The anti-diarrhea activity of red algae-originated sulphated polysaccharides on ETEC-K88 infected mice

Abstract

Polysaccharides from red algae Porphyra haitanensis and Gracilaria lemaneiformis possess various bioactive functions, however, their anti-diarrhea activity remains incompletely defined. In the current study, sulphated polysaccharides were extracted by high pressure treatment plus ethanol precipitation from these two algae, and named PHSP_(hp) and GLSP_(hp), respectively. PHSP_(hp) and GLSP_(hp) showed decreased viscosity and molecular weight. Meanwhile, they have a certain immunomodulatory effect on wound healing and migration of RAW264.7 cells. Moreover, they significantly increased the secretion of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). A BALB/c model infected by enterotoxigenic Escherichia coli (ETEC)-K88 was also established to evaluate the anti-diarrhea activity of PHSP_(hp) and GLSP_(hp). The results showed that PHSP_(hp) and GLSP_(hp) were able to alleviate mice diarrhea symptoms. Meanwhile, they inhibited the release of pro-inflammatory cytokines and suppressed the secretion of immunoglobulin A via reducing the population of B cells. In addition, the nitroblue tetrazolium levels of mouse serum were decreased. Taken together, PHSP_(hp) and GLSP_(hp) alleviated the inflammatory response of ETEC-K88-induced diarrhea through both specific and non-specific immunity. Sulphated polysaccharides from red algae may be used as functional food components for remitting diarrhea.

Fig. 1. SEM and FT-IR spectrum of PHSP_(hp) and GLSP_(hp). (A) SEM of PHSP_(hp) and GLSP_(hp). (B) FT-IR spectra of PHSP_(hp) and GLSP_(hp).

Fig. 2. The influence of PHSP_(hp) and GLSP_(hp) on wound healing of RAW264.7 cells (100×, 24 h). The cells were scratched, and incubated with PBS (as the control group), PHSP_(hp) or GLSP_(hp) followed by a 24 h-recovery period. Microscopic bright field pictures were taken immediately after the scratch and the precise coordinates were reassessed 24 h after incubation.

Fig. 3. The influence of PHSP_(hp) and GLSP_(hp) on RAW264.7 migration (100×, 24 h). (A) RAW264.7 cell migration micrograph. (B) The absorbance values (OD₅₇₀) of eluted crystal violet. Data are present as mean ± SD (n = 3).

Fig. 4. Effects of PHSP_(hp) and GLSP_(hp) on TNF-α and IL-6 production in RAW264.7 cell supernatant. RAW264.7 cells were incubated with PHSP_(hp) and GLSP_(hp) (0, 25, 50, 100, 200 μg mL⁻¹) for 24 h and the cultural supernatants were collected for ELISA. (A and B) Effects of PHSP_(hp) and GLSP_(hp) on the secretion of TNF-α. (C and D) Effects of PHSP_(hp) and GLSP_(hp) on the secretion of IL-6. PBS served as a negative control and 2 μg mL⁻¹ LPS served as a positive control. Data are present as mean ± SD (n = 3). *P < 0.05 versus the PBS group, **P < 0.01 versus the PBS group.

Fig. 5. Effects of PHSP_(hp) and GLSP_(hp) on diarrhea rate and weight changes in ETEC-K88 infected diarrhea mice. (A) Dose lethality curve of ETEC-K88 in BALB/c mice. (B) Variation of diarrhea rate along with infection time. (C) Weight changes along with infection time. Data (A) is present as mean (n = 6), data (B and C) are present as mean ± SD (n = 10).

Fig. 6. Effects of PHSP_(hp) and GLSP_(hp) in mice serum cytokines. (A) MCP-1, (B) TNF-α, (C) IFN-γ, (D) IL-6, (E) IgA. ELISA was carried out in different ETEC-K88-infected sample groups. Data are present as mean ± SD (n = 3). *P < 0.05 versus the PBS group, and **P < 0.01 versus the PBS group. ^#P < 0.05 versus the diarrhea group, and ^##P < 0.01 versus the diarrhea group.

Fig. 7. Effects of PHSP_(hp) and GLSP_(hp) on lymphocyte from mice splenocytes. The infected mice were fed with PHSP_(hp) and GLSP_(hp) for 7 days before they were sacrificed and the splenocytes were obtained. Cells of 1 × 10⁶ were mixed with CD3-APC, CD4-PercP-Cy5.5 and CD19-PE for 30 min at 4 °C, before subject to the flow cytometry. (A) The histogram of B cell FACS analysis. (B) The scatter diagrams of Th cell FACS analysis.