Use of ubc9 overexpression reagent in preparation of a medicament for preventing hypothermia caused by acute cold exposure

Abstract

This invention discloses the application of a UBC9 overexpression reagent in the preparation of drugs for preventing hypothermia caused by acute cold exposure, belonging to the field of biomedical technology. In vitro and in vivo experiments confirmed that acute cold exposure can induce a significant upregulation of UBC9 expression in skeletal muscle. Further, a skeletal muscle-specific UBC9 overexpression mouse model was constructed, revealing that UBC9 overexpression significantly enhances the core body temperature maintenance capacity of mice under acute cold exposure, upregulates the expression of the key thermogenic protein UCP3 and mitochondrial functional proteins TOMM20 and COX4, alleviates mitochondrial structural damage, and reduces mitochondrial oxidative stress levels. This invention reveals for the first time that UBC9 is a key target for the regulation of skeletal muscle thermogenicity under acute cold exposure. Enhancing UBC9 expression can effectively prevent or improve skeletal muscle thermogenic dysfunction caused by cold environments, providing a novel intervention strategy for developing drugs to prevent and treat cold injury.

Description

Technical Field

[0001] This invention belongs to the fields of biology and molecular biology, specifically relating to the application of UBC9 overexpression reagent in the preparation of drugs for preventing hypothermia caused by acute cold exposure. Background Technology

[0002] Cold environments can have various adverse effects on human health, not only interfering with the body's thermoregulation but also increasing the risk of various diseases. When exposed to low temperatures, the human body rapidly experiences peripheral vasoconstriction to reduce heat loss, while simultaneously activating multiple heat-producing organs to initiate heat production processes and maintain core body temperature homeostasis. Prolonged or acute exposure to extreme cold can lead to thermoregulation imbalances, inducing hypothermia and dysfunction of multiple organs, including the respiratory, circulatory, and metabolic systems, which is particularly severe in the elderly and patients with chronic diseases. In my country's vast high-altitude and cold regions, long winters and frequent extreme cold weather pose serious cold-related health threats to residents, outdoor workers, and vulnerable populations. Furthermore, the acute hypothermia-related injuries and deaths caused by extreme cold waves in recent years further highlight the urgency of elucidating the mechanisms of damage and protection after acute cold exposure and identifying key regulatory targets.

[0003] Under acute cold exposure stress, the body primarily maintains core body temperature through both shivering thermogenesis (ST) and non-shivering thermogenesis (NST) mechanisms. Skeletal muscle, as one of the body's largest metabolic organs, boasts a wide distribution and enormous thermogenic potential. It participates in adaptive thermogenesis via both ST and NST pathways, making it a crucial thermogenic tissue that plays an irreplaceable role under acute extreme cold exposure conditions. Skeletal muscle thermogenesis mainly depends on mitochondrial homeostasis, achieving the conversion of energy metabolism into heat energy through various mechanisms such as promoting mitochondrial biogenesis, regulating calcium ion homeostasis, and inducing mitochondrial uncoupling. However, under acute extreme cold exposure conditions, the body's metabolic load increases dramatically, leading to disruption of the skeletal muscle thermogenetic regulatory network and impaired mitochondrial function. The specific molecular mechanisms underlying this decreased thermogenic capacity remain not fully elucidated. Therefore, identifying key regulatory molecules capable of maintaining and remodeling skeletal muscle thermogenetic function under such extreme stress is of significant scientific value for developing targeted drugs to prevent and treat cold injury.

[0004] Ubiquitin-conjugating enzyme 9 (UBC9) is the only E2-binding enzyme in the small ubiquitin-like modifier (SUMOylation) pathway. Containing a SUMO-binding domain, it plays a core role in mediating the SUMOylation cascade. Previous studies have shown that elevated levels of UBC9 and SUMOylation in cells can participate in the regulation of various metabolic processes, including myocardial energy metabolism reprogramming, skeletal muscle fatty acid oxidation, and insulin sensitivity regulation. Furthermore, other studies have reported that SUMOylation can regulate PPARδ transcriptional activity, thereby promoting skeletal muscle fatty acid oxidation and energy metabolism. In skeletal muscle tissue of patients with severe insulin resistance and type 2 diabetes, the expression levels of glucose transporter type 4 (GLUT4) and the key SUMOylation E2-binding enzyme UBC9 were significantly downregulated. In addition, SUMOylation also participates in the regulation of multiple mitochondrial-related signaling pathways, including mitophagy and oxidative stress. However, the role and molecular mechanism of UBC9 in the regulation of skeletal muscle thermogenesis under the specific severe physiological stress of acute cold exposure remain unclear. Summary of the Invention

[0005] Addressing the problems of existing technologies, this invention is the first to demonstrate that UBC9 overexpression can significantly delay the rate of decrease in core body temperature under acute cold exposure. In a 4°C acute cold exposure model, the core body temperature of UBC9-overexpressing mice was significantly higher than that of the control group within 3-6 hours of cold exposure, indicating a significantly reduced risk of developing hypothermia (core body temperature <35°C). Therefore, this invention achieves effective prevention of hypothermia caused by acute cold exposure by preventing skeletal muscle thermogenic dysfunction.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses the use of UBC9 overexpression reagent in the preparation of drugs for the prevention and / or treatment of hypothermia caused by acute cold exposure.

