Application of PAPSS1 agonist and endogenous protein sulfation modification up-regulating agent in preparation and screening of medicine for treating ischemic diseases

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of a PAPSS1 agonist and an endogenous protein sulfation modification up-regulating agent in preparation and screening of medicines for treating ischemic diseases. According to the application, the association between PAPSS1 up-regulation / excitation and histone sulfation modification mediated by PAPSS1 up-regulation / excitation and ischemic diseases is disclosed for the first time, and a new prevention and treatment thought is provided for ischemic cerebral apoplexy and other ischemic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of PAPSS1 agonists and endogenous protein sulfation modification up-regulators in the preparation and screening of drugs for treating ischemic diseases. Background Art

[0002] PAPSS1 (3'-Phosphoadenosine 5'-phosphosulfate synthetase 1, 3'-phosphoadenosine-5'-phosphosulfate synthase 1) belongs to the phosphoadenosine sulfate synthase (PAPS synthase) family. It is an enzyme that plays a key role in the sulfation reaction. It is mainly responsible for the synthesis of 3'-phosphoadenosine-5'-phosphosulfate (PAPS). It can mediate the sulfotransferase catalytic reaction and transfer sulfate ions from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to endogenous protein residues to achieve protein sulfation modification, change chromatin structure and gene expression, and thus regulate the physiological functions of cells.

[0003] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a PAPSS1 agonist and a histone sulfation modification up-regulator in the preparation and screening of drugs for treating ischemic diseases.

[0004] At present, it has been reported that high expression of PAPSS1 is significantly correlated with the age, pathological grade, recurrence and shorter prognosis of patients with brain glioma. In addition, some scholars believe that the PAPSS1 gene is correlated with herpes simplex virus type 1 infection. The above studies show that PAPSS1 plays an important role in human physiology and pathology. Therefore, in-depth research on the function of PAPSS1 and the revelation of related disease mechanisms need to be further deepened to provide new ideas and methods for the diagnosis and treatment of more diseases. Summary of the invention

[0005] The first object of the present invention is to provide a method for upregulating PAPSS1, namely, a PAPSS1 agonist, for use in preventing and / or treating ischemic diseases and in the preparation of a drug for preventing and treating ischemic diseases.

[0006] The applicant of the present application surprisingly discovered a novel target for the prevention and treatment of ischemic diseases, a sulfation-modified substrate synthase PAPSS1. PAPSS1 can mediate the sulfation modification of endogenous proteins. Upregulating / stimulating PAPSS1 can promote the transfer of sulfate ions from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to tyrosine residues on endogenous proteins, thereby regulating cell metabolism by changing chromatin structure and gene expression, thereby providing cells with a stable energy source and effectively alleviating cell damage caused by ischemic injury.

[0007] Therefore, the present application provides the use of a PAPSS1 agonist in preventing and / or treating ischemic diseases and in preparing a drug for preventing and / or treating ischemic diseases.

[0008] Furthermore, the PAPSS1 agonist of the present application prevents and / or treats ischemic diseases by promoting sulfation modification of endogenous proteins, and is used in the preparation of drugs for preventing and / or treating ischemic diseases.

[0009] "Ischemic disease" in this application refers to a disease caused by insufficient blood supply to local tissues due to various reasons, which in turn causes pathological changes such as ischemia and hypoxia, and causes dysfunction of corresponding tissues and organs. Furthermore, the ischemic disease is a disease of severe dysfunction or organ damage caused by tissue hypoxia due to reduced or interrupted blood flow. Preferably, the ischemic disease includes ischemic cerebrovascular disease, myocardial ischemia, liver ischemia, kidney ischemia, intestinal ischemia, ischemic heart disease, ischemic peripheral arterial disease and other diseases. Preferably, the ischemic disease is ischemic cerebrovascular disease, and more preferably includes ischemic stroke and transient ischemic attack.

[0010] Furthermore, the subject suffering from "ischemic disease" is a mammal, such as a rodent, a primate, or a human.

[0011] The PAPSS1 agonist in this application refers to a substance that can promote the activity of the sulfated modified substrate synthase PAPSS1, or a substance that can promote the transcription or expression of the PAPSS1 gene, including but not limited to small molecule compounds, antibodies, exogenous RNA (such as oeRNA), plasmids containing PAPSS1 or exogenous RNA, and viruses loaded with plasmids, etc.

