In Vitro Evaluation of the Antiamoebic Activity of Kaempferol against Trophozoites of Entamoeba histolytica and in the Interactions of Amoebae with Hamster Neutrophils

Entamoeba histolytica (E. histolytica) is a parasite in humans that provokes amoebiasis. The most employed drug is metronidazole (MTZ); however, some studies have reported that this drug induces genotoxic effects. Therefore, it is necessary to explore new compounds without toxicity that can eliminate E. histolytica. Flavonoids are polyphenolic compounds that have demonstrated inhibition of growth and dysregulation of amoebic proteins. Despite the knowledge acquired to date, action mechanisms are not completely understood. The present work evaluates the effect of kaempferol against E. histolytica trophozoites and in the interactions with neutrophils from hamster, which is a susceptibility model. Our study demonstrated a significant reduction in the amoebic viability of trophozoites incubated with kaempferol at 150 μM for 90 min. The gene expression analysis showed a significant downregulation of Pr (peroxiredoxin), Rr (rubrerythrin), and TrxR (thioredoxin reductase). In interactions with amoebae and neutrophils for short times, we observed a reduction in ROS (reactive oxygen species), NO (nitric oxide), and MPO (myeloperoxidase) neutrophil activities. In conclusion, we confirmed that kaempferol is an effective drug against E. histolytica through the decrease in E. histolytica antioxidant enzyme expression and a regulator of several neutrophil mechanisms, such as MPO activity and the regulation of ROS and NO.


Introduction
E. histolytica is a protozoan pathogen in humans and is the causative agent of amoebiasis. Between 40 and 50 million people worldwide are infected with this protozoan parasite, representing a major public health problem. Approximately 100,000 patients die each year worldwide [1]. E. histolytica has been reported to have a high tissue-destroying capacity. According to the World Health Organization (WHO), amoebiasis is the third leading cause of death due to parasitic disease. In Mexico, amoebiasis has a considerable prevalence in both cities and rural areas; the prevalence of this parasite is highest in the states of Morelos (19%) and Chiapas (51%) [2]. Amoebiasis is a gastrointestinal infection that can cause amoebic colitis. The most common symptoms are diarrhea with blood and mucus, and various alterations may occur depending on the location. The most common extraintestinal form is the amoebic liver abscess (ALA), which occurs when amoebae are transported induced mouse mastitis [23] and a mouse model of LPS-induced acute lung injury treated with kaempferol, the MPO activation was reduced [24].
Kaempferol was studied previously in the interaction with E. histolytica trophozoites, and the authors reported the deregulation of amoebic actin and myosin II heavy chain; cortexylin II, a heat shock protein; glyceraldehyde phosphate dehydrogenase; and fructose-1, 6-bisphosphate aldolase (G/FBPA). The authors also showed that at 48 h of kaempferol incubation a concentration of 27.7 µM was able to inhibit 77.1% of amoebic growth compared with trophozoites not treated with this flavonoid [25]. These results are similar to the results reported with (-)-epicatechin [26]. Previous studies analyzed a slight ROS production in 5 × 10 5 E. histolytica trophozoites incubated with kaempferol at 27.7 µM for 48 h compared with trophozoites without kaempferol [27].
In the present work, we analyze the effect of kaempferol on E. histolytica and in the interaction of amoebae with hamster neutrophils for short times, demonstrating that kaempferol has a dual effect on amoeba and neutrophils. On the one hand, it diminished amoebic viability; on the other hand, kaempferol also decreased neutrophils' MPO activity and ROS and NO release. In a hamster susceptible model to ALA, kaempferol could participate in amoebic liver abscess resolution through its anti-inflammatory and antioxidant activities.

