The immunity-promoting activity of porcine placenta in mice as an immunomodulator for functional foods

Zhiwei Zhou, Dn Wng, Wei Liu, Lng He, Pengkun Ling, Junli Ho, Qun Sun,*

a Key Laboratory of Bio-resources and Eco-environment Ministry of the Education, College of Life Sciences, Sichuan University, Chengdu 610064, China

b School of Biomedical Sciences and Technology, Chengdu Medical College, Chengdu 610500, China

ABSTRACT

This study was to explore the immunity-promoting activity of porcine placenta as a potential raw material for functional foods. Porcine placenta was subjected to the analysis for its bioactive substances, and their immunity-promoting activity was determined in mice supplemented with porcine placenta extract (PPE)and freeze-dried porcine placenta powder at high (PPH) and low (PPL) dosage. Results showed that porcine placenta contained placental peptides and 15 free amino acids, and the amounts of estrogen and progesterone in products developed from porcine placenta were within the limit of national standard. Mice model experiment revealed that compared with the control, the PPH treatment significantly improved the spleen index (P < 0.05) by increasing the phagocytic rate of macrophages from 20% to 60% and the conversion rate of T lymphocytes from 8% to 60%. The qPCR analysis disclosed that the porcine placenta powder enhanced mice immunity via promoting the expression of Th1 cytokines of interleukin-2 (IL-2) and IFN-γ, especially the former, by almost 8 times in the spleens of male mice, while inhibited Th2 cytokines of IL-4 and IL-10.This investigation has provided a reference for the development of porcine placenta as a raw material applied in functional foods to improve human immunity.

Keywords:

Porcine placenta

Immunity promoting activity

Phagocytic rate

Cytokines

Functional foods

1. Introduction

Pork, as the most important meat resource in China, produced from 54.3 million pigs according to 2018 Ministry of Agriculture Data in China. With an annual output of two pigs per sow and 1 kg of placenta per birth, about 108 600 t porcine placentas can be produced every year, while such high-yield of porcine placentas were usually discarded as waste, causing huge pollution as well as economic loss.

Human placenta, commonly called Zi He Che in the Chinese Pharmacopoeia “Compendium of Materia Medica”, was believed to have the function of enhancing immunity, proven by the Chinese practice for a long history. Previous studies have shown that human placenta is rich in trace elements [1], amino acids, phospholipids,polysaccharides, as well as interferon, prolactin, gonadotropin, and other bioactive substances [2], so the human placenta was regarded as an ideal immunomodulator.

Due to the ethical issues and the difficulty in obtaining the human placenta, application and research on human placenta have been severely restricted. Therefore, placentas from animals, such as pig, cattle and sheep, have attracted human’s attention. Studies have shown that the sheep placental extract and one component of a small chain peptide with immunoregulatory activity could promote E-rosette formation of human peripheral blood lymphocytes [3]. The main bioactive components in placenta were reported to be the placental peptides that could protect fatigue induced mitochondrial dysfunction through preventing reactive oxygen species and tumor necrosis factor α(TNF-α) generation [4]. Most studies on animal placenta focused on sheep placenta as well as bovine one, however porcine placenta was a cheaper and wider resource for providing raw materials to develop functional foods compared with the other two. It was shown that oral supplement of porcine placenta extract had a beneficial effect on skin photo-aging through activating the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and metalloproteinase-2 (TIMP-2),which potentially inhibited collagen degradation caused by Matrix Metaloproteinase (MMP) [5]. Besides, it was reported that the porcine placenta extract could inhibit the expression of in flammatory factors,thus owing an anti-inflammatory property [6]. However, there was little study on immune regulation of porcine placenta in animal model,which is in need for confirming their bioactivity.

In order to explore the function on immunity enhancement of porcine placenta used as a raw material in functional foods, the main immunomodulatory components in porcine placenta were determined,and the risk of the progesterone and estrogen amount contained was evaluated. The immune function in mice model were determined to evaluate its immunomodulatory capacity, and by examining the expression of immune related cytokine genes, the potential mechanism of immune regulation by porcine placenta was explored.Furthermore, two prototypes of functional foods based on porcine placenta were developed for its application.

2. Materials and methods

2.1 Materials and reagents

The porcine placenta was from Sichuan New Hope Leshan Breeding Base. Kunming mice were purchased from Sichuan Dashuo Biotechnology Co., Ltd. under the Ministry of Science and Technology’s experimental animal production license number of SCXK (Chuan) 2008-24.All the chemicals used were of analytical grade. The R6834 Total RNA Kit I was purchased from Omega company. The PrimeScriptTMRT reagent Kit was purchased from TaKaRa company. The ELISA Kit was purchased from Shanghai ML Bio Co., Ltd. Chinese medicine materials were purchased from local Chinese drugstore in Chengdu.

