CN111214660B - Application of PAX4 gene expression inhibitor in preparation of medicine for inhibiting fibrosis - Google Patents

Abstract

The invention discloses application of a PAX4 gene expression inhibitor in preparation of a medicament for inhibiting fibrosis. Experiments prove that the inhibition effect of the transcription factor PAX4 on downstream genes can be blocked by interfering the expression of the transcription factor PAX4 through small interfering RNA, so that a plurality of downstream fibrosis inhibition factors can play a role; the reduced expression of PAX4 by the small interfering RNA also reduces the expression level of the fibrosis promoting factor and inhibits the fibrosis promoting effect, thereby inhibiting the occurrence of cardiac fibrosis. Thus, PAX4 is a potentially novel important target in the heart for the treatment of cardiac fibrosis to prevent heart failure.

Description

Application of PAX4 gene expression inhibitor in preparation of medicine for inhibiting fibrosis

Technical Field

The invention relates to the field of genes, in particular to application of a PAX4 gene expression inhibitor in preparation of a medicament for inhibiting fibrosis.

Background

Cardiac fibrosis is an important component of most pathological conditions of the heart. Cardiac fibrosis is manifested by excessive deposition of extracellular matrix in cardiac tissue, resulting in destruction of physiological cardiac tissue structures, ultimately leading to heart failure, a serious threat to human health and life. The transdifferentiation of fibroblasts into myofibroblasts is critical for the initiation and maintenance of the cardiac fibrosis response. Myofibroblasts have important contraction and secretion functions and are characterized by expressing alpha-smooth actin (α SMA), fibronectin (fibronectin) and Collagen type I (Collagen I, Col I). Angiotensin II is a polypeptide preparation recognized in present research to regulate vasoconstriction, affect cardiac function, and induce cardiac fibrosis, and is commonly used to treat mice or fibroblasts to mimic the state of cardiac fibrosis. Although researchers in various countries have conducted extensive studies around the mechanism of cardiac fibrosis to date, the specific molecular mechanism is still not well understood. Cardiac fibrosis remains today an important target for clinical treatment of cardiac diseases. Because the treatment of cardiac fibrosis can delay the occurrence and development of heart failure, it is an important problem to be solved to fill the gap in the research mechanism of cardiac fibrosis.

The transcription factor PAX4 is a member of the IV subfamily of the Pairedbox (PAX) family. In the human genome, the PAX4 gene is located in the 3-region 2 band 1 sub-band of the long arm of chromosome 7 and consists of 12 exons and 11 introns. PAX4 is also known as KPD, MODY 9. The PAX4 protein is located in nucleus, and can form transcription factor complex to combine with 5' end specific sequence of target gene to regulate expression of downstream target gene. Current research suggests that members of the PAX family perform important functions in many stages of embryonic development and organogenesis, and also in all aspects of the body after adulthood. The evolution of the sequence of members of the PAX family, from insects, amphibians, birds, mammals, is well conserved. The full-length PAX4 contains 349 amino acids, and its protein structure includes a 128-amino acid bioparate Paired Domain (PD) and a Homology Domain (HD), and its C-terminal not only has a common transcription activation domain of PAX family, but also has a unique negative regulation domain. Current research suggests that members of the PAX family are important regulators of tissue development and cell differentiation. The study of PAX4 was mainly focused on pancreatic islets, cancer and retina-related studies. The current research shows that PAX4 is involved in the differentiation of islet beta cells and delta cells during embryonic development and in the secretion of insulin under normal conditions. The absence of PAX4 induces type one and type two diabetes. Among tumor-related researchers, PAX4 is a potent tumor suppressor of human insulinomas and melanomas. PAX4 can promote migration and invasion of human epithelial cancer. High levels of PAX4 expression in rat retinal photoreceptors have been reported, suggesting that PAX4 may play a role in the retina. However, there is still a gap in the functional studies of PAX4 in the heart.

Disclosure of Invention

The purpose of the present invention is to provide a novel drug for inhibiting cell fibrosis. In order to realize the purpose of the invention, the following technical scheme is adopted: .

One of the purposes of the invention is to provide the application of the PAX4 gene expression inhibitor in the preparation of the drugs for inhibiting fibrosis. The inventor of the application discovers that the expression level of PAX4 is highly expressed in pathological models such as mice, mouse fibroblast fibrosis models and myocardial infarction models by using biochemical, molecular biological and cytological research means. The subsequent technical means of using the small interfering RNA respectively transfects fibroblasts through the small interfering RNA or injects the small interfering RNA into the rat tail vein, and the level of the PAX4 protein in the cells or the heart tissue is reduced through the knock-down of the small interfering RNA, so that the effect of the PAX4 on the heart function of the mouse is determined. Bioinformatics analysis was then used to predict target genes involved in cardiac fibrosis that could be bound and regulated by PAX 4. Thereafter, the regulation and control effect of the PAX4 on downstream genes and the action mechanism are clarified by a method of knocking down the PAX4 by small interfering RNA. This finding was the first finding by the inventors of the present application and was unexpected.

In a preferred embodiment of the present invention, the PAX4 gene expression inhibitor comprises siRNA, PAX4 gene knock-out agent.

