Research method of KAT2A in BV2 microglial cell inflammation mechanism

Abstract

The invention discloses a method for researching an inflammatory mechanism of KAT2A in BV2 microglial cells, and belongs to the technical field of BV2 microglial cell research, and the method comprises the following steps: S1, culturing BV2 microglial cells; s2, constructing a BV2 microglial cell inflammation model; s3, carrying out KAT2A activity inhibition treatment and small interfering RNA cell transfection on the constructed BV2 microglial cell inflammation model; s4, performing western blot analysis; s5, carrying out real-time polymerase chain reaction analysis; s6, carrying out cell electron microscope analysis; s7, detecting and analyzing mitochondrial superoxide and active oxygen; s8, on the basis of an analysis result, researching the effect of knocking down KAT2A in a BV2 microglial cell inflammation mechanism; the invention proves that the inflammatory response of the BV2 microglial cells can be relieved by improving the mitochondrial function by inhibiting the expression of the KAT2A.

Description

Technical Field

[0001] The present invention belongs to the technical field of BV2 microglial cell research, and specifically relates to a method for studying the inflammatory mechanism of KAT2A in BV2 microglial cells. Background Art

[0002] Microglia are intrinsic immune cells of the central nervous system, and their cellular inflammatory responses are widely involved in the occurrence and development of central nervous system diseases.

[0003] Previous studies have shown that inhibiting microglial inflammatory responses can effectively alleviate the progression of depression, anxiety, and neurodegenerative diseases such as Parkinson's disease.

[0004] However, there is currently no research specifically targeting the specific molecular mechanisms of microglial inflammation.

[0005] In view of this, a research method on the inflammatory mechanism of KAT2A in BV2 microglia was designed to solve the above problems. Summary of the invention

[0006] To solve the problems raised in the above background technology, the present invention provides a method for studying the inflammatory mechanism of KAT2A in BV2 microglia, which has the characteristics of proving that inhibiting KAT2A expression can alleviate the inflammatory response of BV2 microglia by improving mitochondrial function.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for studying the inflammatory mechanism of KAT2A in BV2 microglia, comprising the following steps:

[0008] S1: Culture BV2 microglia;

[0009] S2: Construction of BV2 microglial inflammation model;

[0010] S3: The constructed BV2 microglial inflammation model was treated with KAT2A activity inhibition and small interfering RNA cell transfection;

[0011] KAT2A activity inhibition treatments included: treating BV2 microglia with 50 μM concentration of MB-3 for 24 h to inhibit KAT2A activity;

[0012] Small interfering RNA cell transfection includes: using 0.25% trypsin solution to digest BV2 microglia, then resuspending BV2 microglia in DMEM medium and counting the cells at 2×10 per well. 5 BV2 microglia were seeded in 24-well plates at a density of 10 cells and Lipofectamine TM BV2 microglia were treated with 3000 and KAT2A siRNA for 48 h;

[0013] S4: Western blotting analysis of the expression levels of KAT2A and iNOS in the BV2 microglial inflammation model constructed, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0014] S5: Expression levels of inflammatory factors IL-1β and IL-6 in the BV2 microglial inflammation model constructed by real-time polymerase chain reaction analysis, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0015] S6: Mitochondrial structures of the BV2 microglial inflammation model constructed by cell electron microscopy, the BV2 microglial inflammation model after KAT2A activity inhibition, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0016] S7: The levels of mitochondrial mitoSOX signals and ROS signals in the BV2 microglial inflammation model constructed by mitochondrial superoxide and reactive oxygen species detection analysis, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0017] S8: Based on the analyzed expression levels of KAT2A and iNOS, the expression levels of inflammatory factors IL-1β and IL-6, mitochondrial structure, and the levels of mitochondrial mitoSOX signaling and ROS signaling, the role of knocking down KAT2A in the inflammatory mechanism of BV2 microglia was investigated.

[0018] Furthermore, the specific steps of step S1 include:

[0019] The cells were cultured in DMEM medium containing fetal bovine serum and penicillin-streptomycin at 37°C and 5% CO. 2 BV2 microglial cells were cultured in a cell culture incubator.

[0020] Furthermore, the specific steps of step S2 include:

[0021] BV2 microglia cells were treated with 100 ng / mL lipopolysaccharide for 24 h to construct a BV2 microglial inflammation model.

