The saikosaponins were purified on an SPE cartridge and determined on a Waters CORTECTS C18 column  by gradient elution using 0.01% acetic acid aqueous solution and acetonitrile. The results showed good linearity with the r2 values higher than 0.998 for all analytes. The average recoveries at three different concentration levels ranged from 80% to 109% and the intraday and interday precision  were in the range of 1.0%∼1.9% and 1.4%∼2.1%, respectively. The established HPLC-CAD method was subsequently applied to 15 batches of XG to investigate the batch-to-batch consistency and controllability. The proposed method could potentially be used for the quality control of XG and also be helpful in the quality evaluation of Chaihu and its related preparations.Bupleuri Radix  is the principal drug in Xiaochaihu granules (XGs) that is a famous Chinese medicine preparation in China. Since previous analytical methods have not focused on the multiactive saikosaponins of Chaihu, it is difficult to effectively control the quality of XG on the market. In this manuscript, the simultaneous determination of 7 saikosaponins (saikosaponins C, I, H, A, B Xiaochaihu granules (XGs) are derived from a traditional Chinese medicine (TCM) classical formula Xiaochaihu-tang (named Sho-saiko-to in Japanese). It is a modern Chinese medicine preparation composed of 7 herbs including Bupleuri Radix , Scutellaria radix (Huangqin), Codonopsis radix (Dangshen), Glycyrrhiza radix (Ganchao), Pinellia tuber (Banxia), Jujube fruit (Dazhao), and Ginger rhizome (Shengjiang). It has the function of inducing sweat to dispel heat, soothing liver, and harmonizing stomach . Nowaday1 (SSa ⟶ SSb1), saikosaponin I or H (SSc ⟶ SSi or SSh), and saikosaponin B2 (SSd ⟶ SSb2) accordingly. Because of the lacking of UV chromophores, HPLC-UV [2. It is widely acknowledged that the synergistic effects of multiple-active components are responsible for the therapeutic effects of TCM preparation. Therefore, the reported methods and current standards are insufficient to effectively ensure the quality of XG.Chaihu is the principal drug in XG that plays a major therapeutic role in the treatment of the main disease and syndrome. Literature studies reported that saikosaponins have many pharmacology activities, such as antiviral, antibacterial, antihepatitis, and immunomodulating effects –6, and t HPLC-UV  is imper HPLC-UV  and char HPLC-UV  are usua HPLC-UV  compared HPLC-UV , 11 has  HPLC-UV  establis HPLC-UV , 8. The  HPLC-UV , ultraso HPLC-UV , ultraso HPLC-UV , 14, mat HPLC-UV , 16, and HPLC-UV , have be HPLC-UV  only foc2, G, and B1) present in XG were unambiguously identified by comparing with reference standards using LC-Q-Orbitrap HRMS. All analytes were purified and concentrated on a C18 SPE cartridge. The established HPLC-CAD method was proved to be specific, reliable, and practical for the quantitative determination of 7 saikosaponins and successfully applied to the quality evaluation of 15 batched of XG thereof.In this work, 7 saikosaponins , and B2 (SSb2) were purchased from Chengdu Desite Biological Technologies Co., Ltd. . Their structures are shown in Saikosaponin A (SSa) and saikosaponin D (SSd) were purchased from the National Institutes for Food and Drug Control . Saikosaponins C (SSc), I (SSi), H (SSh), G (SSg), B1 (SSb−1), SSi (0.1 mg·mL−1), SSh (0.1 mg·mL−1), SSa (0.3 mg·mL−1), SSb2 (1 mg·mL−1), SSg (0.2 mg·mL−1), and SSb1 (0.5 mg·mL−1) was prepared with methanol. A series of mixed standard solutions were obtained by appropriate dilution of the stock solution for plotting calibration curve. All solutions were stored in the refrigerator at 4°C until analysis.A mix stock standard solution of SSc . The mobile phase was comprised of 0.01% acetic acid (A) and acetonitrile (B) with gradient elution program: 0∼9 min, 5–25% B, 9∼16 min, 25–35% B, 16∼23 min, 35–60% B, and 23∼30 min, 60–80% B. The flow rate was 0.3 mL·min−1. The heated ESI (H-ESI) source was operated, and MS parameters were optimized as follows: spray voltage, 3.5 kV; sheath gas flow, 40 L/min; aux gas flow, 10 L/min; capillary temperature, 320°C; aux gas heater temperature, 350°C. A full MS/dd-MS2 (data dependent MS2) method was used for acquisition. The full scan range was from 100 to 1500 in negative ion mode at a resolution of 70000 and 17,500 for MS2 scan. MS/MS spectra were fragmented by high-energy collision-induced dissociation (HCD) of normalized collision energy (NCE) values at the levels of 10%, 20%, and 30%. Date acquisition and processing were accomplished with Xcalibur software .The qualitative identification of saikosaponins in XG was carried out using a Q Exactive™ hybrid quadrupole-orbitrap mass spectrometer equipped with a Dionex Ultimate 3000 LC system . Analytes were detected at 30°C on a Waters CORTECTS C18 column  consisting of vacuum degasser, quaternary pump, autosampler, thermostatted column compartment, and CAD, connected with Chromeleon software, was employed for quantitative analysis. The column, mobile phase, and column temperature were the same as that of LC-MS condition. The gradient elution program was as follows: 0∼55 min, 30–32% B, 50∼58 min, 32–90% B, and 58∼60 min, 90–30% B, and finally, reconditioning the column with 30% B isocratic for 10 min. The flow rate was 0.8 mL·miny and x represent the logarithm of peak area and logarithm of concentration (μg·mL−1). LOD and LOQ were determined by diluting the reference standard solution with methanol when the signal-to-noise rations (S/N) were about 3 : 1 and 10 : 1, respectively. For intra- and interday precision, the mix standard solution was determined in the same day (n = 6) and in three consecutive days, respectively. Six replicates as per the test method representing a single batch were prepared and analyzed to demonstrate the repeatability. The precision and repeatability were evaluated by calculating the relative standard deviations (RSDs) of each peak area. The recovery was used to express the accuracy of the method, which was performed by spiking known amounts of the sample to three concentration levels  of the mix standard solution.The quantitative HPLC-CAD method was fully validated in terms of specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), intra- and interday precision, repeatability, and accuracy in accordance with ICH guideline Q2(R1) . SpecifiELSD and CAD are both universal detectors and have obvious advantages in detecting those compounds with weak or no UV absorption. The performance comparison between these two detectors was studied by some researchers , 20. It XG is a mixture of multiple herbs and thus has a wide range of complex and diverse chemical components –27. The The HPLC-CAD chromatographic condition described in the reference literature  was atte2 (peak 6) during the production of XG. This result was in accordance with literature reports [7 saikosaponins peaks in XG were unambiguously identified by comparing their retention time and UV and MS spectra with those of reference standard solutions. The extracted ion chromatogram (EIC) and MS/MS data are present in  reports .r2), linearity range, LOD, LOQ, intra- and interday precision, repeatability, and recovery of the 7 analytes are listed in Tables 2, SSg, and SSb1 and NMT 8.0% for SSi and SSh. The average recoveries at three different concentration levels were between 80% and 109%. These obtained values illustrated great repeatability and accuracy.As shown in 2, SSg, and SSb1) in 15 batches of XG. The results  of 4.4%, which demonstrated acceptable batch-to-batch consistency and controllability. Bao et al. [2 (26.9%), SSa (25.8%), SSb1 (22.4%), SSg (14.3%), SSc (6.9%), SSh (5.4%), and SSi (2.9%). The difference between our results and literature data was mainly because of different herb extraction processes of XG and Xiaochaihu-tang (Sho-saiko-to).The validated HPLC-CAD quantitative method was subsequently applied to determine 7 saikosaponins  present in XG were unambiguously identified using LC-Q-Orbitrap HRMS and thus selected as quality control components. A specific, sensitive, and accurate HPLC-CAD method in determining the analytes was described in this manuscript. A C18 SPE cartridge was used to purify and concentrate the analytes by eluting with a 10% methanol aqueous solution containing 5% concentrated ammonia and a 30% methanol aqueous solution, and then by methanol. The peaks of 7 saikosaponins were well-separated, although most of the analytes are isomeric compounds. This quantitative method could potentially meet the requirement for quality analysis for QC laboratories. Also, the proposed method could to be used for quality evaluation of Chaihu and its related preparations.In summary, 7 saikosaponins (SSc, SSi, SSh, SSa, SSb


