Application of Central Polypeptide Angiotensin-(5-7) in the Preparation of Drugs

Abstract

Application of a central polypeptide angiotensin-(5-7) in the preparation of a drug. The present invention demonstrates that Ang-(5-7) in the RVLM of WKY rats can enhance sympathetic output and increase blood pressure, and part of Ang-(5-7) acts on the AT1 receptor to participate in the central regulation of blood pressure. Further exploring the mechanism of action of Ang-(5-7) in the sympathetic center, it is found that Ang-(5-7) in the RVLM of WKY rats can enhance sympathetic output and increase blood pressure through the oxidative stress pathway, while targeting and interfering with the expression of DPP3 in the RVLM endogenously reduces Ang-(5-7) under the pathological state of hypertension, improves oxidative stress, and reduces the blood pressure of SHR rats. The research of the present invention shows that Ang-(5-7) enhances sympathetic output and increases blood pressure through the oxidative stress pathway and participates in the central regulation of the cardiovascular activities of patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of a central small molecule polypeptide angiotensin 5-7 in the preparation of drugs. Background Art

[0002] Angiotensin-(5-7) [Ang-(5-7)] is a newly discovered polypeptide fragment in the renin-angiotensin system. Under the catalysis of dipeptidyl peptidase 3 (DPP3), angiotensin II (Ang II) or angiotensin (1-7) [Ang-(1-7)] can be hydrolyzed into angiotensin-(3-7) [Ang-(3-7)] and Ang-(5-7). Studies have shown that microinjection of Ang-(3-7) into the sympathetic center RVLM can significantly increase blood pressure. Ang-(5-7) is further produced from Ang-(3-7) under the catalysis of DPP3, but the role of Ang-(5-7) in the central regulation of blood pressure and the formation of hypertension has not been clarified. The present invention focuses on studying the cardiovascular effects of Ang-(5-7) and comparing them with the pressor effect of Ang II. It is found that after continuous perfusion of Ang II or Ang-(5-7) into the fourth ventricle for seven days, both blood pressure and heart rate variability increase significantly, and the decline in baroreflex function after perfusion of Ang-(5-7) is more obvious than that after perfusion of Ang II. Summary of the Invention

[0003] The purpose of the present invention is to propose the application of a central polypeptide Ang-(5-7) in the preparation of drugs. The amino acid sequence of Ang-(5-7) is: isoleucine-histidine-proline (I-H-P).

[0004] Essential hypertension is mainly characterized by hyperactivity of sympathetic nerve activity, which is closely related to the abnormal excitation of presympathetic neurons in the RVLM. Ang-(5-7) is a newly discovered polypeptide fragment in the renin-angiotensin system. Under the catalysis of DPP3, Ang II or Ang-(1-7) can be hydrolyzed into Ang-(3-7) and Ang-(5-7). Studies have shown that microinjection of Ang-(3-7) into the sympathetic center RVLM can significantly increase blood pressure. Ang-(5-7) is further produced from Ang-(3-7) under the catalysis of DPP3, but the role of Ang-(5-7) in the central regulation of blood pressure and the formation of hypertension has not been clarified. Therefore, the present invention aims to study the role of Ang-(5-7) in the central regulation of blood pressure and the formation of hypertension in the RVLM.

[0005] Studies have shown that Ang-(5-7) in the RVLM of WKY rats can enhance sympathetic output and increase blood pressure, and part of the effect of Ang-(5-7) acts on the AT1 receptor to participate in the central regulation of blood pressure. Further exploring the mechanism of action of Ang-(5-7) in the sympathetic center, it was found that Ang-(5-7) in the RVLM of WKY rats can enhance sympathetic output and increase blood pressure through the oxidative stress pathway. Under the pathological state of hypertension, targeted interference with DPP3 expression in the RVLM endogenously reduces Ang-(5-7), improves oxidative stress, and reduces the blood pressure of SHR rats. In summary, the research of the present invention shows that Ang-(5-7) enhances sympathetic output and increases blood pressure through the oxidative stress pathway, and participates in the central regulation of cardiovascular activities of patients. Brief Description of the Drawings

[0006] Figure 1 : Acute microinjection of Ang-(5-7) in the RVLM increases renal sympathetic activity and raises blood pressure. A: Original graph of the recordings of mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) after microinjection of Ang-(5-7) in the RVLM of WKY rats; B: Statistical graph of blood pressure, heart rate, and renal sympathetic activity after microinjection of Ang-(5-7) in the RVLM of WKY rats. *P<0.05 vs aCSF, n = 4-8 / group. aCSF: artificial cerebrospinal fluid; ABP: arterial blood pressure; MAP: mean arterial pressure; HR: heart rate; RSNA: renal sympathetic nerve activity; bpm: beats per minute.

