Potential Effect of β-casomorphin-7 on Lymphocyte and TLR\NF-κB Signaling Pathway in Intestinal Mucosa of Aged Mice

The gut plays a vital role in absorbing nutrients and drugs, preventing pathogen invasion and maintaining the health of the body. In many elderly people, gastrointestinal dysfunction, mucosal defense deficiency and oxidative stress increase, influence the ability to absorb nutrients and maintain the balance of normal microbial flora, which leads to lower immunity and an increase in the incidence of inflammation and autoimmunity in the elderly. Experiments show that the gut may become an important target for promoting longevity intervention (Nagura, 1992).
       
The multifunctional properties of biologically active peptides from milk are increasingly acknowledged (Darewicz et al., 2014). β-Casomorphins belong to a family of opioid peptides derived from milk protein. β-Casomorphin-7 (Tyr-Pro-Phe-Pro-Gly-Pro-Ile, β-CM-7) was first isolated from an enzymatic digest of bovine β-casein (Brantl et al., 1979). Current researches show that β-CM-7 can regulate glucose (Yin et al., 2010), antioxidative (Yin et al., 2012 and Wei et al., 2013), immunological (Stanisław et al., 2007), hormonal and neurological responses.
       
Aging would cause the increase of the concentration of inflammatory cytokines in intestinal mucosa, the damage of intestinal tissue structure and the decrease of immune function of small intestinal mucosa (Yin et al., 2019 and Zhang et al., 2016). β-CM-7 can significantly increase the content of SIgA, reduce the content of pro-inflammatory factor TNF-α and significantly improve the activity of antioxidant kinase, such as SOD and CAT in small intestinal mucosa and then reduce the intestinal tissue damage caused by aging, maintain the normal form of intestinal tract and enhance the immunity of small intestinal mucosa. But the mechanism of β-CM-7 reconstruction of intestinal mucosal immune homeostasis and postponing inflammatory aging need further study. The purpose of this study is to further explore the mechanism of β-CM-7 on intestinal mucosal immunity.

Chemicals and reagents
 
β-casomorphin-7 (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) was purchased from Nanjing Peptide Biotech Co., Ltd. (Nanjing, China). All the other chemicals and reagents were of analytical grade.
 
Animals (Gap from the previous line)
 
Forty elderly male KM mice (11 months old) and ten young KM mice (2 months old) were purchased from Nanjing Qinglongshan Animal Center (Nanjing, China). They were housed under controlled environmental conditions of temperature (22 ± 2°C) with a 12-h light/12-h dark cycle and maintained on (unless otherwise stated) standard food pellets and tap water. All animal care and procedures were performed in accordance with Jiangsu Province and institutional policies for animal health and well-being. All mice samples collection and field study were approved by guide for care and use of Laboratory Animals of the Protocol for Animal Study of Animal Management Committee of Jiangsu Province and Yangzhou University. The animals were acclimatized for one week before the study.
 
Experimental design
 
The young mice (n = 10) were the control group (group I) while the elderly mice (n = 40) were randomly divided into four groups (group II to V):
 
Group I (n = 10): Normal control mice with free access to normal diet and intragastric administration of stroke-physiological saline solution for 30 days.
Group II (n = 10): Model control mice with free access to a normal diet and intragastric administration of stroke-physiological saline solution for 30 days.
Group III (L, n = 10): β-CM-7 treated mice; each animal was put on a normal diet and treated with the low dose of β-CM-7 (2×10^-7 mol·d^-1, intragastric administration) for 30 days.
Group IV (M, n = 10): β-CM-7 treated mice, each animal was put on a normal diet and treated with the moderate dose β-CM-7 (1×10^-6mol·d^-1, intragastric administration) for 30 days.
Group V (H, n = 10): β-CM-7 treated mice, each animal was put on a normal diet and treated with the high dose β-CM-7 (5×10^-6 mol·d^-1, intragastric administration) for 30 days.
 
Collection of organ tissues
 
All mice were sacrificed and Post-mortem examination was carried out immediately. Intestinal mucosa were dissected out, washed in phosphate buffer saline. Small intestine samples were taken fixed in 4% neutral-buffered Polyoxymethylene and other intestinal mucosa samples were taken instantly into liquid Nitrogen and stored at -70°C.
 
Analytical methods
 
Histopathological observation
 
Small intestine fixed in 4% neutral-buffered Polyoxymethylene were embedded in paraffin, sliced at a thickness of 5 µm and stained with hematoxylin and eosin (H and E). The histological changes were observed by light microscopic examination at a magnification of 20 X.
 
