CN110478430A - Application of the civil and military soup of a thousand pieces of gold in the drug that retinal pigment epithelium ARPE-19 fibrosis is intervened in preparation - Google Patents

Abstract

The present invention relates to application of the civil and military soup of a thousand pieces of gold in the drug that retinal pigment epithelium ARPE-19 fibrosis is intervened in preparation, belong to technical field of traditional Chinese medicines.The present invention provides application of the civil and military soup of a thousand pieces of gold in the drug that retinal pigment epithelium ARPE-19 fibrosis is intervened in preparation.Compared with model group, the civil and military soup of a thousand pieces of gold can raise E-cad protein expression with dosage-dependent manner, lower α-SMA and COL-I protein expression.

Description

Qianjin Wenwu Decoction Intervenes in the Preparation of ARPE-19 Fibrosis in Retinal Pigment Epithelial Cells application in medicine

technical field

The invention relates to the technical field of traditional Chinese medicines, in particular to the application of Qianjin Wenwu Decoction in the preparation of medicines for intervening ARPE-19 fibrosis of retinal pigment epithelial cells.

Background technique

Qian-Jin-Wen-Wu Decoction (QWD) comes from the Korean medical book "The New Edition of the Four Elephants of Eastern Medicine". Composed of flavored medicines, it is mainly used for the treatment of diabetes and kidney disease in Taiyin people, and it has a remarkable curative effect when used clinically for diabetes and its complications. Previous pharmacological studies have shown that it has an inhibitory effect on the expression of retinol-binding protein, and can slow down or inhibit the incidence and course of retinopathy in diabetic kidney disease (DKD) rats, but its specific mechanism remains to be explored.

Contents of the invention

The purpose of the present invention is to provide the application of Qianjin Wenwu Decoction in the preparation of medicine for intervening ARPE-19 fibrosis of retinal pigment epithelial cells.

The invention provides the application of Qianjin Wenwu Decoction in the preparation of a drug for intervening ARPE-19 fibrosis of retinal pigment epithelial cells.

The present invention also provides the application of Qianjin Wenwu Decoction in the preparation of medicine for inhibiting ARPE-19 fibrosis of retinal pigment epithelial cells.

The present invention also provides the application of Qianjin Wenwu Decoction in the preparation of medicine for inhibiting ARPE-19 fibrosis of retinal pigment epithelial cells induced by high glucose.

Preferably, the raw materials for the preparation of Qianjin Wenwu Decoction consist of kudzu root, scutellaria baicalensis, ligusticum edulis, Chinese yam, schisandra chinensis, bellflower, cohosh, angelica dahurica and Radix Ophiopogon japonicus.

Preferably, the raw materials for the preparation of Qianjin Wenwu Decoction are composed of 10 g of kudzu root, 10 g of scutellaria baicalensis, 10 g of Ligusticum edulis, 10 g of Chinese yam, 5 g of schisandra chinensis, 5 g of bellflower, 5 g of Cimicifuga, 5 g of Angelica dahurica and 5 g of Ophiopogon japonicus.

The invention provides the application of Qianjin Wenwu Decoction in the preparation of a drug for intervening ARPE-19 fibrosis of retinal pigment epithelial cells. The Korean medical prescription "Qianjin Wenwu Decoction" of the present invention can interfere with the fibrosis process of ARPE-19 induced by high glucose. The test results showed that after high glucose induction, compared with the normal group, the cells in the model group were significantly longer and spindle-shaped, and the expression of E-cad protein was down-regulated, and the expression of α-SMA and COL-I proteins were up-regulated; administration (Qianjin Wenwu Decoction) Afterwards, compared with the model group, the protein expression of E-cad was up-regulated in a dose-dependent manner, and the protein expression of α-SMA and COL-I was down-regulated.

