Blood biomarker, kit and medicine for regulating and controlling blood pressure of old people

Abstract

The invention relates to the technical field of biology, in particular to a blood biomarker, a kit and a medicine for regulating and controlling the blood pressure of old people. The blood biomarker is characterized by being at least one of the following non-coding RNA (Ribonucleic Acid) or proteins: LncRNA GAS5, miR-223-3p, NLRP3 and GSDMD. Compared with healthy old people, the expression of LncRNA GAS5 and NLRP3 in blood of old people with hypertension is increased, and the expression of miR-223-3p is reduced. The GSDMD participates in the blood pressure rise of the old mice caused by PM2.5. Therefore, the LncRNA GAS5, the miR-223-3p, the NLRP3 and the GSDMD in the blood of the senile hypertension patients have potential values as the senile hypertension diagnosis biomarkers and the intervention targets.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to blood biomarkers, kits and drugs for regulating blood pressure in the elderly. Background Art

[0002] Population aging is a trend in global demography today. The elderly are at high risk of cardiovascular disease. The incidence and prevalence of cardiovascular disease in my country are clearly on the rise. The current number of people suffering from cardiovascular disease is about 330 million, which is the leading cause of death among residents and seriously threatens the health of the elderly in my country. The 2024 Global Air Quality Report points out that air pollution is the second largest risk factor for death in 2021, causing 8.1 million deaths, of which ambient PM 2.5 It is the largest driver of the global air pollution disease burden, and the elderly population is the largest 2.5 Exposed susceptible population, and PM 2.5 There is a strong causal relationship between exposure and cardiovascular disease.

[0003] Blood pressure is one of the sensitive risk factors in the development of cardiovascular disease, and its regulation is also affected by PM. 2.5 The impact of PM 2.5 The effect on blood pressure may be achieved by damaging endothelial function. Currently, a large number of studies have reported that PM 2.5 The role of oxidative stress and inflammatory response induced by exposure in impaired endothelial function, but PM 2.5 The mechanism of causing inflammatory response is still unclear. With the development of epigenetics, people have found that non-coding RNA plays an important regulatory role in the occurrence and development of hypertension. This paper aims to explore the mechanism of non-coding RNA in the increase of blood pressure in the elderly and find PM 2.5 Biomarkers that cause high blood pressure in the elderly population can be used to discover new intervention targets, providing a reference for further screening of therapeutic targets for lowering blood pressure. Summary of the invention

[0004] In order to screen therapeutic targets for lowering blood pressure, the present invention provides a blood biomarker, a kit and a drug for regulating blood pressure in the elderly.

[0005] The present invention is achieved through the following technical scheme: blood biomarkers for regulating blood pressure in the elderly, wherein the blood biomarkers are at least one of the following characteristic non-coding RNAs or proteins, LncRNA GAS5, miR-223-3p, NLRP3, and GSDMD.

[0006] Among them, LncRNA GAS5 and miR-223-3p are non-coding RNAs, and NLRP3 and GSDMD are proteins.

[0007] As a further improvement of the technical solution of the present invention, the present invention collected peripheral blood from elderly hypertensive patients and healthy elderly control groups, and detected the expression levels of LncRNA GAS5, miR-223-3p, and NLRP3. Compared with the healthy control group, LncRNA GAS5 and NLRP3 in the blood of elderly hypertensive patients were significantly increased, and miR-223-3p was significantly downregulated. Therefore, LncRNA GAS5, miR-223-3p, and NLRP3 in the blood of elderly hypertensive patients have potential value as diagnostic biomarkers and intervention targets for elderly hypertension.

[0008] The present invention also constructs an elderly animal model, PM 2.5 The blood pressure of aged mice increased, the expression of LncRNA GAS5, NLRP3 and GSDMD increased in the aorta tissue of aged mice, and the expression of miR-223-3p decreased. The LncRNA GAS5 / miR-223-3p / NLRP3 axis participates in PM by regulating cell pyroptosis 2.5 The results showed that LncRNA GAS5, miR-223-3p, NLRP3 and GSDMD can be used as new targets for blood pressure regulation in the elderly.

[0009] The present invention further provides primers for DNA amplification of the blood biomarkers that regulate blood pressure in the elderly population.

[0010] Wherein, the primers include an upstream primer and a downstream primer.

[0011] The present invention also provides a blood test kit for the elderly, comprising primers for DNA amplification of the blood biomarkers for regulating blood pressure in the elderly as described above.

[0012] The present invention further provides a method for detecting blood test of the elderly population, using the elderly population blood test kit as described above for detection.

[0013] The present invention also provides a drug for regulating blood pressure in the elderly, including at least one of si-GAS5, miR-223-3p agonist, and DSF. The si-GAS5 can target and interfere with LncRNA GAS5 through small interfering RNA (siRNA), thereby inhibiting the expression of LncRNA GAS5; the miR-223-3p agonist has the same sequence as the target miR-223-3p, and is used to simulate and enhance the biological function of miR-223-3p, upregulate endogenous miR-223-3p, and promote the expression of its downstream target genes; the DSF is determined to be an effective inhibitor of GSDMD pore formation and is found to have a potential role in treating inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The differential expression of LncRNA GAS5, miR-223-3p, and NLRP3 in elderly hypertensive patients and healthy elderly individuals (n=50). Note: control: healthy elderly individuals; hypertension: elderly hypertensive patients. *P<0.05, **P<0.01, ***P<0.001; A is LncRNA GAS5, B is miR-223-3p, and C is NLRP3.