Effect of Kaempferol on the Viability of E. histolytica and VERO Cells
To analyze the effect of kaempferol on the viability of E. histolytica, WST-1 reagent was used. For this assay 15,000 E. histolytica trophozoites were incubated with concentrations of 90, 100, 110, 120, 130, 140, and 150 µM kaempferol or MTZ at 90 min. When analyzing the viability of the trophozoites incubated at concentrations of 90, 100, and 110 µM of kaempferol and MTZ, we did not observe a decrease in the viability of the trophozoites, but when kaempferol was used at concentrations of 130, 140, and 150 µM, we observed a viability of 44.5% with kaempferol at 150 µM. We also observed a significant difference in the viability of kaempferol compared with MTZ at these concentrations ( Figure 1). antioxidant, anti-inflammatory, anticancer, antibacterial, and antiviral activities, as well as against several protozoa [20]. Moreover, kaempferol is an extremely active ROS scavenger [21]. Kaempferol has been reported to have an effect in suppressing ROS production in mouse bone marrow-derived neutrophils [22]. Moreover, in a model of induced mouse mastitis [23] and a mouse model of LPS-induced acute lung injury treated with kaempferol, the MPO activation was reduced [24].
Kaempferol was studied previously in the interaction with E. histolytica trophozoites, and the authors reported the deregulation of amoebic actin and myosin II heavy chain; cortexylin II, a heat shock protein; glyceraldehyde phosphate dehydrogenase; and fructose-1, 6-bisphosphate aldolase (G/FBPA). The authors also showed that at 48 h of kaempferol incubation a concentration of 27.7 µM was able to inhibit 77.1% of amoebic growth compared with trophozoites not treated with this flavonoid [25]. These results are similar to the results reported with (-)-epicatechin [26]. Previous studies analyzed a slight ROS production in 5 × 10 5 E. histolytica trophozoites incubated with kaempferol at 27.7 µM for 48 h compared with trophozoites without kaempferol [27].
In the present work, we analyze the effect of kaempferol on E. histolytica and in the interaction of amoebae with hamster neutrophils for short times, demonstrating that kaempferol has a dual effect on amoeba and neutrophils. On the one hand, it diminished amoebic viability; on the other hand, kaempferol also decreased neutrophils' MPO activity and ROS and NO release. In a hamster susceptible model to ALA, kaempferol could participate in amoebic liver abscess resolution through its anti-inflammatory and antioxidant activities.

Effect of Kaempferol on the Viability of E. histolytica and VERO Cells
To analyze the effect of kaempferol on the viability of E. histolytica, WST-1 reagent was used. For this assay 15,000 E. histolytica trophozoites were incubated with concentrations of 90, 100, 110, 120, 130, 140, and 150 µM kaempferol or MTZ at 90 min. When analyzing the viability of the trophozoites incubated at concentrations of 90, 100, and 110 µM of kaempferol and MTZ, we did not observe a decrease in the viability of the trophozoites, but when kaempferol was used at concentrations of 130, 140, and 150 µM, we observed a viability of 44.5% with kaempferol at 150 µM. We also observed a significant difference in the viability of kaempferol compared with MTZ at these concentrations ( Figure 1).
When we evaluated the VERO cell viability at different times with kaempferol or MTZ at 150 µM using the WST-1 assay, we did not observe any significant differences at any of the assayed times ( Figure 2). p-values were determined with ANOVA (**** p < 0.0001; ** p < 0.01), compared with the control group (Ctrl), while those compared with the MTZ p-values were determined with ANOVA (**** p < 0.0001).
When we evaluated the VERO cell viability at different times with kaempferol or MTZ at 150 µM using the WST-1 assay, we did not observe any significant differences at any of the assayed times ( Figure 2).

Proteins' Overexpression of E. histolytica in the Presence of Kaempferol
After obtaining the total cell lysates of E. histolytica in the presence of kaempferol or MTZ, a protein separation analysis was performed in a 10% SDS-polyacrylamide gel and detected by staining with Coomassie Blue (Figure 3). When analyzing trophozoites incubated with 150 µM kaempferol for 90 min, we observed an overexpression of molecular bands of approximately 67, 59, 27, and 24 kDa (C). On the other hand, we observed a difference in the 67 kDa band in the MTZ samples (B) compared with the Ctrl (A).

Proteins' Overexpression of E. histolytica in the Presence of Kaempferol
After obtaining the total cell lysates of E. histolytica in the presence of kaempferol or MTZ, a protein separation analysis was performed in a 10% SDS-polyacrylamide gel and detected by staining with Coomassie Blue (

Downregulation of E. histolytica-Detoxifying Enzyme Expression in the Presence of Kaempferol
Samples of cDNA obtained from trophozoites of E. histolytica in the presence of kaempferol or MTZ at 150 µM for 90 min were analyzed using qRT-PCR for the quantification of the detoxifying enzyme gene expression ( Figure 4). Using real-time PCR, we

