The effect of sulforaphane on Nrf2 mediated antioxidant capacity in skeletal muscle of mice exposed to acute hypothermia
Skeletal muscle is an important organ for heat production in the body under low temperature conditions. Skeletal muscles maintain body temperature homeostasis through both shivering and non shivering heat production in low-temperature environments. This process requires the hydrolysis of a large amount of ATP to convert its chemical energy into the body’s required thermal energy. Skeletal muscle mainly satisfies heat production needs by increasing the rate of fatty acid oxidation and decomposition in low-temperature environments, while the increase in mitochondrial fatty acid oxidation rate is accompanied by accelerated production of reactive oxygen species (ROS). Previous studies have shown that acute or long-term exposure to low temperatures increases the resting metabolic rate and skeletal muscle ROS levels in mice. The increase in ROS levels can cause oxidative damage to skeletal muscle proteins, nucleic acids, and lipids, thereby affecting the motor performance and recovery of skeletal muscle. Therefore, timely removal of the large amount of ROS produced by skeletal muscles in low-temperature environments and improvement of their antioxidant capacity are crucial for maintaining their normal physiological functions.
The antioxidant defense system mediated by nuclear factor E2 related factor 2 (Nrf2) plays a crucial role in maintaining cellular redox homeostasis. When cells are under oxidative stress, Nrf2 in the cytoplasm is not ubiquitinated or degraded by its negative regulatory protein, Kelch like cyclooxygenase related protein-1 (Keap1). It enters the nucleus and binds to antioxidant response elements (ARE) on numerous gene promoters, upregulating the expression of most antioxidant enzymes and phase II detoxifying enzymes, and exerting a protective effect against oxidative stress in the body. However, studies have found that acute or long-term exposure to low temperatures significantly reduces the expression of Nrf2 mRNA and protein in mouse heart, liver, and lung tissues and organs, indicating that low temperature environments may inhibit the expression of Nrf2 in tissues and organs. However, there is no report at home and abroad on whether acute hypothermia exposure also inhibits Nrf2 expression and antioxidant capacity in skeletal muscle.

Sulforaphane (SFN) is abundant in cruciferous plants and is recognized as a specific activator of Nrf2. Therefore, this study attempts to first observe the effects of acute hypothermia exposure for different durations of 1 hour and 3 hours on the expression of Nrf2 and antioxidant enzymes, as well as antioxidant capacity, in mouse skeletal muscle; Furthermore, based on this, the effects of administering SFN before low-temperature exposure on the Nrf2 mediated antioxidant enzyme system and glutathione redox homeostasis in skeletal muscle were further explored. This study will provide preliminary experimental evidence for exploring the possibility of SFN as a sports nutrition supplement in low-temperature environments.

Nrf2 is a core regulatory factor that maintains the redox homeostasis of skeletal muscle. Previous studies have reported that low temperature exposure inhibits Nrf2 expression in tissues and organs such as the heart, liver, and lungs, leading to a large production of ROS in these organs that cannot be cleared. However, there have been no reports on the effects of acute hypothermia exposure on Nrf2 expression and antioxidant capacity in skeletal muscle. Therefore, this study first investigated the effects of 1 and 3 hours of low temperature exposure on the Nrf2 mediated antioxidant system in skeletal muscle. The results showed that the transcription level of Nrf2 mRNA in skeletal muscle of mice exposed to 3 hours of low temperature was significantly reduced, while the level of ROS was significantly increased. Subsequent experimental results also showed that compared with the PBS+Con group, the expression of Nrf2 protein in skeletal muscle of mice in the PBS+Cold group showed a decreasing trend, and the level of T-AOC was significantly reduced, while the level of ROS showed an increasing trend. It is speculated that 3-hour low-temperature exposure may inhibit the expression of Nrf2 in mouse skeletal muscle and reduce its antioxidant capacity.

Exposure to low temperature for 3 hours reduces the antioxidant capacity of skeletal muscle, which may be related to the inhibition of Nrf2 mediated antioxidant enzyme expression and glutathione redox homeostasis. Previous studies have reported that 3 hours of acute hypothermia exposure can significantly reduce SOD1 expression in mouse kidneys, lung tissues, and brown adipose tissue. Intermittent low-temperature exposure (8 hours per day for 3 days) significantly reduced GPX1 activity and HMOX1 protein expression in rat lung tissue. Long term low-temperature exposure (4 hours per day, 6 days per week, for a total of 2 weeks) significantly reduced SOD1 activity and CAT protein expression in mouse brain tissue. This study found that compared with the 0 and 1h groups, the mRNA transcription levels of antioxidant enzyme genes (Gpx1, Hmox1, Cat, Sod1, Nqo1) in skeletal muscle of mice in the 3h group were significantly reduced. Subsequent experimental results also showed that compared with the PBS+Con group, the mRNA transcription levels of antioxidant enzyme genes (Gpx1, Hmox1, Cat, Sod1, Nqo1) in skeletal muscle of mice in the PBS+Cold group showed a decreasing trend, and the expression of HMOX1 and CAT proteins was significantly reduced. It can be inferred that 3-hour low-temperature exposure may inhibit the transcription and translation of Nrf2 mediated antioxidant enzymes, thereby affecting the antioxidant capacity of skeletal muscle. In addition, studies have shown that acute low-temperature exposure can reduce the GSSG content and GSH/GSSG ratio in human red blood cells, as well as decrease the GSH content in rat liver and stomach tissues. The results of this study showed that compared with the PBS+Con group, the skeletal muscle GSSG content and GSH/GSSG ratio of mice in the PBS+Cold group significantly increased and decreased, respectively, while the GSH content did not change significantly. This indicates that the accumulation of GSSG may be the main reason for the decrease in GSH/GSSG ratio.

