Application of BLT2 antagonist in preparation of medicine for treating hypertension induced by circadian rhythm disorder

Abstract

The invention relates to the technical field of biological medicine. The invention particularly relates to application of a BLT2 antagonist in preparation of a medicine for treating hypertension induced by circadian rhythm disorder. The invention mainly solves the problems of high complication morbidity, poor long-term drug treatment compliance, high drug treatment cost and the like of hypertensive patients caused by circadian rhythm disorder, and provides a method for treating hypertension by targeting a central nervous system BLT2.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of gut microbiota metabolite 12-hydroxyheptadecatrienoic acid (12s-HHT) receptor leukotriene B4 receptor 2 (BLT2) as a target in preparation of a drug for preventing and treating high blood pressure induced by circadian rhythm disorder, and an action mechanism of the drug for regulating sympathetic nerve center through an intestinal-brain axis. BACKGROUND

[0002] Circadian rhythm disorder (such as shift work, abnormal light exposure) is an important inducement of high blood pressure, and about 1.2 billion patients worldwide are affected. Existing studies have shown that circadian rhythm disorder increases blood pressure through mechanisms such as sympathetic nerve activity hyperactivity, intestinal flora disorder and central oxidative stress, but the specific molecular pathway has not been clarified.

[0003] As a key factor in the regulation of circadian rhythm, the intestinal flora can affect cardiovascular function through the intestinal-brain axis by means of metabolites (such as short-chain fatty acids). In recent years, it has been found that circadian rhythm disorder leads to significant enrichment of intestinal flora metabolite 12s-HHT in plasma and cerebrospinal fluid. 12s-HHT is an arachidonic acid derivative that can penetrate the blood-brain barrier and activate its receptor BLT2. The present application highlights the importance of 12s-HHT receptor BLT2 as a target in the preparation of a drug for high blood pressure induced by circadian rhythm disorder.

[0004] At present, the drug for high blood pressure caused by circadian rhythm is similar to the conventional drug for high blood pressure, and lacks innovative specific targeted drugs. The intestinal-brain axis function damage caused by circadian rhythm is an important factor in the occurrence of the disease, and therefore a new intervention strategy based on the intestinal-brain axis is needed. SUMMARY

[0005] The main purpose of the present application is to provide application of a BLT2 antagonist in preparation of a drug for treating high blood pressure induced by circadian rhythm disorder.

[0006] The present application provides application of a BLT2 antagonist in preparation of a drug for treating high blood pressure induced by circadian rhythm disorder, and particularly application of BLT2 as a target in preparation of a drug for high blood pressure induced by circadian rhythm disorder.

[0007] The present application first discloses that 12s-HHT mediates sympathetic nerve hyperactivity and blood pressure elevation by activating the BLT2-AKT-oxidative stress pathway in the hypothalamic paraventricular nucleus (PVN), and provides a new target for preventing and treating circadian rhythm disorder hypertension. High blood pressure caused by circadian rhythm disorder is usually mainly caused by autonomic nervous dysfunction, but the specific mechanism of autonomic nervous dysfunction has not been fully elucidated. Therefore, the present application focuses on elucidating the important role of sympathetic nerve activity hyperactivity caused by circadian rhythm disorder in the occurrence and development of high blood pressure through the mechanism of the intestinal-brain axis.

[0008] Among the clinical evaluation indicators of circadian rhythm disorder hypertension, 24-hour ambulatory blood pressure monitoring (ABPM) is the gold standard. Through ABPM, the blood pressure fluctuation curve of a person throughout the day (including at night when sleeping) can be clearly seen. If the ABPM result shows "non-dipper" or "reverse dipper", and its lifestyle (such as shift work, long-term overtime) clearly points to circadian rhythm disorder, then the clinic determines that its hypertension is strongly related to rhythm disorder.

[0009] The present application mainly solves the problems of high incidence of complications in patients with hypertension caused by circadian rhythm disorder, poor long-term drug treatment compliance, and expensive drug treatment, etc., to provide a method for treating hypertension targeting the central nervous system BLT2. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The schematic diagram of the light regimen for the circadian rhythm disorder model is shown. White represents light, and black represents darkness.

[0011] Figure 2 A and Figure 2 B is a result graph of the influence of circadian rhythm disorder on the spatial learning and memory ability of mice, wherein Figure 2 A is a graph of the movement trajectory of the mouse from the water to the first time finding the platform under the water after the navigation training of the mouse; Figure 2 B is a platform crossing frequency histogram. LD: normal 12h light / dark group; CD: circadian rhythm disorder group. P=0.0276, compared with LD, n=7, the data is represented by mean ± standard error.

