Chronic ketosis provides neuroprotection through HIF-1α-mediated control of the TXNIP/NLRP3 axis by regulating the inflammatory and apoptotic response

doi:10.21203/rs.3.rs-4914710/v1

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Chronic ketosis provides neuroprotection through HIF-1α-mediated control of the TXNIP/NLRP3 axis by regulating the inflammatory and apoptotic response

https://doi.org/10.21203/rs.3.rs-4914710/v1

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We and others have previously demonstrated that hypoxia-inducible factor alpha (HIF-1α) stabilization through diet-induced ketosis plays a vital role during brain ischemic injury. We have recently reported that ketosis-stabilized HIF-1α regulates the inflammatory response and contributes to neuroprotection in a rat stroke model. In the current investigation, we examined the downstream mechanism by which the ketogenic (KG) diet protects against brain damage after stroke in mice. Six-seven-week-old male mice were fed the standard diet (SD) or the KG diet to mimic the metabolic state of chronic ketosis. After four weeks, mice were subjected to photothrombotic ischemic stroke. Behavior analysis was recorded at 24 h, 48h, and 72h post-stroke. After 72h, mice were euthanized for infarction, brain edema, hemorrhage, and molecular analysis. Our results showed that the KG diet significantly alleviated infarction, brain edema, and hemorrhage, improved the neurobehavioral outcomes, and attenuated ischemic stroke-induced oxidative/nitrative stress and apoptotic markers at 72h post-stroke. Further, the KG diet upregulated the HIF-1α and interleukin (IL)-10 expression and inhibited thioredoxin-interacting protein (TXNIP), NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and pro-inflammatory cytokines expression compared to SD-fed mice after stroke. We further showed that the genetic deletion of NLRP3 mediates KG-induced neuroprotection after stroke. Our current study demonstrates that the KG diet exerts neuroprotective effects by inhibiting TXNIP-NLRP3 inflammasome, mainly dependent on heightening the upregulation of IL-10 via HIF-1α stabilization. Thus, the KG diet might be considered a new therapeutic strategy for ischemic patients.

HIF-1α

ischemic stroke

ketogenic diet

NLRP3 inflammasome

Ischemic stroke is the most common kind of damaging cerebrovascular disorders [1]. It is the second major health issue that poses a threat to life, and its burden is rising quickly worldwide [2, 3]. Several immune cells migrate to the ischemic lesion after ischemia and cause the release of a variety of neurotrophic factors, each of which has a positive or negative effect on brain tissue [4–6]. Secondary post-ischemia neuroinflammation causes additional damage and cell apoptosis [7]. The lack of effective therapeutic options is the primary obstacle in the fight against stroke. The only FDA-approved treatments for ischemic stroke are thrombolysis and endovascular thrombectomy; however, these treatments are only available to a small percentage of stroke patients because of their limited effectiveness and the specialized procedures required to perform them [8, 9]. Thus, the primary goal of stroke research is to develop cost-effective and viable treatments that alleviate brain dysfunction caused by ischemic stroke. This can only be done by understanding the underlying molecular events more deeply.

Previous reports have shown the contribution of Inflammation in the pathogenesis of ischemic stroke, whereas NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays a crucial role [10–12]. The cytosolic pattern recognition receptor NLRP3 recruits the adapter protein apoptosis-associated speck-like (ASC) pro-caspase-1 in response to several stroke-induced stimuli. This results in the production of caspase-1 and the subsequent maturation and release of interleukin-1 (IL-1) [13]. In acute stroke, the significance of pro-inflammatory and pro-apoptotic actions of IL-1 β is pretty well supported [14, 15]. Additionally, irrespective of IL-1, the activated caspase-1 results in pyroptotic cell death, which is known to cause a significant release of cytokines through intramembranous pores in glial cells [16]. According to recent studies, the suppression of NLRP3 inflammasome modulates ischemic insult and neurovascular consequences of experimental stroke [17–19]. Yet, because they mostly deal with non-specific neuroprotectants or genetic regulation, they fail to account for the therapeutic benefits. As a result, the need to create novel NLRP3 inhibitors with sufficient biocompatibility for clinical trials has been encouraged.