[0022] Furthermore, the specific steps of step S4 include:

[0023] BV2 microglial cell samples were extracted using RIPA lysis buffer, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was treated with 5% skim milk to block nonspecific binding and then incubated with the primary antibody at 4°C overnight. Subsequently, it was incubated with the secondary antibody labeled with horseradish peroxidase for 1 hour. The immunolabeled bands were displayed using enhanced chemiluminescence reagent, and the expression levels of the target protein bands were quantified using ImageJ software to analyze the expression levels of KAT2A and iNOS.

[0024] Furthermore, the specific steps of step S5 include:

[0025] Total RNA was extracted from BV2 microglial cell samples using VeZol reagent according to the phenol / chloroform method. Subsequently, 200 ng of RNA was reverse transcribed using the reverse transcription reagent HiScript IV RT SuperMix, and the expression levels of target genes were detected using a qPCR detection kit to analyze the expression levels of inflammatory factors IL-1β and IL-6.

[0026] Furthermore, the specific steps of step S6 include:

[0027] BV2 microglia were digested with 0.25% trypsin and centrifuged at 1200rpm for 5min. The supernatant was discarded and the BV2 microglia were resuspended in electron microscopy fixative. The BV2 microglia were rinsed with sucrose-sodium carboxylate solution and incubated in osmium tetroxide-sodium carboxylate for 2h. Subsequently, after being rinsed with water, the BV2 microglia were stained with 2% uranyl acetate water. The samples were dehydrated through a graded ethanol series and then embedded in Epon 812 epoxy resin. Ultrathin sections were made with a diamond knife and images were digitally recorded for analysis of mitochondrial structure.

[0028] Furthermore, the specific steps of step S7 include:

[0029] BV2 microglia were inoculated on cell slides and incubated with mitoSOX fluorescent dye indicator / CM-H2DCFDA probe for 30 min. After the cell slides were washed in the dark, the cell nuclei were stained with DAPI containing anti-fluorescence quencher and the slides were sealed. The mitoSOX / ROS signals were observed under a fluorescence microscope to analyze the levels of mitochondrial mitoSOX signals and ROS signals.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention uses mice as experimental subjects, firstly induces in vitro BV2 microglial inflammatory response by lipopolysaccharide, then inhibits KAT2A level by KAT2A activity inhibition treatment and small interfering RNA cell transfection, and then analyzes the expression levels of KAT2A and iNOS and the expression levels of inflammatory factors IL-1β and IL-6, proving that inhibiting KAT2A expression can alleviate BV2 microglial inflammatory activation, and at the same time analyzes the mitochondrial structure and mitochondrial mitoSOX signal and ROS signal levels, proving that inhibiting KAT2A expression can alleviate mitochondrial structural damage of BV2 microglial cells and reduce oxidative stress levels, that is, proving that inhibiting KAT2A expression can alleviate BV2 microglial inflammatory response by improving mitochondrial function. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure is a protein immunoblotting and real-time polymerase chain reaction experimental result diagram after transfection of KAT2A siRNA in the present invention;

[0033] Figure 2 The results of protein immunoblotting and real-time polymerase chain reaction after MB-3 treatment of the present invention are shown;

[0034] Figure 3 The graphs are the experimental results of electron microscopy, mitochondrial superoxide and reactive oxygen species detection of cells after LPS treatment and transfection with KAT2A siRNA in the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The present invention provides the following technical solution: a method for studying the inflammatory mechanism of KAT2A in BV2 microglia, comprising the following steps:

[0037] S1: Culture BV2 microglia;

[0038] The cells were cultured in DMEM medium (C11995500, Gibco) containing fetal bovine serum (Z7186FBS, Zeta Life) and penicillin-streptomycin (C125C5, NCM Biotech) at 37°C and 5% CO. 2 BV2 microglia cells (Procell, Wuhan, China) were cultured in a cell culture incubator;

[0039] S2: Construction of BV2 microglial inflammation model;

[0040] BV2 microglial cells were treated with 100 ng / mL lipopolysaccharide (LPS) for 24 h to establish a BV2 microglial inflammation model;

[0041] S3: The constructed BV2 microglial inflammation model was treated with KAT2A activity inhibition and small interfering RNA cell transfection;

[0042] KAT2A activity inhibition treatments included: treating BV2 microglia with 50 μM concentration of MB-3 (12095, Cayman) for 24 h to inhibit KAT2A activity;

