Application of butyric acid and HDAC5 in relieving vascular endothelial inflammation caused by PM2.5 and high fat diet combined exposure

Abstract

The invention relates to the technical field of biology, in particular to application of butyric acid and HDAC5 in relieving vascular endothelial inflammation caused by PM2.5 and high fat diet combined exposure. The biomarker is at least one of the following characteristic substances: butyric acid and HDAC5. PM2.5 and HFD combined exposure can lead to reduction of the blood butyric acid level and increase of the aorta HDAC5 level. The sodium butyrate can inhibit expression of vascular inflammation indexes by supplementing butyric acid; the TSA is the trichostatin A, is an effective histone deacetylase inhibitor, and inhibits expression of vascular inflammation indexes by inhibiting HDAC5 (histone deacetylase 5).

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for reducing PM by using butyrate and HDAC5. 2.5 and high-fat diet (HFD)-induced vascular endothelial inflammation. Background Art

[0002] In recent years, the incidence of cardiovascular disease (CVD) has continued to rise worldwide. In 2019, cardiovascular disease caused 18.56 million deaths worldwide, of which 4.58 million were caused by cardiovascular disease in my country, accounting for 43% of total deaths. Hypertension is a major risk factor for CVD. It is estimated that more than 30% (1.38 billion) of adults worldwide suffer from hypertension. The vascular endothelium is a key regulator in the pathogenesis of hypertensive diseases. Its dysfunction and organic damage can lead to the initiation and progression of hypertension. Chronic inflammation is considered to be an important cause of the occurrence and development of hypertension. After the NLRP3 inflammasome is activated, the level of inflammation can be increased, which can lead to changes in the structure and function of the vascular endothelium and cause endothelial damage.

[0003] 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 burden of disease caused by air pollution, and PM 2.5 There is a strong causal relationship between exposure and cardiovascular disease. In addition, lifestyle also plays a very important role in the occurrence and development of cardiovascular disease. Among them, a high-fat diet (HFD) is considered a common risk factor for cardiovascular disease, which can trigger dyslipidemia and obesity in the population through various pathophysiological processes. Obesity is another factor that may increase an individual's exposure to PM. 2.5 Susceptibility to health risk factors.

[0004] Currently for PM 2.5 The mechanism by which HFD causes vascular inflammation is still unclear. With the deepening of intestinal flora research, people have found that intestinal flora imbalance plays an important role in the occurrence and development of cardiovascular diseases, and can affect the occurrence and development of cardiovascular diseases by secreting various metabolites. Gut flora metabolites can affect the activity of histone deacetylase (HDAC). Histone deacetylation modification is involved in the pathophysiological process of various cardiovascular diseases and regulates the expression of downstream target genes through post-transcriptional modification. Deacetylase molecules have potential clinical application value as diagnostic markers and therapeutic targets for cardiovascular diseases. The present invention aims to explore the role of intestinal flora metabolites in PM. 2.5 and HFD-induced vascular inflammation, looking for PM 2.5and biomarkers of HFD-induced vascular inflammation, discover new intervention targets, and provide a reference for further screening of therapeutic targets for lowering blood pressure.

[0005] The above information disclosed in the Background section is only for enhancement of understanding of the background of the present disclosure and therefore it may include information that does not constitute the prior art that is already known to one of ordinary skill in the art. Summary of the invention

[0006] The present invention provides a reference for further screening of therapeutic targets for lowering blood pressure, and provides a method for butyrate and HDAC5 to reduce PM. 2.5 Application in vascular endothelial inflammation caused by combined exposure to HFD.

[0007] The present invention is achieved through the following technical solutions: reducing PM 2.5 A biomarker of vascular endothelial inflammation caused by combined exposure to HFD, wherein the biomarker is at least one of the following characteristic substances, butyrate and HDAC5.

[0008] As a further improvement of the biomarker technical solution of the present invention, PM 2.5 Combined exposure to HFD resulted in decreased blood butyrate levels and increased aortic HDAC5 levels.

[0009] The present invention further provides a method for reducing PM 2.5 Primers for DNA amplification of HDAC5, a biomarker of endothelial inflammation induced by combined HFD exposure.

[0010] As a further improvement of the primer technical solution of the present invention, the primer includes an upstream primer and a downstream primer.

