CN106581151B

CN106581151B - Application of traditional Chinese medicine composition for promoting blood circulation and removing stasis

Info

Publication number: CN106581151B
Application number: CN201611267281.2A
Authority: CN
Inventors: 彭清华; 彭俊; 吴权龙; 潘坤
Original assignee: Hunan University of Chinese Medicine
Current assignee: Hunan University of Chinese Medicine
Priority date: 2016-12-31
Filing date: 2016-12-31
Publication date: 2021-03-19
Anticipated expiration: 2036-12-31
Also published as: CN106581151A

Abstract

The invention provides a traditional Chinese medicine composition for promoting blood circulation and removing stasis and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-10 parts of pseudo-ginseng, 10-30 parts of salvia miltiorrhiza, 15-30 parts of earthworm, 10-30 parts of seaweed, 10-30 parts of kelp and 10-30 parts of turtle shell. The traditional Chinese medicine composition disclosed by the invention plays a synergistic interaction role among specific traditional Chinese medicines, and the obtained traditional Chinese medicine composition can play the effects of promoting blood circulation to remove blood stasis and softening and resolving hard mass, and can dissipate visible substances of blood stasis and phlegm stagnation in eyes, so that the fundus condition of a PVR patient can be obviously improved, and the visual function of the patient is improved.

Description

Application of traditional Chinese medicine composition for promoting blood circulation and removing stasis

Technical Field

The invention relates to the field of traditional Chinese medicine preparations, and in particular relates to a traditional Chinese medicine composition for promoting blood circulation and removing stasis, and a preparation method and application thereof.

Background

Proliferative Vitreoretinopathy (PVR) is a serious complication of fundus diseases such as retinal detachment, diabetic retinopathy, vitreous hemorrhage, and retrobulbar punch through. It is characterized by vitreous cavity, retinal surface and subretinal proliferation membrane, and is easy to cause Tractional Retinal Detachment (TRD), which is an important reason for continuous deterioration of many ocular fundus diseases and also a main reason for failure of the porogenic retinal detachment reduction operation and postoperative recurrence. It was reported that 75% of retinal detachments failed surgery due to PVR. The incidence of PVR in clinical rhegmatogenous retinal detachment is about 5% -10%. The occurrence of PVR is one of the causes of blindness, mild PVR has the symptoms of muscae volitantes, visual deterioration, visual deformation, visual field defect and the like, and the later period of PVR can even lead to light sensation deprivation and blindness, intraocular pressure reduction and eyeball atrophy. Therefore, the intensive exploration of the pathogenesis and treatment of PVR has important clinical significance.

The basic pathophysiological processes of PVR are cell proliferation, synthesis of extracellular matrix (ECM) and contraction of membranes. RPE cells are the basic type involved in PVR cell proliferation and are important mediating cells. Current studies have demonstrated that RPE cells express multiple growth factor receptors via an autocrine pathway, among which the PDGF receptor is one [4 ]. PDGF exhibits a variety of in vitro activities after binding to receptors, not only stimulates mitosis in RPE, fibroblasts and endothelial cells, but also promotes injury repair. Therefore, the PDGF is adopted to intervene in the in vitro culture of rabbit RPE cells to realize the cell model of PVR, and certain theoretical basis and basis are provided.

Current treatments for PVR are surgery-based, with the primary purpose of closing retinal tears, loosening and resisting retinal pulls. The operation includes vitreous cutting, sclera external pad pressing, retina front membrane stripping and intraocular air or silicon oil filling operation, etc. The surgery treatment can only cut off the formed hyperplastic membrane, the retinal anatomical morphology is recovered as much as possible, only 60% -80% of the success rate of anatomical reduction is achieved [7], and the surgery can stimulate the cell migration and differentiation again, which causes the PVR recurrence. Therefore, many scholars propose the use of drugs for preventing the development of PVR, mainly aiming at anti-proliferation and inflammation, and the drugs are in the experimental clinical stage or are not used as clinical conventional drugs for preventing and treating PVR at present due to the drug toxicity, long-term complications, drug concentration and the like.

