Traditional Chinese medicine composition for treating diabetic retinopathy and preparation method thereof

Abstract

The invention relates to the technical field of medicines, and discloses a traditional Chinese medicine composition for treating diabetic retinopathy and a preparation method thereof. The traditional Chinese medicine composition is prepared from the following traditional Chinese medicinal materials in parts by weight: 25-35 parts of astragalus membranaceus, 3-9 parts of rheum officinale, 10-20 parts of salvia miltiorrhiza, 3-9 parts of roasted herba epimedii, 10-20 parts of dried motherwort and 2-4 parts of scalded leech. Research results show that in a db / db mouse DR model, the traditional Chinese medicine composition can enhance the structural integrity of a retina layer, especially INL and ONL. The effect is mediated by the quercetin and the apigenin in the traditional Chinese medicine composition by regulating an HIF-1alpha-VEGFA (High Intensity Factor 1 alpha-vascular endothelial growth factor A) pathway in macrophages, and the HIF-1alpha-VEGFA pathway plays a crucial role in reducing retinal leakage and subsequently slowing down the progress of diabetic retinopathy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a Chinese medicine composition for treating diabetic retinopathy and a preparation method thereof. Background Art

[0002] Diabetic retinopathy (DR) is the consequence of diabetic microangiopathy. Diabetes causes damage to the retinal capillary walls, and the blood is in a hypercoagulable state, which can easily cause thrombosis and blood stasis, and even blood vessel rupture. At present, diabetic retinopathy is the main cause of visual impairment and blindness. In the early stages of the disease, patients do not feel anything. If you check the fundus regularly, the doctor can see if there are any changes in your fundus. This is the best time to prevent it. In the middle stage, retinal edema will occur, and new blood vessels may appear in the eye and bleed into the vitreous cavity. At this time, you will feel blurred vision, black shadows floating in front of your eyes, and distorted vision. In the late stage, vitreous hemorrhage is severe, the retina is detached, and even blindness may occur.

[0003] At present, there are two main methods for treating diabetic retinopathy in clinic: laser treatment and drug treatment. Among them, retinal laser treatment is the basic means of treating diabetic retinopathy. It can directly coagulate and seal new blood vessels, microaneurysms and capillaries with fluorescence leakage. Laser treatment causes part of the retinal tissue to be coagulated by laser, and the remaining retina can get a richer blood oxygen supply, blocking the generation of new blood vessels, thereby preventing the development of retinopathy. However, this treatment method can only delay diabetic retinopathy, and the treatment condition of the patient's condition is poor. In addition, the treatment cost is high and patients cannot afford it. Drug treatment for diabetic patients can effectively reduce the incidence of diabetic fundus lesions, and drug treatment can usually also play a direct therapeutic role in diabetic fundus lesions. Western medicine cannot effectively treat diabetic eye disease, and long-term use of Western medicine has many side effects. Traditional Chinese medicine has a better therapeutic effect. At present, there are many traditional Chinese medicine preparations such as Anxue Granules, Tangwang Decoction, and Paiye Decoction for the treatment of diabetic fundus lesions. They have a certain effect on relieving symptoms such as vitreous hemorrhage and macular edema caused by diabetes, but there are still defects such as unsatisfactory treatment effect and high recurrence rate.

[0004] Therefore, there is still a great demand for effective therapeutic drugs for diabetic eye diseases. The present invention provides a therapeutic drug that can treat both the symptoms and the root cause of diabetic fundus lesions, especially diabetic retinopathy, based on the pathogenesis and clinical symptoms of diabetic eye diseases, and has a low recurrence rate. Summary of the invention

[0005] In view of the shortcomings of current clinical treatment drugs for diabetic fundus lesions, the present invention intends to provide a Chinese medicine composition that can be used to treat diabetic retinopathy based on the pathogenesis and clinical symptoms of diabetic eye diseases. The Chinese medicine composition is used to prepare a therapeutic drug that can treat both the symptoms and the root cause of diabetic retinopathy and has a low recurrence rate.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] On one hand, the present invention provides a traditional Chinese medicine composition for treating diabetic retinopathy. The traditional Chinese medicine composition is prepared from the following traditional Chinese medicine raw materials in parts by weight: 25-35 parts of astragalus, 3-9 parts of rhubarb, 10-20 parts of salvia miltiorrhiza, 3-9 parts of roasted epimedium, 10-20 parts of dried motherwort and 2-4 parts of scalded leech.

[0008] Preferably, the Chinese medicine composition is prepared from the following Chinese medicinal materials in parts by weight: 30 parts of Astragalus, 6 parts of Rhubarb, 15 parts of Salvia miltiorrhiza, 6 parts of roasted Epimedium, 15 parts of dried Leonurus japonicus and 3 parts of scalded Leech.

[0009] Preferably, the Chinese medicine composition is prepared from the following Chinese medicinal raw materials in parts by weight: the Chinese medicine composition is prepared from the following Chinese medicinal raw materials in parts by weight: 32 parts of Astragalus, 5 parts of Rhubarb, 12 parts of Salvia miltiorrhiza, 8 parts of roasted Epimedium, 12 parts of dried Leonurus japonicus and 4 parts of scalded leeches.

