CN114099641A

CN114099641A - Application of STAT6 gene as target in preparation of medicine for treating acute lung injury

Info

Publication number: CN114099641A
Application number: CN202111322249.0A
Authority: CN
Inventors: 陶莎莎; 李倩敏; 杨友静
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2022-03-01

Abstract

The invention discloses an application of a STAT6 gene as a target point in preparation of a medicine for treating acute lung injury, and provides an application of STAT6 as a new target point in development of a medicine for treating acute lung injury caused by lung epithelial cell iron death, wherein STAT6 inhibits lung epithelial cell iron death by negatively regulating a P53-SLC7A11 signal channel, and relieves acute lung injury; in vivo experiments prove that when STAT6 is deleted in vivo, the oxidative stress injury and iron death level of lung epithelial cells are aggravated in an acute lung injury model caused by particulate matters, bacterial lipopolysaccharide and radioactive rays; the unexposed trachea is perfused with the slow virus vector expressing STAT6, so that the iron death level of lung epithelial cells of the mouse is reduced, and the lung injury symptom is relieved. The invention provides a new target and a new idea for developing a medicament for treating acute lung injury.

Description

Application of STAT6 gene as target in preparation of medicine for treating acute lung injury

Technical Field

The invention relates to application of STAT6 gene as a target point in preparation of a medicine for treating acute lung injury, and belongs to the technical field of medicine development.

Background

Acute lung injury is one of the common critical diseases in clinic, and is characterized by lung volume reduction, lung compliance reduction and ventilation/blood flow ratio disorder as pathological features, clinically manifested by progressive hypoxemia and dyspnea, and if the symptoms can not be effectively controlled, the acute lung injury will progress into acute respiratory distress syndrome, even shock, multiple organ dysfunction or failure and the like. In addition, acute lung injury is a common pathological response of many diseases, and the pathogenesis of the novel coronavirus pneumonia is closely related to the occurrence and development of the acute lung injury. The drugs for treating acute lung injury which are clinically used at present are mainly developed aiming at the pathogenesis of acute lung injury such as uncontrolled inflammatory reaction, abnormal thrombofibrinolysis and the like, but the drugs are proved to be incapable of effectively improving the prognosis of patients with acute lung injury in phase II or phase III clinical research, namely, no drug capable of effectively treating acute lung injury exists at present.

Iron death is a novel cell death mode discovered in recent years, and is different from cell death modes such as apoptosis, necrosis and programmed cell death, wherein iron death is a cell death mode in which iron ions in cells are overloaded and depend on lipid peroxidation, and is specifically expressed as ROS metabolism, iron metabolism, amino acid and glutathione metabolism disorder in cells. In recent years, reports have shown that various harmful factors can cause intracellular iron overload and redox imbalance and induce iron death (Ferroptosis) after exposing the endothelial cells of the circulatory system, which suggests that a new damage mechanism may exist for respiratory diseases caused by the exposure of the harmful factors. Recent studies show that iron death is closely related to lung diseases including acute lung injury, chronic obstructive pulmonary disease, pulmonary fibrosis and the like, but the specific action mechanism of iron death is not clear. Wherein, the pathogenesis of the acute lung injury is complex, and no medicament for effectively treating the acute lung injury exists clinically at present. The previous research on pathogenesis of acute lung injury focuses on aspects of autoimmune imbalance, macrophage homeostasis disorder and the like, and the pathogenesis of acute lung injury caused by lung epithelial cell iron death is not clear. At present, the drugs clinically used for treating acute lung injury are concentrated on the regulation and control of the activated alveolar macrophages and inflammatory reaction chains, so that the iron death of the pulmonary epithelial cells is reduced, and the development and clinical medication of the drugs for relieving the acute lung injury are not reported.

The Signal Transducer and Activator of Transcription (STAT) family is composed of a variety of transcription factors that play complex and important roles in the regulation of physiological processes such as cell proliferation, differentiation, apoptosis, and angiogenesis. STAT6 is phosphorylated at the protein level and then transcribed into the nucleus, activating downstream cytokines to exert a series of biological effects. In recent years, STAT6 is found to be closely related to the occurrence and development of various diseases, such as STAT6 for regulating the cell homeostasis of Th1/Th2 to prevent and treat chronic obstructive diseases, asthma and the like, but no research is focused on the effect and the effect mechanism of STAT6 for regulating lung epithelial cell iron death to relieve acute lung injury so as to prevent further development of diseases at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of STAT6 gene in the development of a medicine for relieving acute lung injury by reducing lung epithelial cell iron death as a target point.

The invention aims to provide application of STAT6 gene as a target point in preparation of medicines for treating acute lung injury.

