Method for analyzing correlation between trpc5 and sleep monitoring indicators, echocardiogram and inflammatory factors based on blood biochemical detection

Abstract

The application discloses a TRPC5 and sleep monitoring index, echocardiogram and inflammation factor correlation analysis method based on blood biochemical detection. According to the correlation analysis method, intermittent hypoxia promotes myocardial cell apoptosis and up-regulates the expression of TRPC5, the myocardial cell apoptosis rate induced by intermittent hypoxia is significantly increased after overexpression of TRPC5, which indicates that TRPC5 promotes the myocardial cell apoptosis induced by intermittent hypoxia IH, promotes myocardial cell inflammatory response and cell pyroptosis, the myocardial cell inflammatory response and cell pyroptosis are aggravated after overexpression of TRPC5, which indicates that TRPC5 may promote the myocardial cell damage induced by IH through the inflammatory response, the role mechanism of TRPC5 in myocardial damage is clarified, and it is indicated that TRPC5 can be a promising target for treating myocardial damage caused by OSAHS.

Description

Technical Field

[0001] This invention relates to medical biochemical index detection and analysis technology, specifically a method for correlation analysis of TRPC5 in blood biochemical tests with sleep monitoring indicators, echocardiography, and inflammatory factors. Background Technology

[0002] Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a common sleep-disordered breathing disorder caused by repeated partial or complete collapse of the upper airway during sleep, leading to apnea, hypoxemia, and sleep fragmentation. It is associated with cardiovascular complications such as hypertension, coronary heart disease, and atrial fibrillation, and promotes myocardial damage, seriously threatening human health.

[0003] Intermittent hypoxia (IH) is the most prominent characteristic of OSAHS, which promotes oxidative stress, inflammatory response, excessive activation of the sympathetic nervous system, and calcium deficiency. 2+ Myocardial damage can occur through pathways such as homeostasis imbalance, among which Ca2+ 2+ Homeostasis imbalance plays an important role in myocardial damage caused by OSAHS.

[0004] Canonical transient receptor potential channels (TRPCs) are a class of non-selective, voltage-gated calcium ion channels that participate in calcium metabolism. 2+ Osmosis plays a regulatory role in intracellular calcium ion concentration. TRPC5 is one subtype, mainly expressed in the brain, heart, and kidneys, and is associated with myocardial injury and inflammatory responses. However, the relationship between TRPC5 and myocardial injury and inflammatory responses under intermittent hypoxia remains unclear.

[0005] Therefore, this invention provides a method to investigate the effects of TRPC5 on myocardial injury caused by intermittent hypoxia and its possible mechanisms, with the aim of providing a new therapeutic target for the prevention and treatment of myocardial injury in OSAHS. Summary of the Invention

[0006] The purpose of this invention is to provide a method for correlation analysis of TRPC5 based on blood biochemical tests with sleep monitoring indicators, echocardiography and inflammatory factors, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for correlation analysis of TRPC5 based on blood biochemistry tests with sleep monitoring indicators, echocardiography, and inflammatory factors, the method steps are as follows:

[0009] Step 1: Selection of study subjects. Snoring patients aged 18-75 years were selected and polysomnography was performed. Based on the results of the sleep monitoring, they were divided into OSAHS group and non-OSAHS group, with 50 cases in each group. (1) Central or mixed sleep apnea. (2) Unstable respiratory diseases such as bronchial asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. (3) Diseases that may affect the structure and function of the heart, such as hypertension, diabetes, coronary heart disease, valvular heart disease, cardiomyopathy, congenital heart disease, and severe arrhythmia. (4) Endocrine system diseases: hyperthyroidism and hypothyroidism. (5) Severe liver and kidney dysfunction, acute and chronic inflammation, chronic wasting diseases, and malignant tumors. (6) Others, such as cognitive impairment, sequelae of cerebrovascular disease, and insomnia, were excluded from the selection of subjects.

[0010] Step 2: Clinical data collection, including age, sex, history of alcohol and smoking, body mass index, creatinine, uric acid, aspartate aminotransferase, alanine aminotransferase, N-terminal pro-brain natriuretic peptide, creatine kinase isoenzyme and complete blood count.

[0011] Step 3: Preparation of materials and reagents. Materials include H9C2 cardiomyocytes, fetal bovine serum, DMEM medium, human and rat interleukin-1β, interleukin-18, tumor necrosis factor-α, TRPC5, NLRP3, Caspase-1 antibody, GSDMD antibody, and HRP-labeled goat anti-rabbit / mouse secondary antibody. Reagents include SuperScript III RT reverse transcription kit, qPCR kit, Annexin V-FITC apoptosis detection kit, and ELISA kit. H9C2 cardiomyocytes were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; fetal bovine serum was purchased from Gibco; DMEM medium was purchased from Wuhan Pronosei Biotechnology Co., Ltd.; SuperScript III RT reverse transcription kit and qPCR kit Sybr qpcr mix were purchased from ABI-invitrogen; Annexin... V-FITC apoptosis detection kit was purchased from Beyotime; human and rat interleukin-1β (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor α (TNF-α) ELISA kits were purchased from Linko Biotech; TRPC5, NLRP3, and Caspase-1 antibodies were purchased from Abcam; GSDMD antibody was purchased from Santa Cruz; and HRP-labeled goat anti-rabbit / mouse secondary antibody was purchased from Abcam.

