CN119770660A - Application of PAPSS1 agonists and endogenous protein sulfation modification upregulators in the preparation and screening of drugs for the treatment of ischemic diseases - Google Patents
Application of PAPSS1 agonists and endogenous protein sulfation modification upregulators in the preparation and screening of drugs for the treatment of ischemic diseases Download PDFInfo
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Abstract
本发明属于生物医药技术领域，涉及PAPSS1激动剂、内源性蛋白质硫酸化修饰上调剂在制备及筛选用于治疗缺血性疾病药物中的应用。本申请首次披露PAPSS1上调/激动及其介导的组蛋白硫酸化修饰与缺血性疾病的关联，为缺血性脑卒中等缺血性疾病提供了新的防治思路。
The present invention belongs to the field of biomedicine technology, and relates to the use of PAPSS1 agonists and endogenous protein sulfation modification up-regulators in the preparation and screening of drugs for the treatment of ischemic diseases. This application discloses for the first time the association between PAPSS1 up-regulation/agonism and histone sulfation modification mediated by it and ischemic diseases, providing a new prevention and treatment idea for ischemic diseases such as ischemic stroke.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of PAPSS1 agonists and endogenous protein sulfation modification upregulators in preparation and screening of medicines for treating ischemic diseases.
Background
PAPSS1 (3 '-Phosphoadenosine' -phosphosulfate synthetase 1,3 '-phosphoadenosine-5' -phosphosulfate synthase 1) belongs to the family of phosphoadenosine sulfate synthases (PAPS synthases), is an enzyme playing a key role in sulfation, is mainly responsible for synthesizing 3 '-phosphoadenosine-5' -phosphosulfate (PAPS), can mediate sulfate transferase catalytic reaction, and realizes protein sulfation modification by transferring sulfate ions from 3 '-phosphoadenosine-5' -phosphosulfate (PAPS) to endogenous protein residues, changes chromatin structure and gene expression, thereby regulating physiological functions of cells.
The invention belongs to the technical field of biological medicines, and particularly relates to application of PAPSS1 agonists and histone sulfation modified upregulators in preparation and screening of medicines for treating ischemic diseases.
At present, it has been reported that high expression of PAPSS1 is significantly associated with age, pathological grading, recurrence and shorter prognosis of patients with brain glioma, and moreover, the scholars believe that PAPSS1 gene has a correlation with herpes simplex virus type 1 infection, and the above studies indicate that PAPSS1 has important roles in human physiology. Thus, intensive research into PAPSS1 function and disclosure of related disease mechanisms is needed to provide new ideas and methods for diagnosis and treatment of more diseases.
Disclosure of Invention
A first object of the present invention is to provide the use of an upregulated PAPSS1 formulation, i.e. a PAPSS1 agonist, for the prevention and/or treatment of ischemic diseases, for the preparation of a medicament for the prevention and treatment of ischemic diseases.
The applicant of the application surprisingly found that a brand-new substrate synthetase PAPSS1 for sulfating modification of ischemic disease prevention and treatment targets, wherein PAPSS1 can mediate sulfating modification of endogenous proteins, up-regulation/activation of PAPSS1 can promote sulfate ions to transfer from 3 '-adenosine 5' -phosphosulfate (PAPS) to tyrosine residues on the endogenous proteins, and regulation and control of cell metabolism are realized by changing chromatin structure and gene expression, so that a stable energy source is provided for cells, and cell damage caused by ischemic damage is effectively relieved.
Accordingly, the present application provides the use of a PAPSS1 agonist in the prevention and/or treatment of ischemic diseases and in the preparation of a medicament for the prevention and/or treatment of ischemic diseases.
Furthermore, the PAPSS1 agonist of the application can prevent and/or treat ischemic diseases by promoting the sulfation modification of endogenous proteins and can realize the application in preparing medicines for preventing and/or treating ischemic diseases.
The term "ischemic disease" in the present application refers to a disease in which blood supply to local tissues is insufficient due to various causes, thereby causing pathological changes such as ischemia, hypoxia, etc., and causing dysfunction of the corresponding tissues and organs. Further, the ischemic disease is a disease of serious dysfunction or organ damage caused by hypoxia of tissues due to reduced or interrupted blood flow. Preferably, the ischemic diseases comprise ischemic cerebrovascular diseases, myocardial ischemia, liver ischemia, kidney ischemia, intestinal ischemia, ischemic heart disease, ischemic peripheral arterial disease and the like, preferably, the ischemic diseases are ischemic cerebrovascular diseases, more preferably, ischemic cerebral apoplexy and transient cerebral ischemic attack.
