Hyaluronic acid compound for resisting cell hypoxia injury, preparation method therefor, and use thereof

Abstract

Provided are a hyaluronic acid compound for resisting cell hypoxia injury, a preparation method therefor, and use thereof. The hyaluronic acid compound comprises hyaluronic acid with an average molecular weight in the ten-thousands range and hyaluronic acid with an average molecular weight in the hundred-thousands range according to a volume ratio of 1:3 to 3:1. Hyaluronic acid can inhibit hypoxia-induced overactivation of HIF-1α, reduce the reactive oxygen level in hypoxic cells, and reduce the level of inflammatory factors in the supernatant of hypoxic cells, thereby alleviating hypoxia-induced cell apoptosis. Therefore, hyaluronic acid can be used in the treatment of cell hypoxia injury. Compared with hyaluronic acid of a single molecular weight, the obtained hyaluronic acid compound has a synergistic effect.

Description

A hyaluronic acid complex for resisting cellular hypoxia damage, its preparation method, and its application Technical Field

[0001] This invention belongs to the field of medical aesthetics and cosmetics, specifically relating to a hyaluronic acid compound that resists cell hypoxia damage, its preparation method, and its application. Background Technology

[0002] Hypoxia refers to a pathological process in which insufficient oxygen supply or impaired oxygen utilization in the body's tissues or cells leads to abnormal changes in their morphology, physiological function, and metabolism. The impact of hypoxia on the body's organs depends on the duration of hypoxia, the body's condition, and the degree of hypoxia. In mild hypoxia, the body's tissues can adapt to chronic hypoxia by increasing their ability to utilize oxygen and transport oxygen through the blood, such as by increasing the number of red blood cells, the number of mitochondria in tissue cells, and the surface area of ​​their membranes. In severe hypoxia, the body cannot complete compensatory responses in time, and tissues, cells, and organs will suffer severe hypoxic damage and dysfunction, even leading to death.

[0003] Hypoxia-inducible factor (HIF-1α) is a major transcriptional regulator of cellular responses to hypoxia. Under hypoxic conditions, moderate activation of HIF can reduce cellular sensitivity to hypoxia by stimulating physiological metabolism such as erythropoiesis, angiogenesis, and anaerobic glycolysis. However, long-term chronic hypoxia can lead to excessive activation of HIF-1α, causing irreversible damage to tissues and cells, resulting in adverse disease consequences. Therefore, targeted regulation of HIF-1α expression has become an effective strategy for treating hypoxia-related diseases.

[0004] Hyaluronic acid (HA) is a linear polysaccharide composed of β-D-glucuronic acid and N-acetyl-D-glucosamine, and is an important component of the extracellular matrix. Hyaluronic acid has been reported to possess various biological activities, including inflammation regulation, wound healing, anti-aging, and skin damage repair, and has been widely used in the food, cosmetic, and pharmaceutical fields. Patent CN202010489574.5 discloses a hyaluronic acid compound composition containing different chain segments and molecular fragments, which not only improves skin elasticity but also has a soothing effect on the skin. Patent CN201310149964.8 discloses the synergistic effect of hyaluronic acid compound compositions with different molecular weights in wound healing. These patents all demonstrate that hyaluronic acid has excellent repair capabilities, and that the efficacy of hyaluronic acid with different molecular weights varies significantly.

[0005] However, there are currently no reports on the repair effect of hyaluronic acid on cellular hypoxia damage, and the effects of hyaluronic acid complexes with different molecular weights on hypoxia repair are also unknown. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a hyaluronic acid compound that resists cell hypoxia damage.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a hyaluronic acid complex for resisting cellular hypoxia damage, comprising,

[0010] Hyaluronic acid with an average molecular weight of tens of thousands and hyaluronic acid with an average molecular weight of 100,000 are composed of hyaluronic acid in a volume ratio of 1:3 to 3:1.

[0011] In a preferred embodiment of the preparation method of the hyaluronic acid complex of the present invention, the hyaluronic acid with an average molecular weight in the tens of thousands includes hyaluronic acid with an average molecular weight of 18753 Da.

[0012] In a preferred embodiment of the preparation method of the hyaluronic acid complex of the present invention, the hyaluronic acid with an average molecular weight of 100,000 includes hyaluronic acid with an average molecular weight of 147,671 Da.

