Application of steroid hormone inhibitor in preparation of medicine for treating Parkinson's blood pressure regulation disorder

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of a steroid hormone inhibitor in preparation of a medicine for treating Parkinson's blood pressure regulation disorder. Research finds that TGF-beta1 / SGK-1 signal pathway activation caused by aldosterone increase in PD rats induces carotid body fibrosis, the carotid body fibrosis causes reduction of chemoreceptor sensitivity, and blood pressure regulation disorder is induced. A further research shows that the inhibitor, including a cortisol inhibitor or an aldosterone inhibitor, capable of inhibiting abnormal activation of the HPA axis can effectively reverse abnormal blood pressure regulation of PD rats by improving carotid artery fibrosis of the PD rats and improving the pressure sensing reflex sensitivity, so that the effect of inhibiting abnormal activation of the HPA axis is achieved. The pharmaceutical composition can be used in the related fields of a Parkinson's disease blood pressure regulation disorder mechanism and treatment drugs thereof.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and specifically discloses the application of steroid hormone inhibitors in the preparation of drugs for treating Parkinson's disease and blood pressure regulation disorders. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Its harm is not only manifested in typical motor symptoms such as tremor and rigidity, but also in the severe impact on patients' quality of life caused by various non-motor symptoms such as depression, constipation, sleep disorders, and blood pressure regulation disorders. Among these, non-motor symptoms often appear before motor symptoms, making early diagnosis and treatment of PD crucial.

[0003] Among various non-motor symptoms, blood pressure regulation disorder is particularly prominent, affecting 14%–47% of PD patients to varying degrees, and can appear 5–20 years earlier than motor symptoms. Long-term blood pressure regulation disorder can further impair cognitive function, increase stroke risk, and even raise mortality. Currently, the causes of blood pressure regulation disorder are known to be complex, possibly related to postsynaptic adrenergic receptor hypersensitivity, abnormal urinary sodium excretion, and impaired baroreflex compensation. However, these factors are only preliminary speculations, and the specific pathogenesis remains far from clear, greatly limiting the development of targeted therapeutic drugs.

[0004] The carotid body (CB) is an important peripheral chemoreceptor in the human body. It is primarily composed of type I and type II cells, with type I cells containing tyrosine hydroxylase (TH)-positive neurons being the core functional cells. These cells are highly sensitive to the partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), and hydrogen ion concentration in the blood flowing through the body. Studies have shown a close relationship between the carotid body and blood pressure regulation. For example, specific blocking of P2X3 receptors can lower blood pressure and baseline sympathetic activity through carotid body-related pathways. When arterial blood pressure is too low, the carotid body can exert high-intensity sympathetic nerve stimulation to achieve blood pressure regulation effects such as increased cardiac activity and peripheral vasoconstriction.

[0005] It is important to note that the core pathological feature of Parkinson's disease (PD) is damage to dopaminergic neurons in the substantia nigra, while the carotid body is rich in TH-positive neurons. There is a potential pathophysiological link between the two. However, the specific changes in the carotid body after bilateral substantia nigra dopaminergic neuron damage and its intrinsic connection with blood pressure regulation disorders in PD patients have not yet been elucidated. In summary, current research on the pathogenesis of blood pressure regulation disorders in PD patients is not in-depth, especially regarding the role of the carotid body and related regulatory pathways, which remain unclear. This leads to a lack of targeted clinical treatments. Therefore, elucidating the core mechanisms of blood pressure regulation disorders in PD patients and developing corresponding therapeutic drugs has significant clinical value and research significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention discovers that increased aldosterone levels in PD rats lead to activation of the TGF-β1 / SGK-1 signaling pathway, inducing carotid body fibrosis. Carotid body fibrosis results in decreased chemoreceptor sensitivity, inducing blood pressure regulation disorders. Based on this, this invention further discovers that steroid hormone inhibitors can improve carotid body fibrosis in PD rats, further improving baroreceptor reflex sensitivity and orthostatic hypotension in PD rats.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of steroid hormone inhibitors in the preparation of drugs for treating Parkinson's disease with blood pressure regulation disorders.

[0008] As a first limitation of the above application, the steroid hormone inhibitors include inhibitors that inhibit abnormal activation of the HPA axis.

[0009] As a second limitation on the above application, the steroid hormone inhibitors include steroid receptor antagonists.

[0010] As a third limitation on the above application, the steroid hormone inhibitor includes at least one of cortisol inhibitors or aldosterone inhibitors.

[0011] As a fourth limitation of the above application, the Parkinson's blood pressure regulation disorder is associated with carotid body fibrosis.

[0012] As a further limitation of the fourth limitation of the above application, the Parkinson's blood pressure regulation disorder includes at least one of orthostatic hypotension, supine hypertension, or postprandial hypotension.

[0013] Preferably, the Parkinson's blood pressure regulation disorder is orthostatic hypotension.

