Rewarming rats at 37˚C rescues hypothermia with signicant changes in IL-1β levels in the intestinal tissue and blood

A hypothermia-rewarming SD rat model was established by immersing rats in 15˚C seawater for 5h and then rewarming at 37˚C for 2, 6 and 12 h. The rats were randomly divided into a normal control group (group C), hypothermia group (group H) and rewarming group (group R). The changes in the levels of inammatory factors and pathophysiology of the intestinal tissues of rats were assessed. The blood was collected in test tubes, and the levels of cytokines in the separated plasma were detected using ELISA. The intestinal tissue was ground and lysed, and protein expression proles of 67 inammatory factors were measured using a protein chip. These samples were further subjected to reverse transcription-quantitative (RT-q)PCR analysis and tissue section staining.


Background
Hypothermia is de ned as a central body temperature < 35˚C, and it is a type of clinical syndrome and a major cause of death in individuals who die in the sea [1,2]. Stay in low temperature environment for a long time could result in hypothermia. Shipwrecks and naval battles could cause to people falling into the sea. Since the temperature of seawater is low and the thermal conductivity coe cient of seawater is ~ 23 times that of air, hypothermia after falling into the sea is considerably more likely to occur than on the land [3,4]. With the prevalence of activities such as marine shipping, marine operations and diving increasing, the incidence of shipwrecks and drowning is increasing [5]. Existing research has shown that 2/3 of the people who fall into the sea will develop hypothermia. With the activities in the ocean and expeditions to colder regions of the Earth, such as the north pole, are becoming increasingly popular, the incidence of hypothermia is increasing on a yearly basis [6]. Moreover, the mortality rate of hypothermia is very high. Hundreds of people die of hypothermia in America every year [7]. In hospitals, the mortality rate of hypothermia is 12%, while the mortality rate of moderate to severe cases may be as high as 40% [8].
The tolerance period of the body when immersed at different temperatures of seawater is distinguishable.
The lower the seawater temperature is, the shorter the survival time is, and thus the higher the risk of mortality [9,10]. When the central body temperature is < 32˚C, cardiac dysfunction occurs. When the central body temperature is < 28˚C, dysfunction of the respiratory system, circulatory system, nervous system and coagulation system is observed, which may progress to multiple organ dysfunction syndrome (MODS) [11,12]. The longer the period of immersion in seawater, the greater the extent of deterioration of organs. Under severe hypothermic conditions, the physiology of the body is altered notably, and the so-called 'death triad' including hypothermia, coagulation disorders, and acidosis may occur. If the central body temperature is < 32˚C and suffers trauma at the same time, it could be fatal [13].
Researches showed that hypothermia was associated with alterations in IL-6 and the other proin ammatory cytokines, including IL-1 and TNF-α, which stimulates the release of acute phase proteins [14]. In ammation is correlated with NF-κB activation. Wang et al [15] showed that adenosine 5'monophosphate-induced hypothermia inhibited the activation of NF-κB in endotoxemic rats. Thus, the pro or anti-in ammatory effects of hypothermia may be associated with the activation or inhibition of NF-κB activation.
At present, treatment measures for hypothermia primarily include rewarming, reversing acidosis and treating any dysregulated coagulation [16,17]. Among these, rewarming is the most important treatment measure for hypothermia patients [18]. It has been shown that hypothermia patients may still suffer from 'rewarming shock' after their body temperature returns to the normal levels, that is, improper rewarming may lead to 'secondary damage' [19]. Additionally, it has been shown that hypothermia can also induce dysfunction of the digestive system, including a reduction in intestinal motility. Ulcers of the ileus and pancreatitis may occur when the central body temperature is < 32˚C [13]. The intestinal tract is one of the most likely affected and severely injured internal organs following a traumatic shock, burn or infection.
Intestinal mucosa is vulnerable to ischemia due to the special clamped structure of its blood vessels. Therefore, ischemia-reperfusion injury of intestinal mucosa following trauma may lead to structural and functional changes of the intestinal mucosa [20]. Furthermore, it may also lead to translocation of enteric bacteria, intestinal-derived release of media, cascade reactions of the reticuloendothelial system, release of a large quantities of in ammatory media and cytokines, and thus, systemic in ammatory response syndrome (SIRS), sepsis and even MODS [16]. However, currently, there are only a few studies examining the changes in the intestine following hypothermia. The rewarming methods for hypothermia also require improvement.
In the present study, an improved hypothermia-rewarming model for Sprague-Dawley (SD) rats was established based on a hypothermia model established in our previous study [17,21]. A focus was placed on exploring the changes in the physiological state, structure and the pro le of in ammatory cytokines in the intestinal tissue under hypothermic and rewarming conditions. Additionally, the damage caused by hypothermia to the intestinal tract was assessed.

