CN118001407A - Application of PAD2 in preparation of medicines for treating ischemic hypoxic malignant arrhythmia - Google Patents
Application of PAD2 in preparation of medicines for treating ischemic hypoxic malignant arrhythmia Download PDFInfo
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Abstract
The invention discloses an application of PAD2 in preparing a medicament for treating ischemic-anoxic malignant arrhythmia, belonging to the technical field of biological medicines. The invention provides an application of a reagent for down-regulating or knocking down PAD2 expression in preparing a medicament for regulating arrhythmia susceptibility. The invention also provides application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for recovering the calcium recovery capability of myocardial cells. The invention also provides an application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for reducing interaction between PAD2 and SERCA2a protein. The invention also provides an application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for reducing citrullination of SERCA2a protein. The invention proves that the PAD2 can be down-regulated or knocked out to reduce the susceptibility of malignant arrhythmia of myocardial cells in hemorrhagic shock and improve the survival rate.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of PAD2 in preparation of a medicine for treating ischemic-anoxic malignant arrhythmia.
Background
PAD2 (peptidyl arginine deaminase 2) is a member of the peptidyl arginine deaminase family, which catalyzes the post-translational deamination of proteins by converting arginine residues into citrulline in the presence of calcium ions to regulate the function of the proteins. Family members have different substrate-specific and tissue-specific expression patterns. PAD2 is the most widely expressed family member. Known substrates for this enzyme include myelin basic protein in the central nervous system and vimentin in skeletal muscle and macrophages. This enzyme is believed to play a role in the development and progression of neurodegenerative human diseases, including Alzheimer's disease and multiple sclerosis, which are also associated with the pathogenesis of glaucoma. PAD2 is however blank in critical fields, especially in the field of wound-related diseases.
Disclosure of Invention
The invention aims to provide an application of PAD2 in preparing a medicament for treating ischemic-anoxic malignant arrhythmia, wherein the PAD2 can be down-regulated or knocked out to remarkably reduce the susceptibility and incidence rate of malignant arrhythmia caused by ischemia and hypoxia.
The invention provides an application of a reagent for down-regulating or knocking down PAD2 expression in preparing a medicament for regulating arrhythmia susceptibility.
The invention also provides application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for recovering the calcium recovery capability of myocardial cells.
The invention also provides an application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for reducing interaction between PAD2 and SERCA2a protein.
The invention also provides an application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for reducing citrullination of SERCA2a protein.
Preferably, the agent for down-regulating or knocking down PAD2 expression comprises R29511-siPadi2-2-F with a nucleotide sequence shown as SEQ ID No.1 and R29511-siPadi2-2-R with a nucleotide sequence shown as SEQ ID No. 2.
The beneficial effects are that: according to the invention, a series of animal in-vivo experiments, tissue function experiments and cell function and molecular biology experiments prove that when hemorrhagic shock occurs, PAD2 in nucleus is translocated into cytoplasm due to myocardial tissue ischemia and hypoxia, and interaction with SERCA2a on sarcoplasmic reticulum is increased, and activity of the PAD2 is reduced through citrullination, so that calcium recovery capacity of myocardial cells is disregulated, susceptibility of malignant arrhythmia of cardiac muscle is increased, and the mechanism of PAD2 can be inhibited by downregulation or knockout of PAD 2: PAD2 plays a role in regulating arrhythmia susceptibility by affecting myocardial cell calcium ion recovery, reducing the susceptibility of myocardial cells to malignant arrhythmia in hemorrhagic shock, and increasing survival rate (FIG. 6).