[0012] Figure 3 A to Figure 3 E is a graph showing the changes in cardiovascular function indicators after circadian rhythm disorder; wherein, Figure 3 A is a detection schematic diagram of the mouse; Figure 3 B is a histogram of mean arterial pressure MAP; Figure 3 C is a histogram of heart rate HR; Figure 3 D is a histogram of norepinephrine NE in plasma; Figure 3 E is a histogram of heart rate variability HRV. n=5, the data is represented by mean ± standard error.

[0013] Figure 4 A to Figure 4 E is a graph showing the changes in intestinal flora of mice after circadian rhythm disorder; wherein, Figure 4 A is the alpha diversity index (chao1, shannon and simpson index difference) of the intestinal flora of the mouse; Figure 4 B, Figure 4 C is a beta diversity index (PCoA analysis) of the intestinal flora of the mouse;Figure 4 D、 Figure 4 E LEfSe analysis of mouse gut microbiota. n=10-11.

[0014] Figure 5 A Heatmap of mouse plasma metabolomics; Figure 5 B Volcano plot of mouse plasma metabolomics; Figure 5 C KEGG bubble plot. n=5, data represented as mean ± SEM.

[0015] Figure 6 A to Figure 6 C Results of overexpression of 12s-HHT in PVN to increase central sympathetic activity; wherein Figure 6 A Raw plot of carotid MAP and HR in anesthetized state after overexpression of 12s-HHT in PVN of mice, black arrow indicates the time point of 12s-HHT injection; Figure 6 B Statistical plot of ΔMAP changes after 12s-HHT injection, n=5; Figure 6 C Blue dot map of positioning injection, scale bar is 100 μm.

[0016] Figure 7 A to Figure 7 D Results of changes in blood pressure and plasma NE of mice before and after BLT2 siRNA injection; wherein Figure 7 A Injection schematic diagram and control fluorescent reagent injection diagram; Figure 7 B Statistical plot of blood pressure and plasma NE of mice; Figure 7 C Changes in ROS level before and after BLT2 siRNA injection, left panel is the result of DHE fluorescent staining of PVN nucleus of mice, red fluorescence indicates ROS, scale bar is 100 μm; right panel is the statistical result; Figure 7 D Results of PVN nucleus AKT phosphorylation before and after BLT2 siRNA injection, left side is the original protein band; right side is the statistical plot. n=5, data represented as mean ± SEM.

[0017] Figure 8 A to Figure 8 D Results of changes in mice before and after specific inhibition of BLT2 in PVN nucleus glutamatergic neurons; wherein, Figure 8 A Schematic diagram of virus injection; Figure 8 B Fluorescent positioning map, scale bar is 100 μm; Figure 8 C Statistical plot of blood pressure and heart rate variability; Figure 8 D Expression of BLT2 in PVN nucleus glutamatergic neurons.

[0018] Figure 9The DHE fluorescence staining results of the mouse PVN nucleus are shown in red fluorescence, indicating the change in ROS level before and after virus injection. n=5, the data is expressed as mean ± standard error. DETAILED DESCRIPTION

[0019] In order to enable a clearer understanding of the technical content of the present application, the following examples are described in detail. However, it should be noted that these descriptions are only to further illustrate the features and advantages of the present application, and are not limiting on the claims of the application.

[0020] Unless otherwise specified, the reagents and methods involved in the examples are commonly used reagents and methods in the art.

[0021] The present application provides: Mechanism correlation verification: circadian rhythm disorder significantly increases the level of 12s-HHT in plasma and cerebrospinal fluid; BLT2 receptor expression increases in PVN nucleus under disorder conditions, glutamatergic neurons are highly expressed, and; 12s-HHT activates PVN region BLT2, triggers AKT phosphorylation, and further activates NADPH oxidase, leading to accumulation of reactive oxygen species (ROS) and sympathetic nerve hyperexcitability.

[0022] Functional verification: inhibition of PVN nucleus BLT2 can reduce AKT phosphorylation, reduce oxidative stress and blood pressure elevation; intestinal flora imbalance (such as enrichment of Bacteroides fragilis) is a direct source of 12s-HHT elevation.

