Effect of preserved eggs on the health of SD rats, and anti‐tumor action of HT‐29 cells

Abstract Preserved eggs are traditional alkali‐pickled food in China and have been enjoyed by consumers and extensively studied by researchers for their nutritional tastes and their anti‐tumor, anti‐inflammatory, antioxidant, lipid‐lowering, and blood pressure‐lowering properties. To study the anti‐tumor effects of preserved eggs, this project observed the health on rats, and anti‐tumor effects and separated anti‐tumor active components on HT‐29 cells. SD rats fed for 80 days showed that preserved eggs had no significant effect on weight, food intake, blood pH, liver tissues, or organ indices. Preserved eggs significantly increased blood levels of oxidative stress markers SOD and CAT, decreased MDA levels by 0.46, 0.23, and 0.25 times. Moreover, they also increased the level of IL‐2 from 1233 to 1340 pg/mL. Two water‐soluble bioactive peptide fractions, B1 and B2, with molecular weights ≥10 kDa were further obtained from preserved eggs by ultrafiltration and Superdex Peptide 10/300 GL. The potential mechanism of B1 and B2 is to activate the internal mitochondrial apoptotic pathway and induce apoptosis by up‐regulating the expression of the pro‐apoptotic factors cytochrome C, caspase‐3, and caspase‐9 mRNA in HT‐29 cells.

| 6189   WU et al.   and down-regulating the expression of anti-apoptotic factors interleukin-4 (IL-4) and IL-6 (Batool, Hu, Huang, et al., 2021).In a recent study, Preserved eggs were reported to cause a dramatic increase in reactive oxygen species (ROS) in HepG2 cells.At the same time, they activated the mitochondria-mediated apoptotic pathway by upregulating the expression of the pro-apoptotic gene Bcl-2 and inhibiting the expression of the anti-apoptotic gene Bak.In addition, they reduced the expression of the angiogenic factors Hypoxiainducible factor-1α (HIF-1α) and Vascular endothelial growth factor (VEGF), which may lead to inhibition of cell growth and migration (Wu et al., 2023).The peptides DR-10 and DF-9 isolated from PED can exert anti-inflammatory effects by inhibiting NF-κB and MAPK signaling pathways (Zhao et al., 2017).The peptides smaller than 5 kDa and isolated from preserved egg white reduced the secretion of the anti-inflammatory markers TNFα and IL-8 (Zhang et al., 2020;Zhao et al., 2017).However, the health effects and anti-tumor active components are still unclear and further studies are needed.
To observe the effect of preserved eggs feeding on rats and screen for the anti-tumor active fractions, in vitro and in vivo experiments were performed on Sprague-Dawley (SD) rats and colon cancer cells (HT-29).After 80 days of preserved egg feeding, body weight, food intake, organ index and liver tissue of rats were measured.Moreover, its anti-tumor active fraction was initially isolated and screened.This provides a theoretical basis for the further promotion and high-value utilization of preserved eggs and also lays the foundation for the screening of anti-tumor active peptides of preserved eggs.

| Preparation of preserved egg powders
The duck eggs were purchased from Hubei Shendan Health Food Co., Ltd.Preserved eggs were obtained by following the modified method of (Zhao et al., 2014).Fresh intact duck eggs (50-70 g) were selected and soaked in 4.5% NaOH, 3% NaCl, 0.4% CuSO 4 , and 3.5% Chinese black tea at 25°C for about 5 weeks.Then, they were washed, peeled, homogenized at 10,000 rpm for 30 s by a homogenizer Hsiangtai Machinery Industry Co.,Ltd) and then lyophilized using a lyophilizer Martin Christ Gefriertrocknungsanlagen Co.,Ltd.,Germany).Afterwards, the lyophilized powders was stored at 4°C, for a maximum of 2 weeks.
The water content of the powder was analyzed according to China National Standards CNS GB 5009.3-2010.

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The female Sprague Dawley rats aged 2-3 weeks (100.00 ± 0.13 g) were purchased from Tongji Medical College, Huazhong Science and Technology University.All the rats (n = 40) were housed and treated according to the requirements of the Laboratory Animal Ethics Review Committee.The animals were allowed to take normal rat feed and water (20-25 g/day).A 12 h light/12 h dark room lighting cycle was used in the room.The room temperature and relative humidity were controlled within the range of 25 ± 2°C and 50 ± 5%, respectively.After a seven-day acclimation period, eight rats were sacrificed by decannulation as 0day group.The remaining rats were randomly divided into 4 groups (n = 8 per group), including one control group and three dosed groups: low-dose group (LD, 1.4 g/100 g), middle-dose group (MD, 2.8 g/100 g), and high-dose group (HD, 4.2 g/100 g) as detailed in Figure S.1.Each rat was gavaged with 2 mL/day for 80 days.During the experiment, the body weight and food intake of each rat were measured every 5 days.