The primary feature of ischemic stroke is artery obstruction, which can cause blood flow disturbance or a drop in blood glucose levels, both of which increase the risk of brain damage. Ketone bodies, also known as beta-hydroxybutyrate and acetoacetate, or BHB and AcAc, are alternative energy substrates that the brain is known to use well [20]. Treating drug-resistant pediatric epilepsy with the ketogenic (KG) diet is a well-known non-pharmacological approach that has showed promise in treating other neurological illnesses such as stroke and Alzheimer's disease [21, 22]. We have consistently shown that local intraventricular BHB infusions or the KG diet-induced ketosis are correlated with neuroprotection after ischemic stroke. [23, 24]. Moreover, the KG diet can stop neuronal death brought on by hypoxia or glucose restriction [23]. Although the KG diet has been used in therapeutic settings for more than 70 years, the precise mechanism through which it gives neuroprotection is still unknown. Studies have linked ketosis to altering inflammatory and metabolic pathways [24–27]. Reports have shown that hypoxia-inducible factor-1 alpha (HIF-1α) accumulation elicits a neuroprotective response by modifying inflammatory pathways via modulation of cytokine regulation [28, 29]. Current research links the stabilization of HIF-1α by ketosis as a potential neuroprotective trait in mice via the activation of the Jak1-Stat3 or Akt/Erk pathways by IL-10 [30, 31]. Earlier, we investigated how the KG diet affected inflammatory reactions linked to stabilizing HIF1-α in the preconditioned ketotic rat brain. To identify putative neuroprotective pathways mediated by HIF1-α, we have focused on markers of pro- versus anti-inflammatory cytokines (TNF-α and IL-6 vs IL-10) in the cortical brain of ketotic rats [26, 32].

Based on these previous reports, the present study was designed to investigate the potential mechanisms associated with stabilizing HIF-1α and IL-10-mediated downregulating inflammation in the brains of preconditioned ketotic mice.

Animals and experimental groups

The experimental procedures were approved by both the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines and the Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee Health Science Center in Memphis, Tennessee, USA. Two major experiments were conducted. WT Male CB57BL/6J mice (6–7 weeks old) were obtained from the Jackson Laboratory, Bar Harbor, ME, USA. For four weeks, the mice were fed either the KG (high fat, carbohydrate restriction) or standard lab-chow (STD) diets before the brain tissues were collected, as previously described [26]. The WT mice were randomized and divided into four groups: Sham; pMCAO (mice with standard diet + photothrombotic middle cerebral artery occlusion), KG + pMCAO (mice with KG diet + photothrombotic middle cerebral artery occlusion), and KG (mice with KG diet only). The mice were kept in a standard environment with 45–50% humidity, 22–25°C temperature, and a 12–12 light–dark cycle. They were also given unlimited access to food and drink. Diet protocols were followed, including the standard (STD; 27.5 fat%, 20.0 protein%, 52.6, given by the UTHSC animal facility) and ketogenic (KG; 89.5 fat%, 10.4 protein%, 0.1 CHO%; Research Diets, New Brunswick, NJ diet). In experiment 2, NLRP3 knockout mice were divided into two groups. They were fed either STD or KG diet. All mice were fasted overnight hours before receiving their diets to maintain blood glucose levels and start the ketosis process. A small blood sample from the tail was used to analyze weekly BHB concentrations using a keto-meter (Precision Xtra, Abbott, Alameda, California).