Further, the medicament for treating acute lung injury is used for treating acute lung injury caused by lung epithelial cell iron death.

Further, the acute lung injury is caused by inhalation of particulate matter, infection, or radiation injury.

Furthermore, the drug for treating acute lung injury takes STAT6 gene as a target point, and the expression of STAT6 gene is up-regulated.

Further, the medicine for treating acute lung injury inhibits lung epithelial cell iron death by regulating STAT6 gene expression.

Furthermore, the medicine for treating acute lung injury inhibits lung epithelial cell iron death by regulating and controlling STAT6 gene expression to down-regulate SLC7A11 expression.

Furthermore, the medicine for treating acute lung injury inhibits lung epithelial cell iron death by regulating and controlling STAT6 gene expression negative regulation P53-SLC7A11 signal channel.

Further, the STAT6 gene and CBP act together to inhibit the P53-SLC7A11 signal channel to inhibit iron death of lung epithelial cells.

The second purpose of the invention is to provide the application of the STAT6 gene in preparing a medicament for regulating and controlling the iron death of epithelial cells.

The invention has the beneficial effects that:

the invention discloses and provides an application of STAT6 as a new target point in the development of a medicament for treating acute lung injury caused by lung epithelial cell iron death, wherein STAT6 inhibits lung epithelial cell iron death by negatively regulating a P53-SLC7A11 signal channel, and relieves acute lung injury; in vivo experiments prove that when STAT6 is deleted in vivo, the oxidative stress injury and iron death level of lung epithelial cells are aggravated in an acute lung injury model caused by particulate matters, bacterial lipopolysaccharide and radioactive rays; non-exposed trachea perfusion of a lentivirus vector expressing STAT6 relieved lung injury symptoms of mice. The invention provides a new target and a new idea for developing a medicament for treating acute lung injury.

Description of the drawings:

FIG. 1 shows the result of IHC staining of lung tissue of each group of mice according to the present invention;

FIG. 2 shows the lung tissue Fe of each group of mice of the present invention²⁺Results of level measurement

FIG. 3 shows the H & E staining results of the lung tissues of the mice of each group according to the present invention;

FIG. 4 shows the results of measuring the levels of GSH and MDA in lung tissues of mice of each group according to the present invention;

FIG. 5 shows the expression level of STAT6 and iron death related proteins detected by Western blot on lung tissues of mice in each group;

FIG. 6 shows the results of immunohistochemical staining of lung tissue 8-oxo-dG and PTGS-2 in mice of each group according to the present invention;

FIG. 7 shows WT and STAT6 in accordance with the present invention^cKOMouse lung tissue H&E, dyeing results;

FIG. 8 shows WT and STAT6 in accordance with the present invention^cKOThe immunohistochemical staining result of the mouse lung tissue 8-oxo-dG and PTGS-2;

FIG. 9 shows WT and STAT6 in accordance with the present invention^cKOMouse lung tissue GSH, MDA, Fe²⁺Level ofMeasuring results;

FIG. 10 shows the results of the present invention on the immunofluorescence staining of STAT6, PTGS-2, overexpressed or knocked-down in HBE cells;

FIG. 11 shows that after STAT6 is overexpressed or knocked down in HBE cells, GSH, MDA, and Fe are present in the cells²⁺The results of the level determination;

FIG. 12 is a graph of the results of H & E staining of lung tissue following upregulation of STAT6 in mice in accordance with the present invention;

FIG. 13 shows the IHC staining of lung tissue following upregulation of STAT6 in mice in accordance with the present invention;

FIG. 14 shows that the lung lavage fluid protein content and GSH, MDA, Fe in lung tissue after STAT6 is up-regulated in mice according to the present invention²⁺The results of the level determination;

FIG. 15 shows the level of iron death-related protein detected by Western blot after overexpression or knock-down of STAT6 in HBE cells according to the present invention;

FIG. 16 detection of P53-SLC7A11 signal pathway-related gene levels by Q-PCR without overexpression or knock-down of STAT6 in HBE cells according to the invention;

FIG. 17 shows the results of immunofluorescence staining following overexpression of P53 or STAT6 in HBE cells in accordance with the present invention;

FIG. 18 shows the activity of HBE cells over-expressing P53 or STAT6, and GSH, MDA, and Fe in HBE cells²⁺The results of the level determination;

FIG. 19 shows the results of immunofluorescence assays performed after knocking-down CBPs in HBE cells according to the present invention;

FIG. 20 shows the SLC7A11 promoter activity of the dual-luciferase reporter gene assay performed after over-expression P53, STAT6 or CBP knockdown treatment in HBE cells.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