Ethics statement
The animal experimentation protocol was reviewed and approved by the Ethical Committee of The Sixth Medical Center of Chinese PLA General Hospital (Approval No.MDKN-2020-158), and all rats were handled in accordance with the guidelines described in the Declaration and the National Institutes of Health Guide for Care and Use of Laboratory animals [22]. Implantation of the temperature recorder into the abdominal cavity of rats SD male adult rats (weight, 280-300g; age, 2-3 months) were obtained from the Institute of Zoology, Academy of Military Medical Sciences. All animals were kept under speci c pathogen-free conditions with controlled light/dark cycles and free access to water and standard chow and were fasted for 24 hours before surgical procedures. Rats were anaesthetized with an intraperitoneal injection of 1.5% pentobarbital sodium (40mg/kg body weight), and then xed in the supine position on a plate. A ventral midline incision of 2cm on the right side of the xiphoid process was made, and a temperature recorder wrapped in para n was placed under the abdominal cavity of the rat. Subsequently, the long incision was sutured. The wound was smeared with erythromycin ointment and rats were intraperitoneally injected with penicillin sodium (200,000 u), which lasted for 3 days after operation. After 10 days, the rats which grew regularly and had normal bowel movements were used for further experiments.

Animal preparation and hypothermia-rewarming model
We built a hypothermia-rewarming rats model based on our previous study [17]. A total of 50 SD male rats implanted with a temperature recorder were randomly assigned to three groups: Normal control group (C group, n=10), hypothermia group(H group, n=10) and a hypothermia followed by rewarming group (R group, n=30).The R group included three time-point subgroups: 2, 6 and 12h (R2, R6 and R12, respectively).There were 10 rats in each subgroup. Rats in the C group were xed into the cylindrical upright retainer and placed at room temperature for 5 h. Then, they were anesthetized, and samples of blood and various tissues were obtained. The other rats were xed into the cylindrical upright retainer and then immersed into the 15˚C seawater for 5 h, such that the seawater reached the armpits of the rats. The abdominal temperature and other vital indicators were observed for 5 h, unless the rat died as judged by respiratory and cardiac arrest (Tables SI and SII). After immersion for 5 h, the survived rats in the groups H were anesthetized, and samples of blood and various tissues were obtained. The rats which survived in the 3 R groups were transferred to a 37˚C constant temperature water bath for 2, 6 and 12 h, respectively. After each detection time point, the rats were anaesthetized with an intraperitoneal injection of 1.5% pentobarbital sodium (40mg/kg body weight) to obtain the samples of blood and tissues including the intestinal tissue. The blood was collected via the left ventricle of the rats into test tubes containing citrate solution (1:9 citrate to blood).The blood samples were centrifuged at 1,000 × g for 15min, and the plasma was separated within 1 h and was stored at -80˚C for subsequent use. The small intestinal tissue from the Treitz ligament to the ileocecal junction was rapidly excised from the mesentery and rinsed gently with 5ml ice-cold PBS. A 5cm segment of the ileum, 15cm above the ileocecal junction, was removed for histopathological analysis. The lung, kidney, heart were stored in liquid nitrogen immediately until required for tissue analysis.
Assessment of the expression of in ammatory factors in the intestinal tissues using the protein chip GSR-CAA-67 The frozen small intestinal tissues (~1cm) were xed in 3-fold phosphate-buffer (0.1M, pH7.2), homogenized and centrifugalized (10,000rpm/min for 30 min) for estimation of the levels of in ammatory factors. The protein chip GSR-CAA-67 was used to simultaneously examine the protein levels of 67 different in ammatory factors in the supernatants of the intestinal tissues. Each well of the chip was lled with 100µl tissue lysis supernatant (500μg/ml) and then incubated overnight at 4˚C on a shaker. Well wash Versa(Thermo Fisher Scienti c, Inc.) was used to wash the slides. Subsequently, 80µl detection antibody was added to each well. After washing the slide again, 80µl Cy3-streptavidin was added to each well. The slide was covered with aluminum foil to protect against light and incubated at 37˚C for 1 h on a shaker. Fluorescence signals were detected and observed using a microscope.
Determination of plasma cytokine levels using ELISA Based on the differential analysis, three in ammatory factors (IL-6, IL-10 and IL-1β) were selected for further analysis. To detect the levels of cytokines in the plasma, IL-6(cat.BMS603-2; eBioscience; Thermo Fisher Scienti c, Inc.), IL-10 (cat.BMS629; eBioscience; Thermo Fisher Scienti c, Inc.) and IL-1β (cat.BMS6002; eBioscience; Thermo Fisher Scienti c, Inc.) were measured using a double-antibody sandwich ELISA according to the manufacturer's protocol. The assays were repeated three times for each group and subgroup. Brie y, 100μl plasma and 50μl enzyme conjugate was added to the antibody precoated 96-well ELISA plate and incubated for1h at 37˚C. The ELISA plate was washed ve times with PBS and then incubated for 15 min with a TMB substrate. The absorbance was measured using an ELISA reader.
Reverse transcription-quantitative (RT-q)PCR Small intestinal tissue was harvested and homogenized in 1ml TRIzol ® Reagent (Invitrogen; Thermo Fisher Scienti c, Inc.) per 50-100mg tissue. Total RNA was isolated according to the manufacturer's protocol and was treated with RNeasyMinElute™ Cleanup kit (Qiagen, Inc.). RNA quantity and quality were measured using a NanoDrop 8000 (Thermo Fisher Scienti c, Inc.). RNA (1 μg) was reverse transcribed using an RT kit (Takara Bio, Inc.) according to the manufacturer's protocol. qPCRwas performed using SYBR green technology (Promega Corporation) using an iCycler real-time detection system(Bio-Rad Laboratories, Inc.). Relative mRNA levels were determined following normalization to the housekeeping gene, GAPDH, and the data were analyzed using the 2 -ΔΔCq method. Assays were repeated three times for each group and subgroup.

