Ozone treatment attenuates the ferroptosis induced by cerebral ischemia/reperfusion injury via activation of the Nrf2/Slc7a11/Gpx4 signaling pathway

doi:10.21203/rs.3.rs-3249587/v1

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Research Article

Ozone treatment attenuates the ferroptosis induced by cerebral ischemia/reperfusion injury via activation of the Nrf2/Slc7a11/Gpx4 signaling pathway

https://doi.org/10.21203/rs.3.rs-3249587/v1

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Background

The pathogenesis of brain ischemic/reperfusion (I/R) insult is characterised by the loss of neurons as a result of excessive oxidative stress responses. A form of oxidative cell death known as ferroptosis can be triggered when there is a breakdown in the equilibrium that exists between antioxidants and pro-oxidants in cells. As a natural bioactive molecule with antioxidant/anti-apoptotic and pro-autophagic properties, ozone can enhance the capacity of the antioxidant system and ameliorate oxidative stress. Yet, the mechanism of its role in neuronal ferroptosis remains unclear. Therefore, we investigated the functions and possible mechanisms of ozone in cerebral I/R-induced ferroptotic neuronal death.

Methods

A model of cerebral ischemia-reperfusion injury was created in S-D rats that had been pretreated with ozone. Intraperitoneal administration of the Nrf2 inhibitor ML385, the Slc7a11 inhibitor Erastin, and the Gpx4 inhibitor RSL3 was performed 1h prior to the creation of the model.

Results

According to the findings of our research, ozone preconditioning was able to mitigate neuronal damage caused by cerebral ischemia-reperfusion (I/R), lessen the severity of neurological deficits, lower the volume of cerebral infarcts, and reduce cerebral infarct volume in MCAO rats. One possible mechanism for this protective effect is the suppression of neuronal ferroptosis. Transmission electron microscopy, immunofluorescence, and Western blotting findings all pointed to ferroptosis in the aftermath of MCAO-induced brain damage. The present study found that MCAO caused morphological damage to neuronal mitochondria, enhanced the accumulation of lipid peroxidation, and promoted MDA production. Moreover, MCAO decreased the levels of FTH1 and GPX4, which act as negative regulators of ferroptosis, and increased the levels of ACSL4, which acts as a positive regulator of ferroptosis. Ozone preconditioning has been shown to have a protective impact on neuronal by increasing the nuclear translocation of Nrf2 and the expression of Slc7a11 and Gpx4. Meanwhile, treatment with ML385, Erastin and RSL3 significantly reversed the protective effect of ozone preconditioning on neuronal ferroptosis.

Conclusion

Ozone treatment attenuates the ferroptosis in cerebral ischemia/reperfusion injury rat model via Nrf2/Slc7a11/Gpx4 Pathway, which lays a new theoretical foundation for the use of ozone as a possible therapy to prevent ischemic stroke.

cerebral ischemia reperfusion injury

Ozone

Nrf2

Slc7a11

Gpx4

lipid peroxidation

ferroptosis

Stroke is one of the leading causes of death and physical impairment in adults all over the world[1]. Stroke-related death and disability have put a tremendous strain on society as well as the economy in recent years due to the growing number of senior people[2, 3]. A series of ischemic events occur in the brain when blood supply is cut off, depriving the brain of oxygen and nutrients and causing a stroke[4]. While thrombolytic therapy can restore blood flow to the brain, the process of reperfusing the brain could actually exacerbate the damage already done by the ischemia, leading to cerebral ischemia/reperfusion injuries (CIRI)[5, 6]. Cerebral ischemia-reperfusion injury after stroke is caused by a complex series of pathophysiological changes, including excitotoxicity, oxidative and nitrifying stress, inflammatory response, and cell death[7]. The formation of reactive oxygen species (ROS) is a major contributor, leading to oxidative stress[8]. Cutting off blood supply to the brain alters energy metabolism because brain tissues receive much less blood and oxygen. Rapid formation of reactive oxygen species (ROS) is facilitated by pro-oxidases in mitochondria and oxygen as substrates, both of which become readily available during blood reperfusion[9]. DNA damage, protein malfunction, and lipid peroxidation are all caused by high levels of reactive oxygen species (ROS)[10]. Due to their rapid metabolic rate of oxygen consumption, high amount of polyunsaturated fatty acids (PUFAs), and low activity of antioxidant enzymes, neuronal cells are more prone to lipid peroxidation than other types of cells. Consequently, it is of the utmost importance to investigate alternative methods of mitigating the neuronal death brought on by cerebral ischemia-reperfusion injury.

