Inhibition of SET domain–containing (lysine methyltransferase) 7 alleviates cognitive impairment through suppressing the activation of NOD-like receptor protein 3 inflammasome in isoflurane-induced aged mice

Abstract

Background

As a common postoperative complication to elderly patients, postoperative cognitive dysfunction (POCD) is a central nervous system complication, often taking place after anesthesia and surgery. (Su(var)3-9, enhancer-of-zeste, and trithorax) domain–containing protein 7 (SETD7) plays important roles in metabolic-related diseases, viral infections, tumor formation, and some inflammatory reactions. However, the role and mechanism of SETD7 in POCD have not been previously studied.

Methods

RT-PCR and Western blot were performed to evaluate the efficiency of knockdown of SETD7. The pathological changes of hippocampal neurons in isoflurane-anesthetized mice were detected by HE staining, and the Morris water maze experiment was performed to evaluate the learning and memory abilities of mice. The effect of SETD7 on the hippocampus in isoflurane-induced aged mice was examined by Western blot and TUNEL assay. Then ELISA assay was applied to determine the expression of some inflammatory cytokines, followed by the detection of expression of NOD-like receptor protein 3 (NLRP3) inflammasome through Western blot.

Results

The data of this research revealed that SETD7 knockdown improved cognitive impairment in isoflurane-anesthetized mice, ameliorated cell pyroptosis, inhibited the release of inflammatory cytokines, and suppressed the activation of NLRP3 inflammasome in the hippocampus in isoflurane-induced aged mice.

Conclusion

Collectively, these results provided evidence that the inhibition of SETD7 could alleviate neuroinflammation, pyroptosis, and cognitive impairment by suppressing the activation of the NLRP3 inflammasome in isoflurane-induced aged mice.

Keywords

NOD-like receptor protein 3 inflammasome SET domain–containing (lysine methyltransferase) 7 neuroinflammation pyroptosis postoperative cognitive dysfunction

Introduction

Elderly patients who undergo surgery under general anesthesia are likely to develop postoperative cognitive dysfunction (POCD), which is a common postoperative complication.^1–3 Studies suggest that POCD is caused by temporary impairment of brain function, and it will increase the incidence of Alzheimer’s disease or other complications and hinder a normal recovery of the body, significantly affecting the quality of life of elderly patients after surgery.^4,5

Although the specific pathogenesis of POCD is not clear, several hypotheses could explain the mechanism of POCD. POCD often occurs after surgery, but whether surgical trauma is a direct or indirect cause of POCD is still unknown. Some studies indicated that surgical trauma stress can activate the peripheral immune system, thereby inducing the release various inflammatory factors, including several important pro-inflammatory cytokines IL-6, interleukin 1β (IL-1β), and TNF-α.^6,7 Substantial evidence confirmed that overexpressed pro-inflammatory cytokines, especially in the hippocampus, can lead to the disruption of long-term potentiation and the impairment of hippocampal-mediated cognitive functions.^8–10 The inflammatory response is related to declined neurocognitive function in surgical patients.¹¹ The NOD-like receptor protein 3 (NLRP3) inflammasome has the function of regulating the body’s chronic inflammatory response.¹² NLRP3 is an endogenous or exogenous danger signal cytoplasmic receptor, and it also serves as a molecular platform that activates caspase-1 and regulates the maturation and secretion of pro-inflammatory cytokines, such as IL-1β and interleukin 18 (IL-18).¹³ It has been found that general isoflurane anesthesia can induce the activation of NLRP3 inflammasome, resulting in the cleavage of caspase-1 and secretion of IL-1β and TNF-α in the hippocampus of aged mice.^14–17 Some researchers observed that priming by up-regulation of NLRP3 is likely via the nuclear factor-κB (NF-κB) pathway to increase NLRP3 transcription. The deubiquitination of NLRP3 protein allows the protein to be assembled with the components of the NLRP3 inflammasome, which completes the priming process; therefore, isoflurane might induce NLRP3 inflammasome activation by increasing NLRP3 inflammasome assembly.¹³ Currently, there is a need for a comprehensive understanding of the underlying mechanism of POCD and discovery of molecular mediator to NLRP3 inflammasome.