[0013] In a preferred embodiment of the preparation method of the hyaluronic acid compound of the present invention, the volume ratio of the hyaluronic acid with an average molecular weight of tens of thousands to the hyaluronic acid with an average molecular weight of 100,000 is 3:1.

[0014] As a preferred embodiment of the preparation method of the hyaluronic acid complex of the present invention, the hyaluronic acid complex has the effect of downregulating the expression of hypoxia-inducible factor HIF-1α to exert anti-hypoxia damage and has the effect of repairing hypoxic-damaged keratinocytes.

[0015] As a preferred embodiment of the preparation method of the hyaluronic acid complex of the present invention, the hyaluronic acid complex has a reducing and improving effect on intracellular reactive oxygen species produced by keratinocytes caused by hypoxia, and has a reducing effect on TNF-α, an inflammatory factor produced by keratinocytes caused by hypoxia.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a hyaluronic acid complex, comprising,

[0017] Hyaluronic acid with an average molecular weight of tens of thousands and hyaluronic acid with an average molecular weight of 100,000 are mixed evenly at a volume ratio of 1:3 to 3:1 to obtain a hyaluronic acid compound.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide the application of hyaluronic acid complexes in the preparation of pharmaceuticals or cosmetics.

[0019] As a preferred embodiment of the application described in this invention, the dosage form of the drug includes ointment, patch, injection, oral preparation or lyophilized injection.

[0020] As a preferred embodiment of the application described in this invention, the dosage form of the cosmetic includes toner, essence, lotion, or cream.

[0021] Beneficial effects of this invention:

[0022] (1) The hyaluronic acid of the present invention can inhibit the excessive activation of HIF-1α caused by hypoxia, reduce the level of reactive oxygen species in hypoxic cells, and reduce the level of inflammatory factors in the supernatant of hypoxic cells, thereby alleviating cell apoptosis caused by hypoxia; therefore, hyaluronic acid can be applied to the treatment of cell hypoxia-related damage.

[0023] (2) Compared with single molecular weight hyaluronic acid, the hyaluronic acid compound prepared by the present invention has a synergistic effect. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0025] Figure 1 is an HPSEC diagram of hyaluronic acid of different molecular weights in Example 1 of the present invention.

[0026] Figure 2 shows the effect of hyaluronic acid on the expression level of HIF-1α mRNA in hypoxic HaCaT cells in Example 2 of the present invention.

[0027] Figure 3 shows the effect of hyaluronic acid on the reactive oxygen species level in hypoxic HaCaT cells in Example 3 of the present invention.

[0028] Figure 4 shows the effect of hyaluronic acid on the inflammatory factor TNF-α in hypoxic HaCaT cells in Example 4 of the present invention.

[0029] Figure 5 shows the effect of hyaluronic acid on the apoptosis rate of hypoxic HaCaT cells in Example 5 of the present invention.

[0030] Figure 6 shows the effect of the compound in Example 6 of the present invention on the expression level of HIF-1α mRNA in hypoxic HaCaT cells.

[0031] Figure 7 shows the effect of the compound in Example 6 of the present invention on the reactive oxygen species level of hypoxic HaCaT cells.

[0032] Figure 8 shows the effect of the compound in Example 6 of the present invention on the inflammatory factor TNF-α in hypoxic HaCaT cells.

[0033] Figure 9 shows the effect of the compound in Example 6 of the present invention on the apoptosis rate of hypoxic HaCaT cells. Detailed Implementation

[0034] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0035] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0037] Example 1

[0038] Preparation of hyaluronic acid with different molecular weights:

[0039] Dissolve 1.5g of hyaluronic acid IV (purchased from Xinjiang Fufeng Biotechnology Co., Ltd.) with an average molecular weight of 1322459 Da in 25mL of water, and add 30-3.7×10 4 U hyaluronidase (Sigma) was hydrolyzed at 45°C for 0.5–1.5 h; after hydrolysis, the sample was boiled in water for 10 min to inactivate the enzyme, and then the supernatant of the solution was collected by centrifugation; the supernatant was filtered through a 0.22 μm filter membrane and the molecular weight was determined.

[0040] Based on the molecular weight (M) of the standard w Linear regression analysis was performed on M and its corresponding retention time (T) to obtain the regression equation lg(M) w = -0.8463T + 11.481.

