CN112618536B - Application of taxifolin in treating or preventing fat liquification necrosis after fat transplantation - Google Patents

Abstract

The invention discloses application of taxifolin or pharmaceutically acceptable salt thereof or a composition containing the taxifolin or the pharmaceutically acceptable salt thereof in treating or preventing infection, fat liquefaction or fat necrosis after a fat transplantation operation of an individual.

Description

Application of taxifolin in treating or preventing fat liquification necrosis after fat transplantation

Technical Field

The present invention relates generally to the use of pharmaceutical compositions comprising taxifolin for the treatment and prevention of post-fat transplantation infections, fat liquefaction and fat necrosis.

Background

Fat transplantation (lipopilling) is a transplantation of autologous fat of a patient harvested by liposuction, and fat in a part having a high fat content such as a thigh is extracted by injecting a large amount of physiological saline under aseptic conditions and refilled in a desired part. Patients with congenital defects, trauma or surgical resection, including patients with tertiary burns and women who receive lumpectomy after treatment for breast cancer, which is the most prevalent cancer among women worldwide, may benefit from autologous fat transplantation.

In modern medical care, autologous fat transplantation is increasingly applied, people pay more attention to medicaments for assisting the regeneration and remodeling of fat tissues, French ACMETEA human peptide collagen is sold as a nutritional dietary supplement in the medical and American industry for assisting fat transplantation, but no medicament for promoting the survival of fat transplantation is sold in the market. According to the existing research reports, the traditional Chinese medicine salvia miltiorrhiza bunge injection and the western medicine tamoxifen are proved to have the effect of improving the survival rate of fat transplantation through animal experiments.

Although the salvia miltiorrhiza bunge injection has the effect of improving the survival rate of fat transplantation, the components of the salvia miltiorrhiza bunge injection are complex, the sensitization rate is high, and researchers do not clearly determine that the application is limited due to specific effective chemical components. In addition, although the research shows that tamoxifen has certain efficacy of promoting fat survival, the tamoxifen is a chemotherapeutic drug for breast cancer, has strong cytotoxicity and is limited in application to patients. Therefore, the development of new therapeutic and prophylactic drugs is clinically important.

Disclosure of Invention

In view of the defects and technical problems in the prior art, the invention provides the application of taxifolin or pharmaceutically acceptable salt thereof or a composition containing the taxifolin or the pharmaceutically acceptable salt thereof in treating or preventing infection, fat liquefaction or fat necrosis after fat transplantation operation of an individual.

In order to solve the technical problems, the invention adopts the following technical scheme: use of taxifolin or a pharmaceutically acceptable salt thereof or a composition comprising the taxifolin or the pharmaceutically acceptable salt thereof for treating or preventing infection, fat liquefaction or fat necrosis after a fat transplantation operation in a subject.

Preferably, the pharmaceutically acceptable dosage form is oral liquid, injection, capsule, tablet or granule.

Preferably, the medicament is administered orally, intraperitoneally, subcutaneously or intramuscularly.

Preferably, the subject is a mammal, including non-primates and primates.

Preferably, the administration dosage of the taxifolin or the pharmaceutically acceptable salt thereof is 100-300 mg/Kg of body weight per day by intraperitoneal injection.

Preferably, the administration dosage of the taxifolin or the pharmaceutically acceptable salt thereof is 30mg/Kg body weight per day by intraperitoneal injection.

The invention discloses the application value of taxifolin in adjuvant therapy in fat transplantation, and discovers that the taxifolin can improve the survival rate of fat after transplantation. The invention discovers that the size of the fat cells of the nude mice which are administrated by the taxifolin is smaller than that of the fat cells of a control group, the density of the microvessels is higher than that of the fat cells of the control group, and the nuclear proliferation condition is more obvious than that of the fat cells of the control group. In addition, the invention discovers that the taxifolin can promote the generation of collagen, and the phenomenon explains the increase of fat survival rate after transplantation on one hand and suggests the application value of the taxifolin in the aspects of beauty, health care and skin care on the other hand.

Drawings

FIG. 1 is a 21-day fat visualization chart of a taxifolin injection group and a blank control group after a nude mouse is subjected to human fat transplantation in the embodiment of the invention.

FIG. 2a is a graph showing the body weight of nude mice injected with DHQ group, SSI group and blank control group after human fat transplantation in the example of the present invention for 21 days;

fig. 2b is a statistical chart of fat volumes of nude mice after 21 days of transplantation, which are injected with DHQ group, SSI group and blank control group after the nude mice are subjected to human fat transplantation in the embodiment of the present invention.

FIG. 3a is a comparison graph of HE staining of adipose tissues of a DHQ-injected group, an SSI-injected group and a blank control group after transplantation of human-derived fat in nude mice according to an embodiment of the present invention;

FIG. 3b is a statistical chart of cell numbers of fat tissue HE stained after injecting DHQ group, SSI group and blank control group after transplantation of human fat in nude mice according to the embodiment of the present invention.