SETD7, also known as SET9 or SET7/9, is a 41-kDa lysine methyltransferase which belongs to a family of proteins containing the SET domain. Some studies showed that it cannot only methylate H3K4, but affect the dimethylation of H3K4, change the binding ability of cofactors and histones, and then regulate the state of chromatin, and promote gene expression. SETD7 can also regulate the activity of transcription factors, change the stability of proteins, and activate the promoter region of genes by methylating non-histone proteins. The diversity of histone and non-histone substrates of SETD7 allows it to have multiple functions. SETD7 plays important roles in inflammatory response, cell cycle regulation, DNA damage response, and tumor formation.^18,19 Studies observed that the disorder of SETD7 is related to the pathogenesis of various diseases, including tumorigenesis, pulmonary fibrosis, diabetes, and myocardial ischemia-reperfusion injury.^18–21 In macrophages and human bronchial epithelial cell lines (Beas-2B), knockdown of SETD7 can resist NF-κB–induced oxidative stress and pro-inflammatory cytokine production.²² However, the effect of SETD7 on POCD and the mechanism of action remained unclear. The article aimed to explore whether SETD7 was involved in POCD and the underlying mechanism, hoping to develop a promising molecule target for treating POCD in elderly patients.

Materials and Methods

Animal experiment

Male C57BL/6 mice (15 months old) were purchased from Topbiotech Biotechnology Co, Ltd (Shenzhen, China). All animal experiments were approved by the Animal Investigation Ethics Committee of The Seventh Affiliated Hospital of Sun Yat-Sen University (Shenzhen, China), following the institutional guidelines and national animal welfare. All the mice were housed in an environment with a 12 h light/dark cycle at 22–25°C, and provided with access to enough water and food.²³ Then, the animals were divided into randomly four groups (n = 8 in each group), namely, control, isoflurane, isoflurane + shNC, and isoflurane + shSETD7, and an extra group of isoflurane + MCC950 (an inhibitor of NLRP3) (10 mg/kg, China Peptides Co, Ltd, Shanghai, China). Anesthesia was induced by placing the mice in a plastic container under the condition of 1.5% isoflurane at 600 μg/kg/min in normal air for 2 h, while the mice in the control group were maintained in normal air for 2 h^24,25 The levels of isoflurane and oxygen were detected by a gas monitor. SETD7 shRNA–induced silencing was performed with lipofectamine transfection. We injected shSETD7 or control shNC to the left ventricle which had been previously drilled with a skull microhole using the Hamilton microsurgical syringe. The detailed procedures for left ventricle injection were as follows: isoflurane was used to anesthetize newborn mice for 7 s, followed by left ventricular injection with a 5-μL Hamilton syringe. To locate the injection site, with the lambdoid suture point on the skull surface as the reference point, the injection point was set to 2.0 mm on the head side, 1.5 mm to the right opening, and 2.0 mm deep below the bone surface. The injection was slowly completed within 2 min. After 120 h of transfection, the following experiments were performed. The schema of animal treatment was as shown in Supplementary Figure 1.

Quantitative real-time polymerase chain reaction (RT-PCR)

Total RNA was isolated using RNAiso Plus reagent (Takara, #9109) from the hippocampus of the control group, isoflurane group, isoflurane + shNC group, and isoflurane + shSETD7 group. The first strand cDNA was synthesized using PrimeScript^TM RT Master Mix (Takara, #RR036) with a 10-μL reaction system containing 500 ng total RNA, 5× PrimeScript RT Master Mix, and RNase free ddH₂O. The cDNA was diluted in 1:3 ddH₂O, and then synthesized by SYBR GREEN real-time PCR Master Mix (Roche, #04913914001-1) in the ABI7500 system for PCR reaction. The cDNA was reacted for a total of 40 cycles and then amplified at 95°C for 10 min, at 95°C for 20 sec, 65°C for 20 sec, and eventually at 72°C for 30 sec. ²⁶ Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was considered as an internal reference. Primer sequences used in the article were as follows: Forward primer of SETD7: 5^′-AGTGCACGACGGACTTTCAA-3^′ and reverse primer of SETD7: 5^′-CTCTTTTGCTTAGGTGCGGC-3^′. Forward primer of GAPDH: 5^′-TGCACCACCAACTGCTTAGC-3^′ and reverse primer of GAPDH: 5^′-GGCATGGACTGTGGTCATGA-3^′.

