CN110151769B

CN110151769B - Application of Withaferin a in preparation of medicine for treating fundus ischemic diseases

Info

Publication number: CN110151769B
Application number: CN201910398536.6A
Authority: CN
Inventors: 闫喆一; 曹晓明; 王春芳
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-05-14
Filing date: 2019-05-14
Publication date: 2020-11-13
Anticipated expiration: 2039-05-14
Also published as: CN110151769A

Abstract

The invention discloses application of Withaferin A in preparation of a medicament for treating fundus ischemic diseases. Experiments prove that Withaferin A can inhibit the ischemic diseases of the eyeground from the aspect of pathogenesis: 1. the oxidative stress cell necrosis and apoptosis caused by ischemia are obviously reduced; 2. the targeting property inhibits the vascular endothelial growth factor and the receptor (VEGF-VEGFR2) signal path thereof, and improves the symptoms of pathological microvascular bleeding leakage, abnormal growth of blood vessels and the like. The Withaferin A remarkably relieves the symptoms of fundus ischemic diseases through the dual action of the two mechanisms and inhibits the occurrence and development of the diseases. The Withaferin A serving as a small molecular targeting compound has a stable and safe chemical structure and can permeate blood-eye barriers, so that the Withaferin A has a wide application prospect in fundus ischemic diseases.

Description

Application of Withaferin a in preparation of medicine for treating fundus ischemic diseases

The technical field is as follows:

the invention belongs to the technical field of pharmacy, and particularly relates to application of Withaferin A in preparation of a medicine for treating fundus ischemic diseases.

Background

Retinal and choroidal ischemia, hypoxia, collectively known as ischemic ocular fundus disease, is a significant cause of vision loss and loss, including: 1. acute ischemia: ocular artery occlusion, central retinal artery occlusion, branch retinal artery occlusion, ciliary retinal artery occlusion, distance retinopathy, Purtscher-like retinopathy, etc.; 2. chronic ischemic: central retinal vein occlusion, branch vein occlusion, diabetic retinopathy, ocular ischemic syndrome, macroarteritis retinopathy, retinopathy of prematurity, age-related macular degeneration, etc. Chronic ocular fundus ischemia can also lead to complications such as neovascular glaucoma and corneal neovascularization.

Retinal and choroidal ischemia, hypoxia, can cause deficiencies in a variety of metabolites, but the most urgent and most damaging pathology is due to hypoxia. Thus, this type of disease has a partially common mechanism of impairment: 1. oxidative stress cell necrosis and apoptosis caused by hypoxia, and acute edema and necrosis of tissues are caused. 2. Hypoxia-induced Vascular Endothelial Growth Factor (VEGF) production. VEGF acts through its receptor VEGFR2, increasing vascular permeability, causing significant vascular leakage, leading to persistent severe bleeding and edema in the tissue; long-term VEGF elevation stimulates proliferation of endothelial cells, inducing unhealthy neovascularization, and then severe complications such as massive hemorrhage, fibroplasia, traction retinal detachment, neovascular glaucoma, and the like occur. The incidence of the above diseases is on the rise in the world, and is the leading cause of blindness in children, adults and the elderly in all ages.

Therefore, finding a compound which has the effect of resisting oxidative stress cell death and can target anti-VEGF is a long-term pursuit of clinical workers and scientific researchers in the ophthalmology.

Withaferin a is a small molecule steroidal lactone. Chemical name: 5, 6-epoxy-4, 27-dihydroxy-1-oxo-2, 24-dienolide, formula: c₂₈H₃₈O₆Molecular weight: 470.6, it has stable structure, easy storage, no obvious systemic toxicity even when used in high dose, and the molecular chemical formula is as follows:

previous molecular docking analyses found that Withaferin a could potentially prevent NF- κ B activation by inhibiting the activation of IKK β; it is considered that the Withaferin A has anti-inflammatory and anti-tumor effects, but the dual effects of the Withaferin A on resisting oxidative stress cell death and targeting VEGF resistance in fundus ischemic diseases are not known, so that the application of the Withaferin A in medicaments for fundus ischemic diseases is not reported.

The invention content is as follows:

the invention aims to provide application of Withaferin A in preparation of a medicament for treating fundus ischemic diseases.

In the above application, the fundus ischemic disease is retinopathy or choroidopathy.

