Effect of chronic cold stress on gut microbial diversity, intestinal in�ammation and pyroptosis in mice

Hypothermia is an essential environmental factor in gastrointestinal diseases, but the main molecular mechanisms of pathogenesis remain unclear. The current study sought to better understand how chronic cold stress affects gut damage and its underlying mechanisms. In this work, to establish chronic cold stress (CS)-induced intestinal injury model, mice were subjected to prolonged cold exposure (4°C) for 3 h per day for 3 weeks. Our results indicated that CS led to gut injury via inducing changes of heat shock proteins 70 (HSP70) and apoptosis-related (caspases-3, Bax and Bcl-2) proteins; enhancing expression of intestinal tight-related (ZO-1 and occludin) proteins; promoting releases of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α ), cyclooxygenase-2 (COX-2), high mobility group box 1 (HMGB1), interleukin1 β (IL-1 β ), IL-18 and IL-6 in�ammatory mediators in the ileum; and altering gut microbial diversity. Furthermore, persistent cold exposure resulted in the cleavage of pyroptosis-related Gasdermin D (GSDMD) protein by regulating the NLRP3/ASC/caspase-1 and caspase-11 pathway, and activation of toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-mediated nuclear factor kappa B (NF-κ B) and mitogen-activated protein kinase (MAPK) signaling pathways, which are strongly associated with changes in gut microbiota diversity. Taken together, these investigations provide new insights into the increased risk of intestinal disorders at extremely low temperatures and establish a theoretical foundation for the advancement of novel pharmaceutical interventions targeting cold-related ailments.


Introduction
Environmental stresses are a critical component that largely contributes to high rates of disease and public health issues [38].Particularly noteworthy is the fact that freezing temperatures are substantially to blame for the current burden of overall mortality in many regions [14].The gut, as an organ in touch with the outside world, is especially vulnerable to stresses, which has been related to the etiology of numerous intestinal illnesses [47].Previous studies have demonstrated that cold exposure induced intestinal injury and increased the frequency and severity of diarrhoea in animals [22,44], indicating that cold stress plays a key role in the pathogenesis of intestinal disorders.Therefore, it is essential for exploring the role of cold stress and intestinal damage and its underlying mechanisms.
Intestinal in ammation is a vital risk factor for the development of gastrointestinal diseases, such as in ammatory bowel disease (IBD) [49].Nevertheless, pryoptosis, as a new type of programmed cell death (PCD), is characterized by excessive in ammatory response and cell death, which is closely associated with various intestinal diseases resulted from many stimulus [5,46].Intriguingly, continuous hypothermic exposure can activate many signaling pathways, inducing non-speci c responses, including autophagy, in ammatory responses, and oxidative stress [18,51,53].Few researches have looked at the relationship between cold stress and pyroptosis in intestinal tissue.Abundant researches revealed that the occurrence of pyroptosis is nearly linked to the activation of nucleotide-binding oligomerization domain (NOD)-like receptors protein 3 (NLRP3).The NLRP3 in ammasome recruits and activates caspase-1 directly or via the adaptor protein ASC (apoptosis-associated speckle protein), which is then activated by pro-IL-1/18 and cleaved by Gasdermin D (GSDMD), resulting in severe in ammation and pyroptosis [11,24].
Contrarily, the myeloid differentiation factor 88 (MyD88) adaptor is recruited by the toll-like receptor 4 (TLR4), one of the crucial signaling pathways, to cause the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) which is composed of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 [25].This is one of the key signaling pathways that play a signi cant role in various in ammatory diseases.Meanwhile, NLRP3 activation is intimately related to the TLR4 signaling pathway [26,50].
There is growing evidence has identi ed that the imbalance of intestinal ora is other crucial risk factor for the occurrence and development of intestinal diseases [9].Gut microbial diversity was demonstrated to be in uenced by both host physiology and environmental variables such as season and air temperature [6], while the gut microbiota can modulate host in ammation and immunity through metabolites and bacterial polysaccharides [23,52].Notably, the gastrointestinal system is home to a slew of microorganisms, particularly bacteria, which serve as a source of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [35,45].Conversely, PAMPs and DAMPs interact with the host by evoking responses from membrane-bound pattern recognition receptors (PRRs), like toll-like receptors (TLRs) or NOD-like receptors [3], which lead to in ammation and pyroptosis.However, it is unclear whether chronic cold stress contributes to induce intestinal injury by modulating pyroptosis and in ammation involved in TLR4/MyD88 and NLRP3-related signaling pathway.
Herein, the objectives of this study were to: (i) investigate the pyroptosis and in ammatory response of mouse intestinal tissues under chronic cold stress conditions; (ii) analyze the effects of chronic cold stress on intestinal ora diversity; and (iii) clarify the mechanisms involved in the correlation between intestinal ora imbalance and the activation of signaling pathway associated with in ammation and pyroptosis.