Downregulation of E. histolytica-Detoxifying Enzyme Expression in the Presence of Kaempferol
Samples of cDNA obtained from trophozoites of E. histolytica in the presence of kaempferol or MTZ at 150 µM for 90 min were analyzed using qRT-PCR for the quantification of the detoxifying enzyme gene expression ( Figure 4). Using real-time PCR, we found a downregulation of three genes of trophozoites in the presence of kaempferol, Rr, which had a significant decrease of 84.04-fold compared with the basal control. In the case of Prx, we also observed a decrease of 58.49-fold against the basal control and a decrease of 3.97-fold of TrxR against the basal control. In contrast, we did not observe any difference in the gene expression of Trx in the kaempferol group compared with the basal control. On the other hand, we observed a downregulation of all genes in the kaempferol group compared with the MTZ group.  There is a statistically significant difference between Rr, Prx, and TrxR genes in kaempferol compared with those in the control and the MTZ groups. Statistical analyses were determined with Student's t-test (* p < 0.05; ** p < 0.01). Data represent the mean ± SD (triplicate). p-values were determined using Student's t-test and using Bonferroni's test.

Kaempferol Induces a Decrease in MPO Activity in E. histolytica and Neutrophil Interactions
Supernatants of the interaction of trophozoites of E. histolytica incubated with hamster neutrophils in the presence of 150 µM kaempferol (N+T+kaempferol) or MTZ (N+T+MTZ) at different times (20,40,60, and 90 min) were collected ( Figure 5). The results showed that neutrophil MPO activity decreased significantly in the presence of 150 µM kaempferol at 60 and 90 min of incubation compared with that of the respective control, neutrophils and trophozoites (N+T). We did not observe a significant difference in the DMSO group (N+T+DMSO) compared with the control (N+T); in contrast, when we compared it with the MTZ group (N+T+MTZ), we observed a significant increase at 90 min compared with N+T+kaempferol. We also provided a negative control with an inhibitor of MPO, ABAH (4-amino-benzoic acid hydrazide) (N+T+ABAH). Gene expression of antioxidant enzymes (Rr, Prx, TrxR, and Trx) in trophozoites in the presence of kaempferol at 150 µM for 90 min was determined using qRT-PCR. There is a statistically significant difference between Rr, Prx, and TrxR genes in kaempferol compared with those in the control and the MTZ groups. Statistical analyses were determined with Student's t-test (* p < 0.05; ** p < 0.01). Data represent the mean ± SD (triplicate). p-values were determined using Student's t-test and using Bonferroni's test.

Kaempferol Induces a Decrease in MPO Activity in E. histolytica and Neutrophil Interactions
Supernatants of the interaction of trophozoites of E. histolytica incubated with hamster neutrophils in the presence of 150 µM kaempferol (N+T+kaempferol) or MTZ (N+T+MTZ) at different times (20,40,60, and 90 min) were collected ( Figure 5). The results showed that neutrophil MPO activity decreased significantly in the presence of 150 µM kaempferol at 60 and 90 min of incubation compared with that of the respective control, neutrophils and trophozoites (N+T). We did not observe a significant difference in the DMSO group (N+T+DMSO) compared with the control (N+T); in contrast, when we compared it with the MTZ group (N+T+MTZ), we observed a significant increase at 90 min compared with N+T+kaempferol. We also provided a negative control with an inhibitor of MPO, ABAH (4-amino-benzoic acid hydrazide) (N+T+ABAH).
kaempferol at 60 and 90 min of incubation compared with that of the respective control, neutrophils and trophozoites (N+T). We did not observe a significant difference in the DMSO group (N+T+DMSO) compared with the control (N+T); in contrast, when we compared it with the MTZ group (N+T+MTZ), we observed a significant increase at 90 min compared with N+T+kaempferol. We also provided a negative control with an inhibitor of MPO, ABAH (4-amino-benzoic acid hydrazide) (N+T+ABAH). MPO activity in neutrophils in the presence of trophozoites of E. histolytica incubated with kaempferol or MTZ at 150 µM at different times was determined. DMSO was used as a vehicle, PMA (phorbol 12-myristate 13-acetate) was used as a positive control, and MPO inhibitor 100 mM (ABAH) was used as MPO activity control. Data represent the mean ± SD of the three independent experiments. p-values were determined using two-way ANOVA (**** p < 0.0001; * p < 0.05), comparing N+T with the N+T+kaempferol group, while compared with the MTZ group, p-values were determined using ANOVA (**** p < 0.0001).