Different from that of apoptosis, autophagy, and necroptosis on genetic, morphological, and biochemical levels, ferroptosis is a unique iron-dependent type of RCD[11]. Multiple pathophysiological processes including aging, cancer, liver illness, and kidney ischemia/reperfusion injury, have been linked to ferroptosis[12, 13]. In ferroptosis, oxidation and antioxidation levels are out of whack. Following ferroptosis, the most important mechanism related to plasma membrane damages is lipid peroxidation[14]. Imperfections in the structure and morphology of the mitochondria are one of the typical morphological characteristics of ferroptosis[15]. Damage to the mitochondria cause lipid peroxidation and ferroptosis, which leads to mitochondrial fragmentation, an increase in ROS production, and a slowing down of mitochondrial metabolism. This causes permanent cell death, including the death of neurons caused by cerebral ischemia-reperfusion[12, 16]. Glutathione peroxidase 4 (GPX4) is a key regulator of ferroptosis. This enzyme is required for scavenging lipid ROS and plays a crucial role in the conversion of lipid hydroperoxides to non-toxic lipids[17]. It has been demonstrated that depletion of neuron-specific GPX4 in the forebrain elevates lipid peroxidation, causes ferroptosis, and triggers neurodegeneration[18]. Disrupting the cystine/glutamate antiporter system Xc⁻ also induces ferroptosis. Attenuation of GPX4 activity as a consequence of inhibition of SLC7A11, which is the light chain of system Xc⁻, leads to the accumulation of potentially deadly lipid peroxides and the induction of ferroptosis[19, 20]. The expression of SLC7A11 and GPX4 could be increased by activating nuclear factor-E2-related factor 2 (Nrf2), a major transcription factor that regulates cellular redox homeostasis and inflammation[21, 22].

Ozone (O3) is a highly reactive chemical made up of three oxygen atoms in a triatomic molecule. Ozone has been proven to have dose-dependent effects on the body, with excessive concentrations causing severe oxidative stress, inflammation, and tissue damage, while appropriate quantities causing moderate oxidation and activating antioxidant mechanisms[23]. Ozone preconditioning always can be achieved through autologous blood transfusion of ozone, rectal perfusion, or intra-peritoneal injection before the patient is hypoxic[24]. Research shows that ozone pretreatment can improve antioxidant capacity and reduce oxidative stress[25, 26]. Ozone stimulates the immune system, promotes anti-inflammatory signaling, activates the proteasome, increases blood circulation, releases growth factors, and has antibacterial action[27]. It has been found that ozone is a naturally occurring bioactive molecule that possesses antioxidant, anti-apoptotic, and pro-autophagic capabilities. These characteristics suggest that ozone may be able to represent a novel possible technique to delay neurodegeneration[28].

Our previous in vitro experiments have shown that ozone can attenuate damage in OGD/R cell models. In the present study, we investigated whether ozone attenuates ferroptosis through the Nrf2/Slc7a11/Gpx4 pathway in rats with cerebral ischemia/reperfusion injury by in vivo experiments. This study has the potential to offer fresh understanding into the role that Ozone serves in the ischemic stroke-induced brain injury.

2.1. Animals

Male Sprague-Dawley (S-D) rats (260–300 g) were purchased from the Institute of Zoology, Chinese Academy of Sciences. These rats were maintained in standardized environmental conditions (a 12-hour light/dark cycle at 25 ± 2°C) and were fed a commercial diet (CLEA, Shizuoka, Japan) and water ad libitum. All procedures were carried out in accordance with the National Institutes of Health standards for the care and use of experimental animals (No. 85 − 23, revised in 1996). This study was also approved by the Ethics Committee of the Affiliated Changzhou NO. 2 People’s Hospital of Nanjing Medical University (Jiangsu, China).

