Effect of the oxidative phosphorylation signaling pathway related gene CPT1A on pyroptosis after sevoflurane anesthesia

doi:10.21203/rs.3.rs-4707053/v1

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Effect of the oxidative phosphorylation signaling pathway related gene CPT1A on pyroptosis after sevoflurane anesthesia

https://doi.org/10.21203/rs.3.rs-4707053/v1

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Background

Oxidative phosphorylation signaling pathway plays an important role after anesthesia. However, the link between oxidative phosphorylation related genes (OPRGs) and anesthesia is unclear.

Methods

Transcriptome data was downloaded from the Gene Expression Omnibus (GEO) databases, and 200 OPRGs were obtained from the Molecular Signatures Database (MSigDB). We systematically analyzed the expression profile characteristics of 200 OPRGs and confirmed data quality through tSNE and PCA analysis. Subsequently, the expression level and effect of OPRG carnitine O-palmitoyltransferase 1 (CPT1A) was validated by detected the expression level of inflammation factors. Finally, the activity of pyroptosis pathway was verified after overexpression or knockdown of CPT1A.

Results

We identified that the expression level of OPRGs were significantly regulated after sevoflurane anesthesia. Compared to the control group, the expression of the inflammatory factors IL-1β, IL-6, and TNF-α were increased. Then, the expression level of CPT1A was up-regulated after sevoflurane anesthesia. In addition, the pyroptosis pathway was activated through after upregulating the expression of inflammatory factors.

Conclusion

These findings highlight the effect of OPRG CPT1A during anesthesia, deepen the understanding of OPRGs, inflammation, and pyroptosis for patients in clinic anesthesia.

sevoflurane

oxidative phosphorylation

carnitine O-palmitoyltransferase 1

anesthesia

pyroptosis

More than 150 years ago, William Morton successfully used ether as a general anesthetic. Advances in anesthesia have made many new surgical techniques possible, and deaths directly caused by anesthesia are now rare^[1]. However, further understanding of the molecular mechanisms involved in the anesthesia process could help to significantly reduce the morbidity of anesthesia and improve the quality of perioperative management.

A recently published study investigated the possibility that progesterone attenuates anesthesia-induced developmental neurotoxicity in sevoflurane-exposed neonatal rats^[2]. This protective mechanism is thought to be related to the anti-inflammatory properties of progesterone, which raises important questions about the role of inflammation in mediating neurobehavioral changes in anesthesia-induced developmental neurotoxicity^[3]. The fundamental role of inflammatory cytokines in the control of normal immune function, defense mechanisms, and normal cell growth, and how major surgery and trauma affect these processes. Thus, altering cytokine production and function after major disease and surgery has a significant impact on morbidity and mortality^[4].

Pyroptosis is a new type of programmed cell death discovered and confirmed in recent years, which is characterized by dependence on inflammatory caspases (mainly caspase-1, 4, 5, and 11) and accompanied by the release of a large number of pro-inflammatory factors^[5]. The morphological characteristics, occurrence, and regulatory mechanisms of pyroptosis are different from other cell death modes such as apoptosis and necrosis^[6]. Studies have shown that pyroptosis is widely involved in the occurrence and development of infectious diseases^[7], nervous system related diseases^[8] and atherosclerotic diseases^[9], and plays an important role^[10]. In-depth study of pyroptosis is helpful to understand its role in the occurrence, development, and outcome of related diseases and to provide new ideas for clinical prevention and treatment.

Conducting pathway enrichment analysis on the pre-sequencing data showed that anesthesia mainly causes abnormal changes in the oxidative phosphorylation pathway. In this study, expression levels of oxidative phosphorylation related genes (OPRGs) between pre-sequencing data of the control and anesthesia model groups were performed using bioinformatics analysis. Next, the expression of OPRG was detected after sevoflurane anesthesia. In order to validate the molecular mechanism of the sevoflurane anesthesia model through in vitro cell experiments and elucidate the potential mechanisms that affect the production of inflammatory factors after sevoflurane anesthesia.

