Antimalarial Activity of Brusatol Against Plasmodium Berghei Infected Mice and the Mechanism Revealed by Whole Transcriptome Sequencing (RNA-Seq) Analysis


 BackgroundRecently, artemsinin-resistant malaria strains and clinical cases have appeared in Southeast Asia. Reportedly, there are malaria mutants in Africa that are resistant to artemisinin and its derivatives. Thus, it’s imminent to develop new antimalarial drugs. Brucea javanica is an effective antimalarial drug recorded in Chinese traditional medicine, which has been widely used in the folk for hundreds of years. Brusatol is the main active constituent of Brucea Javanica, thus we studied the effects of brusatol on prevention of malaria infection in vivo. MethodsTo determine the antiplasmodial activity of brusatol, a four-day suppressive test was used by dividing 56 mice into 7 groups of 8 mice each and given 4mg/kg, 3mg/kg, 2mg/kg, 1mg/kg, 0.5mg/kg of brusatol, the standard drug ((artesunate of 140 mg/kg) and the vehicle (normal saline). The best effective dose was used in the following test. The effects of brusatol to plasmodium berghei transcription were tested through RNA-seq and the results were confirmed by RT-qPCR. We also explored the expression of TNF -α, IFN-γ, IL-4, IL-12 to evaluate antimalarial mechanism of brusatol to host by ELISA.ResultsThe results showed that brusatol effectively inhibited plasmodium berghei infection, the best effective dose was 2mg/kg, and the side effects of brusatol to liver and kidney were slight and reversible. The expressions of GSK3β, ATP6A, ATP6B, ATP6M, MSP-2, EMP1, CTCS in plasmodium were significantly lower after brusatol treatment compared with control, while the expression of AMA-1 was significantly increased. The serum concentrations of IFN-γ, TNF-α and IL-4 in artesunate and brusatol group decreased significantly compared with the control group, while there was no statistical difference of the serum concentrations of IL-12.ConclusionsTaken together, these results demonstrated brusatol could be a priority candidate for antimalarial medicine development.


Introduction
Malaria remains a highly fatal infectious disease that seriously threatens human beings' health.
According to the World Health Organization (WHO) report, an estimate of 229 million malaria cases have been reported, including 409,000 deaths worldwide in 2019. Most cases were reported in the aeras of sub-Saharan Africa, accounting for about 93.4% of total malaria cases [1]. Artemisinin-based combination The fragments per kilobase of transcript per million mapped reads (FPKM) method were used to calculate the mRNA levels of genes. Noiseq method (probability ≥ 0.8 & log2 fold-change > 1) was used to screen DEGs. All these DEGs were mapped to the database of KEGG and GO for pathway and GO enrichment analysis.
RT-qPCR validation 8 candidate genes were randomly selected for RT-qPCR validation. Total RNA was extracted from the P. berghei-infected blood samples using RNAiso Plus (Takara, Japan) and reverse transcribed into cDNA using PrimeScript RT reagent kit (Takara, Japan). Quantitative real-time RT PCR (RT-qPCR) was performed with an Mx3000P QPCR system (Agilent Technologies, USA) using SYBR Premix DimerEraser (Takara, Japan). Seryl was selected as housekeeping genes to determine their transcription stability. PCR cycling parameters (30 cycles) were set as denaturation (95℃, 30 s), annealing (59℃, 30 s), and extension (72℃, 1 min). The primer sequences used for qPCR are presented in Table.1. The data are expressed as 2 −ΔΔCt .
Data were expressed as mean ± S.E.M. All statistical analyses were done by GraphPad Prism (version 8.0; GraphPad Software). The difference in mRNA expression level between the control and brusatol group were conducted by using the unpaired T-test. p < 0.05 was considered as statistically signi cant. Table 1 Primer sequences used for RT-qPCR analysis.

