CN112641769A

CN112641769A - Application of rhein in preparation of medicine for promoting diabetic ulcer wound healing

Info

Publication number: CN112641769A
Application number: CN202011546372.6A
Authority: CN
Inventors: 李福伦; 邓禹; 徐凝; 郭冬婕; 郭婉军; 迮侃; 华亮; 段彦娟; 王一飞; 周亚琼; 马天
Original assignee: Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of TCM
Current assignee: Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of TCM
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2021-04-13

Abstract

The invention relates to application of rhein in preparing a medicament for promoting diabetic ulcer wound healing. According to the invention, through the detection of a CCK-8 method, the cell proliferation capacity can be improved after rhein intervenes the immortalized keratinocyte (HaCaT cell) of a human body; the influence of the cell cycle on the cell cycle is researched by using APC BrdU flow cytometry, and rhein is found to increase the proportion of cells in the S phase of the cell cycle and promote the DNA replication of the cells; the effect of rhein on promoting the diabetic wound is detected through an in-vivo animal test, and the rhein on promoting the wound healing is found to be fast and remarkable in effect. Therefore, the rhein, the pharmaceutically acceptable salt or ester thereof, the hydrate thereof or the mixture thereof can be used for preparing the medicine for promoting the healing of the diabetic ulcer wound or preparing the experimental reagent for promoting the proliferation of keratinocytes.

Description

Application of rhein in preparation of medicine for promoting diabetic ulcer wound healing

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of rhein in preparation of a medicine for promoting diabetic ulcer wound healing.

Background

Rhein is an anthraquinone compound extracted from root of Rheum palmatum L of Polygonaceae plant, and has a structural formula

The pharmacological action of the compound is very wide, and the compound is proved to have the pharmacological actions of regulating gastrointestinal function, enhancing immunity, resisting pathogenic microorganisms, resisting tumors, resisting renal fibrosis and the like. In addition, patent document CN1440747A, published japanese 2003.09.10, discloses the use of rhein or rhein salts in the preparation of a medicament for the treatment of obesity; patent document CN101327204A, published japanese patent No. 2008.12.24, discloses the use of rhein active substance in the preparation of a composition for the prevention, control or treatment of fatty liver; patent document CN106344549A, published japanese patent No. 2017.01.25, discloses an application of rhein in the preparation of a medicament for preventing and/or treating hand-foot-and-mouth disease; patent document CN109223748A, published japanese 2019.01.18, also discloses the application of rhein in preparing medicine for preventing and treating hepatic encephalopathy.

Clinically, diabetic skin ulcer is caused by slow healing of wound surfaces due to the fact that skin and blood vessels are in a hyperglycemia environment for a long time. The pathological changes such as peripheral vascular lesion, nerve damage, microcirculation disturbance, local infection and the like can be caused by inflammatory medium release and oxidative stress caused by hyperglycemia, the pathological changes become the basis of ulcer and/or tissue deep damage, the repair and healing capacity of wounds is poor, the number of diabetic foot ulcers is rapidly increased, and the amputation rate is high.

At present, no report of rhein on the aspect of promoting the healing of diabetic ulcer wounds is found.

Disclosure of Invention

The invention aims to provide a new application of rhein aiming at the defects in the prior art.

In a first aspect, the invention provides an application of rhein, a pharmaceutically acceptable salt or ester thereof, a hydrate thereof or a mixture thereof in preparing a medicament for promoting the healing of diabetic ulcer wounds.

As a preferred embodiment, the rhein, its pharmaceutically acceptable salt or ester, its hydrate or a mixture thereof is used as the only active ingredient.

As another preferred embodiment, the medicament further comprises other active ingredients for treating diabetic ulcer wound healing.

As another preferred embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

As another preferred embodiment, the medicament is in the form of powder, ointment, cream, paste, gel, film coating agent, liniment, drop, aerosol, wet application agent or patch.

In a second aspect, the present invention provides the use of rhein, a pharmaceutically acceptable salt or ester thereof, a hydrate thereof, or a mixture thereof, for the preparation of an experimental agent for promoting keratinocyte proliferation.

As another preferred embodiment, the test agent further comprises other active ingredients that promote keratinocyte proliferation.

As another preferred embodiment, the keratinocytes are human immortalized keratinocytes.

It should be noted that, in this document, the "test reagent" is different from a drug, and is a reagent used for performing a research test in vitro.

