Application of paeoniflorin in preparation of medicine for treating diabetic retinopathy

Abstract

The invention provides application of paeoniflorin in preparation of a medicine for inhibiting diabetic retinal vascular hyperplasia. The active ingredients of the medicine are one or more of paeoniflorin, a hydrate of paeoniflorin, pharmaceutically acceptable salt of paeoniflorin, a tautomer of paeoniflorin, a stereoisomer of paeoniflorin and a precursor compound of paeoniflorin. Paeoniflorin has potential to be developed into drugs, health care products or functional food for treating diabetic retinopathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of paeoniflorin in the preparation of a medicament for treating diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) is the most common and severe blinding ocular complication of diabetes. DR seriously affects patients' work and life, and increases the burden on families and society. Retinal microvascular lesions are the most important pathological factors that damage patients' vision and even cause blindness during the progression of DR. The long-term hyperglycemic environment leads to retinal microvascular lesions and retinal inflammation, causing damage to pericytes and endothelial cells in the retina and the disruption of the inner blood-retinal barrier (iBRB), thereby increasing retinal vascular leakage and tissue edema (Morya AK, Ramesh PV, Nishant P, et al. Diabetic retinopathy: A review on its pathophysiology and novel treatment modalities. World J Methodol. 2024, 14(4): 95881.). The early stage of DR is non-proliferative diabetic retinopathy, manifested by the loss of pericytes in retinal microvessels and the damage of retinal microvascular endothelial cells (RMECs), which can increase vascular permeability and disrupt the iBRB. The late stage of DR presents proliferative lesions, characterized by the formation of new and fragile blood vessels in the retina, increasing the instability of the iBRB. Neovascularization can cause preretinal membranes, vitreous hemorrhage, and retinal detachment, ultimately leading to vision loss (Durham JT, Herman IM. Microvascular modifications in diabetic retinopathy. Curr Diab Rep. 2011, 11(4): 253-264.). Inflammation exists throughout the process of DR. The abnormal secretion of inflammatory factors, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), etc., can induce leukocyte activation and migration, trigger leukostasis, and ultimately cause capillary occlusion, retinal non-perfusion, and retinal hypoxia. RMECs and their tight junctions are damaged during this process, causing microaneurysms, hemorrhage, and exudation, resulting in the disruption of the iBRB.During this process, activated white blood cells continue to release inflammatory factors, further damaging RMECs, downregulating tight junctions, and disrupting the iBRB (Yue T, Shi Y, Luo S, et al. The role of inflammation in immune system of diabetic retinopathy: Molecular mechanisms, pathogenetic role and therapeutic implications. Front Immunol. 2022, 13: 1055087.).

[0003] Vascular endothelial growth factor (VEGF) and placental growth factor (PGF) are also considered to be the core promoters of DR disease progression. VEGF-A is one of the subtypes that play a major role in the VEGF family. Under hyperglycemic conditions, VEGF-A induces retinal ischemia and hypoxia, and exacerbates the retinal inflammatory response. After VEGF-A binds to vascular endothelial growth factor receptor 2 (VEGFR2), it activates multiple downstream pathways. On the one hand, it causes the proliferation of RMECs, and on the other hand, it increases the permeability of RMECs and causes neovascularization, both of which can disrupt the tight junctions of RMECs and the iBRB. PGF also belongs to the VEGF family and binds to vascular endothelial growth factor receptor 1 (VEGFR1), and its expression is mainly related to pathological angiogenesis. In addition, PGF activates monocytes in an ischemic and hypoxic environment, increases the expression of inflammation-related factors, and exacerbates the retinal inflammatory environment. In addition, angiogenesis and inflammatory responses under hyperglycemic conditions can be regulated by signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1α (HIF-1α). This pathological pathway is the intervention target of many plant-derived drugs, especially traditional Chinese medicines (Li W, Xing Q, Liu Z, et al. The signaling pathways of traditional Chinese medicine in treating diabetic retinopathy. Front Pharmacol. 2023, 14: 1165649.). Therefore, natural small molecule compounds have therapeutic potential in regulating angiogenesis and inflammatory responses to alleviate diabetic retinopathy. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides the use of paeoniflorin (PAE, C 23 H 28 O 11 ) in the preparation of a drug for inhibiting diabetic retinovascular hyperplasia.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides the use of paeoniflorin in the preparation of a drug for inhibiting diabetic retinopathy vascular hyperplasia. The active ingredient of the drug is one or more of paeoniflorin, the hydrate of paeoniflorin, the pharmaceutically acceptable salt of paeoniflorin, the tautomer of paeoniflorin, the stereoisomer of paeoniflorin, and the precursor compound of paeoniflorin.