Kaempferol Induces Low ROS Release in E. histolytica and Neutrophil Interactions
The quantification of ROS from the interaction of E. histolytica with neutrophils showed that under basal conditions neutrophils and trophozoites produce a scarce amount of ROS ( Figure 6a). As a positive control, we showed a group with LPS (lipopolysaccharide). In this group, we observed an increase in ROS activity compared with neutrophils alone. It is worth mentioning that when neutrophils interacted with the amoebae, an increase in ROS production was observed. In the presence of 150 µM of kaempferol at 90 min with trophozoites and neutrophils, we observed a significant decrease in ROS activity compared with the group in the absence of this drug, and we observed a significant difference compared with the MTZ group.

Kaempferol Induces a Decrease in NO Production in the E. histolytica and Neutrophil Interaction
In the interaction between E. histolytica and neutrophils, an increase in NO concentration was observed compared with neutrophils ( Figure 6b). When kaempferol (150 µM) was incubated in the presence of amoebic trophozoites with neutrophils, we observed a significant decrease in NO production compared with the interactions in the absence of kaempferol. Moreover, we observed a significant difference compared with the MTZ group. ysaccharide). In this group, we observed an increase in ROS activity compared with neutrophils alone. It is worth mentioning that when neutrophils interacted with the amoebae, an increase in ROS production was observed. In the presence of 150 µM of kaempferol at 90 min with trophozoites and neutrophils, we observed a significant decrease in ROS activity compared with the group in the absence of this drug, and we observed a significant difference compared with the MTZ group.  p-values were determined using two-way ANOVA (**** p < 0.0001) to compare the N+T group with the N+T+kaempferol group, while compared with the MTZ group, p-values were determined using two-way ANOVA (**** p < 0.0001). (b) Detection of NO using the Griess method in the interaction of neutrophils in the presence of E. histolytica incubated with kaempferol at 150 µM for 90 min. Data represent the mean ± SD of the three independent experiments. p-values were determined using two-way ANOVA (**** p < 0.0001), comparing the N+T group with the N+T+kaempferol group, while compared with the MTZ group, p-values were determined using ANOVA (**** p < 0.0001).

