Cell Death Dis︱Kong Hui et al reveal the role of P2X7/NLRP3 inflammasome pathway in early diabetic retinopathy

Written by ︱Kong Hui, Chen Tianran, Cui Yan editors︱Wang Sizhen Diabetic retinopathy (DR) is the most common complication of diabetes, which can lead to severe visual impairment and blindness
.

Retinal endothelial cells (RECs) are one of the main cell types involved in DR [1]
.

In the early stages of DR, hypoperfusion may lead to low-grade chronic vascular inflammation (leukocytosis) and progressive and irreversible hypoxia of retinal capillaries, eventually leading to the formation of microaneurysms and acellular capillaries [2]
.

RECs are the main target cells of diabetes-induced vascular injury, and the alteration of RECs plays a crucial role in the development of retinal diseases and is a hallmark of DR
.

The P2X7/NLRP3 pathway plays an important role in amplifying inflammation through an ATP feedback loop, promoting inflammatory response, pyroptosis and apoptosis
.

 Inflammation caused by hyperglycemia and metabolic changes in diabetes damages the retinal neurovascular unit (RNUT), leading to progressive and progressive neuropathy
.

The P2X7/NLRP3 signaling pathway plays an important role in the amplification of neuroinflammation
.

Neuroinflammation is positively associated with the activation of P2X7R through risk-associated molecular patterns (DAMPs), the predominant of which is extracellular ATP, and upon transient ATP stimulation, the P2X7 receptor cation channel opens, resulting in K+ efflux and Na+, Ca2+ influx
.

Under the continuous stimulation of ATP, the P2X7 receptor will form a non-selective membrane pore, allowing some substances with a relative molecular mass of 900 kD to enter the cell, resulting in cell death
.

The CD40-ATP-P2X7 signaling pathway mediates RNUT intercellular signaling crosstalk and promotes the programmed cell death of endothelial cells, which is crucial in the development of DR[2]
.

K+ efflux, calcium dysregulation and glutamine efflux caused by activation of P2X7 are the main mechanisms of NLRP3 inflammasome activation[3]
.

The NLRP3 inflammasome is composed of NLRP3, the adaptor protein ASC, and the effector protein Caspase-1
.

Upon activation, the NLRP3 inflammasome mediates the activation of caspase-1, splitting proIL-1β and proIL-18 into their active forms
.

Therefore, the study of the P2X7/NLRP3 inflammasome signaling pathway can provide new targets for the early intervention and treatment of DR
.

  On April 12, 2022, Dr.
Kong Hui from the Ophthalmology Department of Qilu Hospital of Shandong University, Dr.
Kong Hui and others published a paper entitled "Targeted P2X7/NLRP3 signaling pathway against inflammation, apoptosis, and pyroptosis of retinal endothelial endothelial cells" in the Nature sub-journal Cell Death Disease "cells in diabetic retinopathy" research paper, proposed that the P2X7/NLRP3 inflammasome pathway amplifies the inflammatory response of retinal vascular endothelial cells through the ATP positive feedback loop, promotes cell pyroptosis and apoptosis, and plays a role in endothelial inflammatory damage in early diabetic retinopathy important role
.

Dr.
Kong Hui is the first author of the paper, and Professor Cui Yan is the corresponding author of the paper
.

In this study, the authors first found that a model of diabetes was induced by intraperitoneal injection of streptozotocin (STZ) in male 6-week-old C57BL/6J mice.
The level of LPS in the blood plasma supernatant increased, and the expressions of P2X7 and NLRP3 in the retina as well as downstream inflammatory factors and apoptotic proteins increased.
After adding 3TC (a nucleoside reverse transcriptase inhibitor) treatment, the retinal inflammatory factors IL18, The expression of IL1β and ICAM1 was decreased, and the vascular permeability was decreased (Fig.
1)
.

Figure 1 In STZ-induced diabetic mice, the expression of P2X7/NLRP3 pathway and downstream inflammatory factors increased, and capillary exudation increased
.

Inhibition of P2X7/NLRP3 signaling pathway in DR mice resulted in decreased expression of inflammatory factors, decreased cell-free capillary area, and decreased exudation
.

(Source: Kong H et al.
, Cell Death Dis, 2022) In the in vitro experiments, the authors verified the effect of the P2X7/NLRP3 inflammasome pathway on retinal endothelial cells
.

The apoptosis rate of mRECs cultured in high glucose increased in vitro; compared with high glucose induction alone, the co-application of high glucose induction and LPS/BzATP/TNF-α significantly increased the secretion of IL-18, IL-1β and ATP, as well as cellular apoptosis (Figure 2)
.

After the specific inhibitors of P2X7 and NLRP3, A740003, 3TC and MCC950 were used to inhibit the P2X7/NLRP3 pathway, the secretion of IL-18, IL-1β and ATP, and Casepase1/3/11 were all decreased, which significantly reduced the apoptosis rate of cells.
, reversed the harmful effects of high sugar and LPS
.

These results confirmed the involvement of the P2X7/NLRP3 pathway in the pathogenesis of diabetic retinal dysfunction and the therapeutic potential of 3TC in inhibiting diabetic endothelial damage (Figure 3)
.

Figure 2 In high glucose culture and LPS stimulation, the expression of P2X7/NLRP3 pathway and downstream inflammatory factors increased, and the apoptosis and pyroptosis of mRECs increased
.


(Source: Kong H et al.
, Cell Death Dis, 2022) Figure 3 3TC inhibited the P2X7 and NLRP3 inflammasome pathways attenuating high glucose and LPS-induced apoptosis and pyroptosis of mRECs
.

