Method for evaluating osha-related cognitive impairment based on trpc5 full gene knockout rats and cih model

Abstract

The application discloses an OSAHS related cognitive impairment evaluation method based on a TRPC5 full gene knockout rat and a CIH model, which comprises experimental animal selection, animal grouping, CIH model construction, hippocampal tissue sample collection, hippocampal neuron pathology and apoptosis evaluation, hippocampal neuron subcellular structure evaluation, hippocampal neuron intracellular Ca2+ level detection and data statistical analysis. Through the evaluation method, it is determined that TRPC5 is a key upstream molecule for mediating CIH neurotoxicity, TRPC5 deletion can inhibit pathological calcium influx, reduce calcium overload induced fatal endoplasmic reticulum stress, thereby protecting hippocampal neurons from hypoxic damage; and it is also confirmed that the TRPC5-Ca2+-CHOP pathway is a key mechanism in CIH hippocampal neuron damage, that is, CIH exposure activates the TRPC5 channel to cause intracellular calcium overload, thereby triggering severe endoplasmic reticulum stress and inducing neuron apoptosis, and the deletion of TRPC5 can significantly buffer the pathological cascade reaction triggered by hypoxia without affecting the basic neuron structure.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an OSAHS-related cognitive impairment assessment method based on TRPC5 whole-gene knockout rats and CIH models. Background Technology

[0002] Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a serious public health problem worldwide. Its core pathophysiology is chronic intermittent hypoxia, i.e., repeated cycles of hypoxia and reoxygenation. This process has been proven to be a key driver of damage to the central nervous system. Clinical evidence shows that long-term OSAHS patients often have significant learning and memory impairments and a decline in emotional and cognitive functions. Even with continuous positive airway pressure (CPAP), the "gold standard" treatment, cognitive impairment in some patients is still difficult to completely reverse, suggesting that CIH may initiate some kind of persistent subcellular neuronal damage mechanism.

[0003] At the cellular level, calcium (Ca²⁺) homeostasis imbalance is considered a pivotal event in hypoxic neuronal death. The oxidative stress environment induced by CIH can lead to intracellular Ca²⁺ overload, thereby disrupting mitochondrial function and triggering endoplasmic reticulum stress (ER stress). The ER, as the intracellular calcium reservoir and protein folding factory, is extremely sensitive to Ca²⁺ fluctuations. Sustained Ca²⁺ overload induces the unfolded protein response (UPR) to shift from adaptive protection to lethal execution, activating pro-apoptotic factors such as CHOP, ultimately leading to neuronal apoptosis. The transient receptor potential (TRPC) family is a class of voltage-independent cation channels. Among them, the TRPC5 subtype is highly expressed in the hippocampus of the brain and is crucial for maintaining neuronal synaptic plasticity. TRPC5 also has a unique "redox sensitivity" and is easily activated by reactive oxygen species (ROS) generated by hypoxia / reoxygenation. Studies have confirmed that TRPC5 is involved in CIH-induced inflammatory damage to cardiomyocytes. However, whether TRPC5 acts as a "sensor" and "amplifier" of CIH damage in the central nervous system and mediates hippocampal neuronal death by regulating endoplasmic reticulum stress is currently without direct evidence.

[0004] Currently, while research on OSAHS-related cognitive impairment has proposed that "Ca²⁺ overload—ERS—apoptosis" is a key axis of hypoxic neuronal damage, the specific molecular channels mediating this lethal Ca²⁺ influx under CIH pathological conditions remain unclear. This scientific gap is a key obstacle to blocking the entire pathological cascade. Furthermore, existing treatments for OSAHS primarily rely on CPAP, lacking specific drugs for neuroprotection. There are also no mature evaluation methods to clarify the role and specific molecular mechanisms of TRPC5 in CIH-induced OSAHS-related cognitive impairment, failing to provide effective target screening and mechanism research methods for the prevention and treatment of OSAHS-related cognitive impairment. In addition, existing research focuses primarily on calcium influx pathways such as L-type calcium channels or NMDA receptors, with insufficient research on the role of TRPC5, a non-voltage-dependent cation channel, in OSAHS-related cognitive impairment. There is a lack of systematic evaluation models and detection methods to verify the potential value of TRPC5 as a therapeutic target. Summary of the Invention