[0011] The present invention also provides a detection kit for detecting HDAC5, wherein the detection kit comprises the PM-reducing 2.5 Primers for DNA amplification of HDAC5, a biomarker of endothelial inflammation induced by combined HFD exposure.

[0012] The present invention further provides a method for detecting tissue HDAC5, which uses the detection kit as described above for detection.

[0013] The present invention further provides a method for preventing and controlling PM 2.5 The drug that causes vascular endothelial inflammation when combined with high-fat diet exposure includes at least one of sodium butyrate and TSA. The sodium butyrate can inhibit the expression of vascular inflammation indicators by supplementing butyrate; the TSA, namely trichostatin A, is an effective histone deacetylase (HDAC) inhibitor, which inhibits the expression of vascular inflammation indicators by inhibiting HDAC5.

[0014] The present invention provides a method for reducing PM 2.5 Compared with existing technologies, biomarkers of endothelial inflammation caused by combined exposure to HFD have the following advantages:

[0015] PM 2.5 Combined exposure to HFD can lead to reduced blood butyrate levels and increased aortic HDAC5 levels. Sodium butyrate can inhibit the expression of vascular inflammatory indicators by supplementing butyrate; the TSA, namely trichostatin A, is an effective histone deacetylase (HDAC) inhibitor, which inhibits the expression of vascular inflammatory indicators by inhibiting HDAC5. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] 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.

[0018] Figure 1 The weight and organ coefficients of mice in each group. Note: (A) The weight change trend of mice in each group; (BG) The relative weight changes of heart, liver, spleen, lung, kidney and brain of mice in each group. *P<0.05, **P<0.01.

[0019] Figure 2 The changes of heart rate and blood pressure of mice in each group. Note: (A) heart rate; (B) systolic blood pressure; (C) diastolic blood pressure; (D) mean arterial pressure.

[0020] Figure 3 The changes of vascular ultrasound indexes of mice in each group. Note: (A) vascular diameter; (B) average blood flow velocity; (C) peak systolic blood flow velocity; (D) end-diastolic blood flow velocity; (E) vascular resistance index; (F) vascular pulsatility index.

[0021] Figure 4 The changes of cardiac ultrasound indexes of mice in each group. Note: (A) cardiac output per stroke; (B) ejection fraction; (C) left ventricular short axis shortening rate; (D) cardiac output; (E) left atrial posterior wall thickness during systole; (F) left atrial posterior wall thickness during diastole; (G) left ventricular posterior wall thickness during systole; (H) left ventricular posterior wall thickness during diastole.

[0022] Figure 5HE staining of aorta tissue of mice in each group. Note: The magnification of HE staining result is 400×, scale bar = 20μm.

[0023] Figure 6 EVG staining of aorta tissue of mice in each group. Note: EVG staining result, scale bar = 50μm;

[0024] Figure 7 is the gene expression level of inflammasome-related indicators in mouse aortic tissue.

[0025] Figure 8 The protein expression levels of inflammasome-related indicators in mouse aorta tissue. Note: (A) WB band; (BF) Grayscale analysis of protein bands of inflammasome-related indicators.

[0026] Fig. 9 The levels of short-chain fatty acids in mouse serum. Note: (A) acetic acid; (B) propionic acid; (C) butyric acid; (D) valeric acid; (E) hexanoic acid; (F) isobutyric acid; (G) isovaleric acid.

[0027] Fig.10 For PM 2.5 Effects of combined exposure to a HFD and HFD on HDAC in the aorta of mice.

[0028] Fig.11 For PM 2.5 Effects of combined exposure to FOD and HFD on the expression levels of HDAC5 gene and protein in the aorta of mice.

[0029] Fig.12 The changes of body weight and organ coefficients of mice after sodium butyrate intervention. Note: (A) The change trend of body weight of mice in each group; (BG) The changes of relative weight of heart, liver, spleen, lung, kidney and brain of mice in each group.

[0030] Fig.13 The changes of heart rate and blood pressure of mice after sodium butyrate intervention. Note: (A) heart rate; (B) systolic blood pressure; (C) diastolic blood pressure; (D) mean arterial pressure.

[0031] Fig.14 HE staining of mouse aorta tissue after sodium butyrate intervention. Note: The magnification of the HE staining result is 400×, scale bar = 20μm.

