Evaluation of antiapoptotic potential of Sulforaphane via NRF2 signaling pathway in a primary culture of PBMCs derived from chronic HCV patients

Abstract

Various studies show that on average, Hepatitis C virus (HCV) prevalence in Pakistan by percent in adult population is 11.55%. Most of the people diagnosed with HCV infection are aged around 25-35 with ever increasing incidence, around the globe. Our study highlights critical aspects of chronic HCV pathology and its effect on the Immune system. Peripheral Blood Mononuclear cells, specifically CD8+ T cytotoxic lymphocytes and CD4+ T helper cells endorse a hyper-responsive state owing to the levels of viremia and antigenemia caused by chronic Hepatitis C virus. Consequently, these cells become highly dysfunctional. It is well documented that HCV leads to oxidative stress. It also dysregulates antioxidant defense systems. High levels of reactive oxygen species (ROS) lead to oxidation and damage of cellular proteins and organelles, as a result Peripheral Blood Mononuclear cells (PBMCs) become dysfunctional. HCV specific lymphocytes become exhausted due to constant epitope signaling caused due to HCV specific antigens as well as DNA damage caused by high levels of oxidative stress caused in the chronic HCV infection. This leads to the apoptosis of PBMCs via FAS/FAS-L mediated (extrinsic) and intrinsic pathways respectively. Sulforaphane, which is a naturally derived anti inflammatory and cytoprotective phytochemical can reverse the oxidative stress by activating the NRF2 signaling pathway that can reverse the apoptosis of exhausted virus specific PBMCs. In our study PBMCs were isolated, cultured and treated by optimal doses of Sulforaphane. Cell viability assays were performed to know whether Sulforaphane (SFN) plays a pivotal part in reversing the anti-oxidant stress, cellular exhaustion and apoptosis. The changes at the genetic level are marked by performing RT-PCR, to detect the increased expression of genes involved in anti-oxidant defense systems. The reversal of these stressors leads to enhanced cell survival, reversal of apoptosis, proliferation and improved functionality required to ward off viral load. Cross validating assays were performed using computational biology tools, including molecular docking and GRIND (advanced alignment independent 3D QSAR) to further validate our findings.