2.2. Rat Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) Model

Preparation of rat model of MCAO[29] : The rats were rendered unconscious by injecting 2% pentobarbital sodium intraperitoneally. The rats were positioned supine and inverted on the temperature controller of the operating table. To ensure that the airway was unobstructed, tongues were extracted from the mouth using tweezers. Routinely, the neck was peeled and disinfected. A 2–3 cm median neck incision was made, and the common carotid artery (CCA) and vagus nerve were bluntly separated layer by layer until they were exposed and isolated. The CCA was divided ascendingly to expose the external carotid artery (ECA). To prevent sympathetic nerve stimulation, the internal carotid artery (ICA) was divided. A second 4 − 0 silk thread is knotted at the fork of the ECA near the ICA. Arterial clips obstructed blood flow at the distal end of ICA and the proximal end of CCA. A small incision was made approximately 1 cm from the ICA bifurcation at the ECA, and a thread plug was inserted to tighten the slip-knot. Then, slowly push into the ICA, pause when reaching the ICA at the artery clamp, plug the line to prevent bleeding, and remove the ICA artery clamp. The thread plug was slowly advanced in the direction of the ICA entering the skull (being careful not to insert the pterygopalatine artery), the ICA's small artery clip was removed, and the right ICA was continued until the thread plug reached the mark. Iodophor decontamination and irradiation to maintain warmth. After the blood flow was obstructed for 90 minutes, the thrombus was removed, the ECA was tightened, the arteriolar clamp clamping CCA was removed, the incision was sutured, sterilized, and irradiated to keep the incision warm. Iodophor decontamination and irradiation to maintain warmth. After 120 minutes of blood flow blockage, the thrombus was removed from the ECA for reperfusion, the ECA was tightened, the arteriolar clamp clamping the CCA was removed, and the incision was sutured, sterilized, and irradiated to keep the incision warm.

2.3. Neurological impairment scoring

The neurological impairment score was determined by recording the behavior of the rats after 24 h of reperfusion[30]: 0, no observable deficit; 1, right forelimb flexion; 2, decreased resistance to left lateral push (and right forelimb flexion) without circling; 3, same behavior as grade 2, with circling to the right; 4, severe rotation progressing into barreling, loss of walking or righting reflex. The neurological assessment was performed by a masked investigator and then confirmed by a second investigator blinded to the experimental groups.

2.4. The 2, 3, 5-triphenyltetrazolium chloride (TTC) staining

1% 2,3,5-triphenyltetra-zolium chloride (TTC) solution was used to stain brain tissues for 15 minutes at 37°C in order to evaluate infarct sizes of brain tissues after MCAO operation. Red staining was used to identify non-infarcted brain tissue, while white staining was used to identify infarcts. Images of TTC staining were analyzed using Image J software.

2.5. Nissl staining

Mice were anaesthetized with chloral hydrate twenty-four hours after reperfusion, and then fixed by transcardial perfusion of saline and 4% paraformaldehyde. After 4 hours postfixing in the same solution, the brains were sectioned at 15 m. Two minutes were spent in Nissl staining solution, followed by a series of ethanol concentrations ranging from 50–100%, and finally, five minutes each in xylene. After air drying, the pieces were mounted beneath coverslips in DPX mounting media.

2.6. Transmission electron microscopy (TEM)

TEM was used to assess the ultrastructure of mitochondria in brain neurones. Mice were transcardially perfused with saline and then fixed with 3% glutaraldehyde after 24 hours of reperfusion while under anaesthesia. As soon as the brains were removed, the cortical tissue in the penumbra was sectioned off into 1mm×1mm×1mm cubes. Tissues were soaked in 1% osmic acid in 0.1M phosphate buffer for 30 minutes, dried, and embedded in Araldite after being fixed in 3% glutaraldehyde at 4°C overnight. The H-7650 electron microscope (Hitachi, Tokyo, Japan) was used to look at ultrathin sections (200 nm) that were cut using an ultramicrotome and then stained with uranyl acetate and lead citrate.

2.7. Flameng scoring[31]

Each electron microscope ultra-thin section was divided into 5 fields of vision, with each field containing 20 mitochondria. The following were calculated and scored using the mean and standard deviation of the mitochondrial scores for each field: Normal mitochondrial structure and an intact particle receive a score of 0, normal mitochondrial structure but missing particles receive a score of 1, mitochondrial swelling receives a score of 2, steep rupture receives a score of 3, and incomplete inner and outer mitochondrial membranes receive a score of 4.

