Application of Src tyrosine kinase inhibitor in inhibiting secretion of inflammatory factors
阅读说明：本技术 Src酪氨酸激酶抑制剂在抑制炎症因子分泌中的应用 (Application of Src tyrosine kinase inhibitor in inhibiting secretion of inflammatory factors ) 是由 张慧萍 姜怡邓 徐灵博 王艳华 杨安宁 马胜超 于 2019-12-13 设计创作，主要内容包括：一种Src酪氨酸激酶抑制剂在抑制炎症因子分泌中的应用,属于生物工程技术领域,本发明以人单核细胞源巨噬细胞为研究对象,取细胞培养液并提取细胞的蛋白,同时Elisa检测检测细胞培养液中炎症因子的水平；以Westen-blot检测炎症信号通路相关基因的激活水平；以Westen-blot和酶活性分别检测Src蛋白水平和酶活性。Src酪氨酸激酶抑制剂有望可作为一种药物,在巨噬细胞炎症性疾病的缓解中具有应用价值。(The invention relates to an application of a Src tyrosine kinase inhibitor in inhibiting inflammatory factor secretion, which belongs to the technical field of biological engineering, and takes human monocyte-derived macrophages as a research object, takes cell culture solution and extracts protein of cells, and Elisa detects and detects the level of inflammatory factors in the cell culture solution; detecting the activation level of the genes related to the inflammatory signal pathway by Westen-blot; src protein levels and enzyme activities were measured with Westen-blot and enzyme activities, respectively. The Src tyrosine kinase inhibitor is expected to be a drug and has application value in alleviating macrophage inflammatory diseases.)
1. Use of a Src tyrosine kinase inhibitor for inhibiting the secretion of an inflammatory factor, comprising the steps of:
(1) culturing normal human monocyte-derived macrophages, overexpressing human monocyte-derived macrophages FABP4, and detecting the phosphorylation levels of inflammation-related pathways JAK2/STAT2 by Western-blot;
(2) culturing normal human monocyte-derived macrophages, overexpressing human monocyte-derived macrophages FABP4, and detecting the expression level of inflammatory factors by Elisa;
(3) after overexpression of human monocyte-derived macrophage FABP4, detecting Src and Src phosphorylation level by using Western-blot;
(4) after overexpression, human monocyte-derived macrophage FABP4 is subjected to enzymatic detection on the enzymatic activity of Src;
(5) after overexpression of human monocyte-derived macrophage FABP4, adding a PP2 inhibitor, and detecting the phosphorylation level of JAK2/STAT2 by using a Western-blot detection channel;
(6) after the human monocyte-derived macrophage FABP4 is over-expressed, a PP2 inhibitor is added to detect the secretion level of the human monocyte-derived macrophage inflammatory factor by adopting an Elisa method.
2. The use of a Src tyrosine kinase inhibitor for inhibiting the secretion of inflammatory factors according to claim 1, wherein overexpression of FABP4 results in activation of inflammatory signaling pathways and increased secretion of inflammatory factors.
3. The use of a Src tyrosine kinase inhibitor for inhibiting the secretion of inflammatory factors according to claim 1, wherein Src phosphorylation is increased and kinase activity is increased after overexpression of FABP 4.
4. The use of a Src tyrosine kinase inhibitor for inhibiting the secretion of inflammatory factors according to claim 1, wherein the Src tyrosine kinase inhibitor PP2 is used to decrease the activity of inflammatory pathways and decrease the secretion of inflammatory factors.
