Effect of cold exposure and capsaicin on the expression of histone acetylation and Toll-like receptors in 1,2-dimethylhydrazine-induced colon carcinogenesis

Previous studies have indicated that capsaicin-rich diet and cold weather have shown strong association with tumor incidence. Thus, we investigated the effects of capsaicin and cold exposure in 1,2-dimethylhydrazine (DMH)-induced colorectal cancer as well as the mechanisms underlying capsaicin and cold-induced CRC. Rats were randomly divided into four groups and received cold still water and capsaicin via intragastric gavage until the end of the experiment. The rat’s body weight, thymus weight, and food intakes were assessed. Global levels of histone H3K9, H3K18, H3K27, and H4K16 acetylation and histone deacetylase (HDACs) in colon mucosa were assessed by western blot. Expression levels of Toll-like receptors 2 (TLR2) and Toll-like receptors 4 (TLR4) were measured by western blot and reverse-transcriptase quantitative polymerase chain reaction (qPCR). We found that cold and low-dose capsaicin increased tumor numbers and multiplicity, although there were no differences in tumor incidence. Additionally, rat exposure to cold water and capsaicin display further higher levels of histone H3 lysine 9 (H3K9AC), histone H3 lysine 18 (H3K18AC), histone H3 lysine 27 (H3K27AC), and HDACs compared with the DMH and normal rats. In contrast, a considerable decrease of histone H4 lysine 16 (H4K16AC) was detected in the colon mucosa. Cold and low-dose capsaicin exposure groups were also increased TLR2 and TLR4 protein levels and mRNA levels. These results suggest that chronic cold exposure and capsaicin at a low-dose intervention exacerbate ectopic expression of global histone acetylation and TLR level, which are crucial mechanisms responsible for the progression of colorectal cancer in rats.


Introduction
Colorectal cancer (CRC) has been recognized as a multifactorial malignant disorder and remains its status as the most common cause of cancer-related death in men and women (Siegel et al. 2019). Colon cancer is caused by complex interactions of the immune system with non-modifiable (e.g., genetic and epigenetic alterations) and modifiable (e.g., environmental, diet and lifestyle) risk factors (Grazioso et al. 2019;Lao & Grady, 2011). The fact that known high penetrance gene and epigenetic alterations that are related to colorectal cancer risk explain fewer than 5% of the observed case suggested that environmental factors including diet, alcohol consumption, and ambient environment play a pivotal role in risk (Lichtenstein et al. 2000). Moreover, it is important to note that these modifications of known factors are most associated with cancer death in China (Chen et al. 2019).
Identifying the epigenetic mechanisms and their interaction with diet and Toll-like receptors is crucial to reveal the regulation of molecular pathways leading to colorectal carcinogenesis. Colorectal cancer (CRC) is characterized by the accumulation of genetic and epigenetic alterations (Okugawa et al. 2015). Epigenetic modifications have been implicated in the regulation of gene expression, without affecting the DNA sequence (Strahl and A. C. 2000). Among these modifications, histone acetylation on lysine residues is the most common histone modification. The expression level of histone acetylation is important regarding chromatin states and gene expression. Overall, lysine acetylation generally opens the chromatin and activates gene expression, whereas deacetylated is related to transcriptional repression (Berger 2007). It has previously been reported that aberrant histone acetylation was implicated in the initiation and progression of various solid tumors including CRC (Kaypee et al. 2016). Dynamic levels of reversible acetylation are mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). They are classified into four classes based on cellular localization and functional regulation (Annemieke et al. 2003). More and more research indicated that germline mutations of HDACs increase the risk of breast and lung cancers, and abnormal HDAC overexpression has also been observed in a wide range of malignancies, thereby serving as therapeutic targets for CRC (Glozak and Seto 2007;Singh et al. 2018). Previous study indicated that animal housing at sub-thermoneutral (22-26°C) has a profound effect on obesity, inflammation, cancer, and other metabolic disorders (Ganeshan and Chawla 2017;Hylander and Repasky 2016). For example, studies in broilers revealed that long-term cold stimulation aggravated oxidative stress, ultrastructural cardiac damage, immune dysregulation, and inflammation (Wei et al. 2018). It was also found that the cold environment substantially potentiates the development of malignancy resulting from subthermoneutral laboratory housing temperature cause mild chronic cold stress, which triggers suppression of the antitumor immune response (Kokolusa et al. 2013). Some published reports have described an anti-cancer role for capsaicin, while others have argued that capsaicin is implied as a promoting factor by inducing the growth and progression of preexisting neoplasms (Bode and Dong 2011). In vitro and in vivo experimental of CRC indicated that capsaicin exerts anti-tumor activity through induces cytotoxicity and apoptosis (Lu et al. 2010). On the other hand, Yang et al. reported that human colon carcinoma promotion involved in the Akt/mTOR/STAT-3 signaling pathway (Yang et al. 2013). Toll-like receptors (TLRs) have an important role in the activation of both innate and adaptive immunity, and aberrant activation of the TLR signal can result in disturbance of intestinal immune homeostasis, chronic inflammatory, and therefore an important modulator in the pathogenesis of CRC (Lavelle et al. 2009). It has been also demonstrated that TLR agonists have immune regulatory applications as vaccine adjuvants in cancer therapy (Moradi-Marjaneh et al. 2018). Furthermore, epigenetics has recently been proven to be a new regulator of TLR expression (Xie et al. 2018). However, much less known regarding the mechanism underlying the putative effect of potential chronic cold and capsaicin exposure contribution on the promotion and progression stages of DMH-induced colon carcinoma in rat models.
Therefore, the purpose of this research was to evaluate the expression of aberrant histone modification via the modulation of HDACs as well as the TLR signaling pathway on colon carcinogenesis.