[0005] The purpose of this invention is to provide an assessment method for OSAHS-related cognitive impairment based on TRPC5 knockout rats and CIH models, filling the gap in the existing technology of lacking a clear molecular channel for CIH-induced calcium influx and a mature assessment method for the mechanism of action of TRPC5. At the same time, it provides a means of screening potential therapeutic targets for the prevention and treatment of OSAHS-related cognitive impairment, thereby solving the problems mentioned in the background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution;

[0007] This invention uses TRPC5 knockout rats and wild-type SD rats to construct a CIH model, and sets up four control groups. By integrating morphological, ultrastructural observation and molecular biology techniques, it systematically evaluates the role and molecular mechanism of TRPC5 in CIH-induced OSAHS-related cognitive impairment from multiple dimensions, including hippocampal neuronal morphology, apoptosis, subcellular structure, intracellular Ca²+ levels, and related protein expression. Specifically, SPF-grade healthy male wild-type SD rats and TRPC5- / - SD rats were selected and divided into Control group, CIH group, TRPC5- / -- Control group, and T group. The CIH model was constructed by intermittent hypoxia treatment for 8 weeks in both the RPC5- / --CIH group and the TRPC5- / --CIH group. After synchronous rearing, hippocampal tissue samples were collected. The morphology, apoptosis, subcellular structure, intracellular Ca²+ level, and expression levels of TRPC5, GRP78, and CHOP proteins of hippocampal neurons were detected by HE staining, TUNEL assay, transmission electron microscopy, flow cytometry, and Western blot, respectively. Finally, the differences of each indicator in the four groups were statistically analyzed to clarify the role and molecular mechanism of TRPC5 and evaluate its potential value as a therapeutic target.

[0008] In the evaluation method of this invention, the selection, grouping, and modeling of experimental animals strictly adhere to the ethical requirements for experimental animals. The modeling parameters are intermittent hypoxia treatment from 10:00 to 18:00 daily, with each cycle lasting 8 minutes, for 8 hours daily, for 8 consecutive weeks. These modeling parameters can effectively simulate the pathological characteristics of chronic intermittent hypoxia in OSAHS. During sample testing, the operation steps, reagents, and instrument selection for each experimental method have clear standards. HE staining is used to prepare 5mm hippocampal tissue sections. TUNEL staining is performed by dewaxing, hydration, permeabilization, and blocking. Transmission electron microscopy is used to prepare 8nm ultrathin sections and a double staining method is employed. Flow cytometry is used with Fluo-4AM as a probe to detect Ca²⁺ levels at specific excitation and emission wavelengths. Western blot is used with β-actin as an internal control. The types and dilution ratios of primary and secondary antibodies are clearly defined to ensure the accuracy and reproducibility of the test results. Data statistics are performed using SPSS 23.0 software, with P < 0.05 considered statistically significant, providing a scientific statistical basis for the results analysis.

[0009] Compared with the prior art, the beneficial effects of the present invention are:

[0010] 1. This invention is the first to construct an OSAHS-related cognitive impairment assessment system based on TRPC5 whole-gene knockout rats and CIH models, filling the gap in existing research on the role of TRPC5 in CIH injury of the central nervous system, clarifying that TRPC5 is a key upstream molecule mediating CIH-induced hippocampal neuronal damage, and revealing the key mechanism of the TRPC5-Ca²+-CHOP pathway in CIH hippocampal neuronal damage, providing a new perspective for the study of the molecular mechanism of OSAHS-related cognitive impairment.

[0011] 2. This invention employs multi-dimensional detection indicators and a controlled experimental design to systematically evaluate the role of TRPC5 from five levels: morphology, apoptosis, subcellular structure, ion level, and protein expression. The four controlled experiments can effectively eliminate the influence of TRPC5 deficiency on neurons under basal conditions, accurately reflect the pathological role of TRPC5 under CIH conditions, and provide a comprehensive and convincing detection result, offering a standardized evaluation method for subsequent related research.