Gene consensus clustering analysis of OPRG-related differentially expressed

genes (DEGs)

The gene set related to oxidative phosphorylation pathway was obtained from

the MSigDB website (http://www.gseamsigdb.org/gsea/msigdb/human/geneset/ HALLMARK_OXIDATIVE_PHOSPHORYLATION.html). To identify OPRG-related DEGs The expression level analysis of the OPRG between the control and anesthesia groups was conducted on the GEO website (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141242). R package limma was utilized with |log2-fold change (FC)| ≥ 1 and adjusted p < 0.05. Based on the DEGs, protein-protein interaction network analysis of OPRGs in the STRING database (https://cn.string-db.org/).

Cell culture and drugs

Renal tubular epithelial cell line HK2 (ATCC, Rockville, MD, USA) was cultured with DMEM/F12 medium (Thermo Fisher Scientific, CA, USA), containing 10% fetal bovine serum (Gibco, CA, USA) in a 37℃, 5% CO₂ incubator. Sevoflurane was acquired from Topscience (Shanghai, China).

Plasmid and transfection

Full-length CPT1A was amplified by and cloned into a lentiviral pLVX vector by the Clone Express® Ⅱ One Step Cloning Kit (Vazyme, Nanjing, China). Cells were transfected with pLVX-CPT1A-Flag or vector control using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

qRT-PCR

Total RNA was extracted from tumor cells using Trizol reagent (Invitrogen, CA, USA). The RNA concentration was measured by Nanodrop 2000 (Thermo Fisher Scientific, CA, USA), and then the total RNA was reversely transcribed into cDNA using the TaqMan real-time PCR kit (Thermo Fisher Scientific, CA, USA). QRT-PCR was performed on the Bio-Rad CFX96 real-time PCR detection system. β-actin was used as an internal reference. The relative expression levels were calculated by comparing 2^{− δ δ Ct}. Primers used were: CPT1A, forward, 5'-GATCCTGGACAATACCTCGGAG-3'; CPT1A, reverse, 5'- CTCCACAGCATCAAGAGACTGC-3'; GAPDH, forward, 5'-GGAGCGAGATCCCTCCAAAAT-3'; GAPDH, reverse, 5'-GGCTGTTGTCATACTTCTCATGG-3'.

Western blot analysis

Cell lysates were prepared, and an equal volume of the loading buffer was added and boiled. Protein samples were loaded onto a sodium dodecyl sulfate (SDS)-PAGE gel, electrophoresed, and subsequently electrotransferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was incubated with the primary antibody overnight at 4℃ and then incubated with the corresponding secondary antibody for 1 h at room temperature. After washing with PBS three times, the PVDF membrane was visualized using the ECL substrate (Bio-Rad, Hercules, CA, USA).

TUNEL assay

Add 4% paraformaldehyde for fixation, and fix at room temperature for 20–30 min. Wash with PBS 3 times, 5 min each time. The sample was treated with 1% Triton X-100 and permeated at room temperature for 3–5 min. Wash with PBS 3 times, 5 min each time. The samples were dried with absorbent paper, each sample was dripped with 50 µl TdT enzyme reaction solution, and then placed in a wet box sheltered from light for 60min at 37℃. Wash with PBS 3 times, 5 min each time. DAPI (Hoechst) staining solution restained the nucleus and reacted at room temperature without light for 15 min.

Wash with PBS 3 times, 5 min each time. The excitation wavelength was 543 nm, and the emission wavelength was 571 nm under a fluorescence microscope.

Statistical analysis

R software (version 4.1.2) and related R packages were utilized for statistical analyses. The Pearson correlation analysis was used to compare the expression of the inflammation factor between different groups. p < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.

Differential expression of OPRGs after sevoflurane anesthesia.

