Anti-trypanosomal, anti-inflammatory, and neuroprotective effects of Cichorium intybus sesquiterpene lactones in experimental Trypanosoma evansi infection – Scientific Reports
Hence, the aim of this study was to assess the trypanocidal efficacy of C. intybus sesquiterpene lactones-enriched fraction as well as the antioxidant potential, along with the resultant clinical outcomes in rats experimentally infected with T. evansi. According to our knowledge, this is the first in vivo study that investigates the efficacy of C. intybus sesquiterpene lactones-enriched fraction against T. evansi.
The leaves of C. intybus were purchased from the Harraz market (Cairo, Egypt). The plant material was identified by Dr. Ahmed M. Ayyat (Department of Medicinal and Aromatic Plant Faculty of Agriculture, Beni-Suef University 62521, Egypt; Research Institute of Medicinal and Aromatic Plants (RIMAP), Beni-Suef University 62521, Egypt).
As part of our earlier study, we extracted, fractionated, and analyzed the C. intybus sesquiterpene lactones-enriched fraction using liquid chromatography-mass spectrometry (LC-MS). The previously prepared extract was then used in the present study. The sample was ground into a fine powder (4.5 kg). Ethanol: water (80:20, v/v) was used for the extraction by ultra-sonication for 15 min at room temperature, followed by a 10-min centrifugation at 11,000xg. The collected supernatant was filtered, and the solvent was evaporated till dryness. The residue (420 gm) was extracted with dichloromethane (DCM) till exhaustion, followed by solvent evaporation till dryness yielding 70 gm (15 gm/kg dry wt.). Before LC-MS analysis, 1 g of the extracted fraction was dissolved in methyl alcohol and filtered by a 0.22 μm membrane filter. Rats were given the fraction orally after dissolving it in 2% Tween 80.
Chemicals for assessing oxidative stress biomarkers and AChE activity were bought from Carl Roth-Germany and Loba-chemie Company, Mumbai, India. BATRYNIL (Arabcomed, Arab Company for Medical Products, Egypt), containing diminazene aceturate and phenazone), was used in this study.
Trypanosoma evansi strain was originally isolated from infected camels and cryopreserved in liquid nitrogen using a medium composed of 1× PBS, 1% glucose, and 1% Dimethyl sulfoxide (DMSO). A relatively low concentration of DMSO (1%) was used in the cryopreservation medium because the parasite isolate was stored for maintenance prior to experimental infection rather than for long-term archival preservation. Using this protocol, the T. evansi isolate maintained its viability and infectivity after storage in liquid nitrogen for up to one year. Based on the preliminary study, the used isolate is able to induce an acute infection with 13-15 days-survival period following infection in rats, if not treated. Initially, cryopreserved blood was intraperitoneally injected into two rats to produce a large number of trypanosomes for the experimental groups' subsequent infection in this study.
Eighty healthy male rats weighing between 170 and 200 g were purchased from the Nile Company (El Amyria, Cairo, Egypt) and acclimated for 10 days under controlled environmental conditions, which included a regulated light cycle (12 h day/night cycle), temperature (25 ± 2 °C), and humidity (60 to 70%). The rats were provided with unrestricted access to water and food to assure highest animal welfare conditions.
The experiment was approved by the Research Ethical Committee of Beni-Suef University-Faculty of Veterinary Medicine (approval number 022-296). All methods we carried out in accordance with relevant guidelines and regulations. The study followed the recommendations of the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). The experimental period was one month and the treatment regimens for chicory fraction, commenced 2 weeks before infection and another 2 weeks post-infection. Rats were randomly allocated equally into 4 groups (n = 20/group). Group I was the control negative, which was untreated and uninfected. Group II was control positive and received an intraperitoneal infection with T. evansi (1 × 10 trypanosomes/rat) on the 15 day from the beginning of the experiment. Group III was infected as described and treated intramuscularly with 7 mg/kg body weight of diminazene aceturate (standard chemical drug) as a single dose on the 8 day post-infection (when all the rats, in the infected groups, were parasitemic). The chicory fraction group (IV) was orally treated with 200 mg/kg body weight of sesquiterpene lactones-enriched fraction from C. intybus and infected as described (Supplementary Fig. 1). The chicory fraction was given in a daily 1 ml volume per rat for 4 weeks (2 weeks before infection and 2 weeks after infection). The pre-infection administration of the chicory fraction was designed to evaluate both the prophylactic and early therapeutic potential of the fraction components. The other groups (I, II, III) in the experiment were orally treated with saline (1 ml/rat/day) during the experimental period.
The chicory fraction dosage was determined based on the acute toxicity study, following the Organization for Economic Co-operation and Development (OECD) guideline number 423. A dose of 200 mg/kg body weight of chicory fraction (corresponding to one-tenth of 2000 mg/kg body weight) was chosen as the highest safe dosage that can be daily given for a long time.
A daily hematological assessment was conducted post-infection for all infected rats to detect the appearance of trypanosomes via wet preparation (a glass slide with a drop of blood from a tail vein was covered with a slide cover and microscopically examined). As T. evansi is a monomorphic trypomastigote and has lost the ability to complete its life cycle in insect vectors, its development is locked in the bloodstream form. After the parasite detection in the wet preparation (on the 7 and 8 DPI), the parasitemia degree was estimated using hemocytometer on the 10, 12, and 14 DPI in all infected groups (n = 5/time point) following the previous methodology outlined by Dyary et al..
* N is the total number of trypanosomes in the four large squares.
All parasite counts were performed by the same trained person under standardized conditions to minimize variability.