5. Use of Src tyrosine kinase inhibitors according to claim 1 for inhibiting the secretion of inflammatory factors, characterized in thatThe macrophage cell culture method comprises the following steps: taking out the cryopreserved cells from the liquid nitrogen tank, quickly putting the cells into a beaker or a water bath kettle with 37-DEG C warm water for melting, centrifuging the cells for 5min at 1000r/min of a room-temperature centrifuge, opening the cryopreserved tubes in a superclean bench, completely pouring out the frozen liquid in the cell tubes, completely sucking the residual liquid, adding 10% 1640 culture medium containing serum into the tubes, gently blowing the cells from the bottom of the culture bottle by using a pipettor, slowly re-suspending the cells, and subpackaging the suspended cells to 25cm2Adding a proper amount of 10% serum-containing culture medium into a culture bottle, culturing in a constant-temperature incubator at 37 ℃ and 5% CO2 to enable the mononuclear cells to grow in a suspension manner, centrifuging at 1000rpm for 5min when the cells grow to 80% -90%, and subculturing; control group: administering monocytes PMA to stimulate macrophage formation; Ad-GFP group: the macrophage formed by PMA stimulation of the monocyte is stimulated by Ad-GFP of an adenovirus control group for 6 hours, and then the macrophage is cultured for 48 hours by changing 10% 1640 culture medium; Ad-FABP4 group: the macrophage formed by PMA stimulation of the monocyte is stimulated by Ad-FABP4 adenovirus for 6h, and then the monocyte is cultured for 48h by changing 10% 1640 culture medium; PP2 group: administering monocytes PMA-stimulated macrophages formed with PP2 for 2 h; Ad-FABP4+ PP2 group: the monocytes were stimulated with PMA to form macrophages after 6h with Ad-FABP4 adenovirus and after 48h with 10% 1640 medium for 2h with PP 2.
6. The use of Src tyrosine kinase inhibitors of claim 1 for inhibiting the secretion of inflammatory factors, wherein Elisa measures the levels of inflammatory factors in cell culture by: collecting the cell culture solution into a centrifuge tube, centrifuging at 12000r for 5min, and arranging standard substance holes and sample holes, wherein 50 mu l of standard substances with different concentrations are added into the standard substance holes respectively; adding 10 mul of sample to be detected into the sample hole, and then adding 40 mul of diluent; adding 100 μ l of detection antibody labeled by Horse Radish Peroxidase (HRP) into the standard sample well and the sample well except the blank well, sealing with a sealing plate membrane, and incubating in a 37 deg.C water bath or thermostat for 60 min; discarding liquid, drying on absorbent paper, filling cleaning solution into each hole, standing for 1min, throwing off the cleaning solution, drying on the absorbent paper, and washing the plate for 5 times in this way; adding 50 μ l of substrate A, B to each well, and incubating at 37 deg.C in dark for 5 min; adding 50 mul of stop solution into each hole, and measuring the OD value of each hole at the wavelength of 450nm within 15 min; and (4) calculating a result: the resulting concentration multiplied by the dilution factor is the actual inflammatory factor concentration.
7. The use of a Src tyrosine kinase inhibitor for inhibiting the secretion of inflammatory factors according to claim 1, wherein the enzymatic activity is determined by: the method for kinase determination by using rhodamine 110 peptide as a substrate is shown in a kit (ProFluor Src-family kinase assay, Promega, V1270). briefly, 30 ng of purified c-Src is revived in a kinase solution, incubated with rhodamine 110 peptide at 25 ℃ for 1 hour at 10 mmol/L ATP (adenosine triphosphate for short), then added with a protease solution, incubated at 25 ℃ for 1 hour, and then detected with an enzyme-labeling instrument at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
8. The use of the over-expressed FABP4 in promoting the secretion of inflammatory factors according to claim 1, wherein a Western-blot method for detecting the phosphorylation of JAK2/STAT2 and JAK2/STAT2 and the phosphorylation levels of Src and Src comprises the following steps: collecting the cultured cells into a centrifuge tube, adding a proper amount of protein extracting solution, detecting the protein concentration by using BCA (burst cutting edge), denaturing the protein, loading the protein to gel with a proper concentration, performing electrophoresis, transferring a mold, incubating an antibody, and calculating the protein content according to the gray value after exposure.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a Src tyrosine kinase inhibitor in inhibiting secretion of inflammatory factors.
Background
Inflammation (inflammation): is a defense response of the body to external stimuli. It is divided into infectious inflammation and non-infectious inflammation. Inflammation is generally beneficial and a defense response in humans, but in some cases it is also detrimental and is associated with the development of a number of diseases, such as: heart diseases, neuropathy, diabetes, etc.
