Apparatus comprising carbon fiber sheet-based planar heating element for promoting LDL cholesterol absorption by reducing expression of PCSK9

Abstract

The present invention relates to an apparatus comprising a carbon fiber sheet-based planar heating element for promoting LDL cholesterol absorption by reducing expression of PCSK9. The apparatus has an excellent effect of promoting the absorption of LDL cholesterol in cells and can inhibit the expression of PCSK9, and thus is advantageously applicable for preventing or alleviating diseases such as hyperlipidemia, dyslipidemia, atherosclerosis, cardiovascular diseases, or hypercholesterolemia.

Description

A device that promotes LDL cholesterol absorption by reducing the expression of PCSK9, including a carbon fiber sheet-based planar heating element.

[0001] The present invention relates to a device for promoting LDL absorption by reducing the expression of PCSK9, which includes a planar heating element based on a carbon fiber sheet.

[0002] Cholesterol is essential for various cellular activities, including the biosynthesis of bile acids and hormones, embryonic development, and cell proliferation. It is classified into triglycerides, HDL cholesterol, and LDL cholesterol. Among these, LDL cholesterol (low-density lipoprotein cholesterol; LDL-C) causes excessive cholesterol deposition in blood vessel walls, a major risk factor for coronary artery disease. It can also induce dyslipidemia, increasing the risk of cardiovascular disease, diabetes, hypertension, and stroke.

[0003] With the recent rise in cardiovascular disease due to modern eating habits and growing awareness of cholesterol, treatments aimed at lowering blood LDL-C levels are gaining attention. Conventional treatments known to lower LDL-C include statins, ezetimibe, and fibrates. However, these treatments have limitations, such as the potential for complications like liver toxicity and diabetes. Therefore, there is a growing need for more effective treatments with fewer side effects.

[0004] Proprotein convertase subtilisin / kexin 9 (PCSK9) is an enzyme that plays a crucial role in lipoprotein metabolism. It specifically binds to the low-density lipoprotein receptor (LDLR) in the liver, increasing its degradation and elevating blood LDL-C levels. While PCSK9 is a suitable target for LDL-C-related diseases, monoclonal antibodies targeting PCSK9, such as alirocumab and evolocumab, require subcutaneous injections every two weeks or monthly.

[0005] The present inventors have developed a method utilizing far-infrared (FIR) light as an effective PCSK9 inhibitor with a lower risk of side effects than previously known inhibitors. Far-infrared light is an invisible electromagnetic radiation with a wavelength range of 3–1000 μm, as defined by the International Commission on Illumination. It is known to convert energy into pure heat, which is then recognized as radiant heat by thermoreceptors in skin cells. Far-infrared irradiation also modulates the behavior of water molecules, altering various biological structures and cellular activities. Our recent study demonstrated that far-infrared light affects adipogenesis and osteogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs). Specifically, we demonstrated that far-infrared light regulates adipogenesis in TMSCs by increasing intracellular calcium through activation of transient receptor potential (TRP) 2 channels. However, the effect of far-infrared light on PCSK9, which regulates lipid and cholesterol metabolism, through TRP calcium channels remains unknown.

[0006] Accordingly, the inventors of the present invention designed a planar heating device made of a carbon fiber sheet, taking note of the fact that far-infrared rays emitted from a carbon fiber sheet can activate TRP channels, and when this is used to suppress the expression level of PCSK9 in liver cells, compared to the case of using a conventional ceramic lamp heating device, it provides uniform heat and far-infrared rays over the entire heating area, thereby maintaining an appropriate temperature for a long time, thereby not only minimizing power consumption without causing damage to cells and tissues, but also effectively increasing the absorption of LDL cholesterol by cells and suppressing the expression of PCSK9 by activating TRPV4 channels, thereby completing the present invention.

[0007] The purpose of the present invention is to provide a device for preventing or improving hyperlipidemia, dyslipidemia, atherosclerosis, cardiovascular disease or hypercholesterolemia, which includes a surface heating element made of a carbon fiber sheet.

[0008] One aspect of the present invention provides a device for preventing or improving hyperlipidemia, dyslipidemia, atherosclerosis, cardiovascular disease or hypercholesterolemia, including a surface heating element made of a carbon fiber sheet.

[0009] According to one specific example of the present invention, the target of far-infrared irradiation of the surface heating element may be a hepatocyte. Specifically, the hepatocyte may be a HepG2 cell, but is not limited thereto.

[0010] According to one specific example of the present invention, the far-infrared irradiation temperature of the surface heating element may be 30 to 90°C, preferably 50 to 70°C. Within the above temperature range, the cell viability of hepatocytes may be excellent, and the effect of reducing the level of PCSK9 may be enhanced.

[0011] According to one specific example of the present invention, the far-infrared irradiation time of the surface heating element may be 10 minutes to 10 hours, preferably 30 minutes to 2 hours, and more preferably 30 minutes to 1 hour. In particular, when both the irradiation temperature range of the surface heating element and the irradiation time range of the surface heating element described above are satisfied, the cell viability of liver cells, the promotion of LDL cholesterol absorption, and the effect of reducing the level of PCSK9 may be significantly improved.

[0012] According to one embodiment of the present invention, the device may reduce the level of proprotein convertase subtilisin / kexin type 9 (PCSK9) in hepatocytes.

[0013] A device for promoting LDL cholesterol absorption including a planar heating element based on a carbon fiber sheet is excellent in promoting cellular LDL cholesterol absorption and can suppress the expression of PCSK9, and thus can be usefully utilized in the prevention or improvement of diseases such as hyperlipidemia, dyslipidemia, atherosclerosis, cardiovascular disease, or hypercholesterolemia.

[0014] FIG. 1 is a drawing showing a surface heating element device made of a carbon fiber sheet according to a first embodiment of the present invention.

[0015] Figure 2 is an image actually taken of a surface heating element device made of a carbon fiber sheet according to the first embodiment of the present invention.

[0016] Figure 3 is an exploded view of the heating unit illustrated in Figure 1.