Histopathological (HE) examination
The paraformaldehyde xed small intestinal tissue was embedded in para n, sectioned (5μmthick)using a microtome, and stained with hematoxylin and eosin (H&E) for histological examination by the pathologists.

Statistical analysis
Data were analyzed using SPSS version 20.0 (IBM Corp.)and R software version 3.6.2. All data are presented as the mean ± standard deviation. One-way ANOVA was used under one factor condition while two-way ANOVA was used under two factors condition. Bonferroni method was used to adjust multiple test p-value. A χ2 test was used to compare categorical variables. P<0.05 was considered to indicate a statistically signi cant difference.

Results
Changes in the abdominal temperature of the rats After immersion in 15˚Cseawater for 2 h, compared with the C group, the abdominal temperature of the rats in group H and R dropped signi cantly at all time points (P<0.05). The abdominal temperature decreased sharply in the rst 30 min, and then decreased slowly. After 2 h, the temperature dropped tõ 15.6℃, and then uctuated at this temperature until the 5 th h. During this time, some rats died. There were 30 rats in R group (three sub-groups) and 10 rats in H group. During immersing in 15˚C seawater, 2 rats and 8 rats were dead in H group and R group respectively. Fisher exact test shows that there no difference between the two groups (P = 1, odds ratio = 0.75).The number of rats which remained alive at each time period for each group is summarized in Tables SI and SII. After rewarming in a 37˚Cwater bath for 1 h, the abdominal temperature of the rats in group R increased rapidly to ~38˚C, and then dropped tõ 35˚C. After rewarming for 2 h, the temperature returned to normal until the 12 th h. During the rewarming procedure, no rat died (Table SII). Thus, rewarming in 37˚C water helped the body to warm up quickly and safety. Compared with the 37 0m (Rewarming in 37˚C water for 0 minute)time point, the abdominal temperature of the rats was signi cantly increased at all time points (P<0.05; Fig. 1A).Since the temperature was almost constant after immersion in 15˚C seawater for 2 h, the remaining 3 h are not shown in Fig. 1.