[0014] This invention found that after injecting fluorescent gold into the hypothalamic paraventricular nucleus (PVN) of Parkinson's disease (PD) rats with impaired blood pressure regulation for 7 days, fluorescent gold-labeled neurons appeared in the bilateral substantia nigra (SN), suggesting that neurons in the SN can send fiber projections to the PVN. Compared with the control group, the levels of tyrosine hydroxylase (TH) and dopamine D2 receptor (D2R) in the PVN of PD rats with impaired blood pressure regulation were significantly decreased (P<0.01), while the level of dopamine D1 receptor (D1R) remained relatively stable, and the level of corticotropin-releasing hormone (CRH) was significantly increased (P<0.01). Simultaneously, serum levels of CRH, adrenocorticotropic hormone (ACTH), cortisol (Cort), and aldosterone (ALD) were significantly elevated, indicating that the hypothalamic-pituitary-adrenal (HPA) axis was activated, and this activation state could induce a significant increase in ALD. Furthermore, the carotid body (CB) tissue structure of PD rats showed significant abnormalities, with extensive collagen fiber proliferation and obvious fibrosis characteristics.

[0015] The above results indicate that the core mechanism of blood pressure regulation disorder in PD rats is as follows: increased ALD can activate the TGF-β1 / SGK-1 signaling pathway, thereby inducing CB fibrosis; and CB fibrosis leads to decreased chemoreceptor sensitivity, ultimately inducing blood pressure regulation disorder. Therefore, drugs that reduce ALD levels or inhibit the activation of the TGF-β1 / SGK-1 signaling pathway in PD rats can, to some extent, block the CB fibrosis process in PD rats, thereby improving their blood pressure regulation disorder.

[0016] Therefore, drugs that inhibit abnormal activation of the HPA axis, such as cortisol inhibitors or aldosterone inhibitors, have the potential to improve blood pressure regulation disorders in Parkinson's disease. Aldosterone receptor inhibitors are mineralocorticoid receptor antagonists, including eplerenone, spironolactone, fenelazone, and esaliridone. Cortisol receptor inhibitors are glucocorticoid receptor antagonists, including mifepristone, relacorilant, and exicorilant.

[0017] This invention reveals that cortisol inhibitors improve blood pressure regulation disorders in PD rats, and their mechanism is closely related to the regulatory logic of the HPA axis: cortisol, as a key downstream effector molecule for HPA axis activation, has a synergistic regulatory relationship with the abnormal increase in ALD. During HPA axis activation, elevated CRH sequentially drives increased ACTH secretion. ACTH not only directly promotes ALD secretion from the adrenal cortex but also significantly induces cortisol synthesis and release. Simultaneously, cortisol itself can further enhance HPA axis activation through positive feedback regulation, exacerbating the excessive secretion of CRH and ACTH, forming a vicious cycle of "CRH-ACTH-cortisol / ALD," ultimately leading to a sustained increase in ALD and inducing CB fibrosis.

[0018] Therefore, cortisol inhibitors can directly block the synthesis or action of cortisol. On the one hand, they can break the overactivation cycle of the HPA axis and reduce abnormal ACTH secretion, thereby inhibiting the excessive production of ALD from the source. On the other hand, they can directly reduce the synergistic activation of the TGF-β1 / SGK-1 signaling pathway by cortisol, further alleviating the degree of CB fibrosis. Through these two effects, cortisol inhibitors can restore the normal sensitivity of CB chemoreceptors and ultimately improve blood pressure regulation disorders in PD rats.

[0019] Aldosterone inhibitors act directly on aldosterone, reducing aldosterone levels in PD rats, inhibiting overactivation of the HPA axis, and effectively reversing abnormal blood pressure regulation in PD rats.

[0020] In summary, drugs that inhibit abnormal activation of the HPA axis, including aldosterone inhibitors and cortisol inhibitors, can be used to prepare drugs for treating or improving blood pressure regulation disorders associated with CB fibrosis, providing a new approach for improving blood pressure regulation disorders in PD patients or intervening in the progression of PD disease.

[0021] Secondly, the present invention provides a medicament for treating Parkinson's disease with impaired blood pressure regulation, the medicament comprising a steroid hormone inhibitor that inhibits abnormal activation of the HPA axis.

[0022] Furthermore, the steroid hormone inhibitor includes at least one of a cortisol inhibitor or an aldosterone inhibitor.