Animals
Male C57BL/6 mice, 6 weeks old, 18-22 g, were supplied by Liaoning Changsheng Technology Industrial Co., LTD (Certi cate SCXK2010-0001; Liaoning, China).All mice were maintained for 1 week under controlled environmental conditions (temperature 24°C ± 0.5°C, humidity 40%, light/dark cycle 12 h).All studies were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals, which has been agreed with the Institutional Animal Care and Use Committee of Heilongjiang Bayi Agricultural University

Experimental protocol
To establish chronic cold stress-induced intestinal injury (Fig. 1): all mice acclimatized for one week at ambient conditions and were randomly divided into two groups (n = 6 mice per group), including room temperature (RT, 24°C ± 0.5°C) group and chronic cold stress (CS, 4°C).Firstly, feces sample were rst collected from both groups of mice.Then, mice from the group CS were placed in a climatic chamber at 4°C and kept for 3 h per day, then transferred back to room temperature and lasted for 3 weeks.Before being put to death, feces samples were collected one more following a nal cold stimulation.Meanwhile, intestinal tissues samples were collected for biochemical index determination, hematoxylin-eosin (H & E) staining and western blot analysis.

H & E staining
Fresh intestinal tissues, such as the ileum, jejunum, and colon, were xed with 4% paraformaldehyde, dried with a graded ethanol, embedded in para n wax, and then cut into 5 µm-thick slices to assess the damage to the gut.Hematoxylin-eosin (H & E) was used to stain these sections so that light microscopy could be used to view the pathological alterations to the gut.

Allison Blue (AB)-Periodic Acid Schiff (PAS) staining
Using the AB-PAS staining technique, the characteristics of goblet cells in the intestines were assessed.Brie y, 5 µm intestinal sections were cut using a freezing microtome (Polar DM, Japan), and the sections were then stained with AB-PAS in accordance with the guidelines published by the AB-PAS staining kit's manufacturer (Solarbio, Beijing, China).Photographs were acquired with an optical microscope.

16S rRNA gene sequencing
Fecal samples were collected by using standardized collection procedures.Four samples per group were used for 16S rRNA sequencing.Microbial DNA was extracted and quanti ed.After, the V3-V4 hypervariable region was ampli ed using 338F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3') primers by thermocycler PCR system (GeneAmp 9700, ABI, USA).The resulted PCR products were extracted from a 2% agarose gel and further puri ed using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quanti ed using QuantiFluor™-ST (Promega, USA) according to the manufacturer's protocol.Sequencing was performed on Illumina MiSeq platform by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).The raw data were qualityltered using Usearch or DADA2 software.

Statistical analysis
All data above were calculated as the means ± SEM and analyzed using SPSS19.0(IBM) software.
Comparisons between each group were conducted using one-way ANOVA, whereas the LSD method was used for multiple comparisons analysis.P values of < 0.05, < 0.01 or < 0.001were considered statistically signi cant differences.

Chronic cold stress induced intestinal damage in mice
The pathological features of gut tissues were generated by cold stimulation utilizing HE and AB-PAS staining to see if persistent cold stress may cause intestine damage in mice.When compared to the room temperature (RT) group, the chronic cold stress (CS) group demonstrated severe gut architectural disruption and in ammatory cell in ltration (Fig. 2A and B).Then, using a western blot, the stress-related protein expression of HSP90 and HSP70 was identi ed.Chronic cold stimulation, as seen in Fig. 2C and G, can dramatically increase HSP70 expression, whereas HSP90 protein expression has no signi cance (ns) when compared to the RT group.In addition, under conditions of prolonged exposure to cold, the proteins ZO-1 and occludin, which are the primary elements of intestinal epithelial cells' tight junctions, signi cantly increase.Furthermore, the results showed that persistent cold exposure cloud effectively boost the production of cleaved-caspase-3 and Bax and decrease the Bcl-2 protein expression in gut tissues (Fig. 2H and K), implying that chronic cold exposure resulted in intestinal damage.