In a preferred embodiment of the present invention, the PAX4 gene knock-out agent is siRNA, i.e., small interfering RNA.

In a preferred embodiment of the present invention, the fibrosis is cardiac fibrosis, pancreatic fibrosis or pulmonary fibrosis.

Although the present invention finds preferred application in cellular fibrosis of the human or animal body, the present invention also relates to the use of the PAX4 gene or its expression product in promoting cardiac fibroblast proliferation in vitro.

Although the present invention finds preferred application in cellular fibrosis in humans or animals, the present invention also relates to the use of a PAX4 gene knock-out agent for inhibiting cardiac fibroblast proliferation in vitro.

Although the findings of the present invention are preferably applied to cellular fibrosis of a human or animal body, the present invention also relates to the use of a PAX4 gene knock-out agent for inhibiting or blocking the expression of the fibrosis promoting factor TGF β and promoting the expression of the fibrosis inhibiting factors IL1R2 and CXCL10 in cardiac fibroblasts in vitro.

Although the discovery of the present invention is preferably applied to cellular fibrosis of a human or animal body, the present invention also relates to the use of a PAX4 gene knock-out agent for promoting cellular fibrosis by inhibiting the fibrosis promoting factor TGF β and promoting the fibrosis inhibiting factors IL1R2 and TGIF 2. The experimental results of the application show that after the cardiac fibroblasts are knocked down to PAX4, the level of TGF beta protein is reduced, and the levels of IL1R2 and TGIF2 protein are increased, which indicates that PAX4 plays a role in multi-dimensionally promoting fibrosis by increasing the fibrosis promoting factor TGF beta, and inhibiting the fibrosis inhibiting factors IL1R2 and TGIF 2.

The invention provides the novel application of the PAX4 as a novel important target point for treating the cardiac fibrosis for the first time. Particularly, the invention utilizes biochemical, molecular biological and cytological research means to discover that the expression level of PAX4 is highly expressed in mouse and pathological models such as mouse fibroblast fibrosis model, myocardial infarction model and the like. The subsequent technical means of using the small interfering RNA respectively transfects fibroblasts through the small interfering RNA or injects the small interfering RNA into the rat tail vein, and the level of the PAX4 protein in the cells or the heart tissue is reduced through the knock-down of the small interfering RNA, so that the effect of the PAX4 on the heart function of the mouse is determined. Bioinformatics analysis was then used to predict target genes involved in cardiac fibrosis that could be bound and regulated by PAX 4. Thereafter, the regulation and control effect of the PAX4 on downstream genes and the action mechanism are clarified by a method of knocking down the PAX4 by small interfering RNA. Thus, PAX4 is a potential new important target in the heart for treating cardiac fibrosis and preventing heart failure.

Drawings

FIG. 1: the immunohistochemical experimental analysis shows that the expression level of the PAX4 is increased under the pathological stimulation environment of fibroblasts. FIG. 1A: immunohistochemistry used PAX4 antibody to label PAX4 protein levels and their localization. FIG. 1B: immunohistochemistry the results of IOD analysis quantified using PAX4 antibody labeled PAX 4.

FIG. 2: western blotting confirmed that the transcription factor PAX4 was expressed at elevated levels in a mouse fibrosis model. FIG. 2A: western blot using PAX4 antibody to compare PAX4 protein levels in cardiac tissues, GAPDH as an internal control, in the cardiac fibrosis model and its control. FIG. 2B: and (3) quantitative and statistical analysis results of the protein content detected by the PAX4 western blotting method.

FIG. 3: western blotting detected protein levels of the cardiac fibrosis markers fibronectin, α SMA and Col I in the mouse fibrosis model. FIG. 3A: western blotting was performed using fibrinectin, α SMA and Col I antibodies to compare the levels of fibrinectin, α SMA and Col I proteins in the cardiac tissue in the cardiac fibrosis model and its control group, with GAPDH as the internal control. FIG. 3B: and (3) quantifying and statistically analyzing the protein content detected by the western blotting method of fibrinectin, alpha SMA and Col I.

FIG. 4: protein imprinting measures the levels of PAX4 protein in heart tissue at different time periods in the infarct, border and distant regions of the mouse myocardial infarct model.

FIG. 5: immunofluorescence experiment detects the change of fluorescence intensity of transcription factor PAX4 and myofibroblast markers namely fibrinectin, alpha SMA and Col I after 1 mu M angiotensin II stimulation is given to cardiac fibroblasts for three days. FIG. 5A: the fluorescence intensities of the transcription factor PAX4 and myofibroblast markers namely fibrinectin, alpha SMA and Col I are respectively calculated, the circled fluorescence is cell nucleus, and the rest fluorescence is target genes for detection. FIG. 5B: relative quantification of immunofluorescence and statistical analysis results.

FIG. 6: the western blotting method verifies that the expression level of the transcription factor PAX4 is increased under the stimulation of angiotensin II of fibroblasts. FIG. 6A: western blot experiments tested the protein levels of PAX4 following angiotensin stimulation. FIG. 6B: and (3) quantitative and statistical analysis results of the content of the PAX4 protein detected by the Western blotting method.