[0032] Fig.15 This is EVG staining of mouse aorta tissue after sodium butyrate intervention. Note: EVG staining result, scale bar = 20μm.

[0033] Fig.16 is the gene expression level of inflammasome-related indicators in the aortic tissue of mice after sodium butyrate intervention.

[0034] Fig.17 The protein expression levels of inflammasome-related indicators in mouse aorta tissue after sodium butyrate intervention. Note: (A) WB band; (BF) Grayscale analysis of protein bands of inflammasome-related indicators.

[0035] Fig.18 This is the effect of sodium butyrate intervention on HDAC.

[0036] Fig.19 The changes of body weight and organ coefficients of mice after TSA intervention. Note: (A) The change trend of body weight of mice in each group; (BG) The changes of relative weight of heart, liver, spleen, lung, kidney and brain of mice in each group.

[0037] Fig. 20 The changes of heart rate and blood pressure in mice after TSA intervention. Note: (A) heart rate; (B) systolic blood pressure; (C) diastolic blood pressure; (D) mean arterial pressure.

[0038] Fig.21 HE staining of mouse aorta tissue after TSA intervention. Note: The magnification of HE staining result is 400×, scale bar = 20μm.

[0039] Fig. 22 EVG staining of mouse aorta tissue after TSA intervention. Note: EVG staining result, scale bar = 20μm.

[0040] Fig.23 is the gene expression level of inflammasome-related indicators in the aortic tissue of mice after TSA intervention.

[0041] Fig.24 The protein expression levels of inflammasome-related indicators in mouse aorta tissue after TSA intervention. Note: (A) WB band; (BF) Grayscale analysis of protein bands of inflammasome-related indicators.

[0042] Fig.25 This is the effect of TSA intervention on HDAC. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] The specific embodiments of the present invention are described in detail below.

[0046] Materials and Methods

[0047] 1. Establishment of Animal Model

[0048] Male C57BL / 6J mice (18-22 g, 6 weeks old) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All mice were processed after one week of adaptation. All animal experimental operations in this study were in accordance with the relevant provisions of the Animal Experiment Operation Guide of Shanxi Medical University and were approved by the Animal Ethics Committee of Shanxi Medical University.

[0049] 1.1PM 2.5 And HFD vascular toxicity mouse model:

[0050] Sixty male C57BL / 6J mice were randomly divided into 6 groups: control group, HFD group, PM 2.5 Low dose (PML, 4.5 mg / kg PM 2.5 ) Group, PM 2.5 High dose (PMH, 9 mg / kg PM 2.5 ) group, HFD+PM 2.5 Low dose (HFD+PML) group, HFD+PML group 2.5 High-dose (HFD+PMH) group. After one week of adaptive feeding, the mice were anesthetized with isoflurane and PMH was administered. 2.5 Nasal drip PM in single exposure group and combined exposure group 2.5 The suspension, the Control group, and the HFD-exposed group were all replaced with an equal amount of PBS, once every other day, for a total of 6 weeks. The HFD group and the combined exposure group were fed a high-fat diet, in which 45% of the calories came from fat, 35.2% from carbohydrates, and 19.8% from protein. The Control group and PM 2.5 The single exposure group was fed with normal diet, and body weight was recorded once a week.

[0051] 1.2 Butyrate supplementation mouse model:

[0052] 40 male C57BL / 6J mice were randomly divided into 4 groups: Control group, HFD+PMH group, sodium butyrate (SB) group, HFD+PMH+sodium butyrate (HPB) group. A sodium butyrate solution with a concentration of 200 mg / kg was prepared with normal saline. The SB group and HPB group were given sodium butyrate solution (200 μL per mouse) by gavage every day from the beginning of poisoning, and the Control group and HFD+PMH group were given an equal amount of normal saline by gavage every day. 1.3 TSA intervention mouse model:

[0053] 40 male C57BL / 6J mice were randomly divided into 4 groups: Control group, HFD+PMH group, TSA group, HFD+PMH+TSA (HPT) group. TSA solution with a concentration of 5 μg / mL was prepared, and the TSA solution (100 μL per mouse) was intraperitoneally injected into the TSA group and the HPT group once every other day from the beginning of poisoning, and the same amount of TSA solvent was intraperitoneally injected into the Control group and the HFD+PMH group every other day.

