TWI721274B - Use of a composition in the manufacture of a drug for treating developmental retinal vascular disorder - Google Patents

Abstract

The present invention relates to the use of a composition in the manufacture of a drug for treating developmental retinal vascular disorder, wherein the composition comprises a therapeutically effective amount of a dendrobium nobile lindley (DNL).

Description

Use of composition for preparing medicine for treating developmental retinal vascular disease

The present invention provides a composition for the preparation of a drug for the treatment of developmental retinal vascular diseases, characterized in that the composition contains a Dendrobium nobile, which can effectively improve retinal vascular development abnormalities, thereby avoiding retinal vascular development abnormalities caused by Of ischemic injury.

Norrin-dependent Wnt signaling pathway seems to mediate defects in vasculogenesis (early retinal vascular development). Norrie/Frizzled-4 (Frizzled-4) signal plays an important role in angiogenesis, such as Norrie disease (ND) and familial exudative vitreoretinopathy (FEVR), and ultimately It may progress to retinal ischemia and neovascularization (NV) or angiogenesis. In addition to ND and FEVR, there are other developmental retinal vascular diseases, namely Coats disease (Coats disease) and persistent hyperplastic primary vitreous (PHPV), which have similar fundus pictures, namely peripheral retina There is no peripheral retinal avascularization and subretinal exudation. As mentioned above, these vitreoretinopathy may also cause retinal ischemia and pose a similar threat to the patient’s vision, although they are not like other retinal ischemic diseases (ie central retinal artery occlusion, CRAO), branch retinal artery occlusion (BRAO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), glaucoma, diabetic retinopathy , DR) or neovascular age-related macular degeneration (nvAMD)). As previously reported in the literature, persistent hypoxia is presumed to be one of the main driving forces for the development of these developmental retinal vascular diseases (such as ND).

Retinal ganglion cells (RGCs) and axon cells (amacrine cells) located in the inner retina are susceptible to ischemia/reperfusion (I/R) damage. Furthermore, after ischemia, the immune markers of vimentin and glial fibrillary acidic protein (GFAP) in Müller cells will increase; this is related to the decrease in the number of RGCs. It is known that on ischemic retina, vascular endothelium growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α), pyruvate kinase M2 (PKM2) ) And retinoblastoma-binding protein 2 (RBP2) will be overexpressed at the same time, and further abnormal neovascularization (NV) (late neovascularization) may lead to edema and hemorrhage Visual dysfunction. The up-regulation of HIF-1α and VEGF can also be observed in the retina depleted of Norrin. In addition to VEGF, placental growth factor (PLGF) has been reported to increase in confirmed retinal/choroidal vascular diseases; therefore, the down-regulation of this factor can be used as a result of visual function and a biomarker of treatment.

Dendrobium nobile (DNL) is a member of the orchid family (Orchidae) plant family, which belongs to the herbal medicine for improving eyesight. DNL is also used as a tonic and has been found to have antipyretic/anti-inflammatory effects and anti-angiogenic (such as anti-VEGF/HIF-1α) properties. Dendrobium nobile (DNL) has a variety of active ingredients related to different mechanisms of action, including alkaloids (through the inhibition of p-p38 MAPK and NF-κB pathways to over-express TNFR1), flavonol glycosides ) (α-glucosidase inhibitor), SG-168, polysaccharides (antioxidant). In addition, the anti-angiogenesis or antioxidant moscatilin is the active ingredient of Dendrobium nobile (bibenzyl), and may have different known ingredients (anti-VEGF/HIF-1α, .OH free radical scavenger). , Anti-inflammatory and anti-apoptosis) and unknown mechanism of action.

The present invention uses electroretinogram (ERG), immunohistochemistry (retinal ganglion cells, axonal nerve cells, Mühler cells), histopathology (retinal thickness), cell survival rate and protein measurement analysis (PLGF, HIF) -1α, VEGF-A, PKM2, RBP2 and NDP) confirmed the changes caused by various ischemia/hypoxia (OGD) in the retina or retinal cells. The present invention has found that the protein expression levels of HIF-1α, VEGF, PKM2 and RBP2 are significantly up-regulated in the ischemic retina, but the up-regulation of these proteins can be significantly reduced by administering Dendrobium nobile. When VEGF trap/anti-PLGF Eylea was administered to the ischemic retina in advance, the rise of PLGF induced by ischemia was also significantly reduced; and 1.0g/Kg/day DNL also had this effect. Therefore, its novelty and clinical significance is that DNL may have anti-angiogenesis/VEGF (PLGF) trapping effects. This is inconsistent with the previous report that moscatilin, the bibenzyl component of DNL, is an anti-angiogenic agent by inhibiting HIF-1α and VEGF.

Ischemia may be closely related to developmental retinal vascular diseases (such as FEVR or ND). Although anti-VEGF antibody can effectively remove eye bleeding and macular edema, it is disappointing that some patients have poor vision. Norrin's Wnt signaling pathway plays an important role in the early normal development of retinal blood vessels and the late progression of definite developmental retinal vascular diseases. The latter situation may further aggravate ischemia/hypoxia and form new blood vessels (NV). In agreement, Norrie disease protein (NDP) seems to protect the eye from abnormal angiogenesis and retinal diseases by modulating the Norrin-dependent Wnt signaling pathway. In addition, overexpressed NDP can protect photoreceptors and RGCs from cell death by activating the norrin-dependent Wnt signal pathway. The present invention also confirms that hypoxia (OGD) can lead to a significant decrease in NDP expression and cell survival rate. However, Dendrobium nobile (0.1μM), the bibenzyl component of Dendrobium nobile, can significantly alleviate the damage caused by hypoxia/ischemia-like (OGD). Therefore, the present invention confirms that Dendrobium nobile and/or Dendrobium phenol can activate the NDP-dependent Wnt signal pathway, thereby providing neuroprotection, and resisting the symptoms of retinal ischemia by inhibiting VEGF-A/PLGF and up-regulating NDP.

In addition, after ischemic injury and vehicle administration, the thickness of the inner layer of the retina, the number of RGCs, and the ChAT immunoreactivity on axon-free nerve cells decreased significantly and significantly. Importantly, the present invention proves that these ischemia-induced changes are significant and obviously passivated before and/or after high-dose Dendrobium nobile (1g/kg/day) is administered. In addition, in the ischemic retina before/after administration of vehicle, the overexpression of vimentin/GFAP immunolabel is accompanied by a decrease in b wave. However, the results of the present invention show that these ischemic changes are significantly and significantly offset before/after administration of 1 g/kg/day Dendrobium nobile, which is of clinical importance.

The results of the present invention show that ischemia/hypoxia (ischemia mimic OGD) significantly and obviously affects retinal electrophysiology, morphometrics, immunohistochemistry and retinal molecular biology/cell survival rate. It is clinically important that all these changes due to ischemia/OGD can be effectively reduced by pre-treatment and/or post-treatment with Dendrobium nobile or its bibenzyl component Dendrobium. Therefore, the present invention believes that these protective mechanisms work through the inhibition of HIF-1α, VEGF-A, PKM2, RBP2 and, most importantly, the up-regulation of PLGF and up-regulation of the expression level of NDP (as shown in Figure 9).

In short, Dendrobium nobile and/or Dendrobium phenol can protect or prevent certain retinal ischemia/ischemia-like changes, and it can be achieved by inhibiting PLGF and up-regulating NDP. The treatment of Dendrobium nobile (and/or Dendrobium phenol) is a useful way to prevent and/or manage patients with developmental retinal vascular disease that may develop due to persistent ischemia/hypoxia.

