CN117482098A - Application of ginsenoside Ro in preventing or treating diabetic retinopathy - Google Patents

Abstract

The application provides application of ginsenoside Ro (Ginsenosides Ro) in preventing or treating diabetic retinopathy, in particular to application of ginsenoside Ro (Ginsenosides Ro) in medicaments for preventing or treating diabetic retinopathy by protecting retinal microvascular endothelium. Ginsenoside Ro can effectively enhance the activity of human retina microvascular endothelial cells, enhance autophagy capacity of the cells, and reduce inflammation and oxidative stress of the cells; no toxic or side effect is produced in vitro and in vivo; has the advantages of high safety, small dosage, long-term stability and the like, and is suitable for clinical treatment.

Description

Application of ginsenoside Ro in preventing or treating diabetic retinopathy

Technical Field

The application belongs to the fields of diabetes complications and natural medicines, and particularly provides application of ginsenoside Ro in preparing medicines for preventing or treating diabetic retinopathy.

Background

Diabetic Retinopathy (DR) is the most severe microvascular complication of diabetes and is one of four rough blinding eye diseases. Methods for treating diabetic retinopathy have been directed against vascular proliferation by intravitreal injection of anti-vascular endothelial growth factor and holoretinal photocoagulation. However, the risk of patient compliance is greatly reduced due to the complexity of the disease, and the recurrence and exacerbation of the disease in some patients. Currently, there is no very effective treatment for DR.

Retinal microvascular endothelial cell dysfunction promotes its secretion of adhesion and chemokines, increases the production of oxygen radicals and other reactive oxidative species, and causes oxidative stress. Active oxygen generated in oxidative stress can induce autophagy to generate, and with the help of lysosomes, the dysfunctional component is degraded, which is an important protection mechanism in vivo.

It is reported in the literature that dammarane type ginsenoside Rg1, rg3, rb1 and complex K have pharmacological activity on diabetic complications, but Rg1 and Rb1 are focused on neuroprotection and anti-tumor vascular activity, rg3 is focused on anti-tumor vascular activity, and ginsenoside complex K is focused on rheumatoid arthritis. There is no outstanding advantage in protecting retinal microvasculopathy.

Ginsenoside Ro (Ginsenosides Ro) is oleanane compound extracted from radix Ginseng, is an important active component of radix Ginseng, and has antiinflammatory, immunity regulating, cardiovascular system protecting, antitumor, and antiviral effects. However, no related research and report is available on the regulation of the oleanane-type ginsenoside Ro on diabetic retinopathy.

Disclosure of Invention

In one aspect, the application provides an application of ginsenoside Ro in preparing a medicament for treating diabetic retinopathy.

Further, the medicament is in an oral, injection or external dosage form.

Further, the external preparation is an eye drop preparation.

Further, the medicine also comprises various pharmaceutically acceptable auxiliary materials or excipients.

Further, the drug protects retinal microvascular endothelium or endothelial cells.

Further, the medicament enhances the activity of retinal microvascular vascular endothelial cells.

Further, the drug protects retinal microvascular endothelium or endothelial cells by one or more of the following mechanisms:

(1) Reducing inflammatory response; (2) reducing oxidative stress; (3) decreasing vascular permeability; (4) reducing angiogenesis; (5) enhancing autophagy.

Further, the drug restores reduced retinal thickness in diabetic retinopathy.

Further, the medicament restores vascular proliferation in diabetic retinopathy.

Further, the medicament restores apoptosis in diabetic retinopathy.

Further, the drug restores mitochondrial damage in diabetic retinopathy.

The pharmaceutical dosage forms useful herein include various injectable, oral, and topical dosage forms including, but not limited to, tablets, capsules, oral liquids, water injections, powder injections, eye drops, transdermal formulations, and the like.

The medicaments described herein may also include various pharmaceutically acceptable excipients or excipients including, but not limited to, coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH adjusting agents, flavoring agents, and the like.

The application demonstrates that human saponin Ro has an improving effect on microvascular endothelial dysfunction by enhancing autophagy, reducing inflammation and oxidative stress. Therefore, the Chinese herbal medicine can be used for developing new medicines for preventing/treating diabetic retinopathy, and provides important basis for treating diabetic retinopathy by using traditional Chinese herbal medicines.

