Necrosis is another physiological cellular degradation by traumatic cells. This is not as normal as the apoptosis pathway. The premature cell death is occurred due to autolysis, external and internal factors. Mainly necrosis is activated by some immune components like the complement system, a bacterial toxin, activated NK cells, and macrophages.
Methods for Distinguishing Apoptotic & Necrotic Cell
The two processes are completely different from each other. Inflammatory tissue damage is the main reason for necrosis which may result in more traumatic tissue damage and induce inflammations.
There are several methods on the basis of this apoptotic versus necrotic (necroptotic) cells identified. In both cases, the cellular morphological study is very important. To analyze the morphology of cells some techniques are highly used like, time-lapse microscopy, flow cytometry, and transmission electron microscopy.
Moreover, the biochemical study has equivalent importance to distinguishing in both cells. There are some various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by FACS with the help of annexin V, cationic dyes like propidium iodide, PI & 7amino actinomycin D, 7-AMD), cellular markers such as DNA fragmentation, cellular shrinkage, chromatin condensation, decreased cytosolic pH re-observed by flow cytometry, caspase activation, Bid cleavage, cytochrome c release, expression of apoptotic signal proteins identified by Western blotting.
The periodic process of apoptosis is analyzed by the combination of cationic dyes (PI) with annexin V which helps to discriminant between live cells, early apoptotic cells. Late apoptotic cells and necrotic cells. There has some other important information about the necrotic cells that the primary and secondary necrotic cells are distinguished by analysis of supernatant, HMGB1, and also cytokeratin 18 which released during necrosis.
Difference Between Apoptosis and Necrosis
In three major cell death necrosis cell death induce due to several stressful conditions like hypoxia, ischemic hypoglycemic, physicochemical factors, deprivation of nutrient, and environmental factors like extremely high temperature, pH, etc. This process is characterized by cell swelling, random chromatin fragmentation, loss of membrane integrity, cell rupture, and loss of intracellular content which induce accidental and uncontrolled cell death.
Due to several kinds of stress cells go through a series of physicochemical as well as metabolic changes. As an example, in ischemic reperfusion induce uncontrolled cell death, which began with ischemia followed by restorations end with necrosis. Ischemia occurs due to blood flow obstruction in the tissue which induces a hypoxic environment because of low oxygen and nutrient supply, results in the accumulation of metabolic wastes with low energy supply. The prolonged ischemic induce cell death by hypoxic stress but if this starts to restored then uncontrolled cell death is occurred by reperfusion, also known as reperfusion injury.
Metabolic and Cellular Changes
- During ischemia oxidative phosphorylation is an important metabolic pathway. Help to mateine the energy supply. Due to hypoxia oxidative phosphorylation, impaired and anaerobic respiration induce owing to fulfill the cellular energy supply.
- Hypoxia promotes cellular damage like adenine nuclear translocator (ANT) and many components of ETC which induce ATP hydrolysis by complex V, lead the concentration of free phosphate in the cell.
- Aerobic respiration decreases the cellular pH by producing lactic acid and ionic imbalance by activating Na+/H+ antiporter, induce intercellular [Na+].
- Low concentration of ATP unable to open up the Na+/K+ channel, results in Na+ efflux block which decreases the cellular pH level.
- Additionally, cellular [Ca+] elevated by the opening of Ca+/Na+ antiporter, extracellular Ca+ influx to cell as well as the intracellular [Ca+] increase due to efflux Ca+ from the endoplasmic reticulum.
- The cytosolic Ca+ concentration subsequently enters into mitochondria via a+ uniporter and pump out through Na+/Ca2+ the mitochondrial Ca+ concentration increase dramatically and the Na+/Ca2+ antiporter becomes saturated result mitochondrial matrix hyperpolarization due to excessive [Ca2+].
- During reperfusion ischemic restoration induce necrosis by opulence ROS generation which occurs because of oxygen supply into ischemic tissue.
- Overproduction of ROS promotes mitochondrial dysfunction by disrupting the ratio of NADH/NAD+, which begins due to an extremely high level of Ca2+ that stimulates mitochondrial dehydrogenases. This situation also interrupted in glycolysis and ATP production.
- ROS introduce DNA damage because of PARP1’s higher activation and catalyze poly (ADP) ribosylation of a large amount of protein.
- During restoration cellular aberration try to reverse back by normalizing pH, Ca2+ concentration both in mitochondria and cytosol, PARP1 hyperactivation as well as ATP hydrolysis which collectively helps to open the mitochondrial permeability transition pore (MPTP), induce mitochondrial permeability transition, consequently, promote lysis of mitochondria by depolarization of the mitochondrial inner membrane, mitochondrial matrix swelling followed by rupturing mitochondrial outer all.
- Additionally, nitric oxide (NO) and advanced glycation end (AGE) products are produced which also induce necrosis.
Mechanism of Action
The initiation of necrosis is similar to the extrinsic pathway of apoptosis by the ligand receptors binding mechanism. But both pathways are differing from each other in effector protein action and activation whose are mainly controlled by ligand-receptor interaction.
- TNF induced necrosis initiate after binding of TNF to TNFR, together with TRADD (TNFR1 associated death domain) and RIP1K (receptor-interacting serine/ threonine-protein kinase 1) they form a death domain in the cytosolic part of the receptor.
- After activation of death, the domain induces phosphorylation in RIPK which subsequently activates RIP3K by phosphorylation. During the extrinsic pathway, the RIP1K protein never being phosphorylated but in the case of necrosis, RIP1K phosphorylation is as necessary as cellular respiration. Without phosphorylation of the RIP1K necrosis process never being started.
- After the activation of RIP3K, the heterodimeric formation of RIPK-RIP3K activate MLKL (mixed ligand kinase domain) via phosphorylation which induces translocation of activated MLKL towards the membrane form a channel in the cell membrane by oligomerization, allow cytoplasmic leakage.
- Besides this RIP1 localization into the mitochondrial membrane, induce ATP depletion via diminishing the interaction between CypD and ANT.
- On the other hand, ROS induced lipid peroxidation promote organelle disruption along with decrease cell integrity. ROS formation influenced by ligand via JNK1-mediated ferritin degradation pathway in lysosomes.
Different cellular organelles dysfunction induce uncontrolled cellular death through the necrotic pathway.