Use of tsaigwan cyclic peptide B in the preparation of a drug for treating or alleviating diabetic retinopathy

Abstract

This invention discloses the application of Codonopsis pilosula cyclic peptide B in the preparation of drugs for treating or alleviating diabetic retinopathy, relating to the field of biomedicine. This invention reveals that Codonopsis pilosula cyclic peptide B has a protective function against retinal blood vessels in diabetic retinopathy and plays a positive role in the treatment of diabetic retinopathy, potentially providing new theoretical basis and new drug therapeutic targets for future treatments of diabetic retinopathy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of Codonopsis pilosula cyclic peptide B in the preparation of drugs for treating or alleviating diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is an eye disease caused by chronic, progressive damage to the microvessels of the retina due to long-term high blood sugar, blood pressure, blood lipids, and other abnormalities related to diabetes. It leads to gradual vision loss and is one of the most common and serious complications of diabetes, becoming a leading cause of blindness. With the increasing duration of diabetes and the aging population, the incidence of DR is rising year by year. The pathogenesis of diabetic retinopathy is not yet fully understood, but it is generally believed to be related to damage to the retinal microvessels.

[0003] In diabetic patients, the body's long-term hyperglycemia can cause microvascular complications, initially damaging the capillary network. The pathological process is as follows: thickening of the capillary basement membrane → pericyte damage (accumulation of aldose reductase within the cell) → endothelial cell damage → formation of microaneurysms → capillary occlusion → tissue hypoxia → worsening of capillary occlusion → angiogenesis.

[0004] Early diabetic retinopathy: also known as nonproliferative diabetic retinopathy (NPDR), in which the walls of the microvessels in the retina become fragile and form tiny bumps (microaneurysms). Increased vascular permeability can lead to retinal edema, thickening, and / or exudation. When the central part of the retina (macula) is involved, swelling (macular edema) occurs, which can cause decreased vision.

[0005] Advanced diabetic retinopathy, also known as proliferative diabetic retinopathy (PDR), is characterized by the formation of new blood vessels in the retina. These vessels grow along the inner surface of the retina and penetrate the vitreous humor. These new blood vessels are fragile and prone to bleeding, leading to fibrosis and eventually retinal detachment. In severe cases, it can cause neovascular glaucoma, resulting in permanent vision loss. Traditional treatments include surgical procedures such as laser therapy and vitrectomy, as well as medication. Surgical treatment carries certain risks, while intravitreal injections of anti-VEGF drugs are expensive, and repeated injections may cause retinal neurodegeneration, choroidal capillary atrophy, macular scarring, and even severely impair vision. Therefore, discovering a safe and effective drug is crucial for overcoming this disease.

[0006] *Pseudostellaria heterophylla* (Miq.) Pax ex Pax Hoffm., a plant in the Caryophyllaceae family, is a dried tuberous root with the effects of invigorating qi and strengthening the spleen, promoting body fluid production and moistening the lungs. Clinically, it is generally used to treat diabetes, regulate immune function, and protect the myocardium. *Pseudostellaria heterophylla* cyclic peptide B is one of the main components of this traditional Chinese medicine and is also one of the most studied cyclic peptide components. Modern pharmacological studies have found that *Pseudostellaria heterophylla* has immunomodulatory, hypoglycemic, and myocardial protective effects; however, its application in the treatment of diabetic retinopathy has not yet been studied. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the application of Codonopsis pilosula cyclic peptide B in the preparation of drugs for treating or alleviating diabetic retinopathy. This invention reveals that Codonopsis pilosula cyclic peptide B has a protective effect on retinal vascular endothelial cells in diabetic retinopathy, and is expected to provide new theoretical basis and new drug therapeutic targets for future treatment of diabetic retinopathy.

[0008] The specific technical solution of this invention is as follows:

[0009] On one hand, the present invention provides the use of Codonopsis pilosula cyclic peptide B in the preparation of a drug for treating or alleviating diabetic retinopathy, wherein Codonopsis pilosula cyclic peptide B is cyclic-(glycine-glycine-leucine-proline-proline-proline-isoleucine-phenylalanine).

[0010] On the other hand, the present invention provides the application of Codonopsis pilosula cyclic peptide B in the preparation of drugs that alleviate retinal vascular endothelial cell damage.

[0011] On the other hand, the present invention provides the use of Codonopsis pilosula cyclic peptide B in the preparation of drugs for the prevention and / or treatment of vascular homeostasis disorders following diabetic retinopathy.