The retinopathy is acute retinopathy or chronic retinopathy.

The ischemic disease of the fundus is ocular artery obstruction, central retinal artery obstruction, branch retinal artery obstruction, ciliary retinal artery obstruction, distance retinopathy, Purtscher-like retinopathy, central retinal vein obstruction, branch vein obstruction, diabetic retinopathy, ocular ischemic syndrome, aortoarteritis retinopathy, retinopathy of prematurity, and age-related macular degeneration.

The retinopathy is retinal vascular hemorrhage and leakage or retinal cell necrosis and apoptosis or retinal angiogenesis.

The choroidal disorders are bleeding leakage of choroidal blood vessels or necrotic apoptosis of choroidal cells or neogenesis of choroidal blood vessels.

The invention adopts exogenous Withaferin A to carry out cell experiments and eye acute and chronic ischemic experiments of animal models, and the results show that: the Withaferin A has obvious inhibiting effect on cell necrosis and apoptosis, blood vessel leakage, pathological blood vessel neogenesis and visual function reduction caused by ischemic ocular fundus diseases, normalizes the ocular fundus morphology, structure and function, and thus relieves and inhibits the occurrence and development of diseases. Molecular mechanism experiments show that: the Withaferin A inhibits oxidative stress cell necrosis and apoptosis caused by hypoxia, and directly inhibits the function of a VEGF signal pathway in a targeting way, thereby effectively resisting ischemic fundus oculi lesion and complications thereof from the aspect of pathogenesis. The Withaferin A has definite effect, clear action mechanism and stable and safe chemical structure, can penetrate through the blood-eye barrier, and can be applied to the new clinical era that small-molecule anti-VEGF compounds can treat ischemic ocular fundus diseases in a targeted manner.

Drawings

FIG. 1, inhibition of ischemia reperfusion (I/R) and H in Human Retinal Microvascular Endothelial Cells (HRMECs) by Withaferin A (WFA)₂O₂Induced oxidative stress cell death.

FIG. 2 inhibition of HRMEC by Withaferin As in H₂O₂Induced oxidative stress apoptosis.

Fig. 3 shows the protective effect of the visual function of the Withaferin A mouse after the fundus acute ischemia-reperfusion injury.

Figure 4, Withaferin a upregulates expression of antioxidant proteins and activates ERK, Akt proteins.

Fig. 5, mechanism of Withaferin a inhibiting oxidative stress injury of the fundus: the oxidative stress apoptosis and necrosis in human fundus microvascular endothelial cells are inhibited through Akt/SOD2, SOD3 and Prdx-1 signal paths.

FIG. 6 shows that Withaferin A has an inhibitory effect on VEGF-induced migration and tube formation of ocular fundus microvascular endothelial cells.

FIG. 7, the inhibitory effect of Withaferin A on VEGF-induced pathological increase in ocular fundus microvascular permeability.

FIG. 8, establishment of a typical chronic ocular fundus ischemic lesion-Diabetic Retinopathy (DR) mouse model.

Figure 9, Withaferin a inhibits pathological bleeding and angiogenesis in a typical chronic ocular fundus ischemic lesion-Diabetic Retinopathy (DR) mouse model.

FIG. 10 shows the mechanism of Withaferin A in inhibiting pathological bleeding and angiogenesis of ocular fundus ischemic diseases: the targeting inhibits the vascular endothelial growth factor and the receptor (VEGF-VEGFR2) signal channel.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention is further illustrated with reference to the following figures and examples.

Example 1

Withaferin A (WFA) inhibits ischemia reperfusion (I/R) and H in Human Retinal Microvascular Endothelial Cells (HRMECs)₂O₂Induced oxidative stress cell death.