[0007] In a preferred embodiment, the paeoniflorin is the only active ingredient of the drug.

[0008] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical effects:

[0009] The research results of the present invention show that paeoniflorin can inhibit the proliferation, migration and tube formation of human retinal microvascular endothelial cells (HRMECs) in a high-glucose environment, can inhibit the secretion levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 in HRMECs in a high-glucose environment, improve inflammation, inhibit the expression of vascular endothelial growth factor (VEGF) and placental growth factor (PGF), and reduce abnormal angiogenesis. In a diabetic mouse model, paeoniflorin can reduce retinal angiogenesis and ganglion cell layer edema, thereby protecting the blood-retinal inner barrier.

[0010] In summary, paeoniflorin can reduce vascular hyperplasia and inflammatory responses in a high-glucose environment and has the potential to be developed into a drug for the treatment of diabetic retinopathy. Brief Description of the Drawings

[0011] Figure 1 It is a result diagram of the effect of different concentrations of paeoniflorin on the proliferation of HRMECs in a high-glucose environment;

[0012] Figure 2 It is a result diagram of the effect of different concentrations of paeoniflorin on the migration of HRMECs in a high-glucose environment;

[0013] Figure 3 It is a result diagram of the effect of different concentrations of paeoniflorin on the tube formation of HRMECs in a high-glucose environment;

[0014] Figure 4 It is a result diagram of the effect of different concentrations of paeoniflorin on the secretion of inflammatory factors by HRMECs in a high-glucose environment;

[0015] Figure 5Results showing the effects of paeoniflorin at different concentrations on the expression of PGF, VEGF, and STAT3 / HIF-1α proteins in HRMECs under high glucose conditions (medium glucose concentration: 4.5 g / L).

[0016] Figure 6 Results showing that paeoniflorin at different concentrations can reduce retinal neovascularization and ganglion cell layer edema in diabetic mice. Detailed implementation modes

[0017] The present invention provides the use of paeoniflorin in the preparation of a drug for diabetic retinovascular hyperplasia. The active ingredient of the drug is one or more of paeoniflorin, the hydrate of paeoniflorin, the pharmaceutically acceptable salt of paeoniflorin, the tautomer of paeoniflorin, the stereoisomer of paeoniflorin, and the precursor compound of paeoniflorin. The drug also includes a pharmaceutically acceptable carrier or excipient. Among them:

[0018] The term "pharmaceutically acceptable salt" refers to the salt formed by the compound and a pharmaceutically acceptable inorganic acid or organic acid. The inorganic acids include, but are not limited to: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid; the organic acids include, but are not limited to: formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, asiatatic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids; the "pharmaceutically acceptable" means a substance that is suitable for humans without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance with a reasonable benefit / risk ratio.

[0019] The term "tautomer" refers to functional group isomers generated due to the rapid movement of a certain atom in the molecule between two positions, for example: enol and the corresponding ketone.

[0020] The term "stereoisomer" refers to isomers generated by different arrangements of atoms in space in the molecule, for example: cis-trans isomers, enantiomers, conformational isomers, etc.

[0021] The term "precursor compound" refers to a compound that is inactive in vitro but can be metabolized or chemically reacted in the body to be converted into the active ingredient of the present invention, thereby exerting its pharmacological effect.

[0022] The present invention will be described in detail and specifically below through specific examples to better understand the present invention. However, the following examples do not limit the scope of the present invention.

[0023] Example 1

[0024] This example provides an exploratory experiment on the inhibitory effect of paeoniflorin on the proliferation of HRMECs under high glucose conditions (medium glucose concentration 4.5 g / L). The specific experimental content and results are as follows:

[0025] HRMECs were co-cultured with different concentrations of paeoniflorin for 24 h, 48 h, and 72 h under high glucose conditions. The medium with a glucose concentration of 1 g / L was used as the control group (Con group). The CCK8 method was used to detect cell viability, and the results are as Figure 1 shown.

