Application of angiotensin Ang-(1-7) in prevention, improvement and treatment of rheumatoid arthritis

Abstract

The invention discloses an application of angiotensin Ang-(1-7) in preparation of a medicine for preventing rheumatoid arthritis, an application in preparation of a medicine for improving or treating rheumatoid arthritis and an application in preparation of a medicine for inhibiting generation of nebula of rheumatoid arthritis joints. The invention discloses that angiotensin Ang-(1-7) can inhibit synovial vascularization in rheumatoid arthritis for the first time, and research proves that Ang-(1-7) has an obvious inhibiting effect on activation of human umbilical vein endothelial cells (HUVECs), has an obvious effect on inhibiting synovial vascularization in CIA mice, has no obvious toxicity to mice, and can be used for preparing medicines for treating rheumatoid arthritis. The traditional Chinese medicine composition has a good prospect in prevention, improvement and treatment of rheumatoid arthritis. The invention provides a novel potential optional medicine for treating rheumatoid arthritis, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of angiotensin Ang-(1-7) in preventing, improving and treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease with erosive, symmetrical polyarthritis as the main clinical manifestation. The global prevalence is 0.25%-1%. It can cause disability and malformation and usually requires lifelong treatment. The main pathological characteristics of RA are immune activation causing synovial inflammation and proliferation, synovial neovascularization, and ultimately the formation of pannus, which in turn causes cartilage and bone destruction. Pannus is composed of new microvessels, hyperplastic synovial cells, inflammatory cells and organized cellulose, and has characteristics similar to tumor tissue. It is the main cause and pathological basis for joint lesions and cartilage destruction. Angiogenesis is an important sign of RA disease progression and a sign of the transition from acute inflammation to chronic inflammation. New blood vessels provide oxygen and nutrients to proliferating inflammatory cells, maintaining the chronic inflammatory state of RA. Therefore, inhibiting angiogenesis may be a potential target for the treatment of RA.

[0003] Angiotensin is a class of peptide substances that have extremely strong effects of constricting blood vessels and stimulating the adrenal cortex to secrete aldosterone. It participates in the regulation of blood pressure and body fluids and can be divided into angiotensin I-VII. Angiotensin 1-7 [Angiotensin1-7, Ang-(1-7)] is mainly synthesized by ACE2 hydrolysis of angiotensin II and exerts its effects by binding to the specific receptor MAS. Previous studies have shown that Ang-(1-7) has anti-inflammatory, anti-proliferative, glucose metabolism pattern regulation, and anti-fibrosis effects; the ACE2 / Ang-(1-7) / MAS axis can inhibit the activation of MAPKs and NF-κB signaling pathways in the ankle joint tissue of CIA mice, and improve joint inflammation and bone destruction.

[0004] The Hippo signaling pathway is an important signaling pathway that regulates cell proliferation and differentiation, organ development, and tissue regeneration in mammals. The Hippo pathway is composed of the MAPK family, MST1 / 2, LATS1 / 2, and transcriptional coactivators YAP and TAZ. Studies have shown that YAP and TAZ are upregulated in RA-FLs, and knocking down YAP and TAZ can inhibit the migration and invasion of RA-FLS (rheumatoid arthritis fibroblast-like synoviocytes) by inducing autophagy. Verteporfin (VP) is a YAP inhibitor. VP treatment can significantly inhibit the invasive phenotype of FLS, inhibit synovial hyperplasia in the joints of AIA rats, and improve the severity of arthritis.

[0005] There is no report on the use of Ang-(1-7) for synovial angiogenesis in rheumatoid arthritis. Summary of the invention

[0006] The purpose of the present invention is to provide an application of angiotensin Ang-(1-7) in preventing, improving and treating rheumatoid arthritis in view of the above problems.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of angiotensin Ang-(1-7) in the preparation of a medicament for preventing rheumatoid arthritis.

[0009] The second aspect of the present invention provides the use of angiotensin Ang-(1-7) in the preparation of a medicament for improving or treating rheumatoid arthritis.

[0010] The third aspect of the present invention provides the use of angiotensin Ang-(1-7) in the preparation of a medicament for inhibiting the formation of pannus in rheumatoid arthritis joints.

