Red Algae “Sarcodia suieae” Acetyl-Xylogalactan Downregulate Heat-Induced Macrophage Stress Factors Ddit3 and Hyou1 Compared to the Aquatic Animal Model of Nile Tilapia (Oreochromis niloticus) Brain Arachidonic Acid Expression

Abstract

Anthropogenic climate change is known to be an increased stress that affects aquatic animal behavior and physiological alternations, which can induce the animal's death. In order to known whether the extracted acetyl-xylogalactan function on the regulation of the external high temperature induced death, we first selected the mammalian cell line "RAW 264.7" used in the previous experiment to evaluate the extracted acetyl-xylogalactan function. We aimed to evaluate the effects of the acetyl-xylogalactan on the RAW 264.7 macrophages and Nile Tilapia stress factor expression under the heat environment. In the in vitro cell observation, we assessed the cell survival, phagocytic activity, intracellular Ca²⁺ level, mitochondria potential exchange, apoptotic assay findings, galactosidase activity, RNA-seq by NGS and real-time polymerase chain reaction (QPCR) expression. In the in vivo Nile Tilapia observation aimed to evaluate the blood biochemical indicator, brain metabolites exchange and the liver morphology. In our evaluation of RAW 264.7 macrophages, the RNA sequencing and real-time polymerase chain reaction (PCR) was shown to upregulate the expression of the anti-apoptosis Cflar gene and downregulate the expression of the apoptosis factors Ddit3 and Hyou1 to protect macrophages under heat stress. We already knew the extracted acetyl-xylogalactan function on the mammalian "RAW 264.7" system. Following, we used the aquatic Nile Tilapia model as the anthropogenic climate change high temperature experiment. After feeding the Nile Tilapia with the acetyl-xylogalactan, it was found to reduce the brain arachidonic acid (AA) production, which is related to the NF-κB-induced apoptosis mechanism. Combined with the in vitro and in vivo findings, the acetyl-xylogalactan was able to reduce the heat induced cell or tissue stress.

Keywords: Sarcodia suieae; acetyl-xylogalactan; apoptosis; environment; stress.

Figure 1

RAW 264.7 macrophage survival and phagocytotic activity of: (A) The 30 μg/mL treatment group showed significantly greater survival than the control group (p < 0.01) at 12 and 24 h (p < 0.05) under heat stress; (B) acetyl-xylogalactan treatment was able to significantly induce phagocytic ability (p < 0.05) at 12 h under heat stress, but not at 24 h; (C) the RAW 264.7 cells displayed an oval shape at 37 °C; (D) The RAW 264.7 cells showed at 41 °C (the black bar was shown as 50 μm) (p < 0.05 was marked as *, p < 0.01 was marked as **).

Figure 2

RAW 264.7 macrophage apoptosis detection via Annexin-V: (A) Treatment with 20 µg/mL acetyl-xylogalactan was able to significantly reduce apoptosis (p < 0.05) at 12 h under heat stress. (B) However, apoptosis did not show significant differences among groups at 24 h (p > 0.05). (C) RAW 264.7 macrophage galactosidase detection at a normal temperature (37 °C). (D) 30 μg/mL acetyl-xylogalactan significantly induced galactosidase production (blue-stained cells) at 41 °C (the black bar was shown as 50 μm) (p < 0.05 was marked as *).

Figure 3

RAW 264.7 macrophage intracellular Ca²⁺ concentration and mitochondria membrane potential: (A) There was no significant change (p > 0.05) under pretreatment with various concentrations of acetyl-xylogalactan following incubation at 41 °C. (B) Mitochondrial membrane potential recovery was lower in the treatment groups than in the control group (p < 0.01) (Treatment was presented as 10 μg means the 10 μg acetyl-xylogalactan/mL (culture medium); 20 μg means the 20 μg acetyl-xylogalactan/mL (culture medium); 30 μg means the 30 μg acetyl-xylogalactan/mL (culture medium)) (p < 0.01 was marked as **).