Material and method
Chemicals and preparation 1,2-Dimethylhydrazine (DMH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). DMH was weighed and dissolved in 1Mm EDTA-saline to ensure the stability of the chemical (Gungor et al. 2018). And the pH was adjusted to 7.0 each time with 1 M NaOH solution. 95% purity of capsaicin provided by Xian Ruilin Biotechnology Co (Xian, China).

Animals and treatment
Six-week-old male Wistar rats weighed between 200 and 250g were kept in SPF laboratory animal room generated from Experimental Animal Center in Guangzhou University of Chinese Medicine. The animals were given ad libitum access to water and food and housed in plastic cages in a temperature-controlled (24°C) room with a 12-h light-dark cycle. All the experimental protocols were permitted by the Institutional Animal Ethics Committee of the Guangzhou University of Chinese Medicine (No. 20130001). Based on the previously described protocol [23,24], we made some minor revisions to the established colon carcinoma model induced by 1, 2-dimethylhydrazine (DMH) in rats. Briefly, after a period as long as 3days for adapting to the environment, rats were randomly divided into 4 groups with 10 animals each (normal control group, DMH group, capsaicin exposure group, and cold exposure group). The capsaicin exposure group was given capsaicin at 10mg/kg b.wt. every day. The cold exposure group was treated with cold distilled water (0°C) at 10mg/kg b.wt. until the end of the experiment.
Group A (normal control): Rats received basal diet, drink water, and gavage with the equivalent volume of saline once a day.
Group B (DMH): Rat was administered and subcutaneously injected with DMH (25mg/kg b.wt.) once a week for 12 weeks and also received saline once a day.
Group C (capsaicin combined with DMH): Rats were administered with DMH as in GP B and also given capsaicin (10mg/kg b.wt.) every day throughout the experiment. DMH was weighed and dissolved in normal saline to ensure the stability of the chemical before use with a final concentration of 2 %. And the pH was adjusted to 7.0 each time with 1 M NaOH solution. The preparation of DMH in other groups was the same.
Group D (DMH + cold distilled water): Rats were administered with DMH as in GP B and also given cold distilled water (10mg/kg b.wt.) via intragastric gavage continued till the end of different phases of experiments.
The appearance change of rats was monitored daily. Bodyweight and food intake of rats were recorded weekly throughout the experiment. For food consumption measurement, the model rats were provided with 500g of food in the morning. Food weight was detected at a fixed time to determine daily food intake with the last 24 h. All the rats were anesthetized with chloral hydrate and then sacrificed 30 weeks after the end of DMH administration. The experiment lasted 30 weeks. The experimental protocol is exhibited in Fig. 1.