[0007] Figure 2: Chronic perfusion of Ang-(5-7) in the RVLM enhances sympathetic output, raises blood pressure, and promotes the occurrence and development of hypertension. A: Comparison of the effects of intracerebroventricular perfusion of Ang II and Ang-(5-7) on cardiovascular activities in WKY rats. The upper panel shows the original recordings of blood pressure and heart rate under anesthesia after intracerebroventricular perfusion of Ang II and Ang-(5-7) in WKY rats; the lower panel shows the statistical graphs of blood pressure and heart rate under anesthesia after intracerebroventricular perfusion of Ang II and Ang-(5-7) in WKY rats. *P<0.05 vs aCSF, n = 7 / group. B: Comparison of baroreflex function after intracerebroventricular perfusion of Ang II and Ang-(5-7) in WKY rats. The upper panel shows the original recordings of blood pressure and heart rate after intravenous injection of phenylephrine (40 μg / kg) after intracerebroventricular perfusion of Ang II and Ang-(5-7) in WKY rats; the lower panel shows the statistical graph of baroreflex function after intravenous injection of phenylephrine under anesthesia after intracerebroventricular perfusion of Ang II and Ang-(5-7) in WKY rats. *P<0.05 vs aCSF, n = 7 / group. aCSF: artificial cerebrospinal fluid; ABP: arterial blood pressure; MAP: mean arterial pressure; HR: heart rate; bpm: beats per minute; Ang II: angiotensin II; HRV: heart rate variability; LF / HF: low frequency / high frequency; BRS: baroreflex sensitivity; ΔMAP: change in mean arterial pressure; ΔRR: change in RR interval.

[0008] Figure 3 : Microinjection of Ang-(5-7) after blocking AT1 receptors in the RVLM inhibits sympathetic output and inhibits the increase in blood pressure. The upper panel shows the original recordings of blood pressure, heart rate, and renal sympathetic nerve activity after microinjection of losartan into the RVLM of WKY rats followed by injection of Ang-(5-7); the lower panel shows the statistical graphs of blood pressure, heart rate, and renal sympathetic nerve activity after microinjection of losartan followed by injection of Ang-(5-7) in WKY rats. *P<0.05 vs aCSF, n = 4-8 / group. AT1 receptor: angiotensin II type 1 receptor; Losatan: losartan; ABP: arterial blood pressure; MAP: mean arterial pressure; HR: heart rate; RSNA: renal sympathetic nerve activity; bpm: beats per minute.

[0009] Figure 4: In the RVLM, Ang-(5-7) affects blood pressure through oxidative stress. Above is the original graph of blood pressure, heart rate, and renal sympathetic activity recordings after microinjecting superoxide dismutase mimetic (tempol) into the RVLM of WKY rats and then injecting Ang-(5-7); below is the statistical graph of blood pressure, heart rate, and renal sympathetic activity after microinjecting tempol into WKY rats and then injecting Ang-(5-7). *P<0.05 vs aCSF, n=4-8 / group. Tempol: superoxide dismutase mimetic; ABP: arterial blood pressure; MAP: mean arterial pressure; HR: heart rate; RSNA: renal sympathetic nerve activity; bpm: beats per minute.

[0010] Figure 5 : Effects of intracerebroventricular perfusion of Ang-II and Ang-(5-7) on oxidative stress in the RVLM. A: Fluorescence images of reactive oxygen species (ROS) generation in the RVLM detected by a superoxide anion fluorescence probe (DHE) kit after intracerebroventricular perfusion of Ang-II and Ang-(5-7) in WKY rats; above is the level of ROS in the RVLM after intracerebroventricular perfusion of Ang-II and Ang-(5-7) in WKY rats. The white rectangular box indicates the RVLM. The scale bar in the left picture is 500 μm, and the scale bar in the right picture is 100 μm; B: Statistical graph of ROS content after intracerebroventricular perfusion of Ang-II and Ang-(5-7) in WKY rats, *P<0.05 vs aCSF, n=7 / group. RVLM: rostral ventrolateral medulla; aCSF: artificial cerebrospinal fluid; DHE: superoxide anion fluorescence probe; ROS: reactive oxygen species.