Fatty-type fatty acid binding proteins (a-FABPs), also known as fatty acid binding protein 4 (FABP4), are members of the family of Fatty Acid Binding Proteins (FABPs), bind a variety of hydrophobic compounds, are widely distributed in adipose and ovarian tissue, participate in the anabolism of fatty acids, reversibly bind to intracellular hydrophobic ligands, and transport them to various parts of the cell, thereby playing a role in cellular lipid metabolism and signaling. It is a cytoplasmic protein, which is expressed more in adipocytes and macrophages, and plays an important role in lipid metabolism and inflammation. In adipocytes, FABP4 affects glucose metabolism by mediating lipolysis and lipid re-esterification; in macrophages, FABP4 is involved in lipid metabolism and inflammatory responses, and studies have shown that: the knockout of FABP4 can inhibit inflammatory reaction, reduce endoplasmic reticulum stress, and reduce nuclear factor kappa light chain enhancer of activated B cell activated macrophage. FABP4 can be seen to play an important role in local macrophage responses, but its molecular mechanism is not clear.
Tyrosine Src is activated by phosphorylation at tyrosine sites, which, when activated, causes tyrosine phosphorylation of some compact and adhesive proteins. It has also been shown that tight junction protein aggregation can be inhibited. There is a great deal of evidence that kinase activation is involved in acute injury to multiple organs, including cerebral ischemic injury, myocardial infarction, hepatocellular injury, acute kidney injury, and the like. The c-Src protein, which is one of Tyrosine kinase protein kinase, TPK, is activated at the plasma membrane in the early stage of tumorigenesis and transmits an oncogenic signal. On the one hand, studies have shown that: c-Src binds to the cell membrane by myristoylation, thereby transmitting a signal from outside the cell to inside the cell. On the other hand, c-Src has also been reported to function not only in the plasma membrane but also in the inner membrane. There are more and more researches on using Src kinase as a target of antitumor drugs in malignant tumors, and using Src kinase inhibitors for preventing and treating malignant tumors. However, the relationship between FABP4, c-Src and inflammatory signaling pathways in macrophages is unclear. Src kinase inhibitor PP2 is a pyrazolopyrimidine complex that is effective in inhibiting Src kinase. However, no prior art method for effectively applying Src tyrosine kinase inhibitors in inhibiting the secretion of inflammatory factors has been found so far.
Disclosure of Invention
The invention aims to provide an inhibitor for reducing FABP 4-mediated immune inflammatory response, namely an application method of a Src tyrosine kinase inhibitor in inhibiting inflammatory factor secretion.
The specific technical scheme of the invention is as follows:
use of a Src tyrosine kinase inhibitor for inhibiting the secretion of an inflammatory factor.
The inflammatory response is mediated by FABP 4; the Src inhibitor is
The application process comprises the following steps:
(1) culturing normal human monocyte-derived macrophages, overexpressing human monocyte-derived macrophages FABP4, and detecting phosphorylation levels of JAK2/STAT2 by Western-blot (protein immunoblotting);
(2) after FABP4 is over-expressed, the secretion level of human monocyte-derived macrophage immunoinflammatory factor is detected by an Elisa method (enzyme-linked immunosorbent assay);
(3) after overexpression of human monocyte-derived macrophage FABP4, detecting Src and Src phosphorylation level by using Western-blot;
(4) after overexpression of human monocyte-derived macrophage FABP4, enzymatic detection of the activity level of Src enzyme is adopted;
(5) after overexpression of human monocyte-derived macrophage FABP4, adding a PP2 inhibitor, and detecting the phosphorylation level of JAK2/STAT2 by using a Western-blot detection channel;
(6) after the human monocyte-derived macrophage FABP4 is over-expressed, a PP2 inhibitor is added, and the secretion level of the human monocyte-derived macrophage immunoinflammatory factor is detected by adopting an Elisa method;
furthermore, after FABP4 is over-expressed, the phosphorylation level of human monocyte source JAK2/STAT2 is increased by adopting an Elisa method to detect, and the secretion level of macrophage immune inflammatory factor is also increased.
Further, Src phosphorylation levels and enzyme activity were increased after overexpression of human monocyte-derived macrophage FABP 4.
Furthermore, when a PP2 inhibitor is added after the human monocyte-derived macrophage Ad-FABP4 is over-expressed, compared with an Ad-FABP4 over-expression group, the phosphorylation level of JAK2/STAT2 is reduced, and the secretion level of inflammatory factors is also reduced.
