Effect of Beta-casomorphin-7 on Renal Function and Immunity in Aging Mice

At present, Population aging has become the most serious problem (Rudnicka et al., 2020). Aging is mainly a cellular phenomenon, which can be extended to the individual level. Physiological aging is one of the reasons for the decline of the body’s functional levels. Some researchers proposed that the use of dietary interventions such as bioactive peptides to delay aging is an inevitable trend in gerontological research (Bhullar and Wu 2020).
       
Food-derived bioactive peptides with multiple biological functions. For example, supplementation of marine advanced protein powder is conducive to the growth and development of malnourished protein mice and can improve their immune function (Yang et al., 2020). Royal jelly protein has physiological activities such as anti-aging, antibacterial and anti-fatigue (Ramanathan et al., 2018). However, the study also pointed out that these food-derived biologically active peptides also have some negative effects on human health. The main protein in royal jelly protein is allergens, which can trigger allergic reactions in humans, it can lead to death in extreme cases.
       
At present, most of the international research on BCM-7 focuses on phenotypic identification (Nguyen et al., 2020), digestion and degradation (Asledottir et al., 2019) and a part of the latest research confirms that BCM-7 has improved lactose intolerance (Uscanga-Domínguez et al., 2019). Previous experiments proved that BCM-7 could relieve the damage of small intestinal mucosa caused by free radicals produced by hyperglycemia oxidative stress (Han et al., 2013). However, in another study, BCM-7 can induce intestinal immune disorders (Haq et al., 2014), an earlier study suggested that BCM-7 can reduce renal interstitial fibrosis in diabetic rats (Zhang et al., 2012). Based on these conclusions and phenomenons, we guess how BCM-7 will affect kidney function and immunity in aged mice. With the increase of life expectancy, healthy aging becoming particularly important and stable kidney function is closely related to the physical function and life satisfaction of the elderly (Wuorela et al., 2017). Increasing age also affects skeletal muscle mass and endocrine regulation. Changes in the microscopic anatomy of the kidney, including a decrease in the number of glomeruli, can lead to impaired kidney function. The interaction between chronic kidney disease (CKD) and aging causes premature occurrence of pathological symptoms such as cardiovascular disease, muscle wasting and renal failure in the body (Kooman et al., 2017). In recent decades, global researches on the kidney have mainly focused on IgA nephropathy, diabetic nephropathy, hypertensive nephropathy and otherpathological studies. The experimental modelling is mainly based on disease models. However, there are relatively few studies on animal natural aging models and aging.
       
Hence, we analyze the changes of renal function indexes, cytokines and related proteins in mice through the dose intervention of BCM-7 on naturally aging mice and explore the effects of BCM-7 on the renal function and immunity of naturally aging mice. We hope it can further reveal the impact of BCM-7 on body health and provide more theoretical references for inflammatory aging.

Test animal, experimental equipments and study design
 
Forty-four 11-month-old Male Kunming (KM) mice (SPF, weighed 53-69 g) and eleven 2-month-old KM mice (SPF, weighed 39-50 g) used in this study were purchased from Qinglongshan Animal Center (Jiangsu, China). BCM-7 dry powder was purchased from Nanjing Peptide Biotechnology Co., Ltd. The experimental instrument mainly used the 755S ultraviolet spectrophotometer (Shanghai Baili Biotechnology Co., Ltd.); full-wavelength microplate reader (Thermo Fisher Company, USA); Allegra X-22R benchtop refrigerated centrifuge (Beckman Coulter Company, USA); D1008E vortex instrument (USA SCILOGEX company); ICV-450 electric heating constant temperature incubator (Japan ASONE company).
       
All reagents used in the experimental process were analytically pure. Cystatin C (Cys-c) kit, CK ELISA kit, Adrenocorticotropic hormone (ACTH) biochemical kit all using the manufacturer’s protocol (Elabscience, Wuhan, P.R.C.). Secretory immunity globulin A (sIgA), C-reactive protein (CRP), TGF-β1, TNF-α, IL-1β, IL-6, IL-13 ELISA kits were purchased from Lianke Biotechnology Co. Ltd. (Hangzhou, China). PBS and BCA protein concentration determination kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. A HE staining kit was purchased from Beyotime (Jiangsu, China).
       
All animal care and procedures were performed following Jiangsu Province and institutional policies for animal health and well-being. The mice were randomly and equally divided into five groups and were allowed to free access to a regular diet. Young blank group and Aged control group mice were intragastric administration of normal saline (0.9%; 0.5 mL). Each mouse in BCM-7 treated groups were given at low, medium and high concentrations dose (0.2×10^-6 mol/d, 1×10^-6 mol/d, 5×10^-6 mol/d; 0.1 ml/10 g body weight) of BCM-7, mice were administered for 30 consecutive days.
 