Healthy 6-8 week old male Stat6 Wild Type (WT) mice were purchased from the experimental animals center of the chinese academy of sciences, weighing 18-20g, SPF grade. Stat6^flox/flox、Sftpc^CreMouse buying self-competitionBiotech limited, 18-20g weight, SPF grade, Stat6^flox/floxAnd Sftpc^CreMouse mating to obtain lung epithelial cell STAT6 specific knockouts (STAT 6)^cKO) A mouse. The mice are all raised in SPF level experimental animal center in the school district of Duvilla lake of Suzhou university. The environment of the experimental animal is constant temperature and humidity, the room temperature (23 +/-2 ℃) is 50-60% of relative humidity, the illumination time is according to the circadian rhythm, and the animal can keep drinking water and ingesting food freely.

PBS solution: 10 XPBS solution is prepared, 100ml 10 XPBS solution is added into 900ml deionized water to prepare 1 XPBS solution.

3% sodium pentobarbital: it is used as it is. Accurately weighing 0.3g of pentobarbital sodium crystal by using an analytical balance, adding 10ml of deionized water, sufficiently and uniformly shaking the chloral hydrate solution by using a shaking and mixing device, and storing in dark place.

Iron death inhibitor Ferrostatin-1 (Ferr-1): purchased from TargetMol (usa) and dissolved by addition of 2ml DMSO.

1 × citrate buffer: citrate buffer (100 × concentrate) purchased from kang century, 10ml of 10 × PBS solution was added to 1000ml of deionized water to prepare 1 × citrate buffer.

Example 1:

establishing a model of acute lung injury induced by granular dust (CS), bacterial Lipopolysaccharide (LPS) and radioactive X-ray (X-ray), comprising the following steps:

granular dust group (CS group; 3 mg/mouse), which is an acute lung injury model constructed by non-exposed tracheal perfusion of CS using C57BL/6 mice.

Bacterial lipopolysaccharide group (LPS group, 1mg/kg), which is an acute lung injury model constructed by non-exposed tracheal infusion of LPS using C57BL/6 mice.

A radioactive X-ray group (X-ray, 2Gy/min, irradiation distance: 51cm) was prepared by placing a C57BL/6 mouse in an acute lung injury model constructed by subjecting the mouse to radioactive X-ray irradiation in a miniature animal radioactive irradiation apparatus.

Example 2:

study subjects: c57BL/6 mice; modeling (see example 1)

Grouping: control group (Ctrl group), which was healthy C57BL/6 mice, was intraperitoneally injected with 100. mu.l of physiological saline for 1 week after non-exposed tracheal perfusion with 50. mu.l of physiological saline.

Modeling of CS group referring to example 1, the abdominal cavity was injected with 100. mu.l of saline for 1 week after non-exposed tracheal perfusion of CS.

LPS modeling method referring to example 1, before non-exposed tracheal perfusion LPS 100. mu.l of saline was intraperitoneally injected for two days, and 100. mu.l of saline was re-intraperitoneally injected after LPS treatment.

Ferr-1 intervention CS group (CS + Ferr-1): after C57BL/6 mice were perfused with CS via non-exposed trachea, mice were given daily intraperitoneal injections of ferr-1 (1.25. mu. mol/kg) for 7 days to construct an acute lung injury intervention group model.

Ferr-1 intervention LPS group (LPS + Ferr-1): c57BL/6 mice were intraperitoneally injected with ferr-1 (1.25. mu. mol/kg) two consecutive days prior to LPS treatment, and then an acute lung injury intervention group model was constructed by non-exposed tracheal infusion of LPS and re-intraperitoneal injection of ferr-1 (1.25. mu. mol/kg).

Mice in the CS or CS + Ferr-1 group were treated and sacrificed by feeding for one week, and mice in the LPS or LPS + Ferr-1 group were treated with LPS and sacrificed on day 2. Each group had 6 mice. After the experiment is finished, lung tissues of mice are collected and stained by H & E, IHC, and the relationship between iron death and acute lung injury is determined. The method comprises the following specific steps:

(1) preparation of pathological section of lung tissue

After the lung tissue of the mouse is placed in a 4% paraformaldehyde solution for fixing for 1 week, the tissue is taken out for trimming, paraformaldehyde remained in the tissue is washed away, and gradient dehydration is carried out (85% ethanol solution is used for 2 hours, 95% ethanol solution is used for 1 hour and then is replaced by new 95% ethanol solution for 1 hour, and anhydrous ethanol solution is used for 0.5 hour and then is replaced by new anhydrous ethanol solution for 0.5 hour). After dehydration, the tissue was cleared by treatment with xylene for 30 minutes followed by a 10 minute further treatment with fresh xylene solution. The tissues were cleared and then treated with paraffin wax (paraffin was changed every hour for a total of 3 hours). The waxed tissue is then poured into a container along with melted paraffin and poured into cold water to solidify it immediately into a wax block. After the tissue is embedded, the tissue is sliced, then the tissue is attached to a glass slide, and finally the tissue is dried in an oven at 60 ℃ for 5 hours and then taken out for standby.