[0017] Figure 2 Correlation analysis between LncRNA GAS5 / miR-223-3p / NLRP3 and SBP, DBP (n=50). Note: A: Correlation analysis between LncRNA GAS5 and SBP and DBP; B: Correlation analysis between miR-223-3p and SBP and DBP; C: Correlation analysis between NLRP3 and SBP and DBP. r: correlation coefficient.

[0018] Figure 3 The weight changes of mice in each group at different time points. Note: PBS: PBS control group; LPM 2.5 : Low-dose PM 2.5 Exposure group (2.6 mg / kg·bw); MPM2.5 : Medium dose PM 2.5 Poisoning group (5.2 mg / kg·bw); HPM 2.5 : High dose PM 2.5 Poisoning group (10.4 mg / kg·bw).

[0019] Figure 4 The changes of contraction and relaxation function of thoracic aorta rings in each group of mice (n=6). Note: a: P<0.05 compared with PBS group; b: P<0.05 compared with LPM group 2.5 Compared with P<0.05; a or b is green: represents MPM 2.5 Group with PBS or LPM 2.5 Compared with P<0.05; a or b is blue: represents HPM 2.5 Group with PBS or LPM 2.5 Compared with P<0.05.

[0020] Figure 5 HE staining of aorta tissue of mice in each group (n=3). Note: The magnification of the four pictures on the left is 200×, scale bar=50μm, and the magnification of the four pictures on the right is 400×, scale bar=20μm. Black arrows: intima is incomplete and endothelial cells are damaged; red arrows: endothelial cells fall off; green arrows: vacuolar structures.

[0021] Figure 6 EVG staining of aorta tissue of mice in each group (200×) (n=3). Note: A: EVG staining result, scale bar=50μm; red arrow: elastic fiber disintegration and rupture; B: semi-quantitative analysis result.

[0022] Figure 7 Transmission electron microscopy image of mouse aorta vascular endothelial cells (30000×). Note: Scale bar = 1 μm. Red arrow: The vascular endothelial cell membrane ruptures and protrudes, the membrane pores open, and the contents are released.

[0023] Figure 8 The levels of ET-1, NO and inflammatory factors in the serum of mice in each group (n=6). Note: A: ET-1; B: NO; C: ET-1 / NO; D: IL-1β; E: IL-18.

[0024] Fig. 9 The protein expression levels of NLRP3 inflammasome-related indicators in mouse aorta tissue (n=3). Note: A: WB band; B: Grayscale analysis of NLRP3 inflammasome-related indicators.

[0025] Fig.10The mRNA expression levels of LncRNA GAS5, miR-223-3p, and NLRP3 inflammasome-related indicators in the aorta of mice in each group (n=6).

[0026] Fig.11 The mRNA expression level of GSDMD in mouse aorta tissue (n=6).

[0027] Fig.12 The protein expression levels of GSDMD and GSDMD-N in mouse aorta tissue (n=3). Note: A: WB strips; B: grayscale analysis of GSDMD and GSDMD-N.

[0028] Fig.13 The expression levels of GSDMD, miR-223-3p and LncRNA GAS5 in each group of mice before and after intervention (n=5). Note: vehicle: HPM 2.5 Poisoning (10.4 mg / kg·bw) + solvent control group; DSF: HPM 2.5 Poisoning (10.4 mg / kg·bw) + DSF (50 mg / kg) group; control agomir: HPM 2.5 Poisoning (10.4 mg / kg·bw) + agomir negative control group; miR-223-3p agomir: HPM 2.5 Poisoning (10.4 mg / kg·bw) + miR-223-3pagomir group; si-NC: HPM 2.5 Poisoning (10.4 mg / kg·bw) + siRNA negative control group; si-GAS5: HPM 2.5 Poisoning (10.4 mg / kg·bw) + si-GAS5 group. *P<0.05, **P<0.01, ***P<0.001.

[0029] Fig.14 The weight changes of mice in each group before and after exposure and before and after intervention (n=8 or 5). Note: A: weight changes of mice in each group before exposure and after 7 weeks of exposure; B: weight changes of mice before intervention and after one week of intervention.

[0030] Fig.15 Blood pressure and heart rate of mice in each group before and after poisoning (n=8 or 5). Note: A: HPM 2.5 Blood pressure and heart rate levels of mice in each group before exposure; B: HPM 2.5 Blood pressure and heart rate levels of mice in each group after 7 weeks of exposure; C: Comparison of blood pressure and heart rate of mice in each group before and after exposure.

[0031] Fig.16The blood pressure and heart rate of mice in each group before and after intervention (n=8 or 5). Note: A: blood pressure and heart rate levels of mice in each group before intervention; B: blood pressure and heart rate levels of mice in each group after one week of intervention; C: comparison results of blood pressure and heart rate of mice in each group before and after intervention.

