CN115944737B - Application of MAP-2 inhibitor in preparation of medicine for treating hypertension - Google Patents

Abstract

The invention discloses the application of a MAP-2 inhibitor in the preparation of medicine for treating hypertension. The present invention finds that injection of AAV-retro-MAP-2 virus into perirenal fat can regulate hypertension in SHR rats and DOCA-Salt model rats and reverse kidney and blood vessel damage. This effect can also be achieved by intraperitoneal injection of MAP-2 siRNA in SHR rats. At present, clinically, hypertension is mostly treated with drugs, and patients need to take one or more high-frequency drugs for a long time to control blood pressure. However, the sustained pharmacological effects of the MAP-2 knockdown used in the present invention in the treatment of hypertension may support semi-annual or possibly annual administration to control blood pressure. Less frequent dosing compared with current regimens may help improve medication adherence, an important part of maintaining blood pressure control.

Description

Application of MAP-2 inhibitor in preparation of medicine for treating hypertension

technical field

The invention relates to the application of MAP-2 inhibitors in the preparation of medicines for treating hypertension.

Background technique

Hypertension is an important risk factor for congestive heart failure, stroke, and end-stage renal disease. According to statistics, about 1 in 10 adults in the world suffers from high blood pressure, and the incidence of high blood pressure can reach 100% in some people over the age of 10. It is estimated that by 2010, the number of hypertensive patients worldwide will rise to hundreds of millions of people. Unfortunately, only about one-third of the patients achieved antihypertensive treatment. The main reason is that hypertension is a lifelong disease, and the half-life of all current antihypertensive drugs is relatively short, and the effective time of action is no more than one hour, so it is necessary to take the medicine every day, and it is difficult for patients to persist. In addition, the non-specificity and side effects of drugs also make it difficult to achieve the desired goal in the treatment of hypertension. Therefore, finding a long-term effective way to control hypertension by regulating gene expression in the body, that is, gene therapy, has always been the goal and direction of medical scientists.

Small interfering RNA (small interfering RNA, siRNA) is the initiator of RNA interference, which stimulates the silencing of the complementary target mRNA, which is of great significance to gene regulation and disease treatment.

In the field of neuroscience, labeling or manipulating projection neurons is an essential research tool for studying neural circuits. At present, DiI, a red fluorescent dye, can be used to antegradely label neurons to study the descending projection circuit of the high center; or use Fluoro-Gold to reversely label neurons to study neuronal projections of peripheral muscles at the level of the spinal cord , or the projection loop of the spinal cord conduction tract in the center and so on. However, these fluorescent dye labeling methods can only label conduction bundles, but cannot study the function of conduction bundles by up-regulating or down-regulating gene expression. In this context, AAV2-retro came into being. In 2016, an article published in Neuron, an authoritative journal in the field of neuroscience, described the construction method and application of this AAV2-retro virus.

Perinephric adipose tissue is an atypical visceral fat pad with an intact blood supply, lymphatic drainage, and innervation system. Epidemiological studies have shown that perirenal fat is a risk predictor for hypertension. Our previous study also found that both specific disruption of afferent nerve endings in perirenal adipose tissue and fat clearance can reduce blood pressure, while injection of capsaicin or complete Freund's adjuvant (CFA) in perirenal adipose tissue can increase blood pressure. This suggests that perirenal adipose tissue may be one of the key tissues involved in the regulation of hypertension.

Contents of the invention

The object of the present invention is to provide the application of MAP-2 inhibitor in the preparation of medicine for treating hypertension.

The technical solution adopted by the present invention is: the application of MAP-2 inhibitors in the preparation of drugs for the treatment of hypertension, wherein the GeneID of MAP-2: 25595; the website is https://www.ncbi.nlm.nih.gov/ gene/25595.

Preferably, the MAP-2 inhibitor is AAV-retro-MAP-2 virus.

Preferably, the MAP-2 inhibitor is siRNA of MAP-2.

Preferably, the administration site of the AAV-retro-MAP-2 virus is perirenal fat.

The invention also discloses the application of the AAV-retro-MAP-2 virus in the preparation of medicines for treating kidney or blood vessel injury diseases.

Preferably, the administration site of the AAV-retro-MAP-2 virus is perirenal fat.

The present invention finds that injecting AAV-retro-MAP-2 virus into perirenal fat can regulate hypertension in SHR rats and DOCA-Salt model rats and reverse kidney and blood vessel damage. This effect can also be achieved by intraperitoneal injection of siRNA of MAP-2 in SHR rats. At present, clinically, hypertension is mostly treated with drugs, and patients need to take one or more high-frequency drugs for a long time to control blood pressure. However, the sustained pharmacological effects of the MAP-2 knockdown used in the present invention in the treatment of hypertension may support semi-annual or possibly annual administration to control blood pressure. Less frequent dosing compared with current regimens may help improve medication adherence, an important part of maintaining blood pressure control.