Chronic cold exposure stimulated the production of in ammatory mediators
We measured the expression of TNF-α, iNOS, COX-2, IL-6, and HMGB1 after 3 weeks following prolonged cold exposure because in ammatory mediators are key contributors to many gastrointestinal diseases.In contrast to the RT groups, CS effectively increased the production of HMGB1, IL-6, iNOS, COX-2 and TNFα protein, showing that persistent cold exposure greatly increased the intestinal in ammatory response (Fig. 3).
Chronic cold exposure caused the cleavage of pyroptosis-related GSDMD dependent on the NLRP3/ASC/Caspase-1 and Caspase-11 signaling pathway Due to pyroptosis is closely linked to a variety of gastrointestinal illnesses and in ammatory reactions, western blot is used to see if CS may activate the pyroptosis-related signaling pathway.As shown in Fig. 4, chronic cold exposure increased the expression of NLRP3, ASC, cleaved-caspase1, cleaved-caspase11, GSDMD-N, mature-IL-1β, and mature-IL-18 proteins, suggesting that CS activated the GSDMDmediated pyroptosis signaling pathway by regulating NLRP3/ASC/caspase-1 and caspase-11 signaling pathway.
Chronic cold exposure activated TLR4/MyD88-NF-κB and -MAPK signaling pathway Next, to discover more about how prolonged exposure to cold aggravated the intestinal in ammatory response, western blot was used to determine the traditional NF-κB and MAPK signaling pathways associated with in ammation.The results showed that the phosphorylation of NF-κB (P65), JNK, ERK and p38MAPK, well as the phosphorylation and degradation of IκBα, were all clearly elevated by CS as compared to the RT group, demonstrating that CS could activate the NF-κB and MAPK signaling pathways.Furthermore, TLR4/MyD88, as a critical in ammatory regulator, is also upstream of the NF-κB and MAPK signaling pathways, according to numerous ndings.In contrast to the RT group, continuous cold exposure appeared to result in the TLR4 and MyD88 protein.These ndings implied that the TLR4/MyD88-NF-κB and -MAPK signaling pathways may play an important role in the CS-induced intestinal in ammatory response (Fig. 5).

Chronic cold exposure altered the overall organization of the gut microbiota
To analyze the effect of chronic cold exposure on the composition of gut microbiota, bacterial 16S rRNA sequencing in mice feces samples was performed.The dilution curves of the species richness α index were at and concentrated, indicating that the amount of sequencing data was reasonable and re ected most of the microbial diversity information in the samples (Fig. 6A).Moreover, the Venn diagrams show that there were 506 OTUs in the four groups, 602 OTUs in the CS + 0W and CT + 3W groups, 605 OTUs in the RT + 3W and CT + 3W groups.The number of OTUs unique to the RT + 0W, RT + 3W, CS + 0W and CT + 3W groups was 37, 13, 20 and 17, respectively (Fig. 6B).Then, the principal coordinate analysis (PCoA) based on Unweighted UniFrac showed the CS group of mice had an obvious segregation of their microbiota after 3 weeks of cold stimulation, while there was no signi cant segregation in the RT + 0W and RT + 3W groups (Fig. 6C).Furthermore, the Beta-diversity Ace index was used to assess the number of OTUs of the colony species and was signi cantly different after 3 weeks of cold stimulation, with a signi cant decrease in species richness in the CT + 3W group compared to the RT + 3W and CS + 0W group (Fig. 6D).

Chronic cold exposure resulted in intestinal ora disorders at genus level
To reveal the speci c changes of the intestinal microbial community after chronic cold exposure, we assessed microbial differences at genus level.As illustrated in Fig. 7A, it showed the top 30 gut microbiota at the genus level, of which the most abundant bacteria include six major bacterial genus, norank_f_Muribaculaceae, Lactobacillus, Aerococcus, Alistipes, Acinetobacter, and Akkermansia in all group, accounting for more than 70% of all genus.Moreover, detailed analysis at the genus level showed that chronic cold stress effectively upregulated or down-regulated the relative abundance of some bacteria.When composed to RT + 3W group, Lactobacillus and Carnobacterium were remarkably decreased by cold stimulation for 3 weeks (CS + 3W group), whereas Romboutsia, Corynebacterium, Turicibacter, and Muribaculum, were signi cantly increased (Fig. 7B and C).
Intestinal damage, in ammation and pyroptosis induced by chronic cold exposure are intimately linked to the gut microbiota Increasing evidence has revealed that aberrant changes in a variety of biochemical indicators are common, when an animal is exposed to cold stress.In mouse models of chronic cold stress, redundancy analysis (RDA) was performed to investigate the potential association between gut microbiota and environmental variables.As presented in Fig. 8A, gut indexes (stress, TJs, apoptosis, in ammation, and pyroptosis-related proteins) were examined, suggesting that there is a close correlation between gut microbiota and these indexes.The results of correlation heat map analysis indicated that Lactobacills and ZO-1, NLRP3, ASC, GSDMD, caspase-1, caspase-11, MyD88, p-P65, TNF-α, p-JNK, p-ERK and caspase3 showed signi cant negative correlation, whereas IκBα and Bcl-2 showed signi cant negative correlation.In addition, Jeotgalicoccus, Turicibacter, Corynebacterium, Romboutsia, Aerococcus and these proteins showed signi cant negative correlation, but IκBα and Bcl-2 (Fig. 8B).These investigations unveiled that presisent cold stimulation induced intestinal damage, in ammation and pyroptosis which are closely associated with the gut microbiota change.