Compared with the prior art, the application of the Src tyrosine kinase inhibitor in inhibiting the secretion of inflammatory factors has the following beneficial effects:
the invention discovers the relationship between FABP4 and c-Src in macrophages and an inflammation signal pathway through experimental research, and on the basis, finds an effective application method of a Src tyrosine kinase inhibitor in inhibiting the secretion of inflammatory factors, and has application value in relieving macrophage inflammatory diseases.
Drawings
FIG. 1 is a diagram: a Control normal Control group, an Ad-GFP adenovirus Control group and an Ad-FABP4 overexpression fatty acid binding protein 4 group, and an inflammatory signal pathway JAK2/STAT2 phosphorylation level Control schematic diagram.
FIG. 2 is a Control diagram showing the Control of the content of Control normal Control group, Ad-GFP adenovirus Control group, Ad-FABP4 overexpression fatty acid binding protein 4 group, inflammatory factor IL-1 β -6, and TNF- α.
FIG. 3 is a diagram of: control normal Control group, Ad-GFP adenovirus Control group and Ad-FABP4 overexpression fatty acid binding protein 4 group, and Src phosphorylation level Control schematic diagram.
FIG. 4 is a diagram of: control normal Control group, Ad-GFP adenovirus Control group and Ad-FABP4 overexpression fatty acid binding protein 4 group, and Src kinase activity Control schematic diagram.
FIG. 5 is a diagram: compared with the Ad-FABP4 group, the JAK2/STAT2 phosphorylation level control schematic diagram is shown by adding a PP2 inhibitor after the human monocyte-derived macrophage Ad-FABP4 is over-expressed.
FIG. 6 is a control graph showing the level of inflammatory factor secretion in comparison with Ad-FABP4 group by adding PP2 inhibitor after overexpression of human monocyte-derived macrophage Ad-FABP 4.
As shown in fig. 1, ordinate: protein expression level, abscissa: grouping is respectively Control to represent a Control group, Ad-GFP to represent an empty vector group, Ad-FABP4 to represent a group of over-expressed fatty acid binding protein 4, gray to represent the ratio of non-receptor
As shown in FIG. 2, the ordinate represents the cytokine content, the abscissa represents the Control group, Ad-GFP represents the empty vector group, Ad-FABP4 represents the over-expressed fatty acid binding protein 4 group, IL-1 β represents interleukin 1 β, IL-6 represents interleukin 6, TNF- α represents the tumor necrosis factor α. the Ad-FABP4 group shows IL-1 β -6, TNF- α has increased expression level, and the difference of p less than 0.05 has statistical significance.
As shown in fig. 3, ordinate: ratio of phosphorylated tyrosine kinase to tyrosine kinase. The abscissa: the Control group, Ad-GFP group and Ad-FABP4 represent the Control group, the empty vector group and the fatty acid binding protein over-expression 4 group, respectively. Represents the statistical significance of the increased expression of phosphorylated tyrosine kinases in the Ad-FABP4 group compared to the Ad-GFP group, with p less than 0.01 difference.
As shown in fig. 4, ordinate: tyrosine kinase activity; the abscissa: the Control group, Ad-GFP group and Ad-FABP4 represent the Control group, the empty vector group and the fatty acid binding protein over-expression 4 group, respectively. Represents the increase in tyrosine kinase activity of the Ad-FABP4 group compared to the Ad-GFP group, with a statistical difference of p less than 0.01.