Therefore, we proposed a close correlation between the TNF-α polymorphism  and the disease progress of patients with AECOPD. Comparison of the TNF-α genotypes between the 198 AECOPD diagnosed patients groups and 195 healthy peoples suggested their significant differences of the three genotypes  distribution for TNF-α -308 (P < 0.05), but no differences of that for TNF-α +489. We found that patients with TNF-α -308 GA/AA genotypes showed smaller adjacent arterial diameter, thicker bronchial wall, higher bronchial artery ratio, higher bronchial wall grading, and higher frequency of acute exacerbations than those with TNF-α -308 GG genotype. Patients with TNF-α +489 GA/AA genotypes showed the same AECOPD properties as patients with TNF-α -308 except for the high frequency of acute exacerbations. Further experiment showed that the TNF-α -308 and+489 gene polymorphisms could affect the expression level of TNF-α in macrophages, suggesting the involvement of the macrophage population in disease regulation of AECOPD patients with TNF-α -308G/A and+489G/A genotype heterogeneity. In conclusion, the TNF-α -308 G/A genotype was related to AECOPD susceptibility and progress, while the TNF-α +489G/A genotype was related to AECOPD progress, but not AECOPD susceptibility.Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is becoming a common respiratory disease, leading to increased morbidity and mortality worldwide. Tumor necrosis factor-alpha (TNF- Chronic obstructive pulmonary disease (COPD) refers to a group of chronic inflammatory diseases that cause airflow blockage and breathing-related problems , 2. AlthMacrophages are the most abundant cells in the tumor stroma and exhibit obvious plasticity, which enables them to perform multiple functions in the tumor microenvironment . Tumor-aα, working as a proinflammatory cytokine, plays an important role in an inflammatory response. Many studies show that TNF-α is upregulated in the sputum, broncho alveolar lavage fluid, and bronchial biopsies of patients with COPD [α in the COPD progress. As a well-studied proinflammatory cytokine, many biallelic polymorphisms of TNF-α, including -308G/A (rs1800629), -376G/A (rs1800750), -238G/A (rs361525), and+489 (rs1800610) gene polymorphisms, are discovered. Among these polymorphic sites, -308G/A, -376G/A, and -238G/A polymorphisms are located in the promoter region of the gene, whereas +489 polymorphism is located in the first intron of the gene [α polymorphisms. As the best-studied TNF-α polymorphism [α +489 G/A polymorphism, which is involved in prostate cancer, systemic lupus erythematosus, and rheumatoid arthritis development [α polymorphisms (-308 G/A and+489 G/A) and AECOPD susceptibility of the Shanghai Han Population.Tumor necrosis factor- (TNF-) ith COPD , 14, sug8G/A rs3625, and+4morphism , 17, -30308G/A rs00629, -3morphism , but notmorphism –22. Besielopment , 25. HowThe AECOPD  patients and healthy populations  studied in this work were obtained from the Department of Respiratory Medicine at Minhang Hospital, Fudan University. A total of 198 patients and 195 healthy peoples of the Shanghai Han population were selected from January 2014 to June 2015. The Research Ethics Committees of Minhang Hospital approved the study, and all patients provided written informed consent.1/FVC < 70%; patient respiratory symptoms deteriorated more than the daily variation range, and when there is a need to change the drug regimen, during the disease, patients often had a short-term cough, sputum, shortness of breath and/or exacerbated wheezing, increased sputum, purulent or mucopurulent sputum, fever, and inflammation to significantly exacerbate symptoms. Patients with the genetic predisposition of other diseases, including hypertension, diabetes, coronary heart disease, or rheumatism, were excluded from the sample cohort. As for the control cohort, 195 healthy Han individuals  without disease history of other respiratory or systemic diseases, such as bronchiectasis, pulmonary fibrosis, sarcoidosis, asthma, lung cancer, diabetes, and heart disease, were selected. All of the individuals were confirmed as the normal lung and chest function through high-resolution computerized tomography (HRCT) examination.Specifically, patients were diagnosed as AECOPD according to the criteria described in “Chronic obstructive pulmonary disease treatment guidelines (2013 Revision)” established by the Chinese Medical Association Respiratory Diseases Committee. To be specific, eliminate other disease possibilities, after inhaling bronchodilator, FEVDetailed pathological datum, including medical history, age, gender, smoking index, and specific symptoms  was collected from the patients with AECOPD or the control subjects. Besides, the following two types of COPE classification were considered: Pistolesi et al.  revealedAll patients underwent chest HRCT examination at the end of a calm breath, using a 64-slice helical CT scan, from the apex to the bottom of the lung. Scanning parameters were as follows: voltage 140 kV, thickness 1.25 mm, current 112 mas, pitch 1.375 : 1, and lap speed 12.25 s. Workstation syngo. via was used to read the sections.Two radiologists evaluated the clinical data under double-blinded conditions. The average values of all indicators were taken into analysis. Based on the principles using 960 HU to define the normal lung and low attenuation area (LAA) published by Kitagichi et al. , 29, theα gene polymorphisms located on positions -308 G/A and+489 G/A, which are related to the transcriptional start site (TSS) of the TNF-α gene, were detected in genomic DNA derived from peripheral blood leukocytes. Briefly, 3 mL of blood was drawn from each subject after fasting and stored at -80°C after separation. Gene amplification was conducted using PCR-ABI 2700. Whole blood genomic DNA extraction was performed by Shanghai Yixiang Biotechnology Ltd. DNA was detected after cell lysis, precipitation, protease digestion, DNA adsorption, elution, and agarose gel electrophoresis. The primers for TNF-α -308 were designed as follows: TNF-α-forward (-308): 5′-AGGCCTCAGGACTCAACACA-3′; TNF-α-reverse (-308): 5′-GTTGCTTCTCTCCCTCTT-3′. The primers for TNF-α +489 were designed as follows: TNF-α-forward (+489): 5′-GTGTATGGAGTGAATGAATGAA-3′; TNF-α-reverse (+489): 5′-CCTGAGTGTCTTCTGTGT-3′. The desired gene product was isolated by agarose gel electrophoresis. PCR products were purified and sequenced by Suzhou Genewiz Biotechnology Company. If the result was bimodal at a given position, the position was interpreted as heterozygous; a single peak was interpreted as homozygous.The TNF-α, we downloaded the microarray expression profiles, including GSE71220, GSE42057, and GSE54837, from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi).To gather the expression data of TNF-α concentration assessment using TNF-α (human) EIA Kit .The blood samples from the AECOPD group and control group were obtained in the morning on or before breakfast from the median cubital vein, then immediately centrifuged at 3,000 rpm for 10 minutes. The serum was extracted for TNF-6 cells/mL in RPMI 1640 medium containing 10% FCS and 1% penicillin-streptomycin. Then, the PBMCs were cultured in 24-well plates (500 μl/well) and stimulated for 4 h with phorbol 12-myristate 13-acetate  and ionomycin  in the presence of 1× monensin  to obtain macrophage. The culture supernatants of macrophage were collected, centrifuged for 5 min at 300 g at 4°C, and aliquoted and stored at -80°C until analysis.Whole blood (20-25 mL) collected in tubes containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant was processed between 9 : 00 am and 12 : 00 noon, within 3 h of collection. PBMCs were isolated by Ficoll-Paque  density gradient centrifugation (at 400 g for 30 min at RT with the break turned off) and washed twice with PBS (at 300 g for 10 min at 4°C). PBMCs were resuspended at 2.5 × 10t-test and χ2 test. The Hardy-Weinberg equilibrium was used to calculate the genotypic equilibrium, and the Fisher exact test was used to compare differences between genotype and allele frequency; P < 0.05 was considered as statistically significant.Data were analyzed and graphed using SPSS 19.0 statistical software . Data significance was measured using a The AECOPD cohort consisted of 198 Shanghai Han patients (79.76 ± 8.56 years old). Around 81% of the AECOPD patients were male. A total of 121 (61%) patients were smokers or exsmokers, and 77 patients (39%) stated that they had never smoked.The healthy population consisted of 195 Shanghai Han subjects derived from an anonymous panel of blood donors (81.26 ± 9.29 years old), including 157 males (81%) and 38 females (19%). The statistical analysis revealed no significant difference between the AECOPD cohort and the control subjects in terms of age and gender .α -308G/A and the TNF-α +489G/A gene polymorphisms in the two cohorts were analyzed, respectively  in the AECOPD group were 87.37%, 10.61%, and 2.02%, respectively, while the occurrence rates of the 3 genotypes in the control group were 95.38%, 4.62%, and 0%, respectively. Besides, the frequencies of G and A in the AECOPD group were 92.68% and 7.32%, while the frequencies in the control group were 97.69% and 2.31%, respectively. The results showed a significant difference in genotype frequency of TNF-α between the AECOPD group and control group (P = 0.001), ascribing to the enhanced frequencies of GA and AA in the patient group.For TNF-α +489G/A gene polymorphism, the frequencies of the 3 genotypes  in the AECOPD group were 70.71%, 23.23%, and 6.06%, respectively, while the frequencies of the 3 genotypes in the control group were 75.90%, 20.00%, and 4.10%, respectively. The statistical analysis revealed that there was no significant difference in the occurrence distribution of the 3 TNF-α genotypes between the two groups . Besides, the frequencies of G and A in the AECOPD group were 82.32% and 17.68%, while the frequencies of G and A in the control group were 85.90% and 14.10%, respectively. There was also no significant difference discovered in the occurrence distribution of the 3 TNF-α genotypes between the two groups .For TNF-α-308 or+489 genotypes and AECOPD phenotypes, the adjacent arterial diameter and bronchial wall thickness of AECOPD patients were measured . Two key indexes of the character of AECOPD, the ratio of bronchial wall thickness to adjacent artery diameter  and bronchial wall grading, were calculated. The results showed that the patients with non-GG genotypes (TNF-α-308 and TNF-α +489) had higher bronchial artery ratio and bronchial wall grading, suggesting more serious AECOPD phenotypes (P < 0.05).To investigate the correlation of TNF-measured . As a reα -308, patients carrying non-GG genotypes showed significantly more frequent exacerbations (P = 0.001) and dyspnea (P = 0.007) than patients carrying GG genotype. Unlike TNF-α -308, there was no significant difference between the TNF-α +489AA/GA group and GG group in frequency of acute exacerbations (P = 0.324), while the patients with AA/GA genotype presented a higher ratio of cough to dyspnea than patients with GG genotype (P = 0.001). The detailed data were presented in Furthermore, for TNF-α gene genotypes affect its expression level, we firstly detected the level of TNF-α in blood samples of the AECOPD group and control group. As a result, TNF-α was overexpressed in the AECOPD group  fluid in COPD patients [α in blood was mainly due to the extensive distribution of the macrophage population in the lung, and TNF-α gene genotypes could affect the expression of TNF-α in the lung macrophages. To verify our hypothesis, the PBMCs were isolated from blood samples of AECOPD patients carrying different TNF-α gene genotypes. Then, the cells were cultured and differentiated into macrophage-like cells using PMA. The TNF-α concentration in the culture medium was detected. The result showed that the TNF-α concentration was the highest in the TNF-α -308AA group , bronchial biopsy, and sputum samples of patients with COPD and associated with an elevated risk of acute exacerbation. These findings suggest that TNF-α is potentially involved with a local and systemic inflammatory response in the COPD process and may play an important role in determining the severity of the disease. Our previous study detecting TNF-α in the exhaled breath condensate of patients with AECOPD yielded similar results. Thus, TNF-α is one of the most active inflammatory cytokines in AECOPD. The genetic factor determines the changes in protein structures, which corresponds with pathophysiological changes. Therefore, exploring the relationship of heterogeneity of COPD phenotypes and genotype is necessary and is becoming the future trend for identifying the individualized prevention and treatment programs. The gene coding for TNF-α locates in the histocompatibility complex III region on chromosome 6.It is now widely recognized that COPD is a chronic inflammatory disease, in which multiple inflammatory cells, inflammatory mediators, cytokines in the lung parenchyma, and pulmonary vasculature that ultimately extends to multiple systems in the body are involved. In recent years, TNF-α -308 gene polymorphism in AECOPD susceptibility. Meanwhile, our current study also showed that the TNF-α +489 GA and AA genotypes did not correlate with AECOPD susceptibility in Chinese patients of the Shanghai Han ethnicity, under the results of Hegab et al. [α +489G/A gene polymorphism was correlated to a higher risk of developing breathing problems. This was especially true for patients with COPD without radiographic manifestations of emphysema. Matheson et al. [α +489AA allele had a higher risk of developing breathing problems, which was associated with small airway diseases. Han et al. [α -308 and+489 on macrophages in AECOPD.Our study supported the previous reports about the role of TNF-b et al. , while cb et al. . The maib et al.  yielded n et al.  also desn et al.  found a n et al.  describeα -308 and+489 locus G > A mutation may result in a higher expression value of TNF-α in macrophage. As a proinflammatory factor, TNF-α can induce the onset of COPD. The inflammation in the airway leads to a quick infiltration of neutrophils and promotes neutrophil cell adhesion, elastic peripheral cell dissociation, and proteolytic enzyme activity, which are conducive to the formation of emphysema. TNF-α could also induce the secretion of endothelin-1 by airway smooth muscle cells, resulting in the contraction of airway smooth muscle and airway cell proliferation, causing airway remodeling. TNF-α also promotes the production of IL-6 and IL-8 by bronchial epithelial cells and alveolar macrophages, thereby increasing the damage to lung tissues. In sum, TNF-α -308 and+489 gene polymorphisms are associated with disease severity and poor prognosis of patients with AECOPD in the Chinese Shanghai Han ethnicity. The results laid a theoretical foundation for AECOPD prevention, assessment, and personalized treatment development, thus improving the prognosis of the disease.This study included subjects of the Shanghai Han ethnicity and excluded those with hypertension, diabetes, coronary heart disease, rheumatism, and other genetic diseases. Our hypothesis for the association of the gene polymorphisms and the clinical phenotypes is that the TNF-


There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. in vitro activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of Leishmania major and Leishmania mexicana. Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and Leishmania-infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the in vitro activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of Leishmania-infected macrophages.There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the  Leishmania. The parasite is transmitted between humans and mammalian reservoirs  through the bite of a female phlebotomine sand fly  the alternative pathway, mediated by Th2 cytokines like interleukin-4 (IL-4) and IL-13 in the early stages of infection, forming a favorable environment for Leishmania proliferation , and meglumine antimoniate (Glucantime) have been the standard treatment for CL for the past 70 years . These d species . Second- species –12. Treaspecies –, 14, butspecies –. Two Cocspecies –, 17. Thea of approximately 6.3 and is insoluble at alkaline pH but soluble in weak acidic solvents like acetic acid, where the amino groups become protonated. Many reports have described the antimicrobial activity of chitosan, but the actual mechanism of action has not been fully elucidated  ranging from 70 to 240 μg/ml against L. infantum, L. amazonensis, and Leishmania chagasi promastigotes and amastigotes   investigate chitosan uptake by macrophages to explain its activity against intracellular amastigotes.The aim of our work was to (i) determine the  growth) –37, (ii)Leishmania promastigotes and amastigotes was observed for chitosan and its derivatives, except for carboxymethyl chitosan, which showed no activity against either parasite stage within the experimental parameters tested (pH 7.5 or 6.5 and concentrations up to 400 μg/ml). In the 72-h assays, chitosan and its derivatives (except for carboxymethyl chitosan) were 7 to 20 times more active against L. major and L. mexicana promastigotes and intracellular amastigotes  in culture medium at pH 6.5 than at pH 7.5 (P < 0.05 by t test)  at pH 6.5 (P < 0.05 by an extra-sum-of-squares F test) (L. major promastigotes and amastigotes were significantly more sensitive to chitosan and its derivatives than L. mexicana promastigotes and amastigotes (approximately 1.5 to 2 times more sensitive [P < 0.05 by the extra-sum-of-squares F test]).The antileishmanial activities (against promastigotes and amastigotes) of high-, medium-, and low-molecular-weight  chitosan and its derivatives  were tested. Dose-dependent activity  against  t test)  and 2. H F test)  and 2. A50s were recalculated in terms of molarity, using estimated molecular weights  at pH 6.5. Based on molarity (Tables S4 and S5), HMW chitosan was significantly more active against L. major and L. mexicana promastigotes and amastigotes and, hence, was used in all subsequent studies.To allow like-for-like comparison, ECin vitro activity against L. major amastigotes in three different macrophage types; the EC50s and EC90s in the three different macrophage populations are summarized in P < 0.05 by the extra-sum-of-squares F test). HMW chitosan was significantly more active against intracellular amastigotes in PEMs and BMMs than in differentiated THP-1 cells.We aimed to assess the host cell dependence of the antileishmanial activity of HMW chitosan and amphotericin B (Fungizone) by evaluating the Leishmania-infected and uninfected cells, as shown in P < 0.05 by t test).The activation of M1 macrophages by Th1 lymphocytes plays an important role in the control of CL , 38, 39.L. major-infected BMMs. Less TNF-α was generated when the chitosan concentration was increased to 133.3 μg/ml and above. Lipopolysaccharides (LPS) from Escherichia coli O26:B6 (positive control) stimulated TNF-α production in both uninfected and infected BMMs after a 24-h incubation period at a significantly higher level than did chitosan (P < 0.05 by t test). Our results indicated that HMW chitosan activated M1 macrophages.HMW chitosan at concentrations of 14.8, 44.4, and 133.3 μg/ml stimulated BMMs to produce TNF-α at concentrations of 87 ± 4.5 (mean ± standard deviation [SD]), 712 ± 9, and 48 ± 3 pg/ml, respectively, in uninfected BMMs and 56 ± 3.5, 464 ± 10, and 32 ± 4 pg/ml, respectively, in 2DCFDA  fluorescence) after 4 h of incubation but did not stimulate ROS after 8 h of incubation (Table S1). Other concentrations of HMW chitosan  did not stimulate BMMs to produce ROS after 4 h or 8 h of incubation.ROS plays an important role in the killing of intracellular amastigotes , 38, 39,P < 0.05 by t test) by infecting the cells with L. major, as shown by the results in The ROS dose response in both uninfected and infected BMMs was bell shaped, similar to that seen with TNF-α. Increasing the chitosan concentration from 14.8 to 44.4 μg/ml increased ROS production, after which further increases in concentration reduced ROS production. In addition, ROS production by BMMs was significantly decreased . Scavenging of ROS by the ROS scavenger 5 mM N-acetyl-l-cysteine (NAC) had no significant impact on the activity of chitosan against intracellular amastigotes (P > 0.05 by t test) (L. major amastigotes (data not shown). Even though chitosan stimulated ROS production, ROS did not play a role in the antileishmanial activity of chitosan.We found that  t test) . The ROSNO plays