[0010] On the other hand, the present invention provides a method for preparing a traditional Chinese medicine composition for treating diabetic retinopathy, comprising the following steps: taking astragalus, rhubarb, salvia miltiorrhiza, roasted epimedium and dried motherwort, decocting in water, and filtering to obtain a filtrate; powdering scalded leeches and adding them to the filtrate, further adding auxiliary materials and sterilizing to prepare a finished preparation, wherein the auxiliary materials are medicinal auxiliary materials, and the selection of the auxiliary materials needs to be selected according to the dosage form of the finished preparation.

[0011] Preferably, the astragalus, rhubarb, salvia miltiorrhiza, roasted epimedium and dried motherwort need to be soaked in water for 30-60 minutes before being decocted.

[0012] Preferably, the volume ratio of the total mass of the astragalus, rhubarb, salvia miltiorrhiza, roasted epimedium and dried motherwort to water is 1g:10-15ml.

[0013] Preferably, the decoction is performed 2-3 times, each time for 30-60 minutes.

[0014] Preferably, the decoction is performed twice, the first decoction is for 60 minutes, and the second decoction is for 30 minutes.

[0015] Preferably, the finished preparation is a tablet, powder, capsule, granule or oral liquid.

[0016] Preferably, the finished preparation is a powder.

[0017] Another aspect of the present invention also provides use of the above-mentioned Chinese medicine composition in preparing a drug for treating or preventing diabetic retinopathy.

[0018] Preferably, the drug for treating or preventing diabetic retinopathy is a drug that reduces vascular leakage associated with damage to the blood-retinal barrier (BRB), enhances microcirculation in the limbs, strengthens the structural integrity of the retinal INL and ONL, or regulates HIF-1α-VEGFA to regulate macrophage-mediated inflammatory responses and angiogenesis signals.

[0019] The medicinal materials used in the Chinese medicine composition of this application are as follows:

[0020] Astragalus: It is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of the Leguminosae family. Nature and flavor and meridians: sweet, slightly warm. It enters the lung and spleen meridians. Functions and indications: tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, promoting the production of body fluids and nourishing blood, relieving stagnation and relieving numbness, expelling toxins and expelling pus, and healing sores and promoting tissue regeneration; it is used for qi deficiency and fatigue, poor appetite and loose stools, sinking of qi in the middle, chronic diarrhea and prolapse of the anus, bloody stool and metrorrhagia, spontaneous sweating due to exterior deficiency, edema due to qi deficiency, internal heat and thirst, sallow complexion due to blood deficiency, hemiplegia, pain and numbness in the arthralgia, carbuncle that is difficult to heal, and carbuncle that does not heal for a long time.

[0021] Rhubarb: The dried root and rhizome of Rheum palmatum L., RheumtanguticumMaxim.ex Balf. or Rheum officinale Baill. of the Polygonaceae family. Nature and flavor and meridians: bitter, cold. It enters the spleen, stomach, large intestine, liver, and pericardium meridians. Functions and indications: purgative, clearing away heat and purging fire, cooling blood and detoxifying, removing blood stasis and promoting menstruation, and removing dampness and jaundice. It is used for constipation caused by excess heat accumulation, vomiting and bleeding due to blood heat, red eyes and swollen throat, carbuncle, abdominal pain due to intestinal carbuncle, amenorrhea caused by blood stasis, postpartum blood stasis, traumatic injury, damp-heat dysentery, jaundice and red urine, stranguria, edema; external treatment for burns and scalds. Wine rhubarb is good at clearing heat and toxicity from the upper part of the blood, and is used for red eyes and swollen throat, gingival swelling and pain; cooked rhubarb has a slow purgative effect, clears fire and detoxifies, and is used for fire toxicity sores. Rhubarb charcoal cools blood, removes blood stasis and stops bleeding, and is used for blood heat and bleeding with blood stasis.

[0022] Danshen: The dried root and rhizome of Salvia miltiorrhiza Bge., a plant of the Lamiaceae family. Nature and flavor and meridians: bitter, slightly cold. It enters the heart and liver meridians. Functions and indications: It can promote blood circulation and remove blood stasis, relieve pain during menstruation, clear the heart and relieve vexation, cool blood and eliminate carbuncle; it is used for chest pain, abdominal pain, accumulation of lumps, pain caused by heat, restlessness and insomnia, irregular menstruation, dysmenorrhea and amenorrhea, and sores and swelling.

[0023] Roasted Epimedium: The dried leaves of Epimedium brevicornu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim. or Epimedium koreanum Nakai of the Berberidaceae family. Melt suet oil, add Epimedium shreds, stir-fry over low heat until evenly glossy, remove and cool. Nature and flavor and meridians: pungent, sweet, warm. Enters the liver and kidney meridians. Functions and indications: tonify kidney yang, strengthen tendons and bones, and eliminate rheumatism. Used for kidney yang deficiency, impotence and spermatorrhea, tendon and bone weakness, rheumatic pain, numbness and cramps.