Results
2 mg/kg brusatol effectively inhibit the growth of P. berghei Compared to the control, brusatol reduced parasitemia signi cantly. 2 mg/kg was the most ideal therapeutic dose of brusatol to treat the P. berghei infected mice. At 2 mg/kg dose, the antimalarial effect of brusatol on P. berghei was similar with 140 mg/kg artesunate at the concentration of parasitemia lower than 5% (Fig. 1A, B). Then, we tested the effect of brusatol on malaria anemia, and found that brusatol (2mg / kg) can improve the anemia caused by malaria as well as artesunate.
The hepatic and renal damage caused by brusatol is reversible On Day4, compared with the control and artesunate group, the AST, ALT, CREA and UREA in serum were signi cantly increased in the brusatol group, and the TP, ALB and GLOB were signi cantly decreased in the brusatol group. On Day28 after treatment, there was no statistical difference of these markers between brusatol group and control group or artesunate group (Fig. 2).
On Day4, brusatol treatment in uenced the liver and kidney function of mice, however, when the drug treatment was stopped for 25 days (from Day4 to Day28), all serum concentrations of markers revered to normal levels, showing the abnormal of liver and kidney function caused by brusatol was reversible after stopping the injections of brusatol.
Brusatol regulated immune cytokines to inhibit the P. berghei infection After infection, P. berghei stimulated the host immune system, causing the serum IFN-γ, TNF-α, IL-4 and IL-12 signi cantly increased in P. berghei-infected mice. Compared with the control group, the serum concentrations of IFN-γ, TNF-α and IL-4 in artesunate and brusatol group decreased signi cantly, while there was no statistical difference of the serum concentrations of IL-12. (Fig. 3) The results showed that brusatol could play function against P. berghei by regulating the expression of host IFN-γ, TNF-α and IL-4, whose e ciency was almost equal to artesunate.

RNA-seq analysis and validation of DEGs
Our study identi ed 812 unigenes as DEGs between the control and brusatol group, including 388 upregulated and 424 down-regulated genes (BioProject ID PRJNA699138, Fig. 4A). The results were further validated using the Morpheus online tool, and the DEGs are presented in a hierarchical clustering heat map (Fig. 4B, C). GO and KEGG databases were used to identify the function of DEGs. We found that brusatol had a wide range of effects on various biological pathways of P. berghei, and the distribution of DEGs in 'Biological Process', 'Cellular Component' and 'Molecular Function' was as shown in the gure (Fig. 4B). The majority of the DEGs were demonstrated to be signi cantly enriched in BPs, CCs, MFs, including the 'cellular process' and 'metabolic process' in BPs, the 'membrane' and 'membrane part' in CCs,and the 'catalytic activity' and 'binding' in MFs. KEGG pathway analysis demonstrated that the DEGs were enriched in 'Transport and catabolism', 'cell growth and death', 'signal transduction', 'folding, sorting and degradation', 'energy metabolism' and 'nucleotide metabolism' (Fig. 4C).
8 DEGs (1 up-and 7 down-regulated DEGs), ATP6A, ATP6B, ATP6M, MSP-2, AMA-1, GSK3β, EMP1, CTCS were selected for RT-qPCR analysis. The expression of these eight genes was demonstrated to be signi cantly lower after brusatol treatment compared with control except the expression of AMA-1 (all P < 0.01; Fig. 5A). Consistent with the results of the database analysis, mRNA expression of them was signi cantly lower in brusatol groups compared with control groups (7 of 8 P < 0.05; Fig. 5B), while the expression of AMA-1 was signi cantly increased compared with control groups (P < 0.05; Fig. 5B).

