Co-Delivery of Aceclofenac and Methotrexate Nanoparticles Presents an Effective Treatment for Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) is a common acute inflammatory autoimmune connective tissue arthropathy. The genetic studies, tissue analyses, experimental animal models, and clinical investigations have confirmed that stromal tissue damage and pathology driven by RA mounts the chronic inflammation and dysregulated immune events. Methods: We developed methotrexate (MTX)-loaded lipid-polymer hybrid nanoparticles (MTX-LPHNPs) and aceclofenac (ACE)-loaded nanostructured lipid carriers (ACE-NLCs) for the efficient co-delivery of MTX and ACE via intravenous and transdermal routes, respectively. Bio-assays were performed using ex-vivo skin permeation and transport, macrophage model of inflammation (MMI) (LPS-stimulated THP-1 macrophages), Wistar rats with experimental RA (induction of arthritis with Complete Freund's adjuvant; CFA and BCG), and programmed death of RA affected cells. In addition, gene transcription profiling and serum estimation of inflammatory, signaling, and cell death markers were performed on the blood samples collected from patients with RA. Results: Higher permeation of ACE-NLCs/CE across skin layers confirming the greater "therapeutic index" of ACE. The systemic delivery of MTX-loaded LPHNPs via the parenteral (intravenous) route is shown to modulate the RA-induced inflammation and other immune events. The regulated immunological and signaling pathway(s) influence the immunological axis to program the death of inflamed cells in the MMI and the animals with the experimental RA. Our data suggested the CD40-mediated and Akt1 controlled cell death along with the inhibited autophagy in vitro. Moreover, the ex vivo gene transcription profiling in drug-treated PBMCs and serum analysis of immune/signalling markers confirmed the therapeutic role co-delivery of drug nanoparticles to treat RA. The animals with experimental RA receiving drug treatment were shown to regain the structure of paw bones and joints similar to the control and were comparable with the market formulations. Conclusion: Our findings confirmed the use of co-delivery of drug nanoformulations as the "combination drug regimen" to treat RA.

Mito-Q supplementation of in vitro maturation or in vitro culture medium improves maturation of buffalo oocytes and developmental competence of cloned embryos by reducing ROS production.

Mito-Q is a well-known mitochondria-specific superoxide scavenger. To our knowledge, the effect of Mito-Q on buffalo oocyte maturation and developmental competency of cloned embryos has not been examined. To investigate the effects of Mito-Q on the in vitro maturation (IVM) of buffalo oocytes and the developmental competence of cloned embryos, different concentration of Mito-Q were supplemented with IVM (0, 0.1, 0.5, 1, 2 µM) and in vitro culture (IVC) medium (0, 0.1 µM). Supplementation of IVM medium with 0.1 µM Mito-Q significantly (P ≤ 0.05) increased the cumulus expansion, nuclear maturation, mitochondrial membrane potential (MMP) and antioxidants genes (GPX1 and SOD2) expression and effectively reduced ROS production leading to a significant improvement in the maturation rate of buffalo oocytes. Further, the supplementation of 0.1 µM Mito-Q in IVC medium promotes the cleavage and blastocyst rate significantly over the control. Mito-Q supplementation improves (P ≤ 0.05) MMP, antioxidant gene (GPX1) expression and reduced the ROS level and apoptosis related genes (caspase 9) expression in cloned blastocysts. In conclusion, the present study demonstrated that the supplementation of 0.1 µM Mito-Q in IVM and IVC media exerts a protective role against oxidative stress by reducing ROS production and improving MMP, fostering improved maturation of buffalo oocytes and enhanced developmental competence of cloned embryos. These findings contribute valuable insights into the optimization of assisted reproductive technologies protocols for buffalo breeding and potentially offer novel strategies to enhance reproductive outcomes in livestock species.

Retinoic acid restores the levels of cellular cholesterol in Leishmania donovani infected macrophages by increasing npc1 and npc2 expressions.

Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients.

Preventive as well as therapeutic significances of linoleic acid in the containment of Leishmania donovani infection.

People suffering from malnutrition show compromised levels of ω-6 fatty acid and malnutrition is frequently observed among visceral leishmaniasis (VL) patients as disease inflicts primarily the socioeconomic destitute communities. Dietary linoleic acid (LA, 18:2; ω-6 fatty acid) is the principal source of essential fatty acid and its derivatives i.e. eicosanoids possess immune-modulatory activities. However, its role in VL is not yet established. LA was measured in VL human subjects (serum) as well as in Leishmania(L.)donovani infected hamsters (serum and visceral organs). Organ-specific mRNA expressions of various enzymes of the LA metabolic pathway were measured in visceral organs of infected hamsters. Our findings showed a decrease in the concentrations of LA in the serum samples of VL patients, suggesting malnutrition among these patients. However, in L. donovani infected hamsters, its level was not altered in the early infection (15 days) and then increased at late infection (60 days). Importantly, the supplementation of LA restored the Th-1 type of immune response and significantly reduced the parasite load within infected macrophages in vitro. This protective response of LA was mediated through 5-lipoxygenase pathway not via the cyclooxygenase pathway. Preventive usage of LA to mϕ followed by L. donovani infection also showed the strengthening of Th-1 immune response and significantly fewer parasite loads. Our findings demonstrate the protective role of LA in the containment of the parasite load. Incorporating LA rich oils in daily food habits across highly inflicted regions may be a significant advancement towards the eradication of the disease.