Sample collection and processing
 
The mice were sacrificed by cervical dislocation, dissected, kidney and spleen tissues were immediately removed, rinsed several times in pre-chilled PBS buffer, blotted dry with filter paper and weighed. Part of the tissue was quickly placed in liquid nitrogen and stored in a refrigerator at -70°C until use. Weight the mouse kidney tissue accurately, add 9 times the volume of 0.9% saline to make a 10% homogenate and centrifuge at 4^oC to take the supernatant. Finally, sub-packet in each sterile cryopreservation tubes (200 µl) and stored at -20°C until use.
 
Bodyweight record and organ index of mice during the intervention
 
Within one month of the intervention with BCM-7, the mice were weighed and recorded continuously every week. The animals in fast on the last day of the experiment, renal and spleen were removed, washed in ice-cold normal saline and weighed. The index is calculated using the following formula:
   
Determination of renal function indexes and hormone levels
 
The supernatant of 10% of kidney homogenate was diluted 10 times with 1X PBS. The total protein content of kidney tissue was detected by a microplate reader at 562 nm wavelength using the BCA method. Cys-c, CK, ACTH were measured by double antibody sandwich ELISA, biochemical colorimetry, direct competition ELISA. The specific test strictly follows the instructions of the kit.
 
Observation of renal histopathology
 
Harvested kidney tissues from mice were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 µm thick sections to evaluate the renal pathology of mice in different groups.
 
Determination of cytokines and related proteins in kidney tissue
 
The content of IL-1β, IL-6, IL-13, sIgA, CRP, TGF-β1 and TNF-α was measured by double-antibody sandwich ELISA, the test strictly follows the instructions of the kit.
 
Data processing
 
Use SPSS Statistics (26.0) and GraphPad Prism (8.0) for data analysis and processing, The single-factor continuous variable data conform to the normal distribution and was expressed as x ± s. Single-factor analysis of variance was used for multiple independent samples at the same time point when the variance is homogeneous. The multiple post-mortem comparison uses the LSD-t-test. If the variance were uneven, the non-parametric test (Kruskal-Wallis H test) was used. Repeated measurement analysis of variance was used for multiple measurement data at different time points, p<0.05 considered the difference to be significant and statistically significant.

Within one month, the bodyweight of mice in the low, medium and high BCM-7 treated groups increased slightly with an increasing dose of BCM-7 (1.31%, 1.99%, 2.24%), the bodyweight of Aged control mice and young mice increased by 1.22% and 6.63%, respectively (Fig 1A). The results show that BCM-7 contributes to body weight gain after aging, thisperhaps due to BCM-7 being easily digested and absorbed by the body and enhancing pepsin activity (Asledottir et al., 2019). Around one month. The bodyweight of the Aged control group was significantly lower than that of the young mice (-14.56%), (Fig 1B) and the bodyweight of the mice in the low, medium and high BCM-7 intervention groups were also lower than that of the Aged control group and the degree of weight loss was similar to the intervention dose of BCM-7 (-0.98%, -1.02%, -1.21%), the decrease in body weight with aging may be related to the weakening of the metabolic rate after body aging and the reduction of muscle mass, which are all features of body aging, but the rate of weight gain was greatly influenced by the concentration of BCM-7, too high BCM-7 concentration is not conducive to maintaining a healthy weight in mice. The renal index of the aged control mice increased slightly and the spleen index decreased marginally (Fig 1C and 1D) compared with young mice, those maybe because 11-month-old mice only show reproductive aging and have not reached the state of complete aging (Schneider et al., 2017). The medium-dose BCM-7 intervention lead to a dramatic reduction in the spleen index of aging mice (-33.57%, Fig 1D) compared with the aged control mice, indicating that BCM-7 may cause atrophy and dysfunction of immune organs.
 

       
We found that the content of Cys-C (Fig 2A) was significantly increased in the aging control group compared with the young mice (+13.11%), showing that aging may cause the decrease of glomerular filtration rate in mice, increase the natural aging of the body (Wasén et al., 2003), The ACTH content of aging mice decreases with the increase of the BCM-7 intervention dose, especially the difference between ACTH content in the middle and high BCM-7 intervention groups with young group mice were dramatic (-35.50%; -44.68%), showing that excessive BCM-7 may actively cause endocrine disorders (Fig 2B). The quality of endocrine function affects the body’s hormone levels, development and aging are related to changes in endocrine function in the life cycle. Various diseases caused by age will lead to the decline of hormone negative feedback regulation function and the weakening of cognitive function (Kudielka et al., 2004). There was no evident concentration dependence between Cys-C content of aging mice and BCM-7 intervention dose, the small change range indicated that Cys-C was not affected by the dose of BCM-7. The Cys-C content of middle-dose mice was significantly reduced (-11.11%) compared with the aged control group (Juraschek et al., 2013). Cys-c can be completely filtered by the glomerulus and then absorbed and degraded by the renal tubular cells. Cys-c is not easily affected by inflammation, body muscle mass, diet, body mass, renal tubular secretion. Based on these characteristics, Cys-c is currently an endogenous markerof glomerular filtration rate that is better than creatinine and urea nitrogen (Gompou et al., 2015). CK is one of the key enzymes in energy metabolism. The CK (Fig 2C) content of the aged control group was slightly lower than that of the young blank group (-7.43%), Some studies have found that the CK content is negatively correlated with age (Neidert et al., 2015), which may be affected by the skeletal muscle content of the aging body. Decreased skeletal muscle will lead to insufficient creatine kinase; it is difficult to provide the body’s average energy needs, forcing creatine phosphate to generate creatinine through irreversible non-enzymatic channels. If the glomeruli are damaged, creatinine will not be filtered from the glomeruli. A large amount of enrichment produces toxicity, so creatine kinase and creatinine are closely related and creatine kinase can be used as a marker for early diagnosis of renal function (Ueda and Sakasegawa 2016). The latest research shows that a 4-week high-protein diet can reduce body fat and increase skeletal muscle mass (Ato et al., 2020).
 

