Cell Death Studies: Mechanisms,
Types, and Frontier Advances
I. Introduction
1.1 Background
Cell death is an extremely important process in life activities, which is widely present
throughout the life course from embryonic development to individual aging. From the very
beginning of life, cell death plays an integral role. During embryonic development, cell death
is involved in shaping the morphology and structure of organs. For example, the fingers and
toes of the early human embryo are connected, and it is through the programmed death of
the cells that the excess tissue between the fingers (toes) disappears, and finally the
separated fingers and toes are formed, a process that is precise and orderly, and is essential
for the normal development of the individual.
In adult individuals, cell death maintains a dynamic balance in the number of cells within
tissues and organs. In the case of the skin, a large number of epidermal cells die and fall off
every day, while the stem cells in the basal layer continue to divide and produce new cells to
replenish, thus maintaining the normal structure and function of the skin. If there is an
abnormality in the cell death process, it can lead to various health problems. When the dead
cells do not die normally and continue to proliferate, it may lead to the development of
tumors; On the other hand, excessive death of cells that should not be killed may lead to
neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, which
are closely related to the abnormal death of nerve cells. Therefore, in-depth study of the
mechanism, types and regulation of cell death plays an important role in promoting the
understanding of life processes and overcoming major diseases.
1.2 Purpose of the study
This research report aims to comprehensively dissect cell death, and explore its unique
morphological characteristics, molecular mechanisms, and signaling pathways from the
multiple modes of cell death, such as apoptosis, necrosis, pyroptosis, and autophagic death.
The interrelationship between different types of cell death was sorted out, and their
occurrence under different physiological and pathological conditions was clarified. At the
same time, the application prospects of cell death research in disease diagnosis, treatment
and drug development are discussed, so as to provide theoretical basis and new ideas for
solving related medical problems.
1.3 Implications of the study
From a theoretical perspective, the study of cell death can help deepen the understanding of
the nature of life and fill the knowledge gap in the field of cell biology. By revealing the
molecular mechanism and regulatory network of cell death, we can better understand how
cells play a role in the process of development, differentiation, and aging, and improve the
biological theory system. For example, the study of the regulatory mechanism of apoptosis
genes provides important clues to explain the decision of cell fate during biological evolution.
At the practical application level, the study of cell death has a wide and far-reaching
significance. In the field of medicine, it provides new strategies and targets for overcoming
major diseases such as cancer, neurodegenerative diseases, and cardiovascular diseases.
By regulating the process of cell death, new treatments can be designed, such as drugs that
induce apoptosis in cancer cells, or drugs that inhibit excessive death of nerve cells, bringing
new hope to patients. In agriculture, cell death research can help improve the disease
resistance and quality of crops. Understanding the relationship between plant cell death and
disease resistance mechanisms can lead to the development of more resistant crop varieties
through genetic engineering, reducing the use of pesticides and ensuring food security.
2. The basic concept of cell death
2.1 Definition of cell death
Cell death is biologically defined as the irreversible cessation of cellular life phenomena. This
means that the cell has a permanent loss of various physiological functions, including
metabolic activity, material transport, signaling, etc. When cell death occurs, the structural
integrity of the cell is also disrupted, such as the rupture of the cell membrane, the
disintegration of organelles, the fragmentation of the nucleus, etc. Unlike dying cells, dying
cells are in the transitional stage of death, and there may still be some weak life activities,
such as the slowdown of metabolic activity but not completely stopped, some functions of
the cell membrane are affected but not completely lost, and the cells are still carrying out
some self-help or stress responses in an attempt to maintain their own survival. Cell death is
the end result of this process and is the irreversible endpoint.
2.2 The relationship between cell death and apoptosis and
necrosis
Apoptosis is an active cell death process precisely regulated by genes, also known as
programmed cell death. This process is highly orderly, and during apoptosis, the cell first
undergoes volume reduction, cytoplasmic condensation, endoplasmic reticulum expansion,
and fusion with the cell membrane to form a vesicular structure. Chromatin is highly
condensed, marginalized, and broken into fragments, and the nucleus is lysed. The cell
membrane invaginates the cell into multiple apoptotic bodies, which enclose the complete
organelles and cell contents, and are eventually recognized and engulfed by surrounding
phagocytic cells such as macrophages. Apoptosis plays a key role in the development,
maintenance of tissue homeostasis, and immune regulation of multicellular organisms.
During embryonic development, apoptosis is involved in the morphogenesis of the fingers
and toes, by removing excess cells between the fingers (toes) to create normal limb
structures.
Cell necrosis is a passive form of cell death, usually caused by strong external factors such
as physical damage (high temperature, radiation, mechanical force), chemicals (strong
acids, strong bases, toxins), pathogen infection, or severe ischemia and hypoxia. When cells
die, cells swell rapidly, the integrity of the cell membrane is destroyed, and a large number of
cell contents such as ions, enzymes, metabolites, etc. are released outside the cell,
triggering an inflammatory response in the surrounding tissues. Unlike apoptosis, cell
necrosis lacks an orderly regulatory mechanism and is a passive outcome when cells suffer
unbearable damage. In the acute inflammatory response, the invasion of pathogens may
lead to the necrosis of a large number of cells, causing inflammatory symptoms such as
redness, swelling, and pain in local tissues.