[0023] Therapeutic application: the use of small interfering RNA to target and inhibit PVN nucleus 12s-HHT endogenous receptor BLT2 can block the overactivation of sympathetic center. By interfering with BLT2 in glutamatergic neurons in the PVN nucleus, blood pressure can also be reduced.

[0024] Animal selection Male C57BL / 6 mice weighing 8 weeks were used, purchased from the Naval Special Medical Center Animal Experiment Center. The mice were raised in an animal room with constant temperature (26 ± 2 ℃), humidity control (relative humidity 50%-60%), good ventilation, and water and food could be obtained freely. The normal group was in a 12-hour light: 12-hour dark light-dark cycle, and the model group used the light conditions set for modeling.

[0025] ; Monitoring of arterial blood pressure and heart rate of mice under anesthesia After the mice were weighed, they were anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 0.1 mL / 100 g. After the corneal reflex and toe pinch reflex disappeared, the mice were fixed on a constant temperature pad. The skin on the neck was shaved to expose the skin. The skin was cut along the midline of the neck, and the glands and muscles were bluntly separated to expose the trachea. The surgical suture was pulled out from the back of the trachea, the trachea was cut, the tracheal cannula was inserted, and then the surgical suture was tied and fixed. After fixing, the skin and organ cannula were sutured to prevent the tracheal cannula from falling off during the operation. The carotid artery was bluntly separated to expose the carotid artery. After the artery and vein were carefully separated, the distal end was tied with surgical suture, and the proximal end was pre-knotted. The proximal end of the femoral artery was clamped with an arterial clamp, a small incision was made on the artery with microscissors, and the incision was slightly stretched with microforceps. After stretching, the PE tube filled with heparin solution was inserted, and then the cannula was fixed with suture. After the catheter was inserted, the neck skin was sutured. The arterial catheter was connected to the pressure sensor and PowerLab, and the Labchart software was used to record and analyze the mean arterial pressure (MAP) and heart rate.

[0026] Viral stereotactic injection Draw the electrode: Adjust the heating temperature and gravity of the drawing instrument so that the drawn glass electrode tip is slightly longer, and the tip diameter is about 25 μm.

[0027] Suck the virus: Fix the drawn glass electrode on the electrode holder of the stereotactic instrument, cover the tail end of the electrode with a self-made PE tube, and connect the other end of the PE tube to a 1 ml syringe. Adjust the operating arm of the stereotactic instrument, and lower the electrode into the virus-containing cryopreservation tube. Carefully observe to confirm that the electrode tip is below the liquid level of the cryopreservation tube but cannot touch the bottom of the cryopreservation tube. Slowly withdraw the syringe to form negative pressure and suck the virus into the glass electrode.

[0028] Fixation: Select mice weighing 25-30 g, place the mice in the anesthetic box, and use 3% isoflurane to induce anesthesia for 3 minutes. After anesthesia, shave the mouse's head, keep the left ear bar of the stereotactic instrument fixed, adjust the mouse's head, insert the left ear bar into the mouse's left ear, and then insert the right ear bar into the mouse's right ear. Check if the mouse's head is shaking on both sides, and if the nose is aligned. Then fix the mouse's incisors to the incisor clamp. Shave the top of the mouse's head. Use iodophor and alcohol for disinfection, and then apply aureomycin eye ointment to protect the mouse's eyes.

[0029] Positioning: After the mice are fixed, a heating pad is added under the mouse pad, the anesthesia channel is adjusted, and the concentration is adjusted to 1.5-2%. The skin on the top of the mouse's head is carefully disinfected with iodophor, and then wiped twice with 75% alcohol. The skin on the top of the mouse's head is cut open with scissors to expose the skull, and the surface connective tissue is removed with a cotton swab. Find the bregma and lambda points under the microscope, adjust the height of the incisor bar so that the bregma and lambda are in the horizontal position. Move the glass electrode tip down to the bregma point, and mark the X and Y axis coordinates at this time as zero. First, raise the Z axis, and then move the X and Y axes to move the electrode above the target brain region. In this study, the injection site coordinates are as follows: PVN injection coordinates are as follows: AP: -0.94 mm, ML: ±0.15 mm, DV: -4.43 mm.

[0030] Drilling: Mark the skull above the target brain region with a marker pen, and drop a drop of normal saline to reduce resistance during drilling. Use a 0.5 mm diameter round drill bit to drill a small hole in the skull. After drilling is complete, clean the surface of the skull with a cotton swab.