Disclosure of Invention

One object of the present invention is to provide a Chinese medicinal composition for treating proliferative vitreoretinopathy by a method of activating blood circulation to dissipate stagnation.

Research shows that proliferative vitreoretinopathy, namely PVR, is caused by blood stasis internal stagnation caused by liver and gallbladder qi stagnation, which mostly belongs to qi and blood stasis, yin deficiency and phlegm accumulation caused by spleen and kidney disorder, phlegm dampness accumulation, paste failure caused by the multiple cementation and retardation of the two in the later stage, phlegm accumulation and blood stasis, and finally formation of tangible substances.

The traditional Chinese medicine composition has the effects of promoting blood circulation to remove blood stasis, softening and resolving hard mass, and dissipating visible substances of blood stasis and phlegm in eyes, so that the fundus condition of a PVR patient can be obviously improved, and the visual function of the patient can be improved.

In order to improve the effect of activating blood and resolving masses, the traditional Chinese medicine composition is prepared from the following components in parts by weight: 5-10 parts of pseudo-ginseng, 10-30 parts of salvia miltiorrhiza, 15-30 parts of earthworm, 10-30 parts of seaweed, 10-30 parts of kelp and 10-30 parts of turtle shell.

In order to achieve a better effect of preventing or treating PVR, the oral liquid is further preferably prepared from the following components in parts by weight: 5-9 parts of pseudo-ginseng, 13-20 parts of salvia miltiorrhiza, 13-23 parts of earthworm, 13-19 parts of seaweed, 13-19 parts of kelp and 13-19 parts of turtle shell.

More preferably, the composition is prepared from the following components in parts by weight: 5-7 parts of pseudo-ginseng, 14-17 parts of salvia miltiorrhiza, 16-20 parts of earthworm, 14-17 parts of seaweed, 14-17 parts of kelp and 14-17 parts of turtle shell.

Most preferably prepared from the following components in parts by weight: 6 parts of pseudo-ginseng, 15 parts of salvia miltiorrhiza, 18 parts of earthworm, 15 parts of seaweed, 15 parts of kelp and 15 parts of turtle shell.

In the embodiment of the invention, the active ingredients in the traditional Chinese medicine composition are pseudo-ginseng, salvia miltiorrhiza, earthworm, seaweed, kelp and turtle shell, and the active ingredients in the traditional Chinese medicine composition consist of the seven traditional Chinese medicines.

The weight parts may be μ g, mg, g, kg, etc. known in the art, or multiples thereof, such as 1/10, 1/100, 10, 100, etc.

The traditional Chinese medicine composition of the invention can further comprise a pharmaceutically acceptable carrier and/or excipient. The carriers and excipients herein are understood by those skilled in the art, and can be prepared into various dosage forms as required to select the appropriate carrier or excipient, as will be appreciated by those skilled in the art, and can be selected by those skilled in the art according to the actual requirements of the formulation.

The carrier or excipient used in the Chinese medicinal composition of the present invention may be selected from one or more of diluents, fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, solvents, surfactants or flavoring agents.

The traditional Chinese medicine composition can be prepared into various common pharmaceutical dosage forms by a conventional method, such as tablets, capsules, pills, powder, granules, oral liquid, injection or paste. In the present invention, oral liquid is preferred. The administration mode can be oral, intravenous, subcutaneous, transdermal or topical administration, etc., and is administered to animals or humans in unit administration form.

The traditional Chinese medicine composition is preferably oral liquid, and the concentration of crude drugs is 1g/ml-1.2g/ml, more preferably 1.025g/ml-1.10 g/ml.

The Chinese medicinal composition of the present invention is usually formulated in dosage units. The administration is 1 or more times daily, depending on the clinical dosage amount in the art. Higher or lower doses may also be employed in particular cases, and the dosage suitable for each patient will be ultimately determined by the physician, depending on the mode of administration, age, weight and response of the patient. Generally, it can be determined according to the body weight of the patient, and each administration is 15-30ml per Kg of weight.

