MOLECULAR MECHANISMS OF THE EFFECTS OF NARINGENIN ON DIESEL EXHAUST PARTICLES-INDUCED CARDIOVASCULAR AND HEPATIC DYSFUNCTIONS IN WISTAR RATS

Abstract

One of the major and toxic components of diesel exhaust (emissions) is diesel exhaust particle (DEP). Studies have shown that DEP, a typical air particulate matter (PM), exerts its toxic effects by inducing the generation of free radicals and reactive oxygen species (ROS) which could subsequently lead to oxidative stress and inflammation. This study examined the molecular effects of naringenin on the oxidative and inflammatory effects of DEP on selected tissues of Wistar rats. The DEP collected from a vehicular source in Akure, Nigeria was extracted, fractionated and characterized using methanol as solvent for extraction and Gas Chromatography/Mass Spectrophotometry (GCMS) for characterization. The standard reference material (SRM 2975) DEP obtained from National Institute of Standards and Technology (NIST), USA has been well characterized and was also extracted with methanol (mSRM). The in-vitro oxidative potential of the methanol DEP extract (mDEP) was determined using dithiothreitol oxidation method. The molecular interactions of naringenin with target proteins associated with DEP toxicity were determined using in-silico approach. For animal treatment, forty healthy male albino rats weighing between 85 g and 90 g were divided into eight different groups of five animals per group and treated as follows: group 1 was given dimethylsulfoxide while group 2 and 3 were given 0.064 and 0.640 mg/kg mDEP, respectively; group 4 was given 0.064 mg/kg mSRM; group 5 and 6 were pretreated with 50 mg/kg naringenin for three weeks before given 0.064 and 0.640 mg/kg mDEP respectively; group 7 was pretreated with 50 mg/kg naringenin before 0.064 mg/kg mSRM exposure; group 8 was treated with 50 mg/kg naringenin for three weeks. The mDEP and mSRM were given two times in the last week that preceded sacrifice. All animals had equal access to feeds and water ad libitum and treatments lasted for three weeks. Both naringenin and DEP extracts were administered orally. After the treatments, all animals were sacrificed twenty four (24) hours after the last dose of DEP and blood was collected from the hearts. Serum was prepared and kept at -20oC. Specific organs (liver, heart and aorta) were aseptically removed and stored inside -800C freezer for further biochemical analyses and assays.Aspartate aminotransaminase (AST), alanine aminotransaminase (ALT) and alkaline phosphatase (ALP) were used to assess tissues damage. Serum high density lipoproteins (HDL), low density lipoproteins (LDL), triglycerides (TG) and total cholesterol (TC) were used to assess the effects of DEP on lipideamia. Malondialdehyde (MDA), conjugated dienes (CD), and reduced glutathione (GSH) were used to assess oxidative damage. The genes expressions (at mRNA level) of proteins involved in LDL homeostasis (PCSK-9 & LDL-receptor), inflammation (NF-kB, IkB, IkkB, TNFα, IL-1β, & IL-10) and oxidative stress (Nrf2 & HO-1) were determined by reverse transcription and polymerase chain reactions (PCR) methods. The results show that both mDEP and mSRM caused significant (p<0.001) increase in serum AST, ALT, ALP, LDL, TG, TC, and tissues CD and MDA levels but decrease in serum HDL and tissues GSH levels when compared to controls. The results also show that DEP extracts caused significant (p<0.001) elevation in the expression of pro-inflammatory genes (IL-1β, TNFα, NF-kB, IkB, IkkB) and PCSK-9 gene but significantly (p<0.001) reduced anti-oxidant (Nrf2, HO-1), anti-inflammatory (IL-10) and LDL-receptor mRNA levels when compared with control. Pre-treatment with naringenin significantly (p<0.001) reversed all these DEP-induced adverse effects. In conclusion, naringenin has the potential to protect against DEP-induced oxidative stress and inflammation. Naringeninmay serve as a therapeutic agent against air pollution induced cardiovascular and hepatic damages.