Photothrombotic stroke surgery

The photothrombotic stroke model was chosen for the current research because it has accuracy, repeatability, and consistency of the infarct volume and allows for visual confirmation of middle cerebral artery occlusion and no fatality rate (MCAO) [33]. The photothrombotic procedure was performed as we described, with minor alterations. After being given isoflurane anesthesia (3% for induction and 1.5% during the procedure), pMCAO and KG + pMCAO mice underwent photothrombotic surgery. To expose the artery, the head was shaved, and the left side of the scalp, between the ear and the eye, was cut. Through the retroorbital sinus, 15 mg/kg of Rose Bengal, a photothrombotic dye (RB, Sigma-Aldrich, Cat#330000), was injected. The middle cerebral artery was then exposed to a high intensity photobeam for four minutes. Following surgery, the mice were placed in new cages in groups of 2 to 5 animals each, with free access to food and water. Until the study's last day, researchers looked for any signs of discomfort or distress. As considered necessary, painkillers and fluid food supplements were given. An intraoperative rectal probe and feedback control pad were used to maintain the patient's body temperature at 37 0.5°C. Mice were anesthetized using isoflurane and then transcardially infused with regular cold saline after 72 hours of pMCAO. Body weight changes were recorded weekly and after induction of pMCAO at 0 h, 24 h, 48 h, and 72 h in all animals. Mice were then decapitated, and the brains were removed.

CatWalk Test

Using the CatWalk XT automated analysis device (Noldus Information Technology, Wageningen, The Netherlands), gait analysis was carried out at 24h, 48h, and 72h post-stroke to compute the designated parameters as previously described [10]. Before performing the baseline assessment, the experimental animals were trained to traverse the walkway for five days in a row for 30 minutes. The system's centerpiece is a promenade with a glass surface illuminated by green and red ceiling light sources. The CatWalk XT technology uses a camera underneath a glass walkway to take images of animal footprints and foot force profiles. When an experimental animal walks across the walkway's glass plate, its paw prints are illuminated by the green light source. In contrast, the ceiling light contrasts the animal's body contour. These events were captured by a fully automated camera mounted below the glass plate and then processed by the system's software. A description of parameters is given in (Suppl. Table.S1). Pressure-related parameters, including mean intensity, which shows the complete paw's mean intensity, were measured [34]. We collected three trials of each animal and mean ± s.e.m. was used to calculate and graph the experimental data.

Rotarod test

The rotarod test was performed on experimental animals using rotarod equipment (Med Associates Inc., USA; model ENV-577M). In brief, the experimental animals were trained three days before to the surgery at a speed of 30 rpm/min for five minutes, with an acceleration from 2 to 30 rpm/min within the same time frame. The rotarod was used to evaluate mice's limb motor coordination and stability and the effect of the KG diet following ischemic stroke. The rotarod test was carried out at different time points (24h, 48h, and 72h), and the latency to fall was recorded for two maximum testing times out of 3 when the mice fell off from the rotating rod. Results were expressed as the time (sec) spent on the rotarod.

Infarct size and brain edema

A blinded researcher measured infarction and cerebral edema. The brain was separated following cardiac perfusion with ice-cold normal saline. Six 1-mm thick coronal slices from each brain were then stained for 15 minutes at 37°C using 1% TTC solution (2,3,5-triphenyl tetrazolium chloride-Sigma-Aldrich, St. Louis, MO) and scanned. As we previously reported, Image J software was used to measure the infarct size and both hemisphere regions blindly [35]. The area difference between the contralateral and ipsilateral hemispheres was used to quantify hemispheric edema.

Hemoglobin (Hb) Excess and Hemorrhagic Transformation

A colorimetric hemoglobin detection technique (QuantiChrom Hemoglobin technique Kit, BioAssay Systems, Haywood, CA) was used to assess hemorrhagic transformation following microcarcinomal infarction of the brain and cerebrovascular disruption [36]. In a 10% glycerol Tris-buffer saline solution with 0.5% Tween 20, ipsilateral TTC-brain slices were homogenized. Subsequently, brain samples were subjected to a standard microplate reader (Synergy HT, BioTek equipment) reading at 562 nm for colored hemoglobin. Following the manufacturer's directions, the concentration of hemoglobin was determined and expressed in µg/dL using standard samples.