The traditional Chinese medicine composition is preferably oral liquid, takes water as a diluent, and is preferably prepared by a water decoction method, and the preparation method comprises the following steps: weighing the above components according to a certain proportion, mixing, adding water, boiling, filtering the residue, and concentrating the filtrate to crude drug concentration of 1g/ml-1.2 g/ml.

For better effect, the crude drug concentration is 1.025g/ml-1.10 g/ml.

When not needed immediately, it is refrigerated. The oral liquid prepared by the method has stable performance.

The invention also aims to provide the application of the traditional Chinese medicine composition or the preparation method of the traditional Chinese medicine composition in preparing a medicine for treating proliferative vitreoretinopathy PVR.

Based on the research results of the traditional Chinese medicine theory and modern pharmacology, the traditional Chinese medicine composition summarized by years of clinical tests has reasonable compatibility, has the functions of invigorating blood circulation, softening and resolving hard mass, has the effects of anticoagulation, resisting fiber and inhibiting abnormal cell proliferation, can dissipate the tangible thing of blood stasis in eyes, can effectively prevent and treat proliferative vitreoretinopathy, and has no toxic or side effect.

Drawings

FIG. 1 is a graph showing the results of gel electrophoresis experiments for each group in test example 2;

FIG. 2 is a protein expression pattern of western blot assay of each group in Experimental example 3.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The present invention is described in further detail below with reference to exemplary embodiments. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

All the traditional Chinese medicines referred to in the following examples are known and commercially available products, which are purchased from pharmacy department of the major first subsidiary hospital of traditional Chinese medicine in Hunan, and the unit of the portion is selected as g.

Example 1

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 6g of pseudo-ginseng, 15g of salvia miltiorrhiza, 18g of earthworm, 15g of seaweed, 15g of kelp and 15g of turtle shell.

The embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: mixing the raw materials, adding water 10 times of the total amount of the raw materials, soaking for 30min, decocting for 30min, filtering the residue to obtain mixed medicinal liquid, concentrating the mixed medicinal liquid to crude drug concentration of 1.025g/ml, and storing at 4 deg.C.

Example 2

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 5g of pseudo-ginseng, 13g of salvia miltiorrhiza, 10g of earthworm, 10g of seaweed, 10g of kelp and 10g of turtle shell.

The embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: the embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: mixing the raw materials, adding water 10 times of the total amount of the raw materials, soaking for 30min, decocting for 30min, filtering the residue to obtain mixed medicinal liquid, concentrating the mixed medicinal liquid to crude drug concentration of 1.0g/ml, and refrigerating for storage.

Example 3

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 7g of pseudo-ginseng, 10g of salvia miltiorrhiza, 15g of earthworm, 19g of seaweed, 15g of kelp and 15g of turtle shell.

The embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: the embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: mixing the raw materials, adding water 10 times of the total amount of the raw materials, soaking for 30min, decocting for 30min, filtering the residue to obtain mixed medicinal liquid, concentrating the mixed medicinal liquid to crude drug concentration of 1.1g/ml, and refrigerating for storage.

Example 4

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 6g of pseudo-ginseng, 14g of salvia miltiorrhiza, 20g of earthworm, 13g of seaweed, 13g of kelp and 19g of turtle shell.

The embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: the embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: mixing the raw materials, adding water 10 times of the total amount of the raw materials, soaking for 30min, decocting for 30min, filtering the residue to obtain mixed medicinal liquid, concentrating the mixed medicinal liquid to crude drug concentration of 1.025g/ml, and refrigerating for storage.

Example 5

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 9g of pseudo-ginseng, 20g of salvia miltiorrhiza, 23g of earthworm, 30g of seaweed, 30g of kelp and 13g of turtle shell.

The embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: the embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: mixing the raw materials, adding water 10 times of the total amount of the raw materials, soaking for 30min, decocting for 30min, filtering the residue to obtain mixed medicinal liquid, concentrating the mixed medicinal liquid to crude drug concentration of 1.10g/ml, and refrigerating for storage.

Example 6

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 10g of pseudo-ginseng, 30g of salvia miltiorrhiza, 30g of earthworm, 30g of seaweed, 30g of kelp and 30g of turtle shell.

The embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: the embodiment also provides a preparation method of the oral liquid, which comprises the following specific steps: mixing the raw materials, adding water 10 times of the total amount of the raw materials, soaking for 30min, decocting for 30min, filtering the residue to obtain mixed medicinal liquid, concentrating the mixed medicinal liquid to crude drug concentration of 1.20g/ml, and refrigerating for storage.

Example 7

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 6g of pseudo-ginseng, 17g of salvia miltiorrhiza, 20g of earthworm, 17g of seaweed, 17g of kelp and 17g of turtle shell.

Example 8

The embodiment provides a traditional Chinese medicine composition (oral liquid), which is prepared from the following raw materials by a water decoction method: 6g of pseudo-ginseng, 15g of salvia miltiorrhiza, 16g of earthworm, 14g of seaweed, 14g of kelp and 14g of turtle shell.

The therapeutic effect of the traditional Chinese medicine composition on PVR of the present invention is further illustrated by the following test examples, wherein the composition (oral liquid) in example 1 is taken as an example.

Experimental animals: 10 adult healthy rabbits, male and female, with the weight of 1500 + 2000g, provided by the animal experiment center of the university of traditional Chinese medicine in Hunan, the animal license number: SYXK (xiang) 2015-. Feeding the chicken regularly, controlling the room temperature at 20-26 deg.C, keeping the air circulation, and keeping the relative humidity at about 55%. All animals were acclimatized for 1 week prior to the experiment.

Grouping experiments: 10 adult healthy rabbits are divided into A, B groups of 6 rabbits each by using a random number method. Respectively as follows: a represents blank group, and B represents Chinese medicinal composition group.

Dosage and method of administration: calculated according to the conversion method of the body surface area of the human body and the rabbit body (the human body takes 60Kg of body weight as a standard, and the rabbit takes 1.6Kg of body weight as a standard). Equivalent dose conversion algorithm converts the equivalent clinical dose of the rabbit traditional Chinese medicine composition group into 20.256g/Kg which is approximately equal to 20.5g/Kg, and the traditional Chinese medicine composition oral liquid is administrated by gavage with 20ml/Kg per day, and is administrated by gavage twice in the morning and evening for 5 days continuously. The blank group is administrated by drenching with distilled water 20ml/Kg per day twice in the morning and evening for 5 days.

Test example 1: the effect of the traditional Chinese medicine composition on rabbit RPE cell proliferation under PDGF intervention

Grouping experiments: blank control group (DMEM), normal plasma group (normal plasma), PDGF group (PDGF (10. mu.g.L)^-1) PDGF + AG1296 group (PDGF (10. mu.g. L))^-1)+AG1296(10μmol·L^-1) PDGF + plasma group containing a drug (PDGF (10. mu.g. L))^-1) + containing the blood plasma of the traditional Chinese medicine composition (10 wt%)), the blood plasma containing the traditional Chinese medicine composition is simply called the blood plasma containing the medicine hereinafter.

The experimental steps are as follows:

1. collecting blood plasma containing Chinese medicinal composition from rabbit after administration

Fasting is carried out for 12 hours before blood sampling, after 2 hours of last administration, abdominal cavity injection anesthesia is carried out according to the weight standard of 3.5ml/Kg of chloral hydrate with 10 percent, after the rabbit is fixed, abdominal rabbit hair is cut off, abdominal skin and peritoneum are cut off by tissue scissors, tissues around abdominal aorta blood vessels are separated inactively, abdominal aorta blood is taken under the aseptic condition, the blood is collected in a 1:9 vacuum blood collecting tube containing 3.2 percent of sodium citrate, and the blood is placed after being inverted and mixed evenly. Centrifuging at 3000 rpm for 10min, and collecting supernatant as medicated plasma. Subpackaging with 0.22 μm filter, and storing in-70 deg.C ultra-low temperature refrigerator.

2. CCK method for detecting activity of rabbit RPE cells under PDGF intervention by drug-containing plasma containing traditional Chinese medicine composition

1) The cells in the rabbit RPE log phase were collected, cell suspension concentration was adjusted, 100ul was added per well (96 well plate), plated to a test cell density of 5000 cells/well, and the marginal wells filled with PBS.