[0012] 3. The evaluation method of this invention clarifies that TRPC5 deficiency can alleviate hippocampal neuronal damage induced by chronic intermittent hypoxia by blocking calcium overload and endoplasmic reticulum stress. Furthermore, TRPC5 knockout rats did not show obvious morphological abnormalities under basal conditions, suggesting that inhibiting TRPC5 may not interfere with normal physiological functions and only exerts a protective effect under hypoxic stress. This confirms that TRPC5 is a potential therapeutic target for the prevention and treatment of OSAHS-related cognitive impairment, providing a theoretical basis and experimental support for the development of highly specific, low-side-effect TRPC5 small molecule inhibitors.

[0013] 4. In the evaluation method of this invention, each experimental step, reagent selection, and instrument parameter has clear and specific standards. The operation process is highly repeatable. The breeding and modeling of experimental animals strictly follow the ethical requirements for experimental animals. The detection methods are all mature molecular biology and morphological detection methods. The results are accurate and reliable. It can provide a standardized experimental model and evaluation reference for subsequent target screening and drug development of OSAHS-related cognitive impairment.

[0014] 5. The evaluation method of this invention can not only clarify the role and molecular mechanism of TRPC5 in OSAHS-related cognitive impairment, but also be extended to the study of mechanisms and target screening of other neurological injury diseases related to CIH, calcium overload, and endoplasmic reticulum stress. It has broad application prospects and provides new experimental ideas and methods for the study of hypoxic injury of the central nervous system. Attached Figure Description

[0015] Figure 1 HE staining images of hippocampal tissue from rats in each group of this invention.

[0016] Figure 2This is the TUNEL staining map of the hippocampal tissue of rats in each group of the present invention.

[0017] Figure 3 This is the ultrastructure map of the cells in the hippocampal tissue of rats in each group of the present invention.

[0018] Figure 4 This is the intracellular Ca 2+ level map of the hippocampal tissue of the present invention.

[0019] [[ID=!13]] Figure 5 This is the protein expression map of TRPC5 and endoplasmic reticulum stress-related factors in the hippocampal tissue of rats in each group of the present invention. Detailed implementation manners

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. All experimental operations of the present invention were carried out with the approval of the Animal Experiment Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University, and the approval number is IACUC-20210326-08.

[0021] 1. Experimental materials

[0022] 1.1 Experimental animals: Select 12 SPF-grade healthy male wild-type SD rats aged 8 - 10 weeks with a body weight of 180 - 260 g and 12 SD rats with full gene knockout of TRPC (TRPC5- / -). They are provided by Cyagen Biosciences Inc., and the animal license number is SCXK (Yue) 2020-0055. All animals are raised in accordance with the Guide for the Care and Use of Laboratory Animals.

[0023] Note: There seems to be a formatting issue with the "!" in line ID=13 in the original text. It's not clear if that's a mistake or part of a specific formatting requirement. I've left it as it is in the translation for now.1.2. Main reagents: Hematoxylin staining solution, DAB colorimetric reagent kit (Fuzhou Maixin Biotechnology Development Co., Ltd.); TUNEL apoptosis detection kit-POD (Wuhan Boster Biological Engineering Co., Ltd.); Fluo-4AM calcium ion concentration detection kit (Solepro); SP kit, BCA kit (TransGold); Goat anti-human GRP78 monoclonal antibody (Santa); Anti-TRPC5 antibody [S67-15] (Abcam); Rabbit Anti-GRP78 antibody, Rabbit Anti-DDIT3 antibody (Bioson); Mouse anti-rat β-actin antibody (sinobiological); Goat anti-rabbit IgGH&L (HRP), Goat anti-mouse IgGH&L (HRP) (Abcam); RIPA lysis buffer, SDS-PAGE electrophoresis reagents, transfer reagents, and other conventional molecular biology reagents.

[0024] 1.3 Major Instruments: Transmission electron microscope (HITACHIHT7800, Hitachi, Japan); Microplate reader (Bio-Rad); Chemiluminescence imaging system (Shanghai Qinxiang Scientific Instruments Co., Ltd.); Flow cytometer; Paraffin embedding machine, microtome; Light microscope; Incubator; High-speed centrifuge; Vortex mixer, etc.