2.2 Treatment of the porcine placenta

2.2.1 Porcine placenta extract

The placentas were washed repeatedly with sterile phosphate buffer saline (PBS) to remove residual congestion and tissues. Cut the placenta into pieces and add proper amount of phosphate buffer to fully homogenize. Froze the homogenate at –80 °C for 20 min and immersed it in 42 °C water for 20 min. After repeating this process four times, the homogenate was further centrifuged at 8 000 r/min for 10 min. Collected the supernatant and stored at –20 °C until use.

2.2.2 Porcine placenta freeze-dried powder

The placentas were washed repeatedly with sterile PBS to remove residual congestion and tissues. Then the samples were drained with four layers of skim gauze and crushed for 1 min. The comminuted samples were put into a plate for freeze-drying. The dried samples were immediately vacuum-packed in an aluminum foil bag. The packaged samples were stored at 4 °C until use.

2.3 Animal treatment and experimental design

The effect of porcine placenta on immunological function was evaluated in mice (6–8 weeks of age, weighing (20.0 ± 2.0) g, half male and half female). The mice were randomly divided into control group (C), porcine placenta extract (PPE) group and freeze-dried porcine placenta powder at high (PPH) and low (PPL) dosage group.The PPE, PPH and PPL groups received porcine placenta dissolved in physiological saline, with a dose of 9.5, 28.5 and 9.5 mg/(kg bw·day),respectively, as shown in Table 1. The treatment last for 35 days.During the experiment, the mice were free to eat, drink water and housed in a gnotobiotic isolator under standard laboratory conditions(temperature: 18–22 °C, humidity: 40%–70%, 12 h light-dark cycle).

2.4 Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) method

Preparation of porcine placenta extract and SDS-PAGE was carried out according to the SDS-PAGE method [7]. In order to separate peptides of small molecule well, we increased the crosslinking of gel to 2.6% (the separation gel concentration was 18%).

2.5 High performance liquid chromatography (HPLC) method

Phase A was acetonitrile-water solution (1:1,V/V) and phase B was 0.05 mol/L acetic acid-sodium acetate buffer solution (pH 5.3,containing 0.15% triethylamine). The column temperature was 40 °C,the flow rate was 1.0 mL/min, the detection wavelength was 360 nm,and the injection volume was 20 μL.

2.6 Measurement of organ index

After 35 days of porcine placenta supplementation, the body weight of each mouse was measured and recorded, and then the mice were sacrificed by cervical dislocation. The immune organs such as the spleen and thymus of mice were removed and weighed by electronic balance.

2.7 Measurement of macrophage phagocytic rate

Each mouse was intraperitoneally injected with 1 mL of chicken erythrocyte suspension (20%). Waited for 30 min, the mice were sacrificed by cervical dislocation. Then each mouse was injected with 2 mL of physiological saline. The cell smear was prepared by drawing the washing liquid from the abdominal cavity of mice. Observed and calculated under microscope.

2.8 Measurement of T lymphocyte transformation rate

Each mouse was intraperitoneally injected with 0.3 mL concanavalin (ConA) solution. After 3 days of normal feeding, blood was collected from the tail vein of mice. Preparation of cell smear with blood sample and the number of transformed and untransformed lymphocytes was recorded under the oil mirror.

2.9 RNA extraction and quantitative real-time PCR

RNA from spleen and thymus in mice was isolated using the R6834 Total RNA KitI (Omega) according to manufacturer recommendations. cDNA was obtained using a Reverse the PrimeScriptTMRT reagent Kit (TaKaRa). Real-time PCR was performed on a TaKaRa real-time PCR system (IQ-5 Bio system).Mouse β-actin gene was used as an internal reference and the relative gene expressions of target genes (TNF-α, IFN-γ, Il-2, Il-4, Il-10) were measured according to the 2-ΔΔCtformula [8].

2.10 Determination of major hormone content and prototype development

The amounts of estrogen and progesterone in porcine placenta were determined according to the protocol of ELISA kit (Shanghai ML Bio Co., Ltd). Two prototype products based on porcine placenta were developed for its application. One was the flavored meat paste made of porcine placenta, yam, lotus seed, honey and other additives,with minced placenta in meat paste taking 13.92% of the total weight. The other product was capsule type supplement containing 0.525 g placenta powder and 146 g traditional Chinese medicine powder, including turtle glue, ginseng, Eucommia ulmoides and Achyranthes bidentata.