Morphological study of colon tissues
Colons were longitudinally dissected from the anal to the cecum and then washed it with PBS. The colons were spread out on cleaning tissue paper. The number of colon tumors was recorded for tumor incidence and the size of the tumors was measured using a caliper paper. Tumor volume was calculated using the formula: tumor volume (mm 3 ) = length (mm) × width 2 (mm 2 )/2 as reported by Isha Rani (Isha Rani and Agnihotri 2014).

Histopathological observation
For histology, the colon sections were placed in a 15-ml conical tube filled with a 4% paraformaldehyde solution over 24 h. Colon paraffin sections were prepared by the pathology department at the Guangzhou University of Chinese Medicine. H&E staining sections were visualized through an Olympus TH4-200 (Tokyo Japan) under ×400 magnification. Microscope was classified as tumors in accordance with specific pathological criteria described by Nolte. Colonic neoplasms were classified as adenomas or adenocarcinomas (tubular or mucinous) (Nolte et al. 2016). Tumor incidence is the percentage of rats bearing the indicated type of tumor. Incidence values are represented as a percentage.

Histone extraction
We extracted the total histones from colon tumor samples (n=3/group) using the EpiQuik total histone extraction kit (EpiGentek, Farmingdale, NY, U.S.A.). We prepared histone following the manufacturer's protocol. Briefly, 100mg colon tissues were homogenized in a mortar with liquid nitrogen, adding lysis buffer, and then incubated on ice for 30 min. The homogenized mixture was transferred into a 2-ml vial and centrifuged at 3000g for 5 min at 4°C, and then the supernatant was removed. Balance buffer was added and incubated on ice for 30 min and centrifuged at 3000g for 5 min at 4°C, and the supernatant fraction was transferred into a new vial, adding Balance-DTT buffer immediately. Protein concentrations were quantified by Bradford protein assay kit (KeyGEN, China) and the extracts were stored at −80°C.

Real-time quantitative RT-PCR
Total RNA was extracted from colon tumor tissues (n=6/ group) using a Trizol reagent (Takara, Japan). The concentration and quality of RNA samples were evaluated with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription was carried out to obtain cDNA using the Master Mix kit (Takara, Japan) following the standard protocols. The mRNA levels of TLR2 and TLR4 in colon mucosa were assessed using a Step One Plus realtime PCR system (Thermo Fisher Scientific, CFX384TM Real-time System). β-Actin was used as housekeeping gene to normalize mRNA expression. The relative levels of gene expression were enumerated using the comparative formula 2 -ΔΔCt (C. Liu et al. 2018). Detailed information of the primer sequences was as follows:

Statistical analysis
All the data were summarized as mean±standard deviation (SD) and analyzed by SPSS 23.0. To determine if there were differences between groups, we performed the data with a one-way analysis of variance (ANOVA). Fisher's exact test was performed to compare the tumor incidence between the different groups. We considered differences significant at P< 0.05.

General observation
As shown in Fig. 2A, before 22 weeks, weekly monitoring throughout the experimental period showed no noticeable body weight loss among groups. After 22 weeks, DMHtreated rats showed a decrease in body weight as compared to control rats. Compared with the DMH-treated group, the weight of cold exposure groups was reduced, but the statistical difference among groups was not significant. No difference in food intake was found among groups by the end of weeks 2-6. The food intakes of DMH-treated rats were decreased at the rest of the experimental period. In 28-30 weeks, the food intake was increased in the cold exposure group as compared to the DMH-treated group. Conversely, the food intake was decreased in the capsaicin group in 12-18 weeks compared with that in the DMH group. Figure 2 C and E showed that the thymus weight in the cold and capsaicin exposure groups was lower than that of the normal group, although no significant difference in the thymus weight was observed among DMHtreated, cold exposure, and capsaicin treatment groups.