FIG. 7: the western blot method verifies that the protein levels of the myofibroblast markers fibrinectin, alpha SMA and Col I are increased under the stimulation of angiotensin II of fibroblasts. FIG. 7A: western blot assay was used to detect the protein levels of fibrinectin, α SMA, Col I after angiotensin stimulation. FIG. 7B: and (3) quantifying and statistically analyzing the content of the fibrinectin, the alpha SMA and the Col I proteins detected by the Western blotting method.

FIG. 8: the Western blotting method detects the protein level of PAX4 in heart tissue after rat tail vein injection small interfering RNA transfection and knock-down of mouse PAX4, and verifies the knock-down efficiency of experiment small interfering RNA knock-down PAX 4. FIG. 8A: western blot experiments detected the protein levels of PAX4 after intravenous injection of small interfering RNA from rat tail. FIG. 8B: and (3) quantitative and statistical analysis results of the content of the PAX4 protein detected by the Western blotting method.

FIG. 9: a fibrosis model is constructed by injecting AngII into a mouse buried micro-osmotic pump after PAX4 small interfering RNA is injected into a rat tail vein, and the result of sirius red staining shows that the knockdown PAX4 inhibits the generation of fibrosis. FIG. 9A: results of sirius red stained tissue sections. FIG. 9B: quantitative and statistical analysis of the area of cardiac fibrosis.

FIG. 10: after PAX4 small interfering RNA is injected into a rat tail vein, AngII is injected into a mouse embedded micro-osmotic pump to construct a fibrosis model, and the fluorescence intensity of Col I is detected by an immunofluorescence experiment. The upper panel shows the fluorescence intensity of Col I, and the lower panel shows the co-staining of Col I with the nuclear dye Hoechst.

FIG. 11: after PAX4 small interfering RNA is injected into a rat tail vein, AngII is injected into the rat by a buried micro osmotic pump to construct a fibrosis model, and ultrasonic cardiac detection shows that the knock-down PAX4 has a protective effect on the cardiac function of the mouse. FIG. 11A: left ventricular posterior wall thickness, fig. 11B: EF value, fig. 11C: FS value, FIG. 11D: E/E'.

FIG. 12: the protein level of PAX4 in the transfected cardiac fibroblasts of the small interfering RNA is detected by a western blotting method, and the knocking-down efficiency of the knocked-down PAX4 is verified.

FIG. 13: the immunofluorescence staining method detects the protein expression levels of myofibroblast markers fibronectin (figure 13A), alpha SMA (figure 13B) and Col I (figure 13C) after the transfection of the small interfering RNA to knock down the PAX4 in the fibroblasts, and verifies the inhibition effect of the knock-down PAX4 on fibrosis.

FIG. 14: protein expression levels of myofibroblast markers namely fibrinectin, alpha SMA and Col I after adenovirus infects fibroblasts and overexpresses PAX4 are detected by a western blot experiment, and the promotion effect of PAX4 on fibrosis is verified.

FIG. 15: western blot experiments examined the effect of expression levels of TGF beta, IL1R2 and TGIF2 genes, which may be downstream genes, after transfection of small interfering RNAs to knock down PAX4 in fibroblasts.

Detailed Description

The invention is further described in the following detailed description in conjunction with specific examples, which are intended to be illustrative rather than limiting, and that the methods and reagents used in the invention, as well as related reagents, can be varied and substituted to achieve the same technical results.

The experimental procedures, in which specific conditions are not specified, in the following examples were carried out according to the routine procedures in the art or according to the conditions suggested by the manufacturers.

Example 1, animal pathology model experiment, mouse heart fibrosis model construction, heart tissue sampling, immunohistochemistry and western blot experiment methods, detection of the position and content of PAX4 protein expression, as well as the content of myofibroblast markers fibrinectin and α SMA, extracellular matrix Col I.

Preparation of angiotensin II-induced cardiac fibrosis model in mice: male C57BL/6 mice at 10 weeks of age were randomly divided into two groups, a surgery group and a sham surgery group, and the mice were modeled for fibrosis using angiotensin (3 mg. kg-1. day-1) micro-osmotic pump embedded (Alzet MODEL 1007D, DURECT, Cupertino, CA) for 7 days. Preparation of micro osmotic pressure pump: 1 day before surgery, angiotensin II (dissolved in sterile PBS buffer) was injected into the micro osmotic pump with a 1mL syringe, the micro osmotic pump was soaked in sterile PBS buffer and equilibrated at 37 degrees celsius overnight. During operation, 2-3% isoflurane is used to narcotize mouse, a transverse incision with a length of about 0.7cm is cut at the back neck of the mouse, the mouse is inserted into the subcutaneous part by forceps, subcutaneous tissues are separated bluntly, a micro-osmotic pump is buried, the wound is sutured, and neomycin ointment is coated to prevent infection. The operative group was continuously infused with angiotensin II at a concentration of 3mg/kg/d for 7 days.