Discussion
The drug of choice for amoebiasis is MTZ; however, due to its side effects in long-term treatments, it is essential to look for alternative therapies using new compounds that do not present adverse effects for humans. Kaempferol is a flavonoid whose antiamoebic activity has been described previously in an in vitro assay [20]; however, its effect on E. histolytica trophozoites at short times is unknown. Therefore, it is necessary to test its efficacy. Kaempferol possesses a wide range of biological activities, such as antioxidant, anti-inflammatory, anticancer, antibacterial, and antiviral activities, and it also has effective activity against several protozoa [20].
In our study, the results of the WST-1 assay showed that the OD values, were directly proportional to the viability of 15,000 E. histolytica trophozoites, showing a decrease in their viability by increasing the concentration of kaempferol (130, 140, and 150 µM) at 90 min of incubation compared with MTZ at the same concentrations, considering that MTZ is the drug of choice against amoebiasis. Furthermore, a concentration of 150 µM of kaempferol significantly decreased viability. Additionally, we evaluated the effect of kaempferol at a concentration of 150 µM on the viability of 10,000 VERO cells using the WST assay at 60, 90, 180, and 360 min (Figure 2). In this experiment, we observed that kaempferol had no effect on the viability of mammalian cells at this concentration and at these times.
Kaempferol is obtained from Helianthemum glomeratum, an endemic medicinal herb used to treat diarrhea, abdominal pain, and dysentery in Mexico, and it shows activity against E. histolytica at a concentration of 27.7 µM of kaempferol for 48 h [28]. After obtaining total cell lysates of E. histolytica, protein separation analysis was performed on a 10% SDS-polyacrylamide gel, where we observed overexpression of 67, 59, 27, and 24 kDa protein bands. A previous work reported an overexpression effect of a cytoskeleton protein (actin) in E. histolytica trophozoites incubated with kaempferol at a concentration of 27.7 µM for 48 h [25]. Even though the induction of the protein bands of the weight of 67, 59, 27, and 24 kDa is clearly observed in the presence of kaempferol in the SDS-polyacrylamide gel, due to the limitations of this technique, it is necessary in future studies to carry out two-dimensional gel assays together with the mass spectrometry technique to be able to state with precision which are the amoebic proteins that are being induced by kaempferol.
We also analyzed the relative gene expression of E. histolytica antioxidant enzymes in the presence of kaempferol at a concentration of 150 µM for 90 min, and we found downregulation of genes encoding E. histolytica-detoxifying enzymes such as Rr, Prx, and TrxR compared with the basal control and Rr, Prx, TrxR, and TrxR compared with the MTZ group; these enzymes are responsible for H 2 O 2 detoxification and defense of amoeba against oxidative stress [29]. On the other hand, we did not observe any significant change in genes encoding other enzymes such as Trx compared with the control group. Compared to the MTZ group, Rr, Prx, TrxR, and Trx were downregulated in the presence of kaempferol; these enzymes are also related to the antioxidant capacity of the parasite. The downregulation in the expression of these antioxidant enzymes by kaempferol suggests that the enzymatic activities were depleted due to their participation in the regulation of the oxidative environment. Despite the results achieved on amoebic antioxidant enzymes, it will be necessary to evaluate their distribution and activity in the future. In the case of MTZ, we observed a clear upregulation of these genes. In other studies, it was reported that in E. histolytica trophozoites that were treated with MTZ at a concentration of 50 micromolar for 1, 3, or 5 h, there was an overexpression in the expression of Trx, TRxR, and Prx genes [30].
Importantly, MPO is an enzyme released by neutrophils that binds to E. histolytica trophozoites, causing damage to them [31]. When we performed tests to quantify the MPO in hamster neutrophils and trophozoites with 150 µM of kaempferol at different times, our results showed a significant decrease in MPO activity at 60 and 90 min of incubation compared with the control and MTZ, and the presence of this enzyme was significantly decreased at 90 min. Previous reports showed that in interactions of trophozoites with mouse and hamster neutrophils, MPO activity in the hamster model was lower than in the mouse model [32]. In this study, we demonstrated that kaempferol decreases the MPO activity in hamster neutrophils, which may favor the ALA resolution in a susceptible model where a prolongated and exaggerated inflammatory response exacerbates the amoebic damage [3,32].
In a previous work using Surface Plasmon Resonance (SPR) binding analysis methodology to describe the binding of flavonoids, it was reported that MPO was immobilized on a CM5 sensor chip. Aliquots of 40 µL of kaempferol were injected into the flow cell and it was observed that kaempferol at 25 µM bound the MPO after 120 s. It exhibited significant affinity for MPO. These authors claimed that this affinity could inhibit the activity of this enzyme, which could explain the decrease in MPO activity and the role of kaempferol as an inhibitor of this enzyme in neutrophils [33].
Neutrophils have various mechanisms to damage E. histolytica trophozoites such as ROS and NO. Neutrophils require the respiratory burst to release ROS in the presence of oxygen. This production is catalyzed by means of NADPH oxidase, which, when it is activated, generates two molecules of superoxide anion [34] and NO is synthesized by neutrophil nitric oxide synthase (iNOS) [35]. We reported a significant decrease in ROS and NO concentrations in interactions of trophozoites with hamster neutrophils in the presence of kaempferol, compared with the control group and interactions in the presence of MTZ. Kaempferol was used to inhibit the formation of ROS in mouse bone marrow-derived neutrophils stimulated with PMA at 10 nmol/L for 30 min due to the antioxidant capacity of the flavonoid [22]. In another study in rats treated with 10 mg/kg/day kaempferol, tissue gingival fibroblasts were obtained and homogenized; this drug may exert a profound inhibitory effect against the iNOS [36]. In another study using a model of a biological oxygen monitor, it was reported that a concentration of 92 µM kaempferol inhibited NADPH oxidase activity by decreasing ROS and NO production in human neutrophil stimulated by PMA [37]. A previous report of the modified xanthine/luminol/xanthine oxidase assay was used to evaluate the superoxide scavenging effect of kaempferol, and it was observed that this flavonoid is a potent superoxide anion scavenger that decreases superoxide anion levels [38]. Superoxide is necessary for the normal production of most ROS and NO species involved in oxidative stress [39].
Since neutrophils contribute to host tissue damage in ALA through the massive lysing of these cells and the release of oxidative products (ROS, NO), the scavenger effect of kaempferol may promote the resolution of amoebic abscess. Future studies in vivo are needed to understand the mechanisms of the positive antiamoebic effect of kaempferol in interactions between neutrophils and E. histolytica trophozoites.
Thus, in this study we confirmed kaempferol as an effective drug against E. histolytica, which has a dual effect on trophozoites and hamster neutrophils, through a regulatory effect on MPO, ROS, and NO in these interactions at short times. In the hamster, a chronic susceptible model to ALA, the reduction in the neutrophils' MPO activity as well as the reduction in ROS and NO production could be the mechanisms that could participate in the amoebic liver abscess resolution.