[0012] Step Four: Polysomnography (PSG) was conducted using a Compumedics polysomnography device from Australia for a 7-hour overnight period. Simultaneous monitoring included blood oxygen saturation, pulse, respiratory rate, snoring, and airflow through the mouth and nose. After monitoring, Remlogic software was used for data analysis, sleep report interpretation, and review. The sleep report was interpreted by a professionally trained physician and reviewed by a senior physician. The Apnea-Hypopnea Index (AHI), Mean Oxygen Saturation (MSaO2), and Lowest Oxygen Saturation (LSaO2) were collected.

[0013] Step 5: Echocardiography was performed using a Philips IE33 color Doppler ultrasound diagnostic instrument to measure the left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular posterior wall thickness (LVPWT), interventricular septal thickness (IVST), right ventricular diameter (RVD), right atrial diameter (RAD), E peak, and A peak. The E / A ratio and left ventricular ejection fraction (LVEF) were calculated.

[0014] Step 6: Cell culture. After resuscitation, H9C2 cardiomyocytes were placed in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin and cultured at 37°C with 5% carbon dioxide.

[0015] Step 7: Intermittent hypoxia cell model treatment. When the cell density reaches 70-80%, the cells are treated with IH and modified. H9C2 cells are exposed to IH or normoxic conditions for 24 hours.

[0016] Step 8: Cell transfection and grouping. Seed 10 cells per well in a 6-well plate. 5 H9C2 cells were transfected when the cell density reached 50-60%. TRPC5 overexpression and empty vector plasmid were transfected into the cells, and the cells were cultured for another 48 hours after transfection.

[0017] Step 9: Apoptosis detection. After cell modeling, discard the culture medium and add 500 μL of trypsin per well (the trypsin does not contain EDTA). Centrifuge to collect the suspended cells and perform apoptosis detection using the Annexin V-FITC apoptosis detection kit. Flow cytometry was used to determine the apoptosis rate of each group.

[0018] Step 10: Detection of inflammatory factor concentrations. Serum and cell supernatant IL-1β, IL-18 and TNF-α levels were detected using an ELISA kit.

[0019] Step 11: TRPC5 mRNA relative expression detection. The relative expression of TRPC5 mRNA was detected using an RT-qPCR kit. Cells from each group were collected after treatment. Total RNA was extracted from peripheral blood mononuclear cells and H9C2 cardiomyocytes from each group using the Trizol method. The RNA was reverse transcribed into cDNA. 2 μL of cDNA was used as a template and primers for quantitative real-time PCR. The reaction conditions were 95℃ for 5 min, 95℃ for 10 s, 58℃ for 20 s, and 72℃ for 20 s, for a total of 40 cycles. β-actin was used as an internal control.

[0020] The forward primer for TRPC5 (human) is 5'-TGAGGAGGGCCGATCTGTTA-3'.

[0021] The reverse primer is 5'-TGCAGAAATCCTGAGCCAAGT-3'

[0022] The forward primer for TRPC5 (rat) is 5'-CTACTGGCTTTTGCCAACGG-3'.

[0023] The reverse primer is 5'-AGGGTTTCAAAGAGCGTGGA-3'

[0024] The forward primer for β-actin (human) is 5'-TCCTCCTGAGCGCAAGTACTCC-3'.

[0025] The reverse primer is 5'-CATACTCCTGCTTGCTGATCCAC-3'

[0026] The forward primer for β-actin (rat) is 5'-CTGAACGTGAAATTGTCCGAGA-3'

[0027] The reverse primer is 5'-TTGCCAATGGTGATGACCTG-3';

[0028] Step 12: Cell-related protein detection. Western blot was used to detect cell-related proteins in each group. After extracting total cell protein, protein quantification was performed using the BCA method. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. TRPC5 antibody, NLRP3 antibody, Caspase-1 antibody, and GSDMD antibody were incubated with the membrane overnight. The membrane was then incubated with goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody at room temperature for another 1 hour. After washing the PVDF membrane three times, it was exposed and developed. The gray values ​​of the bands were analyzed using ImageJ software.

[0029] Step Thirteen: Data Processing. Use SPSS 25 statistical software to process the data, and for normally distributed measurement data... This indicates that comparisons between groups were conducted using independent samples t-tests or one-way ANOVA, and non-normally distributed continuous data were analyzed using M(P) tests. 25 P 75 The data indicates that the rank-sum test was used for comparisons between groups, and the frequency (%) was used for count data. The chi-square test was used for comparisons between groups. 2 The correlation between TRPC5 and sleep monitoring indicators, echocardiography, and inflammatory factors was analyzed using the Spearman correlation test.

[0030] As a further aspect of the present invention: in step one, the grouping is based on the apnea-hypopnea index of polysomnography, which is denoted as AHI. If AHI < 5 times / hour, the subjects are divided into the non-OSAHS group; if AHI ≥ 5 times / hour, the subjects are divided into the non-OSAHS group.

[0031] As a further aspect of the present invention: In step two, the body mass index (BMI) is measured. When collecting data on creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), N-terminal pro-brain natriuretic peptide (PPB), creatine kinase isoenzyme, and complete blood count, participants fast for more than 8 hours before blood collection. Creatinine, uric acid, AST, and ALT are measured using Roche methods. The C8000 biochemical analyzer measured creatinine as Cr, uric acid as UA, aspartate aminotransferase as AST, alanine aminotransferase as ALT, N-terminal pro-brain natriuretic peptide as NT-proBNP, and creatine kinase isoenzyme as CK-MB. Complete blood count data collection included white blood cell count, neutrophil count, lymphocyte count, monocyte count, hemoglobin, and platelet count. White blood cell count was recorded as WBC, neutrophil count as NEUT, lymphocyte count as LYC, monocyte count as MONO, hemoglobin as Hb, and platelet count as PLT.