Figure 4

RNA sequencing (transcriptome) analysis: (A) Acetyl-xylogalactan may have affected the counts of the apoptosis gene, as 10 counts were presented in the KEGG analysis. (B) Acetyl-xylogalactan may have regulated the relative cell phenomenon; (C) log two-fold change of the acetyl-xylogalactan various concentration treatment. Totals of 10 μg (D), 20 μg (E), 30 μg (F) Volcano map of the treatments with various concentrations of acetyl-xylogalactan treatment. (Treatment was presented; 10 μg means 10 μg of acetyl-xylogalactan/mL (culture medium); 20 μg means 20 μg of acetyl-xylogalactan/mL (culture medium); 30 μg means 30 μg of acetyl-xylogalactan/mL (culture medium)).

Figure 4

RNA sequencing (transcriptome) analysis: (A) Acetyl-xylogalactan may have affected the counts of the apoptosis gene, as 10 counts were presented in the KEGG analysis. (B) Acetyl-xylogalactan may have regulated the relative cell phenomenon; (C) log two-fold change of the acetyl-xylogalactan various concentration treatment. Totals of 10 μg (D), 20 μg (E), 30 μg (F) Volcano map of the treatments with various concentrations of acetyl-xylogalactan treatment. (Treatment was presented; 10 μg means 10 μg of acetyl-xylogalactan/mL (culture medium); 20 μg means 20 μg of acetyl-xylogalactan/mL (culture medium); 30 μg means 30 μg of acetyl-xylogalactan/mL (culture medium)).

Figure 4

RNA sequencing (transcriptome) analysis: (A) Acetyl-xylogalactan may have affected the counts of the apoptosis gene, as 10 counts were presented in the KEGG analysis. (B) Acetyl-xylogalactan may have regulated the relative cell phenomenon; (C) log two-fold change of the acetyl-xylogalactan various concentration treatment. Totals of 10 μg (D), 20 μg (E), 30 μg (F) Volcano map of the treatments with various concentrations of acetyl-xylogalactan treatment. (Treatment was presented; 10 μg means 10 μg of acetyl-xylogalactan/mL (culture medium); 20 μg means 20 μg of acetyl-xylogalactan/mL (culture medium); 30 μg means 30 μg of acetyl-xylogalactan/mL (culture medium)).

Figure 5

The RNA- seq analysis: (A) The network of the relative RNA gene; (B) the heatmap of the RNA-seq analysis, presented with a log two-fold change; (C) mRNA expression. Acetyl-xylogalactan downregulated apoptosis under heat stress via target genes, such as Nfkbia, Ddit3, Gadd45a, Akt3 and Hyou1, and upregulated Cflar expression. (Treatment was presented; 10 μg means 10 μg of acetyl-xylogalactan/mL (culture medium); 20 μg means 20 μg of acetyl-xylogalactan/mL (culture medium); 30 μg means 30 μg of acetyl-xylogalactan/mL (culture medium)).

Figure 6

Effect of the RAW 264.7 macrophage cytokine IL-6 and IL-17A production of: (A) the blue bar means the RAW 264.7 IL-6 production at 37 °C; red bar means the RAW 264.7 IL-6 production at 41 °C. (B) the blue bar means the RAW 264.7 IL-17A production at 37 °C; red bar means the RAW 264.7 IL-17A production at 41 °C. (* Treatment was presented; 10 μg means 10c of μg acetyl-xylogalactan/mL (culture medium); 20 μg means 20 μg of acetyl-xylogalactan/mL (culture medium); 30 μg means 30 μg of acetyl-xylogalactan/mL (culture medium)) (p < 0.05 was marked as *).

Figure 7

The KEGG analysis of the metabolites pathway. The blue mark denotes p value <0.05. The yellow mark denotes p value > 0.1.

Figure 8

Histological observation of the liver stained by the H&E, PAS and Giemsa stain. The black bar was presented as 50 μm.