Both apoptosis and necrosis fall under the category of cell death and are two different types
of cell death. Apoptosis is a physiological and active death, which has positive significance
for the body and helps to maintain the normal function of cells and tissues; Cell necrosis is
mostly pathological and passive death, which often causes damage to the body and causes
adverse consequences such as inflammation. In some physiological and pathological
situations, apoptosis and necrosis may also transform into each other or coexist. In
ischemia-reperfusion injury, the cells may initially initiate an apoptotic procedure, but if the
injury is too severe, apoptosis may transform into necrosis, exacerbating the degree of
tissue damage.
3. Types and mechanisms of cell death
3.1 Apoptosis
3.1.1 Morphological characteristics of apoptosis
At the onset of apoptosis, the cell volume is significantly reduced, the cytoplasm is
concentrated, and the internal structures are tightly arranged. The microvilli on the surface of
the cell gradually disappear, and the connection between the cell and the surrounding cells
becomes loose, and eventually it detaches from the surrounding cells. With the process of
apoptosis, the endoplasmic reticulum expands continuously, forming many vesicles, which
fuse with the cell membrane, causing many vesicle-like protrusions to appear on the surface
of the cell membrane, that is, the phenomenon of vesicles. The chromatin in the nucleus is
highly condensed, marginally distributed, and tightly attached to the inner side of the nuclear
membrane, showing a half-moon or horseshoe-shaped shape, while the nucleolus is
gradually lysed and disappeared. Subsequently, the cell membrane is concave inward,
dividing the cell into multiple membrane-enclosed bodies, known as apoptotic bodies, which
contain intact organelles and part of the cell's contents. After the formation of apoptotic
bodies, they will be quickly recognized and engulfed by surrounding phagocytic cells such as
macrophages and epithelial cells, and the cell membrane will remain intact throughout the
process, and the cell contents will not leak into the extracellular environment, thus not
triggering an inflammatory response.
3.1.2 Molecular mechanisms of apoptosis
The molecular mechanisms of apoptosis are mainly divided into intrinsic pathway and
extrinsic pathway. The inner initiation pathway, also known as the mitochondrial pathway,
alters the outer membrane permeability of mitochondria when cells are stimulated by internal
stress signals such as DNA damage, oxidative stress, growth factor deficiency, etc. Pro-
apoptotic proteins (e.g., Bax, Bak, etc.) in the Bcl-2 family proteins located in the outer
mitochondrial membrane are activated, and they form pores in the outer mitochondrial
membrane, resulting in a decrease in mitochondrial membrane potential and the release of
cytochrome C from the mitochondria into the cytoplasm. Cytochrome C binds to apoptosis
protease activator-1 (Apaf-1) and ATP to form apoptosomes. Apoptotic bodies recruit and
activate initiating caspase-9, and activated caspase-9 further activates effector caspase-3,
caspase-6, and caspase-7, which cause morphological and biochemical changes in
apoptosis by cleaving multiple substrates within the cell, such as poly (ADP-ribose)
polymerase (PARP), lamin, etc.
The exogenous pathway, also known as the death receptor pathway, has some death
receptors on the cell surface, such as Fas (CD95), tumor necrosis factor receptor 1
(TNFR1), etc. When ligands such as Fas ligand (FasL) and tumor necrosis factor (TNF) bind
to the corresponding death receptor, the intracellular segment of the death receptor
aggregates and recruits the Fas-associated death domain protein (FADD), which then binds
to the inactive initiating Caspase-8 zymogen through the death effector domain (DED) to
form a death-inducing signaling complex (DISC). In DISC, Caspase-8 is activated by self-
cleavage, and activated Caspase-8 can directly activate effector caspase and induce
apoptosis. On the other hand, Caspase-8 can also cleave the Bid protein, making it tBid,
which is transferred to the mitochondria, activating the intrinsic apoptotic pathway, further
amplifying the apoptotic signal.
The Caspase family is a class of cysteine proteases that play a central regulatory role in the
process of apoptosis. Depending on the function, caspase can be divided into initiating
caspases (e.g., Caspase-2, Caspase-8, Caspase-9, Caspase-10, etc.) and effector
caspases (e.g., Caspase-3, Caspase-6, Caspase-7, etc.). The initiating caspase is recruited
and activated in response to the apoptotic signal, which then activates the downstream
effector caspase, which leads to structural and functional disruption of the cell by cleaving
numerous important protein substrates within the cell, ultimately initiating apoptosis.
3.1.3 Biological significance of apoptosis
During embryonic development, apoptosis is involved in the morphogenesis of organs and
tissues. During the formation of the neural tube, excess nerve cells will be removed through
apoptosis, thus ensuring the normal development of the structure and function of the neural
tube. In limb development, apoptosis causes the fingers and toes to gradually separate from
the initial tened state to form independent digital structures. In adult individuals, apoptosis
maintains a dynamic balance in the number of cells within tissues and organs. In the
epidermal layer of the skin, a large number of keratinocytes senescent and die and fall off
every day, while the stem cells in the basal layer continue to divide and produce new cells,
and in the process of migrating these new cells to the epidermal layer, some cells will
undergo apoptosis, so as to maintain the relative stability of the number of epidermal cells
and ensure the normal structure and function of the skin. In the immune system, apoptosis is
involved in the development, activation and maintenance of immune tolerance of immune
cells. During the development of T lymphocytes, immature T lymphocytes undergo positive
and negative selection in the thymus, and apoptosis occurs in those T lymphocytes that do
not recognize their own MHC molecules or have a high affinity for autoantigens, thus
ensuring the tolerance of the mature T lymphocyte pool to autoantigens and avoiding the
occurrence of autoimmune diseases. In the immune response, when the pathogen is
cleared, the activated immune cells are eliminated through apoptosis to prevent the body
from being damaged by an excessive immune response.