[0031] Injection: Rotate the Z axis of the operating arm to lower the glass electrode, and observe the electrode tip under the microscope during the lowering process to prevent the glass electrode from directly piercing the skull due to a shift in the drilling position. When the glass electrode tip contacts the dura mater, mark the Z axis coordinate at this time as zero, and then slowly lower it to the injection site. After reaching the injection site, stay for three minutes, and connect the glass electrode to the pneumatic operation pump. Use the stimulator to control the operation of the air pump, set the stimulator stimulation mode to pulse, the frequency is about 0.6-0.8 Hz, adjust the air pump pressure to make the injection speed about 20 nl / min, inject 50 nl on each side. After injection is complete, the electrode stays at the injection site for 10 minutes to reduce virus diffusion, and then slowly removes the electrode. After removing the electrode, wipe the electrode tip with a wet cotton swab to prevent clogging.

[0032] Suture: After injection is complete, suture the incision and place it in a clean cage for feeding.

[0033] ELISA The 12s-HHT enzyme-linked immunoassay (ELISA) kit is used to determine the level of 12s-HHT in the sample by enzyme-linked immunoassay. The purified 12s-HHT antibody is coated on the microplate to form a solid-phase antibody, and 12s-HHT is added to the coated monoclonal antibody microplate, and HRP-labeled 12s-HHT antigen is added to compete for binding. After thorough washing, add the substrate TMB to develop color. The color intensity of the sample is negatively correlated with the content of 12s-HHT in the sample. The absorbance (OD value) is measured at 450 nm wavelength by an enzyme-labeled instrument, and the content of 12s-HHT in the sample is calculated by the standard curve.

[0034] The specific experimental operation steps are as follows: Specimen processing: after water sampling, freeze-thaw three times at -20°C, and then filter through glass fiber. Tissue samples are extracted with butanol:methanol:water (5:25:70 V:V:V), and the extraction is immediately subjected to the experiment.

[0035] Sample addition: standard wells, blank wells (blank control wells without sample and enzyme-labeled reagent, and the same operation as the other steps), and sample wells are set. Add 50 μl to the standard wells on the enzyme-labeled coating plate, and add 40 μl of sample diluent to the sample wells, then add 10 μl of sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-labeled plate well, try not to touch the well wall, and gently shake to mix.

[0036] Enzyme addition: add 50 μl of enzyme-labeled reagent to each well, except for the blank wells.

[0037] Incubation: after sealing the plate with a sealing film, incubate at 37°C for 60 minutes.

[0038] Liquid preparation: dilute the 30-fold concentrated washing solution with distilled water 30 times for standby use.

[0039] Washing: carefully remove the sealing film, discard the liquid, and shake dry. Add enough washing solution to each well, stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry.

[0040] Color development: add 50 μl of color developing agent A to each well, then add 50 μl of color developing agent B, gently shake to mix, and develop color at 37°C for 10 minutes.

[0041] Termination: add 50 μl of termination solution to each well to terminate the reaction (at this time, the blue color turns yellow).

[0042] Measurement: set the zero with the blank wells, and measure the absorbance (OD value) of each well at 450 nm wavelength in sequence. The measurement should be performed within 15 minutes after adding the termination solution.

[0043] Calculation: plot the standard curve on the coordinate paper with the concentration of the standard as the abscissa and the OD value as the ordinate. According to the OD value of the sample, find the corresponding concentration from the standard curve; multiply by the dilution factor; or calculate the linear regression equation of the standard curve with the concentration and OD value of the standard, substitute the OD value of the sample into the equation, calculate the concentration of the sample, and then multiply by the dilution factor to obtain the actual concentration of the sample.

[0044] Plasma metabolomics detection and analysis Sample room temperature thawing, take the sample 100 μL in the EP tube. 2:1 configuration protein precipitant methanol-acetonitrile, add 2 μg / mL of L-2-chlorophenylalanine, take 300 μL in the EP tube, vortex for 1 minute. Ice water bath ultrasonic extraction for 10 minutes, -40℃ for 30 minutes. 12000 rpm, centrifugal 10 minutes, take 200 μL supernatant in LC-MS sample vial. 1:4 configuration methanol-water 300 μL, reconstitute, vortex for 30 seconds, ice water bath ultrasonic for 3 minutes. -40 ℃, stand for 2 hours, 12000 rpm, centrifugal 10 minutes. Take 150 μL of supernatant, filter through a 0.22 μm organic phase needle filter, then transfer to an LC sample vial, and store at -80 ℃. The quality control sample is prepared by mixing equal volumes of extracts from all samples. Use a high-resolution mass spectrometer and high-performance liquid chromatograph system for analysis. Sample mass spectrum signals are collected using positive and negative ion scanning modes. Chromatographic column: ACQUITY UPLC HSS T3 (100 mm x 2.1 mm, 1.8 um); column temperature: 45℃; mobile phase: water (containing 0.1% formic acid), acetonitrile; flow rate: 0.35 mL / min; sample volume: 5 μL.