[0017] Figure 4 shows an image of a heating unit heated to 60°C by a surface heating element device made of a carbon fiber sheet, taken with a thermal imaging camera, and a graph showing temperature changes according to heating time (A. Heating to 60°C by the CF sheet), and an image of a lower plate heated to 60°C, taken with a thermal imaging camera, and a graph showing temperature changes according to heating time (B. CuttingMat at 60°C).

[0018] Figure 5 is a drawing showing a state in which a culture vessel is placed on a surface heating device made of a carbon fiber sheet as shown in Figure 1.

[0019] Fig. 6 is a side view of a heating unit of a surface heating element device made of a carbon fiber sheet according to a second embodiment of the present invention.

[0020] Figure 7 shows the results of analyzing the effect of far-infrared irradiation on PCSK9 expression in HepG2 cells using a ceramic infrared irradiator.

[0021] Figure 8 shows the results of analyzing the effect on PCSK9 expression according to the incubation time (30 minutes, 1 hour) in a humid 37→ incubator after irradiating HepG2 cells with far-infrared rays for 30 minutes using a ceramic infrared irradiator.

[0022] Figure 9 is an image captured by a thermal imaging camera when irradiated with far infrared rays emitted by a ceramic infrared irradiator.

[0023] Figure 10 shows the results of a comparative analysis of HepG2 cells cultured at 25°C (room temperature, RT) and 37°C without far-infrared irradiation and HepG2 cells irradiated with far-infrared irradiation.

[0024] Figure 11 shows the results of analyzing the effect of far-infrared irradiation on PCSK9 expression using a surface heating device made of carbon fiber sheet.

[0025] Figure 12 shows the results of analyzing the effect of far-infrared rays emitted by a ceramic infrared irradiator (A) or a surface heating device made of a carbon fiber sheet (B) on the absorption of LDLR and LDL by HepG2 cells.

[0026] Figure 13 shows the results of analyzing the effect of far-infrared irradiation on intracellular calcium concentration.

[0027] Figure 14 shows the results of analyzing the effect of intracellular calcium on PCSK9 expression by treating with ionomycin.

[0028] Figure 15 shows the results of analyzing the effect of TRP channels on PCSK9 expression by irradiating far-infrared rays using a ceramic infrared irradiator (A) or a planar heating device made of carbon fiber sheet (B) on HepG2 cells treated with Ruthenium Red.

[0029] Figure 16 shows the results of reverse transcription polymerase chain reaction (RT-PCR) analysis of TRPV channels expressed in HepG2 cells irradiated with far-infrared rays.

[0030] Figure 17 shows the results of analyzing the effects of TRPV1, TRPV2, and TRPV4 channel activators on PCSK9 expression in HepG2 cells.

[0031] Figure 18 shows the results of analyzing the effects of treating HepG2 cells with TRPV1, TRPV2, and TRPV4 channel inhibitors and irradiating them with far-infrared light on PCSK9 expression.

[0032]

[0033] Hereinafter, embodiments of the present invention will be described in more detail with reference to the attached drawings. The embodiments of the present invention may be modified in various ways, and the scope of the present invention should not be construed as being limited to the embodiments described below. These embodiments are provided to more fully explain the present invention to those skilled in the art. Accordingly, the shapes of elements in the drawings have been exaggerated for clarity.

[0034]

[0035] Surface heating element device made of carbon fiber sheet (hereinafter referred to as surface heating element device)

[0036] According to one embodiment of the present invention, the planar heating device made of the carbon fiber sheet may include: a lower plate on which the hepatocytes are arranged; an upper plate spaced apart from the lower plate in an upper region of the lower plate; a guide shaft connected to the upper plate and the lower plate; an elevation stage unit movably connected to the guide shaft and moved in a direction approaching and separating from the culture vessel; a moving unit coupled to the upper plate, connected to the elevation stage unit, and moving the elevation stage unit; and a heating unit coupled to the elevation stage unit and equipped with a carbon fiber sheet planar heating element that applies energy to the hepatocytes.

[0037]

[0038] Hereinafter, a surface heating element device made of a carbon fiber sheet according to a first embodiment of the present invention will be described in detail.

[0039] As shown in FIGS. 1 and 2, a surface heating device made of a carbon fiber sheet according to a first embodiment of the present invention is composed of a lower plate (110), an upper plate (120), a guide shaft (130), an elevation stage unit (140), a moving unit (150), a heating unit (160), and a temperature controller (170).

[0040] The present invention will be described in more detail below through one or more examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0041]

[0042] [Lower plate (110), upper plate (120), guide shaft (130)]

[0043] A culture vessel (S) containing hepatocytes and / or culture medium (or culture solution) is placed on the lower plate (110). One or more culture vessels (S) can be placed on the lower plate (110), and the size and height of the culture vessel (S) can be formed in various ways. For example, the lower plate (110) is manufactured to have a size that can place about four 96-well plates, and for example, the size of the lower plate (110) can be 50x50 mm to 500x1000 mm, but is not limited thereto. As a specific example, 200x200 mm far-infrared irradiation is possible within A4 size.

[0044] A graduation indicator is formed on the upper surface of the lower plate (110) to visually check the size of the culture vessel. The graduation indicator may be formed as a grid-like graduation. In the present embodiment, a cutting mat or a plastic material with graduations (e.g., a ruler) is attached to the lower plate (110) as a graduation indicator, so that the culture vessel (S) can always be maintained in the same position and arrangement (Fig. 5). The types of components attached as the graduation indicator are not limited to those described above.

[0045] The upper plate (120) is spaced apart from the lower plate (110) in the upper region of the lower plate (110).

[0046] In this embodiment, the upper plate (120) and the lower plate (110) are formed into a square plate shape of the same size.

[0047] The guide shaft (130) is connected to the upper plate (120) and the lower plate (110). The upper plate (120) and the lower plate (110) are separated from each other by the guide shaft (130).

[0048]

[0049] [Elevator Stage Unit (140)]

[0050] The lifting stage unit (140) is movably connected to the guide shaft (130) and moves in a direction approaching and away from the culture vessel (S) placed on the lower plate (110).