[0025] 2. Experimental Methods

[0026] 2.1 Animal grouping and CIH model construction: Wild-type SD rats and TRPC5- / -SD rats were divided into four groups: Control group (wild-type normoxic), CIH group (wild-type intermittent hypoxia), TRPC5- / --Control group (knockout normoxic), and TRPC5- / --CIH group (knockout intermittent hypoxia), with 6 rats in each group. The CIH group and TRPC5- / --CIH group rats were placed in an intermittent hypoxia chamber. During the experiment, nitrogen was circulated into the chamber and mixed air was expelled from 10:00 to 18:00 every day for 8 minutes each time, for 8 hours a day, for 8 weeks. The Control group and TRPC5- / --Control group rats were placed in a normoxic chamber and provided with normoxic air, and were fed synchronously for 8 weeks.

[0027] 2.2 Hippocampal tissue sample collection: After 8 weeks of treatment, all rats were sacrificed, and hippocampal tissue was rapidly isolated. Some hippocampal tissue was fixed with 4% paraformaldehyde for HE staining and TUNEL assay. Fresh tissue blocks were excised from some hippocampal tissue for transmission electron microscopy, flow cytometry, and Western blot assay.

[0028] 2.3 Pathological Morphological Evaluation of Hippocampal Neurons – HE Staining Method: Hippocampal tissue fixed in 4% paraformaldehyde was embedded in paraffin and 5mm serial sections were prepared. The sections were then subjected to routine hematoxylin-eosin staining, specifically dewaxing, hydration, hematoxylin staining, bluing, eosin staining, dehydration, clearing, and mounting. The sections were observed and photographed under a light microscope to analyze the morphological characteristics of the hippocampal neurons in the four groups of rats, including the arrangement of neurons, the degree of nuclear condensation, and the number of degenerated and necrotic cells.

[0029] 2.4 Assessment of Hippocampal Neuron Apoptosis – TUNEL Method: Paraffin-embedded hippocampal tissue sections were routinely dewaxed and hydrated; immersed in 1% immunostaining permeabilization buffer, incubated at room temperature for 5 min, rinsed with PBS for 5 min, and repeated 3 times; immersed in 3% H2O2 blocking buffer, incubated at room temperature for 8 min, rinsed with PBS for 5 min, and repeated 3 times; 100 μL TUNEL reaction solution was added, incubated at 37℃ for 1 h, rinsed with PBS, and then the conversion agent-POD was added, incubated at 37℃ for 30 min, rinsed with PBS, and then DAB staining was performed, counterstained with hematoxylin, dehydrated, cleared, and mounted; TUNEL-positive apoptotic cells were observed and counted under a light microscope, and the proportion of positive cells in the hippocampus of the four groups of rats was quantitatively analyzed.

[0030] 2.5 Evaluation of subcellular structure of hippocampal neurons – Transmission electron microscopy: Fresh hippocampal tissue was taken, and three tissue blocks of 1 mm * 5 mm size were cut. The blocks were quickly fixed in pre-cooled 2.5% glutaraldehyde solution and stored at 4°C for at least 24 h. The tissue blocks were removed and rinsed three times with PBS for 5 min each time. The tissue blocks were then fixed in 1% osmium tetroxide solution for at least 1 h and rinsed three times with PBS. The blocks were then dehydrated sequentially with a gradient of 50%, 70%, 80%, 90%, 95%, and 100% alcohol, followed by dehydration with acetone. The dehydrated tissue blocks were then embedded in resin to prepare 8 nm ultrathin sections. The sections were double-stained with 4% uranium acetate and 0.4% lead citrate. After drying, the sections were observed under a transmission electron microscope (HITACHIHT 7800). Images were captured by professionals, and the ultrastructural changes of mitochondria (crista-like structure, swelling, vacuolation) and endoplasmic reticulum (expansion degree, ribosome attachment state) of hippocampal neurons in four groups of rats were analyzed.