Further, the subject suffering from "ischemic disease" is a mammal, such as a rodent, primate, human.
PAPSS1 agonists in the present application refer to substances capable of promoting the activity of the sulfation modification substrate synthase PAPSS1, or substances capable of promoting transcription or expression of the PAPSS1 gene, including but not limited to small molecule compounds, antibodies, exogenous RNAs (e.g., oeRNA), plasmids containing PAPSS1 or exogenous RNAs, and plasmid-loaded viruses, and the like.
Further preferably, the virus is one of a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, and more preferably an adeno-associated virus;
further preferred, the adeno-associated virus comprises any one or more of the following plasmids:
pAAV[shRNA]-CAG>EGFP:T2A:Puro-U6>mPapss1;
pAAV[shRNA]-EGFP-U6>Scramble_shRNA;
pAAV[Exp]-CMV>mPapss1NM_011863.3:P2A:EGFP:WPRE;
and/or pAAV [ Exp ] -CAG > EGFP:WPRE.
The promotion of gene transcription or expression of the sulfated modified substrate synthase PAPSS1 of the present application can be verified by qPCR detection of mRNA levels and Western Blot analysis of protein expression amounts.
The applicant of the present application has further surprisingly found that PAPSS1 up-regulation/agonism promotes the sulphation modification of endogenous proteins, thus achieving a therapeutic effect on ischemic diseases. Accordingly, a second object of the present application is to provide the use of an endogenous protein sulfation modified up-regulator for the prevention and/or treatment of ischemic diseases, for the preparation of a medicament for the prevention and/or treatment of ischemic diseases.
The term "endogenous protein sulfation modified up-regulator" as used herein refers to a substance that promotes protein sulfation modification, and a substance that promotes transcription and expression of genes related to endogenous protein sulfation modified transferase, and includes, but is not limited to, small molecule compounds, antibodies, exogenous RNAs (e.g., oeRNA), plasmids containing exogenous RNAs, and viruses carrying plasmids, etc.
Further, the endogenous protein is a histone, and the corresponding "endogenous protein sulfation modification up-regulator" is preferably "histone sulfation modification up-regulator". More preferably, the histone is H3. More preferably, the site of sulfation modification of histone H3 is Y99, i.e. H3Y99.
The PAPSS1 agonist or endogenous protein sulfation modification up regulator can be used as the only or main active ingredient, can be prepared into medicines for preventing and treating ischemic diseases, and can be prepared into a proper pharmaceutical preparation form. The pharmaceutical formulation forms include, but are not limited to, solid, liquid, gel, semi-fluid, aerosol. Preferably, the pharmaceutical preparation form can be specifically injection (such as liquid injection or powder injection), tablet, capsule, soft capsule, granule, oral liquid, pill, film, inhalant, spray, in situ gel and other dosage forms, and is prepared by a conventional preparation method.
In addition to the active ingredients, the pharmaceutical preparation can contain one or more pharmaceutically acceptable carriers or auxiliary materials according to the dosage form requirement.
A third object of the present invention is the use of PAPSS1 or endogenous protein sulfation modifications mediated by PAPSS1 as targets for screening drugs/active compounds for the prevention and treatment of ischemic diseases, i.e. PAPSS1 agonism or histone sulfation modifications upregulation as targets for the preparation of a product for in vitro screening of candidate substances for the treatment of ischemic diseases.
The screening method provided by the application comprises the following steps:
Verifying whether the PAPSS1 can be up-regulated by the drug to be screened, and determining the PAPSS1 as an ischemic disease prevention and treatment candidate drug/candidate active substance;
And/or verifying whether the drug/active substance to be screened can up-regulate the sulfation modification level, and determining the drug/active substance to be screened as an ischemic disease prevention candidate drug/active substance.
Further, the screening method comprises the following steps:
the drug to be screened is applied to cells in vitro capable of expressing PAPSS1 to determine whether the level of PAPSS1 or histone sulfation modification in the cells is up-regulated.