2) 5% CO2, incubated at 37 ℃ until cell monolayers have plated to the bottom of the wells, and different concentration gradients of drug in serum-free media were added.

3) Culturing at 37 deg.C for 48h with 5% CO 2.

4) 10ul of 7sea-cell counting kit solution in the kit is added into each hole, and zero-setting holes are synchronously arranged. The cultivation was continued for 2 h.

5) The absorbance (OD) at 450nm was measured with a microplate reader.

6) And recording and drawing the result.

3. Results

As shown in Table 1, the results of the activity test of the RPE cells in each group show that the normal plasma group has no obvious inhibition effect on the RPE cells, and has no significant difference (P >0.05) compared with a blank control group. Compared with the PDGF group and the normal plasma group, the cell viability of the PDGF group is obviously increased, the difference of the cell viability between the PDGF group and the normal plasma group has obvious statistical significance (P is less than 0.01), and compared with the PDGF group and the PDGF + AG1296 group, the PDGF + plasma group containing medicines and the PDGF group, the cell viability is obviously inhibited, and the difference has statistical significance (P is less than 0.01). The PDGF is proved to have a certain promotion effect on the activity of the RPE cells, the AG1296 serving as an inhibitor of the PDGF can inhibit the activity of the RPE cells, and the 10 percent of medicated plasma can also inhibit the activity of the RPE cells, has the same effect as the inhibitor, can inhibit the progress of PVR and prevent and treat PVR.

TABLE 1 Effect of drug-containing plasma on RPE cell viability following PDGF intervention

P compared to normal plasma group<0.01; in comparison with the PDGF group,^△△P<0.01

test example 2: the influence of the traditional Chinese medicine composition on the expression of rabbit RPE cell IGF1mRNA under PDGF intervention

The experimental steps are as follows:

2. Cell preparation

1) RPE cells were routinely cultured.

2) Cells in logarithmic phase are collected, cell suspension concentration is adjusted, 300ul of cell suspension is added into each hole (6-hole culture plate), culture medium is filled to 2ml, and cell density to be detected is 75%. Cells were treated as per the intervention mode of table 2 below. 4 multiple wells/set were provided.

TABLE 2 intervention modality of RPE logarithmic phase cell groups

3) After 48h of intervention, cells were harvested for QPCR detection.

3. Primer design

The following gene sequences were downloaded from NCBI and primer design using Primer5.0 software, with the primer sequences shown in Table 3 below. And is delivered to a factory company in advance for synthesis and standby.

TABLE 3 primer sequences

4. RNA extraction and reverse transcription

RNA is extracted from RPE cells and is reversely transcribed to obtain cDNA, and real-time PCR is carried out by taking the cDNA as a template and using a QPCR primer which is designed and synthesized aiming at the target gene to detect the expression of the target gene in the RPE cells.

4.1 Total RNA extraction in RPE cells

1) Adding 300 mu L Trizol into each hole of RPE cells, blowing, standing for 5min at room temperature, and collecting to 1.5ml of an RNase-free EP tube;

2) adding 60 μ L chloroform into each tube, shaking for 15s, and standing at room temperature for 3 min; centrifuging at 12000rpm at 4 deg.C for 15 min;

3) the supernatant from each tube was aspirated into another new 1.5ml lep tube. Adding isopropanol with the same volume, mixing uniformly, and standing at room temperature for 20 min;

4) centrifuging at 4 deg.C and 12000rpm for 10min, and removing supernatant; adding at least 1mL of 75% ethanol precooled at 4 ℃, and washing the precipitate;

5) centrifuging at 4 deg.C and 10000rpm for 5min, and removing supernatant; centrifuging at 4 deg.C and 10000rpm for 5min, sucking off residual liquid, and drying at room temperature (without completely drying);

6) add 20. mu.L RNase-free water to complete dissolution, store at-80 ℃ or perform subsequent experiments.

4.22% agarose gel electrophoresis for identifying RNA purity

Sample application sequence: a1, a2, A3, a4, B1, B2, B3, B4, C1, C2, C3, C4, D1, D2, D3, D4, E1, E2, E3, E4.