[0012] Further preferably, the virus is one of lentivirus, adenovirus, adeno-associated virus, and retrovirus; more preferably adeno-associated virus; Further preferably, 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.

[0013] In the present application, the promotion of gene transcription or expression of the sulfated substrate synthase PAPSS1 can be verified by detecting the mRNA level by qPCR and analyzing the protein expression by Western Blot.

[0014] The applicant of the present application further surprisingly found that PAPSS1 up-regulation / excitation promotes endogenous protein sulfation modification, thereby achieving a therapeutic effect on ischemic diseases. Therefore, the second object of the present invention is to provide an endogenous protein sulfation modification up-regulator for preventing and / or treating ischemic diseases and preparing a drug for preventing and / or treating ischemic diseases.

[0015] The "endogenous protein sulfation modification up-regulator" in this application refers to a substance that promotes protein sulfation modification, as well as a substance that promotes the transcription and expression of endogenous protein sulfation modification transferase-related genes, including but not limited to small molecule compounds, antibodies, exogenous RNA (such as oeRNA), plasmids containing exogenous RNA, and viruses loaded with plasmids.

[0016] Further, the endogenous protein is a histone, and the corresponding "endogenous protein sulfation modification up-regulator" is preferably a "histone sulfation modification up-regulator". More preferably, the histone is H3. More preferably, the sulfation modification site of the histone H3 is Y99, i.e. H3Y99.

[0017] The PAPSS1 agonist or endogenous protein sulfation modification up-regulator in the present application can be used as the sole or main active ingredient to prepare a drug for preventing and treating ischemic diseases, and can be prepared into a suitable pharmaceutical preparation form. The pharmaceutical preparation form includes but is not limited to solid, liquid, gel, semi-fluid, and aerosol. Preferably, the pharmaceutical preparation form can be specifically an injection (such as a liquid injection or a powder injection), a tablet, a capsule, a soft capsule, a granule, an oral liquid, a pill, a film, an inhalant, a spray, an in-situ gel, and the like, and is prepared by a conventional preparation method.

[0018] In addition to the active ingredients, the pharmaceutical preparation may contain one or more pharmaceutically acceptable carriers or excipients according to the dosage form requirements.

[0019] The third purpose of the present invention is to use PAPSS1 or endogenous protein sulfation modification mediated by PAPSS1 as a target for screening drugs / active compounds for preventing and treating ischemic diseases, that is, to use PAPSS1 activation or up-regulation of histone sulfation modification as a target in the preparation of products for in vitro screening of candidate substances for the treatment of ischemic diseases.

[0020] The screening method described in the present application comprises the following steps: Verify whether the drug to be screened can upregulate PAPSS1, and if so, identify it as a candidate drug / candidate active substance for the prevention and treatment of ischemic diseases; And / or, verify whether the drug / active substance to be screened can upregulate the sulfation modification level. If it is upregulated, it will be determined as a candidate drug / candidate active substance for the prevention and treatment of ischemic diseases.

[0021] Furthermore, the screening method comprises: The drugs to be screened are applied to in vitro cells that can express PAPSS1 to determine whether the levels of PAPSS1 or histone sulfation modification in the cells are upregulated.

[0022] Preferably, the screening method can be: culturing cells capable of histone sulfation modification in vitro under conditions suitable for cell growth, setting up two groups of comparative experiments, one group adding the drug to be screened / substance to be screened to the culture dish of the in vitro cells, and the other group adding an equal amount of physiological saline, and the other conditions are the same, incubating, and testing whether the PAPSS1 or histone sulfation modification level in the in vitro cells is upregulated. Western Blot detection or mass spectrometry detection is used to determine the level of PAPSS1 or histone sulfation modification in the cells. If PAPSS1 is upregulated or histone sulfation modification is increased in the cells, the drug to be screened / active substance to be screened can be used as a candidate substance for non-clinical and clinical further verification of whether it has anti-ischemic disease drugs.