3.2 Necrosis
3.2.1 Morphological features of necrosis
When cells die, the integrity of the cell membrane is severely disrupted, resulting in an
imbalance of ions in the cell, and a large amount of water enters the cell, causing the cell to
swell rapidly and increase in size. Intracellular organelles such as mitochondria and
endoplasmic reticulum also swell, the mitochondrial crest structure becomes blurred, and the
endoplasmic reticulum expands into a large vesicle-like structure. The change of the nucleus
is one of the important markers of cell necrosis, which is first manifested as nuclear
condensation, that is, the volume of the nucleus shrinks due to dehydration, chromatin
condensation, and staining deepens. Then the nuclear membrane ruptures, and the
chromatin disintegrates into small fragments, i.e., the nucleus fragments; Finally, under the
action of DNUCLEASE, the DNA of chromatin is broken down, the nucleus loses its affinity
for the basic dye, and the staining fades until the nuclear outline disappears completely, that
is, the nucleolysis. Due to the rupture of the cell membrane, the contents of the cell such as
various enzymes, metabolites, ions, etc. are released outside the cell, which will trigger the
inflammatory response of the surrounding tissues, attract neutrophils, macrophages and
other immune cells to gather at the necrotic site and remove the necrotic tissue, but at the
same time, it will also cause inflammatory symptoms such as redness, swelling and pain of
local tissues.
3.2.2 Molecular mechanisms of necrosis
Traditionally, cell necrosis is a passive, unregulated form of cell death, but recent studies
have found that there is a form of programmed necroptosis, which is a form of necrotosis-like
cell death that is regulated by genes. The key molecules of programmed necrosis are
receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3),
and mixed-lineage kinase domain-like protein (MLKL). When cells are stimulated by tumor
necrosis factor (TNF), Toll-like receptor (TLR) ligands, etc., RIPK1 is recruited into the death
receptor complex and phosphorylated, and the activated RIPK1 further recruits and
phosphorylates RIPK3, which binds to RIPK1 to form necrosomes. RIPK3 in necrosomes
can phosphorylate MLKL, transforming it from a monomeric form to an activated tetrameric
form. Activated MLKL transplaces onto the cell membrane, creating holes in the cell
membrane and disrupting the integrity of the cell membrane, resulting in an outflow of
intracellular material and ultimately triggering cell necrosis. In addition, factors such as
calcium overload and overproduction of reactive oxygen species (ROS) are also involved in
the occurrence of cell necrosis. In ischemia-reperfusion injury, ischemia leads to a decrease
in intracellular ATP, ion pump dysfunction, and a large amount of calcium ions entering the
cell, causing calcium ion overload, activating calcium-dependent proteases and
phospholipases, leading to damage to cell membranes and organelle membranes; At the
same time, a large amount of ROS is produced during reperfusion, which attacks biological
macromolecules such as cell membranes, proteins, and nucleic acids, further exacerbating
cell damage and ultimately leading to cell necrosis.
3.2.3 Biological significance of necrosis
Cell necrosis is usually a passive response of an organism to serious external factors. In the
course of severe infection, pathogens multiply and release toxins that directly damage cells,
leading to cell necrosis, which is a defense mechanism of the body against pathogen
invasion by sacrificing part of the cells to limit the spread of pathogens. During the wound
healing process, the removal of necrotic tissue provides space for the growth of new tissue,
and then fibroblasts and vascular endothelial cells migrate to the site of necrosis to begin
tissue repair and regeneration. However, excessive cell necrosis can cause serious damage
to the body. In myocardial infarction, coronary artery occlusion leads to ischemia and
hypoxia of myocardial cells, necrosis of a large number of myocardial cells, impaired
contraction and diastolic function of myocardium, and in severe cases, it can lead to heart
failure or even life-threatening; In cerebral infarction, the blockage of blood vessels in the
brain causes necrosis of brain tissue, which will lead to the corresponding neurological loss,
such as hemiplegia, aphasia, etc.
3.3 Pyroptosis
3.3.1 Morphological characteristics of pyroptosis
When pyroptosis occurs, the cell first appears to be significantly swollen and continues to
increase in size, which is due to the increased permeability of the cell membrane to ions and
small molecules, resulting in a large amount of water entering the cell. With the progression
of pyroptosis, the cell membrane gradually loses its integrity and ruptures, and the contents
of the cell, including various inflammatory factors and cell debris, are released into the
extracellular environment. These released inflammatory factors, such as interleukin-1β (IL-
1β) and interleukin-18 (IL-18), attract immune cells to the pyroptosis site, triggering a strong
inflammatory response. Under the microscope, you can also see the pyroptosis bodies
formed during the process of pyroptosis, which are small vesicle structures formed by cell
membranes surrounding part of the cell contents, which contain organelle fragments, nucleic
acid fragments and other substances. Pyroptosis bodies are also eventually phagocytosed
and cleared by surrounding phagocytic cells, but pyroptosis triggers a more intense
inflammatory response than apoptosis due to the release of cell contents and the activation
of inflammatory factors.