an important role in the killing of intracellular amastigotes , 38, 39,P < 0.05 by t test) in both uninfected and infected BMMs. The levels of NO produced by L. major-infected BMMs exposed to LPS (positive control) or HMW chitosan were significantly lower than the levels produced by uninfected BMMs (P < 0.05 by t test) (LPS caused significantly higher NO production than did HMW chitosan ( t test) .in vitro stimulatory effect on BMM NO production after 24 h of incubation, we investigated further whether NO has any role in the activity of HMW chitosan against intracellular amastigotes. Inhibition of NO production by the NO inhibitor NG-methyl-l-arginine acetate salt (L-NMMA) at 0.4 mM had no significant influence on the activity of chitosan against intracellular amastigotes (P > 0.05 by t test) (L. major amastigotes (data not shown). Even though chitosan stimulated NO production, it did not play a role in the antileishmanial activity of chitosan.As HMW chitosan had an  t test) , althougP > 0.05 by t test) (P < 0.05 by t test) (L. major amastigotes at the concentrations used. Pinocytosis (CME) played a critical role in the efficacy of HMW chitosan against intracellular amastigotes.We found that the activation of M1 macrophages by HMW chitosan did not play a role in its activity against intracellular amastigotes. Therefore, we investigated whether the antileishmanial effects of HMW chitosan against intracellular amastigotes after 4 h and 24 h of exposure were dependent on the direct activity of chitosan following its entry into the macrophages at pH 6.5. No significant difference was observed in the activity of chitosan against intracellular amastigotes when it was added after prior phagocytosis inhibition with cytochalasin D ( t test) . In cont t test) . The sam t test) . The twoLeishmania-infected macrophages. L. major (L. major-GFP) or L. mexicana-GFP after 4 h and 24 h of exposure to rhodamine-labeled chitosan. There was colocalization of chitosan and intracellular amastigotes after 4 h and 24 h, with normalized mean deviation product (nMDP) color indexes of 0.7 and 1, respectively . The uptake of chitosan increased in a time-dependent manner. The results in Rhodamine-labeled chitosan was used to track the delivery of chitosan to the parasitophorous vacuoles (PVs) of 50s) of ≥750 μg/ml in RPMI 1640 at pH 7.5 or 6.5. These data support previous reports of chitosan’s low cytotoxicity against CCRF-CEM (human lymphoblastic leukemia) and L132 (human embryonic lung) cells that had similar LD50s  of action , 40, 41.ar LD50s , 43.L. major and L. mexicana promastigotes and amastigotes by 7 to 20 times compared to the results at pH 7.5. This higher activity of chitosan at the lower pH of 6.5 could be due to its greater ionization . The greater positive charge could increase the antimicrobial activity of chitosan due to interaction with the negatively charged microbial membrane   , 19. A hhimurium , 45.50s ranging from 70 to 240 μg/ml against L. infantum, L. amazonensis, and L. chagasi promastigotes and amastigotes (–50 of chitosan against L. infantum amastigotes (in PEMs) in RPMI 1640 medium was 100.81 μg/ml, but the pH at which the experiment was conducted was not mentioned  than MMW and LMW chitosan have  of chitosan, we investigated whether the activity of HMW chitosan against the intracellular amastigotes was via direct uptake into the host cell and localization in the parasitophorous vacuole or indirectly via the activation of M1 macrophages, given that the cellular immune responses in cutaneous leishmaniasis play a critical role in self-cure , 52.Leishmania via the triggering of an oxidative burst, and therefore, the host cells increase the production of ROS and NO, which are responsible for killing of the parasite    and LMW le rats) . Anotherle rats) . The belle rats) . This tyer doses . Despitestigotes  and 5. TLeishmania parasites reside, with the labeled chitosan being internalized within 4 h and increasing up to 24 h later. This is consistent with another study where rhodamine isothiocyanate-chitosan (molecular weight of 113 kDa was labelled with RITC) was found to be delivered directly to the U937 macrophage lysosome after 24 h . HMW chitosan demonstrated effective in vitro antileishmanial activity with minimal cytotoxicity, and future work will focus on in vivo studies, formulations, and routes of administration.In summary, our studies indicate that chitosan and its water-soluble derivatives showed antileishmanial activity against both a is approximately 6.3, and therefore, the approximate degrees of ionization of chitosan are 61% and 6% at pH 6.5 and 7.5, respectively.Stocks of amphotericin B deoxycholate (5.2 mM [aqueous])  were prepared, aliquoted, and kept at −20°C until use. Chitosan with three different molecular weights and its derivatives were used and are summarized in All animal work was carried out under a UK Home Office project license according to the Animal (Scientific Procedures) Act 1986 and the new European Directive 2010/63/EU. The project license (70/8427) has been reviewed by the LSHTM Animal Welfare and Ethical Review Board prior to submission and consequent approval by the UK Home Office.g at 4°C for 15 min, and then the pellet was resuspended in RPMI 1640 with 10% (vol/vol) heat-inactivated fetal calf serum .Peritoneal mouse macrophages (PEMs) were obtained from 8- to 12-week-old female CD-1 mice . Two milliliters of a 2% (wt/vol) starch solution in phosphate-buffered saline  was injected intraperitoneally. After 24 h, the animal was sacrificed and the PEMs were harvested by peritoneal lavage with cold RPMI 1640 medium  containing 200 units penicillin and 0.2 mg streptomycin per milliliter . Subsequently, PEMs were centrifuged at 450 × g for 10 min) and resuspended in 10 ml DMEM with 10% (vol/vol) HIFCS and 50 ng/ml human macrophage colony-stimulating factor . After plating out in T175 flasks , BMMs were kept at 37°C, 5% CO2 for 7 to 10 days, after which they were harvested, counted, and used.Bone marrow-derived macrophages (BMMs) were obtained from femurs of 8- to 12-week-old female BALB/c mice (Charles River Ltd.). Briefly, the bone marrow cells were carefully flushed from the bone with Dulbecco’s modified Eagle’s medium  with 10% (vol/vol) HIFCS, 100 U/ml penicillin, and 100 mg/ml streptomycin . Cells were pelleted by centrifugation (450 × l-glutamine and 10% HIFCS. THP-1 cells were incubated in RPMI 1640 plus 10% (vol/vol) HIFCS and 20 ng/ml phorbol 12-myristate 13-acetate  at 37°C and 5% CO2 for 72 h to induce maturation transformation of these monocytes into adherent macrophages (THP-1 cells are a human leukemic monocyte-like-derived cell line. THP-1 cells were cultured in RPMI 1640 medium supplemented with rophages .Human squamous carcinoma (KB) cells are adherent cells derived from epidermal carcinoma in the mouth. KB cells were cultured in RPMI 1640 medium with 10% HIFCS.The numbers of cells and macrophages were estimated by counting with a Neubauer hemocytometer by light microscopy .Leishmania strains were used, two of which  were GFP labeled for the fluorescence microscopy study. They were cultured in Schneider’s insect medium  with 23% (vol/vol) HIFCS, 1× penicillin-streptomycin-glutamine (Gibco-Invitrogen) and supplemented with 700 μg/ml G418 . L. major (MHOM/SA/85/JISH118) and L. mexicana (MNYC/BZ/62/M379) were used for other experiments without the G418. Promastigotes were incubated at 26°C, and the maximum passage number used was 7.Four 4/100 μl) were allowed to adhere to the bottom of a 96-well plate overnight and then exposed to specific concentrations of the tested compounds for 72 h at 37°C and 5% CO2 in an incubator. Podophyllotoxin  was included as a positive control at a starting concentration of 0.05 μM. Cytotoxicity was evaluated by a cell viability assay using resazurin sodium salt solution , which was prepared according to the manufacturer’s instructions. Twenty microliters of the resazurin solution was added to each well of the plates, and fluorescence (cell viability) (x)% viability (mean ± standard deviation [SD] σ). Cytotoxicity was evaluated in RPMI 1640 at two pHs . The pH of RPMI 1640 was reduced from 7.5 to 6.5 by adding 0.05 M acidic buffer 2-N-morpholino ethanesulfonic acid . RPMI 1640 plus MES (0.05 M) at pH 6.5 did not show any cytotoxicity to KB cells.Resuspended KB cells (4 × 10ability)  was meas6 promastigotes/ml and then exposed to different concentrations of HMW, MMW, and LMW chitosan, chitosan derivatives, and amphotericin B (positive control) in sterile 96-well flat-bottom culture plates for 72 h at 26°C. The activity of the compounds against promastigotes was evaluated using the resazurin sodium salt solution  as described above. pH plays a critical role in the solubility and protonation of chitosan, so the activity against promastigotes was evaluated at two different pHs (pH 7.5 and a lower pH of 6.5 obtained by adding MES). The results were expressed as follows: percentage of inhibition = 100% − x% viability (mean ± SD).Promastigotes in RPMI 1640 medium were tested while in the exponential growth phase. The promastigotes were diluted to a density of 5 × 105 cells/ml was dispensed into each well of a 16-well LabTek tissue culture slide  at pH 7.5 or pH 6.5 and incubated for 24 h at 37°C in 5% CO2. After 24 h, the wells were washed with fresh culture medium to remove nonadherent cells. Stationary-phase, low-passage-number Leishmania promastigotes were then added to PEMs at a ratio of 5:1. This infection ratio was previously found to give sufficiently high and reproducible infection levels. Slides were incubated for another 24 h at 34°C to mimic dermal temperatures in 5% CO2. Any free, extracellular parasites were removed by washing the wells with cold culture medium. One slide was fixed with 100% methanol