Protective effect of graphene heating film far-infrared hyperthermia against frostbite in mice

doi:10.12122/j.issn.1673-4254.2025.03.10

Abstract

Abstract:

Objective To investigate the protective effects of graphene heating film far-infrared (FIR) hyperthermia therapy against frostbite in mice and its impacts on microcirculation and coagulation function. Methods Seventy-six C57BL/6J mice were randomized into control, model, graphene-FIR, and carbon fiber-FIR groups. After 7-day FIR intervention (4 h/day), the mice were subjected to acute (4 ℃, 4 h) and intermittent (4 ℃, 4 h/day for 3 days) cold exposure and the changes in rectal temperature were monitored. In liquid nitrogen frostbite experiment, 24 ICR mice were divided into model, graphene-FIR, and carbon fiber-FIR groups, and after a 7-day FIR pretreatment (4 h/day), the liquid nitrogen frostbite models were established and apparent scores of the wounds were assessed on days 3 and 6 after modeling. In carrageenan-induced thrombosis experiment, 40 ICR mice were allocated to control, model, graphene-FIR, carbon fiber-FIR, and prazosin groups to test the effect of a 7-day FIR intervention on thrombosis induced by intraperitoneal carrageenan injection (2.5 mg/kg) by measuring thrombus length, blood perfusion, and serum biomarkers (6-keto-PGF1α, TXB2, t-PA, IL-6, IL-1β, TNF‑α) 24 h after the injection. Results The mice in graphene-FIR group showed significantly elevated rectal temperature in cold exposure tests. In mice with liquid nitrogen-induced frostbite, graphene-FIR treatment significantly reduced the wound scores and reduced frostbite area, producing better effects than carbon fiber. In mice with carrageenan-induced thrombosis, graphene-FIR treatment significantly decreased tail thrombosis length and thrombosis area, increased blood perfusion, lowered serum levels of TXB2, TNF-α and IL-6, and increased the levels of 6-keto-PGF1α and t-PA. Conclusion Graphene heating film FIR therapy can alleviate frostbite injury in mice by improving microcirculation, suppressing thrombosis and inflammatory responses, and reducing coagulation dysfunction.

Key words: graphene heating film, far-infrared therapy, frostbite, microcirculation, inflammation

Jinshui ZHANG, Shuo LI, Dongdong WEI, Xin CHENG, Yun DENG, Youzhi ZHANG. Protective effect of graphene heating film far-infrared hyperthermia against frostbite in mice[J]. Journal of Southern Medical University, 2025, 45(3): 522-530.

Figures/Tables 9

Fig. 1 Procedures of infrared thermal imaging experiment (A), acute and intermittent cold exposure experiment (B-C), liquid nitrogen frostbite experiment (D) and carrageenan-induced thrombosis experiment (E).

Fig. 2 Characterization of properties of graphene and carbon fiber materials. A: Schematic diagram of FIR device. B: Electrothermal properties of graphene and carbon fiber. C: Absorption peak of the human body surface (8.0 µm) and FIR emission peaks of graphene (8 µm) and carbon fiber (7.6 µm). D: Peak comparison of graphene, carbon fiber and human body at 7.5-8.5 µm wavelength (graphene: 1.9 W, carbon ﬁber: 1.9 W). E, F: Surface temperature changes and analysis of graphene-FIR and carbon fiber-FIR mice after irradiation. *P<0.05, **P<0.01 between the two groups.

Fig.3 Rectal temperature of mice after acute (A) and intermittent cold exposure (B). *P<0.05, **P<0.01 vs NC group, ##P<0.01 vs model group; $$P<0.01 vs Car group.

Fig. 4 Frostbite in the dorsal skin of the mice and temperature changes in the frostbite area (A) and comparison of wound temperatures among the 3 groups (B). ##P<0.01 vs model group.

Fig.5 Changes in apparent score of frostbite area in the mice on the 3rd day (A) and 6th day (B) after frostbite. *P<0.05, **P<0.01 vs model group; #P<0.05, ##P<0.01 vs Gra group.

Fig. 6 Thrombus in the tail of the mice (A) and comparison of the length of thrombus among the 4 groups (B). **P<0.01 vs NC group; #P<0.05 vs Model group.

Fig. 7 Blood perfusion images of the mouse tails (A) and comparison of blood perfusion parameters at the root (B) and tail (C) among the 5 groups. **P<0.01 vs NC group; ##P<0.01 vs Model group; $P<0.05, $$P<0.01 vs Car group.

Fig. 8 HE staining of the tissues of mouse tails taken at 2, 4, and 6 cm from the tail tip in each group (Original magnification: ×200). The blue and red dotted lines show the vessel profile and the thrombi formed in the vein, respectively.

Fig. 9 Changes of serum thrombus factors 6-keto-PGF1α, TXB2 and t-PA (A-C) and the inflammatory factors TNF-α, IL-6 and IL-1β (D-F). **P<0.01 vs NC group; #P<0.05, ##P<0.01 vs Model group.