3.3.2 Molecular mechanisms of pyroptosis
Pyroptosis is primarily mediated by inflammatory caspases, including Caspase-1 and
Caspase-11 (mouse)/Caspase-4, Caspase-5 (human). The classical pyroptosis pathway is
mediated by Caspase-1. When cells are stimulated by pathogen-associated molecular
patterns (PAMPs), such as lipopolysaccharides (LPS) of bacteria, nucleic acids of viruses,
etc., or damage-related molecular patterns (DAMPs), such as intracellular ATP, uric acid
crystals, etc., pattern recognition receptors (PRRs) recognize these signals and activate
inflammasomes. Inflammasomes are multiprotein complexes composed primarily of sensor
proteins (e.g., NLRP1, NLRP3, AIM2, etc.), adaptor proteins ASC, and Caspase-1
precursors. A conformational change occurs after the sensor protein recognizes the signal,
recruiting ASCs, which aggregate multiple Caspase-1 precursors together through
homotypic interactions, causing them to self-cleop and activate. Activated caspase - 1 can
cleave gasdermin D (GSDMD) on the one hand, cleaving it into an N-terminal domain
(GSDMD-N) and a C-terminal domain (GSDMD-C), GSDMD-N has membrane punching
activity, it can insert into the cell membrane, forming holes about 10 - 14 nm in diameter,
resulting in increased cell membrane permeability and cell swelling and rupture; On the other
hand, Caspase-1 can also cleave pro-IL-1β and pro-IL-18, allowing them to mature and
release them extracellularly, triggering an inflammatory response. The non-canonical
pyroptosis pathway is mediated by Caspase-11 (mouse)/Caspase-4, Caspase-5 (human).
These caspases directly recognize LPS within the cytosol, undergoing self-activation.
Activated Caspase-11 (mouse)/Caspase-4, Caspase-5 (human) can also cleave GSDMD,
causing pyroptosis. In addition, activated caspase-11 (mice) can also indirectly activate the
NLRP3 inflammasome by inducing potassium efflux, further enhancing the inflammatory
response.
3.3.3 Biological significance of pyroptosis
Pyroptosis plays an important role in the body's defense against infectious diseases. During
bacterial infection, macrophages engulf bacteria, recognize bacterial PAMPs, activate
inflammasomes, trigger pyroptosis, release inflammatory factors, recruit and activate other
immune cells, and jointly remove pathogens. In Mycobacterium tuberculosis infection,
macrophages undergo pyroptosis, which not only kills the bacteria directly, but also presents
bacterial antigens to T lymphocytes, activating the cellular immune response. Pyroptosis
also has potential applications in the treatment of tumors. Some chemotherapy drugs or
immunotherapy methods can induce pyroptosis of tumor cells, activate the body's anti-tumor
immune response by releasing tumor-associated antigens and inflammatory factors, and
enhance the killing effect on tumor cells. Studies have found that some immune checkpoint
inhibitors can induce pyroptosis in tumor cells in addition to regulating the activity of immune
cells when treating tumors, thereby improving the therapeutic effect.
3.4 Other types of cell death
Autophagy was originally thought to be a self-protective mechanism for cells under stress
conditions such as nutrient deficiencies, which provides energy and metabolic substrates for
cells by forming autophagosomes with a double-membrane structure that encapsulates and
degrades damaged organelles, protein aggregates and other substances in cells. But in
some cases, excessive autophagy can also lead to cell death, known as autophagic cell
death. When autophagic cells die, a large number of autophagosomes and autolysosomes
appear in the cell, and these structures continuously degrade important components within
the cell, resulting in impaired cell function and eventual death. The molecular mechanism of
autophagic cell death mainly involves the regulation of autophagy-related genes (ATG).
Stimulated by autophagy-inducible signals, ATG proteins interact to form multiple complexes
involved in the formation and maturation of autophagosomes. The ULK1 complex senses
intracellular trophic status and energy levels and is activated in response to nutrient
deficiencies, initiating the autophagy process; The PI3K-III complex is involved in the
initiation and extension of the autophagosomal membrane; ATG5-ATG12-ATG16L1
complexes and LC3-II, among others, play important roles in the expansion and closure of
autophagosomal membranes.
Ferroptosis is an iron-dependent form of cell death characterized by lipid peroxidation. The
morphological features of ferroptosis are mainly manifested by a decrease in mitochondrial
volume, an increase in membrane density, and a decrease or disappearance of the cristae.
In terms of molecular mechanism, the occurrence of ferroptosis is closely related to the
imbalance of intracellular iron metabolism, lipid peroxidation, and dysfunction of the
antioxidant system. Iron ions in cells catalyze hydrogen peroxide through the Fenton
reaction to produce hydroxyl radicals, which can attack polyunsaturated fatty acids on the
cell membrane and trigger lipid peroxidation. The accumulation of lipid peroxidation products
can lead to damage to the cell membrane and ultimately lead to cell death. Glutathione
peroxidase 4 (GPX4) is a key protein that inhibits ferroptosis, which uses glutathione (GSH)
to reduce lipid peroxides to the corresponding alcohols, thereby preventing lipid peroxidation
from occurring. When the activity of GPX4 is inhibited or expression is reduced, the
sensitivity of cells to ferroptosis increases. In addition, system Xc - is a cystine/glutamate
retrotransporter on the cell membrane that can take up cystine for the synthesis of GSH.
Inhibition of the function of system Xc- leads to a decrease in intracellular GSH levels, which
in turn promotes ferroptosis.
