New application of Rab5a in preparation of medicine for protecting blood vessels

Abstract

The invention provides a novel application of Rab5a in preparation of a medicine for protecting blood vessels, and belongs to the field of scientific medicine. According to the invention, through knockout and overexpression of Rab5a transgenic mice, it is proved that Rab5a plays an important role in occurrence and development of hypertension. Rab5a knockout can aggravate vascular injury of Ang II induced hypertension mice, Rab5a knockout has a remarkable promotion effect on hypertension induced vascular remodeling, and through examination on vascular pathology, Rab5a overexpression inhibits occurrence of hypertension vascular remodeling.

Description

Technical Field

[0001] The invention belongs to the field of medical science, and specifically relates to a new use of Rab5a in the preparation of a vascular protection drug. Background Art

[0002] Hypertension is a disease with high morbidity and mortality among cardiovascular diseases worldwide, which seriously threatens human health and imposes a heavy burden on individuals and society. Accompanying diseases of hypertension include cerebral hemorrhage, chronic renal failure, left ventricular hypertrophy, etc., and vascular damage is the driving link and important basis for hypertension-induced diseases and target organ damage. Vascular wall thickening is one of the main pathological changes of hypertension, which is manifested by the proliferation and enlargement of vascular smooth muscle cells (VSMCs) in blood vessels to resist the increase in vascular wall pressure. However, long-term vascular wall thickening leads to lumen stenosis, further increases blood pressure, and eventually forms refractory hypertension. Therefore, it is of great clinical significance to find new targets that can effectively lower blood pressure and inhibit vascular wall thickening.

[0003] Rab5a is a member of the Rab family of small G proteins and plays an important role in regulating intracellular material transport. Rab5a protein (Uniprot number: P20339), more than 60 mammalian Rab proteins have been identified, and each Rab protein appears to be associated with a specific subcellular compartment. Rab5a is located on the membrane side of coated vesicles and early endosome membranes, regulating the internalization and sorting of GPCRs, including β2-adrenergic receptors and endothelin A and B receptors.

[0004] There are no related research reports on the relationship between Rab5 and hypertension. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of Rab5a in the preparation of a vascular protection drug.

[0006] The present invention provides use of Rab5a or a Rab5a gene expression promoter in preparing a medicine for treating hypertension.

[0007] Furthermore, the hypertension described is hypertension caused by Ang II.

[0008] Furthermore, the Rab5a gene expression promoter includes but is not limited to a Rab5a gene overexpression plasmid or a Rab5a gene overexpression adeno-associated virus; the Rab5a gene overexpression nucleotide sequence is shown in SEQ ID NO.1.

[0009] Furthermore, the drug is a drug for repairing blood vessel wall damage caused by hypertension.

[0010] Furthermore, the drug is a drug that inhibits vascular remodeling caused by hypertension.

[0011] Furthermore, the drug is a drug for reducing high blood pressure.

[0012] Furthermore, the drug is a drug that reduces the thickening of blood vessel walls in hypertensive mice.

[0013] Furthermore, the drug is a drug that reduces the pulse wave velocity of hypertension.

[0014] The present invention also provides a composition for treating hypertension, which is a preparation prepared by taking the Rab5a gene expression factor promoter as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

[0015] Furthermore, the Rab5a gene expression promoter includes but is not limited to a Rab5a gene overexpression plasmid or a Rab5a gene overexpression adeno-associated virus.

[0016] The present invention constructs knockout and overexpression mice of Rab5a by gene induction, and uses AngII sustained-release pump to construct a mouse hypertension model, observes the effect of Rab5a deficiency or increase on blood pressure and vascular function, and proves that Rab5a is a potential target for clinical treatment of hypertension.

[0017] The present invention constructs Rab5a gene knockout mice and combines them with AngⅡ-induced hypertension model. The study finds that after AngⅡ modeling intervention in Rab5a gene knockout mice, their systolic blood pressure, diastolic blood pressure and mean arterial pressure are significantly higher than those of wild-type mice. Rab5a knockout can cause high blood pressure and increase the damage of vascular function. Through vascular ultrasound and vascular pathological detection, Rab5a knockout has a significant promoting effect on vascular remodeling induced by hypertension.