The invention has the advantages that:

1. according to the invention, the CCK-8 method is adopted for detection, and the cell proliferation capacity can be improved after rhein intervenes in HaCaT cells; the influence of APC BrdU flow cytometry on the cell cycle is researched, and rhein is found to increase the proportion of cells in the S phase of the cell cycle and promote the DNA replication of the cells; the effect of rhein on promoting the diabetic wound is detected through an in-vivo animal test, and the rhein on promoting the wound healing effect is obvious. The results suggest that rhein can induce the proliferation of keratinocytes by increasing the proportion of cells in the S phase of the cell cycle, and promote the re-epithelialization process, thereby promoting the healing of the diabetic ulcer wound. Therefore, the rhein, the pharmaceutically acceptable salt or ester thereof, the hydrate thereof or the mixture thereof can be used for preparing the medicine for promoting the healing of the diabetic ulcer wound or preparing the experimental reagent for promoting the proliferation of keratinocytes.

2. The invention proves that rhein promotes the healing speed of the wound surface of the diabetic mouse, has obvious effect and is superior to the basic fibroblast growth factor of the positive medicament.

Drawings

FIG. 1: the effect of rhein on the proliferation capacity of HaCaT cells. P <0.05, P <0.01, P <0.001 compared to DMSO group.

FIG. 2: effect of rhein on HaCaT cell cycle. P <0.05, P <0.01, P <0.001 compared to DMSO group; compared to Rhein 25. mu.M, # P <0.05, # P <0.01, # P < 0.001.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1 Effect of rhein on keratinocyte proliferation

1 materials and methods

1.1 Experimental materials

1.1.1 sources of cells

Human immortalized keratinocytes (HaCaT) were obtained from shanghai ruin hospital.

1.1.2 rhein solution preparation

The rhein (110757-201607) used in the experiment is purchased from China food and drug testing research institute, is a drug identification and content determination reference substance carried in the Chinese pharmacopoeia, and meets the national drug standard. Weighing an appropriate amount, sufficiently dissolving in 100% DMSO by vortex and ultrasonic method to prepare a mother solution with the concentration of 50mM, and storing at-20 ℃ for later use.

1.1.3 Primary reagents

1.1.4 Main Equipment and consumables

1.1.5 solution preparation

1.2 Experimental methods

1.2.1 conventional cell culture and treatment

1.2.1.1 cell recovery

1) The ultra-clean bench operation area is irradiated by ultraviolet for at least 20min before the experiment is started. The cell complete medium was taken out from the 4 ℃ freezer in advance to re-warm at room temperature.

2) Taking out the freezing tube from a refrigerator at minus 80 ℃, wiping the outside of the freezing tube with 70% ethanol, and quickly putting the freezing tube in a cell culture box for rewarming and thawing at 37 ℃. And (3) preparing consumables such as cell culture dishes, centrifuge tubes and pipette guns during cell thawing, and marking the cell culture dishes with information.

3) And after the cell freezing solution is completely thawed, taking out the cell freezing tube from the cell culture box, gently blowing and uniformly mixing the cell freezing solution by using a liquid transfer gun, transferring the cell freezing suspension to a 15mL centrifuge tube, supplementing 2mL of complete culture medium, and centrifuging (800rpm/3 min).

4) The supernatant was pipetted, added 4mL of complete medium and gently pipetted to resuspend the cells, and evenly distributed to 2 new 10cm cell culture dishes, and 8mL of complete medium was added to make up to 10mL of cell culture system (10cm cell culture dishes), respectively.

5) Gently shaking the culture dish to make the cells relatively uniformly distributed, and placing at 37 ℃ with 5% CO₂In a cell culture chamber.

6) Observing the cell growth condition under the microscope every day, changing the cell culture medium every other day, and carrying out subculture in time for subsequent experiments. In order to keep the cell growth state good, the passage operation can be carried out when the cell density reaches about 80%.

7) If the phenol red-free complete culture medium is used for freezing storage, the whole process is replaced by the phenol red-free complete culture medium, and the operation method is the same as the steps 1-6.

1.2.1.2 cell culture with Medium exchange

1) Observing the adherent growth state and density of the cells under a mirror. The ultra-clean bench operation area is irradiated by ultraviolet for at least 20min before the experiment is started. The cell complete medium and sterile PBS solution were removed from the 4 ℃ freezer in advance and allowed to rewet at room temperature.

2) Pipette old medium using pipette gun, gently wash cell surface with sterile PBS solution, pipette wash, repeat 2 times.