2.11 Statistical analysis

All results were expressed as means with their standard errors and analyzed by SPSS 18.0 software. The statistical significance of the differences between the groups was evaluated by the one-way analyses of variance (ANOVAs) with P < 0.05 regarded as significant and P < 0.01 regarded as highly significant.

3. Results

3.1 Evaluation of immunity and nutritional value of porcine placenta

SDS-PAGE was a fast and economic method for the isolation and detection of proteins. Placenta peptide was the main immunomodulatory substance in the placenta and had a specific molecular weight of about 4 kDa. As shown in Fig. 1, one clear protein band of around 4 kDa was detected, which was consistent with that reported by previous studies. So, we speculated that the porcine placental peptide existed in our samples.

Fig. 1 Electrophoretogram of placenta extract. In order to eliminate experimental errors, two electrophoresis strips were made.

In addition, the free amino acids including Asp, Glu, His, Ser,Arg, Gly, Pro, Ala, Val, Met, Ile, Leu, Phe, Cys and Lys were isolated and detected in porcine placenta by HPLC as shown in Fig. 2. Six of these 15 amino acids were essential ones.

Fig. 2 HPLC analysis of amino acids contained. (A) Chromatogram of amino acid of standard. (B) Chromatography curve of free amino acids in placenta peptide extraction.1-Asp, 2-Glu, 3-His, 4-Ser, 5-Arg, 6-CDNB-OH, 7-Gly, 8-Pro, 9-Ala, 10-Val, 11-Met,12-Ile, 13-Leu, 14-Phe, 15-Cys, 16-Lys.

3.2 Effect of porcine placenta on immune organs

Thymus and spleen were the central and important peripheral immune organs [9]. An objective index to evaluate the function of immune organs was the organ index. As shown in Fig. 3, the spleen index of both male and female mice treated with PPH increased about 1.5 times compared with that of the control group. But porcine placenta had little effect on thymus index of mice (P > 0.05). The results suggested that porcine placenta extract had a certain impact on peripheral immune organ index.

Fig. 3 Effect of porcine placenta on immune organ index in mice. (A) Spleen index.(B) Thymus index. *Significant difference P < 0.05.

3.3 Effect of porcine placenta on macrophage phagocytosis

The phagocytosis rate of macrophages re flected the function of it.Chicken erythrocytes (CRBC), macrophages (Mφ), and phagocytic macrophages of CRBC could be clearly distinguished under the microscope (Fig. 4A). As shown in Fig. 4B, porcine placenta could significantly increase the peritoneal macrophage phagocytic activity in both male and female mice. PPE and PPL increased the peritoneal macrophage phagocytic from 20% to 50%. The phagocytosis rate of macrophages reached to 60% in PPH group. The results suggested that the pig placenta effectively enhance the immunity possibly by promoting the phagocytosis of macrophages.

Fig. 4 Effect of porcine placenta on the macrophage phagocytosis in mice.(A) Observation under microscope. (B) Macrophage phagocytosis rate. *Significant difference P < 0.05.

3.4 Effect of porcine placenta on T lymphocyte transformation

T-lymphocyte transformation rate was one of the indicators of immune function. As shown in Fig. 5, the treatment of pig placenta significantly promoted the lymphocyte transformation rate of mice(P < 0.05). The T lymphocyte transformation rate of male control group was only 6%, while that of PPE, PPL and PPH group was 63%,69% and 70%, respectively. In female mice, the transformation rate of T lymphocyte in treatment groups were about 1.7 times higher than that in the control group. Similar to phagocytosis of macrophages,the immune function of T lymphocyte was enhanced. The results suggested that the porcine placenta could effectively enhance the immunity by promoting the proliferation of T lymphocyte.

Fig. 5 Effect of porcine placenta on conversion rate of T lymphocytes in mice.(A) Untransformed T lymphocytes under microscope. (B) Transformed T lymphocytes under microscope. (C) Conversion rate of T lymphocytes. *Significant difference P < 0.05.

3.5 Effects of porcine placenta on expression of immunerelated gene (IL-2, IL-4, IL-10, IFN-γ, TFN-α)

We hypothesized that porcine placenta played a role in immunoregulation by influencing the immune related genes. As shown in Fig. 6, the expressions of Th1 immune factor genes (IL-2 and IFN-γ) in porcine placenta groups were significantly higher than that in control group (P < 0.05). It was worth mentioning that the porcine placenta extract increased the IL-2 mRNA expression in the spleen of male mice by almost 8.0-folds (Fig. 7). The IFN-γ mRNA expression increased about 1.8-folds in porcine placenta treatment group no matter in spleen or thymus.