[0043] Small interfering RNA cell transfection includes: using 0.25% trypsin solution (C125C1, NCM Biotech) to digest BV2 microglia cells, then resuspending BV2 microglia cells in DMEM medium and counting the cells at 2×10 per well. 5 BV2 microglia were seeded in 24-well plates at a density of 10 cells and Lipofectamine TM 3000 (L3000001, ThermoFisher) and KAT2A siRNA were used to treat BV2 microglia for 48 h;

[0044] KAT2A siRNA was purchased from Shanghai Bioengineering, with the sequence 5′-GCUACCUACAAAGUCAAUUAUTT-3′;

[0045] S4: Western blotting analysis of the expression levels of KAT2A and iNOS in the BV2 microglial inflammation model constructed, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0046] BV2 microglial cell samples were extracted with RIPA lysis buffer (P0013B, Beyotime), proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membrane (ISEQ00010, Millipore). The membrane was treated with 5% skim milk to block nonspecific binding, and then incubated with primary antibody at 4°C overnight. Subsequently, it was incubated with horseradish peroxidase-labeled secondary antibody for 1 h. The immunolabeled bands were visualized using enhanced chemiluminescence reagent, and the expression levels of the target protein bands were quantified using ImageJ software (NIH, Bethesda);

[0047] The antibody ratio and product number information are as follows: iNOS (1:1000,18985-1-AP, Proteintech,), KAT2A (1:1000,3305, Cell Signaling), GAPDH (1:3000,60004-1-Ig, Proteintech);

[0048] S5: Expression levels of inflammatory factors IL-1β and IL-6 in the BV2 microglial inflammation model constructed by real-time polymerase chain reaction analysis, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0049] Total RNA was extracted from BV2 microglial cell samples using VeZol reagent (R411-01, Novozyme) according to the phenol / chloroform method, and then 200 ng of RNA was reverse transcribed using the reverse transcription reagent HiScript IV RT SuperMix (R423-01, Novozyme), and the expression level of the target gene was detected using a qPCR detection kit (Q712-02, Vazyme);

[0050] The parameters were set as follows: denaturation at 95°C for 5 min; annealing at 60°C for 30 s and 40 cycles; extension at 72°C for 10 min;

[0051] GAPDH was used as the internal reference gene, and the primer sequences were as follows:

[0052] IL-1β upstream: 5′-GAAATGCCACCTTTTGACAGTG-3′;

[0053] IL-1β downstream: 5′-TGGATGCTCTCATCAGGACAG-3′;

[0054] IL-6 upstream: 5′-TAGTCCTTCCTACCCCAATTTCC-3′;

[0055] IL-6 downstream: 5′-TTGGTCCTTAGCCACTCCTTC-3′;

[0056] GAPDH upstream: 5′-AGGTCGGTGTGAACGGATTTG-3′;

[0057] GAPDH downstream: 5′-GGGGTCGTTGATGGCAACA-3′;

[0058] S6: Mitochondrial structures of the BV2 microglial inflammation model constructed by cell electron microscopy, the BV2 microglial inflammation model after KAT2A activity inhibition, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0059] BV2 microglia were digested with 0.25% trypsin and centrifuged at 1200 rpm for 5 min. The supernatant was discarded and the BV2 microglia were resuspended in electron microscopy fixative (G1102, Seville). The BV2 microglia were rinsed with sucrose-sodium carboxylate solution and then incubated in osmium tetroxide-sodium carboxylate for 2 h. Subsequently, after being rinsed with water, the BV2 microglia were stained with 2% uranyl acetate water. The samples were dehydrated through a graded ethanol series and then embedded in Epon 812 epoxy resin (90529-77-4, SPI Science). Ultrathin sections were made with a diamond knife and images were recorded digitally.

[0060] S7: The levels of mitochondrial mitoSOX signals and ROS signals in the BV2 microglial inflammation model constructed by mitochondrial superoxide and reactive oxygen species detection analysis, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection;

[0061] BV2 microglia were seeded on cell slides and incubated with mitoSOX fluorescent dye indicator (M36007, Invitrogen) / CM-H2DCFDA probe (S0035S, Beyotime) for 30 min. After the cell slides were washed in the dark, the cell nuclei were stained with anti-fluorescence quencher DAPI (0100–20, SouthernBiotech), and the slides were sealed. The mitoSOX / ROS signals were observed using a fluorescence microscope to analyze the levels of mitochondrial mitoSOX signals and ROS signals.