Changes in the physiological state of the rats
In the early stages of immersion in the low-temperature seawater, the stress caused a signi cant increase in heart rate, respiration and amyostasia of rats in the H and R groups. With the core temperature decreasing consistently, the stress exhibited decreased eventually. After immersing in 15˚Ctemperature seawater for 10 min, compared with the C group, the respiratory rate and heart rate of rats in the H and R group increased, although the difference was not statistically signi cant. After immersion in 15˚C temperature seawater for 10-120 min, compared with the C group, the respiratory rate and heart rate of rats in groups R and H decreased signi cantly (P<0.05), where it remained stable. The respiratory and heart rates of rats in group R increased signi cantly after rewarming for 1 h and returned to normal after 2 h. Compared with 37 0m , the respiratory rate and heart rate of rats was signi cantly higher at all time points (P<0.05; Fig. 1B and C).
When immersed in 15˚Cseawater for 10 min, compared with the C group, the rats in groups H and R showed notable amyostasia. After 30 min of immersion, the muscle tremor in the R and H groups peaked. With the core temperature decreasing, the amyostasia of R and H groups also decreased rapidly and was absent after 90 min. However, after rewarming for 30 min, the amyostasia of rats in the R group increased signi cantly, and peaked again after rewarming for 60 min. As the core temperature increased, amyostasia gradually decreased and after 3 h of rewarming, the amyostasia was absent (Fig. 1D).
Based on the above results, rewarming in a 37˚C water bath may recover the vital signs quickly and safely in hypothermic rats.

Expression of intestinal in ammatory factors
The expression levels of 67 in ammatory factors in the small intestinal were detected using a protein chip. Based on the expression of the in ammatory factors, the C group was clustered into a class via hierarchical clustering, which indicated that hypothermia resulted in notable changes to the body (Fig.   S1). The H, R2 and R6 groups could not be clustered into one class; however, the R12 group could almost be clustered into a class, which indicated that rewarming for 12 h could be distinguishable from the H group. Expression of in ammatory cytokines in rats rewarmed for short periods (2 or 6 h) could not be clustered. This suggested that a rewarming period of 12 h was considered su cient to stabilize the physiology in the rats (Fig. S1).
Next, the differentially expressed proteins in the H and R groups (R2, R6 andR12) was compared with the N group (Fig. S2). Compared with the C group, 11 in ammatory factors were differentially expressed, including 7 upregulated and 4 downregulated in ammatory factors, (Fig.S2A). There were 15 and 14 signi cantly differentially expressed in ammatory factors in the R2 and R6 groups, respectively ( Fig. S2B and C).The R12 group exhibited the highest number of differentially expressed in ammatory factors; 15 upregulated and 5 downregulated (Fig. S2D).These changes in in ammatory factors may participate in the development of hypothermia. We selected IL-6, IL-10 and IL-1β for further analysis based on our lab condition (our lab had su cient conditions to study IL-10 because we had studied IL-10 before) and the differential analysis (IL-1β and IL-6 were the most signi cant proteins when compared the H group with the C group (Fig. S2A). These two proteins were also signi cant in the other three comparations (Fig. S2B, C, D).

Plasma levels of IL-6, IL-10 and IL-1β
The expression levels of certain in ammatory factors were altered in the intestine following hypothermia. Thus, whether these changes were also observed in the blood was next assessed. For analysis, three in ammatory factors (IL-6, IL-10 and IL-1β) were selected for further analysis in the blood. Using ELISA, it was shown that following immersion in 15˚C seawater for 5 h, the levels of the three cytokines in the plasma from the H group was signi cantly higher than that of the C group (P<0.05). Compared with the H group, the levels of the three cytokines in the R group was gradually reduced following rewarming in a warm water bath (P<0.05; Fig.2A-C).The ratio of IL-1β:IL-10 and IL-1β:IL-6 ratios were used to measure the balance between pro-and anti-in ammatory cytokines. The ratio of IL-1β:IL-6 and IL-1β:IL-10in the plasma of the H group rats were increased, and they gradually decreased in the R group with time. However, the increase in IL-1β expression was greater than that of IL-10 and IL-6 in the H and R groups (Fig. 2D).Thus, the levels of in ammatory factors in the blood were also altered. The changes in these three in ammatory factors was similar to that observed in the intestine. Thus, rewarming both recovered the vital status and also restored the levels of in ammatory factors in the blood.