[0051] The lifting stage unit (140) is composed of a lifting plate (141) and a guide bush (142).

[0052] The lifting plate (141) is connected to the moving unit (150). In the present embodiment, the lifting plate (141) is made of aluminum. However, it is not limited thereto and may be made of various materials such as acrylic. The lifting plate (141) is formed with a support groove (141a) to which a screw shaft (152) of the moving unit (150), described later, is rotatably coupled.

[0053] The guide bush (142) is coupled to the lifting plate (141) and is slidably connected to the outer surface of the guide shaft (130). A through hole (not shown) through which the guide shaft (130) passes is formed in the guide bush (142).

[0054]

[0055] [Movement Unit (150)]

[0056] The moving unit (150) is coupled to the upper plate (120), connected to the elevation stage unit (140), and moves the elevation stage unit (140). The moving unit (150) can adjust the height of the heating unit (160) from the lower plate (110) within a range of 20 mm to 140 mm. A device for checking the height of the elevation stage unit (140) can be attached between the upper plate (120) and the lower plate (110) (Fig. 5).

[0057] The moving unit (150) is composed of a fixed hub (151), a screw shaft (152), and a rotating member (153).

[0058] The fixed hub (151) is coupled to the central area of ​​the upper plate (120). A screw groove (not shown) is formed in the fixed hub (151) to which a spiral screw (not shown) formed on the outer surface of the screw shaft (152) is connected.

[0059] The screw shaft (152) is screw-connected to the fixed hub (151) and rotatably connected to the lifting plate (141). The lower portion of the screw shaft (152) is rotatably connected to a support groove (141a) formed in the lifting plate (141).

[0060] The rotating member (153) is coupled to the upper end of the screw shaft (152) and rotates the screw shaft (152). In this embodiment, a rotation handle is used as the rotating member (153). If the rotating member (153) is a rotation handle, the screw shaft (152) is rotated manually. Alternatively, a rotation motor may be used as the rotating member (153) to automatically rotate the screw shaft (152).

[0061]

[0062] [Heating unit (160)]

[0063] The heating unit (160) is coupled to the lifting stage unit (140).

[0064] As shown in Fig. 3, the heating unit (160) is composed of a carbon fiber sheet surface heating element (161), an insulating block (162), a first hydrophobic film (163), a second hydrophobic film (164), and a metal electrode (166).

[0065] The carbon fiber sheet surface heating element (161) heats the liver cells and / or culture medium (or culture solution) and emits far infrared rays to the liver cells and / or culture medium.

[0066] Far infrared rays, physically defined as light with a wavelength of 3㎛ to 1,000㎛, are generally known to have various effects, including thermal effects, drying, biological effects, water activation, ripening and growth promotion, penetration, and radiation. Recently, due to these diverse effects, far infrared rays have been recognized as having beneficial effects on the human body, including anti-inflammation, osteoporosis, arthritis, and bone regeneration. Consequently, extensive research is being conducted on the effects of far infrared rays on the human body.

[0067] The carbon fiber sheet surface heating element (161) is made of a highly conductive carbon fiber sheet. This not only maintains the temperature of liver cells and / or culture medium, but also allows for simultaneous testing of the effects of far-infrared radiation on cells using the far-infrared radiation emitted by the carbon fiber sheet.

[0068] In this embodiment, the carbon fiber sheet surface heating element (161) is manufactured to be 200×200 mm. The carbon fiber sheet surface heating element (161) manufactured in this manner can generate heat in a temperature range of, for example, 25°C to 180°C, but is not limited to the above-mentioned temperature range.

[0069] The characteristics of the carbon fiber sheet planar heater (161) manufactured in this example are as shown in Tables 1 and 2 below. Table 1 below shows the electrical characteristics of the carbon fiber sheet planar heater, and Table 2 shows the far-infrared ray emission characteristics of the carbon fiber sheet planar heater.

[0070]

[0071]

[0072] The insulation block (162) is attached to the lower surface of the elevation plate (141) of the elevation stage unit (140). The insulation block (162) insulates heat generated from the carbon fiber sheet surface heating element (161). In the present embodiment, the insulation block (162) is made of cork. However, it is not limited thereto, and may be made of materials such as glass wool, felt, flame-retardant plastic, and urethane board.

[0073] A temperature measuring device for measuring the temperature of the carbon fiber sheet surface heating element (161) may be mounted on the insulation block (162). In this case, a temperature measuring device mounting groove is formed on the lower surface of the insulation block (162) into which the temperature measuring device is mounted. A thermocouple may be mounted as the temperature measuring device.

[0074]

[0075] <First hydrophobic film (163) and second hydrophobic film (164)>

[0076] The first hydrophobic film (163) and the second hydrophobic film (164) are attached to the carbon fiber sheet surface heating body (161).

[0077] The first hydrophobic film (163) has its upper surface bonded to the lower surface of the insulation block (162). The lower surface of the first hydrophobic film (163) is placed on the upper surface of the carbon fiber sheet planar heater (161) and covers the upper surface of the carbon fiber sheet planar heater (161). The first hydrophobic film (163) is formed smaller than the carbon fiber sheet planar heater (161) and does not cover the metal electrodes (166) placed at both ends of the carbon fiber sheet planar heater (161).

[0078] The second hydrophobic film (164) is placed on the lower side of the carbon fiber sheet planar heating element (161) and covers a part of the lower surface of the carbon fiber sheet planar heating element (161), i.e., the central area.

[0079] In this embodiment, the first hydrophobic film (163) and the second hydrophobic film (164) are made of thermoplastic polyurethane (TPU). However, the present invention is not limited thereto, and may be made of films such as polyethylene terephthalate (PET), ethylene vinyl acetate (EVA), etc.

[0080] The first hydrophobic film (163) and the second hydrophobic film (164) prevent moisture evaporated from the culture medium or culture solution from forming on the carbon fiber sheet planar heating element (161) or insulating block (162) or from seeping into the interior.