[0018] At the same time, the present invention constructs Rab5a overexpression mice by injecting adeno-associated virus into the mouse tail vein to verify the effect of Rab5a overexpression on AngⅡ-induced blood pressure increase and vascular pathological state. The results show that Rab5a overexpression can inhibit the increase in blood pressure caused by AngⅡ, and also inhibit the thickening of the vascular wall of AngⅡ-induced hypertensive mice, so that the damage to the vascular wall can be repaired. Rab5a overexpression inhibits the occurrence of hypertensive vascular remodeling. In summary, the present invention proves the important role of Rab5a in the occurrence and development of hypertension by knocking out and overexpressing Rab5a transgenic mice.

[0019] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0020] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 DNA and protein identification for transgenic mice: A.Rab5a + / + type mice (WT) and Rab5a - / - ; Schematic diagram of DNA identification of WT and KO mice; B. DNA identification of blood vessels and tissues of WT and KO mice; C. Protein identification of tissues of WT and KO mice.

[0022] Figure 2 Effect of Rab5a knockout on systolic blood pressure in AngⅡ-induced hypertension model mice, Note: *P<0.05VS WT; #P<0.05VS WT+AngⅡ.

[0023] Figure 3 Effect of Rab5a knockout on diastolic blood pressure in AngⅡ-induced hypertension model mice, Note: *P<0.05VS WT; #P<0.05VS WT+AngⅡ.

[0024] Figure 4 Effect of Rab5a knockout on mean arterial pressure in AngⅡ-induced hypertension model mice, Note: *P<0.05VSWT; #P<0.05VS WT+AngⅡ.

[0025] Figure 5 Effect of Rab5a knockout on body weight in mice.

[0026] Figure 6 Effect of Rab5a knockout on the pulse wave velocity of the abdominal aorta in AngⅡ-induced hypertensive mice. A. Schematic diagram of the pulse wave in the abdominal aorta; B. The pulse wave velocity of the abdominal aorta in each group of mice (Note: *P<0.05, WT+AngⅡvsWT, KO; #P<0.05, KO+AngⅡvs WT+AngⅡ).

[0027] Figure 7 Effect of Rab5a knockout on abdominal aorta wall thickness in AngⅡ-induced hypertensive mice.

[0028] A. Representative images of HE staining of abdominal aorta in each group (40×); B. Thickness of the media of abdominal aorta in each group;

[0029] Note: *P<0.05, WT+AngⅡvs WT,KO; #P<0.05, KO+AngⅡvs WT+AngⅡ.

[0030] Figure 8 QPCR detection of Rab5a mRNA expression in various tissues of C57BL / 6 mice infected with Rab5a-AAV9 (Note: * P<0.05VS NC).

[0031] Fig. 9 Western Blot detection of Rab5a protein expression in vascular tissues of C57BL / 6 mice infected with Rab5a-AAV9; Note: A: Expression of total Rab5a protein; B: Statistical chart of relative expression level of Rab5a, *P<0.05VS NC.

[0032] Fig.10 Effect of Rab5a overexpression on systolic blood pressure in AngⅡ-induced hypertensive mouse model (Note: * P<0.05VSWT; # P<0.05VS WT+AngⅡ).

[0033] Fig.11 Effect of Rab5a overexpression on diastolic blood pressure in AngⅡ-induced hypertensive mouse model (Note: *P<0.05VSWT; #P<0.05VS WT+AngⅡ).

[0034] Fig.12 Effect of Rab5a overexpression on mean arterial pressure in AngⅡ-induced hypertensive mouse model

[0035] (Note: *P<0.05VS WT; #P<0.05VS WT+AngⅡ).

[0036] Fig.13 Effect of Rab5a overexpression on body weight in AngⅡ-induced hypertensive mouse model.