[0054] 2. Mouse Weight and Blood Pressure Monitoring

[0055] The weight of mice was monitored 1 day before exposure and once a week after exposure, for a total of 7 times. The blood pressure of mice was measured at the same time as the weight of mice. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate of mice in the awake state were measured using the BP-2010A intelligent non-invasive sphygmomanometer by the tail-cuff method.

[0056] 3. Small Animal Ultrasound

[0057] One day before the mice were killed, vascular cardiac ultrasound examination was performed using the vevo3100 ultra-high resolution small animal ultrasound imaging system. The chest and abdomen of the mice were depilated with depilatory cream, and the mice were anesthetized and fixed in the supine position. The probe was placed in the chest and abdomen to examine the heart and aorta with ultrasound, including vascular diameter, mean blood flow velocity (MV), peak systolic blood flow velocity (PSV), end-diastolic blood flow velocity (EDV), vascular resistance index (RI), vascular pulsatility index (PI), cardiac stroke volume (SV), ejection fraction (EF), left ventricular fractional shortening (FS), stroke volume (CO), left atrial posterior wall thickness at systole (LVAW; s), left atrial posterior wall thickness at diastole (LVAW; d), left ventricular posterior wall thickness at systole (LVPW; s), and left ventricular posterior wall thickness at diastole (LVPW; d).

[0058] 4. Mouse Organ Coefficient Evaluation

[0059] After the mice were weighed, the left eyeball was removed to collect blood, which was placed at room temperature for 30 minutes, centrifuged at 4000 rpm for 20 minutes, and the upper serum was separated and stored in a -80°C refrigerator. After blood collection, the mice were killed by dislocation, fixed on the experimental table, the chest and abdominal cavity were opened, and the liver, spleen, kidney, heart, lung, aorta, trachea and brain of the mice were removed in turn, the fascia on the surface of the organs were removed, weighed, and the organ coefficient was calculated.

[0060] 5. HE staining of aorta

[0061] After the aorta tissue was fixed and embedded in paraffin, it was sliced ​​and baked for 1 hour; dewaxing: immersing the vascular tissue slices in xylene for 10 minutes, twice; hydration: the dewaxed slices were hydrated in ethanol concentrations and deionized water from high to low, the specific order was: 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 5 minutes each time, and finally washed three times in deionized water, 5 minutes each time; the tissue slices were stained with hematoxylin for 5-10 minutes, rinsed with tap water for 15 minutes; eosin staining for 3-5 minutes, and deionized water was used to wash away the floating color; dehydration and transparency: 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol were placed for 1 minute each, once each, and placed in xylene for 5 minutes, twice; sealing: a small amount of neutral gum was added to the surface of the tissue slice, and the cover glass was contacted with the neutral gum from one side of the slice, and the entire slice surface was carefully covered. Be careful to avoid bubbles during the operation; filming: after the cover glass was fixed, take pictures under a microscope for observation.

[0062] 6. Aorta EVG Staining

[0063] After the aorta tissue was fixed and embedded in paraffin, it was sliced ​​and baked for 1 hour before being dewaxed in xylene for 20 minutes, and then dewaxed in gradient ethanol to distilled water, with each step lasting 5 minutes. The modified VG staining solution was used for 10 minutes, and the excess stain was washed with distilled water for 5-10 seconds; the staining working solution was used for 5 minutes, and the distilled water was used for 5-10 seconds; the differentiation solution was used for 5-10 seconds until the elastic fibers were clear, and the distilled water was used for 5-10 seconds; the gradient ethanol dehydration was started from 75% ethanol, and each step lasted for 3-5 seconds. The xylene was transparentized twice, and each step lasted for 1 minute. The slides were sealed with neutral gum, and the staining results were observed under an optical microscope.

[0064] 7. Detection of short-chain fatty acids in mouse serum by GC-MS / MS

[0065] The serum samples were thawed and vortexed for 1 min before analysis. 50 μL of sample was added to a 1.5 mL EP tube, and 100 μL of phosphoric acid (0.5%, v / v) solution was added, and the mixture was vortexed at 2500 r / min for 3 min; 200 μL of MTBE extractant (containing internal standard) solution was added, and the mixture was vortexed at 2500 r / min for 3 min, ultrasonicated at 4 degrees for 5 min, and then centrifuged at 12000 r / min for 10 min; 100 μL of supernatant was aspirated into the inner liner of the injection bottle for GC-MS / MS analysis.