[0032] Fig.17 The changes of contraction and relaxation function of thoracic aorta rings in each group of mice (n=5).

[0033] Fig.18 HE staining of mouse thoracic aorta (magnification) (n=3). Note: The magnification of the two pictures marked with black frames is 200×, scale bar=50μm, and the magnification of the two pictures without black frames is 400×, scale bar=20μm. Black arrow: intima is incomplete, endothelial cells are damaged; red arrow: endothelial cells fall off; green arrow: vacuolar structure.

[0034] Fig.19 This is an EVG staining image of mouse thoracic aorta (200×) (n=3). Note: A: EVG staining result, scale bar=50μm; red arrow: elastic fiber disintegration and rupture; B: semi-quantitative analysis result.

[0035] Fig. 20 Transmission electron micrograph of mouse thoracic aorta endothelial cells (30000×). Note: Scale bar = 1 μm. Red arrow: The endothelial cell membrane ruptures and protrudes, the membrane pores open, and the contents are released.

[0036] Fig.21 The levels of ET-1, NO and inflammatory factors in mouse serum (n=6). Note: A: ET-1; B: NO; C: ET-1 / NO; D: IL-1β; E: IL-18.

[0037] Fig. 22 The mRNA expression levels of genes in mouse aorta tissue (n=6). Note: A: mRNA levels of each gene in vehicle group and DSF group; B: mRNA levels of each gene in control agomir group and miR-223-3p agomir group; C: mRNA levels of each gene in si-NC group and si-GAS5 group.

[0038] Fig.23 The protein expression levels of NLRP3 inflammasome-related indicators in mouse aorta tissue (n=3). Note: A: WB band; B: Grayscale analysis of NLRP3 inflammasome-related indicators. DETAILED DESCRIPTION

[0039] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0041] The specific embodiments of the present invention are described in detail below.

[0042] Example 1 Differential expression of LncRNA GAS5 / miR-223-3p / NLRP3 in healthy elderly people and elderly patients with hypertension 1.1 Recruitment of research subjects

[0043] A 1:1 matched study design was used to recruit 50 healthy elderly individuals and 50 elderly hypertensive patients from six communities in Taiyuan City, Shanxi Province from July to September 2021 as research subjects. The inclusion criteria for the research subjects were as follows: ① Age > 65 years old; ② Living in Taiyuan City for 1 year or more; ③ Voluntary participation in this study. Exclusion criteria: ① Those with severe cardiovascular and cerebrovascular diseases or other major diseases; ② Those with language or hearing impairments; ③ Those who refused to provide biological samples. The general information of the research subjects is shown in Table 1-1.

[0044] Table 1 General information of elderly hypertensive patients and healthy controls

[0045]

[0046] 1.2 Blood sample collection and processing

[0047] Fasting venous blood was collected from the subjects, who were required to fast for at least 8 hours, and collected by professional nurses. The blood samples were centrifuged at 3000 rpm for 10 min, and the upper plasma was drawn into 1.5 mL EP tubes, which were numbered and frozen at -80°C.

[0048] 1.3 Isolation of mononuclear cells (PBMCs)

[0049] (1) Add 2 mL of PBS to a 5 mL blood collection tube, shake slowly, transfer to a 15 mL centrifuge tube, add 4 mL of PBS to dilute the blood sample, and mix well;

[0050] (2) Pipette 3 mL of human peripheral blood lymphocyte separation solution and add it to the bottom of a new 15 mL centrifuge tube;

[0051] (3) Take the diluted blood sample and slowly add it along the wall of the tube into the centrifuge tube containing human peripheral blood lymphocyte separation medium, making sure that the blood sample remains in the upper layer;

[0052] (4) Centrifuge at 1700 rpm for 20 min (if the centrifugal radius is small, the centrifugal speed can be increased to 2200 rpm). After centrifugation, stratification is visible.

[0053] (5) Aspirate the middle white lymphocyte layer into a new 15 mL centrifuge tube, add 4 mL of PBS stored at 4°C to rinse the cells, centrifuge at 1700 rpm for 10 min, and discard the supernatant;

[0054] (6) If there are red blood cells, add 2 mL of red blood cell lysis buffer, mix the cells, lyse on ice for 5 min, centrifuge at 2500 rpm for 5 min, and discard the supernatant;

[0055] (7) Add 4 mL of PBS stored at 4°C to rinse the cells, centrifuge at 1700 rpm for 10 min, and discard the supernatant;

[0056] (8) Add 0.5 mL of trizol and pipette the cells until there is no obvious precipitation. Transfer the cells to a 1.5 mL RNase-free EP tube and freeze at -80°C.

