Inhibitory Effects of Antimicrobial Peptide JH-3 on Salmonella enterica Serovar Typhimurium Strain CVCC541 Infection-Induced Inflammatory Cytokine Release and Apoptosis in RAW264.7 Cells

Abstract

The antibiotic resistance of Salmonella has become increasingly serious due to the increased use of antibiotics, and antimicrobial peptides have been considered as an ideal antibiotic alternative. Salmonella can induce macrophage apoptosis and thus further damage the immune system. The antimicrobial peptide JH-3 has been shown to have a satisfactory anti-Salmonella effect in previous research, but its mechanism of action remains unknown. In this study, the effects of JH-3 on macrophages infected with Salmonella Typhimurium CVCC541 were evaluated at the cellular level. The results showed that JH-3 significantly alleviated the damage to macrophages caused by S. Typhi infection, reduced the release of lactic dehydrogenase (LDH), and killed the bacteria in macrophages. In addition, JH-3 decreased the phosphorylation level of p65 and the expression and secretion of interleukin 2 (IL-2), IL-6, and tumor necrosis factor-α (TNF-α) by inhibiting the activation of the mitogen-activated protein kinase (MAPK) (p38) signaling pathway and alleviating the cellular inflammatory response. From confocal laser scanning microscopy and flow cytometry assays, JH-3 was observed to inhibit the release of cytochrome c in the cytoplasm; the expression of TNF-αR2, caspase-9, and caspase-8; to further weaken caspase-3 activation; and to reduce the S.-Typhi-induced apoptosis of macrophages. In summary, the mechanism by which JH-3 inhibits Salmonella infection was systematically explored at the cellular level, laying the foundation for the development and utilization of JH-3 as a therapeutic alternative to antibiotics.

Keywords: Salmonella; antimicrobial peptide JH-3; apoptosis; cytokines.

Figure 1

The expression of inflammatory cytokines induced by the infection of RAW264.7 cells by Salmonella enterica Serovar Typhimurium strain CVCC541. (A,C,E): the mRNA expression levels of interleukin 2 (IL-2), IL-6, and tumor necrosis factor-α (TNF-α) after RAW264.7 cells were infected with Salmonella CVCC541 (“*”, p < 0.05; “**”, p < 0.01 ); (B,D,F): the ELISA results of IL-2, IL-6, and TNF-α expression levels after RAW264.7 cells were infected with Salmonella CVCC541 (“*”, p < 0.05; “**”, p < 0.01).

Figure 2

The apoptosis of RAW264.7 cells was induced by Salmonella CVCC541 infection. (A) The RAW264.7 cells infected with Salmonella CVCC541 were observed by scanning electron microscopy. (B) The c-caspase-3 activation in RAW264.7 cells was detected by confocal laser scanning microscopy. (C) The apoptosis rate of the infected RAW264.7 cells was detected by flow cytometry. (D) The caspase-3 and c-caspase-3 activations in RAW264.7 cells were detected by western blot (WB).

Figure 3

JH-3 inhibited the expression of inflammatory cytokines. (A), (C), and (E): JH-3 inhibited the mRNA expression of IL-2, IL-6, and TNF-α by RAW264.7 cells infected with Salmonella CVCC54 (“NS”, p > 0.05; “*”, p < 0.05). (B), (D), and (F): JH-3 inhibited the secretion of IL-2, IL-6, and TNF-α by RAW264.7 cells infected with Salmonella CVCC541 (“NS”, p > 0.05; “*”, p < 0.05).

Figure 4

JH-3 inhibited the activation of the mitogen-activated protein kinase (MAPK) and p65 signaling pathways. (A–C): the effect of JH-3 on the mRNA expression level of JNK, ERK, and p38 assessed by qRT-PCR (“NS”, p > 0.05; “*”, p < 0.05). (D,E): the effect of JH-3 on the expression of p-p38 and p-p65 in RAW264.7 cells assessed by confocal laser scanning microscopy assays.

Figure 5

JH-3 reduced the release of lactic dehydrogenase (LDH) and the survival of bacteria in RAW264.7 cells. (A) The LDH release by RAW264.7 cells at different infection times (“*”, p < 0.05; “**”, p < 0.01). (B) JH-3 could inhibit LDH release by RAW264.7 cells at different multiplicities of infection (MOIs) (“*”, p < 0.05). (C) JH-3 could significantly inhibit the proliferation of CVCC541 and promote the killing of intracellular bacteria (“**”, p < 0.01; “***”, p < 0.001).

Figure 6

JH-3 inhibited Salmonella-CVCC541-induced apoptosis in RAW264.7 cells. (A–C): the effect of JH-3 on the mRNA expression of caspase-3, caspase-8, and caspase-9 (“NS”, p > 0.05; “*”, p < 0.05; “**”, p < 0.01). (D): the effect of JH-3 on cell morphological structure as assessed by transmission electron microscopy. (E): JH-3 reduced the apoptosis rate of RAW264.7 cells as assessed by flow cytometry analysis.

Figure 7

JH-3 downregulated the expression of caspase-9 and TLR4. (A) The expression of caspase-9 in RAW264.7 cells assessed by confocal laser scanning microscopy. (B) The expression of TLR4 in RAW264.7 cells assessed by confocal laser scanning microscopy. (C) The expression of p-p85 in RAW264.7 cells assessed by confocal laser scanning microscopy. (D) JH-3 could significantly reduce the release of cytochrome c in the cytoplasm. (E) JH-3 could also reduce the expression of TNF-αR2 (“NS”, p > 0.05; “*”, p < 0.05).

Figure 8

Signaling pathways regulated by antimicrobial peptide JH-3. On the left side of the figure, the inhibitory effect of JH-3 on the expression of TNF-αR2, caspase-8 (Casp-8), and caspase-9 (Casp-9) is shown. These changes inhibit the apoptosis of Salmonella-infected macrophages. On the right side of the figure, the inhibitory effect of JH-3 on the proliferation of Salmonella CVCC541 is shown (top), as well as the downregulation of TLR4 and the activity of p38 (MAPK) by JH-3 (bottom). The latter changes may inhibit cytokine production by Salmonella-infected macrophages.