Dual role of an essential HtrA2/Omi protease in the human malaria parasite: Maintenance of mitochondrial homeostasis and induction of apoptosis-like cell death under cellular stress.

Members of the HtrA family of serine proteases are known to play roles in mitochondrial homeostasis as well as in programmed cell death. Mitochondrial homeostasis and metabolism are crucial for the survival and propagation of the malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin-like protease activity that can be inhibited by its specific inhibitor, ucf-101. A transgenic parasite line was generated, using the HA-glmS C-terminal tagging approach, for localization as well as for inducible knock-down of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during the asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, the ucf-101 treatment did not affect the parasite growth, suggesting the non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase-like protease as well as parasite cell death, suggesting the involvement of protease activity of PfHtrA2 in apoptosis-like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show the specific roles of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating stress-induced cell death.

A Plasmodium falciparum ubiquitin-specific protease (PfUSP) is essential for parasite survival and its disruption enhances artemisinin efficacy.

Proteins associated with ubiquitin-proteasome system (UPS) are potential drug targets in the malaria parasite. The ubiquitination and deubiquitination are key regulatory processes for the functioning of UPS. In this study, we have characterized the biochemical and functional role of a novel ubiquitin-specific protease (USP) domain-containing protein of the human malaria parasite Plasmodium falciparum (PfUSP). We have shown that the PfUSP is an active deubiquitinase associated with parasite endoplasmic reticulum (ER). Selection linked integration (SLI) method for C-terminal tagging and GlmS-ribozyme mediated inducible knock-down (iKD) of PfUSP was utilized to assess its functional role. Inducible knockdown of PfUSP resulted in a remarkable reduction in parasite growth and multiplication; specifically, PfUSP-iKD disrupted ER morphology and development, blocked the development of healthy schizonts, and hindered proper merozoite development. PfUSP-iKD caused increased ubiquitylation of specific proteins, disrupted organelle homeostasis and reduced parasite survival. Since the mode of action of artemisinin and the artemisinin-resistance are shown to be associated with the proteasome machinery, we analyzed the effect of dihydroartemisinin (DHA) on PfUSP-iKD parasites. Importantly, the PfUSP-knocked-down parasite showed increased sensitivity to dihydroartemisinin (DHA), whereas no change in chloroquine sensitivity was observed, suggesting a role of PfUSP in combating artemisinin-induced cellular stress. Together, the results show that Plasmodium PfUSP is an essential protease for parasite survival, and its inhibition increases the efficacy of artemisinin-based drugs. Therefore, PfUSP can be targeted to develop novel scaffolds for developing new antimalarials to combat artemisinin resistance.

Effect of soya phosphatidylcholine and possible underlying mechanism on ischemia/reperfusion injury in isolated perfused rat heart: an experimental and computational study.

This study was designed to assess the effect of soya phosphatidylcholine (SPC) against ischemia/reperfusion (I/R) injury and the possible underlying mechanism using experimental and computational studies. I/R injury was induced by global ischemia for 30 min followed by reperfusion for 120 min. The perfusion of the SPC was performed for 10 min before inducing global ischemia. In the mechanistic study, the involvement of specific cellular pathways was identified using various inhibitors such as ATP-dependent potassium channel (KATP) inhibitor (glibenclamide), protein kinase C (PKC) inhibitor (chelerythrine), non-selective nitric oxide synthase inhibitor (L-NAME), and endothelium remover (Triton X-100). The computational study of various ligands was performed on toll-like receptor 4 (TLR4) protein using AutoDock version 4.0. SPC (100 µM) significantly decreased the levels of cardiac damage markers and %infarction compared with the vehicle control (VC). Furthermore, cardiodynamics (indices of left ventricular contraction (dp/dtmax), indices of left ventricular relaxation (dp/dtmin), coronary flow, and antioxidant enzyme levels were significantly improved as compared with VC. This protective effect was attenuated by glibenclamide, chelerythrine, and Triton X-100, but it was not attenuated by L-NAME. The computational study showed a significant bonding affinity of SPC to the TLR4-MD2 complex. Thus, SPC reduced myocardial I/R injury in isolated perfused rat hearts, which might be governed by the KATP channel, PKC, endothelium response, and TLR4-MyD88 signaling pathway.

