Application of CpG oligonucleotides in drugs for diabetic cardiac autonomic neuropathy

Abstract

The application of CpG oligonucleotides in drugs for diabetic cardiac autonomic neuropathy: Experiments have confirmed that CpG oligonucleotide 1826 can reduce the activation of satellite glial cells in the stellate ganglion and the expression of inflammatory cytokines, inhibit the expression of purine 2Y12 receptors, and reduce lipid peroxidation damage to alleviate ferroptosis in stellate ganglion neurons, thereby improving cardiac autonomic nerve function. Examples of CpG oligonucleotide 1826 demonstrate that CPG-ODN with anti-inflammatory effects or with anti-damage protective effects on the sympathetic nervous system has preventive and therapeutic effects in diabetic cardiac autonomic neuropathy and sympathetic nerve injury-related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical applications for diabetic cardiac autonomic neuropathy, and particularly to the application of CpG oligonucleotides in the preparation of drugs to reduce diabetic cardiac autonomic neuropathy and in the prevention and treatment of diseases related to sympathetic nerve damage. Background Technology

[0002] Diabetes mellitus (DM) is a group of metabolic clinical syndromes. With social development and improved living standards, the prevalence of diabetes is increasing year by year. Its mortality rate ranks fifth or even fourth among diseases in most high-income countries, making it the fourth leading cause of death after cancer, AIDS, and cardiovascular disease. Diabetes is classified into type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetes. Diabetic cardiac autonomic neuropathy (DCAN) is a common and serious complication of type 2 diabetes, characterized by damage to the autonomic nerve fibers innervating the heart and blood vessels, leading to cardiovascular dysfunction. In particular, regional loss of cardiac sympathetic nerves can lead to remodeling of cardiac sympathetic neurons, thereby increasing susceptibility to ventricular arrhythmias and sudden cardiac death. The cervical sympathetic nervous system is involved in maintaining cardiovascular homeostasis. Cardiac sympathetic nerves originate from the cervical sympathetic ganglia, and nerve fibers from the stellate sympathetic ganglion can distribute to the cardiac plexus, thus affecting cardiac activity. In diabetic autonomic neurofibrillary disease, afferent signals received by stellate ganglion neurons can affect sympathetic efferent activity, thereby influencing the regulation of cardiac function.

[0003] Purine signaling primarily involves the excitation of purine receptors to produce effects. P receptors include P1 and P2 receptors; adenosine and its analogues act on P1 receptors, while adenosine triphosphate (ATP) and its analogues act on P2 receptors. P2 receptors are further divided into ligand-gated non-selective ion channel receptors (P2X receptors) and pro-metabolic G protein-coupled receptors (P2Y receptors). Purinergic receptors have been shown to be associated with cardiovascular disease. The P2Y12 receptor, a member of the P2Y subtype, is a G protein-coupled receptor. P2Y12 receptors are expressed in the cervical sympathetic ganglia. Satellite glial cells (SGCs) tightly surround the neuronal cell bodies in the cervical sympathetic ganglia. Peripheral inflammation leads to SGC activation in the cervical sympathetic ganglia. Subsequently, ATP-induced P2Y12 receptor activation mediates the release of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) from the SGCs, participating in the pathological changes of cardiac autonomic nervous system diseases. Studies have shown that upregulation of P2Y12 expression in the stellate ganglion is involved in the process of diabetic autonomic neuropathy, while downregulation of P2Y12 receptor expression can effectively improve cardiac function by weakening abnormal sympathetic excitation reflexes.

[0004] Diabetic patients experience increased reactive oxygen species (ROS) and oxidative damage. Polyunsaturated fatty acids highly expressed in cell membrane phospholipids are highly susceptible to ROS attack, triggering numerous redox reactions collectively known as lipid peroxidation. Ferraphobia has been described as an iron-dependent form of cell death mediated by the excessive accumulation of lipid peroxides. Glutathione peroxidase 4 (GPX4) is a key regulator of ferrophobia, catalyzing the reduction of lipid peroxides at the expense of glutathione (GSH) and protecting cells from membrane lipid peroxidation. GPX4 inactivation or GSH depletion leads to the accumulation of lipid hydroperoxides, disrupting iron homeostasis and ultimately causing ferrophobia. Morphologically, ferrophobia primarily manifests as mitochondrial alterations, including reduced mitochondrial volume, reduced or even absent mitochondrial cristae, increased double-membrane density, and rupture of the outer mitochondrial membrane. Mitochondrial damage can lead to the release of mitochondrial DNA into the cytoplasm.