2.8. Measurement of brain iron

Following the iron assay kit's (Abcam, Cambridge, MA) instructions, the Fe²⁺ concentration was calculated.

2.9. Measurement of malondialdehyde (MDA) and glutathione (GSH)

The rat cortical tissues were homogenized to measure the Malondialdehyde (MDA) level and the GSH content. Samples were centrifuged at 12,000× g for 10 min, and the supernatants were collected. Using the GSH Assay Kit (Beyotime, Shanghai, China) and the manufacturer's instructions, the content of GSH in the tissue supernatant was found. The MDA level in the supernatants was measured with an MDA Assay Kit (Beyotime, Shanghai, China), following the directions from the maker.

2.10. Lipid peroxidation levels

The lipid peroxidation test kit (Jiancheng, Nanjing, China) was used to determine LPO levels in burned brain tissue and neuronal cultures.

2.11. Immunofluorescence (IF)

For immunofluorescence of sections of brain tissue, the brain was removed, treated overnight with 4% paraformaldehyde in PBS, and then cut into 4 m paraffin slices. After marking with an NRF2 primary antibody and a secondary antibody with a fluorescent tag, the nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI) and coverslips were put on. After washing and fixing the pieces, an Olympus FV3000 confocal laser scanning microscope was used to look at them.

2.12. Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Liquid nitrogen was used to quickly freeze the rat hippocampus tissues. Total RNA was extracted using TRIzol reagent (1 mL; Invitrogen, Carlsbad, CA, USA) per the manufacturer's instructions. SuperScript II Reverse Transcriptase (Invitrogen) was used to convert the isolated total RNA into cDNA. SYBR Green PCR master mix (Thermo Fisher Scientific, Shanghai, China) and the ABI 7500 equipment (Life Technology, Carlsbad, CA, USA) were used to conduct and analyse RT-qPCR. The study was performed using the 2-Ct technique, with GAPDH serving as an internal control. The primer sequences are as follows:

NRF2:

Forward: 5ʹ-GCCTTCCTCTGCTGCCATTAGTC-3ʹ

Reverse: 5ʹ-TGCCTTCAGTGTGCTTCTGGTTG-3ʹ

KEAP1:

Forward: 5ʹ-TGCTCAACCGCTTGCTGTATGC-3ʹ

Reverse: 5ʹ-TCATCCGCCACTCATTCCTCTCC-3ʹ

FTH1:

Forward: 5ʹ-TGCCATCAACCGCCAGATCAAC-3ʹ

Reverse: 5ʹ-AAGTTCTTCAGGGCCACATCATCC-3ʹ

GPX4:

Forward: 5ʹ-CCAGCAACAGCCACGAGTTCC-3ʹ

Reverse: 5ʹ-CACACGCAACCCCTGTACTTATCC-3ʹ

ACSL4:

Forward: 5ʹ-CCATATCGCTCTGTCACGCACTTC-3ʹ

Reverse: 5ʹ-CCAGGCTGTCCTTCTTCCCAAAC-3ʹ

GAPDH:

Forward: 5ʹ-AGACAGCCGCATCTTCTTGT-3ʹ

Reverse: 5ʹ-AGACAGCCGCATCTTCTTGT-3ʹ

2.13. Western blot analysis

Expressions of target proteins were evaluated by western blot assays. The rats were rendered unconscious with 2% pentobarbital before being killed. The brain tissue was extracted from the ice, placed in an EP tube, labeled, and stored at -80°C in the refrigerator. The tissue was ground on ice with RIPA lysate before being centrifuged for 15 minutes at 4℃. The concentration of protein in the supernatant was determined using a BCA protein assay kit. Protein samples were separated by gel electrophoresis employing a 10-200kD protein prepared with a rapid gel preparation kit and then transferred to a polyvinylidene fluoride (PVDF) membrane. Seal at room temperature for one hour with TBST-dissolved 5% skim milk. Primary antibody was incubated at 4 degrees Celsius for 24 hours. The following Primary antibodies are legally obtained from Protein tech Group: anti-NRF2 (16396-1-AP; 1:1000), anti-SLC7A11 (CY7046; 1:1000), anti-GPX4 (14432-1-AP; 1: 750), anti-FTH1 (ab65080; 1ug/ml), anti-ACSL4 (22401-1-AP; 1༚1000), and anti-GAPDH (ab8245; 1:2000). After TBST washing, the strips were incubated at room temperature for 1 h in the presence of secondary antibodies: HRP Goat Anti-Rabbit IgG (1:400). 100–2001 ECL-configured hypersensitive developer (A and B mixed 1:1) was deposited at the location of the target protein, bands were obtained using Protein Simple FluorChemQ2, and band intensity was quantified using ImageJ software.

