescent probe DCFH-DA;
2) preparing a cell climbing sheet according to a cell immunofluorescence method; DCFH-DA at a final concentration of 50. mu. mol/L was added to each treatment group, incubated at 37 ℃ for 50min, and then washed 3 times with PBS; the fluorescence intensity of the cells was examined using the maximum excitation wavelength and emission wavelength of 488nm and 510nm, respectively, and photographed.
(6) The method for researching the influence of high sugar on the expression level of the apoptosis protein in rat islet beta cells by adopting a western blot method comprises the following steps:
1) cell culture, in which RIN-m5F cell strain is subcultured conventionally in RPMI 1640 medium containing 10% fetal calf serum at 37 deg.C under 100% saturation humidity and 5% CO 2;
2) Grouping cells, namely inoculating cells with the density of 1 multiplied by 10 6/mL into a culture plate, adding anthocyanin extract into a culture solution to induce for 8 hours after the cells grow adherent to a monolayer, and then adding H 2 O 2 to induce for 30 minutes under the condition of oxidative stress state, namely preprocessing the anthocyanin extract for 2 hours;
3) Protein recovery: after the cell treatment is finished, removing the culture solution, and washing twice with precooled PBS; add 10% SDS into each well; mixing, transferring into new centrifuge tube, and incubating on ice for 20-30 min; centrifuging at 14000g for 10min, transferring the supernatant into a new centrifuge tube, performing metal bath at 100 ℃ for 10min, adding 6 × loading Buffer, fully oscillating and uniformly mixing, performing metal bath at 100 ℃ for 10min again, and storing in a refrigerator at-80 ℃ for later use;
4) protein quantification: preparing BSA standard substances and working solution with different concentrations according to the BSA kit; adding BSA standard substances with different concentrations and protein samples to be detected into a 96-well plate respectively, incubating for 30min at 37 ℃, and making 5 samples in each group in parallel; measuring the absorbance at 562 nm; calculating the protein concentration in the sample according to the standard curve of the BSA standard substance;
5) and (3) carrying out western blot detection: preparing concentrated glue and separation glue, wherein 10-12% of separation glue is selected as Caspase 3; the formulations of the separation and concentration gums are shown in tables 3 and 4, respectively.
TABLE 3 Release glue formulation
TABLE 4 concentrated gum formulation
a, electrophoresis: filling the prepared gel into an electrophoresis tank, and adding 5-20 mu L of protein to be detected into each lane; the voltage is stabilized at 50-80v according to the molecular weight of Caspase3 protein, and electrophoresis is carried out for 1-2.5 h;
b, film conversion: checking the target protein according to the molecular size, cutting off the gel block, and then putting the protein into a film loading groove according to the sandwich mode of a sandwich;
c. and (3) sealing: sealing the membrane with a sealing solution, and incubating on a shaking table for 1 h;
d. Antibody incubation: diluting primary antibody with a ratio of 1:1000, incubating overnight in a refrigerator at 4 deg.C, removing primary antibody, and cleaning membrane with TPBST for 5-10min three times;
e. Antibody incubation: diluting the secondary antibody with a ratio of 1:5000, incubating for 1h in a shaking table, absorbing the secondary antibody, and washing the membrane with PBST for three times, each time for 5-10 min;
f. Adding chemiluminescence developing solution, developing with an Image Quant LAS 4000mini gel imager, taking pictures, and storing; each band was quantitatively analyzed using Image J analysis software.