[0032] As a further embodiment of the present invention: the IH conditions in step seven include equilibration of 1% O2 + 5% CO2 + N2 for 35 min and equilibration of 21% O2 + 5% CO2 + N2 for 25 min.

[0033] As a further embodiment of the present invention: in step twelfth, the TRPC5 antibody is 1:800, the NLRP3 antibody is 1:600, the Caspase-1 antibody is 1:800, the GSDMD antibody is 1:200, and the goat anti-mouse secondary antibody and the goat anti-rabbit secondary antibody are 1:5000.

[0034] As a further aspect of the present invention: in step thirteen, the difference is statistically significant when P < 0.05.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] According to the technical solution of this invention, intermittent hypoxia promotes cardiomyocyte apoptosis and upregulates TRPC5 expression. Overexpression of TRPC5 significantly increases the apoptosis rate induced by intermittent hypoxia, indicating that TRPC5 promotes cardiomyocyte apoptosis induced by intermittent hypoxia. Serum levels of IL-1β, IL-8, and TNF-α in OSAHS patients are significantly higher than in the non-OSAHS group, and TRPC5 is positively correlated with IL-1β, demonstrating the correlation between TRPC5 and myocardial injury and inflammatory response. Simultaneously, in vitro experimental results show that IH promotes cardiomyocyte inflammatory response and pyroptosis. Overexpression of TRPC5 exacerbates cardiomyocyte inflammatory response and pyroptosis, suggesting that TRPC5 may promote IH-induced cardiomyocyte injury through inflammatory response. This elucidates the mechanism of action of TRPC5 in myocardial injury and suggests that TRPC5 may be a promising target for treating myocardial injury caused by OSAHS. Attached Figure Description

[0037] Figure 1 This is a graph showing the relative expression level of TRPC5 mRNA in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0038] Figure 2 This is a graph showing the concentration of IL-1β in a correlation analysis method based on blood biochemistry tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0039] Figure 3 This is a graph showing the concentration of IL-18 in a correlation analysis method based on blood biochemistry tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0040] Figure 4 This is a graph showing the concentration of TNF-α in a correlation analysis method based on blood biochemistry tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0041] Figure 5 This image shows a normotropic group of flow cytometry-based methods for analyzing the correlation between TRPC5, a blood biochemical assay, and sleep monitoring indicators, echocardiography, and inflammatory factors, to detect cardiomyocyte apoptosis rate.

[0042] Figure 6IH group diagram for flow cytometry detection of cardiomyocyte apoptosis rate in a correlation analysis method based on blood biochemistry test TRPC5 with sleep monitoring indicators, echocardiography and inflammatory factors.

[0043] Figure 7 This is a quantitative graph of cardiomyocyte apoptosis rate in a correlation analysis method based on blood biochemical tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0044] Figure 8 This is a band diagram of TRPC5 protein expression in a method for correlation analysis of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors based on blood biochemistry tests.

[0045] Figure 9 This is a bar chart showing the TRPC5 protein expression in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0046] Figure 10 This is a graph showing the IL-1β level in a correlation analysis method based on blood biochemistry tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0047] Figure 11 This is a graph showing the IL-18 level in a correlation analysis method based on blood biochemistry tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0048] Figure 12 This is a graph showing the TNF-α level in a correlation analysis method based on blood biochemistry tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0049] Figure 13 This is a band diagram of NLRP3 pyroptosis-related protein expression in a correlation analysis method based on blood biochemistry detection of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0050] Figure 14 This is a bar chart showing the expression of Caspase-1 pyroptosis-related proteins in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0051] Figure 15 This is a bar chart showing the expression of pyroptosis-related proteins in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0052] Figure 16This is a bar chart showing the expression of GSDMD pyroptosis-related proteins in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0053] Figure 17 This is a graph showing the TRPC5 expression level in cells of each group detected by RT-qPCR in a method for correlation analysis of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors based on blood biochemistry.

[0054] Figure 18 This image shows a flow cytometry-based assay for the correlation analysis of TRPC5 in blood biochemistry with sleep monitoring indicators, echocardiography, and inflammatory factors, specifically focusing on the detection of normotropic + TRPC5 NC apoptosis in cardiomyocytes.

[0055] Figure 19 This image shows a group of images of IH+TRPC5NC cells detected by flow cytometry in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0056] Figure 20 This image shows a flow cytometry-based correlation analysis of TRPC5 in blood biochemistry with sleep monitoring indicators, echocardiography, and inflammatory factors to detect cardiomyocyte apoptosis using normotropic + TRPC5OE.

[0057] Figure 21 This image shows an IH+TRPC5OE sequence for detecting cardiomyocyte apoptosis using flow cytometry, a method for correlation analysis of TRPC5 based on blood biochemistry tests with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0058] Figure 22 This is a graph showing the quantitative results of cardiomyocyte apoptosis rate in a correlation analysis method based on blood biochemical tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0059] Figure 23 This is a graph showing the IL-1β levels in the NC and OE groups in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0060] Figure 24 This is a graph showing the IL-18 levels in the NC and OE groups in a correlation analysis method based on blood biochemistry tests of TRPC5 with sleep monitoring indicators, echocardiography, and inflammatory factors.