2.14. Statistical analysis

GraphPad Prism 8.0 (GraphPad Software, Inc.) was used for our statistical analysis. At least three measurements are used to calculate the mean and standard deviation of the data. One way analysis of variance and Tukey's HSD test (α = 0.05) were used to examine the data. A statistically significant difference was determined to exist when the p value was less than 0.05.

3.1. Ozone alleviated the brain injury caused by CIRI.

Neuronal injury is the main phenomenon of CIRI-induced brain injury[32]. Our team's previous research has shown that Ozone concentration of 40µg/ml has the best effect on improving brain tissue damage in cerebral ischemia-reperfusion rats^[33]. Therefore, we established the MCAO model of S-D male rats after 5 days of pretreatment with intra-peritoneal injection of ozone (40 µg/ml, 2.5ml/kg/d), and observed the effect of Ozone on neuronal death after cerebral ischemia reperfusion in rats. The results of neurobehavioral evaluation showed that Ozone could significantly reduce the neurological impairment score of MCAO model rats, indicating that Ozone alleviated the CIRI-induced neural function damage (Fig. 1.A). After 24h reperfusion, TTC staining was used to analyze the cerebral infarction volume. As shown in Fig. 1.B, the brain tissue of rats in the Sham group was not significantly damaged. In contrast, the cerebral infarction volume of the rats in the MCAO group was dramatically increased in comparison to that of the rats in the Sham group. The infarct volume of the damaged region of the brain was greatly decreased thanks to pretreatment with Ozone, which did not have any discernible negative effects on the brain tissue itself. Since CIRI-induced brain damage is linked to neuronal damage, the next thing we did was investigate whether or not Ozone may influence the mortality of MCAO/R-induced brain neurons. Nissl staining served as the method via which we determined the effects of morphological alterations in brain tissue. According to the findings, the neurone cells in the Sham group were stained evenly in the cortex and striatum and were large with an abundant cytoplasmic compartment. In contrast, the neurons in the MCAO group were sparsely distributed and exhibited shrunken cell bodies, which showed severe neuronal damage. Pretreatment of Ozone can significantly reduce the damage of neurons, and Ozone itself has no significant damage effect on neurons (Fig. 1.C). These findings demonstrate that Ozone can greatly attenuate CIRI-induced brain damage.

3.2. Ozone inhibited neuronal ferroptosis caused by CIRI.

Prior research has demonstrated that ferroptosis contributes to cerebral ischemia/reperfusion damage[34]. We investigated whether Ozone might impact neuronal ferroptosis. A classic feature of ferroptosis is mitochondrial damage[35]. As shown in Fig. 2.a,b, MCAO/R caused damage to the mitochondrial morphology of cerebral neurones, including the reduction or disappearance of the mitochondrial crista and rupturing of the outer membrane. Notably, pretreatment with Ozone substantially reduced these mitochondrial damages. Another characteristic feature of ferroptosis is excessive lipid peroxidation[36]. In this study, we examined the changes of lipid peroxidation-related factors upon MCAO/R insult. The findings demonstrated that after MCAO injury, the levels of lipid peroxidase (LPO) and the contents of MDA, the byproduct of lipid peroxidation, were significantly higher in the rat brain tissue compared with the Sham group. Ozone pretreatment significantly inhibited the MCAO/R-induced increase of LPO and MDA (Fig. 2.c,d). Reactive free iron (Fe²⁺) buildup in cells is the primary cause of ferroptosis[37]. Ozone appeared to alleviate the increase in cerebral Fe²⁺ that MCAO/R produced (Fig. 2.e). Following that, we used qPCR and immunoblotting assays to investigate the mRNA and protein levels of a ferroptosis-related protein. The results of qPCR and Western blotting showed that MCAO/R could lead to a decrease in the mRNA and protein levels of FTH1 and GPX4, the negative regulator of ferroptosis, as well as an increase in the mRNA and protein levels of ACSL4, a positive regulator of ferroptosis in brain tissue. These changes, which were reversed by ozone pretreatment, are shown in Fig. 2.f-l. The decrease in GSH levels that might occur in the brain as a result of a cerebral stroke may make oxidative stress worse[38]. GSH is a cofactor of the enzyme GPX4 and has the ability to prevent ferroptosis and detoxify lipid peroxidation[39]. The increased amount of GSH in cells as a result of ozone pretreatment can help prevent damage caused by oxidative stress (Fig. 2.m). In conclusion, cerebral I/R generated an overactivation of ferroptosis and lipid peroxidation, both of which point to the possibility that ferroptotic neuronal death is involved in CIRI. It has been shown that pretreatment with ozone can significantly prevent neuronal ferroptosis produced by MCAO.