(2) Immunohistochemistry (IHC) staining:

paraffin sections were routinely dewaxed and hydrated (see hematoxylin-eosin stain dewaxed and hydrated above). Placing the slices on a slice frame, placing the slices into a container filled with 1 Xcitrate buffer solution, boiling the slices on an electromagnetic oven until the slices are boiled, boiling the slices for 8 minutes, closing the electromagnetic oven, and cooling the slices for 5 minutes; this procedure (antigen retrieval) was repeated 3 times. After the antigen retrieval is finished, placing the slices on ice to cool for 20 minutes; cooling and washing with water for 5 minutes; the sections were placed in a staining jar containing 1 XPBS solution and washed on a shaker for 5 minutes. Wiping off water stain on the slices, adding 3% H₂O₂Methanol was applied dropwise to the tissue and allowed to act for 15 minutes (on wet box) to eliminate endogenous peroxidase activity. After washing with water for 5 minutes, the sections were placed in 1 XPBS solution and washed on a shaker for 5 minutes. Expression of PTGS-2 was detected by adding murine PTGS-2(sc-52972), respectively, and after overnight incubation at 4 ℃ PBS was washed three times for 5 minutes each. Adding a secondary antibody provided in the reagent kit (Kangji, SP Rabbit)&Mouse HRP Kit (DAB), rabbit/Mouse Universal Streptavidin-HRP Kit (DAB)), incubated at 37 ℃ for 20 minutes in an incubator, washed three times with PBS, each for 5 minutes. Dripping DAB staining solution for 2 minutes, and washing after a staining effect is achieved; counterstain with hematoxylin for 1 min, and wash with water for 5 min. Gradient dehydration with ethanol (95%, 95%, 100%, 100% for 4 min each), xylene washing three times for 10min each, and oven drying the slices. And finally, sealing the sheet by using neutral resin, and observing the result under a microscope.

The results are shown in FIG. 1, and the IHC results show that: the dry prognosis after administration of Ferr-1 was reduced in brown granules compared to the CS group or LPS group, indicating an increase in expression of iron death marker gene PTGS-2 in the CS or LPS induced acute lung injury model, which was significantly reduced after administration of Ferr-1.

(3) Ferrous ion (Fe) in lung tissue²⁺) And (3) level determination:

grinding lung tissue of each group of mice into homogenate, centrifuging at 12000rpm and 4 deg.C for 15min, collecting supernatant 0.5ml, adding1.5ml iron color developing agent, vortex mixing, boiling water bath for 5min, running water cooling, centrifuging (2300g, 10min), collecting 1.0ml supernatant, measuring absorbance of each tube at 520nm wavelength, and calculating Fe in each group²⁺And (4) horizontal.

The results are shown in FIG. 2, in which Fe is present in the tissue after Ferr-1 dry pretreatment, compared with the CS group or LPS group²⁺The level drops.

(4) Hematoxylin-Eosin (H & E) staining

Dewaxing and hydrating white paraffin slices of mouse lung tissues: soaking the mixture in xylene solution for 10min, taking out the xylene solution and replacing with new xylene, and soaking the xylene solution again for 10 min; soaking the mixture for 4 minutes by using absolute ethyl alcohol, and soaking the mixture for 4 minutes again after replacing new absolute ethyl alcohol; then soaking the mixture for 4 minutes by using 95% ethanol, and soaking the mixture for 4 minutes again by using 80% ethanol; finally, washing the fabric for 5 minutes by running water, drying the fabric in the shade, and waiting for dyeing. Placing the dehydrated slices into a hematoxylin water solution for dyeing for 5 minutes, washing with tap water, and sucking residual water on the slices; putting the slices into hydrochloric acid ethanol for differentiation for several seconds; washing with running water for 2 hours, and then putting into distilled water for a moment; then putting the mixture into 70 percent and 90 percent ethanol for dehydration for 10 minutes respectively; and (5) putting the mixture into alcohol eosin staining solution for staining for 2-3 minutes. Soaking in 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 5min, and dehydrating in gradient. Xylene soak for 5 minutes, and finally neutral gum blocking.