Discussion
Malaria is still one of the most infectious diseases threatening the people's health all over the world. ACTs, as the rst-line antimalarial drug, has shown a resistant trend in the treatment effect of malaria in Southeast Asia recently. The fundamental cause is the emergence of both artemisinin and the combined drug-piperaquine resistance. The thorough solution to the resistant plasmodium strains is to develop new antimalarial drugs. Brucea Javanica is an effective antimalarial herb medicine recorded in traditional Chinese medicine, and the brusatol also exhibited antimalarial effect.
We studied the antimalarial effect of different concentrations of brusatol in parasitemia of P. bergheiinfected BALB/c mice and compared it with artesunate (140 mg/kg). We found that 2 mg /kg of brusatol was the lowest concentration to achieve the ideal therapeutic effect. At this dose, brusatol treatment could control the parasitemia under 5%, which had the same antimalarial effect as artesunate of 140 mg/kg. It is proved that brusatol can play an effective role in the treatment of P. berghei at the dose of 2 mg/kg.
To explore the toxic effects of brusatol on the liver and kidney function in mice, we simulated the treatment process but without P. berghei infection, and then measured the serum concentrations of AST, ALT, TP, ALB, GLOB, CREA and UREA on Day4 and Day28 respectively. AST, ALT, TP, ALB and GLOB are serum markers of liver function; UREA and CREA are serum markers of renal function. On Day4, compared with the control and artesunate group, in the brusatol group, the serum concentrations of AST, ALT, CREA and UREA signi cantly increased, and the serum concentrations of TP, ALB and GLOB signi cantly decreased, which indicated that the liver and kidney functions of brusatol group mice were abnormal. Compared with the artesunate group, the concentration changes of serum markers in brusatol group were more signi cant, which demonstrated that the effect of brusatol on liver and kidney function of mice was more than that of artesunate. To further observation that whether the liver and kidney damage caused by brusatol was reversible, we measured the serum markers above on Day28 after conventional brusatol treatment again. Compared with the control group, the levels of all serum markers returned to normal degree, indicating when the drug has been stopped for a period of time (on Day28), the liver and kidney function can return to normal condition. The damage of liver and kidney function caused by brusatol is slight and reversible.
There are signi cant changes in a variety of cytokines in mice infected with malaria, in which the increase of TNF-α, IFN-γ, IL-4 and IL-12 are signi cant, closely related to the host immune mechanism against plasmodium [19]. IFN-γ is mainly produced by activated T cells and NK cells, and activates erythrocytic speci c cells and antibody-dependent cells to kill the malaria parasites in the erythrocytic stage [20][21][22][23]. TNF-α is released by monocytes and macrophages. The level of TNF-α in cerebral malaria resistant mice infected with P. berghei is higher than that in susceptible mice [24]; the epidemiological studies also suggest that there is a potential protective effect of TNF-α on malaria infection [25]. IL-4 is mainly produced by Th2 cells, activated basophils and mast cells. By inhibiting the activity of monocytes and macrophages, IL-4 weakens their killing effects on the malaria parasites in the erythrocytic stage, and its protection mechanism is also related to its inhibition or down-regulation of in ammatory cytokines secretion [24,26]. IL-12 is produced by activated macrophages, B cells, DC cells, etc. It can help the host to eliminate the malaria parasites by enhancing the killing activity of NK cells and promoting the production of cytotoxic CD8 + T cells. It also plays a speci c role in eliminating the malaria parasites by regulating the production of IFN-γ and TNF-α by T cells and NK cells to induce Th1 cell immunity [27,28]. It is suggested that TNF-α, IFN-γ, IL-4, and IL-12, the four cytokines can re ect the severity of hosts' malaria, so we studied the serum changes of these cytokines after treatment with brusatol. It was found that the concentrations of TNF-α, IFN-γ, IL-4 and IL-12 in the serum of mice infected with P. berghei were signi cantly increased, which were well agreed with that reported in the literature. However, the concentrations of TNF-α, IFN-γ and IL-4 in the serum of mice treated with brusatol were signi cantly decreased (P < 0.01), while there was no statistical difference of the serum concentrations of IL-12. It is suggested that brusatol can exert its antimalarial activity by regulating the expression of TNF-α, IFN-γ, IL-4, and affecting the functions of host T cells, NK cells, monocytes, macrophages and erythrocytes.
To study the antimalarial mechanism of brusatol, we screened 812 DEGs, including 388 up-regulated genes and 424 down-regulated genes, by high throughput screening (HTS) of P. berghei in blood samples of mice in the control group and brusatol group. According to Go analysis, DEGs were demonstrated to be signi cantly enriched in "cellular process", "metabolic process", "membrane", "catalytic activity" and "binding"; KEGG pathway analyses show that DEGs were demonstrated to be associated with "Transport and catabolism", "cell growth and death", "signal transduction", "folding, sorting and degradation", "energy metabolism" and "nucleotide metabolism". The results showed that brusatol could help the host eradicating the malaria parasites by in uencing the metabolism process, the function of the cell membrane and receptor, the catalytic process, and so on. Among these DEGs, we screened several DEGs closely related to the growth, metabolism and invasion of P. berghei: ATP6A, ATP6B, ATP6M, MSP-2, AMA-1, GSK3β, EMP1, CTCS. The results of the RT-qPCR analysis were consistent with the result of HTS.