The present invention uses electroretinogram (müller cells and b-wave changes on bipolar cells), immunohistochemistry (fluorescence immunolabeled retinal ganglion cells (RGC), choline acetyltransferase ( ChAT) immune-positive axonal nerve cells, vimentin/glia fibrillary acidic protein (GFAP) stained Murdoch cells), histopathology (retinal thickness measurement), and/or western blot method (HIF-1α, VEGF, PKM2 and RBP2) to observe various ischemic changes in the rat retina. Importantly, the administration of Dendrobium nobile (DNL) before/after ischemia-reperfusion (I/R) can significantly modulate all these defined ischemic-related characteristics. Most importantly, under the condition of retinal ischemia/ischemia-like (OGD), the protein concentration of placental growth factor (PLGF) is up-regulated (Figure 8), and the protein concentration of Norrie disease protein (NDP) is down-regulated (Figure 2) ), but treatment with Dendrobium nobile (DNL)/moscatilin will offset these changes. In a clinical sense, DNL can prevent or protect the defined retinal ischemia/ischemia-like changes by down-regulating PLGF and up-regulating NDP. Therefore, Dendrobium nobile/Dendrobium can provide another way to prevent or manage developmental vascular diseases related to persistent hypoxia/ischemia by down-regulating the expression level of placental growth factor and up-regulating the concentration of Norri disease protein. ) An alternative method for patients in the course of the disease.

The term "a" or "a" in this text is used to describe the elements and components of the present invention. This term is only for convenience of description and to give the basic idea of the present invention. This description should be understood to include one or at least one, and unless clearly indicated otherwise, the singular number also includes the plural number. When used with the word "including" in the scope of the patent application, the term "a" can mean one or more than one.

The term "or" in this article means the same as "and/or".

The present invention provides a composition for the preparation of a drug for the treatment of developmental retinal vascular disorder (developmental retinal vascular disorder), wherein the composition contains an effective dose of Dendrobium nobile Lindley (DNL).

In a specific embodiment, the ingredients of Dendrobium nobile include bibenzyls, alkaloids, glycosides and polysaccharides. In a preferred embodiment, the Dendrobium nobile contains a bibenzyl compound. In a more preferred embodiment, the bibenzyl compound contains moscatilin.

In a specific embodiment, the structural formula of the moscatilin is:

In a specific embodiment, the treatment of developmental retinal vascular disease is achieved by improving defects in retinal vascular development. The development of retinal blood vessels is divided into two parts: (1) Vasculogenesis: It starts around the 18th week of pregnancy and is completed in the 38th-40th week of pregnancy. Because blood vessels are organized into a network of vascular plexuses, which are formed by the combination of vasculogenesis and angiogenesis; angiogenesis is formed by endothelial precursor cells, which aggregate The cord then forms the lumen, a process of de novo formation of blood vessel development; and (2) angiogenesis: blood vessels are formed by sprouting from existing blood vessels. It is generally believed that angiogenesis forms most of the main retinal vascular plexus of the developing retina, while angiogenesis forms the remaining capillary layer around the fovea and on the deeper and peripheral retina. In a preferred embodiment, the Dendrobium nobile or Dendrobium phenol treats the developmental retinal vascular disease by improving vasculogenesis defects.

As used herein, the term "treatment" includes reducing the severity of developmental retinal vascular disease, delaying the onset of developmental retinal vascular disease, regressing developmental retinal vascular disease, alleviating or stopping developmental retinal vascular disease. Symptoms caused by developmental retinal vascular disease. The term "treatment" includes but is not limited to prophylactic and/or therapeutic treatment.

In another specific embodiment, the developmental retinal vascular disease includes persistent hyperplastic primary vitreous (PHPV), Norrie disease (Norrie disease), and familial exudative vitreoretinopathy (FEVR). ) And Coats disease. In a preferred embodiment, the developmental retinal vascular disease includes Norrie disease and familial exudative vitreoretinopathy. In a more preferred embodiment, the developmental retinal vascular disease is Norrie disease.

Since the developmental retinal vascular disease is a disease of abnormal retinal vascular development, it can cause retinal ischemia and impaired vision of the patient. In another embodiment, the symptoms of the developmental retinal vascular disease include abnormal retinal vascular development, retinal ischemia, and impaired vision.

Because the developmental retinal vascular disease can cause retinal ischemia, and retinal ischemia can cause retinal ischemic damage. In a specific embodiment, the retinal ischemic injury includes thinning of the thickness of the retina, a decrease in the number of retinal ganglion cells (RGC), and related factors (such as hypoxia-inducible factor (HIF), retinoblastoma binding protein 2 (RBP2), pyruvate kinase M2 (PKM2), vascular endothelial growth factor (VEGF)) increased. In another specific embodiment, the Dendrobium nobile or the Dendrobium phenol treats retinal ischemic damage caused by the developmental retinal vascular disease.

The retinal ischemic damage will make the thickness of the retina thinner, so administering the Dendrobium nobile or the Dendrobium phenol can avoid the phenomenon of thinning of the retinal thickness, that is, the Dendrobium nobile or the Dendrobium phenol can treat or increase the retina thickness. In a specific embodiment, the Dendrobium nobile or the Dendrobium phenol treats the thinning of the retinal thickness caused by the developmental retinal vascular disease.

The developmental retinal vascular disease can cause ischemic damage to the retina, which will result in a decrease in the number of retinal ganglion cells (RGC). The administration of Dendrobium nobile or dendrobium can increase the number of retinal ganglion cells (RGC) or Protect retinal ganglion cells from cell death to treat ischemic damage to the retina in this developmental retinal vascular disease. In a specific embodiment, the Dendrobium nobile or the Dendrobium phenol increases the number of retinal ganglion cells (RGC).

In addition, the developmental retinal vascular disease causes ischemic damage to the retina, which in turn promotes hypoxia-inducible factor (HIF), retinoblastoma binding protein 2 (RBP2), pyruvate kinase M2 (PKM2), and vascular endothelial growth in the retina. The expression level of the factor (VEGF) increases, and the administration of the Dendrobium nobile or the Dendrobium phenol can reduce the expression level of the above factors, so as to treat the retinal ischemic damage in the developmental retinal vascular disease. In a specific embodiment, the dendrobium nobile or the dendrobium phenol reduces hypoxia-inducible factor-1α (HIF-1α), retinoblastoma binding protein 2 (RBP2), pyruvate kinase M2 (PKM2) and vascular endothelial growth The expression level of factor (VEGF).

This developmental retinal vascular disease causes angiogenesis. Therefore, in another embodiment, the Dendrobium nobile or the Dendrobium phenol can capture vascular endothelial growth factor (VEGF) to prevent anti-angiogenesis.

The term "expressive quantity" in this article includes but is not limited to the expressive quantity of DNA, RNA or protein.

The retinal ischemic damage caused by the developmental retinal vascular disease will up-regulate the expression level of placental growth factor (PLGF) and down-regulate the expression level of Norrie disease protein (NDP). The administration of Dendrobium nobile or Dendrobium can down-regulate the expression level of placental growth factor (PLGF) and up-regulate the expression level of Nori disease protein (NDP) to treat the developmental retinal vascular disease due to ischemia/deficiency. Retina damage caused by oxygen. Innori disease protein (NDP) can protect the eye from vascular development abnormalities and retinal ischemic damage caused by regulating the norrin-dependent Wnt signal pathway. In a specific embodiment, the Dendrobium nobile or Dendrobium phenol down-regulates the expression level of placental growth factor (PLGF) and up-regulates the expression level of Norrie disease protein (NDP). In a preferred embodiment, the Dendrobium nobile or Dendrobium phenol up-regulates the expression of Norrin disease protein (NDP) to regulate the Norrin-dependent Wnt signal pathway to improve retinal vascular development abnormalities. In a more preferred embodiment, the Dendrobium nobile or Dendrobium phenol up-regulates the expression level of Norrin disease protein (NDP) to regulate the Norrin-dependent Wnt signal pathway to improve retinal ischemia.

The term "effective dose" as used herein refers to a therapeutic dose that can prevent, reduce, prevent, or reverse the development of a symptom in an individual under certain conditions, or partially or completely relieve the individual when the individual starts receiving treatment. It already exists under special circumstances. Symptoms.

In a specific embodiment, the effective dose of Dendrobium nobile is in the range of 0.01 g/kg body weight to 100 g/kg body weight. In a preferred embodiment, the effective dose of Dendrobium nobile is in the range of 0.1 g/kg body weight to 50 g/kg body weight. In a more preferred embodiment, the effective dose of Dendrobium nobile is in the range of 0.5 g/kg body weight to 10 g/kg body weight. In another preferred embodiment, the effective dose of Dendrobium nobile is in the range of 0.1 g/kg body weight to 5 g/kg body weight.