Experimental methods for PAX4 immunohistochemistry: sample preparation: and fixing the paraffin by paraformaldehyde, and slicing the paraffin embedded section. (1) Dewaxing: treating the tissue slices with xylene for 15min 3 times, 100% ethanol for 5min 2 times, 95% ethanol for 5min 2 times, and 80% ethanol for 5min 1 time. And finally washing with distilled water for 2 min. (2) Removal of endogenous catalase: the sections were placed in 3% hydrogen peroxide solution (in 100% methanol) for 10-15min to remove endogenous catalase. Followed by 3 washes with PBS for 5min each. (3) Antigen heat repair: the sections were placed in citrate (PH 6.0) antigen retrieval solution, heat retrieved in autoclave, and epitope was fully developed (2 minutes timed after continuous gassing in autoclave). And (4) after the thermal repairing is finished, placing the wafer at normal temperature, and washing the wafer for 5 minutes by PBS for 3 times when the temperature of the wafer is reduced to the normal temperature. (4) Serum blocking: the sections were placed in a wet box and sealed with 10% goat serum at room temperature for 30 min. (5) Primary antibody incubation: after discarding serum, the prepared primary antibody was added to the sections and incubated overnight in a refrigerator at 4 degrees Celsius (or incubate for 2 hours at 37 degrees Celsius). (6) And (3) secondary antibody incubation: the wet box was taken out of the refrigerator at 4 ℃ and cooled to room temperature, and then washed 3 times with PBS for 5min each time. A horseradish peroxidase-labeled secondary antibody (China fir gold bridge, rabbit two-step method) was then added and incubated at room temperature for 30 min. (7) DAB color development: washed with PBS for 3 times, and developed with DAB developing solution. The DAB working solution preparation method comprises the following steps: 1mLDAB diluent and 50uLDAB developing solution. (8) Counterstaining the nucleus: soaking in distilled water for 2min, adding hematoxylin solution for 30s, and washing with tap water for 3 times. The cells were differentiated in 70% HCl for 5-6s and washed with water for 30 s. The blue color was returned to 60s in 1% ammonia water, and the color floated by washing with distilled water. (whether the nucleus has been blue-stained can be observed under a mirror). (9) Dehydrating, transparent, and sealing with neutral resin sealing agent. Twice with 95% ethanol for 2min, twice with anhydrous ethanol for 2min, and twice with 5min in xylene substitute. (10) Scanning after the slices are dried, and performing positive area statistics by using software.

Extraction of total protein of myocardial tissue: taking myocardial tissue stored in liquid nitrogen, placing into mortarTwo thirds of the mixture (the other third was used for RNA extraction) was ground with liquid nitrogen and added to the tissue lysate (20mmol/L Tris-HCl pH7.4,150mmol/L NaCl,2.5mmol/L EDTA,50mmol/L NaF,0.1mmol/L Na₄P₂O₇，1mmol/L Na₃VO₄1% Triton X-100, 10% Glycerol, 0.1% SDS, 1% deoxycholic acid,1mmol/L PMSF, 1. mu.g/mL aprotinin.) were mixed and allowed to stand on ice for 15 minutes, adding approximately 800 microliters of lysis buffer per 50 milligrams of myocardial tissue. Collecting homogenate, centrifuging at 12000rpm at 4 ℃ for 15 minutes after ultrasonication (45%, 5s on,5s off for 4 cycles), transferring part of the supernatant into a new EP tube, freezing and storing at-80 ℃ after protein quantification, adding one quarter of 5Xloading buffer, mixing uniformly, boiling at 100 ℃ for 5 minutes, freezing and storing, and reserving for subsequent detection of related proteins by western blot.

Western blot experiment: using 10% SDS-PAGE gel to perform electrophoresis, then transferring the nitrocellulose membrane, sealing 5% skim milk at room temperature for 1 hour, and performing cold room overnight incubation at 4 ℃ for the primary antibody, wherein the cargo numbers of the primary antibody are respectively as follows: fibrinectin (ab2413, abcam, Cambridge, MA, USA), α SMA (ab32575, abcam, Cambridge, MA, USA), Col I (203002, MD Biosciences), PAX4(ab101721, abcam, Cambridge, MA, USA), TGF β (10804-MM33, sinobiological, Beijing, China), IL1R2(sc-376247, Santa Cruz Biotech, CA, USA), TGIF2(ab190152, abcam, Cambridge, MA, USA), GAPDH (2118S, CST), TBST washing the membrane three times before exposure to the corresponding species secondary antibody, 1 hour at room temperature, TBST washing the membrane before development, placing the membrane in a developer (then controlled dry luminescence detection). The intensity of the bands was quantified using NIH ImageJ software.

A mode of embedding a pump by using an angiotensin II micro-osmotic pump is adopted, a 10-week male C57BL/6 mouse is selected to construct a mouse heart fibrosis model, the expression level of PAX4 in heart tissues in the heart fibrosis model is detected, and the level of a fibrosis marker is detected to correspond to the fibrosis degree in the heart tissues.

Firstly, cardiac fibrosis model tissues constructed by an angiotensin II embedded micro-osmotic pump and cardiac tissues of a sham operation group are taken and used for carrying out an immunohistochemical experiment by using a PAX4 antibody, the experimental result is shown in figure 1A, the protein level of PAX4 is increased in the fibrotic tissues of the heart, whether in myocardial cells or cardiac fibroblasts, the statistical result is shown in figure 1B, and the statistical analysis of the quantitative result shows that the protein level of PAX4 in the fibrotic tissues is obviously increased compared with the protein level of PAX4 in healthy cardiac tissues.