[0066] 8. qRT-PCR detection of tissue mRNA expression

[0067] The vascular tissue was taken out from -80℃ and placed on ice. About 20mg of tissue was cut and placed in a 1.5mL EP tube. 200μL RNAiso Plus was added. The vascular tissue was crushed and homogenized using an ultrasonic disruptor. 800μL RNAiso Plus was added to the homogenized vascular tissue and allowed to stand on ice for 30min. 200μL chloroform was added and mixed thoroughly by inversion for 15s. After placing at 4℃ for 5min, centrifuged at 12000rpm for 15min. The upper aqueous phase was carefully pipetted into a new EP tube. 500μL isopropanol was added and mixed by inversion for 15s. After placing at 4℃ for 10min, centrifuged at 12000rpm for 10min and the supernatant was discarded. The precipitate was washed 3 times with 1mL pre-cooled 75% ethanol, each time centrifuged at 4℃ for 5min. The supernatant was discarded and the precipitate was dried at room temperature until there was no obvious liquid. 10μL Dissolve the RNA precipitate with DEPC water, take 1 μL to test the RNA purity and concentration; adjust the RNA concentration to 1 μg / μL, perform reverse transcription reaction, and store cDNA at -20℃ for later use; use the fluorescence quantitative kit TB Premix Ex Taq TM For quantitative detection of mRNA, 3 replicate wells were set for each sample and the detection was performed on a fluorescent quantitative PCR instrument. The primer design is as shown in the following table:

[0068] Table 1 mRNA primer sequences

[0069]

[0070]

[0071] 9. Western blotting (WB)

[0072] Aortic tissue (about 20 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.

[0073] 10. Statistical Analysis

[0074] In this embodiment, SPSS22.0 software was used to perform statistical analysis on the experimental data, GraphPad Prism8 was used to draw the graph, and the data were expressed as mean ± standard deviation. When comparing multiple groups, if the data met normality and homogeneity of variance, one-way analysis of variance was used, followed by LSD test; if normality and homogeneity of variance were not met, the nonparametric Kruskal-Wallis rank sum test was used. P < 0.05 indicated that the difference was statistically significant.

[0075] result

[0076] 1.PM 2.5 Overall toxic effects of combined exposure to HFD in mice

[0077] Atmospheric PM 2.5 The body weight of mice was measured 1 day before and after HFD exposure as the baseline value. There was no statistical difference in the baseline value among the groups. Figure 1 As shown in A. Compared with the control group, the weight gain of mice in the HFD and combined exposure groups was faster. From the second week, there was a significant difference between the combined exposure group and the control group (P<0.05), while PM 2.5 There was no significant difference between the treated and control groups. 2.5 The relative weights of the heart and liver of the exposed mice were significantly decreased, and those of the lungs were significantly increased. 2.5 Compared with single exposure, combined exposure significantly increased liver and spleen, and significantly decreased brain. Compared with HFD single exposure, combined exposure significantly increased spleen and lung.

[0078] 2.PM 2.5 Effects of combined exposure to HFD on heart rate and blood pressure in mice

[0079] The blood pressure and heart rate of each group of mice were compared at different time points. Figure 2 As shown in the figure, as early as the second week, the SBP and MAP of the HFD+PMH group were significantly higher than those of the other groups, and they were still higher than the control group until the sixth week of exposure. From the fourth week, the SBP of the other groups was higher than that of the control group. From the second week, the DBP of the HFD+PMH group was significantly higher than that of the control group. From the sixth week, the DBP of the HFD and HFD+PML groups was also significantly increased compared with the control group (P<0.05). Compared with blood pressure, the changes in heart rate were less obvious. After the sixth week, the heart rate of the HFD+PML group increased significantly.

[0080] 3. Ultrasound results of mouse aorta and heart

[0081] We further used an ultra-high-resolution small animal ultrasound imaging system to perform ultrasound examinations of the mouse heart and blood vessels, e.g. Figure 3 As shown, in PM 2.5After single and combined exposure to HFD, the vessel diameter and EDV decreased to varying degrees, while RI showed an increasing trend. 2.5 There was a dose-response relationship after exposure. The combined exposure group was significantly lower than the single exposure group, and the RI was significantly higher than the single exposure group (P<0.05). Figure 4 As shown, SV and CO were significantly lower after combined exposure than those in the individual groups, while the thickness of the left atrial and left ventricular posterior walls increased during both systole and diastole (P<0.05).

