AU2021225272A1 - Use of PAX4 inhibitor in preparation of drug for inhibiting fibrosis - Google Patents

Abstract

Disclosed is the use of a PAX4 inhibitor in the preparation of a drug for inhibiting fibrosis. The inhibiting effect of a transcription factor PAX4 interferes by means of small interfering RNA so as to block the inhibiting effect thereof on a downstream gene, such that multiple fibrosis inhibiting factors thereof in the downstream execute a function. By means of small interfering RNA, both the expression of PAX4 and the expression level of fibrosis promoting factors are reduced and the fibrosis promoting effect thereof is inhibited, thereby inhibiting cardiac fibrosis. Therefore, PAX4 is a potential brand-new important target for treating cardiac fibrosis and further preventing heart failure in the future.

Description

Description

USE OF PAX4 INHIBITOR IN THE MANUFACTURE OF A MEDICAMENT FOR INHIBITING FIBROSIS

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the priority of Chinese invention patent application No.

CN202010115236.5, filed on February 25, 2020, which is hereby incorporated by

reference in its entirety.

FIELD OF THE INVENTION The present invention relates to the field of genes, and in particular to the use of a

PAX4 inhibitor in the manufacture of a medicament for inhibiting fibrosis.

BACKGROUND OF THE INVENTION Cardiac fibrosis is an important constituent part of most cardiac pathological

conditions. Cardiac fibrosis is manifested by excessive deposition of an extracellular

matrix in a cardiac tissue, which leads to the destruction of a structure of a

physiological cardiac tissue and finally leads to heart failure, seriously threatening the

health and life of human beings. The transdifferentiation of fibroblasts into

myofibroblasts is the key to the initiation and maintenance of cardiac fibrosis. The

myofibroblasts have an important contraction and secretion functions and are

characterized by the expression of a-smooth actin (aSMA), fibronectin, and collagen I

(Col I). Angiotensin II is a polypeptide formulation recognized in the current research

that regulates vasoconstriction, affects cardiac functions, and can induce cardiac

fibrosis. This formulation can usually be used for treating mice or fibroblasts to

simulate the state of cardiac fibrosis. Up to now, although researchers from various countries have done a lot of research on the mechanism of cardiac fibrosis, its specific molecular mechanism remains unclear. Cardiac fibrosis is still an important target in the current clinical treatment of heart 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 still existing in the research mechanism of cardiac fibrosis.

A transcription factor PAX4 is a member of a subfamily IV of a Paired box (PAX)

family. In the human genome, a PAX4 gene is located in subband 1 of band 2 of

region 3 on a long arm of chromosome 7 and consists of 12 exons and 11 introns.

PAX4 is also known as KPD and MODY9. A PAX4 protein is in nucleus and can form

a transcription factor complex, which binds with the 5'-terminal specific sequence

of the target genes, and regulates the expression of downstream target genes. Current

research believed that those members of the PAX family play an important role in

many stages of embryonic development and organ formation, and also play a role in

every aspect of the body in adulthood. The members of the PAX family, from insects,

amphibians, birds, and mammals, have a considerably conservative sequence

evolution process. The full-length PAX4 includes 349 amino acids, and its protein

structure includes one bipartite paired domain (PD) of 128 amino acids and one

homeodomain (HD). Its C-terminal not only has a common transcriptional activation

domain of the PAX family, but also has a unique negative regulatory domain. Current

research believed that those members of the PAX family are important regulatory

factors of tissue development and cell differentiation. However, the research on PAX4

mainly focuses on research related to a pancreas islet, cancer, and retina. Current

research has shown that PAX4 is involved in the differentiation of islet beta cells and

delta cells during the period of embryonic development and insulin secretion under

normal conditions. Deletion of PAX4 induces type 1 and type 2 diabetes. In tumor-related research, PAX4 is a potent tumor-inhibiting factor of human insulinoma and melanoma. PAX4 can facilitate the migration and invasion of human epithelial carcinoma. Some researchers have reported that PAX4 is highly expressed in a rat retinal photoreceptor, suggesting that PAX4 may play a role in the retina. However, the research on the PAX4 function in the heart is still unknown.

SUMMARY An objective of the present invention is to provide a novel drug for inhibiting fibrosis.

To realize the objective of the present invention, the following technical solution is

intended to be employed: one of the objectives of the present invention is to provide

use of a PAX4 inhibitor in the manufacture of a medicament for inhibiting fibrosis.

In another aspect of the present invention, the present invention further relates to a

drug for inhibiting fibrosis, including a PAX4 inhibitor.

In another aspect of the present invention, the present invention further relates to a

method for inhibiting fibrosis, which is characterized by including a step of inhibiting

the expression of a PAX4 gene.

The inventor of the present application has found through researches by means of

biochemistry, molecular biology and cytology that PAX4 is highly expressed in a

pathological mouse models such as a mouse fibrosis model, a myocardial infarction

model and the like. Subsequently, a technical means of small interfering RNA is

utilized to knock down and reduce the protein level of PAX4 in a cell or cardiac tissue

by the small interfering RNA, respectively by transfecting fibroblasts with the small

interfering RNA or injection of the small interfering RNA via mouse tail vein, so as

to clarify the effect of PAX4 on the heart function of the mouse. Thereafter, a bioinformatics means is utilized to analyze and predict a target gene involved in cardiac fibrosis that can be bound and regulated by PAX4. Thereafter, the regulation effect of PAX4 on downstream genes is clarified by a method of knocking down

PAX4 with the small interfering RNA, thereby clarifying the mechanism of action.

This discovery is made by the inventor of the present application for the first time and

is unexpected.

