Application of irisin in preparation of medicine for treating blood pressure circadian rhythm disorder

Abstract

The invention discloses an application of irisin in preparation of a medicine for treating blood pressure circadian rhythm disorder, and relates to the technical field of biological medicine.A blood pressure circadian rhythm disorder model rat is constructed, and the blood pressure circadian rhythm normal and blood pressure circadian rhythm disorder rat plasma irisin levels are analyzed and compared, so that the blood pressure circadian rhythm disorder is determined. The blood pressure circadian rhythm disorder rat plasma irisin content is reduced, and circadian rhythm disappears; meanwhile, by constructing an FNDC5 knockout model mouse and a skeletal muscle specific FNDC5 knockout model mouse, blood pressure circadian rhythm disorder of the two mice is found, and skeletal muscle specific overexpression of FNDC5 on the FNDC5 knockout mice can restore normal circadian rhythm of blood pressure, so that the FNDC5 / irisin can be used as a potential medicine for treating the blood pressure circadian rhythm disorder and has a broad application prospect. A new thought is provided for the research of the drug action target of the blood pressure circadian rhythm disorder in the future and the clinical treatment of related diseases of the blood pressure circadian rhythm disorder, so that the pharmaceutical composition has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and more specifically, it relates to the application of irisin in the preparation of drugs for treating circadian rhythm disorders of blood pressure. Background Art

[0002] Irisin is a glycosylated protein mainly secreted by skeletal muscle. It is derived from the precursor protein: FNDC5, which is formed after cleavage by proteolytic enzymes and released into the blood. It is expressed in tissues and organs such as skeletal muscle, liver, and fat. Studies have shown that the secretion of human irisin also has an obvious circadian rhythm, with the lowest level around 6:00 in the morning and reaching the peak around 9:00 in the evening.

[0003] Blood pressure is regulated by both nerves and body fluids to maintain the normal circadian rhythm of the body's blood pressure, which shows an increase in the morning, usually reaching the peak in the afternoon, and a decrease during night rest. This rhythmic change in human blood pressure is of great significance for adapting to the body's activities and protecting important organs such as the heart, liver, and kidneys. The decrease in blood pressure at night is part of the normal circadian rhythm of blood pressure (dipper type). The absence of a decrease in blood pressure at night is more common in hypertensive patients and is associated with more severe end-organ damage and cardiovascular events, including heart failure, chronic kidney disease, etc. On the other hand, hypertensive patients taking antihypertensive drugs before going to bed to lower blood pressure at night can significantly reduce the occurrence of major cardiovascular events. Currently, the regulatory mechanism of blood pressure circadian rhythm is not clear, and there is a lack of effective drugs to restore blood pressure circadian rhythm. Therefore, studying the pathogenesis of circadian rhythm disorders of blood pressure and finding new and effective treatment targets and means for circadian rhythm disorders of blood pressure are urgent problems to be solved at present. In view of this, the inventor of the present invention proposes the application of irisin in the preparation of drugs for treating circadian rhythm disorders of blood pressure to improve the degree of circadian rhythm disorders of blood pressure in patients. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of irisin in the preparation of drugs for treating circadian rhythm disorders of blood pressure.

[0005] The above technical purpose of the present invention is achieved through the following technical solutions: the application of irisin in the preparation of drugs for treating circadian rhythm disorders of blood pressure.

[0006] The present invention is further provided that: the drug contains irisin and pharmaceutically acceptable excipients.

[0007] The present invention is further provided that: the dosage form of the drug includes tablets, liquid preparations or granules.

[0008] On the other hand, the present invention also provides: a biomarker for detecting and / or diagnosing circadian rhythm disorders of blood pressure, and the biomarker is irisin.

[0009] In summary, the present invention has the following beneficial effects:

[0010] (1) By constructing a rat model with disrupted circadian blood pressure rhythm, it was detected that the plasma irisin concentration in the rats decreased, demonstrating that irisin can be used as a potential biomarker for diagnosing disrupted circadian blood pressure rhythm;

[0011] (2) By intervening with the injection of FNDC5 overexpressing virus into skeletal muscle, the degree of disrupted circadian blood pressure rhythm can be effectively improved, demonstrating that irisin can be used as a potential drug for treating disrupted circadian blood pressure rhythm and has important significance in the prevention and treatment of disrupted circadian blood pressure rhythm;