RNA extraction and RT-PCR
 
RNA extraction
 
Total RNA was extracted from the tissue samples using TRIZOL Isolation Reagent (Tian Gen Biochtech Co., Beijing, China). RNA concentration was quantified by measuring absorbance at 260 nm using a Biophotometer (Eppendorf, Hamburg, Germany). Ratios of absorption (260/280 nm) of all preparations were between 1.8 and 2.0. Aliquots of the RNA samples were subjected to 1% denaturing agarose gel electrophoresis with ethidium bromide staining to verify the quality of total RNA.
 
Reverse transcription (RT)
 
Synthesis of first strand complementary DNA (cDNA) was performed with reverse transcriptase and Oligo (dT)18 primer according to the manufacturer’s instructions. The final volume of 20 μL contained 10 U of AMV reverse transcriptase, 1 mM dNTP mixture, 20 U of recombinant RNasin ribonuclease inhibitor and 50 pmol of Oligo(dT)18 primer. All reagents were purchased from TaKaRa, Dalian,China. After incubation (42°C for 60 min), the mixture was incubated at 95°C for 5 min). A negative-RT reaction (RT enzyme replaced with water) was performed to detect DNA contamination.
 
Real-time PCR
 
The nucleotide primer sequences for real-time PCR were showed in Table 1. Quantitative fluorescence real-time RT-PCR analysis was performed with 7300 Real-time PCR system (Applied Biosystems,Foster City, USA). PCR reactions were set up with the Real-time PCR Master Mix SYBR® Green (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. Each sample was run in triplicate.Cycling conditions for both genes were 95°C for 1 min, followed by 45 cycles of 95°C 20 s, 62°C 30 s and 72°C 20 s. The specificity of each PCR product was determined by melting curve analysis. Results (fold changes) were expressed as 2^-ΔΔCt with ΔΔCt=(Ct ij-Ct β-actin j)-(Ct i1-Ct β-actin1), where Ct ij and Ct β-actin j are the Ct for gene i and for β-actin in a sample (named j) and where Ct i1 and Ct β-actin1 are the Ct in sample 1, expressed as the standard (Schmittgen 2001).
 

 
Statistical analysis
 
Data were analyzed statistically using SPSS 16.0 for Windows and expressed as the mean ± SD of 10 mice per group. Experimental results were compared by one-way ANOVA with least significant difference (LSD) post-hoc tests used to compare individual means as appropriate. P<0.05 or P<0.01 were considered statistically significant.

Table 2 shows that the number of duodenal epithelial lymphocytes in the old model group is significantly lower than that in the young control group (P<0.05). Compared with the aged control group, the number of duodenal epithelial lymphocytes in each dose group increased significantly (P<0.05). Compared with the young control group, the number of jejunal epithelial lymphocyte in the old model group was significantly decreased (P<0.05) and the number of jejunal epithelial lymphocyte in each dose group was significantly increased compared with the aged control group (P<0.05). Table 2 shows that the number of epithelial lymphocytes in the ileum of the old model group is lower than that of the young control group, but the difference is non-significant. Compared with the aged control group, the difference between the high dose group and the old model group was statistically significant (P<0.05).
 

       
Table 3 shows that the duodenal mucosal thickness of the aging model group is significantly. (P<0.05) higher than that of the young control group.
 

       
Table 3 shows that the thickness of ileal mucosa in the aging model group was significantly lower than that in the young control group (P<0.05). The thickness of ileal mucosa was increased in each dose group and there was a significant difference between the middle dose group and the model group (P<0.05).
       
As a special component of intestinal related lymphoid tissue, intestinal mucosal intraepithelial lymphocyte is the first immune cell contacted by immune system, foreign antigens and microorganisms. Therefore, the number of intraepithelial lymphocytes in small intestinal mucosa can reflect the integrity of the local mucosal immune barrier and the perfection of the immune defense function of small intestine (Hong et al., 2006). The results showed that the number of epithelial lymphocyte in duodenum, jejunum and ileum of aging model group was less than that of young mice, which indicated that with aging, the ability of intestinal defense against pathogenic microorganism invasion decreased and the mucosal immune function of small intestine also decreased, which was consistent with the results of Ren W Y (Ren et al., 2014). From the results of this study, we can see that each dose of β-CM-7 can increase the number of lymphocyte in each intestinal segment of aging mice and thus play a protective role in the intestinal immune function of aging organisms. In addition, some studies have shown that β-CM-7 can promote the immune function of the digestive tract of weaned piglets (Wang et al., 2016).
       