for 2 min and stained with 10% Giemsa stain for 5 min. The number of PEMs infected with Leishmania amastigotes per 100 macrophages was counted under a microscope. All the experiments were conducted at macrophage infection levels of above 80% prior to the addition of chitosan. Chitosan, its derivatives, and amphotericin B solutions at a range of concentrations (in quadruplicate) were added to the wells (100 μl), and the slides were incubated for 72 h at 34°C in 5% CO2. After 72 h, the slides were fixed with 100% methanol for 2 min and stained with 10% Giemsa stain for 5 min. The slides were examined, and the percentages of macrophages infected were determined. The antileishmanial activities of compounds were expressed as the percentages of reduction in infected macrophages compared to the numbers of infected macrophages in untreated control wells (Leishmania amastigotes.One hundred microliters of PEM culture at 4 × 10ol wells . RPMI 16Leishmania major and the activity of HMW chitosan was assessed. THP-1 cells (cultured in RPMI 1640 plus 10% HIFCS) and BMMs (cultured in DMEM plus 10% HIFCS) were used to assess the host cell dependence of the antileishmanial activity of HMW chitosan  in DMEM at pH 6.5 was dispensed into each well of 96-well plates  and incubated for 24 h at 37°C in 5% CO2. Plates were washed with DMEM to remove nonadherent macrophages. L. major at a 1:5 ratio (5 parasites per host cell) was then added to the wells, and the plates were incubated for 24 h at 34°C in 5% CO2 to allow infection of the adherent macrophages. After 24 h of incubation with macrophages, the infection rate was more than 80%. The effects of HMW chitosan on BMM activation at pH 6.5 were determined by quantifying the release of TNF-α, ROS, and NO as described below.We chose BMMs to evaluate the activation effects of HMW chitosan and to study the cell uptake of chitosan as this macrophage population is more homogenous than those of PEMs and THP-1 cells ; both PEed cells –67. One (i) Measurement of TNF-α. HMW chitosan at concentrations of 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml was added to infected and uninfected macrophages (see “Uptake of chitosan by macrophages” above), and the plates were incubated for 4 or 24 h at 34°C in 5% CO2. Lipopolysaccharides from Escherichia coli O26:B6  were used as a positive control and inducer. TNF-α release by the BMMs was measured using a mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions, using a SpectraMax M3 microplate reader (wavelength 450 nm).(ii) Measurement of ROS. ROS was measured using a 2′,7′-dichlorofluorescein diacetate (DCFDA) cellular reactive oxygen species detection kit . Uninfected and infected macrophages were treated with 25 μM DCFDA in PBS for 45 min at 37°C and then washed once in the buffer. The cells were cultured at 34°C in 5% CO2 for 0.5, 1, 2, 4, 8, and 24 h, with 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml of HMW chitosan or in the presence of H2O2 (25 mM)  as a positive control, in DMEM plus 10% HIFCS (pH 6.5) in quadruplicate wells. In some experiments, cells were pretreated with a selective inhibitor of ROS, N-acetyl-l-cysteine (NAC)  for 2 h before the addition of the inducer or chitosan. At 0.5, 1, 2, 4, 8, and 24 h, the plates were read, using a SpectraMax M3 microplate reader (excitation/emission at 485/535 nm).(iii) Measurement of NO. NO was measured using Griess reagent . HMW chitosan at concentrations of 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml was added to infected and uninfected macrophages, and the plates were incubated at 34°C in 5% CO2 for 4 and 24 h. LPS (100 ng/ml) was used as a positive control. In some experiments, cells were pretreated with the selective inhibitor of nitric oxide NG-methyl-l-arginine acetate salt (L-NMMA)  for 2 h before the addition of LPS. NO was quantified according to the kit protocol. Briefly, 150 μl of the cell culture supernatants (particulates were removed by centrifugation) was mixed gently with 150 μl of the Griess reagent in a 96-well plate and the mixture was incubated for 30 min at room temperature. The absorbance was measured using a SpectraMax M3 plate reader (wavelength 548 nm). Sodium nitrite  at different concentrations was used to create a standard curve , which is a phagocytosis inhibitor, and dynasore , which inhibits pinocytosis (clathrin-mediated endocytosis [CME]) by blocking the GTPase activity of dynamin –71. The –(i) Activity of chitosan after inhibition of the endocytic pathway of BMMs. One hundred microliters of BMM culture (4 × 105/ml) in DMEM at pH 6.5 or pH 7.5 was dispensed into each well of 16-well LabTek culture slides and infected with stationary-phase L. major promastigotes. Some of the infected BMMs were pretreated with dynasore (30 μg/ml) or cytochalasin D (1 μg/ml) for 2 h. Subsequently, HMW chitosan was added to each well at concentrations of 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml and macrophages were incubated for 4 or 24 h at 34°C in 5% CO2. After each time point, the slides were examined as described above for the assay of chitosan activity against intracellular amastigotes of L. major and L. mexicana. The inhibition activity of the uptake (phagocytosis or pinocytosis) of the two inhibitors was evaluated on a fluorescence plate reader using fluorescent latex beads and pHrodo red dextran  after 4 h and 24 h, respectively . dextran . We show(ii) Microscopic imaging of the cellular uptake of rhodamine-labeled chitosan. The qualitative characterization of chitosan uptake of cells was carried out by wide-field microscopy (Nikon Ti-E inverted microscope). Briefly, after deriving BMMs, 500 μl of the BMMs  was seeded in each well of a 4-well LabTek tissue culture slide  and incubated for 24 h at 37°C in 5% CO2. Subsequently, 5 μg/ml of the nuclear dye Hoechst 33342  was added and the slides were incubated for 30 min at 37°C in 5% CO2. The macrophages were washed with PBS, and then L. major-GFP or L. mexicana-GFP parasites were added at a ratio of 10:1 and further incubated for 24 h at 34°C in 5% CO2 . Macrophages were then washed with PBS and 500 μl of LysoTracker deep red  was added to each well. The labeled, infected macrophages were then exposed to 30 μg/ml rhodamine-labeled chitosan  in 500 μl of fresh DMEM plus 10% HIFCS, pH 6.5, and incubated for 4 and 24 h at 37°C with live imaging at each time point. In some experiments, infected BMMs were preincubated with dynasore 30 μg/ml for 2 h before adding rhodamine-labeled chitosan. All the images were collected using a Nikon Ti-E inverted microscope equipped with a 63× objective using Nikon Elements software. Three images for each experiment were then analyzed using ImageJ software. The degree of correlation between pixels in the red and green channels was assessed by using the Colocalization Colormap plugin in the ImageJ software. This plugin enables quantitative visualization of colocalization by calculating the normalized mean deviation product (nMDP) in a color nMDP scale (from −1 to 1): negative indexes (cold colors) refer to no colocalization, while indexes of more than 0 (hot colors) display colocalization and higher numbers refer to greater colocalization (lization , 74.50s were calculated using GraphPad Prism version 7.02 software, and the corresponding sigmoidal dose-response curves were established by using a nonlinear fit with variable slope models. Results are given as mean values ± SD. EC50s were compared by using extra-sum-of-squares F tests. The t test was used to compare differences between means of two or more groups, and P values of 0.05 were considered statistically significant.Dose-response curves and EC


Matricaria chamomilla. The aim of the present study was to investigate the amelioration function of API in APAP-induced hepatotoxicity both in vitro and in vivo and investigate its potential mechanisms. Analysis results of the activities of serum alanine and aspartate aminotransferases (ALT and AST), malondialdehyde, myeloperoxidase (MPO), and reactive oxygen species (ROS) demonstrated therapeutic effects of API. MTT assay results revealed that API attenuated APAP and its metabolic product, N-acetyl-p-benzoquinone imine (NAPQI) induced cytotoxicity in a dose-dependent manner in human liver cells, L-02 cells. Subsequently, metabolomic results of cells and serum analyses demonstrated an aberrant level of carnitine palmitoyltransferase I (CPT1A). We established that API stimulated CPT1A activity in mice liver tissues and L-02 cells. Molecular docking analyses revealed potential interaction of API with CPT1A. Further investigation of the role of CPT1A in L0-2 cells revealed that API reversed cytotoxicity via the AMP-activated protein kinase (AMPK)/GSK-3β signaling pathway and compound C, which is a selective AMPK inhibitor, inhibited activation of CPT1A induced by API. API was bound to the catalytic region of AMPK as indicated by molecular docking results. In addition, compound C suppressed nuclear translocation of nuclear factor erythroid 2–related factor 2 (NRF2) that is enhanced by API and inhibited the antioxidative function of API. In summary, the study demonstrates that API attenuates APAP-induced hepatotoxicity by activating the AMPK/GSK-3β signaling pathway, which subsequently promotes CPT1A activity and activates the NRF2 antioxidant pathway.Overuse of acetaminophen (APAP) is a major cause of drug-induced liver failure at the clinics. Apigenin (API) is a natural flavonoid derived from  Acetaminophen  is a widely used over-the-counter analgesic and antipyretic drug. While APAP has a few toxic side effects at normal doses, prolonged or excessive use of APAP can lead to inflammatory infiltration, degeneration, and necrosis of hepatic lobules in animals and humans (ADDIN EN.CITE). An overdose of APAP is the primary