[0024] Dried motherwort is the fresh or dried aerial part of the plant Leonurusjaponicus Houtt. of the Lamiaceae family. Nature and flavor and meridians: bitter, pungent, slightly cold. It enters the liver, pericardium, and bladder meridians. Functions and indications: promoting blood circulation and regulating menstruation, promoting diuresis and reducing swelling, clearing away heat and detoxifying. It is used for irregular menstruation, dysmenorrhea, amenorrhea, lochia retention, edema, oliguria, sores and swelling.

[0025] Scalded leech: the dried whole body of Whitmania pigra Whitman, Hirudonipponica Whitman or Whitmania acranulata Whitman, animals of the Hirudinidae family; clean leech segments, scald with talcum powder until slightly swollen according to the stir-frying method (General Rule 0213). Nature and flavor and meridians: salty, bitter, flat; slightly toxic. Enters the liver meridian. Functions and indications: break blood and promote menstruation, remove blood stasis and eliminate symptoms. Used for blood stasis, amenorrhea, lumps and masses, hemiplegia caused by stroke, and injuries caused by falls.

[0026] In summary, the present invention has the following beneficial effects:

[0027] The Chinese medicine composition of the present invention uses Astragalus as the main ingredient, Salvia miltiorrhiza, leech powder, and rhubarb as the auxiliary ingredients, and Epimedium and Leonurus japonicus as the auxiliary ingredients, which has the effects of promoting blood circulation, invigorating qi, and anti-inflammatory, and can improve the structure of diabetic retinopathy pathologically to achieve the purpose of treating diabetic retinopathy. And the research results of the present invention show that in the db / db mouse DR model, taking SZF extract can enhance the structural integrity of the retinal layer, especially INL and ONL. This effect is mediated by quercetin and apigenin in SZF by regulating the HIF-1α-VEGFA pathway in macrophages, and the HIF-1α-VEGFA pathway plays a vital role in reducing retinal leakage and subsequently slowing down the progression of diabetic retinopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows the effects of SZF extract on diabetic symptoms in mice (A is the effect of db / db and db / m on the average daily water intake of each mouse; B is the effect of db / db and db / m on 12-hour urine output; C is the fasting blood glucose level of db / db and db / m mice; D is the monthly weighing of db / db and db / m mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0029] Figure 2 is the effect of SZF extract on the thickness of mouse retina (A is the representative OCT images of the retina of five groups of mice and the quantitative analysis results of the GCL, INL, ONL and total retinal thickness; B is the representative retinal HE staining images of five groups and the quantitative analysis of the thickness of retinal layers, including the ganglion cell layer GCL, inner plexiform layer IPL, inner nuclear layer INL, outer plexiform layer OPL, outer nuclear layer ONL and photoreceptor segment PS; *P<0.05, ***P<0.01, ***P<0.001, ****P<0.0001);

[0030] Figure 3 is the effect of SZF extract on diabetic mouse retinopathy (A is five representative mouse fundus images in FP; B is the effect of SZF extract on retinal vascular lesions in DR mice in FFA, the white frame selects the same position to show the expansion of vascular leakage; C is a quantitative analysis of representative images of plantar perfusion area and blood flow velocity of five groups of mice, and the relative perfusion volume is calculated using the ROI average value, and the blood flow velocity and perfusion area are displayed in blue, green, yellow, orange and red from low to high, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0031] Figure 4 are the results of serum cytokine detection and immunofluorescence analysis in mice (A is the serum TNFα level of the five groups of mice; B is the serum STAT3 level of the five groups of mice; C is the immunofluorescence analysis image showing the co-localization of HIF-1α and VEGFA expression in the five groups of mice, scale bar 500 μm, n=3; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0032] Figure 5 is the mass spectrum of the SZF extract of the present invention (A is the negative ion mode, B is the positive ion mode, and the blue numbers represent the compounds determined by comparison with the reference standard; C and D are the high performance liquid chromatography fingerprint reproducibility evaluations of 29 standard compounds and 12 batches of agarose mixtures (S1-S12) of SZF extracts respectively using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).

[0033] Figure 6 It is the regulatory network of the traditional Chinese medicine compound of SZF extract. (A is a Venn diagram of diseases and drug targets; B is the regulatory network of component genes, PPIs and hub targets; C is the PPI network; D is the top ten important targets of PPIs discovered by the Cytohuba plug-in; E) is module 1 discovered by the MCODE plug-in; F is the analysis of Hub genes and their co-expressed genes using GeneMANIA);

[0034] Figure 7 is the functional enrichment analysis (A is the GO analysis result of DEGs; B is the KEGG analysis result of DEGs; C is the co-expression network of DEGs and pathways);

[0035] Figure 8 are the molecular docking results (A is the heat map of the interaction between the five main active ingredients in SZF and the VEGFA and HIF1A targets, B is the binding mode of quercetin and HIF1A, C is the binding mode of kaempferol and HIF1A, and D is the binding mode of apigenin and VEGFA. The middle dotted line represents the hydrogen bond, and the value is the bond length). DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0037] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0038] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0039] Example 1

[0040] A method for preparing a traditional Chinese medicine composition for treating diabetic retinopathy comprises the following steps: taking 30g of astragalus, 6g of rhubarb, 15g of salvia miltiorrhiza, 6g of roasted epimedium and 15g of dried motherwort, soaking them in 750ml water for 30min, decocting them twice, the first decoction is 60min, the second decoction is 30min, filtering; combining the filtrate; taking 3g of scalded leech, grinding it into fine powder and adding it to the filtrate, further adding conventional powder auxiliary materials for sterilization, and freeze-drying to obtain powder.