[0023] Compared with the prior art, this application discloses for the first time the association between upregulation / excitation of PAPSS1 and its mediated histone sulfation modification and ischemic diseases, which is a completely new discovery of its role. Globally, there have been no reports on the use of preparations that upregulate / excite PAPSS1 in the prevention and treatment of ischemic diseases and the preparation of drugs for corresponding uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure shows the dynamic changes of H3Y99sulf in SY5Y and HMC3 cell models; Figure 1 a is the immunoblot image of the OGD / R process of SY5Y and HMC3 cells; Figure 1 b shows the statistical analysis results of western blot of H3Y99sulf in the OGD / R model of SY5Y cells; Figure 1 c is the western blot statistical analysis results of H3Y99sulf in the OGD / R model of HMC3 cells; Figure 2 This is a graph showing the expression changes of H3Y99sulf in the middle cerebral artery occlusion / reperfusion (MCAO / R) mouse model; Figure 2 a Western blot results of the changes of H3Y99sulf during MCAO / R; Figure 2 b is the statistical analysis result of western blot of H3Y99sulf; Figure 3 The expression of PAPSS1 and the changes of H3Y99sulf modification levels in SY5Y and HMC3 cells under OGD and OGDR conditions are shown in Figure 2. Figure 3 a is the western blot result of PAPSS1 and H3Y99sulf in SY5Y and HMC3 cells OGD3h; Figure 3 b is the western blot results of PAPSS1 and H3Y99sulf in SY5Y and HMC3 cells OGD3h / R12h; Figure 4 Figure 2 is a graph showing changes in PAPSS1 expression and H3Y99sulf modification levels in brain tissues of MCAO / R0d and MCAO / R3d mice; Figure 4 a is the western blot result of PAPSS1 and H3Y99sulf in the ischemic contralateral and ischemic brain tissues of MCAO / R0d mice; Figure 4 b shows the statistical analysis results of western blot of PAPSS1 and H3Y99sulf in brain tissue of MCAO / R0d mice; Figure 4 Western blot results of cPAPSS1 and H3Y99sulf in the ischemic contralateral and ischemic brain tissues of MCAO / R0d mice; Figure 4 d is the western blot statistical analysis results of PAPSS1 and H3Y99sulf in the brain tissue of MCAO / R3d mice; Figure 5 This is a graph showing the effect of PAPSS1 overexpression and knockdown on the level of H3Y99sulf; Figure 5 a is the western blot result of H3Y99sulf in PAPSS1 overexpressing cells (SY5Y, HMC3); Figure 5 b is the western blot result of H3Y99sulf in PAPSS1 knockdown cells (HEK293T) and then treated with OGD; Figure 5 c is the statistical analysis result of H3Y99sulf level in PAPSS1 knockdown cells after OGD treatment; Figure 6 This is a diagram showing the effects of AAV-mediated PAPSS1 overexpression and knockdown on histone sulfation levels in animal models; Figure 6 a is a western blot result diagram for analyzing and verifying the overexpression efficiency of PAPSS1 and H3Y99sulf in the PAPSS1 overexpression model; Figure 6b is the statistical analysis of the relative quantitative level of PAPSS1 in the PAPSS1 overexpression model; Figure 6 c is a statistical analysis of the relative quantification level of H3Y99sulf in the PAPSS1 overexpression model; Figure 6 d is the western blot result of using PAPSS1 antibody to verify the knockdown efficiency of PAPSS1 in the PAPSS1 knockdown model. Figure 6 e is a statistical analysis of the relative quantitative level of PAPSS1 in the PAPSS1 knockdown model; Figure 6 f is a statistical analysis of the relative quantification level of H3Y99sulf in the PAPSS1 knockdown model; Figure 7 This is the OGDR experiment diagram of SY5Y and HMC3 cells overexpressing PAPSS1; Figure 7 a is the SY5Y cell counting graph; Figure 7 b is the HMC3 cell counting graph; Figure 7 c is the statistical analysis chart of SY5Y cell counting; 7d is the statistical analysis chart of HMC3 cell counting; Figure 8 This is a diagram of the SY5Y and HMC3 cell experiments in which PAPSS1 was knocked down; Figure 8 a is the SY5Y cell counting graph; Figure 8 b is the HMC3 cell counting graph; Figure 8 c is the statistical analysis chart of SY5Y cell counting; 8d is the statistical analysis chart of HMC3 cell counting; Fig. 9 Schematic diagram of animal experiments to evaluate the effects of regulating PAPSS1 and its mediated H3Y99sulf on the neurological function of MCAO mice; Fig.10 TTC staining images of the cerebral infarction area of ​​mice in different treatment groups; Fig.11 This is the quantitative statistical graph of TTC staining of cerebral infarction volume in mice in different treatment groups; Fig.12 Statistical graph of modified neurological score (mNSS) of mice in different treatment groups after MCAO. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be described clearly and completely below. Obviously, the embodiments described below are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The immunoblot signal intensity and staining images in the following examples were quantitatively analyzed by ImageJ (National Institutes of Health, USA) software. In the following examples, if the specific conditions are not specified, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. Those skilled in the art can refer to the contents of this article and make appropriate improvements to implement and apply the technology of the present invention, but similar replacements and modifications should be deemed to be included in the scope of the disclosure of the present invention.

