Methods for studying the diurnal differences in the cardioprotective effect of PER2 in ISO

Abstract

The present invention discloses a method for studying the circadian difference of myocardial protective effect of PER2 in ISO, which belongs to the field of biomedical technology. The method comprises the following steps: S1: randomly grouping live experimental mice, respectively establishing a pseudo-injury or myocardial ischemia-reperfusion injury I / R model of the experimental mice, and using isoflurane or carrier gas treatment before modeling; S2: culturing H9c2 cells in vitro to ensure circadian rhythm synchronization, and then establishing an in vitro cell-simulated ischemia-reperfusion injury model; S3: performing corresponding functional assays on live animal experimental mice and in vitro cultured cells. The present invention proves that by activating PER2 in a time-dependent manner, ISO has a circadian rhythm in the effect of MI / R-induced injury, and PER2 leads to susceptibility to MI / R injury by reducing ROS and mediating the energy metabolism of CPT1A. This finding is potentially important for ISO as an early therapeutic intervention to alleviate MI / R injury, especially the timing of treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for studying the day-night difference of the myocardial protective effect of PER2 in ISO. Background Art

[0002] Perioperative myocardial injury (PMI) is the most common perioperative complication, which increases the incidence of early and long-term adverse cardiac events after surgery. Although myocardial ischemia can be remedied by timely restoration of blood flow, reperfusion can induce excessive reactive oxygen species (ROS) that can lead to myocardial ischemia-reperfusion injury. The mechanism of myocardial ischemia-reperfusion injury is not fully understood, and there is no acceptable direct treatment for reperfusion injury.

[0003] The frequency and extent of perioperative myocardial injury can be transcriptionally regulated by the patient's biological clock, with more frequent and less perioperative myocardial protection in the morning. The heart's circadian clock affects many aspects of biology and behavior, including metabolism, energy expenditure, and the production of reactive oxygen species. The PER family of genes is central to the regulation of circadian rhythms, of which PER2 is expressed in cardiomyocytes and vascular endothelial tissues and is primarily involved in regulating responses to endogenous myocardial ischemia. The cardioprotective effect of PER2 may be attributed to its antioxidant properties and energy promotion against mitochondrial dysfunction. PER2 knockout mice have larger infarcts at lower levels of oxidative stress during myocardial ischemia, and PER2 prevents myocardial ischemia by promoting efficient glycolysis in cardiomyocytes through the glycolytic pathway.

[0004] Isoflurane (ISO) is a classic inhalation anesthetic that can provide cardioprotection when administered before myocardial ischemia-reperfusion injury. Although the cardioprotective effect of isoflurane has been widely reported, its cardioprotection is not stable and can be enhanced or weakened under different conditions. Many studies have shown that general anesthesia regulates the expression of circadian genes in many organs, such as the brain and heart. Anesthetic drugs can act on the circadian system of animals in a time-dependent manner, and previous studies have found that nocturnal anesthetics do not change biological rhythms. However, whether there are circadian differences in the cardioprotective effect of isoflurane and whether PER2 plays a role in this process remain unknown.

[0005] Therefore, we hypothesized that the cardioprotective effect of isoflurane is circadian by activating PER2 in a time-dependent manner through mediating energy metabolism. These findings have important implications for better use of isoflurane to prevent perioperative myocardial ischemia-reperfusion injury, especially in patients with underlying myocardial injury. Summary of the invention

[0006] In view of the above-mentioned problems, the present invention aims to provide a method for studying the circadian difference of the cardioprotective effect of PER2 in ISO, and proves that ISO activates PER2 in a time-dependent manner, and the effect of ISO on MI / R-induced injury has a circadian rhythm. PER2 leads to susceptibility to MI / R injury by reducing ROS and mediating the energy metabolism of CPT1A. These findings are potentially important for ISO as an early therapeutic intervention to alleviate MI / R injury, especially the timing of treatment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The method for studying the day-night difference of the myocardial protective effect of PER2 in ISO is characterized by comprising the following steps:

[0009] S1: The living experimental mice were randomly divided into groups, and the sham injury or myocardial ischemia-reperfusion injury I / R model was established in the experimental mice. Isoflurane or carrier gas was used for treatment before modeling.