[0057] 1.4 qRT-PCR detection of gene expression levels of LncRNA GAS5 / miR-223-3p / NLRP3 in peripheral blood of the elderly

[0058] 1.4.1 Extraction of total RNA and determination of purity and concentration

[0059] (1) Remove PBMCs from -80°C and place on ice;

[0060] (2) Add 200 μL of chloroform, mix thoroughly by inversion for 15 seconds, let stand at room temperature for 5 minutes, and then centrifuge at 12,000 g for 15 minutes at 4°C;

[0061] (3) Carefully pipette the upper aqueous phase into a new RNase-free EP tube, being sure to avoid pipetting the middle layer;

[0062] (4) Add 500 μL of isopropanol, mix by inversion for 15 seconds, let stand at room temperature for 10 minutes, centrifuge at 12,000 g for 10 minutes at 4°C, and discard the supernatant;

[0063] (5) Wash the pellet three times with 1 mL of pre-cooled 75% ethanol (diluted with DEPC water), centrifuging at 7500 g for 5 min at 4°C each time;

[0064] (6) Discard the supernatant and dry the precipitate at room temperature until no obvious liquid remains;

[0065] (7) Add 10 μL DEPC water to dissolve the RNA precipitate;

[0066] (8) Detection of RNA purity and concentration: Use an ultra-micro spectrophotometer, adjust the concentration to zero with DEPC water, take 1 μL of RNA solution to detect the concentration, and record the RNA concentration and 260 / 280 ratio.

[0067] 1.4.2 Reverse transcription reaction

[0068] Adjust the RNA concentration to 0.5 or 1 μg / μL, and prepare the tailing miRNA reverse transcription reaction system and the common reverse transcription reaction system according to the table below.

[0069] Tailing miRNA reverse transcription reaction system: ① Prepare the mixed solution according to the following reaction system:

[0070]

[0071] ② After mixing, centrifuge for 3 to 5 seconds and run in a PCR instrument at 37°C for 60 minutes and 85°C for 5 minutes. After the run, store at -20°C for later use.

[0072] Common reverse transcription reaction system: ① First remove the genomic DNA and prepare the mixed solution according to the following reaction system:

[0073]

[0074] ② Mix well and run in PCR instrument at 42℃ for 2min and 4℃ forever;

[0075] ③ Reverse transcription reaction: prepare the mixed solution according to the following reaction system, and then add 10 μL to the liquid from which the genomic DNA has been removed;

[0076]

[0077]

[0078] ④Mix well and perform reverse transcription reaction in a PCR instrument according to the program of 37℃15min, 85℃5s, 4℃forever. After the operation, store cDNA at -20℃ for later use.

[0079] 1.4.3 Primer design

[0080] The downstream primer of miR-223-3p and the internal reference U6 primer are provided by the kit. The sequences of other primers are shown in Table 2-1.

[0081] Table 2 mRNA primer sequences

[0082]

[0083] 1.4.4 Amplification reaction

[0084] Fluorescence quantitative kit TB Premix Ex Taq TM For quantitative detection of mRNA, the reaction solution was prepared according to the following reaction system, 3 replicate wells were set for each sample, and the detection was performed on a fluorescent quantitative PCR instrument;

[0085]

[0086] Perform fluorescence quantitative PCR reaction according to the following reaction procedure:

[0087]

[0088] 1.5 Differential expression of LncRNA GAS5 / miR-223-3p / NLRP3 between elderly hypertensive patients and healthy elderly individuals

[0089] The expression levels of LncRNA GAS5 / miR-223-3p / NLRP3 in the two groups were detected by qRT-PCR. Figure 1 As shown in the figure, the expression levels of LncRNA GAS5 and NLRP3 in elderly hypertensive patients were significantly increased (P<0.001), while the expression level of miR-223-3p was significantly decreased (P<0.001).

[0090] 1.6 Correlation between gene expression levels of peripheral blood LncRNA GAS5 / miR-223-3p / NLRP3 and SBP and DBP

[0091] Pearson or Spearman correlation analysis was used to analyze the correlation between the expression levels of LncRNA GAS5 / miR-223-3p / NLRP3 in peripheral blood and SBP and DBP in 50 healthy elderly controls and 50 elderly hypertensive patients. Figure 2 As shown in Figure 2, LncRNA GAS5 and NLRP3 were positively correlated with SBP and DBP, while miR-223-3p was negatively correlated with SBP and DBP. The specific correlation coefficients and P values ​​are shown in Figure 2 .

[0092] In this embodiment, peripheral blood was collected from 50 healthy elderly controls and 50 elderly hypertensive patients, RNA was extracted, and the expression levels of LncRNA GAS5, miR-223-3p and NLRP3 were detected. Compared with the healthy control group, peripheral blood LncRNA GAS5 and NLRP3 in elderly hypertensive patients were significantly upregulated, positively correlated with SBP and DBP, and miR-223-3p was significantly downregulated, negatively correlated with SBP and DBP. Therefore, it is suggested that LncRNA GAS5, NLRP3 and miR-223-3p have potential value as biomarkers and intervention targets for blood pressure regulation in the elderly population.

[0093] Example 2 si-GAS5, miR-223-3p agomir and DSF in PM 2.5 Study on the model of hypertension in aged mice

[0094] 2.1 Establish PM 2.5 Inducing a model of high blood pressure in aged mice, using siRNA, miRNA agomir and DSF to intervene in mice

[0095] Small interfering RNA (siRNA) is a double-stranded RNA molecule of 21-23 nucleotides in length, which mainly acts at the post-transcriptional level. siRNA can target specific mRNA molecules through the RNA interference pathway and guide the RNA-induced silencing complex to degrade them, thereby inhibiting the expression of target genes. miRNA agomir is a class of specially chemically modified miRNA agonists. As an analog of miRNA, agomir can specifically upregulate the expression of miRNA in cells. Disulfiram (DSF), a drug used clinically to treat chronic alcoholism, has recently been identified as an effective inhibitor of GSDMD pore formation and has been found to have potential for the treatment of inflammatory diseases.