[0011] Figure 6 : Effects of intracerebroventricular perfusion of Ang-II and Ang-(5-7) on oxidative stress-related pathways in the RVLM. On the left are the original protein bands of the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (keap1), heme oxygenase 1 (HO1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) in the RVLM detected by Western Blot after intracerebroventricular perfusion of Ang-(5-7) in WKY rats; on the right is the statistical graph of the expression of Nrf2, keap1, HO1, and NQO1. *P<0.05 vs aCSF, n=4 / group. aCSF: artificial cerebrospinal fluid.

[0012] Figure 7: Effects of overexpression of dipeptidyl peptidase 3 (DPP3) in the RVLM on the increase in endogenous Ang-(5-7) and oxidative stress in the RVLM. The upper part is the fluorescence image of the generation of reactive oxygen species (ROS) in the RVLM detected by the DHE kit after overexpression of DPP3 in the RVLM of WKY rats; the lower part is the statistical chart of the ROS level in the RVLM. *P<0.05 vs WKY-GFP, n=6 / group. ROS: Reactive oxygen species. DPP3: Dipeptidyl peptidase 3; DHE: Superoxide anion fluorescent probe; ROS: Reactive oxygen species.

[0013] Figure 8 : Effects of interfering with DPP3 on oxidative stress in the RVLM of spontaneously hypertensive rats (SHRs). The upper part is the fluorescence image of the generation of reactive oxygen species (ROS) in the RVLM detected by the DHE kit after interfering with DPP3 in the RVLM of SHR rats; the lower part is the statistical chart of the ROS level in the RVLM. *P<0.05 vs WKY-GFP; #P<0.05 vs SHR-GFP, n=7 / group. DPP3: Dipeptidyl peptidase 3; DHE: Superoxide anion fluorescent probe; ROS: Reactive oxygen species. Detailed implementation manners

[0014] The present invention will be further described by the following examples in conjunction with the accompanying drawings. Examples

[0015] (I) Experimental steps.

[0016] (1) Animal selection.

[0017] Male Wistar-kyoko (WKY) rats at 12 weeks old and spontaneously hypertensive rats (SHRs) used in this study were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), with body weights between 180-200 grams, and were raised in a 12-hour light room and a 12-hour dark animal room respectively. The experimental rats were allowed to have free access to food and water.

[0018] (2) Microinjection of virus into the RVLM of rats.

[0019] 1) Induction of anesthesia and maintenance: Place the experimental animals in a gas anesthesia induction box, set the oxygen flow rate to 1 ml / min, and the anesthetic concentration to 5 ml / dm 3 , after about 5 minutes of induction of anesthesia in the animals, place them in a stereotaxic apparatus and maintain anesthesia, with the anesthetic concentration of 2 ml / dm 3 .

[0020] 2) Skin preparation: Use an electric clipper to shave the hair on the top of the skull to expose the occipital skin.

[0021] 3) Preoperative disinfection of the surgical area: Use iodophor cotton balls to disinfect the surgical area in a shingle pattern twice, and then deiodinate with 75% medical alcohol.

[0022] 4) Expose the skull surface: Use surgical scissors to cut through the skin and subcutaneous tissue layer by layer, and then use an electric scalpel to excise the tissue on the skull surface to fully expose the skull surface, stop bleeding, and disinfect with alcohol cotton balls.

[0023] 5) Determine the Lambda point: Locate the lambda suture of the posterior fontanelle. The tangent intersection of the three lines is the Lambda point. Mark it with a black signature pen.

[0024] 6) Leveling: Install the microinjection needle into the needle holder groove of the syringe pump, and then move the microinjection needle directly above the Lambda point, about 0.1 ml from the skull surface. Then, according to the rat anatomical atlas and the results of the preliminary experiment, move the stereotaxic instrument (6.5 mm at the tail end from the Lambda point; 0.25 mm at the H angle; 2 mm ± to the side), determine the injection point on the skull surface and make a mark. If the distance between the microinjection needle and the skull surface of the injection point is within 0.2 mm, it indicates that the skull surface is level and the next step can be carried out.

[0025] 7) Craniotomy: According to the injection point position determined in the previous step, use a skull drill to drill through the skull perpendicular to the skull surface. When there is a sense of falling through, it indicates that the skull has been penetrated. Disinfect with alcohol cotton balls.