In one preferred embodiment of the present invention, the PAX4 inhibitor comprises

siRNA and a PAX4 gene knockout reagent.

In one preferred embodiment of the present invention, the PAX4 gene knockout

reagent is siRNA, i.e., small interfering RNA.

In one preferred embodiment of the present invention, the fibrosis refers to cardiac

fibrosis, pancreatic fibrosis, or pulmonary fibrosis.

Although the findings of the present invention are preferably applied to cell fibrosis of

human or animal bodies, the present invention further relates to use of a PAX4 gene or

an expression product thereof in promoting the proliferation of cardiac fibroblasts in

vitro.

Although the findings of the present invention are preferably applied to cell fibrosis of

human or animal bodies, the present invention further relates to use of a PAX4 gene

knockout reagent in inhibiting the proliferation of cardiac fibroblasts in vitro.

Although the findings of the present invention are preferably applied to cell fibrosis of

human or animal bodies, the present invention further relates to use of a PAX4 gene knockout reagent in inhibiting or blocking the expression of a fibrosis promoting factor TGF Pand promoting the expression of fibrosis inhibiting factors IL1R2 and

TGIF2 in cardiac fibroblasts in vitro.

Although the findings of the present invention are preferably applied to cell fibrosis of

human or animal bodies, the present invention further relates to use of a PAX4 gene

knockout agent in inhibiting cell fibrosis by inhibiting a fibrosis promoting factor

TGF Pand promoting fibrosis inhibiting factors IL1R2 and TGIF2. The experimental

results of the present application have confirmed that after PAX4 is knocked down in

cardiac fibroblasts, the level of TGFP protein is decreased, while the levels of IL1R2

and TGIF2 proteins are increased, which suggests that PAX4 plays a

multi-dimensional role in promoting fibrosis by increasing the fibrosis promoting

factor TGFP and inhibiting the fibrosis suppressing factors IL1R2 and TGIF2.

The present invention provides a novel application of PAX4 as a new important target

for treating cardiac fibrosis for the first time. In particular, in the present invention, it

has been found by using biochemical, molecular biological and cytological method

that PAX4 is highly expressed in pathological mouse model such as a mouse

fibroblast fibrosis model, a myocardial infarction model and the like. Subsequently, a

technical means of small interfering RNA is utilized to knock down and reduce the

protein level of PAX4 in a cell or cardiac tissue by the small interfering RNA,

respectively by transfecting fibroblasts with the small interfering RNA or injection of

the small interfering RNA via mouse tail vein, so as to clarify the effect of PAX4 on

the heart function of the mouse. Thereafter, a bioinformatics means is utilized to

analyze and predict a target gene involved in cardiac fibrosis that can be bound and

regulated by PAX4. Thereafter, the regulation effect of PAX4 on downstream genes is

clarified by a method of knocking down PAX4 with the small interfering RNA, thereby clarifying the mechanism of action. Therefore, PAX4 is a potential brand-new important target for treating cardiac fibrosis and thus preventing heart failure.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1: Immunohistochemical assay analysis confirms that PAX4 expression increased

under the pathological stimulation of fibroblasts. Fig. 1A: The protein level and

localization of PAX4 labeled with a PAX4 antibody immunohistochemically. Fig. IB:

Immunohistochemical results of quantitative IOD analysis of PAX4 labeled with the

PAX4 antibody.

Fig. 2: Western blot confirms that the expression level of a transcription factor PAX4

is increased in a mouse fibrosis model. Fig. 2A: Comparison of the protein level of

PAX4 in cardiac tissues of a cardiac fibrosis model and its control group by utilizing a

PAX4 antibody through Western blot, with GAPDH as an internal reference. Fig. 2B:

Quantitative and statistical analysis results of the protein content detected by Western

blot of PAX4.

Fig. 3: Detection of the protein levels of cardiac fibrosis markers fibronectin, aSMA

and Col I in a mouse fibrosis model by Western blot. Fig. 3A: Comparison of the

protein levels of fibronectin, aSMA and Col I in cardiac tissues of a cardiac fibrosis

model and its control group by utilizing antibodies against fibronectin, aSMA and Col

I through Western blot, with GAPDH as an internal reference. Fig. 3B: Quantitative

and statistical analysis results of the protein content detected by Western blot of

fibronectin, aSMA and Col I.

Fig. 4: Detection of the protein level of PAX4 in cardiac tissues of an infarct region, a

border region, and a remote region from the mouse myocardial infarction model at different time points after infarction by Western blot.

Fig. 5: Detection of the changes in fluorescence intensities of transcription factor

PAX4 and myofibroblast markers fibronectin, aSMA and Col I after cardiac

fibroblasts are stimulated with 1 M of angiotensin II for three days by an

immunofluorescence assay. Fig. 5A: The fluorescence intensities of the transcription

factor PAX4 and the myofibroblast markers fibronectin, aSMA and Col I, wherein

circled fluorescence indicates nuclei, and the rest of fluorescence indicates detected

genes of interest. Fig. 5B: Relative quantitative and statistical analysis results of

immunofluorescence.

Fig. 6: Western blot confirms that the expression level of transcription factor PAX4 is

increased under the stimulation of angiotensin II in fibroblasts. Fig. 6A: Detection of

the protein level of PAX4 after stimulation with angiotensin II by a Western blot assay.

Fig. 6B: Quantitative and statistical analysis results of the protein content of PAX4

detected by Western blot.