Sample loading amount: 2 ul/sample +1ul loading buffer

4.3 determination of RNA concentration and purity

Using a ultramicro spectrophotometer, loading: 2ul

4.4 first Strand cDNA Synthesis

1) 1.0. mu.g of Total RNA plus Nuclease-Free Water was added to an RNase-Free EP tube, mixed well, centrifuged, incubated at 70 ℃ for 10min, and immediately placed on ice.

2) The other reagents of the reaction system (ice) were added in the proportions shown in Table 4 below, mixed well and centrifuged briefly.

TABLE 4 amounts of reagents added

3) The above system was reacted at 42 ℃ for 15min, and then reversed Transcriptase was inactivated by water bath in a 70 ℃ water bath for 10 min.

4) The cDNA obtained is stored at-20 ℃ for later use.

5. Real-Time PCR detection of target gene and reference gene expression

1) The reaction system was configured in the following proportions in table 5:

TABLE 5 amounts of reagents added

2) And (3) amplification procedure:

94℃4min；

94℃40sec，60℃30sec，×40cycle

3) obtaining an amplification map

6. Statistical data

And calculating the 2-delta Ct according to the Ct value detected by the instrument.

Calculating the formula: Δ Ct (experiment) ═ Ct target gene (experiment) — Ct reference gene (experiment);

Δ CT (experiment) ═ Δ CT (experiment) - Δct (control);

then, the relative expression level was calculated as 2. DELTA. Ct.

7. Results

1) FIG. 1 shows the purity of RNA in all samples determined by gel electrophoresis. Table 6 shows the results of measuring the RNA concentration and purity in all samples using a ultramicrospectrophotometer. As can be seen from fig. 1 and table 6, all samples were of the appropriate concentration and the complete purity.

TABLE 6 ultra-micro spectrophotometry for measuring RNA concentration and purity

2) IGF1 is a very important influencing factor in the development process of PVR. As shown in Table 7, the PCR detection result shows that the normal plasma group has no influence on the expression of IGF1 gene and has no significant difference (P >0.05) compared with the blank group by taking the normal plasma group as a reference; PDGF intervention can promote the expression of IGF1 gene, and has significant difference (P <0.01) compared with a normal plasma group; the AG1296 and the drug-containing plasma can obviously reduce the expression of IGF1 gene, and the difference is significant in comparison with PDGF group (P < 0.01); wherein, compared with the group containing the drug plasma, the AG1296 group has no significant difference (P > 0.05).

TABLE 7 Effect of drug-containing plasma on IGF1mRNA in RPE cells under PDGF intervention

test example 3: the influence of the traditional Chinese medicine composition on the expression of rabbit RPE cell IGF1 protein under PDGF intervention

Grouping experiments: blank control group (DMEM), normal plasma group (normal plasma), PDGF group (PDGF (10. mu.g.L)^-1) PDGF + AG1296 group (PDGF (10. mu.g. L))^-1)+AG1296(10μmol·L^-1) PDGF + plasma group containing a drug (PDGF (10. mu.g. L))^-1) + contains the traditional Chinese medicine composition plasma (10 wt%)).

The experimental steps are as follows:

2. Western Blotting method for detecting rabbit RPE cell IGF1 protein expression

2.1 cell preparation

Digesting with pancreatin digestive juice and collecting RPE cells in logarithmic growth phase;

1) adjusting the cell suspension density to 5 × 105 cells/ml;

2) taking a 6-well plate, adding 1ml of corresponding cells per well of the cell suspension into each well plate according to the grouping conditions, complementing 2ml of complete culture medium, and gently mixing uniformly;

3) placing in 5% CO₂Culturing in an incubator at 37 ℃;

4) until the cell fusion rate in each well reaches 70-80%, adding corresponding intervention factors according to the intervention mode in the table 1;

5) and after 48h, collecting samples and carrying out western blot detection.