[0037] Fig.14 HE was used to observe the effect of Rab5a overexpression on abdominal aorta vascular pathology in hypertensive mice.

[0038] Fig.15 Effect of Rab5a overexpression on abdominal aorta wall thickness in AngⅡ hypertensive mice. DETAILED DESCRIPTION

[0039] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0040] AngⅡ: angiotensin Ⅱ.

[0041] Example 1: Effect of Rab5a on Vascular Function

[0042] 1. Experimental Methods

[0043] (I) Construction of mice with different Rab5a genotypes

[0044] 1. Construction of mice with different Rab5a genotypes

[0045] (1) Entrusted Saiye (Guangzhou) Biotechnology Co., Ltd. to prepare heritable gene-modified Rab5a mice. The genotype of the C57BL / 6 mice delivered by the company is: Rab5a flox / flox The preparation process is as follows: a targeting vector is designed for the exons of the mouse Rab5a gene. After embryonic stem cell transfection, positive cells are obtained using a positive and negative screening system. The positive cells are injected into blastocysts by microinjection, and then transplanted into C57BL / 6 surrogate mothers for breeding mice. The born chimeric mice are hybridized with wild-type mice to screen for heritable gene-modified Rab5a heterozygous mice (Rab5a flox / + type), Rab5a flox / + The type was self-fertilized to obtain Rab5a flox / flox Type mice.

[0046] (2) Rab5a flox / flox The newborn mice were mated with the Cre-expressing mice. After 21 days, they were separated into male and female cages and identified. The gene identified was Rab5a. + / - mice are Cre-positive heterozygous mice (heterozygous mice carrying the Cre gene have been obtained).

[0047] (3) Rab5a + / - The mice were self-fertilized, and the newborn mice were separated into male and female cages after 21 days and identified. The identified gene was: Rab5a + / - The mice were heterozygous (only one chromosome was knocked out), Rab5a + / + Mice were wild-type mice, Rab5a - / - The mice were whole body knockout mice.

[0048] (4) Rab5a flox / flox , Rab5a + / - , Rab5a - / - , Rab5a + / +Both male and female mice were housed in an SPF animal room on the third floor of the Animal Experiment Center of Fujian University of Traditional Chinese Medicine. The experimental animal use license number is SYXK (Min) 2019-0007 (valid from December 2019 to December 2024, Fujian Provincial Department of Science and Technology). During the experiment, the ambient temperature of the animal room was 20.8-24.3°C, the daily temperature difference did not exceed 4°C, the relative humidity was 47.2-67.2%, the ventilation rate was ≥15 times / h, and the light and dark cycle was 12h lighting / 12h dark. All animals were free to eat and the bedding was changed every 2-3 days.

[0049] 2. Construction of Rab5a overexpression mice

[0050] (1) Rab5a overexpression C57BL / 6 mice were constructed by tail vein injection of adeno-associated virus (AAV): recombinant AAV particles were produced using the AAV Helper-Free System, and the exogenous gene (NM_025887.4|Rab5a[Mouse]|CDS 648 bp (SEQ ID NO.1):

[0051] The recombinant expression plasmid was cloned into a suitable vector, and then co-transfected into AAV-293 cells with pHelper (carrying adenovirus-derived genes) and PAAV-RC (carrying AAV replication and capsid genes). AAV virus particles were collected from the infected cells 2-3 days after transfection, and the supernatant was concentrated and purified after lysis. The titer of the obtained virus was determined by real-time fluorescence quantitative PCR (Quantitative Real-time PCR) after two cesium chloride (CsCl) density gradient centrifugations and one ultrafiltration.

[0052] The virus (AAV-CMV-Rab5a) was injected into C57BL / 6 mice by tail vein injection to obtain mice with overexpression of Rab5a. C57BL / 6 mice with empty virus (AAV-CMV) were injected into the tail vein in the same way as controls. The mice were raised in the SPF animal room on the second floor of the Animal Experiment Center of Fujian University of Traditional Chinese Medicine. The temperature of the animal room was 20.8-24.3℃, the temperature difference during the day was <4℃, the relative humidity was 47.2-67.2%, the ventilation frequency was ≥15 times / h, and the light and dark cycle was 12h / 12h. The experimental animals could eat freely, add food and drinking water at any time, and change the bedding every 2-3 days. The animal experiment was approved by the Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine and approved by the Ethics Committee. All experimental procedures were strictly carried out in accordance with China's "Guiding Opinions on Treating Laboratory Animals Well".