[0010] S2: H9c2 cells were cultured in vitro. To synchronize the circadian rhythm, all cells were first treated with horse serum for 2 h. The end of treatment was taken as "0 o'clock". At this time, all cell culture media were replaced with serum-free DMEM. At "0 o'clock" or "12 o'clock", cells were treated with emulsified ISO, lipid or serum-free DMEM, and then an in vitro cell model of simulated ischemia-reperfusion injury was established.

[0011] S3: Perform corresponding functional assays on the live animal experimental mice in step S1 and the in vitro cultured cells in step S2 to analyze the day-and-night differential effects of PER2 on the cardioprotective effect of ISO.

[0012] Further, in step S1, the experimental mice were randomly divided into eight groups, namely, ZT0 sham operation group, ZT0ISO+sham operation group, ZT12 sham operation group, ZT12ISO+sham operation group, ZT0IR group, ZT0ISO+IR group, ZT12IR group and ZT12ISO+IR group;

[0013] Among them, ZT0 means inhalation of carrier gas at 7 o'clock on the day before modeling, ZT0ISO means inhalation of 1.5% ISO at 7 o'clock on the day before modeling, ZT12 means inhalation of carrier gas at 7 o'clock on the night before modeling, and ZT12ISO means inhalation of 1.5% ISO at 7 o'clock on the night before modeling.

[0014] Furthermore, in step S2, H9c2 cells were cultured in a 5% CO2 humidified incubator at 37°C using DMEM-F12 supplemented with 10% fetal bovine serum and 1% streptomycin / penicillin.

[0015] Further, in step S2, the in vitro experimental cells were divided into 12 groups, namely 0hrC group, 0hrV group, 0hrE1 group, 12hrC group, 12hrV group, 12hrE1 group, 0hrCSIR group, 0hrVSIR group, 0hrEISIR group, 12hrCSIR group, 12hrVSIR group and 12hrE1SIR group;

[0016] Among them, 0hr means within 0h after horse serum treatment, C means serum-free DMEM treatment, V means lipid treatment, El means emulsified ISO treatment; 12hr means 12h after horse serum treatment; SIR means I / R simulated by H2O2.

[0017] Furthermore, the functional assays described in step S3 include: myocardial function assay, cell apoptosis rate assay, myocardial infarction area assay, cell activity assay, free radical generation assay, ATP content assay and oxidative stress assay.

[0018] The beneficial effects of the present invention are:

[0019] The present invention has found through comparative studies of in vivo animal experiments and in vitro cell experiments under different treatment conditions that: (1) ISO has day-night differences in myocardial protection against I / R injury in vivo and in vitro, and the myocardial protection effect is more obvious when ISO is pretreated during the day; (2) ISO affects the cardiac protein PER2 by reducing oxidative stress and promoting energy metabolism, thereby reducing myocardial injury; (3) PER2 regulates CPT1A transcription, which is a key enzyme in fatty acid metabolism and oxidative stress levels. Current findings show that ISO pretreatment of H9c2 cells at 12 hours after serum shock, or ISO pretreatment of mice during the day, confirms the cardioprotective effect, which improves myocardial function or cell activity, reduces myocardial infarction area, reduces myocardial cell apoptosis, reduces oxidative stress, and improves energy metabolism. In contrast, ISO has no significant effect on mice at night or H9c2 cells at the end of serum shock. The cardioprotective effect of ISO is closely related to PER2, and PER2 siRNA eliminates the protective effect of ISO during cardiac injury.

[0020] The present invention demonstrates that ISO activates PER2 in a time-dependent manner, and that the effects of ISO on MI / R-induced injury are circadian, and the results indicate that PER2 contributes to susceptibility to MI / R injury by reducing ROS and mediating CPT1A energy metabolism. These findings are potentially important for ISO as an early therapeutic intervention to mitigate MI / R injury, especially the timing of treatment. Based on the circadian characteristics of PER2 in perioperative myocardial injury and the different effects of different ISO administration times on rhythmic genes and myocardial protection, the application strategy of ISO may take different new directions based on chronotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the day and night differences in the effects of ISO on cardiac function and oxidative stress in the in vivo animal experiment mice of the present invention.

[0022] Figure 2 In the in vitro cell experiment of the present invention, ISO showed the "day and night" difference in protecting H9c2 cells from the effects of simulated ischemia-reperfusion.

[0023] Figure 3 These are the day and night differences in the effects of ISO on myocardial infarction damage in the living animal experiment mice of the present invention.