Fig. 7: Western blot confirms that the protein levels of myofibroblast markers

fibronectin, aSMA and Col I are increased under the stimulation of angiotensin II in

fibroblasts. Fig. 7A: Detection of the protein levels of fibronectin, aSMA and Col I

after stimulation with angiotensin II by a Western blot assay. Fig. 7B: Quantitative

and statistical analysis results of the protein contents of fibronectin, aSMA and Col I

detected by Western blot.

Fig. 8: The protein level of PAX4 in a cardiac tissue is detected by Western blot after

PAX4 is knocked down in mice transfected with small interfering RNA by tail vein

injection to verify the PAX4 knock-down efficiency. Fig. 8A: Detection of the protein level of PAX4 after injection of the small interfering RNA into the tail vein of the mouse by a Western blot assay. Fig. 8B: Quantitative and statistical analysis results of the protein content of PAX4 detected by Western blot.

Fig. 9: Small interfering RNA of PAX4 was injected into mice via tail vein and then

fibrosis model was established by implanting a micro-osmotic pump infused with

AnglI. And it is found that knocking down PAX4 inhibits fibrosis by staining with

Picrosirius red. Fig. 9A: Results of tissue sections stained with Picrosirius red. Fig. 9B:

Quantitative and statistical analysis results of cardiac fibrosis area.

Fig. 10: Small interfering RNA of PAX4 was injected into mice via tail vein and then

fibrosis model was established by implanting a micro-osmotic pump infused with

AnglI. the fluorescence intensity of Col I is detected by an immunofluorescence

staining. The upper figure shows the fluorescence intensity of Col I, and the lower

figure shows co-staining of Col I with a nuclear dye Hoechst.

Fig. 11: Small interfering RNA of PAX4 was injected into mice via tail vein and then

fibrosis model was established by implanting a micro-osmotic pump infused with

AnglI. and it is found that knocking down PAX4 has a protective effect on the heart

function of the mice by echocardiography. Fig. 11A: left ventricular posterior wall

thickness, Fig. 1IB: ejection fraction (EF) value, Fig. 1IC: fraction shortening (FS)

value, and Fig. I1D: the ratio of the peak flow velocities during early diastole and the

peak early diastolic velocities of the mitral valve ring E/E'.

Fig. 12: The protein level of PAX4 in cardiac fibroblasts transfected with small

interfering RNA is detected by Western blot to verify the knock-down efficiency in

knocking down PAX4.

Fig. 13: The protein expression levels of myofibroblast markers fibronectin (Fig. 13A),

aSMA (Fig. 13B) and Col I (Fig. 13C) are detected by immunofluorescence staining

after PAX4 is knocked down infibroblasts transfected with small interfering RNA to

verify the inhibitory effect of knocking down PAX4 on fibrosis.

Fig. 14: The protein expression levels of myofibroblast markers fibronectin, aSMA,

and Col I are detected by Western blot after PAX4 is overexpressed infibroblasts

infected with adenovirus to verify the promoting effect of PAX4 onfibrosis.

Fig. 15: Detection of the effect on the protein expression levels of possible

downstream genes TGFP, ILR2 and TGIF2 after PAX4 is knocked down in

fibroblasts transfected with small interfering RNA by a Western blot assay.

DETAILED DESCRIPTION The present invention will be described in further detail below with reference to

specific examples, which are explanations rather than limitations of the present

invention. The methods and related reagents used in the present invention can have

other optional and alternative solutions, as long as the same technical results can be

achieved.

The experimental methods in the following examples which are not specified with

specific conditions shall be carried out according to conventional operations in the art

to which the present invention belongs or the conditions suggested by a manufacturer.

Example 1 Assay of animal pathological model: a mouse cardiac fibrosis model was

established, a cardiac tissue was taken, and the expression location and content of

PAX4 protein, as well as the content of myofibroblast markers fibronectin and aSMA,

and the content of an extracellular matrix Col I were detected by utilizing

immunohistochemistry and a Western blot assay.

Preparation of angiotensin II-induced mouse cardiac fibrosis model: 10-week-old

male C57BL/6 mice were randomly divided into two groups, an operation group, and

a sham operation group. The mice were implanted with a micro-osmotic pump (Alzet

MODEL 1007D, DURECT, Cupertino, CA) injected with angiotensin (3 mg kg-I day-1)

for 7 days to establish the fibrosis model. Preparation of the micro-osmotic pump: 1

day before operation, angiotensin II (dissolved in a sterile PBS buffer) was injected

into the miniosmotic pump with a 1 mL syringe, and the micro-osmotic pump was

soaked in the sterile PBS buffer and equilibrated at 37°C overnight. During the

operation, the mice were anesthetized with 2%-3% isoflurane, and a transverse

incision of about 0.7 cm in length was cut in a back neck of the mouse. A tweezer was

inserted into the skin for blunt dissection of a subcutaneous tissue. The miniosmotic

pump was implanted, the wound was sutured, and neomycin ointment was applied

onto the wound to prevent infection. The operation group received continuous

infusion of angiotensin IIat a concentration of 3 mg kg-1 day-' for 7 days.

Assay method of immunohistochemistry of PAX4: a sample was fixed with

paraformaldehyde, embedded in paraffin, and then sliced. (1) Dewaxing: tissue

sections were treated with xylene for 15 min 3 times, with 100% ethanol for 5 min

twice, with 95% ethanol for 5 min twice, and with 80% ethanol for 5 min once.

Finally, the tissue sections were washed with distilled water for 2 min. (2) Removal of

endogenous catalase: the sections were placed in a 3% hydrogen peroxide solution

(formulated with 100% methanol) for 10-15 min to remove endogenous catalase.