[0053] (2) After 15 days, three mice were randomly selected and anesthetized with isoflurane. The mice were killed by cervical dislocation, and the blood vessels and tissues were quickly removed. The RNA and protein were extracted, and QPCR and Western Blot experiments were performed to further confirm the successful overexpression of the mice.

[0054] (II) Construction of Ang II-induced hypertensive mouse model

[0055] 1. Grouping

[0056] (1) Grouping of Rab5a knockout mice (Rab5a KO):

[0057] Take 8-10 week old Rab5a + / + and Rab5a - / - There were 12 mice of each type and the mice were divided into four groups, with 6 mice in each group:

[0058] WT+PBS group: Rab5a + / + +PBS,

[0059] WT+AngⅡ group: Rab5a + / + +AngⅡ,

[0060] Rab5a KO+PBS group: Rab5a - / - +PBS,

[0061] Rab5a KO+AngⅡ group: Rab5a - / - +AngⅡ.

[0062] (2) Rab5a overexpression (OV) mice (Rab5a OV) grouping:

[0063] Twelve AAV-CMV and AAV-CMV-Rab5a mice aged 8-10 weeks were selected and divided into four groups, with 6 mice in each group:

[0064] NC+Saline group: AAV-CMV+Saline,

[0065] NC+AngⅡ group: AAV-CMV+AngⅡ,

[0066] Rab5a OV+Saline group: AAV-CMV-Rab5a+Saline,

[0067] Rab5a OV+AngⅡ group: AAV-CMV-Rab5a+AngⅡ.

[0068] 2. Modeling steps

[0069] (1) Weigh all mice one day in advance and take the average. Prepare AngⅡ at a dose of 500ng / kg / min. Place the Alzet Osmotic Pump (Model: 2004) upright, insert the matching needle into a 1mL syringe, draw 220μL of the prepared AngⅡ, and inject AngⅡ into the pump. While injecting, slowly lift the needle until a small amount of liquid overflows from the pump port, then stop injecting and insert the pump cap into the pump. Draw an equal volume of saline into the Alzet Osmotic Pump and perform the same operation as above.

[0070] (2) Anesthetize the mouse with 2% sodium pentobarbital (0.225 mL / 100 g). After the mouse is anesthetized, make an incision of about 1 cm in the middle of the shoulder blade on the back of the mouse. Use forceps to free the subcutaneous tissue to the back, bury the pre-installed micropump under the incision, and suture the skin incision with needle and thread.

[0071] (3) After the operation, place the mouse on a plate covered with bedding and keep it warm with a heating lamp. Observe the mouse's vital signs at all times until the mouse wakes up from anesthesia and then put it back into the cage.

[0072] (III) Measurement of blood pressure and body weight and pathological examination of mice in different groups

[0073] 1. Determination of blood pressure and body weight of mice in different groups

[0074] Before surgery, baseline blood pressure and body weight were measured, and blood pressure was measured 0, 7, 14, 21, and 28 days after surgery.

[0075] Before measuring blood pressure each time, weigh each mouse and record the weight. The blood pressure test steps are as follows:

[0076] (1) Install the Coda blood pressure measurement system (Kent Scientific), including the heating plate, differential pressure sensor device, computer with Coda software, etc., and turn on the switch and power.

[0077] (2) Check whether there is any leakage in each channel of the blood pressure monitor. If there is any leakage, the leather cover should be replaced in time and tested again until there is no leakage in each channel.