[0024] Figure 4 These are the day and night differences in the effects of ISO on oxidative stress and energy metabolism in the present invention.

[0025] Figure 5 The results of paired-end RNA sequencing of RNA isolated from the left ventricle of mice treated with or without ISO at ZT0 / 12 in the present invention.

[0026] Figure 6 This is the test result of whether PER2 knockdown of the present invention eliminates the protective effect of ISO in SIR-treated H9c2 cells. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0028] The specific experimental procedures in the present invention were approved by the National Animal Protection Committee (Zhujiang Hospital of Southern Medical University, Guangzhou, China), including in vivo experiments on living animals and in vitro experiments.

[0029] The in vivo animal experiment process is as follows: adult male C57BL / 6J mice (age 8-10 weeks) were housed in a specific pathogen-free facility with temperature and humidity control, with a 12-hour light / dark cycle, and allowed free access to water and standard laboratory rodent food, as shown in Table 1. The mice were randomly divided into eight groups and treated with isoflurane or carrier gas (air containing 30% oxygen) before sham injury or ischemia-reperfusion injury. The specific treatment conditions of the eight groups of mice are shown in Table 1 below.

[0030] Table 1 Experimental animal grouping and treatment design

[0031]

[0032] Ischemia-reperfusion was performed after two weeks of environmental feeding in advance. Mice were anesthetized with pentobarbital (50 mg / kg, intraperitoneal), connected to a mechanical ventilator (VentEtilated, Harvard Apparatus, Holliston, MA, USA) and endotracheal intubation for ventilation. Next, a left anterior thoracotomy was performed to expose the heart, the pericardium was cut open, the left coronary artery was identified with the naked eye, and an 8.0 nylon suture was placed around the blood vessel. Ischemia was induced by pulling in the suture, and it was confirmed that the color of the blood vessel changed immediately from light red to dark purple, and the myocardium supplied by the blood vessel changed from bright red to white. At the same time, ECG immediately showed ST segment elevation. After 30 minutes, the suture was released, the left coronary artery was reperfused, and the myocardium was reperfused for 24 hours. In the sham operation, only the thorax and pericardium were opened, and the left anterior descending branch was not ligated. After the air chest was evacuated, the skin wound was sutured. The mice were kept warm by a heating plate, and the breathing of the mice was detected until spontaneous breathing appeared. 125ug / Kg of buprenorphine was given for analgesia after surgery. After 24 hours of myocardial reperfusion, myocardial function, cell apoptosis rate, infarct size and oxidative stress were measured.

[0033] The specific in vitro experimental process is as follows: H9c2 cells were cultured in a 37°C 5% CO2 humidified incubator with DMEM-F12 supplemented with 10% fetal bovine serum and 1% streptomycin / penicillin solution. In order to synchronize the circadian rhythm, all cells were first treated with horse serum for 2 hours, and the end of treatment was taken as "0 o'clock". At this time, all cell culture media were replaced with serum-free DMEM. At "0 o'clock" or "12 o'clock", the cells were treated with emulsified ISO, lipids or serum-free DMEM, and then an in vitro cell model of ischemia-reperfusion injury was established. The specific treatment conditions of different groups of cells are shown in the attached figure. Figure 2 As shown in A.

[0034] The specific method for measuring myocardial function and myocardial infarct size was as follows: After 24 hours of myocardial reperfusion, the mice were re-anesthetized and fixed on the experimental table. The left ventricular ejection fraction (EF) and left ventricular contraction fraction (FS) were calculated using a Vevo2100 echocardiography machine (VisualSonics, Toronto, Canada). The ligature around the coronary artery was re-tied, and the infarct size was evaluated by staining with 1% Evans blue and 2% triphenyltetrazolium chloride (TTC).

[0035] Furthermore, the specific method for determining the cell apoptosis rate in the present invention is: using TUNEL mixture to measure the cell apoptosis rate. Myocardial slices are incubated in 50uL TUNEL mixture at 37°C for 1 hour. After the TUNEL reaction is completed, DAPI staining is performed to observe the cell nucleus. Cell apoptosis rate = number of apoptotic cells (green) / total number of cells (blue) × 100%.

[0036] The specific method of cell activity determination is: the cell activity is determined by CCK-8 colorimetry. After culturing with graded concentrations of EI and washing with PBS, the cells are treated according to the manufacturer's instructions. The cell activity is calculated by determining the ratio of the optical density of the experimental well to the optical density of the control well at 450 nm using an ELISA reader (BioTek, W).