Because of the homology to the retinal and choroidal blood supply, primary human retinal microvasculature was used in this projectSkin cells (HRMECs); administering cellular ischemia reperfusion (I/R) to simulate acute ischemic state of ocular fundus, and administering H₂O₂Treatment can mimic oxidative stress cell damage caused by acute and chronic ischemia. Primary HRMECs cells are purchased from Angio-Proteomie, cells are recovered by Endothelial Growth Medium (EGM) containing 5% FBS, 1% streptomycin and 1% EGF, after the cells are attached to the wall, the cells are replaced, the cells are propagated to 70% of the confluent bottom surface for passage, the cell suspension is centrifuged at 1,000rpm for 5 minutes, and the general passage ratio is 1: 3, the cells were planted in culture dishes on average, and 7 generations of the cells were used for the experiment. Different doses of WFA (0,12.5,25,50 or 100nmol/ml) were administered to cells for 15min pretreatment, followed by ischemia (3 h)/reperfusion (2h) injury, followed by conventional MTT staining to detect cell viability and conventional lactate dehydrogenase detection kit (LDH) to detect cell mortality. The results show that Withaferin a significantly improves cell viability (fig. 1A) and significantly reduces cell death rate (fig. 1B). WFA (0,12.5,25,50 or 100nmol/ml) was given to cells for 15min pretreatment, followed by H₂O₂Mimicking oxidative stress injury of cells, H₂O₂After 2 hours of treatment (100. mu. mol/ml), cell viability was still measured by the conventional MTT method and cell mortality by the conventional LDH method. The results show that Withaferin a also significantly improved cell viability (fig. 1C), and decreased cell death rate (fig. 1D). As can be seen from FIG. 1, Withaferin A (WFA) inhibits ischemia reperfusion (I/R) and H in HRMECs₂O₂The induced oxidative stress cell death, namely the Withaferin A inhibits the oxidative stress cell necrosis and apoptosis of the cells caused by acute and chronic ischemia of the eyeground, and the effect has obvious dose dependence. In fig. 1, n is 5-6. P<0.05，**p<0.01，***p<0.01。

Example 2

Withaferin A inhibits H in HRMECs₂O₂Induced oxidative stress apoptosis.

This experiment employed H₂O₂Treatment mimicked oxidative stress cell injury caused by acute and chronic ischemia. Culturing HRMECs by conventional method, taking 7-generation inner cells, pretreating with WFA (50nmol/ml) for 15min, and culturing₂O₂(100. mu. mol/ml) treatment for 2 hours in vitro experiments to simulate oxidative stress injuryEvaluation of apoptosis by Annexin V-FITC staining assay kit and counting of early apoptotic cells (Annexin V)⁺/PI^-) And late apoptotic cells (Annexin V)⁺/PI^-) The results show that: h₂O₂The resulting induced oxidative stress increased both early apoptotic cells and late apoptotic cells, while the pretreatment with WFA significantly decreased apoptotic cells, and the histogram showed apoptosis of each group of counted HRMECs (fig. 2A). The results demonstrate that Withaferin A inhibits H₂O₂Induced oxidative stress apoptosis. Cell protein samples of each group were prepared routinely, assayed, run on SDS-PAGE, trans-membrane, blocked, anti-overnight, incubated secondary antibodies, and apoptotic end-of-life protein Caspase3 and its activated form, clear Caspase3, were detected by Western analysis in cell tissues of each group to detect apoptosis, and histograms show the clear Caspase3/Caspase3 ratio in cells of each group (FIG. 2B). The result shows that the Withaferin A pretreatment can reduce H₂O₂The resulting clear Caspase3/Caspase3 was elevated. Both experimental results in FIG. 2 demonstrate that Withaferin A inhibits H in HRMECs₂O₂The induced oxidative stress apoptosis, namely the Withaferin A inhibits the oxidative stress apoptosis of the cells caused by acute and chronic ischemia of the eyeground. In fig. 2, n is 2-3. # p<0.05，##p<0.01。

Example 3

Withaferin A for protecting mouse fundus acute ischemia reperfusion injury (I/R) rearview function