Relative gene expression levels of the cytokines in the gut
Differentially expressed in ammatory factors were found in the hypothermic rats using a protein chip. To verify the change in these factors, IL-1β, IL-10 and IL-6were selected for qPCR analysis. The results showed that the expression levels of IL-1β in the H group was signi cantly increased ~4.5-fold compared with the C group (P<0.05; Fig. 3A). When rewarming for 2 h, the levels of IL-1β mRNA decreased to normal. However, after rewarming for 6 h, the levels of IL-1β were increased signi cantly, ~3.5-fold higher than that of the C group. As the rewarming time increased, the expression levels of IL-1β gradually decreased (Fig.3A).Compared with group C, the mRNA levels of IL-10 in group R2, R6 and R12 increased(P<0.05; Fig.3B).The mRNA expression levels of IL-6 in the R2 group increased, whereas it decreased in the H, R6 and R12groups, although the differences were not statistically signi cant (Fig.  3C).The ratio of IL-1β:IL-6 and IL-1β:IL-10 were analyzed. Compared with the C group, IL-1βexpression was higher than that of IL-10 in the H group and all the R groups (P<0.05; Fig.3D).

Pathological changes in the intestinal tissues
The histological analysis showed that the morphology of intestinal mucosa in the C group was normal (Fig. 4). The villi of the intestinal mucosa and the epithelial cells were neatly arranged. They were free of edema in the interstitial tissue and the villi structure was intact. However, a mass of necrosis was observed in the epithelium of intestinal mucosa villi in the H group, with a large degree of neutrophil and in ammatory cell in ltration, and the shed villi interstitial structure was lost. In the R group, the tight junction structure of the intestinal tissue and the permeability was altered. At the top of the villi, there was increased neutrophil in ltration. These ndings suggest that severe in ammation occurred in the intestine following hypothermia.

IL-1βexpression in intestinal tissues based on immunohistochemistry (IHC) analysis
To investigate the expression and distribution of IL-1β in intestinal tissues, IHC was performed. As shown in Fig.5, positive IHC staining, which was considered as brownish yellow or brown particles that could be distinguished from the background cells, was primarily localized in the cytoplasm of the intestinal tissue cells. Compared with the C group, the proportion of positive cells was higher in the H group. In the R2 group, the staining of IL-1βwas decreased compared with the H group. However, the number of positive cells was increased in the intestinal tissue of the R6 group. With a longer rewarming period, the intensity of IL-1β expression gradually decreased. This phenomenon was observed with regard to the amyostasia.
When hypothermic or rewarming, amyostasia was rst increased and then subsequently decreased (Fig.  1D). However, for IL-1β, there was only one time-point and the end of immersion. A possible explanation for this is that when the temperature is decreasing, the attempts to correct this to maintain the normal body temperatures. However, as the temperature decreases further, the body is unable to maintain a physiological temperature. Thus, perhaps the lower temperature suppresses certain physiological functions. Additionally, this observation may be the result of evolution. When the temperature is too low, the body stops trying to increase the core temperature, instead reserving energy, thus resulting in damage to the body from the colder temperatures.