Discussion
Intestinal diseases are extremely correlated with low temperature, for example, cold exposure exacerbates diarrhea in children and some young stock [10,22].Disturbance of intestinal ora, in ammation and pyroptosis are recognized to play essential roles in the occurrence and development of various intestinal disorders, such as IBD [1,4,36], however, few have investigated the underlying mechanisms of their cold stress and intestinal damage.Consequently, the current study's objective is to investigate how intestinal ora, in ammation, pyroptosis, and their interactions change in mice that are subjected to chronic cold stress.The ndings indicated that chronic cold exposure could alter the gut microbiome, cause pyroptosis, and trigger in ammation reaction in intestinal tissue of mice by activation of TLR4/MyD88-NF-κB/-MAPK and NLRP3/ASC/caspase-1-and caspase-11-mediated GSDMD signaling pathway, which are intimately associated with the change of intestinal ora.
Numerous studies have demonstrated that cold stress can harm a number of mammalian tissues, including the lungs, colon, liver, and hippocampus [7,15,28,33].In this study, chronic cold stress led to structural damage and in ammatory cell in ltration in the ileum, jejunum and colon of mice.Because they are important for the protection and alteration of epithelial barrier function, ZO-1 and occludin are frequently employed as markers of tight junctional barrier function and permeability function of intestinal epithelial cells [8,12].The results showed that cold exposure induced the increase of ZO-1 and occludin proteins expression in the ileum.Moreover, heat shock proteins (HSPs), which include HSP70 and HSP90, are critical stress proteins that play crucial regulatory functions in in ammation and oxidative stress, as well as a vital role as molecular chaperones in stress protection [29,39].The ndings showed that continuous cold stimulation induces an increase in HSP70 protein expression but not HSP90.It has been suggested that an essential indicator of intestinal injury is the apoptosis of intestinal epithelial cells [37].
In the present study, the expression of apoptosis-related proteins in the ileum tissues, such as cleavedcaspase-3, Bcl-2, and Bax, was shown to be considerably increased in response to cold exposure.These investigations demonstrated that intestinal stress from persistent cold stress results in impaired intestinal barrier function, bowel stress, and intestinal epithelial cell apoptosis, all of which result in intestine.
Intestinal in ammation can start and progress more easily when the intestinal epithelial barrier is disrupted, and that pro-in ammatory cytokines and mediators play a critical role in the beginning of in ammation [34].Meanwhile, intestinal in ammation is also an important causative factor for intestinal illnesses, such as IBD [20,30].Zhuang et al. revealed that acute cold water-immersion restraint (CWIR) stress signi cantly induced intestinal in ammation [54].Our team's previous ndings revealed that cold exposure provoked hippocampal neuroin ammation, liver in ammation and colon in ammation [48].In the present study, western blot analysis showed that chronic hypothermia exposure resulted in the increase level of iNOS, COX-2, HMGB1, IL-1β, IL-18, IL-6, and TNF-α in the ileum tissues, consistent with previous studies.Furthermore, pyroptosis, as a newly discovered pro-in ammatory type of programmed cell death, has been demonstrated to result in serials of gastrointestinal disease, including IBD and intestinal ischemia-reperfusion injury [46] [17].The conventional pathway on caspase-1-dependent and the noncanonical pathway on caspase-4/5/11-dependent are the two common pathways of pyroptosis [40,42].Caspases-1/11 is found in mouse cells, while caspases-4/5 are present in human cells.caspase-1 and caspase-11 (or caspase-4 and caspase-5) are both processed by Gasdermin D (GSDMD), which results in an N-terminal fragment GSDMD-N that forms a pore in the plasma membrane and causes pyroptosis and a severe in ammatory response [21].Our ndings initially showed that an obvious release of mature-IL-1β and IL-18 and formation of GSDMD-N was induced by cold stress.Given that in mice, the essential pyroptosis executor GSDMD serves as a shared substrate for caspase-1 and caspase-11 [31], the effect of cold exposure on caspase-1 and caspase-11 activation was further explored, and it was shown that caspase-1/11 was highly activated.