[0031] 2.6 Detection of Intracellular Ca²⁺ Levels in Hippocampal Neurons – Flow Cytometry: Fresh rat hippocampal tissue was collected and washed twice with PBS. The tissue was placed on a 200-mesh sieve and gently ground with a syringe needle core to prepare a single-cell suspension. The suspension was transferred to a 1.5 ml centrifuge tube and centrifuged at 1500 rpm for 5 min, discarding the supernatant. 1 mL of Fluo-3 / AM staining solution with a final concentration of 5 μM was added to the centrifuge tube, vortexed, and incubated at 37°C for 15 min. The cells were collected by centrifugation at 1500 rpm for 5 min, washed twice with PBS, and centrifuged for 5 min each time. The cells were resuspended in 500 μl PBS, and the cell suspension was analyzed using flow cytometry. The excitation wavelength was set to 488 nm and the emission wavelength to 529 nm. Fluorescence intensity was detected to reflect the intracellular Ca²⁺ level of hippocampal neurons. The fluorescence intensity values ​​of the four groups were recorded and statistically analyzed.

[0032] 2.7 Detection of Related Protein Expression Levels – Western Blot Method: Total protein was extracted from hippocampal tissue using RIPA lysis buffer, and protease inhibitors were added to prevent protein degradation; protein concentration was detected using a BCA protein quantification kit, and the protein concentrations of each sample were adjusted to be consistent; SDS-PAGE electrophoresis was performed, with a sample loading of 20 μg. After electrophoresis, the protein was transferred to a PVDF membrane; the PVDF membrane was placed in 5% skim milk and blocked at room temperature for 2 hours; primary antibodies were added, including Anti-TRPC5 antibody, RabbitAnti-GRP78 antibody, RabbitAnti-DDIT3 antibody, and mouse anti-rat β-actinib antibody. Antibody was prepared at dilutions of 1:400, 1:400, 1:400, and 1:1000, and incubated overnight at 4°C on a shaker. The next day, the membrane was washed three times with TBST for 10 min each time. Secondary antibodies (goat anti-rabbit IgGH&L (HRP) and goat anti-mouse IgGH&L (HRP)) were added and incubated at room temperature for 1 h. The membrane was washed three times with TBST for 10 min each time. Color development and imaging were performed using a chemiluminescence imaging system. The gray values ​​of the bands were quantified using ImageLab software. The relative expression level of the protein was expressed as the ratio of the gray value of the target protein (TRPC5, GRP78, CHOP) to the gray value of the internal control β-actin. The relative expression levels of each protein in the four groups of rats were statistically analyzed.

[0033] 2.8 Statistical Analysis: SPSS 23.0 statistical software was used to analyze all test data. Normally distributed measurement data were expressed as x±s. The t-test was used to compare the two groups, and P<0.05 was considered statistically significant. Based on the statistical analysis results, the differences of various indicators among the four groups of rats were compared to analyze the role and molecular mechanism of TRPC5 in CIH-induced hippocampal neuronal damage.

[0034] 3. Experimental Results

[0035] 3.1 TRPC5 deficiency significantly improved CIH-induced histopathological damage to neurons in the CA1 region of the hippocampus: HE staining results showed ( Figure 1 In the Control group and the TRPC5- / --Control group, hippocampal neurons showed full morphology, clear structure, and uniform intercellular spaces, with no obvious pathological changes. In the CIH group, neurons showed disordered arrangement, pyknosis of some nuclei, and an increase in degenerated and necrotic cells. Compared with the CIH group, neurons in the TRPC5- / --CIH group were more regularly arranged, and the number of necrotic / pyknosis cells was significantly reduced, suggesting that TRPC5 deficiency can alleviate CIH-induced morphological damage to hippocampal neurons.

[0036] 3.2 TRPC5 deficiency inhibits CIH-triggered hippocampal neuronal apoptosis: TUNEL assay results showed ( Figure 2 In the Control group and the TRPC5- / --Control group, only occasional TUNEL-positive cells were observed, with no statistically significant difference between the two groups (P>0.05). Eight weeks of CIH exposure led to a sharp increase in the proportion of TUNEL-positive cells in the hippocampus of SD rats (P<0.05), indicating severe DNA breaks and apoptosis. The number of TUNEL-positive cells in the TRPC5- / --CIH group was significantly lower than that in the CIH group (P<0.05), indicating that TRPC5 is a key mediator of CIH-induced programmed cell death in hippocampal neurons.