[0045] Mouse water maze experiment The spatial learning and memory ability of mice is evaluated by water maze experiment, thereby reflecting the degree of circadian rhythm disorder of mice. It mainly includes two stages of positioning navigation experiment and spatial exploration experiment. In the positioning navigation experiment, first, the mice are trained to navigate, and the mice are placed in the water with their heads facing the pool wall (random quadrant), and the time for the mice to find the underwater platform in the water is recorded. After the mouse stays on the platform for 10 seconds, it is removed and the water on the hair is wiped off, and it is placed back in the cage. Each mouse is trained in this way for 5 days, 4 times a day, with a 30-minute interval between two training sessions. Then, in the spatial exploration experiment, on the 6th day after the end of training on the 5th day, the underwater platform is removed, and the time for the mouse to find the underwater platform for the first time and the number of platform crossings are observed and recorded.

[0046] Heart rate variability (HRV) monitoring Fixation: The mouse is put to sleep and fixed on the operating table in a supine position.

[0047] Electrode connection: Two leads are used, with acupuncture needles inserted subcutaneously into the mouse, and electrode end clamps clamped on the needle. The right upper limb of the mouse is connected to the negative electrode (NEG), the left lower limb is connected to the positive electrode (POS), and the right lower limb is connected to the ground (EARTH).

[0048] HRV detection: Connect powerlab and computer, set up software, select mouse-specific parameter settings: sampling frequency 2k; range: 5mV. Start recording.

[0049] Save: After recording is completed, press the software pause recording button to save the file. Remove the electrodes, stop anesthesia, and observe the mouse recovery state until it is awake.

[0050] Injection of 12s-HHT into PVN nucleus of mice The mouse is operated as described above in the ambulatory blood pressure monitoring experiment under anesthesia, and the powerlab is connected to continuously monitor the blood pressure of the mouse. The injection is positioned as described above in the virus positioning injection operation, and the 12s-HHT solution with the prepared concentration is injected into the PVN nucleus at an amount of 50 nl per side. Immediately after the injection is completed, the arterial blood pressure value is observed and recorded.

[0051] Statistical method All data in this experiment were statistically analyzed and plotted using GraphPad Prism 8.0. The experimental data are expressed as mean ± standard error (Mean ± SEM), the P value of the experiment under two conditions is calculated by unpaired t test, and the P value of the experiment before and after administration in the same animal is calculated by paired t test. The P value of the experiment under multiple conditions is calculated by analysis of variance, and P<0.05 represents that the difference is statistically significant.

[0052] Example 1 Model establishment and verification All experimental animals were first placed in an animal room with appropriate temperature and humidity for a one-week adaptation period, during which they could freely take food and water. The experimental group and the control group were both under a 12-hour light: 12-hour dark light condition, with the light turned on at Circadian Time (CT) CT8 and turned off at CT20. CT8 was set as Zeitgeber Time (ZT) ZT0; CT20 was set as ZT12.

[0053] Model establishment Normal light group mouse (LD group) model: After the mice were adapted to the environment, they were randomly divided into groups and placed in a 12h light / 12h dark environment (Light / Dark=12h:12h) with a light intensity of 180 Lux, the light was turned on at ZT0 and turned off at ZT12, and sufficient water and food were provided for 4 weeks.

[0054] Circadian rhythm disorder group mouse (CD group) model: After the mice were adapted to the environment, CT8 was set as ZT0 and CT20 was set as ZT12. The time-controlled light box was set to turn on the light at ZT0 and turn off the light at ZT12, so that the mice were under a 12-hour light: 12-hour dark feeding condition every day, and then the light was turned on at ZT18 and turned off at ZT6 every 2 days. That is, the light cycle feeding lasted for 4 weeks.