(7) Cell immunofluorescence method for detecting effect of anthocyanin for inducing rat islet beta cells to generate autophagosomes
inoculating 1 × 105 cell suspensions into 12-hole culture plates paved with slides respectively to culture for 12 hours under normal conditions and in a high-sugar rat islet beta cell apoptosis model;
adding 50 mu M anthocyanin solution into each hole, and culturing for 8 hours;
thirdly, absorbing the culture solution by using a liquid transfer gun, and washing for 2-3 times by using PBS; adding 4% paraformaldehyde, fixing at room temperature for 10min, removing paraformaldehyde, and washing with PBS for 3 times; removing PBS, adding 10% goat serum to obtain sealing solution, and sealing at room temperature for 30 min;
Adding 0.1% saponin into 10% confining liquid prepared from goat serum to obtain diluent, mixing the diluent with an antibody (LC3, MBL, PM036) according to the ratio of 1:1000, coating the climbing sheet on an anti-dilution solution, and incubating for 1h at 37 ℃ in a dark place;
sixthly, washing the climbing tablet for three times by using a sealing liquid;
Seventhly, incubating a secondary antibody (Southern Biotech,4050-05) by the same method, and using a climbing film for three times by using a sealing solution;
dropping proper amount of sealing tablet (southern biotech DAPI fluorescence-G, 0100-20) on glass slide, covering the sealing tablet with the climbing tablet facing downwards (without generating air bubbles), and drying in dark;
Ninthly, observing the fluorescent point-like aggregation number by using a laser confocal microscope, wherein the fluorescent point-like aggregation number is the number of autophagosomes, and the point-like concentration degree can preliminarily evaluate the autophagosomes generating capability of the rat islet beta cells; after autophagy occurs, LC3-I which is dispersed in cytoplasm is subjected to ubiquitination processing modification and is combined with Phosphatidylethanolamine (PE) on the surface of an autophagosome membrane to form LC 3-II. The cell autophagy can be preliminarily evaluated by observing the number of point-shaped aggregates by using a laser confocal method, namely the number of autophagosomes, and the point-shaped density degree.
photograph of the red.
(8) Establishment of RFP-GFP-RIN Stable cell line
Construction of lentiviral vectors
the RFP-GFP-LC3 fragment was amplified from the PCMV-RFP-GFP-LC3 plasmid by PCR technique. The amplification product is transformed by 10g/L agar, and recombinant plasmids are screened out to obtain RFP-GFP-LC3-GV374 plasmid. And (4) confirming the construction of the lentiviral vector through sequencing comparison. (this step is made by Shanghai Jikai Gene science and technology Co., Ltd.)
② packaging preparation of lentivirus
293FT cells are cultured to about 90 percent, trypsinized, passaged to a cell plate at the density of 1 multiplied by 10 6/mL for 24h after being digested, the logarithmic phase of cell growth is carried out according to the instruction operation of a Lipo3000 transfection kit, cell supernatant is collected after 24h of culture, 3000r/min is carried out, centrifugation is carried out for 10min, and the supernatant is taken.
(iii) screening of Stable cell lines
the collected lentiviral particles were added to RIN-m5F cells and cultured for 48 h. During the period, new medium was observed and replaced, and after the culture was completed, puromycin at a volume ratio of 6. mu.g/mL was added to select infected RIN-m5F cells. After 7 days of culture, cells were screened for red and green fluorescence by fluorescence microscopy. Selecting positive clone cells, transferring the positive clone cells to a new culture dish, and continuing culturing to obtain the RFP-GFP-LC3 islet beta stable cell strain.
(9) Detection of autophagy flow effect of anthocyanin inducing rat islet beta cells by using cell immunofluorescence method
inoculating a suspension containing a stable cell strain of 1 multiplied by 10 5/mL RFP/GFP-LC3 in a culture plate paved with a slide in a high-sugar rat islet beta cell apoptosis model;
adding 50 mu M anthocyanin and 20 mu M chloroquine into each hole, culturing for 2 hours, and adding 30mM glucose to culture for 36 hours;
Thirdly, removing the culture medium after the reaction is finished, and washing the slide for 2-3 times by PBS; adding 4% paraformaldehyde, fixing at room temperature for 10min, removing paraformaldehyde, and washing with PBS for 3 times;
Dripping a proper amount of the sealing tablet on a glass slide, covering the sealing tablet with the climbing sheet facing downwards (no air bubbles are generated), and drying in a dark place;
And fifthly, adopting a laser confocal microscope to observe the ratio of red fluorescence to green fluorescence to judge autophagy flow and taking a picture.
3. results
3.1 construction of model of apoptosis of rat islet beta cells induced by high sugar
3.1.1 Effect of high sugar on the viability of rat islet beta cells
the effect of glucose at various concentrations on the viability of rat islet beta cells (RIN-m5F) was studied using the CCK-8 method and the results are shown in FIG. 2. Compared with a control group, the cell activity of the rat islet beta cells cultured by adding 10-30mM glucose in the culture medium for 24h has no obvious difference; however, 36h and 48h after 30mM glucose treatment of rat islet beta cells, the cell activity was significantly reduced compared to the control group, wherein 76.03% and 53.60% respectively. The result shows that 30mM high sugar can obviously inhibit the activity of rat islet beta cells in 36h and 48h, so that the invention preliminarily determines the condition that the concentration is 30mM and the time is shorter for 36h as a high sugar apoptosis model.