Cuproptosis is a newly discovered modality of cell death in 2022 and is mediated by copper
ions. When copper dies, intracellular copper ions bind to key enzymes in the TCA cycle,
such as lipoylated proteins, resulting in impaired function of these enzymes and obstruction
of the TCA cycle. At the same time, copper ions also promote the aggregation and
inactivation of mitochondrial respiratory chain complex I, inhibit mitochondrial respiration,
and lead to cellular energy metabolism disorders. In addition, copper-induced protein toxicity
stress activates the unfolded protein response (UPR), further exacerbating cell damage and
ultimately leading to cell death.
Sodium-overload necrosis is cell death caused by an abnormally high concentration of
sodium ions in cells. When cells are damaged or ion transport is abnormal, the sodium-
potassium pump on the cell membrane malfunctions and is unable to maintain a normal
sodium ion concentration gradient, resulting in a large amount of sodium ions entering the
cell. Intracellular sodium overload will cause a series of changes such as cell swelling,
mitochondrial dysfunction, and increased production of reactive oxygen species, which will
eventually lead to cell membrane rupture and cell necrosis.
Disulfidptosis is a type of cell death triggered by intracellular disulfide bond stress. Under
certain conditions, the disulfide bond formation and reduction balance within the cell is
disrupted, and excessive disulfide bonds accumulate in the protein, resulting in abnormal
protein structure and function. This protein toxic stress activates a series of signaling
pathways, such as endoplasmic reticulum stress response, mitochondrial dysfunction, etc.,
and ultimately leads to cell death.
Fourth, the research methods of cell death
4.1 Morphological detection methods
Light microscopy is a fundamental tool for the morphological detection of cell death. By
conventional hematoxylin-eosin (HE) staining, the apoptotic cells showed the characteristics
of nuclear condensation, chromatin condensation, marginalization, etc., and the nucleus was
darkened, the morphology became smaller and irregular, and the cell volume was also
reduced. Necrotic cells are characterized by swollen cells, sparse and irregular distribution of
chromatin in the nucleus, blurred boundaries, rupture of cell membranes, outflow of cell
contents, and infiltration of inflammatory cells in surrounding tissues. Using acridine
orange/ethidium bromide (AO/EB) double staining, AO can make the nuclei of normal cells
and early apoptotic cells fluoresce green, while EB can only enter late apoptotic cells and
necrotic cells with damaged cell membranes, making their nuclei fluoresce orange-red,
which can clearly distinguish cells in different states by fluorescence microscopy.
Electron microscopy provides higher resolution images of cell structures and is the gold
standard for determining apoptosis. Under transmission electron microscopy, the chromatin
of apoptotic cells is consolidated, gathered around the nuclear membrane, and presents
crescent-shaped or ring-shaped bodies, the cytoplasm is condensed, the endoplasmic
reticulum expands and fuses with the cell membrane to form vacuoles, the mitochondrial
structure is relatively intact in the early stage of apoptosis, and the nucleus is lysed into
fragments in the late stage, resulting in apoptotic bodies. The chromatin of necrotic cells is
sparse and finely granular, irregularly distributed, the boundaries are unclear, the cytoplasm
is swollen, the organelle structure is severely damaged, and the cell membrane is
incomplete. Scanning electron microscopy is mainly used to observe the changes in cell
surface morphology, such as the reduction or disappearance of microvilli on the surface of
apoptotic cells, and the appearance of vesicular protrusions. The surface of necrotic cells is
rougher and irregular, with a large amount of cell contents spilling.
4.2 Biochemical testing methods
During apoptosis, endogenous endonucleases are activated to sever chromosomal DNA
between nucleosomes, producing oligonucleotide fragments that are integer multiples of 180
to 200 bp, and characteristic DNA ladders can be observed by agarose gel electrophoresis.
The TUNEL (Terminal - deoxynucleotidyl Transferase Mediated dUTP Nick - End Labeling)
method uses terminal deoxynucleotidyl transferase (TdT) to attach labeled dUTPs such as
biotin, digoxigenin or fluorescein to the 3'-OH end of DNA fragments, and then label and
detect apoptotic cells in situ through corresponding detection systems, such as fluorescence
microscopy and flow cytometry. This method is highly sensitive and can detect a small
number of apoptotic cells, but necrotic cells may also be false-positive due to DNA breaks.
The Caspase family plays a key role in apoptosis, and detection of Caspase activity by
colorimetric or fluorometric methods is a commonly used biochemical assay. The
colorimetric method utilizes a caspase-specific substrate, such as Ac-DEVD-pNA, which
releases p-nitroaniline (pNA) from the caspase-cleavaged substrate with an absorption peak
at 405 nm, which can be detected by detecting absorbance values. The fluorescence
method uses a fluorescently labeled substrate, such as Ac-DEVD-AMC, where Caspase
cleavage releases 7-amino-4-methylcoumarin (AMC), which fluoresces upon excitation at a
specific wavelength and determines Caspase activity by fluorescence intensity. In addition,
Western blot can also be used to detect the splice body of Caspase, such as Caspase-3 is
cleaved by inactive 32kDa zymogen into 17kDa and 19kDa subunits after activation, so as to
determine the occurrence of apoptosis.