[0027] Example 1 Dynamic regulation of PAPSS1-mediated histone sulfation modification (H3Y99sulf) during cerebral ischemia-reperfusion In this example, oxygen glucose deprivation-reperfusion (OGD / R) was used as a cell model to study the dynamic changes of H3Y99sulf in a human neuroblastoma cell line (SY5Y) and a human microglial cell line (HMC3); and mice treated with MCAO / R were used as an animal model to study the dynamic changes of H3Y99sulf in a mouse MCAO model.

[0028] (I) Dynamic changes of H3Y99sulf in the OGD / R cell model Methods: Establishment of oxygen-glucose deprivation-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 at a ratio of 94% N2, 5% CO2, and 1% O2. After 15 minutes of equilibrium, the incubator was sealed and then placed in the incubator for 3 hours of oxygen-glucose deprivation (OGD). After the treatment, the cells were reperfused (OGD / R), that is, they continued to be cultured in DMEM medium, and samples were collected at 0, 3, 6, 12, 24, and 48 hours for subsequent experimental analysis.

[0029] To evaluate the changes of H3Y99sulf in the OGD / R cell model, we analyzed the expression level of H3Y99sulf in SY5Y and HMC3 cells by Western blot. The experiment was divided into a control group and different treatment groups after 3 hours of OGD treatment and 0h, 3h, 6h, 12h, 24h and 48h of reperfusion. Figure 1 As shown in the figure, compared with the control group, the expression level of H3Y99sulf in SY5Y and HMC3 cells was significantly upregulated after OGD treatment, and its expression gradually decreased during the subsequent reperfusion process. As the reperfusion time increased, the expression level of H3Y99sulf gradually recovered. Figure 1a is the western blot result of the changes of H3Y99sulf during OGD / R in SY5Y and HMC3 cells. Histone H3 was used as an internal reference to verify the consistency of protein loading. Figure 1 b is the statistical analysis result of western blot of H3Y99sulf in SY5Y cells (n=3, P<0.05, P<0.01); Figure 1 c is the western blot statistical analysis results of H3Y99sulf in HMC3 cells (n=3, P<0.05, P<0.01).

[0030] The above results indicate that histone sulfation modification (H3Y99sulf) undergoes dynamic regulation during the OGD / R process.

[0031] (II) Dynamic changes of H3Y99sulf in the MCAO / R mouse model The thread occlusion method was used to establish a mouse model of middle cerebral artery ischemia-reperfusion (MCAO / R): referring to the thread occlusion method of Longa et al., the middle cerebral artery was occluded for 1.5 hours and then reperfused to simulate the process of cerebral ischemia-reperfusion; the mice were anesthetized and the skin was prepared. The skin was incised about 1 cm along the midline of the neck, and the muscles, blood vessels and nerves on both sides were freed to avoid damage and fixed. The right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were exposed. The distal end of the ECA was ligated, and the blood flow at the proximal end of the CCA and the bifurcation of the ICA was temporarily blocked. The nylon thread was inserted through the stump of the ECA, entered the ICA through the bifurcation of the CCA and ICA, and finally reached the middle cerebral artery (MCA). When resistance was encountered, the insertion was stopped, and the thread insertion length was about 9-11 mm. After 1.5 hours of ischemia, the mice were anesthetized again, the neck incision was cut to expose the neck blood vessels, the blood flow of ECA was temporarily blocked, the suture was pulled out to restore blood flow, and the reperfusion process was started. Samples were collected at 0h, 8h, 24h, 3d and 7d after reperfusion for subsequent experimental analysis. The sham operation group was anesthetized and a midline incision was made in the neck. The right common carotid artery and the internal and external carotid arteries were found and carefully separated. No suture was inserted and the skin was sutured directly.