[0012] (3) The present invention also provides the application of irisin in drugs for treating disrupted circadian blood pressure rhythm. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the evaluation of the model establishment of rats with disrupted circadian blood pressure rhythm and the effects on blood pressure and circulating irisin (wherein, A. Original 24-hour recording data of body temperature and activity of normal rats and rats with disrupted circadian blood pressure rhythm, 1 rat in each group; B. Plasma melatonin levels in rats of each group at six time points of ZT0, 4, 8, 12, 16, and 20h; C. Plasma cortisol levels in rats of each group at six time points of ZT0, 4, 8, 12, 16, and 20h; D. Changes in systolic blood pressure monitored by remote sensing in rats of each group for 48h; E. Changes in heart rate monitored by remote sensing in rats of each group for 48h; F-G. Circadian rhythm amplitude (E) and cycle length (F) of systolic blood pressure, *P<0.05 vs. LD, n = 5; H-I. Circadian rhythm amplitude (E) and cycle length (F) of heart rate, *P<0.05 vs. LD, n = 5);

[0014] Figure 2Effect of circadian rhythm disorder of rat blood pressure on circulating irisin and neutralizing FNDC5 on blood pressure circadian rhythm (wherein, A. Changes in plasma irisin levels in rats of each group at different time points in the awake state, n = 5; B. Schematic diagram of intraperitoneal injection of anti-FNDC5 monoclonal antibody or IgG; C. Changes in systolic blood pressure and amplitude of systolic blood pressure circadian rhythm in rats of each group monitored by remote sensing for 48 h, black arrow represents intraperitoneal injection of anti-FNDC5 monoclonal antibody or IgG, *P<0.05 vs. LD+IgG, n = 5; D. Changes in diastolic blood pressure and amplitude of diastolic blood pressure circadian rhythm in rats of each group monitored by remote sensing for 48 h, black arrow represents intraperitoneal injection of anti-FNDC5 monoclonal antibody or IgG, *P<0.05 vs. LD+IgG, n = 5; E. Changes in heart rate and amplitude of heart rate circadian rhythm in rats of each group monitored by remote sensing for 48 h, black arrow represents intraperitoneal injection of anti-FNDC5 monoclonal antibody or IgG, *P<0.05 vs. LD+IgG, n = 5);

[0015] Figure 3 Changes in blood pressure circadian rhythm of FNDC5 knockout mice (wherein, A. Changes in systolic blood pressure and amplitude of systolic blood pressure circadian rhythm in mice of each group monitored by remote sensing for 48 h, *P<0.05 vs. WT, n = 5; B. Changes in diastolic blood pressure and amplitude of diastolic blood pressure circadian rhythm in mice of each group monitored by remote sensing for 48 h, *P<0.05 vs. WT; C. Changes in heart rate and amplitude of heart rate circadian rhythm in mice of each group monitored by remote sensing for 48 h, *P<0.05 vs. WT);

[0016] Figure 4 Changes in FNDC5 expression and irisin content in plasma, skeletal muscle, liver and adipose tissue of rats with normal blood pressure circadian rhythm (wherein, A. FNDC5 expression levels at different time points in gastrocnemius muscle, liver and inguinal adipose tissue of rats with normal blood pressure circadian rhythm, *P<0.05 vs. ZT4h, n = 5; B. Irisin content at different time points in plasma, gastrocnemius muscle and liver tissue of rats with normal blood pressure circadian rhythm, *P<0.05 vs. ZT4h, n = 5);

[0017] Figure 5 Changes in blood pressure circadian rhythm of skeletal muscle-specific FNDC5 knockout mice (wherein, A. Breeding schematic diagram of skeletal muscle-specific FNDC5 knockout mice; B. Changes in systolic blood pressure and amplitude of systolic blood pressure circadian rhythm in mice of each group monitored by remote sensing for 48 h, *P<0.05 vs. WT, n = 5; C. Changes in diastolic blood pressure and amplitude of diastolic blood pressure circadian rhythm in mice of each group monitored by remote sensing for 48 h, *P<0.05 vs. WT; D. Changes in heart rate and amplitude of heart rate circadian rhythm in mice of each group monitored by remote sensing for 48 h, *P<0.05 vs. WT);