GlmS mediated knock-down of a phospholipase expedite alternate pathway to generate phosphocholine required for phosphatidylcholine synthesis in Plasmodium falciparum.

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from the catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with up-regulation of phosphocholine and SAM levels; these results show up-regulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights the presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite.

An essential vesicular-trafficking phospholipase mediates neutral lipid synthesis and contributes to hemozoin formation in Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum is the pathogen responsible for the most devastating form of human malaria. As it replicates asexually in the erythrocytes of its human host, the parasite feeds on haemoglobin uptaken from these cells. Heme, a toxic by-product of haemoglobin utilization by the parasite, is neutralized into inert hemozoin in the food vacuole of the parasite. Lipid homeostasis and phospholipid metabolism are crucial for this process, as well as for the parasite's survival and propagation within the host. P. falciparum harbours a uniquely large family of phospholipases, which are suggested to play key roles in lipid metabolism and utilization. RESULTS: Here, we show that one of the parasite phospholipase (P. falciparum lysophospholipase, PfLPL1) plays an essential role in lipid homeostasis linked with the haemoglobin degradation and heme conversion pathway. Fluorescence tagging showed that the PfLPL1 in infected blood cells localizes to dynamic vesicular structures that traffic from the host-parasite interface at the parasite periphery, through the cytosol, to get incorporated into a large vesicular lipid rich body next to the food-vacuole. PfLPL1 is shown to harbour enzymatic activity to catabolize phospholipids, and its transient downregulation in the parasite caused a significant reduction of neutral lipids in the food vacuole-associated lipid bodies. This hindered the conversion of heme, originating from host haemoglobin, into the hemozoin, and disrupted the parasite development cycle and parasite growth. Detailed lipidomic analyses of inducible knock-down parasites deciphered the functional role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Further, exogenous fatty-acids were able to complement downregulation of PfLPL1 to rescue the parasite growth as well as restore hemozoin levels. CONCLUSIONS: We found that the transient downregulation of PfLPL1 in the parasite disrupted lipid homeostasis and caused a reduction in neutral lipids essentially required for heme to hemozoin conversion. Our study suggests a crucial link between phospholipid catabolism and generation of neutral lipids (TAGs) with the host haemoglobin degradation pathway.

Spray dried formulation of mesalamine embedded with probiotic biomass for the treatment of ulcerative colitis: in-vitro and in-vivo studies.

This study is using the targeted approach and anti-inflammatory action of the probiotic biomass to lessen the side effects of therapeutic agents of ulcerative colitis. The aim of the present study is to prepare mesalamine loaded eudragit S-100 with probiotic microparticles by spray drying method. The in-vitro release of the optimized formulation was 90.55 ± 2.42 in 24 hr, which display controlled drug release of mesalamine at a particular region. Mesalamine loaded eudragit S-100 with probiotic microparticles (F12) presented average particle size of 4.91 µm. The statistical analysis was done by one way ANOVA and then comparison test of Bonferroni was done and p values <.05 were considered as significant. The effects of spray dried microparticles over inflamed Caco-2 cell were also evaluated by determining the concentration of IL-8. From in-vivo study it was seen that pretreatment of mesalamine with probiotic prevents DNBS (Dinitrobenzenesulfonic acid) induced colitis in rats and represents protective action against ulcerative colitis because of its antioxidant and anti-inflammatory actions. The results give the foundation for a combination of targeted approach along with the anti-inflammatory potential of the probiotic which might help to decrease the problems which are seen with the traditional cure and management of ulcerative colitis.

An anti-asthmatic activity of natural Toll-like receptor-4 antagonist in OVA-LPS-induced asthmatic rats.