3.1.2 Effect of high sugars on the apoptosis Rate of rat islet beta cells
To further determine the optimal conditions for the high-glucose apoptosis model, the flow cytometry was used to analyze the effect of glucose at different concentrations on the apoptosis rate of rat islet beta cells after 36h treatment, and the results are shown in fig. 3. After the rat islet beta cells are induced for 36h by 0-30mM glucose, the apoptosis rates are found to be 11.82%, 14.63%, 17.31% and 22.49%, respectively. The apoptosis rate in the 30mM glucose treated group was significantly increased (P < 0.01) compared to the control group, with an early apoptosis rate of 10.21%, a late apoptosis rate of 12.28%, and a necrosis rate of only 0.04%. As can be seen, the treatment of 30mM glucose for 36h has obvious apoptosis-promoting effect on rat islet beta cells. Therefore, the optimal conditions for the finally screened high-glucose apoptosis model are that the glucose concentration is 30mM and the treatment time is 36 h.
3.1.3 Effect of high sugars on apoptotic protein expression in rat islet beta cells
In the constructed high-sugar apoptosis model, the effect of high sugar on the expression level of apoptosis protein in rat islet beta cells was studied by western blotting method, and the results are shown in fig. 4. The protein expression of the apoptotic protein Caspase3 cleaved into clear-Caspase 3 after glucose treatment at 30mM for 36h was increased compared to the control group, while the Bcl 2 protein expression was not significantly changed. Studies have reported that the mechanisms of islet beta cell apoptosis are largely divided into the extrinsic, intrinsic and granzyme B pathways. However, these pathways ultimately activate the Caspase (Caspase) family by activating, transporting associated kinases and proteins, whereas Caspase3 is one of the most important apoptotic performers in the Caspase family, responsible for cleavage of all or part of the key proteins during the execution phase of apoptosis. In the invention, the protein expression of Caspase3 is activated in a high-glucose apoptosis model, so that the rat islet beta cell is subjected to apoptosis. The B-cell lymphoma/leukemia-2 (Bcl-2) protein family is closely related to the endogenous apoptotic pathway, particularly the mitochondrial pathway. Anti-apoptosis proteins such as Bcl-2, Bcl-xl, Mcl-l and the like are inhibited under stress conditions, pro-apoptosis proteins such as Bax, Bak, Bid and the like are activated and recruited to a mitochondrial membrane, so that the permeability of the mitochondrial membrane is changed, Cytochrome C (Cytochrome C, Cyto-C) is released, Caspase 9 is activated, and further Caspase3, Caspase 6 and Caspase 7 are activated, thereby inducing apoptosis. However, the expression level of Bcl-2 protein in the present text has not changed significantly, and it was preliminarily speculated that the induction of rat islet beta cell apoptosis by high glucose might not lead to apoptosis through the mitochondrial pathway. Therefore, the experimental result shows that 30mM glucose activates caspase3 to form a cascade reaction to cause apoptosis, so that the successful construction of a high-glucose apoptosis model is evaluated at the expression level of apoptotic protein.
3.1.4 Effect of high sugars on reactive oxygen radicals in rat islet beta cells
in the constructed hyperglycogenic apoptosis model, the effect of high sugar on the expression level of Reactive Oxygen Species (ROS) in rat islet β cells was studied, and the results are shown in fig. 5. The effect of high sugars on the intracellular ROS levels of RIN-m5F was confirmed by observation of fluorescent DCF. After 30mM glucose was induced in rat islet beta cells for 36h, the glucose group was found to significantly increase intracellular ROS levels compared to the control group. In rat islet beta cells, ROS produced by the oxidative metabolism of sugars by normoglycemic persons can be eliminated by superoxide dismutase, while at high sugars the ROS expression level is increased and cannot be completely eliminated, resulting in the damage of intracellular components. When intracellular ROS are accumulated to a certain degree, the ROS can cause stress reaction of mitochondria and endoplasmic reticulum organelles, so that apoptotic proteins (such as Caspase-3,6 and 7) are activated to cause apoptosis. Therefore, the experiment shows that 30mM glucose can obviously increase the ROS expression level in rat islet beta cells, so that the successful construction of a high-glucose cell apoptosis model can be evaluated at the active oxygen free radical expression level.