FIG. 4a is a PCNA immunohistochemical staining comparison graph of an injected DHQ group, an SSI group and a blank control group after a nude mouse is subjected to human fat transplantation in an embodiment of the present invention;

FIG. 4b is a statistical chart of the number of PCNA positive cells in the DHQ-injected group, SSI-injected group and blank control group after the transplantation of human-derived fat in nude mice according to the present invention.

FIG. 5a is a graph comparing the CD31 immunohistochemical staining of mice injected with DHQ, SSI and placebo after transplantation of human fat in nude mice according to the present invention;

fig. 5b is a statistical chart of the number of CD31 positive blood vessels in the DHQ, SSI and placebo groups injected after transplantation of human fat in nude mice according to the present invention.

FIG. 6a is a graph showing the comparison of oil red O staining of an injected DHQ group, an SSI group and a blank control group after a nude mouse is subjected to human fat transplantation in accordance with an embodiment of the present invention;

fig. 6b is a statistical chart of oil red O staining regions of nude mice after injection of oil red O staining of DHQ group, SSI group and blank control group after human fat transplantation in the embodiment of the present invention.

FIG. 7a is a graph comparing sirius red staining of mice injected with DHQ, SSI and blank control groups after transplantation of human fat in nude mice according to the present invention;

FIG. 7b is a statistical chart of sirius red staining areas of mice injected with DHQ, SSI and blank control groups after transplantation of human fat in nude mice according to the present invention.

Detailed Description

The present invention is further illustrated by the following figures and specific examples, which are to be understood as illustrative only and not as limiting the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalent modifications thereof which may occur to those skilled in the art upon reading the present specification.

Unless otherwise indicated, all terms used in this application have the meanings commonly understood by those skilled in the art. The composition containing the taxifolin or the pharmaceutically acceptable salt thereof is prepared into pharmaceutically acceptable dosage forms including injections, oral liquid, capsules, tablets or granules together with pharmaceutically acceptable carriers.

The "pharmaceutically acceptable carrier" refers to a carrier that does not interfere with the effectiveness of the biological activity of the active ingredient, and may be a solid or a liquid, and includes pharmaceutically acceptable excipients, buffers, emulsifiers, stabilizers, preservatives, diluents, encapsulating agents, and the like. For example, the buffer is phosphate, acetate, borate, carbonate, or the like. The pharmaceutical compositions of the present application can be prepared by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, packaging, entrapping or lyophilizing processes.

Taxifolin dihydrate, also known as Taxifolin, is referred to as DHQ for short, has a molecular weight of 304.25, is easily dissolved in hot water, and can be prepared into 10mM/L DHQ by using deionized water.

The preparation of the taxifolin group medicament (DHQ group) of the application comprises the following steps: weighing 3.14mgTAX, adding 10ml deionized water to prepare a liquid medicine with the concentration of 10mmol/L, heating the liquid in 70 ℃ water bath to fully dissolve, uniformly mixing, transferring to a new EP tube, subpackaging into 1ml/EP tube, and storing in a refrigerator at-20 ℃ for later use.

Preparing SSI group in proportion: taking tanshinone IIA sodium sulfonate injection, diluting to 2mg/ml, and storing in a refrigerator at-20 ℃ for later use.

Blank control (Ctrl group): physiological saline.

Pharmacological test methods and test results of examples of the present application

Transplanting human-derived fat to nude mouse

Evenly dividing 30 nude mice into 6 groups, namely a Control group, transplanting 0.5ml of human adipose tissue on the back of the nude mice, and injecting 0.2ml of normal saline into the abdominal cavity; in the DHQ group, 0.5ml of human adipose tissue is transplanted on the back of a nude mouse, and 0.2ml of taxifolin is injected into the abdominal cavity; in SSI group, 0.5ml of human adipose tissue and 0.2ml of tanshinone IIA sodium sulfonate were transplanted to the back of nude mice, and administration was continued for 21 days. The adipose tissues were then taken out for imaging and the volume of fat was observed.

As shown in figure 1, the DHQ group fat is increased to different degrees through the taxifolin group medicament, and the phenomena of fat liquefaction and necrosis are not generated.

As shown in FIGS. 2a and 2b, the human adipose tissues were transplanted to the back of the nude mice, and the administration was carried out for 21 days, during which there was no significant difference in the body weight of the nude mice, and the volume of the transplanted adipose mass in DHQ group was significantly larger than that in the control group (p < 0.05).

② HE dyeing

Sequentially immersing the baked tissue slices into xylene I, dewaxing the xylene II for 10min respectively, sequentially immersing the dewaxed tissue slices into absolute ethyl alcohol I, absolute ethyl alcohol II, 95% alcohol, 80% alcohol and 70% alcohol for 2min respectively, and washing with PBS for 2 times, 5min each time. Staining with hematoxylin for 3min, washing with tap water for 3min, separating with 1% hydrochloric acid alcohol for 2s, washing with tap water for 2min, sequentially soaking in 50%, 70%, and 80% alcohol for 2min, respectively, soaking in eosin for 5s, and washing with tap water for 3 min. Then sequentially immersing the tissue slices in 95% alcohol, absolute ethyl alcohol I and absolute ethyl alcohol II for 3min respectively, and finally sequentially immersing xylene I and xylene II for 5min respectively. And sealing the neutral gum into pieces, and performing microscopic examination. Photographs were taken under 200X, 400X glasses, respectively.