Western blot

Total protein was separated from tissues, and lysed by a mixture of buffer containing protease inhibitor and phenylmethanesulfonyl fluoride (PMSF). Protein samples were well mixed, fully lysed by ultrasonic waves for several times, and then centrifuged at 13,000g for 15 min at 4°C. After collecting the supernatant, the protein concentration was detected by a BCA assay kit (Thermo fisher, #23225). The remaining supernatant was gently mixed with 6× loading buffer at 4:1 and then boiled in a water bath at the 100°C for 5 min. The fresh protein was electrophoretically separated by 10% SDS-PAGE and transferred to the PVDF membranes, followed by blocking with 5% non-fat milk for 2 h at room temperature. After washing the membranes in 1× TBST, the membranes were then incubated with the primary antibody, which was diluted in 1× TBS buffer at different ratios at 4°C overnight. After another round of washing with 1× TBST for three times, the secondary antibody for goat-anti-mouse HRP (Beyotime, Cat. No. A0216) or goat-anti-rabbit HRP (Beyotime Cat. No. A0208) at a dilution of 1:1000 was incubated with the membranes for 2 h at room temperature.²⁷ Then, the protein expression was detected by the ECL kit (Thermo fisher, #34580). The image was displayed in the GS800 densitometer scanner (Bio-Rad). The primary antibodies used in this section were SETD7 (Sigma, #SAB2701317, rabbit, 1:1000), GSDMD (CST, #93709, rabbit, 1:1000), GSDMD-NT (CST, #36425, rabbit, 1:1000), GAPDH (CST, #5174, rabbit, 1:1000), NLRP3 (Sigma, #SAB1410191, 1:1000), ASC (Sigma, #SAB4501315, rabbit, 1:1000), caspase-1 (CST, #3866, rabbit, 1:1000), cleaved caspase-1 (CST, #89332, rabbit, 1:1000), and GAPDH (CST, #5174, rabbit, 1:1000).

Short hairpin RNA (shRNA) and cell transfection

SETD7 shRNA–induced silencing was constructed with the pCMV-Tag5B vector. The mice were transfected with SETD7 shRNA and shRNA control by using Lipofectamine 2000 reagent (cat. no. 11668019; Invitrogen; Thermo fisher Scientific, Inc.).²⁸ The sequence of RNA duplexes for SETD7 shRNA interference was 5^′-CACCGCTCTTCTTCCAGATCCTTATTTCAAGAGAATAAGGATCTGGAAGAAGAGCTTTTTTG-3^′, and its shRNA control was designed and synthesized by Invitrogen/Thermo Fisher Scientific, Inc. The scrambled shRNA vectors were negative controls. The expression level of SETD7 was determined by Western blot and RT-PCR.

Hematoxylin and eosin (HE) staining

The tissue samples were obtained from isoflurane-anesthetized mice. The tissues were sliced into 4-μm thick sections after they were fully fixed in 4% neutral formalin for 48 h and then embedded in paraffin. The sections were rinsed in xylene I for 5 min, and then in xylene II for 5 min, and then immersed in ethanol with a concentration gradient from 100% to 70% for 2 min for deparaffinization. Each section was then washed in ddH₂O twice for 2 min. Next, the sections were stained in hematoxylin solution for 10 min, followed by color separation with 1% ethanol hydrochloride for several seconds. After washing the sections in running water for 1 h to remove excess color, the sections were stained in eosin solution for 3 min and then soaked in ethanol with a concentration gradient from 70% to 100% for dehydration (2 min) and xylene I and xylene II (each for 5 min). Eventually, the sections were mounted by neutral balsam.²⁹ The image was analyzed by IPLab 4.0 imaging software (Scanalytics, Inc.)