We first performed differential expression analysis of 200 OPRGs after sevoflurane anesthesia. We obtained 33 DEGs with the adjusted p < 0.05 and |log2(FC)|>1, of which COX4l1, CS, CYB5A, DLST, ECH1, ECHS1, ISCA1, OPA1, CPT1A, ETFB, and TIMM10 were highly expressed after sevoflurane anesthesia, and the remaining DEGs were highly expressed after sevoflurane anesthesia (Fig. 1A). Protein-protein interaction network analysis based on the String database revealed a close linkage between most genes (Fig. 1B).

Data quality was analyzed through tSNE and PCA analysis between the control and anesthesia groups.

To explore the characteristics of OPRGs expression profiles after sevoflurane anesthesia, we performed unsupervised clustering analysis to identify anesthesia subtypes based on OPRGs expression levels. The results showed that two clusters were clearly classified, and samples were classified into cluster A (control) and cluster B (anesthesia) (Fig. 2A). The PCA results further demonstrate the excellent grouping effect (Fig. 2B). In addition, we mapped OPRGs expression heatmaps and found that OPRGs were expressed at higher levels in cluster B (anesthesia) compared to cluster A (control).

The correlation between the expression of CPT1A and inflammatory factors.

To explore the correlation between CPT1A and inflammatory factors, we performed Pearson analysis between two groups. The results showed that patients in cluster B (anesthesia) had higher expression of CPT1A and inflammatory factors (IL-1β, IL-6 and TNF-α) (Figs. 3A–C), suggesting that groups after sevoflurane anesthesia have higher inflamation activity.

The expression of inflammatory factors was lower under the condition of low expression of CPT1A.

After treatment with the anesthetic drug sevoflurane, the mRNA and protein level of CPT1A in HK2 cells were detected. It was found that the expression level of CPT1A was up-regulated after sevoflurane anesthesia (Fig. 4A-B). Then, CPT1A overexpression and knockdown HK2 cells were established (Fig. 4C-D). In order to verify the effect of CPT1A in sevoflurane treatment. QRT-PCR assay was used to detect the mRNA expression level of inflammatory factors (Fig. 4E-G), and western blot was used to detect the expression level of inflammatory factors. It was verified that the expression of inflammatory factors was lower under the condition of low expression of the OPRG gene CPT1A (Fig. 4H).

CPT1A affects renal cell activity through the pyroptosis pathway.

Pyrotosis is a type of programmed cell death that has gradually been defined in recent years. Unlike apoptosis, pyroptosis is an inflammatory cell death that primarily involves the release of inflammatory mediators during cell death, leading to an inflammatory response. Considering the abnormal expression of inflammation factors, pyroptosis indicators (Gasdermin D, caspase-1) were detected. It was verified that the expression of pyroptosis indicators was lower under the condition of low expression of the OPRG gene (Fig. 5A-C). Pyroptosis was detected by TUNEL assay, which indicated that CPT1A induced pyroptosis in renal cells (Fig. 5D). The above results showed that CPT1A is the critical gene that affects renal cell activity through the pyroptosis pathway.

Numerous studies have shown that the OPRG signaling pathway plays an essential role after sevoflurane anesthesia^[11]. However, fewer studies focus on relationship between the OPRG signaling pathway and inflammation. Recent studies have shown the critical role of inflammation factor induced pyroptosis in renal function^[12]. The in-depth study of pyroptosis after sevoflurane anesthesia is helpful in reducing in anesthetic morbidity, and improving the quality of perioperative management, and providing new ideas for clinical prevention and treatment.