Toll-like receptor-4 (TLR4) is a key component of the innate immune system and activation of TLR4 signaling has a significant role in the pathogenesis of asthma. Therefore, our objective was to identify the natural TLR4 antagonist and evaluate its activity in experimentally induced asthma. Soya lecithin origin phosphatidylcholine (soya PC) was identified as a natural TLR4 antagonist by computational study. Based on the computational study, TLR4 antagonist activity of soya PC was confirmed in in vitro lipopolysaccharide (LPS)-induced neutrophil adhesion assay. In the in vivo study, rats were sensitized with ovalbumin (OVA) (100 µg/kg, i.p.) on the 7th, 14th and 21st days and challenged intranasally with OVA (100 µg/100 µL) and LPS (10 ng/100 µL), 4 days/wk for 3 weeks. At the end of the experiment, we performed lung function parameters (respiratory rate, tidal volume, airflow rate), inflammatory cytokines (interleukin [IL]-4, IL-5, IL-13), total and differential leukocytes in blood as well as bronchoalveolar lavage fluid (BALf) and histological examinations. The computational study indicated that TLR4 antagonist activity of soya PC is due to linoleic acid (18:2) fatty acid chain. Soya PC significantly suppressed the LPS-induced neutrophil adhesion in a concentration-dependent manner to 1 µg/mL. The treatment of soya PC (5 and 10 mg/kg, 18 days, i.p.) significantly improved the lung function parameters, total and differential leukocyte counts in blood and BALf in asthmatic rats. This efficacy of soya PC was in extent similar to dexamethasone (2.5 mg/kg, 18 days, i.p.). However, soya PC was superior to dexamethasone in terms of benefits. The protective action of soya PC may be due to TLR4 antagonist activity and linoleic acid composition.

Development and characterization of morin hydrate-loaded micellar nanocarriers for the effective management of Alzheimer's disease.

The aim of this study was to prepare and characterise oral delivery of morin hydrate-loaded micellar nanocarriers using Pluronic P127 and Pluronic F123 for the effective management of Alzheimer's disease. After administration of formulation brain and blood drug concentration were found to be highest for optimised morin hydrate-loaded micellar nanocarriers as compared to plain morin hydrate. Significant (p < 0.05) reduction in assessed pharmacodynamic parameters was observed after administration of morin hydrate-loaded micellar nanocarriers as compared to disease control group. Chronic treatment with morin-loaded micelles significantly increased the memory in AlCl3 induced Alzheimer's disease in Wistar rats.

A novel class of Plasmodial ClpP protease inhibitors as potential antimalarial agents.

The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria parasite, represents a potential drug target. In the present study, we utilized in silico structure-based screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC50 9.0±0.2µM), a 9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P. falciparum cultures with compound 33 caused morphological and developmental abnormalities in the parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of apicoplast.

Eps15 homology domain containing protein of Plasmodium falciparum (PfEHD) associates with endocytosis and vesicular trafficking towards neutral lipid storage site.

The human malaria parasite, Plasmodium falciparum, takes up numerous host cytosolic components and exogenous nutrients through endocytosis during the intra-erythrocytic stages. Eps15 homology domain-containing proteins (EHDs) are conserved NTPases, which are implicated in membrane remodeling and regulation of specific endocytic transport steps in eukaryotic cells. In the present study, we have characterized the dynamin-like C-terminal Eps15 homology domain containing protein of P. falciparum (PfEHD). Using a GFP-targeting approach, we studied localization and trafficking of PfEHD in the parasite. The PfEHD-GFP fusion protein was found to be a membrane bound protein that associates with vesicular network in the parasite. Time-lapse microscopy studies showed that these vesicles originate at parasite plasma membrane, migrate through the parasite cytosol and culminate into a large multi-vesicular like structure near the food-vacuole. Co-staining of food vacuole membrane showed that the multi-vesicular structure is juxtaposed but outside the food vacuole. Labeling of parasites with neutral lipid specific dye, Nile Red, showed that this large structure is neutral lipid storage site in the parasites. Proteomic analysis identified endocytosis modulators as PfEHD associated proteins in the parasites. Treatment of parasites with endocytosis inhibitors obstructed the development of PfEHD-labeled vesicles and blocked their targeting to the lipid storage site. Overall, our data suggests that the PfEHD is involved in endocytosis and plays a role in the generation of endocytic vesicles at the parasite plasma membrane, that are subsequently targeted to the neutral lipid generation/storage site localized near the food vacuole.

Rootstock scion interaction studies on various horticultural attributes of pomato grafts under protected structures.