[0061] Figure 25 This is a graph showing the TNF-α levels in the NC and OE groups compared to a correlation analysis method based on blood biochemistry tests for TRPC5 and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0062] Figure 26 This is a Western blot image showing the correlation between TRPC5 based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0063] Figure 27 This is a bar chart showing the TRPC5 protein expression in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0064] Figure 28 This is a bar chart showing the expression of NLRP3 protein in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0065] Figure 29 This is a bar chart showing the expression of Caspase-1 protein in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors.

[0066] Figure 30 This is a bar chart showing the expression of GSDMD protein in a correlation analysis method based on blood biochemistry tests and sleep monitoring indicators, echocardiography, and inflammatory factors. Detailed Implementation

[0067] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0068] Please see Figures 1-3 In this embodiment of the invention, a method for correlation analysis of TRPC5 based on blood biochemical tests with sleep monitoring indicators, echocardiography, and inflammatory factors is described below:

[0069] Step 1: Selection of study subjects. Snoring patients aged 18-75 years were selected and polysomnography was performed. Based on the results of the sleep monitoring, they were divided into OSAHS group and non-OSAHS group, with 50 cases in each group. (1) Central or mixed sleep apnea. (2) Unstable respiratory diseases such as bronchial asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. (3) Diseases that may affect the structure and function of the heart, such as hypertension, diabetes, coronary heart disease, valvular heart disease, cardiomyopathy, congenital heart disease, and severe arrhythmia. (4) Endocrine system diseases: hyperthyroidism and hypothyroidism. (5) Severe liver and kidney dysfunction, acute and chronic inflammation, chronic wasting diseases, and malignant tumors. (6) Others, such as cognitive impairment, sequelae of cerebrovascular disease, and insomnia, were excluded from the selection of subjects.

[0070] Step 2: Clinical data collection, including age, sex, history of alcohol and smoking, body mass index, creatinine, uric acid, aspartate aminotransferase, alanine aminotransferase, N-terminal pro-brain natriuretic peptide, creatine kinase isoenzyme and complete blood count.

[0071] Step 3: Preparation of materials and reagents. Materials include H9C2 cardiomyocytes, fetal bovine serum, DMEM medium, human and rat interleukin-1β, interleukin-18, tumor necrosis factor-α, TRPC5, NLRP3, Caspase-1 antibody, GSDMD antibody, and HRP-labeled goat anti-rabbit / mouse secondary antibody. Reagents include SuperScript III RT reverse transcription kit, qPCR kit, Annexin V-FITC apoptosis detection kit, and ELISA kit. H9C2 cardiomyocytes were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; fetal bovine serum was purchased from Gibco; DMEM medium was purchased from Wuhan Pronosei Biotechnology Co., Ltd.; SuperScript III RT reverse transcription kit and qPCR kit Sybr qpcr mix were purchased from ABI-invitrogen; Annexin... V-FITC apoptosis detection kit was purchased from Beyotime; human and rat interleukin-1β (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor α (TNF-α) ELISA kits were purchased from Linko Biotech; TRPC5, NLRP3, and Caspase-1 antibodies were purchased from Abcam; GSDMD antibody was purchased from Santa Cruz; and HRP-labeled goat anti-rabbit / mouse secondary antibody was purchased from Abcam.

[0072] Step Four: Polysomnography (PSG) was conducted using a Compumedics polysomnography device from Australia for a 7-hour overnight period. Simultaneous monitoring included blood oxygen saturation, pulse, respiratory rate, snoring, and airflow through the mouth and nose. After monitoring, Remlogic software was used for data analysis, sleep report interpretation, and review. The sleep report was interpreted by a professionally trained physician and reviewed by a senior physician. The Apnea-Hypopnea Index (AHI), Mean Oxygen Saturation (MSaO2), and Lowest Oxygen Saturation (LSaO2) were collected.

[0073] Step 5: Echocardiography was performed using a Philips IE33 color Doppler ultrasound diagnostic instrument to measure the left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular posterior wall thickness (LVPWT), interventricular septal thickness (IVST), right ventricular diameter (RVD), right atrial diameter (RAD), E peak, and A peak. The E / A ratio and left ventricular ejection fraction (LVEF) were calculated.

[0074] Step 6: Cell culture. After resuscitation, H9C2 cardiomyocytes were placed in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin and cultured at 37°C with 5% carbon dioxide.

[0075] Step 7: Intermittent hypoxia cell model treatment. When the cell density reaches 70-80%, the cells are treated with IH and modified. H9C2 cells are exposed to IH or normoxic conditions for 24 hours.

[0076] Step 8: Cell transfection and grouping. Seed 10 cells per well in a 6-well plate. 5 H9C2 cells were transfected when the cell density reached 50-60%. TRPC5 overexpression and empty vector plasmid were transfected into the cells, and the cells were cultured for another 48 hours after transfection.

[0077] Step 9: Apoptosis detection. After cell modeling, discard the culture medium and add 500 μL of trypsin per well (the trypsin does not contain EDTA). Centrifuge to collect the suspended cells and perform apoptosis detection using the Annexin V-FITC apoptosis detection kit. Flow cytometry was used to determine the apoptosis rate of each group.

[0078] Step 10: Detection of inflammatory factor concentrations. Serum and cell supernatant IL-1β, IL-18 and TNF-α levels were detected using an ELISA kit.