[0082] 4. Histopathological Results of Mouse Aorta

[0083] 4.1 HE staining results

[0084] In order to more intuitively observe the damage of mouse blood vessels and vascular endothelium, we performed HE staining on paraffin sections of thoracic aorta tissue. Figure 5 As shown, the aorta wall of the control group was uniform, the endothelial cells on the intima were tightly connected and arranged in an orderly manner, and there was no inflammatory cell infiltration. In the single exposure group, necrotic endothelial cells were seen in the intima of the blood vessels, and the cell gaps in the middle membrane of the blood vessel wall were enlarged to varying degrees. In the combined exposure group, a large number of necrotic endothelial cells were seen in the intima, the cells were disorderly arranged, the nuclei were condensed into clusters, and part of the intima was peeled off.

[0085] 4.2 EVG staining results

[0086] In order to observe the changes in mouse vascular elastic fibers, we performed EVG staining on the paraffin sections of mouse thoracic aorta tissue. EVG staining can make the elastic fibers appear black. Figure 6 As shown, the elastic fibers in the aorta of the control group were arranged in an orderly and regular manner, the elastic fiber lines were clear and round, and the entire media elastic fiber layer was dense and complete. The elastic fiber lines in the single exposure group became thinner and rougher, while the elastic fibers in the combined exposure group were broken and defective, and the distance between the elastic fibers in each layer became larger.

[0087] 5.PM 2.5 Effects of combined exposure to HFD on vascular inflammation in mice

[0088] The results of inflammasome-related indicator genes and protein expression levels in mouse aorta tissue are as follows Figure 7 and 8 As shown in the figure, compared with the control group, the gene expression levels of Caspase-1 and IL-18 in the single exposure group were significantly increased, the protein expression levels of NLRP3 and ASC were significantly increased, and the IL-1β gene and protein in the PMH group were significantly higher than those in the control group. Compared with the single exposure group, the expression levels of each index in the combined exposure group increased to varying degrees, proving that PM 2.5There was a synergistic effect with HFD. The gene expression levels of NLRP3, IL-1β and IL-18 and the protein expression levels of IL-1β and IL-18 in the combined exposure group showed a dose-response relationship.

[0089] 6.PM 2.5 Combined exposure to HFD leads to abnormal short-chain fatty acid metabolism in serum of mice

[0090] We used GC-MS / MS to detect the levels of short-chain fatty acids in mouse serum. Fig. 9 As shown in the figure, compared with the control group, the levels of butyrate and isobutyrate in the HFD, PMH, and HFD+PMH groups were significantly higher than those in the control group, and the difference was statistically significant. Acetic acid, valeric acid, and caproic acid were significantly increased in the combined exposure group. Studies have found that butyrate can regulate the occurrence and development of hypertension, so we chose sodium butyrate intervention to explore the effect of butyrate on PM 2.5 and possible mechanisms of vascular inflammation induced by high-fat diet exposure.

[0091] 7.PM 2.5 Effects of combined exposure to HFD on vascular HDAC in mice

[0092] To observe PM 2.5 To investigate the effects of combined exposure to HFD and HFD on HDAC in mouse blood vessels, we used qRT-PCR to detect HDAC1-11 in mouse aorta. Fig.10 As shown. Combined exposure caused significant changes in HADC3, HADC5, HADC8, and HADC9 in aortic tissue (P<0.05). Among them, HDAC5 was most highly expressed in heart and skeletal muscle, and HDAC5 could inhibit angiogenesis in endothelial cells.

[0093] 8.PM 2.5 Effects of combined exposure to HFD on vascular HDAC5 in mice

[0094] The results of HDAC5 gene and protein expression levels in mouse aorta tissue are as follows Fig.11 As shown in the figure, compared with the control group, the gene expression levels of the HFD and PMH single exposure groups were significantly increased, and there was a dose-response relationship between the combined exposure groups, and the protein expression levels of the combined exposure groups were also significantly increased. 2.5 and HFD-induced vascular toxicity.