Preferably, the screening mode comprises the steps of culturing cells capable of carrying out histone sulfation modification in vitro under the condition of suitable cell growth, setting two groups of comparison experiments, adding the medicine to be screened/the substance to be screened into a culture dish of the cells in vitro, adding an equal amount of physiological saline into the other group, incubating under the same conditions, and testing whether the PAPSS1 or histone sulfation modification level in the cells in vitro is up-regulated. Determination of PAPSS1 or histone sulfation modification levels in cells was detected using Western Blot detection or mass spectrometry. If PAPSS1 up-regulation or histone sulfation modification in cells is improved, the drug to be screened/active substance to be screened can be used as a candidate substance for non-clinical and clinical continuous verification of whether the drug has anti-ischemic disease drugs.
Compared with the prior art, the application discloses the up-regulation/excitation of PAPSS1 and the association of histone sulfation modification mediated by the up-regulation/excitation and ischemic diseases for the first time, which is a brand new effect discovery, and no report on the preparation of up-regulation/excitation of PAPSS1 in preventing and treating ischemic diseases and preparing medicines with corresponding purposes is seen in the global scope.
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FIG. 1 is a dynamic change chart of H3Y99sulf in SY5Y and HMC3 cell models, wherein FIG. 1a is an immunoblotting chart of the SY5Y, HMC3 cell OGD/R process, FIG. 1b is a western blot statistical analysis result of a SY5Y cell OGD/R model H3Y99sulf, and FIG. 1c is a western blot statistical analysis result of an HMC3 cell OGD/R model H3Y99 sulf;
FIG. 2 is a graph showing the change in expression of a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model H3Y99sulf, wherein FIG. 2a shows the result of a western blot of changes in the MCAO/R process of H3Y99 sulfur, and FIG. 2b shows the result of a western blot statistical analysis of H3Y99 sulf;
FIG. 3 is a graph showing changes in PAPS 1 expression and H3Y99sulf modification levels in the case of OGD and OGDR for SY5Y and HMC3 cells, wherein FIG. 3a shows the western blot results of PAPS 1 and H3Y99sulf in SY5Y and HMC3 cells OGD3H, and FIG. 3b shows the western blot results of PAPS 1 and H3Y99sulf in SY5Y and HMC3 cells OGD 3H/R12H;
FIG. 4 is a graph showing changes in PAPSS1 expression and H3Y99sulf modification levels in brain tissue of MCAO/R0d and MCAO/R3d mice, wherein FIG. 4a shows the western blot results of PAPSS1 and H3Y99sulf in brain tissue of the opposite and opposite ischemia sides of the MCAO/R0d mice, FIG. 4b shows the western blot statistical analysis results of PAPSS1 and H3Y99sulf in brain tissue of the MCAO/R0d mice, FIG. 4cPAPSS1 and H3Y99sulf show the western blot results of PAPSS1 and H3Y99sulf in brain tissue of the opposite and opposite ischemia sides of the MCAO/R0d mice, and FIG. 4d shows the western blot statistical analysis results of PAPSS1 and H3Y99sulf in brain tissue of the MCAO/R3d mice;
FIG. 5 is a graph showing the effect of PAPSS1 overexpression and knockdown on the level change of H3Y99sulf, wherein FIG. 5a is a western blot of H3Y99sulf in PAPSS1 overexpressing cells (SY 5Y, HMC 3), FIG. 5b is a western blot of H3Y99sulf from knockdown PAPSS1 cells (HEK 293T) after OGD treatment, and FIG. 5c is a statistical analysis of H3Y99sulf levels from knockdown PAPSS1 cells after OGD treatment;
FIG. 6 is a graph showing the effect of AAV-mediated PAPSS1 overexpression and knock-down on histone sulfation in an animal model, wherein FIG. 6a is a graph showing the western blot results of analysis and verification of PAPSS1, H3Y99sulf overexpression efficiency in a PAPSS1 model, FIG. 6b is a graph showing the statistical analysis of the relative quantization level of PAPSS 1in a PAPSS1 overexpression model, FIG. 6c is a graph showing the statistical analysis of the relative quantization level of H3Y99sulf in a PAPSS1 overexpression model, FIG. 6d is a graph showing the western blot results of verification of the knock-down efficiency of PAPSS1 using PAPSS1 antibodies in a PAPSS1 knock-down model, FIG. 6e is a graph showing the statistical analysis of the relative quantization level of PAPSS 1in a PAPSS1 knock-down model, and FIG. 6f is a graph showing the statistical analysis of the relative quantization level of H3Y99sulf in a PAPSS1 knock-down model;