       
As is shown in Fig 3, we observed the swelling of individual renal tubular epithelium and the degeneration of renal tubular epithelium in low-dose aging mice. Middle-dose aging mice showed noticeable renal tubular epithelial turbidity and vacuolar degeneration. The renal tubular cortex and epithelial cells of the high-dose aging mice were swollen, the epithelium was slightly vacuolated, the interstitial cells were infiltrated by the inflammatory cells and the individual renal tubular epithelial cells were shed, a small amount of protein cast was seen. The cortical structure of the mice in the aging control group was clear and a small number of lymphocytes were scattered and infiltrated in the renal cortical interstitium. In the young control group, the cortical structure was clear, the number of nephrons was normal and there was no degeneration, swelling, or inflammation of tubular epithelial cells. Whereas, there was slight vacuolation in the renal cortical tubular epithelium.
 

       
In the final experiment, the results show that the sIgA content of the aged control mice was significantly increased compared with the young mice (+126.09%). The sIgA content of the BCM-7 group mice were proportional to the BCM-7 intervention dose (Fig 4A), the results found that the low dose BCM-7 can dramatically reduce the sIgA content ofaging mice (-58.08%), indicating that aging causes the mice humoral immunity to strengthen and low dose BCM-7 can effectively reverse this trend. However, the production levels of pro-inflammatory and anti-inflammatory factors vary abnormally (Fig 4B; 4C). To compared with young mice, the levels of IL-1β, IL-6, IL-13 and TGF-β1 all decreased in the aged control mice, especially the levels of IL-6 and TGF-β1 were significantly lower than those of young mice (-25.20%; -32.72%), only TNF-a level in the aged control group was slightly higher than that in young mice. In the BCM-7 intervention group, IL-1β levels were proportional to the BCM-7 dose, TNF-a and TGF-β1 levels were inversely proportional to the BCM-7 dose (Fig 4C). The middle-dose group BCM-7 can significantly increase the levels of IL-6 and IL-13 in aging mice compared with the low-dose group (+37.29%; +98.41%), high-dose BCM-7 can significantly increase IL-1β in aging mice compared with aged control mice (+46.40%). Finally, we observed changes in the level of inflammation marker CRP (Fig 4D), The CRP level of aged control group mice was slightly lower than that of young mice and the CRP level of mice in the BCM-7 intervention group showed an increasing trend with the rise of the BCM-7 intervention dose, the high-dose BCM-7 can significantly increase the CRP level of aging mice compared with the aged control group (+71.55%). These results show us that inflammatory cells infiltrate in the aging mouse combined with the pathological section of kidney tissue in this experiment.
 

       
To repel harmful stimuli and restore the body to a healthy state, the immune system will cause a series of pathological manifestations such as tissue damage (Mazzaferro et al., 2020). The body’s aging process is accompanied by dysregulation of cytokines and changes in the quality and quantity of the immune system, This process is called immune aging (Alvarez-Rodriguez et al., 2012). Plasma CRP is moderately elevated and can be used to predict mortality (Iannitti and Palmieri 2011). After taking an equal gradient concentration of BCM-7 to gavage the aging mouse, as the intervention dose increases, BCM-7 can Significantly induce kidney inflammation and gradually increase kidney damage (Huang et al., 2008), physiological aging and immune aging occur almost simultaneously. We observed that the changes between pro-inflammatory and anti-inflammatory factors were disordered and that the mice showed dysregulation of cytokines under the dual influence of BCM-7 and aging. For the inflammatory marker CRP, the experimental result further indicates that the intervention of BCM-7 can trigger inflammatory stress in aging mice and the degree of stress is proportional to the concentration of BCM-7 intervention.

To explore the effect of BCM-7 on renal function and immunity in aging mice, we have tested renal function indexes, cytokines, IgA and CRP, It was found that the medium concentration of BCM-7 is most conducive to theweight gain of aging mice and can significantly reduce the Cys-C content of aging mice, improve glomerular filtration function, Proper intake of BCM-7 may be beneficial to the energy supply of the elderly. However, excessive intake of BCM-7 will aggravate the inflammatory stress in aging mice, leading to disorders of the endocrine and immunity, It is noticed that we should take in milk and dairy food reasonably.

This project was sponsored by grants from the National Natural Science Fund (81803235) and Research and innovation project of Yangzhou University (No: X20200907).