[0005] CpG oligonucleotides (CpG ODNs) are synthetic short-chain DNA molecules that contain unmethylated CpG dinucleotides (CpG motifs) in specific sequence settings. Summary of the Invention

[0006] The first objective of this invention is to provide the application of CPG-ODN in the preparation of drugs for the prevention and treatment of diabetic cardiac autonomic neuropathy.

[0007] The second objective of this invention is to provide the application of CPG-ODN in the preparation of drugs for the prevention and treatment of diseases related to sympathetic nerve damage.

[0008] The effects of CPG-ODN involve inhibiting the activation of satellite glial cells in the stellate ganglion of a type 2 diabetic rat model, reducing the expression of inflammatory cytokines, decreasing the expression of purine 2Y (P2Y)12 receptors, and improving ferroptosis pathological changes, thereby alleviating diabetic cardiac autonomic neuropathy. The results indicate that CPG-ODN can prevent and treat diabetic cardiac autonomic neuropathy and sympathetic nerve damage-related diseases through these effects. CPG-ODN 1826 inhibits the activation of satellite glial cells in the stellate ganglion of a type 2 diabetic rat model, reduces the expression of inflammatory cytokines, decreases the expression of purine 2Y (P2Y)12 receptors, and improves the pathological changes of ferroptosis in nerve cells. CPG-ODN 1826, as an example, demonstrates that CPG-ODN with anti-inflammatory effects or with anti-damage protective effects on the sympathetic nervous system has pharmacological effects in preventing and treating diabetic cardiac autonomic neuropathy and sympathetic nerve damage-related diseases. Attached Figure Description

[0009] Figure 1The effect of CpG ODN 1826 on sympathetic nerve discharge (SND) activity in a type 2 diabetic rat model was investigated. The SND level in diabetic rats was significantly higher than that in control rats (p<0.001). Figure 1 (A, B). CpG ODN 1826 treatment significantly improved abnormal neck spondylosis (SND) in diabetic rats compared to the untreated group. The results indicate that CpG 1826 treatment effectively reduced the activation of neck SND in diabetic rats (p<0.001). Figure 1 A, B).

[0010] Figure 2 The expression level of P2Y12 receptor in the stellate sympathetic ganglion of diabetic rats was significantly higher than that in control rats (p<0.001). After treatment with CpG 1826, the expression level of P2Y12 receptor was significantly reduced compared with the untreated DM group (p<0.01).

[0011] Figure 3 The expression level of P2Y12 receptor in the stellate ganglion of diabetic rats was significantly higher than that in control rats (p<0.01). CpG 1826 treatment significantly reduced the expression level of P2Y12 receptor compared with the untreated DM group (p<0.01).

[0012] Figure 4 This study aimed to detect the co-expression of P2Y12 receptor and glial fibrillary acidic protein (GFAP), a satellite glial cell marker, in the stellate ganglion (SG) of type 2 diabetic rats with neuropathic pain using double immunofluorescence staining. Immunofluorescence analysis showed co-expression of P2Y12 receptor and GFAP in the SG, suggesting the presence of PY12 receptor in SG satellite glial cells. The co-expression of P2Y12 receptor and GFAP in the SG of diabetic model (DM) rats was increased compared to the control group. CpG ODN 1826 treatment reduced the upregulated co-expression of P2Y12 receptor and GFAP in the SG of diabetic model rats.

[0013] Figure 5 To detect IL-1β mRNA expression using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that IL-1β mRNA expression in the SG of diabetic model rats (DM) was significantly increased compared with the control group (p<0.001); IL-1β mRNA expression after CpG ODN1826 treatment was decreased compared with the untreated model group (p<0.01).