Description of drawings

Figure 1. Spontaneously hypertensive rats (SHR) were intraperitoneally injected with MAP-2 RNAi every 3 days, and the arterial blood pressure was monitored continuously once a week for 38 days. Systemic knockdown of MAP-2 in SHR rats can significantly reduce blood pressure levels. A: Statistical chart of changes in systolic blood pressure (SBP). B: Statistical chart of changes in diastolic blood pressure (DBP). C: Statistical graph of mean arterial pressure (MAP) changes. D: Comparison of mRNA levels of MAP-2 gene in dorsal root ganglia of two groups of rats. (4 in each group, P<0.05)

Figure 2. Monitoring blood pressure and heart rate for 16 weeks (1 time/week) after injection of AAV-retro-MAP-2 virus into the perirenal fat of SHR rats. Injection of AAV-retro-MAP-2 virus into the perirenal fat of SHR rats knocked down the level of MAP-2 in the dorsal root ganglion (DRG) and significantly reduced the blood pressure. A: Statistical chart of changes in systolic blood pressure (SBP). B: Statistical chart of changes in diastolic blood pressure (DBP). C: Statistical graph of mean arterial pressure (MAP) changes. D: Statistical chart of heart rate (Heartrate) changes. (10 rats in each group, P<0.05)

Fig. 3. The renal hypertension model was established after unilateral nephrectomy+DOCA-Salt (DOCA salt) induction of SD (Sprague Dawley) rats for 5 weeks. After modeling, compared with the control group, the blood pressure of DOCA-Salt rats was significantly increased, and the renal function was significantly decreased. A: Creatinine (CREA) content in the blood of the two groups. B: Statistical chart of changes in systolic blood pressure (SBP). C: The blood urea nitrogen (BUN) content of the two groups. D: Statistical graph of mean arterial pressure (MAP) changes. (10 rats in each group, P<0.05)

Figure 4, A: Dorsal root ganglia (L1 segment) of DOCA-Salt rats were stained with MAP-2 antibody by immunofluorescent staining. The expression of MAP-2 in the dorsal root ganglion (L1 segment) of DOCA-Salt rats was significantly enhanced. B: Statistical graph based on the MAP-2 positive rate in A. (n=3 for each group, P<0.05)

Figure 5. Blood pressure was monitored for 6 weeks (once a week) after AAV-retro-MAP-2 virus was injected into the perirenal fat of DOCA-Salt rats. Injecting AAV-retro-MAP-2 virus into the perirenal fat of DOCA-Salt rats can significantly reduce the renal hypertension induced by DOCA-Salt. A: Statistical chart of changes in systolic blood pressure (SBP). B: Systolic blood pressure (SBP) levels of the four groups at week 6. C: Statistical graph of mean arterial pressure (MAP) changes. D: Mean arterial pressure (MAP) levels of the four groups at week 6. (8 rats in the control group (Sham); 10 rats in the DOCA-Salt group; 9 rats in the AAV-retro-MAP-2 group; 10 rats in the DOCA-Salt+AAV-retro-MAP-2 group, P<0.05)

Figure 6, A: Immunofluorescent staining of MAP-2 on the 5 segments of dorsal root ganglion (T11-L2) of these four groups of rats. B: Make statistics on the staining results. AAV-retro-MAP-2 virus knocked down the dorsal root ganglia at the T12-L1 segment, suggesting that these three segments may be projection segments of the perirenal fat. (3 in each group, P<0.05)

Figure 7, A: The blood urea nitrogen (BUN) content of the four groups. B: Creatinine (CREA) content in the blood of the four groups. (8 rats in the control group (Sham); 10 rats in the DOCA-Salt group; 9 rats in the AAV-retro-MAP-2 group; 10 rats in the DOCA-Salt+AAV-retro-MAP-2 group, P<0.05). Injecting AAV-retro-MAP-2 virus into the perirenal fat of DOCA-Salt rats can significantly improve the renal function decline caused by DOCA-Salt. C: Masson and HE staining of kidney tissues of four groups of rats. D: Statistical map of Masson's area. (3 in each group, P<0.05). Perinephric fat injection of AAV-retro-MAP-2 virus in rats can significantly improve renal fibrosis induced by DOCA-Salt.