3.2 Effect of anthocyanidins on rat islet beta cytotoxicity
toxicity of six anthocyanins against rat islet beta cells under normal conditions was observed by the method using CCK-8, and the results are shown in Table 5. Research shows that compared with a control group, the inhibition rate of the six anthocyanidins with the concentration of 25-50 mu M on cell growth is below 10.95 percent, which indicates that the six anthocyanidins are non-toxic to cells; the inhibition rate of the five anthocyanidins of Dp, Pn, Pg, Cy and Pt with the concentration of 50-100 mu M on the cell growth is not obviously different from that of a control group; however, the inhibition rate of the cell growth by the Mv at a concentration of 100. mu.M was significantly increased. At a concentration of 200 μ M of six anthocyanins, the cells died in large numbers with significant cytotoxicity, 45.25% after Cy treatment and 42.44% next with Mv. The result shows that the anthocyanin concentration is 200 mu M, the cytotoxicity is shown, the concentration of Dp, Pn, Pg, Cy and Pt is suitable for treating RIN-M5F cells by five kinds of anthocyanidins of 25-100 mu M, and the suitable concentration of Mv is lower than 100 mu M.
TABLE 5 Effect of different concentrations of anthocyanins on rat islet beta cytotoxicity
3.3 Effect of anthocyanin on rat islet beta cell autophagosome under Normal conditions
the effect of different concentrations of anthocyanins on rat islet β cell autophagosomes under normal conditions was observed by means of cellular immunofluorescence, as shown in figure 6 and table 6. According to the research result, the four anthocyanidins of Pt, Pn, Pg and Mv with the concentration of 25 mu M induce the rat islet beta cell autophagosome, but the effect is not obviously different compared with the control group, and the two anthocyanidins of 25 mu M Dp and Cy induce the rat islet beta cell autophagosome with relatively obvious effect; compared with a control group, the number of rat islet beta cell autophagosomes induced by the five anthocyanidins of Dp, Cy, Pt, Pn and Pg with the concentration of 50-100 mu M is increased remarkably and in a dose gradient manner, wherein the number of rat islet beta cell autophagosomes induced by the five anthocyanidins is respectively Dp, Cy, Pt, Pn and Pg from large to small; however, Mv induced no significant difference in the number of autophagosomes for rat islet cells compared to the control group, with Dp, Cy, Pt, Pn and Pg induced autophagosomes for rat islet beta cells at a concentration of 50 μ M of 9.74, 9.68, 8.8, 8.35, 7.35 per cell, respectively. Whereas, Mv at 25-80. mu.M induced no significant difference in the number of islet beta cell autophagosomes in rats compared to the control group. Therefore, in subsequent experiments, 50 μ M was selected as the concentration of anthocyanin that induces autophagosomes of rat islet β cells under normal conditions.
TABLE 6 anthocyanidin induces autophagosome counts in rat islet beta cells under normal conditions
3.4 Effect of anthocyanins on rat islet beta cell autophagosome in a high-carbohydrate apoptosis model
To study the effect of anthocyanins on rat islet β cell autophagosomes in a hyperglycocyte apoptosis model, it was first determined whether hyperglycosemia itself induced rat islet β cell autophagosomes, and second the ability of different anthocyanins to induce rat islet β cell autophagosomes in a hyperglycocyte apoptosis model was analyzed by cytoimmunofluorescence and western blot methods. The effect of different concentrations of glucose to induce autophagosome numbers of rat islet beta cells was found by the method of cellular immunofluorescence, as shown in fig. 7. The 0-30mM glucose induced no significant difference in autophagosome numbers of rat islet beta cells compared to the control group, whereas the 40mM treated group showed significant difference. This indicates that 0-30mM glucose by itself does not induce autophagosomes in rat islet beta cells, consistent with the ChieEbato study that 30mM high sugar does not induce autophagy in islet beta cells. Thus, it was determined that a high sugar concentration of 30mM did not induce autophagosomes in rat islet beta cells.