[0041] Example 2

[0042] A method for preparing a traditional Chinese medicine composition for treating diabetic retinopathy comprises the following steps: taking 32g of astragalus, 5g of rhubarb, 12g of salvia miltiorrhiza, 8g of roasted epimedium and 12g of dried motherwort, soaking them in 750ml of water for 30min, decocting them 3 times, decocting them for 45min for the first time, 30min for the second time, filtering and combining the filtrate; taking 3g of scalded leech, grinding it into fine powder and adding it to the filtrate, further adding conventional granule auxiliary materials for sterilization, and preparing granules according to a conventional method.

[0043] Example 3

[0044] A method for preparing a traditional Chinese medicine composition for treating diabetic retinopathy comprises the following steps: taking 25g of astragalus, 3g of rhubarb, 10g of salvia miltiorrhiza, 3g of roasted epimedium and 10g of dried motherwort, soaking them in 650ml of water for 60min, decocting them twice, the first decoction being 45min and the second decoction being 30min, filtering and combining the filtrate; taking 2g of scalded leech, grinding it into fine powder and adding it to the filtrate, further adding conventional tablet auxiliary materials for sterilization, and preparing tablets according to a conventional method.

[0045] Example 4

[0046] A method for preparing a traditional Chinese medicine composition for treating diabetic retinopathy comprises the following steps: taking 35g of astragalus, 9g of rhubarb, 20g of salvia miltiorrhiza, 9g of roasted epimedium and 20g of dried motherwort, soaking them in 1300ml water for 45min, decocting them twice, the first decoction being 45min and the second decoction being 45min, filtering and combining the filtrate; taking 4g of scalded leech, grinding it into fine powder and adding it to the filtrate, further adding conventional oral liquid auxiliary materials for sterilization, and preparing oral liquid according to conventional methods.

[0047] Test Example 1: Animal Pharmacology Experiment

[0048] 1. Materials and Methods

[0049] 1.1 Animal research experiment of the Chinese medicine composition (SZF extract) of Example 1

[0050] The mature spontaneous model of type 2 diabetes was studied in 40 male, C57BL / KsJdb / db mice, and the control group was 10 male, non-diabetic db / m mice; these mice were all 7 weeks old at the time of the experiment and were from Changzhou Kevins Experimental Animal Co., Ltd., with animal license number SCXK (Su) 2021-0013. The housing conditions of mice strictly followed the experimental requirements. All experimental procedures were strictly reviewed and approved by the Laboratory Animal Ethics Committee of Changchun University of Chinese Medicine. The ethical approval number of this invention is 2024743, which reflects the compliance with high standards of animal welfare and research integrity.

[0051] After a week of adaptive feeding of the animal model, the fasting blood glucose level at the tail tip of the mice was evaluated. The results showed that the blood glucose concentration in the db / db group was significantly increased compared with the db / m group. According to the blood glucose level and body weight, the mice were divided into five different groups: normal group (db / m), model group (db / db), low-dose group, medium-dose group and high-dose group. According to the established daily regimen, the Chinese medicine composition was a powder prepared without adding excipients in Example 1 (named SZF extract). Before administration, the SZF extract was diluted with sterile water for oral administration. Based on the body surface area, the dose conversion of mice followed the method outlined by Professor Xu Shuyun in the book "Pharmacological Experimental Methodology": mouse equivalent dose (mg / kg) = 9.1*human clinical dose (mg / kg). Therefore, the low-dose group was orally administered 1.06 mg / g per day, the medium-dose group was gavaged 2.12 mg / g per day, and the high-dose group was gavaged 4.24 mg / g per day. This dosing regimen was once a day for 12 consecutive weeks. During this period, provide db / m and db / db mice with an equal amount of sterile drinking water.

[0052] Food and water intake was monitored every six days for each cage containing five mice, and the mean values ​​were calculated. Body weight was assessed monthly, and urine was collected over a 12-hour period using metabolic cages. Prior to this, mice were euthanized after an overnight fast. Fasting serum samples were subsequently collected for analysis. Blood glucose levels were quantified using an automated biochemical analyzer and a GLU kit.

[0053] 1.2 Color fundus photography (FP) and fluorescein fundus angiography (FFA)

[0054] The morphology and pathological changes of the fundus of db / db mice were examined using a Micron IV retinal imaging microscope. Continuous anesthesia was maintained using isoflurane, and mydriasis was performed with compound tropicamide eye drops (0.5%; 0.05 mL). Each mouse was placed on a microscope platform and OmniVision produced by Neuhausen SA, Switzerland, was applied to its eyes. After color fundus photography FP, FFA was performed by intraperitoneal injection of sodium fluorescein solution (10%; 0.05 mL).