[0026] 8) Microinjection: Install the microinjection needle into the needle holder groove of the syringe pump, and then move the microinjection needle directly above the Lambda point, about 0.1 ml from the skull surface. Then, adjust the stereotaxic instrument to make the tip of the needle above the injection point. Then slowly insert the needle and stop at RVLM (depth Z: +9.5 mm) for 1 min. Start the microinjection pump and slowly inject the virus suspension into the nucleus (about 5 min). After injection, stay still for 5 min. Wait for the virus to spread sufficiently, then the needle can be withdrawn. For the contralateral side, the steps are similar.

[0027] 9) Suture: After injecting the virus into the bilateral nuclei, suture the incision intermittently, disinfect with iodophor cotton balls twice, and deiodinate with alcohol cotton balls once. Finally, inject antibiotics intramuscularly and place on a warming pad until it wakes up.

[0028] (3) Monitoring of arterial blood pressure in rats under anesthesia.

[0029] 1) Anesthesia and skin preparation: After weighing the experimental animals, anesthetize with a mixed urethane at 0.5 ml / 100 g. Use a hemostatic forceps to squeeze the animal's toes. If the animal does not move, it indicates that the anesthesia is completed, and then fix it. Then supplement 1 / 3 of the initial dose of the mixed anesthetic every hour to maintain anesthesia. Use an electric clipper to shave the hair on the neck and lower abdomen to expose the skin.

[0030] 2) Tracheal intubation: Longitudinally incise the skin, gradually expose the trachea, pass a silk thread through the dorsal side of the trachea, make an inverted "T" incision at 5 mm below the cricoid cartilage, and finally insert the tracheal cannula into the trachea for about 1 cm.

[0031] 3) Femoral artery and vein intubation: Lift the skin in the iliac region and make an incision about 3 cm long along the direction of the blood vessels. After exposing the muscles, bluntly separate the artery and vein with a curved forceps. Pass a silk thread through the dorsal side of the artery, ligate the distal end, tie a slipknot at the proximal end, and clamp the proximal end with an artery clip. Between the artery clip and the artery of the distal end thread, make a small incision at the distal end with a micro-scissors, and then expand the opening with a micro-forceps. Finally, hold the arterial wall with a micro-forceps in the left hand and rotate the catheter into the femoral artery with the right hand, and then connect the arterial catheter to the Powerlab blood pressure monitoring system. The method of venous intubation is the same as that of arterial intubation.

[0032] (4) Monitoring of rat RSNA.

[0033] 1) Anesthesia and tracheal, arterial and venous intubation Anesthesia and tracheal, arterial and venous intubation: The steps are the same as described above.

[0034] 2) Isolation of renal sympathetic nerve: Incise the subcutaneous fascia through a longitudinal incision on the left lumbar region of the rat, expose the left kidney, renal artery and renal nerve along the retroperitoneal path, carefully isolate the renal sympathetic nerve near the abdominal aorta and the renal hilum of the renal artery and vein, and carefully place it on the silver wire electrode with a glass microprobe. The electrical signals are amplified with high-frequency and low-frequency cut-off frequencies of 1000 and 100 Hz respectively.

[0035] 3) Recording and calculation of renal sympathetic nerve activity: Use PowerLab (ADInstruments, Australia) to record and integrate the rectified output. Then observe the waveform of renal sympathetic nerve discharge in the oscilloscope to judge whether the nerve discharge signal is renal sympathetic nerve discharge. When it is determined that the separated nerve is the renal sympathetic nerve, use the Wacker glue mixture to electrically isolate the nerve, electrode and surrounding tissues. After the waveform of the renal sympathetic nerve electrical signal is stable, record the baseline value of RSNA. Then quickly inject 2 ml of mixed anesthetic intravenously to euthanize the rat, and the maximum value of rat RSNA can be induced. When the renal sympathetic nerve discharge of the rat completely disappears, it is the background noise of RSNA. Therefore, RSNA level = (baseline value - background noise) / (maximum value - background noise).

[0036] (5) Microinjection of drugs into rat RVLM.

[0037] 1) Anesthesia and tracheal, arterial and venous intubation: The same as the operation steps for monitoring arterial blood pressure in rats under anesthesia.

[0038] 2) Set the parameters of the animal ventilator and connect: Set the ventilator to assist / control ventilation mode, respiratory rate: 60 - 85 bpm, tidal volume: 2 - 2.5 ml, peak inspiratory pressure (PIP): 15 cmH2O, positive end-expiratory pressure (PEEP): 1 cmH2O. Inject gallamine triethiodide solution intravenously at a rate of 0.1 ml / 100 g (supplement every hour), and finally connect the tracheal intubation to the ventilator.