3) After 10mL of complete medium was added, the cell culture dish was returned to the incubator.

4) If the phenol red-free complete culture medium is used, the whole process is replaced by the phenol red-free complete culture medium, and the operation method is the same as the steps 1-3.

1.2.1.3 cell subculture

1) Observing the adherent growth state and density of the cells under a mirror, and performing cell digestion and passage operation when the cell density reaches about 80 percent, thereby preventing the conditions of turbid culture medium, cell contact inhibition and the like caused by overhigh density of the cells from influencing the normal growth and proliferation of the cells. The ultra-clean bench operation area is irradiated by ultraviolet for at least 20min before the experiment is started. The cell complete medium, sterile PBS solution and pancreatin were taken out from the 4 ℃ freezer in advance to be rewarmed at room temperature.

2) Pipette the old media using a pipette gun, add sterile PBS solution slowly along the dish wall, gently shake the dish to wash the cell surface, pipette the PBS solution, repeat 2 times.

3) After 1mL of pancreatin was added, the petri dish was gently shaken and left at room temperature for about 5min, and cells were observed under a microscope to become round and fall off in a quicksand shape.

4) Add 2mL of complete medium quickly to the dish and shake gently to cover the cells evenly to stop the trypsinization reaction and prevent over-digestion. And (4) slightly blowing and beating the cells by inclining the culture dish until the bottom of the culture dish is bright, namely the cells are completely separated from the wall.

5) Gently flicked to resuspend the cells, pipetted into a 15mL centrifuge tube and centrifuged (800rpm/3 min).

6) Cell culture dish information labeling was performed during centrifugation and the supernatant was pipetted after centrifugation was complete.

7) Add 2mL of complete medium and gently blow to resuspend the cells, evenly distribute and move to 2 new 10cm cell culture dishes, add 9mL of complete medium to make up to 10mL of cell culture system (10cm cell culture dishes) respectively. The generation ratio is based on specific conditions, and is generally more than 1:2 to 1: 3.

8) If the phenol red-free complete culture medium is used, the whole process is replaced by the phenol red-free complete culture medium, and the operation method is the same as the steps 1-7.

1.2.1.4 cell cryopreservation

1) The adherent growth state and density of the cells are observed under a mirror, the cells are in good growth state, the density is about 80%, and the cell cryopreservation operation is suitable for preventing the unsatisfactory recovery culture condition of the subsequent cells caused by poor cell state, over-low or over-high density and the like. The ultra-clean bench operation area is irradiated by ultraviolet for at least 20min before the experiment is started. The cell complete medium, sterile PBS solution, pancreatin and fetal calf serum were removed from the 4 ℃ freezer in advance and re-warmed at room temperature.

6) During centrifugation, information marking of the cryopreserved tubes is carried out, and after the centrifugation is finished, supernatant liquid is pipetted.

7) Adding the prepared freezing liquid, gently blowing and beating to resuspend cells, evenly distributing the cells to the marked freezing tubes according to 1 mL/tube of the freezing liquid, placing the cells in a gradient cooling box, placing the cells in a refrigerator at minus 80 ℃ for storage, storing the cells overnight, and then transferring the cells to a liquid nitrogen tank for storage for later use.

1.2.1.5 cell Deestrogenic cultures

The adherent growth state and density of the cells are observed under a mirror, and the culture operation of removing estrogen from the cells can be carried out when the growth state of the cells is good or the density reaches about 80 percent. Because the subject research relates to estrogen-like action of estrogen receptors and rhein, a complete culture medium for cell de-estrogen treatment is replaced and used for cell culture 48 hours before the beginning of a formal experiment, and then phenol-red-free pancreatin is used for digestion treatment in the whole process of subculture, namely, the interference of endogenous hormones of cells is avoided while the influence of estrogen or estrogen-like action of serum, phenol red and the like is eliminated, the sensitivity of experimental cells on the estrogen action is enhanced to a certain extent, and the cells grown and subcultured under the de-estrogen culture condition are not more than 4 generations at most so as to guarantee the scientificity and the rigidness of the experiment to the greatest extent.

1.2.2 Effect of rhein on keratinocyte proliferation

1.2.2.1CCK-8 method for detecting influence of rhein on keratinocyte proliferation capacity

1) Planting cells: observing the adherent growth state and density of the HaCaT cells cultured by removing estrogen under a mirror, and taking the cells in logarithmic growth phase. The ultra-clean bench operation area is irradiated by ultraviolet for at least 20min before the experiment is started. The cell complete medium and sterile PBS solution were removed from the 4 ℃ freezer in advance and allowed to rewet at room temperature.