[0062] S8: Based on the analyzed expression levels of KAT2A and iNOS, the expression levels of inflammatory factors IL-1β and IL-6, mitochondrial structure, and the levels of mitochondrial mitoSOX signaling and ROS signaling, the role of knocking down KAT2A in the inflammatory mechanism of BV2 microglia was investigated.

[0063] The results of protein immunoblotting after LPS treatment are shown in the attached figure. Figure 1 Shown are: (A) KAT2A expression in BV2 microglia after LPS treatment;

[0064] By the attached Figure 1 (A) It can be seen that: after LPS treatment, the expression of KAT2A in BV2 microglia was upregulated, i.e., increased;

[0065] The results of protein immunoblotting and real-time polymerase chain reaction after MB-3 treatment are shown in the Appendix. Figure 2 As shown, (A) represents the expression of iNOS after MB-3 treatment, (B) represents the expression of inflammatory factor IL-1β after MB-3 treatment, and (C) represents the expression of inflammatory factor IL-6 after MB-3 treatment;

[0066] By the attached Figure 2 (A) It can be seen that MB-3 treatment inhibited the upregulation of iNOS induced by LPS;

[0067] By the attached Figure 2 (B) It can be seen that MB-3 treatment inhibited the upregulation of inflammatory factor IL-1β induced by LPS;

[0068] By the attached Figure 2 (C) It can be seen that after MB-3 treatment, the upregulation of inflammatory factor IL-6 induced by LPS was inhibited;

[0069] The results of protein immunoblotting and real-time polymerase chain reaction after transfection of KAT2A siRNA are shown in the attached figure. Figure 1 As shown, (B) represents the expression of iNOS and KAT2A after transfection with KAT2A siRNA, (C) represents the expression of inflammatory factor IL-1β after transfection with KAT2A siRNA, and (D) represents the expression of inflammatory factor IL-6 after transfection with KAT2A siRNA;

[0070] By the attached Figure 1 (B) It can be seen that after transfection with KAT2A siRNA, the upregulation of KAT2A expression caused by LPS was reversed, and the upregulation of iNOS expression caused by LPS was inhibited;

[0071] By the attached Figure 1 (C) It can be seen that after transfection with KAT2A siRNA, the upregulation of the inflammatory factor IL-1β induced by LPS was inhibited;

[0072] By the attached Figure 1 (D) It can be seen that after transfection with KAT2A siRNA, the upregulation of the expression of inflammatory factor IL-6 induced by LPS was inhibited;

[0073] The above results indicate that knocking down KAT2A expression can alleviate the inflammatory level of BV2 microglia;

[0074] The results of cell electron microscopy are shown in the attached Figure 3 As shown, (A) represents the mitochondrial structure of BV2 microglia after LPS treatment and after LPS treatment and transfection with KAT2A siRNA;

[0075] By the attached Figure 3 (A) It can be seen that after LPS treatment, the mitochondrial structure of BV2 microglia was destroyed, the cristae disappeared, and the outer membrane ruptured. Knockdown of KAT2A can alleviate mitochondrial damage;

[0076] The results of mitochondrial superoxide and reactive oxygen species detection are shown in the attached Figure 3 As shown, (B) represents the mitoSOX signal of BV2 microglia after LPS treatment and after LPS treatment and transfection of KAT2A siRNA, (C) represents the ROS signal of BV2 microglia after LPS treatment and after LPS treatment and transfection of KAT2A siRNA;

[0077] By the attached Figure 3 (B) It can be seen that the mitoSOX signal of BV2 microglia was enhanced after LPS treatment, and knockdown of KAT2A could reduce the mitoSOX signal;

[0078] By the attached Figure 3 (C) It can be seen that the ROS signal of BV2 microglia is enhanced after LPS treatment, and knockdown of KAT2A can reduce the ROS signal;

[0079] The above results indicate that knocking down KAT2A expression can inhibit mitochondrial structural damage and oxidative stress levels in BV2 microglia;

[0080] That is, knocking down KAT2A expression can alleviate the inflammatory response of BV2 microglia by improving mitochondrial function.