[0014] Figure 6Western blotting was used to detect the protein expression level of IL-1β. The results showed that the protein expression level of IL-1β in the SG of diabetic model rats (DM) was significantly increased compared with the control group (p<0.01); the protein expression level of IL-1β after CpG ODN 1826 treatment was decreased compared with the untreated model group (p<0.05).

[0015] Figure 7 To quantitatively detect TNF-α mRNA expression by RT-PCR. The results showed that TNF-α mRNA expression in the SG of diabetic model rats (DM) was significantly increased compared with the control group (p<0.001); TNF-α mRNA expression after CpG ODN 1826 treatment was decreased compared with the untreated model group (p<0.001).

[0016] Figure 8 Western blotting was used to detect the protein expression level of TNF-α. The results showed that the protein expression level of TNF-α in the SG of diabetic model rats (DM) was significantly increased compared with the control group (p<0.01); the protein expression level of TNF-α after CpG ODN1826 treatment was decreased compared with the untreated model group (p<0.05).

[0017] Figure 9 Western blotting was used to detect the protein expression level of NF-κB p65. The results showed that the protein expression level of NF-κB p65 in the SG of diabetic model rats (DM) was significantly increased compared with the control group (p<0.01); the protein expression level of NF-κB p65 after CpG ODN 1826 treatment was decreased compared with the untreated model group (p<0.05).

[0018] Figure 10 This study investigated the protein expression changes of glutathione peroxidase 4 (GPX4) in the SG (sigmata) of a type 2 diabetic rat model. Western blot results showed that GPX4 expression in the SG of diabetic model (DM) rats was significantly lower than that in the control group (p<0.01); GPX4 expression was upregulated after CpG ODN 1826 treatment compared with the untreated model group (p<0.01).

[0019] Figure 11 This study investigated the changes in reactive oxygen species (ROS) content in the SG (sigmata) of a type 2 diabetic rat model. Chemiluminescence assay results showed that the ROS content in the SG of the diabetic model group (DM) rats was significantly increased compared to the control group (p<0.001); the ROS content after CpG ODN1826 treatment was decreased compared to the untreated model group (p<0.001).

[0020] Figure 12 Fe in SG, a type 2 diabetic rat model 2+ Content changes. Colorimetric detection results showed that Fe in the SG of diabetic model rats (DM) was... 2+ The content was significantly increased compared to the control group (p<0.01); Fe was significantly increased after CpG ODN 1826 treatment. 2+ The content of [a substance] was lower than that in the untreated model group (p<0.01).

[0021] Figure 13 The study investigated the changes in malondialdehyde (MDA) content in the SG (sigmata) of a type 2 diabetic rat model. Chemiluminescence assay showed that the MDA content in the SG of the diabetic model group (DM) rats was significantly increased compared to the control group (p<0.001); the MDA content after CpG ODN 1826 treatment was decreased compared to the untreated model group (p<0.001).

[0022] Figure 14 This study investigated the changes in reduced glutathione (GSH) content in the SG (sigmata) of a type 2 diabetic rat model. Microplate assay results showed that the GSH content in the SG of the diabetic model group (DM) rats was significantly lower than that in the control group (p<0.01); however, GSH content was upregulated after treatment with CpG ODN 1826 compared to the untreated model group (p<0.01).

[0023] Figure 15 The effect of CpG 1826 on mitochondrial morphology in type II diabetic rats was investigated. In the diagram, a represents the control group, b represents the diabetic group, c represents the diabetic model treated with CpG ODN 1826, and d represents the diabetic model treated with CpG ODN control. Transmission electron microscopy analysis showed that compared with the control group, diabetic rats exhibited reduced mitochondrial volume, increased membrane density, decreased mitochondrial cristae, and ruptured outer mitochondrial membranes. CpG ODN 1826 treatment improved the abnormal mitochondrial structural changes in diabetic rats. Mitochondria (arrows indicate mitochondria). Scale bar: 2 μm. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the embodiments and the accompanying drawings.