by means of cellular immunofluorescence, Dp, Cy, Pt, Pn, Pg and Mv induced islet β cell autophagosome profiles in rats in a high glucose apoptosis model as shown in fig. 8. According to research results, the four anthocyanidin of Dp, Cy, Pt and Pn with the concentration of 50 mu M induces the number of rat islet beta cell autophagosomes in a high-glucose apoptosis model to be greatly different from that of a control group, and Pg is remarkably different, wherein the number of the autophagosomes induced in the high-glucose apoptosis model is Dp, Cy, Pt, Pn and Pg from high to low, and 50 mu M Mv induces the number of rat islet beta cell autophagosomes in the high-glucose apoptosis model to be not remarkably different from that of the control group. And the five kinds of anthocyanin with the concentration of 50 mu M can obviously induce autophagosomes of rat islet beta cells, wherein Dp and Cy have strong capacity of inducing autophagosomes, but Mv does not induce autophagosomes of rat islet beta cells in a high-glucose apoptosis model. The four anthocyanins Dp, Cy, Pt and Pn are used as follow-up research objects because the numbers of autophagosomes inducing rat islet beta cells in a high-carbohydrate apoptosis model are very different and the autophagosomes inducing ability is strong.
In order to further determine the capacity of four anthocyanins Dp, Cy, Pt and Pn in inducing rat islet beta-cell autophagy in a high-glucose apoptosis model, the expression level of autophagic marker protein LC3 in the high-glucose apoptosis model is detected by a western blot method, and the result is shown in FIG. 9, and the result shows that the expression level of LC 3-II/LC 3-I in the Dp, Cy, Pt and Pn treatment groups is enhanced compared with that in the control group, and Dp, Cy, Pt and Pn are sequentially from strong to weak, which is consistent with the experimental result of cellular immunofluorescence. The Dp is shown to have the strongest capacity of inducing rat islet beta cell autophagy under normal conditions and in a high-glucose apoptosis model, so that the Dp is used as a target of subsequent research.
the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. a method for detecting the autophagy effect of anthocyanin on rat islet beta cells comprises the following steps:
Step one, dissolving paeoniflorin, pelargonidin, malvidin, cyanidin, delphinidin and petunidin into dimethyl sulfoxide respectively, configuring the mixture into a stock concentration of 400mM, and storing the mixture at the temperature of minus 20 ℃ in a dark place;
Step two, culturing rat islet beta cells by using RPMI 1640 culture solution containing 10% fetal calf serum in an incubator at 37 ℃ and 5% carbon dioxide;
Step three, screening the toxic concentration range of the rat islet beta cells by the anthocyanin;
fourthly, constructing a high-sugar rat islet beta cell apoptosis model by using a flow cytometry technology;
fifthly, verifying a high-glucose rat islet beta cell apoptosis model by a reactive oxygen species ROS level experiment;
step six, verifying a high-sugar rat islet beta cell apoptosis model by using the expression level of the apoptosis protein;
seventhly, detecting the autophagosome effect of the rat islet beta cells induced by the anthocyanin by using a cell immunofluorescence method;
step eight, establishing an RFP-GFP-RIN stable cell strain: constructing a lentivirus vector, packaging and preparing lentivirus, and screening a stable cell strain;
Step nine, detecting the autophagy flow effect of the rat islet beta cells induced by the anthocyanin by using a cell immunofluorescence method;
and step ten, judging the autophagosome and autophagy flow effects generated by inducing the rat islet beta cells by combining the anthocyanin.
2. the method for detecting the autophagy effect of the anthocyanin on rat islet beta cells, as claimed in claim 1, wherein the screening of the anthocyanin for the rat islet beta cell cytotoxicity concentration range in the step III comprises the following steps:
1) Inoculating 2X 10 4 cell suspensions into a 96-well plate, taking 200 mu L of each well, and placing the well in an incubator at 37 ℃ for 12 h;
2) After the cell monolayer grows in an adherent manner, adding six kinds of anthocyanin according to the final concentration of 25-200 mu M, and continuously culturing for 8h in a 37 ℃ culture box;
3) after the culture is finished, adding 20 mu LCCK-8 solution into each hole according to the proportion of 1:10 to continue culturing for 2 h;
4) Measuring the OD value of each sample at 450nm by using a microplate reader;
5) simultaneously setting a blank group, and only adding a culture medium and a CCK-8 reagent; the negative group is not added with the drug cell hole to be detected, and each group is provided with 5 multiple holes; calculating the cell survival rate according to a formula;
Cell viability (%) × 100% (treatment OD-blank OD)/(negative OD-blank OD), and cytotoxicity (%) × 100% (negative OD-treatment OD)/(negative-blank OD).