[0095] 9. Sodium butyrate supplementation affects PM 2.5 and vascular inflammation caused by HFD exposure

[0096] 9.1 Effect of Sodium Butyrate on PM 2.5 and overall toxicity due to HFD exposure

[0097] Atmospheric PM 2.5 The body weight of mice was measured on the day before and after exposure as the baseline value. There was no statistical difference in the baseline value among the groups. Fig.12 As shown in A. Compared with the control group, the weight of mice in the combined exposure group increased faster. From the first week, there was a significant difference between the mice in the combined exposure group and the control group and the SB group (P<0.05), which lasted until the end of the exposure. From the second week, the difference between the mice in the combined exposure group and the HPB group began to show statistical significance. It can be seen from the organ coefficient results that after the combined exposure, the relative weight of the heart and brain decreased significantly, and the liver increased significantly. After the intervention of butyrate, the relative weight of the heart and brain increased significantly, and the liver decreased significantly. This shows that butyrate has an effect on the heart, liver and brain of mice.

[0098] 9.2 Effects of sodium butyrate intervention on heart rate and blood pressure in mice

[0099] The blood pressure and heart rate of each group of mice were compared at different time points. Fig.13 As shown in the figure, at the second week, SBP and MAP in the HFD+PMH group were significantly higher than those in the control group, and remained significant until the sixth week of exposure. From the fifth week, SBP and MAP in the HFD+PMH group were significantly higher than those in the HPB group. From the fourth week, DBP in the HFD+PMH group was significantly higher than that in the control group, and from the fifth week, DBP in the HFD+PMH group was significantly higher than that in the HPB group (P<0.05). Compared with blood pressure, there was no obvious trend in the change of heart rate.

[0100] 9.3 Histopathological results after sodium butyrate intervention

[0101] HE staining was performed on paraffin sections of thoracic aorta tissue. Fig.14 As shown in the figure, the aorta wall of the control group and the SB group was uniform, and the endothelial cells on the intima were tightly connected and arranged in an orderly manner. In the combined exposure group, a large number of necrotic endothelial cells were seen in the intima, the cells were arranged in a disordered manner, the nuclei were condensed into a mass, part of the intima was peeled off, and inflammatory cell infiltration was seen. After the intervention of butyrate on the basis of combined exposure, the necrotic endothelial cells were reduced, the cells were arranged in a more orderly manner, and the inflammatory cells were reduced.

[0102] EVG staining was performed on paraffin sections of mouse thoracic aorta tissue. Fig.15 As shown in the figure, the elastic fibers of the aorta in the Control group and the SB group were arranged in an orderly and regular manner, and the elastic fiber lines were clear and round. The elastic fibers in the combined exposure group were rough, broken, and defective, and the spacing between the elastic fibers in each layer became larger. After the combined exposure and butyrate intervention, the elastic fibers were arranged in an orderly and regular manner, and the media layer became dense and complete.

[0103] 9.4 Effect of Sodium Butyrate on PM 2.5 and the effects of HFD exposure on vascular inflammation

[0104] The results of inflammasome-related indicator genes and protein expression levels in mouse aorta tissue are as follows Fig.16 and 17 As shown in the figure, compared with the control group, the expression levels of inflammasome-related genes and proteins in the aorta tissue of mice in the combined exposure group were significantly increased. After the intervention of butyrate, the expression of inflammatory indicators was significantly decreased. Except for the gene levels of ASC and IL-1β, the rest were not significantly different from the SB group. This shows that butyrate plays an important role in the prevention and treatment of PM 2.5 and HFD-induced endothelial inflammation.

[0105] 9.5 Effect of sodium butyrate on HDAC

[0106] The results of HDAC expression levels in mouse aorta tissue after sodium butyrate intervention are as follows Fig.18 As shown in Figure 2, after the intervention of sodium butyrate on the basis of combined exposure, the expression levels of HDAC5 and HDAC8 decreased significantly, indicating that sodium butyrate can alleviate PM by inhibiting the expression of HDAC5. 2.5 and HFD combined exposure leading to vascular endothelial inflammation.