Morphological study of colon tumor and histopathological analysis
Seven months after the experiment started, no visible colon tumor was found in normal control. Most of the colonic tumors were developed in the distal colon than in the proximal colon of Wistar rats. As shown in Fig. 2 D and F, the average volume tumor of the DMH-treated group was 15.24 ± 12.45 mm 3 and that of the cold exposure and capsaicin treatment groups was 34.58± 27.76 mm 3 and 22.70 ± 18.02 mm 3 , respectively. These data suggest that there was a bigger volume in rats of the cold exposure and capsaicin treatment than the DMH treatment. There was no difference in tumor incidence among groups (Table 1). The tumor multiplicity was higher among the cold exposure and capsaicin groups as compared to the DMH group due to the increased number of tumors. A trend towards the increase of invasive tumors was observed in the cold and capsaicin group. Furthermore, the pathological type of the cold group was the most serious. Histopathological analysis showed that most tumors were either tubular adenocarcinomas or poorly differentiated mucinous adenocarcinomas (Fig. 3).

Effect of cold exposure and capsaicin on HDAC activity
The expression of HDAC1, 2, 3, and 8 in colonic mucosa is shown in Fig. 4 A and B. The protein expression levels of HDAC 1, 2, 3, and 8 were significantly elevated in colonic tissue from DMH-treated rats. Compared to those in the DMH group, the HDAC 1, 2, 3, and 8 expressed in the cold and capsaicin exposure groups were further increased. These findings implicated that the activation of the HDACs may be involved in the CAC induced by DMH and cold exposure and capsaicin treatment could exacerbate the expression levels of HDACs.
Effect of cold exposure and capsaicin on colonic tissue H3K9ac, H3K18ac, H3K27ac, and H4K16ac protein levels The effect of cold exposure and capsaicin H3K9ac, H3K18ac, H3K27ac, and H4K16ac in colon mucosa is shown in Fig. 5. Compared to those in the control group, H3K9ac, H3K18ac, and H3K27ac in the DMH group were increased. In contrast, exposure of rats with cold water and capsaicin resulted in a loss of H4K16 acetylation in colon mucosa during colorectal carcinogenesis. Compared with the DMH group, the cold and capsaicin exposure group further increased the levels of H3K9ac and H3K27ac but had no effect on H3K18ac levels. However, the protein level of H4K16ac was lower than that in the DMH-treated group, but the statistical difference among the groups was not significant.
Effect of cold exposure and capsaicin on colonic mucosa TLR2 and TLR4 protein levels The expression of TLR2 and TLR4 protein levels and mRNAs in colonic mucosa are shown in Fig. 6. The cold and capsaicin exposure group had significantly higher levels of TLR2 and TLR4 protein and mRNAs in colon mucosa as compared with control. Compared to those in the DMH group, expression of Fig. 2 Bodyweight, food intake, thymus weight, and tumor volume in normal rats, cold exposure and capsaicin-treated rats. A Effects of cold exposure and capsaicin on the bodyweight of rats. B Effects of cold exposure and capsaicin on the food intake of rats. C, E Change in thymus weight and thymic morphology. D, F Macroscopic image of the colonic tumors and tumor volumes in different treatment groups. Data were expressed as the mean ± S.D. Comparisons: # control compared with DMH, * DMH compared with cold exposure and capsaicin-treated group. # P < 0.05, ## P < 0.01, * P < 0.05, ** P < 0.01 TLR2 and TLR4 protein levels and mRNAs in cold and capsaicin exposure groups were further increased.