Pomato is a horticultural wonder plant, as tomato and potato can be produced from a single plant. This experiment explored the influence of diverse graft combinations of tomato scions grafted onto potato rootstocks on various growth and yield-attributing traits. The investigation outcomes confirmed the significantly positive influence of scion grafted onto rootstock on various yielding attributes of tomato and potato harvested from pomato grafts. Scion "Rakshita" grafted onto the rootstock of Kufri Himalini had the maximum fruit length. In contrast, the fruits harvested from the graft combination of Avtar grafted onto Kufri Khyati had the maximum number of fruits per cluster and the number of fruits per plant. The highest average fruit weight, fruit yield per meter square, and total fruit yield quintal per hectare were recorded with control "Avtar. The longest harvest duration was noticed with the graft combination of Heemsohna grafted onto Kufri Himalini. Moreover, on, rootstock Kufri Himalini with scion Rakshita resulted in maximum tuber length, and average tuber weight, while Kufri Himalini with Avtar had maximum fruit width. The maximum number of tubers per plant was also noticed with Kufri Pukhraj with Palam Tomato hybrid -1. The potato harvested from the rootstock of Kufri Pukhraj with Avtar had the highest tuber yield per plant, total tuber yield quintal per hectare, and tuber equivalent yield. The highest survival percentage of grafted plants was noted in Heemsohna onto Kufri Jyoti. In context to the cumulative yield of tomato fruits and potato tubers obtained from the pomato graft was found to be incremented in grafts of Avtar grafted onto Kufri Pukhraj followed by Rakshita grafted onto Kufri Rakshita, which also resulted in the maximum benefit-cost ratio with highest net return and gross return. The graft combination of scion Avtar and Rakshita onto Rootstock Kufri Pukhraj resulted in a positive increment in yield attributing traits of the pomato plant than of control of un-grafted tomato and potato.

Captivating actions of pomological crops waste as biosorbents for environmental remediation: a comprehensive review.

Water pollutants are an emerging environmental hurdle for crop production and human health risks. In recent decades, the removal of contaminants from water using a cutting-edge approach like biosorbents is a strategy that is both cost-efficient and sustainable. For instance, since biowaste from fruit crops implies the frequent occurrence of average annual waste, it is imperative to formulate strategic initiatives to mitigate this emerging problem while simultaneously recognizing the potential for reutilization and reintroduction of such waste into the industrial sector. Fruit crops such as peels, seeds, skins, branches and stalks can be altered into biosorbents for water treatment. Partially mitigating the adverse impacts of biowaste that estimate to incur costs of billions of dollars around the world would be achieved with this engineering application. This review provides a perspective on the existing literature and brings up-to-date information and findings in the field of pomological crop waste as biosorbents for environmental remediation. In this way, we review the detrimental impact of environmental contaminants on biological organisms and different types of fruit crop waste and their utilization for wastewater treatment, with special emphasis on the formulation of biowaste sorbents (removal efficiency is > 80%) and their application for capturing pollutants such as heavy metals, organic and inorganic dyes and oils. Besides, the newly invented techniques for the characterization of fruit-based biosorbents, the parametric evaluation of biosorbents and their comparison with other available biosorbents are discussed. This review will be helpful for remediating contaminants in wastewater and a panacea for practical engineering solutions.

A Plasmodium falciparum lysophospholipase regulates host fatty acid flux via parasite lipid storage to enable controlled asexual schizogony.

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis of Plasmodium falciparum. To generate such phospholipids, the parasite extensively scavenges, recycles, and reassembles host lipids. P. falciparum possesses an unusually large number of lysophospholipases, whose roles and importance remain to be elucidated. Here, we functionally characterize one P. falciparum lysophospholipase, PfLPL3, to reveal its key role in parasite propagation during asexual blood stages. PfLPL3 displays a dynamic localization throughout asexual stages, mainly localizing in the host-parasite interface. Inducible knockdown of PfLPL3 disrupts parasite development from trophozoites to schizont, inducing a drastic reduction in merozoite progenies. Detailed lipidomic analyses show that PfLPL3 generates fatty acids from scavenged host lipids to generate neutral lipids. These are then timely mobilized to allow schizogony and merozoite formation. We then identify inhibitors of PfLPL3 from Medicine for Malaria Venture (MMV) with potent antimalarial activity, which could also serve as pertinent chemical tools to study parasite lipid synthesis.