Drawings

FIG. 1A shows changes in hRMECs cell viability caused by AGEs;

FIG. 1B is a graph showing that Ro causes a change in the viability of hRMECs cells;

FIG. 1C shows that co-treatment of AGEs and Ro resulted in changes in hRMECs cell viability;

FIG. 1D is the effect of Ro on lactate dehydrogenase content in cell supernatants;

FIG. 1E is the effect of Ro on endothelial cell injury status;

FIG. 2A is the effect of Ro on advanced glycation end product (AGEs) -induced expression of hRMECs cells ICAM-1 and VCAM-1;

FIG. 2B is the effect of Ro on advanced glycation end product (AGEs) -induced expression of hRMECs cells TNF- α and IL-1β;

FIG. 2C is Ro versus advanced glycation end products (AGEs) induced hRMECs cells 8-OhdG, NAD ⁺ Effects of NADH, ROS;

FIG. 2D is the effect of Ro on advanced glycation end product (AGEs) -induced expression of hRMECs fine VEGF;

FIG. 3A is the effect of Ro on expression of advanced glycation end products (AGEs) induced expression of hRMECs cells Beclin-1, LC3, P62;

FIG. 3B is the effect of Ro on expression levels of advanced glycation end product (AGEs) induced hRMECs cells Beclin-1, LC3, ATG5, ATG12, P62, P-DRP-1 proteins;

FIG. 3C is the effect of Ro on advanced glycation end products (AGEs) induced hRMECs cells under transmission electron microscopy;

FIG. 4A is the effect of Ro treatment on total retinal thickness (OCT) in STZ-induced diabetic model mice;

FIG. 4B is the effect of Ro treatment on STZ-induced retinal vascular proliferation in mice model for diabetes (FAA);

FIG. 4C is the effect of Ro treatment on total retinal thickness (HE) in STZ-induced diabetic model mice;

FIG. 4D is the effect of Ro treatment on total retinal thickness in STZ-induced diabetic model mice (data comparison);

fig. 4E is the effect of Ro treatment on STZ-induced diabetic model mouse retinal microvascular endothelial cell mitochondria.

Detailed Description

Example 1 in vitro test

Cell culture:

human retinal microvascular endothelial cells (hRMECs) were purchased from Shanghai Boltd Biotech (cat# BES2536 HC). hRMECs cells were cultured in DMEM medium supplemented with 10% FBS in an incubator at 37℃with 5% carbon dioxide. When hRMECs cells grew to 90% confluence, the stock culture was discarded, pancreatin was added for digestion, the cell suspension was transferred into a centrifuge tube, centrifuged at 1000rpm for 5min, the above was discarded, fresh DMEM medium containing 10% FBS was added, and the cell single cell suspension was gently blown for 1:3 passages and inoculated into a new T25 flask. In all experiments, hRMECs cells were used in exponential phase.

Preparing a solution:

ginsenoside Ro was purchased from Beijing Ruida Heng Hui technology development Co., ltd (product number: HB 21068), and stock solution prepared by adding DMSO to 50mM was frozen. AGEs modeling agent (50 mg/mL) was dissolved in PBS. Indicated Ro and AGEs concentrations were prepared immediately prior to use, and AGEs-induced cell damage models were prepared. Then, the correlation experiments were performed sequentially.

CCK-8 cells were seeded at 8000 cells/well in 96-well plates, and after adherence, the drug concentration was diluted with serum-free medium, added to 96-well plates, and incubated in a constant temperature incubator. Cell viability of different groups of hrecs cells was assessed by chemosensitivity of CCK-8 using an enzyme-labeled assay.

LDH detection: the cell supernatants were assayed for the level of LDH using a Lactate Dehydrogenase (LDH) kit (cat# A020-1) from Nanjing, and the apoptosis of the cells of the different treatment groups was assessed.

ROS detection: the active oxygen detection was performed using a fluorescent probe DCFH-DA using an active oxygen detection kit (cat No. S0033S) purchased from Biyun, and the fluorescence intensity was detected using a flow cytometer.

Form of transmission electron microscope observation: mitochondrial autophagy and autophagy lysosomes were visualized by transmission electron microscopy.

Westernblot: lysing cells with lysate containing protease inhibitor and phosphatase inhibitor, extracting protein, ultrasonic crushing on ice for 3min, centrifuging at 4deg.C and 12000r for 30min to remove insoluble impurities, detecting protein concentration with BCA protein quantification kit, adding appropriate amount of Loadingbuffer, decocting in boiling water for 10min, and cooling. Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate the proteins, and transferred to nitrocellulose membranes. The mixture was blocked with 5% skim milk at room temperature for 2h, and the primary antibody was added and incubated overnight at 4 ℃. The next day, TBST was washed 3 times, secondary antibody incubated 1h at room temperature, TBST was washed 3 times, and blots were visualized by enhancing chemiluminescence using eBLOT imaging system.

RT-PCR: 1ml Trizol was added and the cells after the administration treatment were harvested. Nucleic acid concentration was extracted and determined. Reverse transcription was performed using the Takara reverse transcription kit (RR 036A). Fluorescence quantification was performed using a fluorescence quantification kit (RR 820A), and primer sequences for fluorescence quantification were as follows:

the statistical analysis method comprises the following steps:

statistical analysis was performed using SPSS statistical software. All data are expressed as mean ± standard deviation. Statistical analysis of the metrology data was performed using unpaired t-test and One-way ANOVA, with P < 0.05 being statistically different.