[0023] Based on the above mechanistic studies and animal experimental results, the steroid hormone inhibitor provided by this invention, which can inhibit abnormal activation of the HPA axis, can effectively reverse abnormal blood pressure regulation in PD rats by improving carotid body fibrosis and enhancing baroreceptor sensitivity. Therefore, inhibitors that inhibit abnormal activation of the HPA axis, including cortisol inhibitors or aldosterone inhibitors, can be developed into drugs for PD-related blood pressure regulation disorders, providing new ideas for the clinical intervention of this type of neurodegenerative disease. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is the immunofluorescence staining result after 6-OHDA was injected into SN for 6-8 weeks in Example 1 of the present invention; Figure 2 The statistical results of the relative MFI and mRNA content of TH, D1R and D2R in different groups in Real-time PCR in Example 1 of this invention; Figure 3 The statistical results of the relative expression levels of TH, D1R, and D2R in different groups in the Western blot experiment of Example 1 of the present invention; Figure 4 The results of the measurement of changes in baroreflex function in PD rats in Example 1 of this invention; wherein, Figure 4 A is a schematic diagram of an in vitro perfusion experiment of the carotid artery body; Figure 4 B represents the changes in the carotid artery baroreceptor reflex function curve in 6-OHDA rats and under the influence of 5mM DNP; Figure 4 C is the original recording of the blood pressure reflex response after intrasinus perfusion with KH solution and 5 mM DNP; Figure 5 The results of HE staining and Masson staining of the carotid body of rats in different groups in Example 1 of this invention; Figure 6 The above are the statistical results of the relative expression levels of HIF-1α, SGK-1, TGF-β1 and Vimentin in the CB tissues of rats in the same group in Example 1 of this invention; Figure 7 The images show fluorescence images of rats in different groups after 7 days of PVN injection with fluorescent gold in Example 1 of this invention. Figure 8 Representative fluorescence images of CRH-positive cells, c-Fos-positive cells, and CRH / c-Fos double-labeled cells in the PVN of rats from different groups in Example 1 of this invention; Figure 9 This is the statistical result of the expression levels of Th, D1R and D2R in different groups of rat PVN determined by Real-time PCR in Example 1 of the present invention; Figure 10This is the statistical result of the relative expression levels of CRH, D1R and D2R proteins in the PVN of different groups of rats in the Western blot experiment of Example 1 of the present invention; Figure 11 The above are the statistical results of the relative expression levels of CRH, ACTH, Cort and ALD in the serum of different groups in Example 1 of the present invention. Figure 12 The results of H&E staining and Masson staining of the carotid body of rats in different groups in Example 2 of this invention; Figure 13 The statistical results of the relative expression levels of Vimentin, HIF-1α, SGK-1 and TGF-β1 in rat CB of different groups in Example 2 of the present invention; Figure 14 This is a statistical result of the blood pressure changes of different groups of rats in Example 2 of the present invention as perfusion pressure increased; Figure 15 This is the statistical result of the blood pressure of different groups of rats in Example 2 of the present invention dropping to the lowest level at the maximum perfusion pressure; Figure 16 The above are the statistical results of various functions of the baroreceptor reflex in different groups of rats in Example 2 of this invention; Figure 17 This is the original curve of blood pressure changes in different groups of rats under different body positions in Example 2 of the present invention; Figure 18 This is a statistical result of the blood pressure recovery time of rats in different groups after changes in body position in Example 2 of the present invention; Figure 19 The results show the statistical results of the mean arterial pressure at the lowest position for rats in different groups in Example 2 of this invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, cell culture, and related fields.

[0028] All experimental animals used in this invention were purchased from reputable companies and possessed the corresponding certificates of conformity. Specifically, the RIPA lysis buffer was purchased from Hebei Ruipat Biotechnology Co., Ltd.; CRH antibody and β-actin antibody were purchased from Abclonal; TH antibody, D1R antibody, and TGF-β1 antibody were purchased from Proteintech; c-Fosantibody was purchased from Synaptic System (Germany); D2R antibody was purchased from Santa Cruz Biotechnology; HIF-1α antibody was purchased from Gentex; SGK-1 antibody was purchased from Huaan Biotechnology; Vimentin antibody was purchased from Jingjie Biotechnology; Alexa Fluor 488 and Cy3 were purchased from Jackson ImmunoResearch (USA); the cDNA synthesis kit was purchased from Vazyme; ChamQ Universal SYBR QPCR and Master Mix were purchased from Zhongshi Tongchuang; the high-purity RNA extraction kit was purchased from Sigma-Aldrich (USA); AAV9 virus was purchased from Brinkes (Shenzhen, China); Mifepristone (a cortisol inhibitor) was purchased from Aladdin; and Eplerenone (an aldosterone inhibitor) was purchased from Aladdin.

[0029] This invention uses GraphPad Prism v8.0 to analyze the obtained data. Data are expressed as mean ± SEM. A t-test was used to assess the difference between the experimental and control groups, with P < 0.05 considered statistically significant. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[0030] Table 1 shows a statistical table of the English and Chinese abbreviations and their corresponding full English and Chinese names in this invention.

[0031] Table 1

[0032] To better illustrate the embodiments provided by the present invention, further examples are given below.

