Baicalein Ameliorates Streptococcus suis-Induced Infection In Vitro and In Vivo

Abstract

As an important zoonotic pathogen, Streptococcus suis (S. suis) infection has been reported to be a causative agent for variety of diseases in humans and animals, especially Streptococcal toxic shock-like syndrome (STSLS), which is commonly seen in cases of severe S. suis infection. STSLS is often accompanied by excessive production of inflammatory cytokines, which is the main cause of death. This calls for development of new strategies to avert the damage caused by STSLS. In this study, we found for the first time that Baicalein, combined with ampicillin, effectively improved severe S. suis infection. Further experiments demonstrated that baicalein significantly inhibited the hemolytic activity of SLY by directly binding to SLY and destroying its secondary structure. Cell-based assays revealed that Baicalein did not exert toxic effects and conferred protection in S. suis-infected cells. Interestingly, compared with ampicillin alone, Baicalein combined with ampicillin resulted in a higher survival rate in mice severely infected with S. suis. At the same time, we found that baicalein can be combined with meropenem against MRSA. In conclusion, these results indicate that baicalein has a good application prospect.

Keywords: Streptococcus suis; anti-hemolysin; antibacterial; baicalein; suilysin.

Figure 1

Baicalein inhibits the hemolytic activity of SLY: (A) The growth curve of SC19. SC19 was cultured with 5% newborn bovine serum and treated with different concentrations of baicalein; (B) Hemolytic activity of supernatants from SC19 and baicalein co-culture system; ** p < 0.01, *** p < 0.001 vs. 0 μg/mL. (C) Effect of baicalein on the hemolytic activity of purified SLY (100 ng/mL). ** p < 0.01, *** p < 0.001 vs. 0 μg/mL.

Figure 2

Baicalein reduced SS2-mediated cytokine production at the cellular level: (A) TNF-α; (B) IL-6, and (C) IL-1β. Cells were incubated with SS2 (MOI = 10:1) and different concentrations of baicalein for 6 h. ELISA was used to determine the concentrations of TNF-α, IL-1 β, and IL-6. “NC” represents no treatment with SC19. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. SS2 alone; (D) Hemolytic activity of ampicillin to the RBCs in the absence or presence of baicalein; (E) The addition of baicalein exerts a negligible effect on the cytotoxicity of ampicillin in VERO cells.

Figure 3

Baicalein protects J774 cells from SC19-mediated cell damage: (A) The cells not infected with SC19; Scale bar, 10 µm. (B) SC19-infected cells; (C) SC19-infected cells treated with 32 μg/mL baicalein; (D) LDH release from SC19-infected cells treated with different concentrations of baicalein (0 to 32 μg/mL). The results are presented as mean ± SD (n = 3). *** p < 0.001 vs. 0 μg/mL.

Figure 4

Baicalein destroys hemolytic activity based on the direct binding to the SLY; (A) Baicalein is docked into the binding site of the SLY (Total view); (B) Baicalein and SLY binding site (detailed view). The baicalein is represented with green sticks; the hydrogen bond is shown in a black dotted line; (C) ITC analysis of the interaction between SLY and baicalein; 0.5 mmol/L of baicalein was dropped into 0.02 mmol/L of SLY in PBS buffer at 25 °C. Calculated thermodynamic parameters, including the equilibrium dissociation constant (KD = 8.651 × 10⁻⁷ mol/ L).

Figure 5

The survival rates and tissue pathological changes of SC19-infected mice; (A) The survival rates of baicalein + ampicillin and ampicillin cured severely infected mice model; (B) Blood levels of AST, ALT, and CK at 6 h post-infection (two-tailed, unpaired t-tests, n = 5); *** p < 0.001 vs SS2 alone. (C) Pathological changes of lung and brain tissue after baicalein and ampicillin treatment. Baicalein alleviated tissue damage of infected mice. The H&E images (40×) of tissue lesions.