Experimental results:

as shown in FIG. 1, the effect of AGEs on hRMECs cells was first detected. Treatment with AGEs at concentrations of 100, 200 and 400 μg/ml for 24h resulted in a significant decrease in cell viability in a time-dependent manner (FIG. 1A, P < 0.05). In contrast, ro (1.5625, 3.125, 6.25, 12.5, 25, 50. Mu.M) treatment for 24h had no effect on cell viability (FIG. 1B, P > 0.05). Incubation of Ro (6.25, 12.5. Mu.M) and AGEs (400. Mu.g/ml) for 24h gave significant protection to the cells (FIG. 1C, P < 0.05). After Ro treatment, the lactate dehydrogenase content in the cell supernatant was significantly reduced (FIG. 1D, P < 0.05). After Ro administration, the damaged status of endothelial cells was significantly reduced (fig. 1E).

As shown in FIG. 2, the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion factor 1 (VCAM-1) genes was significantly increased (FIG. 2A, P < 0.01) in the model group, and significantly decreased after Ro treatment, compared to the normal control groupLow (FIG. 2A, P < 0.01) indicates that Ro is able to reverse AGEs-induced endothelial activation and injury. Compared with the normal control group, the expression of tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) genes of the model group is obviously increased (figure 2B, P < 0.01), and the expression of the tumor necrosis factor is obviously reduced after the treatment of Ro (figure 2B, P < 0.01), which shows that the Ro can obviously reduce the inflammatory injury induced by AGEs. Model group 8-hydroxydeoxyguanosine (8-OhdG), NAD compared to normal control group ⁺ The expression of NADH and Reactive Oxygen Species (ROS) was significantly increased (FIG. 2C, P < 0.01), and the expression of Ro was significantly decreased after treatment (FIG. 2C, P < 0.01), indicating that Ro was able to significantly decrease the oxidative stress induced by AGEs. The expression of Vascular Endothelial Growth Factor (VEGF) was significantly increased in the model group (FIG. 2D, P < 0.01) compared to the normal control group, and significantly decreased after treatment with Ro (FIG. 2D, P < 0.01), indicating that Ro was able to significantly inhibit vascular permeability as well as vascular neovascularization. The above phenomenon suggests that Ro can protect vascular endothelial cells by reducing inflammatory responses, oxidative stress, vascular permeability, and angiogenesis.

FIG. 3 shows that the model groups Beclin-1 and LC3 autophagy gene expression was significantly reduced, P62 gene expression was significantly increased (FIG. 3A, P < 0.01) and Ro treatment was significantly increased (FIG. 3A, P < 0.01) compared to the normal control group. Compared with the normal control group, the expression level of the Beclin-1, LC3, ATG5 and ATG12 proteins of the model group is obviously reduced (shown in figure 3B, P < 0.01), the level of the P62 and P-DRP-1 proteins is obviously increased (shown in figure 3B, P < 0.05 and P < 0.01), the expression level of the Beclin-1, LC3, ATG5 and ATG12 proteins after Ro treatment is obviously increased (shown in figure 3B, P < 0.01), and the level of the P62 and P-DRP-1 proteins is obviously reduced (shown in figure 3B, P < 0.05 and P < 0.01). Under a transmission electron microscope, compared with a normal control group, the mitochondria of the model group are swollen, mitochondrial cristae threads are dissolved, and an inner membrane is locally ruptured; aggregation of a large number of autophagosomes, showing that autophagy flow was blocked (fig. 3C), mitochondrial morphology recovered after Ro treatment, clear outer membrane border, tidy alignment of mitochondrial cristae lines, and clarity; small amounts of autophagosomes and autophagosomes showed gradual restoration of autophagic flow. It was shown that Ro is able to protect retinal endothelial cells by enhancing autophagy levels.

Example 2 in vivo test

Experimental materials:

streptozotocin (Sigma, lot number: V900890-1 g); ginsenoside Ro (Ruida Henghui, batch number: HB 21068); calcium dobesilate tablet (Wanhao pharmaceutical industry, lot number: 2210212). The streptozotocin is dissolved in sodium citrate buffer before use, is prepared for use, and is placed on ice for being preserved in a dark place. Ginsenoside Ro and calcium dobesilate are dissolved in sodium carboxymethyl cellulose. 32 6 week old male and female half C57BL/6J mice were purchased from Siro (Suzhou) biotechnology Co., ltd, license number SCXK (Su) 2022-0016; animal feeding is performed at animal centers of medical plant institute of Chinese medical sciences. All mice were kept in an alternating dark and bright environment at 22-26 ℃, 40-70% humidity and 12 hours, with free water and food intake. The use of experimental animals followed the guidelines for care and use of experimental animals issued by the national institutes of health and the committee for care and use of animals by the national institutes of medical and plant research of the national academy of medical science.