[0037] The specific methods of apoptotic cell analysis and cell morphology analysis are as follows: the cells were measured for apoptosis rate by flow cytometry, the harvested cells were resuspended in 1× annexin V binding buffer and propidium iodide binding buffer at room temperature for 15 minutes, and the apoptotic ratio was measured by CytoFLEX flow cytometer system. The cell morphology was observed and the images were captured by Axiovrt. optical microscope.

[0038] The specific method for quantitative determination of free radical generation is as follows: at the end of reperfusion, mitochondria of mouse myocardial tissue were extracted from the whole heart using a kit (KeyGENBiotech). Fresh mouse myocardial tissue samples were washed with ice-cold saline to remove blood and ground with ice-cold buffer in a small-volume glass homogenizer. After grinding, the ground tissue was transferred to mitochondrial extraction, gently mixed and centrifuged at 4°C, and the mitochondrial precipitate was collected.

[0039] A specific fluorescent probe (BestBio, Shanghai, China) was used to detect the level of reactive oxygen species in myocardial mitochondria. Fresh purified mitochondrial suspension was fully mixed in the staining solution, incubated in the dark at 37 degrees Celsius for 1 hour, and the fluorescence intensity was measured using a multifunctional microplate analyzer. The intracellular ROS level was evaluated by measuring the intensity of the fluorescence of 2',7'-dichlorofluorescein diacetate (DCFH-DA) in the cells. After drug treatment, the cells were incubated in 10UmDCFH-DA for 20 minutes. The cells were washed three times with DMEM without phenol red to remove the remaining DCFH-DA, and the fluorescence intensity was measured spectrophotometrically using a multifunctional microplate analyzer in time. The amount of emitted fluorescence is related to the number of ROS in the cells. Each measurement was performed in duplicate, and the cell fluorescence intensity was expressed as the fold change relative to the control group.

[0040] Specific method for ATP content determination: ATP staining is used to determine the energy metabolism level of myocardial tissue and H9c2 cell mitochondria. After all background ATP is exhausted, the sample or standard is added to the test well and quickly mixed with the enzyme solution. The RLU value is determined using a multifunctional microplate reader, and the ATP content of the sample is determined according to the standard curve.

[0041] In the present invention, in order to reduce bias during statistical analysis, the personnel who perform or evaluate the results do not know the results of the grouping. All data are expressed as mean ± SEM. After checking normality using the Shapiro-Wilk test, when the data pass the normality test, the t-test is used to determine the difference between the two groups. Comparison was made by analysis of variance with Bonferroni correction, and a post hoc t test was performed. The number of samples for the in vivo experiment was determined based on previous experiments. The sample size was calculated a priori using power analysis software G*PowerforIR. After calculating the effect size d as 2, the α error was 0.05, and the β error was 0.2 (efficacy was 0.8), a total of 6 mice and 4 well cells were used in each group in the experiment. All analyses were performed using GraphPadPrism version 8.2.0. P<0.05 was considered statistically significant, and the significance test was a two-tailed test.

[0042] Test results:

[0043] Diurnal differences in the effects of isoflurane on cardiac function and oxidative stress

[0044] The results of the day-night differences in the effects of ISO on cardiac function and oxidative stress in mice in vivo are shown in the attached figure. Figure 1 As shown, A is the treatment of experimental mice, B is a representative M-mode ultrasound image of the sham operation group, C is the left ventricular ejection fraction (EF) and contraction fraction (FS) of mice at ZT0 (P=0.0025) and FS (P<0.0001) were lower than those at ZT12. Compared with the sham operation at ZT0, ISO treatment at ZT0 increased EF and FS, P<0.0001. Compared with the sham operation at ZT12, ISO treatment at ZT12 reduced EF (P=0.0003) and FS (P=0.0148); D is the expression of PER2 mRNA in mouse heart, Per2 mRNA expression at ZT12 was higher than that at ZT0, and ISO treatment at ZT0 could increase Per2 mRNA expression, but ISO treatment at ZT12 was ineffective; E is the reactive oxygen species (ROS) in myocardial mitochondria of sham operation mice. ROS expression was higher at ZT0 than at ZT12 (P<0.0001). Compared with the ZTO sham operation group, the expression of reactive oxygen species in the ZTOISO+sham operation group was decreased (P<0.0001). Data are expressed as SEM±mean.