[0055] 1.3 Optical Coherence Tomography (OCT) and Thickness Analysis

[0056] The OCT module integrated into the Micron IV retinal imaging microscope is used to obtain high-resolution images, revealing the complex structure of the retinal layers. The entire field of view of the retina was segmented using InSight 2D software, and a comprehensive analysis was performed using a hierarchical statistical method, focusing on measuring three different layers of the retina: the ganglion cell layer (GCL), which is key to transmitting visual information; the inner nuclear layer (INL), which contains the cell bodies of multiple types of neurons; and the outer nuclear layer (ONL), which is where the photoreceptor cell bodies are located. In addition, the thickness of the retina was evaluated to further understand the integrity of the overall retinal structure.

[0057] 1.4 Hematoxylin and eosin (H&E) staining

[0058] The mice underwent a series of procedures, including injections, dissection and fixation of the eyes. The eyes were then stained to improve visibility and subsequently analyzed to assess retinal structure and measure thickness, aiming to provide detailed insights into retinal morphology.

[0059] 1.5 Regional blood flow velocity and perfused blood vessel area detection

[0060] During the experiment, mice were anesthetized with isoflurane. The regional blood flow velocity and perforated vessel area of ​​the plantar skin were measured using a laser speckle imaging system (RFLSI III, RWD), and the relative perfusion was calculated using the ROI average.

[0061] 1.6 Multiplex immunofluorescence

[0062] After the paraffin sections were dried, antigens were extracted, and the sections were rinsed three times in a PBS solution at pH 7.4, stirred, and purified. After removing moisture, 3% hydrogen peroxide solution was dripped onto the slides, incubated for 25 minutes at room temperature and in the dark, and then washed three times in a PBS solution at pH 7.4, 5 minutes / time. The slides were placed flat in a sealed box where serum was injected dropwise, and the slides were covered with a lid and incubated at room temperature for 30 minutes to allow the serum to reach the tissue normally. The primary antibody was blocked with a mixture of 10% goat rabbit serum and 3% BSA. After removing the blocking solution, the specified amount of primary antibody was mixed with sterile PBS. The antibodies used included VEGFA and HIF-1α, with dilutions of 1:100 and 1:50, respectively. In a dark and humid environment, the slides were placed at 4°C overnight, and the samples were applied in turn and incubated overnight. The slides were placed in PBS at pH 7.4 and washed three times on a rocking shaker, 5 minutes / time. Then, the slides were placed flat in a humid and dark box, and the secondary antibody linked to HRP was applied cyclically and incubated at room temperature for 50 minutes. After completion, the slides were washed three more times in PBS at pH 7.4, 5 minutes each time. After drying, the cooling reagent was slowly added in a circular direction. The specific TSA reagent used was VEGFAiF488 Tyramide. After the incubation, the slides were transferred to TBST, washed three times within five minutes, and then stained with DAPI, and incubated in the dark at room temperature for 10 minutes. After that, the slides were rinsed three times with PBS at pH 7.4, 5 minutes each time. After rapid drying, the autofluorescence quencher was applied to the slides for five minutes. The slides were then rinsed three times with pH 7.4 PBS, with a five-minute interval between each time. After the slides were completely dry, a layer of anti-fluorescence quencher was applied to their surface to maintain the staining effect. The prepared slides were imaged using CaseViewer software.

[0063] 2. Experimental results

[0064] 2.1 Effects of SZF extract on basic indicators of normal and model groups of mice

[0065] After 12 weeks of treatment, a significant reduction in average daily food consumption was observed in the groups receiving medium and high doses of SZF extract. These doses were also effective in significantly reducing the amount of water consumed per mouse on day 108. There was no significant difference in urine volume between the db / db and db / m groups before the start of gavage. However, starting from the eighth week of treatment, the urine volume within 12 hours in the db / db group and in the mice receiving low, medium, and high doses of SZF increased significantly compared with the db / m group (Figure 1A). At the 12th week of treatment, both low and medium doses of SZF extract significantly reduced the urine volume of db / db mice (Figure 1B). In addition, although the low dose of SZF extract significantly improved the fasting blood glucose levels of db / db mice, it had no substantial effect on reducing their body weight (Figures 1C and Figure 1D ).

[0066] 2.2SZF extract can alleviate retinopathy in db / db mice

[0067] Retinal thickness measurements at week 12 after oral administration of mice showed that retinal thickness was significantly reduced in db / db mice compared with db / m mice. Treatment with a high dose of SZF extract significantly increased the overall thickness of the retina. Detailed analysis of the retinal layers, especially the GCL, INL, and ONL, showed that the INL and ONL were severely thinned in db / db mice compared with db / m mice. In contrast, administration of a high dose of SZF extract resulted in a significant increase in the thickness of these cell layers. In addition, low and medium doses of SZF extract also significantly increased the thickness of the ONL in a dose-responsive manner. No other abnormalities were observed, such as obvious retinal edema ( Figure 2 A). H&E staining confirmed that SZF extract enhanced the thickness of the upper, inner, and lower retinal layers in a dose-dependent manner, especially at the medium and high doses. In the db / db group, there was a slight decrease in ganglion cells and some disorganization of the cells within the retina ( Figure 2 B). In conclusion, the results of OCT and H&E staining indicated that SZF extract improved DR mainly by thickening the retinal structures of INL and ONL.