Discussion
Recent several epidemiological studies have associated cold temperatures and capsaicin consumption with an increased risk of several cancers (Du et al. 2020;Michal Freedman et al. 2015;Voskarides 2019). Our findings demonstrated that cold exposure and low-dose capsaicin administration aggravate the ectopic expression of histone acetylation level and histone-modifying enzymes through the structure of chromatin, making rats induced by DMH more susceptible to CRC. Moreover, cold and capsaicin exposure further increased TLR2 and TLR4 expression as well as increased histone H3 acetylation of TLR2 and TLR4 in the colonic mucosa of DMH-induced rats but not normal rats. These results suggest that higher HDAC expression in the colonic mucosa and ectopic expression of histone acetylation may be involved in chromatin remodeling, which might play a fundamental role in the pathogenesis of CRC.
More recently, evidence supports an emerging view that environmental factors such as diet, lifestyle, and environment are among the top risk factors that predispose to cancer. For example, males exposed to a high concentration of toxic contaminants, sperm nuclear basic proteins (SNBP), were involved in DNA oxidative damage Lettieri et al. 2020aLettieri et al. , 2020b. DNA damage contributes to the initiation and progression of cancer by increasing cell proliferation, genetic alterations, and genomic instability (Akshay Bhat et al. 2018;Chatterjee and Walker 2017). In addition, it is also demonstrated that environmental contaminants can have transgenerational inherited effects on spermatozoa, DNA, and the molecular alterations of SNP (Lettieri et al. 2020a(Lettieri et al. , 2020b. The presence of high levels of DNA damage in Fig. 3 Representative sections stained with H&E showing the histopathology of the colonic neoplastic lesions. A Adenoma, characterized by non-invasive polypoid tumor projecting into the intestinal lumen, B invasive tubular adenocarcinoma, and C invasive mucinous adenocarcinoma. Both featuring invasion of abnormal tubular or mucinous structures through the muscularis mucosa or deeper layers of the intestinal wall human spermatozoa has been correlated with infertility and childhood cancer (Bisht et al. 2017. Furthermore, it has been reported that cold environmental temperature can be potential cancer-causing factor, and people who live in the cold environment have a high risk of cancer incidence and mortality (Sharma et al. 2015). Kokolus and colleagues found that when tumor-bearing mice are housed at thermoneutral temperature (30-31°C), they observed reduced tumor formation, growth, and metastasis (Kokolusa et al. 2013). Capsaicin is the major pungent alkaloid of chili peppers (Naves et al. 2019). Conflicting epidemiological and basic research studies suggest that capsaicin exerts a role in either a protective effect or carcinogenic effect (Bode and Dong 2011;Friedman et al. 2019). Recent studies indicate the protective effect of capsaicin against various types of cancer. It has been shown that capsaicin at 50mg/kg reduced cancer risk in DMH-induced CRC rat models by inhibiting the cytotoxicity, genotoxicity, proliferation, and apoptosis of cancer cell/tissue (Caetano et al. 2018). They also showed that high-dose capsaicin decreases the proportion of tubular adenocarcinoma and tumor size in DMH-induced CRC (Caetano et al. 2021). Additionally, capsaicin suppresses the growth of numerous types of cancer cell, suggesting that it has chemopreventive activities Zheng et al. 2016). However, various studies have suggested a potential pro-carcinogenic effect of capsaicin use. Epidemiologic studies suggested the consumption of hot peppers, which contain variable levels of capsaicin, might increase the risk of CRC (Zhivotovskiy et al. 2012). It was reported that capsaicin has tumor-promoting activities in skin, lung, and colon cancer in different chemically induced Semi-quantitative analysis of immunoblots was normalized by corresponding histone 3 or histone 4 bands. Data were presented as the mean ± S. D form three separate experiments. Comparisons: # control compared with DMH, *DMH compared with cold exposure and capsaicintreated group. # P < 0.05, ## P < 0.01, * P < 0.05, ** P < 0.01 carcinogenesis models (Geng et al. 2016;Liu et al. 2015;Nalinia et al. 1998). Another study indicated that capsaicin could significantly accelerate tumor formation and growth in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin carcinogenesis through the inflammation-dependent mechanism (Z. Liu et al. 2015). In vitro study showed that low concentrations of capsaicin enhanced the migratory and invasive capability of CRC cells by upregulating the expression of tumor-associated NADH oxidase (Liu et al. 2012). The results of the present study showed that chronic cold exposure and long-term application of capsaicin at 10mg/kg promote the development and progression of CRC in animal experimental models. We accordingly speculated that the anti-cancer activity of capsaicin may depend on its concentration. Further study is needed to precisely delineate the effects of capsaicin on CRC.