Description of drawings

Fig. 1 is the cell morphology observation result figure of different concentrations of glucose serum-free medium provided by the present invention;

Fig. 2 is the MTT experiment provided by the present invention to detect Qianjin Wenwu decoction to ARPE-19 cytotoxic effect result figure;

Fig. 3 is the western blot of E-cad, α-SMA and COL-I protein expression in ARPE-19 cells provided by the present invention after being given drugs;

Fig. 4 is a histogram of protein expression of E-cad, α-SMA and COL-I in ARPE-19 cells provided by the present invention after being given drugs.

Detailed ways

The invention provides the application of Qianjin Wenwu Decoction in the preparation of a drug for intervening ARPE-19 fibrosis of retinal pigment epithelial cells. The Qianjin Wenwu Decoction of the present invention is "Qianjin Wenwu Decoction" of the Korean medical prescription. In the present invention, the raw materials for the preparation of the Qianjin Wenwu Decoction are Pueraria lobata, Scutellaria baicalensis, Ligusticum edulis, Chinese yam, Schisandra chinensis, platycodon grandiflorum, Cimicifuga, Angelica dahurica and More preferably, the raw materials for the preparation of Qianjin Wenwu Decoction are composed of Pueraria 10g, Scutellaria baicalensis 10g, Ligusticum edodes 10g, Chinese yam 10g, Schisandra 5g, Platycodon grandiflorum 5g, Cimicifuga 5g, Angelica dahurica 5g and Ophiopogon japonicus 5g. The present invention has no special limitation on the source of Qianjin Wenwu Decoction. Specifically, the preparation of Qianjin Wenwu Decoction of the present invention preferably includes: extracting and mixing the composition of raw materials twice with 10 times and 8 times of 30% ethanol respectively, then concentrating to a solid-liquid ratio of 1:1; g.mL ^-1 is the concentration of the sample, and it is adsorbed by macroporous adsorbent resin (MAR); finally, the purified medicinal solution is concentrated again to a solid-liquid ratio of 1:1, and freeze-dried to obtain Qianjin Wenwu Decoction.

The present invention also provides the application of Qianjin Wenwu Decoction in the preparation of medicine for inhibiting ARPE-19 fibrosis of retinal pigment epithelial cells.

The present invention also provides the application of Qianjin Wenwu Decoction in the preparation of medicine for inhibiting ARPE-19 fibrosis of retinal pigment epithelial cells induced by high glucose. After high glucose induction, compared with the normal group, the cells in the model group were significantly longer and spindle-shaped, and the expression of E-cad protein was down-regulated, and the expression of α-SMA and COL-I proteins were up-regulated; after administration, compared with the model group, the dose E-cad protein expression was up-regulated, and α-SMA and COL-I protein expression were down-regulated in a dependent manner.

The application of Qianjin Wenwu Decoction of the present invention in the preparation of drugs for intervening ARPE-19 fibrosis of retinal pigment epithelial cells will be further described in detail below in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

Example 1

Materials and instruments are shown in Table 1 and Table 2.

Table 1 Medicinal materials and reagents

Table 2 Main instruments and equipment

cell culture

1. Configuration of culture medium

Fetal bovine serum was taken and inactivated at 55°C. Heat DMEM in a 37°C water bath. Prepare DMEM medium containing 10% FBS.

2. Cell Recovery

Heat the prepared medium in a 37°C water bath, take out the human retinal pigment epithelial ARPE-19 cells from the -80°C refrigerator, and place them in a 37°C water bath to melt for use. In an ultra-clean bench with good ventilation and lighting equipment, wipe the test bench with 75% alcohol, light the alcohol lamp, sterilize the bottle mouth of the medium, use a pipette gun to suck out 5ml of the medium, and place it into a 15ml test tube , use a pipette gun to transfer the frozen cell liquid to a test tube and centrifuge for 3 minutes (1000r·min ^-1 ). Use an aspirator to suck out the centrifuged supernatant, take an appropriate amount of medium and blow the cells until they are evenly distributed, and finally transfer to the culture medium. Into the dish, add 10ml medium. Put the culture dish into a 37°C, 5% CO ₂ incubator and incubate for 48h.