[0033] Example 1 In this invention, SD rats were used as experimental subjects and randomly divided into a control group (Sham group) and an experimental group (6-OHDA group), with 10 rats in each group. The 6-OHDA group was used to establish a PD rat model by injecting 2 μL of 6-OHDA (3 mg / mL) into the bilateral substantia nigra (SN) using stereotactic brain imaging. The Sham group received an equal volume of saline. The rats' motor coordination ability after model establishment was assessed using a rotarod test. 24-hour ambulatory blood pressure monitoring in the conscious state and upright blood pressure in the anesthetized state were monitored using a DSI wireless telemetry device to determine if there was blood pressure regulation disorder. Carotid sinus isolation perfusion technology and arterial blood pressure monitoring technology were used to detect changes in CB function and BP in both groups of rats, and baroreceptor reflex function curves were plotted. Real-time PCR and Western blot were used to determine the mRNA and protein expression levels of tyrosine hydroxylase (TH), dopamine D1 receptor (D1R), and dopamine D2 receptor (D2R) in the SN brain region. The mRNA and protein expression levels of corticotropin-releasing hormone (CRH), D1R, and D2R in the hypothalamic paraventricular nucleus (PVN) were also measured. The protein expression levels of hypoxia-inducible factor-1α (HIF-1α), serum and glucocorticoid-inducible kinase-1 (SGK-1), transforming growth factor-β1 (TGF-β1), and vimentin in CB tissue were determined. The expression levels of PVN-CRH neurons, D1R, and D2R were determined using immunofluorescence colocalization. Serum levels of CRH, adreno-corticotropic hormone (ACTH), corticosterone (Cort), and aldosterone (ALD) were measured using ELISA. Fibrosis in CB cells was assessed using hematoxylin-eosin staining (HE staining) and Masson staining.

[0034] I. Experimental Methods Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all adopt conventional methods in this technical field.

[0035] 1. Method of the rotator bar experiment Using a rotundiform training device, rats were trained on the rotundiform for three consecutive days (days 55, 56, and 57 post-lesion). During the testing phase (day 58 post-lesion), rats adapted to an initial rotation speed of 4 r / min for 10 seconds, then accelerated to 40 r / min within 3 minutes and maintained this speed for 5 minutes. During this period, rats fell off the rotundiform due to motor dysfunction. The computer automatically recorded the time the rats spent on the rotundiform before falling. The movement time of the Sham group and the 6-OHDA group was compared to determine the presence of motor dysfunction. Each rat was tested three times, and the average time spent on the rotundiform was used for statistical analysis.

[0036] 2. Carotid sinus area isolation perfusion The experimental rats were anesthetized, and a midline incision was made in the neck for endotracheal intubation. The bilateral carotid sinus regions were fully exposed, and the bilateral depressor nerves and the right sinus nerve were separated and severed. The left occipital artery, superior thyroid artery, and the proximal end of the common carotid artery were ligated, and a cannula was inserted into the distal end of the common carotid artery as the inflow tract. The distal end of the external carotid artery was then ligated, and a cannula was inserted into its proximal end as the outflow tract. A peristaltic pump connected to 37°C oxygenated KH solution was used to isolate and perfuse the left carotid sinus region of the rats. A sensitive pressure transducer (XHYP200) was connected to the inflow tract to monitor the intrasinus pressure (ISP) of the carotid sinus. The KH solution contains 118.0 mmol / L NaCl, 25.0 mmol / L NaHCO3, 4.7 mmol / L KCl, 1.2 mmol / L KH2PO4, 1.2 mmol / L MgSO4, 2.5 mmol / L CaCl2, and 5.6 mmol / L glucose, with a pH of 7.35 to 7.45.

[0037] Experimental procedure for sloping perfusion: The functional parameters of the carotid sinus were determined by isolating the carotid sinus region with KH solution. Isolation perfusion was performed with the intravascular coagulation (ISP) maintained at 100 mmHg. During the experiment, the perfusion pressure was first reduced to 0 mmHg, then automatically and uniformly increased to 250 mmHg in a sloping manner, then rapidly reduced to 0 mmHg, and subsequently maintained at 100 mmHg, a process lasting 90 s. A pressure-sensing function curve was plotted with mean arterial pressure (MAP) on the ordinate and ISP on the abscissa, to determine the pressure reflex function of the carotid sinus region and the reflexive decrease in PS and MAP. The ISP value at which a reflexive decrease in systemic MAP of 3 mmHg was just achieved was defined as TP; the ISP value at which the reflexive decrease in MAP ceased was defined as SP; and the pressure value at which MAP equaled ISP was defined as EP. The difference between SP and TP was defined as the pressure reflex's OR.

[0038] The effects of 2,4-dinitrophenol (DNP), a chemical that can cause a sharp increase in HIF-1α levels, on the function of the chemoreceptor receptor (CB) were observed, and CB function was then tested. According to experimental requirements, DNP (5 mM) was added to the perfusion fluid, and then the ISP (Intensity Sensitive Point) was repeatedly increased and decreased, and the changes in the reflectance curve were observed. After the effect appeared, the fluid was immediately flushed with KH solution, and the above reflectance process was repeated to try to restore the function to the pre-drug level.