[0018] Figure 6 Verification of the transfection efficiency of adeno-associated virus transfected with FNDC5 overexpressed in skeletal muscle and the effect of skeletal muscle-specific overexpression of FNDC5 on the circadian rhythm of blood pressure in FNDC5 knockout mice (wherein, A. Schematic diagram of transfection of AAV-GFP or AAV-FNDC5 in the gastrocnemius muscle of mice; B. Immunofluorescence detection of virus transfection in frozen sections 4 weeks after transfection of 1011 vg / ml AAV-FNDC5 in the gastrocnemius muscle of mice; C. PCR detection of the expression level of FNDC5 after transfection of AAV-FNDC5 in the gastrocnemius muscle of mice, *P<0.05 vs. AAV-GFP, n = 5; D. WB detection of the expression level of FNDC5 after transfection of AAV-FNDC5 in the gastrocnemius muscle of mice, *P<0.05 vs. AAV-GFP, n = 5; E. Changes in systolic blood pressure and the amplitude of circadian rhythm of systolic blood pressure monitored by telemetry for 24 hours in each group of mice, *P<0.05 vs. AAV-GFP, n = 5; F. Changes in diastolic blood pressure and the amplitude of circadian rhythm of diastolic blood pressure monitored by telemetry for 24 hours in each group of mice, *P<0.05 vs. AAV-GFP, n = 5; G. Changes in heart rate and the amplitude of circadian rhythm of heart rate monitored by telemetry for 24 hours in each group of mice, *P<0.05 vs. AAV-GFP, n = 5). Detailed implementation manners

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to the conditions described in "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al. (Science Press, 2002) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0020] Experimental animals

[0021] The Sprague-Dawley (SD) rats used in this study were male, weighing between 180-200 g, and the C57BL / 6J mice were male, weighing between 20-25 g. They were provided by the Animal Experiment Center of the Naval Medical Center of China and purchased from Shanghai Bikai Biotechnology Co., Ltd. The experimental animals were kept in an environment with a relative humidity of 50%-60%, a temperature of 26±2°C, and good ventilation. The control group was raised under a 12h:12h light-dark cycle, and the model group was raised according to the set light conditions, with unrestricted access to water and food. Fndc5 Knock-out (KO) mice were constructed by Jiangsu Jicui Yakang Biotechnology Co., Ltd., and the breeding conditions were the same as those of the rats. The experimental animal design and operations were carried out in accordance with the relevant regulations of the Ethics Committee of the Naval Medical University and the Animal Experiment Center. Experimental materials

[0022]

[0023]

[0024] Experimental instruments

[0025]

[0026]

[0027]

[0028] Example 1: Analyze the circadian rhythms of blood pressure and circulating irisin in rats with normal blood pressure circadian rhythm and rats with disrupted blood pressure circadian rhythm

[0029] Preparation of an animal model with disrupted blood pressure circadian rhythm

[0030] In this study, an animal model with disrupted blood pressure circadian rhythm was prepared by changing the light cycle (abnormal Bmal1 circadian transcriptional mechanism), mainly to test the regulatory and improvement effects of exogenous supplementation of irisin and increasing endogenous irisin through exercise training on disrupted blood pressure circadian rhythm. The method of creating the model by changing the light cycle is as follows: An animal model with disrupted blood pressure circadian rhythm was prepared by changing the light cycle (abnormal Bmal1 circadian transcriptional mechanism) as follows: Normal light group (LD group): SD rats were placed in a 12h light / 12h dark environment (Light / Dark = 12h:12h), the illumination intensity of the fluorescent lamp was 180 Lux, the light was turned on at 8:00 am and turned off at 8:00 pm, and sufficient drinking water and feed were provided. Circadian rhythm disruption group (CD group): The rats were placed in the light environment shown below. After the animals adapted to the environment, the light was turned on 6 hours earlier every 2 days and the time was adjusted. Compared with the control group, only the light rhythm was changed, and other conditions remained the same. The rats in each group were continuously intervened for 32 days (for the specific protocol, refer to J Biol Rhythms. 2012;27(1):59-69).

[0031] Except for the animals that need to detect the expression of clock genes in the RVLM at six time points of ZT 0, 4, 8, 12, 16, and 20 h, the cardiovascular function of the remaining animals was monitored under anesthesia, the last blood collection was completed, and subsequent overdose anesthesia for euthanasia was also completed.