3. Cell passage

First take the DMEM medium and trypsin out of the refrigerator, and preheat to 37°C in a water bath. Take out the culture dish that has been overgrown with ARPE-19 cells from the incubator, and use a microscope to observe whether the number of cells and the cell shape meet the requirement of covering 80% of the bottom of the culture dish. Transfer the petri dish that meets the requirements to a well-ventilated and well-lit ultra-clean bench that has been wiped by 75%, use an aspirator to suck away the medium in the upper layer of the petri dish, and then rinse the petri dish with the medium to wash away floating dead cells. Take 3ml of trypsin in a petri dish, shake evenly up and down, left and right for about 1 min to fully contact the cells with trypsin, place the petri dish in an incubator for incubation, and take it out after timing for 3 min. Remove the Petri dish from the incubator and add culture medium. Take out two brand new media, mark them as ARPE-19 cells with a marker pen, record the date of experiment and the name of the experimenter, and transfer an appropriate amount of pre-prepared media. Take out the culture dish in the incubator, tap it to make the cells slide off, observe whether the cells are flowing under the light, use an optical microscope to observe the dispersion of the cells, and then transfer to the test tube. Put the test tube containing the cells into a high-speed centrifuge for 3 minutes (1000r·min ^-1 ), and take it out after the time is up. Use an aspirator to suck away the supernatant, add the medium with a pipette gun, and blow up the cells at the bottom, up and down thirty times. Transfer the complete cells after pipetting to a new Petri dish, then perform cross-shaking, and place them in an incubator for cultivation. Incubate for 48 hours at 37°C, 5% CO ₂ .

4. Cell cryopreservation

Freeze the cells in the dish when they are about 80% full. Take out the DMEM medium and trypsin from the refrigerator, and arrange to preheat it in a 37°C water bath in advance. Put on new disposable sterile gloves, click the button to turn off the ultraviolet lamp in the ultra-clean bench, turn on the exhaust system and fluorescent lamp, turn on the alcohol lamp, and wipe the ultra-clean bench with 75% alcohol. Sterilize the DMEM medium and trypsin at about 37°C, transfer to a new 15ml tube and sterilize the same. Wash off dead cells by adding new medium with a pipette, then aspirate. Then transfer 2ml of trypsin with a pipette gun, and shake about 20 times in a cross shape. Put the petri dish in the incubator for 3min to digest, then take out and beat the cells, transfer into 5-6ml DMEM medium, blow and beat the cells until uniform, centrifuge at 1000r·min ^-1 for 3min with a centrifuge. Take out the cell freezing tube, and mark the cell type (ARPE-19 cells), freezing date and other information. Take out the cryogen and sterilize the top of the bottle with an alcohol burner. Take out the centrifuged cells, pump out the supernatant, transfer 1.5ml of freezing liquid, pipette until the cells are evenly dispersed, transfer the cells into the cryopreservation tube, tighten the cap of the cryopreservation tube, wrap the sterilized cryopreservation tube with parafilm, Put it in a refrigerator at -80°C, wait for 48 hours, and move it into a liquid nitrogen tank.

Toxicity of Qianjin Wenwu Decoction to ARPE-19 cells detected by MTT assay

MTT method was used to measure the toxicity of each drug concentration to cells, so as to determine the optimal drug concentration.