In another embodiment, the effective dose of the dendrobium phenol ranges from 0.01 g/kg body weight to 100 g/kg body weight. In a preferred embodiment, the effective dose of the dendrobium phenol is in the range of 0.1 g/kg body weight to 50 g/kg body weight. In a more preferred embodiment, the effective dose of the dendrobium phenol ranges from 0.5 g/kg body weight to 10 g/kg body weight. In another preferred embodiment, the effective dose of the dendrobol is in the range of 0.1 g/kg body weight to 5 g/kg body weight.

In a specific embodiment, the effective dose is administered once a day. In a preferred embodiment, the effective dose is administered two or more times a day.

The drug can be administered on a routine schedule. The routine schedule used in this article refers to the predetermined and designated period. The routine timetable may cover some periods, which are the same or different in length, as long as the timetable is predetermined. For example, a routine schedule may involve dosing once a day, every day, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis, or any set number of days in between. The number of weeks. Alternatively, the predetermined routine schedule may involve dosing on a once-daily basis, for the first week, and then on a daily basis, for several months, etc. In other specific embodiments, the present invention provides that the drug can be taken orally, and the timing is dependent on or independent of food intake. Thus, for example, the drug can be taken every morning and/or every night, regardless of when the patient has eaten or will eat.

The medicament of the present invention may further comprise a pharmaceutically acceptable carrier, and can be administered to a body through many different ways in the conventional treatment methods in the relevant field of the present invention. In some embodiments, the composition (including Dendrobium nobile or Dendrobium phenol) and a pharmaceutically acceptable carrier will be administered via external, intravenous, intramuscular, subcutaneous, topical, oral or inhalation administration. The drug will be delivered to the target through the digestive and circulatory system. In a preferred embodiment, the route of administration of the drug is oral administration.

In another embodiment, the individual is an animal, preferably a mammal, and more preferably a human.

The term "pharmaceutically acceptable carrier" as used herein is determined by the specific combination of administration and the specific method of administration of the composition. As used herein, the term "carrier" includes but is not limited to any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, etc. penetration and absorption delaying agents, buffers, carrier solutions, suspensions , Colloid, etc. These media and agents for pharmaceutically active substances are well known in the art. Unless any conventional medium or agent is incompatible with the active ingredient, its therapeutic combination needs to be considered. Supplementary active ingredients can also be incorporated into the composition. The term "pharmaceutically acceptable" means that molecular entities and compositions do not produce allergic or similar adverse reactions when administered to a subject. The preparation of water compositions with protein as the active substance is well known in the art. Usually, the composition is prepared as a liquid solution, lozenge, capsule or suspension injection; it can also be prepared as a solid form that can be used for injection which can be dissolved or suspended.

The preparation of the composition (including Dendrobium nobile or Dendrobium phenol) and a pharmaceutically acceptable carrier may be through sterile aqueous solutions or dispersions, water suspensions, oil emulsions, water in oil emulsions, and emulsification at specific points. Liquid, long-stay emulsion, viscous emulsion, microemulsion, nanoemulsion, liposomes, microparticles, microspheres, nanospheres, nanoparticle, micromercury, and several sustainable-release natural or synthetic polymers . The pharmaceutically acceptable carrier and the Dendrobium nobile (or Dendrobium phenol) can also be formulated into aerosols, tablets, pills, capsules, sterile powders, suppositories, lotions, creams, ointments, pastes, gels , Hydrogel, or other formulations that can be used for composition delivery.

When the composition is prepared into a medicine, the unit dose of Dendrobium nobile or Dendrobium phenol contained in each unit of medicine is 0.5-50 grams (g), preferably 0.1-10 grams (g), more preferably 0.01- 1 gram (g).

Therefore, the composition (including Dendrobium nobile (DNL) or Dendrobium phenol) provided by the present invention can treat developmental retinal vascular diseases, including but not limited to repairing/treating abnormal retinal vascular development or defects in vascular development; in addition, Due to abnormal retinal vascular development, it can cause retinal ischemia, so the composition of the present invention can further improve the adverse effects and damage caused by ischemia/hypoxia to the retina, that is, protect the retina from ischemia/hypoxia damage.

In Figure 1, (a) to (e) are observations of retinal ganglion cell-5 (RGC-) treated by moscatilin (Mos) on oxygen glucose deprivation (OGD) under an optical microscope. 5) Study on the effect of cell viability. Compared with the normal control group (cells cultured in DMEM medium and pre-administered DMSO, DMSO+DMEM), DMSO (Vehicle) followed by OGD (DMSO+OGD) was pre-administered Later, the number of cells is small and some will be deformed (as indicated by the arrow). Pre-administration of dendrobium (Pre-OGD Mos 0.1μM) 1 hour before OGD treatment will alleviate the changes induced by OGD. (f) Quantitative analysis of the influence of Mos on OGD-treated cells using MTT analysis. **: There is a significant difference between the normal control group (DMSO+DMEM) and the DMSO+OGD group (P<0.01). †: There is a significant difference between the DMSO+OGD group and the Pre-OGD Mos 0.1μM group (P=0.04). The experimental results are expressed as mean±standard error (SEM) (the number is 6). The scale bar is 50μm. DMSO+DMEM: DMEM medium containing DMSO; DMSO+OGD: DMSO was administered 1 hour before OGD treatment, followed by OGD; Pre-OGD Mos 0.1μM: 0.1μM dendrobium (Mos) was administered 1 hour before OGD treatment, and then Then OGD; During OGD Mos 0.1 μM: 0.1 μM dendrobol (Mos) was administered during OGD; and Post-OGD Mos 0.1 μM: 0.1 μM dendrobol (Mos) was administered 1 hour after OGD treatment. N: number.

Figure 2 shows the effect of moscatilin (Mos) on the protein expression of Norrie disease protein (NDP) relative to β-actin. Top image: a series of representative images of immune ink dots; bottom image: histogram. In the normal control group (DMSO+DMEM: retinal ganglion cell-5 (RGC-5) cells cultured in DMEM medium, and pre-administered with dimethylsulfoxide (DMSO)), the expression level of β-actin protein adjusted by NDP in the normal control group (DMSO+DMEM: retinal ganglion cell-5 (RGC-5)) Is 100%. **: There is a significant difference between the DMSO+DMEM group and the DMSO+OGD group (P<0.01). †: There is a significant difference between the DMSO+OGD group and the Pre-OGD Mos 0.1μM group (P=0.048). The experimental results are expressed as mean±standard error (SEM) (the number is 3). OGD: oxygen glucose deprivation; RGC: retinal ganglion cells. DMSO+DMEM: DMEM medium containing DMSO; DMSO+OGD: DMSO was administered 1 hour before OGD treatment, followed by OGD; Pre-OGD Mos 0.1μM: 0.1μM dendrobium (Mos) was administered 1 hour before OGD treatment, and then Then OGD; During OGD Mos 0.1 μM: 0.1 μM dendrobol (Mos) was administered during OGD; and Post-OGD Mos 0.1 μM: 0.1 μM dendrobol (Mos) was administered 1 hour after OGD treatment.

Figure 3 shows the electroretinogram (ERG) analysis. (a) and (b): Compared with the retina of the control group (sham), the vehicle was administered before the retinal ischemia reperfusion (I/R) induced by high intraocular pressure (HIOP) After vehicle (a) or later administration of vehicle (b), the b-wave amplitude of ERG drops sharply. And this reduction can be done by pre-administering Dendrobium nobile (DNL) (DNL1.0+I/R; DNL0.5+I/R, a) or post-administering Dendrobium nobile (I/R+DNL1.0) , B) will produce a dose-dependent offset. (c): Compared with the control group (sham), the b-wave ratio of ERG in the Vehicle+I/R group was significantly reduced after retina ischemia-reperfusion (I/R) (**; P<0.01) . Pre-administered high-dose (DNL1.0+I/R) and low-dose (DNL0.5+I/R) DNL showed a dose response and significantly offset this ischemic-induced reduction (††; P< 0.01). (d): The b-wave amplitude of ERG decreased significantly on the 1, 3, 5, or 7 days after the administration of the vehicle after the I/R retina and ischemia (**; P<0.01). Post-administration of DNL (I/R+DNL1.0) can significantly alleviate the decrease in the b-wave amplitude of ERG (†/††; P<0.05/0.01). The experimental results are expressed as mean ± standard error (SEM); the number of experiments is 10-12. Sham (control group): sham procedure operation experiment (the number is 12); Vehicle+I/R: vehicle is administered before retinal ischemia-reperfusion (I/R), followed by I/R surgery (the number is 12); DNL0.5+I/R: 0.5g/kg/day of DNL before retinal ischemia-reperfusion (I/R), followed by I/R surgery (the number is 12); DNL1.0+I /R: 1.0g/kg/day DNL before retinal ischemia-reperfusion (I/R), followed by I/R surgery (12 in number); I/R+Vehicle D7: after I/R surgery The 7th day after vehicle (vehicle); I/R+DNL1.0 D7: The 7th day after I/R surgery with 1.0g/kg/day DNL; I/R+Vehicle: I/ Vehicle (vehicle) (quantity 10) after R surgery; I/R+DNL1.0: 1.0g/kg/day DNL (quantity 10) after I/R surgery; Pre-ischemia: I /R before surgery; Post-ischemia D1: the first day after I/R surgery; Post-ischemia D3: the third day after I/R surgery; Post-ischemia D5: the fifth day after I/R surgery; Post-ischemia D7: The 7th day after I/R surgery.