[0081]

[0082] <Metal electrode (166)>

[0083] In order to connect power to the carbon fiber sheet planar heater (161), a metal electrode (166) is attached to the carbon fiber sheet planar heater (161). The metal electrode (166) is attached to both ends of the carbon fiber sheet planar heater (161). In the present embodiment, the metal electrode (166) is attached to the upper surface of the carbon fiber sheet planar heater (161), but alternatively, it may be attached to the lower surface of the carbon fiber sheet planar heater (161) or to both the upper and lower surfaces.

[0084] The metal electrode (166) may be made of a metal including copper, aluminum, silver, nickel, tin, etc., or an alloy thereof. The metal electrode (166) may be in the form of a ribbon or a wire. In the present embodiment, copper is used as the metal electrode (166). Copper tape is attached to both ends of the carbon fiber sheet-surface heating element (161) and a power source is connected.

[0085] The four sides of the heating unit (160) made of a carbon fiber sheet surface heating element (161), an insulating block (162), a first hydrophobic film (163), a second hydrophobic film (164), and a metal electrode (166) are wrapped with an insulating film. Polyimide (PI), polypropylene (PP), polyethylene terephthalate (PET), ethylene vinyl acetate (EVA), etc. can be used as the insulating film.

[0086]

[0087] <Temperature controller (170)>

[0088] A temperature controller (170) controls the power connected to the carbon fiber sheet planar heater (161) to control the heating temperature of the carbon fiber sheet planar heater (161). Specifically, the temperature controller (170) is connected to a temperature measuring device that measures the temperature of the carbon fiber sheet planar heater (161), and can control the temperature and time of the carbon fiber sheet planar heater (161).

[0089]

[0090] Hereinafter, the operation of a heating device manufactured from a carbon fiber sheet according to the first embodiment of the present invention will be described in detail.

[0091] The temperature of the culture medium (or culture solution) containing the above-mentioned hepatocytes is approximately 35 to 40°C and must be maintained at this temperature. Therefore, before placing the culture vessel (S) containing the above-mentioned culture medium (or culture solution) in the above-mentioned surface heating device, a process of preheating the lower plate (110) is necessary.

[0092] In this embodiment, power is connected to the metal electrode (166) of the heating unit (160) to heat the temperature of the carbon fiber sheet surface heating element (161) to about 60°C. The heating temperature can be adjusted to 60°C to 80°C depending on the height of the heating unit (160) and the size of the culture vessel (S) so as to maintain the liver cells and / or culture medium at 37°C. The rotating member (153) of the moving unit (150) is operated to lower the lifting plate (141) of the lifting stage unit (140) to the lower plate (110).

[0093] In this embodiment, when the gap between the heating unit (160) and the lower plate (110) is about 40 mm, the temperature of the carbon fiber sheet surface heating element (161) is maintained at about 60°C, and after about 1 minute, the temperature of the lower plate (110) becomes about 35°C to 40°C (average 37°C) (Fig. 4).

[0094] The lower plate (110) requires a preheating process of about 30 minutes, and after the preheating process is completed, the elevating plate (141) is raised to increase the gap between the heating unit (160) and the lower plate (110), and then a culture vessel (S) containing hepatocytes and / or culture medium (or culture solution) is placed in the central area of ​​the lower plate (110).

[0095] As shown in Fig. 5, the elevating plate (141) is lowered until the gap between the heating unit (160) and the lower plate (110) becomes about 1 to 50 cm or 25 to 35 cm. The liver cells and / or culture medium are cultured for an appropriate period of time (about 30 minutes to 24 hours).

[0096] Hereinafter, a surface heating body device manufactured from a carbon fiber sheet according to a second embodiment of the present invention will be described in detail. Reference will be made primarily to FIGS. 1 and 6.

[0097] A planar heating device made of a carbon fiber sheet according to a second embodiment of the present invention differs from the first embodiment in the heating unit (260). The heating unit (260) according to the second embodiment includes a plurality of thin carbon fiber sheet planar heating elements. When a plurality of thin carbon fiber sheet planar heating elements are used, the amount of heat and far-infrared rays emitted from the carbon fiber sheet planar heating element can be controlled more precisely and easily than when a single thick carbon fiber sheet planar heating element is used. Accordingly, the temperature range can be configured more diversely, and the effect of the amount of far-infrared rays emitted on cells can be tested under various conditions.

[0098] In the second embodiment, the heating unit (260) includes two carbon fiber sheet planar heating elements, and a hydrophobic film is attached to the upper and lower surfaces of each carbon fiber sheet planar heating element. However, the present invention is not limited thereto, and the number of carbon fiber sheet planar heating elements may be formed in greater numbers, and the number of hydrophobic films may be increased accordingly.

[0099] Since the remaining configuration is identical to the first implementation example, a detailed description of the identical configuration is omitted. The same symbols are used for identical configurations.

[0100]

[0101] [Heating unit (260)]

[0102] The heating unit (260) is coupled to the lifting stage unit (140).

[0103] As illustrated in FIG. 6, the heat generating unit (260) is composed of a first carbon fiber sheet surface-type heat generating body (261a), a second carbon fiber sheet surface-type heat generating body (261b), an insulation block (262), a first hydrophobic film (263), a second hydrophobic film (264), a third hydrophobic film (265), and a metal electrode (266). The same contents as the heat generating unit (160) according to the first embodiment will be omitted for detailed description, and the distinguishing features will be described.

[0104]

[0105] <Insulating block (262)>

[0106]

[0107] The insulation block (262) is identical to the insulation block (162) of the heating unit (160) of the first embodiment.

[0108]

[0109] <First hydrophobic film (263), second hydrophobic film (264), third hydrophobic film (265)>

[0110] The first hydrophobic film (263) and the second hydrophobic film (264) are attached to the upper and lower surfaces of the first carbon fiber sheet planar heating element (261a), respectively, and the second hydrophobic film (264) and the third hydrophobic film (265) are attached to the upper and lower surfaces of the second carbon fiber sheet planar heating element (261b), respectively.

[0111] The upper surface of the first hydrophobic film (263) is bonded to the lower surface of the insulation block (262). The lower surface of the first hydrophobic film (263) is placed on the upper surface of the first carbon fiber sheet planar heating element (261a), thereby covering the upper surface of the first carbon fiber sheet planar heating element (261a).