Drawings
FIG. 1 is a graph of the results of analysis of serum samples from patients with hemorrhagic shock (n=12) collected at clinical diagnosis, wherein A represents patients dying from hemorrhagic shock with severe ventricular arrhythmias; b represents serum PAD2 protein levels in healthy control groups and hemorrhagic shock patients; c represents a correlation (n=19) analysis of serum PAD2 protein and lactate concentration in a group of hemorrhagic shock patients, the line in the figure representing a linear fit; d represents that peak levels of serum PAD2 protein occur within 24 hours after admission to hospital;
FIG. 2 is a graph showing the protective effect of Padi2 inhibition or knockdown on hemorrhagic shock-induced malignant arrhythmia in a hemorrhagic shock mouse model, where A represents electrocardiographic monitoring after hemorrhagic shock in wild-type mice (n=4) and Pad2 knockdown mice (n=4); b represents a statistical graph of PP interval, PR interval, QT interval, RR interval and heart rate; c represents the trace of normalized sarcomere length and average sarcomere contraction amplitude of adult cardiomyocytes in response to 15V and 0.5 to 7Hz electronic pacing in WT (n=20 from 5 mice) and Padi2 -/- (n=18 from 4 mice), along with statistics;
FIG. 3 is a graph showing the results of Pad2 in affecting the intake of Ca 2+ by myocardial cells in the occurrence of malignant arrhythmia following hemorrhagic shock; panel A shows representative adult mouse cardiomyocyte recordings of cytosolic Ca 2+ at 15V and 0.5-7Hz electronic pacing; b represents the average Ca 2+ attenuation Tau; n=20 cells from 5 wild-type mice and n=18 cells from 4 Pad2 knockout mice; PCA panels (C), volcanic panels (D), heat panels (E), GO enrichment analysis panels (F) and KEGG enrichment analysis panels (G) of Co-ip-MS were performed using Pad2 and IgG antibodies, respectively, after over-expression of Pad 2;
FIG. 4 is a graph of the results of hypoxia-induced transfer of PAD2 from the nucleus to the sarcoplasmic reticulum and increased interaction of PAD2 and SERCA2a, where A represents representative confocal images and quantification results (blue, DAPI; red, phalloidin; green, PAD2; scale bar, 25 μm) of hypoxic (1%O 2,5%CO2, 1 h) or normoxic (5% CO 2) cardiomyocytes; b represents PAD2 protein expression in the nucleus and cytoplasm of cardiomyocytes after hypoxia; c represents representative confocal images and quantitative results (blue, DAPI; green, PAD2; red, PDI) of hypoxic cardiomyocytes (1%O 2,5%CO2, 1 h); d represents CoIP (co-immunoprecipitation) that after hypoxia was found, cardiomyocyte Pad2 and SERCA2a protein interactions were increased;
FIG. 5 is a graph showing the results of increasing the interaction of PAD2 with SERCA2a and decreasing the enzymatic activity thereof by citrullination modulation after hypoxia of cardiomyocytes, wherein A indicates a significant decrease in SERCA2a enzymatic activity after hypoxia of cardiomyocytes; b represents PAD2 expression on the sarcoplasmic/endoplasmic reticulum of cardiomyocytes after hypoxia; c represents the expression of SERCA2a protein on the sarcoplasmic reticulum/endoplasmic reticulum of cardiomyocytes after hypoxia; d represents the level of primary cardiomyocyte SERCA2a citrullination following hypoxia;
FIG. 6 is a schematic diagram of increased susceptibility to malignant arrhythmia of cardiomyocytes due to severe hemorrhagic shock;
FIG. 7 is a gene editing flow chart of a mouse model;
FIG. 8 is a diagram showing the result of PCR verification of a knockout mouse, wherein P represents a positive control; w represents a wild-type control; b represents a blank; m is a DNA standard gradient indicator.
Detailed Description
The invention provides an application of a reagent for down-regulating or knocking down PAD2 expression in preparing a medicament for regulating arrhythmia susceptibility.