2) Pipet the old media, add sterile PBS solution slowly along the dish wall, shake the dish gently to wash the cell surface, pipet PBS solution, repeat 2 times.

6) After centrifugation, the supernatant was pipetted and gently vortexed to resuspend the cells by adding de-estrogenic complete medium.

7) And (3) completing cell counting in a cell counting instrument by using a counting plate, seeding HaCaT cells into a 96-well plate according to the cell suspension with the density of 5000 cells/well and 100 mu L per well, standing for a while until the cells slowly sink, and placing the 96-well plate into a cell culture box for conventional culture.

8) And (3) medicine adding intervention: after the cells grow completely adherent, the cells are treated by liquid changing and medicine adding treatment by using a completely estrogen-removed culture medium containing rhein, wherein the concentration is set to be 6.25 mu M, 12.5 mu M, 25 mu M, 50 mu M and 100 mu M, each hole is 100 mu L, and each concentration is provided with 3 multiple holes; meanwhile, the medium containing 0.1% DMSO and completely removed with estrogen is used as a control solution for treatment, each well is 100 μ L, 3 multiple wells are arranged, and the wells are returned to the cell culture box for continuous culture.

9) CCK-8 assay: the primary medium was pipetted 24h after the dosing intervention and the medium was changed with 100. mu.L per well in a de-estrogenic complete medium containing 10% CCK-8. Shaking gently in cross direction, mixing, and incubating in incubator for 1h10 min.

10) And (3) measuring the absorbance under the condition of 450nm wavelength of the microplate reader to obtain the Optical Density (OD) value. Cell survival (%) ═ OD value of experimental group/OD value of control group × 100%.

1.2.2.2 Effect of rhein on keratinocyte proliferation and cell cycle

1) Planting cells and medicine adding intervention: observing the adherent growth state and density of the HaCaT cells cultured by removing estrogen under a mirror, and taking the cells in logarithmic growth phase. The ultra-clean bench operation area is irradiated by ultraviolet for at least 20min before the experiment is started. The cell complete medium and sterile PBS solution were removed from the 4 ℃ freezer in advance and allowed to rewet at room temperature.

7) Cell counting was performed using a counting plate in a cytometer with HaCaT cells at a density of 1 × 10⁸Each 6cm culture dish is seeded with 3.5mL of cell suspension per dish into the 6cm culture dish, after the culture dish is slightly stood, the cells slowly sink to the bottom, and the culture dish is placed in a cell culture box for conventional culture.

8) After cells grow completely adherent to the wall, the solution changing and dosing treatment is carried out by using a de-estrogen complete culture medium with rhein final concentration of 25 mu M and 50 mu M, each dish is 3.5mL, each concentration is provided with 3 multiple holes, meanwhile, the de-estrogen complete culture medium containing 0.1% DMSO is used as a control group for changing solution treatment, each dish is 3.5mL, and 3 multiple holes are arranged. And (5) returning the culture medium to the cell culture box for continuous culture.

9) 5-Bromodeoxyuridine (5-bromo-2' -deoxyuridine, BrdU) labeling: and after the medicine is added for intervention for 23h, BrdU labeling is carried out, 35 mu L of BrdU solution is added into each dish, and the operation is carried out in a dark place. And lightly shaking in the cross direction, mixing uniformly, and immediately returning to the cell culture box.

10) After BrdU labeling for 1h, cells are digested and collected and centrifuged, and the method is the same as the steps 2-5.

11) Fixing and membrane rupture: cells were resuspended in 100. mu.L of Cytofix/Cytoperm Buffer per tube and incubated on ice for 15-30 min.

12) The cells were washed with 1mL of 1 XPerm/Wash Buffer, centrifuged (200g/5min) and the supernatant discarded.

13) And (3) strengthening membrane rupture: cells were resuspended in 100. mu.L of Cytoperm Permeabilization Buffer Plus per tube and incubated on ice for 10 min.

14) The cells were washed with 1mL of 1 XPerm/Wash Buffer, centrifuged (200g/5min) and the supernatant discarded.

15) Cell re-fixation and rupture of membranes: cells were resuspended in 100. mu.L of Cytofix/Cytoperm Buffer per tube and incubated on ice for 5 min.

16) The cells were washed with 1mL of 1 XPerm/Wash Buffer, centrifuged (200g/5min) and the supernatant discarded.