In this study, we first analyzed the differential expression levels of OPRGs using the dataset. Most were highly expressed and closely related. Subsequently, we identified data from two groups: cluster A (control) and cluster B (anesthesia). Compared to cluster A (control), higher expression level of OPRGs, and higher levels of inflammation factors (IL-1β, IL-6, and TNF-α) were found in cluster B (anesthesia). The above results imply that cluster B (anesthesia) has a more active inflammation effect. Next, we identified the DEGs between the two groups and further identified the CPT1A gene. The expression of CPT1A gene showed significant difference between the two groups. In addition, the expression of CPT1A was positively related to inflammation factors. This demonstrated a close association between CPT1A gene and inflammation. Next, we constructed a CPT1A knockdown or up-regulated renal cell line to verify the effect of CPT1A. We first detected the pyroptosis in renal cells and the results showed that down-regulated CPT1A ameliated pyroptosis induced after sevoflurane anesthesia in renal cells. Next, anesthesia related proteins were detected, and we found that down-regulated CPT1A rescued the activation of the pyroptosis pathway after sevoflurane anesthesia.

Our study also has some limitations. Data used for bioinformatics was performed by a study from another team. Further study was needed to be conducted by using the newly sequence technique. Second, sequence data was conducted by using rats samples, while clinic samples would be more solid. In addition, in vivo and in vitro experiments are needed to further explore the specific mechanisms of pyroptosis.

In this study, we found that OPRGs play an important role after sevoflurane anesthesia. Secondly, we verified that OPRG CPT1A is the critical gene to induce pyroptosis after sevoflurane anesthesia.

OPRGs

oxidative phosphorylation related genes

GEO

Gene Expression Omnibus

MSigDB

Molecular Signatures Database

CPT1A

carnitine O-palmitoyltransferase 1

fold change

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The expression level analysis of the OPRG between the control and anesthesia groups was conducted on the GEO website (https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE141242). The gene set related to oxidative phosphorylation pathway was obtained from the MSigDB website (http://www.gseamsigdb.org/gsea/msigdb/human/geneset/HALLMARK_OXIDATIVE_PHOSPHORYLATION.html). Based on the DEGs, protein-protein interaction network analysis of OPRGs in the STRING database (https://cn.string-db.org/).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

The present study was supported by the research topic of Guangdong Provincial Bureau of Traditional Chinese Medicine (Grant No.202304291028164200).

Authors' contributions

Baoyi Han and Yue Zhang: designed the experiments, performed the execution, writing and editing of the original draft. Zurong Hu, Chuanhua Zhou, Jingru Yang and Jie Jia: collected the data, performed statistical analyses, and data curation. Kunwei Li: reviewed the manuscript. Liangcheng Zhang:, supervised, reviewed, and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

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PEREZ-ZOGHBI J F NEUDECKERV, BRAMBRINK AM. Does inflammation mediate behavioural alterations in anaesthesia-induced developmental neurotoxicity? [J]. Br J Anaesth. 2022;128(4):602–5.
SHEERAN P, HALL GM. Cytokines in anaesthesia [J]. Br J Anaesth. 1997;78(2):201–19.
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KETELUT-CARNEIRO N, FITZGERALD KA. Apoptosis, Pyroptosis, and Necroptosis-Oh My! The Many Ways a Cell Can Die [J]. J Mol Biol. 2022;434(4):167378.
ZHENG X, CHEN W, GONG F, et al. The Role and Mechanism of Pyroptosis and Potential Therapeutic Targets in Sepsis: A Review [J]. Front Immunol. 2021;12:711939.
WANG S, YUAN Y H, CHEN N H, et al. The mechanisms of NLRP3 inflammasome/pyroptosis activation and their role in Parkinson's disease [J]. Int Immunopharmacol. 2019;67:458–64.
YANG Z, SHI J. Role of Pyroptosis and Ferroptosis in the Progression of Atherosclerotic Plaques [J]. Front cell Dev biology. 2022;10:811196.
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No competing interests reported.

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You are reading this latest preprint version

Effect of the oxidative phosphorylation signaling pathway related gene CPT1A on pyroptosis after sevoflurane anesthesia

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Gene consensus clustering analysis of OPRG-related differentially expressed

genes (DEGs)

Cell culture and drugs

Plasmid and transfection

qRT-PCR

Western blot analysis

TUNEL assay

Statistical analysis

Results

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1