References 30

1	Indo HP, Yen HC, Nakanishi I, et al. A mitochondrial superoxide theory for oxidative stress diseases and aging[J]. J Clin Biochem Nutr, 2015, 56(1): 1-7.
2	Goglia F, Liverini G, De Leo T, et al. Thyroid state and mitochondrial population during cold exposure[J]. Pflugers Arch, 1983, 396(1): 49-53.
3	Hess KL, Wilson TE, Sauder CL, et al. Aging affects the cardiovascular responses to cold stress in humans[J]. J Appl Physiol, 2009, 107(4): 1076-82.
4	Duncko R, Johnson L, Merikangas K, et al. Working memory performance after acute exposure to the cold pressor stress in healthy volunteers[J]. Neurobiol Learn Mem, 2009, 91(4): 377-81.
5	苗艳丰, 卫贺, 李诚, 等. 干姜干热药性活性分子的筛选与抗小鼠低温冻伤的甲状腺氧化应激保护作用机制[J]. 中草药, 2022, 53(17): 5379-88.
6	Sheridan RL, Goverman JM, Gregory Walker T. Diagnosis and treatment of frostbite[J]. N Engl J Med, 2022, 386(23): 2213-20.
7	Hsu YH, Chen YC, Chen YW, et al. Far-infrared radiation prevents decline in β-cell mass and function in diabetic mice via the mitochondria-mediated Sirtuin1 pathway[J]. Metabolism, 2020, 104: 154143.
8	Sharma N, Shin EJ, Kim NH, et al. Protective potentials of far-infrared ray against neuropsychotoxic conditions[J]. Neurochem Int, 2019, 122: 144-8.
9	Yu TT, Hu YM, Feng GP, et al. A graphene-based flexible device as a specific far-infrared emitter for noninvasive tumor therapy[J]. Adv Ther, 2020, 3(3): 1900195.
10	Zhang JS, Li S, Cheng X, et al. Far-infrared therapy based on graphene ameliorates high-fat diet-induced anxiety-like behavior in obese mice via alleviating intestinal barrier damage and neuroinflammation[J]. Neurochem Res, 2024, 49(7): 1735-50.
11	Li S, Miao XY, Zhang JS, et al. Far-infrared therapy promotes exercise capacity and glucose metabolism in mice by modulating microbiota homeostasis and activating AMPK[J]. Sci Rep, 2024, 14(1): 16314.
12	Lee H, Choi TK, Lee YB, et al. A graphene-based electrochemical device with thermoresponsive microneedles for diabetes monitoring and therapy[J]. Nat Nanotechnol, 2016, 11(6): 566-72.
13	Inoue SI, Emmett MJ, Lim HW, et al. Short-term cold exposure induces persistent epigenomic memory in brown fat[J]. Cell Metab, 2024, 36(8): 1764-78.e9.
14	苗明三, 项丽玲, 白明, 等. 冻伤动物模型制备规范(草案)[J]. 中国中药杂志, 2018, 43(2): 410-4.
15	Meng ZW, Wang SN, Chen FR, et al. Discovery of highly selective, potent, covalent, and orally bioavailable factor XIIa inhibitors for the treatment of thrombo-inflammation[J]. J Med Chem, 2024, 67(13): 10946-66.
16	Qin B, Fu SJ, Xu XF, et al. Far-infrared radiation and its therapeutic parameters: a superior alternative for future regenerative medicine?[J]. Pharmacol Res, 2024, 208: 107349.
17	Kowaltowski AJ. Cold exposure and the metabolism of mice, men, and other wonderful creatures[J]. Physiology, 2022, 37(5): 0.
18	Cheng YC, Lung CW, Jan YK, et al. Evaluating the far-infrared radiation bioeffects on micro vascular dysfunction, nervous system, and plantar pressure in diabetes mellitus[J]. Int J Low Extrem Wounds, 2020, 19(2): 125-31.
19	EricDrinkwater. Thermoregulation and Human Performance [M]. Basel: KARGER, 2008: 74-88.
20	王洪瑾, 李毅, 冯艳萍, 等. 红景天苷对慢性低氧后冻伤大鼠血管内皮细胞的保护作用[J]. 中国应用生理学杂志, 2022, 38(6): 664-9.
21	Geng Q, Holmér I, Hartog DEA, et al. Temperature limit values for touching cold surfaces with the fingertip[J]. Ann Occup Hyg, 2006, 50(8): 851-62.
22	宋晓丹, 成秀梅, 周湘, 等. 寒凝血瘀证动物模型研究现状[J]. 中国实验方剂学杂志, 2022, 28(15): 267-74.
23	Sun SW, Ding CB, Liu XL, et al. Silk protein/polyvinylpyrrolidone nanofiber membranes loaded with puerarin accelerate wound healing in mice by reducing the inflammatory response[J]. Biomater Adv, 2022, 135: 212734.
24	Li MQ, Tang XP, Liao ZY, et al. Hypoxia and low temperature upregulate transferrin to induce hypercoagulability at high altitude[J]. Blood, 2022, 140(19): 2063-75.
25	Sumner DS, Boswick JA Jr, Doolittle WH. Prediction of tissue loss in human frostbite with xenon-133[J]. Surgery, 1971, 69(6): 899-903.
26	Weatherley-white RC, Sjostrom B, Paton BC. Experimental studies in cold injury. ii. the pathogenesis of frostbite[J]. J Surg Res, 1964, 4: 17-22.
27	Li Q, Chen Y, Zhao D, et al. LongShengZhi Capsule reduces carrageenan-induced thrombosis by reducing activation of platelets and endothelial cells[J]. Pharmacol Res, 2019, 144: 167-80.
28	Wang TB, Guan RM, Xia F, et al. Curcumin promotes venous thrombi resolve process in a mouse deep venous thrombosis model via regulating miR-499[J]. Microvasc Res, 2021, 136: 104148.
29	Li Q, Chen Y, Zhao D, et al. NaoXinTong capsule inhibits carrageenan-induced thrombosis in mice[J]. J Cardiovasc Pharmacol, 2018, 72(1): 49-59.
30	Lu GJ, Guo HT, Zhang Y, et al. Graphene far-infrared irradiation can effectively relieve the blood pressure level of rat untr-HT in primary hypertension[J]. Int J Mol Sci, 2024, 25(12): 6675.

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