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. 2024 Jan 23;13(2):136.
doi: 10.3390/antiox13020136.

Mulberry Leaf Polysaccharides Attenuate Oxidative Stress Injury in Peripheral Blood Leukocytes by Regulating Endoplasmic Reticulum Stress

Wenqiang Jiang  1 , Yan Lin  2 , Linjie Qian  1 , Siyue Lu  2 , Huaishun Shen  1   2 , Xianping Ge  1   2 , Linghong Miao  1   2
Affiliations

Mulberry Leaf Polysaccharides Attenuate Oxidative Stress Injury in Peripheral Blood Leukocytes by Regulating Endoplasmic Reticulum Stress

Wenqiang Jiang et al. Antioxidants (Basel). .

Abstract

The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H2O2)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of Megalobrama amblycephala. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 μM of H2O2 for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 μg/mL) for 3 h, and then stimulated with 100 μM of H2O2 for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs' antioxidant capacities. The 200 μg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H2O2-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (p < 0.05). The over-production of reactive oxygen species, depletion of nicotinamide adenine dinucleotide phosphate, and collapse of the mitochondrial membrane potential were significantly inhibited in the 200-MLP pre-treatment group (p < 0.05). The expressions of endoplasmic reticulum stress-related genes (forkhead box O1α (foxO1α), binding immunoglobulin protein (bip), activating transcription factor 6 (atf6), and C/EBP-homologous protein (chop)), Ca2+ transport-related genes (voltage-dependent anion-selective channel 1 (vdac1), mitofusin 2 (mfn2), and mitochondrial Ca2+ uniporter (mcu)), and interleukin 6 (il-6) and bcl2-associated x (bax) were significantly lower in the 200-MLP pre-treatment group than in the HP group (p < 0.05), which rebounded to normal levels in the NC group (p > 0.05). These results indicated that MLP pre-treatment attenuated H2O2-induced PBL oxidative damage in the M. amblycephala by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for M. amblycephala, as they protect against oxidative damage.

Keywords: Megalobrama amblycephala; endoplasmic reticulum stress; mulberry leaf polysaccharides; oxidative damage; peripheral blood leukocytes.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Physicochemical properties of mulberry leaf polysaccharides. Molecular weight distribution of mulberry leaf polysaccharides (A). Monosaccharide composition of mulberry leaf polysaccharides (B).
Figure 2
Figure 2
The effect of MLPs on the antioxidant capacity of H2O2-exposed PBLs. (A) Visualizing LDH cytotoxicity, MDA and NADPH contents, and CAT and SOD activities on a pair heatmap. In the graphical presentation of data, numerical values are displayed by colors. The dendrogram for strain clustering is shown on the top and left sides of the heatmap. The width of the cluster merged from the two sides represents the distance of the two clusters. LDH cytotoxicity (B), MDA content (C), CAT activity (D), SOD activity (E), NADPH content (F). Data are expressed as the mean ± SEM (n = 6). Different little letters above the bars indicate significant differences (p < 0.05, Tukey’s test) among the HP group and MLP (50-MLP, 100-MLP, and 200-MLP) groups. * indicates a significant difference between the NC and HP, MLP (50-MLP, 100-MLP, and 200-MLP) groups (p < 0.05, independent t-test); ** indicates an extremely significant difference between the NC and HP, MLP (50-MLP, 100-MLP, and 200-MLP) groups (p < 0.01, independent t-test). ‘−’ indicates no reagents added; ‘+’ indicates reagents added.
Figure 3
Figure 3
Effect of MLPs on ROS generation (A,B) and MMP status (C,D) in H2O2-induced PBLs. Data are expressed as the mean ± SEM (n = 6); ns indicates no significant difference (independent t-test, p > 0.05); * indicates a significant difference (independent t-test, p < 0.05); ** indicates an extremely significant difference (independent t-test, p < 0.01). ‘−’ indicates no reagents added; ‘+’ indicates reagents added.
Figure 4
Figure 4
Assessment of gene expressions: foxO1α (A), bip (B), atf6 (C), chop (D), mfn2 (E), grp75 (F), vdac-1 (G), mcu (H), bax (I), and il-6 (J). Data are expressed as the mean ± SEM (n = 9); ns indicates no significant difference (independent t-test, p > 0.05); * indicates a significant difference (independent t-test, p < 0.05); ** indicates an extremely significant difference (independent t-test, p < 0.01). Heatmap illustrating the relationship between genes (K). Rows and columns correspond to genes; each cell contains the corresponding correlation and p value. Pearson’s R-values are color-coded according to the color legend. The edge width corresponds to Mantel’s r statistic for the corresponding distance correlations between the different diets and genes. * indicates p-value < 0.05, ** indicates p-value < 0.01, *** indicates p-value < 0.001 (K).
Figure 5
Figure 5
Schematic diagram depicting the effects and possible mechanisms of H2O2 stress on antioxidant capacity, endoplasmic reticulum stress, apoptosis, and Ca2+ transport-related gene expression. Notes: the blue arrow represents the HP group, and the red arrow represents the 200-MLP group; the upward arrows are representative of significant up-regulation or improvement, and the downward arrows indicate significant down-regulation or inhibition.
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