[0068] Furthermore, FP analysis showed that the retinal lesions in db / db mice resembled cotton wool spots. These lesions were characterized by whitish color, blurred borders, irregular outlines, and a cloudy appearance, indicating localized microvascular occlusion in the retina. However, these manifestations disappeared after administration of SZF extract ( Figure 3 A). In addition, FFA was used to examine vascular abnormalities and pathological conditions. It was observed that db / db mice showed more obvious vascular leakage compared with the db / m control group. Treatment with SZF extract alleviated this abnormal leakage to a large extent. No obvious vascular dilation or microaneurysms were observed in any group ( Figure 3 B).

[0069] 2.3SZF extract improves foot microcirculation in db / db mice

[0070] Using a laser Doppler imaging system, it was observed that the foot perfusion of db / db mice was significantly lower compared with the control group. After administration of SZF extract, there was a significant dose-responsive increase in foot perfusion. In particular, the high dose of SZF extract significantly increased the perfusion, and the data showed a statistically significant difference ( Figure 3 C). These findings suggest that SZF extract can effectively enhance microcirculatory function in the feet of mice.

[0071] 3.4SZF extract can reduce serum inflammation

[0072] In addition, the levels of TNF-α and STAT3 in serum were quantitatively evaluated using specific ELISA kits. The results of the analysis showed that the administration of SZF extract significantly reduced inflammatory markers, as evidenced by the reduction of these cytokines ( Figure 4 A and 4B).

[0073] 3.9SZF extract delays DR progression by regulating the HIF-1α-VEGFA pathway

[0074] Immunofluorescence experiments determined the expression levels of HIF-1α and VEGFA. The experiments confirmed that treatment with SZF extract significantly reduced the levels of HIF-1α and VEGFA. The experiments also found that these proteins may affect each other ( Figure 4 C).

[0075] In summary, SZF extract significantly alleviated vascular leakage associated with BRB injury in the db / db mouse model and enhanced microcirculation in the limbs, thereby slowing down the progression of DR. Comprehensive analysis using retinal OCT and HE staining techniques showed that SZF extract prevented the development of DR mainly by strengthening the structural integrity of retinal INL and ONL. Immunofluorescence experiments further verified the involvement of key targets such as HIF-1α and VEGFA, demonstrating their important roles in regulating disease progression and confirming their effectiveness in therapeutic applications.

[0076] Test case 2: Mechanism bioinformatics analysis

[0077] 1 Experimental methods

[0078] 1.1 Qualitative analysis of active ingredients in SZF extracts--UPLC-Q-TOF-MS / MS analysis

[0079] Sample solution: Take an appropriate amount of the powder (SZF extract) of Example 1 without adding auxiliary materials, add an appropriate amount of methanol to dissolve by ultrasonication, and then cool to a fixed volume for later use.

[0080] UPLC-Q-TOF-MS / MS conditions

[0081] SZF extracts were analyzed using Waters ACQUITY UPLC I-Class Plus and Xevo G2-XS QTOF Systems.

[0082] Column: ACQUITY UPLC BEH C18 column; mobile phase: 0.1% formic acid and acetonitrile; 30-minute gradient elution; flow rate: 0.3 ml / min, column temperature: 30 °C; mass spectrometry detection had a mass range of m / z 50 to 1500, using positive and negative ion modes at voltages of -2.0 kV and 3.0 kV, respectively. Mass spectrometry scans included a low collision energy of 6 V and a variable high collision energy of 40 to 60 V, which facilitated the acquisition of MS1 and MS2 spectra.

[0083] 1.2 Prediction of disease targets for diabetic retinopathy

[0084] The obtained ingredients were entered into the Swiss Target Prediction database (http: / / www.swisstargetprediction.ch / ) to identify their potential targets. A table was organized to display the results according to the following structure: "TCM-ingredient code-ingredient name-target". Disease target data specifically related to the keyword "diabetic retinopathy" were collected using the GeneCards database (https: / / www.genecards.org / ). Through this systematic approach, a comprehensive understanding of the connections between the identified ingredients and their potential therapeutic effects in diabetic retinopathy was obtained.

[0085] 1.3 Establishing a regulatory network for traditional Chinese medicine compound prescriptions

[0086] By analyzing the potential targets of SZF extracts, we found their intersections with related genes and created Venn diagrams using the R software package to visually represent this overlap. The regulatory network of the Chinese medicine compound was constructed using Cytoscape version 3.8.2, with the intersection genes and active ingredients as nodes in the network. This helps to better understand the interactions between compounds and their target genes, highlighting the intricate relationships behind the therapeutic effects of traditional Chinese medicine.

[0087] 1.4 Construction of PPI protein interaction network and screening of central genes

[0088] The STRING database (https: / / www.string-db.org / ) is a resource for constructing protein-protein interaction (PPI) networks using common differentially expressed genes. To improve clarity, irrelevant nodes in the network with a confidence score lower than 0.4 were filtered out. For visualization purposes, Cytoscape 3.8.2 software was used, and the CytoHubba plugin helped to determine the top 10 hub targets based on node scores. In addition, the MCODE plugin was used to pinpoint key modules in the PPI network, and the following criteria were used for screening: degree cutoff of 2, node score cutoff of 0.2, K-score of 2, and maximum depth of 100. The modules with the highest scores were considered as the final functional modules. The intersections of these elements represent the core targets of the network. To gain insight into the biological features associated with these core targets, we constructed and analyzed a network consisting of core genes and their co-expressed counterparts using the GeneMANIA platform.

