Abstract
Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide-induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW) was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-SOD, Mn-SOD, GPx and GR) were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(P)H and GSH (56% and 74% respectively; P<0.001), increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT) followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c). It also significantly activated caspase-9 and caspase-3 and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P<0.05). A combination of camphene and geraniol (CG; 1∶1), when pre-administered in rats (10 mg/kg BW), accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(P)H and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore CG, a combination of natural terpenes prevented nimesulide induced cellular damage and ensuing hepatotoxicity.
Highlights
Mitochondria play a central role in regulating cell survival and death signaling in liver cells
Mitochondrial dysfunction is one of the crucial events involved in cell death by apoptosis
Oxidative stress makes mitochondria more susceptible to untoward effects of a drug resulting into cellular malfunctioning which is undetectable until a critical threshold is achieved [9,34]
Summary
Mitochondria play a central role in regulating cell survival and death signaling in liver cells. Rising evidences suggest that apoptotic pathways converge at the mitochondria, where signaling is initiated through a series of molecular events culminating in the release of death factors [1] This triggers either caspase-dependent or independent apoptosis. Apoptosis-inducing factor (AIF) and endonuclease G (EndoG), along with other important mitochondrial proapoptotic proteins, are reported to translocate to the nucleus and cause oligonucleosomal DNA fragmentation during mitochondria mediated caspase-independent cell death [1,2] Release of these mitochondrial death effectors is tightly regulated by the Bcl-2 family proteins during membrane permeability transition (MPT). It is clearly a redox sensitive process in which membrane protein thiols (Cys-56 of adenine nucleotide translocator; ANT) get oxidized and crosslinked; leading to increased MPT pore opening [3,4]. Threshold mitochondrial damage with oxidative stress causes oxidative DNA damage and increased 8oxoguanine-DNA glycosylase (OGG; DNA repair enzyme) to overcome cellular damage [6]
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