[0112] The second hydrophobic film (264) is arranged with its upper surface below the first carbon fiber sheet planar heating element (261a), thereby covering the lower surface of the first carbon fiber sheet planar heating element (261a). The lower surface of the second hydrophobic film (264) is arranged with its upper surface above the second carbon fiber sheet planar heating element (261b), thereby covering the upper surface of the second carbon fiber sheet planar heating element (261b).

[0113] The third hydrophobic film (265) is arranged with its upper surface below the second carbon fiber sheet planar heating element (261b) to cover the lower surface of the second carbon fiber sheet planar heating element (261b).

[0114] The first hydrophobic film (263) and the second hydrophobic film (264) are formed to be shorter in length than the first carbon fiber sheet planar heater (261a) and the second carbon fiber sheet planar heater (261b), so that the metal electrodes (266) attached to both ends of the first carbon fiber sheet planar heater (261a) and the second carbon fiber sheet planar heater (261b) are not covered by the first hydrophobic film (263), the second hydrophobic film (264), and the third hydrophobic film (265).

[0115] In this embodiment, the first hydrophobic film (263), the second hydrophobic film (264), and the third hydrophobic film (265) are made of thermoplastic polyurethane (TPU). However, the present invention is not limited thereto, and may be made of films such as polyethylene terephthalate (PET), ethylene vinyl acetate (EVA), etc.

[0116]

[0117] The first hydrophobic film (263), the second hydrophobic film (264), and the third hydrophobic film (265) prevent moisture evaporated from the liver cells and / or the culture medium from forming on or seeping into the first carbon fiber sheet planar heating element (261 a), the second carbon fiber sheet planar heating element (261 b), or the insulation block (262).

[0118]

[0119] <Metal electrode (266)>

[0120] In order to connect power to the first carbon fiber sheet planar heater (261a) and the second carbon fiber sheet planar heater (261b), metal electrodes (266) are attached to the first carbon fiber sheet planar heater (261a) and the second carbon fiber sheet planar heater (261b), respectively. The metal electrodes (266) are attached to both ends of the first carbon fiber sheet planar heater (261a) and the second carbon fiber sheet planar heater (261b). In this embodiment, the metal electrode (266) is attached to the upper surfaces of the first carbon fiber sheet planar heater (261a) and the second carbon fiber sheet planar heater (261b), respectively. However, alternatively, the metal electrode (266) may be attached to the lower surfaces of the first carbon fiber sheet planar heater (261a) and the second carbon fiber sheet planar heater (261b), respectively, or may be attached to both the upper and lower surfaces.

[0121] In this embodiment, copper is used as the metal electrode (266). Copper tape is attached to both ends of the first carbon fiber sheet planar heating element (261a) and the second carbon fiber sheet planar heating element (261b), and power is connected to each metal electrode (266).

[0122]

[0123] Hereinafter, the operation of a surface heating element device manufactured from a carbon fiber sheet according to a second embodiment of the present invention will be described in detail. Reference will be made primarily to FIGS. 1 and 6.

[0124]

[0125] Basically, the operation is the same as that of the surface heating element device made of carbon fiber sheet according to the first embodiment, so the operation that is different from that of the first embodiment will be explained.

[0126] When power is connected to the metal electrode (266) of the heating unit (260), when testing with the same temperature range and far-infrared ray emission amount as in the first embodiment, power is supplied only to the second carbon fiber sheet surface heating element (261b).

[0127] In contrast, when it is desired to emit heat in a range greater than the temperature range and far-infrared ray emission amount of the first embodiment, power is supplied to the first carbon fiber sheet planar heating element (261a) and the second carbon fiber sheet planar heating element (261b).

[0128]

[0129] The above description has explained the technical idea of ​​the present invention using one embodiment, and those skilled in the art to which the present invention pertains will be able to make various modifications and variations without departing from the essential characteristics of the present invention. Therefore, the embodiments described in the present invention are not intended to limit the technical idea of ​​the present invention but to explain it, and the scope of the technical idea of ​​the present invention is not limited by these embodiments. The protection scope of the present invention should be interpreted by the claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

[0130]

[0131] Hereinafter, the present invention will be described in more detail through examples.

[0132]

[0133] Manufacturing Example 1. Cell Culture

[0134] HepG2 cells were obtained from the Korean Organoid Bank (Seoul, Republic of Korea). They were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC, VA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin (100 U / ml; Sigma-Aldrich, St. Louis, MO, USA), and 25 mM HEPES (Gibco, Grand Island, New York) at 37°C in a 5% CO2 atmosphere. HepG2 cells at passages 68 to 76 were used.

[0135]

[0136] Example 1. Far-infrared irradiation using a surface heating element device made of carbon fiber sheet.

[0137] Far infrared rays were irradiated using a surface heating element device according to the present invention, which controls the temperature of carbon fiber sheets (CFS) that emit far infrared (FIR) radiation.

[0138] Specifically, the carbon fiber sheet was preheated for 1 hour, and the distance between the lower plate (110) containing the culture vessel (S) containing HepG2 cells and the carbon fiber sheet surface heating element (161) was set to 3 cm, and the far infrared ray (FIR-CFS) emitted from the carbon fiber sheet was irradiated at 60°C or 70°C for 1 or 2 hours. After the FIR-CFS irradiation, the HepG2 cells were additionally cultured in an incubator at 37°C and 5% CO2 for 1 hour.

[0139]

[0140] Comparative Example 1. Far-infrared irradiation using a ceramic infrared irradiator

[0141] Far-infrared radiation was irradiated using a ceramic infrared irradiator used in a previous study. Specifically, a screw-base ceramic infrared irradiator (Model No. SO.TM 9H, Saeik Medical Co., Ltd., Bucheon, Republic of Korea) with an emission wavelength of 3 to 10 μm (Model No. IOT / 90-250W, Elstein-Werk M. Steinmetz GmbH & Co. KG, Northeim, Germany) was used. The ceramic infrared irradiator was preheated for 1 hour, and far-infrared radiation was irradiated for 30 minutes at a distance of 30 cm from the ceramic infrared irradiator to a 6-well plate containing HepG2 cells. After far-infrared irradiation, HepG2 cells were additionally cultured in an incubator at 37°C and 5% CO2 for 1 hour.