17) DNase treatment exposed the BrdU site: resuspend cells (i.e., 30. mu.g DNase/10) with 100. mu.L DNase dilution (diluted to 300. mu.g/ml with Dulbecco's phosphate buffered saline, DPBS) per tube⁶cells), incubated for 1h in an incubator at 37 ℃.

18) The cells were washed with 1mL of 1 XPerm/Wash Buffer, centrifuged (200g/5min) and the supernatant discarded.

19) BrdU fluorescent antibody staining: cells were resuspended in 50. mu.L of Perm/Wash Buffer containing diluted BrdU fluorescent antibody and incubated at room temperature for 20 min.

20) The cells were washed with 1mL of 1 XPerm/Wash Buffer, centrifuged (200g/5min) and the supernatant discarded.

21)7-AAD staining of Total DNA: cells were resuspended in 20. mu.L of 7-Aminoactinomycin D (7-Aminoactinomycin D,7-AAD) solution.

22) Resuspending cells, testing and analyzing on a machine: after resuspending the cells in 1mL stationary buffer, the cells were placed on ice, examined by flow cytometry, and analyzed for cell proliferation using Flowjo software.

1.3 statistical analysis

Experimental data were statistically analyzed using SPSS 24.0 software. Data are measured and normally distributed as mean + -standard deviation

Represents; the data is metering data, if the data obeys normal distribution and has uniform variance, the overall mean value of the comparison among a plurality of groups adopts single-factor variance analysis, and the comparison in the group uses pairing t test; if the normal distribution is not obeyed, nonparametric testing is performed. When P is present<At 0.05, the difference was considered statistically significant.

2 results of the experiment

2.1CCK-8 method for detecting influence of rhein on keratinocyte proliferation ability

The results of CCK-8 detection are shown in figure 1 after intervention of rhein for 24h at 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M and 100. mu.M with HaCaT cells as the study objects. Rhein can improve the proliferation capacity of HaCaT cells under the concentration of 6.25 muM, 12.5 muM, 25 muM and 50 muM, and compared with a DMSO group, the difference has statistical significance (P is less than 0.05).

2.2 Effect of rhein on keratinocyte proliferation and cell cycle

The result of APC BrdU flow cytometry after 25. mu.M and 50. mu.M intervention of rhein for 24h with HaCaT cells as the study object is shown in FIG. 2. The proportion of S-phase cells in the DMSO group is (35.87 +/-1.94)%, the proportion of S-phase cells in the rhein 25 mu M group is (48.87 +/-0.97)%, and the proportion of S-phase cells in the rhein 50 mu M group is (58.90 +/-3.699)%. The rhein 25 mu M and 50 mu M groups can increase the S phase ratio of the cell cycle, and compared with the DMSO group, the difference has statistical significance (P is less than 0.05).

Example 2 Effect of rhein on ulcer wound healing in diabetic mice

1 Material

1.1 instruments

YP-6001N electronic balance (Shanghai precision scientific instruments Co., Ltd.); model P4-2 constant temperature magnetic stirrer (shanghai meipu instruments manufacturing ltd); model RE52CS rotary evaporator (shanghai yangrong biochemical instruments ltd); DHG-9140A type electric heating constant temperature air-blast drying oven (shanghai essence macro experimental facilities limited); model XW-80A vortex mixer (Haiman Linbel instruments, Inc.).

1.2 reagents and drugs

Rhein (110757-.

Bei Fu Xin (recombinant bovine basic fibroblast growth factor gel, Zhuhai Yisheng biopharmaceutical Co., Ltd., batch No. 20160714, specification: 21000 IU (5g) per bottle^-1) (ii) a 95% ethanol (national chemical group, Ltd., batch No. 20131114); carbomer (Guangzhou Kangqiao Hanpu pharmaceuticals, Inc., lot number 20130001); glycerol (Fengyi oleochemical (Shanghai) Co., Ltd., Lot No. 20110010).

1.3 animal and feed

db/db mice are derived from the C57BL/KsJ inbred strain autosomal recessive inheritance, and belong to the type II diabetes mellitus model.

21 SPF-grade mice of 8 weeks (female) db/db were fed with normal feed.

1.4 pharmaceutical preparation

Rhein base: weighing 3mg of rhein, fully dissolving the rhein in 100% DMSO by a vortex and ultrasonic method, mixing with the swelled carbomer, adding a proper amount of triethanolamine to adjust the pH value to 6-8, preparing 12.5g of gel, sticking a label, and storing in a refrigerator at 4 ℃.