Figure 4 shows the thickness analysis of the whole or inner retina marked with cresyl violet. (a), (b) and (e) are respectively receiving sham procedure (Sham) (a), via ischemia reperfusion (I/R) and pre-administration of vehicle (vehicle) (b) or post-administration of vehicle Agent (e) of the retina. (c), (d) and (f) are respectively ischemia plus reperfusion and pre-administered 0.5g/kg/day (c, DNL0.5+I/R), 1.0g/kg/day (d, DNL1 .0+I/R) or 1.0g/kg/day (f, I/R+DNL1.0) of retinal sections of Dendrobium nobile (DNL). (g) and (h) are the morphological analysis of the thickness of the inner layer and the entire retinal slice with the same eccentricity. The experimental results are expressed as mean±SEM, and the number of experiments is 10-12. **: There is a significant difference between the retina of the fake program (P<0.01). † or ††: There is a significant difference compared with vehicle+I/R or I/R+vehicle (P<0.05 or P<0.01). ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell Layer (ganglion cell layer). The scale bar is 50μm.

Figure 5 shows Fluorogold staining. Microscopic photographs show the sham procedure (a, Sham), the administration of vehicle before ischemia and reperfusion (I/R) (b, Vehicle+I/R) or the administration of 1.0g/before/after ischemia The density of retinal ganglion cell (RGC) in kg/day of Dendrobium nobile (DNL) plus ischemia reperfusion (c, DNL1.0+I/R; d, I/R+DNL1.0) . (e) is the quantitative analysis of RGC density, the number is shown in each column, and the experimental result is expressed as the mean ± standard error (SEM) (the number is 4). **: Significantly different from the sham procedure retina (P<0.01; Sham group vs. Vehicle+I/R group); †† or †: Significantly different from Vehicle+I/R group (P<0.01 or P<0.05 ; Vehicle+I/R group to DNL1.0+I/R group or I/R+DNL1.0 group). The scale bar is 50μm.

Figure 6 shows the immunohistochemistry experiment of Choline acetyltransferase (ChAT; red). (a) Sham retina (Sham), the nucleus is 4',6-diamidine-2-phenylindole dihydrochloride (4,6-diamidine-2-phenylindole dihydrochloride, DAPI; blue ) Perform counterstaining. Axonal cell bodies (Sham; short arrow) are located in the inner nuclear layer (INL) and ganglion cell layer (GCL), and their neuronal processes (long arrow) are located in the inner core layer (INL) and ganglion cell layer (GCL). The inner plexiform layer (IPL) shows the appearance of double bands. (b) and (e) are respectively the retina after ischemia-reperfusion (I/R) and vehicle (Vehicle+I/R or I/R+Vehicle) administered before/after ischemia; and can be observed The immunoreactivity in the IPL is significantly reduced with a significant decrease in the number of axonal neuronal cell bodies. (c), (d) and (f) are respectively via ischemia reperfusion (I/R) and pre-administered 0.5g/kg/day (c, DNL0.5+I/R) or 1.0g/kg/ Dendrobium nobile (DNL) on day (d, DNL1.0+I/R), or after ischemia-reperfusion (I/R) and 1.0g/kg/day (f, I/R+DNL1. 0) Retina section of Dendrobium nobile. In these groups, when 0.5 and 1.0 g/Kg/day of Dendrobium nobile were administered to the ischemic retina in advance, these ischemia-induced changes were significantly reduced in a dose-dependent manner. The subsequent administration of 1.0g/Kg/day of Dendrobium nobile also significantly reduced these changes caused by ischemia. The scale bar is 50μm.

Figure 7 shows the immunohistochemical staining of vimentin. (b) After the sham procedure (Sham), the end of the müller cells (müller cells) cell can be observed in the ganglion cell layer (GCL) anti-vimentin (green) immunoreactivity (arrow; in (c) ) And (f) can also be observed); and the processes of the Mühl cells in the inner plexiform layer (IPL) (arrows; can also be observed in (c) and (f)), the inner plexiform layer (INL) ) And the outer nuclear layer (ONL) were also immunostained. (c) and (f) are respectively compared with the sham procedure retina, when vehicle (Vehicle+I/R or I/R+Vehicle) is administered before/after ischemia, the anti-vimentin immunolabeling on the retina is significant Enhanced. (d), (e), and (g) are by pre-administering 0.5g/kg/day (DNL0.5+I/R) or 1g/kg/day of Dendrobium nobile (DNL) (DNL1.0 +I/R), or after 1g/kg/day (I/R+DNL1.0) of Dendrobium nobile, the above-mentioned enhancement phenomenon will be significantly reduced. (h) to (n) are immunohistochemical staining of glial fibrillary acidic protein (GFAP). After the sham procedure (Sham, i), the end of Mühle cells showed GFAP immunoreactivity in the ganglion cell layer (GCL) (arrow; can also be observed in (j) and (m)), and the end of Mühle cells The processes of the inner plexiform layer (IPL) (arrows; can also be observed at (j) and (m)), the inner core layer (INL) and the outer core layer (ONL) also show GFAP immunoreactivity. Compared with the sham-programmed retina, the anti-GFAP immunolabeling is enhanced after ischemia and pre/post vehicle administration (Vehicle+I/R, j; I/R+Vehicle, m). By pre-administering 0.5g/kg/day (DNL0.5+I/R, k) or 1g/kg/day (DNL1.0+I/R, l) of Dendrobium nobile (DNL), or administering later 1g/kg/day (I/R+DNL1.0, n) Dendrobium nobile can reduce the above-mentioned enhancement degree. (a) and (h) are 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue) for counterstaining the nucleus in the retina of the pseudo-procedure. The scale bar is 25μm.