2.2 sample preparation

Adding cell sample of protein to be extracted into RIPA lysate containing protease and phosphatase inhibitor, fully lysing at 4 deg.C, scraping into 1.5ml EP tube, heating at 95 deg.C above for 10min, centrifuging at 12000g for 10min, collecting supernatant, quantifying protein, and storing in refrigerator at-80 deg.C.

2.3 protein quantification

(1) Drawing a standard curve: an ELISA plate was prepared and the reagents were added as in Table 8 below

TABLE 8 kinds and amounts of reagents added

(2) Preparing a proper amount of BCA working solution from the BCA reagent A and the reagent B according to the volume ratio of 50:1 according to the number of samples, and fully and uniformly mixing;

(3) 160 mul of BCA working solution is added into each well;

(4) the microplate was placed on a shaker for 30sec, left at 37 ℃ for 30 minutes, and then the absorbance was measured at 562 nm. Drawing a standard curve by taking the light absorption value as an abscissa and the protein concentration (mu g/mu l) as an ordinate;

(5) mu.l of the protein to be assayed and 18. mu.l of PBS (diluted 10 times) were added to the microplate, 160. mu.l of BCA working solution was added, the microplate was placed on a shaker for 30sec, left at 37 ℃ for 30 minutes, and then the absorbance was measured at 562 nm.

(6) According to the absorbance value of the tested sample, the corresponding protein concentration (mug/mul) can be found on the standard curve, and the actual concentration (unit: mug/mul) of the sample is obtained by multiplying the dilution factor (10) of the sample.

2.4 preparation of PAGE gels

(1) Preparation of lower layer separation gel

Selecting different gel concentrations according to the molecular weight of the target protein, separating the high molecular weight protein by using low-concentration glue and the low molecular weight protein by using high-concentration glue, preparing the separation glue with different concentrations according to the following table, and preparing the concentrated glue at the last time after the lower layer is gelled and solidified. The present experiment used 10% separation gel.

(2) Preparation of upper layer concentrated glue

The following Table 9 was used to prepare concentrated gels as required, and a comb was inserted

TABLE 9 sample volume for each component

2.5 Loading and electrophoresis

(1) Loading: the loading amount of each hole is about 40 mug of protein, and the loading amount can be increased according to the experimental requirements. And (3) adding a proper amount of loading buffer solution into the required protein according to the protein quantitative result, carrying out boiling water bath for 10min, and centrifuging to obtain a supernatant for loading. And placing the prepared PAGE gel into an electrophoresis tank, adding a proper amount of electrophoresis buffer solution, taking down a comb, and lightly blowing and punching a sample adding hole by using a gun to avoid the influence of residual gel in the hole on sample loading. The prepared sample is slowly added into the corresponding hole by using a sample adding gun, and the sample adding hole is prevented from overflowing.

(2) Electrophoresis: the gel is concentrated for 80V 20 minutes generally, the gel is separated for 120V 60 minutes, and the voltage and time can be adjusted according to the specific experimental requirements. And cutting off the power supply when the dye reaches the bottom of the gel, stopping electrophoresis, and performing next membrane conversion.

2.6 transfer film

(1) 6 pieces of filter paper having the same size as the transfer area were cut out. Then, they were soaked in an electrotransfer buffer. Cutting a PVDF membrane with the same size, soaking in methanol for 2min to be semitransparent, washing with clear water, placing in electrotransformation buffer solution, and balancing for 10 min.

(2) 3 pieces of filter paper are padded on the PVDF membrane, then the gel is laid on the PVDF membrane, 3 pieces of filter paper are placed on the PVDF membrane, a glass rod is used for rolling on the surface of the filter paper to remove air bubbles on each layer, then the PVDF membrane is placed into a transfer tank, an electrotransfer buffer solution is poured into the transfer tank, the glue faces to a cathode, the membrane faces to an anode, the temperature is 4 ℃, and 380mA transfer is carried out for 1.5 hours.

2.7 blocking of membranes and antibody incubation

(1) And (3) sealing: 5% skim milk powder (BSA for detection of phosphorylated proteins) was blocked at room temperature for 1 hour or overnight at 4 ℃.