[0012] This invention, through administering Codonopsis pilosula cyclic peptide B to diabetic model mice and retinal vascular endothelial cells cultured in high glucose, found that Codonopsis pilosula cyclic peptide B can protect retinal vessels; simultaneously, it enhances the activity of retinal vascular endothelial cells under high glucose conditions and ameliorates high glucose-induced retinal vascular endothelial cell damage. Currently, there are no related reports internationally; therefore, the "protective effect of Codonopsis pilosula cyclic peptide B on retinal vascular endothelial cells in diabetic retinopathy" proposed in this invention is expected to provide new theoretical basis and new drug therapeutic targets for future treatment of diabetic retinopathy.

[0013] Specifically, this invention has found that Codonopsis pilosula cyclic peptide B can alleviate diabetic retinopathy by inhibiting inflammation and angiogenesis. This inhibitory effect is exerted by regulating the ATF4-induced inflammatory pathway.

[0014] Preferably, the drug is an injectable or oral formulation. The dosage is 10–40 mg / ml.

[0015] In this embodiment of the invention, the administration group was given an example of intraperitoneal injection of 25 mg / ml of Codonopsis pilosula cyclic peptide B.

[0016] The present invention also provides a medicament for treating or alleviating diabetic retinopathy, said medicament being either Codonopsis pilosula cyclic peptide B itself or a pharmaceutically acceptable salt thereof.

[0017] The Codonopsis pilosula cyclic peptide B is cyclic-(glycine-glycine-leucine-proline-proline-proline-isoleucine-phenylalanine).

[0018] Preferably, the drug is an injectable or oral formulation. The dosage is 10–40 mg / ml.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention reveals that Codonopsis pilosula cyclic peptide B has a protective function on retinal blood vessels in diabetic retinopathy and plays a positive role in the treatment of diabetic retinopathy. It is expected to provide new theoretical basis and new drug therapeutic targets for the future treatment of diabetic retinopathy. Attached Figure Description

[0021] Figure 1 The changes in body weight and blood glucose in diabetic mice after treatment with Codonopsis pilosula cyclic peptide B were shown; among them, ***p<0.001.

[0022] Figure 2 Fundus images of diabetic mice after treatment with Codonopsis pilosula cyclic peptide B and retinal angiography fluorescein intensity of each group of mice; among them, **p<0.01 model group compared with control group, #p<0.05 treatment group compared with model group.

[0023] Figure 3 The images show retinal patch and capillary density maps of diabetic mice after treatment with Codonopsis pilosula cyclic peptide B; **p<0.01 indicates the model group compared to the control group; #p<0.05 indicates the treatment group compared to the model group.

[0024] Figure 4 The image shows the cell viability of human umbilical vein endothelial cells treated with Codonopsis pilosula cyclic peptide B under high glucose conditions; **p<0.01 model group compared with control group, #p<0.05, ##p<0.01 treatment group compared with model group.

[0025] Figure 5 The study aimed to assess the lumen formation in human umbilical vein endothelial cells under high glucose conditions after treatment with Codonopsis pilosula cyclic peptide B; *p<0.05 compared to the control group, and ##p<0.01 compared to the treatment group compared to the model group.

[0026] Figure 6 The study investigated the cell migration of human umbilical vein endothelial cells under high glucose conditions after treatment with Codonopsis pilosula cyclic peptide B; among them, **p<0.01 was used to compare the model group with the control group, and ##p<0.01 was used to compare the drug-treated group with the model group. Detailed Implementation

[0027] Example 1

[0028] 1. Materials and Instruments

[0029] Experimental animals: Male mice, housed in an SPF environment at the Animal Center of Zhejiang University of Traditional Chinese Medicine (ethics approval number 12022).

[0030] Experimental instruments and reagents: balance, Codonopsis pilosula cyclic peptide B (cyclic-(glycine-glycine-leucine-proline-proline-proline-isoleucine-phenylalanine), Chengdu Efbio Biotechnology Co., Ltd., AB1547), physiological saline.

[0031] 2. Animal model preparation

[0032] To establish a mouse model of diabetes induced by intraperitoneal injection of streptozotocin (STZ): After fasting for 8 hours each day, mice were injected intraperitoneally with freshly prepared STZ dissolved in citrate buffer at a concentration of 55 mg / kg for 5 consecutive days. Mice whose blood glucose levels exceeded 16.5 mM after 1 week were considered to have successfully established the model.

[0033] 3. Grouping and Dosing

[0034] Untreated male mice were used as the normal control group (Contorl). Mice that successfully developed the model were randomly divided into a model group (DM) and a treatment group (DM+HB). The treatment group received an intraperitoneal injection of ginseng cyclic peptide B2 5 mg / ml, while the normal control group and the model group received an equal volume of physiological saline. The weight and blood glucose of the mice were measured and recorded daily.