[0045] Attached Figure 2The results of the day-night difference of ISO in protecting H9c2 cells from simulated ischemia-reperfusion injury in an in vitro cell experiment, where A is the design of the in vitro cell experiment; B is the expression of Per2 mRNA in undamaged H9c2 cells (n=4 in each group); C is the expression of ROS in undamaged H9c2 cells (n=4 in each group). ROS expression was higher at 12h than at 0h (P<0.0001). Compared with the 12hV group, emulsified ISO (EI) led to a significant reduction in ROS production at 12h (P<0.0001). D is the evaluation of cell activity by CCK assay, and cell activity is expressed as optical density. EI increased the viability of SIR-injured H9c2 cells in a dose-dependent manner. The pro-survival effect of 2.24mM emulsified ISO was the most obvious (n=6 in each group). E is the cell morphology observed under an inverted / phase contrast microscope and the images obtained (200×) (n=6 per group); F is the level of apoptosis of cells injured by SIR, and representative flow cytometry images (left) quantitatively analyzed the apoptosis rate. Compared with the 12hV group, in the 12hEI group, EI treatment reduced H9c2.

[0046] As attached Figure 1 As shown in B and C, typical M-mode ultrasound results show that the EF and FS of the left ventricle of mice at ZT12 were higher compared with ZT0. ZT0+ISO improved cardiac function by increasing EF and FS. Isoflurane treatment at ZT12 decreased EF and FS compared with sham surgery at ZT12.

[0047] Oxidative stress in myocardial mitochondria and H9c2 cells was measured at several time points without injury, and compared with ZT12 (see Appendix Figure 1 E) and 0 (as shown in the attached Figure 2 In the in vivo experiments at ZT0 and in vitro experiments at 12 h, isoflurane treatment led to a significant reduction in ROS production, while at ZT12 (as shown in the attached figure). Figure 1 E) and 0h (as shown in the attached Figure 2 No difference was observed in (C).

[0048] Diurnal differences in the effects of isoflurane on circadian gene expression

[0049] mRNA samples were collected from mice and cells at rhythmic time points without injury. Per2 mRNA levels showed significant rhythmic differences, with low levels of Per2 mRNA in cells in the sham group at ZT0 and at 12h without injury. In addition, mPer2 expression increased in mice at ZT0 and cells at 12h after ISO treatment, but was not significantly increased at ZT12 and 0h (see Appendix). Figure 1 D and attached Figure 2 B) did not increase.

[0050] Diurnal differences in ISO effects in myocardial infarction

[0051] In vivo animal experiments, ISO pretreatment at different time periods during the day and night had different effects on alleviating myocardial infarction damage. Figure 3 Figure 1 shows the representative M-mode ultrasound images after IR injury; B shows the EF and FS of the left ventricle. Compared with the ZT0IR group, the EF and FS were increased in ZT0ISO treatment (P<0.0001). C shows the representative images of cardiac sections stained with eosin blue and TTC double staining, the blue staining part represents the non-ischemic normal area; the red staining part represents the area at risk (AAR); the unstained part represents the infarct area (IA). ZT0ISO treatment reduced the infarct size (P<0.0001); there was no difference in infarct size between the ZT12IR group and the ZT12ISO+ISO group (P=0.64); D shows the representative images of cells for myocardial apoptosis determined by TUNEL staining (green) (left), and the cell nucleus was detected by DAPI (blue), scale bar=200um; the apoptotic rate of cells in the ZT0IR group was higher (P=0.0003, <0.0001 compared with the ZT12IR and ZTOISO+IR groups). No difference was observed between the ZT12IR group and the ZT12ISO+IR group (P=0.98).

[0052] As attached Figure 3 As shown in A and B, typical M-mode ultrasound results showed that the EF and FS of the left ventricle of mice were higher after ischemia / reperfusion at ZT12. ISO treatment at ZT0 promoted the recovery of cardiac function after ischemia and improved EF and FS compared with the ZT0IR group.