[0081] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for studying the inflammatory mechanism of KAT2A in BV2 microglia, characterized in that: The following steps are involved: S1: Culture BV2 microglia; S2: Construction of BV2 microglial inflammation model; S3: The constructed BV2 microglial inflammation model was treated with KAT2A activity inhibition and small interfering RNA cell transfection; KAT2A activity inhibition treatments included: treating BV2 microglia with 50 μM concentration of MB-3 for 24 h to inhibit KAT2A activity; Small interfering RNA cell transfection includes: using 0.25% trypsin solution to digest BV2 microglia, then resuspending BV2 microglia in DMEM medium and counting the cells at 2×10 per well. 5 BV2 microglia were seeded in 24-well plates at a density of 10 cells and Lipofectamine TM BV2 microglia were treated with 3000 and KAT2A siRNA for 48 h; S4: Western blotting analysis of the expression levels of KAT2A and iNOS in the BV2 microglial inflammation model constructed, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection; S5: Expression levels of inflammatory factors IL-1β and IL-6 in the BV2 microglial inflammation model constructed by real-time polymerase chain reaction analysis, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection; S6: Mitochondrial structures of the BV2 microglial inflammation model constructed by cell electron microscopy, the BV2 microglial inflammation model after KAT2A activity inhibition, and the BV2 microglial inflammation model after small interfering RNA cell transfection; S7: The levels of mitochondrial mitoSOX signals and ROS signals in the BV2 microglial inflammation model constructed by mitochondrial superoxide and reactive oxygen species detection analysis, the BV2 microglial inflammation model after KAT2A activity inhibition treatment, and the BV2 microglial inflammation model after small interfering RNA cell transfection; S8: Based on the analyzed expression levels of KAT2A and iNOS, the expression levels of inflammatory factors IL-1β and IL-6, mitochondrial structure, and the levels of mitochondrial mitoSOX signaling and ROS signaling, the role of knocking down KAT2A in the inflammatory mechanism of BV2 microglia was investigated.

2. The method for studying the inflammatory mechanism of KAT2A in BV2 microglia according to claim 1, characterized in that: The specific steps of step S1 include: BV2 microglia were cultured in DMEM medium containing fetal bovine serum and penicillin-streptomycin in a cell culture incubator at 37°C and 5% CO2.

3. The method for studying the inflammatory mechanism of KAT2A in BV2 microglia according to claim 1, characterized in that: The specific steps of step S2 include: BV2 microglia cells were treated with 100 ng / mL lipopolysaccharide for 24 h to construct a BV2 microglial inflammation model.

4. The method for studying the inflammatory mechanism of KAT2A in BV2 microglia according to claim 1, characterized in that: The specific steps of step S4 include: BV2 microglial cell samples were extracted using RIPA lysis buffer, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was treated with 5% skim milk to block nonspecific binding and then incubated with the primary antibody at 4°C overnight. Subsequently, it was incubated with the secondary antibody labeled with horseradish peroxidase for 1 hour. The immunolabeled bands were displayed using enhanced chemiluminescence reagent, and the expression levels of the target protein bands were quantified using ImageJ software to analyze the expression levels of KAT2A and iNOS.

5. The method for studying the inflammatory mechanism of KAT2A in BV2 microglia according to claim 1, characterized in that: The specific steps of step S5 include: Total RNA was extracted from BV2 microglial cell samples using VeZol reagent according to the phenol / chloroform method. Subsequently, 200 ng of RNA was reverse transcribed using the reverse transcription reagent HiScript IV RT SuperMix, and the expression levels of target genes were detected using a qPCR detection kit to analyze the expression levels of inflammatory factors IL-1β and IL-6.

6. The method for studying the inflammatory mechanism of KAT2A in BV2 microglia according to claim 1, characterized in that: The specific steps of step S6 include: BV2 microglia were digested with 0.25% trypsin and centrifuged at 1200rpm for 5min. The supernatant was discarded and the BV2 microglia were resuspended in electron microscopy fixative. The BV2 microglia were rinsed with sucrose-sodium carboxylate solution and incubated in osmium tetroxide-sodium carboxylate for 2h. Subsequently, after being rinsed with water, the BV2 microglia were stained with 2% uranyl acetate water. The samples were dehydrated through a graded ethanol series and then embedded in Epon 812 epoxy resin. Ultrathin sections were made with a diamond knife and images were digitally recorded for analysis of mitochondrial structure.

7. The method for studying the inflammatory mechanism of KAT2A in BV2 microglia according to claim 1, characterized in that: The specific steps of step S7 include: BV2 microglia were inoculated on cell slides and incubated with mitoSOX fluorescent dye indicator / CM-H2DCFDA probe for 30 min. After the cell slides were washed in the dark, the cell nuclei were stained with DAPI containing anti-fluorescence quencher and the slides were sealed. The mitoSOX / ROS signals were observed under a fluorescence microscope to analyze the levels of mitochondrial mitoSOX signals and ROS signals.

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