[0078] (3) Place the mouse in a holder and heat it on a heating plate for 3 minutes. Put a blocking ring and a blood pressure detection ring of appropriate size on the tail of the mouse. After setting up the Coda software on the computer, start the measurement. Each mouse is measured for 3 cycles, and 15 blood pressure data are recorded for each cycle. The systolic blood pressure, diastolic blood pressure, and mean arterial pressure data of all qualified mice are recorded, and the average value is taken as the blood pressure value of the mouse.

[0079] 2. Small animal ultrasound for mouse abdominal aorta and pathological examination

[0080] (1) Small animal ultrasound examination of the mouse abdominal aorta

[0081] 1) Ultrasound examination was performed on mice 4 weeks after modeling and administration. The mice were fixed on the ultrasound examination table under continuous isoflurane inhalation anesthesia, and the chest and abdominal hair was gently removed with depilatory cream.

[0082] 2) Use color Doppler ultrasound diagnostic instrument to detect, first use B mode to observe the overall pulsation of the aorta, then switch to M mode to record the pulse wave at the distal and proximal ends of the aorta, and finally calculate the pulse wave conduction velocity of the abdominal aorta. The pulse wave conduction velocity of the abdominal aorta is the distance between the distal end and the proximal end of the abdominal aorta / the conduction time from the distal end to the proximal end of the abdominal aorta. The changes in the index are statistically analyzed by Doppler ultrasound analysis software.

[0083] (2) Pathological examination

[0084] 1) Animal materials

[0085] After the ultrasound examination of mice, the animals were collected. The mice were anesthetized with isoflurane, and after anesthesia, they were painlessly killed by cervical dislocation. The abdominal aorta was taken, and part of it was fixed in 4% paraformaldehyde for HE staining to observe morphological changes, Sirius red method to observe vascular collagen deposition, and immunohistochemistry to detect protein expression; part of it was put into liquid nitrogen and stored at -80℃. At the same time, blood was taken from the abdominal aorta, placed in a 4℃ refrigerator for four hours, and then centrifuged at 3000 rpm for 20 minutes to take the supernatant, which was stored at -80℃ and used for ELISA detection method to detect the level of inflammatory factors.

[0086] 2) HE staining to observe the pathological changes of the abdominal aorta

[0087] ①Dehydration and embedding

[0088] The abdominal aorta was fixed in 4% formaldehyde for 24 h, dehydrated with graded alcohol, made transparent with xylene, and embedded after three passes of paraffin wax dehydration.

[0089] ② Dehydration of slices

[0090] The vascular tissue was cut into 4 μm slices, flattened in 37°C pure water, and picked up with a glass slide. The slices were placed in a constant temperature box at 60°C for 6 hours, dewaxed in xylene I and II for 15 minutes each, then placed in 100%, 95%, 90%, 80%, 70% and other alcohol solutions for 5 minutes each, and then placed in distilled water for 3 minutes.

[0091] ③ Dyeing

[0092] After washing with ddH2O, transfer the slices into hematoxylin solution for 5 to 10 minutes to color the cell nuclei. Wash off the residual hematoxylin stain on the slices with running water. Place the slices into 1% hydrochloric acid ethanol solution (hydrochloric acid: 70% ethanol = 1:99) to fade until the slices turn red and the color is lighter, about a few seconds to ten seconds. Soak in tap water to turn blue. Soak in ddH2O for 1 to 2 seconds, transfer to 0.5% eosin alcohol solution for staining for 3 to 5 minutes. Naturally dry the slides, seal the slides with neutral resin glue, and place them in a ventilated place to dry. Observe the morphological changes of the abdominal aorta under an optical microscope.

[0093] (3) Data analysis and statistics

[0094] The experimental data were statistically analyzed using SPSS 20.0. The data of the present invention are all quantitative data. The independent sample t test was used for comparison between two samples, and one-way ANOVA was used for comparison between multiple samples. P < 0.05 was considered statistically significant. All experiments were repeated at least 3 times.