[0039] 3. Method for reverse labeling from PVN to SN Normal Sham rats and 6-OHDA rats were anesthetized with pentobarbital (60 mg / kg, ip). Fluorogold was microinjected bilaterally into the PVN (peripheral brain region) via a cranial drill at the ventricular surface site. Fluorogold was injected twice, 50 nL each time, into the PVN through a glass microtube connected to a Nanoject II microinjector. The wounds were sutured after injection, and the animals were returned to their cages for approximately 7 days to allow the tracer to be transported retrogradely from the PVN to the upstream brain regions. The animals' activity, signs of infection, and food and water intake were monitored daily. After modeling, perfusion and tissue sampling were performed on a portion of the model animals for immunofluorescence analysis to observe the expression of fluorescent gold in the SN (single brain region).

[0040] 4. Detection of serum aldosterone, CRH, ACTH, Cort, and other indicators. The levels of CRH, ACTH, and Cort in serum were detected using enzyme-linked immunosorbent assay (ELISA), and the procedure was strictly followed according to the kit instructions.

[0041] 5. Western Blot Rats in the Sham and 6-OHDA groups were anesthetized with 2%–3% isoflurane. The entire brain tissue was removed by decapitation, including the cerebellum, brainstem, and anterior third of the distal brain. The remaining brain tissue, along with the brainstem, was rapidly placed in 0.9% saline at low temperature (0–4°C). Using a vibratory microtome (VT1000S, Leica, Germany), coronal sections containing the PVN region were precisely excised, each slice being 600 μm thick. The PVN was then excised according to the atlas location and placed in cryovials for rapid freezing in liquid nitrogen. The tissue was used immediately or stored at -80°C. The SN (snail) was obtained in the same manner.

[0042] Rats were euthanized by decapitation after anesthesia. The bifurcation of both carotid arteries was removed and placed in a sterile KH solution saturated with 95% O2-5% CO2 and kept cold. The carotid artery tissue was then separated from the bifurcation under a stereomicroscope.

[0043] Total protein was extracted from each group of samples, and protein concentration was measured. Depending on the detection index, 30-50 μg of protein samples were selected for electrophoresis. After 10% SDS-PAGE gel electrophoresis, the protein was transferred to a PVDF membrane by electroporation. Subsequently, the membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4℃ with primary antibodies (CRH, 1:500; TH, 1:1000; D2R, 1:1000; D1R, 1:1000; SGK-1, 1:1000; TGF-β1, 1:1000; Vimentin, 1:1000; HIF-1α, 1:1000; β-actin). The following day, the membrane was washed three times with TBST for 10 minutes each time, then goat anti-rabbit IgG (1:5000) was added and incubated at room temperature for 1 hour. After washing with TBST again, ECL luminescent agent was added, and the membrane was developed using a ChemiScopes6 imaging system to obtain bands. Quantitative analysis of the bands was performed. The ratio of the gray values ​​of the target band to the β-actin band was used as the final result, and statistical analysis was conducted.

[0044] 6. Real-time PCR 6.1 After extracting RNA from brain tissue, the RNA was washed and eluted, and the RNA concentration and purity were determined. The absorbance (OD) values ​​of the RNA solution at 260 nm and 280 nm were measured respectively. If the ratio was between 1.8 and 2.0, the extracted RNA could be used for reverse transcription.

[0045] The reverse transcription system for cDNA is shown in Table 2.

[0046] Table 2

[0047] Reverse transcription reaction procedure: 50℃, 5 min, to complete the synthesis of the first strand; 95℃, 1 min, to inactivate MuLV. 6.2 Real-time PCR amplification The real-time PCR amplification system is shown in Table 3.

[0048] Table 3

[0049] The real-time PCR procedure is shown in Table 4.

[0050] Table 4

[0051] The specific RNA primer sequences are as follows: The TH primer sequence is as follows: Upstream primer: 5'-CTACTGTCCGCCCGTGATTT-3' Downstream primer: 3'-ACCAGTACACCGTGGAGAGT-5' The D1R primer sequence is: Upstream primer: 5'-CCAGATCGGGCATTTGGAGA-3' Downstream primer: 3'-CGTAGCAATCCAAGCCATGC-5' The D2R primer sequence is: Upstream primer: 5'-GGAGGGAGCGACAGTTCTTA-3' Downstream primer: 3'-TGGGGCCCTTGAGGATTAGA-5' The GAPDH primer sequences are: Upstream primer: 5'-GGCTCATGACCACAGTCCAT-3' Downstream primer: 3'-ACATTGGGGGTAGGAACACG-5'.

[0052] After amplification, the melting curves of the PCR products were analyzed, showing good overlap, indicating good primer specificity. GAPDH was used as an internal control to detect fluorescence signal intensity, and the experimental data were calculated using the ΔΔCT method to determine gene expression. -ΔΔCT value.