[0055] Model validation Circadian rhythm disorder mice behavior abnormalities. In the field of circadian rhythm research, mouse behavior experiments are an indispensable research method. In order to explore whether the experimental conditions can cause mice to have circadian rhythm disorders, the spatial learning and memory ability of mice was evaluated through the water maze experiment, so as to reflect the degree of biological rhythm disorder of mice. After the mice were adapted to the environment for one week, they were placed in LD and CD environments for four weeks. Then the mice were given navigation training. Subsequently, the spatial exploration experiment was carried out, and the trajectory of the mouse from entering the water to finding the underwater platform for the first time and the number of platform crossings were observed and recorded, as shown in Figure 2 A. As shown in Figure 2 B, it was found that the number of platform crossings of the CD group mice increased (P = 0.0276). This provides behavioral abnormality data support for the success of the establishment of the circadian rhythm disorder mouse model.

[0056] Example 2 Circadian rhythm disorder leads to sympathetic nerve activity hyperactivity and blood pressure elevation experiment The results of Example 1 show that the light conditions set can cause the circadian rhythm of mice to be disordered, and the expression of SCN clock genes is changed. In order to explore the effect of circadian rhythm disorder on blood pressure and sympathetic nerves of mice, and to determine whether circadian rhythm disorder will lead to sympathetic nerve activity hyperactivity and blood pressure elevation, after the success of the circadian rhythm disorder modeling, the carotid artery blood pressure, heart rate, plasma norepinephrine content and heart rate variability of the LD and CD group mice under anesthesia were detected.

[0057] As shown in Figure 3 B to Figure 3 E, it was found that the blood pressure of the CD group mice increased (P < 0.0001), the heart rate accelerated (P = 0.0034), the plasma norepinephrine content increased (P = 0.0248), and the heart rate variability increased (P = 0.01). Therefore, it is proved that after the success of the modeling, the circadian rhythm disorder leads to sympathetic nerve activity hyperactivity and blood pressure elevation.

[0058] Example 3 Circadian rhythm disorder induces intestinal flora disorder and blood 12s-HHT production increase experiment The feces of the circadian rhythm disorder CD group mice were subjected to 16S rRNA and metagenomic detection, and the species classification, diversity (α / β diversity), significantly different flora, flora gene function, etc. of the intestinal flora were subjected to sequencing analysis. The specific analysis methods and steps are as follows: The sequencing raw reads data volume distribution is between 78454~81927, the cleantags data volume distribution is between 47786~59364 after quality control, the valid tags (i.e. the final data used for analysis) data volume distribution is between 36558~51466 after removing chimeras, and the ASV number of each sample is distributed between 326~589. The sample difference is statistically analyzed, and the T_test algorithm: the difference ASV number is 116, the difference genus number is 13, and the difference phylum number is 2; the Wilcoxon algorithm: the difference ASV number is 195, the difference genus number is 15, and the difference phylum number is 2. Among them, the alpha diversity analysis is to evaluate the species richness and uniformity within the sample by calculating different alpha diversity indices. On the basis of the diversity index, the alpha diversity violinplot analysis (Kruskal Wallis / Wilcoxon algorithm) can be used to calculate the significance of the diversity index in different groups; the beta diversity analysis evaluates the similarity and difference of the community in different biological environments (different groups). The PCoA or NMDS analysis based on the Bray Curtis distance matrix algorithm can be used to evaluate the significance of the difference between the sample community distribution in different groups, combined with the difference significance p value in the Adonis and Anosim analysis (also based on the Bray Curtis distance matrix algorithm). The difference species (ASV or door class level) between different groups is calculated by the statistical algorithm (ANOVA / Kruskal Wallis / T test / Wilcoxon), and the difference species heatmap is drawn; among them, 1. ANOVA analysis, i.e. one-way ANOVA, is suitable for comparison of >=3 groups, and is suitable for data that meet the normal distribution; 2. Kruskal Wallis test, suitable for comparison of >=3 groups, suitable for data that do not meet the normal distribution; 3. T test, suitable for comparison of 2 groups, suitable for data that meet the normal distribution; 4. Wilcoxon test, suitable for comparison of 2 groups, suitable for data that do not meet the normal distribution. Finally, the correlation analysis between species and species or between environmental factors and species, such as RDA / CCA, correlation heatmap, correlation network diagram, and Indicator, random forest analysis for correlation and prediction model analysis.