[0039] 3) Set the parameters of the intravenous infusion pump and connect: Connect a 50 ml syringe filled with 0.9% sodium chloride injection to the intravenous catheter, and set the infusion rate to 1 ml / h.

[0040] 4) Expose the surface of the skull and the foramen magnum: Use an electrosurgical knife to gradually dissect the posterior neck muscles layer by layer until the occipital bone and the foramen magnum are exposed. Finally, dissect the tissue on the surface of the skull caudal to the posterior fontanelle as well.

[0041] 5) Craniotomy: First, thin the edge of the skull with a skull drill, then use a rongeur to dissect the skull, and finally use a rongeur to modify the surrounding bone spurs. Pay attention to observing the blood pressure at all times during the process of thinning the skull to prevent too low blood pressure.

[0042] 6) Incise and unfold the meninges: Use a micro-scissors to incise the meninges from bottom to top and from left to right, and then use micro-forceps to unfold the meninges and cover the edge of the skull. This operation is carried out under a microscope and the bleeding is minimized as much as possible.

[0043] 7) Aspirate part of the cerebellar tissue: Use an electric aspirator to aspirate the cerebellar tissue from bottom to top until the obex point and the brainstem area above the RVLM are exposed. After the aspiration, cover the brain tissue surface and other muscle tissues with a saline-soaked cotton ball to prevent excessive body water loss.

[0044] 8) Microinjection: Inject the drug into the glass micropipette with a 1 ml syringe, and flick the bubbles in the tube with your finger. Then fix the glass electrode on the stereotaxic apparatus and connect the air pump. Then, according to the atlas, with the obex point as the reference, move the head end 2.5 mm, 2 mm to the left and right, and 3.5 mm deep, and position the glass micropipette to the RVLM. Finally, according to the microscope scale, start the air pump and slowly inject 100 nl of the drug, wait for 5 minutes and then slowly withdraw the glass micropipette. After the injection, cover the surface of the brainstem with a standard artificial cerebrospinal fluid-soaked cotton ball.

[0045] (6) Perfusion of the fourth ventricle.

[0046] Assemble the micro-osmotic pump: Under sterile conditions in a biosafety cabinet, perfuse and assemble the flow regulator and the ALZET micro-osmotic pump with the drug according to the instructions.

[0047] Induction anesthesia and maintenance: Place the experimental animals in a gas anesthesia induction box, set the oxygen flow rate to 1 ml / min, and the anesthetic concentration to 5 ml / dm 3 , after about 5 minutes of induction anesthesia in the animals, place them in a stereotaxic apparatus and maintain anesthesia, with the anesthetic concentration at 2 ml / dm 3 .

[0048] Skin preparation: Use an electric clipper to shave the hair on the back of the neck to expose the occipital skin.

[0049] Preoperative disinfection of the surgical area: Use iodophor cotton balls to disinfect the surgical area in a shingle pattern twice, and then deiodize with 75% medical alcohol.

[0050] Expose the dura mater of the foramen magnum: Use surgical scissors to cut through the skin layer by layer and bluntly separate the subcutaneous tissue of the neck until the ALZET micro-osmotic pump can be placed. Then use an electrocautery to cut through the neck muscles layer by layer until the surface of the dura mater of the foramen magnum is fully exposed, and disinfect with alcohol cotton swabs.

[0051] Fix the micro-osmotic pump: Use the tip of a 1 ml syringe to pierce the dura mater of the foramen magnum. After seeing clear cerebrospinal fluid flowing out, place the micro-osmotic pump in the subcutaneous tissue of the neck, and insert the liquid outlet end of the flow regulator into the cerebral ventricle through the needle hole of the dura mater of the foramen magnum. Then use tissue glue to fix the micro-osmotic pump and the flow regulator.

[0052] Suture: Carefully align the incision, suture intermittently, and disinfect with iodophor cotton balls twice and deiodize with alcohol cotton balls once. Finally, inject antibiotics intramuscularly, place on a warming pad, and wait for it to wake up. Inject antibiotics intramuscularly continuously for three days after surgery.

[0053] (7)RVLM tissue extraction.

[0054] ①RVLM tissue extraction.

[0055] 1) Specimen collection: Use a guillotine to separate the head of the euthanized rat, then quickly use a rongeur to carefully remove the skull completely from the foramen magnum. Finally, use a curved forceps to break the cranial nerves of the brain and peel off the cerebellum to expose the Obex point of the brainstem. Dip the blood with a cotton ball and place it in a 35 mm cell culture dish. Wrap it well with tin foil and carefully place it in a liquid nitrogen tank. If for preservation, transfer it to an -80°C refrigerator.