It is crucial to note that in ammation and pyroptosis may arise from pro-caspase-1 cleavage and the recruitment of an adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), by the NLRP3 in ammasome after the initial identi cation of various stimuli [31].In this work, we also discovered that chronic cold stress dramatically increased NLRP3 activation and ASC protein expression.These ndings indicated that persistent cold stress had the ability to cause pyroptosis and in ammation, which may be linked to the caspase-11-dependent atypical pathways and caspase-1-dependent typical pathway that is regulated by NLRP3.Numerous studies have shown that the TLR4/MyD88-mediated the NF-κB and MAPK signaling pathways are implicated in a number of in ammation-related disorders, including acute lung injury and acute ulcerative colitis, and they play a signi cant role in the control of in ammatory responses [19,32].Currently, it is generally accepted that the transcription factor NF-κB mediated by TLR4 is one of primary pathway which results in the NLRP3 in ammasome activation [16].
In the current study, the ndings of the western blot analysis demonstrated that long-term cold exposure stimulated the TLR4/MyD88 signal, which in turn activated the NF-κB and MAPK signaling pathways.
TLR4 and NLRP3 can be activated by a variety of PAMPs and DAMPs, leading to in ammatory reaction and pyroptosis, which in turn contribute to the onset and progression of diseases [24].Nevertheless, the gut microbiota is increasingly recognized as one of the major sources of PAMPs and DAMPs and has been reported to be intimately linked to a variety of chronic disorders, including IBD [2,13].There is strong evidence to support the idea that a myriad of acute or chronic stresses, such as the dietary habit and environmental conditions, can have an impact on gut microbial diversity and even gut microbiota dysbiosis [43,55].In our study, mice exposed to persistent cold exposure had intestinal ora abnormalities at the genus level, with Lactobacillus and Carnobacterium showing notable decreases and Romboutsia, Corynebacterium, Turicibacter, and Muribaculum displaying apparent increases.More critically, recent researches have revealed that abnormalities in the composition of the intestinal ora are directly linked to the activation of signaling pathways that promote in ammation and pyroptosis, such as TLR4/MyD88/NF-κB and NLRP3 in ammsome signaling pathways [27,41].Thus, under persistent cold stimulation, the crosstalk between intestinal indicators and gut microbiome were analyzed.Interestingly, our ndings showed that, in contrast to Lactobacills, the relative abundance of Jeotgalicoccus, Turicibacter, Corynebacterium, Romboutsia, and Aerococcus positively correlated with not only the activation of the TLR4/MyD88/NF-κB, MAPK, NLRP3/ASC, and caspase-1/11/GSDMD signaling pathway, but also the expression of ZO-1, HSP70 and apoptosis-related protein.
In summary, as presented in Fig. 9, the data could implicate that chronic cold stress triggers intestinal injury, cell apoptosis, in ammation and pyroptosis, with possible mechanisms related to NLRP3-mediated caspase-1-dependent canonical pathway and caspase-11-dependent noncanonical pathway, as well as TLR4/MyD88-NF-κB/-MAPK signaling pathway, which is closely correlation with changes in gut microbiota diversity.When taken as a whole, these observations offer fresh understanding of the increased risk of intestinal disease at extremely low temperatures and lay the groundwork for the development of innovative pharmaceutical interventions aimed at addressing cold-related maladies.

Declarations
Data availability The original data analyzed during the current study are available from the corresponding author upon reasonable request.
Con icts of interest The authors declare that they have no con ict of interest.

Figures
Figure 1 The protocol of the experimental design.The mice of control group were housed at a temperature of 24 ± 0.5 °C, and cold group was stayed at 4 °C.Tissue samples were collected from each group at 3 weeks.In addition, samples of feces were taken during weeks 0 and 3 of cold stress stimulation.Effect of chronic cold exposure on gut microbiota abundance at genus level in mice.C57BL/6 mice were kept at room temperature (RT) or cold stress (CS, 4 o C) for 3 weeks, 3 hours per day.In the last week, feces samples were collected and examined using 16S rRNA Gene Sequencing.(A) The relative abundance of the top thirty abundant bacteria at the genus level.(B) Heat mapping was used to assess microbiota species in the level of genus.(C) Linear discriminant analysis (LDA) scores were computed for features (at the genus level) that were differentially abundant between the RT+3W and CS+3W groups without treatment.* p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2 Effect
Figure 2

Figure 3 Effect
Figure 3

Figure 4 Effect
Figure 4

Figure 5 Effect
Figure 5