Understanding The Mechanisms Of Anti-Inflammatory Activities Of Cryptolepine
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Date
2020-10
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University Of Ghana
Abstract
Although inflammation coordinates host immunity against infectious pathogens and alarmins, its dysregulation results in severe pathological conditions. Dysregulated inflammatory responses come about as a result of many factors including hyperactivity of pro-inflammatory signaling pathways. Hyperactivity of the Toll like receptor (TLR) - nuclear factor kappa B (NF-κB) signaling pathway is reported to be associated with many inflammatory diseases, which makes the pathway a good therapeutic target. Although cryptolepine, an alkaloid obtained from Cryptolepis sanguinolenta, has been demonstrated to possess anti-inflammatory properties in vivo, its mechanisms of action are not fully understood. This study sought to determine the anti-inflammatory effects of cryptolepine and its underlying mechanisms of action in a murine macrophage cell line (RAW Blue cells) stably transfected with secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. The cytotoxic effect of cryptolepine on the cells was determined by 3-(4, 5Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. The activity of the TLR1/TLR2-NF-κB signaling pathway was induced with Pam3CSK4 (100 ng/mL) in the absence or presence of cryptolepine (0.5 - 1 μM) for 24 hours. Afterwards, culture supernatants were harvested and the activity of the pathway measured by assessing the levels of SEAP using
Quanti-Blue assay. In addition, the effect of cryptolepine on the transcript levels of Tnf-α, Il-6, Il-1β, Il-23, Ccl2, Cxcl2, Ikbkb, Nfkb1, Rela, Tlr2 and Tlr1 were assessed by RT-qPCR and the levels of TNF-α, IL-6, IL-1β, IL-23, MIP-2α and MCP-1 determined using multiplex ELISA technique. Compared to untreated cells, cryptolepine-treated cells showed a dose-dependent decline in viability with 1 μM being the maximum non-toxic concentration of cryptolepine.
Cryptolepine (0.5 – 1 μM) also dose-dependently inhibited the TLR1/TLR2-NF-κB signaling pathway activity. Additionally, the transcript levels of Tnf-α, Il-6, Il-1β, Il-23, Mip-2α, Mcp1,Tlr1, Tlr2, Rela, Ikkβ
and Nf-κb1 were all attenuated by cryptolepine. Cryptolepine also suppressed the protein levels of TNF-α, IL-6, IL-23, MCP-1 but not IL-1β and MIP-2α. In summary, the results from this study demonstrated that cryptolepine inhibited the activity of the TLR1/TLR2 signaling pathway, attenuated the transcript levels of key signaling molecules and suppressed the levels of key pro-inflammatory cytokines and chemokines, which suggest that cryptolepine may be a good anti-inflammatory therapeutic agent.
Description
PhD. Molecular Cell Biology Of Infectious Diseases
Keywords
Anti-Inflammatory, Cryptolepine