[0056] 2) Specimen rewarming: Pre-cool the cryostat to -20°C in advance, then place the brain specimen in the cryostat for about 10 minutes of rewarming until the brain tissue turns pink, which is convenient for sectioning.

[0057] 3) Extraction of RVLM tissue: Use a blade to excise the anterior half of the brain along the coronal plane, and embed the posterior half of the brain and the brainstem vertically on the base with OCT embedding medium. After the tissue is fixed, install the base into the fixing slot of the microtome. Then, cut the brainstem tissue to the Obex point at a section thickness of 50 μm first, and continue to cut forward 20 times to reach the tail end of RVLM. Finally, use a syringe with a needle inner diameter of 1 mm to puncture the brain tissue in RVLM at a depth of about 5 mm according to the rat brain atlas position, and quickly transfer it to a pre-cooled 1.5 ml centrifuge tube.

[0058] 4) Lysis: Add 100 μl of the pre-prepared mixed lysis buffer dropwise to the centrifuge tube containing RVLM brain tissue. Set the power of the ultrasonic cell disruptor to 10%, the working time to 3S, the working interval to 9S, and perform tissue ultrasonic fragmentation on ice for 5 cycles. Finally, let it stand and lyse on ice for 10 min. After the lysis is completed, place the tissue lysate in a low-temperature centrifuge and centrifuge at 12,000 rpm at 4 °C for 20 min. After centrifugation, carefully aspirate the supernatant with a 100 μl pipette to obtain the protein supernatant after lysis.

[0059] ② Protein concentration determination and denaturation.

[0060] 1) Protein concentration determination: First, prepare a 96-well plate and the prepared 0.5 mg / ml BCA protein standard. The specific steps are as follows: ① Add 0, 2, 4, 8, 12, 16, 20 μl of protein standard to the first vertical column of wells in sequence. In the wells for the protein to be measured, add 1 μl of protein supernatant in sequence. Make 2 duplicate wells for each protein sample. Finally, add PBS dilution to supplement the liquid volume in each well to 20 μl in sequence. ② Prepare the BCA working solution freshly at a ratio of A reagent: B reagent of 50:1, and add 200 μl of the working solution to each well. ③ Transfer the 96-well plate to a 37 °C constant temperature incubator and incubate for about 25 min. ④ Use a multifunctional microplate reader to measure the protein absorbance value at a wavelength of 560 nm, and calculate the standard curve (correlation coefficient R 2 > 0.99), and substitute the average protein absorbance value of each tissue sample into the formula to calculate the sample protein concentration.

[0061] 2) Protein denaturation: Calculate the volumes of 5X protein loading buffer and ddH2O to be added to the protein supernatant in each sample according to a protein concentration of 5 μg / μl, and dilute each protein sample to the same protein concentration. Then mix and centrifuge the samples in each centrifuge tube, and incubate in a 100 °C metal bath for 10 min. Finally, after cooling the protein samples to room temperature, store them at -20 °C.

[0062] (8) Western Blot.

[0063] 1) Gel preparation (two 1.5-mm mini gels): ① First, prepare 2 sets of cleaned and matched long and short glass plates in advance, install them in the card slots, and check for leaks with ddH2O. If the airtightness is good, dry them for standby. ② Mix 8 ml each of the lower-layer gel solutions A and B in the 10% SDS-PAGE Gel Rapid Preparation Kit (Shanghai YaMei) in a beaker, add 160 μl of the coagulation promoter, and blow and mix well. ③ Pour the lower-layer gel from one end of the short glass plate, avoiding the generation of air bubbles during the process. Then, seal it with ddH2O for about 15 minutes. Prepare the comb teeth and 1X electrophoresis buffer during the solidification of the lower-layer gel. ④ When a refractive bright line appears between the lower-layer gel and ddH2O, it indicates that the lower-layer gel has solidified. Carefully pour off the upper-layer ddH2O. ⑤ Take 2 ml each of the upper-layer gel solutions A and B in a beaker, add 40 μl of the coagulation promoter, and blow and mix well. ⑥ Pour the upper-layer gel into the glass sandwich plate, insert the comb teeth, and wait for the upper-layer gel to solidify. Prepare the protein samples during this period.