3.3. The improvement effect of Ozone on CIRI can be achieved by activating NRF2.

In ischemic stroke, the maintenance of redox homeostasis is the responsibility of Nrf2, which serves as a master transcription factor ^[40]. TTC staining showed that Ozone reduced infarct volume in MCAO brain tissue, while ML385 reversed this effect (Fig. 3.A). Immunofluorescence was utilised to examine where Nrf2 was located inside the cytoplasm and nucleus of neurons. According to the findings, Ozone boosted the nuclear translocation of Nrf2 in the brain tissue neurons of the MCAO model, which in turn triggered the antioxidant action and thus reduced neuronal damage (Fig. 3.B-D). Keap1, which is a repressor of the Nrf2 pathway, is an essential component in the process of regulating the activity of Nrf2^[41]. The findings of the qPCR experiment showed that MCAO induced an increase in the level of KEAP1 mRNA and a decrease in the level of Nrf2 mRNA in brain tissue. These changes, which were reversed by Ozone pretreatment, were shown in the Fig. 3.E, F. In addition to the ozone pretreatment, we also used an NRF2 specific inhibitor called ML385 in the MCAO model. This was done so that we could determine whether or not Nrf2 had a role in the protective effect of ozone against CIRI-induced ferroptosis. Results from Western blotting demonstrated that MCAO reduced protein levels of NRF2 and the negative regulator of ferroptosis FTH1 in brain tissue, and that these changes were restored by pretreatment with Ozone (Fig. 3.G-I). The findings demonstrated that ozone pretreatment reduced the amount of damage caused to the brain tissue by CIRI. This was accomplished by increasing the nuclear translocation and level of Nrf2 in neurons, which in turn inhibited neuronal ferroptosis.

3.4. The improvement effect of Ozone on CIRI can be achieved by activating SLC7A11.

The cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11; also known as xCT) promotes cystine uptake and glutathione biosynthesis, resulting in protection from oxidative stress and ferroptotic cell death^[19]. To investigate whether the protective effect of ozone on CIRI-induced ferroptosis was related to Slc7a11, we also used the particular inhibitor of Slc7a11, Erastin, on the basis of ozone pretreatment in the MCAO model. This allowed us to determine whether or not Slc7a11 was involved. The findings of the TTC staining demonstrated that Ozone pretreatment decreased the cerebral infarction volume of the rats in the MCAO group. Yet Erastin counteracted this tendency (Fig. 4.A). The inhibitory impact of ozone on MCAO-related ferroptosis was eliminated by Erastin, as shown by Western blot examination of SLC7A11 and FTH1 protein expression (Fig. 4.B-D). This provides more evidence that SLC7A11 is crucial to the curative effects of Ozone preconditioning in cerebral ischemia/reperfusion injury.