FIG. 7 is an experimental view of SY5Y and HMC3 cells OGDR over-expressing PAPSS1, wherein FIG. 7a is a SY5Y cell count plot, FIG. 7b is an HMC3 cell count plot, FIG. 7c is a SY5Y cell count statistical analysis plot, and FIG. 7d is an HMC3 cell count statistical analysis plot;
FIG. 8 is a schematic diagram of SY5Y and HMC3 cell experiments with PAPSS1 knockdown, wherein FIG. 8a is a SY5Y cell count diagram, FIG. 8b is a HMC3 cell count diagram, FIG. 8c is a SY5Y cell count statistical analysis diagram, and FIG. 8d is a HMC3 cell count statistical analysis diagram;
FIG. 9 is a schematic diagram of an animal experiment evaluating the effects of regulatory PAPSS1 and its mediated H3Y99sulf on MCAO mouse neurological function;
FIG. 10 is a graph of TTC staining of cerebral infarct areas in mice from different treatment groups;
FIG. 11 is a graph showing quantitative statistics of TTC staining of cerebral infarction volumes of mice in different treatment groups;
FIG. 12 is a statistical plot of modified neurological score (mNSS) of mice in different treatment groups following MCAO.
Detailed Description
The technical scheme of the present invention will be clearly and completely described below. It will be apparent that the examples described below are only some, but not all, embodiments of the invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Immunoblot signal intensity and staining images in the following examples were all quantitatively analyzed by ImageJ (national institutes of health) software. The following examples, in which no specific conditions are noted, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products available commercially without the manufacturer's knowledge. Those skilled in the art can make appropriate modifications to the technology described herein to make and use the invention, but similar substitutions and modifications are intended to be included within the scope of the present disclosure.
Example 1 PASS1-mediated dynamic modulation of histone sulfation modification (H3Y 99 sulf) during cerebral ischemia reperfusion
In this example, the dynamic changes of H3Y99sulf in human neuroblastoma cell line (SY 5Y) and human microglial cell line (HMC 3) were studied using hypoxia glucose deprivation-reperfusion (OGD/R) as a cell model, and the dynamic changes of H3Y99sulf in a mouse MCAO model were studied using mice treated with MCAO/R as an animal model.
Dynamic changes in H3Y99sulf in OGD/R cell model
The method comprises the following steps of establishing an anoxic and ischemic reperfusion (OGD/R) model:
SY5Y cells and HMC3 cells were cultured in a sugar-free medium and placed in an aerated incubator, the gas in the incubator was continuously introduced in a ratio of 94% N2, 5% CO2, 1% O2, equilibrated for 15 minutes, and the incubator was sealed, and then placed in the incubator for 3 hours of anaerobic glucose-deficient treatment (OGD). After the end of the treatment, the cells were subjected to reperfusion (OGD/R), i.e. continued culture in DMEM medium, and samples were collected at 0, 3, 6, 12, 24 and 48 hours, respectively, for subsequent experimental analysis.
To assess the changes in the OGD/R cell model of H3Y99sulf, we analyzed the expression levels of H3Y99sulf in SY5Y and HMC3 cells by Western blot. The experiments were divided into control and different treatment groups with reperfusion for 0h, 3h, 6h, 12h, 24h and 48h after 3 hours of OGD treatment. As shown in fig. 1, the expression level of H3Y99sulf in SY5Y and HMC3 cells was significantly up-regulated after OGD treatment compared to control, while its expression was gradually reduced during subsequent reperfusion. The expression level of H3Y99sulf gradually rises with the extension of the recharging time. FIG. 1a shows the result of western blot of H3Y99sulf during the OGD/R of SY5Y and HMC3 cells, histone H3 was used as an internal control to verify protein load consistency. FIG. 1b shows the result of a western blot analysis of H3Y99sulf in SY5Y cells (n=3,P<0.05,P < 0.01), fig. 1c shows the result of western blot statistical analysis of H3Y99sulf in HMC3 cells (n=3,P<0.05,P<0.01)。
The above results indicate that histone sulfation modifications (H3Y 99 sulf) undergo dynamic regulation during OGD/R.