[0026] Compared with the HG group, each paeoniflorin administration group could significantly reduce the proliferation ability of HRMECs (in the three time periods of 24 h, 48 h, and 72 h, the HG+PAE 5 μM group, HG+PAE 15 μM group, and HG+PAE 25 μM group, P<0.001). And within the experimental concentration range, paeoniflorin inhibited the proliferation of HRMECs in a dose-dependent manner under high glucose conditions.

[0027] Example 2

[0028] This example provides an exploratory experiment on the inhibitory effect of paeoniflorin on the migration of HRMECs under high glucose conditions (medium glucose concentration 4.5 g / L). The specific experimental content and results are as follows:

[0029] HRMECs were plated in 6-well plates at a density of 2×10 5 cells per well and cultured in 5% FBS DMEM medium for 24 h. The old medium was discarded and replaced with serum-free DMEM medium. After 6 h of starvation treatment, a scratch was made perpendicular to the plate surface with a pipette tip, and 3 scratches were made in each well. The floating cells and cell debris were washed away with PBS. According to the groups of Con group, HG group, HG+PAE 5 μM group, HG+PAE 15 μM group, and HG+PAE 25 μM group, the corresponding media were added. Then, at 0 h and 12 h of culture, the scratch healing was observed under an inverted microscope (magnification 200×), and the results are as Figure 2 shown.

[0030] Compared with the HG group, the proliferation ability of cells in each paeoniflorin administration group was inhibited (in the HG+PAE 5 μM group, P<0.01 at 24 h, P<0.001 at 48 h and 72 h; in the HG+PAE 15 μM group, P<0.001; in the HG+PAE 25 μM group, P<0.001); compared with the HG+PAE 5 μM group, there were significant differences in the degree of inhibition of HRMECs migration between the HG+PAE 15 μM group and the HG+PAE 25 μM group (P<0.001 in the HG+PAE 15 μM group, P<0.001 in the HG+PAE 25 μM group).

[0031] Example 3

[0032] This example provides an exploratory experiment on the inhibitory effect of paeoniflorin on the tube formation of HRMECs under a high-glucose environment (the sugar concentration in the culture medium is 4.5 g / L). The specific experimental content and results are as follows:

[0033] The 96-well plate was placed on an ice box for operation. 50 μL of Matrigel matrix gel was added to each well and left in the incubator for 30 min to solidify. HRMECs were seeded into the 96-well plate at a density of 5×10 4 cells per well. After the cells adhered, the corresponding culture media were added according to the groups of Con group, HG group, HG+PAE 5 μM group, HG+PAE 15 μM group, and HG+PAE 25 μM group. Three replicate wells were set for each group. After 48 h, the lumen formation status of the cells in each group was observed under a microscope and images were collected (magnification 200×). The results are as Figure 3 shown.

[0034] Compared with the HG group, the lumen formation ability of each paeoniflorin administration group was inhibited (P<0.01 for the HG+PAE 5 μM group, P<0.001 for the HG+PAE 15 μM group, and P<0.001 for the HG+PAE 25 μM group); compared with the HG+PAE 5 μM group, there were significant differences in the degree of inhibition of HRMECs lumen formation between the HG+PAE 15 μM group and the HG+PAE 25 μM group (P<0.001 for the HG+PAE 15 μM group and P<0.001 for the HG+PAE 25 μM group).

[0035] Example 4

[0036] This example provides an exploratory experiment on the inhibitory effect of paeoniflorin on the secretion of inflammatory factors by HRMECs under a high-glucose environment (the sugar concentration in the culture medium is 4.5 g / L). The specific experimental content and results are as follows:

[0037] HRMECs were seeded into the 6-well plate at a density of 2×10 5 cells per well and cultured with 5% FBS DMEM medium for 24 h. After the cells adhered, the corresponding culture media were added according to the groups of Con group, HG group, HG+PAE 5 μM group, HG+PAE 15 μM group, and HG+PAE 25 μM group, and then cultured in the incubator for another 48 h. The supernatant in each well was collected, and the expression levels of TNF-α, IL-1β, IL-6, and IL-8 factors in the supernatant were detected by ELISA. The results are as Figure 4 shown.