[0025] Using methods known in the art, oral or injectable CPG-ODN formulations suitable for the treatment of diabetic cardiac autonomic neuropathy are prepared.

[0026] Using methods known in the art, oral or injectable CPG-ODN formulations suitable for the treatment of diseases related to sympathetic nerve damage are prepared.

[0027] Application of CpG oligodeoxynucleotides in the preparation of oral, injectable, lozenge, or other local or systemic dosage forms for the prevention and treatment of the above-mentioned diseases.

[0028] To better understand the essence of this invention, the following example illustrates the role of CPG-ODN 1826 in inhibiting the activation of satellite glial cells in the cervical sympathetic ganglion, reducing the expression of inflammatory cytokines, decreasing the expression of purine 2Y (P2Y)12 receptors, and improving the pathological changes of nerve cell ferroptosis induced by lipid peroxidation damage, thereby alleviating diabetic cardiac autonomic neuropathy. This demonstrates the application of CPG-ODN in diabetic cardiac autonomic neuropathy and the prevention and treatment of sympathetic nerve damage-related diseases.

[0029] I. Materials and Methods

[0030] 1. Type II Diabetes Model Establishment and Grouping

[0031] Healthy adult male SD rats were housed in separate cages in a quiet, well-ventilated environment with a room temperature of 20-25℃ and a relative humidity of 40%-60%. A 12-hour / 12-hour light / dark cycle was implemented, and rats had free access to water. Cages and bedding were changed every other day. Male SD rats (clean grade) were selected for this experiment. After one week of housekeeping at room temperature with standard feed and free access to water, they were randomly divided into a normal control group and a type II diabetes model group (diabetic group). The control group was fed a standard feed throughout the experiment, while the model group was fed a high-sugar, high-fat diet (containing 77.8% standard rat feed, 10% lard, 10% sucrose, 2% cholesterol, and 0.2% sodium cholate) for four weeks.

[0032] At the end of the fifth week, rats with type 2 diabetes mellitus (T2DM) were intraperitoneally injected with a low dose (30 mg / kg) of streptozotocin (STZ, 7.5 mg / mL, freshly dissolved in 0.1 M pH 4.4 citrate buffer). Simultaneously, control rats were intraperitoneally injected with the same dose of the aforementioned citrate-sodium citrate buffer. One week after streptozotocin (STZ) administration (at the end of the sixth week), rats with fasting blood glucose levels >7.8 mM or postprandial blood glucose levels >11.1 mM were considered to have successfully established a type 2 diabetes model.

[0033] After successful modeling, rats were divided into four groups: a control group, a type 2 diabetes mellitus (T2DM) model group, a T2DM model group treated with CpG ODN 1826, and a T2DM model group treated with CpG ODN control. T2DM model rats were intraperitoneally injected with either CpG ODN 1826 or a CpG ODN control (200 μg / kg body weight). CpG ODN 1826 and the CpG ODN control were dissolved in sterile water. Throughout the experiment, control group rats were fed standard laboratory food. Diabetic group rats continued to be fed a high-sugar, high-fat diet. At the end of week 7 (one week after daily treatment), all animals were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital and euthanized after fasting for 8–10 hours. Stellate ganglion and whole blood samples were then collected for analysis.

[0034] 2. Measurement of blood pressure, heart rate variability, and sympathetic nerve discharge.

[0035] Blood pressure and heart rate were measured using a non-invasive tail cuff technique (Softron BP-98A, Softron Co., Tokyo, Japan). The sensor was placed on the rat's tail, and blood pressure was obtained by inflating and deflating the cuff. Heart rate variability (HRV) was analyzed using a frequency domain method based on electrocardiogram (ECG) recordings. The power of the variation in the RR interval (distance between two R waves) across the entire spectral frequency range (total power frequency, TP, 0–0.5 Hz) and at very low frequencies (VLF, 0.003–0.04 Hz), low frequencies (LF, 0.04–0.15 Hz), and high frequencies (HF, 0.15–0.40 Hz) was calculated from a short 5-minute recording. Postganglionic cervical sympathetic discharges (SND) in the SG were recorded and analyzed using an RM6240 biosignal analysis system (Chengdu Instrument Factory, China). Briefly, after anesthetizing rats with sodium pentobarbital (50 mg / kg, ip), the left cervical sympathetic nerve was isolated and gently placed on a bipolar gold wire electrode. The neuroelectrode connector was electrically isolated from the surrounding tissue using sterile cotton soaked in paraffin oil. The data was recorded and integrated using an RM-6240B multichannel physiological signal acquisition and processing system (Chengdu Instrument Factory, China). A reference electrode was then attached to a skin fold.