The experimental method comprises the following steps:

the preparation method of the model comprises the following steps: mice were randomly divided into normal control and model groups. Normal control mice were fed normal diet and model mice were fed KK diet. After 6 weeks of feed induction, all mice were fasted for 12 hours without water withdrawal, mice of the model group were intraperitoneally injected with a solution of streptozotocin of 55mg/ml, and mice of the normal control group were injected with an equal dose of sodium citrate. Mice in each group were tested for fasting glycemia (FBG) using a rogowski glucometer after one week for 5 days of continuous injection. FBG is more than or equal to 11.1mmol/L, which is a diabetes mouse. Diabetic retinopathy often occurs in mice at 20 weeks of age, and administration will begin from the 20 th week of age.

Grouping and administration methods: the diabetic model animals are randomly divided into a diabetes model group, a ginsenoside Ro group and a calcium dobesilate group, sodium carboxymethyl cellulose is given to normal control group mice and model group mice, and the other groups of mice are respectively and gastrectly administered according to doses once a day for 4 weeks.

The processing and sample collection method comprises the following steps: at week 25 of the experiment, relevant in vivo index assays were performed. Detecting retina functions of each group of mice, after the mice are anesthetized, expanding pupils by using 1% topiramate, coating carbomer gel on eyeballs of the mice to keep the eyeballs moist, preventing acute cataract, and detecting retina thicknesses of each group of mice by an optical coherence tomography (iso OCT,4D-ISOCT microscope imaging system, optoprobe, canada); the retinal vascular changes of each group of mice were detected by Fundus Fluorescence Angiography (FFA) by intraperitoneal injection of a fluorescein sodium contrast agent; HE staining detects pathological changes in the retina of mice; TUNEL staining detects apoptosis of retinal cells; ultrastructural results of retinal microvascular endothelial cells were obtained using transmission electron microscopy analysis.

Experimental results:

visual function, retinal thickness and changes in retinal vasculature were measured by OCT and FFA. OCT images showed a significant decrease in total retinal thickness in model mice compared to normal control (fig. 4A, D, P < 0.01); FFA images showed a significant increase in retinal vascular proliferation in model mice compared to normal control (fig. 4B, D, P < 0.01). The retinal thickness recovery of the mice after Ro treatment (fig. 4A, B, D, P < 0.01) and the vascular proliferation conditions were improved (fig. 4b, P < 0.01).

HE stained images showed a significant decrease in total retinal thickness in model mice (fig. 4C, D, P < 0.01) compared to normal control mice, and retinal thickness recovery in Ro treated mice (fig. 4C, D, P < 0.01) and significant decrease in retinal apoptosis (fig. 4C, D, P < 0.01).

The mitochondria in the retinal microvascular endothelial cells of the model group mice were extremely swollen, the crest lines were dissolved and disappeared, and cavitation was severe, as shown by transmission electron microscopy, compared to the normal control group. After Ro treatment, mitochondrial morphology was restored, the adventitia was clearly defined and cristae lines were aligned (fig. 4E).

Claims (11)

1. Application of ginsenoside Ro in preparing medicine for treating diabetic retinopathy is provided.

2. The use according to claim 1, wherein the medicament is in an oral, injectable or topical dosage form.

3. The use of claim 2, wherein the topical dosage form is an eye drop dosage form.

4. The use according to any one of claims 1-3, wherein the medicament further comprises pharmaceutically acceptable excipients or excipients.

5. The use according to any one of claims 1-4, wherein the medicament protects retinal microvascular endothelium or vascular endothelial cells.

6. The use according to any one of claims 1-4, wherein the medicament enhances retinal microvascular vascular endothelial cell viability.

7. The use of claim 5, wherein the medicament protects retinal microvascular vascular endothelial cells by one or more of the following mechanisms: (1) reducing inflammatory response; (2) reducing oxidative stress; (3) decreasing vascular permeability; (4) reducing angiogenesis; (5) enhancing autophagy.

8. The use of any one of claims 1-4, wherein the medicament restores reduced retinal thickness in diabetic retinopathy.

9. The use according to any one of claims 1-4, wherein the medicament restores vascular proliferation in diabetic retinopathy.

10. The use of any one of claims 1-4, wherein the medicament restores apoptosis in diabetic retinopathy.

11. The use of any one of claims 1-4, wherein the medicament restores mitochondrial damage in diabetic retinopathy.