Discussion
Hypothermia is becoming increasingly prevalent, and when caused by seawater, it has a high rate of mortality. Longer periods of immersion may result in worse outcomes [24]. Appropriate early treatment is very important for reversing and limiting hypothermia [25]. In the present study, a hypothermia-rewarming model was established via immersion of rats in 15˚C seawater for 5h followed by rewarming in a 37˚C water bath. Physiological changes, changes in the in ammatory response and damage of the intestinal tissue structure was observed. When rats were immersed in 15˚C seawater, the heart rate, respiratory frequency, and amyostasia were initially increased and subsequently decreased. This may be an early response to cold stimulation. As the time the rats were immersed in the cold water increased, the damage to the thermoregulatory center increased, and the more the body's aerobic metabolism was reduced. The rat's consciousness deteriorated as the respiratory rate and heart rate reduced. In addition, muscle tremor disappeared. During immersion in 15˚C seawater, some rats died. No rewarming shock was observed during the rewarming period and there was no death during this period either. Thus, rewarming at 37˚C is a good choice for early treatment of hypothermia. After rewarming, the temperature of the abdomen in the rats rst increased to 38˚C and then decreased to 35˚C,and the temperature increased to normal body temperatures.
It was found that the intestine could initiate a 'second attack' when the level of stress experienced increased [26], resulting in a release of in ammatory factors, platelet-activation factor and tumor necrosis factor [27]. Hypothermia is associated with white blood cell activation, increased levels of cytokines and SIRS [10,28].
In the present study, the expression of certain in ammatory factors, including IL-1β, IL-6 and IL-10 was altered in the intestinal tissue and blood, particularly for IL-1β. IL-1β is a pro-in ammatory cytokine, whereas IL-6 may serve as a pro-or anti-in ammatory cytokine [11,29]. In addition, according the ratios of IL-1β to IL-10 and IL-1β to IL-6 ratios, IL-1βexpression was signi cantly higher than that of IL-10 and IL-6 in the intestine following hypothermia, and it returned to around normal expression levels following rewarming for 2 h. Thus, it is speculated that IL-1β is a hypothermia speci c in ammatory factor that could be used to assess the severity of hypothermia.
IL-1β is a common in ammatory factor that is produced by monocytes, endothelial cells, broblasts and other types of cells in response to infection [30,31]. IL-1β could stimulate the production of colonystimulating factor, and platelet-derived growth factor and also could induce T cells to produce IL-2, which serves an important role in immune response and tissue repair. As a pre-in ammatory regulatory factor, IL-1βmay be used to re ect the severity of intestinal damage during the early phase, and activate NF-κB to increase the expression of IL-6 and TNF-αin an autocrine manner [32,33]. It has been reported that the expression of in ammatory factors is upregulated in the plasma of hypothermia patients [34]. The high ratios of IL-1β to IL-10 and IL-1β to IL-6 in the present study supported the hypothesis that IL-1β was the predominant pro-in ammatory factor that mediated the initial in ammatory response following ischemia reperfusion mediated by hypothermia [23]. The in ammatory response following immersion in lowtemperature seawater and rewarming is complex. According to Cryeret al [35], the exaggerated proin ammatory response possibly coexists with an exaggerated counter-in ammatory response. According to Moldaweret al [36] the situation is more complicated than a pro-in ammatory response followed by an anti-in ammatory response. After rewarming for 2 h, the ratios returned to around normal levels, and IHC analysis of the intestinal tissue indicated that IL-1β expression was higher in the intestinal tissue of the hypothermic rats compared with the control group. This may be related to the increase in the levels of pro-in ammatory factors stimulated by the stress response in the intestine under hypothermic conditions. After rewarming for 2h, IL-1βexpression was signi cantly downregulated, which indicated that rewarming may be a useful method of reversing hypothermia. The above results together show that IL-1β is a speci c in ammatory factor in intestinal tissues expressed following immersion at low-temperatures followed by rewarming.
The present study has some limitations. First, only 67 in ammatory associated genes were detected in the present study. Some in ammatory modulators, such as IL-8 and cyclooxygenase-2, were not included.
Other genes may also serve important roles in response to hypothermia, and these may have been missed. Additionally, a larger range of rewarming temperatures should be assessed to determine the optimal temperature. Third, knockdown and overexpression of IL-1β should be performed to better determine its role in hypothermia. Forth, more time-points of immersion at 15˚C seawater is required to observe the extent of changes over time, particularly with regard to IL-1β. Finally, it will be more rigorous if the immersion of rats in water at room temperature (for ve hours) as blank control, and the rats which died during immersion in 15˚C seawater should also have been analyzed. In the future, the mechanism of IL-1β in hypothermia will be further studied.

Conclusions
The present study established a hypothermia-rewarming rat model. The results showed that: i) Rewarming in 37˚C may serve as a candidate method for early treatment of hypothermia. ii) Expression of certain in ammatory factors, including IL-1β, IL-6 and IL-10 is altered notably under hypothermic conditions. iii) IL-1β may serve as a biomarker and/or therapeutic target for assessing the severity and management of hypothermia, respectively. The other in ammatory factors assessed, such as IL-10 and IL-6 may not be of particular relevance with regard to hypothermia. Abbreviations IHC,immunohistochemistry; MODS,multiple organ dysfunction syndrome; SIRS,systemic in ammatory response syndrome  HE staining of the intestinal tissue in each group. C: normal control group; H: 15˚C seawater immersion for 5 h; R2, R6, R12: 15˚C seawater immersion for 5 h, and rewarming in 37˚C warm water bath for 2, 6, 12 h respectively. Comparison of IHC of IL-1β (" * " indicated the comparison between group N and other groups with pvalue < 0.05; " #" indicated the comparison between H and R2, R6, R12 group with p-value < 0.05). C: normal control group; H: 15˚C seawater immersion for 5 h; R2, R6, R12: 15˚C seawater immersion for 5h, and rewarming in 37˚C water for 2, 6, 12 h respectively.

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