[0095] 2. Experimental results:

[0096] (I) Identification of Rab5a whole-body knockout mice

[0097] The preparation of transgenic mice for the study of disease-related models has become one of the irreplaceable means to study whether a specific gene is involved in regulating the occurrence and development of diseases. The present invention uses ES targeting technology combined with microinjection to prepare transgenic mice with whole-body knockout of Rab5a, which is used to study the regulatory effect of Rab5a knockout on blood pressure and vascular remodeling in AngⅡ-induced hypertensive mice. After breeding, the transgenic mice are extracted from their tail tissues, and PCR amplification is performed using specific primers. The mouse gene is preliminarily identified by the size of the product band.

[0098] like Figure 1As shown in A, there is only one band, and the band size is 361bp, which is Rab5a - / - Mouse type (KO); only one band, and the band size is 168bp, which is Rab5a + / + type mice (WT); these two genotypes of mice were used to construct the subsequent AngⅡ-induced hypertension mouse model.

[0099] In addition, to further identify the WT and KO mice, their blood vessels and other tissues were collected and identified. Figure 1 As shown in B: In each tissue, the bands of WT and KO mice are both 168 bp and 361 bp, respectively. At the same time, some tissues were selected to detect the protein expression of Rab5a by Western Blot technology. The results are shown in Figure 1 As shown in C: WT mice express Rab5a protein normally, while KO mice do not express Rab5a protein. These results confirm that WT and KO mice have been successfully constructed and can be used for subsequent experimental studies.

[0100] The experimental results showed that Rab5a was successfully constructed - / - (KO type), Rab5a + / + (WT type) mouse model.

[0101] (II) Effects of Rab5a knockout

[0102] 1. Effect of Rab5a knockout on systolic blood pressure in AngⅡ-induced hypertensive mice

[0103] The present invention uses the successfully constructed WT and KO mice as test subjects, and constructs an AngⅡ-induced hypertensive mouse model by subcutaneously burying an implantable capsule osmotic pump (Alzet Osmotic Pump) to continuously release AngⅡ (500ng / kg / min) for 28 days.

[0104] The mice were divided into four groups: WT+PBS group, Rab5a KO+PBS group, WT+AngⅡ group and Rab5a KO+AngⅡ group. Figure 2 , Figure 3 and Figure 4 As shown: When measuring the basal blood pressure, it was found that there was no significant difference in the systolic blood pressure, diastolic blood pressure and mean arterial pressure among the four groups. On the 7th, 14th, 21st and 28th days after AngⅡ modeling, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of the WT+AngⅡ group and Rab5a KO+AngⅡ group were significantly higher than those of the WT+PBS and KO+PBS groups (P<0.05), proving that the AngⅡ-induced hypertensive mouse model was successfully established.

[0105] At 7, 14, 21, and 28 days, the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of the Rab5a KO+AngⅡ group were significantly higher than those of the WT+AngⅡ group, but there was no difference between the WT+PBS group and the KO+PBS group. This indicates that Rab5a knockout can significantly increase the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of AngⅡ-induced hypertensive mice. Figure 5 It can be seen that the weight of the four groups of mice did not change much.

[0106] The experimental results showed that Rab5a knockout would further increase the systolic blood pressure, diastolic blood pressure and mean arterial pressure of AngⅡ-induced hypertensive mice, and had almost no effect on the weight of mice.

[0107] 2. Effect of Rab5a knockout on pulse wave velocity in the abdominal aorta of AngⅡ-induced hypertensive mice

[0108] The above experiments confirmed that Rab5a knockout promoted further increase in blood pressure of AngⅡ-induced hypertensive mice. One of the pathological bases of major target organ damage is the change in vascular elasticity. Vascular elasticity, also known as vascular compliance, mainly reflects the vasodilation state and endothelial function of blood vessels. Long-term hypertension has a serious impact on vascular elasticity. The decrease in vascular elasticity will negatively feedback and aggravate the increase in blood pressure, causing a vicious cycle. The decrease in vascular elasticity is a sign of early vascular damage and can be used as an indicator for clinical evaluation of cardiovascular disease. Clinically, ultrasound technology is used to measure the conduction velocity of the aortic pulse wave to evaluate vascular elasticity and reflect the function of blood vessels, which has been widely used. The present invention measures the conduction velocity of the abdominal aorta pulse wave of mice by a small animal ultrasound instrument to evaluate whether Rab5a knockout will aggravate vascular damage in AngⅡ-induced hypertensive mice.