In the experiment, an ischemia reperfusion injury (I/R) mouse model is selected to simulate the acute ischemia and hypoxia injury of the eyeground. Since the clinical manifestations of acute ocular fundus ischemic diseases are acute visual function impairment, the experiment detects the visual function changes of the mice in each group. Healthy wild type male C57 mice, age 6-8 weeks. All mice have bright hair color and luster, no depilation, no abnormality is found in eye examination, refraction interstitium is clear, eyeground is normal, and the random classification is as follows: 1. a Control group; 2. I/R injury group; 3. I/R injury + WFA (10nmol/g/d) intraperitoneal injection group; 4. I/R lesion + WFA (5nmol/g/d) paraeyeball (periocular) injection group. 20 in each group. The corresponding drug pretreatment was given 2 hours before the injury model was performed. Stable anesthesia is carried out by using 2% isoflurane, a needle connected with a physiological saline infusion tube is inserted into the right anterior chambers of the three groups of mice, the intraocular pressure of about 110mmHg can be formed in the eyes by increasing the 150cm of the infusion bottle, and pale bulbar conjunctiva and fundus blood vessel disconnection can be observed. After lasting for l hours, the height of the infusion bottle is reduced to the level of the animal, the anterior chamber infusion needle is pulled out, the conjunctiva congestion can be seen, and the blood supply is restored. After 7 days of injury, mice were examined for visual electrophysiological changes in vivo, including the flashing visual evoked potential (fVEP) and the flashing electroretinogram (fhrg). The results show that: the amplitudes of the flashing visual evoked potential (fVEP) groups N1-P1, P1-N2 and N2-P2 after the acute ischemia-reperfusion injury of the mouse fundus are all obviously reduced compared with the amplitudes of the normal groups; WFA significantly reduced the P1-N2, N2-P2 down-regulation of fVEP amplitude both by intraperitoneal and periocular administration, but the difference between the two modes of administration was not statistically significant (fig. 3A). The fERG experimental result shows that the component amplitudes of the a wave and the b wave of the I/R damage group are obviously reduced compared with the normal group; WFA administered intraperitoneally and periocularly significantly reduced ERG B wave amplitude downregulation, and the difference in effect between the two modes of administration was not statistically significant (fig. 3B). The two results show that the Withaferin A can inhibit the mouse acute ischemia-reperfusion injury (I/R) rearview function damage, the Withaferin A can pass through the blood-eye barrier, and the action of local administration and systemic administration is not obviously different. In fig. 3, n is 5-6. P <0.05, p <0.01, NS is undifferentiated, ip, i.p.i. i.p.; po, periocular injection.

Example 4

The mechanism of Withaferin a in inhibiting oxidative stress-induced ocular fundus injury: the oxidative stress apoptosis and necrosis in human fundus microvascular endothelial cells are inhibited through Akt/SOD2, SOD3 and Prdx-1 signal paths.

In order to clarify the mechanism of action of Withaferin a in inhibiting oxidative stress injury caused by hypoxia, H was used in this experiment₂O₂Treating and simulating oxidative stress cell injury caused by acute and chronic ischemia, culturing HRMECs regularly, taking 7-generation cells, treating HRMECs with WFA (50nmol/ml) for 15min or 30 min, preparing each group of cell protein samples, determining content, performing SDS-PAGE electrophoresis, transferring membranes, sealing, performing first-antibody overnight, incubating second antibody, and detecting AMPK, ERK1/2(Thr202/Tyr204) and Western in cell tissuesAkt (Serine473) three housekeeping proteins and their activated forms, and the histogram shows the amounts of the corresponding proteins in each group of cells. The results show that WFA significantly enhanced the phosphorylation levels of two housekeeping proteins, ERK1/2(Thr202/Tyr204) and Akt (Serine473) for a short period of time (FIG. 4A). It is speculated that WFA may have antioxidant stress effects. Culturing HRMECs routinely, taking 7 generations of cells, treating HRMECs with WFA (50nmol/ml) for 30 minutes, extracting total RNA, dissolving, reversely transcribing the RNA into cDNA, performing PCR amplification on a Bio-Rad PCR instrument by using real-time PCR kit, and displaying the results: WFA (50nmol/ml) incubation of HRMECs significantly enhanced the RNA expression levels of all antioxidant proteins tested (fig. 4B). The experimental result confirms that WFA has the function of resisting oxidative stress. Extracting each group of cell protein samples, and further analyzing Western blot detection results and histograms to show that: WFA significantly enhanced the protein expression levels of three antioxidant proteins, SOD2, SOD3, Prdx-1 (FIG. 4C). FIG. 4 shows the results that Withaferin A up-regulates the expression of antioxidant proteins SOD2, SOD3, Prdx-1 and activates ERK, Akt protein as housekeeping protein, ERK, Akt, in oxidative stress cell injury caused by acute and chronic ischemia of ocular fundus, all n-4-6, # p in the figure are likely to act on the upstream of antioxidant proteins SOD2, SOD3, Prdx-1<0.05，##p<0.01，###p<0.001。