[0032] We monitored the blood pressure of rats in the awake state by wireless telemetry technology and found that the decrease in blood pressure during the day in the disordered rats was reduced. To clarify the effect of the decreased activity of the disordered rats on the circulating irisin level, we simultaneously detected the plasma irisin levels of the two groups of rats at six time points within 24 hours. The experimental results are as Figure 1 shown. It was found that the content of irisin in normal rats was the highest at ZT4 and the lowest at ZT16. Therefore, in subsequent experiments, the differences in irisin or FNDC5 at these two time points were compared. The circulating irisin level in the disordered rats decreased during the day, and the circadian rhythm was abnormal. It was proved that the abnormal circadian rhythms of blood pressure and circulating irisin in rats were successfully induced by changing the light rhythm.

[0033] Among them, the specific experimental protocol for wireless remote sensing monitoring is as follows:

[0034] After the rats were anesthetized by inhalation (2% isoflurane), they were fixed in the supine position. The skin of the left groin was prepared, the skin was disinfected, a hole towel was laid, the skin was incised along the midline of the groin, the fascia and muscles were bluntly dissected, the femoral artery was exposed, about 3 - 5 cm of the femoral artery was dissected free, the distal end was ligated with a silk thread, the proximal end was clamped with an arterial clip, a small incision was made with a micro-scissors, the connecting tube of the implant (Emka easyTEL implant, France) was inserted into the femoral artery, and after ligation, it was checked for no bleeding. The electrocardiogram electrodes were fixed subcutaneously at the apex of the heart and under the skin of the right upper limb, the implant was fixed under the skin of the back, the incision was sutured, the skin of the incision was disinfected with alcohol, and penicillin was intramuscularly injected continuously for 3 days to prevent infection. The gas anesthesia was stopped and the animals were awake. One week before the end of the blood pressure circadian rhythm disorder modeling, the blood pressure implant was implanted, and on the last day, the EMKA Receiver telemetry system was used to continuously monitor the blood pressure for 48 h, and the systolic blood pressure (SBP) and heart rate data were collected. After the mice were anesthetized by inhalation, they were fixed in the supine position, the left common carotid artery was exposed and dissected free, and the implant (DSI implant) was inserted into the carotid artery. After 7 days of recovery, the SBP and heart rate data for 48 h were collected.

[0035] Among them, the specific experimental protocol for ELISA is as follows:

[0036] The processing methods for plasma, skeletal muscle, adipose, and liver tissue samples are as follows: For plasma, blood was collected from rats via the tail vein and from mice via orbital blood collection at specific time points. 1% heparin was added for anticoagulation, and the mixture was incubated for 20 min and then centrifuged (2000 - 3000 revolutions per minute) to collect the supernatant. Since the levels of irisin in the circulation of rats with normal circadian rhythms of blood pressure are at the highest and lowest levels of the day at ZT4h and ZT16h, respectively, for skeletal muscle, adipose, and liver tissue sampling, it was performed at ZT4h and ZT16h. After the animals were euthanized, the gastrocnemius muscle, liver, and inguinal adipose tissue were quickly removed. For skeletal muscle and liver tissue, RIPA lysis buffer was added for homogenization, while for adipose tissue, a total protein extraction kit for animal adipose tissue and cells (purchased from English Biotechnology Co., Ltd., AT - 022) was used to extract the protein supernatant. ELISA kits for NE (purchased from Shanghai Xitang Biotechnology Co., Ltd., F16311 for rats and F11235 for mice) and irisin (purchased from R&D Systems, DY9420 - 05) were used to detect the levels of NE and irisin in the samples. The irisin ELISA kit was verified using plasma from Fndc5 knockout mice, and the specific steps were carried out according to the instructions.

[0037] Among them, the specific experimental protocol for Western Blot is as follows:

[0038] After the total protein of skeletal muscle, adipose, and liver tissue samples was extracted, the protein concentration was detected by the BCA method. After denaturing the samples, a conventional Western Blot experiment (Am J Physiol Regul Integr Comp Physiol. 2022, 323(6):R861 - R74) was performed to detect the protein expression levels of FNDC5 in skeletal muscle, adipose, and liver tissues.

[0039] Example 2: Neutralizing FNDC5 leads to abnormal circadian rhythm of blood pressure in normal mice

[0040] To clarify the effect of irisin on the circadian rhythm of blood pressure, in this study, FNDC5 monoclonal antibody was intraperitoneally injected into normal mice to neutralize FNDC5, and the experimental results are as Figure 2 shown. It was found that the daily fluctuation amplitudes of systolic blood pressure, diastolic blood pressure, and heart rate decreased, and the 24 - hour average heart rate increased. These results indicate that neutralizing FNDC5 leads to abnormal circadian rhythm of blood pressure in normal mice.