Western Blot Analysis

The cortical brain's nuclear protein extracts were immunoblotted with anti-Hif1α and anti-PARP antibodies, normalized to β-actin. Meanwhile, whole cell RIPA® lysates were immunoblotted for other proteins. Briefly, 50µg protein was loaded into each lane, separated, and then transferred to PVDF membranes. The membranes were blocked with non-fat dry milk (5% in TBST) for 2 h at room temperature to avoid the nonspecific binding and probed with primary antibodies against NLRP3, cleaved Caspase-1, ASC (1:1000; AG-20B-0014; AG-20B-0042; AG-25B-0006 Adipogen Life Sciences), TXNIP (1:1000; NBP1–54578SS; Novus Biologicals), Cl.PARP, (BD Bioscience Pharmingen, San Diego, CA), IL-6, IL-10, IL-1β, TNF-α, Caspase-3 (1:1000; 12912; 12163; 12242; 11948; 9664; Cell Signaling Technology), and β-actin (1:1000, #A5316 Sigma) at 4°C overnight. Next day, Following twice TBST washes for 10 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody and anti-rabbit antibody (1:10000; A9044; A9169; Sigma USA) for 1 h at room temperature, as previously described [37]. The bands were visualized using an enhanced chemiluminescent substrate solution (Thermo Fisher Scientific). The optical density of samples was analyzed using ImageJ software and normalized to the loading controls.

Slot Blot

By using slot blot analysis, nitrotyrosine (NT) immunoreactivity was assessed as a commonly used indication of lipid peroxidation, 4-hydorxynonenal (4-HNE), and superoxide-dependent peroxynitrite production [38]. A slot blot microfiltration device was used to immobilize 20 µg of peri-infarct (penumbral) sample homogenates onto a nitrocellulose membrane. The membranes were then blocked with 5% nonfat milk and treated with either an anti-nitrotyrosine or anti-4 hydroxynonenal antibody (1:1000; SMC-511; Stress Marq Biosciences; 05–233; Millipore). Next, goat anti-mouse IgG that had been labeled with peroxidase was added, and the membranes were detected using a Pierce Super Signal Kit. Using densitometry Image J software, the optical density of several samples was measured.

Statistical Analysis

Results were presented as mean ± SEM. Tukey's post-hoc test was used to assess differences between the experimental groups after the students' t-test or ANOVA. All statistical analyses were performed using GraphPad Prism 9.0 software (San Diego, CA, USA). Significance was defined by a p < 0.05.

KG diet modulated metabolic panel in the pMCAO group

Stroke led to body weight loss in pMCAO group animals but was not significant (Supplementary material; Table-S2). After KG supplementation, body weight loss was significantly reserved following pMCAO at 72 h. We did not see any significant changes in body weight at earlier time points between the pMCAO and KG-supplemented pMCAO animals. Blood ketone levels in KG + pMCAO and KG group mice spiked in the first week of diet to 3.9 mM compared to sham and pMCAO mice and remained stabilized throughout the experiment to an average of 2.4 mM (Supplementary material; Figure-S1A). Interestingly, the KG diet did not change fasting blood glucose levels (data not shown). At the same time, blood glucose concentrations were significantly lower in KG + pMCAO compared to the pMCAO group (Supplementary material; Fig-S1B).

KG diet improved sensory-motor performance in the pMCAO group.

Motor coordination was evaluated by the maximum time spent on a rotating rod. pMCAO group animals showed motor impairment compared to the sham (Fig. 1). However, KG preconditioning in the KG + pMCAO group significantly improved motor coordination (p < 0.05). Our catwalk findings showed a marked difference in speed-dependent parameters, including average run speed and body speed in pMCAO and KG + pMCAO mice. pMCAO mice significantly (p < 0.01) showed less run average rate than sham following injury. Meanwhile, KG diet-fed mice showed remarkably increased run average speed at different time points post-pMCAO (Fig. 2A, B & C). Furthermore, pMCAO mice markedly walked slower than sham, whereas KG + PMCAO significantly (p < 0.05) recovered and restored their speed in the right front paw (RF) (Fig. 2D, E & F). This result exhibits that pMCAO mice were poorly performed due to the brain injury, and it took the system more than 2 seconds to record the run. The mean intensity was significantly higher in KG + pMCAO compared to pMCAO mice for the left hind paw (LH) and right hind paw (RH) (Fig. 2G, H, I, J, K & L) at each time point post-injury. This result suggests that pMCAO mice avoided putting high pressure on their paws while running in the walkway. We also detected there is a slight increase in coupling in pMCAO mice in left front paw to right hind paw (LF->RH) in comparison to sham and KG + pMCAO mice markedly reduced the coupling post-injury at different time points (Fig. 2M, N & O).