As shown in fig. 5, ordinate: and (4) protein expression. The abscissa: grouping is respectively Control to represent a Control group, Ad-GFP to represent an empty vector group, Ad-FABP4 to represent a group over-expressing fatty acid binding protein 4, PP2 to represent a tyrosine kinase inhibitor group, and Ad-FABP4+ PP2 to represent a group over-expressing fatty acid binding protein 4 and adding a tyrosine kinase inhibitor. Grey represents the ratio of phosphorylated
As shown in FIG. 6, the ordinate represents the cytokine content, the abscissa represents the Control group, Ad-GFP represents the empty vector group, Ad-FABP4 represents the overexpressed fatty acid binding protein 4 group, PP2 represents the tyrosine kinase inhibitor group, Ad-FABP4+ PP2 represents the overexpressed fatty acid binding protein 4 group and the tyrosine kinase inhibitor group is added, the light gray column IL-1 β is interleukin 1 β, the dark gray IL-6 is interleukin 6, the black TNF- α is tumor necrosis factor α, which represents the decrease in the expression levels of IL-1 β, IL-6 and TNF- α compared to the Ad-GFP group, the increase in the expression levels of IL-1 β, IL-6 and TNF- α in the Ad-FABP4 group, the difference of p less than 0.05 is statistically significant, and the # represents the decrease in the expression levels of IL-1 β, IL-6 and TNF- α compared to the Ad-FABP4 group, the difference of p less than 0.05 is statistically significant.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
1 Material
1.1 Primary reagents
The inflammatory factor detection kit is purchased from Chinese enzyme immunoassay, the holoprotein extraction kit is purchased from Chinese bebo, the Western-blot gel kit is purchased from American Bio-Rad, the ECL developer is purchased from Chinese Kaiky, and the kinase activity detection kit is purchased from American abcam.
1.2 Main instruments
The microplate reader was purchased from Bio-TEK, the gel imager was purchased from BIO-RAD, the cryorefrigerator was purchased from Sanyo, Japan, the precision electronic balance was purchased from Sartorius, Germany, and the low-temperature high-speed centrifuge was purchased from Heraeus, Germany; the desktop centrifuge was purchased from Eppendorf, Germany, and the constant temperature water bath was purchased from Hualida, Taicang, China; water purifier-ZYUPH-I-10 was purchased from TMillicore, USA; ice making machines are available from BILON corporation, china; the microscope was purchased from Olympus ltd, japan.
2 method
2.1 macrophage cell culture and grouping
Taking out the cryopreserved cells from the liquid nitrogen tank, quickly putting the cells into a beaker or a water bath kettle with 37-DEG C warm water for melting, centrifuging the cells for 5min at 1000r/min of a room-temperature centrifuge, opening the cryopreserved tubes in a superclean bench, completely pouring out the frozen liquid in the cell tubes, completely sucking the residual liquid, adding 10% 1640 culture medium containing serum into the tubes, gently blowing the cells from the bottom of the culture bottle by using a pipettor, slowly re-suspending the cells, and subpackaging the suspended cells to 25cm2Adding a proper amount of 10% serum-containing culture medium into the culture bottle, and culturing in a constant-temperature incubator at 37 ℃ and 5
Control group (normal Control group): administration of monocytes PMA stimulates the formation of macrophages.
Ad-GFP group (empty vector group): after administration of PMA (stimulated monocytes) forming macrophages, Ad-GFP adenovirus control group was stimulated for 6h and then cultured in 10% 1640 medium for 48 h.
Ad-FABP4 panel (over-expression fatty acid binding protein 4 panel): after administration of monocytes stimulated by PMA to form macrophages, Ad-FABP4 adenovirus was added for 6h and cultured in 10% 1640 medium for 48 h.
PP2 group: after administration of monocytes PMA to stimulate formed macrophages, PP2 was used for 2 h.
Ad-FABP4+ PP2 group: after administration of monocytes stimulated by PMA to formed macrophages, Ad-FABP4 adenovirus was administered for 6h, followed by culture in 10% 1640 medium for 48h and PP2 for 2 h.
2.2 Elisa detection of inflammatory factor levels in cell culture
Collecting the cell culture solution into a centrifuge tube, centrifuging at 12000r for 5min, and arranging standard substance holes and sample holes, wherein 50 mu l of standard substances with different concentrations are added into the standard substance holes respectively; adding 10 mul of sample to be detected into the sample hole, and then adding 40 mul of diluent; adding 100 μ l of detection antibody labeled by Horse Radish Peroxidase (HRP) into the standard sample well and the sample well except the blank well, sealing with a sealing plate membrane, and incubating in a 37 deg.C water bath or thermostat for 60 min; discarding liquid, drying on absorbent paper, filling cleaning solution into each hole, standing for 1min, throwing off the cleaning solution, drying on the absorbent paper, and washing the plate for 5 times in this way; adding 50 μ l of substrate A, B to each well, and incubating at 37 deg.C in dark for 5 min;
adding 50 mul of stop solution into each hole, and measuring the OD value of each hole at the wavelength of 450nm within 15 min; and (4) calculating a result: the resulting concentration multiplied by the dilution factor is the actual inflammatory factor concentration.