In order to further clarify a specific mechanism of inhibiting oxidative stress cell necrosis and apoptosis by Withaferin A in oxidative stress cell injury caused by acute and chronic ischemia of eyeground, HRMECs cells are pretreated for 2 hours by an Akt inhibitor IV (1 mu M) or an ERK inhibitor U1026(10 mu M), and then are treated by WFA (50nmol/ml), so that cell protein samples of each group are extracted, and the Western blot detection result shows that: akt inhibitor IV significantly blocked WFA-mediated upregulation of SOD2, SOD3 and Prdx-, while U1026 was null, and the histogram shows the amount of the corresponding protein in each group of cells (fig. 5A). This result demonstrates that Withaferin A inhibits oxidative stress injury caused by hypoxia through Akt/SOD2, SOD3, Prdx-1B, but not ERK/SOD2, SOD3, Prdx-1 signaling pathways. After the same treatment given to the cells, the results of staining with ROS fluorescent probe Dihydroethidium (DHE) show that: WFA significantly inhibited H₂O₂Resulting in increased active oxygen, Akt inhibitor IV blocked the action of WFA, while U1026 was ineffective, bar chart showingThe corresponding staining results in each group of cells are shown (FIG. 5B). This result further illustrates that WFA inhibits oxidative stress damage in human eye fundus cells by Akt/SOD2, SOD3, Prdx-1. Extracting each group of cell protein samples, and displaying a Western blot detection result: WFA Pre-processing can reduce H₂O₂The resulting increase in clear Caspase3/Caspase3, while Akt inhibitor IV blocked this effect of WFA, the histogram shows the clear Caspase3/Caspase3 levels in each group of cells (FIG. 5C). This result is further illustrated: the Withaferin A inhibits oxidative stress apoptosis in human eye bottom cells through Akt/SOD2, SOD3 and Prdx-1/Caspase3 signal channels. MTT detection can be seen: WFA Pre-processing can reduce H₂O₂Resulting in cell necrosis and apoptosis, whereas Akt inhibitor IV blocked this effect of WFA (fig. 5D). Further shows that WFA inhibits oxidative stress cell necrosis and apoptosis through Akt/SOD2, SOD3 and Prdx-1 signal pathways. All the results in the above fig. 5 collectively illustrate that Withaferin a inhibits oxidative stress cell death and apoptosis in vascular endothelial cells in the fundus of the human eye through Akt/SOD2, SOD3, Prdx-1 signaling pathway in acute and chronic ischemia of the fundus oculi, where n is 5-6 in fig. 5. P<0.05，**p<0.01，***p<0.01, no difference in NS.

Example 5

The Withaferin A has inhibition effect on VEGF-induced fundus microvascular endothelial cell migration, vascularization and pathological microvascular permeability increase.

Long-term ocular fundus hypoxia due to chronic hematological disorders of the ocular fundus induces Vascular Endothelial Growth Factor (VEGF) production. VEGF can increase the permeability of blood vessels to cause obvious blood vessel leakage and pathological angiogenesis, so VEGF is directly used in the experiment to induce the migration of ocular fundus microvascular endothelial cells, the canalization and the pathological increase of the permeability of the microvasculature.

To study the effect of Withaferin a on VEGF-induced migration and tube formation of ocular fundus microvascular endothelial cells, i used three experiments:

1. scratch test: HRMECs cells are digested and inoculated into the middle Insert of Culture Insert, after the cells grow over the Insert area, the Insert is removed by tweezers, and the scratch with the width of 500 μm can be generated. WFA (0,10,20,50 or 100nmol/ml) was given for pretreatment 15 minutes and VEGF (10ng/ml) was given for incubation for 8 hours, photographed and recorded, and the picture data was collected to analyze the experimental results. The results of this experiment can be seen: VEGF (10ng/ml) culture for 8 hours significantly increased migration of HRMECs, whereas Withaferin A administration inhibited migration of HRMECs with a dose-dependent increase, bar graph showing the results of various cell scratch experiments (FIG. 6A).