The method for down-regulating or knocking down PAD2 expression is not particularly limited, preferably comprises Crisper-Cas 9 technology, wherein Crisper-Cas 9 technology is adopted to construct PAD2 whole-body gene knockout mice in the embodiment, the survival rate of the PAD2 whole-body gene knockout mice after hemorrhagic shock is obviously higher than that of WT (wild type) mice, the incidence rate and susceptibility of malignant ventricular arrhythmia are obviously lower than those of the WT mice, and the susceptibility of arrhythmia can be regulated and controlled after down-regulating or knocking down PAD2 expression is confirmed.
In the embodiment of the invention, the primary myocardial cells of the wild mice and the PAD2 gene knockout mice are extracted for electric stimulation, and the result shows that the 2Hz and above high-frequency electric stimulation can induce the myocardial cells of the wild mice to generate strong direct contraction, the myocardial cells of the PAD2 whole-body gene knockout mice can endure the high-frequency electric stimulation of up to 7Hz and still can maintain regular contraction, and the synchronous detection of the calcium ion signals in the myocardial cells shows that the calcium ion Tau value of the myocardial cells of the PAD2 whole-body gene knockout mice is lower than the calcium ion Tau value of the myocardial cells of the WT mice, thus indicating that the calcium ion recovery rate is faster, and suggesting that the PAD2 possibly plays a role in regulating arrhythmia susceptibility by influencing the calcium ion recovery of the myocardial cells; according to the invention, PAD2 adenovirus is over-expressed through myocardial cell infection, then Co-ip experiments are carried out by using PAD2 specific antibodies, proteomics high-throughput screening is carried out on experimental products, and as a result, molecules with obvious protein interactions with PAD2 in myocardial cells are mainly concentrated on cell contraction, tight connection and calcium ion channel proteins, among molecules with PAD2 protein interactions, SERCA2a is obviously related to PAD2, and Co-ip experiments prove that protein interactions exist between PAD2 and SERCA2a, and after hypoxia stimulation, the interaction between PAD2 and SERCA2a is increased. However, the detection of separated sarcoplasmic reticulum/endoplasmic reticulum proteins shows that PAD2 protein on the sarcoplasmic reticulum/endoplasmic reticulum is increased after hypoxia stimulation, but SERCA2a protein is unchanged; after hypoxia stimulation, citrullinated SERCA2a is increased, which fully demonstrates that PAD2 is enriched towards the sarcoplasmic/endoplasmic reticulum following hypoxia stimulation, and calcium channel function is regulated by citrullinated SERCA2a, mediating a corresponding change in arrhythmia susceptibility.
According to the embodiment of the invention, in-vivo experiments, tissue function experiments and cell function and molecular biology experiments of the animals prove that when hemorrhagic shock occurs, PAD2 in nucleus is translocated into cytoplasm due to myocardial tissue ischemia and hypoxia, and the interaction with SERCA2a on sarcoplasmic reticulum is increased, and the activity of the PAD2 is reduced through citrullination, so that the calcium recovery capacity of myocardial cells is reduced, the susceptibility of malignant arrhythmia of cardiac muscle is increased, and the mechanism of the PAD2 can be inhibited by downregulation or knockout, so that the susceptibility of malignant arrhythmia of myocardial cells in the hemorrhagic shock is reduced, and the survival rate is improved. The invention also provides application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for recovering the calcium recovery capability of myocardial cells.
The present invention preferably reduces PAD2 expression in cells by constructing siPAD2, wherein siPAD sequences used are as follows:
R29511-siPadi2-2-F（SEQ ID No.1）：GCUGCAUGAAGGACAAUUATT
R29511-siPadi2-2-R（SEQ ID No.2）：UAAUUGUCCUUCAUGCAGCTT。
Based on siPAD sequences, adenoviruses of Ad-shPAD2 can also be constructed for cell and animal experiments.
The invention also provides an application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for reducing interaction between PAD2 and SERCA2a protein.
The application of the present invention is the same as that described above, and will not be described here again.
The invention also provides an application of the reagent for down-regulating or knocking down PAD2 expression in preparation of the reagent for reducing citrullination of SERCA2a protein.