[0008] Furthermore, the UBC9 overexpression reagent reduces the risk of hypothermia by preventing and / or improving skeletal muscle thermogenic impairment caused by acute cold exposure.

[0009] Furthermore, in any of the above applications, the UBC9 overexpression reagent exerts a protective effect by upregulating the expression of UCP3, TOMM20 and / or COX4 in skeletal muscle, and / or by reducing oxidative stress in skeletal muscle mitochondria and maintaining mitochondrial structural integrity.

[0010] Furthermore, in any of the above applications, the UBC9 overexpression reagent is an expression vector containing the UBC9 encoding gene.

[0011] Preferably, the expression vector is a eukaryotic expression vector.

[0012] Preferably, the eukaryotic expression vector is a plasmid expression vector or a viral expression vector.

[0013] Preferably, the viral expression vector is an adenovirus vector, a lentivirus vector, or an adeno-associated virus vector.

[0014] This invention also discloses the application of reagents for detecting the expression level of UBC9 protein or gene in the preparation of kits for assessing the risk of skeletal muscle thermogenic impairment caused by acute cold exposure.

[0015] This invention also discloses the application of reagents for detecting the expression level of UBC9 protein or gene in the preparation of kits for predicting the risk of hypothermia caused by acute cold exposure.

[0016] The "acute cold exposure-induced skeletal muscle thermogenic disorder" described in this invention is the core upstream mechanism leading to acute cold exposure-related hypothermia. Preventing or improving this disorder can effectively reduce the risk of hypothermia. However, the pharmaceutical use of this invention does not directly cover the emergency treatment of patients who have already developed hypothermia.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] This invention can effectively delay or prevent the drop in core body temperature by preventing or improving skeletal muscle heat production disorders caused by acute cold exposure, thereby reducing the incidence and severity of acute cold exposure-related hypothermia and providing a new intervention strategy for the clinical prevention and treatment of cold environment injuries.

[0019] This invention elucidates for the first time the intrinsic link between UBC9 and acute cold exposure injury. Experimental results show that after 24 hours of acute cold exposure at 4°C, both the mRNA and protein expression of UBC9 in mouse skeletal muscle were significantly upregulated; similarly, after 24 hours of cold stimulation at 32°C, UBC9 expression in skeletal muscle cells was significantly increased. These results confirm that UBC9 can serve as a novel biomarker for skeletal muscle thermogenic activation under acute cold exposure, providing an innovative molecular target for the early prediction, treatment, and prognosis of skeletal muscle thermogenic impairment caused by acute cold exposure.

[0020] This invention further confirms through in vivo and in vitro functional experiments that UBC9 overexpression can significantly enhance the thermogenic capacity of skeletal muscle under acute cold exposure: Animal experiments show that under continuous cold exposure at 4°C for 24 hours, UBC9 overexpression can significantly increase the expression levels of key thermogenic proteins UCP3, mitochondrial marker proteins TOMM20 and COX4 in mouse skeletal muscle, effectively maintaining mitochondrial homeostasis of skeletal muscle cells and enhancing the thermogenic effect; Cell experiments show that UBC9 overexpression can also significantly upregulate the expression of the above-mentioned thermogenic and mitochondrial-related proteins, enhance cellular thermogenic function, and clarify that UBC9 has a clear protective effect against skeletal muscle thermogenic impairment caused by acute cold exposure.

[0021] This invention fills the research gap in UBC9 regulation of skeletal muscle adaptive thermogenesis, verifies the feasibility of UBC9 as a drug target for preventing and treating skeletal muscle thermogenic disorders caused by acute cold exposure, and constructs a targeted intervention strategy with UBC9 overexpression as the core. This not only provides new theoretical support for elucidating the molecular mechanism of skeletal muscle thermogenic disorders caused by acute cold exposure, but also provides new ideas and directions for the innovative drug development of metabolic-related diseases such as diabetes and obesity. Attached Figure Description

[0022] Figure 1 UBC9 expression was upregulated in mouse skeletal muscle tissue after acute cold exposure at 4°C. A. Western blot analysis of UBC9 and UCP3 protein expression in mouse skeletal muscle tissue after 24 hours of cold exposure at 4°C. B. qPCR analysis of UBC9 transcriptional expression in mouse skeletal muscle tissue after 24 hours of cold exposure at 4°C. C. Immunofluorescence analysis of co-localization expression of UBC9 and UCP3 in mouse skeletal muscle tissue after 24 hours of cold exposure at 4°C (n=3).