[0032] To evaluate the changes of H3Y99sulf in the MCAO / R mouse model, we analyzed the expression level of H3Y99sulf in the ischemic brain tissue of mice by Western blot. The experiment was divided into a sham operation group and different treatment groups after 1.5 hours of MCAO treatment and 0h, 8h, 24h, 3d and 7d of reperfusion. Figure 2As shown in the figure, compared with the sham operation group, the H3Y99sulf modification level was significantly upregulated after 1.5h of cerebral ischemia, and its modification level gradually decreased during the subsequent reperfusion process. As the reperfusion time prolonged, the expression level of H3Y99sulf gradually recovered. Figure 2 a is the western blot result of the change of H3Y99sulf during MCAO / R. Histone H3 was used as an internal reference to verify the consistency of protein loading: Figure 2 b is the statistical analysis result of western blot of H3Y99sulf in the ischemic brain tissue of mice (n=5, P<0.05, P<0.01).

[0033] The above results indicate that, consistent with the cell experiments, H3Y99sulf undergoes dynamic regulation during mouse MCAO / R.

[0034] (III) Association between histone sulfation modification (H3Y99sulf) and PAPSS1 In order to verify that the dynamic regulation of histone sulfation modification (H3Y99sulf) during cerebral ischemia-reperfusion is mediated by PAPSS1, the expression of H3Y99sulf in cells ( Figure 3 a, 3b) PAPSS1 expression and H3Y99sulf modification level during ischemia (OGD3h) and reperfusion (OGD3h / R12h) periods; Animals ( Figure 4 a, 4b) PAPSS1 expression and H3Y99sulf modification levels during the ischemic period (MCAO1.5h) and reperfusion period (MCAO1.5 / R3d).

[0035] Figure 3 The expression of PAPSS1 and the level of H3Y99sulf modification in SY5Y and HMC3 cells under OGD and OGDR conditions are shown. Figure 3 a is the western blot result of PAPSS1 and H3Y99sulf in SY5Y and HMC3 cells OGD3h, Tubulin and Histone H3 were used as internal references to verify the consistency of protein loading; Figure 3 b shows the western blot results of PAPSS1 and H3Y99sulf in SY5Y and HMC3 cells OGD3h / R12h. Tubulin and H3 were used as internal references to verify the consistency of protein loading.

[0036] Figure 3The results showed that in SY5Y and HCM3 cells, OGD treatment upregulated the expression level of PAPSS1 protein, while OGDR treatment downregulated PAPSS1 expression, which was consistent with the change trend of H3Y99sulf in cell experiments.

[0037] Figure 4 The expression of PAPSS1 and the level of H3Y99sulf modification in the brain tissue of MCAO / R0d and MCAO / R3d mice are shown. Figure 4 a is the western blot result of PAPSS1 and H3Y99sulf in the ischemic contralateral and ischemic brain tissues of MCAO / R0d mice. Tubulin and Histone H3 were used as internal references to verify the consistency of protein loading; Figure 4 b is the western blot statistical analysis results of PAPSS1 and H3Y99sulf in the brain tissue of MCAO / R0d mice (n=6, P<0.05, P<0.01); Figure 4 c is the western blot result of PAPSS1 and H3Y99sulf in the ischemic contralateral and ischemic brain tissues of MCAO / R0d mice. Tubulin and H3 were used as internal references to verify the consistency of protein loading. Figure 4 d is the western blot statistical analysis results of PAPSS1 and H3Y99sulf in the brain tissue of MCAO / R3d mice (n=6, P<0.05, P<0.01).

[0038] Figure 4 The results showed that compared with the contralateral ischemic side, MCAO treatment upregulated the expression level of PAPSS1 protein in the ischemic side of the mouse brain tissue, and PAPSS1 expression was downregulated after MCAO / R treatment, which was consistent with the change trend of H3Y99sulf in animal experiments, indicating that the dynamic regulation of H3Y99sulf during cerebral ischemia-reperfusion may be mediated by PAPSS1.