Then the sections were washed with PBS for 3 times, with 5 minutes each time. (3)

Antigen thermal repair: the sections were placed in a citrate (pH 6.0) antigen repair

solution, and subjected to thermal repair in a pressure cooker, such that an antigen

epitope was fully unfolded (timing for 2 minutes after the pressure cooker

continuously emitted gas). After the thermal repair was completed, the sections were

placed at room temperature, and when the temperature of the sections was reduced to

room temperature, the sections were washed with PBS for 3 times, with 5 minutes

each time. (4) Serum blocking: the sections were placed in a wet box and blocked

with 10% goat serum at room temperature for 30 min. (5) Incubation with primary

antibody: the serum was discarded, and then a formulated primary antibody was

added onto the sections, which was kept overnight in a refrigerator at 4°C (or

incubated in an incubator at 37°C for 2 hours). (6) Incubation with secondary

antibody: the wet box was taken out from the refrigerator at 4°C, and naturally raised

to room temperature, and then the sections were washed with PBS for 3 times, with 5

minutes each time. Then, the second antibody labeled with horseradish peroxidase

(Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd., a rabbit

two-step kit) was added, and incubation was carried out at room temperature for 30

min. (7) DAB color development: the sections were washed with PBS for 3 times and

subjected to color development with a DAB color developing solution. Formulation

method of a DAB working solution: 1 mL of a DAB diluent + 50 L of the DAB color

developing solution. (8) Re-staining of nucleus: the sections were first soaked in

distilled water for 2 min, then placed in a hematoxylin solution for 30 s, and rinsed

with tap water for 3 times. Differentiation was performed in 70% hydrochloric acid in

alcohol for 5-6 s, and the sections were washed with water for 30 s. The sections were

performed in 1% ammonia water for 60 s, and unfixed dyes were washed off in

distilled water. (It could be observed under a microscope whether the nucleus was

blued). (9) Dehydrating, permeabilizing, and mounting with a neutral resin mounting

medium. The sections were treated with 95% ethanol twice for 2 min, with absolute ethanol twice for 2 min, and finally in a xylene substitute twice for 5 min. (10) After dried in the air, the sections were scanned to conduct statistics of a positive area by software.

Extraction of total protein from myocardial tissue: a myocardial tissue stored in liquid

nitrogen was taken, put into a mortar and grounded with liquid nitrogen, and two

thirds (the other third was used for extracting RNAs) of the grounded myocardial

tissue was added into a tissue lysis buffer (20 mmol/L Tris-HCl pH 7.4, 150 mmol/L

NaCl, 2.5 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L Na4 P20 7 , 1 mmol/L Na3VO 4

, 1% Triton X-100, 10% Glycerol, 0.1% SDS, 1% deoxycholic acid, 1 mmol/L PMSF,

and lg/mL aprotinin), mixed uniformly, and allowed to stand on ice for 15 minutes,

wherein 800 microliters of the lysis buffer was added per 50 mg of the myocardial

tissue. The resulting homogenate was collected, ultrasonically crushed (45%, 5 s on, 5

s off for 4 cycles), and then centrifuged at 4°C and 12,000 rpm for 15 minutes. A part

of supernatant was transferred into a new EP tube and cryopreserved at -80°C after

protein quantification, and a quarter of the volume of a 5x loading buffer was added to

another part of supernatant, mixed evenly, boiled at 100°C for 5 minutes, and

cryopreserved for later detection of related proteins by Western blot.

Western blot assay: after electrophoresis with a 10% SDS-PAGE gel, protein was

transferred onto a nitrocellulose membrane, blocked with 5% fat free milk at room

temperature for 1 hour, and incubated with primary antibodies in a cold room at 4°C

overnight. The catalogue number of the primary antibodies were as follows:

Fibronectin (ab2413, abeam, Cambridge, MA, USA), aSMA (ab32575, abeam,

Cambridge, MA, USA), Col 1 (203002, MD Biosciences), PAX4 (ab101721, abeam,

Cambridge, MA, USA), TGFP (10804-MM33, Sino biological, Beijing, China),

IL1R2 (sc-376247, Santa Cruz Biotech, CA, USA), TGIF2 (ab190152, abeam,

Cambridge, MA, USA), and GAPDH (2118S, CST). The membrane was washed with

TBST for three times, and then was incubated with secondary antibodies of

corresponding species at room temperature for 1 hour, and the membrane was washed

with TBST again, and then put into a Chemiluminescent HRP substrate (Millipore

Corporation). Then the membrane was put into a luminescence detection machine for

exposure. The band intensity was quantified by using NIH ImageJ software.

A mouse cardiac fibrosis model was established with 10-week-old male C57BL/6

mice in the manner of implanting a micro-osmotic pump infused with angiotensin II.

The expression level of PAX4 in the cardiac tissue of the cardiac fibrosis model was

detected, and meanwhile the levels of fibrosis markers were detected to correspond to

the degree of fibrosis in the cardiac tissue.

Firstly, cardiac tissues were taken from the cardiac fibrosis model established by

implanting the micro-osmotic pump infused with angiotensin II and the sham

operation group and subjected to an immunohistochemical assay with a PAX4

antibody. The assay results were shown in Fig. 1A, and in the tissue in which cardiac

fibrosis occurred, the protein level of PAX4 was increased in both cardiac myocytes

and cardiac fibroblasts. The statistical results were shown in Fig. IB, and the

statistical analysis of the quantitative results showed that the protein level of PAX4 in

the fibrotic cardiac tissue was significantly increased compared with that in a healthy

cardiac tissue.

