Leupeptin maintains redox homeostasis via targeting ROS-autophagy-inflammatory axis in LPS-stimulated macrophages and cytokines dichotomy in Con-A challenged lymphocyte.

Information regarding cellular anti-inflammatory and immunomodulatory attributes of leupeptin with respect to modulation of perturbed macrophage function and lymphocytes has not yet been delineated, particularly in the context of ROS-cytokines-autophagy-inflammatory signalling cascades. Therefore, the present study identified the attributes and mechanisms of leupeptin, from actinomycetes, in relation to excessive oxidative stress mediated disrupted immune homeostasis and inflammatory mechanism in activated macrophages and lymphocytes. Results revealed that leupeptin treatment showed noticeable inhibition in the production of NO, ROS, mitochondrial membrane potential and phagocytosis activity in LPS-stimulated macrophages. These findings were accompanied by reduction in TNF-α, IL-1β, IL-6, IFN-γ/IL-10 ratio, endopeptidases, oxidative effectors (Cox-2, IL-5, IL-15, IL-17, COX-2), iNOS with concomitant increase in Arg 1, Msr 1 and Mrc - 1exprssion in leupeptin treatment. Additionally, compared to LPS-challenged cells, marked alleviation in MDC, lysotracker staining, beclin-1, LC3B expression, and enhanced p62 levels in leupeptin exposed cells indicate the reversal of impaired autophagy flux. Subsequently, oxi-inflammatory signalling analysis demonstrated p-PTEN, p-NF-κB, p-PI3K, p-Akt, p-p38, and ERK1/2 upregulation decisively thwarted by leupeptin administration. In silico analysis further implied its target selectivity to these cascades. Furthermore, decreased proliferation index and Th1, Th2/IL-10 cytokines ratio in mitogen-challenged splenic lymphocytes confers its role in mitigating unwarranted inflammation mediated by disrupted regulation of adaptive immune cells. Together, these findings signify the attributes of leupeptin as an alternative anti-inflammatory strategy and affirm it as a promising natural entity to modulate immune-mediated response during inflammatory disorder.