[0142]

[0143] Example 2. Western blot analysis

[0144] For Western blot analysis, HepG2 cells for each experimental trial were gently washed twice with DPBS (Welgene Inc., Gyeongsan, Korea) and lysed using lysis buffer (pH 7.5, 1 mM Tris-HCl 20 mM, Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA) containing 1 X Protease Inhibitor Cocktail™, 1 mM β-glycerophosphate, 1 mM phenylmethanesulfonyl fluoride, 1 mM NaF, and 1 mM Na3VO4. All samples were prepared at a concentration of 50 ug / ul, and equal amounts of protein (20 μg) were loaded using a BCA assay kit (Sigma-Aldrich, Massachusetts, USA), separated using SDS gel, and transferred to nitrocellulose blotting membranes with a pore size of 20 μm (Amersham™ Protran™ 0.2 μm NC; GE Healthcare Life science, Chicago, Illinois). After transferring the proteins to the membranes using a Western blot transfer kit (Biorad, California, USA), the membranes were incubated overnight with primary antibodies (PCSK9, LDLR, SREBP1, and SREBP2 in Table 3) at a 1:1000 dilution in 1 X TBST containing 5% skim milk (BD Life Sciences, New Jersey, USA) or with β-actin at a 1:3000 dilution in 5% skim milk. Next, the blots were washed three times for 10 min each with 1 X TBST and incubated for 1 h at room temperature in 5% skim milk containing secondary antibodies diluted 1:3000.The blots were washed three times with 1 X TBST for 10 min each, and the blots of target proteins were visualized using Amersham™ImageQuant™800 (Cytiva, Marlborough, Massachusetts, USA) according to chemiluminescence using ECL solution (Biorad, California, USA). Protein levels were quantified and analyzed using Image Lab software (Biorad, California, USA).

[0145]

[0146] AntibodySpeciesDilutionCompany (Catalog#)PCSK9Mouse1:1000Santa cruz(sc-515082)LDLRRabbit1:1000Abcam(EP1553Y)β-actinRabbit1:3000Abfrontier(LF-PA0207)

[0147]

[0148] Example 3. RT-PCR analysis

[0149] For reverse transcription polymerase chain reaction (RT-PCR) analysis, total RNA was purified from far-red irradiated HepG2 cells using the PureLink RNA Mini Kit (Invitrogen™, Massachusetts, USA). cDNA was synthesized using 5X Reverse Transcription Premix (Elpis Biotech, Daejeon, Republic of Korea) containing M-MLV reverse transcriptase, dNTPs, reaction buffer, and oligo dT15. The distinct primers in Table 4 were used to amplify specific target PCR products. The PCR conditions were 94 °C for 2 min, followed by 40 cycles of three sections: 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 5 min. PCR products were separated using an electrophoresis device on a 2% agarose gel (TAE buffer 0.5%), and the separated PCR products were visualized with Davinchi-Gel™ (Model No. CG550, YOUNG IN Labplus Co., Ltd., Gangnam, Republic of Korea). GAPDH was used as a reference gene.

[0150]

[0151] PrimersSequences (5' → 3')Amplicon size (bp)TRPV1F- CAG GCT CTA TGA TCG CAG GAGR- TTT GAA CTC GTT GTC TGT GAG G121TRPV2F- TCA GGT TGG AGA CAT TAG ATG GAR- TCG GTA GTT GAG GTT GAC TCT T155TRPV3F- AGG CTT CTA CTT CGG TGA GACR-AGG GCG TGA AGG ATG TTG TTG138TRPV4F- GAT GGG CGA CCA AAT CTG CR- GAG GAC TCA TAT AGG GTG GAC TC98TRPV5F- GGT TTT CTA CCT AAG GCA GAA GGR- CTC GAA GCA GTG GAG ACT CT148TRPV6F- GTG AGC CTC AAG TGG AAG CR- GTG CGG TTA TTG GTC CTG GG131GAPDHF- GTC AGT GGT GGA CCT GAC CTR- AGG GGT CTA CAT GGC AAC TG420

[0152]

[0153] Example 4. Statistical Analysis

[0154] Data values ​​are expressed as mean ± standard deviation (SD). Statistical significance was determined using a t-test for comparisons between two groups. Multiple comparisons were performed using one-way analysis of variance (ANOVA). All statistical analyses were performed using the statistical software provided by GraphPad Prism software version 7 (GraphPad Software, San Diego, CA). In all analyses, P <0.05 was considered statistically significant.

[0155]

[0156] Experimental Example 1. Confirmation of the PCSK9 expression inhibition effect of far-infrared rays emitted from carbon fiber sheets.

[0157] 1-1. Confirmation and optimization of the PCSK9 expression inhibition effect when irradiated with far-infrared rays using a ceramic infrared irradiator.

[0158] 1-1-1. Confirmation of the PCSK9 expression inhibition effect according to the far-infrared irradiation time.

[0159] To determine the effect of far-infrared irradiation with a ceramic infrared irradiator on the expression of PCSK9 in HepG2 cells, far-infrared irradiation was performed on HepG2 cells using the method of Comparative Example 1, and then Western blot was performed using the method of Example 2. As a result of the analysis, it was confirmed that the expression of PCSK9 protein in HepG2 cells was significantly reduced when far-infrared irradiation was performed with a ceramic infrared irradiator (p < 0.01, Fig. 7).

[0160] Furthermore, after irradiating the cells with far-infrared rays for 30 minutes using a ceramic infrared irradiator, the cells were incubated for 30 minutes and 1 hour. A significant decrease in PCSK9 protein levels was observed in the 1-hour incubation group (Fig. 8). Based on these results, the experimental conditions were irradiated with far-infrared rays for 30 minutes using a ceramic infrared irradiator, followed by an additional 1-hour incubation.

