CN115120665B

CN115120665B - Traditional Chinese medicine composition with efficacy of treating diabetic retinopathy

Info

Publication number: CN115120665B
Application number: CN202210684780.0A
Authority: CN
Inventors: 孙利; 丁永芳
Original assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Current assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2023-06-02
Anticipated expiration: 2042-06-17
Also published as: CN115120665A

Abstract

The invention belongs to the field of the effect of traditional Chinese medicines in treating diabetic retinopathy, and particularly relates to a specific application of the traditional Chinese medicines in treating diabetic retinopathy caused by the change of blood retinal barrier permeability. The technical scheme adopted is that the traditional Chinese medicine composition with the efficacy of treating diabetic retinopathy consists of the following components in parts by weight: 500-4000 parts of astragalus membranaceus, 100-1000 parts of shizandra berry, 500-3000 parts of medlar, 500-3000 parts of radix trichosanthis, 500-3000 parts of rhizoma anemarrhenae, 500-3000 parts of radix puerariae, 500-3000 parts of allium macrostemon, 200-900 parts of rhizoma acori graminei, 500-3000 parts of angelica sinensis and 500-3000 parts of radix salviae miltiorrhizae. Furthermore, the traditional Chinese medicine composition can be prepared into medicines with different dosage forms such as tablets, capsules, granules, oral liquid or injection with pharmaceutically acceptable auxiliary materials. The Chinese medicinal composition increases the expression of occludin by down regulating the expression of VEGF in diabetic rat retina, thereby playing the roles of protecting blood retina barrier, reducing BRB permeability and delaying diabetic retinopathy process.

Description

Traditional Chinese medicine composition with efficacy of treating diabetic retinopathy

Technical Field

The invention belongs to the field of the effect of traditional Chinese medicines in treating diabetic retinopathy, and particularly relates to a specific application of the traditional Chinese medicines in treating diabetic retinopathy caused by the change of blood retinal barrier permeability.

Background

Diabetic Retinopathy (DR) is one of the most common, most severe and therapeutically troublesome microvascular complications of diabetes, which results in irreversible blindness, an important cause of blindness in patients with disability. The basic pathological change in diabetic retinopathy is a change in blood retinal barrier (blood dbrb) permeability. The pathogenesis is the result of the combined action of multiple links, namely, the hyperglycemia causes vascular disorder, the coagulation system is hyperfunctional, the fibrinolysis function is low, and retinal capillaries are blocked, so that retinal ischemia and hypoxia are caused, the abnormal secretion of VEGF by retinal pigment epithelial cells is induced, endothelial cell injury is caused, BRB is destroyed and new blood vessels are generated, therefore, in the occurrence and development processes of diabetic retinopathy, VEGF is a key factor, the stability of blood retinal barriers is closely related, and the over-expression of VEGF is inhibited, so that the progress of the diabetic retinopathy can be delayed.

Disclosure of Invention

Object of the Invention

The Chinese medicinal composition is used for treating diabetic retinopathy by searching a Chinese medicinal composition in a treasury of traditional Chinese medicine.

Adopts the technical proposal that

A traditional Chinese medicine composition with the efficacy of treating diabetic retinopathy consists of the following components in parts by weight: 500-4000 parts of astragalus membranaceus, 100-1000 parts of shizandra berry, 500-3000 parts of medlar, 500-3000 parts of radix trichosanthis, 500-3000 parts of rhizoma anemarrhenae, 500-3000 parts of radix puerariae, 500-3000 parts of allium macrostemon, 200-900 parts of rhizoma acori graminei, 500-3000 parts of angelica sinensis and 500-3000 parts of radix salviae miltiorrhizae.

Further, the mixture ratio is 1500-2500 parts of astragalus root, 300-600 parts of shizandra berry, 800-1200 parts of medlar, 800-1200 parts of radix trichosanthis, 800-1200 parts of rhizoma anemarrhenae, 800-1200 parts of kudzuvine root, 800-1200 parts of allium macrostemon, 500-700 parts of grassleaf sweelflag rhizome, 800-1200 parts of angelica and 800-1200 parts of red sage root.

Furthermore, the traditional Chinese medicine composition can be prepared into medicines with different dosage forms such as tablets, capsules, granules, oral liquid or injection with pharmaceutically acceptable auxiliary materials.