[0107] 10. TSA intervention on PM 2.5 and the effects of HFD exposure on vascular inflammation

[0108] 10.1TSA vs PM 2.5 and overall toxicity due to HFD exposure

[0109] Atmospheric PM 2.5 The body weight of mice was measured 1 day before and after HFD exposure as the baseline value. There was no statistical difference in the baseline value among the groups. Fig.19 As shown in A. Compared with the control group, the weight of mice in the combined exposure group increased faster. From the first week, there was a significant difference between the mice in the combined exposure group and the control group (P<0.05), which lasted until the end of the exposure. In the second week, there was a statistically significant difference between the mice in the combined exposure group and the HPB group. From the organ coefficient results, it can be seen that after the combined exposure, the relative weight of the heart and brain decreased significantly, and after the intervention of TSA, the relative weight of the heart and brain increased significantly. This shows that TSA intervention has an effect on the heart and brain of mice.

[0110] 10.2 Effects of TSA intervention on heart rate and blood pressure in mice

[0111] The blood pressure and heart rate of each group of mice were compared at different time points. Fig. 20As shown in the figure, starting from the 4th week, SBP, DBP and MAP in the HFD+PMH group were significantly higher than those in the control group, and remained significant until the 6th week of exposure. Starting from the 5th week, SBP and MAP in the HFD+PMH group were significantly higher than those in the HPT group (P<0.05). Compared with blood pressure, there was no obvious trend in the change of heart rate.

[0112] 10.3 Histopathological Results after TSA Intervention

[0113] HE staining was performed on paraffin sections of thoracic aorta tissue. Fig.21 As shown in the figure, the aortic wall of the control group and TSA group was uniform, and the endothelial cells on the intima were tightly connected and arranged in an orderly manner. In the combined exposure group, a large number of necrotic endothelial cells were seen in the intima, the cells were arranged in a disordered manner, part of the intima was exfoliated, and inflammatory cell infiltration was seen. After TSA intervention on the basis of combined exposure, the number of necrotic endothelial cells decreased, the cells were arranged in a more orderly manner, and the number of inflammatory cells decreased.

[0114] EVG staining was performed on paraffin sections of mouse thoracic aorta tissue. Fig. 22 As shown in the figure, the elastic fibers of the aorta in the Control group and TSA group were arranged in an orderly and regular manner. The elastic fibers in the combined exposure group were rough and broken, and the distance between the elastic fibers in each layer became larger. After TSA intervention based on combined exposure, it can be seen that the elastic fibers became orderly and regular.

[0115] 10.4TSA vs PM 2.5 and the effects of HFD exposure on vascular inflammation

[0116] The results of the expression levels of inflammasome-related indicator genes and proteins in the aorta tissue of TSA mice were shown in Figure 2. Fig.23 and 24 As shown in the figure, compared with the control group, the expression levels of inflammasome-related genes and proteins in the aorta tissue of mice in the combined exposure group were significantly increased. After TSA intervention, the expression levels of inflammatory indicators decreased significantly (P<0.05). This indicates that TSA is effective in preventing and treating PM. 2.5 and HFD-induced endothelial inflammation.

[0117] 10.5 Effect of TSA on HDAC

[0118] The results of HDAC expression levels in mouse aorta tissue after TSA intervention were as follows Fig.25 As shown in the figure, after TSA intervention on the basis of combined exposure, the expression levels of HDAC5 and HDAC8 also decreased significantly, indicating that TSA can alleviate vascular inflammation by inhibiting the expression of HDAC5.

[0119] 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. Reduce PM 2.5 Biomarkers of endothelial inflammation caused by combined exposure to HFD, characterized by: The biomarker is at least one of the following characteristic substances, butyrate and HDAC5.

2. The method for reducing PM according to claim 1 2.5 Biomarkers of endothelial inflammation caused by combined exposure to HFD, characterized by: PM 2.5 Combined exposure to HFD resulted in decreased blood butyrate levels and increased aortic HDAC5 levels.

3. The method for reducing PM according to claim 1 2.5 Primers for DNA amplification of HDAC5, a biomarker of endothelial inflammation induced by combined HFD exposure.

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

5. A detection kit for detecting HDAC5, characterized in that: The detection kit comprises the PM reduction method according to claim 3 2.5 Primers for DNA amplification of HDAC5, a biomarker of endothelial inflammation induced by combined HFD exposure.

6. A method for detecting HDAC5 in a tissue, characterized in that: The detection is performed using the detection kit as described in claim 5.

7. Used to prevent PM 2.5 Combined exposure to a high-fat diet and a drug that causes endothelial inflammation, characterized in that Includes at least one of sodium butyrate and TSA.

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