Curcumin nanoparticles in heat stroke management.

OBJECTIVE: The exacerbation of extreme high-temperature events due to global climate change poses a significant challenge to public health, particularly impacting the central nervous system through heat stroke. This study aims to develop Poly(amidoamine) (PAMAM) nanoparticles loaded with curcumin (PAMAM@Cur) to enhance its therapeutic efficacy in hypothalamic neural damage in a heat stroke model and explore its potential mechanisms. METHODS: Curcumin (Cur) was encapsulated into PAMAM nanoparticles through a hydrophobic interaction method, and various techniques were employed to characterize their physicochemical properties. A heat stroke mouse model was established to monitor body temperature and serum biochemical parameters, conduct behavioral assessments, histological examinations, and biochemical analyses. Transcriptomic and proteomic analyses were performed to investigate the therapeutic mechanisms of PAMAM@Cur, validated in an N2a cell model. RESULTS: PAMAM@Cur demonstrated good stability, photostability, cell compatibility, significant blood-brain barrier (BBB) penetration capability, and effective accumulation in the brain. PAMAM@Cur markedly improved behavioral performance and neural cell structural integrity in heat stroke mice, alleviated inflammatory responses, with superior therapeutic effects compared to Cur or PAMAM alone. Multi-omics analysis revealed that PAMAM@Cur regulated antioxidant defense genes and iron death-related genes, particularly upregulating the PCBP2 protein, stabilizing SLC7A11 and GPX4 mRNA, and reducing iron-dependent cell death. CONCLUSION: By enhancing the drug delivery properties of Cur and modulating molecular pathways relevant to disease treatment, PAMAM@Cur significantly enhances the therapeutic effects against hypothalamic neural damage induced by heat stroke, showcasing the potential of nanotechnology in improving traditional drug efficacy and providing new strategies for future clinical applications. SIGNIFICANCE: This study highlights the outlook of nanotechnology in treating neurological disorders caused by heat stroke, offering a novel therapeutic approach with potential clinical applications.

Curcumin alleviates heatstroke-induced liver injury in dry-heat environments by inhibiting the expression of NF-κB, iNOS, and ICAM-1 in rats.

we aimed to monitor liver injury in rat model during heat stress and heatstroke in dry-heat environment and investigate the effects of curcumin on heatstroke-induced liver injury and the underlying mechanisms. Sprague-Dawley (SD) rats were randomly divided into four groups: normal saline (NS), and 50 (50-cur), 100 (100-cur), and 200 mg/kg curcumin (200-cur) groups. They were administered the indicated doses of curcumin by gavage once daily for 7 days. On day 8, the rats were transferred to a simulated climate cabin, At 0, 50, 100, and 150 min, the core temperature (Tc) was measured respectively. After sacrificing the rats, tissue samples were collected, measure histology indices, serum enzymes, lipopolysaccharides (LPSs), cytokines, nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1). The Tc increased with time in all groups. Curcumin alleviation of symptoms and improvement in pathological scores. The level of enzymes, LPS, and cytokines increased during heatstroke in the NS group, but curcumin decreased the levels of these indicators. The differences of the indicators between NS and 200-cur groups at 150 min were significant (P < 0.05). The expression of NF-κB p65, iNOS, and ICAM-1 was upregulated in the NS group at 150 min, but their expression was relatively lower in the curcumin groups (P < 0.05). Thus, our findings indicate acute liver injury during heat stress and heatstroke. The mechanism involves cascade-amplification inflammatory response induced by the gut endotoxin. Furthermore, curcumin alleviated heatstroke-induced liver injury in a dose-dependent manner by downregulating NF-κB, iNOS, and ICAM-1.