[0053] ISO treatment at ZT0 reduced infarct size compared with the ZT0IR group (mean difference = 22.05 vs reperfusion group; 95% confidence interval CI: 17.32-26.77; P < 0.0001) (see Supplementary Figure 2). Figure 3 =(C) , but there was no significant difference between the ZT12 + reperfusion group and the ZT12 isoflurane treatment + reperfusion group (P = 0.64). TUNEL staining showed that there was a higher apoptosis rate in the ZT0 reperfusion group compared with the ZT12 reperfusion group. ISO treatment significantly reduced the apoptotic index in the ZT0 ISO + IR group compared with ZT0 (mean difference = 34.08 vs IR group; 95% confidence interval CI: 27.72-40.45; P < 0.0001), while no difference was observed in the ZT12IR and ZT12ISO + IR groups (P = 0.98), as shown in Supplementary Figure 5 Figure 3 As shown in D.

[0054] Diurnal differences in the effects of ISO on protecting H9H2 cells from simulated ischemia-reperfusion

[0055] H9c2 cells were incubated in 1.12, 1.40, 1.68, 1.96, 2.24, 2.52 or 2.8 mmol / L EI for 6 h, treated with 1.98 Ul / mL fat emulsion as a vehicle for 6 h, and treated with H2O2 for 2 h. Cell viability was measured. The activity of H9c2 cells simulating ischemia-reperfusion injury increased in a dose-dependent manner with EI (e.g., Figure 2 The prosurvival effect was most pronounced at 2.24 mM EI; therefore, in subsequent experiments, cells were exposed to 1.98 Ul / mL lipid and 2.24 mmol / L EI.

[0056] Compared with the 0hCSIR group, the 12hCSIR group showed reduced contraction and cell attachment. Compared with the 12hV SIR group, EI treatment at 12h reduced morphological damage. Differently, no differences in cell morphology were observed between the 0h V and 0hEISIR groups. Compared with the 0hCSIR group, the 12hCSIR group had a higher level of cell apoptosis. While the 12hEI treatment significantly reduced the apoptotic rate of H9c2 cells, there was no difference in apoptosis between the 0hV and 0hEISIR groups, as shown by the attachment Figure 2 As shown in F.

[0057] Diurnal differences in the effects of ISO on oxidative stress and energy metabolism

[0058] The day-night differences in the effects of ISO on oxidative stress and energy metabolism are shown in the attached figure. Figure 4 As shown, A is the expression of ROS in myocardial mitochondria of mice in the risk zone. Compared with the ZT0 IR group, ROS expression was reduced in the ZT0ISO+IR group (P=0.0003). B is the ATP content of myocardial mitochondria of mice. ZT0ISO treatment increased ATP content (P<0.0001, vs. ZT0IR group). No differences in ROS expression (P=0.05) and ATP (P=0.15) were observed between the ZT12IR group and the ZT12ISO+IR group. C is the expression of ROS in H9c2 cells. Compared with the 12hV SIR group, E1 treatment at 12h significantly reduced the level of ROS (P<0.0001). D is the ATP content of H9c2 cells. 12h E1 treatment increased ATP concentration (P<0.0001, vs. 12hV group). There was no significant difference in ROS expression (P=0.93) and ATP (content) between the 0hVSIR group and the 0hEISIR group.

[0059] After 24 hours of reperfusion, the effect of ISO on oxidative stress in myocardial mitochondria treated with ischemia-reperfusion was investigated. Compared with the ZT12IR group, the level of ROS in myocardial mitochondria in the ZT0IR group was higher, as shown in the attached Figure 4 As shown in Figure A. The level of ROS in the mitochondria of the ZT0ISO+IR group was lower than that of the ZT0IR group. ATP assay showed that without ISO pretreatment, the ATP level in the myocardium of ZT12 mice was higher than that of ZT0 mice. After ISO treatment, the ATP concentration increased more significantly in ZT0. In contrast, there was no significant difference in the ROS and ATP levels between the ZT12IR group and the ZT12ISO+IR group, as shown in the attached Figure. Figure 4 As shown in B.

[0060] The effects of EI on oxidative stress and ATP concentration in SIR-treated H9c2 cells were determined in vitro. The ROS levels in H9c2 cells treated with H2O2 were lower at 0 h than at 12 h after serum shock. Compared with the 12 h VSIR group, the 12 h application of EI resulted in a significant decrease in ROS levels, as shown in the attached Figure 4 As shown in C. The ATP concentration was measured. The 0hC group produced more ATP than the 12hC group. Compared with the 12hVSIR group, the 12h EI treatment increased the ATP concentration, but there was no significant difference at 0h, as shown in the attached Figure 4 As shown in D.