[0064] 2) Loading samples: ① After the upper-layer gel has fully solidified, carefully remove the glass sandwich plate and install it in the electrophoresis tank. ② Pour the electrophoresis buffer into the electrophoresis tank until it is full, and slowly and vertically pull out the comb teeth. ③ Blow and mix the melted protein samples and protein marker well, and sequentially take 5 μl and add it vertically into the sample wells.

[0065] 3) Electrophoresis: Connect the electrophoresis device correctly according to the positive and negative markings, and set the electrophoresis parameters to a constant voltage of 80 V and an electrophoresis time of 2 h.

[0066] 4) Blotting: ① Before the electrophoresis ends, soak the PVDF membrane of the appropriate size in methanol for at least 1 minute, prepare the rapid blotting solution, and soak the blotting filter paper and sponge thoroughly with the blotting solution. ② After the electrophoresis ends, take out the glass sandwich plate, carefully lift the short glass plate and cut off the upper-layer gel, and transfer the lower-layer gel to the filter paper in the original electrophoresis direction. ③ Completely cover the activated PVDF membrane on the lower-layer gel, and use a roller to remove the air bubbles between the two. ④ Continue to cover the PVDF membrane with filter paper and sponge to form a sandwich structure, install the blotting clip in the blotting tank, and pour the blotting solution until it is full. ⑤ Set the blotting parameters to a constant current of 400 mA and a blotting time of 30 minutes.

[0067] 5) Blocking: Prepare 5% skim milk or BSA in advance. After the blotting is completed, put the PVDF membrane into it and incubate it at room temperature on a horizontal shaker for 2 h.

[0068] 6) Primary antibody incubation: After the PVDF membrane is blocked, wash the membrane with TBST solution for 5 minutes and repeat 3 times. Prepare the primary antibody dilution solution with TBST solution according to the recommended optimal ratio in the product manual. Then, put the PVDF membrane into a sealed bag containing the primary antibody solution and incubate it overnight at 4°C.

[0069] 7) Secondary antibody incubation: After the primary antibody incubation, wash the membrane with TBST solution for 5 min and repeat 3 times. Prepare the secondary antibody dilution with TBST solution. In an incubation box, allow the PVDF membrane to fully contact the secondary antibody and incubate at room temperature for 2 h. Finally, wash the membrane with TBST for 5 min and repeat 3 times.

[0070] 8) Luminescence imaging: Prepare the developing solution in advance. Lay the PVDF membrane flat in the center of a black luminescence plate. After uniformly and quickly pipetting the developing solution, place it in the imager. Use the Tanon imaging system to perform luminescence development on the PVDF membrane and save the photos.

[0071] (9) Detection of reactive oxygen species in the RVLM region of rats.

[0072] 1) Sample collection and cryosection: ① The sample collection steps are the same as above. ② When performing cryosection of the RVLM, set the section thickness to 20 μm. After flattening the brain sections, attach them to the adhesive glass slides.

[0073] 2) Incubation: This operation is carried out in the dark throughout. Drop 50 μl of DHE staining solution on each brain section. After incubating at 37 °C for 30 minutes, rinse 3 times in a glass dish containing PBS solution, 1 minute each time. After the slides are dried, drop about 50 μl of anti-fluorescence quenching mounting medium (containing DAPI). Finally, drop a small amount of nail polish on the edge of the coverslip for fixation and store in a humid box.

[0074] 3) Fluorescence imaging: Use a fluorescence microscope to image and statistically analyze the fluorescence intensity in the RVLM region.

[0075] (10) Statistical methods.

[0076] All values are expressed as mean ± standard error (Mean ± SEM). Statistical analysis is performed using Graphpad Prism 8.0 version (GraphPad software, San Diego, CA, USA). For each index after microinjection of Ang-(5-7) into the RVLM of rats, one-way ANOVA is used to analyze the data; for each index after interfering with DPP3 in the RVLM of rats, two-way ANOVA (or mixed model) is used for testing. When the P value < 0.05, the present invention considers the difference to be statistically significant.

[0077] (II) Experimental analysis:

[0078] 1. The effect of Ang-(5-7) in the RVLM on the regulation of cardiovascular activity.

[0079] 1.1 Microinjection of Ang-(5-7) into the RVLM increases renal sympathetic activity and raises blood pressure.