3. The method for detecting the autophagy effect of the anthocyanin on rat islet beta cells, as claimed in claim 1, wherein the step four of constructing the model of the high-sugar rat islet beta cell apoptosis by using the flow cytometry comprises the following steps:
1) inoculating 5 × 10 5 cell suspensions into 12-well cell plates, and culturing for 12 h;
2) adding glucose with final concentration of 10, 20 and 30mM into each well, and culturing for 24, 36 and 48 h;
3) after the culture is finished, respectively sucking the culture medium in each hole into a centrifuge tube (1), washing each hole for 1 time by using PBS and transferring the hole into the corresponding centrifuge tube (1);
4) Then 200-300ul pancreatin without EDTA is added into each hole, 2mL of RPMI 1640 culture solution of 10% fetal bovine serum is directly added after the cells are digested for 30s and is lightly blown to make the adherent cells suspended in the culture solution, and then the adherent cells are moved into a new centrifuge tube (2);
5) centrifuging the centrifugal tube (2) for 8min at 1000rpm, transferring the supernatant into the corresponding centrifugal tube (1), centrifuging for 8min at 3000rpm, and removing the supernatant;
6) Adding 2ml of LPBS into the centrifuge tubes (1) and (2) respectively, and gently resuspending the cells by using a pipette gun;
7) Centrifuging the suspended cells in the centrifugal tube (1) once again at 1000rpm multiplied by 8min respectively, transferring the supernatant into the corresponding centrifugal tube (2), centrifuging at 3000rpm multiplied by 8min, and removing the supernatant;
8) Adding 300-500ul of Bindbuffer to resuspend the cells, and mixing the centrifuge tube (1) and the corresponding cell (2);
9) after being mixed gently and evenly, the mixture is sieved into a centrifuge tube through a 200-mesh cell sieve to obtain a cell suspension to be detected;
10) before using a flow cytometry instrument, adding 5uL annexin-V-FICT into 500uL of the solution each time, incubating for 10min at room temperature, and mixing uniformly;
11) then adding 5ul PI, mixing uniformly, and incubating for 5min at room temperature;
12) Detecting the apoptosis condition of the rat islet beta cells by using a flow cytometry instrument with an excitation wavelength Ex being 488nm and an emission wavelength Em being 530 nm;
13) according to the apoptosis condition of the rat islet beta cells, the rat islet beta cell apoptosis model condition is determined to be 30mM and 36 h.
4. the method for detecting the effect of anthocyanin on inducing autophagy of rat islet beta cells as claimed in claim 1, wherein the ROS level test of reactive oxygen species in the fifth step comprises the following steps:
1) Labeling intracellular ROS levels by fluorescent probe DCFH-DA;
2) preparing a cell climbing sheet according to a cell immunofluorescence method; DCFH-DA at a final concentration of 50. mu. mol/L was added to each treatment group, incubated at 37 ℃ for 50min, and then washed 3 times with PBS; the fluorescence intensity of the cells was examined using the maximum excitation wavelength and emission wavelength of 488nm and 510nm, respectively, and photographed.
