Malaria is still one of the most infectious diseases threatening the people’s health all over the world. ACTs, as the first-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. berghei-infected 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 significantly increased, and the serum concentrations of TP, ALB and GLOB significantly 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 significant, 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 significant 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 significant, closely related to the host immune mechanism against plasmodium . IFN-γ is mainly produced by activated T cells and NK cells, and activates erythrocytic specific cells and antibody-dependent cells to kill the malaria parasites in the erythrocytic stage [20–23]. 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 ; the epidemiological studies also suggest that there is a potential protective effect of TNF-α on malaria infection . 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 inflammatory 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 specific 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 reflect 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 significantly 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 significantly 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 significantly 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 influencing 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 confirmed 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 . Compared with the control group, the expression of GSK3β in P. berghei treated with brusatol decreased significantly (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 Ca2+-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 . 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 significantly (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 . 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 significantly (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 . After treatment with brusatol, the expression of P. berghei EMP1 in mice increased significantly (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 . After treatment with brusatol, the expression of P. berghei AMA-1 in mice increased significantly (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 significantly, 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 significantly, which may also be the target of brusatol against malaria, but their specific 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.