Figure 8, A: TUNEL staining of kidney tissues in four groups. B: Statistical diagram of TUNEL staining positive cells. C: Western Blot for fibrosis indicators (COL-1, TGF-β, α-SMA) and apoptosis indicators (Caspase-3, Bax, BCL-2). D: Western Blot statistics. (3 in each group, P<0.05). Injection of AAV-retro-MAP-2 virus into the perirenal fat of DOCA-Salt rats can significantly improve the apoptosis and fibrosis of renal cells induced by DOCA-Salt.

Figure 9, A: Kidney body weight ratio of four groups of rats. (6 rats in each group, P<0.05) B: Kidney blood flow speckle experiment of rats in four groups. C: Statistical graph of the blood flow map. D: Electron micrographs of renal cortex in four groups of kidneys. (White arrows: basement membrane; black arrows: foot processes; red stars: apoptotic cells.) (3 rats in each group, P<0.05). AAV-retro-MAP-2 was injected into the perirenal fat of DOCA-Salt rats The virus can significantly improve renal hypertrophy, glomerular basement membrane thickening, foot process fusion, and apoptosis and insufficiency caused by DOCA-Salt.

Fig. 10: Ex vivo contraction and relaxation experiments of the mesenteric arteries of four groups of rats. A: Endothelium-dependent vasoconstriction experiment. B: Endothelium-independent vasoconstriction test. C: Endothelium-dependent vasodilation assay. D: Endothelium-independent vasodilation experiment. (3 in each group, P<0.05).

Figure 11: A: Blood flow speckle experiments were performed on the mesenteric arteries of four groups of rats. B: Statistical graph of the blood flow map. C: Western Blot western blot of mesenteric artery fibrosis indicators (COL-1, TGF-β, α-SMA). D: Western Blot statistics. (3 in each group, P<0.05)

Detailed ways

In order to further explain the technical means and effects of the present invention to achieve the intended purpose of the invention, the specific implementation, structure, features and effects of the present invention will be described in detail below in conjunction with the accompanying drawings and preferred embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

As shown in Figure 1, we intraperitoneally injected MAP-2 RNAi (Ribobio, Nanjing, Jiangsu) into 8-week-old male spontaneously hypertensive rats (SHR, Victoria Lihua) once every 3 days, and performed weekly Continuous tail artery blood pressure monitoring for 38 days. After blood pressure statistics, we found that systemic knockdown of MAP-2 in SHR rats could significantly reduce blood pressure levels (Fig. 1A, B and C). Then we euthanized the rats, took the dorsal root ganglion, extracted tissue RNA, and detected the mRNA level of MAP-2 gene in the dorsal root ganglion by fluorescent real-time quantitative PCR (RT-PCR), and found that compared with the control group The mRNA level of MAP-2 in the DRG of SHR rats injected with MAP-2 RNAi was significantly decreased (Fig. 1D).

This suggests that knockdown of MAP-2 can reduce spontaneous hypertension.

Example 2

The construction method of neuroretrograde virus AAV-retro-MAP-2 is as follows: first establish an empty vector H12663pAAV-U6-spgRNAv2.0[shRNA]-CMV-EGFP-WPRE-spolyA (Hanbio, Shanghai), and then insert the target Gene MAP2 (target: GCATAACAGTACCTAGCAT), the upstream and downstream cloning restriction sites are BsmBI and NheI, respectively, so that the original copy is generated after construction: pAAV-U6-shRNA(Map2)-CMV-EGFP-WPRE-spolyA. As shown in Figure 2, the neuroretrograde virus AAV-retro-MAP-2 was also injected in the perirenal fat of SHR rats. The aim was to knock down the expression of the MAP-2 gene in the dorsal root ganglia projected by the perirenal fat. We monitored blood pressure and heart rate for 16 weeks (1 time/week), and found that the hypertension level (Fig. 2A, B and C) and heart rate (Fig. 2D) were significantly reduced in SHR rats. Blood pressure and heart rate remained stable for as long as 16 weeks.

This suggests that knockdown of MAP-2 in dorsal root ganglia projected from perirenal fat can reduce spontaneous hypertension.

Example 3

As shown in Figure 3, in order to further explore whether the knockdown of MAP-2 gene in the DRG projected by the perirenal fat has the same effect on other types of hypertension, we constructed SD rats (8 weeks old, Weitongli Hua) model of renal hypertension. After unilateral nephrectomy, SD rats were fed with 1% saline and subcutaneously injected with deoxycorticosterone acetate (DOCA, 10 mg/2 times/week, Sigma, USA) for a total of five weeks. Kidney function was monitored by monitoring blood creatinine and blood urea nitrogen levels (Fig. 3A and C), and blood pressure was monitored weekly (Fig. 3B and D). We found that the blood pressure, serum creatinine and blood urea nitrogen levels of rats in the model group were significantly higher than those in the control group.