[0041] Among them, the specific experimental protocol for neutralizing FNDC5 is as follows:

[0042] Rats with normal circadian blood pressure rhythm were intraperitoneally injected with FNDC5 monoclonal antibody or control IgG (5 μg) at ZT 0h (the dosage was referred to Nat Med. 2019; 25(1): 165-175).

[0043] Example 3: Abnormal circadian blood pressure rhythm in FNDC5 knockout mice

[0044] To verify the effects of endogenous irisin deficiency on the body, we used FNDC5 knockout mice and observed their 24-hour blood pressure. The experimental results are as Figure 3 shown. It was found that the amplitude of systolic blood pressure circadian fluctuation decreased, and the 24-hour average heart rate increased. The above results suggest that irisin plays an important role in the regulation of circadian blood pressure rhythm.

[0045] Example 4: Circadian rhythm changes of FNDC5 in the skeletal muscle of rats with normal circadian blood pressure rhythm

[0046] Studies have shown that circulating irisin mainly comes from muscle, liver and adipose tissue. To clarify which tissue the circadian rhythmically fluctuating irisin in the blood is mainly related to, we detected the expression changes of FNDC5 in three tissues of rats and found that only the skeletal muscle tissue showed circadian rhythm changes. We further detected the changes in the content of irisin in the plasma, liver and skeletal muscle tissues of two groups of rats. The experimental results are as Figure 4 shown. It was found that the content of irisin in the plasma and skeletal muscle tissues showed circadian rhythm changes. These results suggest that the circadian rhythm fluctuations in the level of circulating irisin mainly originate from skeletal muscle.

[0047] Example 5: Abnormal circadian blood pressure rhythm in skeletal muscle-specific FNDC5 knockout mice

[0048] To further verify, this study bred skeletal muscle-specific FNDC5 knockout mice. The experimental results are as Figure 5 shown. It was observed that the waveforms of systolic and diastolic blood pressure changed within 24 hours, and the daily fluctuation amplitude decreased significantly. The heart rate increased, and the circadian rhythm change of the heart rate decreased significantly. These results are the same as those of FNDC5 knockout mice, indicating that the lack of irisin derived from skeletal muscle will lead to abnormal circadian blood pressure rhythm.

[0049] Example 6: Restoration of circadian blood pressure rhythm by skeletal muscle-specific overexpression of FNDC5 in FNDC5 knockout mice

[0050] After that, to clarify the effects of skeletal muscle-specific overexpression of FNDC5 on knockout mice, we first verified the transfection efficiency of the overexpression virus. First, the FNDC5 overexpression virus was injected into the skeletal muscle of wild-type mice. Four weeks after injection, the samples were taken. Through frozen section immunofluorescence, it was found that the virus was successfully transfected and successfully verified at the gene and molecular levels. The experimental results are as Figure 6As shown, remote blood pressure monitoring found that the circadian difference in systolic blood pressure in FNDC5 knockout mice was restored, and the 24-hour average heart rate decreased significantly, suggesting that irisin derived from skeletal muscle plays an important role in the regulation of blood pressure circadian rhythm.

[0051] In summary, by constructing a rat model with disrupted blood pressure circadian rhythm and analyzing and comparing the plasma irisin levels in rats with normal and disrupted blood pressure circadian rhythm, it was found that the plasma irisin content in rats with disrupted blood pressure circadian rhythm decreased and the circadian rhythm disappeared. At the same time, by constructing FNDC5 knockout model mice and skeletal muscle-specific FNDC5 knockout model mice, it was found that the blood pressure circadian rhythm was disrupted in both types of mice, while skeletal muscle-specific overexpression of FNDC5 in FNDC5 knockout mice could restore the normal circadian rhythm of blood pressure, proving that FNDC5 / irisin can be used as a potential drug for treating disrupted blood pressure circadian rhythm, providing new ideas for future research on drug action targets for disrupted blood pressure circadian rhythm and the clinical treatment of related diseases, and thus having important clinical application value.

[0052] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. 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 perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of irisin in the preparation of a drug for treating circadian rhythm disorder of blood pressure.

2. The application according to claim 1, characterized in that: The drug contains irisin and a pharmaceutically acceptable excipient.

3. The application according to claim 1, characterized in that: The dosage form of the drug includes tablets.

4. A biomarker for detecting and / or diagnosing circadian blood pressure rhythm disorders, characterized in that: The biomarker is irisin.

Images