Jiawei Bai-Hu-decoction ameliorated heat stroke-induced brain injury by inhibiting TLR4/NF-κB signal and mitophagy of glial cell.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Bai-Hu-Decoction (JWBHD), a prescription formulated with seven traditional Chinese medicinal material has demonstrated clinical efficacy in mitigating brain injury among heat stroke (HS) patients. AIM OF THE STUDY: This study aimed to evaluate the therapeutic efficacy of JWBHD on rat model of HS and to explore its therapeutic mechanisms by integrating network pharmacology and pharmacodynamic methodologies, which major components were analyzed by using UPLC-MS/MS. MATERIALS AND METHODS: The network pharmacology analysis was firstly conducted to predict the potential active ingredients and therapeutic targets of JWBHD. The anti-HS effectiveness of JWBHD was then evaluated on rats experienced HS. Rat brain tissues were harvested for a comprehensive array of experiments, including Western blot, PCR, H&E staining, Nissl staining, ELISA, transmission electron microscope, flow cytometry and immunofluorescence to validate the protective effects of JWBHD against HS-induced brain damage. Furthermore, the inhibitory effects of JWBHD on TLR4/NF-κB signal and mitophagy of glial were further verified on HS-challenged F98 cell line. Finally, the chemical compositions of the water extract of JWBHD were analyzed by using UPLC-MS/MS. RESULTS: Network pharmacology has identified fifty core targets and numerous HS-related signaling pathways as potential therapeutic targets of JWBHD. Analysis of protein-protein interaction (PPI) and GO suggests that JWBHD may suppress HS-induced inflammatory signals. In experiments conducted on HS-rats, JWBHD significantly reduced the core temperature, restored blood pressure and alleviated neurological defect. Furthermore, JWBHD downregulated the counts of white blood cells and monocytes, decreased the levels of inflammatory cytokines such as IL-1ß, IL-6 and TNF-α in peripheral blood, and suppressed the expression of TLR4 and NF-κB in the cerebral cortex of HS-rats. Besides, JWBHD inhibited the apoptosis of cortical cells and mitigated the damage to the cerebral cortex in HS group. Conversely, overactive mitophagy was observed in the cerebral cortex of HS-rats. However, JWBHD restored the mitochondrial membrane potential and downregulated expressions of mitophagic proteins including Pink1, Parkin, LC3B and Tom20. JWBHD reduced the co-localization of Pink1 and GFAP, a specific marker of astrocytes in the cerebral cortex of HS-rats. In addition, the inhibitory effect of JWBHD on TLR4/NF-κB signaling and overactive mitophagy were further confirmed in F98 cells. Finally, UPLC-MS/MS analysis showed that the main components of JWBHD include isoliquiritigenin, liquiritin, dipotassium glycyrrhizinate, ginsenoside Rb1, ginsenoside Re, etc. CONCLUSIONS: JWBHD protected rats from HS and prevented HS-induced damage in the cerebral cortex by suppressing TLR4/NF-κB signaling and mitophagy of glial.

Hypoxia-inducible factor prolyl hydroxylase inhibitor alleviates heatstroke-induced acute kidney injury by activating BNIP3-mediated mitophagy.

Hypoxia-induced inflammation and apoptosis are important pathophysiological features of heat stroke-induced acute kidney injury (HS-AKI). Hypoxia-inducible factor (HIF) is a key protein that regulates cell adaptation to hypoxia. HIF-prolyl hydroxylase inhibitor (HIF-PHI) stabilizes HIF to increase cell adaptation to hypoxia. Herein, we reported that HIF-PHI pretreatment significantly improved renal function, enhanced thermotolerance, and increased the survival rate of mice in the context of HS. Moreover, HIF-PHI could alleviate HS-induced mitochondrial damage, inflammation, and apoptosis in renal tubular epithelial cells (RTECs) by enhancing mitophagy in vitro and in vivo. By contrast, mitophagy inhibitors Mdivi-1, 3-MA, and Baf-A1 reversed the renoprotective effects of HIF-PHI. Mechanistically, HIF-PHI protects RTECs from inflammation and apoptosis by enhancing Bcl-2 adenovirus E18 19-kDa-interacting protein 3 (BNIP3)-mediated mitophagy, while genetic ablation of BNIP3 attenuated HIF-PHI-induced mitophagy and abolished HIF-PHI-mediated renal protection. Thus, our results indicated that HIF-PHI protects renal function by upregulating BNIP3-mediated mitophagy to improve HS-induced inflammation and apoptosis of RTECs, suggesting HIF-PHI as a promising therapeutic agent to treat HS-AKI.

Effects of isorhamnetin on liver injury in heat stroke-affected rats under dry-heat environments via oxidative stress and inflammatory response.