② blank matrix: 0.375g carbomer and 0.5g DMSO were weighed, 11.5mL water was added to swell overnight to serve as a blank gel matrix, and the label was placed in a refrigerator at 4 ℃.

2 method

2.1 establishment of diabetic wound model

By self-energizingThe physical contrast method is divided into rhein group, model group and positive drug group according to random numbers. Under aseptic condition, wound surface modeling is performed, after mouse is anesthetized and depilated by a depilatory knife, punches with diameters of 0.6cm are used for manufacturing the area of 0.28cm on two sides of the spine²The depth of the wound surface reaches the position under fascia, the wound is bound, and the skin is raised in a single cage and marked among groups respectively.

2.2 dosing regimens

After the model is established, after the wound surface of a mouse is disinfected in a wound periphery, the rhein group is externally coated with rhein matrix, the positive medicines are all coated with positive medicines, and the model group is treated by blank matrix. Dressing change is performed 1 time a day.

2.3 Observation index

2.3.1 general observations

The wound surface is visually observed for the presence or absence of exudate, infection, wound color, edema condition, granulation tissue growth condition, etc. when changing the dressing every day.

2.3.2 wound area ratio

The wound surface is marked by a ruler and photographed by a camera on days 3, 7, 9 and 11 after the mouse wound surface is molded, the area data is obtained by analyzing with Image analysis software Image J, and the percentage (A/%) of the wound surface area is calculated. A (%) ═ wound area at observation time point/original wound area × 100%.

2.4 statistical methods

Statistics are carried out by SPSS 21.0 software, and data are used

And (4) showing. And (3) performing comparison among groups conforming to normal distribution by adopting One-Way ANOVA analysis, performing pairwise comparison by adopting an LSD test, and taking alpha as 0.05 as a test standard.

3 results

3.1 general observations

The diabetic mouse wound surface is not infected after molding, and edema is generated around the back wound. Research results show that the wound surface of the model group is crusted into yellow brown scabs, bleeding phenomenon exists, healing is slow, secretion is more, the blood scabs cover the wound surface, and granulation regeneration is less; the wound surface of the positive medicine group has a little effusion, is orange red, has granulation tissue generation and is better repaired; the healing speed of the rhein group wound surface is fast, the exudate is less, the wound surface is orange red, and granulation tissues are generated.

3.2 area ratio of wound surface

On the 3 rd day of administration, the wound area of rhein group is significantly smaller than that of the model group and the positive drug group (P <0.05), and the difference between the wound area of the positive drug group and the wound area of the model group has no statistical significance (P > 0.05); on the 7 th day of administration, the wound area of the rhein group is significantly smaller than that of the model group and the positive drug group (P <0.05 or P <0.01), and the difference of the wound area of the positive drug group compared with the model group is not statistically significant (P > 0.05); the wound area of the positive drug group and rhein group was significantly smaller than the model group (P <0.01) and the wound area of rhein group was significantly smaller than the positive drug group (P <0.01) on days 9 and 11 of administration. See table 1.

TABLE 1 area ratio of wound surface at different time points in each group

Note: in comparison with the set of models,^*P<0.05，^**P<0.01; compared with the positive drug group,^#P<0.05，^##P<0.01。

the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. Rhein, pharmaceutically acceptable salt or ester thereof, hydrate thereof or mixture thereof in preparing medicament for promoting healing of diabetic ulcer wound.

2. The use according to claim 1, wherein rhein, its pharmaceutically acceptable salts or esters, its hydrates or mixtures thereof is used as the sole active ingredient.

3. The use according to claim 1, wherein the medicament further comprises other active ingredients for treating diabetic ulcer wound healing.

4. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.

5. The use of claim 1, wherein the medicament is in the form of a powder, ointment, cream, paste, gel, film, liniment, drops, aerosol, wet-dressing or patch.

6. The application of rhein, pharmaceutically acceptable salts or esters thereof, hydrates thereof or mixtures thereof in preparing experimental reagents for promoting keratinocyte proliferation.

7. The use according to claim 6, wherein rhein, its pharmaceutically acceptable salts or esters, its hydrates or mixtures thereof is used as the sole active ingredient.

8. The use according to claim 6, wherein the test agent further comprises an additional active ingredient that promotes keratinocyte proliferation.

9. Use according to claim 6, wherein the keratinocytes are human immortalised keratinocytes.