Baicalin inhibits the lethality of Shiga-like toxin 2 in mice

Abstract

Shiga-like toxins (Stxs), produced by pathogenic Escherichia coli, are a major virulence factor involved in severe diseases in human and animals. These toxins are ribosome-inactivating proteins, and treatment for diseases caused by them is not available. Therefore, there is an urgent need for agents capable of effectively targeting this lethal toxin. In this study, we identified baicalin, a flavonoid compound used in Chinese traditional medicine, as a compound against Shiga-like toxin 2 (Stx2). We found that baicalin significantly improves renal function and reduces Stx2-induced lethality in mice. Further experiments revealed that baicalin induces the formation of oligomers by the toxin by direct binding. We also identified the residues important for such interactions and analyzed their roles in binding baicalin by biophysical and biochemical analyses. Our results establish baicalin as a candidate compound for the development of therapeutics against diseases caused by Stxs.

FIG 1

Inhibitory effect of baicalin against rStx2. (A) Chemical structure of baicalin. (B) Inhibition of cell death by baicalin. rStx2 was added to HeLa cells treated with the indicated concentrations of baicalin for 72 h. Cytotoxicity was evaluated by measuring extracellular LDH 72 h after addition of the toxins. (C) rStx2 premixed with baicalin can protect HeLa cells. rStx2 mixed with the indicated concentrations of baicalin was added to HeLa cells, and the LDH release was measured after incubating for 72 h. (D) Effects of baicalin on the inhibition of protein synthesis by rStx2. Baicalin was added to in vitro protein synthesis with rStx2, and the expression of luciferase was measured. All data are means and standard errors from three independent experiments. *, P < 0.05; **, P < 0.01.

FIG 2

Baicalin protects mice against lethality induced by rStx2. (A) Groups (n = 10) of mice injected with rStx2 were treated with PBS solution or with BAI 6 h after toxin injection, and survival of mice was monitored for 6 days. The death of mice was followed up to 6 days after rStx2 injection, with no additional mortalities shown in the figure. The curves for BAI-treated mice are statistically significantly different from those for rStx2-injected mice, as evaluated by the log rank test (P = 0.0002 for 100 mg/kg of BAI against rStx2). (B) Effects of BAI on body weight caused by sublethal doses of rStx2. Groups (n = 10) of mice receiving Stx2 or a control solution were administered BAI or PBS 6 h after toxin injection. The body weight was monitored at 24-h intervals for 8 days. Similar results were obtained from more than three independent experiments. *, P < 0.05 for rStx versus rStx plus BAI and PBS at day 6; **, P < 0.01 for rStx2 versus PBS at day 7 and for rStx2 versus rStx plus BAI and PBS at day 8.

FIG 3

Treatment with baicalin alleviated the pathologies induced by rStx2. (A) Baicalin reduced tissue damage in kidney. Sections of kidneys from representative rStx2-treated mice receiving BAI or control PBS solution are shown. Note the swelling and casts in the lumen and the smaller glomerulus in the tissue of untreated mice (left). In contrast, the morphology of tissues from mice receiving Stx and treated with BAI (middle) was similar to that of tissues from control mice (right). Each group contained 10 sections, and 5 fields were observed in each section. (B and C) Effects of baicalin on blood urea nitrogen (BUN) (B) and creatinine (Cr) (C) induced by rStx2. The blood of mice in the relevant groups was evaluated for BUN and Cr at 72 h after treatment. Each circle represents the BUN or Cr level from one mouse (n = 5). (D) Baicalin treatment reduced the level of hemoglobin in the serum. Sera of relevant mice were evaluated for hemoglobin spectrophotometrically. Similar results were obtained in multiple independent experiments. (E) Baicalin reduced the production of several cytokines induced by rStx2. Kidney tissues from relevant mice were measured for cytokines by ELISA. Note the significant reduction of cytokine production after baicalin treatment. **, P < 0.01.

FIG 4

Overlay of structures of ricin subunit A (cyan) and Shiga-like toxin 2 subunit A1 (purple). The crystal structures of RTA and Stx2A1 are displayed in ribbon representation, with coils for α-helices and arrows for β strands. BAI is superimposed in stick mode, with carbon and oxygen atoms in yellow and red, respectively.

FIG 5

Structure-based sequence alignment of the A subunit of ricin and the A1 subunit of Shiga-like toxin 2. Identical residues are highlighted in red, and similar residues are shown in red font. Secondary structures are shown for both proteins above and below their respective sequences, with coils representing α/η-helices and arrows representing β-strands.

FIG 6

Validation of the involvement of rStx2 residues in its interaction with baicalin. (A) Purified rStx2 mutants were measured for their toxicity to HeLa cells by determining LDH release upon treatment of the cells with recombinant holotoxin. (B) Ability of the mutants to inhibit protein synthesis in a cell-free system. The effects of baicalin were evaluated by including this compound in parallel reactions. The results are expressed as the percentage of protein synthesis in control reactions without toxin or baicalin. All data are means and standard errors from three independent experiments. *, P < 0.05; ** P < 0.01.