1. Seed board

Heat the DMEM medium and trypsin in a water bath at 37°C. Turn off the ultraviolet lamp of the ultra-clean table, turn on the lighting and ventilation, put on disposable gloves, spray 75% alcohol for disinfection, and continue to spray 75% alcohol on the surface of the ultra-clean table for wiping and disinfection. Take out the culture medium full of ARPE-19 cells in the incubator, put it under a microscope to observe the growth of ARPE-19, and take the grown ARPE-19 cells to the ultra-clean bench. Light the alcohol lamp, and disinfect the mouth of the medium and trypsin bottles. Wash the dead cells with PBS first, and suck up the PBS with an aspirator. Add 3ml of trypsin to the Petri dish, shake it crosswise for 30 times, and put it in the incubator for 3 minutes. Take out the petri dish, add DMEM medium to stop the digestion, and transfer the cells to the test tube. Centrifuge in an ultra-high-speed centrifuge for 3 minutes (1000r·min ^-1 ), take out the test tube, suck off the supernatant, add medium to blow off the cells at the bottom, take 15 μl of the liquid into a counting plate, and count under a microscope, according to the settings in advance The seed board density begins to dilute. The diluted cell suspension was poured into a 96-well plate with a volume of 200 μl per well. Put the 96-well plate into the incubator for 18h.

2. To investigate the effect of a certain concentration of drugs on ARPE-19 cells

Add the extract of Qianjin Wenwu Decoction to the 96-well plate, add 100 μl of drug to each well, (each group is given sequentially 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 500 μg, 1000 μg, 2000 μg) into the incubator for 18 hours. After 18 hours, 20 μl of MTT reagent (5 mg·ml ^-1 ) was added and stored in the dark. Active cells can reduce MTT to water-insoluble blue crystalline formazan, and then put it into the incubator for further incubation for 4 hours. After 4 hours, add 100 μl of dissolving solution to dissolve the formazan crystals, put them in the incubator for 4 hours, and wrap them with tin foil.

3 data detection

Turn on the spectrophotometer and set the parameters to detect the absorbance at 570nm.

When APRE-19 cells were given different concentrations of Qianjin Wenwu Decoction, the results are shown in Figure 2. The cell survival rate in the 500 μg group was 84.76%, indicating that there was no obvious toxicity when administered with 500 μg (concentration: 5 μg·μl ^-1 ). And the cells grow well.

cell modeling

Heat the DMEM medium and trypsin in a water bath at 37°C. Digest ARPE-19 cells with trypsin, put them in the incubator for 3 minutes, take them out to observe the digestion status of the cells after the time is up, add 3ml medium, blow and beat the cells that have not been digested, and remove the ARPE-19 cells that have stopped digesting in the culture dish Add the suspension into a test tube, put the test tube into a centrifuge, and set it at 1000r·min ^-1 for 3 minutes. After the time is up, take out the test tube, suck away the supernatant, add 3ml medium to blow up the bottom details, and then plant the cells into a 6-well plate, and then put them in a 37°C, 5% CO ₂ incubator for culture ; Serum-free medium containing different concentrations of glucose was added to each culture dish (the final concentration of glucose was 25 μmol L ^-1 , 5 μmol/L ^-1 , 30 μmol ^/ L ^-1 , 40 μmol L ^-1 , 50 μmol L -1 ¹ ) Treat for 72 hours. Cell morphology was observed under a microscope, and the optimal dosage concentration was obtained through modeling experiments for drug treatment of cells.

APRE-19 cells were given 5 μmol·L ^-1 and 50 μmol·L ^-1 glucose serum-free medium, respectively. Compared with the 5 μmol L ^-1 group, the cells in the 50 μmol L ^-1 group showed a significant spindle shape, the intercellular space became larger, and the degree of fusion decreased, indicating that the ARPE-19 cells had better model indicators when the glucose concentration was 50 μmol L ^-1 . Obviously, 50 μmol·L ^-1 was determined as the glucose concentration for modeling. The results of cell modeling observed under an inverted phase-contrast microscope are shown in Figure 1, in which, A: 5 μmol·L ^-1 glucose was administered; B: 50 μmol·L ^-1 glucose was administered.