Protein kinases related to growth and metabolism of P. berghei: Glycogen synthase kinase 3β (GSK3β) is a Ser/ Thr protein kinase commonly distributed in eukaryotic cells, activated by tyrosine phosphorylation and inhibited by serine phosphorylation. Plasmodium falciparum Glycogen synthase kinase 3 (PfGSK3) was con rmed to be necessary for the growth of P. falciparum, so it is considered to be an important target of new anti-malarial drugs. Recently, sensitive PfGSK3 inhibitors were considered as potential new anti-malarial drugs [29]. Compared with the control group, the expression of GSK3β in P. berghei treated with brusatol decreased signi cantly (P < 0.01), indicating that GSK3β is an important target of brusatol in anti-malarial treatment. Plasmodium falciparum calcium ATPase 6 (PfATP6) is a kind of Sarco/endoplasmic reticulum Ca 2+ -ATPase. It regulates the intracellular calcium concentration of P. falciparum by consuming ATP, thus maintaining the stability of calcium concentration in the malaria parasites. PfATP6 was known as one of the effective targets of artesunate against malaria [30]. Artesunate and its derivatives inhibit PfATP6, which leads to an increase of intracellular calcium concentration and plays a role in eradicating P. falciparum. The malaria Parasites also showed drug resistance through PfATP6 gene mutation [31,32]. After treatment with brusatol, the expression of several typical ATP6 protein kinases: ATP6A,ATP6B, ATP6M in P. berghei decreased signi cantly (P < 0.01), indicating that brusatol can kill the malaria parasites by inhibiting the expression of ATP6, and it may be a new anti-malarial drug to solve artesunate resistance.
3 proteins among them, MSP-2, EMP1 and AMA-1, are closely related to the invasion and immune escape of P. berghei. Glycosylphosphatidylinositol anchored protein (GPI-AP) MSP-2, is the second abundant protein on the merozoite surface of P. falciparum. It may participate in the adhesion process of the malaria parasites to host red blood cells and play an important role in its invasion to red blood cells [33].
MSP-2 is a potential target for anti-malaria vaccines or drugs. The candidate vaccine based on MSP-2 had an obvious anti-malaria effect on the invasion of P. falciparum into red blood cells [34,35]. After treatment with brusatol, the expression of MSP-2 of P. berghei in mice decreased signi cantly (P < 0.01), indicating that brusatol can inhibit the invasion of P. berghei into the host red blood cells by reducing the expression of MSP-2. PfEMP1 is a variable antigen expressed by P. falciparum, which exists on the surface of infected host red blood cells and mediates the combination of infected red blood cells and vascular endothelial cells so that the malaria parasites can avoid spleen clearance [36,37]. Also, PfEMP1 regulated the host's immune response by binding the CD36 receptor on antigen-presenting cells, while inhibited the production of IFN-γ in human peripheral blood mononuclear cells (PBMCs) in the early stage of P. falciparum infection [38]. After treatment with brusatol, the expression of P. berghei EMP1 in mice increased signi cantly (P < 0.01), which indicated that brusatol could help host immune system recognize malaria parasites by reducing the expression of EMP1, thus promoting host clearance of malaria parasites. The Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) is synthesized in the erythrocytic stage of P. falciparum. In recent years, studies have reported AMA-1 of Plasmodium species featured functional conservation, providing the theories foundation for the development of cross-species inhibitors against malaria [39]. After treatment with brusatol, the expression of P. berghei AMA-1 in mice increased signi cantly (P < 0.01), which indicated that brusatol could promote the recognition and immune clearance of the host to the malaria parasites by increasing the expression of the parasite AMA-1. After treatment with brusatol, the expression of CTCS of P. berghei also decreased signi cantly, while the results of RT-qPCR demonstrated that its expression was very low, sometimes even could not be detected. It is speculated that this protein is an important target of brusatol against malaria. However, there are few studies on CTCS, and the role of CTCS in the parasites' growth, development, or invasion is still unclear, which needs further study.
Besides, after treatment with brusatol, the expression of many genes in P. berghei increased or decreased signi cantly, which may also be the target of brusatol against malaria, but their speci c mechanisms are still unknown. Further studies will be needed. As more and more malaria parasites show resistance to artesunate and its derivatives, protein kinases that regulate parasites growth and differentiation have become new targets of antimalarial drug development. Brusatol is a new and effective antimalarial drug, which acts on GSK3β, ATP6A, ATP6B, ATP6M, MSP-2, EMP1, AMA-1, CTCS and many other proteins.

Conclusion
This study showed that the 2mg/kg brusatol was effective on the inhibition of the growth of parasitemia in P. berghei-infected mice and there was little but reversible effect on host liver and kidney function.

Consent for publication
No applicable Availability of data and materials The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.

Competing interests
The authors declare that they have no competing interests.  Figure 1 Effect of different doses of brusatol and artesunate on parasitemia in P. berghei-infected mice. A: The percentage of plasmodium infected red blood cells after different treatments. B: Observation of plasmodium infected red blood cells after different treatments under microscope (a: control, parasitemia was near 17%; b: artesunate of 140 mg/kg, parasitemia was near 2%; c: brusatol of 0.5 mg/kg, parasitemia was near 6%; d: brusatol of 2 mg/kg, parasitemia was near 1%). The red arrows refer to the schizont stage; the yellow arrows refer to the trophozoite stage; the blue arrows refer to the ring stage; the green arrows refer to normal red blood cells. C: The HGB and RBC statistic of normal, control, 140 mg/kg artesunate and 2 mg/kg brusatol groups. Asterisks (*) indicate signi cant differences between groups (*P<0.05, **P<0.01, ***P<0.001 vs. adjacent). NS indicates no statistical differences between groups.