The specific activation effect of SFN on Nrf2 has been widely confirmed. A study found that a 12 week SFN dietary intervention activated the Nrf2 regulated antioxidant enzyme system in the extensor digitorum longus muscle of elderly mice, improving muscle strength and exercise endurance. In addition, rats were intraperitoneally injected with SFN three days before exhaustion exercise, resulting in a decrease in plasma lactate dehydrogenase and creatine phosphokinase activity, as well as an increase in Nrf2 and antioxidant enzyme (NQO1, GST, GSR) protein expression and activity in the lateral thigh muscle. The time and distance from exercise to exhaustion also increased. The above research results indicate that SFN activation of Nrf2 plays an important role in enhancing its mediated antioxidant capacity and exercise endurance. Therefore, to improve the decrease in skeletal muscle antioxidant capacity during 3 hours of low-temperature exposure, we supplemented mice with SFN before 3 hours of low-temperature exposure. The experimental results showed that compared with the PBS+Cold group, the SFN+Cold group mice showed a significant increase in skeletal muscle Nrf2 mRNA and protein expression, as well as T-AOC, and a significant decrease in ROS levels. Reminder: SFN activation of Nrf2 can increase the T-AOC of skeletal muscle exposed to low temperature for 3 hours, eliminate excessive ROS, and have a positive effect on maintaining the redox homeostasis of skeletal muscle.

Supplementing SFN enhances the antioxidant capacity of skeletal muscle in mice exposed to low temperatures, which is closely related to the activation of Nrf2 mediated antioxidant enzyme system by SFN. The endogenous antioxidant enzymes regulated by Nrf2 are the main executors of ROS clearance. For example, SOD1 catalyzes the dismutation of superoxide anion radicals to generate oxygen and hydrogen peroxide; CAT can dismutation hydrogen peroxide into water and oxygen; GPX1 catalyzes the generation of water and corresponding alcohols from hydrogen peroxide or organic peroxides using GSH as a substrate; NQO1 catalyzes the double electron reduction reaction of quinone to form hydroquinone, promotes quinone excretion, and prevents quinone from generating ROS through single electron reduction reaction; HMOX1 catalyzes the decomposition of toxic free hemoglobin to produce biliverdin, CO, and ferrous ions with antioxidant damage functions. The results of this study showed that administering SFN before 3 hours of low temperature exposure significantly increased the mRNA transcription levels of skeletal muscle antioxidant enzyme genes (Gpx1, Hmox1, Cat, Sod1, Nqo1) and the protein expression of HMOX1 and SOD1 in SFN+Cold group mice compared to PBS+Cold group. The above results indicate that SFN activation of Nrf2 enhances the transcription and translation of these antioxidant enzyme genes.

In addition, supplementing SFN enhances the antioxidant capacity of skeletal muscle in mice exposed to low temperatures. In addition to increased expression of antioxidant enzymes, it is also closely related to SFN activation of the Nrf2 mediated glutathione redox system. Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine, and is an important small molecule active oligopeptide in the antioxidant defense system of organisms. Glutathione mainly exists in the form of GSH in cells. The active thiol group of cysteine in its molecule can provide electrons to ROS, and then GSH is converted into a stable dimer GSSG to prevent ROS from continuously grabbing electrons, thus protecting proteins, lipids, and nucleic acids from oxidative damage and maintaining the redox homeostasis of cells. The generation of GSH in the body is mainly through two pathways: synthesis and reduction. Firstly, glutamic acid and cysteine are catalyzed by GCLC and GCLM to form glutamylcysteine, followed by glutamylcysteine and glycine being catalyzed by GSS to form GSH; Secondly, GSSG can be reduced to GSH under the action of NADPH and GSR. Therefore, Gclc, Gclm, Gss, and Gsr are key enzyme genes that regulate the generation of GSH and are downstream target genes of Nrf2. We found that after SFN supplementation, the mRNA transcription levels of glutathione synthase genes Gclm, Gss, and Gsr in skeletal muscle of SFN+Cold group mice were significantly increased compared to PBS+Cold group, and the contents of GSH and GSSG were significantly decreased. It is speculated that this may be acute low-temperature stress, and SFN supplementation can enhance the de novo synthesis and reduction pathways of GSH. However, GSH plays an important role in the elimination of a large amount of ROS production caused by acute low-temperature exposure, ultimately leading to a significant consumption of GSH. From the key indicator of evaluating the redox balance of the body, the GSH/GSSG ratio, it can be seen that the GSH/GSSG ratio in the SFN+Cold group is significantly increased.

Overall, this study indicates that 3-hour acute hypothermia exposure inhibits Nrf2 mediated antioxidant activity. Prior to exposure to low temperatures, administration of sulforaphane activated Nrf2 mediated antioxidant enzymes and glutathione antioxidant systems, enhancing skeletal muscle antioxidant capacity.