Abstract:

Objective To investigate the protective effects and mechanisms of Echinococcus granulosus antigen B (AgB) and calcium-binding protein 1 (CBP1) in immune thrombocytopenia (ITP) mouse models through TLR4/NF-κB/NLRP3 signaling pathway. Methods Forty-two healthy female BALB/c mice were randomly divided into six groups: control, ITP, AgB, AgB + ITP, CBP1 and CBP1 + ITP, with seven mice in each group. Mice in the AgB and AgB + ITP groups received daily intraperitoneal injection of 100 μg (200 μl) of AgB, while those in the CBP1 and CBP1 + ITP groups received 100 μg (200 μl) of CBP1, mice in the control and ITP groups received 200 μl of PBS, all for five consecutive days. Subsequently, mice in the ITP, AgB + ITP and CBP1 + ITP groups received daily intraperitoneal injection of 3 μg (200 μl) of anti-CD41 monoclonal antibody (Ab) to establish ITP model, while those in the control, AgB, and CBP1 groups received 200 μl of PBS daily, all for five consecutive days. Platelet counts in peripheral blood were measured one day before modeling (D0), during modeling (D1-D5), and one day after modeling (D6). On the day after modeling, the mice were euthanized to collect spleens and livers, which were weighed for calculation of organ index. The mRNA relative transcription levels of Toll-like receptor 4 (TLR4), NOD-like receptor pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and IL-1β in spleen tissues were detected by qRT-PCR. The relative expression levels of TLR4, inducible nitric oxide synthase (iNOS), nuclear factor κB (NF-κB), and caspase-1 in spleen tissues were detected by Western blotting. One-way ANOVA was used for comparisons between groups, and the LSD-t test was used for pairwise comparison between multiple groups. Results From the D1 to D6, the platelet counts in the ITP group mice were (102.1 ± 6.8) × 10⁹/L, (234.7 ± 18.1) × 10⁹/L, (229.7 ± 45.8) × 10⁹/L, (316.7 ± 26.8) × 10⁹/L, (320.6 ± 60.5) × 10⁹/L, (179.1 ± 22.2) × 10⁹/L, which were lower than those of (526.6 ± 90.4) × 10⁹/L, (679.3 ± 58.5) × 10⁹/L, (828.0 ± 61.0) × 10⁹/L, (855.3 ± 101.9) × 10⁹/L, (784.1 ± 177.7) × 10⁹/L, (877.4 ± 107.5) × 10⁹/L in the control group (LSD-t = -4.2, -5.5, -6.9, -6.3, -3.9, -4.8; all P < 0.05). Platelet counts in the mice of AgB + ITP group and CBP1 + ITP group on D6 were (512.6 ± 100.5) × 10⁹/L and (511.1 ± 114.8) × 10⁹/L, which were higher than those in the ITP group (LSD-t = 2.3, 2.3; both P < 0.05). The spleen index of the ITP group was 12.1 ± 1.2, higher than that of the control group (6.3 ± 0.4) (LSD-t = 6.8, P < 0.01). The spleen index in the AgB + ITP group was 9.0 ± 0.3, which was lower than that in the ITP group (LSD-t = -3.6, P < 0.01). The results of qRT-PCR showed that the relative transcription levels of TLR4, NLRP3, ASC, IL-1β in spleen tissue of the ITP group were 7.5 ± 2.1, 5.3 ± 1.5, 3.6 ± 0.7, 4.0 ± 0.9, respectively, which were higher than those of 1.3 ± 0.3, 1.2 ± 0.2, 1.2 ± 0.3, 1.0 ± 0.1 in the control group (LSD-t = 4.8, 4.5, 4.2, 5.2; P < 0.01). The mRNA relative transcription levels of the AgB + ITP group were 1.7 ± 0.3, 0.6 ± 0.1, 1.0 ± 0.3 and 0.7 ± 0.1, while those of CBP1 + ITP group were 1.7 ± 0.1, 1.0 ± 0.3, 1.1 ± 0.4 and 0.4 ± 0.1, all were lower than those of ITP group (LSD-t = -4.6, -5.1, -4.5, -5.9, -4.5, -4.7, -4.4, -6.3; all P < 0.01). Western blotting results showed that the relative expression levels of TLR4, iNOS, NF-κB p65, caspase-1 in spleen tissue of the ITP group were 0.7 ± 0.1, 0.5 ± 0.0, 1.4 ± 0.2, 1.5 ± 0.2, all were higher than those of 0.2 ± 0.0, 0.3 ± 0.0, 0.9 ± 0.2, 0.8 ± 0.2 in the control group (LSD-t = 8.6, 6.5, 3.1, 3.5; all P < 0.01). The relative expression levels of AgB + ITP group were 0.4 ± 0.0, 0.4 ± 0.0, 0.9 ± 0.1, 1.0 ± 0.1, while those of CBP1 + ITP group were 0.2 ± 0.1, 0.2 ± 0.0, 0.6 ± 0.1, 0.7 ± 0.1, all were lower than those of ITP group (LSD-t = -6.1, -2.8, -3.1, -2.3, -8.9, -7.1, -4.9, -4.2; all P < 0.05). Conclusion Both AgB and CBP1 showed protective effect in mouse ITP, and their mechanisms are associated with the regulation of the TLR4/NF-κB/NLRP3 pathway.

Key words: Echinococcus granulosus, Immune thrombocytopenia, TLR4/NF-κB/NLRP3 pathway, Antigen B, Calcium binding protein 1