5. the method for detecting the autophagy effect of the anthocyanin on rat islet beta cells, as claimed in claim 1, wherein the verification of the apoptosis protein expression level in the sixth step of the hyperglycosemia rat islet beta cell apoptosis model comprises the following steps:
1) Cell culture, in which RIN-m5F cell strain is subcultured conventionally in RPMI 1640 medium containing 10% fetal calf serum at 37 deg.C under 100% saturation humidity and 5% CO 2;
2) protein recovery: after the cell treatment is finished, removing the culture solution, and washing twice with precooled PBS; add 10% SDS into each well; mixing, transferring into new centrifuge tube, incubating on ice for 20-30min, performing metal bath at 100 deg.C for 10min, adding 6 × loadingBuffer, mixing, performing metal bath at 100 deg.C for 10min, and storing in-80 deg.C refrigerator;
3) protein quantification: preparing BSA standard substances and working solution with different concentrations according to the BSA kit; adding BSA standard substances with different concentrations and protein samples to be detected into a 96-well plate respectively, incubating for 30min at 37 ℃, and making 5 samples in each group in parallel; measuring the absorbance at 562 nm; calculating the protein concentration in the sample according to the standard curve of the BSA standard substance;
4) and (3) carrying out western blot detection: preparing concentrated glue and separation glue, wherein 10-12% of separation glue is selected as Caspase 3;
a. Electrophoresis: filling the prepared gel into an electrophoresis tank, and adding 5-20 mu L of protein to be detected into each lane; the voltage is stabilized at 50-80v according to the molecular weight of Caspase3 protein, and electrophoresis is carried out for 1-2.5 h;
b. Film transfer: checking the target protein according to the molecular size, cutting off the gel block, and then putting the protein into a film loading groove according to the sandwich mode of a sandwich;
c. And (3) sealing: sealing the membrane with a sealing solution, and incubating on a shaking table for 1 h;
d. antibody incubation: diluting primary antibody with a ratio of 1:1000, incubating overnight in a refrigerator at 4 deg.C, removing primary antibody, and cleaning membrane with TPBST for 5-10min three times;
e. Antibody incubation: diluting the secondary antibody with a ratio of 1:5000, incubating for 1h in a shaking table, absorbing the secondary antibody, and washing the membrane with PBST for three times, each time for 5-10 min;
f. adding chemiluminescence developing solution, developing with an Image Quant LAS 4000mini gel imager, taking pictures, and storing; each band was quantitatively analyzed using Image J analysis software.
6. The method for detecting the autophagy effect of the anthocyanin on rat islet beta cells as claimed in claim 1, wherein the step seven for detecting the autophagy effect of the anthocyanin on rat islet beta cells by using a cellular immunofluorescence method comprises the following steps:
1) under normal conditions and in a high-glucose rat islet beta cell apoptosis model, cell suspensions containing 1 × 10 5 cells are respectively inoculated into 12-hole culture plates paved with slides for culture for 12 h;
2) Adding 50 mu M anthocyanin solution into each hole, and culturing for 8 h;
3) absorbing the culture solution by using a liquid transfer gun, and washing for 2-3 times by using PBS; adding 4% paraformaldehyde, fixing at room temperature for 10min, removing paraformaldehyde, and washing with PBS for 3 times; removing PBS, adding 10% goat serum to obtain sealing solution, and sealing at room temperature for 30 min;
4) Adding 0.1% saponin into 10% confining liquid prepared from goat serum to obtain diluent, mixing the diluent with one antibody LC3, (MBL, PM036) according to the ratio of 1:1000, coating the climbing sheet on an anti-dilution solution, and incubating for 1h at 37 ℃ in a dark place;
5) washing the climbing sheet with sealing liquid for three times;
6) Incubating a secondary antibody, Goatanti-Rabbit IgG (Southern Biotech,4050-05), against the same antibody, and applying the slide three times with blocking solution;
7) dropping a proper amount of the sealing tablet on a glass slide, covering the sealing tablet with the front face of the climbing tablet downwards, and drying in a dark place;
8) the number of fluorescent point-like aggregation is observed by a laser confocal microscope, namely the number of autophagosomes, and the point-like concentration degree can preliminarily evaluate the autophagosomal capacity of the rat islet beta cells; after autophagy occurs, LC3-I which is dispersed in cytoplasm is subjected to ubiquitination processing modification and is combined with phosphatidylethanolamine PE on the surface of an autophagosome membrane to form LC 3-II; the number of the point-shaped aggregates is observed by adopting laser confocal focusing through a cell immunofluorescence method, namely the number of autophagosomes, and the point-shaped concentration degree can preliminarily evaluate the cell autophagy;
9) and (6) taking a picture.