[0061] PER2 plays a cardioprotective role in ISO and participates in circadian rhythm differences through oxidative stress and energy metabolism

[0062] To gain molecular insights into the regulation of ISO on I / R injury, RNA was isolated from the left ventricle of mice treated with or without ISO at ZT0 / 12 and RNA-seq was performed. Figure 5 As shown, A and B are heat maps of circadian rhythm and metabolism related genes; C is the expression of Per2 mRNA in mouse heart; D is the expression of mPer2 in H9c2 cells; E is the expression of PER2 protein in mouse heart; F is the expression of Ctpa mRNA in mouse heart; G is the expression of Ctpa mRNA in H92c cells; H is the expression of CPT1A protein in mouse heart.

[0063] According to the heat map of circadian rhythm genes (see Figure 5(A and B), there are significant differences in the expression levels of circadian genes between ZT0 and ZT12. ISO treatment at ZT0 leads to significant changes in circadian gene expression, including an increase in night-time "dominant" genes (Per2, Cyrl, and Per1) and a decrease in day-time "dominant" genes (Bmal1 and Clock). Protein and mRNA were collected from mice and cells after 24 hours of reperfusion. PER2 is an important circadian gene, and its transcript levels show low expression in mice at ZT0 and cells at 12h, and high expression in mice at ZT12 and cells at 0h. ISO treatment of mice at ZT0 and cells at 12h increased Per2 expression; however, it did not increase after ISO treatment at ZT12 and 0h, as shown in the attached figure. Figure 5 As shown in C and D. The changes in PER2 protein levels were consistent with the mRNA results, as shown in Supplementary Figure 2. Figure 5 As shown in E.

[0064] Furthermore, the present invention also tested whether Per2 knockdown abolished the protective effect of ISO in SIR-treated H9c2 cells. The results are shown in the attached figure. Figure 6 As shown, A is the efficacy of PsiRNA-Per2 confirmed by qRT-PCR; B is the image obtained by observing cell morphology under an inverted / phase contrast microscope (200×); C is the level of cell apoptosis, representative images of flow cytometry (left), quantitative analysis of cell apoptosis rate (right); D is the ROS expression of H9c2 cells; E is the ATP content of H9c2 cells: Per2siRNA transfection exacerbated oxidative stress and reduced ATP levels (P<0.0001VS0hEI12hSIR group); F is the expression of Cpt1a mRNA after siRNA-Per2 treatment. After siRNA-Per transfection, the expression of Cpt1a mRNA was significantly reduced (P<0.0001, <0.0001, =0.04, and =0.0005vs 0hr VSIR, 0hr EISIR, 12hr VSIR, and 12hr EISIR groups); G is the electrophoresis result of chromatin immunoprecipitation PCR products.

[0065] From the attached Figure 6 As can be seen in Figure A, siRNA-Per2 significantly reduced the expression of Per2. Figure 6 As can be seen in Figure B, siRNA-Per2 reduced the cardioprotective effect of ISO, as shown by changes in cell viability and morphology. After siRNA-Per2 treatment, apoptosis of H9c2 cells increased at 0 and 12 h, as shown in the attached Figure 6 As shown in Figure 2C, it is noteworthy that after siRNA-Per2 treatment, oxidative stress was aggravated, as shown in Figure 2C. Figure 6 As shown in D; ATP level was significantly decreased after siRNA-Per2 transfection, as shown in the attached Figure 6 As shown in E.

[0066] CPT1A is a target gene of PER2

[0067] From the attached Figure 5 As can be seen in B, the heat map of metabolism-related genes shows that there are diurnal differences in Cptla levels. CPT1A is a rate-limiting enzyme for the oxidation of long-chain fatty acids from the cell matrix to the mitochondrial matrix. The expression of Cptla mRNA and protein was measured. We found that there were differences in the expression of metabolism-related genes treated with ISO at ZT0 / 12. Cptla had similar fluctuations to Per2. Under the same treatment conditions, the mRNA and protein expression of PER2 and CPT1A were similar, as shown in the attached figure. Figure 5 As shown in Figures FH. After siRNA-Per2 transfection, CPT1A was significantly reduced, as shown in Figure 4. Figure 6 As shown in G. Therefore, we investigated the targeting relationship between PER2 and CPT1A.