[0053] II. Experimental Results 1. The rotarod test results showed that, compared with the Sham group, the 6-OHDA group rats had a significantly shorter time spent on the rotarod, proving that the 6-OHDA group rats exhibited motor dysfunction. Blood pressure monitoring results showed that, after changing the rats from a supine to an upright position, compared with the Sham group, the 6-OHDA group rats exhibited delayed blood pressure recovery and symptoms of blood pressure regulation disorder, indicating that the 6-OHDA group rats experienced orthostatic hypotension. These results indicate that 6-OHDA induced motor dysfunction and blood pressure regulation disorder in rats, successfully establishing the model.

[0054] 2. TH, as the rate-limiting enzyme in dopamine synthesis, is recognized as a marker of dopaminergic neurons. Most TH-immunoreactive (TH-IR) neurons in the SN are dopaminergic neurons; therefore, observing TH-IR can indirectly detect dopaminergic neurons in the SN. Immunofluorescence staining results after injecting 6-OHDA into the SN for 6-8 weeks are as follows: Figure 1 As shown. By Figure 1 It can be seen that, compared with the Sham group, the number of TH-IR neurons in the 6-OHDA group rats was reduced, D2R was reduced, and D1R did not show significant changes.

[0055] The statistical results of the relative MFI and mRNA content of different groups of TH, D1R, and D2R in real-time PCR are as follows: Figure 2 As shown.

[0056] The statistical results of the relative expression levels of TH, D1R, and D2R in different groups in the Western blot experiment are as follows: Figure 3 As shown.

[0057] Real-time PCR and Western blot experiments showed that, compared with the Sham group, the TH content of the 6-OHDA group was significantly reduced (P<0.01), the expression of D2R was reduced (P<0.05), and the expression of D1R did not show significant changes.

[0058] 3. The sensitivity of the carotid body in Parkinson's disease rats was investigated by in vivo isolating and perfusing the carotid sinus region with KH solution. The results of the baroreflex function measurement in PD rats are as follows: Figure 4 As shown. Among them, Figure 4 A is a schematic diagram of an in vitro perfusion experiment of the carotid artery body; Figure 4 B represents the changes in the carotid artery baroreceptor reflex function curve in 6-OHDA rats and under the influence of 5mM DNP; Figure 4 C is the original recording of the blood pressure reflex response after intrasinus perfusion with KH solution and 5 mM DNP. Table 5 shows the results of the measurement of CB baroreflex function parameters in different groups of rats.

[0059] Table 5

[0060] Depend on Figure 4 It can be seen that, compared with the Sham group, the 6-OHDA group rats showed decreased sinus sensitivity, causing the functional curve to shift to the upper right, the slope of the curve to decrease significantly, and the magnitude of the reflexive decrease in blood pressure to decrease. As shown in Table 5, in the 6-OHDA group, the PS of Sham+DNP and 6-OHDA+DNP decreased from 0.31±0.02 in the control group to 0.25±0.04, 0.19±0.03, and 0.19±0.03, respectively. In the 6-OHDA group, the RD of Sham+DNP and 6-OHDA+DNP decreased from 38.19±1.85 mmHg in the control group to 33.1±1.79, 23.6±3.19, and 24.19±2.22 mmHg, respectively. In the 6-OHDA group, the threshold pressure (TP) of Sham+DNP and 6-OHDA+DNP increased from 77.08±1.1 mmHg to 89.2±1.13, 96.7±1.55, and 98.36±0.94 mmHg, respectively; the EP of Sham+DNP and 6-OHDA+DNP increased from 100.79±3 mmHg to 110.03±4.6, 102.47±6.44, and 112.15±6.35 mmHg, respectively; and the SP of Sham+DNP and 6-OHDA+DNP decreased from 189.59±1.64 mmHg to 199.59±1.36, 201.89±1.85, and 200.21±1.38 mmHg, respectively.

[0061] Since carotid sinus baroreceptors may also affect blood pressure through baroreflexes, the above procedure was repeated after adding DNP to the perfusion fluid. Changes in the sinus baroreflex under hypoxic conditions were observed to determine the role of CB in the baroreflex process. The results showed a significant decrease in the sinus baroreflex, with differences in response to hypoxia between the normal group and Parkinson's disease rats. This indicates that CB plays a role in the isolated perfusion system, and that CB sensitivity is reduced in PD rats, resulting in weakened blood pressure regulation.

[0062] 4. To investigate whether fibrosis occurred in the carotid body (CB) in some way, we performed HE and Masson staining on the CBs of both groups of rats. The results of HE and Masson staining of the carotid body in different groups of rats are shown below. Figure 5 As shown. By Figure 5 It was found that, compared with the Sham group, the CB tissue structure of rats in the 6-OHDA group was disordered, and significant fibrosis was also observed. Western blot results also showed that the expression of the fibrosis marker Vimentin was significantly increased in the 6-OHDA group.

