Powerful solution for experimental cerebral malaria treatment: artesunate and tetramethylpyrazine

Background Cerebral malaria (CM) is a kind of serious neurological complication caused by the acute Plasmodium falciparum infection. About 300000 patients including children under 5 years old died from this disease every year. Even intravenous artesunate (Art) is employed as the most effictive drug in the treatment of CM, high incidence of death and neurological sequelae are still inevitable. Therefore, we assessed the combination of Art and tetramethylpyrazine (TMP), to treat experimental CM (ECM) in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). A non-biased whole brain quantitative proteomic analysis was also conducted to get some insight of the mechanism of the combinational treatment. Results Treatment of (ECM)-C57BL/6 mice with the combination of Art and TMP increased the survival, improved clinical signs and prevented neurological manifestations. These effects were related to reduction of parasitised red blood cells (pRBC) adhesion, sequestration, maintaining brain microvascular integrity, increasing nerve growth factor, neurotrophin levels, and alleviating hippocampal neuronal damage and astrocyte activation. The pharmacological effects of Art-TMP combination therapy were analyzed by ECM mice brain proteomic function enrichment. Based on an isobaric tag for relative and absolute quantitation (iTRAQ) fold-change of 1.2 (P-value < 0.05), 217 down-regulated and 177 up-regulated proteins were identified, presenting a significantly altered proteome profile of the combined Art and TMP group as compared to the group treated with Art or TMP alone. These results suggested that the Art-TMP combination could be used as a powerful solution for CM and its neurologic damage. Conclusions An effictive therapy for CM with low mortality rate and protect against ECM-induced neurocognitive impairment has been proposed through the combination of Art and TMP, which can provide an effective adjuvant treatment in the clinic. iTRAQ proteomics provide a resource for further mechanistic studies to examine the synergistic effects of Art and TMP and their potential to serve as an adjunctive treatment method and intervention targets. Author Summary Cerebral malaria (CM) is the most serious neurological complication caused by Plasmodium falciparum infection. Even after antimalarial treatment, severe neurological sequelae still exist. We used tetramethylpyrazine (TMP), the main ingredient of the traditional Chinese medicine Chuanxiong, and artesunate (Art) as a combination of drugs. We found that Art-TMP combination could improve the clinical symptoms of CM and protect the nervous system. At the same time, proteomics was used to analyze the protective mechanism of Art-TMP combination administration on ECM mice. This study suggests that the combination of Art and TMP may be used as an adjuvant therapy for clinical CM and iTRAQ proteomics provides resources for further study of Art-TMP combination and provides potential prognostic biomarkers for this therapeutic intervention.

injuries [9,10]. TMP has been demonstrated to increase cerebral blood flow, improve 112 microcirculation, inhibit the production of pro-inflammatory factors and protect 113 learning and memory functions [11,12]. Recently, clinical studies have reported that 114 TMP exerts beneficial effects on the nervous system that can promote functional 115 recovery from nerve injury. Adjunctive therapy is defined as an additional treatment 116 that modifies the pathological processes caused by malaria to improve its clinical  of ECM [17].  The open field test 175 We used the open field test to observe the spontaneous activity characteristics of 176 mice entering a new environment [19]. Here, we used normal mice of the same age as 9 177 control. Briefly, mice were placed in four drums of an empty field activity test box (an 178 area with a central radius of 7.5 cm in a barrel is considered as the central area). Mice 179 were placed in the empty field and spontaneous activities of the animals were 180 observed within 5 minutes and recorded by the software automatically. 182 Spontaneous alternation in the Y-maze was performed as previously described 183 [20]. A computer-controlled infrared camera system was installed directly above the 184 Y maze to track the location of mice. Animals were placed in a fixed position of the Y 185 maze in turn and allowed to explore freely for 8 minutes; normal mice were also used 186 as a control. The total number of entries into each arm was recorded during the 187 experiment.   201 Mice in four groups were imaged at d7 p.i. using the following protocol. Each 202 mouse was anesthetised with 2,2,2-tribromoethanol (0.32 mg/kg -1 ). Vessel integrity 203 and patency was scanned using TOF-2D-FLASH employing a magnetic resonance  The levels of cytokines including brain-derived neurotrophic factor (BDNF), 219 neurotrophic factor-3 (NT-3) and tumour necrosis factor (TNF-α) were determined in 220 the mice brain using commercial enzyme-linked immunosorbent assay (ELISA) kits    For simple western analysis by Wes TM , brain homogenate samples were prepared, 257 and protein concentration was determined using the bicinchoninic acid kit. The brain 258 homogenate samples were diluted to a final concentration of 1 μg/µL as required by 259 Wes TM . Capillary electrophoresis immunoassay was performed using Wes-specific    that resulted in death between d8 and 12 p.i. (Fig. 1A, B). In contrast, only 286 approximately 30% mice died in the Art group; deaths were observed from d10 p.i.