2. Migration experiment: HRMECs (1.0X 10) were added to the inner chamber of the Transwell chamber ⁵100 μ l/well), WFA (0,10,20,50 or 100nmol/ml) for 15 minutes, VEGF (10ng/ml) for 8 hours, removing the inner chamber of the Transwell, wiping the cells on the side of the PVPF membrane close to the inner chamber with a cotton swab, fixing the cells on the other side with formaldehyde at room temperature for 30 minutes, crystal violet staining for 20 minutes, washing with clear water for more than 3 times, observing the migration of the cells under a microscope, and counting. The results show that: VEGF (10ng/ml) incubation for 8 hours significantly increased migration of HRMECs in the Transwell chamber, whereas Withaferin A administration inhibited migration of HRMECs with a dose-dependent increase, and the histogram shows the results of the cell migration experiments for each group (FIG. 6B).

3. Tube forming experiment: mixing HRMECs (1 x 10)⁴Per well, 50 μ l) were plated on a Matrigel-plated 96-well plate, and the plated cells were treated with WFA (0,10,20,50 or 100nmol/ml) for 15 minutes and then with VEGF (10ng/ml) for 8 hours of culture, as shown: VEGF (10ng/ml) culture for 8 hours significantly increased angiogenesis of HRMECs, whereas Withaferin A administration inhibited angiogenesis of HRMECs with a dose-dependent increase in effect, the histogram shows the tube-forming experimental results (FIG. 6C).

As shown in fig. 6, these three experiments illustrate: the Withaferin A has an inhibiting effect on VEGF-induced migration and tube formation of ocular fundus microvascular endothelial cells, and the effects are increased in a dose-dependent manner. In the figure, n is 5, and the difference has statistical significance, x: p <0.01 compared to Control group; ***: p <0.001 compared to Control group; #: p <0.05 compared to VEGF group; # #: p <0.01 compared to VEGF group; # ##: p <0.001 compared to VEGF group.

To investigate the effect of Withaferin a on VEGF-induced increase in microvascular permeability, i used two experiments:

X-CELLigence cell transendothelial cell resistance experiment

Taking HRMECs from 3-5 generations after passage, 3 × 10 per well⁴The cells were seeded on an E-16 plate and transferred to the X-CELLigence System. The resistance across endothelial cells was monitored for each group of cells, measured every 15 minutes. The higher the transendothelial cell resistance the lower the membrane permeability. After the cell index curve reached the highest peak and stabilized, WFA (50nmol/ml) was given for pretreatment for 15 minutes, and VEGF (10ng/ml) was given for 8 hours of incubation, the results are shown: the curve shown in FIG. 7A is 1 and a Vehicle group from top to bottom; 2. withaferin a; 3. VEGF lesion + withferin a group; 4. VEGF injury group. Histograms show the transendothelial electrical resistance of the groups, demonstrating that VEGF culture in the X-celgene system significantly increased the permeability of the monolayer of HRMECs membrane, while administration of Withaferin a pretreatment inhibited the increase in permeability of the monolayer of HRMECs membrane (fig. 7A).

Transwell chamber monolayer cell membrane permeability assay.

This experiment used a Transwell chamber (Coning corporation) of 0.4um pore size, and HRMECs from the 3 rd to 5 th generations after passage were taken, 1.0x10⁵Perwell, inoculated into a Transwell chamber, when the permeability is stabilized for three consecutive days after the cells are grown and fused, treated with WFA (50nmol/ml) pretreatment for 15 minutes followed by VEGF (10ng/ml) incubation for 8 hours, 1mg/ml FITC-Dextran solution is added to the chamber of the Transwell chamber, and the absorbance measured by a microplate reader (490nm) after half an hour is representative of the cell membrane permeability. Histograms show the permeability of the cell membranes of the various groups of monolayers, with the results being seen: in the Transwell chamber, VEGF significantly increased the permeability of the monolayer of HRMECs membrane to the tracer, while Withaferin a administration inhibited the passage of the tracer (fig. 7B).

The two above experiments in fig. 7 illustrate the inhibitory effect of Withaferin a on VEGF-induced microvascular permeability increase, where n is 4-6, with statistical differences: p <0.01 compared to Control group; #: p <0.05 compared to VEGF group; # #: p <0.01 compared to VEGF group.

Fig. 6 and fig. 7 together illustrate that the ocular fundus ischemic disease causes VEGF to induce the migration, tube formation and pathological increase of permeability of ocular fundus microvascular endothelial cells, and in a cell experiment, Withaferin a clearly and remarkably inhibits the effect.