[0037] 3.3 TRPC5 deficiency alleviates CIH-induced mitochondrial damage and endoplasmic reticulum dilatation: Transmission electron microscopy results show ( Figure 3 In the Control group, neurons showed intact nuclear membranes, uniform chromatin distribution, clear and dense mitochondrial cristae, neatly arranged rough endoplasmic reticulum, and good ribosome attachment. The CIH group exhibited severe organelle stress characteristics, including mitochondrial swelling, decreased matrix electron density, vacuolation, mitochondrial cristae breakage or disappearance, significant expansion of the endoplasmic reticulum cisterns, and ribosome shedding. The TRPC5- / --CIH group showed significant reduction in the above ultrastructural lesions, with only mild mitochondrial swelling, relatively preserved mitochondrial cristae structures, and significantly improved endoplasmic reticulum expansion compared to the CIH group, suggesting that TRPC5 deficiency protects neurons from CIH at the organelle level.

[0038] 3.4 TRPC5 deficiency blocks CIH-induced intracellular calcium overload: Flow cytometry results showed that the Fluo-4 fluorescence intensity in the Control group was 11733.333±1279.882, while it was significantly increased to 22197.000±3112.336 in the CIH group (P<0.05 compared with the Control group); the Fluo-4 fluorescence intensity in the TRPC5- / --Control group was 10576.857±1104.753 (P<0.05 compared with the CIH group); and the Fluo-4 fluorescence intensity in the TRPC5- / --CIH group was 15154.250±2085.822, which was significantly lower than that in the CIH group (P<0.05), but still higher than that in the TRPC5- / --Control group (P<0.05). Figure 4 Table 1 shows that the TRPC5 channel is a key "gateway" mediating pathological calcium influx under CIH conditions, and knocking out this channel can effectively reduce calcium overload.

[0039]

[0040] 3.5. TRPC5 deficiency alleviates endoplasmic reticulum stress by downregulating GRP78 and CHOP expression: Western blot results showed ( Figure 5 (Table 2) Compared with the Control group, the relative expression levels of TRPC5, GRP78, and CHOP proteins in the CIH group were significantly upregulated (P<0.05). In the Control group, TRPC5 was 0.667±0.063, GRP78 was 0.541±0.040, and CHOP was 0.632±0.053; in the CIH group, TRPC5 was 0.972±0.051, GRP78 was 0.881±0.029, and CHOP was 1.149±0.146. In the TRPC5- / --Control group, the expression levels of TRPC5, GRP78, and CHOP proteins were significantly decreased (P<0.05 compared with the CIH group). The expression levels of TRPC5, GRP78, and CHOP proteins in the TRPC5- / --CIH group were 0.266±0.042, 0.145±0.002, and 0.192±0.016, respectively. The expression levels of TRPC5, GRP78, and CHOP proteins in the TRPC5- / --CIH group were significantly reversed compared to the CIH group (P<0.05), at 0.420±0.076, 0.316±0.023, and 0.356±0.059, respectively. The intergroup F values ​​for the expression levels of each protein in the four groups were 108.776, 548.915, and 100.715, respectively, with P=0.001 for all of them. This indicates that blocking TRPC5-mediated calcium influx can effectively reduce the protein folding load of the endoplasmic reticulum and inhibit the lethal endoplasmic reticulum stress response mediated by CHOP.

[0041]

[0042] 4. Experimental Conclusions

[0043] The evaluation method of this invention clarifies that TRPC5 is a key upstream molecule mediating CIH neurotoxicity. TRPC5 deficiency protects hippocampal neurons from hypoxia damage by inhibiting pathological calcium influx and alleviating fatal endoplasmic reticulum stress induced by calcium overload. At the same time, it confirms the key mechanism of the TRPC5-Ca²+-CHOP pathway in CIH hippocampal neuronal injury, namely, CIH exposure leads to intracellular calcium overload by activating TRPC5 channels, thereby triggering severe endoplasmic reticulum stress and inducing neuronal apoptosis, while TRPC5 deficiency can significantly buffer the pathological cascade response triggered by hypoxia without affecting the basic neuronal structure.