The application of the present invention is the same as that described above, and will not be described here again.
For further explanation of the present invention, the application of PAD2 provided in the present invention in the preparation of a medicament for treating ischemic hypoxic malignant arrhythmia is described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the examples of the present invention, unless specifically stated otherwise, materials, instruments and consumables used in the present invention are conventional commercial products, and methods are also common in the art of molecular biology.
The materials used in the invention are as follows: experimental animals (C57 BL/6J mice, S-D rats), microsurgery equipment, body vision mirrors, small animal arterial cannulas, 1mL insulin needles, blood pressure monitors, electrocardiographs and other life and biochemical monitoring equipment; western Blot related experimental equipment and reagents; immune tissue fluorescence experiment and Confocal experiment related experimental equipment and reagent; single cell contraction, and a calcium ion signal detection system.
Example 1
In the diagnosis and treatment data of trauma patients collected by the emergency and trauma treatment center and the intensive care unit in the TONG state area of Beijing university Hospital in 2023, 1 month to 2023, 12 months, the applicant found that severe acute hemorrhagic shock patients usually die due to severe and rapid malignant ventricular arrhythmias (A in FIG. 1), and that the concentration of peripheral blood PAD2 of the hemorrhagic shock patients was significantly increased by # (C in FIG. 1) compared with that of healthy volunteers by biochemical detection of the hemorrhagic shock patients and the significantly increased concentration of lactic acid in FIG. 1) within 24 hours after admission (E in FIG. 1).
* Patient demographic information is shown in table 1.
Table 1 demographic statistics for trauma patients
The severity of hemorrhagic shock is evaluated by adopting a lactic acid value, and blood lactic acid of more than 2mmol/L is clinically used as an index of severe hemorrhagic shock, and blood lactic acid of more than 4mmol/L is more an index of critical hemorrhagic shock.
ELISA Kit (CAYMAN PAD human ELISA Kit 501450) is used for detecting the#PAD2
Example 2
1. Constructing PAD2 whole-body gene knockout mice (C57 BL/6J strain) by Crisper-Cas 9 technology;
Gene editing was performed according to the strategy shown in fig. 7: wild type mice: ① The PCR reaction comprises a WT band; ② The PCR reaction contained a WT band.
Heterozygous mice: ① The PCR reaction comprises a WT band and a KO band; ② The PCR reaction contained a WT band.
Homozygous mice: ① The PCR reaction comprises a KO band; ② No product was present in the PCR reaction.
Primer information is shown in table 2:
Table 2PCR validates the primers used
The results of PCR validation of the construction of the knockout mice are shown in fig. 8, in which: p represents a positive control; w represents a wild-type control; b represents a blank; m is a DNA standard gradient indicator.
The PCR verification reaction procedure was as follows: 25. Mu.L of reaction system: 2X TAQ MASTER Mix, dye Plus, (Vazyme P112-03) 12.5. Mu. L, ddH 2 O9.5. Mu.L, primer F/R (10 pmol/. Mu.L each) 1. Mu.L each, template (. Apprxeq.100 ng/. Mu.L) 1. Mu.L;
The procedure is as follows: 95 ℃ for 5min;98℃for 30s,65℃for 30s (0.5℃minus each cycle), 72℃for 45s,20 cycles; 98 ℃ for 30s,55 ℃ for 30s,72 ℃ for 45s,20 cycles; 72 ℃ for 5min; preserving at 10 ℃.
2. A hemorrhagic shock model mouse is constructed by a way of blood loss of a left lower limb femoral artery cannula of the mouse, and the electrocardio and blood pressure of the mouse are monitored in the operation process.
The survival rate of PAD2 whole body gene knockout mice after hemorrhagic shock was significantly higher than WT (wild type) mice, and the incidence and susceptibility to malignant ventricular arrhythmias were significantly lower than WT mice (a and B in fig. 2).