Thereafter, a Western blot assay was carried out by using the total proteins of the

cardiac tissues from the cardiac fibrosis model and the sham operation group. The

assay results of detecting the protein level of PAX4 were shown in Fig. 2. The

obtained results were consistent with those of the immunohistochemical assay, and the protein level of the transcription factor PAX4 was significantly increased in the fibrotic cardiac tissue (Fig. 2A). The statistical analysis of the quantitative results (Fig.

2B) showed that, the protein level of PAX4 in the fibrotic cardiac tissue was

significantly increased compared with that in a healthy cardiac tissue.

Meanwhile, the protein levels of the fibrosis markers fibronectin, aSMA and Col I in

the cardiac tissue were also detected by the Western blot assay. The assay results were

shown in Fig. 3A, the protein levels of thefibrosis markersfibronectin, aSMA and

Col I in the fibrotic cardiac tissue were significantly increased than those in a normal

cardiac tissue. The statistical analysis of the quantitative results (Fig. 3B) showed that

the protein levels of fibronectin, aSMA and Col I in the fibrotic cardiac tissue was

significantly increased. However, whether there was a causal relationship between the

increase of PAX4 and the increase of the fibrosis markers fibronectin, aSMA and Col

I still needed to be verified by subsequent experiments.

Example 2 Detection of the protein levels of PAX4 in an infarct region and a remote

region of a cardiac tissue of a mouse myocardial infarction model at different time

points after operation.

-week-old male C57BL/6 mice were used for establishing a myocardial infarction

model. The mice were randomly divided into a myocardial infarction group and a

sham operation control group. The myocardial infarction group was subjected to left

coronary artery stenosis to induce the occurrence of myocardial infarction. The

control group was subjected to sham operation. The operation was performed under

gas anesthesia, and the mice were subjected to gas anesthesia by inhaling 2%

isoflurane. In the assay group, an infarct region, a border region, and a remote region

of cardiac tissues of the mice were taken to collect the cardiac tissues on days 1, 4 and

7 after the operation, respectively.

The protein level of an endogenous transcription factor PAX4 in the cardiac tissue in

the infarct region, the border region, and the remote region on days 1, 4 and 7 after the

operation of the mouse myocardial infarction model was detected by Western blot. A

left side of Fig. 4 showed the specific locations of the infarct region, the border region,

and the remote region of heart infarction. The assay results showed that, compared

with the sham operation group, the protein levels of PAX4 in all areas of myocardial

infarction operation were increased in different degrees. The results of the myocardial

infarction model in combination with the results of the pathological conditions of

fibrosis in Example 1, suggested that the protein level of PAX4 in the cardiac tissue

was increased when pathological changes occurred in the heart, and the specific

function of this increase was verified in the following subsequent assays.

Example 3 Detection of the protein levels of PAX4, and fibrosis markers fibronectin,

aSMA and Col I by immunofluorescence and a Western blot assay after cardiac

fibroblasts were treated with 1 M angiotensin for three days.

Isolation and culture of cardiac fibroblasts from adult mice: male 8 weeks old

C57/BL6 micewere sacrificed by cervical dislocation, and quickly soaked in 75%

alcohol for about half a minute, and immediately chests of the mice were opened to

take out hearts on an ultra-clean workbench. The hearts were put into a PBS buffer at

4°C to be washed twice, then blood vessels in the atrium and the heart bottom were

cut off, and then ventricles were chopped into small pieces and washed with PBS once

to wash away some residual blood. 0.1% typeII collagenase (330U, Worthington,

Columbia, NJ, USA/Sigma, St. Louis, MO, USA) formulated in a PBS balanced salt

solution was added for digestion. The whole digestion process was carried out at a constant temperature of 36-37°C under stirring. A supernatant of the resulting digestion solution was taken every 8 minutes of the digestion and added into the same amount of a DMEM culture medium containing 10% FBS, followed by uniform mixing. This process was repeated for about 7-8 times until the tissue pieces were completely digested. Several tubes of collected cells were centrifuged at room temperature at 1,000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended with a DMEM culture medium containing 10% FBS. The suspension of cardiac myocytes obtained each time was mixed, seeded in a culture dish with a diameter of 100 mm, and placed in a 37°C incubator with 5% C02 for 2 hours to make the fibroblasts basically adherent. The culture medium in the culture dish was pipetted and discarded, and a fresh DMEM culture medium containing 10%

FBS was added to continue the culture. After 3 days, the cells were fully grown and

passaged for subsequent assays.

A method of extracting protein from cardiac fibroblasts: the cells were first digested

from a base gel with pancreatin and centrifuged, and then the supernatant was taken,

washed with cold PBS for three times, subsequently subjected to cell lysis with a cell

lysis buffer (20 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 2.5 mmol/L EDTA, 50

mmol/L NaF, 0.1 mmol/L Na4 P20 7 , 1 mmol/L Na3VO 4 , 1% Triton X-100, 10%

glycerol, 0.1% SDS, 1% deoxycholic acid, 1 mmol/L PMSF, and 1 mg/mL aprotinin),

subjected to ultrasonic crushing, and then centrifuged at 12,000 g under the condition

of 4°C for 15 minutes. The supernatant was collected. 5 microliters of the supernatant

were taken for protein quantification, and a 5x gel loading buffer was added into the

rest of the supernatant to be kept at 100°C for 5 minutes to ensure protein

denaturation.

Immunofluorescence staining assay: the cells were fixed with warm 4% paraformaldehyde of 37°C under the condition of 37°C for 15 minutes, then washed with warm PBS for 3 times, and subsequently permeabilized with 0.2% Triton X-100 for 20-30 minutes. The cells were washed with warm PBS for 3 times, and then a blocking solution (5% BSA) was added for blocking for 30 minutes. Thereafter, the cells were incubated with primary antibodies against aSMA (ab32575, abeam,

Cambridge, MA, USA), fibronectin (ab2413, abeam, Cambridge, MA, USA) and

PAX4 (ab101721, abeam, Cambridge, MA, USA) under a condition of 4°C overnight.