Figure 8: (a1) Western blotting analysis shows β-actin (β-actin), hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), acetone The expression levels of acid kinase M2 (PKM2) and retinoblastoma binding protein 2 (RBP2). Columns 1 or 2 are respectively sham retina (Sham) or ischemic retina (Vehicle+I/R) administered with vehicle in advance. Column 3 shows the retina that received ischemia and was pre-administered 1 g/kg/day of Dendrobium nobile (DNL) (DNL1.0+I/R) before ischemia. Columns 4, 5, and 6 are for receiving ischemia and pre-administering 10μM/5μl JIB-04 (RBP2 inhibitor), 4μM/5μl shikonin (PKM2 inhibitor) and 125μg/5μl before ischemia, respectively The retina of avastin (anti-VEGF). Each bar in (a2) shows the ratio of RBP2, HIF-1α, PKM2 and VEGF to β-actin. **: There is a significant difference between the sham retina (Sham) and the ischemic retina (Vehicle+I/R) administered with vehicle in advance (P<0.01). †Or††: There was a significant difference between the Vehicle+I/R group and the ischemic retina pre-administered DNL1, JIB-04, Shikonin or Aisiting (P<0.05 or P<0.01). The experimental results are expressed as mean±standard error (SEM) (numbers range from 4 to 10). (b) is the analysis result of enzyme immunosorbent assay (ELISA). Measure the concentration of placental growth factor (PLGF) in the retina in different groups (ie, sham program (Sham), Vehicle+I/R, DNL1.0+I/R or Eylea+I/R). **: There is a significant difference between the retina of the sham procedure and the Vehicle+I/R group (P<0.01). †Or††: There is a significant difference between Vehicle+I/R group and DNL1.0+I/R group or Eylea+I/R group (P<0.05 or P<0.01). The experimental results are expressed as mean±standard error (SEM) (the number is 4). Sham (control group): sham procedure operation experiment (the number is 10); Vehicle+I/R: intravitreal administration of vehicle before retinal ischemia-reperfusion (I/R), followed by I/R surgery ( The number is 10); DNL1.0+I/R: 1.0g/kg/day of Dendrobium nobile is administered intravitreally before retinal ischemia-reperfusion (I/R), followed by I/R surgery (the number is 10) ; JIB-04+I/R: JIB-04 administered intravitreally before retinal ischemia-reperfusion (I/R), followed by I/R surgery (number of 4); Shikonin+I/R: retinal ischemia-reperfusion (I/R) Shikonin was administered intravitreally before perfusion (I/R), followed by I/R surgery (number 7); Avastin+I/R: intravitreal administration before retinal ischemia-reperfusion (I/R) Avastin was given, followed by I/R surgery (the number is 4); Eylea+I/R: the retina received ischemia-reperfusion (I/R) plus pre-administration of 200μg/5μl before ischemia Eylea (quantity is 4).

Figure 9 shows the path analysis of Dendrobium nobile (DNL)/moscatilin in the treatment of retinal ischemia. NDP: Norrie disease protein; PKM2: pyruvate kinase M2; RBP2: retinoblastoma binding protein 2; HIF-1α: hypoxia-inducible factor-1α; VEGF-A: vascular endothelial growth factor-A; VEGF-B: Vascular endothelial growth factor-B; PLGF: placental growth factor.

The present invention includes but is not limited to the above and the following descriptions. The implementation is shown in the following example.

Method:

Chemical and drug administration

Dendrobium nobile (DNL) was purchased from Keda Company (Taipei, Taiwan) and dissolved in water (ddH ₂ O). For electrophysiology, immunohistochemistry and molecular biology research, drug administration will be conducted for 7 days and involves various experimental design groups, that is, post-ischemic administration (1g/kg/day high-dose Dendrobium nobile) (DNL), I/R+DNL1.0) or pre-administered before ischemia (daily high-dose DNL, DNL1.0+I/R; low-dose DNL 0.5g/kg/day, DNL0.5+I/ R). Rats in the ischemic vehicle (vehicel) group were administered (I/R+Vehicel) or pre-administered with a similar volume of vehicle (Vehicel+I/R) as in the DNL group.

Compared with the sham procedure group (sham), vehicle plus ischemia group (vehicle+I/R) or DNL1.0+I/R group, the ischemic eyes of other groups were first treated with 1% cycloplegia (tropicamide). ) And 2.5% phenylephrine to dilate the pupils, and then use a 30-gauge needle connected to a 25μl syringe for intravitreal injections. Specifically, the eyes undergoing ischemia surgery were administered intravitreal inhibitors/antibodies, namely 10μM/5μl JIB-04 (Sigma-Aldrich), 4μM/5μl Shikonin (Shikonin) one day before ischemia. (S7576; Sigma-Aldrich), 100mg/5μl Avastin (Hoffmann-La Roche) or 200μg/5μl eylea (Regeneron Pharmaceuticals Inc.). Animals were sacrificed one day after retina ischemia-reperfusion (I/R) and administration of related compounds or sham procedure (Sham) operation. Collect retinal samples and measure hypoxia-inducible factor-1α (HIF-1α), retinoblastoma binding protein 2 (RBP2), Western blot analysis or enzyme-linked immunoassay (ELISA) analysis The protein expression levels of pyruvate kinase M2 (PKM2), vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PLGF).

Moscatilin was purchased from EMMX Biotechnology (EN10271, CA, USA) and dissolved in DMSO (vehicle). Previous reports indicated that higher concentrations of dendrobol (1.25-20μM) would decrease the cell viability of two cell lines with 7.0 and 6.7μM, respectively, _{of IC 50 after} 24 hours of treatment in a dose- and time-dependent manner; therefore, 0.1μM is currently selected To evaluate its protective effect on oxygen glucose deprivation (OGD). Dendrobium (0.1 μM) was administered 1 hour before OGD, during OGD, or 1 hour after OGD. The therapeutic effect was evaluated by cell viability analysis (MTT assay) and Western blot method (Western blot).

In vitro experiments

Oxygen glucose deprivation (OGD) and cell processing

Retinal ganglion cell-5 (RGC-5) does not transform into rat retinal ganglion cell (RGC), but mouse retinal neuronal precursor cell. Oxygen glucose deprivation (OGD) is defined as under hypoxic (similar to ischemic) conditions (ie 1% oxygen (monitored by an analyzer; Penguin Incubator: control range 1~89%; Astec Company, Kukuoka, Japan), 94 % Nitrogen, 5% carbon dioxide), the cells were maintained in DMEM medium without glucose at 37°C. This experiment has different groups, including (i) DMSO-administered cells in DMEM medium (control group cells; DMSO+DMEM); (ii) DMSO is given 1 hour before OGD, followed by OGD-treated cells (DMSO+OGD); (iii) Administer moscatilin (Mos) (0.1μM in DMEM medium) 1 hour before OGD, followed by OGD-treated cells (Pre-OGD Mos 0.1μM); (iv) ) Cells that were given Dendrobium during OGD (During OGD Mos 0.1 μM); and (v) Cells that were given Dendrobium (DMSO+OGD) (Post-OGD Mos 0.1 μM) 1 hour after OGD. At the end of the 1-day OGD period, the cell culture will be returned to the DMEM medium for another 24 hours. Then proceed to cell viability analysis (for cell survival rate) and Western blot analysis (for Norrie disease protein (NDP)).

MTT cell survival rate analysis

(blue formazan)。因此，深紫色甲

的數量增加係對應於更高的細胞存活率。在37℃下，將MTT(0.5mg/mL；Sigma-Aldrich)加入至含有原始100μL細胞的96孔盤中3小時。然後藉由加入100μL DMSO溶解還原的MTT。攪動平板後，使用酵素連結免疫分析法(ELISA)讀取器(Synergy H1 Multi-Mode Reader BioTek Instruments)在562nm下測量溶解的甲

的光密度(OD)。細胞存活率以相對於對照組(100%)的OD值 作為表示。 Mitochondrial nicotine adenine dinucleotide phosphate hydrogen (NADPH)-dependent redox enzyme can reduce MTT to form formazan

(blue formazan). Therefore, the dark purple armor

An increase in the number of cells corresponds to a higher cell survival rate. At 37°C, MTT (0.5 mg/mL; Sigma-Aldrich) was added to a 96-well dish containing the original 100 μL of cells for 3 hours. Then dissolve the reduced MTT by adding 100 μL of DMSO. After agitating the plate, use an enzyme linked immunoassay (ELISA) reader (Synergy H1 Multi-Mode Reader BioTek Instruments) to measure the dissolved formazan at 562 nm.

The optical density (OD). The cell survival rate is expressed as the OD value relative to the control group (100%).

In vivo experiment

Animals

The present invention uses 6-week-old Wistar rats.

Establishment of retinal ischemia

Anesthesia and euthanasia

The animals were anesthetized by intraperitoneal injection with 100 mg/kg of ketamine (Pfizer) and 5 mg/kg of xylazine (Sigma-Aldrich). In addition, at least 140 mg/kg pentobarbital (SCI Pharmtech) was administered intraperitoneally to sacrifice animals in a humane manner.

Ischemia induced

Rats (200-250g) were anesthetized with the above-mentioned anesthetic and placed in a stereotaxic frame. A 30-gauge needle connected to a 0.9% saline bottle was used to puncture the anterior chamber of the rat's eye to increase the intraocular pressure (IOP) to 120 mm mercury column and maintain it for 1 hour. The establishment of ischemic damage is confirmed by the whitening of the retina. The sham procedure of the above-mentioned ischemia-inducing process was performed, but the saline bottle connected to the rat's eye was not raised, as a control. The animal was placed on a heating pad at 37°C and maintained a normal body temperature during the period of ischemia and the subsequent 3 hours of reperfusion.