[0039] Example 2 Effects of PAPSS1 overexpression and knockdown on histone sulfation modification levels 1. Cell experiments: 1. Plasmid Construction The coding sequence of human PAPSS1 was amplified by PCR and cloned into pHAGE / NEO(+)-Flag vector. The oligonucleotide sequence for knocking down the protein expression of the corresponding gene was inserted into the pLKO.1 / Puro(+) vector, the specific sequence is: PAPSS1 (human) TGGATCGAGTTTATTGGAATG.

[0040] 2. Lentivirus Packaging and Infection 2×10^6 HEK293T cells were plated in 100-mm cell culture dishes and transfected with PEI according to the manufacturer's instructions. To express the target protein, we co-transfected psPAX2, pMD2.G, and core plasmid (pHAGE-Flag-PAPSS1) to generate lentivirus; to knock down the target protein, we co-transfected pMDL, pVSVG, pREV, and pLKO.1 (sh-Scramble, sh-PAPSS1) to generate lentivirus. For lentiviral infection, 500 µl of virus-containing medium was added to 5×10^4 cells and 1% polybrene was added.

[0041] Figure 5 It was shown that overexpression of PAPSS1 could upregulate H3Y99sulf levels, while knockdown of PAPSS1 significantly attenuated the effect of OGD treatment on H3Y99sulf levels.

[0042] in, Figure 5 a is the western blot result of H3Y99sulf in PAPSS1-overexpressing cells (SY5Y, HMC3). Tubulin and Histone H3 were used as internal references to verify the consistency of protein loading amount.

[0043] Figure 5 b is the western blot result of H3Y99sulf in PAPSS1 knockdown cells (HEK293T) and then treated with OGD. Tubulin and Histone H3 were used as internal references to verify the consistency of protein loading. It can be seen that knockdown of PAPSS1 will significantly reduce the effect of OGD treatment on the level of H3Y99sulf in cells.

[0044] Figure 5 c is statistical analysis using two-sided t test ( , P < 0.05), and the data are presented as mean ± standard deviation (sd), and the error bars represent the range of three independent experiments. Figure 5The results showed that overexpression of PAPSS1 upregulated the level of cellular H3Y99sulf, while knockdown of PAPSS1 significantly attenuated the effect of OGD treatment on the level of H3Y99sulf in cells.

[0045] 2. Animal Experimentation In this example, the influence of histone sulfation modification level was verified by adeno-associated virus (AAV)-mediated PAPSS1 gene knockdown and overexpression. The specific operation method is as follows: 1. Construction of plasmid: Adeno-associated virus was constructed using 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.

[0046] 2. Virus injection: Each mouse was injected at four sites in the cortex and hippocampus (0.5 µL per site, injection rate 0.25 µL / min): site 1, 0.4 mm from bregma, 2.3 mm lateral, 1.8 mm deep; site 2, 0.4 mm from bregma, 2.3 mm lateral, 3.5 mm deep; site 3, 2.2 mm from bregma, 1.8 mm lateral, 1.5 mm deep; site 4, 2.2 mm from bregma, 3.0 mm lateral, 1.5 mm deep; all target sites were located in the left hemisphere (i.e., ipsilateral to MCAO). Virus injections were performed using a stereotaxic apparatus, with 0.5 µL of virus injected at each site at an injection rate of 0.25 µL / min. The needle was slowly withdrawn over 10 minutes. Animal experiments were performed on mice 21 days after virus injection.

[0047] Figure 6 Shown are the effects of AAV-mediated PAPSS1 overexpression and knockdown on histone sulfation (H3Y99sulf) levels in animal models.

[0048] Specifically, Figure 6 a is a western blot result diagram for analyzing and verifying the overexpression efficiency of PAPSS1 and H3Y99sulf in the PAPSS1 overexpression model. Tubulin and Histone H3 were used as internal references to verify the consistency of protein loading. The intensity value of H3Y99sulf has been normalized to the intensity value of histone H3 in the same sample.

[0049] Figure 6 b and 6c are statistical analysis diagrams of the relative quantitative levels of PAPSS1 and H3Y99sulf in the PAPSS1 overexpression model, respectively. The statistical analysis was performed using a two-sided t test ( , P<0.01; , P < 0.05), and the data are presented as mean ± standard deviation (sd), and the error bars represent the range of three independent experiments.