Cell grouping and dosing

The cells were transferred to a 6-well plate, and after 18 hours of growth, the growth of the cells was observed. Serum-free medium with glucose concentration determined in cell modeling was added to each well for 72 hours. The cells were divided into blank group, model group, low-dose group, middle ^- dose group and high-dose group. The blank group and the model group were not given drugs. The concentration was 2μg·μl ^-1 , and the administration concentration of the high dose group was 5μg·μl ^-1 . After administration, culture and observe for 72 hours. Aspirate the drug in each well, then give 130 μl of lysate to each well, and freeze overnight.

Western blotting

1. Extract protein

Prepare the cell scraper, PBS buffer, scrape the six-well plate vigorously with the cell scraper, scrape off the protein sticking to the six-well plate, and rinse the cell scraper with PBS buffer after each scraping group. Transfer the protein suspension in the six-well plate to a 2ml tube with a pipette gun. Put the tube into a refrigerated centrifuge and centrifuge at 4°C for 10 min at a speed of 13,000. Transfer the centrifuged supernatant to a 1.5ml tube with a pipette gun, and store the tube at -20°C. 13000r·min ^-1 . Collect the supernatant and store it at -20°C.

2. Glue making

Select a 1cm glass plate for glue mixing, clean it, clamp it with clips, and check for leaks with three-distilled water; take out AA and APS from the refrigerator, and prepare 8% and 12% separating gel and stacking gel according to the glue mixing table ( Do not add TEMED first), shake and mix well, make sure that the glass plate does not leak, pour out triple distilled water and dry the water with paper, add TEMED to the separating gel and shake and mix well to keep it fully covered, on the premise of ensuring the height of the separating gel Next, use a 1ml pipette gun to inject into the glass plate, leaving the space required for the stacking gel, then fill it with methanol, shake it left and right to make the gel surface smooth, and let it stand. After the separation gel is solidified (about 1 hour), pour out the methanol, wash with triple distilled water three times, blot dry with paper, and test the comb to ensure that the comb adheres to the glass plate. Add the concentrated gel to TEMED and oscillate to mix well, quickly pour into the glass plate, and then quickly insert the comb to avoid the generation of air bubbles; wait until the concentrated gel is completely solidified, pull out the comb, and clean the sample well to ensure that there is no concentration in the sample well Glue, wipe off the water and set aside. If the prepared plastic sheet is not used immediately, it can be wrapped in tin foil or plastic wrap and placed in a 4°C refrigerator for refrigeration.

3. Electrophoresis

Clamp the gel plate with clips and put it into the electrophoresis tank with the side of the small glass plate facing inward; add new 1×running buffer between the two plates, cover the inner small glass plate, and add recovered running buffer around it; pipette with 10μl Add 1 μl mark to the gun first, and then add samples in sequence; electrophoresis, run the gel at a constant current (30mA), and turn off the power when the protein sample reaches the bottom 1cm of the gel (about 1h).

4. Transfer printing

After electrophoresis, prepare two PVDF membranes and soak them in appropriate amount of methanol; pour transbuffer into a suitable container, put in clips, cotton for transfer, and PVDF membrane in turn. Pry open the rubber plate, scrape off the concentrated gel, scratch the surrounding of the separation gel, quickly put the glue on the PVDF membrane, cover with cotton, and clip the clip; put the clip into the transfer tank, pour the conversion buffer, and transfer Place jars in ice. Start transfer printing, transfer with constant voltage 65V for 1h, and transfer with constant voltage 35V for 1h.

5. Wash the membrane and paste the antibody

Measure 100ml of 0.05% PBS-T20, then weigh 5g of membrane washing milk and dissolve it in it, mix evenly, put the transferred PVDF membrane into milk to seal for 1 hour; then take out the PVDF membrane and wash it with 0.05% PBS-T20 Four times, followed by 15min, 5min, 5min, 5min; incubate the corresponding primary antibody (see the instructions for the ratio of the antibody, diluted with PBS-T20 buffer containing 5% w·v ^-1 skimmed milk powder or BSA), shake at room temperature Shake for 4 hours or incubate at 4°C for 12 hours; after the primary antibody is attached, wash with 0.05% PBS-T20 for 15 minutes, 5 minutes, 5 minutes, and 5 minutes; %w·v ^-1 skimmed milk powder in PBS-T20 buffer), incubated on a shaker at room temperature for 1h; after the secondary antibody was shaken, the PVDF membrane was removed and washed with 0.05% PBS-T20 for 15min, 15min, and 15min in sequence.