At the same time, we used fluorescent real-time quantitative PCR (RT-PCR) to detect the mRNA level of MAP-2 gene in the dorsal root ganglia of the two groups of rats, and found that compared with the control group, the model group significantly increased (Figure 4A and B ).

We randomly selected half of the rats in these two groups to inject AAV-retro-MAP-2 into their perirenal fat, and continuously monitored blood pressure for 6 weeks. We found that the simple injection of AAV-retro-MAP-2 virus does not affect the blood pressure of rats, and if the virus is injected into renally hypertensive rats, the blood pressure can be significantly reduced in the first week, and it has been stable. Six weeks (Figure 5).

In order to verify the DRG segment of perirenal fat projection and the knockout level of MAP-2 in each segment, we took the T11 to L2 segments of four groups of rats, and performed immunofluorescence staining on them for MAP-2 gene. We found that the DRG of perirenal fat projection mainly consisted of three segments: T12, T13 and L1. The knockout of MAP-2 was most significant in T13 and L1 (Fig. 6).

We examined the serum creatinine and urea nitrogen levels in four groups of rats, and found that injection of AAV-retro-MAP-2 into the perirenal fat significantly reduced the serum creatinine and urea nitrogen levels in the renohypertensive rats (Fig. 7A and B) . Masson and HE staining also demonstrated that perirenal fat injection of AAV-retro-MAP-2 could significantly reduce renal fibrosis in renally hypertensive rats (Fig. 7C and D).

TUNEL staining on kidney tissue (Beyond, Shanghai) showed that perirenal fat injection of AAV-retro-MAP-2 could significantly reduce renal cell apoptosis in renally hypertensive rats (Figure 8A and B). The results of Western Blot on some protein indicators of fibrosis and apoptosis also verified the above results (Fig. 8C and D).

We measured and calculated the kidney weight-to-body weight ratio of the four groups of rats, and the knockdown of MAP-2 in DRG could also significantly improve the renal hypertrophy in the rats with renal hypertension (Fig. 9A). Blood flow speckle experiments showed that knockdown of MAP-2 in DRG could significantly increase renal blood flow in renally hypertensive rats (Fig. 9B and C). By electron microscopy, we observed that knockdown of MAP-2 in DRG could improve the thickening of glomerular basement membrane, fusion of podocytes and apoptosis in renal hypertensive rats (Fig. 9D).

As shown in Fig. 10, we conducted isolated vascular ring experiments on the mesenteric arteries of four groups of rats to detect their contraction and relaxation abilities. We used endothelin (ET-1) (Fig. 10A) and norepinephrine (NA) (Fig. 10C) and acetylcholine (Ach) (FIG. 10D) to verify endothelium-independent and endothelium-dependent vasodilation capabilities, respectively. We found that renohypertension impaired the endothelium-dependent relaxation capacity of mesenteric arteries, whereas knockdown of MAP-2 in DRG improved its function (Fig. 10D).

Blood flow speckle experiments on mesenteric arteries showed that knockdown of MAP-2 in DRG could significantly increase the blood flow of mesenteric arteries in reno-hypertensive rats (Fig. 11A and B). The results of Western Blot on some protein indicators of fibrosis also showed that the knockdown of MAP-2 in DRG can significantly improve the fibrosis of mesenteric artery in rats with renal hypertension (Fig. 11C and D).

In summary, knockdown of MAP-2 in DRG can significantly reduce the blood pressure of renohypertensive rats, and also improve the damage of renal function and vascular function in renohypertensive rats.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art , without departing from the scope of the technical solution of the present invention, when the technical content disclosed above can be used to make some changes or be modified into equivalent embodiments with equivalent changes, but as long as it does not depart from the technical solution of the present invention, the technical content of the present invention In essence, any brief modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solution of the present invention.

Claims (2)

1. Use of a MAP-2 inhibitor in the manufacture of a medicament for the treatment of a hypertensive disorder, said MAP-2 inhibitor being the vector AAV-retro-MAP-2 virus loaded with shRNA of MAP-2; or the MAP-2 inhibitor is siRNA of MAP-2, and the sequence of the MAP-2 inhibitor is GCACAGAGACTCCGGATAT.
2. 2. The use according to claim 1, characterized in that: the administration site of the AAV-retro-MAP-2 virus is perirenal fat.