USE OF HAPTOGLOBIN SUBUNIT FOR PROMOTING WOUND HEALING

Abstract

Provided is a method for promoting wound healing, which comprises: administering a haptoglobin subunit to a subject in need thereof. Also provided is a method for promoting wound healing, which comprises: administering a modified haptoglobin subunit to a subject in need thereof, the modified haptoglobin subunit comprising an amino acid sequence selected from one of SEQ ID NOs: 1-3.

Claims

1. A method for promoting wound healing, comprising: administering a haptoglobin subunit to a subject in need thereof.

2. The method as claimed in claim 1, wherein the haptoglobin subunit is present in form of a haptoglobin 1-1 protein, a haptoglobin 2-1 protein, or a haptoglobin 2-2 protein.

3. The method as claimed in claim 1, wherein the haptoglobin subunit is a haptoglobin 1 subunit, a haptoglobin 2 subunit, or a haptoglobin subunit.

4. The method as claimed in claim 2, wherein the haptoglobin 1-1 protein, the haptoglobin 2-1 protein, and the haptoglobin 2-2 protein are individually obtained via purification from blood, gene engineering, or chemical synthesis.

5. The method as claimed in claim 1, wherein the haptoglobin subunit is obtained via purification from blood, gene engineering, or chemical synthesis.

6. The method as claimed in claim 1, wherein the haptoglobin subunit has an anti-oxidant property and/or an anti-bacterial property.

7. The method as claimed in claim 1, wherein the wound is a chronic wound.

8. The method as claimed in claim 7, wherein the chronic wound is a pressure ulcer, a decubitus ulcer, a leg ulcer, or a diabetic foot ulcer.

9. A method for promoting wound healing, comprising: administering a modified haptoglobin subunit to a subject in need thereof, the modified haptoglobin subunit comprising an amino acid sequence selected from one of SEQ ID NOs: 1-3.

10. The method as claimed in claim 9, wherein the modified haptoglobin subunit is obtained via gene engineering or chemical synthesis.

11. The method as claimed in claim 9, wherein the haptoglobin subunit has an anti-oxidant property and/or an anti-bacterial property.

12. The method as claimed in claim 9, wherein the wound is a chronic wound.

13. The method as claimed in claim 12, wherein the chronic wound is a pressure ulcer, a decubitus ulcer, a leg ulcer, or a diabetic foot ulcer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a curve diagram illustrating the anti-oxidant effect of various Hp subunits;

[0020] FIG. 2 is a picture showing the wound healing effect of an Hp 1 subunit in vitro;

[0021] FIG. 3A is a curve diagram illustrating the wound area percentage of STZ-injected mice treated with various wound dressings;

[0022] FIG. 3B is a picture showing the wounds of STZ-injected mice treated with various wound dressings;

[0023] FIG. 4A is a curve diagram illustrating the wound area percentage of HFD-fed mice treated with various wound dressings;

[0024] FIG. 4B a picture showing the wounds of HFD-fed mice treated with various wound dressings;

[0025] FIG. 5 is a curve diagram illustrating the effect of various Hp phenotypes against Escherichia coli;

[0026] FIG. 6 is a curve diagram illustrating the effect of various Hp phenotypes in different concentration against Clostridium difficile; and

[0027] FIG. 7 is a curve diagram the survival rate of Clostridium difficile incubated with various Hp phenotypes in different concentration.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The detailed description and preferred embodiment of the invention will be set forth in the following content, and provided for people skilled in the art so as to understand the characteristic of the invention.

Example 1

Cell Culture

[0029] Human umbilical vein endothelial cells (HUVECs) were purchased from the Bioresource Collection and Research Center (BCRC). All cells were cultured in a 10-cm culture dish coated with a layer of gelatin (0.25 mg/mL), and these cells were maintained in M199 medium supplemented with 20% fetal bovine serum (FBS), 100 U/mL of penicillin, 0.1 mg/mL of streptomycin, and 2 mmol/L L-glutamine at 37 C.