KG diet reduced infarction, brain edema, and hemorrhage in the pMCAO group.

Coronal brain slices stained with TTC revealed white infarcted areas following 72 hours of pMCAO, as illustrated in Fig. 3A. The pMCAO group's dead brain tissue and ipsilateral edema were correlated with the stroke animals' visible infarction regions, which suggested brain injury and neuronal cell death. Nevertheless, supplementation with the KG diet effectively reduces infarct size and edema, improving functional outcomes and providing neuroprotective effects against ischemic stroke, as shown in Fig. 3B & C. To examine the effects of the KG diet on hemorrhage, brain tissue Hb content was estimated as an index for the incidence of intracerebral hemorrhage in perfused brains at 72 h h after pMCAO (Fig. 3D). The Hb content was significantly (p < 0.01) reduced in the KG + pMCAO group compared to pMCAO.

KG diet modulated HIF1α-mediated inflammatory response in the pMCAO group.

Immunoblotting results (Fig. 4) show significantly (p < 0.001) higher protein levels of HIF1α in the cortical brain of KG + pMCAO mice compared to the pMCAO group. In contrast, its level was diminished significantly (p < 0.01) in pMCAO compared to the sham group. IL-10 levels were significantly higher in the cortical brain of KG preconditioning mice (KG + pMCAO) compared to stroke-induced mice maintained on an STD diet (pMCAO). We used western blotting analyses to detect the expression of NLRP3 inflammasome-associated proteins in the ipsilateral brain tissue. These proteins include NLRP3, TXNIP, cleaved-caspase-1, apoptosis-associated speck-like protein (ASC), and IL-1β. It is thought that NLRP3 signaling proteins contribute to the development of ischemic brain injury (Figs. 5 & 6). Stroke triggered NLRP3 inflammasome activation by upregulating NLRP3 inflammasome-associated proteins, as shown in Fig. 6A-C. However, these proteins were significantly downregulated in KG diet-fed animals. Although, we found a non-significant downregulation of cleaved caspase 1 (Fig. 6B). The effect of the KG diet on TNF-α and IL-6, both pleiotropic cytokines that rapidly upregulate in the brain after injury, was also examined. Our immunoblotting showed that TNF-α and IL-6 were highly expressed in the pMCAO group compared to the sham (Fig. 7). In the KG diet preconditioning group (KG + pMCAO), the TNF-α and IL-6 levels were significantly (p < 0.01; p < 0.05 respectively) lower when compared to the pMCAO group. Our results showed that the KG diet fed reduced inflammation by inhibiting the activation of NLRP3 inflammasomes against ischemic stroke.

KG diet attenuates the activation of caspase-3 and PARP in the pMCAO group

To examine the effect of the KG diet on neural apoptotic pathways, we next estimated activation of the pro-apoptotic PARP and caspase-3 at 72 h after pMCAO (Fig. 8A & B). In the ischemic condition, the activation of PARP following DNA damage might contribute to caspase-3 activation. The expression of cleaved PARP and cleaved caspase-3 were significantly (p < 0.001) increased after pMCAO compared to shams. Supplementation of the KG diet significantly reduced activation of caspase-3 (p < 0.05) expression, parallel with a marginal decrease in activation of PARP in the KG + pMCAO group.

KG diet ameliorated the oxidative damage in the pMCAO group.

Slot blotting results showed significantly (p < 0.0001) higher expression of the nitrotyrosine and 4-HNE in the pMCAO compared to the sham-operated group, whereas the KG diet showed a stronger inhibitory effect of both proteins compared with the pMCAO group (Fig. 9A & B). This might be explained by the KG diet, which could have antioxidant effects and reduced oxidative damage following ischemic stroke.

Effect of genetic manipulation of NLRP3 on metabolic panel, cerebral infarction, edema, and sensory-motor performance following PMCAO.