[0063] Western blot analysis was used to determine the expression levels of HIF-1α, SGK-1, TGF-β1, and Vimentin in rat CB tissues from different groups. The statistical results of the relative expression levels of HIF-1α, SGK-1, TGF-β1, and Vimentin in rat CB tissues from different groups are shown below. Figure 6 As shown. Further investigation into the fibrosis mechanism of CB revealed that increased ALD can promote organ fibrosis in multiple ways. Since CB does not secrete ALD, this invention hypothesizes that ALD may act on the mineralocorticoid receptor (MR) and downstream effector mediator SGK-1 to induce inflammatory cell infiltration and extracellular matrix deposition. Figure 6 It can be seen that, compared with the Sham group, the 6-OHDA group showed a significant increase in the levels of SGK-1, TGF-β1, Vimentin, and HIF-1α proteins in the CB tissue.

[0064] 5. Fluorescence patterns of rats in different groups 7 days after PVN injection of fluorescent gold are shown below. Figure 7 As shown. By Figure 7 It can be seen that 7 days after PVN injection of fluorescent gold, fluorescent gold-labeled neurons were found in the SN brain region, indicating that SN neurons can send fiber projections to PVN.

[0065] 6. To evaluate the impact of dopaminergic neuronal degeneration in the SN on dopamine receptors in the PVN and its relationship with CB fibrosis, this invention used immunofluorescence, real-time PCR, and Western blot to detect the expression of D1R, D2R, and CRH in the PVN. Representative fluorescence images of CRH-positive cells, c-Fos-positive cells, and CRH / c-Fos double-labeled cells in the PVN of different rat groups are shown below. Figure 8 As shown, Figure 8 Green represents CRH-positive cells, and red represents c-Fos-positive cells. The statistical results of Th, D1R, and D2R expression levels in PVNs of different rat groups determined by real-time PCR are shown below. Figure 9 As shown in the figure. The statistical results of the relative expression levels of CRH, D1R, and D2R proteins in the PVN of different groups of rats in the Western blot experiment are as follows: Figure 10 As shown.

[0066] Depend on Figure 8-10 The results showed that after dopaminergic neurons in the SN were destroyed, the expression of inhibitory receptor D2R in the PVN was significantly reduced (P<0.001), CRH was significantly increased (P<0.01), and the expression of D1R remained almost unchanged.

[0067] 7. The HPA axis includes CRH released from the PVN, ACTH released from the pituitary gland, and Cort released from the adrenal glands. These three hormones are released into the bloodstream and exert their effects throughout the body. ACTH and angiotensin promote the formation of ALD, and ALD and the mineralocorticoid receptor (MR) play important roles in the fibrosis process. To investigate the role of the HPA axis in CB fibrosis in 6-OHDA group rats, this invention collected blood samples and performed ELISA detection. The statistical results of the relative expression levels of CRH, ACTH, Cort, and ALD in serum of different groups are as follows: Figure 11 As shown.

[0068] Depend on Figure 11 It was found that, compared with the Sham group, the relative expression levels of CRH, ACTH, Cort and ALD in the blood of rats in the 6-OHDA group were significantly increased (P<0.001).

[0069] In summary, this invention creatively discovers that increased aldosterone in PD rats leads to activation of the TGF-β1 / SGK-1 signaling pathway, which in turn induces carotid body fibrosis. Carotid body fibrosis further reduces the sensitivity of chemoreceptors, inducing blood pressure regulation disorders.

[0070] Example 2 Based on the results of Example 1, this invention investigates the effects of cortisol inhibitors and aldosterone inhibitors on blood pressure regulation disorders in PD rats. The specific details are as follows: Forty SD rats were randomly divided into four groups according to the method described in Example 1. One group served as the control group (Sham group), and the other three groups were used to establish a rat model of PD accompanied by blood pressure regulation disorder and motor dysfunction (denoted as PD rats). The cortisol inhibitor Mifepristone and the aldosterone inhibitor Eplerenone were dissolved in corn oil, respectively. The PD + Mifepristone group (denoted as PD+Mif group) was fed with 50 mg / kg BD Mifepristone daily; the PD + Eplerenone group (denoted as PD+Epl group) was fed with 50 mg / kg BD Eplerenone daily. The Sham group and PD group had normal diet and water. The different groups were fed for 30 days, and the carotid body fibrosis index, baroreceptor reflex sensitivity, and orthostatic hypotension were measured in different groups.

[0071] 1. After 30 days of feeding, the H&E staining and Masson staining results of the carotid body of rats in different groups are as follows: Figure 15 As shown.

[0072] Depend on Figure 12It can be seen that, compared with the PD group, the cell structure arrangement of the carotid body of rats in the PD+Mif group and the PD+Epl group was restored, the cell volume was reduced, and the number of cells increased in H&E staining; at the same time, Masson staining results showed that the fibrosis of the carotid body in these two groups was improved.