The overall disruption of the epigenetic landscape including alteration in DNA methylation and histone modification pattern is the most common feature of all human tumors (Nebbioso et al. 2018). DNA methylation is central to the control of retroelements in the human genome, ontogeny, and phylogeny (Yoder et al. 1997). Transcriptional regulatory sequences from retroelements, typically from LINEs, have changed with regard to methylation and activity (Schulz 2006). Many studies suggest hypomethylation of LINEs may be essential in the pathophysiology of various diseases and cancer, contributing to chromosomal instability and chromatin structure alteration (Jin et al. 2009;Vafadar-Isfahani et al. 2017). It was reported that the DNA methylation expression level of two retrotransposon sequences-LINE1 and L1 was decreased after cortical spreading depression (CDS) induction, suggesting the essential role of DNA methylation of retrotransposon in the pathological state of ischemia (Drongitis et al. 2016). By now, retroelements in diseases are implicated in a much wider range of afflictions, such as atherosclerosis and schizophrenia Kan et al. 2004;Tabaei and Tabaee 2019). Recent research has shown that regulation of specific HDAC isoforms and aberrant histone modification alterations imposed by diet might not be the only mechanisms responsible for neoplastic cell transformation but also be implicated in the development of cancer (Nebbioso et al. 2018). The overexpression of class I HDACs has been reported in many cancer types including colon carcinoma (Weichert et al. 2008). Furthermore, the expression was enhanced in strongly proliferating and poorly differentiated tumors and upregulation of HDAC correlated with poor prognosis of CRC (Ashktorab et al. 2009;Y. Li and Seto 2016). In addition, H3K9ac, H3K18ac, and H3K27ac were reported to be significantly upregulated in colorectal adenomas and cancers as compared to their normal counterparts (Karczmarski et al. 2014). Tamagawa et al. reported a decreased methylation of H3K27me2 in liver metastasis CRC in comparison with primary tumors (Tamagawa et al. 2013). Moreover, several studies have suggested that global patterns of histone modification provide additional independent information for histological subtype, prognosis, and cancer recurrence (David B. Seligson et al. 2009;Tamagawa et al. 2013). For instance, in breast cancer, low global H3K27me3 methylation levels have Fig. 6 Effect of cold and capsaicin exposure on the expression of TLR2 and TLR4 proteins in different treatment groups. A Representative Western immunoblot images are shown. B Semi-quantitative analysis of immunoblots was normalized by corresponding β-actin bands. C, D The mRNA expression levels of TLR2 and TLR4 in the colon tissues of rats in different groups. Data were presented as the mean ± S. D from three independent experiments. Comparisons: # control compared with DMH, *DMH compared with cold exposure and capsaicintreated group. # P < 0.05, ## P < 0.01, * P < 0.05, ** P < 0.01 been described using genome-wide analyses whose expression correlated with aggressive subtypes of breast cancer (Hsieh et al. 2020). Loss cell levels of H3K4me2 and H3K9me3 demonstrated correlations with poor differentiation, shorter disease-free survival (DFS), and metastases in pancreatic cancer (Manuyakorn et al. 2010). The hypoacetylation of H3K9, H3K18, and H4K16 was strongly relevant to the clinical outcome of prostate cancer (D. B. Seligson et al. 2005). While elevated global histone acetylated histone H3 (H3ac) in colon cancer tissues was reported to predict poor overall survival in patients (Hashimoto et al. 2013), our results showed that the expression of H3K9ac, H3K18ac, and H3K27ac in the colons of DMH-induced CRC rats was significantly increased, and cold exposure and capsaicin treatment further increased this phenomenon. As compared with the DMH group, the acetylation of H4K16 was clearly downregulated in the cold exposure and capsaicin treatment groups. We postulated that upregulated HDACs lead to abnormal patterns of histone acetylation, which is associated with the deregulation of gene transcription, thereby contributing to the CRC progression. Understanding the complex biology of histone modification and its regulators is thus essential for CRC pathogenesis and treatment.