2.3 enzyme Activity assay
The rhodamine 110 peptide is used as a substrate, and a method adopted by kinase determination is shown in a kit (ProFluor Src-family kinase assay, Promega, V1270). Briefly, 30 ng of purified c-Src was reconstituted with kinase solution and incubated with rhodamine 110 peptide at 25 ℃ for one hour at 10 mmol/L ATP (adenosine triphosphate for short). Then adding protease solution, incubating for one hour at 25 deg.C, and detecting excitation wavelength of 485 nm and emission wavelength of 530 nm with enzyme labeling instrument.
2.4 Western-blot detection of protein level expression of JAK2, p-JAK2, STAT2, p-STAT2, Src, p-Src.
Preparing glue: separating gel and concentrating gel with proper size and concentration are selected according to the molecular weight. The components are prepared according to the mixture ratio. After preparation, pouring separation glue into the glass plate to the position indicated by the glass plate, adding a proper amount of distilled water to the glue surface, flattening the glue surface, and removing bubbles; after the separation gel solidified, the distilled water was poured out and sucked dry with filter paper. The concentrated gum was then added and the comb was immediately and rapidly inserted into the concentrated gum.
Loading and electrophoresis: assembling the prepared glue and the electrophoresis tank, checking whether the upper glass plate and the lower glass plate are aligned and flat, putting the glass plates into the electrophoresis tank after the assembly is finished, adding the electrophoresis liquid which just passes through the upper edge of the electrophoresis tank into the electrophoresis tank, and then sequentially and respectively adding a Marker and samples of corresponding groups into the holes (paying attention to vertical sample adding, suspending, avoiding puncturing the sample holes, avoiding bubbles or enabling the samples to flow into other holes); electrophoresis: after the sample runs through the concentrated gel at the voltage of 80V, the voltage is increased to 120V, and the whole process of separating the gel is finished. And stopping electrophoresis when the Marker runs to a green line at the bottom of the glass plate clamp, and preparing to transfer the film.
Film transfer: a. preparing a film transferring article: PVDF film, thick filter paper, absolute ethyl alcohol, milk sealing liquid and the like which are suitable for molecular size are selected.
b. Completely soaking the filter paper prepared in advance with an electrotransfer solution, shearing a PVDF membrane with a proper size, and activating the PVDF membrane in advance for 5min by using absolute ethyl alcohol.
c. And (3) transferring the film by adopting semi-dry transfer: slightly prying the glass plate, cutting out a target strip according to the prompt of a Marker, cutting the target strip by using a rubber plate, putting the glass plate into a glass vessel with electrotransformation liquid, sequentially placing an article on an electrotransformation instrument by using tweezers according to the sequence of filter paper-PVDF (polyvinylidene fluoride membrane) -glue-filter paper, taking care to remove air bubbles in the middle of each layer, inserting electrodes, and starting electrotransformation.
And (3) sealing: the PVDF membrane after membrane transfer is put into 5 percent of skimmed milk powder for sealing, and is shaken on a shaking table at room temperature for 4h (according to different incubation times at room temperature).
Incubating the primary antibody: the primary antibody was diluted with PBST to the appropriate concentration and the blocked membrane was washed three times with PBST for 10min each. And adding the diluted primary antibody into an incubation box, putting the cleaned PVDF membrane into the incubation box, and incubating on a shaking table at 4 ℃ overnight or at room temperature for about 2 hours.
Incubation of secondary antibody: washing with PBST for 5min at room temperature on a shaker for 3 times, placing the membrane in a clean incubation box, adding diluted appropriate ratio of corresponding secondary antibody of murine or rabbit origin (dilution ratio is usually 1: 5000), and incubating at room temperature for 2 h.
Exposure and gel image analysis: washing with PBST for 10min on a shaker at room temperature was repeated 3 times. And (3) under the condition of keeping out of light, mixing the developing solution according to the ratio of A: b =1:1 and mix it evenly with a pipette, place the membrane flat with the protein side facing up on the development plate, add the prepared developer evenly drop by drop, protected from light, and expose immediately. The optical density of the bands was analyzed by a gel image processing system to reflect the relative expression of the protein in terms of the ratio of the objective to the corresponding internal reference.
