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Z-VAD-FMK

 Z-VAD-FMK, which is referred to as (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoro methyl ketone) is the type of inhibitory molecule that can block the process of apoptosis by inhibiting the caspase enzyme. This caspase inhibitor influences the three types of cellular deaths. First, it can inhibit apoptosis. Moreover, it can also sensitize the cells to undergo necrosis along with the induction of autophagy. Studies also explained the involvement of kinase RIP1, cyclophilin-D complex, and ANT. The exact mechanism of Z-VAD-FMK regarding the sensitization to necrosis includes the inhibition of the caspase-8 enzyme, which mediates proteolysis of receptor-interacting protein kinase 1 along with the disruption of the interaction between ANT and cyclophilin-D. Along with necrotic cell death, RIP1 also plays an important role in the autophagy of the cells. This caspase inhibitor has a negative role for the caspase-8 enzyme in the autophagic form of cellular death. These two concepts regarding the positive effects of RIP-1 and the negative role of caspase-8 are major indications of the interconnection of these two types of cellular death.

Z-VAD-FMK

Figure 1::Structural representation of Z-VAD-FMK.

 It also can trigger the process of necroptosis which is defined as programmed necrosis or cell death via inflammatory processes. Studies explained that necroptosis is regulated in the form of a signaling pathway that depends upon RIP3, and RIP1 along with the MLKL. These signaling molecules are regulated via the caspase pathway, especially RIP1 which induces the apoptosis by caspase 8 signaling pathway. This caspase 8 is stimulated with an inflammatory stimulus like TLR 3-4 agonist (Toll-Like Receptors), and tumor necrosis factor-α. Z-VAD-FMK block the caspase 8 activity that in return induces the process of necroptosis. Some studies suggest that this type of necroptosis (mediated by Z-VAD) affects different inflammatory diseases. Meanwhile, it also stated that necroptosis has an anti-inflammatory role and thus can prevent bacterial and viral infections. But these statements remained controversial like Z-VAD has a negative role in pneumovirus diseases in mice and a positive role in SAP (severe acute pancreatitis model) in lung injury. One of the studies explained its use in the alleviation of endotoxic shock with the help of necroptosis which is mediated through the regulation of macrophage activation. These macrophages produce an unrestrained amount of pro-inflammatory cytokines. The pathogenesis of the endotoxin shock is unclear but it is found that the Z-VAD-FMK can remarkably reduce the level of mortality in lipopolysaccharides-challenged mice. So, any substance or immunoregulatory cell that can inhibit the pro-inflammatory responses induced by LPS can easily alleviate the level of endotoxic shock. During the onset of the endotoxin shock, there is the accumulation of the myeloid cells that play an important role in both innate and adaptive immunity. This reduces the level of pro-inflammatory molecules, thus alleviating the level of inflammation by inhibiting M1 macrophages.  Also, it can act as an immune suppressor because it releases immune suppressive mediators like Arg-1 and IL-10. They found that the intraperitoneal injections of the Z-VAD-FMK cause the necroptosis of the macrophages along with inhibition of their activation. Furthermore, it stimulates the accumulation of the MDSC, which also inhibits the M1 macrophage activation. Together these two mechanisms cause the alleviation in the pathogenesis of endotoxic shock, so can be used as a treatment agent for endotoxic shock.

Z-VAD-FMK ameliorates the endotoxin shock

Figure 2: Z-VAD-FMK ameliorates the endotoxin shock by promoting macrophage necroptosis and MDSCs accumulation.

Studies also explained its use to prevent the loss of follicles induced by the ischemic injury after the transplantation.  During the ovarian tissue transplantation, before the revascularization, the hypoxic period, which lasts for 3 to 5 days results in the ischemia of the follicles.  These hypoxic conditions lead to injury caused by the production of oxygen-derived reactive oxygen species (ROS). These ovarian tissues after the transplantation undergo apoptosis which is defined as programmed cell death. The main signaling pathway involved in the initiation of apoptosis includes the activation of the caspase enzyme. Upon inhibition of the caspase enzyme, there will be a disruption in the signaling pathway of apoptosis. Several studies give us a clue regarding the caspase inhibitors which can be widely used to prevent apoptosis in different tissue like the brain, pancreas, intestine, myocardium, etc. These caspase inhibitors like Z-VAD bind to the catalytic site of the enzyme and inhibit it. Studies also observed that the treatment before the cryopreservation of the ovarian tissue improves the quality of the follicles and the survival ability of the tissues. Moreover, z-vad treatment can also increase the number of proliferative cells and reduce the delay to continue the estrous cycle. Results also explained its effectiveness to improve the preservation of any biological entity before organ transplantation.

It can also reduce the level of apoptosis induced by cryopreservation by inhibition of the caspase 3 enzyme. In a broader view, it disrupts the apoptotic pathway by inhibiting the activation of caspase 8 and 9 which act as the initiator, and the inhibition of caspase 3, which acts as an effector for the apoptotic pathway. Indeed, it increase the level of resistance to cryopreservation that indirectly enhanced the hatching rates by increasing its survivability and development after post-warming cultures (24 and 48 hours). Along with this, it does not change the number of the blastocyst which are required for embryonic development. 

 Similarly, one of the studies conducted on the investigation of caspase inhibitors Z-vad suggested its use for lung injuries that are induced by the apoptosis of the pulmonary cells or tissues as we have above discussed the lung injuries in severe acute pancreatitis model in rats. In this study, they divided their sample rats into different groups including sham, severe acute pancreatitis, and SAP+z-vad. After the establishment of the SAP, they sacrificed these rats at specific intervals and stained them with HE for the assessment of lung injury. Along with this assessment, they used ELISA to detect myeloperoxidase (MPO), cytokines concentration (IL-1β ), and tumor necrosis factor α. they also used western blotting analysis for the detection of caspase-3 within the tissues of the lungs. They found that the rats which are categorized under SAP show evident lung injury. They also explained that the pre-treatment can cause the inhibition of cytokines release and MPO and Caspase-3. so, it can be used as a therapeutic approach for lung injury associated with acute pancreatitis.



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