关键词: 疟疾, 地塞米松, 脾脏, 促炎症反应

Abstract:

Objective To explore the immunoprotective effect of dexamethasone (DEX) on experimental cerebral malaria (ECM) in mice. Methods C57BL/6 mice were randomly assigned into infection group, DEX treatment group and control group with 20 mice in each group. The mice in the infection group and DEX treatment group were injected intraperitoneally with 1 × 10⁶ Plasmodium berghei ANKA-infected red blood cells, while mice in the control group did not receive any treatment. The mice in the DEX treatment group were treated with intraperitoneal dexamethasone sodium phosphate (80 mg/kg) on the 1st and 2nd days post-infection, while the control and infection groups were injected with an equal volume of normal saline. From the 3rd day on post-infection, the tail vein blood of infected mice was collected to prepare blood smears, and the infected erythrocytes were counted after Giemsa staining to observe the changes of parasitemia. The survival rate and ECM-related neurological symptoms of the mice were recorded daily. On the 3rd and 5th days post-infection, mice spleen cell suspensions were prepared, and flow cytometry was used to detect spleen Th1 cells, regulatory T cells (Tregs), the expression level of programmed death receptor-1 (PD-1), toxic T lymphocyte-associated antigen-4 (CTLA-4) on CD4⁺ T cells, M1 and M2 macrophages. The spleen cell suspensions were cultured for 48 hours, and the culture supernatant was tested by ELISA for secretion pro-inflammatory/anti-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10. On the 6th day post-infection, Evans blue (EB) staining was used to evaluate the effect of DEX treatment on the blood-brain barrier in mice. One-way ANOVA test was used to compare the results from flow cytometry and cytokine detection, independent sample t-test was used to compare the mice parasitemia, and Log-Rank test was used to compare survival rate. Results Plasmodium-infected red blood cells began to appear in the peripheral blood of mice in the infection group and DEX treatment group from the 3rd day post-infection, and the parasitemia was (25.89 ± 1.97)% and (22.91 ± 3.21)% on the 9th day post-infection, respectively. The difference was not statistically significant (t = 1.12, P > 0.05). Mice in the infection group began to die on the 6th day post-infection, accompanied by ECM neurological symptoms, and all died by the 9th day post-infection. Mice in the DEX treatment group began to die on the 8th day post-infection, accompanied by ECM neurological symptoms, and all died by the 10th day post-infection. Compared to the infection group, DEX treatment delayed the onset of cerebral malaria (χ² = 4.31, P < 0.05). On the 3rd and 5th days post-infection, the proportion of spleen CD4⁺ T cells expressing PD-1 in the DEX treatment group was (18.10 ± 0.52)% and (39.13 ± 0.91)%, respectively, higher than the control group [(11.53 ± 1.00)% and (13.72 ± 1.78)%] and the infection group [(14.87 ± 1.02)% and (35.87 ± 0.74)%]. The differences were statistically significant (F = 41.41, 378.00; P < 0.05, 0.01). The proportion of spleen CD4⁺ T cells expressing CTLA-4 were (3.05 ± 0.12)% and (8.70 ± 1.01)%, respectively, higher than the control group [(0.28 ± 0.02)% and (0.45 ± 0.10)%] and the infection group [(0.44 ± 0.13)% and (0.68 ± 0.13)%]. The differences were statistically significant (F = 730.20, 191.80; P < 0.01). On the 3rd and 5th days post-infection, the proportion of Th1 in the spleen cells of the mice in the DEX treatment group was (0.14 ± 0.04)% and (0.63 ± 0.06)%, respectively, which were lower than the infection group [(0.30 ± 0.06)% and (0.80 ± 0.04)%]. The differences were statistically significant (F = 10.93, 56.58; P < 0.05, 0.01). On the 5th day post-infection, the proportion of Tregs cells in the spleen cells of the DEX treatment mice was (1.30 ± 0.08)%, which was higher than the control group (1.12 ± 0.08)% and the infection group(0.86 ± 0.03)% (F = 36.51, P < 0.01). On the 3rd day post-infection, the proportion of M1 macrophages in the spleen cells of the mice in the DEX treatment group was (0.43 ± 0.12)%, which was lower than the infection group (1.03 ± 0.14)% (F = 29.81, P < 0.01). On the 5th day post-infection, the proportion of M2 macrophages in the spleen cells of the DEX treatment mice was (1.56 ± 0.20)%, which was higher than the control group (0.49 ± 0.04)% and the infection group (0.87 ± 0.06)% (F = 60.21, P < 0.01). On the 3rd and 5th days post-infection, the levels of TNF-α in the culture supernatant of spleen cells in the DEX treatment group were (140.18 ± 27.38) pg/ml and (328.23 ± 75.70) pg/ml, which were lower than those in the infection group [(247.34 ± 35.34) pg/ml and (650.88 ± 43.56) pg/ml] (F = 11.70, 32.09; P < 0.01). IL-6 levels were (40.03 ± 9.09) pg/ml and (1 270.52 ± 83.75) pg/ml, which were lower than the infection group (511.97 ± 41.70) pg/ml and the control group (4 131.31 ± 645.16) pg/ml. The difference was statistically significant (F = 299.40, 93.11; P < 0.01). The level of IL-10 was (283.06 ± 44.83) pg/ml and (592.63 ± 51.69) pg/ml, higher than (118.82 ± 8.47) pg/ml and (424.49 ± 51.44) pg/ml in the infection group (F = 36.04, 73.95; P < 0.01). On the 6th day post-infection, the blood-brain barrier of the mice in the infection group was dramatically damaged, and EB penetrated into the brain tissue, showing a dark blue stain. The blood-brain barrier of the mice in the control group was intact, and there was no obvious infiltration of EB. The mice in the DEX treatment group only had a small amount of EB infiltrated into the brain tissue. Conclusion In ECM mice, DEX regulates the activation and response intensity of Th1 and Tregs cells through enhancing the expression levels of immunosuppressive molecules PD-1 and CTLA-4 on the surface of CD4⁺ T cells, promotes the polarization of M1 to M2 macrophages, and inhibits the secretion levels of pro-inflammatory factors, alleviates blood-brain barrier damage in ECM mice, thereby reducing the pathogenicity of ECM.

Key words: Malaria, Dexamethasone, Spleen, Proinflammatory response