According to the invention, quantitative blood loss is carried out through femoral artery intubation of the mice, the mice are judged to generate hemorrhagic shock through blood pressure monitoring, myocardial tissue ischemia and hypoxia, and electrocardiographic monitoring shows that severe ventricular arrhythmias including malignant ventricular tachycardia and ventricular fibrillation are simultaneously and simultaneously concurrent when the mice generate lethal hemorrhagic shock, and then the mice die rapidly (within 1h after operation), while PAD2 knockout mice can maintain higher survival rate under the same conditions and have no malignant arrhythmia; by extracting the whole-body gene knockout mice of PAD2 and the myocardial cells of the WT (wild-type) mice, marking intracellular calcium ions by using Fura-2 af dye and then giving electric stimulation, observing myocardial contraction condition and calcium ion signals, the high-frequency electric stimulation of 2Hz and above can induce the myocardial cells of the wild-type mice to generate tonic contraction, the whole-body gene knockout mice of PAD2 can endure the high-frequency electric stimulation of up to 7Hz and still maintain regular contraction (C in fig. 2), and calcium ion signal detection shows that the calcium ion Peak value of the whole-body gene knockout mice of PAD2 is obviously higher than that of the myocardial cells of the WT mice, the Tau value is obviously lower than that of the myocardial cells of the WT mice, and the calcium uptake rate is obviously increased, and the calcium reservoir content of sarcoplasmic reticulum is increased (A and B in fig. 3), so that the PAD2 possibly plays a role in regulating arrhythmia susceptibility by influencing the calcium ion recovery of the myocardial cells.
3. The experimental products are subjected to proteomics high-throughput screening by infecting myocardial cells with over-expressed PAD2 adenovirus and then carrying out Co-ip experiments by using PAD2 specific antibodies.
After the primary myocardial cells of the milk rats overexpress PAD2, co-ip-MS (Co-immunoprecipitation proteomics) experiments show that after the PAD2 is overexpressed, proteins with increased interaction with the PAD2 are mainly concentrated on key functions such as myocardial contraction/relaxation functions, calcium ion regulation, tight connection, energy metabolism and the like (D to G in fig. 3); this was also confirmed by fluorescence labeling of intracellular PAD2, cytoskeleton, the transfer of PAD2 from nucleus to cytoplasm after hypoxia stimulation, and isolation of cytoplasmic nucleoprotein for Western Blot (FIGS. 4A and B); by fluorescent labeling of intracellular PAD2, organelles, co-localization of PAD2 with sarcoplasmic/endoplasmic reticulum was found to increase following hypoxia stimulation (D in fig. 4); co-Ip experiments demonstrated that PAD2 has a protein interaction with SERCA2a (C in FIG. 4); enzyme activity detection shows that after hypoxia stimulation, PAD2 enzyme activity in myocardial cells is enhanced, and SERCA2a enzyme activity is reduced (Figure 5A); and PAD2 expression on sarcoplasmic/endoplasmic reticulum increased after hypoxia stimulation, SERCA2a expression was unchanged, and SERCA2a citrullination increased after hypoxia (B to D in fig. 5).
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (5)
1. An application of an agent for down-regulating or knocking down PAD2 expression in preparing a medicament for regulating arrhythmia susceptibility.
2. Use of an agent that down regulates or knocks down PAD2 expression in the preparation of an agent that restores calcium recovery from cardiomyocytes.
3. Use of an agent that down regulates or knocks down PAD2 expression in the preparation of an agent that reduces interaction between PAD2 and a SERCA2a protein.
4. Use of an agent that down regulates or knocks down PAD2 expression in the preparation of an agent that reduces citrullination of a SERCA2a protein.
5. The use according to any one of claims 1 to 4, wherein the agent for down-regulating or knocking down PAD2 expression comprises R29511-siPadi2-2-F having a nucleotide sequence shown in SEQ ID No.1 and R29511-siPadi2-2-R having a nucleotide sequence shown in SEQ ID No. 2.
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