[0080] AsFigure 1 As shown in the figure, in the present invention, different concentrations (0.1, 1, 10, 100 ng / 100 nl) of Ang-(5-7) were acutely microinjected into the bilateral RVLM, and its effects on cardiovascular activities (blood pressure BP, heart rate HR, and renal sympathetic nerve activity RSNA) were observed. Figure 1 A), the results showed that after microinjecting 1, 10, 100 ng / 100 nl of Ang-(5-7) into the RVLM, the blood pressure increased significantly; after microinjecting 10, 100 ng / 100 nl of Ang-(5-7) into the RVLM, both the heart rate and renal sympathetic nerve activity increased significantly. Figure 1 B).

[0081] 1.2 Effects of Ang-(5-7) in the central nervous system on the occurrence and development of hypertension.

[0082] As Figure 2 shown, in the fourth ventricle of WKY rats, Ang-(5-7) (24 μg / d) was continuously perfused for seven days using an ALZET micro-osmotic pump (1007D), and its effects on cardiovascular activities were observed. The results showed that after continuously perfusing Ang-(5-7) in the fourth ventricle for seven days, both the blood pressure and heart rate increased significantly. Figure 2 A). To clarify the efficacy of Ang-(5-7) in regulating blood pressure in the central nervous system, Ang II and Ang-(5-7) were respectively perfused into the fourth ventricle of WKY rats, and their effects on blood pressure BP, heart rate HR, heart rate variability HRV, and baroreflex BRS were observed. The results showed that after continuously perfusing Ang II or Ang-(5-7) in the fourth ventricle for seven days, both the blood pressure and heart rate variability increased significantly. Figure 2 A), the baroreflex function decreased significantly. Figure 2 B), and there was no statistical difference between the two groups. Perfusing Ang-(5-7) into the ventricle increased renal sympathetic nerve activity and elevated blood pressure.

[0083] 1.3 Ang-(5-7) in the RVLM partially acts on the AT1 receptor to affect blood pressure.

[0084] As Figure 3 shown, after pre-injecting losartan (1 nmol) into the RVLM of WKY rats to block the AT1 receptor and then injecting Ang-(5-7), it was found that the pressor effect of Ang-(5-7) and renal sympathetic nerve activity could be partially blocked, while there was no obvious statistical difference in heart rate.

[0085] 2. Mechanism of the role of Ang-(5-7) in the RVLM in regulating cardiovascular activities.

[0086] 2.1 Ang-(5-7) in the RVLM affects blood pressure through oxidative stress.

[0087] To further clarify the regulatory mechanism of the blood pressure increase by Ang-(5-7) in the RVLM, after pre-microinjecting tempol into the RVLM of WKY rats to block the synthesis of reactive oxygen species (ROS), and then injecting Ang-(5-7), it was found that the pressor effect of Ang-(5-7) and renal sympathetic activity could be partially blocked ( Figure 4 ), while there was no significant statistical difference in heart rate. Using an ALZET micro-osmotic pump (1007D) to continuously perfuse Ang-(5-7) (24 μg / d) into the fourth ventricle of WKY rats for seven days, the effect on oxidative stress in the RVLM was observed by Western Blot and a reactive oxygen species detection kit. The results showed that after continuously perfusing Ang II or Ang-(5-7) into the fourth ventricle for seven days, the levels of reactive oxygen species (ROS) in both groups increased significantly ( Figure 5 A and 5B), while there were no significant differences in the expression levels of Nrf2, keap1, HO1, and NQO1 ( Figure 6 ).

[0088] 2.2 Endogenous overexpression of DPP3 in the RVLM increases Ang-(5-7) and leads to enhanced oxidative stress.

[0089] After overexpressing DPP3 in the bilateral RVLM of WKY rats, the reactive level in the RVLM was detected using a reactive oxygen species detection kit. The results showed that the ROS level increased significantly after DPP3 overexpression ( Figure 7 ).

[0090] 2.3 Interfering with DPP3 endogenously in the RVLM to reduce Ang-(5-7) can alleviate oxidative stress in hypertensive rats.

[0091] After interfering with DPP3 in the bilateral RVLM of SHR rats, the reactive level in the RVLM was detected using a reactive oxygen species detection kit. It was found that the ROS level decreased significantly after endogenous reduction of Ang-(5-7) by DPP3 interference ( Figure 8 ).

[0092] Although the preferred embodiments of the present invention are disclosed for illustration, those of ordinary skill in the art understand that various improvements, additions, and substitutions are possible without departing from the scope and spirit defined by the appended claims of the present invention, and all are within the protection scope of the present invention.

Claims

1. Use of central angiotensin-(5-7) in the preparation of a hypertensive drug, characterized in that The amino acid sequence of angiotensin-(5-7) is: isoleucine - histidine - proline.

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