TUNEL staining

The apoptosis of hippocampal neuron was detected by TUNEL staining.²⁹ The samples were embedded in paraffin, conventionally deparaffinized and hydrated, and then carefully sectioned. Following the instruction of the TUNEL assay kit (Roche Ltd, Basel, Switzerland), the sections were treated by proteinase K for 20 min at room temperature. After washing by 1× PBS twice, the sections were added with 50 μL mixture of TUNEL solution and incubated in a wet box at 37°C for 1 h away from light. After washing with 1× PBS twice, the sections were incubated with 50 μL coverter-POD at 37°C for 30 min in the dark. Next, the sections were further washed with 1× PBS twice, incubated with 50 μL DAB solution, and reacted for 10 min at room temperature. The sections were subsequently re-stained by hematoxylin, dehydrated, and permeabilized. Eventually, the sections were mounted in neutral balsam. The image was analyzed by IP lab7.0 imaging software. TUNEL-positive cells were calculated at random in the same area, and the number of positive cells per unit was also quantified.

Morris water maze test

The previously treated mice were administered with the MWM test to record the number of times of crossing the platform, the distance traveled in the quadrant, the escape latency time, the time of staying in platform quadrants, and the swimming speed.³⁰ The aim of the test was to study the learning and memory abilities of mice. The classic test contains the position navigation test and space exploration test. In the position navigation test, each mouse facing the pool wall was put into the water from four different points randomly, and the time for finding the fixed underwater platform was recorded. In the space exploration test, the original fixed platform was removed and the mice were put into water at a random point. Each mouse was allowed to swim for 60 sec to test their memory.

Enzyme-linked immunosorbent assay

Tissue samples were obtained from the hippocampus of the previously treated mice. The samples were fully homogenated by a homogenizer and ultrasonic breaker, then further lysed in a buffer containing 1 mm PMSF, and centrifuged at 5000g for 5 min to collect the supernatant. The concentration of the supernatant was detected by the BCA assay kit. The expression levels of IL-1β, IL-18, TNF-α, and IL-6 were determined by using the corresponding ELISA kits (Wuhan Boster Biological Technology, Ltd) according to the instruction of the assay kit.²⁹ The results of the 96-well plate were read by a microplate reader (450 nm).

Statistical analysis

The mean values of the data obtained from three independent experiments were presented as the mean ± SEM. SPSS 19.0 version software (United States) was applied for data analysis. The difference between different groups was conducted by one-way analysis of variance (ANOVA), followed by the Bonferroni test. Comparisons for the spatial training sessions of MWM were performed by repeated two-way ANOVA followed by the LSD test. A p-value < 0.05 was considered statistically significant.

Results

Knockdown of SETD7 improved cognitive impairment in isoflurane-anesthetized mice

To explore the underlying function of SETD7, the adenovirus knockdown of SETD7 was constructed and transfected into mice exposed to isoflurane. RT-PCR and Western blot were employed to determine the efficiency of knockdown of SETD7. The results showed that knockdown of SETD7 was successful at the mRNA and protein levels (Figures 1(a) to (c)). To uncover the function of SETD7 on the isoflurane-induced cognitive impairment in the mice, we detected the pathological changes of neurons in the hippocampus of isoflurane-anesthetized mice by HE staining. Here, irregular changes in isoflurane-induced neuron morphology were observed when compared with the control group, specifically the neurons were disorderly and loosely arranged, the cells became smaller, and the nuclear condensation and the cytoplasm were reduced. However, knockdown of SETD7 improved isoflurane-induced neuronal morphological damage, and there were a few cell morphology changes; some normal neurons could still be seen, and the neurons were neatly and tightly arranged than those in the group treated by only isoflurane (Figure 1(d)). The Morris water maze test was used to evaluate the learning and memory abilities of the mice. The result showed that as compared with the control group from 1 to 4 days, the escape latency time to reach the fixed platform increased significantly in the isoflurane group and isoflurane with shRNA control group. Moreover, the number of times of crossing the platform, time staying in the platform quadrant and distance traveled in the target quadrant were all obviously reduced in the isoflurane group and isoflurane with shRNA control group, as compared with the control group. However, there was no significant difference in swimming speed in the four groups. Isoflurane with shRNA SETD7 group showed the recovered learning and memory abilities, previously damaged by the isoflurane (Figures 2(a) to (f)). Collectively, our data revealed that isoflurane exposure could cause cognitive dysfunction in mice, but knocking down SETD7 may effectively improve the side effects of isoflurane anesthesia.