Fig. 6 Effect of porcine placenta on Th1 cytokine mRNA expression in mice. (A) IL-2 mRNA expression in spleen. (B) IL-2 mRNA expression in thymus.(C) IFN-γ mRNA expression in spleen. (D) IFN-γ mRNA expression in thymus. *Significant difference P < 0.05, **highly significant difference P < 0.01.

Fig. 7 Effect of porcine placenta on Th2 cytokine mRNA expression in mice. (A) IL-4 mRNA expression in spleen. (B) IL-4 mRNA expression in thymus. (C) IL-10 mRNA expression in spleen. (D) IL-10 mRNA expression in thymus. β-actin was used as an internal control to normalize the data. *Significant difference P < 0.05.

Fig. 8 Effect of porcine placenta on TNF-α mRNA expression in mice. (A) TNF-α mRNA expression in spleen. (B) TNF-α mRNA expression in thymus. β-actin was used as an internal control to normalize the data. *Significant difference P < 0.05.

On the other hand, porcine placenta significantly down regulated the expression of IL-10 gene, which negatively related to immunity,compared with the control group in spleen (P < 0.05). In PPH treatment group, the expression of IL-10 gene was only 60% of the control group. Besides, the expression of IL-4 gene in treatment groups was also decreased in both spleen and thymus.

Additionally, the results in Fig. 8 showed that the high concentration of porcine placenta freeze-dried powder reduced the expression of TNF-α mRNA in male mice to 70% of the control group but little effect on the expression of TNF-α mRNA in female mice.All these data suggested that porcine placenta enhance immunity possibly via in fluencing immune related genes.

3.6 Hormone content of porcine placenta and prototype developed

The amount of estrogen and progesterone in the porcine placenta and products developed was shown in Table 2. It was clear that the levels of these two hormones in the freeze-dried powder were enriched to a certain extent compared with the fresh one, however, they should not be consumed as thefinal products. The contents of progesterone in meat paste and capsule developed were 2.25 and 2.06 μg/kg,respectively, and estrogen 1.07 and 0.98 μg/kg, respectively, which both were within the limits of 10 μg/kg and 3 μg/kg, respectively, as defined by TITLE 21 (Food and Drug) of Code of Federal.

Table 2Hormone levels in the porcine placenta and products developed.

4. Discussion

It was well known that placental peptide played an important role in immune regulation. In this study, a clear protein band was detected near 4 kDa, which was consistent with that reported. Since free amino acids also showed effect on promoting immunity, this study measured the free amino acids in porcine placenta. There were 15 free amino acids detected in the placenta, 6 of which were essential amino acids.This was similar to that reported in the previous study [10].

The atrophy, quality decline and function decline of immune organs will impair body immunity [11]. Therefore, the index of thymus and spleen was measured to estimate the function of immune organs [12]. In this study, high dosage porcine placenta treatment could significantly improve the spleen index of mice, suggesting that the porcine placenta had a certain impact on spleen function. The thymus index did not increase significantly, possibly due to the short treatment time and low dosage, and its specific mechanism needed further study.

Macrophages were important immune cells in the body and had the functions of anti-infective, anti-tumor and immune regulation.When T cells were exposed to the antigenic peptide, the macrophages were activated quickly, thereby activating lymphocyte immune response [13]. The results of this study showed that porcine placenta could significantly promote the phagocytosis of mice macrophages to exogenous antigens, thus enhancing the resistance to exogenous pathogens. We speculated that the function of porcine placenta in promoting macrophage phagocytosis was related to the expression of immune cytokines, and further experiments confirmed this hypothesis.

T cells could be divided into several subgroups, including the cytotoxic T cell, regulatory/suppressor T cell, memory T cell and helper T cell. Among them, helper T cells which could secrete various immune factors, had functions of assisting humoral immunity and cellular immunity [14]. T lymphocyte was a key cell that performed a specific cellular immune response and could re flect the cellular immune status of the body. Therefore, the proliferation and transformation of T lymphocytes was one of the important indicators re flecting the cellular immune function [15]. In this study, after ConA stimulation, peripheral blood T lymphoblast transformation rate in lyophilized powder experimental groups significantly increased compared to the control group. It indicated that the porcine placenta could promote the proliferation of T lymphocytes effectively and enhance the body immunity.