Example 6

Withaferin a inhibits pathological bleeding and angiogenesis in a typical chronic ocular fundus ischemic disorder-Diabetic Retinopathy (DR) animal model.

Chronic ischemic ocular fundus diseases include: the experiment selects a Diabetic Retinopathy (DR) animal model with the most representative chronic fundus ischemic diseases to detect the effect of Withaferin A in an animal body, because the clinical main manifestations of the chronic fundus ischemic diseases are fundus hemorrhage leakage and pathological angiogenesis, the central retinal vein occlusion, branch vein occlusion, diabetic retinopathy, ocular ischemic syndrome, macroarteritis retinopathy, retinopathy of prematurity, age-related macular degeneration and the like. Healthy wild type male C57 mice were selected and aged 6-8 weeks. All mice had bright hair color and no depilation, no abnormality was found in eye examination, clear refraction interstitium, normal fundus, which were: 1. a Control group; 2. withaferin group a; 3. HFD/STZ-DR (high fat diet + streptozotocin induced diabetic retinopathy) group; 4. HFD/STZ-dr (Withaferin a) (high fat diet + streptozotocin induced diabetic retinopathy + Withaferin a treatment) group, 20 each. Mice in the HFD/STZ-DR, HFD/STZ-DR (withaferin a) groups were first given High Fat Diet (HFD) (60% fat caloric diet) to induce obesity, impaired glucose tolerance and insulin resistance for 12 weeks (fig. 8B, C, D). Thereafter, mice were given a single injection of low-dose Streptozotocin (STZ) citrate buffer (50mg/kg) into the abdominal cavity after fasting for 12 hours. Blood glucose levels were checked after 3 days using a check, and mice were considered to have elevated blood glucose above 12mmol (fig. 8E); after determining the blood sugar level of the mice was elevated, the mice were fed a High Fat Diet (HFD) for 8 weeks, the blood sugar level of the mice was significantly increased (FIG. 8F, H), there was no significant difference in body weight (FIG. 8G), and microvascular leakage occurred in the fundus (FIG. 8I), confirming the success of the DR model by HFD/STZ. The molding process is shown in fig. 8A. In fig. 8, p <0.05, p <0.01, and NS are not different. Thereafter, the HFD/STZ-DR (Withaferin A) group was intraperitoneally implanted with an Alzet implantable drug delivery pump Withaferin A10nmol/g/d for 7 days, and the remaining mice were given control treatment for 7 days. After the treatment, the mice in each group were anesthetized, and 10ml/kg of a mixture of FITC-Dextran and FRITC-Concanavalin A1: 2 was slowly injected into the superior vena cava to stop bleeding by cotton ball compression. After 5 minutes, the mice were sacrificed and the eyes were quickly harvested. The eyeball specimens were placed in 4% PFA-PBS precooled at 4 ℃ and fixed on ice for 3 hours. Conventional retinal patches, hard mounting patches. Stained retinal sections were photographed by confocal microscopy, FRITC-Concanavalina for blood vessel delineation, and FITC-Dextran for blood vessel leakage. ImageJ software calculates the extent of vascular leakage. An increase in microvascular permeability is suggested by FITC-Dextran leakage; when FRITC-ConcanavalinA appeared in the non-perfusion area, and FITC-Dextran leakage was severe, it was suggested that new blood vessels appeared. The results show that: retinal vascular leakage was not seen in Control and Withaferin a mice. Compared with the Control group, the DR mouse group induced by HFD/STZ has obviously increased retinal vascular leakage and has retinal neovascularization, which proves that the DR mouse model is successfully induced. The HFD/STZ-DR (Withaferin A) group significantly reduced retinal vascular leakage and inhibited the appearance of retinal neovasculature in DR mice. This result suggests that Withaferin a can also inhibit DR-induced pathological microvascular bleeding, leakage and angiogenesis in animals and that Withaferin a can act across the blood-ocular barrier (fig. 9A). The histogram shows retinal vascular leakage in each set of retinal patches. n-4, the difference was statistically significant (fig. 9B), fig. 9: ***: p <0.001 compared to Control group; # #: p <0.01 compared to the HFD/STZ-DR group. The two leftmost gray arrows in the third graph from top to bottom on the right represent retinal neovascularization, and the remaining white arrows represent retinal microvascular bleeding leaks.