Figure 1.

Knockdown of SETD7 improved hippocampal neuronal damage in isoflurane-anesthetized mice. (A) The mice were pretreated with shRNA SETD7 and shRNA control for 120 h, and anesthetized by isoflurane. Total mRNA was acquired from four groups of mice (n = 8): control, isoflurane, isoflurane + shNC, and isoflurane + shSETD7, respectively. The expression of SETD7 was detected by RT-PCR. (B and C) The protein level of SETD7 was detected by Western blot. (D) Pathological sections in the hippocampal neurons of the mice were analyzed by HE staining. **p < 0.01.

Figure 2.

Knockdown of SETD7 improved cognitive impairment in isoflurane-anesthetized mice. (A to F) The mice were pretreated with shRNA SETD7 and shRNA control for 120 h, and anesthetized by isoflurane. The Morris water maze experiment was performed to record the number of times the mice crossed the platform, the distance covered in the quadrant, the escape latency time, the time of staying in platform quadrants, and the swimming speed. **p < 0.01.

Knockdown of SETD7 improved pyroptosis in the hippocampus of isoflurane-anesthetized aged mice

For exploring the mechanism of SETD7 in isoflurane-induced cognitive impairment in aged mice, we studied the effect of SETD7 on the damage to neuronal cells in the hippocampal region of mice exposed to isoflurane. Some evidence showed that cell pyroptosis was related to the pathogenesis of different kinds of diseases. It has been reported that pyroptosis, which is a new form of programmed cell death, can be triggered by two kinds of pathways, including the classic active caspase-1 inflammasome pathway and atypical caspase-11 inflammasome pathway. The two cytokines digest GSDMD into two fragments (N domain and C domain). The N-terminal cleavage product GSDMD-NT forms pores in the lipid membrane and then induces cell pyroptosis by cell membrane destruction. Western blot result showed that GSDMD-NT rather than GSDMD was highly expressed in the isoflurane group than in the control group, but knockdown of SETD7 reduced GSDMD-NT expression to nearly the control level (Figure 3(a) and (b)). Subsequently, TUNEL staining was performed to assess neuronal cell death in the hippocampus; the results showed that isoflurane aggravated neuron death than control group, which was opposite to the knockdown of SETD7 group (Figure 3(c) and (d)). These results indicated that isoflurane activated pyroptosis, but knockdown of SETD7 reversed the cell death to some extent. We speculated that knockdown of SETD7 improved cognitive impairment via ameliorating nerve cell pyroptosis.

Figure 3.

Knockdown of SETD7 improved pyroptosis in the hippocampus of isoflurane-anesthetized aged mice. (A and B) The mice were pretreated with shRNA SETD7 and shRNA control for 120 h, and anesthetized by isoflurane. The protein levels of GSDMD-NT and GSDMD were detected by Western blot. (C and D) The TUNEL assay was used to detect the neuronal cell death in the hippocampus of isoflurane-anesthetized aged mice. **p < 0.01.