Dynamic changes of H3Y99sulf in (two) MCAO/R mouse model
A cerebral middle artery ischemia reperfusion (MCAO/R) mouse model is established by adopting a line plug method (Thread occlusion method), namely, a Longa equal line plug method is used for simulating a cerebral ischemia reperfusion process by adopting reperfusion after the middle cerebral artery is occluded for l.5 hours, and the mouse is anesthetized and skin-prepared and then is cut along the median line of the neck for about 1cm, so that muscles, blood vessels and nerves on the left side and the right side are dissociated, and damage is avoided and fixation is realized. The right Common Carotid Artery (CCA), internal Carotid Artery (ICA), and External Carotid Artery (ECA) were exposed. Ligating the ECA distal end and temporarily blocking blood flow at the proximal CCA end and ICA bifurcation. The Middle Cerebral Artery (MCA) is finally reached by inserting the nylon Long Xianshuan through the ECA stump and entering the ICA through the CCA and the ICA bifurcation. When resistance is encountered, insertion is stopped and the plug insertion length is about 9-11mm. After 1.5 hours of ischemia, the mice were again anesthetized, the neck incision was cut, the neck blood vessel was exposed, the blood flow of ECA was temporarily blocked, the plug was pulled out to restore blood flow, and the reperfusion process was started, and samples were collected at 0h, 8h, 24h, 3d and 7d of reperfusion, respectively, for subsequent experimental analysis. The false operation group carries out the central incision of the neck after anesthesia, finds the right common carotid artery and internal and external carotid arteries, separates carefully, does not insert a wire plug, and directly sews the skin.
To assess the change in H3Y99sulf in the MCAO/R mouse model, we analyzed the expression level of H3Y99sulf in mouse ischemic side brain tissue by Western blot. The experiments were divided into sham surgery groups and different treatment groups with MCAO 1.5 hours later, reperfusion for 0h, 8h, 24h, 3d and 7 d. As shown in fig. 2, the level of H3Y99sulf modification was significantly up-regulated after 1.5H of cerebral ischemia compared to sham, while during subsequent reperfusion, the level of modification gradually decreased and the expression level of H3Y99sulf gradually increased with prolonged reperfusion time. Wherein FIG. 2a is a western blot result of H3Y99sulf varied in MCAO/R process, histone H3 was used as an internal reference to verify protein loading consistency FIG. 2b is a western blot statistical analysis result of H3Y99sulf in mouse ischemia side brain tissue (n=5,P<0.05,P<0.01) 。
The above results indicate that, consistent with the cellular experiments, H3Y99sulf underwent dynamic regulation during the mouse MCAO/R process.
Association of (III) histone sulfation modification (H3Y 99 sulf) with PAPSS1
To verify that the dynamic regulatory properties exhibited by histone sulfation modifications (H3Y 99 sulf) during cerebral ischemia reperfusion are mediated by PAPSS1, the expression of PAPSS1 and the level of H3Y99sulf modifications at the level of ischemia (OGD 3H) and reperfusion (OGD 3H/R12H) in cells (FIG. 3a,3 b) and the expression of PAPSS1 and the level of H3Y99sulf modifications at the level of ischemia (MCAO1.5H) and reperfusion (MCAO 1.5/R3 d) in animals (FIG. 4a,4 b) were examined.
FIG. 3 shows PAPSS1 expression and H3Y99sulf modification levels in OGD and OGDR for SY5Y and HMC3 cells. Wherein FIG. 3a is a graph showing the result of western blot of PAPSS1 and H3Y99sulf in SY5Y and HMC3 cells OGD3H, and Tubulin and Histone H3 are used as an internal reference to verify the consistency of the protein loading, and FIG. 3b is a graph showing the result of western blot of PAPSS1 and H3Y99sulf in SY5Y and HMC3 cells OGD3H/R12H, and Tubulin and H3 are used as an internal reference to verify the consistency of the protein loading.