Example 2

Anti-Oxidant Test

[0030] In this experiment, production level of thiobarbituric acid-reactive substances (TBARS) was measured to determine oxidization level of low-density lipoproteins (LDL). Specifically, 1004, of each test article was mixed with 5 M copper (II) sulfate (CuSO.sub.4) and 20 g of LDL, and then incubated at 37 C. for 2 hours. 250 L of 20% tricholoroacetic acid was added to the precipitated protein. After that, 250 L of 0.67% 2-thiobarbituric acid was added to the obtained mixture and incubated at 80 C. for 30 minutes. The final resultant was separated to give a supernatant by centrifugation at 3,000 g for 5 minutes. 300 L of the supernatant was taken to a 96-well plate to measure its absorbance under 540 nm.

[0031] The result is shown in FIG. 1. Probucol is used as control, which is used for treatment of xanthoma and is a well-known powerful oxidant. An inhibitory concentration (IC.sub.50) for anti-oxidant activity of an Hp 1 subunit is the greatest in all the test articles, and an Hp subunit, an Hp 1-1 protein, and probucol follow it sequentially. That is, anti-oxidant activity of each Hp phenotype and each Hp subunit prevails against that of probucol.

Example 3

Scratch Assay

[0032] A scratch assay was used to determine wound healing. Specifically, HUVECs were seeded into a 6-well culture dish containing 0.5% FBS. When these cells grew to a confluent cell monolayer, a cell-free zone was formed by scratching the monolayer with the tip of a micropipette so as to mimic a wound. At the 0th, 2nd, 4th, and 8th hours after the cell culture at 37 C., an inverted microscope (Nikon TE 2000) was introduced to observe cell condition in each well.

[0033] The result is shown in FIG. 2. Compared with control HUVECs, Hp 1 subunit-treated HUVECs have a relatively obvious effect on wound healing at the 2nd, 4th, and 8th hours. In other words, each Hp subunit can promote cell migration and wound healing.

Example 4

Animal Experiment

[0034] In this experiment, two animal models of diabetes were established. For the first one, experimental mice were intraperitoneally injected with streptozotocin (STZ) at a dosage of 65 mg/kg per body weight. Since STZ has cytotoxicity to insulin-producing cells, it can induce these mice to suffer from diabetes. For the second one, experimental mice were fed with high-fat diets (HFD) to elevate their blood glucose concentrations. As such, diabetes was mimicked in these HFD-fed mice.

[0035] After three weeks, the blood glucose concentration of each mouse was measured by a blood glucose meter to confirm the STZ-injected mice and the HFD-fed mice indeed suffer from diabetes (TABLE 1). After anesthesia administration by injection with nembutal at a dosage of 65 mg/kg per body weight, a full thickness skin of each mouse's back was excised to form a wound. After which, different dressings were applied to the wounds, respectively. On the 0th, 4th, 7th, 11th, and 14th days after this application, each wound was photographed and its area size was calculated with ImageJ software.

TABLE-US-00001 TABLE 1 Control STZ HFD Blood glucose (mg/dL) 110 117 167 124 199 206 224 208 220 197 251 249

[0036] As shown in FIGS. 3A and 3B, the dressing containing 2 mg/mL of Hp al subunits can promote wound healing in the STZ-induced diabetic mouse model. In another aspect, the dressing containing 0.2 mg/mL of Hp 1 subunits can promote wound healing in the HFD-induced diabetic mouse model.

Example 5

Anti-Bacterial Test

[0037] This in vitro experiment was used to identify efficacy of test articles against Clostridium difficile, a gram-positive bacterium, and Escherichia coli, a gram-negative bacterium. Various Hp phenotypes were added to LB culture medium in various concentrations (100 g/mL-200 g/mL) so that the finally obtained medium has a volume of 1 mL. Ampicillin or penicillin/streptomycin was used as control, which is a well-known antibiotic. After incubation at 37 C. for various periods, absorbance under 600 nm of the medium was measured.

[0038] As shown in FIGS. 5-7, all Hp phenotypes possess ability against a gram-positive bacterium and a gram-negative bacterium, and the ability is nearly equal to that of the conventional antibiotic, ampicillin or penicillin/streptomycin.

[0039] While the invention has been described in connection with what is considered the most practical and preferred embodiments, it is understood that this invention is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.