[0050] The above results indicate that AAV-mediated PAPSS1 overexpression upregulates H3Y99sulf levels.

[0051] Figure 6 d is the western blot result of using PAPSS1 antibody to verify the knockdown efficiency of PAPSS1 in the PAPSS1 knockdown model. Tubulin and Histone H3 were used as internal references to verify the consistency of protein loading and the intensity value of H3Y99sulf.

[0052] Figure 6 e and 6f are statistical analysis diagrams of the relative quantitative levels of PAPSS1 and H3Y99sulf in the PAPSS1 knockdown model, respectively. The statistical analysis was performed using a two-sided t test ( , P<0.01; , P < 0.05). Data are presented as mean ± standard deviation (SD), and the error bars represent the range of three independent experiments.

[0053] The above results indicate that AAV-mediated PAPSS1 knockdown downregulates H3Y99sulf levels.

[0054] From Examples 1 and 2, it can be seen that PAPSS1-mediated H3Y99sulf is dynamically regulated in ischemia-reperfusion injury, and its temporal expression pattern reveals that it plays an important role in cell damage and repair. It can be used as a potential target and has application prospects in the preparation and screening of drugs for the prevention and treatment of ischemic diseases.

[0055] Example 3 Effects of PAPSS1 overexpression and knockdown on ischemia-induced cell damage This example shows that overexpression of PAPSS1 can reduce cell damage caused by ischemia, and knockdown of PAPSS1 can aggravate cell damage caused by ischemia.

[0056] SY5Y and HMC3 cells overexpressing PAPSS1 were treated with OGDR. Cells were imaged and quantified using a cell counter ( Figure 7 a, 7b).

[0057] The data were statistically analyzed using a two-sided t test ( , P < 0.05). Data are presented as mean ± standard deviation (sd), and error bars represent the range of four independent experiments ( Figure 7 c, 7d).

[0058] The results showed that PAPSS1 overexpression increased the survival rate of SY5Y and HMC3 cells under OGDR stress.

[0059] PAPSS1 knockdown SY5Y and HMC3 cells were treated with OGDR. Cells were imaged using a cell counter and quantitatively analyzed using a cell counter ( Figure 8 a, 8b).

[0060] The data were statistically analyzed using a two-sided t test ( , P<0.05). The results are expressed as mean ± standard deviation (sd), and the error bars are the range of four independent experiments ( Figure 8 c, 8d).

[0061] The results showed that knockdown of PAPSS1 reduced the survival rate of SY5Y and HMC3 cells under OGDR stress.

[0062] Example 4 Effects of knocking down and upregulating the expression level of PAPSS1 on neurological damage in MCAO mice In this example, in order to verify whether the neuroprotective effect of PAPSS1 regulation on cells can be reflected in animal models, the effect of supplementation of H3Y99sulf complement (Na 2 SO 4 )、Na 2 SO 4 The effects of combined negative regulation of PAPSS1 and single positive regulation of PAPSS1 on the MCAO mouse model.

[0063] 1. Experimental grouping and intervention (see the flowchart for the operation flow chart) Fig. 9 , outlining the overall design and process of the experiment) Healthy male C57BL / 6 mice were divided into the following 5 groups according to the random number table method, including 5 mice in the sham operation + NaCl group and 8 mice in each of the other groups. All mice in each group were scored with mNSS neurological function and TTC staining was performed to calculate the cerebral infarction volume: 1. Sham + NaCl group: After anesthesia, a midline incision was made in the neck, and the right common carotid artery and the internal and external carotid arteries were found and carefully separated. No suture was inserted and the skin was sutured directly. After MCAO surgery, 2.06 mg / kg NaCl (with 2.5 mg / kg NaCl) was injected into the tail vein every day. 2 SO 4The sodium ion concentration in the experiment was kept consistent to eliminate the influence of sodium ions on the experiment), once a day for 3 consecutive days.

[0064] 2. MCAO+NaCl group: The left middle cerebral artery of mice was blocked for 1.5h by suture occlusion method, and then reperfused for 3 days. After successful modeling, mNSS scores were tested daily. After MCAO surgery, 2.06 mg / kg of NaCl was injected into the tail vein once a day for 3 consecutive days.