Scintillation electroretinogram measurement

Before sham procedure or retinal ischemia/reperfusion (day 0), and after sham procedure or retinal ischemia/reperfusion and one day after pre-administration of the specified drug, the flash electroretinogram (Flash ERG) of all animals was recorded. ). In the post-administration group, all animals were recorded before ischemia (day 0) and after ischemia (administration of appropriate compounds after ischemia and ERG recording on days 1, 3, 5, or 7 after ischemia). ERG data. The mice need to adapt to the dark for 8 hours, and then anesthetize to record the ERG when the pupils dilate. A strobe was placed 2 cm in front of the animal’s eyes to induce a 0.5 Hz stimulus. Collect 15 consecutive records every 2 seconds at 10kHz; maximize the amplitude and calculate the average by the amplifier P511/stabilized power supply RPS107/stimulator PS22 (Grass-Telefactor). In order to compare the different groups, calculate the ratio of the b-wave amplitude of one eye (sham procedure or ischemia) to the untreated normal eye.

Cresyl violet staining

In all groups, rats were sacrificed and intracardial perfusion was performed with saline. Mark the eyeballs with silk sutures in the direction of the cornea at 12 o'clock, then remove the eyeballs and fix them in 4% paraformaldehyde at 4°C for 24 hours. They are dehydrated through a series of gradient-designed ethanol. Buried in paraffin (Tissue-Tek TEC 5). Cut 5μm thick slices along the vertical meridian. The sections were marked with cresol purple and observed under an optical microscope (Leica). Retinal slices were taken at the same magnification, and the thickness of the retina of different layers was measured from the photos (Ilford Pan-F plus film, 50ASA). In order to quantify the degree of retinal ischemic damage, the thickness of the entire retina (from the inner limiting membrane (ILM) to the retinal pigment epithelium (RPE) layer) and the thickness of the inner retina (from the inner limiting membrane to the inner core) The thickness of the inner nuclear layer (INL) is measured.

RGC retrograde staining

When the animal is under anesthesia, a 2 cm incision is made on the animal's scalp, and two small holes are drilled to penetrate into the skull. In addition, 10 μl of 5% fluorogold (Sigma-Aldrich) was injected into the cranium at depths of 3.8, 4.0, and 4.2 mm through a micropipette. In different groups, fluorescein gold was injected 3 days before animal sacrifice. The retinal sample is removed, fixed, dissected, and processed in the previous manner. The density of retinal ganglion cells (RGC) is the ratio of the total number of RGCs divided by the total area of the retinal sample.

Immunofluorescence analysis

After the animal was sacrificed, its heart was perfused with saline (w/v); then, the rat’s eyeballs were taken out, fixed with 4% (w/v) paraformaldehyde for 45 minutes, dehydrated in the previous way and embedded in paraffin wax in. Samples were taken one day after the administration of DNL or vehicle before or after the sham procedure or induction of retinal ischemia and ischemia. The 5μm retinal section was combined with primary antibody (goat anti-acetylcholine transferase (ChAT) multi-strain antibody (1:100; AB144p; Chemicon), mouse anti-vimentin monoclonal antibody (1:100; V6630; Sigma-Aldrich) ), or multiple rabbit anti-glia fibrillary acidic protein (GFAP) antibodies (Millipore)) and incubated overnight. Next, the retinal section and the appropriate secondary antibody (rhodamine (rhodamine) conjugated rabbit anti-goat antibody (1:500; AP106R; Chemicon), fluorescent isothiocyanate (FITC) conjugated goat anti-small Mouse IgG (1:500; AP124F; Millipore)/anti-rabbit IgG (Millipore). At the same time, the nucleus was stained and labeled with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes). Finally, Observe the retinal slices with a fluorescent microscope (Olympus BX61, Göttingen, Germany). In order to compare the thickness of the retina (cresyl purple staining) or the degree of immunoreactivity of the sliced retinas between different groups, the researchers did not know the conditions of the sliced samples , To measure the thickness of the retina or to evaluate the degree of immunolabeling of different groups to the control group (sham procedure).

Analysis of Western Ink Spot Method

The removed retina/cell sample is further subjected to ultrasonic processing in a lysis buffer (Mammalian Protein Extraction Reagent (MPER; HyCell)). The same amount of denatured protein (40 μg/30 μl/well) was processed on sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad). After separation, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane, and the PVDF membrane was further immersed in various primary antibodies (rabbit monoclonal anti-β-actin antibody (AC-15; 1:2000) at 4°C. ;Ab6276)/anti-HIF-1α antibody (1:200; H1alpha67-ChIP Grade; Abcam Inc.), rabbit multi-strain anti-VEGF antibody (A-20; 1:200; sc-152)/anti-PKM2 antibody (1: 500; ab38237), or rabbit monoclonal anti-RBP2 monoclonal antibody (ab177486; 1:1000; Abcam Inc.) for 12 hours. Next, dip the ink dot into the relevant secondary antibody, namely horseradish peroxidase (HRP). Conjugated goat anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology Inc.) or goat anti-mouse IgG (1:5,000; sc-2005) at 37°C for 1 hour. Dilute the primary/secondary antibody with 5% skim milk Finally, use the enhanced chemiluminescence analysis system (HyCell) to scan the membrane, and expose it to X-ray film (Fujifilm). Then, transmit the density scan to evaluate the quantity of each protein.

Enzyme immunosorbent assay (ELISA)

ELISA was used to determine the expression level of placental growth factor (PLGF). After one day of ischemia, the retina was separated from the removed eye cup, dissociated, and then lysed by incubating with mammalian protein extraction reagent (Sigma-Aldrich) for 30 minutes, and then at 13,000 rpm Centrifuge for 30 minutes. The bicinchoninic acid protein kit (Thermo Fisher Scientific) was used to determine all the proteins in each sample. PLGF ELISA kit (CSB-E07400; Cusabio Life Scince) was used to measure the expression level of PLGF in the supernatant. First spread the anti-PLGF antibody on the microwells. Wash each microwell twice with 200μL washing buffer for more than 15 minutes, then combine various concentrations (100μL) of PLGF or PLGF standard protein in the sample with the antibody coated in the microwell, and combine them at room temperature. Shake on a shaker (75 rpm) for 2 hours. After washing each microwell twice with 200 μL washing buffer (phosphate buffered saline (PBS) containing 0.05% Tween 20), PBST, 100 μL of biotin-conjugated anti-PLGF antibody (with Assay buffer dilution: PBST and 0.5% bovine serum albumin (BSA)) were added to each microwell and shaken (75 rpm) for 1 hour to bind the PLGF captured by the coated antibody. After two washes to remove unbound biotin-conjugated anti-PLGF, 100 μl avidin-horseradish peroxidase (HRP; diluted with assay buffer) was added and combined with biotin-conjugated anti-VEGF -A antibody, finally shake on a shaker (75 rpm). After incubating on a shaker for 1 hour, the unbound avidin-HRP was removed after washing twice. Finally, 90μL of 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine) (100μM) solution (which can be oxidized by HRP) was added to each well 20 minutes. Then add 50 μL stop solution (sulfuric acid solution, 100 μM) to make the color change to yellow. A spectrophotometer (Synergy H1 Hybrid Multi-Mode Reader, Biotek ELx800) was used to immediately detect the maximum absorbance (OD) at 450 nm. The PLGF concentration of each sample was determined by establishing a standard curve using different amounts of PLGF (200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 pg/mL). Zero the instrument with 100 μL of sample diluent (as blank). The experimental results are expressed as OD value relative to the OD value of the control group (normalized to 100%).

Statistical Analysis

The comparison between the two groups uses unpaired Student’s t-tests. A one-way covariate analysis (ANOVA) was performed to compare 3 or more independent groups. After single factor covariate analysis, the Dunnett test was used to compare the control group (e.g. vehicle-treated ischemic retina, Vehicle+I/R) with all other groups (e.g. DNL-treated ischemic retina, DNL1.0+I /R). The experimental results are expressed as mean±standard error. P<0.05 represents a significant difference.

