MOLECULAR EFFECTS OF HESPERETIN IN DIESEL EXHAUST PARTICLES-INDUCED CARDIO-PULMONARY DYSFUNCTION IN ALBINO RATS

Abstract

Studies have linked the cardiovascular-pulmonary and atherosclerosis effect of air pollution to ambient particulate matter (PM) such as diesel exhaust particles (DEP), causing increase in oxidative stress and inflammatory response. This present study examines whether Hesperetin can protect DEP-induced oxidative and inflammatory response in the cardio-vascular and pulmonary tissue in albino rats. DEP was extracted, fractionated using n-hexane as solvent and characterized by Gas Chromatography/Mass Spectrophotometry (GC/MS). Previously characterized DEP standard reference material (SRM 2975) was also extracted with n-hexane. In vitro redox (oxidative) potential was done by dithiothreitol (DTT) assay. Forty male albino rats (6 weeks old) were divided into 8 groups of 5 rats/group: control group was given DMSO and CMC-Na vehicle orally twice in the last week of treatment; group(s) 2, 3, 4 received (0.064, 0.64, 0.064 mg/kg) of hDEP and hSRM orally twice in the last week of treatment respectively; group(s) 5, 6, 7 were pretreated with 200 mg/kg HESP for 2 weeks while hDEP and hSRM was given orally twice in the last week of treatment respectively; group 8 received 200 mg/kg HESP alone orally for 2 weeks. The rats were sacrificed 24 hrs after the last treatment and serum was collected for enzyme activity; Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), as well as lipid profiles; cholesterol, Triglyceride, High Density Lipoprotein (HDL)-Cholesterol, Low Density Lipoprotein (LDL)-Cholesterol was used to assess tissues damage and lipideamia. Heart, aorta and lungs were excised aseptically then, oxidative stress markers such as lipid peroxidation (LPO), conjugated dienes (CD) and reduced glutathione (GSH) levels were determined as indices of oxidative damage. The gene expressions of the following pathways involved in inflammation (NF-kB, IkB, IkkB, TNFα, IL-1β, & IL-10), oxidative stress (Nrf2, HO-1) as well as LDL homeostasis (PCSK- 9 & LDL-receptor) were determined by reverse transcription and polymerase chain reactions (PCR). The molecular interaction of hesperetin with target proteins associated with DEP-induced toxicity was determined using in-silico method. The results showed that DEP caused 0.4367μm/min and 0.2911μm/min reductions in DTT concentration. It also contains non-polar PAH with Benzo(b)flouoranthene and Benzo(ghi)perylene having the highest and lowest PAHs concentration. The results showed that DEP caused significant increase in the serum biomarkers, ALT and AST which was attenuated by hesperetin. The DEP caused toxicity to the heart, aorta and lungs by increasing CD and MDA levels while it decreased serum HDL and GSH levels when compared with control. However, pre-treatment with hesperetin significantly (p< 0.001) reversed these effects. Diesel exhaust particles also caused significant increase in the expression of PCSK- 9 in the heart, TNF-α, IL-1β as well as the expression of IkKB, IkB and NF-kB genes in the heart, aorta and lungs but decreased IL-10 and LDL-R gene expressions. Pre-treatment with hesperetin reversed all these effects while enhancing DEP-induced Nrf2 and HO-1 levels. Molecular docking result of HESP with NF-kB and PCSK-9 gave a binding energy of - 6.4 and - 7.6 Kcal/mol respectively. In summary, this study shows that hesperetin has the potency to protect against DEPinduced cardio-pulmonary dysfunction.