[0079] Step 11: TRPC5 mRNA relative expression detection. The relative expression of TRPC5 mRNA was detected using an RT-qPCR kit. Cells from each group were collected after treatment. Total RNA was extracted from peripheral blood mononuclear cells and H9C2 cardiomyocytes from each group using the Trizol method. The RNA was reverse transcribed into cDNA. 2 μL of cDNA was used as a template and primers for quantitative real-time PCR. The reaction conditions were 95℃ for 5 min, 95℃ for 10 s, 58℃ for 20 s, and 72℃ for 20 s, for a total of 40 cycles. β-actin was used as an internal control.

[0080] The forward primer for TRPC5 (human) is 5'-TGAGGAGGGCCGATCTGTTA-3'.

[0081] The reverse primer is 5'-TGCAGAAATCCTGAGCCAAGT-3'

[0082] The forward primer for TRPC5 (rat) is 5'-CTACTGGCTTTTGCCAACGG-3'.

[0083] The reverse primer is 5'-AGGGTTTCAAAGAGCGTGGA-3'

[0084] The forward primer for β-actin (human) is 5'-TCCTCCTGAGCGCAAGTACTCC-3'.

[0085] The reverse primer is 5'-CATACTCCTGCTTGCTGATCCAC-3'

[0086] The forward primer for β-actin (rat) is 5'-CTGAACGTGAAATTGTCCGAGA-3'

[0087] The reverse primer is 5'-TTGCCAATGGTGATGACCTG-3';

[0088] Step 12: Cell-related protein detection. Western blot was used to detect cell-related proteins in each group. After extracting total cell protein, protein quantification was performed using the BCA method. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. TRPC5 antibody, NLRP3 antibody, Caspase-1 antibody, and GSDMD antibody were incubated with the membrane overnight. The membrane was then incubated with goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody at room temperature for another 1 hour. After washing the PVDF membrane three times, it was exposed and developed. The gray values ​​of the bands were analyzed using ImageJ software.

[0089] Step Thirteen: Data Processing. Use SPSS 25 statistical software to process the data, and for normally distributed measurement data... This indicates that comparisons between groups were conducted using independent samples t-tests or one-way ANOVA, and non-normally distributed continuous data were analyzed using M(P) tests. 25 P 75 The data indicates that the rank-sum test was used for comparisons between groups, and the frequency (%) was used for count data. The chi-square test was used for comparisons between groups. 2 The correlation between TRPC5 and sleep monitoring indicators, echocardiography, and inflammatory factors was analyzed using the Spearman correlation test.

[0090] In step one, the grouping was based on the apnea-hypopnea index of polysomnography, which is denoted as AHI. If AHI < 5 times / hour, the subjects were divided into the non-OSAHS group; if AHI ≥ 5 times / hour, the subjects were divided into the non-OSAHS group.

[0091] In step two, body mass index (BMI) was recorded. For the collection of creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), N-terminal pro-brain natriuretic peptide (NT-proBNP), creatine kinase isoenzyme, and complete blood count data, participants fasted for more than 8 hours before blood collection. Creatinine, uric acid, AST, and ALT were measured using a Roche C8000 biochemical analyzer. Creatinine was recorded as Cr, uric acid as UA, aspartate aminotransferase as AST, alanine aminotransferase as ALT, N-terminal pro-brain natriuretic peptide (NT-proBNP) as NT-proBNP, and creatine kinase isoenzyme as CK-MB. Complete blood count data collection included white blood cell count, neutrophil count, lymphocyte count, monocyte count, hemoglobin, and platelet count. White blood cell count was recorded as WBC, neutrophil count as NEUT, lymphocyte count as LYC, monocyte count as MONO, hemoglobin as Hb, and platelet count as PLT.

[0092] In step three, human and rat interleukin-1β is abbreviated as IL-1β, and interleukin-18 is abbreviated as IL-18.

[0093] Step 7 involves IH conditions including equilibration of 1% O2 + 5% CO2 + N2 for 35 min and equilibration of 21% O2 + 5% CO2 + N2 for 25 min.

[0094] In step 12, the TRPC5 antibody was diluted 1:800, the NLRP3 antibody was diluted 1:600, the Caspase-1 antibody was diluted 1:800, the GSDMD antibody was diluted 1:200, and the goat anti-mouse secondary antibody and the goat anti-rabbit secondary antibody were diluted 1:5000.

[0095] In step thirteen, a p-value < 0.05 indicates a statistically significant difference.

[0096] Baseline data analysis and statistics:

[0097] Compared with the non-OSAHS group, the OSAHS group had a higher proportion of males, higher age, higher hypertension, higher diabetes, higher AHI, higher ODI, lower IL-6, and lower LAD, while lower MSaO2, lower LSaO2, and lower E / A, with statistically significant differences (P<0.05).

[0098] Baseline data table

[0099]

[0100]

[0101]

[0102] Statistical analysis of TRPC5 and inflammatory factor expression levels in peripheral blood of subjects:

[0103] RT-qPCR results showed that the relative expression level of TRPC5 mRNA in the peripheral blood of patients in the OSAHS group was significantly higher than that in the non-OSAHS group, and the difference was statistically significant (P<0.05). Figure 1 ELISA results showed that serum IL-1β, IL-18, and TNF-α levels in the OSAHS group were higher than those in the non-OSAHS group, and the differences were statistically significant (P<0.05). Figure 2-3 ), Figures 1-4 *** indicates P < 0.001, and **** indicates P < 0.0001.

[0104] Correlation analysis of TRPC5 with sleep apnea monitoring, echocardiographic indicators and inflammatory factors in OSAHS patients.