[0011] In any of the above-mentioned application technical solutions, the Ang-(1-7) improves joint damage in rheumatoid arthritis.

[0012] In the above-mentioned application technology scheme, the Ang-(1-7) improves rheumatoid arthritis joint damage by inhibiting synovial angiogenesis.

[0013] In any of the above-mentioned application technical solutions, the Ang-(1-7) inhibits HUVECs cell proliferation, migration, invasion, and tube-forming ability.

[0014] In the above-mentioned application technology scheme, the Ang-(1-7) improves rheumatoid arthritis joint damage by regulating the Hippo signaling pathway.

[0015] In the above-mentioned application technology scheme, the Ang-(1-7) promotes the expression of p-LATS and p-YAP proteins.

[0016] The beneficial effects of the present invention are as follows: the present invention discloses for the first time that angiotensin Ang-(1-7) can inhibit synovial angiogenesis in rheumatoid arthritis, and studies have confirmed that Ang-(1-7) has a significant inhibitory effect on the activation of human umbilical vein endothelial cells (HUVECs), has a significant effect on inhibiting synovial angiogenesis in CIA mice, and has no obvious toxicity to mice, and has good prospects for application in the prevention, improvement and treatment of rheumatoid arthritis. The present invention provides a new potential optional drug for the treatment of rheumatoid arthritis, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effects of Ang-(1-7) on joint damage in CIA mice are shown.

[0018] Figure 2 This is a HE staining image of mouse knee joint.

[0019] Figure 3 Representative images and statistical analysis results of CD31 immunohistochemical staining of knee joints of CIA mice.

[0020] Figure 4 These are representative images and statistical analysis results of the Edu experiment.

[0021] Figure 5 These are the images of HUVEC cells migrating at 0 hours and 24 hours and the statistical analysis results of the migration area.

[0022] Figure 6 Representative images and statistical analysis results of Transwell experiments.

[0023] Figure 7 These are representative images and statistical analysis results of the HUVEC tube formation experiment.

[0024] Figure 8 These are the results of protein expression and phosphorylation levels of LATS and YAP in HUVECs. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] The research route of the present invention is: using tumor necrosis factor α (TNF-α) to induce HUVEC cell activation, constructing a type II collagen-induced arthritis (CIA) mouse model, intervening with Ang-(1-7), and detecting the effect of Ang-(1-7) on inhibiting HUVEC cell activation and improving synovial angiogenesis in CIA mice by regulating the activation of the Hippo pathway.

[0028] The specific research is as follows:

[0029] Example 1

[0030] 1. Main experimental reagents

[0031] Chicken type II collagen was purchased from Chondrex (USA); angiotensin Ang-(1-7) was purchased from APExBIO (USA); YAP, p-YAP, LATS, and p-LATS antibodies were purchased from CST (USA); and CCK-8 reagent was purchased from Dojindo Chemical Co., Ltd. (Japan).

[0032] 2. Implementation Methods

[0033] 1. Establishment and intervention of CIA model

[0034] The collagen-induced arthritis (CIA) model was established using DBA / 1 mice by immunization on days 1 and 21. Chicken collagen type II was mixed with an equal volume of complete Freund's adjuvant to form an emulsion-like mixture, and 0.1 ml was injected intradermally at the base of the tail of DBA / 1 mice. On day 21, a secondary immunization was performed using chicken collagen type II emulsified in the same volume of incomplete Freund's adjuvant. The control group was given a subcutaneous injection of normal saline into the tail as a control. Starting from day 28, the CIA+Ang-(1-7) group received intraperitoneal injection of Ang-(1-7) (2.0 mg / kg) once a day for 2 weeks; the control group and CIA group received intraperitoneal injection of normal saline as a control.

[0035] 2. Cell culture

[0036] Add RPMI-1640 medium containing 10% serum and 1% penicillin-streptomycin double antibody to human umbilical vein endothelial cells, culture in a 5% CO2 37°C incubator, and subculture the cells when the cell density grows to about 90%. When the cells show logarithmic growth, the experiment can be carried out.