The H & E staining results are shown in fig. 3, where solid arrows indicate nodule formation of inflammatory cells in lung tissue, dashed arrows indicate increased inflammatory cell infiltration and increased lung septal thickening, and the results in fig. 3 show: compared with Ctrl mice, the infiltration of inflammatory cells in lung tissues of CS group and LPS group is increased, and the lung interval is thickened; and after the intervention of Ferr-1, the pathological changes of acute lung injury caused by CS or LPS can be improved.

(5) Determination of GSH and MDA levels in lung tissue

Grinding lung tissues of mice in each group into homogenate, taking 100 mu l of tissue suspension, adding 100 mu l of reagent I, uniformly mixing by vortex, centrifuging (3500rpm,10min), taking 100 mu l of supernatant, sequentially adding reagent II 100 mu l and reagent III 25 mu l, measuring absorbance of each tube at 405 wavelengths, calculating GSH level in each group, further, centrifuging the cell suspension left after homogenization (12000rpm,4 ℃ for 15min), taking 100 mu l of supernatant, adding MDA detection working solution (TBA storage solution 50ul, TBA diluent 150ul and antioxidant 3ul) prepared in advance, uniformly mixing by vortex, boiling water bath for 15min, cooling by running water, centrifuging (3000g, 10min), taking 200ul of supernatant, measuring absorbance at 532nm wavelength, and calculating the MDA generation amount in each group.

The results of the GSH, MDA assay are shown in fig. 4, where after Ferr-1 dry pretreatment administration, GSH levels in tissues are elevated, and MDA production is reduced, compared to CS or LPS groups, indicating that CS or LPS results in increased levels of oxidative stress in lung tissues. The above results indicate that iron death is involved in CS or LPS induced acute lung injury.

Example 3:

study subjects: the grouping and construction method is the same as that in the embodiment 1, and after the model is successfully constructed, mouse lung tissues are collected for Western blot and IHC staining.

(1) Western blot: the gel was formulated according to the reagent instructions. Putting the prepared SDS-PAGE gel into an electrophoresis device, slowly pouring electrophoresis liquid, pulling out a comb in the gel, and adding a sample and a protein marker into gel pores, wherein each pore is 10 mu L. And adjusting the voltage to 75V, running the glue at constant voltage, and adjusting the voltage to 180V when a protein marker strip appears. Stopping electrophoresis when the sample runs to the bottom of the electrophoresis. And (5) turning off the power supply, taking out the gel plate, and flushing redundant glue running liquid by tap water. The PVDF membrane is activated by methanol for 15 seconds in advance, and is put into a membrane transferring solution together with filter paper and sponge required by membrane transferring. And (3) starting the gel, buckling the gel into a membrane transferring solution, and placing the gel according to the thick sponge, the thick filter paper, the gel, the PPVDF membrane, the thin filter paper and the thin sponge in sequence to prepare the membrane transferring. The voltage was adjusted to 75V and the membranes were spun for 2 hours. And taking out the transferred film, activating by methanol, rinsing by using triple distilled water, displaying protein after 5 minutes of ponceau dyeing, photographing and recording original data, and cutting a small corner at the upper right corner of the film according to the sample adding direction to show a mark. Primary antibody incubation was performed overnight at 4 ℃ and PBST washed 4 times for 5 minutes each. After incubation for 1 hour, the PBST was washed 4 times for 5 minutes each. Finally, the membrane is swept.

Western blot antibody information: primary antibody purchased from Santa cruz: STAT6(sc-374021), p-STAT6(sc-136019), PTGS-2(sc-52972), GAPDH (sc-32233); HRP secondary antibodies were purchased from immunology: plano, TX RS0001 (mouse), RS0002 (rabbit).

The result of Western blot is shown in figure 5, and the expression levels of STAT6, p-STAT6 and PTGS-2 in lung tissues of mice are increased after the treatment of CS, LPS and X-ray, which indicates that STAT6 is activated, the phosphorylation level is increased, and the level of the iron death marker protein PTGS-2 is increased.

The IHC results are shown in FIG. 6, and the results show that compared with Ctrl group, after CS, LPS and X-ray treatment, brown particles are increased in the lung tissues of the mice, which indicates that STAT6 expression is increased and PTGS-2 level is increased in the lung tissues of the mice. The above results indicate that exposure to multiple factors promotes the onset of iron death and stress up-regulates STAT6 expression.

Example 4:

study subjects: the grouping and construction method was the same as in example 1, except that the study was replaced with WT or STAT6^cKOA mouse. Mice lung tissue was harvested for H&E, IHC staining, GSH, MDA, Fe²⁺And (4) measuring the level.