In normal cells, phosphatidylserine (PS) is located on the inner side of the cell membrane,
whereas in the early stage of apoptosis, PS is flipped from the inner to the outer side of the
cell membrane. Annexin V is a Ca² -dependent phospholipid-binding protein with high ⁺
affinity for PS that is detected by flow cytometry or fluorescence microscopy using
fluorescein (e.g., FITC, PE) or biotinylated Annexin V in combination with PI (propidium
iodide). Normal cells were negative for both Annexin V and PI; Early apoptotic cells were
Annexin V positive, PI negative; Late apoptotic and necrotic cells were positive for both
Annexin V and PI. This method can effectively distinguish between apoptotic cells and
necrotic cells at different stages.
4.3 Molecular biology detection methods
Real-time PCR (qPCR) detects the expression levels of genes associated with cell death.
Taking apoptosis as an example, Bcl-2 family genes are crucial in the regulation of
apoptosis, with Bcl-2 having anti-apoptotic effects and Bax promoting apoptosis. Total RNA
was extracted, reverse transcribed into cDNA, specific primers for Bcl-2 and Bax genes were
designed, PCR amplification was monitored by fluorescent dyes (e.g., SYBR Green) or
fluorescent probes (e.g., TaqMan probes) in qPCR reactions, relative expression of genes
was calculated based on Ct values (cycle thresholds), and differences in Bcl-2 and Bax gene
expression between experimental and control groups were compared, and the regulation of
apoptosis could be analyzed. In addition, qPCR can also be used for expression analysis of
pyroptosis-related genes such as NLRP3 and Caspase-1, as well as ferroptosis-related
genes such as GPX4 and SLC7A11.
Western blot is used to detect the expression and modification of cell death-related proteins.
Taking Caspase-3 protein in apoptosis as an example, the total protein of cells was
extracted, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate
proteins of different molecular weights in the gel, and then the proteins were transferred to
nitrocellulose membrane or PVDF membrane by transfer, the non-specific binding site on the
membrane was blocked with 5% skimmed milk powder or BSA, primary antibody against
Caspase-3 was added, incubated overnight at 4°C, the corresponding secondary antibody
was added after washing the membrane, and incubated at room temperature for 1-2 hours.
Protein bands are colored using chemiluminescent substrates (e.g., ECL) or chromogenic
substrates (e.g., DAB), and the presence and strength of the bands can be used to
determine the level of expression of the caspase-3 protein and whether it is activated
(cleaved). Similarly, other cell death-related proteins, such as autophagy-related proteins
LC3 and p62, ferroptosis-related protein FTH1, can also be detected by Western blot.
5 Research status and frontier progress of cell death
5.1 Current status of research on cell death in disease
In the field of cancer research, the mechanism of cell death is closely related to the
occurrence, development and treatment of cancer. Cancer cells often have the ability to
evade apoptosis, which is one of the important causes of cancer. Inactivating mutations in
the tumor suppressor gene p53 prevent cancer cells from initiating apoptosis normally,
resulting in indefinite cell proliferation. Inducing apoptosis of cancer cells is an important
strategy in cancer treatment. Many chemotherapy drugs, such as cisplatin and paclitaxel,
activate the apoptosis signaling pathway and promote cancer cell death by damaging the
DNA of cancer cells and interfering with microtubule assembly. In recent years,
immunotherapy has become a new hot spot in cancer treatment, and one of the
mechanisms of action of immune checkpoint inhibitors is to activate T lymphocytes by
relieving immunosuppression, enabling them to recognize and kill cancer cells, and induce
apoptosis in cancer cells. Novel cell death modalities such as pyroptosis and ferroptosis are
also attracting attention in cancer research. Studies have found that some chemotherapy
drugs or immunotherapy methods can induce pyroptosis of tumor cells, activate the body's
anti-tumor immune response by releasing tumor-related antigens and inflammatory factors,
and enhance the killing effect on tumor cells. Inducing ferroptosis in tumor cells has also
become a potential anti-cancer strategy, which provides a new idea for cancer treatment by
inhibiting the activity of glutathione peroxidase 4 (GPX4), increasing the level of lipid
peroxidation in tumor cells, and inducing ferroptosis.
The pathogenesis of neurodegenerative diseases such as Alzheimer's disease and
Parkinson's disease is closely related to the abnormal death of nerve cells. In Alzheimer's
disease, a large number of amyloid plaques and neurofibrillary tangles appear in the brain,
and these pathological changes lead to an increase in apoptosis of nerve cells . Aggregation
of amyloid β protein (Aβ) can activate the death receptor pathway and induce apoptosis in
neuronal cells; At the same time, Aβ also causes mitochondrial dysfunction, leading to nerve
cell death through the intrinsic apoptotic pathway. In Parkinson's disease, abnormal
aggregation of α-synuclein forms Lewy bodies, which damage nerve cells, leading to
apoptosis . In addition, necrosis and autophagic death of nerve cells are also involved in the
pathogenesis of neurodegenerative diseases. In cerebral ischemia-reperfusion injury,
ischemia leads to nerve cell energy metabolism disorders, cell membrane ion pump
dysfunction, and a large amount of calcium influx, causing cell necrosis. The large amount of
reactive oxygen species (ROS) produced during reperfusion can further damage nerve cells,
leading to apoptosis and autophagic death.
Autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, etc., in
which abnormal cell death plays a key role in the pathogenesis. In systemic lupus
erythematosus, apoptosis is abnormally increased, and the clearance of apoptotic cells is
impaired, resulting in a large number of apoptotic cells and their contents accumulating in
the body. The components of these apoptotic cells, such as nucleic acids and proteins, are
recognized by the immune system as autoantigens, triggering an autoimmune response that
produces a large number of autoantibodies to attack the body's own tissues and organs.