[0023] Figure 2 UBC9 expression was upregulated in C2C12 skeletal muscle cells after 32°C cold stimulation. A. Western blot analysis of UBC9 and UCP3 protein expression in C2C12 skeletal muscle cells after 0, 6, 12, and 24 hours of 32°C cold stimulation. B. qPCR analysis of UBC9 transcriptional expression in C2C12 skeletal muscle cells after 0, 6, 12, and 24 hours of 32°C cold stimulation. C. Immunofluorescence analysis of co-localization expression of UBC9 and UCP3 in skeletal muscle cells after 32°C cold stimulation (n=3).

[0024] Figure 3A successful mouse model of UBC9 skeletal muscle specific overexpression was achieved. A. Schematic diagram of AAV-UBC9 overexpression mouse virus construction. B. Western blot analysis of UBC9 protein levels in skeletal muscle tissue of mice in the AAV-NC group and the AAV-UBC9 adeno-associated virus overexpression group. C. qPCR analysis of UBC9 transcriptional levels in skeletal muscle tissue of mice in the AAV-NC group and the AAV-UBC9 adeno-associated virus overexpression group. Data are expressed as mean ± SEM, n=3.

[0025] Figure 4 Mice with UBC9 skeletal muscle-specific overexpression showed upregulated body temperature, thermoproduction, and mitochondrial function during acute cold exposure at 4°C. A. Core body temperature changes in AAV-UBC9 adeno-associated virus (AAV-UBC9) overexpression and control mice. B. Western blot analysis of UBC9, UCP3, TOMM20, and COX4 protein levels in skeletal muscle tissue of AAV-UBC9 adeno-associated virus (AAV-UBC9) overexpression and control mice. C. qPCR analysis of UBC9, UCP3, TOMM20, and COX4 transcriptional levels in skeletal muscle tissue of AAV-UBC9 adeno-associated virus (AAV-UBC9) overexpression and control mice. D. Electron microscopy analysis of mitochondrial morphological changes in skeletal muscle tissue of AAV-UBC9 adeno-associated virus (AAV-UBC9) overexpression and control mice. Data are expressed as mean ± SEM, n=6.

[0026] Figure 5 UBC9 overexpression in C2C12 skeletal muscle cells upregulated thermoproduction and mitochondrial function under 32°C cold stimulation. A. Western blot analysis of changes in the expression of the thermogenic gene UCP3 and mitochondrial genes TOMM20 and COX4 in C2C12 cells after UBC9 overexpression. B. Immunofluorescence analysis of changes in mitochondrial membrane potential JC-1 in C2C12 cells after UBC9 overexpression. C. Immunofluorescence analysis of changes in the mitochondrial oxidative stress marker Mitosox in C2C12 cells after UBC9 overexpression. Data are expressed as mean ± SEM, n=6. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention. Unless otherwise specified, all reagents and materials used in the present invention are commercially available. All experimental data in the present invention are percentages. The chi-square test was used to compare the two sample rates. Statistical analysis was performed using GraphPad Prism 9.5 software, with P < 0.05 considered statistically significant.

[0028] Example 1: UBC9 expression was upregulated in mouse skeletal muscle tissue after acute cold exposure at 4°C.

[0029] 1. Laboratory animals and their care.

[0030] Laboratory animal species, sex, age, and origin: C57BL / 6J mice, male, 10 weeks old. C57BL / 6J mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Mice were housed in a specific pathogen-free (SPF) grade animal laboratory at a controlled temperature of 25℃ and relative humidity of 45%–70%, using a 12h light / 12h dark cycle. They had free access to food and water.

[0031] 2. Establishment of an acute cold exposure model in mice.

[0032] To establish an acute cold exposure model, a 4°C cold stimulation model was used. Mice were randomly divided into a Control group and a 4°C acute cold exposure group. The Control group was given a 25°C stimulus, while the 4°C acute cold exposure group was given a 4°C cold stimulus for 6h, 12h, and 24h. Both groups were given food and water. Mice were then sacrificed, and skeletal muscle tissue was extracted.

[0033] 3. Western blot was used to detect the expression of UBC9 and UCP3 in skeletal muscle tissue of mice in the room temperature group and the acute cold exposure group.

[0034] (1) Extraction of skeletal muscle tissue protein: Take an appropriate amount of mouse skeletal muscle tissue, cut it into small tissue pieces, place it in a 1.5 mL centrifuge tube, add 200 µL RIPA lysis buffer, and then add an appropriate amount of grinding beads to the tube; place the centrifuge tube in a tissue homogenizer and grind it at 60 Hz for 60 sec to fully break the tissue, and then place it on ice for lysis for 30 min, vortexing as needed during the process.

[0035] (2) Protein concentration determination: ① The protein concentration of each sample was determined using the BCA method. Gradient concentrations of BCA standards were prepared at 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0 mg / mL for establishing the standard curve. ② The standards and the target protein sample were added to each well of a 96-well plate. 25 µL of the standards were added to each well; the target protein sample was diluted with 5 µL of target protein to 20 µL of distilled water, and then added to each well at a ratio of 25 µL. ③ 200 µL of a freshly prepared mixture of BCA reagent kit solution A and solution B (A:B = 200:4, volume ratio) was added to each well. ④ The 96-well plate was thoroughly mixed and incubated in a 37°C incubator in the dark for 30 min to ensure the colorimetric reaction proceeded fully. ⑤ After incubation, the absorbance (OD value) of the standards and the target protein sample was measured at 570 nm using a microplate reader. ⑥ Establish a standard curve with the concentration of BCA standard as the x-axis and the corresponding OD value as the y-axis. Calculate the actual protein concentration of each protein sample to be tested based on the standard curve fitting equation.

[0036] (3) Western blot: ① Protein sample preparation: Take 40µg of protein sample from each group, adjust the loading volume to 20µL, add 5µL of 4× loading buffer, and make up the remaining volume with distilled water. After thorough shaking and mixing, boil at 100℃ for 10min to fully denature the protein, and take the supernatant for subsequent electrophoresis after centrifugation. ② SDS PAGE gel preparation: Prepare separating gel and stacking gel of appropriate concentration according to the molecular weight of the target protein. Generally, the larger the molecular weight of the target protein, the lower the concentration of the separating gel should be; the smaller the molecular weight, the higher the concentration of the separating gel should be. In this study, a 12% separating gel and the corresponding stacking gel were used for electrophoretic separation. ③ Electrophoresis: Load 20µL of protein sample and perform SDS PAGE electrophoresis at a constant voltage of 100V. ④ Transfer membrane: Cut the PVDF membrane to 7.5cm×5.5cm and activate it with methanol for 30sec. Place filter paper, PVDF membrane, gel and filter paper in the order from negative electrode to positive electrode, and transfer the membrane at a constant voltage of 90V for 2h. ⑤ Blocking: Place the PVDF membrane in 5% skim milk blocking solution and block at room temperature for 2 hours. ⑥ Primary antibody incubation: Incubate the membrane with a 1:1000 diluted primary antibody at 4°C for 12 hours, then wash three times with TBST for 5-15 minutes each time. ⑦ Secondary antibody incubation: Incubate the membrane with a 1:5000 diluted secondary antibody of the corresponding species on a shaker at room temperature for 1 hour, then wash three times with TBS for 15 minutes each time. ⑧ Development: Mix equal volumes of ECL chemiluminescence solutions A and B, cover the PVDF membrane in the dark, and image using manual or automatic mode and save the image. ⑨ Result analysis: Quantitatively analyze the grayscale values ​​of the target bands using ImageJ software.

[0037] The results showed that, compared with the control group, the expression of UBC9 protein and the expression of the thermogenic marker protein UCP3 were significantly upregulated in the skeletal muscle tissue of mice in the 4℃ acute cold exposure group. (See attached figures.) Figure 1 A. This indicates that the expression level of UBC9 is significantly positively correlated with the progression of acute cold exposure.

[0038] 4. qPCR was used to detect the expression of UBC9 in the skeletal muscle tissue of mice in the room temperature group and the acute cold exposure group.

[0039] (1) RNA extraction from mouse skeletal muscle tissue. ① Take an RNase-free centrifuge tube, put in 3 RNase-free grinding beads, add mouse skeletal muscle tissue, then add 1 mL of Trizol reagent, place in a tissue homogenizer, and homogenize 3 times at a frequency of 60 Hz, 60 sec each time. ② Place the above EP tube at room temperature for 5 min, add 200 µL of chloroform, invert the EP tube by hand to mix thoroughly, and continue to stand at room temperature for 15 min; then centrifuge at 4℃ and 12000 rpm for 15 min. ③ After centrifugation, carefully transfer the supernatant to another new RNase-free EP tube, add an equal volume of isopropanol to the supernatant, invert to mix, and stand at room temperature for 10 min, then centrifuge at 4℃ and 12000 rpm for 15 min.

[0040] ④ Discard the supernatant after centrifugation, add 1 mL of 75% ethanol to the EP tube, gently invert to suspend the precipitate, and then centrifuge at 4℃ and 12000 rpm for 15 min, discarding the supernatant. ⑤ Place the EP tube at room temperature to air dry the precipitate. After the precipitate is dry, add 10 µL of RNase-free water and gently pipette to fully dissolve the RNA.

[0041] (2) Reverse transcription reaction.

[0042] Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser manufactured by Takara. Following the kit instructions strictly, the reaction mixture was prepared with gDNA Eraser, 5×gDNA Eraser Buffer, total RNA, and RNase-free water in the specified proportions. Genomic DNA removal was performed first at 42°C for 2 minutes. After genomic DNA removal, PrimeScript Buffer, RNase-free water, RT Enzyme Mix I, and RT Primer Mix were added to the reaction mixture in the specified proportions. The mixture was thoroughly mixed before reverse transcription, with the following reaction program: incubation at 37°C for 15 minutes, followed by heating at 85°C for 5 seconds to terminate the reaction.

[0043] (3) qPCR reaction.

[0044] First, the coding sequence of the target mouse gene was retrieved from the NCBI database, and primers were designed using this database. Real-time PCR (qPCR) was performed using the SYBR Premix Ex Taq II kit. The reaction mixture consisted of: 0.5 µL forward primer, 0.5 µL reverse primer, 1 µL cDNA template, 3 µL RNase-free water, and 5 µL TB Green. The qPCR program was: 95℃ pre-denaturation for 30 sec; 95℃, 5 sec, 40 cycles; followed by sequential reactions at 95℃ for 30 min (40 cycles), 95℃ for 15 min, 60℃ for 30 min, 95℃ for 15 min, and so on. The 18S gene was used as an internal control gene, and 2... -ΔΔCt The method was used to analyze the expression level of the target gene. The primer sequences are as follows: .

[0045] The results showed that, compared with the control group, the transcriptional expression of UBC9 in the skeletal muscle tissue of mice in the 4℃ acute cold exposure group was significantly upregulated. (See attached results). Figure 1 B. This indicates that the expression level of UBC9 is significantly positively correlated with the progression of acute cold exposure.

[0046] 5. Immunofluorescence was used to detect the colocalization of UBC9 and UCP3 in the skeletal muscle tissue of mice in the room temperature group and the acute cold exposure group.

[0047] (1) Paraffin section preparation steps: ① Tissue sampling and fixation: Take the target tissue and place it in 4% paraformaldehyde fixative for 12-24 hours to ensure sufficient fixation and maintain cell morphology integrity. ② Tissue dehydration: Place the fixed tissue in a Thermo automated tissue dehydrator and perform gradient dehydration according to the standard dehydration program to remove water from the tissue. ③ Tissue clearing: Place the dehydrated tissue in xylene I and xylene II in sequence, soaking for 45 minutes each time, to make the tissue clear and facilitate subsequent paraffin infiltration. ④ Tissue paraffin infiltration: Transfer the cleared tissue to melted paraffin I for overnight paraffin infiltration, and then place it in paraffin II and paraffin III in sequence, soaking for 1 hour each time to ensure that the paraffin fully penetrates into the tissue. ⑤ Tissue embedding: Place the fully paraffin-infiltrated tissue in an embedding cassette, add melted paraffin for embedding, embed for 5-6 hours, and store at room temperature for later use after the paraffin has completely solidified. ⑥ Paraffin sectioning: The embedded tissue was serially sectioned using a Leica paraffin microtome to a thickness of 4µm. The sectioned tissue sections were then evenly attached to glass slides treated to prevent detachment. ⑦ Baking: The glass slides with the sections attached were first placed in a 60 ℃ oven for 1-2 hours to remove residual moisture from the surface of the sections, and then transferred to a 65 ℃ oven for 48 hours to enhance the adhesion between the sections and the glass slide and prevent the sections from falling off during subsequent staining.

[0048] (2) Antigen retrieval: The slide was placed in 1× antigen retrieval solution prepared by 203 mL of double distilled water and 7 mL of 30× antigen retrieval solution, heated in a water bath at 100°C for 40 min, and then cooled naturally to room temperature.

[0049] (3) Immunofluorescence staining: ① Cell permeabilization: Permeabilize with 0.2% Triton X 100 at room temperature for 5 min. ② Blocking: Block with goat serum at room temperature for 30 min. ③ Primary antibody incubation: Discard the blocking serum, do not wash the slides, add 50 µL of primary antibody working solution diluted 1:100 to each slide, and incubate overnight at 4℃. ④ Warming: Remove and place at room temperature for 30 min. ⑤ Secondary antibody incubation: Wash 3 times with PBS, dilute secondary antibody working solution 1:100, and incubate at room temperature in the dark for 1 h. ⑥ Mounting: After washing 3 times with PBS, air dry and mount with anti-fade anti-fluorescence quenching mounting medium.

[0050] The results showed that UBC9 and the thermogenic marker protein UCP3 co-localized in mouse skeletal muscle tissue, and their expression was enhanced in the 4°C acute cold exposure group. (See attached figures.) Figure 1 C. This indicates that UBC9 and UCP3 are co-located.

[0051] Example 2: UBC9 expression was upregulated in C2C12 skeletal muscle cells subjected to 32°C cold stimulation.

[0052] 1. Western blot was used to detect the expression of UBC9 and UCP3 proteins in skeletal muscle cells of the control group (37℃) and the 32℃ cold stimulation group (32℃ for 6h, 12h, and 24h).

[0053] (1) Extraction of total protein from skeletal muscle cells: Skeletal muscle cells from the control group and the 32℃ cold stimulation group were collected, washed with PBS, and then lysed with an appropriate amount of RIPA lysis buffer on ice for 30 min. The supernatant was obtained by centrifugation at 12000 rpm / s for 20 min at 4℃. The supernatant was the extracted total protein from the cells.

[0054] (2) Determine protein concentration and Western blot: The specific operation steps are the same as in Example 1.

[0055] The results showed that, compared with the control group, the expression of UBC9 protein and the expression of the thermogenic marker protein UCP3 were significantly upregulated in the 32℃ cold stimulation group. (See attached figures.) Figure 2 A. This indicates that the expression level of UBC9 is significantly positively correlated with the progression of acute cold exposure.

[0056] 2. qPCR was used to detect the transcriptional expression of UBC9 in skeletal muscle cells of the control group (37℃) and the 32℃ cold stimulation group (32℃ for 6h, 12h, and 24h).

[0057] (1) Extraction of total RNA from cells: Skeletal muscle cells from the control group and the 32℃ cold stimulation group were collected, washed with PBS, and then lysed with 1 mL of Trizol. The subsequent RNA extraction steps were the same as in Example 1.

[0058] (2) qPCR detection of UBC9 transcriptional expression in skeletal muscle cells of the control group and the 32℃ cold stimulation group: the specific steps are the same as in Example 1.

[0059] The results showed that, compared with the control group, the UBC9 transcription level in skeletal muscle cells was significantly upregulated in the 32℃ cold stimulation group. (See attached figures.) Figure 2 B. This indicates that the expression level of UBC9 is significantly positively correlated with the progression of acute cold exposure.

[0060] 3. Immunofluorescence was used to detect the co-localization expression of UBC9 and UCP3 in skeletal muscle cells of the control group (37℃) and the 32℃ cold stimulation group (32℃ for 6h, 12h, and 24h).

[0061] (1) Preparation of cell crawling slides: Collect skeletal muscle cell crawling slides from the control group and the 32℃ cold stimulation group, wash with PBS, and fix in 4% paraformaldehyde fixative for 30 min.

[0062] (2) Immunofluorescence staining: Immunofluorescence detection of co-localization expression of UBC9 and UCP3 in skeletal muscle cells of the control group and the 32℃ cold stimulation group: The specific steps are the same as in Example 1.

[0063] The results showed that UBC9 and the thermogenic marker protein UCP3 co-localized in mouse skeletal muscle cells, and their expression was enhanced in the 32℃ cold stimulation group. (See attached figures.) Figure 2 C. This indicates that UBC9 and UCP3 are co-located.

[0064] Example 3: Successful establishment of a mouse model with UBC9 skeletal muscle-specific overexpression.

[0065] To determine whether skeletal muscle-specific overexpression of UBC9 has a protective effect against acute cold exposure-induced thermogenic impairment, an adeno-associated virus (pAAV-tMCK-) specifically overexpressing UBC9 was first constructed. UBC9 The virus vectors pAAV-tMCK-MCS-EGFP-3xFLAG-WPRE (abbreviated as AAV-UBC9) and control virus pAAV-tMCK-MCS-EGFP-3xFLAG-WPRE (abbreviated as AAV-NC) were both constructed at Heyuan Biotechnology (Shanghai) Co., Ltd. A schematic diagram of the viral vector construction is shown below. Figure 3 A. Skeletal muscle-specific UBC9 overexpression mice were constructed in C57BL / 6J mice by spot injection of adeno-associated virus specifically overexpressing UBC9 into the skeletal muscle. After 3 weeks, the UBC9 expression level was detected by Western blot and qPCR experiments to verify the overexpression efficiency of UBC9. Specific experimental methods for Western blot and qPCR experiments are detailed in Example 1.

[0066] The results showed that, compared with the AAV-NC group, the expression of UBC9 in skeletal muscle tissue was significantly increased in the AAV-UBC9 group. (See attached figures.) Figure 3 BC. The results indicate that skeletal muscle-specific overexpression of UBC9 was successfully established in mice.

[0067] Example 4: UBC9 skeletal muscle-specific overexpression mice showed upregulated body temperature, thermoproduction, and mitochondrial function during acute cold exposure at 4°C.

[0068] 1. Acute cold exposure test and core body temperature measurement of AAV-NC and AAV-UBC9 mice.

[0069] AAV-NC and AAV-UBC9 mice were randomly divided into groups and placed in an environment of 4°C for acute cold exposure stimulation. Body temperature was measured before and after the experiment. During the experiment, rectal temperature was measured and recorded every 1 hour using a four-channel thermocouple probe gently inserted into the mouse anus.

[0070] The results showed that, compared with AAV-NC, AAV-UBC9 mice showed no statistically significant difference in core body temperature at 2 hours of cold exposure, but their core body temperature was significantly higher than that of the control group from 3 hours onwards, and remained higher until 6 hours of cold exposure. (See attached results). Figure 4 A. The results showed that mice with skeletal muscle-specific overexpression of UBC9 had significantly enhanced core body temperature maintenance.

[0071] 2. Western blot was used to detect the expression of UBC9, the thermogenic gene UCP3, and the mitochondrial genes TOMM20 and COX4 proteins in the skeletal muscle tissue of mice in the AAV-NC and AAV-UBC9 groups.

[0072] (1) Extraction of skeletal muscle tissue protein: Take an appropriate amount of mouse skeletal muscle tissue, cut it into small tissue pieces, place it in a 1.5 mL centrifuge tube, add 200 µL of RIPA lysis buffer, and then add an appropriate amount of grinding beads to the tube; place the centrifuge tube in a tissue homogenizer and grind it at 60 Hz for 60 sec to fully break the tissue, and then place it on ice for lysis for 30 min, vortexing as needed during the process.

[0073] (2) Determine protein concentration and Western blot: The specific operation steps are the same as in Example 1.

[0074] The results showed that, during acute cold exposure at 4°C, compared with the AAV-NC group, the expression of UBC9 protein in the skeletal muscle tissue of mice in the AAV-UBC9 group was significantly upregulated, as were the expression of the thermogenic gene UCP3 and the mitochondrial genes TOMM20 and COX4. (See attached figures.) Figure 4 B.

[0075] 3. qPCR was used to detect the transcriptional expression of UBC9, the thermogenic gene UCP3, and the mitochondrial genes TOMM20 and COX4 in the skeletal muscle tissue of mice in the AAV-NC and AAV-UBC9 groups.

[0076] (1) Extraction of RNA from skeletal muscle tissue: Skeletal muscle tissue from AAV-NC and AAV-UBC9 mice was collected, cut into small tissue pieces, placed in 1.5 mL centrifuge tubes, and 1 mL of Trizol was added for lysis. Then, an appropriate amount of enzyme-free grinding beads were added to the tubes. The centrifuge tubes were placed in a tissue homogenizer and homogenized at 60 Hz for 60 sec to fully break down the tissue. The tissue was then placed on ice for lysis for 30 min, with vortexing as needed during the process. The subsequent RNA extraction steps were the same as in Example 1.

[0077] (2) qPCR detection of UBC9 transcriptional expression in skeletal muscle tissue of AAV-NC and AAV-UBC9 groups mice: the specific steps are the same as in Example 1.

[0078] The results showed that, during acute cold exposure at 4°C, compared with the AAV-NC group, the transcriptional expression of UBC9 in the skeletal muscle tissue of mice in the AAV-UBC9 group was significantly upregulated, as were the transcriptional expressions of the thermogenic gene UCP3 and the mitochondrial genes TOMM20 and COX4. (See attached figures.) Figure 4 C.

[0079] 4. Electron microscopy was used to examine the morphological changes of mitochondria in the skeletal muscle tissue of mice in the AAV-NC and AAV-UBC9 groups.

[0080] (1) Sample fixation: Fresh skeletal muscle tissue from mice was immediately placed in electron microscopy fixation solution to maintain the integrity of the tissue and mitochondria's ultrastructure.

[0081] (2) Washing and dehydration: The fixed sample was washed three times with sodium dimethylarsinate buffer, each time thoroughly; then the alcohol-acetone gradient dehydration method was used to perform density gradient dehydration to ensure thorough dehydration and prepare for subsequent embedding.

[0082] (3) Sectioning and staining: The dehydrated sample was sectioned into ultrathin sections with a thickness of 60-80 nm. After staining, the sections were left to air dry at room temperature overnight.

[0083] (4) Electron microscopy imaging: The samples were observed and images were acquired using a JEM-2000 EX transmission electron microscope from Servicebio, Japan.

[0084] (5) Mitochondrial morphological analysis: The number and size of mitochondria were quantitatively analyzed using iTEM software. The number of mitochondria was counted using a 100µm² square field of view at the same magnification. For the mitochondrial diameter measurement, samples from 3 mice in each group were selected for each experiment, and the diameter of mitochondria in each group was quantitatively detected to ensure the reliability of the analysis results.

[0085] The results showed that, during acute cold exposure at 4°C, compared with the AAV-NC group, the AAV-UBC9 group mice exhibited increased mitochondrial numbers, reduced mitochondrial swelling, and a significantly decreased number of abnormal mitochondria in skeletal muscle tissue. (See attached figures). Figure 4 D. This indicates that overexpression of UBC9 can alleviate mitochondrial damage caused by acute cold exposure.

[0086] Example 5: UBC9 overexpression of C2C12 skeletal muscle cells upregulated thermoproduction and mitochondrial function under 32°C cold stimulation.

[0087] 1. Western blot was used to detect the expression of UBC9, UCP3, TOMM20, and COX4 proteins in skeletal muscle cells of the adNC and adUBC9 groups.

[0088] (1) Extraction of total protein from skeletal muscle cells: Skeletal muscle cells from the adNC group and the adUBC9 group were collected, washed with PBS, and then lysed with an appropriate amount of RIPA lysis buffer on ice for 30 min. The supernatant was obtained by centrifugation at 12000 rpm / s for 20 min at 4℃. The supernatant was the extracted total protein from the cells.

[0089] (2) Determine protein concentration and Western blot: The specific operation steps are the same as in Example 1.

[0090] The results showed that, under 32℃ cold stimulation, compared with the adNC group, the expression of UBC9 protein in skeletal muscle cells was significantly upregulated in the adUBC9 group, and the expression of the thermogenic marker protein UCP3 was also significantly upregulated. (See attached figures.) Figure 5 A. This indicates that UBC9 overexpression can enhance mitochondrial function in skeletal muscle cells.

[0091] 2. Immunofluorescence was used to detect changes in mitochondrial membrane potential (JC-1) in skeletal muscle cells of the adNC and adUBC9 groups.

[0092] (1) Preparation of cell crawling slices: collect skeletal muscle cell crawling slices from adNC group and adUBC9 group.

[0093] (2) Immunofluorescence staining: JC-1 dye was incubated at 37°C for 10 min to detect changes in JC-1 expression in skeletal muscle cells of the adNC group and adUBC9 group: the specific steps are the same as in Example 1.

[0094] The results showed that, under 32℃ cold stimulation, JC-1 expression was enhanced in skeletal muscle cells of mice in the adUBC9 group compared with adNC. (See attached figures). Figure 5 B. This indicates that overexpression of UBC9 can enhance mitochondrial activity.

[0095] 3. Immunofluorescence was used to detect changes in mitochondrial oxidative stress (Mitosox) in skeletal muscle cells of the adNC and adUBC9 groups.

[0096] (1) Preparation of cell crawling slices: collect skeletal muscle cell crawling slices from adNC group and adUBC9 group.

[0097] (2) Immunofluorescence staining: Mitosox dye was incubated at 37°C for 15 min to detect changes in Mitosox expression in skeletal muscle cells of the adNC group and adUBC9 group: the specific steps are the same as in Example 1.

[0098] The results showed that, under 32℃ cold stimulation, compared with adNC, the expression of Mitosox in the skeletal muscle cells of mice in the adUBC9 group was reduced. (See attached figures.) Figure 5 C. This indicates that overexpression of UBC9 can reduce mitochondrial oxidative stress levels.

[0099] In summary, this invention experimentally revealed that UBC9 expression was significantly upregulated in a mouse model of acute cold exposure at 4°C. Mice overexpressing UBC9 showed increased thermogenesis and mitochondrial function; at the cellular level, UBC9 overexpression enhanced thermogenesis and mitochondrial function in skeletal muscle cells. These results indicate that UBC9 plays an important protective role in acute cold exposure and may be a novel target for the prevention or treatment of skeletal muscle thermogenic dysfunction caused by acute cold exposure, providing new theoretical basis for further exploration of key preventive and therapeutic targets for skeletal muscle thermogenic dysfunction caused by acute cold exposure.

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of UBC9 overexpression reagent in the preparation of drugs for the prevention and / or treatment of hypothermia caused by acute cold exposure.

2. The application according to claim 1, characterized in that, The UBC9 overexpression reagent reduces the risk of hypothermia by preventing and / or improving skeletal muscle thermogenic impairment caused by acute cold exposure.

3. The application according to claim 1, characterized in that, The UBC9 overexpression reagent exerts a protective effect by upregulating the expression of UCP3, TOMM20 and / or COX4 in skeletal muscle, and / or by reducing oxidative stress in skeletal muscle mitochondria and maintaining mitochondrial structural integrity.

4. The application according to claim 1, characterized in that, The UBC9 overexpression reagent is an expression vector containing the UBC9 encoding gene.

5. The application according to claim 4, characterized in that, The expression vector is a eukaryotic expression vector.

6. The application according to claim 5, characterized in that, The eukaryotic expression vector is a plasmid expression vector or a viral expression vector.

7. The application according to claim 6, characterized in that, The viral expression vector is an adenovirus vector, a lentivirus vector, or an adeno-associated virus vector.

8. Application of reagents for detecting UBC9 protein or gene expression levels in the preparation of kits for assessing the risk of skeletal muscle thermogenic impairment caused by acute cold exposure.

9. Application of reagents for detecting UBC9 protein or gene expression levels in the preparation of kits for predicting the risk of hypothermia caused by acute cold exposure.

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