Inhibition of hemozoin-induced neuroin ammation by medicinal plants used for the treatment of malaria in Southwest Nigeria: implications for cerebral malaria


 This study aimed to investigate some antimalarial plants for effects on neuroinflammation. Freeze-dried infusions of the plants were investigated for effects on hemozoin-induced neuroinflammation in BV-2 microglia and levels of inflammatory mediators measured. Alstonia boonei (stem bark), Anacardium occidentale (stem bark), Azadiractha indica (leaves), Enantia chlorantha (stem bark), Khaya senegalensis (stem bark), Mangifera indica (stem bark), and Nauclea latifolia (stem bark) produced significant (p<0.05) reduction in TNFα, IL-6, IL-1β, MCP-1, RANTES and iNOS/NO production in BV-2 microglia stimulated with a synthetic hemozoin (400 µg/mL). Further experiments showed that pre-treatment with 50 µg/mL of A. boonei, A. indica, A. occidentale, E. chlorantha and M. indica prior to hemozoin stimulation resulted in inhibition of NF-κB activation by >40%, while E. chlorantha, K. senegalensis and N. latifolia produced weak activities. Pretreatment with A. indica (50 µg/mL) produced the highest inhibition (58.6%) of hemozoin-induced increased NLRP3 protein expression, while A. occidentale (50 µg/mL), M. indica (50 µg/mL) and A. boonei (50 µg/mL) reduced expression by 54.1%, 49.2% and 47.1%, respectively. Hemozoin-induced increased caspase-1 activity was reduced by A. boonei, A. occidentale, A. indica, E. chlorantha, and M. indica. These results suggest that A. boonei, A. occidentale, A. indica and M. indica produced strong inhibition of hemozoin-induced neuroinflammation through mechanisms involving NFκB and NLRP3 inflammasome activation, while moderate activities were produced by E. chlorantha, K. senegalensis and N. latifolia. The outcome of the study provides pharmacological evidence for the potential benefits of the plants as herbal treatments of cerebral malaria symptoms.


Introduction
According to the World Health Organisation (WHO), malaria was responsible for approximately 409,000 deaths in 2019, with Africa accounting for more than 94% of global fatalities (WHO 2020). The most severe neurological complication of acute Plasmodium falciparum malaria is cerebral malaria (CM), a condition that is responsible for the majority of deaths in African children. In spite of effective antimalarial treatments and intensive care, cerebral malaria causes 15-20% mortality, and survivors may develop long-term neurological deficits (Carter et al. 2005;Boivin et al. 2007;John et al. 2008). Consequently, adjunctive treatments are needed to reduce the mortality of this condition.
Several investigations have now established that in malaria, including CM, there is formation of a metabolic product hemozoin as a result of infection of erythrocytes by Plasmodium falciparum. Hemozoin is known to induce inflammation and morphological changes in microvascular endothelium (Medana and Turner 2006;Prato et al. 2011), leading to an increase in blood brain barrier (BBB) permeability, neuroinflammation and neurological sequelae in survivors (Basilico et al. 2003;Tripathi et al. 2009). Similar to these reports, our investigations have revealed that a synthetic form of hemozoin is in fact able to induce neuroinflammation in BV-2 microglia (Velagapudi et al. 2019). Consequently, reducing the CNS effects of hemozoin in CM is a potential adjunctive strategy in reducing the neurological outcomes of the disease.
Plants have a long history of use Traditional African Medicine (TAM) for treating malaria and its complications such as CM. Some of these plants are potential interventions in treating CM due to their ability to reduce processes involved in the activation of neuroinflammation in the brain. For instance, Terminalia albida has been shown to improve survival in experimental CM through its anti-inflammatory action (Camara et al. 2019). Similarly, an extract of Azadiractha indica was shown to reduce neuroinflammation and severity of brain oedema in an experimental model of CM (Bedri et al. 2013). Recently, we reported that an extract of Zanthoxylum zanthoxyloides attenuated neuroinflammation induced with synthetic hemozoin in BV-2 microglia (Ogunrinade et al. 2021). These reports are encouraging developments, and suggest that further pharmacological evaluation of plants used in TAM for treating malaria and/or CM is warranted.
In this study, we conducted a survey of plants used in the treatment of malaria and/or CM in southwest Nigeria and evaluated freeze-dried infusions of plants with the highest fidelity levels for effects on the increased production of pro-inflammatory mediators in BV-2 microglia stimulated with a synthetic form of hemozoin (HZ).

Materials
Synthetic hemozoin was purchased from InvivoGen (France) and prepared fresh in sterile distilled water.

Collection of plant samples
Field surveys were conducted in Agbegi village, Ikire (Southwest Nigeria), Ile-Igbon (Southwest Nigeria), Beere/Oje area (Ibadan, Southwest Nigeria), Ilesa (Southwest Nigeria) and Ile-Ife (Southwest Nigeria). Traditional medicine practitioners in these communities were approached and informed of the purpose of the study, including research objectives, methods of data collection, and intention to publish data.
Thereafter, semi-structured interviews were conducted to gather information on the use, preparation, application and properties of herbs used for treating symptoms of malaria and/or cerebral malaria were obtained from each traditional medicine practitioner. Healers were asked specifically for herbal preparations used in treating the usual symptoms of malaria (fever, aches, pains), in addition to cerebral malaria specific symptoms such as coma and seizures.
The Fidelity Level (FL) which is the ratio between the number of informants who independently suggested the use of a plant for malaria/cerebral malaria and the total number of informants who mentioned the plant for any use was calculated using the formula: FL = × 100 Np: is the number of informants that report a use of a plant species to treat malaria and/or cerebral malaria N: the number of informants that used the plants as a medicine to treat any given disease (Friedman et al. 1986).
Plants with FL>50% for the treatment of malaria/cerebral malaria symptoms in these surveys were collected and samples authenticated by a botanist in the Herbarium of Forestry Research Institute of Nigeria, Ibadan. Voucher specimens were prepared and numbers assigned. measurements were carried out according to the manufacturer's instructions and absorbance measured at 450nm in a Tecan Infinite M Nano microplate reader.

Nitrite production
BV-2 microglia were seeded out in a 24-well plate at 5 x 10 5 cells/ml and treated with 25 and 50 µg/ml of plant infusions for 30 min, followed by stimulation with HZ (400 µg/ml) for 24 h. At the end of the stimulation period, culture supernatants were collected, and levels of NO were determined using the Griess assay kit (Promega).
BV-2 cells were seeded in 96-well plates and pre-treated with 25 and 50 µg/ml of plant infusions 30 min prior to activation with HZ (400 μg/ml) for a further 24 h.
Activity of caspase-1 in cells was thereafter measured according to the manufacturer's instructions. Luminescence was read with FLUOstar OPTIM reader (BMG LABTECH).

In cell western assays for iNOS and NLRP3 proteins
BV-2 microglia were seeded into 96-well plates and treated with 25 and 50 µg/ml of plant infusions and incubated for 30 min prior to stimulation with HZ (400 μg/ml) for a further 24 h. At the end of each experiment, cells were fixed with 8% paraformaldehyde solution (100 µL) for 15 min., followed by washing with PBS. The cells were then incubated with rabbit anti-iNOS (Abcam) and rabbit anti-NLRP3 (Abcam) antibodies overnight at 4°C. Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature.
Then, 100 µL avidin HRP substrate was added to each well and absorbance measured at 450nm with a Tecan Infinite M Nano microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).

NF-B transcription factor assay
Effects of plant infusions on DNA binding of NF-B following stimulation by hemozoin was quantitatively evaluated using the NF-B transcription factor assay kit (Abcam).
Following treatment of BV-2 microglia with 25 and 50 µg/ml of plant infusions for 30 min, the cells were stimulated with HZ (400 μg/ml) for 60 min. This was followed by preparation of nuclear extracts, which were then used in DNA binding assays according to the manufacturer's instructions. Absorbance was measured at 450nm with a Tecan Infinite M Nano microplate reader.

Phospho-p65 NF-B and phospho-IB ELISAs
BV-2 microglia cells were seeded out into 6-well plates and treated with 25 and 50 µg/ml of freeze-dried plant infusions and incubated for 30 min prior to stimulation with HZ (400 μg/ml) for a further 60 min. At the end of the experiments, plates were washed with PBS, followed by the addition of cell lysis buffer (Cell Signalling Technology). The plate was then incubated on ice for 5 min and cells scraped.
Extracts were sonicated and centrifuged at 14,000 × g at 4C for 10 min. Protein levels of phospho-p65 in the resulting cell lysates were measured using PathScan ® phospho-NF-B p65 (Ser536) sandwich ELISA kit. Absorbance (protein expression) was measured at 450nm with a Tecan Infinite M Nano microplate reader.

Statistical analysis
Data are expressed as mean ± SEM for at least 3 independent experiments (n=3) and analysed using one-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparison test. Statistical analysis were conducted using the GraphPad Prism software.

Ethnopharmacological field surveys
Ethnopharmacological field surveys conducted in 5 locations within southwest Nigeria revealed that 12 of the plant species mentioned by traditional medicine practitioners had >50% fidelity level for treating symptoms of malaria and cerebral

Anti-inflammatory effects of freeze-dried plants infusions were mediated through mechanisms involving NF-B activation
Encouraged by results showing varying degrees of anti-inflammatory activity by the plants, we then investigated their effects on HZ-induced activation of the NF-B transcription factor. Interestingly, we observed that incubating BV-2 microglia with 50 µg/mL of A. boonei, A. indica, A. occidentale, E. chlorantha and M. indica prior to stimulation with HZ (400 µg/mL) produced >40% reduction in the levels of phospho-p65 protein measured using ELISA ( Figure 4A).
We further used a transcription factor assay to evaluate the impact of pre-treatment with freeze-dried plants on nuclear DNA binding of NF-B following stimulation of BV-2 microglia with HZ (400 µg/mL). Results in Figure 4B shows that 50 µg/mL of freeze-dried A. boonei, A. occidentale, A. indica, M. indica and N. latifolia inhibited HZ-induced increased DNA binding by 50.5%, 48.8%, 53.4%, 49.6%, and 43.4%, respectively ( Figure 4B).
Following results showing inhibition of HZ-induced increased IL-1β production by freeze-dried plants, we investigated their effects of protein levels of NLRP3 inflammasome, as well as caspase-1 activity in HZ-stimulated BV-2 microglia. Figure 5A show that increased protein expression of NLRP3 inflammasome following stimulation with HZ (400 µg/mL) was significantly reduced Vernonia amygdalina (leaves) were reported to score an FL greater than 50%.
In preliminary pharmacological investigations on freeze-dried infusions of the plants,
The effects of these plants in reducing the production of pro-inflammatory cytokines and chemokines is remarkable considering the roles of these mediators in the pathogenesis of cerebral malaria. Increased levels of TNF, IL-6 and IL-1β have been reported to be associated with the pathogenesis of cerebral malaria (Idro et al. 2010). Also, studies have shown that chemokines such as MCP-1 and RANTES/CCL5 contribute to the pathogenesis of this malaria complication (Pacher et al. 2007). Furthermore, we have shown that a synthetic form of the malaria metabolic product hemozoin increased pro-inflammatory cytokine production in BV-2 microglia (Velagapudi et al. 2019), confirming their roles in CNS immune responses to malaria infection.
Excessive production of nitric oxide (NO) by brain microglia during neuroinflammation has been linked to damage to adjacent neurons. In cerebral malaria, iNOS-mediated NO production has been postulated to be cytotoxic to neurons through different mechanisms (Pacher et al. 2007). Results from this study show strong inhibition of iNOS-mediated increased production of NO by freeze-dried infusions of Alstonia boonei, Anacardium occidentale, Azadiractha indica, and Mangifera indica in hemozoin-stimulated BV-2 microglia, while Enantia chlorantha, Khaya senegalensis and Nauclea latifolia produced modest effects. These results further demonstrate the potential benefits of these plants as herbal remedies in the treatment of cerebral malaria, at least due to their ability to reduce inflammatory responses in the brain. Previous studies have reported anti-inflammatory effects for Activation of the NF-B is known to regulate the production of pro-inflammatory cytokines, chemokines and mediators such nitric oxide. Activation of this transcription factor has also been implicated in the pathology of cerebral malaria (Kumar et al. 2003;Tripathi et al. 2006;Tripathi et al. 2009), and has been shown to be one of the critical mechanisms involved in hemozoin-induced neuroinflammation (Velagapudi et al. 2019  There have been suggestions in scientific literature that malaria hemozoin activates NLRP3 inflammasome/caspase-1 pathway to release IL-1β (Dostert et al. 2009;Shio et al. 2009;Velagapudi et al. 2019;Ogunrinade et al. 2021). In this study, we have demonstrated significant inhibition of hemozoin-induced NLRP3 protein expression by all plants investigated. However, Khaya senegalensis and Nauclea latifolia did not have effect on hemozoin-induced caspase-1 activity.
Results of this study have shown that freeze-dried samples of Alstonia boonei, Anacardium occidentale, Azadiractha indica and Mangifera indica produced strong activity in reducing synthetic hemozoin-induced neuroinflammation in BV-2 microglia through mechanisms involving NF-B and NLRP3 inflammasome activation.

Moderate activities have been shown by Enantia chlorantha, Khaya senegalensis
and Nauclea latifolia. The outcome of this study provides pharmacological evidence for the use of these plants in Traditional African Medicine for treating symptoms and complications of malaria, including cerebral malaria. It is not yet clear how these plants may be acting in vivo to overcome the blood-brain barrier, and should be investigated in human intervention studies involving patients presenting with malaria and/or cerebral malaria.

Acknowledgement
This work was supported by the Ekhagastiftelsen [grant number 2019-72].

Data Availability
All data used during this study are available from the corresponding author.

Conflicts of Interest
The authors declare that they have no conflicts of interest.