Studies have found that the complement system plays an important role in the process of
apoptotic cell clearance, and defects or abnormalities in complement components can lead
to impaired apoptotic cell clearance and increase the risk of autoimmune diseases. In
rheumatoid arthritis, the resistance to apoptosis and excessive proliferation of synovial cells
in the joints, as well as the infiltration and activation of inflammatory cells, lead to joint
inflammation and tissue damage . Pyroptosis is also involved in the pathogenesis of
rheumatoid arthritis, in which macrophages in the synovial membrane of joints are stimulated
by inflammation and activate inflammones, triggering pyroptosis, releasing a large number of
inflammatory factors, and aggravating joint inflammation.
5.2 Cutting-edge breakthroughs in cell death research
In recent years, a series of important breakthroughs have been made in the study of the
molecular mechanism of cell death. Xu Daichao's team from the Interdisciplinary Research
Center for Biology and Chemistry at the Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, and Gu Jinyang's team from Union Hospital of Tongji Medical College
of Huazhong University of Science and Technology found that palmitoylation regulates the
activation of RIPK1 kinase, which can be used as a broad-spectrum initiation mechanism for
programmed cell death (apoptosis or programmed necrosis). Palmitoylation of RIPK1 was
induced by TNF stimulation for a short period of time, and the modification site was located
at a conserved cysteine residue C257 in its kinase domain. DHHC5 is the primary
palmitoyltransferase that mediates palmitoylation of RIPK1, and DHHC5 is dependent on
K63 ubiquitination of RIPK1 for its recruitment. When cells sense TNF, RIPK1 is rapidly
recruited to a TNF receptor complex adjacent to the cell membrane, where RIPK1
undergoes a ubiquitination modification of K63 linkage in response to the E3 enzyme
cIAP1/2. These K63 ubiquitin chains recruit DHHC5 localized to the cell membrane, bringing
it closer to RIPK1. Subsequently, DHHC5 catalyzes palmitoylation on the RIPK1 kinase
domain, increasing the hydrophobic properties of the kinase domain and promoting its
homologous interactions. In the presence of cell death checkpoint blockage, the above
processes can enhance the trans-self-activation of RIPK1, ultimately leading to downstream
RIPK1-mediated cell death, including apoptosis and programmed necrosis.
In terms of the discovery of new cell death methods, sodium overload cell necrosis is a
newly recognized cell death mode in recent years. When cells are damaged or ion transport
is abnormal, the sodium-potassium pump on the cell membrane malfunctions and is unable
to maintain a normal sodium ion concentration gradient, resulting in a large amount of
sodium ions entering the cell. Intracellular sodium overload will cause a series of changes
such as cell swelling, mitochondrial dysfunction, and increased production of reactive
oxygen species, which will eventually lead to cell membrane rupture and cell necrosis.
Studies have also found that some drugs can intervene in the occurrence of sodium
overload cell necrosis by regulating the function of sodium ion channels or sodium-
potassium pumps, providing potential targets for the treatment of related diseases.
5.3 Challenges and prospects for cell death research
The complex cell death signaling pathway, with a large number of intersections and
regulatory nodes, makes it a great challenge to comprehensively elucidate the mechanism of
cell death. Taking apoptosis as an example, there is an intercorrelation and regulation
between the internal and external apoptotic pathways, and they are also regulated by
various factors such as Bcl-2 family proteins and IAP family proteins. There are differences
in the activation and regulation of cell death signaling pathways under different cell types
and physiological and pathological conditions, which increases the complexity of research.
The signaling pathways of other cell death modes, such as cell necrosis, pyroptosis, and
autophagic death, are similarly complex, and there are interactions and influences between
them. In ischemia-reperfusion injury, apoptosis, necrosis, and autophagic death may occur
simultaneously, and the intertransformation and regulatory mechanisms between these cell
death modes are not fully understood.
At present, most of the research on cell death is based on in vitro cell models and animal
models, which are different from human physiological and pathological states. In vitro cell
models lack the complex microenvironment and cell-cell interactions in vivo, and animal
models cannot fully simulate the occurrence and development of human diseases. In the
study of neurodegenerative diseases, the commonly used mouse models are still different
from the pathogenesis and course of human neurodegenerative diseases, although they can
simulate some pathological features. In addition, there may be differences in cell death
mechanisms between different species, which makes it difficult to translate basic research
results into clinical applications.
With the deepening of research, it is expected to further reveal the molecular mechanism
and regulatory network of cell death, and provide more theoretical basis and therapeutic
targets for overcoming major diseases. In terms of cancer treatment, based on the in-depth
understanding of the mechanism of cell death, more precise and efficient anti-cancer drugs
and treatments will be developed to improve the survival rate and quality of life of cancer
patients. In the field of neurodegenerative disease treatment, we are looking for drugs or
treatment strategies that can inhibit the abnormal death of nerve cells, delay the progression
of the disease, and improve the symptoms of patients. Combined with multi-omics
technology (such as genomics, transcriptomics, proteomics, metabolomics, etc.), the
changes of genes, proteins and metabolites in the process of cell death are comprehensively
and systematically studied, and the molecular mechanism and regulatory network of cell
death are deeply explored. Using single-cell sequencing technology, we will study the
mechanism of cell death at the level of individual cells and reveal the heterogeneity of
different cell types in the process of cell death.