[0073] 2. The relative expression levels of Vimentin, HIF-1α, SGK-1, and TGF-β1 in rat CBs from different groups were detected by Western blot. The statistical results are shown below. Figure 13 As shown. By Figure 16 It was found that, compared with the PD group, the relative expression levels of HIF-1α, Vimentin, SGK-1 and TGF-β1 in the carotid body of rats in the PD+Mif group and PD+Epl group were significantly reduced.

[0074] 3. The carotid sinus region of rats in different groups was isolated and perfused, and blood pressure was measured using wireless telemetry. The statistical results of blood pressure changes in different groups of rats with increasing perfusion pressure are shown below. Figure 14 As shown in the figure, the statistical results of the lowest blood pressure in different groups of rats at the maximum perfusion pressure are as follows: Figure 15 As shown in the figure. Statistical results of various functions of the baroreceptor reflex in rats from different groups are as follows. Figure 16 As shown in the figure. The original curves of blood pressure changes due to positional changes in rats from different groups are shown in the figure. Figure 17 As shown in the figure, the statistical results of blood pressure recovery time after body position changes in rats of different groups are as follows: Figure 18 As shown in the figure, the statistical results of mean arterial pressure at the lowest body position in different groups of rats are as follows: Figure 19 As shown.

[0075] Depend on Figure 14-19 The results showed that the stress-induced reflex sensitivity was restored in both the PD+Mif and PD+Epl groups, and the blood pressure response after postural changes also returned to normal. After 30 days of treatment with Mif and Epl blockers in Parkinson's rats, the baroreflex curves of the PD+Mif and PD+Epl groups showed a downward shift compared to the PD group. At maximum perfusion, there was no statistically significant difference in blood pressure between the PD+Mif and PD+Epl groups and the Sham group. Furthermore, this invention also found that TP, EP, SP, OR, PS, and RD levels could all be restored to the levels of the Sham group. Administration of Mif and Epl to PD rats restored blood pressure regulation during postural changes to the levels of the sham-operated group.

[0076] In summary, treatment with the cortisol inhibitor mifepristone (Mif) and the aldosterone inhibitor eplerenone (Epl) for 30 days significantly improved blood pressure regulation in PD rats. At the molecular level, the abnormally high expression of HIF-1α, Vimentin, SGK-1, and TGF-β1 in the rat carotid body (CB) was significantly inhibited; morphologically, the cellular structure of the CB was restored, and the degree of fibrosis was improved. Regarding blood pressure regulation, baroreceptor reflex-related indicators (TP, EP, SP, etc.) in both treatment groups returned to the levels of the sham-operated group (Sham), and the blood pressure response after postural changes and stress-induced reflex sensitivity returned to normal. Blood pressure at maximum perfusion pressure was not statistically different from that in the Sham group. In conclusion, Mif and Epl can effectively reverse blood pressure regulation abnormalities in PD rats by regulating the expression of related molecules and improving the pathological state of CB tissue.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a steroid hormone inhibitor in the manufacture of a medicament for treating a Parkinsonian dysregulation of blood pressure.

2. Use of a steroid hormone inhibitor according to claim 1 for the manufacture of a medicament for the treatment of a dysregulation of blood pressure in Parkinson's disease. The steroid hormone inhibitor includes an inhibitor of abnormal activation of the HPA axis.

3. Use of a steroid hormone inhibitor according to claim 2 for the manufacture of a medicament for the treatment of a dysregulation of blood pressure in Parkinson's disease. The steroid hormone inhibitor includes a steroid receptor antagonist.

4. Use of a steroid hormone inhibitor according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of a dysregulation of blood pressure in Parkinson's disease, characterized in that, The steroid hormone inhibitor includes at least one of a cortisol inhibitor or an aldosterone inhibitor.

5. Use of a steroid hormone inhibitor according to claim 1 for the manufacture of a medicament for the treatment of a dysregulation of blood pressure in Parkinson's disease. The Parkinsonian dysregulation of blood pressure is associated with carotid body fibrosis.

6. Use of a steroid hormone inhibitor according to claim 4 for the manufacture of a medicament for the treatment of a dysregulation of blood pressure in Parkinson's disease. The Parkinsonian dysregulation of blood pressure includes at least one of orthostatic hypotension, supine hypertension, or postprandial hypotension.

7. Use of a steroid hormone inhibitor according to claim 6 for the manufacture of a medicament for the treatment of a dysregulation of blood pressure in Parkinson's disease. The Parkinsonian dysregulation of blood pressure is orthostatic hypotension.

8. A medicament for treating a Parkinson's blood pressure regulation disorder, characterized by, The steroid hormone inhibitor includes an inhibitor of abnormal activation of the HPA axis.

9. The medicament for treating a Parkinson's blood pressure regulation disorder according to claim 8, wherein The steroid hormone inhibitor includes at least one of a cortisol inhibitor or an aldosterone inhibitor.

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