[0105] Spearman correlation analysis showed that TRPC5 was positively correlated with AHI (r = 0.415, P = 0.003), ODI (r = 0.360, P = 0.014), LAD (r = 0.293, P = 0.041), and IL-1β (r = 0.305, P = 0.033), and negatively correlated with E / A (r = 0.354, P = 0.013) and MSaO2 (r = -0.350, P = 0.015), with statistically significant differences (P < 0.05).

[0106]

[0107]

[0108] Statistics on IH-induced cardiomyocyte apoptosis and TRPC5 expression:

[0109] Flow cytometry results showed that the apoptosis rate in the IH group was significantly higher than that in the normoxia group, and the difference was statistically significant (P<0.05). Figure 5-6 Western blot results showed that TRPC5 expression in cardiomyocytes of the IH group was higher than that of the normoxic group, and the difference was statistically significant (P<0.05). Figure 7-8 ), Figure 5-8 *** indicates P < 0.001, and **** indicates P < 0.0001.

[0110] Statistics on IH promoting cardiomyocyte inflammatory response and pyroptosis:

[0111] ELISA was used to measure the concentrations of inflammatory factors in cell supernatant. The results showed that the concentrations of IL-1β, IL-18, and TNF-α in the IH group were higher than those in the normoxia group, and the differences were statistically significant (P<0.05). Figure 9-11 Western blot results showed that the expression of NLRP3, Caspase-1, and GSDMD in cardiomyocytes of the IH group was higher than that of the normoxia group, and the difference was statistically significant (P<0.05). Figure 12-15 ), Figure 9-15 *** indicates P < 0.001, and * indicates P < 0.05.

[0112] Statistics on how TRPC5 promotes IH-induced cardiomyocyte damage through inflammatory response:

[0113] To further investigate the effect of TRPC5 on cardiomyocyte injury under intermittent hypoxia, we constructed TRPC5-overexpressing cardiomyocytes using lentivirus. RT-qPCR results showed that the relative expression level of TRPC5 mRNA was higher than that of the control group (H9C2 cells) and the empty vector group (…). Figure 16 ).

[0114] Flow cytometry analysis of cardiomyocyte apoptosis showed that the apoptosis rate of cardiomyocytes in the IH+TRPC5OE group was higher than that in the normoxia+TRPC5OE group and the IH+TRPC5NC group. Figure 17-22 ELISA results showed that the levels of IL-1β, IL-18, and TNF-α in the IH+TRPC5OE group were higher than those in the normoxia+TRPC5OE group and the IH+TRPC5NC group. Figure 23-25 ).

[0115] Figure 17-25 The asterisk (*) indicates P < 0.05, the ** indicates P < 0.01, the *** indicates P < 0.001, and the **** indicates P < 0.0001.

[0116] Westren blot analysis of pyroptosis-related proteins showed that the levels of NLRP3, Caspase-1, and GSDMD proteins in cardiomyocytes of the IH+TRPC5OE group were higher than those of the normoxia+TRPC5OE group and the IH+TRPC5NC group. Figure 26-30 ).

[0117] Figure 26-30 * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0118] The etiology of OSAHS is complex and diverse, and its occurrence is associated with multiple factors, including obesity, upper airway anatomical abnormalities, and genetic factors. The interaction of these factors can lead to myocardial damage, which is often difficult to detect in its early stages. Chronic intermittent hypoxia is a characteristic change in OSAHS, promoting myocardial damage through oxidative stress, systemic inflammatory response, and excessive activation of the sympathetic nervous system, ultimately leading to heart failure and seriously threatening human health. Therefore, the prevention and treatment of myocardial damage in OSAHS is of great significance.

[0119] Myocardial injury is a common complication of obstructive ventricular dysfunction (OSAHS). Previous studies have shown that OSAHS is associated with myocardial injury and diastolic dysfunction, with the severity of OSAHS positively correlated with diastolic dysfunction. Furthermore, left atrial volume (LAD) is positively correlated with the severity of OSAHS. Baseline data in this study indicate that OSAHS patients have larger LADs and lower E / A ratios, suggesting the occurrence of myocardial injury, consistent with previous research. However, the mechanism by which OSAHS leads to myocardial injury remains unclear.

[0120] IH-induced oxidative stress not only promotes the production of reactive oxygen species, but also regulates Ca2+. 2+ Concentration. Ca 2+ It is a second messenger, participating in various physiological processes such as apoptosis, proliferation, and energy metabolism. Intracellular Ca... 2+ Concentration changes are involved in many cardiovascular diseases and play an important role in myocardial injury. TRPC5 is a class of non-selective cation channels with a tetrameric structure, mainly involved in calcium metabolism. 2+TRPC5 plays a crucial role in calcium ion signal transduction. Studies have shown that TRPC5 is involved in myocardial injury. TRPC5 levels are increased in the cardiac tissue of patients with end-stage heart failure. TRPC5 gene knockout can alleviate cholinergic-induced tachycardia, myocardial fibrosis, and myocardial hypertrophy. However, there are currently no studies on the association between TRPC5 and myocardial injury in patients with obstructive angina pectoris (OSAHS). This study assessed the relationship between OSAHS, myocardial injury, and peripheral blood TRPC5 levels. The results showed that TRPC5 levels in PBMCs of OSAHS patients were associated with myocardial injury, suggesting that TRPC5 may be involved in myocardial injury in OSAHS patients. Simultaneously, this study also explored the relationship between TRPC5 and intermittent hypoxia-induced myocardial injury at the cellular level. According to this study, intermittent hypoxia promotes cardiomyocyte apoptosis and upregulates TRPC5 expression. Overexpression of TRPC5 significantly increased the rate of intermittent hypoxia-induced cardiomyocyte apoptosis, indicating that TRPC5 promotes intermittent hypoxia-induced cardiomyocyte apoptosis.