Advantageous effects

In the development and development process of diabetic retinopathy, VEGF is a key factor, and is closely related to the stability of blood retinal barrier, and inhibiting the overexpression of VEGF can delay the progress of diabetic retinopathy. The Chinese medicinal composition increases the expression of occludin by down regulating the expression of VEGF in diabetic rat retina, thereby playing the roles of protecting blood retina barrier, reducing BRB permeability and delaying diabetic retinopathy process.

Drawings

FIG. 1 is a graph showing the effect of a composition on blood glucose in diabetic retinopathy rats.

FIG. 2 is a graph showing the effect of the composition on retinal permeability in diabetic rats.

FIG. 3 is an effect of the composition on retinal Occudin expression in diabetic rats.

Detailed Description

In the embodiment listed in the specification, the specific proportion of 20g of astragalus, 5g of schisandra chinensis, 10g of medlar, 10g of radix trichosanthis, 10g of rhizoma anemarrhenae, 10g of radix puerariae, 10g of allium macrostemon, 6g of rhizoma acori graminei, 10g of angelica and 10g of radix salviae miltiorrhizae is adopted. Experimental data are used for showing the effect of the traditional Chinese medicine composition for preventing and treating diabetic retinopathy in vivo.

1. Test agent

The raw materials are weighed according to the proportion for 7 days, and distilled water with the amount of 10 times is respectively added for extraction for 3 times, and each time lasts for 1.5 hours. Filtering, mixing the extractive solutions, and concentrating to 1g crude drug per ml.

2. Experimental animal

2.1 building animal model Male SD rats were selected, after no water retention for 12h, the animals except for the normal control group were injected intraperitoneally with freshly prepared and precooled STZ sodium citrate buffer (60 mg/kg) according to body weight, and after preparation, the injection was completed within 15min to induce diabetes model. Normal control rats were intraperitoneally injected with an equal volume of citrate buffer. Rats were observed for no abnormalities 2h after injection and were given a free diet. After 72h of molding, the tail vein is taken for blood measurement of fasting blood sugar, and the blood sugar is more than 16.5mmol/L as a standard for molding success.

2.2 group administration

Rats successfully modeled were randomly divided into model groups, positive drug groups and high, medium, and low dose groups of composition, 24 per group. The administration groups were administered by intragastric administration at doses of 55.0, 27.5 and 13.75g crude drug/kg, respectively, with a volume of administration of 10ml/kg. The normal control and model groups were given equal volumes of distilled water. The administration was performed once daily for 8 weeks.

2.3 detection index

2.3.1 Conventional index: rats were blood glucose was monitored by orbital blood sampling at 8W after dosing, respectively, before grouping (i.e., 72h after molding).

2.3.2 retinal permeability (evans blue method): to examine the blood-retinal barrier lesions of post-dose model rats, after the last dose (i.e., after 8W of continuous administration), a portion of animals (8 animals per group) were anesthetized by intraperitoneal injection with 10% chloral hydrate (3 ml/kg), the right jugular vein was exposed, and an evans blue solution was slowly injected at a dose of 45mg/kg over 1min, and after 120min of circulation, the right common carotid artery was cannulated and perfused continuously for 2min with a 1% paraformaldehyde solution (ph=3.5 sodium citrate buffer formulation) pre-heated to 37 ℃. One side eyeball was extracted, retina was separated, and after 8 hours of vacuum drying, it was weighed, 150. Mu.l formamide was added, extraction was performed at 70℃for 18 hours, centrifugation was performed at 17500r/min at 4℃for 120 minutes, and the supernatant was taken and its absorbance at wavelengths of 620nm and 740nm, respectively, was measured. The penetration of evans blue was determined by a standard curve and evans blue (ng) was normalized with the dry weight of the retina (mg).