Example 7

The mechanism of the Withaferin A for inhibiting pathological bleeding and exudation and angiogenesis of the fundus ischemic disease is as follows: the targeting inhibits the vascular endothelial growth factor and the receptor (VEGF-VEGFR2) signal channel.

In order to clarify the action mechanism of Withaferin A for inhibiting pathological bleeding and angiogenesis of ischemic diseases of eyeground, HRMECs are cultured conventionally, 7 generations of cells are taken, WFA (0,10,50 or 100nmol/mL) with different doses is given to the cells for 15min for pretreatment, HG (High Glucose, High sugar treatment, 25 mu mol/mL D-Glucose) is given for 8 hours to simulate Diabetic Retinopathy (DR) injury, the expression of secretory VEGF in a culture medium is slightly increased after HG is given for 8 hours by gel electrophoresis of the conventional culture medium, and the expression of secretory VEGF in the culture medium which is given to the Withaferin A for pretreatment is not obviously changed (figure 10A). Media ELISA analysis also showed that secreted VEGF expression in the media increased 8 hours after HG administration, with no significant change in secreted VEGF expression in the media after Withaferin a pretreatment (fig. 10B). These two results illustrate that: withaferin a did not affect secreted VEGF expression. Extracting each group of cell protein samples, and displaying a Western blot detection result: intracellular VEGF expression increased 8 hours after HG administration, whereas intracellular VEGF expression did not change significantly after Withaferin a pretreatment; however, intracellular VEGF downstream receptors VEGFR2 and VEGFR2, activated form pvgfr 2, were stacked (fig. 10C). This result illustrates that: withaferin a also did not affect cellular VEGF expression, but Withaferin a affected VEGF binding to its receptor VEGFR 2. In order to research the specific mechanism that Withaferin A influences the combination of VEGF-VEGFR2, HRMECs are cultured conventionally, and 7 generations of cells are taken and divided into 1 and Control groups; 2. WFA group; 3. HG group; 4. HG + WFA group. WFA (50nmol/ml) is used for carrying out pretreatment on cells for 15min, HG is used for simulating Diabetic Retinopathy (DR) damage for 8 hours, and the result of detection by a conventional CO-immunoprecipitation method (CO-IP) shows that a small amount of VEGF-VEGFR2 is combined in the Control group; VEGF-VEGFR2 binding was significantly reduced in the WFA group; HG group VEGF-VEGFR2 bound more; the HG + WFA group VEGF-VEGFR2 binding was significantly reduced (fig. 10D), which suggests that Withaferin a could target VEGF binding to its receptor VEGFR2, thereby blocking VEGF signaling pathway conduction, and thus inhibiting VEGF-induced fundus microvascular endothelial cell migration, tubulogenesis, and pathological increase in microvascular permeability caused by long-term ocular fundus hypoxia. In fig. 10, n is 4-6, p is 0.05, and NS is not different.

The in vitro, in vivo and molecular mechanism experimental results show that the Withaferin A can obviously reduce oxidative stress cell necrosis and apoptosis caused by fundus ischemia; the targeting inhibits vascular endothelial growth factor and receptor (VEGF-VEGFR2) signal path, and improves pathological microvascular bleeding and leakage and angiogenesis and other symptoms. Thereby effectively resisting acute and chronic ischemic fundus diseases from the aspect of pathogenesis. The Withaferin A has clear effect and action mechanism, can penetrate through the blood-eye barrier, and can be applied to the new clinical era that the anti-oxidation small-molecule medicament can be used for treating ischemic ocular fundus diseases in a targeted manner.

The complications of the fundus ischemic diseases mainly refer to neovascular glaucoma and corneal neovascularization.

Claims

The application of Withaferi in preparing the medicine for treating the ischemic diseases of the eyeground; the fundus ischemic disease is retinopathy.
2. Use according to claim 1, characterized in that: the retinopathy is acute retinopathy or chronic retinopathy.
3. Use according to claim 1, characterized in that: the retinopathy is central retinal artery occlusion, branch retinal artery occlusion, ciliary retinal artery occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration.
4. Use according to claim 1, characterized in that: the retinopathy is retinal vascular hemorrhage and leakage or retinal cell necrosis and apoptosis or retinal angiogenesis.