Defining ER-mitochondria contact dynamics in Plasmodium falciparum by targeting component of phospholipid synthesis pathway, phosphatidylserine synthase (PfPSS).

The malaria parasite completes the asexual cycle inside the host erythrocyte, which requires extensive membrane biogenesis for its development and multiplication. Metabolic pathways for the synthesis of membrane phospholipids (PL), including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), are crucial for parasite survival. Here, we have studied the P. falciparum enzyme responsible for PS synthesis, Phosphatidylserine synthase (PfPSS), GFP targeting approach confirmed it to be localized in the parasite ER as well as in ER-protrusions. Detailed high resolution microscopy, using these transgenic parasites expressing PfPSS-GFP, redefined the dynamics of ER during the intraerythrocytic life cycle and its association with the mitochondria. We report for the first time presence of ER-mitochondria contact (ERMC) in Plasmodium; ERMC is formed by PfPSS containing ER-protrusions, which associate with the mitochondria surface throughout the parasite growth cycle. Further, ERMC is found to be stable and refractory to ER and mitochondrial stresses, suggesting that it is formed through strong tethering complexes. PfPSS was found to interact with other major key enzyme involved in PL synthesis, choline/Etn-phosphotransferase (CEPT), which suggest that ER is the major site for PL biosynthesis. Overall, this study defines the morphological organisation of ERMC which mediates PL synthesis/transport in the Plasmodium.

Heme Detoxification Protein (PfHDP) is essential for the hemoglobin uptake and metabolism in Plasmodium falciparum.

Hemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP); a protein localized in the parasitophorus vacuole, parasite food vacuole, and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using GFP trap beads showed the association of PfHDP with Hb as well as with the members of PTEX translocon complex. Association of PfHDP with Hb or Pfexp-2, a component of translocon complex was confirmed by protein-protein interaction and immunolocalization tools. Based on these associations, we studied the role of PfHDP in Hb uptake using the PfHDP-HA-GlmS transgenic parasites line. PfHDP knockdown significantly reduced the Hb uptake in these transgenic parasites in comparison to the wild-type parasites. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Transient knockdown of a member of translocon complex, PfHSP101 in HSP101-DDDHA parasites also showed a decreased uptake of Hb inside the parasite. Together, these results advocate an interaction between PfHDP and the translocon complex at the parasitophorus vacuole membrane and also suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

Systems biology analyses reveal enhanced chronic morphine distortion of gut-brain interrelationships in simian human immunodeficiency virus infected rhesus macaques.

Background: Commonly used opioids, such as morphine have been implicated in augmented SIV/HIV persistence within the central nervous system (CNS). However, the extent of myeloid cell polarization and viral persistence in different brain regions remains unclear. Additionally, the additive effects of morphine on SIV/HIV dysregulation of gut-brain crosstalk remain underexplored. Therefore, studies focused on understanding how drugs of abuse such as morphine affect immune dynamics, viral persistence and gut-brain interrelationships are warranted. Materials and methods: For a total of 9 weeks, rhesus macaques were ramped-up, and twice daily injections of either morphine (n = 4) or saline (n = 4) administered. This was later followed with infection with SHIVAD8EO variants. At necropsy, mononuclear cells were isolated from diverse brain [frontal lobe, cerebellum, medulla, putamen, hippocampus (HIP) and subventricular zone (SVZ)] and gut [lamina propria (LP) and muscularis (MUSC) of ascending colon, duodenum, and ileum] regions. Multiparametric flow cytometry was used to were profile for myeloid cell polarity/activation and results corroborated with indirect immunofluorescence assays. Simian human immunodeficiency virus (SHIV) DNA levels were measured with aid of the digital droplet polymerase chain reaction (PCR) assay. Luminex assays were then used to evaluate soluble plasma/CSF biomarker levels. Finally, changes in the fecal microbiome were evaluated using 16S rRNA on the Illumina NovaSeq platform. Results: Flow Cytometry-based semi-supervised analysis revealed that morphine exposure led to exacerbated M1 (CD14/CD16)/M2 (CD163/CD206) polarization in activated microglia that spanned across diverse brain regions. This was accompanied by elevated SHIV DNA within the sites of neurogenesis-HIP and SVZ. HIP/SVZ CD16+ activated microglia positively correlated with SHIV DNA levels in the brain (r = 0.548, p = 0.042). Simultaneously, morphine dependence depleted butyrate-producing bacteria, including Ruminococcus (p = 0.05), Lachnospira (p = 0.068) genera and Roseburia_sp_831b (p = 0.068). Finally, morphine also altered the regulation of CNS inflammation by reducing the levels of IL1 Receptor antagonist (IL1Ra). Conclusion: These findings are suggestive that morphine promotes CNS inflammation by altering receptor modulation, increasing myeloid brain activation, distorting gut-brain crosstalk, and causing selective enhancement of SHIV persistence in sites of neurogenesis.