[0036] 3. Quantitative real-time polymerase chain reaction (real-time PCR)

[0037] Total RNA was extracted from SGs using the Eastep™ Total RNA Extraction Kit (Promega Biotech Co., Ltd., Beijing, China). cDNA templates were synthesized using a reverse transcription system kit (Promega Biotech Co., Ltd., Beijing, China). Primers used were designed and synthesized by Shanghai Sangong Biotechnology Co., Ltd.: β-actin forward 5'-tgtcacaactgggacgata-3' and reverse 5'-ggggtgttgaagtcaaa-3'; P2Y12 forward 5'-CTTCGTCCCTTCCATTTG-3' and reverse 5'-AGGTGCTCTCTTCACGTA-3'; IL-1β forward 5'-CCTATGTTGCCCGTGGAG-3' and reverse 5'-CACACTACTAGCGGTCGTCA-3'; TNF-α forward 5'-CACGTCGTAGCAAACCACCAA-3' and reverse 5'-GTTGGTTGTCTTTGAGATCCAT-3'. GreenMasterMix and ABI StepOnePlus performs quantitative RT-PCR TM Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression levels were measured using 2- ΔΔCT Quantitative analysis was performed. β-actin was used as an internal reference.

[0038] 4. Protein blotting

[0039] The harvested SG was homogenized in a homogenizer equipped with RIPA lysis buffer (Applygen, Beijing, China). The homogenate was placed in an ice bath for 20 minutes and then centrifuged at 12,000 rpm for 10 minutes at 4°C. The total protein concentration in the supernatant was determined using a BCA protein quantification kit (Boster Biological, Wuhan, China). Samples containing an equal amount of protein (20 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. At room temperature, the membrane was blocked for 2 hours with 5% skim dry milk in 25 mM Tris buffered saline (pH 7.2) containing 0.05% Tween 20 (TBST), and then cultured with primary antibodies: rabbit monoclonal P2Y12 antibody (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α) (1:800, Abcam, Cambridge, MA, USA), rabbit anti-glutathione peroxidase 4 (GPX4) (1:500, Affinity, Cincinnati, OH, USA), β-actin (1:1000, ZSGB-BIO, Beijing, China), and rabbit anti-nuclear transcription factor-κB (NF-κB) p65 (1:500, Abcam-, Cambridge, MA and USA) overnight at 4°C. Subsequently, the membrane was incubated with a secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG, 1:2000, ZSGB-BIO, Beijing, China) bound to horseradish peroxidase for 1 hour at room temperature. The intensity of the resulting chemiluminescent bands was analyzed using Image Pro Plus software.

[0040] 5. Double-labeled immunofluorescence

[0041] For double-labeled immunofluorescence staining, frozen SG sections were cut to 8 μm thickness using a cryostat. Sections were washed with PBS and incubated for 2 hours at room temperature in blocking solution containing 3% bovine serum albumin and 0.3% Triton X-100 in PBS. Tissues were mixed with primary antibodies: rabbit anti-P2Y12 (1:100, Alomone Laboratory, Jerusalem, Israel) and mouse anti-glial fibrillary acidic protein (GFAP) (1:100; Millipore). Subsequently, secondary antibodies (goat anti-rabbit TRITC and goat anti-mouse FITC, ZSGB Bio, Beijing, China; Alexa Fluor® 488 bound to goat anti-rabbit and Cy5 bound to goat anti-mouse, Abcam, Cambridge, MA, USA) were diluted to 1:200 in PBS at room temperature. Nuclear staining was performed using 4',6-diamino-2-phenylindole (DAPI). All images were taken under a fluorescence microscope (Olympus Tokyo, Japan) and viewed by a single-blind method.