[0068] The specific binding region of PER2 and CPT1A was predicted by an online analysis website (http: / / bioinfo.life.hust.edu.cn / AnimalTFDB / ), and the ChIP results showed that the PCR products of the PER2IP group were enriched in agarose gel electrophoresis, which revealed the targeting relationship between PER2 and CPT1A compared with IgG immunoprecipitation. CPT1A is the target gene of PER2, as shown in the attached Figure 6 As shown in H.

[0069] In summary, the research method of the present invention ultimately revealed that: (1) ISO's myocardial protection against I / R injury in vivo and in vitro varies with circadian rhythm; (2) ISO affects cardiac protein PER2 by reducing oxidative stress and promoting energy metabolism, thereby reducing myocardial injury; (3) PER2 regulates the transcription of CPT1A (a key enzyme in fatty acid metabolism and oxidative stress levels) to exert a cardioprotective effect. Current findings show that ISO pretreatment of ZT0 mice or H9c2 cells 12h after serum shock exhibits a significant cardioprotective effect, which improves myocardial function or cell activity, reduces myocardial infarction area, reduces myocardial cell apoptosis, reduces oxidative stress, and improves energy metabolism. In contrast, there was no significant effect on mice at night or H9c2 cells at the serum shock endpoint. ISO's cardioprotective effect is closely related to PER2, and PER2 siRNA eliminates ISO's protective effect during cardiac injury.

[0070] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for studying the diurnal differences in the myocardial protective effect of PER2 in ISO, characterized in that: The following steps are included: S1: The living experimental mice were randomly divided into groups, and the experimental mice were respectively established with sham injury or myocardial ischemia-reperfusion injury I / R models, and treated with isoflurane or carrier gas before modeling; the carrier gas was air containing 30% oxygen; S2: H9c2 cells were cultured in vitro. To synchronize the circadian rhythm, all cells were first treated with horse serum for 2 h. The end of treatment was taken as "0 o'clock". At this time, all cell culture medium was replaced with serum-free DMEM. At "0 o'clock" or "12 o'clock", the cells were treated with emulsified ISO, lipid or serum-free DMEM, and then an in vitro cell model of simulated ischemia-reperfusion injury was established. S3: Perform corresponding functional assays on the live animal experimental mice in step S1 and the in vitro cultured cells in step S2 to analyze the day-night differential effects of PER2 on the myocardial protection of ISO; In step S1, the experimental mice were randomly divided into eight groups, namely, ZT0 sham operation group, ZT0ISO+sham operation group, ZT12 sham operation group, ZT12ISO+sham operation group, ZT0IR group, ZT0ISO+IR group, ZT12IR group and ZT12ISO+IR group; Among them, ZT0 means inhalation of carrier gas at 7 o'clock on the day before modeling, ZT0ISO means inhalation of 1.5% ISO at 7 o'clock on the day before modeling, ZT12 means inhalation of carrier gas at 7 o'clock on the night before modeling, and ZT12ISO means inhalation of 1.5% ISO at 7 o'clock on the night before modeling.

2. The method for studying the day-night difference of the myocardial protective effect of PER2 in ISO according to claim 1, characterized in that: In step S2, H9c2 cells were cultured in a 5% CO2 humidified incubator at 37°C using DMEM-F12 supplemented with 10% fetal bovine serum and 1% streptomycin / penicillin.

3. The method for studying the day-night difference of the myocardial protective effect of PER2 in ISO according to claim 2, characterized in that: In step S2, the in vitro experimental cells are divided into 12 groups, namely, 0hrC group, 0hrV group, 0hrE1 group, 12hrC group, 12hrV group, 12hrE1 group, 0hrCSIR group, 0hrVSIR group, 0hrEISIR group, 12hrCSIR group, 12hrVSIR group and 12hrE1SIR group; Among them, 0hr means within 0h after horse serum treatment, C means serum-free DMEM treatment, V means lipid treatment, El means emulsified ISO treatment; 12hr means 12h after horse serum treatment; SIR means I / R simulated by H2O2.

4. The method for studying the day-night difference of the myocardial protective effect of PER2 in ISO according to claim 1, characterized in that: The functional assays described in step S3 include: myocardial function assay, cell apoptosis rate assay, myocardial infarction area assay, cell activity assay, free radical generation assay, ATP content assay and oxidative stress assay.

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