The results in FIG. 3 show that OGD treatment up-regulates the expression level of PAPSS1 protein in SY5Y, HCM cells, respectively, and that PAPSS1 expression is down-regulated after OGDR treatment, consistent with the trend of H3Y99sulf in cell experiments.
FIG. 4 shows PAPSS1 expression and H3Y99sulf modification levels in brain tissue of MCAO/R0d and MCAO/R3d mice. Wherein, FIG. 4a shows the western blot results of PAPSS1 and H3Y99sulf in brain tissue of MCAO/R0d mice on the opposite ischemic side and on the ischemic side, tubulin and Histone H3 were used as internal references to verify the consistency of the protein loading amounts, FIG. 4b shows the western blot statistical analysis results of PAPSS1 and H3Y99sulf in brain tissue of MCAO/R0d mice (n=6,P<0.05,P < 0.01), FIG. 4c shows the western blot results of PAPSS1 and H3Y99sulf in brain tissue of the opposite and opposite ischemic side of MCAO/R0d mice, tubulin and H3 were used as internal controls to verify the consistency of protein loading, FIG. 4d shows the western blot statistical analysis results of PAPSS1 and H3Y99sulf in brain tissue of MCAO/R3d mice (n=6,P<0.05,P<0.01)。
The results in FIG. 4 show that MCAO treatment upregulated the expression level of PAPSS1 protein in mouse ischemic brain tissue compared to the opposite side, and that MCAO/R treatment down-regulated PAPSS1 expression, consistent with the trend of H3Y99sulf in animal experiments. It was shown that dynamic regulation of H3Y99sulf during cerebral ischemia reperfusion may be mediated by PAPSS 1.
Example 2 Effect of PASS1 overexpression and knock-down on the level of histone sulfation modification
Cell assay (one):
1. Plasmid construction
The coding sequence of human PAPSS1 was amplified by PCR and cloned into pHAGE/NEO (+) -Flag vector. The oligonucleotide sequences for knocking down the expression of the corresponding gene proteins were inserted into the pLKO.1/Puro (+) vector with the specific sequence PAPSS1 (human) TGGATCGAGTTTATTGGAATG.
2. Lentivirus packaging and infection
2X 10≡6 HEK293T cells were seeded in 100-mm cell culture dishes and transfected with PEI, the transfection procedure was performed as per the manufacturer's instructions. To express the target protein, we co-transfected psPAX, pMD2.G, and core plasmid (pHAGE-Flag-PAPSS 1) to generate lentiviruses, and to knock down the target protein, we co-transfected pMDL, pVSVG, pREV and pLKO.1 (sh-Scramble, sh-PAPSS 1) to generate lentiviruses. For lentiviral infection, 500. Mu.l of virus-containing medium was added to 5X 10. Sup.4 cells and 1% polybrene was added.
Fig. 5 shows that over-expression of PAPSS1 can up-regulate H3Y99sulf levels, while knock-down of PAPSS1 significantly reduces the effect of OGD treatment on H3Y99sulf levels.
FIG. 5a shows the western blot results of H3Y99sulf in PAPSS1 overexpressing cells (SY 5Y, HMC 3), with Tubulin and Histone H3 used as internal controls to verify the consistency of protein loading.
FIG. 5b shows the western blot results of H3Y99sulf from knock-down PAPSS1 cells (HEK 293T) after OGD treatment, tubulin and Histone H3 were used as internal controls to verify the consistency of protein loading. It can be seen that knockdown of PAPSS1 significantly reduces the effect of OGD treatment on H3Y99sulf levels in cells.
FIG. 5c shows a statistical analysis using a double-sided t-testP < 0.05), data are presented as mean ± standard deviation (s.d.), error bars represent the range of three independent experiments. The results in FIG. 5 show that over-expression of PAPSS1 will up-regulate the level of H3Y99sulf in the cells, while knock-down of PAPSS1 significantly reduces the effect of OGD treatment on H3Y99sulf levels in the cells.
(II) animal experiments:
in this example, the effect of histone sulfation modification levels was verified by adeno-associated virus (AAV) -mediated PAPSS1 gene knockdown and overexpression. The specific operation method is as follows:
1. Constructing a plasmid:
adeno-associated virus was constructed with the following plasmids:
pAAV[shRNA]-CAG>EGFP:T2A:Puro-U6>mPapss1;
pAAV[shRNA]-EGFP-U6>Scramble_shRNA;
pAAV[Exp]-CMV>mPapss1NM_011863.3:P2A:EGFP:WPRE;
and pAAV [ Exp ] -CAG > EGFP:WPRE.