Thereafter, western blotting was performed using heart fibrosis model tissue and total protein of heart tissue of sham operated group, and the results of the experiment for detecting the protein level of PAX4 are shown in fig. 2, and the experiment resulted in the same results as the immunohistochemical experiment, in which the protein level of the transcription factor PAX4 was significantly increased in the fibrotic heart tissue (fig. 2A). Quantitative results statistical analysis (fig. 2B) showed that PAX4 protein levels were significantly elevated in fibrotic cardiac tissue compared to healthy cardiac tissue.

Meanwhile, protein levels of fibrosis markers namely fibrinectin, alpha SMA and Col I in heart tissues are detected by using a Western blotting experiment. The experimental results are shown in fig. 3A, and the experiments show that the protein levels of the fibrosis markers fibrinectin, alpha SMA and Col I in the fibrotic heart tissue are obviously increased compared with the content in normal heart tissue. Quantitative results statistical analysis (fig. 3B) showed that the protein levels of fibrinectin, α SMA and Col I were significantly elevated in fibrotic cardiac tissue. However, whether the increase of PAX4 is related to the increase of fibrosis markers fibrinectin, alpha SMA and Col I or not is still verified by subsequent experiment results.

Example 2 levels of PAX4 protein were measured in the infarct zone and distal compartment at different time points of heart tissue surgery in the mouse myocardial infarction model.

10 week male C57BL/6 mice were used to perform the myocardial infarction model building surgery. Mice were randomly grouped into myocardial infarction groups and sham operated control groups. The myocardial infarction group induced the development of myocardial infarction using left coronary stenosis. Sham surgery was performed on the control group. The procedure was performed under gas anesthesia, and mice were gas anesthetized by inhalation of 2% isoflurane. The experimental group collected heart tissue from the infarct area, border area and distant area of mouse heart tissue at 1 day, 4 days and 7 days after the operation.

Protein levels of transcription factor PAX4 endogenous to cardiac tissues in infarct, border and distant regions were detected by Western blotting at 1 day, 4 days, 7 days after the operation of mouse myocardial infarct model. The specific locations of the infarcted, border, distant regions of the heart infarct are shown on the left side of figure 4. The experimental results showed that the levels of PAX4 protein were increased to different extents in each region of myocardial infarction surgery compared to the sham surgery group. The results of this myocardial infarction model in combination with the results of the fibrotic conditions of example 1 suggest that the level of PAX4 protein in the heart tissue increases when the heart undergoes pathological changes, and the specific function of this increase is verified in the experiments shown later below.

Example 3 protein levels of PAX4, fibrosis markers fibrinectin, α SMA and Col I were measured using immunofluorescence and western blot experiments three days after treatment of cells with 1 μ M angiotensin using cardiac fibroblasts.

Isolation and culture of adult mouse cardiac fibroblasts: male C57/BL6 mice, about 8 weeks old, were sacrificed by cervical dislocation, rapidly soaked in 75% alcohol for about half a minute, immediately placed in an ultraclean bench to open the chest and remove the heart, placed in 4 ℃ PBS buffer solution to wash twice, the blood vessels in the atrium and the fundus of the heart were cut off, and then the ventricle was cut into small pieces and washed once with PBS to wash off part of the blood residues. Digestion was performed by adding 0.1% collagenase type II (330U, Worthington, Columbia, NJ, USA/Sigma, St. Louis, MO, USA) in PBS balanced salt solution. The whole digestion process is carried out under the condition of constant-temperature stirring at 36-37 ℃, supernatant digestive juice is taken after digestion is carried out for 8 minutes, and the supernatant digestive juice is added into the DMEM culture solution containing 10% FBS in equal amount and is uniformly mixed. Repeating the process for about 7-8 times until the tissue mass is completely digested, centrifuging the collected tubes of cells at room temperature and 1000rpm for 5 minutes, discarding the supernatant, resuspending the cells in DMEM (DMEM) culture solution containing 10% FBS (fetal bovine serum), combining the cardiomyocyte suspensions obtained each time, inoculating the combined cells into a culture dish with the diameter of 100mm, and culturing at 37 ℃ and 5% CO₂The culture box is placed for 2 hours to ensure that the fibroblasts are basically attached to the wall. The culture solution in the culture dish is removed by suction, and a new DMEM culture solution containing 10% FBS is added for continuous culture. After 3 days the cells were confluent, passaged and subjected to subsequent experiments.

The extraction method of the cardiac fibroblast protein comprises the following steps: cells were first digested with pancreatin from the background gel, centrifuged and the supernatant was washed three times with cold PBS, followed by lysis with cell lysate (20mmol/LTris-HCl PH7.4,150mmol/L NaCl,2.5mmol/L EDTA,50mmol/L NaF,0.1mmol/L Na4P2O7,1mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxyglycolic acid,1mmol/L PMSF, and 1mg/ml aprotinin), sonicated and centrifuged at 12000g for 15min at 4 ℃. And (6) collecting the supernatant. After taking 5 microliters for protein quantification, the remaining supernatant was added to 5 Xgel loading buffer at 100 ℃ for 5 minutes to ensure protein denaturation.