[0041] As shown in Figure 1, calculations based on the above equations show that adding 6.8 × 10⁻⁶ hyaluronidase... 3 Hyaluronic acid I with a molecular weight of 2762 Da can be prepared by hydrolyzing U for 1.5 hours.

[0042] Add 3.5 × 10⁻⁶ hyaluronidase 4 Hyaluronic acid II with a molecular weight of 18753 Da can be prepared by hydrolyzing U for 1 hour;

[0043] Adding 30U of hyaluronidase and hydrolyzing for 1 hour can prepare hyaluronic acid III with a molecular weight of 147671 Da.

[0044] Example 2

[0045] RT-qPCR detection of the effect of hyaluronic acid on HIF-1α mRNA in hypoxic HaCaT cells:

[0046] Human keratinocytes HaCaT (purchased from BeiNa ChuangLian Biotechnology Research Institute) were cultured in DMEM medium containing 10% FBS and 1% double antibiotics in an incubator at 37°C and 5% CO2.

[0047] Cells in the logarithmic growth phase were selected, and the cell density was adjusted to 2 × 10⁻⁶. 5 2 mL / well was inoculated into 6-well plates;

[0048] Cells were divided into a blank group (Control, DMEM medium), a hypoxia group (Hypoxia, DMEM medium containing 1500 μM CoCl2), and a hyaluronic acid administration group with different molecular weights (DMEM medium containing 250 μg / mL hyaluronic acid and 1500 μM CoCl2). Each group had 3 replicates.

[0049] After culturing for 24 hours, the culture medium was removed, and DMEM solution containing 250 μg / mL hyaluronic acid was added and cultured for 12 hours. Then, CoCl2 solution was added to make the final concentration 1500 μM CoCl2.

[0050] After culturing for another 6 hours, the original culture medium was discarded, and the cells were washed twice with PBS. 1 mL of Trizol was added to each well to extract RNA. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit, and finally, RT-qPCR was performed using a fluorescence detection kit.

[0051] Use 2 -ΔΔCt The expression level of HIF-1α mRNA was calculated using a method with β-actin as the internal control.

[0052] Primer sequences are shown in Table 1.

[0053] Table 1

[0054] As shown in Figure 2, the mRNA level of HIF-1α in hypoxia-induced HaCaT cells increased significantly, while the mRNA level of HIF-1α in hypoxic HaCaT cells treated with hyaluronic acid of different molecular weights was significantly downregulated, with hyaluronic acid II and III showing the best inhibitory effects.

[0055] Example 3

[0056] Flow cytometry detection of reactive oxygen species in cells:

[0057] Cells were plated and drug-treated according to the method described in Example 2;

[0058] After the culture was completed, the old culture medium was discarded, and the cells were washed twice with PBS. Then, the probe was incubated according to the instructions of the reactive oxygen species (ROS) detection kit (Beyotime Biotechnology Co., Ltd.). Specifically, the DCFH-DA probe was diluted 1000 times with serum-free DMEM medium, and 1 mL of the diluted probe solution was added to each 6-well plate and incubated in a cell culture incubator at 37°C for 25 min.

[0059] The cells were then washed twice with serum-free DMEM medium, digested with 0.25% trypsin + 0.03% EDTA, and incubated for 3-5 minutes. The digestion was then terminated with DMEM medium containing serum, and the cells were collected by centrifugation at 1200 r / min for 5 minutes.

[0060] Finally, flow cytometry was used to detect the intensity of intracellular reactive oxygen species.

[0061] Figure 3 shows that hypoxia leads to a sharp increase in intracellular reactive oxygen species (ROS) levels, while hyaluronic acid treatment significantly reduces ROS levels in hypoxic cells. Hyaluronic acid II and III showed the best inhibitory effect on ROS, indicating that hyaluronic acid can alleviate the damage to cells caused by hypoxia.

[0062] Example 4

[0063] ELISA detection of the inflammatory factor TNF-α:

[0064] Cells were plated and drug-treated according to the method described in Example 2;

[0065] After culture, the cell supernatant was collected into a 2 mL centrifuge tube and centrifuged at 10,000 r / min for 10 min. The supernatant was then used to determine the content of inflammatory factors according to the instructions of the corresponding ELISA kit.

[0066] Figure 4 shows that, compared with the control group, the inflammatory factors in the cell supernatant of the hypoxia group were significantly increased, indicating that hypoxia induced an intracellular inflammatory response. Compared with the hypoxia group, the inflammatory factors in the hypoxia cell supernatant were inhibited to varying degrees after treatment with hyaluronic acid, with hyaluronic acid II and III showing the best inhibitory effects.