2. Virus injection:
Each mouse was injected at four sites of cortex and hippocampus (0.5. Mu.L per site at 0.25. Mu.L/min) at point 1, bregma 0.4 mm, lateral 2.3 mm, depth 1.8 mm, point 2, bregma 0.4 mm, lateral 2.3 mm, depth 3.5 mm, point 3, bregma 2.2 mm, lateral 1.8 mm, depth 1.5 mm, point 4, bregma 2.2 mm, lateral 3.0mm, depth 1.5 mm, all points located in the left hemisphere (i.e., ipsilateral to MCAO). Virus injection was performed using a stereotactic apparatus, injecting 0.5 μl of virus per spot, at a rate of 0.25 μl/min. The needle slowly withdraws within 10 minutes. Mice were subjected to animal experiments 21 days after virus injection.
FIG. 6 shows the effect of AAV-mediated PAPSS1 overexpression and knock-down on histone sulfation (H3Y 99 sulf) levels in animal models.
Specifically, FIG. 6a is a graph of western blot results of analytical verification of PAPS 1, H3Y99sulf overexpression efficiency in PAPS 1 overexpression model. Tubulin and Histone H3 were used as internal controls to verify that the consistent protein load H3Y99sulf intensity values were normalized to the intensity values of histone H3 in the same sample.
FIGS. 6b and 6c are graphs of statistical analyses of relative quantitative levels of PAPSS1 and H3Y99sulf in a PAPSS1 overexpression model, respectively, using a two-sided t-test,P<0.01;P < 0.05), data are presented as mean ± standard deviation (s.d.), error bars represent the range of three independent experiments.
The results indicated that AAV-mediated overexpression of PAPSS1 upregulated H3Y99sulf levels.
FIG. 6d shows the western blot results of PAPS 1 knockdown efficiency using PAPS 1 antibodies in PAPS 1 knockdown model. Tubulin and Histone H were used as internal controls to verify consistent protein loading H3Y99sulf intensity values.
FIGS. 6e and 6f are graphs of statistical analyses of PAPS 1 relative quantification levels and H3Y99sulf relative quantification levels, respectively, in the PAPS 1 knockdown model, using a two-sided t-test,P<0.01;P < 0.05). Data are presented as mean ± standard deviation (s.d.), error bars represent the range of three independent experiments.
The results indicated that AAV-mediated PAPSS1 knockdown down H3Y99sulf levels.
According to the embodiment 1 and the embodiment 2, the PAPSS1 mediated H3Y99sulf presents dynamic regulation and control in ischemia reperfusion injury, and the time expression mode reveals that the PAPSS1 mediated H3Y sulf has important roles in cell injury and repair process, can be used as a potential target, and has application prospect in preparing and screening medicines for preventing and treating ischemic diseases.
Example 3 Effect of PASS1 overexpression and knockdown on ischemia-induced cell injury
This example shows that over-expression of PAPSS1 reduces ischemia-induced cell damage and knocking down PAPSS1 aggravates ischemia-induced cell damage.
SY5Y and HMC3 cells overexpressing PAPSS1 were treated with OGDR, respectively. Imaged by a cytometer and analyzed quantitatively using a cytometer (fig. 7a, 7 b).
Statistical analysis of data using double-sided t-testP < 0.05). Data are presented as mean ± standard deviation (s.d.), error bars represent the range of four independent experiments (fig. 7c, 7 d).
The results indicate that PAPSS1 overexpression increases the survival of SY5Y and HMC3 cells under OGDR stress.
SY5Y and HMC3 cells using knockdown PAPSS1 were treated with OGDR. Imaged by a cytometer and quantitatively analyzed using a cytometer (fig. 8a, 8 b).
Statistical analysis of data using double-sided t-testP < 0.05). Results are expressed as mean ± standard deviation (s.d.), error bars are the range of four independent experiments (fig. 8c, 8 d).
The results indicate that knock-down of PAPSS1 reduced the survival of SY5Y and HMC3 cells under OGDR stress.