[0109] The results are as follows Figure 6 As shown: The conduction velocity of the abdominal aorta pulse wave in the WT+PBS group and the Rab5a KO+PBS group was 2.83±0.41mm / ms and 3.00±0.39mm / ms, respectively, and there was no significant difference between the two. After 28 days of AngⅡ modeling, the conduction velocity of the abdominal aorta pulse wave in the WT+AngⅡ group was 4.77±0.62mm / ms, which was significantly higher than that in the WT+PBS group and the Rab5a KO+PBS group, indicating that 28 days of AngⅡ modeling caused damage to the mouse blood vessels. After AngⅡ modeling, the conduction velocity of the abdominal aorta pulse wave in Rab5a knockout mice was 6.14±0.62mm / ms, which was higher than that in the WT+AngⅡ group. This once again verified that Rab5a knockout would aggravate vascular damage in AngⅡ-induced hypertensive mice.

[0110] The experimental results showed that Rab5a knockout increased the pulse wave conduction velocity of the abdominal aorta in AngⅡ-induced hypertensive mice, indicating that Rab5a knockout would aggravate vascular damage in AngⅡ-induced hypertensive mice.

[0111] 3. Effect of Rab5a knockout on abdominal aorta wall thickness in AngⅡ-induced hypertensive mice

[0112] Vascular disease is the initiating factor for the development of hypertension, and long-term hypertension can also cause thickening of the vascular wall, leading to lumen stenosis, thrombosis, and ultimately vascular occlusion. Therefore, HE staining was used to evaluate the effect of Rab5a knockout on abdominal aortic pathology in AngⅡ-induced hypertensive mice.

[0113] The results are as follows Figure 7 As shown: Compared with the WT group and the KO group, the abdominal aorta wall of the WT+AngⅡ group was significantly thickened, and that of the KO+AngⅡ group was thicker than that of the WT+AngⅡ group (P<0.05), indicating that Rab5a knockout significantly increased the thickness of the abdominal aorta wall in AngⅡ-induced hypertensive mice. Because the increase in the thickness of the aortic wall causes the lumen to narrow, the aortic pulse wave conduction velocity will increase, which is consistent with the above-mentioned result that Rab5a knockout accelerated the abdominal aorta pulse wave conduction velocity in AngⅡ-induced hypertensive mice.

[0114] The experimental results showed that Rab5a knockout significantly increased the thickness of the abdominal aorta wall in AngⅡ-induced hypertensive mice, once again verifying that Rab5a knockout would aggravate the increase in blood pressure and vascular damage in AngⅡ-induced hypertensive mice.

[0115] 3. Effects of Rab5a overexpression

[0116] 1. Rab5a overexpression inhibits the increase in blood pressure in AngⅡ-induced hypertensive mice

[0117] The present invention constructs Rab5a overexpression mice to study the effect of Rab5a overexpression on the blood pressure of AngⅡ-induced hypertensive mice and the influence on vascular remodeling.

[0118] The results are as follows Figure 8 As shown in the figure, the mRNA levels of Rab5a in the abdominal aorta and liver of the Rab5a overexpression mice (Rab5a OV) constructed by tail vein injection of adeno-associated virus AAV2 / 9 coated with Rab5a overexpression plasmid were significantly increased; while there was no statistical difference in the expression of Rab5a in the brain, kidney, and quadriceps. At the same time, the total protein expression of Rab5a in vascular tissue was detected, and the results were as follows Fig. 9As shown in Figure 2, the protein level of Rab5a in the vascular tissue of Rab5a OV mice was significantly increased. The above results indicate that the tissue-specific overexpression mouse of Rab5a was successfully constructed.