Result:

(a) MTT cell survival rate determination

First, examine the changes in the cell morphology and number of RGC-5 cells under an optical microscope. Changes in cell morphology may imply the severity of ischemic injury. The cells cultured in the DMEM medium (DMSO+DMEM; Figure 1a) pre-administered DMSO showed a cone shape and showed characteristic neuronal morphology. Compared with the DMSO+DMEM group, the cells undergoing oxygen glucose deprivation (OGD) and pre-administered DMSO (DMSO+OGD) will be deformed (as indicated by the white arrow; Figure 1b); in addition, the number of cells is also significant cut back. Administration of moscatilin (0.1 to 100μM) to treat cells suffering from oxygen and glucose deprivation; in addition, the present invention proves that the protective effect of pre-administration of moscatilin in OGD before OGD is 0.1μM (62.65%±4.35%). ; Quantity is 6) or 1μM (66.36%±9.35%; quantity is 4) has better cell survival rate; however, at 10μM (15.43±3.09%; quantity is 4) and 100μM (12.49%; quantity is 1) However, cytotoxic effects (decreased cell survival rate) occur at times. Therefore, the concentration of the latter may exceed the degree of pharmacological protection. However, this result is not contradictory, because previous studies also pointed out that Dendrobium has cytotoxic effects in time-dependent (1~3 days) and dose-dependent (1.25~20μM), which may be related to the concentration of 20 or 50μM and It is related to the ability to induce G2 arrest in mitosis after 15 hours of treatment. However, dendrobin is proved to be non-toxic and effective when the concentration is equal to or less than 1μM. Scavenger of OH free radicals. Compared with the DMSO+OGD group (Figure 1b), 0.1 μM moscatilin was administered to assess cell protection 1 hour before OGD (Figure 1c), during OGD (Figure 1d), and 1 hour after OGD (Figure 1e) The degree of cytoprotection against OGD. One hour before OGD, the effect of 0.1μM dendrobium is the most effective (Figure 1c), the second is the effect of administration during OGD (Figure 1d), and the last one hour after OGD has no effect (Figure 1e).

Compared with the DMSO+DMEM group (normal control group: 100%; the number is 6), pre-administered DMSO and received OGD, the cell survival rate in the DMSO+OGD group was significantly reduced (53.66±2.67%) (P<0.001) (Figure 1f). In addition, compared with the DMSO+OGD group, administration of 0.1μM dendrobium 1 hour before OGD produced a significant cytoprotective effect against OGD (Figure 1f; 62.65±4.35%; P=0.04). However, during OGD (Figure 1f; 56.03±4.08%; P=0.31) or 1 hour after OGD (Figure 1f; 52.61±4.16%; P=0.41) administration of 0.1 μM dendrobium did not have a significant cytoprotective effect against resistance OGD.

(b) The effect of dendrobium on the expression of NDP relative to β-actin in vitro

In order to study the changes of Norrie disease protein (NDP) related to vasculogenesis, representative western blot images and analysis bar graphs are shown at the top and bottom of Figure 2, respectively. Compared with the DMSO+DMEM group (normal control: 100%; the number is 3), the administration of vehicle before OGD treatment followed by OGD (DMSO+OGD; P<0.001) significantly reduced the amount of NDP to 44.54±3.15%. When the DMSO+OGD group was compared with the pre-OGD Mos 0.1μM group, the number of NDP increased significantly (108.38±29.33%) (P=0.048). When dendrobium phenol was administered 1 hour before OGD, the increase in protein expression was the greatest; then it was administered 1 hour after OGD (54.36±3.88%), and finally it was administered during OGD (48.99±). 9.89%).

(c) The influence of Dendrobium nobile on the b wave of ERG

The present invention then tests the electrophysiological function of the retina. In the retina under the sham procedure (Sham, Fig. 3), the b-wave amplitude of the electroretinogram (ERG) was measured to be 0.41 mV. After retinal ischemia, the b-wave amplitude decreases drastically, and it is not affected by vehicle (vehicel) treatment before or after ischemia (Vehicel+I/R: 0.03mV, Figure 3a; I/R +Vehicel: 0.07mV, Figure 3b). However, before ischemia (DNL0.5+I/R; DNL1.0+I/R, Figure 3a) or after ischemia with Dendrobium nobile (DNL) treatment (I/R+DNL1.0 D7, Figure 3b ) Alleviate the decrease of b wave caused by ischemia, and the amplitude increased to 0.18, 0.22 and 0.15mV respectively. In addition, treatment with DNL before ischemia attenuated the decrease in amplitude in a dose-dependent manner.

As shown in Figure 3c (the number is 12), compared with the control group (Sham) (1.00±0.08), the b-wave ratio (0.10±0.03) of the Vehicle+I/R group was significantly decreased (P=0.002). Importantly, DNL treatment before ischemia can significantly reduce the decrease in b-wave ratio caused by ischemia after I/R (DNL1.0+I/R: 0.57±0.06; DNL0.5+ I/R: 0.45±0.03 (P<0.001)).

In Figure 3d (the number is 10), compared with the control group (Sham), in the I/R+Vehicle group (vehicle administered after I/R ischemia surgery), the b-wave ratio was significantly reduced ( Day 1: 0.13±0.04; Day 3: 0.10±0.02; Day 5: 0.09±0.03; Day 7: 0.06±0.03) (P<0.001). Importantly, the administration of Dendrobium nobile (DNL) (1.0g/kg/day, I/R+DNL1.0) after ischemia significantly reduced the decrease in b-wave ratio induced by ischemia (Day 1: 0.14 ±0.04; Day 3: 0.26±0.05 (P=0.02); Day 5: 0.34±0.05 (P=0.003); Day 7: 0.40±0.04 (P<0.001)). The b-wave ratios before ischemia (day 0) were 1.00±0.09 (I/R+Vehicle) and 1.00±0.08 (I/R+DNL1.0), respectively. When comparing the b-wave ratios (0.99±0.06, 0.97±0.07, 0.98±0.06, 1.02±0.01, and 0.99±0.07) of the ERG on days 0, 1, 3, 5, and 7 of the sham program (sham) After comparing them, they did not find any significant differences.

(d) The effect of DNL on the thickness of the retinal layer marked by cresol purple

The evaluation of retinal thickness is based on retinal samples (10-12, Figure 4) cut out at the same distance (1.5mm) from the optic disc for all different groups. Compared with the retina that received the sham procedure (Sham, Figures 4a and 4g: the whole retina is 225.50±3.26μm, the inner retina is 112.08±2.58μm), the vehicle is pre-administered and The thickness of the retina of the animals receiving I/R was significantly reduced (Vehicle+I/R, Figures 4b and 4g: the whole retina was 110.83±1.85μm, and the inner retina was 62.50±3.06μm) (P<0.001). In addition, when animals receive I/R and pre-administer Dendrobium nobile (DNL), the above reduction will be dose-dependent and significantly offset (DNL1.0+I/R, Figures 4c and 4g: the entire retina is 190.08 ±4.48μm, the inner retina is 94.92±2.27μm; DNL0.5+I/R, Figure 4d and 4g: the whole retina is 148.58±2.80μm, the inner retina is 78.25±1.53μm) (P<0.001).

Compared with the retina that received the sham procedure (Sham, Fig. 4a), the thickness of the retina in rats that received I/R and the subsequent administration of vehicle (vehicle) was significantly reduced (I/R+vehicle, Figs. 4e and 4h: the entire retina It is 115.00±2.04μm, and the inner retina is 63.92±3.30μm) (P<0.001). In addition, post-administration of Dendrobium nobile can significantly reduce the reduction induced by ischemia (I/R+DNL1.0, Figures 4f and 4h: the whole retina is 125.25±2.66μm (P=0.006); the inner retina is 71.50 ±1.51μm (P=0.048)).

(e) The effect of DNL on the density of retrograde fluorescent gold immunolabeled RGC

Figure 5 shows the density analysis of retinal ganglion cells (RGC) (the number is 4). The density of RGCs in the sham program group (Sham, Figures 5a and 5e) is 363.23±2.84 cells/field. Compared with the sham program group, the RGC density was significantly reduced (192.06±23.53 cells/field) in the animals (Vehicle+I/R, Figures 5b and 5e) that received retinal ischemia and pre-administration of vehicle (Vehicle) ( P<0.001). In addition, when animals received retinal ischemia and pre-administration of Dendrobium nobile (DNL) (DNL1.0+I/R, Figures 5c and 5e: 295.15±7.14 cells/field) or administered Dendrobium nobile (DNL) after ischemia ( I/R+DNL1.0, Figure 5d and 5e: 256.26±9.46 cells/field), the above reduction phenomenon was significantly reduced (P=0.006 or 0.045).