[0037] 3. EdU cell proliferation assay

[0038] HUVEC were seeded into 96-well plates. After the cells adhered to the wall, the cells were intervened with Ang-(1-7) or with or without TNF-α for 24 hours according to the grouping; then an equal volume of EDU working solution was added for labeling and culture was continued for 4 hours. Then the culture medium was removed, the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and then washed three times with BSA washing solution. Then the Click-iT reaction mixture was prepared, and the Click-iT reaction mixture was added to each sample and incubated at room temperature in the dark. After 30 minutes, the reaction mixture was removed and the cells in the wells were washed with BSA washing solution for 5 minutes. Finally, the electron fluorescence microscope was used to take pictures and count.

[0039] 4. Cell Migration

[0040] When the monolayer cells in the 6-well plate reached 100% confluence, scratches were made with a 200 μL sterile pipette tip. The cells were rinsed several times with PBS to remove detached and damaged cells. The cells were then placed in serum-free medium and continued to be cultured for 0 hours and 24 hours. The scratches at fixed positions were photographed, and the cell migration area was analyzed using Image J software to calculate the cell migration ability.

[0041] 5. Transwell assay

[0042] The cells were treated with medium containing different drugs for 24 h, and then suspended in serum-free medium and cultured at 1 × 10 5 The cells were placed in a Transwell chamber pre-coated with Matrigel at a density of 10 cells / well. The cell chamber was placed in complete medium and cultured for 36 hours. The migrating cells were fixed in methanol and stained with 0.5% crystal violet. The migrating cells in three randomly selected areas were counted and photographed under a microscope.

[0043] 6. Tube Formation Experiment

[0044] 50 μl of matrix gel was added to a 96-well plate and incubated at 37°C for 1 hour. HUVECs (2×10 5 Cells / ml) were seeded in 96-well plates pre-coated with matrix gel and incubated for 6 hours. Images of tubular morphology were captured using a microscope, and the ability to quantify tubular formation was analyzed by counting the number of nodes using Image J analysis software.

[0045] 7. Western blot

[0046] Total protein was extracted from HUVECs (human umbilical vein endothelial cells) using RIPA lysis buffer containing protease inhibitors, phosphatase inhibitors, and PMSF, and the protein concentration was determined using a BCA protein concentration assay kit. The samples were boiled in SDS loading buffer. Equal amounts of protein were separated using SDS-PAGE and transferred to a PVDF membrane. Next, the PVDF membrane was blocked for 1 hour at room temperature and stained with primary antibodies overnight at 4°C. The next day, the corresponding species-specific secondary antibodies were incubated for 60 minutes. Finally, the protein-antibody complex was visualized using an ECL enhancement kit.

[0047] 8. Immunohistochemical staining

[0048] After fixation and decalcification of the knee joint, the tissue was dehydrated in gradient alcohol, embedded in paraffin, and made into 5μm sections. The sections were dewaxed in xylene, dehydrated in gradient ethanol, microwaved for 15 minutes to extract antigens, blocked with serum for 30 minutes, and then the primary anti-CD31 antibody was added. The mixture was incubated at 4°C overnight. The next day, the biotin-labeled secondary antibody was incubated at room temperature for 30 minutes, rinsed three times with PBS, and the horseradish enzyme-labeled drop was added with streptavidin working solution, rinsed three times with PBS, stained with DBA, stained with hematoxylin, dehydrated with gradient alcohol, and sealed with neutral resin. Finally, the tissue staining results were observed under an optical microscope.

[0049] 9. Statistical analysis

[0050] Statistical analysis was performed using commercial software (GraphPadPrism 10.0.0; GraphPad Software, San Diego, CA, USA). Each experiment was repeated at least 3 times. Data are presented as mean ± standard deviation (SD). The comparison between two groups was performed using t-test, and the comparison between multiple groups was performed using one-way analysis of variance. A p value of 0.05 was considered statistically significant.