Ovalbumin/lipopolysaccharide induced vasculitis in rats: a new predictive model.

OBJECTIVES: Currently, there are several animal models for vasculitis. Ovalbumin and lipopolysaccharide (OVA, LPS) are well established for causing inflammation and used as an adjunct in the vasculitis induction. However, to date, none has established the effect of OVA and LPS in disease induction. Therefore, in the present study, an attempt has been made to develop a new animal model for vasculitis using OVA/LPS in rats. METHODS: A total of 42 Wistar rats were divided randomly into seven groups (n=6/group), normal control, and three different doses (0.5, 1, and 5 mg/kg) of OVA and LPS treated groups. Half of the rats in each group received only intraperitoneal sensitization, while the remaining half rats were additionally subjected to a one-week intranasal challenge. RESULTS: Results showed that both OVA/LPS in their respective groups have significantly increased circulating inflammatory cells, C-reactive protein (CRP), Inflammatory cytokines (IL-1ß, IL-6, TNF-α), Kidney damage markers (BUN, Creatinine), and liver function enzymes (AST, ALT) in a dose-dependent manner. CONCLUSIONS: OVA/LPS induced vascular inflammation in a dose-dependent manner. However, the higher (5 mg/kg) dose of ovalbumin and lipopolysaccharide has contributed to severe vascular inflammation through increasing inflammatory cytokines. These findings suggest that OVA/LPS may contribute as a possible model for vasculitis in rats.

Embelin Alleviates Severe Airway Inflammation in OVA-LPS-Induced Rat Model of Allergic Asthma.

BACKGROUND: Asthma is a chronic lung disease, which causes wheezing, tightness in the chest, shortness of breath and coughing. In the wake of coronavirus disease 2019 (COVID-19), which affect the lungs, asthma patients are at high risk. Embelin, a natural benzoquinone obtained mainly from Embelia ribes Burm, has excellent biological properties, including protection against acute asthma. However, since asthma is a chronic and multi-factorial inflammatory disease, asthma conferred by a single allergen in an animal may not be clinically significant. Therefore, the purpose of the current study was to evaluate the effectiveness of embelin against ovalbumin (OVA)-lipopolysaccharide (LPS)-induced severe airway inflammation in experimental animals and to investigate the plausible mechanism of action. METHODS: Rats (n=36) were divided into six groups. Group I served as a normal control. Groups II-VI were sensitised with severe allergens (OVA and LPS) on day 7, 14 and 21, followed by OVA and LPS challenge for 30 min three times/week for 3 weeks. Group II acted as an asthmatic disease control and received only vehicle. On the other hand, groups III-V received embelin (12.5, 25 and 50 mg/kg, P.O. respectively) while group VI received a standard dexamethasone (2.5 mg/kg, P.O.) for 15 days from day 27. Lung function parameters, including the respiratory rate, tidal volume and airflow rate were measured at the end of the experiment (day 42). The total and differential counts of leukocytes in the blood and bronchoalveolar fluid (BALF) were calculated. Th2-mediated serum pro-inflammatory cytokines such as interleukin (IL)-4, IL-5 and IL-13 levels were analyzed. At the end of the study protocol, the lung tissues were removed for a histopathology study. Additionally, a molecular docking simulation on embelin and standard dexamethasone was applied to support the in vivo findings. RESULTS: Significant inhibition of eosinophils, neutrophils, lymphocytes and monocytes in the blood and the BALF was seen in the groups, which received embelin (25 and 50 mg/kg) and dexamethasone (2.5 mg/kg). Moreover, the lung function parameters were normalised by embelin (25 and 50 mg/kg) treatment significantly. The lung histopathological changes confirmed the protective effect of embelin against severe airway inflammation. The docking findings indicated good binding efficacy of embelin to IL-13. CONCLUSION: Overall, our findings indicate that embelin can alleviate severe airway inflammation in OVA-LPS-induced model of allergic asthma occurring by suppression of Th2-mediated immune response. Due to its promising anti-asthmatic effect, it is recommended that embelin should be investigated in clinical trials against asthma. It should also be further explored against COVID-19 or COVID-like diseases due to its ameliorative effects on cytokines and immune cell infiltration.