[0089] 1.5 Functional enrichment analysis

[0090] GO provides functional annotations for genes, allowing people to understand their biological roles. On the other hand, KEGG can be used to analyze and map various signaling pathways associated with these genes. For visualization, we use R packages to create informative and easy-to-understand graphical representations of these data.

[0091] 1.6 Molecular docking

[0092] Molecular docking studies were performed on the key targets identified in the PPI network and the core genes in the "pathway genes" network diagram using the main components of the SZF extract. The protein structures of the 18 core targets were retrieved from the Protein Data Bank (https: / / www.rcsb.org / ) in three-dimensional format. After downloading, various preprocessing steps were performed using PyMOL 2.5 software, including removal of water molecules, addition of hydrogen atoms, and extraction of ligands. Molecular docking was then performed using AutoDockVina version 1.1.2.

[0093] 1.7 Statistical analysis

[0094] GraphPad Prism software was used for statistical analysis in this study, and the results are presented as mean and SE. A critical value of P value less than 0.05 indicated statistical significance, indicating that there was a meaningful difference in the data analyzed.

[0095] 2 Experimental results

[0096] 2.1 Qualitative analysis and quality control of active ingredients in SZF extract

[0097] Figure 5A and 5B depict the extracted ion chromatograms representing all identified chemical entities in the formula. Among the 152 compounds that were directly identified or indirectly suggested. The identification results showed that each compound was related to one of the herbal ingredients in the formula, mainly including Astragalus, Rheum officinale, and Salvia miltiorrhiza. The identified substances have a variety of chemical compositions, including well-known compounds such as cryptotanshinone, danshensu, protocatechuic aldehyde, ferulic acid, aloe-emodin-8-O-β-D-pyranoglucopyranoside, emodin-3-methyl ether, calcein 7-O-glucoside, scrophulariaceae, huperzine, astragaloside, 2-propenoic acid, 3-[2-[(1E)-2-(3,4-dihydroxyphenyl)vinyl]-3,4-dihydroxyphenyl]- and many other substances. In addition, 12 different batches of SZF extracts were analyzed and each batch was compared with a standard reference spectrum. As Figure 5 C and 5D, the recorded similarity indices ranged from 0.886 to 0.995, indicating that the quality of SZF was highly consistent and controllable.

[0098] 2.2 Target gene screening and SZF regulatory network construction

[0099] The key compounds in the important composition of the present invention identified by ULTRA HPLC / Q-TOF-MS analysis were imported into the SwissTarget prediction website to identify potential drug targets. 4023 DR-related targets were retrieved using the GeneCards database. By comparing the targets of SZF extract and DR, 344 shared targets were found ( Figure 6 A).

[0100] A network graph named "Active Ingredient Cross-Gene" was constructed using Cytoscape 3.8.2 software, which included 395 nodes and 1457 edges. These nodes and their interconnections highlight the functional interactions in the network, and the nodes with the most interconnections indicate that the target or compound is more important in the network. Notable active ingredients include magnolamine, apigenin, isorhamnetin, quercetin, and kaempferol, all of which show high connectivity ( Figure 6 B).

[0101] In addition, after importing 344 differentially expressed genes (DEGs) into STRING, a relationship network with 18 nodes and 7392 edges was formed ( Figure 6 C). The Cytohuba plugin identified the top 10 key targets in the protein-protein interaction (PPI) network, namely STAT3, GAPDH, BCL2, CASP3, HIF1A, MTOR, CTNNB1, JUN, TNF, and ESR1 ( Figure 6D). Cluster analysis using the MCODE plug-in revealed 11 functional modules, including key targets such as STAT3, GAPDH, BCL2, CASP3, HIF1A, MTOR, CTNNB1, JUN, TNF, and ESR1 ( Figure 6 E).

[0102] The network generated by GeneMANIA contained a total of 30 genes, 20 related genes, and 265 links. Analysis of the 10 hub genes in this complex PPI network showed that physical interactions accounted for 52.34%, co-expression accounted for 26.87%, co-localization accounted for 6.30%, predicted interactions accounted for 5.84%, genetic interactions accounted for 4.33%, and pathway participation accounted for 4.31%. The network functional information showed that these targets were related to the regulation of miRNA by RNA polymerase II ( Figure 6 F).

[0103] 2.3 Functional enrichment analysis

[0104] GO functional analysis was performed on 344 targets shared by SZF extract and diabetic retinopathy. This analysis revealed a wide range of biological processes, with a total of 2679 BP categories, 117 CC categories, and 232 MF categories enriched, showing the broad biological significance of these targets ( Figure 7 A). Further exploration through KEGG pathway enrichment analysis revealed 180 pathways that may be regulated by these targets ( Figure 7 B). We also constructed a co-expression network to illustrate the interactions between differentially expressed genes (DEGs, represented by gray nodes) and the pathways they affect (yellow nodes), and the color-coded lines describe the specific pathways in which different genes participate ( Figure 7 C).