Amoebic Cultures
E. histolytica trophozoites of the HM-1: IMSS strain were cultured axenically at 37 • C in Diamond's trypticase yeast iron extract (TYI-S-33) culture medium supplemented with 10% bovine serum (Gibco, BRL, Grand Island, NY, USA). The trophozoites were cooled, detached, and harvested at the end of the logarithmic growth phase (48 h) by chilling to 4 • C. The trophozoites were concentrated via centrifugation at 300× g for 5 min and used immediately [40].

Real-Time qPCR of Antioxidant Enzymes of E. histolytica
For the extraction of RNA from 15,000 E. histolytica trophozoites incubated with 150 µM of kaempferol or MTZ for 90 min at 37 • C, the TRIzol-Chloroform (Thermo Scientific, Waltham, MA, USA) method was used. At the end of this process, the cells were resuspended in RNAse-free water. RNA was stored at −80 • C until use. RNA quantification was performed using a Nanodrop Lite (Thermo Scientific, Waltham, MA, USA). The isolated RNA was treated with RQ1 RNase-Free DNase (PROMEGA, Fitchburg, WI, USA) to avoid genomic DNA contamination and cDNA was synthesized using the First Strand cDNA synthesis kit (Thermo Scientific, MA, USA) according to the manufacturer's protocol. RT-qPCR was performed with a Step One Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) by monitoring the increase in fluorescence in real time using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA). Melting curve protocols were performed to ensure the specificity of the amplification products. The primers were designed using Primer Express 3.0.1 software (Applied Biosystems, Foster City, CA, USA) and synthesized commercially (IDT Integrated DNA Technologies, Coralville, IA, USA). The size of all amplicons was designed of 150 pb. (Table 1). For E. histolytica trophozoites, primers specific for Rr, Prx, TrxR, and Trx were used; as a control, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from E. histolytica was used. The application of the comparative Cycle Threshold (CT) method was conducted to validate the effect of treatment on the expression of two endogenous controls 18S subunit ribosomal and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We selected GAPDH because no statistically significant relationship was found between the treatment and basal expression of this gene. The relative quantification of antioxidant enzymes was calculated using the CT method by applying the comparative cycle threshold CT, which uses the arithmetic formula 2 −∆∆CT [45]. To validate the method, we verified that the amplification efficiency for the target genes and the endogenous gene GAPDH were nearly equal, examined CT variations with serial of cDNA template dilutions, and a plot of log cDNA concentrations versus CT was made and efficiency was calculated using the equation E = −1 + 10 (−1/slope) . The statistical significance between the untreated and treated trophozoites was calculated using Bonferroni's test with GraphPad Prism statistical software (GraphPad, San Diego, CA, USA).

Neutrophil Purification
For the isolation of neutrophils, we used male hamsters (Mesocricetus auratus), an amoebiasis-susceptible model weighing approximately 100 g of six to eight weeks of age. Neutrophils were extracted from an enriched fraction, which was obtained from hamsters via cardiac puncture, using a 3 mL insulin syringe with heparin (Pisa, MEXICO, Ciudad de México, México). The heparinized blood was placed in a 15 mL conical tube. Subsequently, 6 mL of PolymorphoPrep (Sigma-Aldrich, MO, USA) was placed in another 15 mL tube, and 6 mL of blood was added into 15 mL tube. The tube was centrifuged at 758× g for 35 min at 20 • C. The supernatant was discarded, the pellet containing the neutrophil enriched fraction was obtained, and the neutrophil layer was collected. In both treatments, the remaining red blood cells were subjected to hypotonic shock with sterile 0.2% NaCl for 30 s and 1.6% NaCl for 1 min. Finally, neutrophils were resuspended in RPMI + SFB 10% (Sigma, MO, USA) or DMEM/F-12/Media + SFB 10%, incubated at 37 • C, and counted with the Türk staining method; a representative image of inactivated neutrophils is shown in Figure S1 in Supplementary Material [46]. The number of neutrophils was counted in a Neubauer chamber, and their viability (viable cells excluding trypan blue) and purity (morphology) ascertained. Briefly, ≥95% of the purified cells were neutrophils, and the cells were adjusted to 300,000 neutrophils/mL [32]. The animals were handled in accordance with Mexican Federal Regulations for Animal Experimentation and Care (NOM-062-ZOO-1999, Ministry of Agriculture, México City, México). The institutional Animal Care and Use Committee from Escuela Superior de Medicina acted as the regulatory office for approving research protocols and approved the animal care and handling of the hamsters (protocol number ESM-CICUAL 07/26-08-2019).