3. Statistical treatment
Data are averaged. + -. standard deviation: () And (4) showing. The mean comparison between two samples adopts two-sample independent t test, the mean comparison between multiple samples adopts One-way ANOVA test, and the pairwise comparison between the groups adopts Student-Newman-Keuls test, so as to obtain the final productPThe difference is more than or equal to 0.05, and the difference is significant.
4 results
4.1 Western-blot detected protein levels of JAK2, p-JAK2, STAT2, p-STAT2
The Western-blot is adopted to detect the protein level changes of JAK2, p-JAK2, STAT2 and p-STAT2, compared with a Control group, the level changes of p-JAK2/JAK2 and p-STAT2/STAT2 of an AD-GFP group are not different, compared with the Ad-GFP group, the level changes of p-JAK2/JAK2 and p-STAT2/STAT2 of an AD-FABP4 group are increased, and the difference has significance (the* P<0.05,# P<0.05) (see fig. 1).
4.2 Elisa to detect changes in inflammatory factor levels
The change of the levels of the inflammatory factors IL-1 β, IL-6 and TNF- α is detected by Elisa, compared with the Control group, the levels of the inflammatory factors IL-1 β, IL-6 and TNF- α in the Ad-GFP group are not different, compared with the Ad-GFP group, the levels of the inflammatory factors IL-1 β, IL-6 and TNF- α in AD-FABP4 are increased, and the difference is significant (the level of the inflammatory factors IL-1 8938, IL-6 and TNF- α in the Ad-GFP group is detected by Elisa* P<0.05) (see fig. 2).
4.3 Src, p-Src protein levels by Western-blot
Western blot (Western blot) is adopted to detect the expression level of Src and p-Src protein, compared with a Control group, the p-Src/Src level of an Ad-GFP group is not different, compared with the Ad-GFP group, the p-Src/Src level of an AD-FABP4 group is increased, and the difference is significant (the p-Src/Src level of the AD-FABP4 group is higher than that of the Ad-GFP group)** P<0.01) (see fig. 3).
4.4 detection of enzyme Activity Src enzyme Activity
Using enzymesActivity detection of the change of the Src enzyme activity, compared with the Control group, the Src enzyme activity of the Ad-GFP group is not different, compared with the Ad-GFP group, the Src enzyme activity of the AD-FABP4 group is increased, and the difference has significance (A)** P<0.01) (see FIG. 4)
4.5 Western-blot detection of P-JAK2/JAK2, P-STAT2/STAT2 protein levels after PP2 intervention
Expression of JAK2, p-JAK2, STAT2 and p-STAT2 was detected by Western-blot. Compared with the Control group, the Ad-GFP group has no difference in the level changes of p-JAK2/JAK2 and p-STAT2/STAT2, and compared with the Ad-GFP group, the Ad-FABP4 group has higher levels of p-JAK2/JAK2 and p-STAT2/STAT2, and the difference has significance (the* P<0.05'), decreased levels of p-JAK2/JAK2, p-STAT2/STAT2 in the AD-FABP4+ PP2 group compared to the AD-FABP4 group, with statistical significance for the differences (A) (0.05))# P<0.05) (see fig. 5).
4.6 Elisa to detect changes in inflammatory factor levels
To investigate whether Src is involved in FABP 4-induced macrophage inflammatory response, changes in inflammatory factors were detected by Elisa following addition of Src inhibitors. The results showed no difference in Ad-GFP group compared to control group; compared with the Ad-GFP group (overexpression FABP4 group) and the Ad-FABP4 group, the level of the inflammatory factors is increased, and the difference has statistical significance (P<0.01) and the levels of inflammatory factors IL-1 β, IL-6 and TNF- α in the Ad-FABP4+ PP2 group are reduced compared with the Ad-FABP4 group, and the difference is significant (P<0.05) (see fig. 6).
5 conclusion
The Src tyrosine kinase inhibitor PP2 can reduce macrophage inflammatory factor secretion mediated by FABP 4.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
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