cause (over 60%) of drug-induced liver injury (DILI), which is a leading cause of acute liver failure (ALF) in developed countries .via the activity of CYP450 enzyme (mainly CYP2E1 in human body), which induces hepatotoxicity  toxicity . Althougtoxicity , once antoxicity . The suptoxicity , which ctoxicity . The pritoxicity . Howevertoxicity . TherefoMatricaria chamomilla with high antioxidant capacity. Previous studies have revealed that flavonoids can protect against different types of hepatopathy through anti-inflammatory and antioxidant mechanisms, especially hepatopathy that is associated with AP-induced liver damage (Apigenin (API) or versulin  is a natural flavonoid derived from r damage . In addir damage , lipopolr damage , and iscr damage . In the in vivo  was subsequently administered orally 4 h after treatment with APAP, and liver tissues and serum were collected 4 h after treatment with API. Afterward, a series of cellular damage and oxidative stress injury parameters were examined. APAP increased alanine aminotransferase and aspartate aminotransferase activities, liver malondialdehyde (MDA), myeloperoxidase (MPO), and reactive oxygen species (ROS) levels, whereas treatment with API reversed APAP-induced hepatotoxicity in vivo . H&E stain vivo . Besidesin vivo . To explin vivo .in vivo and in vitro.Subsequently, a cellular APAP hepatocyte toxicity model was constructed by incubating different concentrations of API in normal human liver cells, L-02, for 15 min, followed by incubation of APAP or its metabolite, NAPQI, for 24 h. Cell viability of L-02 cells was evaluated using MTT assay, and the results revealed that API not only enhanced hepatotoxicity caused by APAP and its metabolites but also enhanced hepatotoxicity caused solely by NAPQI, both in a dose-dependent manner . ROS res2X and Q2Y values for Principal Component Analysis (PCA) performed on cell and serum samples in positive and negative modes were over 0.741 and 0.781, respectively  was used for assessment. Finally, 77 different metabolites in serum samples and 51 metabolites in cell samples were identified by searching MS libraries. The treatment trend of API is presented in OPLS-DA was performed using APAP and control groups to determine potential differential metabolites, and variable importance in projection (VIP >1) and p < 0.01) were analyzed by MetaboAnalyst to enrich potential pathways. Results of the analyses revealed that carnitine synthesis and oxidation of fatty acids played crucial roles in APAP-induced liver injury  were examined to verify the relationship between API and CPT1A. Results of the analyses revealed that API enhanced CPT1A activity, mRNA expression of CPT1A, and protein expression of CPT1A in liver tissues, although treatment with APAP alone had no effect , which din vitro in a dose-dependent manner. Therefore, we established that API could significantly increase the expression of CPT1A.Furthermore, CPT1A activity, mRNA expression, and protein expression in L-02 cells were evaluated, and the results are presented in A computational protein/molecule docking analysis was performed to further investigate the relationship between API and CPT1A. A three-dimensional (3D) interaction map revealed that API interacted with the active conformation of CPT1A. API can interact with the active site of CPT1A with a binding energy of −7.6 kcal/mol to form four strong hydrogen bonds with amino acid residues including GLY 244, GLY 246, TYR 254, and TYR 241 of CPT1A ; Table1.The results have demonstrated that API can directly bind to CPT1A and subsequently promote its activity. Previous studies have demonstrated that AMP-activated protein kinase (AMPK) can activate CPT1A to protect the liver. in vitro and in vivo.Western blot results revealed that API can activate the AMPK pathway in mice liver and activate phosphorylation of AMPK and GSK, which is also part of the pathway. Further experiments on L-02 cells demonstrated that API activated p-AMPK in a dose-dependent manner, which suggested that API activated the AMPK pathway both in vivo and in vitro activities.Molecular docking analyses further demonstrated that API interacted with the active site of AMPK with a binding energy of −9.0 kcal/mol . API forCompound C, an inhibitor of AMPK , was useNuclear factor erythroid 2–related factor 2 (NRF2) is a transcription factor that regulates cellular defense by inducing the expression of various detoxiﬁcation and antioxidant genes , and AMPin vitro data. Compound C decreased the phosphorylation level of AMPK and GSK-3β, as well as inhibited the levels of CPT1A and nuclear translocation of NRF2 . During the period, APAP could have been completely metabolized to form NAPQI in mice , RPMI1640, and fetal bovine serum (FBS) were purchased from Life Technology . NE-PER nuclear, cytoplasmic extraction reagents, and a Pierce BCA Protein Assay Kit were purchased from Thermo Fisher Scientific . A whole-cell protein extraction and enhanced chemiluminescence kits were obtained from Millipore . PrimeScript RT Master Mix and SYBR Premix Ex Taq were purchased from TaKaRa . Antibodies for immunoblotting, including anti-actin, -CPT1A, -Phospho-AMPKα1, -AMPKα1, -Phospho-GSK-3β, -GSK-3β, -NRF2, and -LaminB were purchased from Cell Signaling Technology . Methanol and acetonitrile (HPLC grade) were purchased from Thermo Fisher Chemicals . APAP, NAPQI, and compound C were purchased from Sigma-Aldrich . Fenclonine was purchased from Aladdin .API (purity >98.5%) was purchased from Meilune . Kits for ROS, MDA, and MPO were purchased from Nanjing Jiancheng Bioengineering Institute . Lipofectamine RNAiMAX, 2′-7′-dichlorodihydrofluorescein diacetate (HC57BL/6J mice (20 g ± 2) were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences . Experimental animals were given water and fed on ad libitum diet. Animals were housed in rooms maintained at a constant temperature of 20–25°C and a humidity of 65 ± 5% with a 12-h light–dark cycle. All animals received humane care in compliance with the institutional animal care guidelines approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine. The protocol was reviewed and approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine (Permit Number: PZSHUTCM200327006). All surgeries were performed under sodium pentobarbital anesthesia, and efforts were made to minimize suffering.Twenty-four mice were divided randomly into four groups: 1) control group (vehicle), 2) model group (APAP = 300 mg/kg), 3) treatment group (APAP = 300 mg/kg + API = 20 mg/kg), and 4) APAP (300 mg/kg) + API (80 mg/kg) group. APAP or saline (vehicle) was orally administered to the mice after depriving them of food and water for 12 h, which was followed by administration of API (20 or 80 mg/kg) after 4 h. Mice were euthanized 8 h after APAP administration (4 h after API administration), and blood and liver samples collected.To evaluate the role of time course in APAP-induced hepatotoxicity, mice were divided randomly into three groups: Twenty-four mice were divided randomly into four groups: 1) control group (vehicle), 2) model group (APAP = 300 mg/kg), and 3) treatment group (APAP = 300 mg/kg + API = 80 mg/kg). APAP or saline (vehicle) was orally administered to the mice after depriving them of food and water for 12 h, which was followed by administration of API (80 mg/kg) after 4 h. Mice were euthanized 4 and 16 h after APAP administration (0 and 12 h after API administration), and blood and liver samples were collected.To evaluate the role of AMPK in regulating APAP-induced hepatotoxicity, mice were divided randomly into six groups: 1) DMSO, 2) DMSO + APAP (300 mg/kg), 3) DMSO + APAP (300 mg/kg) + API (80 mg/kg), 4) compound C (10 mg/kg), 5) compound C (10 mg/kg) + APAP (300 mg/kg), and 6) compound C (10 mg/kg) + APAP (300 mg/kg) + API (80 mg/kg). Mice were injected intraperitoneally with compound C after depriving them of food and water for 12 h, which was followed by administration of APAP or saline 1 h after administration of compound C. Afterward, API was administered 5 h after treatment with compound C. Mice were euthanized 9 h after compound C administration, and blood and liver samples were collected.μM) for 15 min and finally incubated with APAP or NAPQI for 48 h. After treating cells with 500 μg/ml MTT for 4 h, the resulting formazan blue was dissolved in 10% SDS, 5% isobutanol, and 0.01 M HCl, and plates were scanned using a microplate reader  at a wavelength of 570 nm, with 630 nm as a reference wavelength. Cell viability was standardized as a percentage of the control.The L-02 cell lines were purchased from the Shanghai Institute of Cell Biology  and cultured in DMEM with FBS and 1% antibiotics. Cells were placed into 96-well plates at an initial density of 5,000 cells per well. After attachment, cells were pretreated with API or compound C . Protein concentrations of the supernatants were quantified using BCA protein assay kits, calculated as units of fluorescence per microgram of protein, and concentrations presented as percentages of controls (% control).ROS in cells was measured using an H2DCFDA probe as described previously . To meashttps://www.swissmodel.expasy.org/), and ligand binding sites of the proteins were predicted by POCASA 1.1 in which the principle was consistent with AutoDock software . One-way ANOVA with least significant difference The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All animals received humane care in compliance with the institutional animal care guidelines approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese MedicineJZ, XL, JL, and HY performed the experiments. CH and LL designed the research study. JZ and FL collected the data. JZ and CH analyzed the data. JZ and XL wrote the manuscript.This work was financially supported by the Natural Science