The Inhibitory Effect of Early Pregnancy Factor on Red Meat Neu5Gc-Mediated Antibody Production in CMAH-/- Mice

Abstract

The meat derived from mammals such as cows, sheep, and pigs is commonly referred to as red meat. Recent studies have shown that consuming red meat can activate the immune system, produce antibodies, and subsequently develop into tumors and cancer. This is due to the presence of a potential carcinogenic compound in red meat called N-ethanol neuraminic acid (Neu5Gc). Neu5Gc is a common sialic monosaccharide in mammals, synthesized from N-acetylneuraminic acid (Neu5Ac) in the body and typically present in most mammals. However, due to the lack of the CMAH gene encoding the cytidine 5'-monophosphate Neu5Ac hydroxylase, humans are unable to synthesize Neu5Gc. Compared to primates such as mice or chimpanzees, the specific loss of Neu5Gc expression in humans is attributed to fixed genome mutations in CMAH. Although Neu5Gc cannot be produced, it can be introduced from specific dietary sources such as red meat and milk, so it is necessary to use mice or chimpanzees that knock out the CMAH gene instead of humans as experimental models. Further research has shown that early pregnancy factor (EPF) has the ability to regulate CD4⁺T cell-dependent immune responses. In this study, we established a simulated human animal model using C57/BL6 mice with CMAH gene knockout and analyzed the inhibitory effect of EPF on red meat Neu5Gc-induced CMAH^-/- C57/BL6 mouse antibody production and chronic inflammation development. The results showed that the intervention of EPF reduced slow weight gain and shortened colon length in mice. In addition, EPF treatment significantly reduced the levels of anti Neu5Gc antibodies in the body, as well as the inflammatory factors IL-6 and IL-1β, TNF-α and the activity of MPO. In addition, it also alleviated damage to liver and intestinal tissues and reduced the content of CD4 cells and the expression of B cell activation molecules CD80 and CD86 in mice. In summary, EPF effectively inhibited Neu5Gc-induced antibody production, reduced inflammation levels in mice, and alleviated Neu5Gc-induced inflammation. This will provide a new re-search concept and potential approach for developing immunosuppressants to address safety issues related to long-term consumption of red meat.

Keywords: CMAH; EPF; N-glycolylneuraminic acid; Neu5Gc; early pregnancy factor; red meat.

Figure 1

Animal experiment group and implementation plan.

Figure 2

The effect of EPF on colon length and body weight in red meat Neu5Gc-induced inflammation mice. (A): effect of EPF on colon length in inflammatory mice. (B): statistical results of colon length. (C): effect of EPF on Neu5Gc-induced body weight in mice. 1. Blank group 2. Control group 3. Inflammation group 4. Pro-inflammation group 5. EPF intervention group. The data are presented as mean ± SD, and statistical analysis was performed using one-way analysis of variance (ANOVA). Significance levels were indicated as *** p < 0.001, **** p < 0.0001 and ^### p < 0.001 compared to each respective group.

Figure 3

EPF reduced the levels of anti-Neu5Gc antibodies in red meat Neu5Gc-induced inflammation mice. 1. Blank group 2. Control group 3. Inflammation group 4. Pro-inflammation group 5. EPF intervention group. The data are presented as mean ± SD, and statistical analysis was performed using one-way analysis of variance (ANOVA). Significance levels were indicated as ** p < 0.01 and **** p < 0.0001 compared to each respective group.

Figure 4

The administration of EPF resulted in a reduction in the levels of inflammatory factors in the serum of mice with inflammation. After blood collection, the serum was separated, and the levels of inflammatory factors in the serum were analyzed. (A): the levels of IL-1β; (B): the levels of IL-6; (C): the levels of TNF-α. (D): MPO activity in tissues. 1. Blank group 2. Control group 3. Inflammation group 4. Pro-inflammation group 5. EPF intervention group. The data are presented as mean ± SD and analyzed using one-way analysis of variance. Significance levels were indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to each respective group.

Figure 5

The administration of EPF mitigates histopathological damage in mice with Neu5Gc-induced inflammation. After euthanizing the mice, liver, colon, small intestine, kidney, and lung specimens were collected from each experimental group for subsequent determination of tissue and organ indices through weighing. Subsequently, histopathological analysis was conducted using HE staining to examine the mice in each group. (A): effects of EPF on tissue and organ index in Neu5Gc-induced inflammatory mice. 1. Blank group 2. Control group 3. Inflammation group 4. Pro-inflammation group 5. EPF intervention group. (B): effects of EPF on histopathology in mice with Neu5Gc-induced inflammation. Significance levels were indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to each respective group. The red boxes represent the location of the lesion.

Figure 6

After undergoing EPF treatment, the CD4 cell count in Neu5Gc-induced inflammatory mice exhibited a significant decrease. After euthanizing the mice, the intact spleens were delicately excised and meticulously rinsed with distilled water. Subsequently, the CD4 cell population within the spleens was assessed via immunohistochemistry. (A): effect of EPF on the CD4 cell content in the spleen of mice with Neu5Gc-induced inflammation. (B): alterations in CD4 cell numbers in the spleen of mice with Neu5Gc-induced inflammation. 1. Blank group 2. Control group 3. Inflammation group 4. Pro-inflammation group 5. EPF intervention group. The data are presented as mean ± SD, and statistical analysis was performed using one-way analysis of variance (ANOVA). Significance levels were indicated as * p < 0.05 and **** p < 0.0001 compared to each respective group.

Figure 7

The expression of key molecules involved in B cell activation was downregulated by EPF in Neu5GC-induced inflammatory mice. We assessed the transcriptional levels of CD19, CD80, CD86, and CD138, which are crucial for B cell activation in inflammatory mice, following EPF intervention using fluorescent quantitative PCR. (A). Transcriptional status of CD80 mRNA molecule. (B). Transcriptional status of CD86 mRNA molecule. (C). Transcriptional status of CD19 mRNA molecule. (D). Transcriptional status of D138 mRNA molecule. The experimental groups included: 1. Blank group 2. Control group 3. Inflammation group 4. Pro-inflammation group 5. EPF intervention group. The data are presented as mean ± SD, and statistical analysis was performed using one-way analysis of variance (* p < 0.05, *** p < 0.001 and **** p < 0.0001 indicate significance compared to each group). “ns” denotes no significance.