Immunofluorescence staining experiment: cells were fixed with 37 ℃ warm 4% paraformaldehyde for 15 minutes at 37 ℃, washed 3 times with warm PBS, and then disrupted with 0.2% Triton X-100 for 20-30 minutes. After 3 washes with warm PBS, blocking was added (5% BSA) for 30 min. Thereafter, primary anti- α SMA (ab32575, abcam, Cambridge, MA, USA), fibrinectin (ab2413, abcam, Cambridge, MA, USA), PAX4(ab101721, abcam, Cambridge, MA, USA) were used for overnight incubation at 4 degrees celsius. After recovery of the primary antibody, PBS washes were 3 times followed by incubation of the secondary antibody Alexa Fluor4881 hours at room temperature. Nuclei were stained with Hoechst (Invitrogen, Carlsbad, Calif., USA) for 8 minutes at room temperature. Fluorescence intensity was counted and analyzed using the morpholinology Explorer BioApplication module of the high content screening imaging system Cellomics array Scan VTI HCS Reader (Thermo Fisher Scientific, Rockford, IL, USA).

At cellular level, we extracted mouse primary cardiac fibroblasts, cultured to P2 passages in 12-well plates, and harvested three days after stimulating the cells with angiotensin II at a concentration of 1 μ M. The samples were first fixed and their endogenous PAX4 and fibrosis markers fibrinectin, α SMA and Col I protein levels were detected using immunofluorescence. The fluorescence circled in FIG. 5A is the nuclear location, and the remaining fluorescence indicates the location and amount, respectively, of the specific protein of interest (PAX4, fibronectin, Col I, and. alpha. SMA) recognized by the specific antibody. The experimental result can see that the transcription factor PAX4 is mainly expressed in the nucleus. The experimental results suggest that the fluorescence intensities of PAX4, fibrinectin, alpha SMA and Col I are all enhanced to different degrees under the stimulation of angiotensin II. The quantitative results in fig. 5B show a significant increase in the levels of transcription factor PAX4 and the common markers fibronectin, α SMA and Col I when cardiac fibrosis occurs three days after angiotensin II stimulation.

Thereafter, Western blot experiments were performed using total protein of cardiac fibroblasts cultured under the same treatment conditions, and the protein level of PAX4 was first detected. The experimental results are shown in fig. 6, the experiment obtained results similar to those of the immunofluorescence experiment, the protein level of the transcription factor PAX4 is increased after angiotensin II stimulation (fig. 6A), and the quantitative result statistical analysis shows that the protein level increase degree is significant. The protein levels of the myofibroblast markers namely fibrinectin, alpha SMA and Col I are also detected (figure 7), the experimental result shows that the angiotensin II stimulates the cardiac fibroblasts for three days to promote the protein levels of the fibrosis markers namely fibrinectin, alpha SMA and Col I (figure 7A), and the statistical analysis of the quantitative result shows that the protein level of the fibrosis marker is obviously increased compared with that of a control group after the angiotensin II is stimulated.

Example 4 knock down of PAX4 levels in mice by means of intravenous small interfering RNA from rat tail, followed by the construction of a fibrosis model, the effect of PAX4 on cardiac function and on the protein levels of extracellular matrix Col I, myofibroblast markers fibrinectin and alpha SMA, was examined by methods of echocardiography, comparison of cardiac-to-body ratios, sirius red (PSR) staining, western blot experiments, immunofluorescence experiments.

Rat tail intravenous small interfering RNA: wild-type C57BL/6 adult mice (11 weeks old, male, approximately 27g in weight) were selected and administered 10nmol of PAX4 knockdown interfering RNA sequences (chemically modified sequences, including knockdown and nonsense control sequences) (Ribobio Co., Ltd., Guangzhou, China) daily for 3 days at a volume of 0.12mL per injection (dissolved in saline). On the fourth day, infusion of angiotensin II was started by pump-embedding using the pump-embedding method described above. Mice were injected with control and knockdown interfering RNA sequences every 1 day from day five until 7 days of continuous infusion of angiotensin II. Measuring ultrasonic index, body weight and heart weight, and collecting myocardial tissue for subsequent detection.

Performing ultrasonic cardiac: the mouse is placed in an anesthesia box and is anesthetized by isoflurane (2.5 percent isoflurane, 0.8L/min), after the anesthesia is finished, the mouse is taken out of the anesthesia box, is rapidly placed on a heating plate in a supine position, a nose mask connected with anesthetic is worn, the four limbs of the mouse are fixed by rubber strips, and the concentration of the isoflurane is adjusted to 1 percent to maintain the anesthesia. The breast was depilated with depilatory cream (Nail, Canada). Mouse echocardiography measurements were performed using a Vevo 2100 sonicator (Fujifilm visual sonic, Canada).

Heart weighing: after the blood collection of the mouse is finished, the thorax of the mouse is cut off rapidly, the heart is irrigated by an indwelling needle with ice PBS (0.8% NaCl, 0.02% KCl, 0.02% KH2PO4 and 0.4% Na2HPO4), then the whole heart is taken out, tissues such as blood vessels, fat and the like on the heart are cut off in the ice PBS, water is sucked by filter paper, the weight of the whole heart (heart weight, HW) is weighed, the auricle on the heart is cut off after weighing, and the whole ventricle is left. The transection part of the papillary muscle in the middle of the heart is reserved by a surgical blade, the transection part is placed in 4% paraformaldehyde (W/V%, prepared by PBS) and fixed to be used as a tissue section, and the rest myocardial tissues are placed in a freezing tube and frozen in liquid nitrogen.