| Blood pH, IL-2 level and visceral indexes
Rat blood was taken from the retro-orbital plexus, then blood pH and IL-2 levels were measured in rats using a blood gas analyzer and IL-2 kit, respectively.The rat spleen, kidney, liver, and perisplenic adipose were weighed, and the visceral indexes were calculated using following equation.
where W i,l,j,k is the visceral weight (i, l, j, k are spleen, kidney, liver, and perisplenic adipose weights of rats, respectively), g; and W 0 is weight of rats, g.

| Analysis of oxidative stress markers
According to the ratio of liver tissue weight (g): volume (mL) = 1: 9, nine times volume of 0.9% saline was added to liver tissue and (1) Visceral index ( % ) = W i,l,j,k ∕ W 0 × 100 tissues were homogenized in an ice-water bath.Then, the tissue was centrifuged at 2500 rpm for 10 min at 4°C and the supernatant was taken for determination.Then, the activities of oxidative stress markers, catalase (CAT), malondialdehyde (MDA), and superoxide dismutase (SOD) in the supernatant were determined using colorimetric microplate assay according to the instructions (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) by using a microplate reader (iMark, Lenovo Biological Technology Co., Ltd, China).The CAT, MDA, and SOD activity were expressed as mmol g −1 protein.

| Histopathological analysis
Fresh liver tissues were fixed in 4% paraformaldehyde solution for more than 24 h.Subsequently, the tissues were embedded in paraffin and cut to a thickness of 4 μm by a rotary machine, followed by HE staining.The histopathological images of the liver were taken under a microscope.

| Cell culture
HT-29 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China).HT-29 cells were cultured using the Tao et al. method (Tao et al., 2021) as described in Supporting information S.1.

| Preparation of PED
The PED preparation procedure was executed by Liang's method with a slight modification (Liang et al., 2020) as highlighted in Supporting information S.2.

| Separation of PED
The water-soluble and lipid-soluble components in the PED were separated by ethyl acetate (EtOAc) extraction.400 mg PED were mixed with 50 mL distilled water and 16 mL ethyl acetate.The mixture was transferred to a separating funnel and gently shaken to mix the two phases.It is then left to stand and once the two phases were completely separated, slowly rotated the piston to release the lower liquid, and then poured out the upper liquid from the separating funnel.The ethyl acetate in the extraction product was removed using a rotary evaporator (RE-52AA, Shyarong, Shanghai, China).The watersoluble and lipid-soluble resultants were lyophilized and stored at 4°C for subsequent studies.

| Primary screening of active ingredients
The HT-29 cells were seeded in a 96-well plate at a density of 5 × 10 4 cells/well, and incubated in 100 μL McCoy's 5A medium for 24 h.
Then, the cells were treated with 100 μL/well of water-soluble and lipid-soluble components, respectively, 5-FU (2 mg/mL) were used as positive control.Ten microliter CCK-8 solution was added to each well and incubated for 2 h, the absorbance was then measured at 450 nm using a microplate reader.The morphology of the cells was observed by fluorescence microscopy (OLYMPUS IX71, Tokyo, Japan).The components with the best effect to inhibit the proliferation of HT-29 cells were selected and recorded as component A.
The following equation was used to calculate the cell proliferation inhibition rate (CPIR) where A and B are the OD 450 of the control and sample, respectively.

| Separation and screening of component A
The component A was separated using 3 and 10 kDa ultrafiltration tubes.During the separation, the component A was mixed with sterile water at a ratio of 1:1 (w/v) and added to ultrafiltration centrifuge tubes with a cut-off MW of 3 kDa by centrifugation at 4000 rpm for 40 min at 4°C (Sigma3-30 k, Sigma Aldrich, St, Louis, USA).The ≥3 kDa fraction was then added to a 10 kDa centrifuge tube, centrifuged at 4000 rpm for 40 min at 4°C, and repeated twice.The three components were collected and marked as A 1 (MW <3 kDa), A 2 (3 kDa ≤ MW < 10 kDa), and A 3 (MW ≥10 kDa).The inhibitory effects of A 1 , A 2 , and A 3 on HT-29 tumor cells were determined by the CCK-8 method.The component having the best effects among A 1 , A 2 ,and A 3 was then marked as component B.

| Purification of component B
Component B was separated using Superdex peptide 10/300GL GE gel filtration columns via the ÄKTA™ pure 25 system (Agilent Technologies, Inc.).The component B was dissolved in deionized water at a concentration of 20 mg/mL, filtered through a 0.22 μm microporous membrane, and then injected into the machine with a 2 mL injection volume.The elution was performed at a flow rate of 0.5 mL/min using deionized water as the mobile phase, and meanwhile, the components with different peaks were collected using 280 nm as the detection line.Under this condition, the elution was repeated several times for bulk preparation.The elution peak sample solutions were collected and lyophilized, then label as B 1 and B 2 and store at −20°C. (2) WU et al.

| RT-qPCR analysis
The genes involved in cell apoptosis metabolism were analyzed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) (Zhong et al., 2018), as underlined in Supporting information S.3.

| Statistics and analysis
One-way ANOVA was performed for each experiment, and statistical significance was obtained by using Duncan's multiple range test on IBM SPSS Statistics 20.0 (SPSS Inc, Chicago, IL, USA), where p < .05 was considered to be significant.

| Effect of preserved eggs on body weight and food intake
SD rats are fast-growing and have reliable breeding performance, so they are widely used in safety and nutrition-related tests.In pathophysiology, overweight or marked weight loss is considered as an indicator of health problems (Poirier et al., 2006).As shown in Figure 1A, after 80 days of feeding with PED (water content of 1.32%), the rats gained weight normally from 100.00 to 290.71 g, not significantly different from the control group (p > .05).Furthermore, the daily intake of each rat was between 42.31 and 66.00 g after feeding with preserved eggs (Figure 1B), and the significance analysis showed no significant difference in the intake of rats (p > .05).
Therefore, preserved eggs did not adversely affect the eating behavior of rats.Table 1 shows the effect of preserved eggs on the blood pH of rats.After 80 days of feeding, the blood pH still remained in the normal range of 7.00-7.40(Zausinger et al., 2002) and no statistical difference (p > .05)was found.This suggested that long-term intake of preserved eggs did not affect the eating behavior of the rats or the acid-base balance of their blood.

| Changes in visceral indexes
The viscera index refers to the ratio of organ weight to body weight and is a direct assessment of the extent of lesions in the internal organs (Yan et al., 2021).Table 2 showed that after 80 days of preserved eggs intervention, the visceral indexes of spleen, kidney, liver, and perisplenic adipose in each group were not significantly different compared with the control group (p > .05),indicating that long-term intake of preserved eggs did not affect the normal growth of organs in SD rats.In the current animal studies, it has been demonstrated that preserved eggs significantly reduced triglyceride and total cholesterol levels, as well as low-density lipoprotein (LDL-C)/high-density lipoprotein (HDL-C) levels in the liver of rats, which is related to lipid metabolism in rats (van der Made et al., 2014).

| Effects of preserved eggs on the CAT, MDA, and SOD values
The three primary antioxidant enzymes, namely CAT, SOD, and GPx contained in the cells were thought to be necessary for life in all F I G U R E 1 Effects of preserved eggs on body weight (A) and food intake (B) of rats.Control: 0 g/100 g; LD: low-dose group, 1.4 g/100 g; MD: middle-dose group, 2.8 g/100 g; HD: high-dose group, 4.2 g/100 g.
oxygen metabolizing cells (Bertero et al., 2019).MDA is an important indicator of membrane system injuries and deterioration of cellular metabolism caused by environmental stress (Fan et al., 2012).
SOD and CAT can play an antioxidant role by quenching free radicals and inhibiting the production of harmful products such as MDA (Shi et al., 2021).Furthermore, the SOD, CAT, and MDA are key to redox homeostasis in the gastrointestinal body (Bertero et al., 2019).
Figure 2 shows the changes in SOD, CAT and MDA in the liver of rats.The liver SOD activity was increased (p < .05)and dosedependent after treatment, and the HD group was approximately 1.5 times larger than the control group.Meanwhile, CAT levels were increased (p < .05) to about 1.2 times of the control group.
However, there was no significant difference in the CAT expression between LD, MD, and HD groups (p > .05).Furthermore, MDA levels decreased consistently in a dose-dependent manner from 0.699 to 0.559 mmol/gprot.These results suggested that preserved eggs exhibited their antioxidant activity by increasing the levels of SOD and CAT activities and decreasing the levels of MDA.

| Effect of preserved eggs on the histopathology of liver tissue
The liver is an organ with mainly metabolic functions and has functions such as de-oxidation, storage of liver sugar, and secretory protein synthesis (Boby et al., 2021).Figure 3A shows the liver tissue anatomy of each group.The liver tissue of rats in the control group was bright red in color, smooth on the surface, soft and elastic in texture without visible particles.There was no significant difference in the treatment group compared to the control group.

TA B L E 1
Changes in blood pH of rats fed preserved eggs (n = 8).

| Effect of preserved eggs on IL-2 content
IL-2 induces biological effects in lymphokine-activated killer cells (LAK) and cytotoxic CD8 T lymphocytes (CTL), enhancing their ability to lyse tumor cells, and has been found in numerous studies to play an important role in anti-tumor (Abbas et al., 2018).Figure 4 shows the changes of IL-2 in serum after 80 days of feeding.As shown in the figure, the level of IL-2 in the control group was 1175 pg/mL.
In the LD, MD, and HD groups, the intake of preserved egg significantly increased the expression of IL-2 in rat serum, from 1233 pg/ mL to 1290 pg/mL and 1340 pg/mL (p < .05), in a dose-dependent manner.This indicates that the preserved eggs can increase the expression of IL-2 in rat serum within a certain dose range and exert anti-tumor effects.A recent study confirmed that preserved eggs induced apoptosis in kidney cancer cells Caki-1, ACHN and 786-O.
The possible mechanism was through upregulation of the levels of the pro-apoptotic genes Bax and IL-10 and reduction in the expression of the anti-apoptotic factors IL-4 and IL-6 (Batool, Hu, Huang, et al., 2021).

| The effects of lipid-and water-soluble components on HT-29 cells
To explore the anti-tumor mechanism, the effects of lipid-soluble and water-soluble components on the CPIR and morphology of HT-29 cells were studied.As shown in Figure 5A, the control group had the smallest CPIR of 0.20%, while the values of the lipid-soluble and water-soluble fractions were 47.00% and 84.20%, respectively.The water-soluble fraction was less effective than the positive anti-tumor drug 5-FU (92%) in inhibiting the growth of HT-29 cells.Morphology in Figure 5B showed that untreated cells displayed a normal polygonal structure without significant damage.The cells treated with the lipid-soluble

| Effect of different molecules of component a on HT-29 cells
It was found that HT-29 cells activity was significantly inhibited at a PED concentration of 2 mg/mL.Simultaneously, HT-29 cells underwent contraction, plasma membrane blistering, and chromatin condensation associated with DNA cleavage into steps.In addition, PED induced apoptosis, with the apoptosis rate of HT-29 cells in the high concentration group being 16.5%, which was 5.8 times higher than that of the control group (Liang et al., 2020).This suggested that PED could effectively promote apoptosis in HT-29 tumor cells, but the exact mechanism needs to be further investigated.
Figure 6A compares the effects of different fractions on CPIR of HT-29 cells.The CPIRs of A 1 , A 2 and PED on HT-29 cells were 83%, 85%, and 81%, respectively.A 3 had the highest CPIR of 90% and was not significantly different (p > .05)from the 5-FU group (95%).This indicated that A 3 had the best tumor suppression effect. of nuclei, indicating cell death due to apoptosis.Therefore, A 3 was selected as component B.

| The ÄKTA™ pure system analysis component B
The separation of component B by gel chromatography is shown in Figure 7, where two peaks with MW ≥10 kDa were found and named B 1 and B 2 .The basic principle of gel chromatography, also known as molecular sieving, is to separate substances according to their molecular size by the distribution of pore sizes in gel molecular sieves (Worsztynowicz et al., 2020).Therefore, B 2 had a smaller MW and  mediates cellular mitochondrial apoptosis, plays an important role in mitochondrial energy metabolism by performing electron transfer between respiratory chain complex enzyme III and respiratory chain complex enzyme IV (Liu et al., 2007).When apoptotic signals reach the mitochondria, pro-and anti-apoptotic proteins of the Bcl family can act together to release CytC, which then activates caspase-9 by forming an apoptotic complex and eventually activates caspase-3 to induce apoptosis (Shao et al., 2018).
It was reported that PED can induce apoptosis in HT-29 and HepG2 cells by upregulating the expression of Bax mRNA and downregulating the expression of Bcl-2 mRNA and COX-2 mRNA to promote the release of caspase-3 and caspase-9 (Liang et al., 2020).
As shown in Figure 8, compared with the control group, B 1 upregulated the expressions of caspase-3 caspase-9 and CytC to 1.04, 1.36, and 0.82 times, and B 2 up-regulated those expressions to 0.97, 1.00, and 0.60 times, respectively (p < .05).The expressions in PED were superior to that of the control but less than those in both B 1 and B 2 .