Knockdown of SETD7 inhibited the release of inflammatory cytokines in the hippocampus of isoflurane-anesthetized aged mice

Cell pyroptosis is an inflammatory signaling pathway, which is related to classic inflammatory cytokines, such as IL-1β, IL-18, TNF-α, and IL-6. To examine the function of SETD7 in the release of inflammatory cytokines in the hippocampus of isoflurane-anesthetized aged mice, the ELISA assay was applied to determine the expression levels of IL-1β, IL-18, TNF-α, and IL-6. The result showed that in the isoflurane and isoflurane with shRNA control groups, the four inflammatory cytokines had high levels of increase than those in the control group, but the treatment of isoflurane followed by knocking down SETD7 reduced the expression levels of the four cytokines to nearly those of the control group’s level (Figures 4(a) to (d)). These findings demonstrated that knockdown of SETD7 could effectively inhibit the release of inflammatory cytokines in the hippocampus of isoflurane-induced aged mice.

Figure 4.

Knockdown of SETD7 inhibited the release of inflammatory cytokines in the hippocampus of isoflurane-anesthetized aged mice. ((a) to (d)) The mice were pretreated with shRNA SETD7 and shRNA control for 120 h, and anesthetized by isoflurane. The expression levels of IL-1β, IL-18, TNF-α, and IL-6 were analyzed by the ELISA assay from tissues in the hippocampus of isoflurane-anaesthetized aged mice. **p < 0.01.

Knockdown of SETD7 inhibited the activation of the NLRP3 inflammasome in the hippocampus of isoflurane-anesthetized aged mice

Cell pyroptosis has been recently identified as the NLRP3 inflammasome–dependent signaling pathway. Therefore, the relationship of SETD7 and NLRP3 inflammasome was determined by performing Western blot. For a more accurate comparison, we added an extra control group treated with isoflurane and MCC950 (an inhibitor of NLRP3). The expression levels of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-IL-1β, IL-1β, and IL-18 were detected by Western blot, and the data revealed that the expression levels of above cytokines were all increased in the isoflurane and isoflurane with shRNA control group, as compared with the control group and extra control group. However, in the isoflurane with shRNA SETD7 group, the levels of these cytokines were reduced, and this was consistent with the data from the isoflurane with MCC950 group (Figure 5(a) and (b)). These findings indicated that knockdown of SETD7 inhibited the activation of the NLRP3 inflammasome in the hippocampus of isoflurane-anesthetized aged mice.

Figure 5.

Knockdown of SETD7 inhibited the activation of the NLRP3 inflammasome in the hippocampus of isoflurane-anesthetized aged mice. (A and B) The mice were pretreated with shRNA SETD7 and shRNA control for 120 h, and anesthetized by isoflurane. The mice were pretreated with MCC950 (10 mg/kg), and anesthetized by isoflurane. Western blot was employed to detect the protein expression levels of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-IL-1β, IL-1β, and IL-18. **p < 0.01.

Discussion

This study found that knockdown of SETD7 inhibited neuronal pyroptosis in the hippocampus and improved cognitive impairment in the isoflurane-anesthetized aged mice. Importantly, we further elucidated that knockdown of SETD7 alleviated neuroinflammation, pyroptosis, and cognitive impairment through suppressing the activation of NLRP3 inflammasome in isoflurane-induced aged mice. SETD7 has been found to play an indispensable role in isoflurane general anesthesia–induced POCD, which offers a new strategy to treat general anesthesia–induced POCD.

It was widely recognized that POCD is one of the common postoperative complications among elderly patients with neurotoxicity and cognitive dysfunction. The most important cause of POCD was neuroinflammation induced by general anesthesia.³¹ Interestingly, SETD7 has been proved to regulate the transcription of inflammatory genes.^32,33 Moreover, knockdown of SETD7 in cardiomyocytes can effectively suppress the apoptosis induced by hypoxia/reoxygenation and significantly reduce the production of reactive oxygen species.²¹ SETD7 mediates the vascular invasion in articular cartilage and chondrocytes apoptosis in osteoarthritis.³⁴ MiR-345-5p inhibits tumorigenesis of papillary thyroid carcinoma by targeting SETD7.³⁵ Some reports showed that increased expression of SETD7 promotes cell proliferation by regulating cell cycle and indicates poor prognosis in hepatocellular carcinoma.³⁶ However, the relationship between SETD7 and POCD remains unknown. In the present research, our finding revealed that knockdown of SETD7 noticeably improved isoflurane-induced cognitive impairment in the aged mice. Although little is known about the role of pyroptosis in neurotoxicity and cognitive deficits mediated by general anesthesia,^15,17 our results showed that knockdown of SETD7 improved pyroptosis in the hippocampus of isoflurane-anesthetized aged mice. All these findings strongly supported that pyroptosis may participate in the isoflurane-induced neuroinflammation and cognitive dysfunction.