(2) A first antibody: the antibodies were diluted according to the instructions, added to the blocking solution to dilute to the desired concentration, and incubated at membrane chamber temperature for 2 hours or at 4 ℃ overnight.

(3) Secondary antibody: the membrane incubated with the primary antibody was washed 3 times with TBST for 5min each. The secondary HRP-labeled antibody was then diluted 1:2000 according to the amount and incubated with the membrane for 1h at 37 ℃. Wash 3 times with TBST for 5min each time.

2.8ECL chemiluminescence detection

Mixing the ECL solution SolA and SolB at a ratio of 1:1 according to kit specification, and spreading on a membrane for reaction for 5 min. (the film is taken out of TBST, a washing solution is absorbed on filter paper, the filter paper is placed on a preservative film, the front side is upward according to marked marks, when an ECL solution reacts for about 4min in the front side, tweezers are used for clamping the film on the water absorption paper to filter the ECL solution, the film is laid on another preservative film, the front side is downward at this time, the preservative film is reversely turned after being wrapped, transparent glue is used for fixing, crease is removed as much as possible, and a developing clamp is immediately closed and placed in a dark room for developing after 5 min). Exposure: 1min, 5min and 45min, then developing, fixing and drying.

2.9 gel image analysis

The film was scanned and the image was grey scale analyzed using the image analysis software IPP 6.0.

3. Results

The results are shown in Table 10 and FIG. 2. And (3) displaying a Western blot detection result: normal plasma had no effect on IGF1 protein expression, no significant difference compared to the blank control group (P > 0.05); PDGF intervention can promote the expression of IGF1 protein, and has significant difference compared with a normal plasma group (P < 0.01); the AG1296 and the drug-containing plasma can obviously reduce the expression of IGF1 protein, and compared with a PDGF group, the difference is significant (P < 0.01); wherein, compared with the group containing the drug plasma, the AG1296 group has no significant difference (P > 0.05).

TABLE 10 Effect of drug-containing plasma on IGF1 protein expression in RPE cells under PDGF intervention

P compared to normal plasma group<0.01; in comparison with the PDGF group,^□□P<0.01。

in the test examples herein, statistical analysis was performed on all data using SPPS17.0 statistical software, with the mean. + -. standard deviation of the measured data

And (4) showing. Carrying out homogeneity of variance test on the samples, when the variances are uniform, carrying out One-Way ANOVA test, and carrying out multiple comparison among groups; when the variance was not uniform, the total difference was compared by Kruskal-Wallis Htest and the comparison between two groups was performed by Mann-Whitney U, using a nonparametric rank sum test. Counting data adopts complete random design of rank sum test of multi-sample comparison, significance is expressed by P<And (5) judging by 0.05.

Finally, the method of the present application is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of the traditional Chinese medicine composition for promoting blood circulation and removing stasis in preparing the medicine for treating or preventing proliferative vitreoretinopathy is characterized in that the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight:

5-10 parts of pseudo-ginseng, 10-30 parts of salvia miltiorrhiza, 15-30 parts of earthworm, 10-30 parts of seaweed, 10-30 parts of kelp and 10-30 parts of turtle shell.

2. The application of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight:

5-9 parts of pseudo-ginseng, 13-20 parts of salvia miltiorrhiza, 15-23 parts of earthworm, 13-19 parts of seaweed, 13-19 parts of kelp and 13-19 parts of turtle shell.

3. The application of claim 2, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight:

6 parts of pseudo-ginseng, 15 parts of salvia miltiorrhiza, 18 parts of earthworm, 15 parts of seaweed, 15 parts of kelp and 15 parts of turtle shell.

4. The use of any one of claims 1-3, wherein the Chinese medicinal composition further comprises a pharmaceutically acceptable carrier and/or excipient.

5. The use of any one of claims 1-4, wherein the composition is in the form of a tablet, capsule, pill, powder, granule, oral liquid, injection, or ointment.

6. The use of claim 5, wherein the Chinese medicinal composition is an oral liquid with a crude drug concentration of 1g/ml to 1.2 g/ml.

7. The use of claim 6, wherein the crude drug concentration is 1.025g/ml to 1.10 g/ml.