[0065] 3. MCAO+H3Y99sulf complement (Na 2 SO 4 ) group: The left middle cerebral artery of mice was blocked by suture for 1.5 h, and then reperfused for 3 days. After successful modeling, mNSS scores were measured daily. After MCAO surgery, 2.5 mg / kg of Na was injected into the tail vein every day. 2 SO 4 , once a day for 3 consecutive days.

[0066] 4. MCAO + Na 2 SO 4 +AAV-shPAPSS1 group: The left middle cerebral artery of mice was blocked by suture for 1.5 h, and then reperfused for 3 days. After successful modeling, mNSS scores were measured daily. AAV-shPAPSS1 was microinjected into the brain 21 days before MCAO surgery; 2.5 mg / kg of Na was injected into the tail vein every day after MCAO surgery. 2 SO 4 , once a day for 3 consecutive days.

[0067] 5. MCAO+ NaCl + AAV-oePAPSS1 group: The left middle cerebral artery of mice was blocked for 1.5h by suture occlusion method, and reperfusion was performed for 3 days. After successful modeling, mNSS scores were tested daily. AAV-oePAPSS1 was microinjected into the brain 21 days before MCAO surgery; 2.06 mg / kg of NaCl was injected into the tail vein once a day for 3 consecutive days after MCAO surgery.

[0068] 2. Key experimental methods 1. 2,3,5-Triphenyltetrazolium chloride (TTC) staining The infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Coronal brain sections were immersed in 2% TTC solution and incubated at 37°C for 20 minutes. Non-infarcted areas appeared in red, while infarcted areas appeared in white. Images of the infarcted and non-infarcted hemispheres were analyzed using ImageJ software.

[0069] 2. Use modified neurological score (mNSS) The scoring system has a total of 18 points, including motor, sensory and reflex functions. Motor function (0-8 points) mainly evaluates the gait, coordination and motor ability of mice; sensory function (0-6 points) evaluates the response of mice to external stimuli such as touch and pain; reflex function (0-4 points) is scored based on plantar reflex, tail reflex and other reactions. The scoring is performed by two blind assessors, and the higher the score, the more severe the neurological deficit.

[0070] (III) Experimental results Intravenous injection of H3Y99sulf complement sulfate showed that it could significantly reduce the infarct volume in MCAO mice ( Fig.10 ), the area decreased from 31.6±5.0% to 16.8±6.3% ( Fig.11 ). In addition, Na 2 SO 4 It also improved the neurological impairment score (mNSS) after MCAO in mice ( Fig.12 ).

[0071] The above results further verified that upregulating H3Y99sulf can effectively improve neurological damage in ischemic mice.

Claims

1. Use of a histone sulfation modification upregulator in the preparation of a drug for preventing and / or treating ischemic diseases, characterized in that: The histone sulfation modification up-regulator is a substance that promotes sulfation modification, and the ischemic disease is ischemic cerebrovascular disease.

2. The use according to claim 1, wherein the histone sulfation modification up-regulator is a small molecule compound, an antibody, an exogenous RNA, a plasmid or a virus.

3. The use according to claim 1 or 2, characterized in that: The ischemic cerebrovascular disease includes ischemic stroke and transient ischemic attack.

4. Application of up-regulation of histone sulfation modification as a target in screening products for candidate substances for treating ischemic diseases, wherein the ischemic disease is ischemic cerebrovascular disease.

5. Use of a PAPSS1 agonist in the preparation of a drug for preventing and / or treating ischemic diseases, wherein the PAPSS1 agonist achieves the use of the drug by upregulating histone sulfation modification, characterized in that: The PAPSS1 agonist is a substance that promotes the activity of the sulfation-modified substrate synthase PAPSS1, and the ischemic disease is ischemic cerebrovascular disease.

6. The use according to claim 5, wherein the PAPSS1 agonist is a small molecule compound, an antibody, an exogenous RNA, a plasmid or a virus.

7. The use according to claim 6, wherein the plasmid is a plasmid comprising PAPSS1.

8. The use according to any one of claims 5 to 7, characterized in that: The ischemic cerebrovascular disease includes ischemic stroke and transient ischemic attack.

9. Application of PAPSS1 agonism as a target in the preparation of a product for in vitro screening of candidate substances for the treatment of ischemic diseases, wherein the ischemic disease is ischemic cerebrovascular disease.

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