[0035] 4. Detection indicators

[0036] (1) Record and analyze changes in mouse body weight and blood glucose.

[0037] Data processing and analysis were performed using GraphPad software. All results are expressed as mean ± variance. One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant. Figure 1 Changes in body weight and blood glucose levels in diabetic mice after treatment with Codonopsis pilosula cyclic peptide B. Figure 1 It can be seen that the weight and blood glucose of diabetic mice treated with Codonopsis pilosula cyclic peptide B did not change significantly, and Codonopsis pilosula cyclic peptide B had no significant effect on the weight and blood glucose of mice.

[0038] (2) Fundus photography of mice and observation of retinal vascular fluorescein intensity

[0039] Four weeks later, mice in each group were sacrificed for fundus photography and retinal vascular fluorescein intensity was observed. Data were processed and analyzed using GraphPad software. All results are expressed as mean ± variance. One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0040] Figure 2 Fundus photography was performed on diabetic mice after treatment with Codonopsis pilosula cyclic peptide B. The intensity of fluorescein in the retinal vessels of each group was compared. **p<0.01: Vascular leakage was more severe in the model group compared to the control group; #p<0.05: Vascular leakage was reduced in the treatment group compared to the model group. Figure 2 It was found that diabetic mice had more severe retinal vascular leakage compared to normal mice; the retinal leakage in diabetic mice treated with Codonopsis pilosula cyclic peptide B was significantly reduced, and Codonopsis pilosula cyclic peptide B had no significant effect on mouse body weight or blood glucose. This indicates that Codonopsis pilosula cyclic peptide B can alleviate retinal vascular leakage in diabetic mice.

[0041] (3) Measurement of capillary density in mice

[0042] After sacrifice, eyeballs from mice in each group were fixed in 4% paraformaldehyde. The retina was dissected under a microscope, made into four radial incisions resembling a four-leaf clover, washed with PBS, and incubated in 3g / L Triton X-100 at 4°C for 1 hour. After removal from Triton X-100, goat serum was added to block the antigen at room temperature for 2 hours. The blocking solution was removed without washing. α-SMA monoclonal antibody (1:100, diluted in 1g / L Triton X-100) was incubated in a water bath at 37°C for 3 hours. A small amount of anti-fluorescence quencher was added for mounting, and the slides were observed under a fluorescence microscope at 490nm. Data were processed and analyzed using GraphPad software. Results are expressed as mean ± variance. One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0043] Figure 3 Capillary density in diabetic mice treated with Codonopsis pilosula cyclic peptide B; **p<0.01: capillary density was significantly reduced in the model group compared to the control group; #p<0.05: capillary density was increased in the treatment group compared to the model group. Figure 3 It was found that diabetic mice had decreased capillary density compared to normal mice; however, diabetic mice treated with Codonopsis pilosula cyclic peptide B showed increased retinal capillary density. This indicates that Codonopsis pilosula cyclic peptide B can improve retinal capillary density in diabetic mice.

[0044] Example 2

[0045] 1. Materials and Instruments

[0046] Experimental cells: Human umbilical vein endothelial cells (HUVECs, purchased from ATCC cell bank).

[0047] Experimental instruments and reagents: Cell incubator (Thermo 240i), PBS (Zhejiang Senrui Biotechnology Co., Ltd.), high glucose DMEM medium (Gibco 11965092), newborn fetal bovine serum (Biological Industries), Codonopsis pilosula cyclic peptide B (Chengdu Efa Biotechnology Co., Ltd., AB1547), physiological saline, Matrigel, Transwell chambers.

[0048] 2. Cell model preparation

[0049] A cell model was obtained by treating HUVECs with 30 μM glucose for 24 hours.

[0050] 3. Grouping and Dosing

[0051] Untreated human umbilical vein endothelial cells were used as the control group (Contorl). The cell models prepared in step 2 were randomly divided into the model group (HG) and the Codonopsis pilosula cyclic peptide B administration group (HB). The Codonopsis pilosula cyclic peptide B administration group was further divided into 1μM, 5μM, 10μM, 20μM, 50μM and 100μM dose groups for treatment.

[0052] 4. Detection indicators

[0053] (1) Cell viability assay

[0054] Cell count 5×10 4 Cells were seeded at a density of 10 cells / ml in 96-well plates (treated for 24 hours). Cells were observed under a microscope. Each group (n≥6 replicates) included a culture medium blank group, a model group, and a group treated with Codonopsis pilosula cyclic peptide B. After adding 10 μl of Cck-8 reagent to the cell supernatant, cells were placed in a full-wavelength microplate reader for readings. Cell viability was calculated by measuring absorbance at 450 nm and 630 nm, using these as reference wavelengths for dual-wavelength measurement. Cell viability was recorded for each group. Data were processed and analyzed using GraphPad software. Results are expressed as mean ± variance. One-way ANOVA was used for comparisons between groups, with p < 0.05 indicating statistical significance.