Protective Effect of Hemin Against Experimental Chronic Fatigue Syndrome in Mice: Possible Role of Neurotransmitters.

Chronic fatigue syndrome (CFS) is a disorder characterized by persistent and relapsing fatigue along with long-lasting and debilitating fatigue, myalgia, cognitive impairment, and many other common symptoms. The present study was conducted to explore the protective effect of hemin on CFS in experimental mice. Male albino mice were subjected to stress-induced CFS in a forced swimming test apparatus for 21 days. After animals had been subjected to the forced swimming test, hemin (5 and 10 mg/kg; i.p.) and hemin (10 mg/kg) + tin(IV) protoporphyrin (SnPP), a hemeoxygenase-1 (HO-1) enzyme inhibitor, were administered daily for 21 days. Various behavioral tests (immobility period, locomotor activity, grip strength, and anxiety) and estimations of biochemical parameters (lipid peroxidation, nitrite, and GSH), mitochondrial complex dysfunctions (complexes I and II), and neurotransmitters (dopamine, serotonin, and norepinephrine and their metabolites) were subsequently assessed. Animals exposed to 10 min of forced swimming session for 21 days showed a fatigue-like behavior (as increase in immobility period, decreased grip strength, and anxiety) and biochemical alteration observed by increased oxidative stress, mitochondrial dysfunction, and neurotransmitter level alteration. Treatment with hemin (5 and 10 mg/kg) for 21 days significantly improved the decreased immobility period, increased locomotor activity, and improved anxiety-like behavior, oxidative defense, mitochondrial complex dysfunction, and neurotransmitter level in the brain. Further, these observations were reversed by SnPP, suggesting that the antifatigue effect of hemin is HO-1 dependent. The present study highlights the protective role of hemin against experimental CFS-induced behavioral, biochemical, and neurotransmitter alterations.

An experimental model of asthma in rats using ovalbumin and lipopolysaccharide allergens.

Asthma is chronic and multi-factorial inflammatory disease hence single allergen induced asthma in an animal is not identical to clinical asthma. Therefore, we developed a novel experimental model of asthma in rats using ovalbumin (OVA) and lipopolysaccharide (LPS) allergens. Rats were divided into four groups; normal (NC), OVA, LPS, and OVA-LPS treated. Rats were sensitized with OVA (100 µg/kg, adsorbed in 100 mg/mL aluminum hydroxide, i.p.), LPS (10 µg/kg, i.p.) and both (OVA-LPS) on 7th, 14th, 21st days and was followed by challenge with OVA (1%w/v), LPS (1%w/v), OVA (0.5%w/v) and LPS (0.5%w/v) for 30 min thrice/week for three weeks in the OVA, LPS and OVA-LPS groups, respectively. On 41 day, lung function parameters (respiration rate, tidal volume, and airflow rate), total and differential leukocytes count in the blood as well as BALf and inflammatory cytokines (IL-4, IL-5, and IL-13) in serum were measured. Histology of lungs was performed. The results suggested that the tidal volume and airflow rate were significantly decreased while respiration rate, total and differential leukocytes count in blood as well as BALf and serum cytokines level were significantly increased in the OVA-LPS as compared to NC, OVA, and LPS. In conclusion, the combination of OVA and LPS induced phenotypes of severe asthma with eosinophilic, neutrophilic and lymphocytic inflammation.