Example 4 effects of knockdown and upregulation of PAPSS1 expression levels on impaired nerve function in MCAO mice
In this example, to verify whether the neuroprotective effects of regulatory PAPSS1 on cells could be demonstrated in animal models, the effects of supplementation with H3Y99sulf complement (Na 2SO4）、Na2SO4 in combination with negative regulatory PAPSS1 and positive regulatory PAPSS1 alone on MCAO mouse models were evaluated.
Experimental grouping and intervention (operational flow chart see FIG. 9, summarizing the overall design and flow of the experiment)
Healthy male C57BL/6 mice were divided into the following 5 groups according to the random number table method, 5 of which were sham surgery + NaCl group, 8 of the other mice each, and all mice of each group were subjected to mNSS neurological function scoring and TTC staining to calculate cerebral infarction volumes:
1. False operation + NaCl group, namely, after anesthesia, going through the median incision of the neck, finding the right common carotid artery and internal and external carotid arteries, carefully separating, directly suturing the skin without inserting a wire plug. MCAO was injected daily at the tail vein with 2.06 mg/kg NaCl (consistent with the sodium ion concentration in Na 2SO4 at 2.5mg/kg, excluding the effect of sodium ions on the experiment) once daily for 3 consecutive days.
2. MCAO+NaCl group, blocking left middle cerebral artery of mice by using a thrombus method for 1.5h, reperfusion for 3d, and detecting mNSS score every day after successful modeling. MCAO was injected by tail vein of 2.06 mg/kg of NaCl once daily for 3 consecutive days.
Mcao+h3y99sulfur complement (Na 2SO4) group mNSS scores were measured daily after successful molding by blocking the left middle cerebral artery of mice for 1.5h with a wire-plug method, reperfusion for 3 d. MCAO was injected with 2.5 mg/kg of Na 2SO4 by tail vein once daily for 3 consecutive days.
MCAO+Na 2SO4 +AAV-shPAPSS group, wherein the left middle cerebral artery of the mice is blocked by a wire bolt method for 1.5h, reperfusion is carried out for 3d, and mNSS score is detected every day after successful modeling. The brain was subjected to AAV-shPAPSS1 microinjection 21 days before MCAO surgery, and 2.5. 2.5 mg/kg of Na 2SO4 was injected into the tail vein every day after MCAO surgery, once a day for 3 consecutive days.
MCAO+NaCl+AAV-oePAPSS groups, blocking left middle cerebral artery of mice by using a wire-plug method for 1.5h, reperfusion for 3d, and detecting mNSS score every day after successful modeling. AAV-oePAPSS1 was microinjected into the brain 21 days before MCAO surgery, and 2.06 mg/kg of NaCl was injected into the tail vein of the brain once a day for 3 consecutive days after MCAO surgery.
(II) Critical Experimental methods
1.2, 3, 5-Triphenyltetrazolium chloride (TTC) staining
The determination of cerebral infarct volume was performed by 2,3, 5-triphenyltetrazolium chloride (TTC) staining. The coronal brain sections were immersed in 2% TTC solution and incubated at 37 ℃ for 20 min. The non-infarcted area was shown as red, while the infarcted area was white. Images of infarcted and non-infarcted hemispheres were analyzed by ImageJ software.
2. Improved neurological scoring (mNSS)
The scoring system is generally divided into 18 points, including three aspects of motor, sensory and reflex functions. Motor function (0-8 points) mainly evaluates gait, coordination and motor ability of mice, sensory function (0-6 points) evaluates responses of mice to external stimuli such as touch sense and pain sense, and reflex function (0-4 points) scores according to responses such as plantar reflex and tail reflex. Scoring was performed by two blind evaluators, with higher scores indicating more severe neurological deficit.
(III) results of experiments
The results show that the cerebral infarction volume of the MCAO mice can be significantly reduced by intravenous injection of H3Y99sulf complement sulfate (figure 10), and the area is reduced from 31.6+/-5.0% to 16.8+/-6.3% (figure 11). In addition, na 2SO4 also improved the neurological impairment score (mNSS) following MCAO in mice (fig. 12).
The above results further demonstrate that up-regulation of H3Y99sulf is effective in ameliorating impaired neurological function in ischemic mice.
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