[0044] The evaluation method of this invention confirms that TRPC5 provides a new potential therapeutic target for the prevention and treatment of OSAHS-related brain injury, and provides a theoretical basis for the development of targeted neuroprotective drugs.

[0045] 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 assessing OSAHS-related cognitive impairment based on TRPC5 whole-gene knockout rats and a CIH model, characterized in that, It includes the following steps: S1. Selection of experimental animals: Select SPF-grade healthy male wild-type SD rats and TRPC5 gene knockout SD rats (TRPC5- / -) at 8-10 weeks old and with a body weight of 180-260 g; S2. Animal grouping and construction of CIH model: Divide the rats into Control group, CIH group, TRPC5- / - - Control group and TRPC5- / - - CIH group, with 6 rats in each group; Place the rats in the CIH group and the TRPC5- / - - CIH group in an intermittent hypoxia chamber. From 10:00 to 18:00 every day, nitrogen is circulated into the chamber and the mixed air is exhausted. Each cycle is 8 minutes, and it lasts for 8 hours every day for 8 consecutive weeks; Place the rats in the Control group and the TRPC5- / - - Control group in a normoxia chamber to provide normoxic air and raise them synchronously for 8 weeks; S3. Collection of hippocampal tissue samples: Sacrifice all rats after 8 weeks of treatment and collect hippocampal tissue samples; S4. Evaluation of hippocampal neuron pathology and apoptosis: Use HE staining method to evaluate the morphological changes of hippocampal neurons, and use TUNEL method to detect the apoptosis of hippocampal neuron cells; S5. Evaluation of subcellular structure of hippocampal neurons: Use transmission electron microscopy (TEM) to observe the ultrastructural changes of mitochondria and endoplasmic reticulum in hippocampal neurons; S6. Detection of intracellular Ca²⁺ level in hippocampal neurons: Use flow cytometry and use Fluo-4AM as a calcium fluorescent probe to detect the intracellular Ca²⁺ level in hippocampal neurons; S7. Detection of relative expression levels of related proteins: Use Western blot method to detect the relative protein expression levels of TRPC5, endoplasmic reticulum stress (ERS) marker protein GRP78, and pro-apoptotic factor CHOP in hippocampal tissue; 2. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: S8. Data statistical analysis: Use SPSS 23.0 statistical software to analyze the above detection data. The measurement data conforming to the normal distribution are expressed as x±s. The t-test is used for comparison between two groups. P<0.05 is considered to have statistically significant differences. According to the detection results of each index and the statistical analysis conclusions, evaluate the role and molecular mechanism of TRPC5 in CIH-induced OSAHS-related cognitive impairment.

3. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: The TRPC5 gene knockout SD rats and wild-type SD rats in S1 are provided by Cyagen Biosciences Inc., and the animal license number is SCXK (Yue) 2020-0055. The feeding and use of experimental animals comply with the Guide for the Care and Use of Laboratory Animals and are approved by the Animal Experiment Ethics Committee.

4. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: The procedure for the TUNEL method in S4 is as follows: Take paraffin-embedded hippocampal tissue sections, perform routine dewaxing and hydration, immerse in 1% immunostaining permeabilization buffer and let stand at room temperature for 5 minutes, then wash with PBS for 5 minutes. The operation process of the HE staining method in S4 is as follows: Take the hippocampal tissue of rats, fix it with 4% paraformaldehyde, embed it in paraffin, prepare 5 mm sections, stain with hematoxylin-eosin, observe and photograph under a light microscope, and analyze the morphological characteristics such as the arrangement of hippocampal neurons, nuclear pyknosis, degeneration and necrosis. 3 times; Immerse in a quality 2 O 2 fraction of 3% H blocking solution and let it stand at room temperature for 8 minutes, and rinse with PBS for 5 minutes Three stainings were performed, followed by staining according to the TUNEL apoptosis detection kit-POD instructions, to detect and quantify the number of TUNEL-positive apoptotic cells.

5. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: The sample processing procedure for transmission electron microscopy observation in S5 is as follows: Fresh hippocampal tissue from SD rats is cut into 1mm*5mm tissue blocks, which are quickly placed in pre-cooled 2.5% glutaraldehyde solution for fixation. After being stored at 4°C for at least 24 hours, the tissue is washed three times with PBS and fixed in 1% osmium tetroxide solution for at least 1 hour. After being washed with PBS, the tissue is dehydrated by a gradient of alcohol and acetone, embedded in resin, and 8nm ultrathin sections are prepared. The sections are then stained with 4% uranium acetate and 0.4% lead citrate. The mitochondrial cristae structure, mitochondrial swelling, endoplasmic reticulum expansion, and ribosome attachment status are observed under a transmission electron microscope.

6. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: The specific procedure for detecting intracellular Ca²⁺ levels using flow cytometry in S6 is as follows: Fresh rat hippocampal tissue is taken, washed twice with PBS, and gently ground on a 200-mesh sieve using a syringe needle core to prepare a single-cell suspension. The suspension is centrifuged at 1500 rpm for 5 min and the supernatant is discarded. 1 mL of Fluo-3 / AM staining solution with a final concentration of 5 μM is added, vortexed, and incubated at 37°C for 15 min. Cells are collected by centrifugation at 1500 rpm for 5 min, washed twice with PBS, and resuspended in 500 μl of PBS. The fluorescence intensity is detected at an excitation wavelength of 488 nm and an emission wavelength of 529 nm using flow cytometry. The fluorescence intensity reflects the intracellular Ca²⁺ level.

7. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: The Western blot procedure in S7 is as follows: Total protein is extracted from hippocampal tissue using RIPA lysis buffer, and protein concentration is detected using a BCA protein quantification kit; after SDS-PAGE electrophoresis, membrane transfer and blocking, primary antibodies are added for incubation. The primary antibodies include anti-TRPC5 antibody, anti-GRP78 antibody, anti-CHOP antibody, and anti-β-actin antibody, and the membrane is incubated overnight at 4°C; after washing with TBST, secondary antibodies are added for incubation at room temperature, color development and imaging are performed, and the gray values ​​of the bands are quantified using ImageLab software. The relative protein expression level is expressed as the ratio of the gray value of the target protein to the gray value of the internal control β-actin.

8. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 7, characterized in that: The primary antibodies were diluted as follows: anti-TRPC5 antibody, anti-GRP78 antibody, and anti-CHOP antibody were diluted at a ratio of 1:400, and anti-β-actin antibody was diluted at a ratio of 1:1000; the secondary antibodies were goat anti-rabbit IgGH&L (HRP) and goat anti-mouse IgGH&L (HRP).

9. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: The reagents used in S4 to S7 include: hematoxylin staining solution, DAB colorimetric reagent kit, TUNEL apoptosis detection kit-POD, Fluo-4AM calcium ion concentration detection kit, RIPA lysis buffer, BCA protein quantification kit, anti-TRPC5 antibody [S67-15], goat anti-human GRP78 monoclonal antibody, RabbitAnti-GRP78 antibody, RabbitAnti-DDIT3 antibody, mouse anti-rat β-actin antibody, goat anti-rabbit IgGH&L (HRP), and goat anti-mouse IgGH&L (HRP); the instruments used include: transmission electron microscope (HITACHIHT7800), microplate reader (Bio-Rad), chemiluminescence imaging system, and flow cytometer.

10. The method for assessing OSAHS-related cognitive impairment based on TRPC5 whole-genome knockout rats and CIH models according to claim 1, characterized in that: Based on the statistical analysis results of S8, by comparing the differences in the degree of morphological damage, apoptosis level, subcellular structural integrity, intracellular Ca²+ level, and expression levels of TRPC5, GRP78, and CHOP proteins in the hippocampal neurons of the four groups of rats, the molecular mechanism by which TRPC5 participates in CIH-induced OSAHS-related cognitive impairment through mediating calcium overload and endoplasmic reticulum stress was clarified. At the same time, the potential value of TRPC5 as a therapeutic target for OSAHS-related cognitive impairment was evaluated.

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