There are many molecular pathways involved in CRC development and different levels of evidence support that chronic inflammation plays an essential role in cancer development and progression (Diakos et al. 2014;Monteleone et al. 2012;Grivennikov et al. 2010). Aberrant activation of TLRs has been shown to increase the risk of colorectal cancer caused by disruption of chronic inflammation, immune response, and epithelial barrier homeostasis, which predisposes individuals to develop CRC (Cario 2010;Li et al. 2014;Xiang et al. 2012). Thus, its aberrant activation can be implicated in the pathogenesis of intestinal diseases, such as inflammatory bowel disease (IBD), colitis-associated cancer (CAC), and CRC. The abnormal activation of Toll-like receptors (TLRs) leads to an impairment of immune homeostasis, which contributes to CRC development. It is reported that TLR2 and TLR4 are upregulated in CRC and correlated with a poor prognosis in patients with CRC (Nihon-Yanagi et al. 2012;Xu et al. 2011). Additionally, it has been shown that genetic variation in TLR2 and TLR4 gene interaction with dietary factors increased susceptibility to CRC and multiple singlenucleotide polymorphisms (SNPs) in these genetic profiles, associated with CRC prognosis (Kopp et al. 2018;Slattery et al. 2012). Furthermore, recent research investigating the role of TLRs in diseases indicates that DNA methylation and histone modification epigenetic have emerged as new mechanisms involved in the regulation of TLRs (Hennessy and McKernan 2016). Takahashi et al. (Takahashi et al. 2009) suggested that epigenetic modification, including histone acetylation and DNA methylation, acts as a negative regulator of TLR4 gene transcription when associated with ZNF160, a repressive-associated transcription factor in human intestinal epithelial cells. TLR2 was reported to decrease methylation of the proximal human TLR2 promoter, resulting in an upregulation of TLR2 (Haehnel et al. 2002). Here we observed that cold and capsaicin exposure further increased Fig. 7 Schematic diagram depicting the role of histone acetylation and Toll-like receptors in promoting colorectal cancer pathogenesis. In comparison with the normal rats, rats exposed to cold and capsaicin have higher levels of HDACs activity in the colon mucosa, which results in abnormal expression of histone H3-K9, H3-K18, H3-K27, and H4-K16 acetylation. The innately higher levels are associated with the relaxation of chromatin of TLR2 and TLR4 genes. These results implicate a crucial role of histone acetylation-induced chromatin remodeling and can also positively regulate the expression of TLRs, thereby exacerbating the CRC progression the expression of TLR2 and TLR4 in the colon mucosa and this could be related to the epigenetic mechanism. Histone modification might be one of the regulators of TLR2 and TLR4. Further studies are necessary to identify the potential genetic locus regulated by histone modification upon cold exposure and administration of capsaicin in DMH-induced CRC through chromatin immunoprecipitation (ChIP) and next-generation sequencing (ChIP-seq) assay.

Conclusion
In summary, as shown in Fig. 7, the present study indicated that cold exposure and long-term administration of capsaicin at a low dose further aggravate the abnormal expression of HDACs and global histone modification as well as TLRs and that such changes are strongly correlated with the development and progression of CRC. A better understanding of the epigenetics and TLRs on cold exposure and capsaicin treatment rats may help to clarify the mechanism of tumorigenesis which could afford a novel therapeutic approach for CRC patients. Lastly, further investigations are required to understand the epigenetic mechanisms of TLR regulation and the role of TLRs in the pathogenesis, prevention, and treatment of CRC.
Author contribution Jingchun Qin and Huixuan Li wrote the first draft of the manuscript. Bin Wen designed the study. Jingchun Qin, Huixuan Li, and Weitao Yu conducted the experiment and analyzed the data. Li Wei contributed to review and edit the paper. Bin Wen revised the manuscript. All the authors have read and agreed to the published version of the manuscript.
Funding This work was supported by the National Natural Science Foundation of China (Grant No. 81673944).
Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval This study was approved by the institution's Ethics Review Board (S2017037).

Consent for publication Not applicable
Conflict of interest The authors declare no competing interests.