During aging, the intestinal mucosa shrinks, its thickness decreases and bacteria contact with epithelium directly too much, which makes the intestinal mucosa as a physical barrier of epithelium ineffective and the barrier function deteriorates gradually, resulting in a series of changes in immune response in the elderly. The results showed that the thickness of duodenum and jejunum mucosa in the aged model mice was increased compared with that in the young mice and the thickness of ileum mucosa was decreased. It was reported that the ileum was more prone to structural damage in the age-related histological changes of the small intestine (Omotoso et al., 2012). After feeding β-CM-7, the mucosal thickness of the whole small intestine segment was increased at medium dose. In conclusion, β-CM-7 has a certain beneficial effect on the intestinal mucosal thickness of senile organisms.
       
TLR is an important innate immune recognition receptor. TLR2 and TLR4 participate in intestinal mucosal immune response. The mechanism of TLR signal transduction pathway in intestinal mucosa is to defend pathogens. Studies have shown that TLR can be involved in the protection of Salmonella infection, which indicates that TLR4 can interact with other TLRs to prevent intestinal infections of pathogenic bacteria (Alvesalo et al., 1977). In this study, we found that the relative expression of TLR4 in intestinal mucosa of aged mice was lower than that of young mice, while β-CM-7 could effectively increase the expression of TLR4 in intestinal mucosa of mice (in Fig 1), promote the high expression of TLRs-related signals MyD88 and TRIF, activate NF-κB and regulate the immune function of intestinal mucosa of mice through MyD88 dependent/independent dual pathways. Recent studies have found that MyD88-dependent pathway mediated by TLRs is the main signal pathway for activating NF-κB in cells (Miao et al., 2011 and Wang et al., 2017 and Takaesu et al., 2003). When pathogens invade, TLRs act on the cell membrane and activate it. TLRs transduction pathways include MyD88 dependent pathway and MyD88 independent pathway (or TRIF pathway). TLR4 is mediated by both MyD88 and TRIF pathways.
 

       
NF-κB signaling pathway is an important way to link the body’s antioxidant system with the immune system. NF-κB is a transcription factor sensitive to oxidative stress, which is involved in inflammation, congenital immunity, cell differentiation and apoptosis (Cummins et al., 2010 and Garg et al., 2012). In addition, NF-κB is located in the downstream signaling pathway of TLRs, which can facilitate gene transcription and expression by combining the specific locations of multiple gene promoters and enhancers. The results showed that the relative expression of NF-κB gene in intestinal mucosa of aged mice was lower than that of young mice. After the intervention of β-CM-7, the relative expression of NF-κB gene in intestinal mucosa of aged mice could be up-regulated in each dose group (Fig 2), which indicated that β-CM-7 could activate the transcription of NF-κB and promote the release of NF-κB protein. According to the results of this study, it can be inferred that the mechanism of β-CM-7 improving intestinal mucosal immunity may be through activating the transcription of NF-κB, promoting the release of NF-κB, further activating the specific immune system, inducing the proliferation of lymphocytes and the expression of SIgA and cytokines, so as to alleviate the intestinal mucosal immunity caused by aging.
 

       
Studies have shown that the regulation of NF-κB mainly involves TRAF6 and that NF-κB and TRAF6 are the major signal transduction genes downstream of TLR4 (Verstak et al., 2009 and Ma et al., 2018). TLRs signal triggers MyD88/TRAF6-dependent signaling pathway by dimeric Toll receptor mediated by extracellular domain, activates NF-κB protein in cytoplasm and participates in the regulation of large-scale inflammatory response. TRAF6 pathway produces cytokines and adhesion molecules to regulate acquired immunity and enhance cellular immunity. It is the necessary pathway for downward transduction of TLRs signal such as TLR1, TLR2, TLR4, TLR5, TLR7 and TLR9. The results showed that the relative expression of TRAF6 gene in intestinal mucosa of aged mice was lower than that of young mice. After the intervention of β-CM-7, the relative expression of TRAF6 gene in intestinal mucosa of aged mice could be up-regulated in each dose group (Fig 3). It indicated that β-CM-7 could activate TRAF6 and regulate the activation of NF-κB, thus regulating the intestinal immune function.
 

       
In conclusion, aging can lead to the decrease of relative expression of TLR4, TRAF6 and NF-κB genes in intestinal mucosa, while β-CM-7 can significantly. (P<0.05) increase the expression of TLR4 and activate NF-κB in intestinal mucosa of mice. Then, the relative expression of TRAF6 gene in intestinal mucosa of aged mice was up-regulated to regulate the activation of NF-κB, so as to regulate the intestinal and overall immune function. It is suggested that the protective mechanism of β-CM-7 on intestinal mucosa of aged animals is related to the expression of TLR4, TRAF6 and NF-κB genes.

This project was sponsored by grants from The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No:16KJB330011) and Yangzhou University Overseas Study Program (No: 2019).