H&The results of E staining are shown in FIG. 7, which shows STAT6 compared to WT group^cKOThe group mice are treated by CS, LPS or X-ray, inflammatory cell infiltration of lung tissue is increased, lung intervals are thickened, and the result shows that STAT6 is compared with the WT group^cKOThe lung injury was more severe in the group mice.

IHC staining results are shown in FIG. 8, which shows STAT6 compared to WT group^cKOThe increase in lung tissue brown particles following administration of CS, LPS or X-ray treatment to group mice indicates a lack of upregulation of 8-oxo-dG and PTGS-2 levels by STAT6 compared to the WT group.

GSH、MDA、Fe²⁺The results of the level measurements are shown in FIG. 9, STAT6 compared to WT group^cKOAfter CS, LPS or X-ray treatment is given to the mice in the group, GSH level in lung tissue is obviously reduced, MDA and Fe²⁺The levels appeared to increase to various degrees. The above results indicate that STAT6 deficiency exacerbates the degree of oxidative stress injury and promotes the onset of iron death.

Example 5:

after HBE cells over-express or knock down STAT6, indirect immunofluorescent staining, cell activity, GSH and MDA are collected、Fe²⁺The level determination comprises the following specific steps:

(1) live cell indirect immunofluorescence staining: taking out cells from the incubator, discarding the culture solution, adding 1ml of 1 XPBS to wash away the residual culture solution; after discarding PBS, 1ml of cold methanol was added for fixation, and after standing on ice for 15 minutes, the remaining liquid was washed off twice with 1ml of 1 XPBS, and then on the 2 nd washing, it was placed on a shaker for 3 minutes. The round cover glass is taken out of the culture dish, 30-100 mu l of primary anti-PTGS-2 (sc-52972) is added, the mixture is subjected to room-temperature light-shielding action for 50 minutes, washed with PBS three times, added with secondary antibodies (Kangji, SP Rabbit & Mouse HRP Kit (DAB), Rabbit/Mouse universal Streptavidin-HRP Kit (DAB)), and subjected to room-temperature light-shielding action for 1 hour, washed with PBS three times, added with 70-80 mu l of DAPI for cell nucleus staining, and the result is observed under a fluorescence microscope after standing for 15 minutes.

The indirect immunofluorescence results of fig. 10 show: compared with Ctrl group, after CS and LPS treatment, the intensity of red fluorescence signals is enhanced, after cell over-expression of STAT6, the intensity of red fluorescence signals is weakened, and STATA6 is knocked down, after CS or LPS treatment, the intensity of red fluorescence signals is enhanced compared with sh-Ctrl group, and the result shows that the STAT6 is up-regulated or knocked down, and PTGS-2 expression can be effectively inhibited or promoted.

(2) Cell activity assay

Inoculating HBE cell suspension into 96-well plate, culturing for 24 hr, adding CS suspension, adding 10ul MTT solution (5mg/ml) per well the next day, and returning the cells to CO₂And continuously culturing for 3 hours in the incubator, then removing culture solution in the pores, adding 150ul DMSO in each pore, placing on a shaking table, shaking at a low speed for 10min, measuring the light absorption value by an enzyme-labeling instrument at 575nm, and calculating the activity of each group of cells.

Cell Activity (A, E), GSH (B, F), MDA (C, G) and Fe²⁺(D, H) the results of the level measurement are shown in FIG. 11, which shows that after the cells are exposed to CS and LPS, the activity of the cells is obviously reduced, the GSH content is reduced, and MDA and Fe are contained²⁺The level is remarkably increased; after STAT6 is over-expressed, the cell activity and GSH content are higher than those of Veh group, MDA and Fe²⁺The levels were significantly lower than in the Veh group. The results show that the overexpression of STAT6 is effective in improving the oxidative stress and redox associated with the cell iron death caused by CS and LPSIs out of balance.

Example 6:

four groups are set:

the empty load group (Veh group), which was healthy C57BL/6 mice, was subjected to non-exposed tracheal perfusion of 50 μ l of blank lentiviral vector, i) 7 days later, followed by re-non-exposed tracheal perfusion of blank lentiviral vector and CS (3mg/50 μ l) or ii) 13 days later, followed by non-exposed tracheal perfusion of blank lentiviral vector and LPS (1mg/kg) to construct a model of acute lung injury.

STAT6 lentiviral vector group (STAT6 group), which was healthy C57BL/6 mice, was perfused with STAT6 lentiviral vector 50. mu.l via unexposed trachea, iii) 7 days later, again perfused with STAT6 lentiviral vector and CS (3 mg/50. mu.l) or iv) 13 days later, and then perfused with STAT6 lentiviral vector and LPS (1mg/kg) via unexposed trachea to construct STAT6 intervention acute lung injury model.