[0121] Inflammatory response induced by intraepithelial hemorrhage (IH) is another important pathophysiological change in obstructive sleep apnea-hypopnea syndrome (OSAHS). IH can not only activate various immune cells through HIF-α and promote the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α, but also activate the pro-inflammatory transcription factor NF-κB, promoting the synthesis of inflammatory mediators, leading to changes such as myocardial remodeling and cardiomyocyte apoptosis, and promoting myocardial injury. Studies have shown that TRPC5 is closely related to the inflammatory response. In rats with spinal cord ischemia-reperfusion injury, downregulating TRPC5 expression can significantly increase the expression of angiogenic proteins, alleviate the inflammatory response, and promote injury repair. TRPC5 is involved in the inflammatory response, neuronal cell death, and cognitive impairment in the hippocampus of rats with traumatic brain injury. TRPC5 gene knockout can downregulate the expression of inflammatory factors in the myocardial tissue of diabetic mice and reduce myocardial injury. This study shows that the serum levels of IL-1β, IL-8, and TNF-α in OSAHS patients are significantly higher than those in the non-OSAHS group, and TRPC5 is positively correlated with IL-1β, indicating the correlation between TRPC5 and myocardial injury and inflammatory response. Meanwhile, in vitro experimental results showed that IH promoted the inflammatory response and pyroptosis of cardiomyocytes. Overexpression of TRPC5 aggravated the inflammatory response and pyroptosis of cardiomyocytes, indicating that TRPC5 may promote IH-induced cardiomyocyte damage through inflammatory response.

[0122] The relative expression of TRPC5 mRNA in peripheral blood and the levels of IL-1β, IL-18, and TNF-α in serum were higher in the OSAHS group than in the non-OSAHS group (P<0.05). Spearman correlation analysis showed that TRPC5 was positively correlated with AHI (r=0.415, P=0.003), ODI (r=0.360, P=0.014), LAD (r=0.293, P=0.041), and IL-1β (r=0.305, P=0.033), and negatively correlated with E / A (r=0.354, P=0.013) and MSaO2 (r=-0.350, P=0.015). The apoptosis rate of H9C2 cells, and the levels of TRPC5, IL-1β, IL-18, TNF-α, NLRP3, Caspase-1, and GSDMD in the IH group were all higher than those in the normoxia group (all P<0.05). The apoptosis rate, levels of IL-1β, IL-18, TNF-α, NLRP3, Caspase-1, and GSDMD in the IH+TRPC5OE group were significantly higher than those in the IH+TRPC5NC group and the normotropic+TRPC5OE group (all P<0.05). Conclusion: IH promotes cardiomyocyte injury and inflammatory response; overexpression of TRPC5 may exacerbate IH-induced cardiomyocyte injury through inflammatory response.