NLRP3 knock-out mice showed significant restoration of body weight in KG group animals (Suppl. material Fig. 2S A). After KG supplementation, body weight was significantly reserved following pMCAO at 72 hrs. We also observed significant changes in body weight before pMCAO in KG-supplemented animals compared to the STD group. Blood ketone level was found to be non-significant in KG group mice compared to STD after 72 hrs (Suppl. Material Fig. 2S B). Interestingly, the KG diet did not change fasting blood glucose levels (data not shown) before PMCAO in KG group animals. At the same time, blood glucose concentration was significantly lower in the KG group compared to the STD group after 72 hrs (Suppl. material Fig. 2S C).

To confirm the role of NLRP3 in ischemic stroke in our findings, we checked whether the genetic knocking down of NLRP3 positively affects the ischemic stroke outcome in the STD or KG diet-fed mice group. According to our TTC sectioning results (Figure. 10A), NLRP3 knockout mice exhibited non-significant cerebral infarct size and edema in either STD or KG group after photothrombotic stroke (Figure. 10B). Our data also showed non-remarkable changes in cerebral edema in KG group compared to 72 hrs after stroke (Figure. 10C). Our catwalk findings represented that there was no marked difference in several speed-dependent, pressure related and coupling parameters including average run speed, body speed, mean intensity and coupling in STD or KG group animals (Fig. 10D, E, F, G & H). Our data indicate that NLRP3 knockout mice not only had less ischemic infarct but also maintained better sensory-motor performance.

An early occurrence in an ischemic stroke is inflammation-mediated cell dysfunction, which is a complex consequence of the emergence of neurological impairments [39]. Moreover, hypoxia and the formation of hypoxic signaling intermediates are known to accompany inflammatory conditions, which in turn initiate inflammatory responses by activating cytokines and inflammatory cells [40]. The current study's findings demonstrated that hypoxia-ischemia insult activated the NLRP3 inflammasome in the photothrombotic stroke model in mice, resulting in brain damage. Preconditioning with the KG diet suppressed NLRP3 inflammasome activation through HIF-1α-mediated downregulation of pro-inflammatory cytokines via IL-10. We also found that these effects were associated with restoring neurobehavioural outcomes and improving brain injury in NLRP3 knockout mice.

The KG diet is a high-fat, extremely low-carbohydrate diet that causes the liver to produce more ketone bodies due to increased fat beta-oxidation. Ketone bodies (BHB, AcAc) are well used by the brain as energy substrates, particularly when there is a shortage of glucose, like when someone is chronically fasting or eating a restricted diet. Increased blood levels of ketone bodies resulted from ketosis, an alternative energy source to glucose and one that the brain is known to utilize efficiently [20]. When glucose metabolism is disrupted, such as when ischemia-reperfusion damage causes oxidative stress, ketones are advantageous substrates [41]. The KG diet is a well-known non-pharmacological strategy for treating drug-resistant epilepsy in children, and it has demonstrated potential in treating other neurological conditions such as Alzheimer's and stroke [21].

We and others have consistently reported that the ketogenic state induced by the KG diet or calorie restriction or by infusions of BHB provides neuroprotection following cerebral ischemia in an experimental stroke model [42, 26, 32, 43–45]. The current study further shows that a 4-week KG diet improved the mice's brain ischemia tolerance to MCAO, as evidenced by improvements in the volume, edema, and Hb content of infarcts. In several mouse models of ischemic stroke, it is well-known that ischemic brain injury results in brain tissue destruction and neurobehavioral abnormalities [46]. In the present study, we demonstrated that photothrombotic stroke-induced neurobehavioral deficits and body weight loss. KG diet pre-supplementation improved the behavioral outcomes and body weight following photothrombotic stroke. Our findings are consistent with as previously reported [47]. In addition, our results showed that genetic deletion of NLRP3 provides protection against ischemic brain damage. Moreover, in the present study, NLRP3 deficiency significantly reduced the infarct size and improved neurobehaviour outcomes following pMCAO.