[0161]

[0162] 1-1-2. Confirmation of the PCSK9 expression suppression effect of far-infrared irradiation.

[0163] During far-infrared irradiation under the conditions of Comparative Example 1, temperature changes over time were captured using a thermal imaging camera and analyzed using the FLIR Thermal Studio program. As a result, it was confirmed that the ceramic infrared irradiator had a high radiant heat of 400 to 500 ℃, and that the temperature of the medium increased to approximately 37 ℃ when irradiated with far-infrared ray (Fig. 9). To confirm the potential thermal effect of ceramic far-infrared irradiation on PCSK9, the PCSK9 protein expression of HepG2 cells exposed to ceramic far-infrared irradiation was compared with that of cells maintained in a humidified CO2 incubator at 25 ℃ (room temperature, RT) and 37 ℃ without being exposed to far-infrared ray.

[0164] As a result of the analysis, there was no significant difference in the expression of PCSK9 protein in HepG2 cells not irradiated with far-infrared rays between 37°C and RT, but PCSK9 was significantly reduced only in HepG2 cells exposed to far-infrared rays (Fig. 10).

[0165] These results confirmed that the inhibition of PCSK9 by far-infrared rays was independent of the thermal effect.

[0166]

[0167] 1-2. Confirmation and optimization of the PCSK9 expression inhibition effect when carbon fiber sheets are irradiated with far-infrared radiation (CFIR).

[0168] To determine the effect of carbon fiber sheet far-infrared (CFIR) irradiation on PCSK9 expression in HepG2 cells, far-infrared irradiation was performed on HepG2 cells using the method of Example 1, and then Western blot was performed using the method of Example 2.

[0169] The analysis results showed that the temperature of the medium was maintained at 34-36℃, showing a lower temperature increase than the ceramic. In the case of PCSK9 protein expression of HepG2 cells, it was confirmed that it decreased more at a temperature of 60℃ than at 70℃, and when irradiated at 60℃ for 2 hours, the expression of PCSK9 was found to decrease further (p < 0.01, Fig. 11). Based on these results, the subsequent far-infrared irradiation of the carbon fiber sheet was conducted under the experimental conditions of irradiation at 60℃ for 2 hours.

[0170]

[0171] Experimental Example 2. Analysis of the Effects of Far-Infrared Rays Emitted from Carbon Fiber Sheets on LDLR and LDL Absorption in Liver Cells

[0172] Low-density lipoprotein (LDL) is known as a molecule that transports cholesterol from the liver and intestines to tissues via the bloodstream. To determine the effect of far-infrared irradiation on inhibiting PCSK9 expression on the low-density lipoprotein receptor (LDLR) and LDL uptake in HepG2 cells, an LDL uptake assay kit was used to analyze the effects of far-infrared irradiation on LDLR and LDL uptake.

[0173] Specifically, intracellular low-density lipoprotein uptake was assessed using the LDL Uptake Assay kit (ab133127, Abcam, Cambridge, UK) according to the manufacturer's protocol. HepG2 cells were counterstained with hematoxylin and seeded at a density of 3.0 × 10 in 6-well plates for 3 days. 5Cells were cultured at a density of 10 cells / well. The culture medium was changed 24 hours before far-infrared irradiation and placed in an incubator prior to the LDL staining protocol. After exposure to far-infrared irradiation, the cells were cultured with LDL-Dylight™ 550 (10 μg / ml) in a humidified 37°C incubator for 4 hours, and then washed three times with DBPS (Disinfection by-products). The cells were fixed with 4% PFA for 10 minutes and washed three times with 1 x TBST for 5 minutes each. The cells were then incubated with 3% BSA blocking buffer for 30 minutes at room temperature. LDL receptor expression was visualized by incubating with rabbit anti-LDL receptor primary antibody (Abcam, 1:500) for 1 hour, followed by incubation with FITC-conjugated secondary antibody (1:500) for 1 hour. The cells were washed three times with DPBS and counterstained with DAPI for 1 minute. Glass coverslips with stained cells were mounted on slides and sealed with nail polish. LDLR, LDL, and nuclei were visualized with FITC (LDLR, green), Texas red (LDL, red), and DAPI (nuclei, blue), respectively, using a confocal microscope (LSM5 Pascall, Cal ZEISS, Oberkocen, Germany).

[0174] As shown in Fig. 12A, the analysis results confirmed that far-infrared irradiation using a ceramic infrared irradiator increased the expression of LDLR (green) and thus the intracellular uptake of LDL (red). In addition, it was confirmed that far-infrared irradiation using a planar heating device made of carbon fiber sheets also increased the expression of LDLR (green) and thus the intracellular uptake of LDL (red) (Fig. 12B).

[0175] Through these results, it was confirmed that the far-infrared rays emitted from the ceramic infrared irradiator and the surface heating element device made of carbon fiber sheet were effective in increasing LDL absorption in HepG2 cells.

[0176]

[0177] Experimental Example 3. Analysis of the Effect of Far-Infrared Rays Emitted from a Carbon Fiber Sheet on the Expression of PCSK9 in Hepatocyte Calcium Channels

[0178] 3-1. Analysis of the effect on intracellular calcium concentration

[0179] Previous studies have shown that far-infrared irradiation increases intracellular calcium in tonsil-derived stem cells and bovine aorta endothelial cells (BAECs), leading to inhibition of adipogenesis and vasorelaxation, respectively (Kim et al., 2021; Park et al., 2013). To determine whether these changes persisted in HepG2 cells after far-infrared irradiation, intracellular calcium concentrations were measured using Fluo-4 AM (Thermo Fisher Science, Waltham, MA, USA), a fluorescent indicator of high-level calcium.

[0180] Specifically, intracellular calcium in HepG2 cells was stained with Fluo4-AM (0.5 μM), and the cultured HepG2 cells were exposed to far-red light for 30 min, followed by immediate washing with DPBS. Subsequently, the HepG2 cells were fixed with 4% paraformaldehyde (4% PFA, Tech&Innovation, Gangwon, Republic of Korea) for 15 min at room temperature. After washing twice with DPBS, the nuclei of the fixed HepG2 cells were stained with DAPI (5 mg / ml) for 1 min. Fluorescence intensity images were captured with a confocal microscope (LSM5 Pascall, Cal ZEISS, Oberkochen, Germany), and the fluorescence intensities were analyzed using Image J software.