As shown in fig. 3a and 3 b: HE staining results showed that the number of fat per unit area was greater in the DHQ group than in the control group (p < 0.05).

③ immunohistochemical staining

The sample wax block was mounted on a microtome, cut into 5 μm slices, and the slices were then spread in a warm water dish. The developed tissue section is transferred to a glass slide, placed in an incubator at 60 ℃ for 2h, and dried. The slice racks were immersed in grade I and grade II xylenes for 15min each. The slices were taken out and immersed in 95%, 85%, 75% ethanol for 1min each in turn. The sections were removed and soaked in PBS for 5min × 3 times. Taking out the slices, placing in antigen repairing solution, and repairing at high temperature and low fire for 10 min. The sections were removed and soaked in PBS for 5min × 3 times. Each section was wiped dry with absorbent paper and an immunohistochemical pen was used to draw a circle around the tissue to prevent spillage when liquid was added. Dropping goat serum until the tissue is completely covered, and heating the inner chamber of the wet box for 15 min. Primary antibody was diluted 1:200 with PBS, added dropwise to completely cover the tissue, and wet-boxed overnight at 4 ℃. The slices were taken out, placed on a slicing rack, and immersed in PBS for 5min × 3 times. The fluorescent secondary antibody was added dropwise, diluted with PBS1:200 (care was taken to avoid light hereinafter), added dropwise until the tissue was completely covered, and incubated at room temperature for 60 min. The slices were taken out, placed on a slicing rack, and immersed in PBS for 5min × 3 times. Peripheral fluid was wiped dry and DAPI was added dropwise until complete coverage of the tissue to counter stain the nuclei. Placing on a slicing rack, and soaking in PBS for 5min × 3 times. And taking out the slices in sequence, wiping the surrounding liquid, dripping an anti-fluorescence quenching agent, and sealing the slices by a cover glass. The staining effect was observed under a fluorescent microscope. Photographs were taken under 200X, 400X glasses, respectively.

As shown in fig. 4a and 4b, and 5a and 5 b: PCNA immunohistochemical results show that the positive area of PCNA in the DHQ group is larger than that of the control group, and the number of cells with stronger nuclear proliferation capacity in unit area of the DHQ group is larger than that of the control group (p is less than 0.05); the CD31 immunohistochemistry results showed that the microvascular density in the DHQ group was greater than that in the control group (p < 0.05).

Dye with oil red O

Drying the frozen section (thickness of 4-8um) at room temperature for 15-20 min. Incubate with 100% isopropanol for 5 minutes (avoid bringing water into oil red O). 0.5% oil red O solution was incubated for 7-8 minutes (in an oven at 60 ℃) and washed with 85% isopropanol solution for 3 minutes, double dehydrated. Hematoxylin staining for 1-1.5 min. And (5) double dehydration and washing. The sealing agent seals the sheet. As a result, the lipid was red or the orange nucleus was light blue.

As shown in fig. 6a and 6 b: the results of oil red O staining showed that the area of oil red O staining was larger (p <0.05) per unit area in the DHQ group than in the control group, suggesting that the number of adipocytes per unit area in the DHQ group was larger than in the control group.

Dyeing with sirius red

Tissues were fixed in 10% formalin and routinely dehydrated for embedding. The slices were 6 μm thick and were dewaxed conventionally to water. The sirius red staining solution was stained for 1 hour. And (5) flushing with running water. And (4) conventional dehydration, transparency and neutral gum sealing. And (3) dyeing results: the collagen fiber I is orange yellow or red and the collagen fiber III is green when observed by a polarized light microscope.

As shown in fig. 7a and 7 b: the sirius red staining result shows that the sirius red staining area under the unit area of the DHQ group is larger than that of the control group (p is less than 0.05), and the DHQ is suggested to promote the generation of connective tissues among adipose tissues and protect the adipose tissues by promoting the generation of collagen fibers.

Claims (6)

1. Use of taxifolin or a pharmaceutically acceptable salt thereof or a composition comprising the taxifolin or the pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing fat liquefaction or fat necrosis after a fat transplantation operation of an individual.

2. The use according to claim 1, wherein the medicament is in the form of oral liquid, injection, capsule, tablet or granule.

3. The use according to claim 1, wherein the medicament is administered orally, intraperitoneally, subcutaneously or intramuscularly.

4. The use of claim 1, wherein the subject is a mammal, including a non-primate and a primate.

5. The use of any one of claims 1 to 4, wherein the taxifolin or the pharmaceutically acceptable salt thereof is administered at a dose of 20 to 100mg/Kg body weight per day by intraperitoneal injection.

6. The use of any one of claims 1 to 4, wherein the taxifolin or the pharmaceutically acceptable salt thereof is administered at a dose of 30mg/Kg body weight per day by intraperitoneal injection.

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