Foundation of Shanghai (19ZR1451800), Budget of Experiment Center for Science and Technology (18LK022), and Guoyiqiangyou Foundation of Shanghai Hongkou (No. HGY-KY-2018–03 and HGY-MGB-2018–01–05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9–Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019–1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9–Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9–Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control. Neospora caninum, an obligate intracellular protozoan, belongs to Apicomplexa phylum, such as Plasmodium spp and Toxoplasma gondii. It causes neosporosis, a disease strongly correlated to abortion and decreased fertility in cattle, costing billions of dollars worldwide3. There is no effective commercial treatment for neosporosis, despite the efforts of several chemotherapy-focused groups6. For example, Artemisinins10, Miltefosine11, plant extracts14, Diamidines16, Buparvaquone17, organometallic ruthenium complexes18, Thiazolides20 were evaluated in in vitro and in vivo models with encouraging results. Moreover, polyether ionophore antibiotics21, Triazinones24, bumped kinase inhibitors25 have also been tested in farm ruminants26.Plasmodium spp, the etiologic agent of malaria, our group determined the efficacy of this molecule on N. caninum, either alone or combined with Pyrimethamine (Pyr)27. MB, a phenothiazinium dye, was the first synthetic drug described to cure a patient affected by malaria in the XIX century, pioneering work performed by Paul Ehrlich29. Indeed, MB was used against Plasmodium until the use of Chloroquine and other drugs (massively utilized after the Second World War), which lack some of the reversible MB side effects (blue urine and sclera)31. During the later 20th/early 21st Centuries, the intense use of Chloroquine, Artemisinin, and Pyrimethamine has led to proven cases of Plasmodium resistance, demanding novel therapeutic candidates32. Interestingly, no reports of Plasmodium resistance have been reported in experimental MB-based therapies, reviving the dye as an antimalarial drug33. Moreover, phenothiazinium dyes such as New Methylene blue and Toluidine Blue O also demonstrated antimicrobial properties35, inspiring us to test these compounds against N. caninum.Based on the promising effects of Methylene Blue (MB) against N. caninum, using in vitro and in vivo models.Ehrlich’s brilliant observations, concerning Methylene Blue suggest that other phenothiazinium derivatives  may also provide safe, low-cost candidates to treat parasitic diseases. Since the livestock industry is particularly sensitive to costs, inexpensive formulations based on phenothiazinium dyes have the potential to improve the outcomes in cattle affected by neosporosis. To this end, we have investigated the anti-parasitic effects of the phenothiaziums Methylene Blue, New Methylene Blue, 1,9-Dimethyl Methylene Blue and Toluidine Blue O on 36. MB inhibits N. caninum proliferation, with an IC50 of 0.349 μM  probably improves the selective pressure, generating resistant parasites. On the other side, phenothiazinium dyes have multiple targets of inhibition in Apicomplexa, as demonstrated in Plasmodium33. Although the complete mechanism of action of MB and analogs in Plasmodium is unclear, the dye inhibits the Glutathione Reductase enzyme38, the polymerization of heme into hemozoin39 and also acts as a subversive substrate of antioxidant disulfide reductases40. Nevertheless, future assays related to the mechanism of action of dyes in N. caninum will be mandatory to elucidate the lower inhibitory/resistance induction of NMB compared to MB, TBO, and DMMB. Once all tested compounds induced parasite resistance, the use of single-drug therapies is potentially unfeasible for N. caninum using phenothiazinium dyes or Pyr. Indeed, for T. gondii, the use of drug combinations is a current and efficient strategy to minimize parasite resistance and recrudescence41.Compared to the phenothiazinium dyes (excepting NMB), Pyr demonstrated a delayed resistance induction Fig. , which min vitro clearance of N. caninum. TBO was excluded from the clearance assay due to its high inhibition dose: a toxic effect to the host cells would affect the interpretation of the results. For example, concerning the toxic values in cells (CC50), the concentrations applied in clearance assays (4 × IC50) were lower for MB (1.6 μM), NMB (0.2 μM) and DMMB (0.08 μM) compared to TBO (~ 7.5 μM). Moreover, the value of CC50/4 × IC50 for TBO was 3.3 whereas, for MB, NMB and DMMB were 39, 109.4 and 106.5, respectively.The phenothiazinium dyes demonstrated different patterns for the 27, DMMB and NMB eliminated all extracellular tachyzoites after the fourth cycle  is strongly recommended for malaria treatment due to the emergence of Art-resistant parasites42. Likewise, a similar approach may be considered for N. caninum. Assays related to the mechanism of action of phenothiazinium dyes and Pyr are necessary, as there is a clear difference when compounds are tested in proliferation and clearance assays. Moreover, an adequate description of the mechanism of action of phenothiaziniums is also important to establish future formulations against neosporosis, prepared to act in a specific phase of the disease .As observed previously for MBcle Fig. . Howevercle Fig. . The comely Fig. . No toxiely Fig. , despite45. However, the inhibitory concentrations for yeast, bacteria or mammalian cells are usually above 1 μM46, whereas against N. caninum the oxidant effects were observed at nanomolar levels  when incubated with phenothiazinium dyes at 4 × IC50. Vero cells incubated with tested compounds at 4 × IC50 doses proliferate normally as verified in compound-free controls (data not shown).For nds Fig. . The higtly Fig. , but pronum Fig. . The comlls Fig. , indicatN. caninum infected or non-infected cells with phenothiazinium dyes demonstrated a different pattern in mitochondrial potential compared to Pyr  altered the parasite organization in vacuoles, indicating a widespread effect in the parasite/vacuole structure  with deleterious effects usually observed in long term treatments (five weeks) using doses >100 mg/Kg/day53. However, no studies have ever been performed for assessing the in vivo toxic effects of DMMB, demanding complementary toxicological assays. In a murine model of malaria, it was estimated that a regimen using MB 45.77 mg/Kg/day eliminates 90% of Plasmodium berghei36. Moreover, the effectiveness of intraperitoneal MB (50 and 100 mg/Kg/day) to control the blood stage of Plasmodium yoelii in BALB/c mice was also demonstrated54. Thus, for the initial treatment of animals acutely infected with N. caninum, a dose of 50 mg/Kg/day (MB and/or DMMB) represented a rational choice. We used cerebral neosporosis as a first approach once the main features of the compounds  may be assessed before the use of more complex models.The clearance assay is a more suitable method to guide a rational strategy for the treatment of neosporosis in animal models. The clearance assay reveals the elimination of parasites from cell culture, whereas proliferation measures the capacity of tachyzoite multiplication in the presence of the compound. Therefore, the tes Fig. . TBO andN. caninum infection, 2 out of 6 animals treated with PBS died. Under treatment with DMMB 1 out of 6 died after the seventh and eighth days. The DMMB + Pyr combination demonstrated a similar result, with 2 animals succumbing after the eighth day. However, all animals (6/6) were protected by the combination of DMMB with MB  demonstrated superior performance compared to combinations based on compounds belonging to different categories (DMMB and Pyr), indicating possibly more than one mechanism of action among phenothiazinium dyes against N. caninum. Once the synergistic phenomena observed in the proliferation (NMB + MB) and in vivo approaches (DMMB + MB) have been not observed in Apicomplexa, further studies will be required. No toxic effects were observed  in treated animals for 30 days. However, complementary assays such as dose-dependent treatment, use of alternative routes of administration and/or infection will be required to determine a safe application level of these compounds/combinations for clinical use. The data from the acute model of infection, as applied in this study, is an initial approach and usually will be useful for future testing in chronic and congenital models.On the sixth day after  MB Fig.  and no e MB Fig. . The comin vitro or in vivo respectively, which is usually observed when effective compounds from different families are combined58. Thus, further work will be performed to investigate the common and exclusive targets of phenothiazinium dyes. Moreover, the effectiveness, pharmacodynamics, half-life, and clearance of each compound (and the combinations) are fundamental points for the design of future clinical strategies. Despite the absence of toxic effects in the host cell in all assays using MB, NMB and DMMB, complementary strategies to clarify the side effects of these compounds and formulations will be demanded. Since there is no data concerning the use of phenothiazinium dyes for the treatment of food animals, our study describes a new potential form to control neosporosis. The use of low-cost compounds, such as phenothiazinium dyes against neosporosis, represents a suitable form for livestock management, improving the productivity of the cattle and impacting the economy of beef and dairy industry worldwide.The combinations of MB with NMB or DMMB demonstrated synergism N. caninum Nc1 expressing β-galactosidase (NcLacZCAT)59 was used in proliferation and clearance assays. For oxidative stress assays, Nc1 purified tachyzoites (using 5 μm syringe filtration) were employed. In the in vivo model of