Five rats were selected randomly (ensuring unbiased representation) from each group and underwent deep anesthesia by isoflurane on zero day (prior to the challenge) and on the tenth- and fourteenth-day post infection (DPI) in order to collect blood samples. Following the blood collection, the animals were euthanized humanely via isoflurane overdose.
A portion of the blood specimens was collected on EDTA, as an anticoagulant, for analyzing gene expression of IL-6, IL-1β, TGF-β, and IL-10 and relevant hematological parameters. For conducting biochemical analysis, the remaining portion of blood specimens was collected in tubes without an anticoagulant for the separation of the serum. The serum specimens were kept at -20℃ till analysis.
Following the animals' euthanasia, on the 14 DPI, spleen and brain were collected post-mortem. The brain tissues underwent a saline wash and allocated into 2 parts; the first one was homogenized to estimate oxidant/antioxidant parameters and the activity of AChE. The second part of the brain and the whole spleen were kept in formalin solution (10%) for histopathological analysis (Supplementary Fig. 1).
On zero day as well as the 10, and 14 DPI, the total erythrocyte and leucocyte counting was conducted via haemocytometer method. The blood Hb and packed cell volume (PCV) were estimated as described by Drabkin and Austin and Thrall et al., respectively. Blood indices involving mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), as well as mean corpuscular hemoglobin concentration (MCHC), were calculated following Dacie and Lewis. The absolute differential leucocytic count was calculated and conducted in accordance with Jain.
The concentrations of triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and serum glucose were estimated using commercial kits, in accordance with the manufacturer's protocol on zero day, 10, and 14 DPI. The levels of very low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) were calculated by Friedewald's formula (VLDL-C = Triglycerides/5, and LDL-C = TC – (VLDL-C + HDL-C)).
In phosphate buffer saline (pH 7.4), the brains were homogenized at a 10% (w/v) concentration, and centrifuged at 1000 xg at 4 °C for 15 min. The resulting supernatant was isolated and utilized for the oxidant/antioxidant parameters and AChE activity measurements.
The malondialdehyde (MDA) content was estimated calorimetrically, in brain homogenate, following the method of Albro et al.. The methodology depends on the thiobarbituric acid (TBA) reagent reacting with MDA in an acidic medium, forming a red color. A 1.25 ml of 10% trichloroacetic acid (TCA) was mixed with 0.25 ml of brain homogenate in a glass tube and incubated in boiling water for 20 min. After incubation, the tubes were cooled in tap water and 0.5 ml D.W was added to each tube, then the tubes were centrifuged at 1800 xg for 10 min. 1 ml of the supernatant was mixed with 0.5 ml of 0.67% TBA, then the tubes were incubated in boiling water for 20 min. The tubes were cooled in tap water and the optical density was determined at 532 nm against a TBA blank. The content was expressed as nM/100 mg tissue.
By using DTNB (5,5'-dithiobis-2-nitrobenzoic acid), the reduced glutathione (GSH) content and glutathione peroxidase (GPx) activity were determined as reported by Sedlak and Lindsay and Rotruck et al., respectively. For estimation of GSH content, the technique involved in protein precipitation by TCA. 250 µl of 10% TCA was mixed with 250 µl of tissue homogenate in a glass tube for 10 min. The tubes were centrifuged at 1800 xg for 10 min. The supernatant (250 µl) was mixed with 125 µl of Ellman's reagent (19.8 mg DTNB in 100 mL 0.1% sodium nitrate) and 750 µl of phosphate buffer and the optical density was read immediately at 412 nm against the blank. The content was expressed as nM/100 mg tissue. For GPx activity, the reaction mixture contained 100 µL 0.4 M phosphate buffer (pH 7.0), 50 µL 10 mM sodium azide, 100 µL tissue homogenate, 100 µL 2 mM reduced glutathione and 50 µL 0.2 mM HO. The contents were incubated for 10 min at 37 °C, 200 µL 10% TCA was added to stop the reaction and centrifuged at 1800 xg for 10 min. The supernatant was assayed for glutathione content using Ellman's reagent. The activity was expressed as nM of GSH consumed/min/100 mg tissue.
The activity of AChE was measured using DTNB as the procedure of Ellman et al. and the modification mentioned by Gorun et al.. Brain homogenates (0.01 ml) were incubated with 0.05 ml of acetylthiocholine solution for 30 min and the enzyme activity was stopped by the addition of 1.8 ml of DTNB reagent in ethanol (12.4 mg DTNB dissolved in 120 ml of 96% ethanol, 80 ml of D.W and 50 ml of 0.1 M phosphate buffer, pH 7.6). The absorbance was measured immediately at 412 nm. The principle is based on measurement of the thiocholine production rate as a result of acetylthiocholine hydrolysis and the result was expressed as nM thiocholine produced/min/100 mg tissue.
The Qiagen RNeasy kit was used to extract total RNA from blood samples following the manufacturer's instructions. The RevertAid First Strand cDNA Synthesis Kit was used to convert the isolated RNA to cDNA, and a real-time PCR system (Applied Biosystems, USA) was used to quantify the levels of IL-6, IL-1β, TGF-β, and IL-10 gene expression using SsoAdvancedTM Universal SYBR Green Supermix following the manufacturer's guidelines. For the normalization of gene expression data by the f0 method, the beta-actin gene (Actb) was utilized as a housekeeping gene. Beta-actin was used as the internal control due to its stable expression under the experimental conditions. The primers used in the present study are presented in Supplementary Table 1.
The data analysis was conducted utilizing IBM SPSS Version 26. To elucidate significant intergroup differences at the same time point, a one-way analysis of variance complemented by the Tukey post-hoc test was employed. Data are presented as means accompanied by standard error (SE), with statistical significance established at P < 0.05.
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