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The TMP treatment group did not improve the survival or reduce parasitemia and 288 death occurred on d9 to 12 p.i.. Art + TMP treatment significantly improved the 289 outcome in ECM mice; none of the mice in this group died and parasitaemia was 290 reported only in 10.12% mice on d12 p.i. In addition, malaria parasites could still be 291 observed in the Art group. A decrease in late trophozoites was observed in the blood 292 smears of the Art + TMP group as compared to the blood smears of the Infected group 293 and TMP alone treatment group on d7 p.i. (Fig. 1C).

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Art-TMP combination reduces clinical symptoms of ECM 296 We next utilized rapid murine coma and behaviour scale (RMCBS) to assess 297 ECM manifestations after drug therapy. We found that ECM mice developed a score 298 less than 4 points, consistent with their symptoms of reduced exploratory behaviour, 299 decreased reflex, self-preservation, and finally coma and epilepsy ( Fig. 2A). The score 300 in the Art-treated mice was significantly higher than that in the ECM mice in later 301 stages of infection. Particularly, mice in the Art + TMP group presented distinctly 302 higher RMCBS values as compared to ECM mice ( Fig. 2A). Results also showed 303 significant differences between the Art + TMP and the Art groups at 9 to 11 days p.i.

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The value was higher than 12 points in the Art + TMP group on d12 p.i. (Fig. 2A).

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ECM mice (15.68 g) and TMP-treated mice (14.82 g) showed significantly lower 306 weight on d7 p.i. as compared with the weight of mice in the Art (18.72 g) or Art + 307 TMP treatment group (18.10 g), even until on d12 p.i. (Fig. 2B). The weight of mice 308 in the Art + TMP group (18.05 g) was found to be higher than that of mice in the Art 15 309 group (17.63 g) from d8 p.i. to d12 p.i. (Fig. 2B). 310 There was no significant difference in the body temperature of mice between the 311 TMP group and the Infected group. However, on d9 p.i. and d11 p.i., the body  for Art + TMP), which is consistent with our assessment of mice status using 326 RMCBS. Notably, the results showed significant differences between mice in the Art 327 and Art + TMP groups (Fig. 3A). Similarly, the total movement time that mice in the  Art-TMP combination reduces cerebrovascular pathology and increases vessel 346 integrity and patency 347 We next checked the hypothesis that the Art-TMP combination had a more 348 profound effect than the Art and TMP alone on brain pathology. We found 349 significantly fewer adherent leukocytes in mice in the Art + TMP group than in the 350 untreated mice on d7 p.i. (Fig. 4A). In addition, MRI is considered as a valuable    Interestingly, these were significantly higher than those in the Art group (Fig. 6C, 6D, 392 p < 0.001). In addition, levels of pro-inflammatory factor TNF-α were also 393 significantly lower in mice in the Art and Art + TMP groups than those in mice in the 394 Infected group (Fig. 6E, p < 0.01).  Art + TMP groups can be found in the S3 Table . We then compared the brain 412 proteins that increased in mice in Art, TMP, and Art + TMP groups relative to those       Art-TMP combination therapy, demonstrating this pathway to be involved in the 690 molecular mechanism of action of drugs. Similarly, the PPI analysis revealed the 691 down-regulated proteins specific to the Art-TMP combination group to be centred on 692 the blood-brain transport, which plays important roles in the pathological 693 improvement of ECM. Together, the bioinformatics studies predicted that the 694 neuroprotective effects of the combined Art and TMP therapy may be associated with 695 axon development or blood-brain transport. 696 We identified two down-regulated proteins, hemopexin and Ngp and three 697 up-regulated proteins, namely Slit2, Tiam2 and Syntenin in the ECM mice brain 698 tissues treated with drugs. Our WB validation analyses showed that the protein levels  This may also explain why neurological signs improved in the Art-and TMP-treated 720 ECM mice. In addition to immune-related pathways, Slit-Robo signalling pathway 721 also governs axon growth and angiogenesis. Interestingly, Slit2 protein was also 722 up-regulated in the ECM mice brain after administration of Art and TMP, providing 723 further evidence for the mechanism of action of these drugs that involves axon growth 724 and angiogenesis.

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Intranasal administration offers several advantages including high bioavailability, 726 no liver first-pass effect, rapid absorption, and rapid onset of action of drugs that can 34 727 easily enter the cerebrospinal fluid in the CNS, thus specifically targeting the brain. In 728 ECM mice, a previous study reported the intranasal delivery of the anti-malarial drug 729 Art to be an efficient way to contribute to decreasing malaria-related mortality [51]. 730 Similarly, we treated ECM mice using Art or TMP or Art-TMP combination 731 intranasally and observed the same results as reported by the above study; however, 732 we also observed TMP to play a neuroprotective role in the ECM mice as a result of 733 the targeted delivery to the brain. proteome, such as axon growth, angiogenesis, and blood-brain transport. To the best 760 of our knowledge, present study is the first comprehensive study to describe the brain 761 proteomic alterations in ECM mice treated with Art, TMP and Art-TMP combination.

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This proteomic study not only provides the basis for further studies on mechanism of 763 action of drugs, but also assists in identifying potential biomarkers for monitoring