The mechanism through which diet-induced ketosis confers neuroprotection remains unclear despite its many emerging pre-clinical or clinical investigations [26]. Data from previous studies have demonstrated that the KG diet exerts a neuroprotective effect by alleviating inflammatory pathways [24–27]. Puchowicz et al. (2008) reported the HIF-1α level was elevated after the KG diet because increased succinate inhibited the prolyl hydroxylase, an enzyme responsible for the degradation of HIF. In our study, we also found upregulation of HIF-1α and enhanced expression of IL-10 in mice pre-conditioning with KG diet. Recent studies combine ketosis-mediated stabilization of HIF-1α as a potential neuroprotective phenotype in mice via IL-10-mediated activation of Jak1-Stat3, Akt/ Erk pathways [30, 31]. We also hypothesize other inflammatory responses, especially NLRP3 inflammasome, may be crucial in post-ischemic damage. Nevertheless, the effects of KG on NLRP3 inflammasome in the brain damage through HIF-1α remain unclear.

The inflammatory responses to brain damage in the etiology of neurodegenerative disease and stroke have been implicated to be mediated by the NLRP3 inflammasome and its activation/related products, TXNIP, ASC specks, caspase-1, and IL-1β [48, 19]. Although NLRP3 deficiency decreased brain injury in and in an animal model of stroke, mounting evidence suggests that the levels of NLRP3-inflammasome and IL-1 were elevated in the brain injury. As an indirect inhibitor of the NLRP3 inflammasome, resveratrol has been shown to have protective benefits against ischemic stroke injury in earlier studies [19, 49]. This study further explored the possibility of a connection between the reduction of the NLRP3-mediated inflammatory response and the neuroprotective benefits of KG diet preconditioning. According to our findings, increased TXNIP/NLRP3 inflammasome activation after pMCAO mice exhibited markedly elevated ASC and caspase-1 activity. By downregulating the expression of NLRP3, TXNIP, ASC, and Cl-caspase-1, the KG diet significantly decreased the activation of the NLRP3-inflammasome. According to Luheshi et al. (2011), early production of IL-1β in areas of local neuronal injury underlines it as the major form of IL-1, adding to inflammation early after ischemia [50]. Such a connection between NLRP3 inflammasome and cytokine release in the acute phase may help explain why the KG diet had such striking effects in our investigations. Earlier studies demonstrated that inhibition of the NLRP3 inflammasome is the primary reason for diminishing both TXNIP and stroke-induced injury [17, 11, 51]. Consistent with earlier reports, our hypothesis supports that reduced NLRP3 inflammasome activation coincident with neuroprotective modulations. Furthermore, we found a significant increase in TNF-α and IL-6 in pMCAO stroke mice, which was significantly suppressed in KG diet pre-treated mice following PT stoke consistent with the previous studies [19]. The inverse relationship we observe with HIF1α, and IL-6 levels is similar to previously reported. They reported that HIF1α protein levels were higher under sustained hypoxia with a significant reduction in both mRNA and protein levels of IL-6 [15]In the present pMCAO model, we could track a remarkable caspase-1/IL-1β repression followed by slightly diminished PARP and caspase-3 cleavage, addressing confined inflammation and degeneration in KG diet-supplemented mice.

Inflammatory pathways that are mediated by IL-10 have been thoroughly researched for their significance in the development of tailored strategies intended to regulate harmful inflammation in the brain [52]. IL-10 receptor activation has been shown to trigger the JAK1-STAT3-mediated downregulation of pro-inflammatory cytokines, specifically [53]. Additionally, overexpression of IL-10 in mice has been linked with a resistance to ischemic injury [54, 30]. According to a previous study [55], IL-10 regulates the immune system by inhibiting the production of pro-inflammatory cytokines like IL-6 and TNF-α through attenuation of the NLRP3 inflammasome, which is consistent with our findings.