[0096] Specifically, after one week of adaptive feeding, SAMP8 mice were anesthetized with isoflurane, and 20 μL of blank poison solution was dripped into the nasal cavity of the PBS group. 2.5 The exposed groups were given 20 μL of PM of different concentrations in the nasal cavity. 2.5The poison solution was used once every other day for a total of 8 weeks. Preparation of DSF intervention agent: 40 mg of DSF powder was dissolved in 1.2 mL of DMSO solution, and after thorough mixing, 4.8 mL of PEG300 was added. After mixing and clarifying, 600 μL of Tween 80 was added. After mixing and clarifying, 5.4 mL of sterile deionized water was added. si-NC and si-GAS5, control agomir and miR-223-3p agomir were packaged and stored, 10 nmol per tube. 200 μL of sterile PBS dissolution reagent was added before each injection, and then tail vein injection was performed. On days 50-56, mice were intraperitoneally injected with 600 μL DSF (50 mg / kg) and solvent 1 hour before exposure; on days 52, 53, and 56, mice were injected with si-GAS5 and si-NC, miR-223-3p agomir and control agomir via tail vein 1 hour before exposure, 10 nmol / 200 μL per mouse each time. On day 57, after removing the eyeballs to collect blood, the mice were killed by dislocation of the neck and samples were collected.

[0097] 2.2 Statistical analysis

[0098] In this embodiment, SPSS26.0 software was used to perform statistical analysis on the experimental data, and GraphPad Prism8 was used for drawing. When comparing between two groups, if the data met normality and homogeneity of variance, an independent sample t test was used; if normality was met but the variance was unequal, a t' test was used; if normality was not met, a nonparametric Mann-Whitney U rank sum test was used. When comparing between multiple groups, if the data met normality and homogeneity of variance, a one-way analysis of variance was used, and a pairwise comparison was performed using the SNK method; if normality and homogeneity of variance were not met, a nonparametric Kruskal-Wallis rank sum test was used. P<0.05 (bilateral) indicated that the difference was statistically significant.

[0099] 2.3PM 2.5 Effects on various indicators of SAMP8 mice

[0100] 2.3.1PM 2.5 Changes in body weight of mice in the exposure groups

[0101] like Figure 3 As shown, the body weight of mice in each group increased slightly with the extension of exposure time, but no statistical difference was found.

[0102] 2.3.2PM 2.5 Effects on blood pressure and heart rate in SAMP8 mice

[0103] The blood pressure and heart rate of mice in the awake state were measured using the BP-2010A intelligent non-invasive sphygmomanometer by the tail-cuff method. The blood pressure and heart rate of mice in each group were compared at different time points, as shown in Table 2-1. As early as the 24th day, MPM 2.5 and HPM 2.5 The SBP of the HPM group was significantly higher than that of the PBS group. 2.5 The SBP of the group was significantly higher than that of the MPM group after day 40. 2.5 Group. Description of exposure to PM 2.5 Causes SBP to increase in aged mice, and with PM 2.5 Compared with the DBP and MAP in the PBS group, the blood pressure increased with the increase of PM concentration from the 24th day. 2.5 Compared with blood pressure, HR changes occurred later. After the 40th day, MPM 2.5 The heart rate of the group was higher than the baseline and LPM 2.5 group (P<0.05).

[0104]

[0105]

[0106] 2.3.3 Systolic and diastolic function of mouse thoracic aorta

[0107] The vascular contraction and relaxation experiment (ex vivo vascular ring experiment) is to separate various blood vessels (including aorta, coronary artery, mesenteric artery, renal artery, etc.) and cut the blood vessels into 3-5mm ex vivo vascular rings after cleaning the surrounding tissues of the blood vessels. The ex vivo vascular rings are suspended on the vascular tension sensor with tungsten wire or other metal wire, and the tension transducer is connected. The vascular tension is observed after different intervention treatments, and the contraction and relaxation responses of the vascular rings to various interventions are evaluated.

[0108] The thoracic aorta of each group of mice was taken to measure the vasoconstriction and relaxation function. Figure 4 As shown in Figure 3, there was no significant difference in the contraction response induced by phenylephrine (PE) and the relaxation response induced by sodium nitroprusside (SNP) among the groups of vascular rings. 2.5 Acetylcholine chloride (ACH) (10 -5 mol / L and 10 -4 mol / L) caused a weakened relaxation response (P<0.05); MPM 2.5 Group ACH in 10 -4 mol / L caused a weakened relaxation response (P<0.05). This indicates that medium and high concentrations of PM 2.5 Exposure reduced vasodilation in mice.