After the primary antibodies were recovered and stored, the cells were washed with

PBS for 3 times, and then incubated with a secondary antibody Alexa Fluor 488 at

room temperature for 1 hour. The cells were subjected to nuclear staining with

Hoechst (Invitrogen, Carlsbad, CA, USA) under the condition of room temperature

for 8 minutes. Statistics and analysis of the fluorescence intensity were conducted by

using a Morphology Explorer BioApplication module of a high-content screening

imaging system Cellomics ArrayScan VTI HCS Reader (Thermo Fisher Scientific,

Rockford, IL, USA).

At the cellular level, primary mouse cardiac fibroblasts were extracted, cultured in a

12-well plate to a P2 generation, and stimulated with angiotensin II at a concentration

of 1 M for three days, and then samples were collected. Firstly, the samples were

fixed, and detected for the protein levels of endogenous PAX4 and fibrosis markers

fibronectin, aSMA and Col I by immunofluorescence. The fluorescence circled in Fig.

A were positions of nuclei, and the rest of fluorescence indicated the locations and

contents of specific proteins of interest (PAX4, fibronectin, Col I and aSMA)

identified by specific antibodies, respectively. It could be seen from the assay results

that the transcription factor PAX4 was mainly expressed in the nucleus. The assay

results indicated that under the stimulation of angiotensin II, the fluorescence

intensities of PAX4, fibronectin, aSMA and Col I were enhanced in different degrees.

The quantitative results in Fig. 5B showed that the contents of the transcription factor

PAX4, and common cardiac fibrosis markers fibronectin, aSMA and Col I were

increased significantly after three days of stimulation with angiotensin II.

Thereafter, a western blot assay was conducted with the total protein of cardiac

fibroblasts cultured under the same conditions, and the protein level of PAX4 was

detected first. The assay results were shown in Fig. 6. The assay obtained results close

to those of the immunofluorescence assay, and the protein level of the transcription

factor PAX4 was increased after the stimulation with angiotensin II (Fig. 6A). The

statistical analysis of the quantitative results showed that the increase degree of the

protein level was significant. Meanwhile, the protein levels of the myofibroblast

markers fibronectin, aSMA and Col I were also detected (Fig. 7). The assay results

showed that the stimulation of the cardiac fibroblasts with angiotensin II for three

days could promote the protein levels of the fibrosis markers fibronectin, aSMA and

Col I (Fig. 7A). The statistical analysis of the quantitative results showed that after

stimulation with angiotensin II, the protein levels of the fibrosis markers were

significantly increased compared with those of the control group.

Example 4 The level of PAX4 in mice was knocked down in the manner of injecting

small interfering RNA into tail veins of mice, and then a fibrosis model was

established. The effects of PAX4 on the heart function and on the protein levels of

myofibroblast markers fibronectin and aSMA, and an extracellular matrix Col I were

detected by echocardiography, comparison of a heart weight/body weight ratio,

staining with Picrosirius red (PSR), a Western blot assay and an immunofluorescence

assay.

Tail vein injection of small interfering RNA into mice: wild C57BL/6 adult mice (11 weeks old, male, about 27 g) were selected, and administrated with 10 nmol of small interfering RNA sequence for knock down of PAX4 (a chemically modified sequence, including a knock-down sequence and a nonsense control sequence) (Ribobio Co., Ltd

(Guangzhou, China)) by tail vein injection every day, at an injection volume of 0.12

mL (dissolved in normal saline) each time for 3 days. On day 4, the infusion of

angiotensin II was started with the aforementioned pump implantation method. From

day 5, the control sequence and the small interfering RNA sequence for knock down

were injected into tail veins of the mice every other day until angiotensin II had been

continuously infused for 7 days. The ultrasonic indexes, body weights and heart

weights of the mice were measured, and myocardial tissues were collected for

subsequent detection.

Echocardiography: the mice were put into an anesthesia box and given isoflurane

(2.5% isoflurane, 0.8 L/min) for anesthesia. After anesthesia was completed, the mice

were taken out of the anesthesia box, quickly put on a heating plate in a supine

position and worn a nasal mask connected with an anesthetic. The limbs of the mice

were fixed with an adhesive tape, and the isoflurane concentration was adjusted to 1%

to maintain anesthesia. Hair removal was performed on the chest with depilatory

cream (Nail, Canada). An echocardiogram of the mice was detected by a Vevo 2100

ultrasonic instrument (Fujifilm Visual sonics, Canada).

Weighing of the heart: after blood sampling was completed, the chest of each mouse

was quickly cut open, and the heart was lavaged with cold PBS (0.8% NaCl, 0.02%

KCl, 0.02% KH 2 PO 4 , and 0.4% Na2HPO 4) through an indwelling needle. After that, a

whole heart was taken out, tissues such as blood vessels, fat and the like on the heart

were cut off in cold PBS, the heart was dried with filter paper, and a whole heart

weight (HW) was weighed. After weighing, the auricle on the heart was cut off, leaving a whole ventricle. A transection part of the papillary muscle in the middle of the heart was taken with a surgical blade and fixed in 4% paraformaldehyde (W/V%, formulated in PBS) for preparation of tissue sections. The rest of the myocardial tissues was cryopreserved in liquid nitrogen in a cryopreservation tube.