7. The method for detecting the autophagy effect of the anthocyanin on rat islet beta cells, as claimed in claim 1, wherein the step eight for establishing the RFP-GFP-RIN stable cell strain comprises the following steps:
1) amplifying an RFP-GFP-LC3 fragment from a PCMV-RFP-GFP-LC3 plasmid by a PCR technology; the amplification product is transformed by 10g/L agar, and recombinant plasmids are screened out to obtain RFP-GFP-LC3-GV374 plasmids; confirming the construction of the lentiviral vector through sequencing comparison;
2) culturing 293FT cells to 90%, digesting with pancreatin, subculturing in a cell plate at the density of 1 × 10 6/mL for 24h, performing operation according to the instruction of a Lipo3000 transfection kit after the logarithmic phase of cell growth, collecting cell supernatant after culturing for 24h, performing 3000r/min, centrifuging for 10min, and taking the supernatant;
3) adding the collected lentivirus particles into RIN-m5F cells for culturing for 48 h; during the period, new culture medium is observed and replaced, and after the culture is finished, puromycin with the volume ratio of 6 mug/mL is added for screening infected RIN-m5F cells; after culturing for 7 days, screening out cells with red and green fluorescence under a fluorescence microscope; selecting positive clone cells, transferring the positive clone cells to a new culture dish, and continuing culturing to obtain the RFP-GFP-LC3 islet beta stable cell strain.
8. the method for detecting the autophagy effect of the anthocyanin on rat islet beta cells, as claimed in claim 1, wherein the step nine of detecting the autophagy effect of the anthocyanin on rat islet beta cells by using a cellular immunofluorescence method comprises the following steps:
1) In a high-glucose rat islet beta cell apoptosis model, a suspension containing 1 × 10 5/mL RFP/GFP-LC3 stable cell line was seeded into a culture plate that had been plated on a slide;
2) adding 50 mu M anthocyanin and 20 mu M chloroquine into each well, culturing for 2h, and adding 30mM glucose for culturing for 36 h;
3) After the reaction is finished, removing the culture medium, and washing the climbing film for 2-3 times by PBS; adding 4% paraformaldehyde, fixing at room temperature for 10min, removing paraformaldehyde, and washing with PBS for 3 times;
4) dropping a proper amount of the sealing tablet on a glass slide, covering the sealing tablet with the front face of the climbing tablet downwards, and drying in a dark place;
5) And (3) observing the ratio of red fluorescence to green fluorescence by adopting a laser confocal microscope observation microscope to judge autophagy flow and taking a picture.
9. use of the method for detecting the autophagy effect of the rat islet beta cell induced by anthocyanin according to any one of claims 1-8 in screening the autophagy effect of the rat islet beta cell induced by anthocyanin.
10. the use of any one of claims 1-8 for detecting the autophagy of rat islet beta cells induced by anthocyanin for preventing diabetes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980772A (en) * | 2021-03-09 | 2021-06-18 | 广东医科大学 | Method for detecting autophagy effect of anthocyanin-induced mouse islet beta cells |
| CN113621579A (en) * | 2021-08-11 | 2021-11-09 | 华南师范大学 | Bifluorescent S2 cell line for indicating autophagy of cells and preparation method thereof |
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| BO ZHANG等: "Anthocyanins from Chinese bayberry extract activate transcription factor Nrf2 in beta cells and negatively regulate oxidative stress-induced autophagy", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| CHONG-DE SUN等: "Cyanidin-3-Glucoside-Rich Extract from Chinese Bayberry Fruit Protects Pancreatic β Cells and Ameliorates Hyperglycemia in Streptozotocin-Induced Diabetic Mice", 《J MED FOOD》 * |
| PON VELAYUTHAM ANANDHBABU等: "Recent advances in understanding the anti-diabetic actions of dietary flavonoids", 《THE JOURNAL OF NUTRITIONAL BIOCHEMISTRY》 * |
| TIAN GUO等: "Sonodynamic therapy inhibits palmitate induced beta cell dysfunction via PINK1/Parkin-dependent mitophagy", 《CELL DEATH AND DISEASE》 * |
| ZHENQI ZHOU等: "Estrogen Receptor α Protects Pancreatic β-cells from Apoptosis by Preserving Mitochondrial Function and Suppressing Endoplasmic Reticulum Stress", 《J BIOL CHEM》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980772A (en) * | 2021-03-09 | 2021-06-18 | 广东医科大学 | Method for detecting autophagy effect of anthocyanin-induced mouse islet beta cells |
| CN113621579A (en) * | 2021-08-11 | 2021-11-09 | 华南师范大学 | Bifluorescent S2 cell line for indicating autophagy of cells and preparation method thereof |
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