[0109] 2.3.4 HE staining of mouse aorta tissue

[0110] In order to more intuitively observe the damage of mouse blood vessels and vascular endothelium, HE staining was performed on the paraffin sections of mouse aorta tissue. Figure 5 The aorta wall of the control group was smooth, the intimal epithelial cells were tightly connected and arranged in an orderly manner, and there was no inflammatory cell infiltration; low and medium concentrations of PM 2.5 The vascular wall of the group showed incomplete intima, endothelial cell damage, and the intercellular space in the middle membrane increased to varying degrees. 2.5 The intima was obviously incomplete in the group, the endothelial cells were arranged in disorder, there was obvious endothelial cell shedding, the smooth muscle cells were disordered, and there were vacuolar structures.

[0111] 2.3.5 EVG staining of mouse aorta tissue

[0112] like Figure 6 As shown in the figure, in order to observe the changes in mouse vascular elastic fibers, EVG staining was performed on mouse thoracic aorta tissue sections. EVG staining can make the elastic fibers appear black. Figure 6 -A shows that the elastic fiber layer of the aorta in the control group was dense and intact. 2.5 The elastic fibers in the thoracic aorta of the mice in the group A became thinner, the distance between the elastic fibers in each layer became larger, the internal elastic layer was damaged, and obvious elastic fiber breakage and disintegration were observed. Semi-quantitative analysis was performed, and the results were as follows Figure 6 -B, HPM 2.5 The elastic fibers in the group were significantly lower than those in the control group.

[0113] 2.3.6 Transmission electron microscopy observation of mouse aortic endothelial cells

[0114] like Figure 7 As shown, it was found that the vascular endothelial cells of the control group mice had regular morphology, complete cell structure, and continuous cell membrane without protrusions; PM 2.5 In the group, it was observed that the endothelial cells lost their original cell structure, the cell nuclei condensed, and the cell membrane lost its integrity; when exposed to different concentrations of PM 2.5 The endothelial cell membranes of the blood vessels underneath are ruptured to varying degrees, forming protrusions, opening membrane pores, and releasing contents.

[0115] 2.3.7 Levels of inflammatory factors and ET-1 / NO in mouse serum

[0116] Serum IL-1β and IL-18 levels Figure 8 -A and Figure 8 -B, HPM 2.5 The levels of IL-1β and IL-18 in the MPM group were significantly increased. 2.5The levels of ET-1 and NO in serum were also significantly higher than those in the PBS group. Figure 8 Compared with the PBS group, the 2.5 The level of ET-1 in the serum of mice was significantly increased (P<0.01). 2.5 The ET-1 level in the group was significantly higher than that in the MPM 2.5 group (P<0.05); HPM 2.5 Groups and MPMs 2.5 The NO content in the serum of the mice in the HPM group was significantly lower than that in the PBS group. 2.5 The NO content in the group was significantly lower than that in the LPM 2.5 group (P<0.05); comparing the ratio of ET-1 to NO, it was found that MPM 2.5 Groups and HPM 2.5 The ET-1 / NO ratio in the PBS group was significantly higher than that in the LPM group. 2.5 group (P<0.01), and HPM 2.5 The ET-1 / NO ratio of the group was significantly higher than that of MPM 2.5 group (P<0.001).

[0117] 2.3.8PM 2.5 Effects on the LncRNA GAS5 / miR-223-3p / NLRP3 axis

[0118] Western Blot was used to detect the protein expression levels of NLRP3 inflammasome in mouse aorta tissue, including NLRP3, ASC, Pro-Caspase-1 and Caspase-1. Fig. 9 As shown, MPM 2.5 Groups and HPM 2.5 The NLRP3 level in the HPM group was significantly higher than that in the PBS group (P<0.05). 2.5 The expression of the 2.5 Groups and MPMs 2.5 group, indicating high concentration of PM 2.5 Exposure induced activation of the NLRP3 inflammasome.

[0119] The mRNA levels of LncRNA GAS5, miR-223-3p, and NLRP3 in mouse aorta tissues Fig.10 HPM 2.5 The mRNA levels of LncRNA GAS5 and NLRP3 in the group were significantly higher than those in the control group and LPM 2.5 Groups and MPMs 2.5 Group, MPM 2.5 Groups and HPM2.5 The mRNA level of miR-223-3p in the group was significantly lower than that in the control group and LPM 2.5 Group.

[0120] 2.3.9PM 2.5 Effects on GSDMD gene and protein expression levels

[0121] like Fig.11 and Fig.12 As shown, compared with PBS and LPM 2.5 Groups and MPMs 2.5 Compared with the HPM group 2.5 The expression level of pyroptosis-related protein GSDMD in the MPM group was significantly increased (P<0.05). 2.5 Groups and HPM 2.5 The protein expression of GSDMD-N in the pyroptosis group was significantly higher than that in the control group (P<0.05). 2.5 Exposure may induce pyroptosis of vascular endothelial cells.

[0122] 2.4 Effects of intervention on various indicators of SAMP8 mice

[0123] 2.4.1 Evaluation of intervention effects

[0124] like Fig.13 As shown in Figure 2A, compared with the vehicle group, the expression level of GSDMD was significantly decreased after intraperitoneal injection of DSF (P<0.01), indicating that DSF can inhibit the expression of GSDMD to a certain extent in vivo; Fig.13 As shown in Figure 2B, the expression level of miR-223-3p in the aorta of mice injected with miR-223-3p agomir in the tail vein was significantly higher than that in the control agomir group (P<0.001), indicating that miR-223-3p agomir can effectively upregulate the expression of miR-223-3p; Fig.13 -C, the expression level of LncRNA GAS5 in the aorta of mice in the tail vein injection si-GAS5 group was significantly lower than that in the si-NC group (P<0.01), indicating that si-GAS5 can effectively downregulate the expression of LncRNA GAS5.