[0181] As shown in Figure 13, the analysis results showed that far-infrared irradiation from a ceramic infrared irradiator significantly increased the intracellular calcium concentration of HepG2 cells. These results confirmed that the inhibition of PCSK9 expression by far-infrared irradiation may be related to changes in intracellular calcium concentration.

[0182]

[0183] 3-2. Analysis of the effect of intracellular calcium on PCSK9 expression

[0184] To determine whether intracellular calcium is involved in PCSK9 expression, HepG2 cells were treated with ionomycin (AG Scientific, CA, USA), an ionophore known to increase intracellular calcium. Ionomycin was used at various doses (2.5, 5, and 10 μM).

[0185] As a result of the analysis, it was confirmed that, similar to the results of Experimental Example 4-1, ionomycin reduces PCSK9 protein expression in HepG2 cells (Fig. 14).

[0186]

[0187] 3-3. Analysis of the Effect of TRP Channels on PCSK9 Expression

[0188] It is known that a representative form of intracellular calcium regulation is through the activation of TRP channels, which are calcium channels. Through previous studies, the present inventors have confirmed that the TRP channel TRPV2 plays a mediating role in the inhibition of adipogenic differentiation in tonsil-derived stem cells induced by ceramic far-infrared irradiation (Kim et al., 2021, 2019). Accordingly, to determine whether TRP channels are involved in determining intracellular calcium and PCSK9 expression levels in HepG2 cells after far-infrared irradiation, we measured the effect on PCSK9 using various concentrations of Ruthenium Red (Sigma, Massachusetts, USA), a known TRP channel inhibitor.

[0189] Specifically, HepG2 cells were treated with Ruthenium Red at concentrations of 2.5, 5, and 10 μM, and irradiated with far-infrared rays according to the conditions optimized in Experimental Example 1 and Comparative Example 1.

[0190] Our analysis revealed that ruthenium red concentration-dependently restored the decreased expression of PCSK9 induced by far-infrared irradiation from a ceramic infrared irradiator (Fig. 15A) and a planar heating device made of carbon fiber sheets (Fig. 15B). These results confirmed that the inhibition of PCSK9 protein expression by far-infrared irradiation was partially mediated through the activation of TRP calcium channels, which increased the intracellular calcium concentration in HepG2 cells.

[0191]

[0192] 3-4. Analysis of the Effect of TRPV4 Channels on PCSK9 Expression

[0193] The gene expression presented in Table 4 was investigated through reverse transcription polymerase chain reaction (RT-PCR) analysis in Example 3. As a result of the analysis, the expression of TRPV1, TRPV2, TRPV4, and TRPV6 genes was confirmed in HepG2 cells irradiated with far-infrared light (Fig. 16). Among these, TRPV1, TRPV2, and TRPV4 have been reported to be involved in lipid metabolism and lipogenesis (Dhakal and Lee, 2019), and were selected as potential candidates for regulating PCSK9 protein expression by far-infrared light irradiation.

[0194] To identify the TRPV channel that regulates PCSK9 among the above candidates, HepG2 cells were treated with the corresponding channel activators and inhibitors and then irradiated with far-infrared light. Specifically, when capsaicin (10, 25 μM) was treated as a TRPV1 activator, probenecid (1, 2, 4 mM) as a TRPV2 activator, and 4α-PDD (5, 10, 20 μM) as a TRPV4 activator, the effect of far-infrared irradiation was simulated in all cases, significantly reducing the protein expression of PCSK9 (Fig. 17).

[0195] In addition, treatment with RN1734, a TRPV4 inhibitor, at concentrations of 20, 40, and 60 μM significantly restored the decrease in PCSK9 protein expression induced by far-infrared irradiation (Fig. 18). On the other hand, inhibition of TRPV1 and TRPV2 by capsazepine (10, 20 μM) and tranilast (50, 100, and 200 μM) did not restore the decrease in PCSK9 protein expression induced by far-infrared irradiation, but rather further reduced PCSK9 protein expression. These results confirmed that TRPV4 is an important calcium channel activated by far-infrared irradiation and suppresses the protein expression of PCSK9.

[0196]

[0197] The present invention has been described above, focusing on specific embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0198]

[0199] (Explanation of symbols)

[0200] 110: Lower plate

[0201] 120: Top plate

[0202] 130: Guide shaft

[0203] 140: Elevator stage unit

[0204] 141: Elevator plate

[0205] 141a: Support groove

[0206] 142: Guide Bush

[0207] 150: Mobile Unit

[0208] 151: Fixed hub

[0209] 152: Screw shaft

[0210] 153: Rotating member

[0211] 160, 260: Heating unit

[0212] 161: Carbon fiber sheet surface heating element

[0213] 162, 262: Insulating blocks

[0214] 163, 263: First hydrophobic film

[0215] 164, 264: Second hydrophobic film

[0216] 165: Third hydrophobic film

[0217] 166, 266: Metal electrode

[0218] 170: Thermostat

[0219] 261a: First carbon fiber sheet surface heating element

[0220] 261b: Second carbon fiber sheet surface heating element

[0221] S: culture vessel

Claims

1. Contains a surface heating element made of carbon fiber sheet A device for preventing or improving hyperlipidemia, dyslipidemia, atherosclerosis, cardiovascular disease or hypercholesterolemia.

2. In paragraph 1, A device in which the target of far-infrared irradiation of the above-mentioned surface heating element is liver cells.

3. In paragraph 1, A device in which the far-infrared ray irradiation temperature of the above surface heating element is 30 to 90°C.

4. In paragraph 1, A device in which the far-infrared irradiation time of the above surface heating element is 10 minutes to 10 hours.

5. In the first paragraph, the device is a device that reduces the level of PCSK9 in hepatocytes.

Images