Abstract:

Objective To understand the effects of artesunate (ART) combined with recombinant human erythropoietin (rhEPO) on the expression of cerebral malaria-associated factors in the brain of Plasmodium berghei-infected mice and the underlying mechanism. Methods Forty female C57BL/6 mice were divided into 5 groups (n = 8 in each group). Four groups of mice were infected with P. berghei ANKA strain by intraperitoneal injection of 1 × 10⁶ P. berghei-infected mouse erythrocytes. Two to four days post infection, mice in infection control group were not treated; Mice in ART group were each treated orally with 40 mg/(kg·d)of ART in a total volume of 200 μl by garvage; Mice in rhEPO group were each treated with 50 U rhEPO by intravenous injection; Mice in ART+rhEPO group were each treated with both ART and rhEPO at above mentioned dosage and route. The last group of mice was given with PBS only as normal control. The treatment was performed for consecutive 3 days. The parasitemia in blood of each mouse was observed by Giemsa-stained thin blood smears and the mortality was monitored 4 days post infection. The sera were collected from eyeball blood 5 days post infection. The levels of IFN-γ, TNF-α and IL-10 in the serum of each mouse were measured by ELISA. Four mice in each group were sacrificed by cervical dislocation 5 days post infection and the brain samples were collected. The relative expression levels of NF-κB, granzyme B, IFN-γ, VCAM-1 and ICAM mRNA in the brains were detected by quantitative real-time PCR (qRT-PCR). The percentage and absolute numbers of CD4⁺ and CD8⁺ T cells in brain were counted by flow cytometry. Results In infection control group, mice exhibited neurological symptoms and began to die around 6 days post infection and all mice died 12 days post infection, while mice in ART group and rhEPO group began to die 8 and 10 days post infection, respectively, and all mice died 26 days post infection. However, the significant improvement of mortality in combination group of ART+rhEPO was observed with prolonged occurrence of death (12 day post infection) and survival rate of 50% at 26 days post infection. The blood smear results showed that the ART+rhEPO group had significantly lower parasitemia(3.14% on day 6 to 7.10% on day 12) than group treated with rhEPO alone (4.96%-15.50%) or group without treatment (4.98%-19.90%) during day 7 to 12 post-infection. ELISA results showed that the levels of pro-inflammatory cytokines IFN-γ and TNF-α in the sera of ART+rhEPO treated mice were (378.94 ± 145.18) pg/ml and (109.89 ± 27.05) pg/ml, respectively, which were significant lower than that in infection control group [(726.09 ± 35.40) pg/ml and (345.97 ± 42.70) pg/ml, P < 0.05 or P < 0.01, respectively]. The level of IFN-γ in the ART+rhEPO group was also significantly lower than that in ART group [(795.46 ± 64.48) pg/ml, P < 0.01]. Meanwhile, the anti-inflammatory cytokine IL-10 in ART+rhEPO group [(224.18 ± 22.93) pg/ml] was significantly higher than that in infection control group [(90.72 ± 6.30) pg/ml] and in ART group [(103.99 ± 18.04) pg/ml] (P < 0.01). The relative mRNA expression levels of NF-κB, granzyme B, VCAM-1, ICAM-1 and IFN-γ in the brains of ART+rhEPO group (0.89 ± 0.59, 17.23 ± 4.39, 1.63 ± 0.21, 1.48 ± 0.16, and 11.68 ± 2.63, respectively) were significantly lower than that in the mice of infection control group (3.62 ± 0.36, 88.54 ± 21.13, 2.65 ± 0.50, 3.13 ± 0.62, and 60.56 ± 4.19, respectively) (P < 0.05 or P < 0.01). The relative mRNA expression levels of NF-κB, granzyme B, ICAM-1 and IFN-γ were significantly lower than that in rhEPO group (2.00 ± 0.59, 58.55 ± 1.11, 2.68 ± 0.29, and 44.69 ± 1.17, respectively) (P < 0.05 or P < 0.01). At the same time, the percentage of CD4⁺ T and CD8⁺ T cells in the brains of ART+rhEPO group [(1.29 ± 0.06)% and(1.68 ± 0.28)%] was significantly lower than that in the mice of infection control group [(2.77 ± 0.26)%, and (5.30 ± 0.35)%, P < 0.01, respectively] and ART group [(2.04 ± 0.66)% and (3.65 ± 0.14)%, P < 0.01, respectively]. Conclusion Combined treatment of ART with rhEPO significantly inhibit the infection level of P. beighei and reduce the expression of cerebral malaria associated factors in mice. The mechanism underlying the inhibition is possibly related to the down-regulation of pro-inflammatory cytokines/chemokines and reduced filtration of CD4⁺/CD8⁺ T cells in the brain, and up-regulation of anti-inflammatory cytokine IL-10.

Key words: Cerebral malaria, Erythropoietin, Artesunate, Associated factors