[0067] Example 5

[0068] Flow cytometry detection of apoptosis:

[0069] Cells were plated and treated with drugs according to the method described in Example 2; after culture, the cells were stained according to the Annexin V-FITC apoptosis detection kit (Beyotime Biotechnology Co., Ltd.), and finally the apoptosis rate was detected by flow cytometry.

[0070] Figure 5 shows that the apoptosis rate of cells in the hypoxia group was much higher than that in the control group, while the apoptosis rate of cells treated with hyaluronic acid decreased significantly. Similarly, hyaluronic acid II and III showed better repair effects.

[0071] Example 6

[0072] The repair effect of the compound on cellular hypoxia damage:

[0073] (1) Preparation of the compound: First, hyaluronic acid II and III with a concentration of 250 μg / mL were prepared using DMEM medium;

[0074] Then, the compound compositions of hyaluronic acid II and III with the same mass concentration but different volume ratios were thoroughly mixed evenly, and the specific composition ratios are shown in Table 2 below.

[0075] Table 2

[0076] (1) The effect of the compound composition on HIF-1α mRNA in hypoxic HaCaT cells was detected according to the method described in Example 2;

[0077] The effect of the compound composition on reactive oxygen species in hypoxic cells was tested according to Example 3.

[0078] The effect of the compound composition on the inflammatory factor TNF-α in hypoxic cells was tested according to Example 4, and the effect of the compound composition on apoptosis in hypoxic cells was tested according to Example 5.

[0079] Experimental results showed that, compared with hyaluronic acid alone, the compound composition of hyaluronic acid had a better inhibitory effect on HIF-1α mRNA in hypoxic-damaged cells (Figure 6), and also significantly reduced the levels of reactive oxygen species (Figure 7) and inflammatory factors (Figure 8) in hypoxic cells. The hypoxic cell apoptosis rate was lowest when the ratio of hyaluronic acid II to III was 1:3 (Figure 9), indicating that the composition had the best hypoxia repair effect.

[0080] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A hyaluronic acid complex for protection against cell hypoxic damage, characterized in that: Comprising, The hyaluronic acid with an average molecular weight of the order of ten thousand and the hyaluronic acid with an average molecular weight of the order of one hundred thousand are mixed in a volume ratio of 1:3 to 3:

1.

2. The hyaluronic acid complex of claim 1, wherein: The hyaluronic acid with an average molecular weight of the order of ten thousand comprises hyaluronic acid with an average molecular weight of 18753 Da.

3. The hyaluronic acid complex of claim 1 or 2, characterized in that: The hyaluronic acid with an average molecular weight of the order of one hundred thousand comprises hyaluronic acid with an average molecular weight of 147671 Da.

4. The hyaluronic acid complex of claim 3, wherein: The volume ratio of the hyaluronic acid with an average molecular weight of the order of ten thousand and the hyaluronic acid with an average molecular weight of the order of one hundred thousand is 3:

1.

5. The hyaluronic acid complex of any one of claims 1, 2 or 4, wherein: The hyaluronic acid complex has the effect of down-regulating the expression of hypoxia-inducible factor HIF-1α and plays a role in resisting hypoxic injury, and has a repairing effect on keratinocytes damaged by hypoxia.

6. The hyaluronic acid complex of claim 5, wherein: The hyaluronic acid complex has a reducing and improving effect on the intracellular reactive oxygen species produced by keratinocytes damaged by hypoxia, and has a reducing effect on the inflammatory factor TNF-α produced by keratinocytes damaged by hypoxia.

7. Process for the preparation of the hyaluronic acid complex according to any one of claims 1 to 6, characterized in that: Comprising, The hyaluronic acid with an average molecular weight of the order of ten thousand and the hyaluronic acid with an average molecular weight of the order of one hundred thousand are mixed in a volume ratio of 1:3 to 3:

1.

8. The use of the hyaluronic acid complex as claimed in claims 1 to 6 in the preparation of a medicine or a cosmetic.

9. Use according to claim 8, wherein: The dosage form of the medicine comprises ointment, patch, injection, oral preparation or freeze-dried needle.

10. The use according to claim 8, characterized in that: The dosage form of the cosmetic comprises skin toner, essence, emulsion or cream.

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