[0123] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for correlation analysis of TRPC5 based on blood biochemical tests with sleep monitoring indicators, echocardiography, and inflammatory factors, characterized in that: The method steps are as follows: Step 1: Selection of study subjects. Snoring patients aged 18-75 years were selected and polysomnography was performed. Based on the sleep monitoring results, they were divided into OSAHS group and non-OSAHS group, with 50 patients in each group. Step 2: Clinical data collection, including age, sex, history of alcohol and smoking, body mass index, creatinine, uric acid, aspartate aminotransferase, alanine aminotransferase, N-terminal pro-brain natriuretic peptide, creatine kinase isoenzyme and complete blood count. Step 3: Preparation of materials and reagents. Materials include H9C2 cardiomyocytes, fetal bovine serum, DMEM medium, human and rat interleukin-1β, interleukin-18, tumor necrosis factor α, TRPC5, NLRP3, Caspase-1 antibody, GSDMD antibody, and HRP-labeled goat anti-rabbit / mouse secondary antibody. Reagents include SuperScript III RT reverse transcription kit, qPCR kit, Annexin V-FITC apoptosis detection kit, and ELISA kit. Step 4: Polysomnography. A 7-hour overnight polysomnography was conducted using a Compumedics polysomnography device from Australia. Simultaneous monitoring of blood oxygen saturation, pulse, respiratory rate, snoring, and airflow through the mouth and nose was performed. After the monitoring, Remlogic software was used to analyze the data, interpret the sleep report, and review it. Apnea-hypopnea index (AHI), mean blood oxygen saturation (MSaO2), and lowest blood oxygen saturation (LSaO2) were collected. Step 5: Echocardiography was performed using a Philips IE33 color Doppler ultrasound diagnostic instrument to measure the left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular posterior wall thickness (LVPWT), interventricular septal thickness (IVST), right ventricular diameter (RVD), right atrial diameter (RAD), E peak, and A peak. The E / A ratio and left ventricular ejection fraction (LVEF) were calculated. Step 6: Cell culture. After resuscitation, H9C2 cardiomyocytes were placed in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin and cultured at 37°C with 5% carbon dioxide. Step 7: Intermittent hypoxia cell model treatment. When the cell density reaches 70-80%, the cells are treated with IH and modified. H9C2 cells are exposed to IH or normoxic conditions for 24 hours. Step 8: Cell transfection and grouping. Seed 10 cells per well in a 6-well plate. 5 H9C2 cells were transfected when the cell density reached 50-60%. TRPC5 overexpression and empty vector plasmid were transfected into the cells, and the cells were cultured for another 48 hours after transfection. Step 9: Apoptosis detection. After cell modeling, discard the culture medium and add 500 μL of trypsin per well (the trypsin does not contain EDTA). Centrifuge to collect the suspended cells and perform apoptosis detection using the Annexin V-FITC apoptosis detection kit. Flow cytometry was used to determine the apoptosis rate of each group. Step 10: Detection of inflammatory factor concentrations. Serum and cell supernatant IL-1β, IL-18 and TNF-α levels were detected using an ELISA kit. Step 11: TRPC5 mRNA relative expression detection. The relative expression of TRPC5 mRNA was detected using an RT-qPCR kit. Cells from each group were collected after treatment. Total RNA was extracted from peripheral blood mononuclear cells and H9C2 cardiomyocytes from each group using the Trizol method. The RNA was reverse transcribed into cDNA. 2 μL of cDNA was used as a template and primers for quantitative real-time PCR. The reaction conditions were 95℃ for 5 min, 95℃ for 10 s, 58℃ for 20 s, and 72℃ for 20 s, for a total of 40 cycles. β-actin was used as an internal control. The forward primer for TRPC5 (human) is 5'-TGAGGAGGGCCGATCTGTTA-3'. The reverse primer is 5'-TGCAGAAATCCTGAGCCAAGT-3' The forward primer for TRPC5 (rat) is 5'-CTACTGGCTTTTGCCAACGG-3'. The reverse primer is 5'-AGGGTTTCAAAGAGCGTGGA-3' The forward primer for β-actin (human) is 5'-TCCTCCTGAGCGCAAGTACTCC-3'. The reverse primer is 5'-CATACTCCTGCTTGCTGATCCAC-3' The forward primer for β-actin (rat) is 5'-CTGAACGTGAAATTGTCCGAGA-3' The reverse primer is 5'-TTGCCAATGGTGATGACCTG-3'; Step 12: Cell-related protein detection. Western blot was used to detect cell-related proteins in each group. After extracting total cell protein, protein quantification was performed using the BCA method. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. TRPC5 antibody, NLRP3 antibody, Caspase-1 antibody, and GSDMD antibody were incubated with the membrane overnight. The membrane was then incubated with goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody at room temperature for another 1 hour. After washing the PVDF membrane three times, it was exposed and developed. The gray values ​​of the bands were analyzed using ImageJ software. Step Thirteen: Data Processing. Use SPSS 25 statistical software to process the data. For normally distributed measurement data... This indicates that comparisons between groups were conducted using independent samples t-tests or one-way ANOVA, and non-normally distributed continuous data were analyzed using M(P) tests. 25 P 75 The data indicates that the rank-sum test was used for comparisons between groups, and the frequency (%) was used for count data. The chi-square test was used for comparisons between groups. 2 The correlation between TRPC5 and sleep monitoring indicators, echocardiography, and inflammatory factors was analyzed using the Spearman correlation test.

2. The method for correlation analysis of TRPC5 based on blood biochemical detection with sleep monitoring indicators, echocardiography, and inflammatory factors according to claim 1, characterized in that: In step one, the grouping is based on the apnea-hypopnea index of polysomnography, which is denoted as AHI. If AHI < 5 times / hour, the subjects are divided into the non-OSAHS group; if AHI ≥ 5 times / hour, the subjects are divided into the non-OSAHS group.

3. The method for correlation analysis of TRPC5 based on blood biochemical detection with sleep monitoring indicators, echocardiography, and inflammatory factors according to claim 1, characterized in that: In step two, body mass index (BMI) was used. For the collection of creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), N-terminal pro-brain natriuretic peptide (NT-proBNP), creatine kinase isoenzyme, and complete blood count data, participants fasted for more than 8 hours before blood collection. Creatinine, uric acid, AST, and ALT were measured using a Roche C8000 biochemical analyzer. Creatinine was recorded as Cr, uric acid as UA, AST as AST, ALT as ALT, NT-proBNP as NT-proBNP, and CK-MB as creatine kinase isoenzyme. Complete blood count data collection included white blood cell count, neutrophil count, lymphocyte count, monocyte count, hemoglobin, and platelet count. White blood cell count was recorded as WBC, neutrophil count as NEUT, lymphocyte count as LYC, monocyte count as MONO, hemoglobin as Hb, and platelet count as PLT.

4. The method for correlation analysis of TRPC5 based on blood biochemical detection with sleep monitoring indicators, echocardiography, and inflammatory factors according to claim 1, characterized in that: The IH conditions in step seven include equilibration of 1% O2 + 5% CO2 + N2 for 35 minutes and equilibration of 21% O2 + 5% CO2 + N2 for 25 minutes.

5. The method for correlation analysis of TRPC5 based on blood biochemical detection with sleep monitoring indicators, echocardiography, and inflammatory factors according to claim 1, characterized in that: In step 12, the TRPC5 antibody is diluted 1:800, the NLRP3 antibody is diluted 1:600, the Caspase-1 antibody is diluted 1:800, the GSDMD antibody is diluted 1:200, and the goat anti-mouse secondary antibody and the goat anti-rabbit secondary antibody are diluted 1:5000.

6. The method for correlation analysis of TRPC5 based on blood biochemical detection with sleep monitoring indicators, echocardiography, and inflammatory factors according to claim 1, characterized in that: In step thirteen, a difference of P < 0.05 is considered statistically significant.

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