3.5. The improvement effect of Ozone on CIRI can be achieved by activating GPX4.

Glutathione peroxidase 4 (GPX4) uses glutathione to protect cells from ferroptosis by eliminating phospholipid peroxides[42]. In the MCAO model, in addition to the pretreatment with Ozone, we also employed RSL3, which is a particular inhibitor of Gpx4. This was done so that we could determine whether or not the protective effect of ozone against CIR-induced ferroptosis was connected to Gpx4. TTC staining showed that Ozone reduced infarct volume in MCAO brain tissue, while RSL3 reversed this effect (Fig. 5.A). By using immunoblotting, we detected the expression of the GPX4 and FTH1 proteins. These findings demonstrated that the inhibitory impact of Ozone on the ferroptosis that was caused by MCAO was nullified by the presence of RSL3 (Fig. 5.B-D). This shows that Ozone prevents neuronal ferroptosis and lessens the damage caused by ischemia and reperfusion of the brain thanks to the antioxidant activity of Gpx4.

Ozone is an important oxidant gas that has a role in a variety of processes, including the reduction of pain, suppression of inflammation, and modification of the immune system[43, 44]. To this point, ozone has seen widespread use in the management of pain, as well as the treatment of gastrointestinal disorders, lung diseases, diabetes, ischemia injury, cancer, infectious diseases, dentistry, and a variety of other medical and non-medical conditions[45–48]. In addition, it has been found that ozone is a natural bioactive molecule that possesses antioxidant, anti-apoptotic, and pro-autophagic capabilities. These features suggest that ozone may be able to represent a fresh possible method for delaying the progression of neurodegeneration[28]. In this research, we investigated the preventive effect of ozone and its mechanism against cerebral ischemia-reperfusion (I/R) injury in rats. The findings demonstrated that ozone had the ability to greatly lessen the volume of cerebral infarction, improve neuronal damage, and lower neurological scores in MCAO model rats, all of which verified that ozone had the capacity to lessen the amount of brain damage inflicted by MCAO model rats. (Fig. 1)

Ferroptosis appears to play a role in a number of pathological illnesses, including neurodegenerative diseases, traumatic brain injuries, and strokes, according to the evidence that is accumulating[49–51]. Ferroptosis is a type of programmed cell death characterized by lipid peroxidation dependent on generation of reactive oxygen species and severe iron overload[52, 53]. Ferroptosis and neuronal injury can occur when large levels of Fe²⁺ bind to polyunsaturated fatty acids, which then sets off the Fenton reaction and releases reactive oxygen species (ROS) from lipids[54]. MDA is the principal end product of lipid peroxidation, and lipid peroxide (LPO) is a marker of the extent to which this process has progressed in neurons[55]. The extent of cerebral ischemia/reperfusion injury and ferroptosis is correlated with the levels of ferroptosis-related markers such as MDA, LPO, and Fe²⁺. The findings demonstrated that a pretreatment with ozone prevented an increase in the amount of Fe²⁺, LPO, and MDA that was present in the brain tissue of MCAO/R model rats. Disruption of mitochondrial morphology is a typical feature of ferroptosis, these disruptions included reductions in the number of mitochondrial cristae, hypodense matrix, as well as shorter length and swollen mitochondrial morphologies[15, 56]. The results of transmission electron microscopy demonstrated that the morphology of mitochondria in neurons of MCAO/R rats was considerably damaged. Furthermore, the results showed that ozone pretreatment could effectively suppress the morphological alterations of mitochondria linked to ferroptosis in the brain tissue of MCAO/R model rats. The process of ferroptosis is a complicated one that is controlled by a number of different proteins. FTH1 is a ubiquitous intracellular protein that stores iron in a form that is readily available, non-toxic, and soluble. It is also one of the most essential negative regulators of the process known as ferroptosis[53]. Lipid peroxidation is minimised thanks to GPX4, which uses reduced glutathione as a cofactor to convert lipid hydroperoxides to lipid alcohols[57]. ACSL4 is an important enzyme that has a role in lipid composition regulation and ferroptosis[58]. Thus, in the brain tissue of MCAO/R model rats, ACSL4 expression was up whereas GPX4 and FTH1 expression was decreased. Ozone was shown to considerably attenuate the MCAO/ R-induced modifications to ACSL4, GPX4, and FTH1. (Fig. 2)