2.3.3 measurement of retinal Occudin expression: after the experiment is finished (i.e. after the last administration), animals are sacrificed, the double eyeballs of the rats are immediately picked up, retinal tissues are separated, the rat is placed into a 1.5ml centrifuge tube without RNase, and the rat is quickly placed into liquid nitrogen for storage for later use. Adding 1ml of TRIzol which is cooled into 50mg of retina, and lysing retinal tissue; extracting total RNA of retina; reverse transcription into cDNA, and amplification of corresponding gene with beta-actin as reference. Occlutin upstream primer sequence: 5'-CCTGTCTATGCTCGTCATCG-3', downstream primer sequence: 5 'TAGCCGTACCGGTA-3'; beta-actin upstream primer sequence: 5'-TGGCACCCAGCACAATGAA-3', downstream primer sequence: 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'. The number of cycles that the fluorescence value experiences when reaching the threshold, i.e. the Ct value, is recorded. Delta ct=objective gene Ct value-reference gene Ct value, the 2-delta Ct method calculates the relative expression amount of Occludin mRNA, and each specimen is repeated 3 times.

2.3.4 Determination of retinal VEGF expression: after the last administration, the remaining rats (8 per group) were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 ml/kg), the right common carotid artery was isolated, the proximal end was ligated, cannulated, the left jugular vein was cut, pre-washed with physiological saline, and after 4% paraformaldehyde was washed until the eyeball was whitish, and the eyeball was removed and fixed in 4% paraformaldehyde for 24 hours. The iris is cut off in a ring shape at a position 0.5mm away from the tooth edge, a rear eye cup is cut into 3 pieces by taking a nipple as a center orange-leaf sample, retina is gently separated, 0.01mmol/L PBS (pH 7.4) is rinsed and then put into 3% trypsin for digestion for 2-4 h at 37 ℃, a layer of transparent retina vascular network is repeatedly blown, the transparent retina vascular network is paved on a glass slide, the transparent retina vascular network is naturally dried, VEGF immunohistochemical detection is carried out, the retina structure is observed under a microscope after the neutral resin is sealed, and an image is acquired. Each slice was selected under a high power microscope for 3 different fields of view, each field of view not overlapping each other, image-Pro Plus 6.0 Image analysis software analyzed the images. The average A value of the brown-yellow or tan positive reaction sites in the image was determined.

3. Experimental results

3.1 In general.

Rats in the normal control group grow rapidly, weight gradually increases, hair is glossy, and eating is normal; after the model group animals are successfully molded, the food consumption, the water intake and the urine consumption of the model group animals start to increase, the animals are emaciated, the hair color is sallow, and obvious three more and one less symptoms appear; by the 8 th week of the experiment, rats in the model group move slowly, are extremely emaciated, have turbid crystals and have abdominal distension in individual animals; animals in the administration group had good conditions, slightly emaciated, shiny hair, and individual rats found cloudy crystals.

3.2 effects on biochemical indicators in serum.

Blood glucose was significantly elevated in all animals 72h after STZ injection (fig. 1). Animal blood Glucose (GLU) was maintained at higher levels (P < 0.01) throughout the experimental period model group; following continuous administration of 8w, the blood glucose level of the composition in the high dose group animals can be significantly reduced (P < 0.01). The results are shown in figure 1, namely the effect of the traditional Chinese medicine composition on blood sugar of diabetic retinopathy rats, and the results are that delta P is less than 0.01 compared with a normal control group; p <0.01 compared to model group.

3.3 effects on retinal permeability.

After model group rats were modeled by 8w, the permeation amount of evans blue was significantly increased, compared with normal control group (P < 0.01), retinal permeability was significantly increased in diabetic retinopathy rats, and blood-retinal barrier was damaged. The high and medium doses of the composition can obviously reduce the permeation quantity (P < 0.05-0.01) of the Evan blue and lighten the lesions of the blood-retina barrier of diabetic animals. The results are shown in figure 2, namely the effect of the traditional Chinese medicine composition on the retinal permeability of diabetic rats, and the result is that delta P is less than 0.01 compared with the normal control group; p <0.05, P <0.01 compared to model group.

3.4 effects on retinal Occidin expression.

Compared with the normal control group, the expression level of the Occidin mRNA of the retina of the animal in the model group is obviously reduced (P < 0.01), and after the animals in the traditional Chinese medicine composition group are continuously dosed by 8w, the expression level of the Occidin mRNA of the rats in the high and medium dose groups is obviously increased, and compared with the model group, the Occidin mRNA of the rats in the high and medium dose groups is obviously different (P < 0.05-0.01). The results are shown in FIG. 3, which shows the effect of the Chinese medicinal composition of the present invention on the expression of the retina Occidin in diabetic rats, and the result is that DeltaP <0.01 compared with the normal control group; p <0.05, P <0.01 compared to model group.