Tissue sectioning and staining: the horizontal cross section of the papillary muscle of the heart is fixed in 4% paraformaldehyde solution (W/V% in PBS) for 6-8 hours, then the paraformaldehyde is discarded, and 20% sucrose solution (W/V% in PBS) is added for dehydration. Then the mixture is put into 70 percent (3 hours) ethanol solution and 80 percent (3 hours) ethanol solution in turn for gradient dehydration, and finally the mixture is put into 90 percent ethanol plus n-butanol solution (the volume ratio is 1: 1) overnight. The next day, 95% ethanol plus n-butanol solution (45 min 2 times), n-butanol (30 min), butanol (20 min) were sequentially added, surface fluid was blotted with filter paper, and tissue blocks were embedded with paraffin. Then, a heart was sliced with a microtome, and paraffin was cut into sections of 5 μm thick, and the sections were transversely cut at the papillary muscle level, and then stained with sirius red to detect collagen deposition. Dewaxing was first carried out with xylene for 3 min, 3 min for 2min with 100% ethanol, 3 min for 2min with 95% ethanol, 3 min for 1 min with 80% ethanol, and 3 min for 1 min with 70% ethanol. Finally, the mixture was washed with distilled water 3 times. Then, sirius red dyeing is carried out, water is dipped to be dry firstly, the solution is placed into the sirius red solution for dyeing for 1 minute, the loose color is washed off in distilled water (3 times), the solution is quickly washed with 95% ethanol for 1 time, then the solution is placed into 100% ethanol for 1 minute and 2 times (the yellow-dyed color is not washed off), finally, 80% dimethylbenzene is used for carrying out transparent treatment (10 minutes and 2 times), the surface of the slice is covered with neutral resin, and the slice is sealed and stored by a cover glass. And finally, observing and quantitatively analyzing the tissue section. Quantitative collagen fiber area (sirius red stain) was analyzed using a nanoboomer-SQ (Hamamatsu, Japan) image analysis system. The sirius red stained sections were observed, and the collagen fibrosis area (red stained portion) was measured for each specimen, and the total heart cross sectional area was measured by dividing the fibrosis area by the total heart area, i.e., the percentage of fibrosis.

After indexes such as ultrasonic heartbeat and the like are detected, weighing the weight, collecting the weighed weight of the heart tissue, taking part of the heart tissue to carry out tissue embedding of a frozen section and a paraffin section to prepare a subsequent immunohistochemical experiment, and collecting a protein sample of the heart tissue to carry out a western blot experiment.

The protein knockdown efficiency of PAX4 was first examined (fig. 8), along with the effect of angiotensin II stimulation on PAX4 protein levels. The experimental result shows that the protein knockdown efficiency of PAX4 is good, and the myocardial PAX4 protein expression of mice injected with nonsense sequences is increased after angiotensin II is administered, which is consistent with the experimental result in the previous example 1. Quantitative results statistical analysis indicated that angiotensin II significantly promoted PAX4 protein levels, with the protein levels after knockdown being one-third of those before knockdown (fig. 8B).

Next, as shown in fig. 9, the results of sirius red staining indicate that the area of collagen representing cardiac fibrosis increases after administration of angiotensin II to mice injected with nonsense sequences. There was no significant change in the area of cardiac fibrosis between mice injected with PAX4 small interfering RNA and mice injected with the nonsense sequence. The mice injected with PAX4 small interfering RNA were given angiotensin II, and the area of cardiac fibrosis was increased compared to mice injected with PAX4 small interfering RNA without angiotensin II; compared to mice given angiotensin II after injection of nonsense small interfering RNA, cardiac fibrosis area decreased (fig. 9A). Quantitative results statistical analysis of the area of cardiac fibrosis in mice indicated that knockdown PAX4 was able to significantly inhibit cardiac fibrosis caused by angiotensin II (fig. 9B).

The immunofluorescence experiment of Col I was carried out using frozen sections of heart tissue (fig. 10), and the experimental results were consistent with the results of sirius red staining, and after administration of angiotensin II to mice injected with nonsense sequences, the Col I red fluorescence intensity was increased in heart tissue, and after injection of PAX4 small interfering RNA, the Col I fluorescence intensity was significantly decreased in heart tissue of angiotensin II mice administered with angiotensin II.

Echocardiographic results as shown in fig. 11, knock-down of PAX4 significantly reduced the angiotensin II-induced left ventricular posterior wall thickness (LVPWD; D) (fig. 11A), significantly improved the decrease in EF due to angiotensin II (fig. 11B), improved the tendency toward decrease in FS due to angiotensin II (fig. 11C), and significantly improved the increase in E/E' due to angiotensin II (fig. 11D). The ultrasonic cardiac results indicate that the knockdown PAX4 has a protective effect on the contraction function and the relaxation function of the heart of the mouse.