[0042] 6. Determination of iron content, lipid peroxidation, and antioxidant activity levels

[0043] Malondialdehyde (MDA) is considered a biomarker for lipid peroxidation. The main endogenous antioxidant against lipid peroxidation is GPX4, which is considered a key regulator of iron accumulation-induced reactive oxygen species (ROS) chain reactions. The functional activity of GPX4 depends on the synthesis of GSH. Iron, MDA, and glutathione levels were determined using a colorimetric assay kit (Nanjing Jiancheng Biotechnology Institute, China) according to the manufacturer's instructions. GPX4 levels were determined using an ELISA kit (Nanjing Camelot Biotechnology Co., Ltd., China) using a double-antibody sandwich technique. Tissue homogenates were prepared in an ice bath and then centrifuged at 2500 rpm for 10 min. The supernatant was used for the following tests. ROS levels were determined using a chemiluminescence assay kit (Nanjing Jiancheng Biotechnology Institute, China). Fresh tissue was used to prepare single-cell suspensions using enzymatic digestion for detection. The procedures were strictly followed according to the instructions. A multi-functional microplate reader was used to detect the absorbance values ​​of each group at the corresponding wavelengths. The results were calculated according to the corresponding formulas in the manual.

[0044] 7. Transmission electron microscope.

[0045] Fresh DRG tissue was cut into 1mm pieces 3 The fragments were rapidly fixed in 2.5% glutaraldehyde. After rinsing three times for 15 minutes with 0.1M PBS, the samples were dehydrated at 4°C, followed by three times with 100% acetone at room temperature. Selected images were observed under a transmission electron microscope after embedding, curing, sectioning, and staining.

[0046] 8. Statistical Analysis

[0047] Data were analyzed using SPSS 21.0 software (SPSS, Chicago, IL, USA). One-way ANOVA and Bonferroni's post hoc test were used to determine statistical significance for multiple comparisons. p < 0.05 was considered statistically significant.

[0048] II. Results

[0049] (I) Effects of CpG ODN 1826 on blood pressure, heart rate and cervical sympathetic nerve activity in type II diabetic rats

[0050] Table 1 shows that blood pressure and heart rate were higher in the diabetic group than in the control group, but decreased significantly after treatment with CpG ODN 1826. Sympathetic nerve discharge (SND) activity was as follows: Figure 1 As shown, the SND level in diabetic rats was significantly higher than that in control rats (p<0.001). Figure 1 (A, B) This indicates increased excitability of the cervical sympathetic nerves in patients with type 2 diabetes. However, treatment with CpG ODN 1826 significantly improved abnormal cervical sympathetic nerve dissociation (SND) in diabetic rats (p<0.001). Figure 1 B). There was no significant difference between the DM+CpG ODN control group and the diabetes mellitus (DM) group (p>0.05). Figure 1 B). These results indicate that CpG ODN1826 treatment can effectively reduce the activation level of SND in the neck of diabetic rats.

[0051] Table 1. Effects of CpG ODN 1826 on heart rate (HR) and blood pressure (BP) in type 2 diabetic (T2DM) rats.

[0052]

[0053] *p<0.05, **p<0.01 and ***p<0.001 compared with the control group; ###p<0.001 compared with the diabetes group.

[0054] (II) Effects of CpG ODN 1826 on heart rate variability in type II diabetic rats

[0055] Heart rate variability (HRV) is reported in Table 2. Compared with the control group, the frequency domain variables such as TP, VLF, LF, and HF were significantly reduced in the type 2 diabetes group, indicating a decrease in both sympathetic and parasympathetic tone. Furthermore, an imbalance between sympathetic and parasympathetic tone was observed in diabetic rats with a significant increase in the LF / HF ratio. However, compared with diabetic rats, rats treated with CpG ODN 1826 showed significantly increased TP, VLF, LF, and HF (p<0.01). In addition, CpG treatment significantly improved the LF / HF ratio in the DM group (p<0.001). There was no significant difference between the DM+CpG ODN control group and the DM group. These data suggest that CpG ODN 1826 improves the autonomic imbalance in diabetic rats.