Tissue sections and staining: a horizontal cross section of the papillary muscle of the

heart was fixed in a 4% paraformaldehyde solution (W/V%, formulated in PBS) for

6-8 hours, then paraformaldehyde was discarded, and the horizontal cross section was

added into a 20% sucrose solution (W/V%, formulated in PBS) for dehydration. Then

the dehydrated horizontal cross section was sequentially put into 70% (3 hours) and

% (3 hours) ethanol solutions for gradient dehydration, and finally put into a

solution of 90% ethanol plus n-butanol (a volume ratio of 1:1) overnight. On the next

day, the dehydrated horizontal cross section was sequentially put into a solution of

% ethanol plus n-butanol (45 minutes twice), n-butanol (30 minutes) and butanol

(20 minutes). The surface liquid of the dehydrated horizontal cross section was

absorbed by filter paper, and a tissue mass was embedded with paraffin. After that,

paraffin heart sections with a thickness of 5 m were made with a microtome by

transecting horizontally at the papillary muscle, and the sections were stained with

Picrosirius red to detect collagen deposition. First, dewaxing was conducted in xylene

for 10 minutes for 3 times, in 100% ethanol for 3 minutes twice, in 95% ethanol for 3

minutes twice, in 80% ethanol for 3 minutes once, and in 70% ethanol for 3 minutes

once. Finally, the sections were washed with distilled water for 3 times. Thereafter

staining with Picrosirius red was conducted, wherein water was removed by dipping

first, and the sections were put into a Picrosirius red solution for staining for 1 minute,

washed with distilled water to remove unfixed dyes (for 3 times), quickly washed

once with 95% ethanol, then put into 100% ethanol to be washed for 1 minute twice

(be careful not to wash off yellow stained color), and finally, subjected to permeabilization treatment with 80% xylene (for 10 minutes twice), and covered with neutral resin on the surface thereof, followed by covering with a cover slip for mounting. Finally, observation and quantitative analysis of the tissue sections were conducted. A collagen fibrosis area (stained with Picrosirius red) was analyzed and quantified with a NanoZoomer-SQ (Hamamatsu, Japan) image analysis system. The sections stained with Picrosirius red were observed, and each specimen was measured for the collagen fibrosis area (red stained parts) and a cross-sectional area of the whole heart, and the percentage of fibrosis was obtained by dividing the fibrosis area by a total heart area.

After the indexes such as echocardiography were detected, the body weight was

weighed, and the cardiac tissue was collected and weighed. Part of the cardiac tissues

was taken to conduct tissue embedding of frozen sections and paraffin sections for

preparation for the subsequent immunohistochemical assay, and a protein sample of

the cardiac tissue was collected for a Western blot assay.

Firstly, the protein knock-down efficiency of PAX4 was detected (Fig. 8), and

meanwhile the effect of angiotensin II stimulation on the protein level of PAX4 was

detected. The assay results showed that the protein knock-down efficiency of PAX4

was good, and the expression of PAX4 protein in the myocardium of the mice injected

with the nonsense sequence was increased after administration of angiotensin II,

which was consistent with the assay results in the Example 1 above. The statistical

analysis of the quantitative results showed that angiotensin II significantly promoted

the PAX4 protein level, and the protein level after knocking down was one third of

that before knocking down (Fig. 8B).

Secondly, the results of staining with Picrosirius red were shown in Fig. 9. In the mice injected with the nonsense sequence, after administration of angiotensin II, a collagen area representing cardiac fibrosis was increased. There was no significant change in the cardiac fibrosis area between the mice injected with the small interfering RNA of

PAX4 and the mice injected with the nonsense sequence. The cardiac fibrosis area of

the mice injected with the small interfering RNA of PAX4 and then administrated

with angiotensin II was increased compared with that of the mice injected with the

small interfering RNA of PAX4 and not administrated with angiotensin II; and was

decreased compared with that of the mice injected with the nonsense small interfering

RNA and then administrated with angiotensin II (Fig. 9A). The statistical analysis of

the quantitative results of the cardiac fibrosis area of the mice showed that knocking

down PAX4 could significantly inhibit cardiac fibrosis induced by angiotensin II (Fig.

9B).

An immunofluorescence assay of Col I was carried out with frozen sections of the

cardiac tissues (Fig. 10), and the assay results were consistent with those of staining

with Picrosirius red. The red fluorescence intensity of Col I in the cardiac tissues of

mice injected with the nonsense sequence was increased after administration of

angiotensin II, and the fluorescence intensity of Col I in the cardiac tissues of the mice

injected with the small interfering RNA of PAX4 and then administrated with

angiotensin II was decreased significantly.

The results of echocardiography were shown in Fig. 11. Knocking down PAX4 could

significantly reduce the increase of the left ventricular posterior wall thickness

(LVPWD; d) induced by angiotensin II (Fig. 11A), could significantly improve the

decrease of EF caused by angiotensin II (Fig. 11B), had the trend of improving the

decrease of FS caused by angiotensin (Fig. 11C), and could significantly improve the

increase of E/E' caused by angiotensin II (Fig. 11D). The aforementioned echocardiographic results suggested that knocking down PAX4 could have a protection effect on both the systolic and diastolic functions of the heart of the mouse.

In summary of the aforementioned assay results, it was suggested that knocking down

PAX4 could have the effects of relieving the increase of the extracellular matrix,

inhibiting the occurrence of fibrosis and improving the heart function.

Example 5 Interfering the protein level of PAX4 at the level of cardiac fibroblasts.

The effect on the protein levels of myofibroblast markers fibronectin, aSMA and Col I

was detected by an immunofluorescence assay or a Western blot assay.