The combination of cell death research with emerging technologies such as artificial
intelligence and big data will bring new opportunities in this field. By constructing databases
and models related to cell death, and using artificial intelligence algorithms to analyze
massive research data, we can explore potential cell death regulatory mechanisms and
therapeutic targets. Use artificial intelligence technology to design novel cell death inducers
or inhibitors to accelerate the drug development process.
VI. Conclusions
6.1 Summary of the study
This study comprehensively describes the key biological process of cell death, covering a
variety of cell death types and their mechanisms. As a typical representative of programmed
cell death, apoptosis is precisely regulated by the Caspase family through the internal and
external initiation pathways, and plays an indispensable role in embryonic development,
tissue homeostasis maintenance, and immune regulation. From passive death in the
traditional understanding to the discovery of programmed necrosis, a form of genetically
regulated form, the molecular mechanism of cellular necrosis involves key molecules such
as RIPK1, RIPK3 and MLKL, which is of great significance in resisting pathogen invasion
and wound healing, but excessive necrosis can also cause serious damage to the body.
Pyroptosis, mediated by inflammatory caspases, has shown unique value in infectious
disease defense and tumor therapy through classical and non-classical pathways. In
addition, new cell death modalities such as autophagic death, ferroptosis, copper thorptosis,
sodium overload cell necrosis, and disulfide death have their own unique morphological
characteristics, molecular mechanisms, and biological significance, which greatly enrich our
understanding of cell death.
In terms of research methods, morphological detection methods, such as optical microscopy
and electron microscopy, show us the morphological changes in the process of cell death
from different resolution levels. Biochemical detection methods, such as DNA ladder band
analysis, Caspase activity assay, and Annexin V/PI double staining, provide a basis for the
determination of cell death from molecular and biochemical perspectives. Molecular biology
assays, such as real-time PCR and Western blot, go down to the gene and protein level to
help us study the expression and regulation of genes and proteins associated with cell
death.
At present, significant progress has been made in the field of cell death research, and
inducing apoptosis, pyroptosis and ferroptosis of cancer cells has become an important
therapeutic strategy in cancer. In neurodegenerative diseases, abnormalities such as
apoptosis, necrosis, and autophagic death are involved in the pathogenesis. In autoimmune
diseases, abnormal cell death triggers an autoimmune response. At the same time, there are
also breakthroughs in the study of molecular mechanisms and the discovery of new cell
death methods, such as palmitoylation modification to regulate the activation of RIPK1
kinase to initiate programmed cell death, and sodium overload cell necrosis has been
discovered as a new cell death mode. However, the complexity of cell death signaling
pathways and the differences between research models and humans remain challenges for
current research.
6.2 Research outlook
Looking forward to the future, cell death research has broad application prospects in disease
treatment. In cancer treatment, based on the in-depth understanding of the mechanism of
cell death, it is expected that more targeted anti-cancer drugs with fewer side effects will be
developed, such as designing drugs that specifically induce ferroptosis or pyroptosis in
cancer cells, while reducing the damage to normal cells. In the treatment of
neurodegenerative diseases, effective neuroprotective strategies are found to inhibit the
abnormal death of nerve cells and delay disease progression by intervening in cell death-
related signaling pathways.
The fusion of multi-omics techniques will provide a more holistic view of cell death research.
By integrating data from genomics, transcriptomics, proteomics, and metabolomics, it is
possible to more systematically unravel molecular events and regulatory networks in the
process of cell death, and discover potential therapeutic targets and biomarkers. The
application of single-cell sequencing technology will enable us to deeply study the
mechanism of cell death at the level of individual cells, reveal the heterogeneity of different
cell types in the process of cell death, and provide a basis for precision treatment.
The combination with emerging technologies will breathe new life into cell death research.
Using artificial intelligence and big data technology, we can analyze and mine massive cell
death research data to accelerate the research and development process of cell death-
related drugs. For example, machine learning algorithms can be used to predict the structure
and activity of novel cell death inducers or inhibitors, improving the efficiency and success
rate of drug development. With the continuous deepening of research, the field of cell death
will surely bring more breakthroughs and hope for overcoming major diseases and protecting
human health.
Thanks
In the process of completing this research report on cell death, I have received the help of
many teachers, colleagues, relatives and friends, and I would like to express my sincerest
gratitude to them.
I would like to express my heartfelt gratitude to my supervisor for his careful guidance and
valuable advice in determining the research direction, collecting and collating the data, and
writing the report. Your rigorous academic attitude, profound professional knowledge and
persistent pursuit of scientific research have always inspired me to continue to move
forward, giving me a deeper knowledge and understanding in the field of cell death research,
and laying a solid foundation for the completion of this report.
I would like to thank my colleagues for sharing their research experiences and insights in the
daily exchanges and discussions, which have provided me with new ideas and inspiration. In
particular, I would like to thank my partners who have given me specific help in experimental
techniques and data analysis, which has allowed me to avoid many detours in the research
process and successfully complete various research tasks.
My family has been my most solid support, giving me meticulous care and support during the
long research and writing period, understanding my busyness and stress, and creating a
safe research environment for me to devote myself to the study of cell death.
At the same time, I would also like to thank those researchers who have worked hard in the
field of cell death research, and it is your research results that have provided rich materials
and theoretical basis for my report, and promoted the development and progress of the
entire field.
In the future, I will continue to work hard to contribute to the research in the field of cell
death, and live up to everyone's expectations and help.
(Note: Portions of the document may be AI-generated)