3.5 Effects on expression of rat retinal VEGF.

The retina VEGF positive expression cytoplasm is brown yellow and is mainly distributed in the inner core layer, the inner slave layer and the nerve fiber layer. The normal control group has little VEGF expression; the VEGF expression of the retina of the animal in the model group is obviously enhanced, and the whole retina has positive expression; after 8 weeks of continuous administration, the expression level was significantly reduced (P < 0.01) compared with the model group.

TABLE 1 Effect of the inventive Chinese medicinal composition on retinal VEGF expression (x+ -s)

Note that Δp <0.01 compared to normal control and P <0.01 compared to model.

Discussion: in the development and development process of diabetic retinopathy, VEGF is a key factor, and is closely related to the stability of blood retinal barrier, and inhibiting the overexpression of VEGF can delay the progress of diabetic retinopathy. The experimental result shows that the traditional Chinese medicine composition increases the expression of occludin by down regulating the expression of VEGF in the retina of a diabetic rat, thereby playing the roles of protecting blood retina barrier, reducing BRB permeability and delaying diabetic retinopathy process.

The diabetic complications are the main cause of death disability of diabetics, how to control the diabetic complications and reduce the death rate, and the combination of traditional Chinese medicine and western medicine is adopted to comprehensively prevent and treat the diabetic complications, so that the clinical symptoms of the patients can be improved, the life quality can be improved, and the progress of the complications can be lightened and delayed. The traditional Chinese medicine composition provided by the invention is used as a traditional Chinese medicine compound with definite clinical curative effect and action mechanism, and has wide clinical application prospect.

Summary

Animal experiments show that the composition can stabilize the blood-retina barrier by reducing the permeability of the blood-retina barrier, and has a certain blood sugar reducing effect. The composition of the invention can be further prepared into medicines with different dosage forms such as tablets, capsules, granules, oral liquid or injection with pharmaceutically acceptable auxiliary materials.

SEQUENCE LISTING

<110> Jiangsu province Chinese medical institute

<120> A Chinese medicinal composition for treating diabetic retinopathy

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Occludin forward

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cctgtctatg ctcgtcatcg 20

<210> 2

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Occludin reverse

<400> 2

tagccgtaac cgtagccgta 20

<210> 3

<211> 19

<212> DNA

<213> Artificial

<220>

<223> β-acti forward

<400> 3

tggcacccag cacaatgaa 19

<210> 4

<211> 25

<212> DNA

<213> Artificial

<220>

<223> β-acti reverse

<400> 4

ctaagtcata gtccgcctag aagca 25

Claims

1. The traditional Chinese medicine composition with the efficacy of treating diabetic retinopathy is characterized by comprising the following raw materials in parts by weight: 500-4000 parts of astragalus membranaceus, 100-1000 parts of shizandra berry, 500-3000 parts of medlar, 500-3000 parts of radix trichosanthis, 500-3000 parts of rhizoma anemarrhenae, 500-3000 parts of radix puerariae, 500-3000 parts of allium macrostemon, 200-900 parts of rhizoma acori graminei, 500-3000 parts of angelica sinensis and 500-3000 parts of radix salviae miltiorrhizae.

2. The traditional Chinese medicine composition with the efficacy of treating diabetic retinopathy according to claim 1, which is characterized by 1500-2500 parts of astragalus membranaceus, 300-600 parts of schisandra chinensis, 800-1200 parts of medlar, 800-1200 parts of radix trichosanthis, 800-1200 parts of rhizoma anemarrhenae, 800-1200 parts of radix puerariae, 800-1200 parts of allium macrostemon, 500-700 parts of rhizoma acori graminei, 800-1200 parts of angelica sinensis and 800-1200 parts of radix salviae miltiorrhizae.

3. The Chinese medicinal composition for treating diabetic retinopathy according to claim 1 or 2, wherein the Chinese medicinal composition can be prepared into tablets, capsules, granules, oral liquid or injection with pharmaceutically acceptable auxiliary materials.