[0051] (P.Value<0.05 is indicated by *; P.Value<0.01 is indicated by **; P.Value<0.001 is indicated by ***)

[0052] 3. Results

[0053] 1. Effects of Ang-(1-7) on joint damage in CIA mice

[0054] Macroscopic observation showed that Ang-(1-7) reduced the redness and swelling of the hind paw joints of mice ( Figure 1 HE staining showed that the synovial tissue of the CIA model group mice had obvious hyperplasia and invaded the joint cavity. The inflammatory cells in the synovial tissue increased significantly, and angiogenesis increased, accompanied by collagen deposition and cartilage destruction. Ang-(1-7) improved the synovial hyperplasia, inflammatory cell infiltration, angiogenesis and cartilage destruction in the joints of CIA mice ( Figure 2 ).

[0055] 2. Effect of Ang-(1-7) on synovial angiogenesis in CIA mice

[0056] In the CIA group, immunohistochemical staining showed that the endothelial cell marker CD31 was highly expressed in the synovium, while the expression of CD31 in the synovium was significantly decreased after Ang-(1-7) intervention ( Figure 3 ).

[0057] 3. Effect of Ang-(1-7) on HUVEC cell proliferation

[0058] CCK-8 assay was used to detect the proliferation ability of HUVEC cells. The results showed that the proliferation ability of HUVECs increased after TNF-α induction, and the proliferation of HUVECs decreased after Ang-(1-7) intervention ( Figure 4 ).

[0059] 4. Effect of Ang-(1-7) on HUVEC migration

[0060] Compared with the Control group, TNF-α induced an enhanced migration ability of HUVECs, while Ang-(1-7) intervention significantly reduced the migration ability of HUVECs ( Figure 5 ).

[0061] 5. Effect of Ang-(1-7) on HUVEC invasion

[0062] The invasion ability of HUVEC cells was detected by Transwell assay. The results showed that TNF-α induced an enhanced invasion ability of HUVECs, while Ang-(1-7) intervention decreased the invasion ability of HUVECs. Figure 6 ).

[0063] 6. Effect of Ang-(1-7) on HUVEC tube-forming ability

[0064] To evaluate the effect of Ang-(1-7) on the angiogenesis ability of HUVECs, we performed a matrix gel tube formation assay and evaluated the effect of Ang-(1-7) on the tube formation ability of HUVECs by the number of nodes. The results showed that Ang-(1-7) inhibited the tube formation ability enhanced by TNF-α ( Figure 7 ).

[0065] 7. Effect of Ang-(1-7) on the expression of Hippo pathway in HUVEC

[0066] We used Western blot to detect the expression of Hippo-YAP pathway-related proteins in HUVECs. Compared with the Control group, TNF-α and Ang-(1-7) intervention had no effect on the expression of LATS1 and YAP total proteins. However, after TNF-α intervention, the expression of p-LATS and p-YAP decreased significantly, and Ang-(1-7) restored the protein expression of p-LATS and p-YAP ( Figure 8 ).

[0067] The above results demonstrate that Ang-(1-7) can improve rheumatoid arthritis joint damage by inhibiting synovial angiogenesis, which may be achieved by regulating the Hippo signaling pathway.

Claims

1. Application of angiotensin Ang-(1-7) in the preparation of drugs for preventing rheumatoid arthritis.

2. Use of angiotensin Ang-(1-7) in the preparation of drugs for improving or treating rheumatoid arthritis.

3. Use of angiotensin Ang-(1-7) in the preparation of a drug for inhibiting pannus formation in rheumatoid arthritis joints.

4. The use according to any one of claims 1 to 3, characterized in that: The Ang-(1-7) improves rheumatoid arthritis joint damage.

5. The use according to claim 4, characterized in that: The Ang-(1-7) improves rheumatoid arthritis joint damage by inhibiting synovial angiogenesis.

6. The use according to any one of claims 1 to 3, characterized in that: The Ang-(1-7) inhibits HUVECs cell proliferation, migration, invasion, and tube-forming ability.

7. The use according to claim 6, characterized in that: The Ang-(1-7) improves rheumatoid arthritis joint damage by regulating the Hippo signaling pathway.

8. The use according to claim 7, characterized in that: The Ang-(1-7) promotes the expression of p-LATS and p-YAP proteins.

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