(f) The effect of DNL on ChAT immunoreactivity

The choline acetyltransferase (ChAT) immunoreactivity in the sham-proceded retina (Sham, Figure 6a) is shown in the axonal neuronal cell bodies (amacrine) in the inner nuclear layer (INL) and ganglion cell layer (GCL) Cell bodies) (short arrow) appear choline acetyltransferase (ChAT) (red) immunolabel; this also confirms that there are 2 obvious strata (long arrow) in the inner plexiform layer (IPL) ). In the retina (Vehicle+I/R, Figure 6b; I/R+Vehicle, Figure 6e) that received ischemia and pre-administration/post-administration of vehicle, the number of ChAT immunolabeled axon-free neurocyte bodies was significantly reduced In addition, the immune markers of the inner plexiform layer (IPL) of axonal neuron cell bodies are significantly reduced. It is clinically important that when Dendrobium nobile (DNL) (DNL0.5+I/R, Figure 6c; DNL1.0+I/R, Figure 6d) is pre-administered to the ischemic retina, these The change will be reduced in a dose-dependent manner. In addition, post-administration of Dendrobium nobile (I/R+DNL1.0, Figure 6f) also significantly reduced the changes caused by ischemia. The combined images of the ChAT immunoreaction marker and the cell nucleus stained with 4',6-diamidino-2-phenylindole (DAPI) immunolabeling are shown in all pictures.

(g) The effect of DNL on vimentin and GFAP immunoreactivity

Perform immunohistochemical studies to study vimentin and GFAP immunoreactivity.

Immunohistochemical staining of vimentin

In the control retina (Sham, Fig. 7b), the processes of müller cells were immunolabeled with vimentin at the ends (arrows, Figs. 7c and 7f) on the ganglion cell layer (GCL) and protruded Extends to the inner plexiform layer (IPL), inner core layer (INL) and outer core layer (ONL) (arrows, Figures 7c and 7f). Compared with the control retina (Sham, Fig. 7b), the anti-vimentin immune response of the retina that received I/R and pre-administration/post-administration of vehicle was enhanced (Vehicle+I/R, Fig. 7c; I/R+ Vehicle, Figure 7f). By pre-administering Dendrobium nobile (DNL), the above enhancement will be significantly and dose-dependently weakened (DNL0.5+I/R, Figure 7d; DNL1.0+I/R, Figure 7e). In addition, post-administration of Dendrobium nobile (I/R+DNL1.0, Figure 7g) significantly eliminated the changes induced by ischemia.

Immunohistochemical staining of GFAP

Compared with the control retina (Sham, Fig. 7i), it was observed that the anti-glial fibrillary acidic protein (GFAP) immunolabeling in the ischemic retina and the increase of pre/post vehicle administration (Vehicle+I/R, Fig. 7j; I/R+Vehicle, Figure 7m). In addition, when the ischemic retina is pre-administered Dendrobium nobile, the above changes will be obvious and eliminated in a dose-dependent manner (DNL0.5+I/R, Figure 7k; DNL1.0+I/R, Figure 71). Post-administration of Dendrobium nobile also significantly eliminated the above-mentioned changes induced by ischemia (I/R+DNL1.0, Figure 7n). 4',6-diamidino-2-phenylindole (DAPI) (blue; Figures 7a and 7h) was used to stain the nuclei of the sham retina.

(h) The effect of DNL1.0 on the expression of various proteins in the rat retina

0.001)。此外，當缺血性視網膜預先施予1.0g/Kg天的金釵石斛(DNL)時，會顯著抑制這種上升現象(P<0.001；HIF-1α：56.08±6.76；VEGF：51.87±9.89；PKM2：71.99±3.05；RBP2：50.64±1.48)。另外，在預先施予各自的抑制劑/抗體：JIB-04(RBP2抑製劑)、紫草素(shikonin)(PKM2抑制劑)和癌思停(avastin)(VEGF抗體)後，其能顯著抵銷缺血所誘發的HIF-1α(JIB-04：53.98±2.29；shikonin：42.65±0.76；avastin：84.61±3.96(P=0.07))、VEGF(JIB-04：27.82±1.21；shikonin：57.55±9.40；avastin：5.38±2.51)、PKM2(JIB-04：60.36±7.59； shikonin：44.94±10.91；avastin：84.44±4.53(P=0.01))和RBP2(JIB-04：5.83±1.43；shikonin：3.40±0.23；avastin：78.35±3.29(P=0.02))；P<0.001)的表現量增加(P<0.002，除癌思停(avastin)組外)。 Measure the expression level of various proteins in the control retina (Sham, the number is 4-10), and the results are shown in Figure 8a1 and 8a2 (HIF-1α=51.17±5.14%; VEGF=59.72±6.94%; PKM2=52.93±7.01% ; RBP2=12.81±0.55%). On the contrary, after receiving I/R and pre-administration of vehicle (Vehicle), it was observed that the expression levels of HIF-1α, VEGF, PKM2 and RBP2 (normalized to 100%) increased significantly (P

0.001). In addition, when the ischemic retina was pre-administered 1.0g/Kg day of Dendrobium nobile (DNL), it would significantly inhibit this increase (P<0.001; HIF-1α: 56.08±6.76; VEGF: 51.87±9.89; PKM2: 71.99±3.05; RBP2: 50.64±1.48). In addition, after pre-administering the respective inhibitors/antibodies: JIB-04 (RBP2 inhibitor), shikonin (PKM2 inhibitor) and avastin (VEGF antibody), it can significantly resist HIF-1α induced by pin ischemia (JIB-04: 53.98±2.29; shikonin: 42.65±0.76; avastin: 84.61±3.96 (P=0.07)), VEGF (JIB-04: 27.82±1.21; shikonin: 57.55± 9.40; avastin: 5.38±2.51), PKM2 (JIB-04: 60.36±7.59; shikonin: 44.94±10.91; avastin: 84.44±4.53 (P=0.01)) and RBP2 (JIB-04: 5.83±1.43; shikonin: 3.40 ±0.23; avastin: 78.35±3.29 (P=0.02)); P<0.001) increased performance (P<0.002, except for the avastin group).

As shown in Figure 8b (the number is 4), compared with the control retina (Sham: 15.11±1.58pg/ml), after receiving I/R and pre-administered vehicle, the expression level of placental growth factor (PLGF) is significant Increase (Vehicle+I/R=39.53±5.25) (P=0.004). In addition, when pre-administration of Dendrobium nobile (DNL) (DNL1.0+I/R=19.93±2.24) or anti-PLGF antibody takes eylea (Eylea+I/R=6.44±0.60) for ischemic In the retina, this increase was significantly worse (P=0.01 or P<0.001).

The proper description of the present invention can be implemented under elements or limitations not specifically disclosed herein. The terms that have been used for description are not limiting. There is no difference between using these terms and any other equivalent expressions and descriptions, but it should be recognized that the rights in the present invention may be modified. Therefore, although the present invention has described embodiments and other situations, the content disclosed herein can be modified and changed by those skilled in the art, and such modifications and changes are considered to be within the scope of the rights of the present invention.

Claims (4)

A composition for the preparation of a medicine for the treatment of developmental retinal vascular diseases, wherein the composition comprises an effective dose of Dendrobium nobile Lindley, wherein the developmental retinal vascular disease is Norrie disease Retinopathy. The use described in item 1 of the scope of patent application, wherein the symptoms of the developmental retinal vascular disease include abnormal retinal vascular development, retinal ischemia, and impaired vision. The use as described in item 1 of the scope of patent application, wherein the Dendrobium nobile is used to treat the retinal ischemic damage caused by the developmental retinal vascular disease. The use described in item 1 of the scope of patent application, wherein the Dendrobium nobile is used to treat the thinning of the retinal thickness caused by the developmental retinal vascular disease.

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