Pyroptosis has recently been identified as NLRP3 inflammasome–dependent and involves two different inflammasome signaling pathways.^37–39 The NLRP3 inflammasome participates in the cleavage of caspase-1, and regulates the maturation and secretion of IL-1β and IL-18.^40,41 Besides, AIM2 assembles a multiprotein complex called the inflammasome upon binding to DNA, which drives pyroptosis and proteolytic cleavage of the pro-inflammatory cytokines pro–IL-1β and pro–IL-18.^42,43 In addition, knockdown of SETD7 before CC1 could prevent spinal microgliosis and reduce neuropathic pain by suppressing the expression levels of inflammatory genes (Ccl2, IL-6, and IL-1β).⁴⁴ Consistent with previous studies, knockdown of SETD7 inhibited the release of inflammatory cytokines (IL-1β, IL-18, IL-6, and TNF-α) in the hippocampus of isoflurane-anesthetized aged mice. It has been reported that the activation of the NLRP3 inflammasome triggers pyroptosis, further leading to neuronal impairment, cognitive dysfunction, and eventually even death.^45–47 Therefore, we detected the effect of SETD7 on NLRP3 inflammasome and pro-inflammatory cytokine production, and the result showed that knockdown of SETD7 inhibited the activation of the NLRP3 inflammasome in the hippocampus of isoflurane-anesthetized aged mice and simultaneously decreased the expression levels of inflammatory cytokines. In summary, all these findings confirmed the hypothesis that SETD7 may act as a vital regulator on NLRP3 inflammasome-dependent pyroptosis in reaction to isoflurane-induced neuronal damage in the aged mice with hippocampus and cognitive dysfunction.

There were still several limitations in the article: the effect of SETD7 on primary cells in isoflurane-anesthetized aged mice was not investigated; moreover, overexpression of SETD7 should be constructed to further study its function; related clinical data should be considered during the research.

This research provided evidence that NLRP3 inflammasome–dependent pyroptosis participated in neuroinflammation and cognitive impairment in isoflurane-anesthetized aged mice and that the inhibition of SETD7 alleviated neuroinflammation, pyroptosis, and cognitive dysfunction in the mice. Thus, SETD7 has the potential to be further explored for treatment of elder patients with POCD.

Supplemental Material

sj-pdf-1-het-10.1177_09603271211061497 – Supplemental Material for Inhibition of SET domain–containing (lysine methyltransferase) 7 alleviates cognitive impairment through suppressing the activation of NOD-like receptor protein 3 inflammasome in isoflurane-induced aged mice

Supplemental Material, sj-pdf-1-het-10.1177_09603271211061497 for Inhibition of SET domain–containing (lysine methyltransferase) 7 alleviates cognitive impairment through suppressing the activation of NOD-like receptor protein 3 inflammasome in isoflurane-induced aged mice by Chao Ma, Xianjun Yu, Dong Li, Youwen Fan, Yajun Tang, Qiang Tao, Hanbin Xie and Lei Zheng in Human & Experimental Toxicology

Footnotes

Authors' contributions

CM and XY performed the experiments and conducted data analysis. LZ designed the experiment and revised the manuscript. DL and YF wrote the first version of the manuscript. YT and QT contributed to literature research and animal experiment.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethics approval and consent to participate

All animal experiments were approved by the Animal Investigation Ethics Committee of The Seventh Affiliated Hospital of Sun Yat-Sen University (Shenzhen, China).

Data availability

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

ORCID iD

Lei Zheng

Supplemental Material

Supplemental material for this article is available online.

Appendix

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