Neutrophils and E. histolytica Trophozoite Interactions
A total of 300,000 neutrophils were incubated with 15,000 E. histolytica trophozoites for 90 min at 37 • C in RPMI + SFB 10% or DMEM/F-12/Media + FBS 10% medium in a 24-well plate in the presence or absence of 150 µM of kaempferol or MTZ for 90 min at 37 • C. Neutrophils in the presence of LPS (20 µg/mL) (Sigma-Aldrich, MO, USA) and interactions in the presence of ABAH, an inhibitor of MPO at 100 µM/L (Sigma, St. Louis, MO, USA) and DMSO, were used. LPS was a positive control of the production of ROS and NO. On the other hand, we used ABAH as negative control, as it inhibited MPO activity.

MPO Activity
MPO quantification was performed on the supernatants of the interactions for 20, 40, 60, and 90 min in RPMI + SFB 10% medium, from which 50 µL was transferred to 96-well plates, and 50 µL of 3,3 ,5,5 -tetramethylbenzidine (TMB) (Sigma-Aldrich, MO, USA) was subsequently added followed immediately by 50 µL of H 2 O 2 (5 mM/L in water, high purity) (Sigma-Aldrich, MO, USA). The mixture was allowed to react for 10 min until a color change was visualized. After, the reaction was stopped with 50 µL of 2.5 M/L sulfuric acid (Sigma-Aldrich, MO, USA). The plates were centrifuged at 600× g for 10 min, 200 µL of the supernatant from each well was transferred to another plate, and the optical density (OD) in each well was determined at 405 nm using a microplate reader. PMA was used as a positive control of MPO activity [32].

ROS and NO Production
ROS and NO production were determined using a ROS kit (Abcam, MA, USA) [47] and a NO assay kit (Abcam, MA, USA) [48]. The production of ROS and NO was quantified from the interaction of 300,000 neutrophils with 15,000 E. histolytica trophozoites with 150 µM of kaempferol or MTZ for 90 min at 37 • C in DMEM/F12 (Sigma-Aldrich, MO, USA) + 5% SFB (Sigma-Aldrich, MO, USA) for 90 min. LPS (20 µg/mL) was used as a positive control, and trophozoites in the absence of kaempferol and trophozoites with 0.05% DMSO were used as controls. At the end of the interactions, the supernatant was removed. Samples were previously deproteinized for the nitric oxide assay. For ROS and NO determination, the manufacturer's specifications were followed. The plates were read in a microplate reader; ROS was read at a wavelength of 480 nm excitation/530 nm and the production of NO was read at a wavelength of 540 nm.

Statistical Analysis
All the data were processed using GraphPad Prism 8.0 software. Statistical analyses were performed using one-way ANOVA, two-way ANOVA, and Student's t-test. If a significant (p < 0.05) main effect or association was identified, the respective group means were compared using the Bonferroni test.

Conclusions
In conclusion, in this study we confirmed that kaempferol is an effective drug against E. histolytica compared with MTZ at the same concentration, because kaempferol decreased its viability. In addition, we observed an overexpression of some E. histolytica proteins in the presence of kaempferol and a downregulation in the expression of Pr, Rr, and TrxR genes, which are antioxidant enzymes of the parasite. Interestingly, kaempferol acts both on E. histolytica trophozoites and on hamster neutrophils, regulating the production of ROS and ON as well as the activity of neutrophil MPO.
Further studies are necessary in clinical assays because kaempferol could be effective for patients with amoebiasis, as this flavonoid does not present adverse effects like MTZ does.