[0055] Figure 4 The cell viability of HUVECs under high glucose environment after treatment with Codonopsis pilosula cyclic peptide B. **p < 0.01:** The model group showed decreased cell viability compared to the control group. After treatment with Codonopsis pilosula cyclic peptide B, the cell viability of the treatment group (p < 0.01) was significantly increased compared to the model group, with the 20 μM dose showing the most significant effect. Figure 4It was found that HUVECs cultured in high glucose showed decreased cell viability compared to the control group; however, HUVECs treated with Codonopsis pilosula cyclic peptide B showed significantly enhanced cell viability compared to the model group, with a 20 μM dose showing the best effect. This indicates that Codonopsis pilosula cyclic peptide B can improve the viability of vascular endothelial cells under high glucose conditions.

[0056] (2) Lumen formation

[0057] Take a 96-well plate, add 100 μl of liquid Matrigel to each well, and count the cells at 5 × 10⁶ cells / well. 4 Cells were added at a density of 1 / ml to 96-well plates (treatment time 24 hours). Cells in each group were observed under a microscope. Each group (n≥6 replicates) included a culture medium blank group, a model group, and a group treated with Codonopsis pilosula cyclic peptide B (20 μM). The formation of the tubules was recorded by photograph and counted, and the average value was taken. Data were processed and analyzed using GraphPad software. Results are expressed as mean ± variance. One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0058] Figure 5 This study describes the lumen formation in human umbilical vein endothelial cells treated with Codonopsis pilosula cyclic peptide B under a high-glucose environment. *p<0.05 Lumen formation was increased in the model group compared to the control group; ##p<0.01 Lumen formation was significantly reduced in the treatment group compared to the model group. Figure 5 The results showed that HUVECs cultured in high glucose exhibited increased lumen formation compared to the control group; while HUVECs treated with Codonopsis pilosula cyclic peptide B showed reduced lumen formation compared to the model group. This indicates that Codonopsis pilosula cyclic peptide B can inhibit lumen formation in vascular endothelial cells.

[0059] (3) Cell migration status

[0060] Adjust the cell concentration to 2×10 5 Cells were suspended in serum-free culture medium at a concentration of 150 μl / ml. 24-well plates and Transwell chambers were prepared. 800 μl of culture medium containing 10% serum was added to the bottom of the 24-well plate, and 150 μl of cell suspension was added to each chamber. Each group (n≥6 replicates) included a culture medium blank group, a negative control group, a model group, and a group treated with Codonopsis pilosula cyclic peptide B (20 μM). Cells were incubated for 24 h. The lower surface of the chambers was fixed by immersing in 70% methanol solution for 30 min, stained with crystal violet, examined under a microscope, and the cell count was counted. Data were processed and analyzed using GraphPad software. Results are expressed as mean ± variance. One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0061] Figure 6This study describes the cell migration of human umbilical vein endothelial cells under high glucose conditions after treatment with Codonopsis pilosula cyclic peptide B. **p < 0.01: Cell migration was significantly increased in the model group compared to the control group; ##p < 0.01: Cell migration was significantly decreased in the treatment group compared to the model group. Figure 6 The results showed that HUVECs cultured in high glucose exhibited increased cell migration compared to the control group; however, HUVECs treated with Codonopsis pilosula cyclic peptide B showed decreased cell migration compared to the model group. This indicates that Codonopsis pilosula cyclic peptide B can inhibit vascular endothelial cell migration.

Claims

1. The use of Codonopsis pilosula cyclic peptide B in the preparation of drugs for treating or alleviating diabetic retinopathy, wherein Codonopsis pilosula cyclic peptide B is cyclic-(glycine-glycine-leucine-proline-proline-proline-isoleucine-phenylalanine), and its use is independent of lowering blood glucose levels.

2. The application of Codonopsis pilosula cyclic peptide B in the preparation of drugs that reduce retinal vascular endothelial cell damage, wherein Codonopsis pilosula cyclic peptide B is cyclic-(glycine-glycine-leucine-proline-proline-proline-isoleucine-phenylalanine), and its application is independent of lowering blood glucose levels.

3. Use according to claim 1 or 2, characterized in that, The drug is an injectable or oral formulation.

4. The use according to claim 3, wherein the compound is ###0002### The dosage is 10~40mg / ml.

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