[0119] In order to observe the effect of Rab5a overexpression on hypertension, the above Rab5a overexpression mice were used to construct an AngⅡ-induced hypertension model. Fig.10 , 11 As shown in Figure 12, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of the Rab5a overexpression hypertensive mouse model (Rab5aOV+AngⅡ) were significantly lower than those of the empty virus (NC+AngⅡ) group, and there was no effect on the weight of the mice ( Fig.13 ). The above results indicate that overexpression of Rab5a can inhibit the increase in blood pressure caused by AngⅡ.

[0120] HE detection of vascular pathological damage in mice found that compared with the NC+AngⅡ group, the vascular wall thickness of the Rab5aOV+AngⅡ group was significantly reduced, indicating that Rab5a overexpression inhibited the vascular wall thickening induced by AngⅡ in hypertensive mice ( Fig.14 and Fig.15 ), and the vascular wall thickness of the NC+Saline group and the Rab5aOV+Saline group were compared. There was no difference between the two groups, indicating that Rab5a overexpression has no effect on vascular function under normal conditions.

[0121] The experimental results show that the present invention constructed Rab5a overexpression mice, and the study found that Rab5a overexpression can reduce the blood pressure of AngⅡ-induced hypertensive mice, inhibit the thickening of the vascular wall of AngⅡ-induced hypertensive mice, repair the vascular wall damage, and inhibit the vascular remodeling caused by hypertension.

[0122] The present invention studies the correlation between Rab5a and hypertension for the first time by constructing Rab5a knockout mice and combining them with the AngⅡ-induced hypertension model. The experimental results show that after the AngⅡ modeling intervention of Rab5a knockout mice, their systolic blood pressure, diastolic blood pressure, and mean arterial pressure are significantly higher than those of wild-type mice. Rab5a knockout can cause high blood pressure and increase the damage of vascular function. Through vascular ultrasound and vascular pathological detection, Rab5a knockout has a significant promoting effect on vascular remodeling induced by hypertension.

[0123] At the same time, the present invention constructs Rab5a overexpression mice by injecting adeno-associated virus into the mouse tail vein to verify the effect of Rab5a overexpression on AngⅡ-induced blood pressure increase and vascular pathological state. The results show that Rab5a overexpression can inhibit the increase in blood pressure caused by AngⅡ, and also inhibit the thickening of the vascular wall of AngⅡ-induced hypertensive mice, so that the damage to the vascular wall can be repaired. Rab5a overexpression inhibits the occurrence of hypertensive vascular remodeling. In summary, the present invention proves the important role of Rab5a in the occurrence and development of hypertension by knocking out and overexpressing Rab5a transgenic mice.

Claims

1. Use of Rab5a or a Rab5a gene expression promoter in the preparation of a drug for treating hypertension.

2. The use according to claim 1, characterized in that The hypertension reported is hypertension caused by AngⅡ.

3. The use according to claim 1 or 2, characterized in that The Rab5a gene expression promoter includes a Rab5a gene overexpression plasmid or a Rab5a gene overexpression adeno-associated virus; the Rab5a gene overexpression nucleotide sequence is shown in SEQ ID NO.

1.

4. The use according to claim 1 or 2, characterized in that: The medicine is a medicine for repairing damage to blood vessel walls caused by high blood pressure.

5. The use according to claim 1 or 2, characterized in that: The drug is a drug that inhibits vascular remodeling caused by hypertension.

6. The use according to claim 1 or 2, characterized in that: The drug is a drug for reducing high blood pressure.

7. The use according to claim 1 or 2, characterized in that: The drug is a drug that reduces the thickening of blood vessel walls in hypertensive mice.

8. The use according to claim 1 or 2, characterized in that: The drug is a drug for reducing the pulse wave velocity of hypertension.

9. A composition for treating hypertension, characterized in that: The invention is a preparation prepared by taking Rab5a gene expression factor promoter as active ingredient and adding pharmaceutically acceptable auxiliary materials.

10. The composition according to claim 9, characterized in that: The Rab5a gene expression promoter includes a Rab5a gene overexpression plasmid or a Rab5a gene overexpression adeno-associated virus.

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