Immune cytokines were divided into Th1 and Th2 type. Th1 cytokines positively regulated immune function, while Th2 cytokines were different, negatively related to immune regulation. It can be seen from the results that the Th1 type cytokines expressions in all experimental groups were significantly higher than that in the control group (P< 0.05). IL-2 was the main cytokine that caused T cell proliferation and promoted T cell division. Therefore, pig placenta may improve the T cell transformation rate by promoting the expression ofIL-2gene. Besides, with the rise ofIL-2gene expression, pig placenta could regulate macrophages by affecting the polarization type of macrophage. There were two polarization types of macrophages: M1 and M2 [16]. M1 cells had the function of promoting in flammatory response and antigen presentation, while M2 existed in tumor microenvironment, inhibiting in flammatory response,promoting tissue repair andfibrosis [17]. IL-2 regulated macrophage polarization through the Jak3-Stat5 signaling pathway, promoted macrophage polarization from M0 toward M1, and enhanced macrophage phagocytosis [18]. IFN-γ was also a Th1 cytokine secreted by helper T cells [19]. The regulation of macrophages by IFN-γ was similar to that of IL-2. It could also promote the differentiation of macrophages to M1 type.

On the opposite, Th2 cytokines showed a negative correlation to the immune regulation. IL-4 could inhibit the secretion of IL-2, TNF-α and other factors from Th1 cells and induce immune tolerance [20].Unlike IL-2, IL-4 activated the Jak-Stat6 pathway and induced macrophage M2 polarization [21]. IL-4also inhibited phagocytic ability of macrophages due to its inhibition of inflammatory response [22].IL-10 was currently recognized as an immunosuppressive factor [23].IL-10 reduced the expression level of major histocompatibility complex II (MHC II) molecules on the surface of monocytemacrophage and impaired the ability of antigen presenting cell to present antigen. Therefore, IL-10 could affect the phagocytosis of macrophages, and then inhibit cellular immunity [24]. In addition,IL-10 was also called cytokine synthesis inhibitor (CSIF), it could inhibit the production of cytokines including IL-1, TNF-α and IFN-γ,thus inhibiting the cellular immune response [25]. Since IL-4 and IL-10 were both negatively correlated with immune regulation, the inhibition of Th2 type cytokines by porcine placenta, on the contrary,could enhance the body immunity.

TNF-α was an important cytokine that participated in inflammatory reactions, it was secreted by macrophages and could activate lymphocytes, promote the proliferation of T lymphocytes as well as enhance the function of phagocytic cells. Therefore, we expected the expression ofTNF-αgene could increase in porcine placenta treatment group. However, the results showed that porcine placental lyophilized powder inhibited the expression ofTNF-αgene in the thymus and spleen. Previous report has reported that placental peptide extract can reduce the expression ofTNF-αgene by promoting the secretion of IL-6 [4]. It might be the reason to explain the decrease of TNF-α in porcine placenta treatment group.

According to the immunoregulation ability of porcine placenta,two kinds of functional foods have been made based on it. One of them was the flavored meat paste made of porcine placenta yam and other materials, and the other was the capsule made of porcine placenta and other food medicine materials. Porcine placenta was rich in hormones, so it might cause some side effects when estrogen and progesterone intake was excessive. Accordingly,the hormone content in porcine placenta was measured, and by adjusting the proportion of porcine placenta in functional foods according to its hormone content, the hormone levels of both products were within the limits of related standards.

It was worth mentioning that the regulation of porcine placenta on the body immunity of male mice was significantly stronger than on female mice in this research, so we put forward some conjectures on this situation that the immune response between male and female might not be the same [26]. It has been found that the thymus function of male mice degenerated faster than female mice, therefore male immune organs might be more sensitive to external regulation [27].The specific mechanism needed further experiments to explore.

5. Conclusion

This study revealed that the two porcine placenta raw materials(extract and freeze-dried powder) could significantly improve mice immunity by in fluencing the cytokines expression. Therefore, porcine placenta can be a new choice in functional food development because it is rich in nutrients while the hormone content contained can be controlled under the limit in its prototype. Furthermore, the specific pathway of porcine placenta that affects body immunity and the gender difference of its immunoregulation need further investigation.

Competing interest

The authors all declare that they have no competing interests.

Acknowledgements

This work was supported by grants from the National Key Research and Development Projects (2019YFE0103800), Scientific Research Project of Sichuan Provincial Health Department (No.18PJ586) and Research and Innovation Team Project of Chengdu Medical College (No. CYTD16-04).