[0105] 2.4 Molecular docking

[0106] The present invention evaluates the potential of a series of small molecule ligands (i.e., magnolamine, apigenin, isorhamnetin, quercetin, and kaempferol) to interact with protein receptors such as HIF1A and VEGFA. This study performed a detailed calculation of the binding energy, and the results are as follows Figure 8 As shown in A, negative values ​​of binding energy indicate that the ligand has an intrinsic tendency to spontaneously bind to the receptor, thus affirming the stability and feasibility of the interaction. Docking analysis highlights that the lower the value of binding affinity, the stronger and more durable the interaction between the ligand and its respective protein target. This is particularly evident in the case of quercetin and kaempferol, which have a significant affinity for HIF1A, highlighting their potential as modulators of cellular oxygen sensing pathways ( Figure 8B and 8C). In addition, apigenin also has a significant affinity for vascular endothelial growth factor (VEGFA), suggesting that there is a strong interaction between them that can affect the angiogenesis process ( Figure 8 D).

[0107] In summary, the present invention determines that HIF1A participates in the pathogenesis of diabetic retinopathy by directly regulating the expression of VEGFA. The two together constitute the key pathway for regulating retinal neovascularization, and clarify the complex network involving macrophages, HIF-1α and VEGFA in the pathogenesis of DR. In a hyperglycemic environment, oxidative stress and inflammatory responses synergistically activate macrophages. This activation, together with the effects of TNFα, STAT3, HIF-1α and VEGFA, constructs an intricate network that enhances vascular permeability and leakage, which is the main pathological feature of DR. In other words, DR is a multifactorial disease that requires a comprehensive approach to consider immune response, hypoxia and angiogenesis.

[0108] At the same time, the present invention also proves the rational interaction between quercetin and HIF1A, and apigenin and VEGFA. The molecular basis of the efficacy of SZF extract is clarified, and it can be positioned as a promising treatment for DR. Compared with traditional treatment methods, the ability of the Chinese medicine composition of the present invention to target this key pathogenic pathway has multiple advantages. SZF extract regulates macrophage-mediated inflammatory response and angiogenesis signals by regulating HIF-1α-VEGFA, providing a new method for solving the complex pathophysiological problems of DR.

[0109] The above-described embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A Chinese medicine composition for treating diabetic retinopathy, characterized in that: The Chinese medicine composition is prepared from the following Chinese medicinal materials in parts by weight: 25-35 parts of astragalus, 3-9 parts of rhubarb, 10-20 parts of salvia miltiorrhiza, 3-9 parts of roasted epimedium, 10-20 parts of dried motherwort and 2-4 parts of scalded leech.

2. A Chinese medicine composition for treating diabetic retinopathy according to claim 1, characterized in that: The Chinese medicine composition is prepared from the following Chinese medicinal materials in parts by weight: 30 parts of astragalus, 6 parts of rhubarb, 15 parts of salvia miltiorrhiza, 6 parts of roasted epimedium, 15 parts of dried motherwort and 3 parts of scalded leech.

3. A Chinese medicine composition for treating diabetic retinopathy according to claim 1, characterized in that: The Chinese medicine composition is prepared from the following Chinese medicinal materials in parts by weight: 32 parts of astragalus, 5 parts of rhubarb, 12 parts of salvia miltiorrhiza, 8 parts of roasted epimedium, 12 parts of dried motherwort and 4 parts of scalded leech.

4. A method for preparing the Chinese medicine composition for treating diabetic retinopathy according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: taking astragalus, rhubarb, salvia miltiorrhiza, roasted epimedium and dried motherwort, boiling with water, filtering to obtain filtrate; grinding scalded leeches into powder and adding the powder into the filtrate, further adding auxiliary materials and sterilizing to prepare a finished preparation.

5. The method for preparing a Chinese medicine composition for treating diabetic retinopathy according to claim 4, characterized in that: The astragalus, rhubarb, salvia miltiorrhiza, roasted epimedium and dried motherwort need to be soaked in water for 30-60 minutes before being decocted in water.

6. The method for preparing a Chinese medicine composition for treating diabetic retinopathy according to claim 4, characterized in that: The volume ratio of the total mass of the astragalus, rhubarb, salvia miltiorrhiza, roasted epimedium and dried motherwort to water is 1g:10-15ml.

7. The method for preparing a Chinese medicine composition for treating diabetic retinopathy according to claim 4, characterized in that: The decoction is specifically performed 2-3 times, each time for 30-60 minutes.

8. A method for preparing a Chinese medicine composition for treating diabetic retinopathy according to any one of claims 4 to 7, characterized in that: The finished preparation is a tablet, powder, capsule, granule or oral liquid.

9. Use of the Chinese medicine composition for treating diabetic retinopathy as claimed in any one of claims 1 to 3 in preparing a drug for treating or preventing diabetic retinopathy.

10. The use according to claim 9, characterized in that: The drug for treating or preventing diabetic retinopathy is a drug that reduces vascular leakage associated with damage to the blood-retinal barrier (BRB), enhances microcirculation in the limbs, strengthens the structural integrity of the retinal INL and ONL, or regulates HIF-1α-VEGFA to regulate macrophage-mediated inflammatory responses and angiogenesis signals.

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