infection, the virulent N. caninum Liverpool strain (NcLiv) was used. NcLiv was cultivated in Vero cell monolayers for 30 passages when it was inoculated in C57Bl/6 mice to improve its virulence. The in vivo assay was performed using NcLiv cultures at the 20th passage.in vitro use. For in vivo application, DMMB, MB and Pyr were diluted (2.5 mg/ml) in PBS.Methylene Blue ([7-(dimethylamino)phenothiazin-3-ylidene]-dimethylazanium;chloride, catalogue number: 1428008), New Methylene Blue (ethyl-azanium;chloride, catalogue number: R313718), Toluidine Blue O ((7-amino-8-methylphenothiazin-3-ylidene)-dimethylazanium;chloride, catalogue number: T3260), 1,9 – Dimethyl-Methylene Blue (-dimethylazanium;chloride, catalogue number: 341088) and Pyrimethamine (5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine, catalogue number: 1589007) were purchased from Sigma-Aldrich and diluted (5 mg/ml each) in PBS  or DMSO (Pyr) for 27. Briefly, purified N. caninum tachyzoites were distributed (1 × 103/well) in a Vero cell culture in a 96 well plate and incubated for 2 h to allow cell invasion. After the invasion process, seven NMB, TBO or DMMB dilutions  were added, in triplicate, to the infected cells and the plates were incubated for 72 h, at 37 °C with 5% CO2. For the evaluation of the drug combinations, after parasite invasion, seven fractional inhibitory concentrations , of each compound alone or combined were applied on proliferating tachyzoites under the same conditions. The 2 × IC50 concentrations for MB, Pyr, NMB, and DMMB were 0.7, 0.7, 0.125, and 0.05 μM, respectively. The drug combination was composed of a solution containing an equal concentration of each compound. As a control, the same concentration of every single compound was used. After incubation, the wells were washed with PBS and lysed with 125 μl CPRG lysis buffer (100 mM 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 8.0; 1 mM CaCl2; 1% Triton X-100, 0.5% SDS; 5 mM DTT) for 1 h at 50 °C. The lysates were incubated with 125 μl of CPRG buffer  for 1 h and the plates read at 570 nm in an ELISA reader . Three independent assays were performed.The proliferation assay was performed as described in60 adapted for Vero cells27. Briefly, 5 × 103 Vero cells/well were cultivated in 96 well plates until cell confluence. The cells were cultured with dilutions of NMB, TBO, and DMMB (starting from 100 μM) for 72 h, at 37 °C, with 5% CO2. The treated wells were incubated with MTT solution (500 μg/ml) for 4 h, 37 °C, 5% CO2 followed by formazan dilution within acidified isopropanol (HCl 50 mM). The plates were read at 570 nm in an ELISA reader  and cytotoxicity estimated as previously described27. Three independent assays were performed.The deleterious effects of the phenothiazinium dyes to the host cells were evaluated by MTT assay5/well) were distributed in Vero cell monolayers (cultivated in 24 well plates) and incubated for 2 h, 37 °C, 5% CO2 for cell invasion. After the invasion, the cultures were washed with PBS and treated with the IC50 concentration of phenothiazinium dyes and Pyr  for 72 h, 37 °C, 5% CO2. The control was incubated with RPMI with 0.05% DMSO (similar to Pry treated samples), under the same conditions. The treated samples and the non-treated control were pooled in 15 mL tubes, washed with PBS and resuspended in 6 ml of RPMI without phenol red. The suspension was divided into three parts. The first part of the suspension (1 ml/tube) was centrifuged  and applied for the parasite burden evaluation by CPRG assay (24 h of incubation at 37 °C). Likewise, the second part of the suspension was applied in proliferation assays as described in item 3.3. The selected parasites (125 μl/well) were inoculated in duplicate in 96 well plates containing Vero cell monolayers. The plates were incubated for 2 h, 37 °C, 5% CO2 for cell invasion and treated with seven serial dilutions of dyes and Pyr. The higher concentrations of MB, NMB, TBO, DMMB, and Pry applied in proliferation assays were 5 μM, 1 μM, 10 μM, 0.1 μM, and 5 μM respectively. The plates were incubated for 72 h, 37 °C, 5% CO2 and analyzed by CPRG assay, as described in item 3.3. From CPRG assays, the percentage of inhibition in comparison to the non-treated samples was calculated and plotted. Finally, the third part of the suspension of 3 ml (1 ml/well) was inoculated in fresh Vero cell monolayers, as performed in the first round of selection. In the second round of selection, the cultures were treated with 2 x IC50 of phenothiazinium dyes and Pyr. After the second round of selection, the samples were analyzed as described for the first one. Finally, the third round of selection was also performed under the same conditions using 4 × IC50 of phenothiazinium dyes and Pyr and analyzed as previously described. Two independent assays were performed.The resistance assay was schematically described in Supplementary Fig. 27. Briefly, purified tachyzoites  were allowed to invade Vero cells monolayers, cultured in 24 well plates, for 2 h, at 37 °C, with 5% CO2. After the invasion, NMB, DMMB, and combinations of these compounds with MB or Pyr (4 × the IC50 of each one) were added and incubated for 72 h, at 37 °C, with 5% CO2. After this, the cultures were detached and the extracellular tachyzoites counted in a hemocytometer. The remaining tachyzoites were incubated in a new Vero cell culture under the same conditions. Four consecutive cycles of incubation were performed and the inhibition percentage and the absolute number of tachyzoites were calculated. Finally, the final parasite burden was measured after incubation with CPRG for 24 h as described in the proliferation assay.Clearance assays were performed as previously described6/tube) or cells , in RPMI without phenol red, were incubated with 4 × IC50 of MB (1.6 μM), 4 × IC50 of NMB (0.25 μM) and 4 × IC50 of DMMB (0.1 μM) for 30 min at 37 °C with 5% CO2. Additionally, these dyes were combined  and applied to parasites or cells under the same conditions. After the incubation, tachyzoites, and cells were washed twice with PBS and processed for flow cytometry analysis. Cells were incubated with 0.05% trypsin in PBS (Life Technologies) supplemented with 2 μM DCFDA (Sigma) for 30 min, at 37 °C, with 5% CO2 in the dark. Tachyzoites were incubated under the same conditions; however without a trypsinization step. The median fluorescence intensity (from gates representing cells or tachyzoites) was measured in a BD FACS-Canto (BD Biosciences) flow cytometer (Ex.488/Em.535 nm) with the FACSDiva (BD) 6.1.3 software. The fluorescence percentage was calculated in relation to the non-treated group. Two independent experiments were performed for each assay.Tachyzoites (Nc1 strain) and Vero cells were used in oxidative stress assays. Purified tachyzoites (1 × 105/well) over Vero cell monolayers in 24-well plates with coverslips at the bottom and incubated during 48 hours for invasion and proliferation. After incubation, the cultures were treated with IC50 and 10 × IC50 of MB (0.5 μM / 5 μM), NMB (0.06 μM / 0.6 μM), TBO (2 μM / 20 μM), DMMB (0.02 μM / 0.2 μM) and Pyr (0.5 μM / 5 μM) for 24 h at 37 °C and 5% CO2. The supernatants were discarded and 400 nM Mitotracker  were added to the wells and incubated for 20 minutes at 37 °C and 5% CO2. The supernatants were discarded and the wells were washed twice with PBS. Then, the cells were fixed with 3.7% formaldehyde, followed by permeabilization (0.2% Triton X-100) and blocking . The nuclei were observed using 300 nM 4′,6-diamidino-2-phenylindole . N. caninum was detected with serum anti-N. caninum  and anti-mouse conjugated with Alexa 488 . The anti-N. caninum serum was generated in BALB/c animals , detailed in the Supplementary Material , according to the local Animal Ethics Committee, approved by the animal research committee at Universidade Federal de Uberlândia (UFU) (Comitê de Ética na Utilização de Animais da UFU – CEUA/UFU), under protocol number 109/16. The treatment strategy was based on previous studies performed with N. caninum61. The animals were intraperitoneally (i.p.) infected with 1 × 107 NcLiv tachyzoites at day 0 and after 12 h the animals were treated with each compound and their combinations. Single doses of DMMB  or combinations of this dye with Pyr  or MB (50 mg/Kg/day i.p.) were administered during five days. A dose of Pyr was chosen which is insufficient to cure the animal against the disease caused by T. gondii62 and was suitable as a parameter for the drug combination studies. The non-treated control received only PBS. Animals were observed daily for clinical signs , and mortality during 30 days post-infection. After 30 days of challenge, surviving animals were euthanized and the brain tissues (20 mg) were collected for parasite quantification using real-time PCR , as previously described64. The genomic DNA was extracted using  and the concentration normalized to 40 ng/μl . N. caninum DNA was amplified  using primers Nc5  and normalized using primers for mouse GAPDH . The cerebral parasite burden was calculated by interpolation from a standard curve established from serial dilutions of DNA extracted from tachyzoites.For the 27. The IC50 and the combination index (CI) were obtained using Compusyn software (http://www.combosyn.com/) and the selective index (CC50/IC50) was also determined. From CI values, synergism (CI < 1) and antagonism (CI > 1) interactions were generated65. For clearance, oxidative stress and in vivo (parasite burden) assays, data were analyzed by one-way ANOVA followed by a Tukey’s post hoc test (compared to non-treated groups) on GraphPad 5.0 software.The percentages of proliferation and toxicity were calculated using the mean absorbance of the compound-free control and the absorbance from each treatment as described previouslySupplementary Information.