Cerebral ischemia sets off a series of intricate biochemical and cellular reactions that lead to an overproduction of reactive species and oxidative damage, which is a key factor in the pathophysiology of stroke [35, 56]. It has been shown that excessive ROS/RNS generation during an acute ischemic stroke can overwhelm the body's antioxidant defenses, harm macromolecules, including lipids, proteins, and nucleic acids, and ultimately result in neuronal damage [57]. Increased levels of 4-HNE, an index of lipid peroxidation, and nitrotyrosine, a nitrosative stress biomarker, were also among the indicators. Thus, in this study, to explore the neuroprotective mechanisms of HIF-1α, we measured the expression level of these oxidant indicators by western blot. We used western blot to assess the expression level of these oxidant markers in the present study to investigate the neuroprotective mechanisms of HIF-1α. The findings demonstrated that cerebral ischemia induced oxidative damage and weakened the antioxidant defense system, but KG diet supplementation prevented these effects following ischemic stroke. Additionally, it has been suggested that the complex TXNIP can sense an increase in ROS during ischemia and cause this complex to dissociate [58]. TXNIP is normally maintained in the reduced state in normal cells. When ROS generation is elevated, TXNIP dissociates from this complex and binds to the LRR region of NLRP3, activating the NLRP3 inflammasome [59]. The current study found that pre-treatment with the KG diet decreased the production of ROS following stroke and could lower the activation of the TXNIP/NLRP3 inflammasome, suggesting that the KG diet may protect brain damage from ROS-mediated NLRP3 activation. Thus, the neuroprotective effects of the KG diet may be attributed to the stabilization of HIF-1α or/and activation of pro-survival pathways and inhibition of oxidative stress in the early stage of stroke.

The results of this study show that IL-10 overexpression was linked to HIF-1α stabilization in KG-pre-treated animals. Additionally, the KG diet could prevent cerebral ischemic injury by reducing apoptosis, oxidative stress, and the neuroinflammatory response and enhancing behavioral outcomes. Moreover, KG treatment deactivates NLRP3 inflammasomes, which could be used as a novel therapeutic target for the clinical management of ischemic stroke. The molecular mechanism of the KG diet against cerebral ischemic injury is intricate and encompasses several molecular pathways. These protective benefits are probably due to the suppression of TXNIP/NLRP3 response via IL-10, mediated by HIF-1α. However, there is still insufficient information about the specific molecular mechanism of the KG diet. The underlying mechanisms need to be further revealed before the KG diet can be used in clinical settings.

Author contributions

Kehkashan Parveen conducted all experiments, performed result analysis, wrote the first draft, and prepared the manuscript. Mohd. Salman Helped with surgical procedures and neurobehavior analysis. Golnoush Mirzahosseini performed the NLRP3 KO experiment. Arshi Parveen Helped with experiments and reviewed the manuscript. Michelle Puchowicz and Tauheed Ishrat Designed and oversaw the whole project, including experimental design, managing resources, and critically reviewing the manuscript.

Supplementary file: The full uncropped blots in the supplementary file Figure 3S- Figure 7S.

Funding

The UTHSC Bridge funding and the Department of Pediatrics startup fund supported this work.

Acknowledgments

The UTHSC Bridge funding and the Department of Pediatrics startup fund supported this work.

Data Availability

The data generated and analyzed in this study are available from the corresponding author upon reasonable request.

Declaration of Conflict of Interest

The authors declare that they have no conflicts of interest.

Compliance with Ethical Standards

The institutional animal committee at UTHSC approved all animal studies procedures in accordance with the ethical guidelines of the National Institutes of Health for the care and use of laboratory animals.

Consent to Participate

Not applicable

Consent for Publication: All authors have agreed to publish this manuscript.

Competing Interests: The authors declare no competing interests.

Code availability

Not applicable

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Chronic ketosis provides neuroprotection through HIF-1α-mediated control of the TXNIP/NLRP3 axis by regulating the inflammatory and apoptotic response

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Abstract

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Introduction

Methods

Animals and experimental groups

Photothrombotic stroke surgery

CatWalk Test

Rotarod test

Infarct size and brain edema

Hemoglobin (Hb) Excess and Hemorrhagic Transformation

Western Blot Analysis

Slot Blot

Statistical Analysis

Results

KG diet modulated metabolic panel in the pMCAO group

KG diet attenuates the activation of caspase-3 and PARP in the pMCAO group

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1