[0125] 2.4.2 Effects of intervention on mouse body weight

[0126] like Fig.14 As shown in the figure, the body weight of mice in both groups increased after 7 weeks of exposure, but there was no statistical significance (P>0.05). Compared with before intervention, the body weight of mice in both groups decreased slightly after intervention, but there was no statistical significance (P>0.05).

[0127] 2.4.3 Effects of intervention on blood pressure and heart rate in mice

[0128] First, the tail-cuff method was used to non-invasively measure the changes in blood pressure and heart rate of mice before and after 7 weeks of exposure. Fig.15 Compared with before exposure, PM 2.5 After exposure, SBP, MAP and heart rate were significantly increased in all groups (P<0.01). 2.5 There were different degrees of increase after exposure.

[0129] The tail-cuff method was then used to non-invasively measure the blood pressure and heart rate changes of mice before and after the last week of intervention. Fig.16 As shown. The SBP of the vehicle group and the control agomir group increased significantly, while the SBP and DBP of the DSF, miR-223-3p agomir group and the si-GAS5 group decreased significantly. The MAP of the control agomir group increased significantly, while the MAP of the DSF, miR-223-3p agomir group and the si-GAS5 group decreased significantly. In order to exclude the influence of the intervention reagents and intervention methods, the blood pressure and heart rate of the DSF group, the miR-223-3p agomir group and the si-GAS5 group were compared with those of the corresponding negative control group. The results are shown in Fig.16 -B, after the three intervention treatments, the SBP, DBP and MAP of mice decreased significantly (P<0.001).

[0130] 2.4.4 Effects of intervention on vasoconstriction and relaxation of the thoracic aorta in mice

[0131] The thoracic aorta was taken to measure the vasoconstriction and relaxation function. Fig.17 As shown, compared with the control agomir group, the miR-223-3p group had an increased effect on vascular rings and PE (10 -5 Compared with the vehicle group, the DSF group had a weaker contractile response to ACH (10 -4 mol / L) caused an enhanced relaxation response (P<0.05).

[0132] 2.4.5 Effect of intervention on HE staining of mouse aortic tissue

[0133] from Fig.18It can be seen that the vascular intima of the vehicle group lost its integrity, and vacuolar structures were seen in many places. After DSF intervention, the vascular wall became smooth, and no obvious cell damage was observed. The vascular inner wall of the control agomir group was obviously uneven, with endothelial cell shedding and vacuolar structures. After miR-223-3p agomir intervention, the intima became relatively smooth, and endothelial cells were neatly attached to the vascular inner wall. The si-NC group showed obvious incomplete intima, with overflow of cell contents. After si-GAS5 intervention, the intima became relatively smooth, and no obvious endothelial cell shedding was observed. The above results show that DSF, miR-223-3p agomir and si-GAS5 intervention can improve the intima of the blood vessels exposed to PM. 2.5 Subsequent vascular endothelial damage.

[0134] 2.4.6 Effect of intervention on EVG staining of mouse aortic tissue

[0135] from Fig.19 As can be seen from the figure, the mice in the vehicle group, control agomir group, and si-NC group were exposed to high concentrations of PM 2.5 Afterwards, the elastic fibers in the aorta became sparse and thin, and the elastic fiber layer was incomplete in many places, with obvious ruptures and disintegrations. After intervention with DSF, miR-223-3p agomir, and si-GAS5, the elastic fiber layer became complete, and the distance between the elastic fibers in each layer became smaller. Semi-quantitative analysis found that compared with the vehicle and control agomir groups, the elastic fibers in the mouse aorta increased significantly after intervention with DSF and miR-223-3pagomir. The above results suggest that intervention with DSF, miR-223-3p agomir, and si-GAS5 can reduce PM 2.5 Resulting in damage to vascular elastic fibers.

[0136] 2.4.7 Transmission electron microscopy observation of mouse aortic endothelial cells after intervention

[0137] like Fig. 20 As shown, the mice in the vehicle, control agomir group, and si-NC group were exposed to high concentrations of PM 2.5After that, the original cell structure was lost, the cell nucleus condensed, the cell membrane became incomplete, and there were ruptures of varying degrees in multiple places, which led to the opening of membrane pores, the release of cell contents, and the formation of protrusions, which had characteristics similar to cell membrane blistering, which was consistent with the typical characteristics of cell pyroptosis. After DSF, miR-223-3p agomir and si-GAS5 intervention in mice, the structure of their vascular endothelial cells became significantly intact, the cell membrane did not show obvious ruptures, and no cell contents were released. The above results show that DSF, miR-223-3pagomir and si-GAS5 intervention can significantly improve PM 2.5 Resulting in endothelial cell pyroptosis.