Transfection of small interfering RNA into cardiac fibroblasts: the cardiac fibroblasts

of P2 generation were passaged from a P1 generation into a 6-well plate on the night

before the assay of transfection of the small interfering RNA and cultured overnight in

an environment of 37°C and 5% C02 to ensure that the morphology of the fibroblasts

was spread and any extracellular matrix that affected the transfection efficiency had

not been produced. On the day of transfection, the fibroblasts in the 6-well plate were

washed with warm PBS at 37°C gently and repeatedly for three times to ensure that

the medium was thoroughly washed away. Then, 500 L of OPTI-MEM (Opti-MEM I

Reduced Serum Medium, 31985070, Life) was added into each well, and then 80

nmol/L of small interfering RNA of PAX4 (sc-152040, Santa Cruz Biotech, CA, USA)

or control small interfering RNA (sc-37007, Santa Cruz Biotech, CA, USA) and 3 L

of a HiPerFect transfection reagent (301705, QIAGEN, Beijing, China) were added

respectively. 6 hours after the transfection, a DMEM medium containing 10% fetal

bovine serum was added for culture. The assay of protein detection used cell samples

that had been transfected with the small interfering RNA for three days.

PAX4 of cardiac fibroblasts infected with adenoviruses: the cardiac fibroblasts of P2

generation were passaged from a P1 generation into a 6-well plate on the night before

the assay of adding virus for infection, and cultured overnight in an environment of

37°C and 5% C02 to ensure that the morphology of the fibroblasts was spread and

any extracellular matrix that affected the transfection efficiency had not been

produced. When the cells were grown to a density of 60%, cells were infected with

2MOI PAX4 or control adenoviruses under a serum-free condition. After 6 hours, the

medium was replaced by DMEM containing 10% fetal bovine serum, and culture was

conducted for three days for subsequent assays.

To further determine the role of PAX4 in the cardiac fibroblasts, firstly, an assay was

designed to knock down PAX4 by transfecting the small interfering RNA of PAX4

into the cardiac fibroblasts, and the Western blot assay had confirmed that knock

down of PAX4 in the cardiac fibroblasts was effective (Fig. 12). Thereafter, the

fluorescence intensities of the myofibroblast markers fibronectin, aSMA and Col I

after knocking down of PAX4 were detected by an immunofluorescence assay. The

assay results showed that the fluorescence of the myofibroblast markers fibronectin

(Fig. 13A), aSMA (Fig. 13B) and Col I (Fig. 13C) was decreased after knocking

down of PAX4.

Three days after over-expression of PAX4 in the fibroblasts, a Western blot assay was

carried out. The assay results were shown in Fig. 14. After the over-expression of

PAX4, all the protein levels of the myofibroblast markers fibronectin, aSMA and Col

I were increased in different degrees.

All the aforementioned assays suggested that PAX4 could promote the occurrence of

cardiac fibrosis, and knocking down PAX4 could reduce the contents of the fibrosis markers fibronectin, aSMA and Col I.

Example 6 In the cardiac fibroblasts, the protein level of PAX4 in the cells was

knocked down, and thus the expression level of its downstream regulatory gene was

detected by means of a Western blot assay.

Both the animal experiment and the cell assay suggested that knocking down PAX4

could have a protection effect on the heart function. However, the mechanism of

action was still unclear. Therefore, we used bioinformatics analysis to find three

possible effect targets that affected cardiac fibrosis and might be regulated by PAX4:

TGFP (promoting fibrosis), and IL1R2 and TGIF2 (fibrosis inhibiting factors).

TRANSFAC predicted that PAX4 could bind to the promoters of TGFP, IL1R2 and

TGIF2, so we detected the protein levels of these three genes upon knock down of

PAX4 by the method of a Western blot assay.

As shown in Fig. 15, after PAX4 was knocked down in the cardiac fibroblasts, the

protein level of TGFP was decreased, while the protein levels of IL1R2 and TGIF2

were increased, which suggested that PAX4 played a multi role in promoting fibrosis

by promoting the fibrosis promoting factor TGFP and inhibiting the fibrosis inhibiting

factors IL1R2 and TGIF2.

The aforementioned specific embodiments are only preferred examples of the present

invention, rather than limiting the present invention, and any modification, equivalent

substitution and improvement within the spirit and principle of the present invention

should be included in the protection scope of the present invention. The above

descriptions are preferred examples of the present invention. It should be noted that a

person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present invention, but such improvements and modifications shall also be deemed as the protection scope of the present invention.

Claims (8)

Claims

1. Use of a PAX4 inhibitor in the manufacture of a medicament for inhibiting fibrosis.

2. The use according to claim 1, wherein the PAX4 inhibitor comprises siRNA and a

PAX4 gene knockout reagent.

3. The use according to claim 2, wherein the PAX4 gene knockout reagent is siRNA.

4. The use according to any one of claims 1-3, wherein thefibrosis refers to cardiac

fibrosis, pancreatic fibrosis or pulmonary fibrosis.

5. Use of a PAX4 gene or an expression product thereof in promoting the proliferation

of cardiac fibroblasts in vitro.

6. Use of a PAX4 gene knockout reagent in inhibiting the proliferation of cardiac

fibroblasts in vitro.

7. Use of a PAX4 gene knockout reagent in inhibiting or blocking the expression of a

fibrosis promoting factor TGF Pand promoting the expression of fibrosis inhibiting

factors IL1R2 and TGIF2 in cardiac fibroblasts in vitro.

8. Use of a PAX4 gene knockout agent in inhibiting cell fibrosis by inhibiting a

fibrosis promoting factor TGF Pand promoting fibrosis inhibiting factors IL1R2 and

TGIF2.