Non-exposed tracheal perfusion CS mice were treated, sacrificed by feeding for one week, and LPS mice were treated with LPS and sacrificed on day 2. Each group had 6 mice. Collecting mouse lung lavage liquid to determine protein content after experiment, collecting lung tissue, and performing H&E. IHC staining and GSH, MDA, Fe²⁺Level determination, the effect of up-regulating STAT6 on acute lung injury was explored at the animal level.

H & E staining of lung tissue was performed on each group of mice, and the results are shown in fig. 12, with solid arrows indicating nodule formation of inflammatory cells in lung tissue, dashed arrows indicating increased inflammatory cell infiltration and thickening of lung spaces, and the results in fig. 12 show: compared with Veh mice, STAT6 group has reduced inflammatory cell infiltration of lung tissues and improved lung interval thickening.

The results of IHC staining of lung tissue of each group of mice are shown in FIG. 13, and the results of IHC staining show that brown particles are reduced, DNA damage by CS or LPS is reduced, and iron death is suppressed in lung tissue of STAT6 group compared with Veh group mice.

(1) Lung lavage fluid protein assay

And (3) taking out the collected mouse lung lavage fluid, accurately sucking 50ul of sample solution into a 96-well plate, adding 0.01% Coomassie brilliant blue 200ul prepared in advance, standing for 10min, measuring absorbance of each group at 595nm by using an enzyme labeling instrument, and calculating the protein concentration in each group.

Lung lavage fluid protein (A, E), lung tissue GSH (B, F), MDA (C, G), Fe²⁺(D, H) the results of the level measurement are shown in FIG. 14, and compared with the Veh group mice, the content of protein in lung lavage fluid of STAT6 group mice was decreased, indicating that the degree of lung tissue damage was decreased, the content of GSH in lung tissue of STAT6 group mice was increased, MDA and Fe were contained in lung tissue of STAT6 group mice²⁺The generation is reduced. The results show that the in vivo up-regulation of STAT6 can effectively inhibit the occurrence of iron death and relieve acute lung injury caused by exposure of various factors.

Example 7:

study subjects: grouping and construction methods and example 5, cells were collected and subjected to Western blot assay.

The Western blot result is shown in figure 15, the over-expression of STAT6 in the cells inhibits the expression of PTGS-2, promotes the expression of SLC7A11, increases the expression of PTGS-2 after the STAT6 is knocked down, and inhibits the expression of SLC7A 11. The results show that STAT6 can remarkably up-regulate the SLC7A11 level and play a role in inhibiting the occurrence of cell iron death.

(1) The Quantitative real time polymerase chain reaction (Q-PCR/qPCR/rt-qPCR) procedure was as follows:

absorbing the culture solution, washing with PBS once, directly adding a proper amount of TRI zon into the culture dish, standing for 2 minutes, and repeatedly blowing and absorbing with a gun head to fully lyse the cells. The lysed cells were transferred to a 1.5ml EP tube, added with an appropriate amount of chloroform, shaken vigorously 50 times and then allowed to stand for 2 minutes. Centrifuging at 12000rpm at 4 deg.C for 15min, separating the sample into three layers, collecting the upper colorless aqueous layer, adding equal volume of isopropanol, and standing at room temperature for 10 min. Centrifuge at 12000rpm for 20 minutes at 4 ℃ and discard the supernatant. Washing with 75% ethanol for 3 times, each 1 time at 4 deg.C and 12000rpm, centrifuging for 5min, and discarding the supernatant. Air-drying at room temperature, adding 30-100 microliter of RNase-free water, and fully dissolving RNA. After being placed in a water bath tank at 57 ℃ for 8 minutes, the mixture was allowed to stand on ice for 10 minutes.

RT reactions were prepared on ice using PrimeScript RT Master Mix Perfect Real Time kit, as indicated. Setting reverse transcription conditions: 15 minutes at 37 ℃; 5 seconds at 85 ℃; finally, the mixture was placed at 4 ℃. And adding the obtained RT reaction liquid into a Real Time reaction system in the next step. The Real time PCR reaction was performed according to SYBR Premix Ex TaqTM Specification II (Perfect Real time) (Takara code: DRR 081). A PCR reaction solution was prepared as described using a Thermal Cycler Dice Real Time System (Takara Code: TP800) amplification apparatus, and a Real Time PCR reaction was carried out.