[0138] 2.4.8 Effects of intervention on inflammatory factors and ET-1 / NO in mouse serum

[0139] Serum IL-1β and IL-18 levels Fig.21 As shown, compared with the vehicle, control agomir and si-NC groups, the levels of IL-1β and IL-18 in the mouse serum were significantly decreased after intervention with DSF, miR-223-3p agomir and si-GAS5. Compared with the vehicle and control agomir groups, the level of ET-1 in the mouse serum was significantly decreased after intervention with DSF and miR-223-3p agomir; compared with the vehicle, control agomir and si-NC groups, the level of NO in the mouse serum was significantly increased and the ratio of ET-1 to NO was significantly decreased after intervention with DSF, miR-223-3p agomir and si-GAS5. The above results suggest that DSF, miR-223-3p agomir and si-GAS5 intervention can inhibit the release of inflammatory factors and alleviate PM. 2.5 Exposure-induced endothelial damage.

[0140] 2.4.9 Effects of intervention on gene mRNA expression levels in mouse aortic tissue

[0141] Total RNA was extracted from mouse aorta tissue, reverse transcribed, and subjected to fluorescence quantitative PCR reaction. β-actin was used as an internal reference, and the relative expression of mRNA was calculated using the ΔΔCT method. The downstream primers of miR-223-3p and the internal reference U6 primer were provided by the kit, and the sequences of other primers are shown in Table 2-2.

[0142] Table 2-2mRNA primer sequences

[0143]

[0144]

[0145] The results are as follows Fig. 22 As shown in the figure, compared with the si-NC group, after interfering with LncRNA GAS5, the expression of miR-223-3p was significantly upregulated, and the expression of GSDMD and ASC was significantly downregulated. After overexpression of miR-223-3p, the expression levels of GSDMD, NLRP3, ASC and Caspase-1 were significantly lower than those in the control agomir group (P<0.05). Further intervention of the pyroptosis-related index GSDMD, compared with the vehicle group, the gene expression levels of GSDMD, NLRP3 and ASC were significantly decreased after DSF intervention (P<0.05).

[0146] 2.4.10 Effect of intervention on protein expression levels of NLRP3 inflammasome and pyroptosis-related indicators in mouse aortic tissue

[0147] Aortic tissue (about 30 mg) was taken from a -80°C refrigerator and placed in an EP tube. An appropriate amount of protein lysis buffer containing 1% PMSF and 1% phosphatase inhibitor was added. The tissue was broken by an ultrasonic disruptor and then lysed on ice for 30 minutes. The tissue was centrifuged at 4°C and 12,000 rpm for 15 minutes. The supernatant was transferred to a new EP tube. After BCA protein quantification, Western blot was used to detect the expression of related proteins in the aortic tissue.

[0148] like Fig.23 As shown in the figure, after interfering with LncRNA GAS5, the levels of N-terminal proteins GSDMD-N and ASC, Pro-Caspase-1, and Caspase-1 with pore-forming activity in the process of cell pyroptosis were significantly decreased. After overexpression of miR-223-3p, the levels of GSDMD, GSDMD-N, and NLRP3, ASC, Pro-Caspase-1, and Caspase-1 in mouse aortic tissues were decreased. After DSF intervention, the levels of pyroptosis-related proteins GSDMD and GSDMD-N, as well as ASC, Pro-Caspase-1, and Caspase-1 were also decreased.

[0149] In this example, LncRNA GAS5, NLRP3 and GSDMD were expressed in the aorta tissue of aged mice, while miR-223-3p was expressed in the aorta tissue of aged mice. LncRNA GAS5 / miR-223-3p / NLRP3 axis participates in PM by regulating cell pyroptosis 2.5 Exposure-induced increased blood pressure. si-GAS5, miR-223-3p agomir and DSF can improve endothelial cell pyroptosis, while reducing inflammatory response, restoring endothelial function and lowering blood pressure. This further illustrates that LncRNA GAS5, miR-223-3p, NLRP3 and GSDMD can serve as new targets for blood pressure regulation in the elderly.

[0150] The above is only a specific implementation of the present invention, which enables those skilled in the art to understand or implement the present invention. Although detailed descriptions are given with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments, and they should all be covered by the protection scope of the claims.

Claims

1. A blood biomarker for regulating blood pressure in the elderly, characterized in that: The blood biomarker is at least one of the following characteristic non-coding RNAs or proteins: LncRNA GAS5, miR-223-3p, NLRP3, and GSDMD.

2. The blood biomarker for regulating blood pressure in the elderly according to claim 1, characterized in that: Compared with healthy elderly people, the expression of LncRNA GAS5 and NLRP3 in the blood of elderly hypertensive people was increased, and the expression of miR-223-3p was decreased.

3. Primers for DNA amplification of blood biomarkers for regulating blood pressure in the elderly as claimed in claim 1.

4. The primer according to claim 3, characterized in that The primers include an upstream primer and a downstream primer.

5. A blood test kit for the elderly, characterized in that: The invention comprises primers for amplifying DNA of the blood biomarker for regulating blood pressure in the elderly as claimed in claim 1.

6. A method for blood testing of the elderly, characterized in that: The blood test kit for the elderly population as described in claim 5 is used for detection.

7. A drug for regulating blood pressure in the elderly, characterized in that: Including at least one of si-GAS5, miR-223-3p agonist, and disulfiram.

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