[0038] Compared with the HG group, the secretion of TNF-α, IL-1β, IL-6, and IL-8 factors in each paeoniflorin administration group was inhibited (P < 0.01 in the HG + PAE 5 μM group, P < 0.001 in the HG + 15 μM paeoniflorin group, and P < 0.001 in the HG + PAE 25 μM group); compared with the HG + PAE 5 μM group, there were significant differences in the degree of inhibition of TNF-α, IL-1β, IL-6, and IL-8 factor secretion between the HG + PAE 15 μM group and the HG + PAE 25 μM group (P < 0.001 in both the HG + PAE 15 μM group and the HG + PAE 25 μM group).

[0039] Example 5

[0040] This example provides that paeoniflorin inhibits the expression of PGF, VEGF, and STAT3 / HIF-1α proteins in HRMECs under a high-glucose environment (medium sugar concentration 4.5 g / L). The specific experimental content and results are as follows:

[0041] Plate HRMECs in a 6-well plate at a density of 2×10 5 cells per well and culture them in 5% FBS DMEM medium for 24 h. After the cells adhere, add the corresponding medium according to the groups of the Con group, HG group, HG + PAE 5 μM group, HG + PAE 15 μM group, and HG + PAE 25 μM group, and continue to culture them in an incubator for 48 h. Extract proteins using RIPA lysis method and detect the expression of PGF, VEGF, and STAT3 / HIF-1α proteins by WB method. The results are as Figure 5 shown.

[0042] The expression of PGF, VEGF, STAT3, and HIF-1α in each paeoniflorin administration group was inhibited (P < 0.001 in the HG + PAE 5 μM group, P < 0.001 in the HG + PAE 15 μM group, and P < 0.001 in the HG + PAE 25 μM group for PGF, STAT3, and HIF-1α; P < 0.01 in the HG + PAE 5 μM group, P < 0.001 in the HG + PAE 15 μM group, and P < 0.001 in the HG + PAE 25 μM group for VEGF), and compared with the HG + PAE 5 μM group, there were significant differences in the degree of inhibition of protein expression between the HG + PAE 15 μM group and the HG + PAE 25 μM group (both P < 0.001).

[0043] Example 6

[0044] This example provides that paeoniflorin alleviates retinal neovascularization and ganglion cell layer (GCL) edema in diabetic mice. The specific experimental content and results are as follows:

[0045] A type II diabetes mouse model was constructed by feeding a high-fat diet and injecting a small dose of streptozotocin (STZ) intraperitoneally. A total of 30 six-week-old C57 male mice were divided into 5 groups. Among them, 6 were in the control group (Negative control, NC), and the remaining 24 were fed a high-fat diet for 4 weeks and then injected with STZ at 40 mg / kg intraperitoneally. After continuing to feed a high-fat diet for 1 week, the random blood glucose was measured. If it was above 11.1 mmol / L, the model was considered successfully established, and paeoniflorin intervention was given. The type II diabetic mice were divided into a model group (Diabetes mellitus, DM), low, medium, and high-dose paeoniflorin groups (DM + PAE 20 mg / kg, DM + PAE 40 mg / kg, DM + PAE 80 mg / kg). The administration method of paeoniflorin was once a day by gavage for a total of 4 weeks. After sampling, the eyeballs of the mice were paraffin-embedded, sectioned, and stained with H&E to evaluate retinal neovascularization and ganglion cell layer edema. The results are as Figure 6 shown.

[0046] Compared with the DM group, the number of neovascularization and the thickness of the GCL in the NC group were significantly lower than those in the model group (P < 0.001). The number of neovascularization and the thickness of the GCL in the low, medium, and high-dose paeoniflorin groups (DM + PAE 20 mg / kg, DM + PAE 40 mg / kg, DM + PAE 80 mg / kg) were also significantly lower than those in the model group (P < 0.05), and showed a dose-dependent trend.

[0047] As can be seen from the above examples, paeoniflorin can reduce vascular proliferation and inflammatory responses in a hyperglycemic environment, which provides a strategy for the preparation of drugs for the treatment of diabetic retinopathy.

[0048] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Use of paeoniflorin in the preparation of a drug for inhibiting diabetic retinopathy vascular proliferation, characterized in that, The active ingredient of the drug is one or more of paeoniflorin, the hydrate of paeoniflorin, the pharmaceutically acceptable salt of paeoniflorin, the tautomer of paeoniflorin, the stereoisomer of paeoniflorin, and the precursor compound of paeoniflorin.

2. The application according to claim 1, characterized in that, The paeoniflorin is the only active ingredient of the drug.

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