(Credit: Kong H et al.
, Cell Death Dis, 2022) ATP is a potent inducer of NLRP3 activation and IL-1β maturation, and is the only physiological agonist of P2X7
.

Diabetic hyperglycemia and high LPS levels activate P2X7 to induce REC death through a dual mechanism of pro-inflammatory cytokines and ATP release and macropore formation
.

After activation of P2X7, the NLRP3 inflammasome is activated through K+ efflux, ROS and glutathine efflux, etc.
, while the stimulation of LPS mediates the NLRP3 inflammasome-induced pyroptosis through the Caspase-1 canonical pathway and the Caspase-11 non-canonical pathway
.

The card domain of Caspase11 can recognize LPS to form an oligomeric complex and be activated.
The activated Caspase11 cleaves the carboxyl terminus of GSDMD to form a GSDMD-NT active fragment, and multiple GSDMD-NT fragments migrate to the cell membrane to form a stable annular pore.
The inner and outer diameters were 15 nm and 32 nm, respectively, leading to pyroptosis
.

Pannexin1 (PANX1) is a channel that mediates ATP release.
The activation of PANX1 requires C-terminal cleavage mediated by Caspase11.
Caspase11 can activate pannexin-1 channel leading to ATP release, which further amplifies P2X7-mediated cytotoxicity [4]
.

Activated Caspase11 simultaneously activates the NLRP3/ASC-Casp-1 pathway, leading to the maturation and secretion of proinflammatory cytokines IL-1β and IL-18
.

Figure 4 Summary of the article: P2X7 mediates typical and atypical activation of NLRP3 to induce pyroptosis
.


(Source: Kong H et al.
, Cell Death Dis, 2022) The conclusion and discussion of the article, inspiration and prospect ATP may be the most primitive extracellular messenger.
Under physiological conditions, ATP exists in the extracellular space in a small amount (nmol/ l) [5]
.

Second, they are stored intracellularly in large quantities (from 5-10 µmol/l) [6]
.

Extracellular ATP may act as a "danger" signal under pathological conditions
.

Almost all cells can release ATP extracellularly upon specific stimuli
.

ATP can be released after cell injury and death, and it can also be released from living cells through different channels [7]
.

Its release mechanisms include secretory exocytosis, connexin or ubiquitin hemichannels, ATP-binding cassette (ABC) transporters, calcium homeostasis regulator (CALMH) channels, ATP-gated P2X7R, and two types of channel maximal anion channels ( MACs) and volume-regulated ion channels (VRACs)
.

The team's study confirmed that high glucose and high LPS environment during DR induced the opening of the macropore of RECsP2X7 in mice, and mediated the ATP autocrine feedback loop to activate the inflammasome pathway, triggering the assembly and continued release, amplification and continuation of the NLRP3 inflammasome Inflammation and induce P2X7 and NLRP3-dependent pyroptosis, while 3TC, a nucleoside reverse transcriptase inhibitor first used to treat HIV, can inhibit the P2X7 receptor, protect the endothelium by targeting the P2X7/NLRP3 signaling pathway Cells were protected from apoptosis, reduced retinal inflammation, and had a therapeutic effect on early DR (Figure 4)
.

The team proposed that the P2X7/NLRP3 inflammasome pathway is involved in early diabetic retinopathy and plays a crucial role in the inflammation, apoptosis and pyroptosis of endothelial cells, which provides a direction for the study of the inflammatory mechanism of DR.
It also provides a new target for early DR drug therapy (Figure 4)
.

The study has limitations, and the molecular underlying mechanism for the transformation of P2X7 from ion channels into macropores is unclear
.

K+ efflux is widely regarded as a standard upstream signal for NLRP3 inflammasome activation; it has also been shown that extracellular ATP can lead to the formation of the P2X7 receptor-paxillin-NLRP3 complex
.

The construction of this complex promotes the assembly of the NLRP3 inflammasome complex through deubiquitination of NLRP3, a mechanism that plays an important role in activating NLRP3 inflammatory activation, and these specific mechanisms are involved in cell apoptosis and pyroptosis.
Its role needs further study
.

Link to the original text: https://doi.
org/10.
1038/S41419-022-04786-W The first author Kong Hui (left), the corresponding author Cui Yan (right) (Photo provided by: Cui Yan Research Group of Ophthalmology Department of Qilu Hospital of Shandong University) Kong Hui, doctor of ophthalmology, attending physician of ophthalmology, research direction cataract, fundus disease
.

The master's degree is from Fan Xianqun, Academician of the Department of Ophthalmology of Shanghai Jiaotong University, and the doctor's degree is from Professor Cui Yan of Qilu Hospital of Shandong University
.

 Cui Yan, Doctor of Ophthalmology, Doctoral Supervisor, Chief Physician of Ophthalmology Department of Qilu Hospital of Shandong University, Professor of Ophthalmology of Shandong University
.

Currently specializing in vitreoretinal diseases
.

He presided over a number of national and provincial natural science funds, and published dozens of SCI papers as the first author and corresponding author
.

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18~4.
30) References (swipe up and down to read) [1] Bharadwaj AS, Appukuttan B, Wilmarth PA, Pan Y, Stempel AJ, Chipps TJ, et al.
Role of the retinal vascular endothelial cell in ocular disease.
Prog Retin Eye Res.
2013;32:102–80.
[2] Subauste CS.
The CD40-ATP-P2X7 receptor pathway: