Mechanism of microglia ferroptosis in smoke inhalation-induced brain injury

doi:10.11958/20231628

Abstract

Abstract:

Objective To investigate the underlying mechanism of microglia ferroptosis in smoke inhalation-induced (SII) brain injury. Methods Twenty SPF-grade male C57BL/6 mice were randomly divided into the control group, the SII group, the ferrostatin-1 group (Fer-1, 2.5 mmol/kg) and the deferoxamine group (DFO, 200 mg/kg), with 5 mice in each group. Mice in the Fer-1 group and the DFO group were intraperitoneally injected with Fer-1 and DFO 1, 3 and 5 day after smoke exposure, respectively. The pathological changes of brain tissue were examined by HE staining and Prussian blue staining on the 6th day after smoke exposure. RT-qPCR was used to detect levels of inflammatory factors, brain tissue damage markers and ferroptosis markers. The contents of iron in mouse brain tissue were determined by double pyridine colorimetric assay. The contents of malondialdehyde (MDA) and lipid peroxide (LPO) in mouse brain tissue were determined by thiobarbituric acid (TBA) colorimetric assay. Superoxide dismutase (SOD) activity in mouse brain tissue was measured by xanthine oxidase assay kit. The contents of glutathione (GSH) in mouse brain tissue were determined by direct method of dithiodinitrobenzoic acid (DTNB) assay. BV2 cells were cultured in complete DMEM medium and divided into the control group, the erastin group (10 μmol/L), the Fer-1 group (erastinc stimulation combined with 5 mmol/L Fer-1 treatment) and the DFO group (erastinc stimulation combined with 50 mmol/L DFO treatment). After 24 h, cell viability was detected by CCK-8 assay, cell apoptosis was detected by Annexin V-FITC/PI flow cytometry, intracellular reactive oxygen species (ROS) production was detected by DCFDA staining, and mitochondrial membrane potential was detected by MitoTracker Red CMXRos staining. Results Compared with the control group, enhanced iron deposition and inflammation in brain tissue, elevated mRNA expression of inflammatory markers and damage markers in brain tissue, up-regulated ACSL4 and NCOA4 mRNA levels, down-regulated GPX4 and SLC7A11 mRNA levels, decreased GSH and SOD contents, and increased LPO and MDA contents were observed in brain tissue of the SII group (P<0.05). The mRNA expression level of 7 member of solute carrier family 11 (SLC7A11) was decreased in mice of the SII group. The contents of GSH and SOD were decreased, and the contents of LPO and MDA were increased (P<0.05). Compared with the SII group, all the above parameters were reversed in the Fer-1 group and the DFO group, and the damage of mouse brain tissue was alleviated (P<0.05). In BV2 cell experiments, compared with the control group, decreased survival rate of BV2 cells and increased apoptosis rate were found in the erastin group (P<0.05), and increased intracellular ROS level and decreased mitochondrial membrane potential were also observed in the erastin-stimulated BV2 cells. The above parameters were opposite to those of the erastin group in the Fer-1 group and the DFO group (P<0.05), and the oxidative damage of BV2 cells was alleviated. Conclusion The ferroptosis inhibitors Fer-1 and DFO can inhibit microglia ferroptosis and alleviate smoke inhalation-induced brain injury.

Key words: smoke inhalation injury, microglia, ferroptosis, deferoxamine, ferrostatin-1

CLC Number:

R363.21，R644

Figures/Tables 9

References 29

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基因名称	引物序列（5'→3'）	产物大小/bp
IL-1β	上游：ATCCAGCACTCTGCTTTCAGG 下游：TGCATCTGTAGGAGTCCCTGT	180
IL-6	上游：CTCTGGGAAATCGTGGAAAT 下游CCAGTTTGGTAGCATCCATC	134
IL-8	上游：ACCCGCTCGCTTCTCTGT 下游：AAGGGAGCTTCAGGGTCAAG	233
TNF-α	上游：ATGAGAAGTTCCCAAATGGC 下游：CTCCACTTGGTGGTTTGCTA	125
Zo-1	上游TCTTGCAAAGTATCCCTTCTGT 下游CAGAAATCGTGCTGATGTGCC	102
VE-Cadherin	上游CACCACAGCTATCACGTTTTGC 下游：CCAAGTCTCCCCTGGTTAGG	132
Occludin	上游：CCGGCCGCCAAGGTTC 下游：ACATGCATCTCTCCGCCATAC	183
Claudin-5	上游：CACACCTCGTCGCTAGTGC 下游GCTCCCAAGATAAGCGAACCT	168
ACSL4	上游：TCAAGGGAGGCTTTAGGCGA 下游：GCCTCCGCTCACTAAGGAC	117
NCOA4	上游：GCCAGAGCAGAAGTCAGCAT 下游：GTCCTGTGGGTTGGTACTGG	118
GPX4	上游：GATGAGCTGGGGCCGTCTG 下游：TGACGATGCACACGAAACCC	200
SLC7A11	上游：AATACGGAGCCTTCCACGAG 下游：CTCCAGGGGCAGTCAGTTAG	86
GAPDH	上游：CCCCATACACAGTGTTAGCC 下游：GAGTGATTTTCCCGTCC	96

基因名称	引物序列（5'→3'）	产物大小/bp
IL-1β	上游：ATCCAGCACTCTGCTTTCAGG 下游：TGCATCTGTAGGAGTCCCTGT	180
IL-6	上游：CTCTGGGAAATCGTGGAAAT 下游CCAGTTTGGTAGCATCCATC	134
IL-8	上游：ACCCGCTCGCTTCTCTGT 下游：AAGGGAGCTTCAGGGTCAAG	233
TNF-α	上游：ATGAGAAGTTCCCAAATGGC 下游：CTCCACTTGGTGGTTTGCTA	125
Zo-1	上游TCTTGCAAAGTATCCCTTCTGT 下游CAGAAATCGTGCTGATGTGCC	102
VE-Cadherin	上游CACCACAGCTATCACGTTTTGC 下游：CCAAGTCTCCCCTGGTTAGG	132
Occludin	上游：CCGGCCGCCAAGGTTC 下游：ACATGCATCTCTCCGCCATAC	183
Claudin-5	上游：CACACCTCGTCGCTAGTGC 下游GCTCCCAAGATAAGCGAACCT	168
ACSL4	上游：TCAAGGGAGGCTTTAGGCGA 下游：GCCTCCGCTCACTAAGGAC	117
NCOA4	上游：GCCAGAGCAGAAGTCAGCAT 下游：GTCCTGTGGGTTGGTACTGG	118
GPX4	上游：GATGAGCTGGGGCCGTCTG 下游：TGACGATGCACACGAAACCC	200
SLC7A11	上游：AATACGGAGCCTTCCACGAG 下游：CTCCAGGGGCAGTCAGTTAG	86
GAPDH	上游：CCCCATACACAGTGTTAGCC 下游：GAGTGATTTTCCCGTCC	96

组别	IL-1β	IL-6	IL-8	TNF-α
Control组	1.00±0.18	1.00±0.19	1.00±0.17	1.00±0.21
SII组	2.74±0.42^a	2.61±0.49^a	3.64±0.67^a	5.00±1.11^a
Fer-1组	1.57±0.25^b	1.34±0.20^b	1.57±0.36^b	2.77±0.47^b
DFO组	1.46±0.27^b	1.53±0.29^b	1.69±0.37^b	2.73±0.75^b
F	31.563^＊＊	24.594^＊＊	35.215^＊＊	26.198^＊＊

组别	IL-1β	IL-6	IL-8	TNF-α
Control组	1.00±0.18	1.00±0.19	1.00±0.17	1.00±0.21
SII组	2.74±0.42^a	2.61±0.49^a	3.64±0.67^a	5.00±1.11^a
Fer-1组	1.57±0.25^b	1.34±0.20^b	1.57±0.36^b	2.77±0.47^b
DFO组	1.46±0.27^b	1.53±0.29^b	1.69±0.37^b	2.73±0.75^b
F	31.563^＊＊	24.594^＊＊	35.215^＊＊	26.198^＊＊

组别	Claudin-5	Occludin	VE-Cadherin	Zo-1
Control组	1.00±0.13	1.00±0.19	1.00±0.13	1.00±0.14
SII组	2.88±0.30^a	3.06±0.38^a	3.47±0.71^a	3.11±0.53^a
Fer-1组	1.36±0.20^b	1.79±0.28^b	1.79±0.38^b	1.60±0.44^b
DFO组	1.54±0.31^b	1.69±0.22^b	1.90±0.21^b	1.72±0.27^b
F	53.833^＊＊	47.648^＊＊	30.353^＊＊	28.411^＊＊