[0056] Table 2. Effects of CpG ODN 1826 on heart rate variability in type 2 diabetic rats

[0057]

[0058] *p<0.05 and **p<0.01 compared with the control group; #p<0.05 and ##p<0.01 compared with the diabetes group.

[0059] (III) Effects of CpG ODN 1826 on P2Y12 expression in the stellate ganglion

[0060] The expression of P2Y12 receptor in the stellate ganglion of diabetic rats at the mRNA level was significantly higher than that in control rats (p<0.001); Figure 2 Compared with the untreated DM group, CpG ODN 1826 treatment significantly reduced the mRNA expression level of the P2Y12 receptor (p<0.01). Figure 2 ).

[0061] The expression of P2Y12 receptor in the stellate ganglion of diabetic rats was significantly higher at the protein level than that in control rats (p<0.01). Figure 3 Compared with the untreated DM group, CpG ODN 1826 treatment significantly reduced the protein expression level of the P2Y12 receptor (p<0.01). Figure 3 ).

[0062] like Figure 4As shown, in dual immunofluorescence analysis, the co-expression levels of P2Y12 and GFAP were high in the stellate ganglion (SG) of diabetic rats. CpG ODN 1826 treatment significantly reduced the co-expression level. GFAP is a marker of satellite glial cells (SGCs). Upregulation of GFAP revealed SGC activation. These data suggest that the P2Y12 receptor and GFAP are co-localized in the SGC of the SG. The P2Y12 receptor may be involved in SGC activation. CpG ODN 1826 may inhibit nociceptive signaling and SGC activation by reducing P2Y12 receptor expression.

[0063] (iv) Effects of CpG ODN 1826 on the expression of IL-1β and TNF-α in the stellate ganglion

[0064] Activated satellite glial cells lead to an increase in pro-inflammatory factors. Quantitative RT-PCR was used to detect IL-1β mRNA expression. Compared with control rats, diabetic rats showed significantly increased IL-1β mRNA expression (p<0.001). Figure 5 However, CpG ODN1826 treatment significantly reduced IL-1β mRNA expression (p<0.01). Figure 5 There was no significant difference between the DM group and the DM+CpG ODN control group. Western blotting was used to detect IL-1β protein expression. Compared with control rats, diabetic rats showed significantly increased IL-1β protein expression (p<0.011). Figure 6 However, CpG ODN 1826 treatment significantly reduced the upregulation of IL-1β protein expression (p<0.05). Figure 6 There was no significant difference between the DM group and the DM+CpG ODN control group. CpG ODN1826 treatment exerted a protective effect by inhibiting the pro-inflammatory cytokine IL-1β.

[0065] Quantitative RT-PCR was used to detect TNF-α mRNA expression. Compared with control rats, diabetic rats showed significantly increased IL-1β mRNA expression (p<0.001). Figure 7 However, CpG ODN 1826 treatment significantly reduced TNF-α mRNA expression (p<0.001). Figure 7 There was no significant difference between the DM group and the DM+CpG ODN control group. Western blotting was used to detect TNF-α protein expression. Compared with control rats, diabetic rats showed significantly increased TNF-α protein expression (p<0.01). Figure 8 However, CpG ODN 1826 treatment significantly reduced the upregulation of TNF-α protein expression (p<0.05). Figure 8 There was no significant difference between the DM group and the DM+CpG ODN control group. CpG ODN 1826 treatment exerted a protective effect by inhibiting the pro-inflammatory cytokine TNF-α.

[0066] (V) Effects of CpG ODN 1826 on NF-κB p65 expression in type II diabetic rats

[0067] The nuclear factor-κB signaling pathway links metabolism and inflammation. Experimental results showed that the NF-κBp65 protein level in diabetic rats was significantly higher than that in control rats (p<0.01). Figure 9 However, after treatment with CpG ODN 1826, the expression of NF-κB p65 in diabetic rats was significantly reduced (p<0.05). Figure 9 There was no significant difference between the DM group and the DM+CpG ODN control group.

[0068] (VI) Effects of CpG ODN 1826 on glutathione peroxidase 4 (GPX4) expression in type II diabetic rats

[0069] The main function of glutathione peroxidase 4 (GPX4) is to reduce excess reactive oxygen species (ROS) in cells into non-toxic compounds. Inhibition of GPX4 leads to ROS accumulation and the production of lipid peroxides, resulting in ferroptosis in tissue cells. Experimental results showed that the GPX4 protein level in diabetic rats was significantly lower than that in control rats (p<0.01). Figure 10 However, after treatment with CpG ODN ODN1826, GPX4 expression in diabetic rats was significantly increased (p<0.01). Figure 10 There was no significant difference between the DM group and the DM+CpG ODN control group.

[0070] (VII) Effects of CpG ODN 1826 on iron content, lipid oxidation, and antioxidant activity in type II diabetic rats

[0071] Compared with the control group, the reactive oxygen species level in the DM group was significantly increased (p<0.001); Figure 11 Administration of CpG ODN1826 resulted in a decrease in reactive oxygen species levels in diabetic rats (p<0.001). Figure 11 ).

[0072] Compared with the control group, the iron level in the DM group was significantly higher (p<0.01); Figure 12 Administration of CpG ODN 1826 resulted in a decrease in iron levels in diabetic rats (p<0.01). Figure 12 ).

[0073] Compared with the control group, the malondialdehyde level in the DM group was significantly higher (p<0.001); Figure 13 Administration of CpG ODN1826 resulted in a decrease in malondialdehyde levels in diabetic rats (p<0.001). Figure 13 ).

[0074] In addition, compared with the control group, the DM group had significantly lower glutathione (GSH) levels (p<0.01); Figure 14 CpG1826 treatment significantly increased (p<0.01); Figure 14 ).

[0075] Treatment with CpG ODN 1826 improved the pathological changes in iron content, lipid oxidation, and antioxidant activity in diabetic rats.

[0076] (VIII) Effects of CpG 1826 on mitochondrial morphology in type II diabetic rats

[0077] Figure 15 Transmission electron microscopy revealed morphological changes in mitochondria of the stellate ganglion in each group. Compared with the control group, diabetic rats showed reduced mitochondrial volume, increased membrane density, decreased mitochondrial cristae, and ruptured outer mitochondrial membranes. CpG ODN1826 treatment improved these abnormal mitochondrial structural changes.

[0078] The inventors discovered that CpG ODN 1826 maintains the balance of the autonomic nervous system by attenuating the increased sympathetic nerve activity associated with diabetes and plays a positive protective role in type 2 diabetic cardiac autonomic neuropathy. Its mechanism may involve regulating P2Y12 receptors in satellite glial cells (SGCs) of the stellate ganglion by reducing NF-κB expression. Activation of SGCs releases a large number of inflammatory factors, leading to abnormal neuronal-glial cell communication. CpG ODN 1826 inhibits P2Y12 receptors, reducing SGC activation and the expression of inflammatory cytokines, thereby improving cardiac autonomic function. Furthermore, CpG ODN 1826 treatment alleviates ferroptosis in stellate ganglion neurons by reducing lipid peroxidation damage. These effects of CpG ODN 1826 can serve as examples of the preventive and therapeutic effects of CpG ODN on diabetic cardiac autonomic neuropathy and sympathetic nerve damage-related diseases.

Claims

1. Use of CpG oligodeoxynucleotide CpG ODN 1826 in the preparation of a drug for preventing and treating diabetic cardiac autonomic neuropathy.

2. Use of CpG oligodeoxynucleotide CpG ODN 1826 in the preparation of a drug for preventing and treating diabetic cardiac autonomic neuropathy, and diseases related to sympathetic nerve injury.

3. Use according to claim 1 or 2, characterized in that, The drug is in the form of oral or injection.

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