6. Imaging

Put the washed transfer film in a dark room, take 1ml of liquid A and liquid B, and mix evenly with a pipette; prepare developer and water, turn on the infrared light and turn off the fluorescent light, pay attention not to expose, cut the film; use tweezers After taking out the membrane, use paper to absorb the mixed liquid on the membrane, put the membrane in the mixture, and rinse it with a pipette gun; put the rinsed membrane into the plastic film in the splint, use paper to remove air bubbles and fix it, and observe whether There is fluorescence; if the fluorescence is obvious, the cut membrane is placed on the film, and after closing the splint for a while, take out the film immediately (be careful not to misplace it, so as to prevent the blurred bands; if the fluorescence is not obvious or no fluorescence is observed , then close the splint and place it in a dark place for a period of time before performing subsequent operations (close the clamp to prevent the film from drying); put the film in the developer from the bottom and soak it completely, when you see clearer bands When there are spots, you can take out the film and put it in water for cleaning, and then put it into the fixer for fixing after cleaning, as shown in Figure 3.

The results of the expression of E-cad, α-SMA and COL-I proteins in ARPE-19 cells after being given drugs are shown in Figure 4 (###: P<0.001vs blank group, ***: P<0.001**: P<0.01vs model group), as can be seen from Figure 3 and Figure 4, compared with the blank group, the expression level of E-cad in the model group was significantly reduced, and the expression levels of COL-I and α-SMA were significantly increased, indicating that Model building. After administration of various concentrations of the drug, the drug down-regulated the expression of α-SMA and COL-I, and up-regulated the expression of E-cad in a dose-dependent manner. These results indicated that Qianjin Wenwu Decoction could interfere with the fibrosis process of ARPE-19 cells induced by high glucose.

The experimental results of the present invention show that after induction by high glucose, compared with the normal group, the cells in the model group are obviously elongated and spindle-shaped, and the expression of E-cad protein is down-regulated, and the expression of α-SMA and COL-I protein is up-regulated; Compared with the model group, the expression of E-cad protein was up-regulated in a dose-dependent manner, and the protein expression of α-SMA and COL-I was down-regulated.

The Korean medical prescription "Qianjin Wenwu Decoction" can interfere with the fibrosis process of ARPE-19 in retinal pigment epithelial cells induced by high glucose.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (5)

1. application of the civil and military soup of a thousand pieces of gold in the drug that retinal pigment epithelium ARPE-19 fibrosis is intervened in preparation.

2. application of the civil and military soup of a thousand pieces of gold in the drug that preparation inhibits retinal pigment epithelium ARPE-19 fibrosis.

3. the civil and military soup of a thousand pieces of gold answering in the drug that preparation inhibits high glucose induction retinal pigment epithelium ARPE-19 fibrosis With.

4. described in any item applications according to claim 1~3, which is characterized in that the standby raw material processed of the civil and military soup of a thousand pieces of gold It is made of pueraria lobata, radix scutellariae, rhizoma ligustici, Chinese yam, Schisandra chinensis, campanulaceae, cimicifugae foetidae, the root of Dahurain angelica and Radix Ophiopogonis.

5. application according to claim 4, which is characterized in that the standby raw material processed of the civil and military soup of a thousand pieces of gold by pueraria lobata 10g, Radix scutellariae 10g, rhizoma ligustici 10g, Chinese yam 10g, Schisandra chinensis 5g, campanulaceae 5g, cimicifugae foetidae 5g, root of Dahurain angelica 5g and Radix Ophiopogonis 5g composition.