The experimental results are combined, and the fact that the knocking-down PAX4 can play the roles of relieving the increase of extracellular matrix, inhibiting the occurrence of fibrosis and improving the cardiac function is suggested.

Example 5 intervention at the PAX4 protein level at the cardiac fibroblast level, the effect on the protein levels for the myofibroblast markers fibronectin, α SMA and Col I was examined using immunofluorescence or Western blot experiments.

Cardiac fibroblasts transfected with small interfering RNA: the passage of cardiac fibroblasts, P2, from P1 to 6-well plates was overnight at 37 ℃ in a 5% carbon dioxide environment the evening before experimental transfection of small interfering RNA, ensuring that the fibroblasts are morphologically spread and no extracellular matrix affecting transfection efficiency is produced. Transfection the fibroblasts in the 6-well plates were gently washed in warm PBS at 37 ℃ on the day and the washes were repeated three times to ensure thorough washing of the Medium, after which 500. mu.l OPTI-MEM (Opti-MEM I Reduced Serum Medium,31985070, Life) was added to each well, followed by 80nmol/LPAX4 small interfering RNA (sc-152040, Santa Cruz Biotech, Calif., USA) or control small interfering RNA (sc-37007, Santa Cruz Biotech, Calif., USA) and 3. mu.l HiPerFect transfection reagent (301705, QIAGEN, Beijing, China), respectively. After 6 hours of transfection, DMEM medium containing 10% fetal bovine serum was added for culture. The test for detecting proteins uses a sample of cells transfected with small interfering RNA for three days.

Adenovirus cardiac fibroblast infection PAX 4: the cardiac fibroblasts, P2, were passaged from P1 to 6-well plates overnight before experimental viral infection, and cultured overnight at 37 ℃ in a 5% carbon dioxide atmosphere, ensuring that the fibroblasts were morphologically spread and no extracellular matrix affecting transfection efficiency was produced. When the cells grow to 60% density, the cells are infected by using 2MOI PAX4 or control adenovirus under serum-free conditions, the cells are changed after 6 hours, and the cells are cultured by using DMEM containing 10% fetal bovine serum for three days for subsequent experiments.

To further determine the role of PAX4 in cardiac fibroblasts, experiments were first designed to knock down PAX4 by means of small interfering RNA-transfected PAX4 in cardiac fibroblasts, and western blot experiments confirmed that knock down PAX4 in cardiac fibroblasts was effective (fig. 12). Thereafter fluorescence intensity of myofibroblast markers fibronectin, α SMA and Col I after knock-down of PAX4 was measured using immunofluorescence experiments. The results of the experiment show that the myofibroblast markers fibronectin (fig. 13A), α SMA (fig. 13B) and Col I (fig. 13C) have reduced fluorescence after knock-down of PAX 4.

Western blot experiments were performed three days after PAX4 was overexpressed in fibroblasts, and the results are shown in fig. 14, where the myofibroblast markers fibronectin, α SMA and Col I protein levels were increased to different extents after PAX4 was overexpressed.

The experiments all indicate that the PAX4 can promote the occurrence of cardiac fibrosis, and the knock-down PAX4 can reduce the contents of fibrosis markers, namely fibrinectin, alpha SMA and Col I.

Example 6 in cardiac fibroblasts, the protein level of PAX4 in cells was knocked down to detect the expression level of its downstream regulatory genes by means of western blot experiments.

Both animal experiments and cell experiments suggest that the knockdown PAX4 can protect the heart function. However, the mechanism of action is not clear. Therefore, the bioinformatics hand analysis is utilized to find three action targets which are possibly regulated by PAX4 and influence cardiac fibrosis, namely TGF beta (fibrosis promotion), IL1R2 and TGIF2 (fibrosis inhibition factor). TRANSFAC predicts that PAX4 binds to the promoters of TGF β, IL1R2 and TGIF2, so we tested the protein levels of these three genes at the time of knocking down PAX4 using Western blot.

As shown in fig. 15, TGF β protein levels decreased and IL1R2 and TGIF2 protein levels increased after cardiac fibroblast knockdown of PAX4, suggesting that PAX4 plays a role in multi-dimensionally promoting fibrosis by promoting fibrosis promoting factor TGF β, inhibiting fibrosis inhibiting factors IL1R2 and TGIF 2.

The above detailed description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention. While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (5)

1. The application of PAX4 gene expression inhibitor in preparing medicine for inhibiting fibrosis, wherein the fibrosis refers to cardiac fibrosis, and the PAX4 gene expression inhibitor comprises siRNA.
2. The application of the PAX4 gene expression inhibitor in preparing a medicament for inhibiting fibrosis, wherein the fibrosis refers to cardiac fibrosis, and the PAX4 gene expression inhibitor comprises a PAX4 gene knockout reagent.
3. 3. The use according to claim 2, wherein the PAX4 knock-out agent is siRNA.
4. Use of a PAX4 knock-out agent for inhibiting proliferation of cardiac fibroblasts in vitro.
5. The application of the PAX4 gene knockout reagent in inhibiting or blocking the expression of a fibrosis promoting factor TGF beta and promoting the expression of fibrosis inhibiting factors IL1R2 and TGIF2 in vitro cardiac fibroblasts.

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