Hi Shriniwas! I believe your cascade could start by the reactive oxygen species production. That could be the primary effect of your herbal extract. The increasing in ROS will trigger the production of antioxidant enzymes. But if that is not enough and the cell cannot recover, mitochondria metabolism will be affected by ROS, and then the intrinsic pathway of apoptosis will be activated. A dose response experiment could be a nice idea to find the concentration where antioxidant enzymes production are not enough anymore (or if they are at some point) to rescue the cell. But the main issue in my opinion is that you are working with extract, and not a purified compound. So you can see some biological effect of your extract, would be nice if you could purify the active compounds. Good luck!

Dear Shriniwas;

I think your justification should start from reactive oxygen species (ROS). For example in fish, apoptosis and oxidative damage of fish tissues are induced by ROS, and could be restrained by antioxidant molecules and enzymes. As we know, apoptotic pathways are mainly classified into intrinsic (mitochondrial) pathway and the extrinsic (death ligand) pathway, and are regulated by caspase-9 and -8, respectively. For instance, a study in mammals reported that oxidative damage and apoptosis were closely related to depression of antioxidant defense (Armstrong et al., 2002). In human epithelial ovarian cancer cells, up-regulation of TOR signaling factor gene expression repressed caspase-3, -8 and -9 expressions (Alvero et al., 2009). In fish, a dietary indispensable amino acid (IAA) deficiency has been documented to up-regulate caspase-3, -8 and -9 mRNA levels, and thus promote DNA fragmentation and apoptosis, which might be partially related to the inhibition of TOR signaling pathway (Jiang et al., 2016). In our study, we found elevated levels of TOR and antioxidants enzymes gene expression in response to optimum dietary IAA (Threonine) in fish (Habte-Tsion et al., 2015, 2016), while the reverse was found in the expression of tumor necrosis factor alpha, TNF-α (Habte-Tsion et al., 2016). Several studies have been reported that TOR signalling pathway could regulate the inflammatory responses. For example, the inhibition of mTOR with rapamycin blocked the production of anti-inflammatory cytokines, whereas production of TNF-α was enhanced in human monocytes (Weichhart et al., 2011). In our study, we obtained higher activities of SOD, CAT and GPx in fish fed optimum dietary threonine (Habte-Tsion et al., 2016). Therefore, I think the correlation starts from ROS, followed by TOR-cytokines-apoptosis signalling pathway and then antioxidants genes and their enzymes, respectively.

Hope it helps!

Good luck!

References:

Alvero AB, Montagna MK, Chen R, Kim KH, Kyungjin K, Visintin I, et al. NV-128, a novel isoflavone derivative, induces caspase-independent cell death through the Akt/mammalian target of rapamycin pathway. Cancer-Am Cancer Soc 2009; 115:3204-16.

Armstrong JS, Steinauer KK, Hornung B, Irish JM, Lecane P, Birrell GW, et al. Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ 2002; 9:252-63.

Habte-Tsion HM, Ren MC, Liu B, Ge XP, Xie J, Chen R. Threonine modulates immune response, antioxidant status and gene expressions of antioxidant enzymes and antioxidant-immune-cytokine-related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephala). Fish Shellfish Immunol 2016; 51:189-99.

Habte-Tsion HM, Ren MC, Liu B, Xie J, Ge XP, Chen R, et al. Threonine affects digestion capacity and hepatopancreatic gene expression of juvenile blunt snout bream (Megalobrama amblycephala). Br J Nutr 2015c; 114:533-43.

Jiang WD, Feng L, Qu B, Wu P, Kuang SY, Jiang J, et al. Changes in integrity of the gill during histidine deficiency or excess due to depression of cellular anti-oxidative ability, induction of apoptosis, inflammation and impair of cell-cell tight junctions related to Nrf2, TOR and NF-κB signaling in fish. Fish Shellfish Immunol 2016; 56:111-22.

Dear Shriniwas, it is like two-edged sword, try to narrow down the exposure time to confirm which one activated first. 

I obtained similar results recently where there was a reduction in the levels of the GSH level but increased activities of GPx, GST, CAT SOD and XO when some drugs were used to treat a disease but all the same, there was increase in lipid peroxidation (MDA level), DNA fragmentation, caspases 3 and 9 activity, mATPase activity anda large amplitude MMPT pore opening both in vitro and in vivo. The curative effect of the drugs with concomitant increase in the antioxidant enzyme activities could not rescue the cells from mitochondrial-mediated apoptosis. Thank you