Primer information is shown in table 1:

the QPCR result is shown in figure 16, after the STAT6 gene is overexpressed, the P53 gene is not influenced, the downstream gene P21 is inhibited, the level of the SLC7A11 gene related to iron death is increased, and after the STAT6 gene is knocked down, the level of P21 is increased, and the expression of the SLC7A11 is inhibited. The above results indicate that STAT6 plays a role in inhibiting iron death by regulating the P53-SLC7A11 signal pathway.

Example 8:

after HBE cells over-express P53 or STAT6, cells are collected for indirect immunofluorescence staining and cell activity, GSH, MDA, Fe²⁺And (4) measuring the level.

The results are shown in FIGS. 17-18, which show that after the P53 is over-expressed in the cells, the red fluorescence intensity is obviously enhanced, the cell activity is reduced, the GSH level is obviously reduced, and MDA and Fe are added²⁺The level is increased, after P53 and STAT6 are over-expressed, the red fluorescence intensity is weakened, the GSH content is higher than that of the over-expressed P53 group, MDA and Fe²⁺Resulting in a less than over-expressed P53 group. The above results indicate that up-regulation of STAT6 significantly inhibits the occurrence of iron death in cells caused by overexpression of P53.

Example 9:

after HBE cells are knocked down for CBP, P53 and STAT6 or over-expressed for P53 and STAT6, the cells are collected for indirect immunofluorescence staining and dual-luciferase reporter gene tests.

The results of indirect immunofluorescence staining are shown in FIG. 19, which shows that CBP is knocked down and red fluorescence intensity is not enhanced after CS treatment compared to sh-Ctrl group.

(1) Dual luciferase reporter: inserting Ctrl (Non-target) or specific gene SLC7A11 into a reporter gene vector to construct a reporter gene plasmid, transfecting cells with the plasmid, specifically, respectively mixing the corresponding plasmid combinations with the corresponding transfection reagents, adding 20 μ l of serum-free culture medium, incubating at normal temperature for 20min, adding corresponding 12-well plate, transfecting for 6 hr, replacing fresh culture medium, co-transfecting for 36-48 hr, discarding culture medium, washing cells with 100 μ l PBS, adding 50 μ l1xPLB into each well, placing in shaking table for 20-30min to ensure that lysis buffer solution completely lyses cells, finally, adding 10 μ l of the supernatant into each hole of a white opaque 96-hole enzyme label plate, adding 100 μ l of Luciferase Assay Reagent I, detecting the Luciferase reaction intensity by using an enzyme label instrument, reading the first numerical value, after adding 100. mu.l of Luciferase Assay Reagent II to each well, the reaction intensity of internal reference Renilla Luciferase was measured.

The results of the dual-luciferase reporter gene test are shown in fig. 20, and the results in fig. 20A show that after the P53 is knocked down, SLC7a11 transcription is activated, STAT6 is deficient, SLC7a11 transcription is inhibited, and simultaneously after CBP and P53 or CBP and STAT6 are knocked down, the SLC7a11 transcription level is unchanged; the results in FIG. 20B show that SLC7A11 promoter activity was inhibited after overexpression of P53, SLC7A11 transcription was activated after overexpression of STAT6, and that P53 inhibition of SLC7A11 promoter transcription disappeared after simultaneous knockdown of CBP and overexpression of P53. The above results indicate that STAT6 inhibits the onset of iron death by CBP inhibiting the P53-SLC7a11 signaling pathway.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. An application of STAT6 gene as a target point in preparing a medicament for treating acute lung injury.

2. The use according to claim 1, wherein the medicament for the treatment of acute lung injury is the treatment of acute lung injury caused by iron death of lung epithelial cells.

3. The use of claim 2, wherein the acute lung injury is caused by inhalation of particulate matter, infection, or radiation injury.

4. The use according to claim 2, wherein the agent for the treatment of acute lung injury targets STAT6 gene and upregulates the expression of STAT6 gene.

5. The use according to claim 2, wherein the medicament for the treatment of acute lung injury inhibits lung epithelial iron death by modulating STAT6 gene expression.

6. The use of claim 2, wherein the medicament for the treatment of acute lung injury inhibits lung epithelial iron death by down-regulating SLC7a11 expression via modulation of STAT6 gene expression.

7. The use according to claim 2, wherein the medicament for the treatment of acute lung injury inhibits iron death in lung epithelial cells by negatively regulating the P53-SLC7a11 signaling pathway by regulating STAT6 gene expression.

8. The use according to claim 2, wherein the STAT6 gene, in combination with CBP, inhibits the P53-SLC7A11 signaling pathway which inhibits iron death in lung epithelial cells.

9. An application of STAT6 gene in preparing medicine for regulating and controlling the iron death of epithelial cells.