New research reveals that low levels of glutathione (GSH) and glutathione peroxidase 4(Gpx4) contribute to iron accumulation, lipid peroxidation, and stroke. Gpx4 has been shown to reduce lipid peroxidation in neurons. Therefore, blocking Gpx4 activity causes ferroptosis. The executioner of ferroptosis within the neuronal cell is the deadly metabolic imbalance caused by GSH depletion or inactivation of Gpx4[59, 60]. Lipid peroxidation of polyunsaturated fatty acids (PUFAs) and the accumulation of PUFAs-O-OH, which can form lipid ROS, are caused by damage to the cystine/glutamate antiporter system (system Xc-), which consists of solute carrier family 3 member 2 (SLC3A2) and solute carrier family 7 member 11 (SLC7A11)[61]. The expression of Slc7a11 and Gpx4 is controlled by Nrf2, which is an essential antioxidant transcription factor[21]. By binding to the antioxidant response element Are, Nrf2 controls the transcription of many antioxidant enzymes, protecting cells from the damage caused by oxidative stress. Keap1 acts as Nrf2's primary regulator[62]. By oxidising cysteine residues of keap1 that function as REDOX sensors, REDOX signalling directly breaks the Keap1-Nrf2 connection in the classical pathway, allowing Nrf2 to detach from its binding to Keap1 and stably translocate to the nucleus for continued transcription[63–65]. Thus, Nrf2, Slc7a11, and Gpx4 levels were drastically reduced in the brain tissue of MCAO model rats, and Nrf2 nuclear translocation was blocked. We demonstrated that ozone could promote Nrf2 nuclear translocation and significantly increase the levels of Nrf2, Slc7a11 and Gpx4 in brain tissue, thereby inhibiting neuronal ferroptosis and reducing brain infarct volume in MCAO rats. To provide additional evidence that the inhibitory effect of ozone on neuronal ferroptosis is connected to the Nrf2/Slc7a11/Gpx4 pathway, we used some specific inhibitors include ML385, which blocks the activity of Nrf2, Erastin, which blocks the activity of Slc7a11, and RSL3, which blocks the activity of Gpx4[66–68]. Our research shown that the injection of these inhibitors might further reduce the level of FTH1 in the brain tissue and increase the infarct volume of brain tissue in MCAO model rats, thereby reversing the inhibitory effect of ozone on neuronal ferroptosis. (Fig. 3–5)

Taken together, our findings give evidence that ozone has the ability to reduce the severity of cerebral I/R injury by preventing neuronal ferroptosis via the Nrf2/Slc7a11/Gpx4 pathway. This finding establishes a novel theoretical foundation for the use of ozone as a possible therapeutic for the prevention of ischemic stroke.

Author Contributions FRZ, YL conceived and designed the study. FRZ, LY and XLW operated the experiment and collected the data. FRZ analyzed the data. FRZ and LY wrote the paper. ZQW reviewed and edited the manuscript. All authors read and approved the final manuscript.

Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data Availability All data included are available reasonable demand to the corresponding authors in this study.

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

Ethical Approval This study was approved by the Ethics Committee of the Affiliated Changzhou NO. 2 People’s Hospital of Nanjing Medical University (Jiangsu, China).

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No competing interests reported.

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Ozone treatment attenuates the ferroptosis induced by cerebral ischemia/reperfusion injury via activation of the Nrf2/Slc7a11/Gpx4 signaling pathway

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1. Animals

2.2. Rat Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) Model

2.3. Neurological impairment scoring

2.4. The 2, 3, 5-triphenyltetrazolium chloride (TTC) staining

2.5. Nissl staining

2.6. Transmission electron microscopy (TEM)

2.7. Flameng scoring[31]

2.8. Measurement of brain iron

2.9. Measurement of malondialdehyde (MDA) and glutathione (GSH)

2.10. Lipid peroxidation levels

2.11. Immunofluorescence (IF)

2.12. Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

2.13. Western blot analysis

2.14. Statistical analysis

3. Results

3.1. Ozone alleviated the brain injury caused by CIRI.

3.2. Ozone inhibited neuronal ferroptosis caused by CIRI.

3.3. The improvement effect of Ozone on CIRI can be achieved by activating NRF2.

3.4. The improvement effect of Ozone on CIRI can be achieved by activating SLC7A11.

3.5. The improvement effect of Ozone on CIRI can be achieved by activating GPX4.

4. Discussion

Declarations

References

Additional Declarations

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