[0059] It was found that the intestinal flora alpha diversity of the CD group mice did not change significantly, the beta diversity changed significantly, indicating that the structure of the flora changed significantly, and the abundance did not change significantly. And, as shown in Figure 4 A to Figure 4 E, the Desulfovibrionaceae of the CD group mice was significantly enriched. It is proved that the biological rhythm disorder leads to the change of the composition of the intestinal flora.

[0060] Through plasma metabolomics, 12s-HHT was screened in the plasma of the LD and CD group mice. As shown in the heat map of Figure 5 A and the volcano plot of Figure 6 B, the significant difference of 12s-HHT can be found.

[0061] ELISA detection was used to verify the higher content of 12s-HHT in the plasma and cerebrospinal fluid of the CD group mice. The correlation analysis of 12s-HHT and norepinephrine NE verified the relationship between them and the sympathetic nervous system.

[0062] Example 4 12s-HHT in the PVN nucleus group of the blood pressure raising effect experiment Different doses of 12s-HHT were injected into the PVN nucleus group of the mice under anesthesia, and the MAP and HR of the mice were recorded through carotid cannulation, as shown in Figure 6 A, which shows that after the increase of 12s-HHT in the PVN, the MAP of the mice increases to different degrees, and the increase is different with the concentration gradient.

[0063] ;

[0064] Example 5 The role of the endogenous receptor BLT2 of the intestinal flora metabolite 12s-HHT in the induction of sympathetic nervous activity and blood pressure increase by circadian rhythm disorder BLT2 is involved in the regulation of blood pressure by circadian rhythm disorder. BLT2 siRNA (BLT2 small interfering RNA, Shanghai Zuorun Biotechnology Co., Ltd.) was injected into the PVN nucleus group of the LD and CD mice, and the blood pressure and NE content in the plasma were detected, and it was found that, as shown in Figure 7 B, the blood pressure of the CD group mice decreased after the injection of small interfering RNA (P<0.0001), and the NE value in the plasma decreased (P<0.0001). The reduction of BLT2 in the PVN nucleus group changes the ROS level.

[0065] The ROS level of the PVN tissue of the mice before and after the injection of BLT2 siRNA was detected, and it was found that, as shown in Figure 7As shown in Fig. C, the ROS level of the CDBLT2 siRNA group was lower than that of the CD CON siRNA group (P<0.0001).

[0066] AKT phosphorylation in the PVN tissue was detected before and after the injection of BLT2 siRNA in mice, and it was found that, as shown in Fig. Figure 7 As shown in Fig. D, the AKT phosphorylation of the CDBLT2 siRNA group was lower than that of the CD CON siRNA group (P<0.0001). The reduction of BLT2 changed the AKT phosphorylation in the PVN tissue.

[0067] As shown in Fig. Figure 8 As shown in Fig. A, the BLT2 in the PVN glutamatergic neurons was inhibited, and the blood pressure and heart rate variability were observed, which proved that the BLT2 in the PVN glutamatergic neurons was involved in the regulation of blood pressure in circadian rhythm disorder.

[0068] It was found that, as shown in Fig. Figure 8 As shown in Fig. C, the blood pressure (P=0.0026) and heart rate variability (P=0.0014) of the CD group of mice decreased significantly after the inhibition of BLT2, while the LD group showed no change.

[0069] The injection of virus (rAAV-EF1a-DIO-EGFP-5' miR30-shRNA(BLT2)-3' miR30-WPRE-hGH polyA, Wuhan Zhimibao Science and Technology Co., Ltd.) reduced the BLT2 in the PVN glutamatergic neurons, and it was found that, as shown in Fig. Figure 8 As shown in Fig. D, the expression of BLT2 on the glutamatergic neurons decreased after the injection of virus, which proved that the virus was effective. And, as shown in Fig. Figure 9 The reduction of BLT2 in the PVN glutamatergic neurons changed the ROS level.

[0070] In this specification, the present application has been described with reference to its specific embodiments. However, it is obvious that various modifications and changes can be made without departing from the spirit and scope of the present application. Therefore, the specification should be considered as illustrative rather than limiting.

Claims

1. Use of a BLT2 antagonist in the preparation of a medicament for the treatment of high blood pressure induced by circadian rhythm disorder.

2. Use according to claim 1, characterized in that, The BLT2 antagonist is a PVN nucleus BLT2 antagonist.

3. Use according to claim 1, characterized in that, The BLT2 antagonist is an antagonist of the 12s-HHT endogenous receptor BLT2.

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