| Effect of B 1 and B 2 on apoptosis
The effects of B 1 and B 2 on the apoptosis of HT-29 cells are shown in Table 3 and Figure 9, where the early and late apoptosis characterized by DNA fragmentation after activation of the caspase pathway were in the lower right and upper right corners, respectively.The apoptosis rates of HT-29 cells in B 1 and B 2 were 4.61% and 3.19% respectively, slightly higher than 2.86% in the PED group, and much higher than 0.32% in the control group (P < 0.05).Therefore, we supposed that the anti-tumor effect was achieved through peptide fractions action and was closely related to the mitochondrial pathway (So et al., 2009).
With the animal care and use protocol approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, the study was performed following the Regulations for the Administration of Affairs Concerning Experimental Animals.(Animal Welfare No. 00140424, Animal Quality Certificate No. Same letters in the same column indicate no significant difference in the visceral indexes for LD,MD and HD compared with the control group (p > .05).TA B L E 2 Changes in visceral indexes of rats fed preserved eggs (n = 8).F I G U R E 2 Effects of preserved eggs on oxidation indicators of rats.(a) SOD; (b) CAT; (c) MDA.The letters in the figure indicate significant differences (p < .05).The histopathological results of the liver are shown in Figure3B.The control group showed no obvious abnormalities under light microscopy, with normal cell size, neat arrangement, clear margins, and intact nuclei morphology.In the meantime, the cell plasma was evenly stained, and no inflammatory cells were observed.No significant difference was found among the LD group, the MD group, and the control group.A small number of round fat vacuoles appeared in the HP group, but they did not reach the extent of liver lesions, and no inflammatory cell infiltration and hepatocyte necrosis were observed.Therefore, long-term intake of preserved eggs did not lead to liver tissue damage or risk of fatty liver disease.

F
I G U R E 3 Liver dissection (A) and histopathological findings (HE, 100×) (B).(a, e) Control; (b, f) LD; (c, g) MD; (d, h) HD.F I G U R E 4 Effects of preserved eggs on IL-2 in blood of rats in 80 days.The letters in the figure indicate significant differences (p < .05).components underwent slight changes with bleaching of the cell membrane and chromatin condensation (Figure 5C), while those treated with water-soluble components were easily observed with blurred cell edges, disorganized cell structure, and disappearance of visible nuclei (Figure 5D).Therefore, the water-soluble component which disrupted the structure of tumor cells and inhibited their growth and proliferation was chosen to investigate the effects on HT-29 cell apoptosis.

Figure
Figure 6B-G compares the morphological changes of HT-29 cells in different treatments.The commonly known apoptosis characteristics are cell distortion, cell shrinkage, cell membrane blebbing,and chromatin condensation, which may be linked with cleavage of DNA(Lee et al., 2002).Untreated cells in the control were tightly arranged with a normal polygonal structure.Compared with A 1 and A 2 , which had little effect on the HT-29 cell structure, the cells in A 3 were mostly distorted with defocused cell edges and disappearance significantly higher content than B 1 (p < .05).Although the ÄKTA™ Pure 25 System allows easy and fast differentiation of the peptide fractions, it executes preliminary separation and purification.Better purification can be achieved by combining different methods, such as solid-phase metal affinity chromatography IMAC coupled with mass spectrometry (MALDI-TOF-MS)(Gagnaire et al., 2009).Further studies should focus on the analytical identification of B 1 and B 2 to determine their molecular weight and specific structure.

3. 9 |
Effect of B 1 and B 2 on Caspase-3, Caspase-9, and CytC mRNA expressionCaspase-3 and caspase-9 are important regulatory proteases in the mitochondrial pathway, with caspase-3 mainly involved in the late stages of apoptosis(Jeon et al., 2017;Micucci et al., 2015;Wang et al., 2017).CytC, located in the outer mitochondrial membrane and F I G U R E 7 Chromatogram of gel filtration for component B by ÄKTA Pure 25 using Superdex 200 10/300 GL.F I G U R E 8 The expression of gene mRNA exposed to B 1 and B 2 in HT-29 cells.(A) Caspase-3; (B) Caspase-9; (C) CytC.The letters in the figures indicate significant differences (p < .05).
Cell apoptosis in HT-29 cells exposed to B 1 and B 2 were significantly different compared with the control group (p < .05).TA B L E 3 Cell apoptosis in HT-29 cells exposed to B 1 and B 2 .F I G U R E 9 The effect of B 1 and B 2 on apoptosis of HT-29 cells.(A) Control; (B): B 1; (C): B 2; (D) PED; (E) 5-FU.