Isorhamnetin is a natural flavonoid compound, rich in brass, alkaloids, and sterols with a high medicinal value. This study investigated the effects of isorhamnetin on liver injury and oxidative and inflammatory responses in heat-stroke-affected rats in a dry-heat environment. Fifty Sprague Dawley rats were randomly divided into five groups: normal temperature control (NC, saline), dry-heat control (DHC, saline), low-dose isorhamnetin-pretreated (L-AS, 25 mg/Kg), medium-dose isorhamnetin-pretreated (M-AS, 50 mg/Kg), and high-dose isorhamnetin-pretreated (H-AS, 100 mg/Kg) group. Saline was administered to the NC and DHC groups and corresponding concentrations of isorhamnetin were administered to the remaining three groups for 1 week. Blood and liver tissue were analyzed for oxidative stress and inflammation. The liver histopathological injury score, serum liver enzyme (alanine transaminase, aspartate transaminase, and lactate dehydrogenase), liver oxidative stress index (superoxide dismutase [SOD], catalase [CAT], and malondialdehyde), and inflammation index (tumor necrosis factor α [TNF-α], interleukin [IL]-1ß, IL-6, and lipopolysaccharides) were significantly higher in the DHC group than in the NC group (P < 0.05). These index values in the L-AS, M-AS, and H-AS groups were significantly lower than those in the DHC group (P < 0.05). The index values decreased significantly with an increase in the concentration of isorhamnetin (P < 0.05), while the index values of CAT and SOD showed the opposite tendency (P < 0.05). The expression of liver tissue nuclear factor kappa B (NF-κB), caspase-3, and heat shock protein (HSP-70) was higher in the DHC group than in the NC group (P < 0.05). Comparison between the isorhamnetin and DHC groups revealed that the expression of NF-кB and caspase-3 was decreased, while that of HSP-70 continued to increase (P < 0.05). The difference was significant for HSP-70 among all the isorhamnetin groups (P < 0.05); however, the NF-кB and caspase-3 values in the L-AS and H-AS groups did not differ. In summary, isorhamnetin has protective effects against liver injury in heat-stroke-affected rats. This protective effect may be related to its activities concerning antioxidative stress, anti-inflammatory response, inhibition of NF-кB and caspase-3 expression, and enhancement of HSP-70 expression.

Thromboinflammation in acute injury: infections, heatstroke, and trauma.

Tissue microcirculation is essential for the maintenance of organ homeostasis. Following acute infections, activation of coagulation and inflammation, which are critical interconnected responses, lead to thromboinflammation and microthrombosis, thereby contributing to multiorgan dysfunction. Sepsis is the most common underlying disease and has been extensively studied. However, the COVID-19 pandemic further illustrated the pathomechanisms of diseases in which thromboinflammation plays a critical role. During thromboinflammation, injury to monocytes, neutrophils, platelets, and endothelial cells, along with coagulation and complement activation, was further characterized. Thrombin is pivotal in orchestrating thrombosis and inflammation and has long been considered a potential therapeutic target in sepsis. Although thromboprophylaxis for venous thromboembolism with heparins is part of standard management for COVID-19, it also potentially attenuates organ dysfunction due to thrombotic sequela. In contrast, the effectiveness of anticoagulation with heparin, antithrombin, or thrombomodulin to reduce mortality has not conclusively been proven in sepsis. Nonetheless, thromboinflammation has also been reported as an important pathophysiologic mechanism in other critical illnesses, including heatstroke, trauma, and ischemia/reperfusion injury, and may provide a potential therapeutic target for future clinical studies.

Shikonin prevents mice from heat stroke-induced death via suppressing a trigger IL-17A on the inflammatory and oxidative pathways.

Heat stroke (HS) is the deadliest disease. Due to the complex pathogenesis of HS, lack of effective therapeutic drugs for clinical treatment. Shikonin (SK) is the main active compound of Radix Arnebiae, which was evaluated on the HS model (temperature: (41 ± 0.5) ℃, relative humidity: (60 ± 5) %) via pathological and biochemical approaches in vivo and in vitro. Upon the dose of 10 mg.kg-1, SK delays the rising rate of core temperature, prolongs the survival time of mice, and improves organ injury and coagulation function markedly. Serum HS biomarkers interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were decreased significantly by SK, which contribute to liver and lung protection in the models. Three pathways' responses to heat-stress were found to have a close connection with the IL-17 pathway via RNA sequencing and network analysis. WB and IHC results showed that the nuclear factor-κB (NF-κB) p65 in the SK group was down-regulated (P < 0.05). The expressions of nuclear factor erythroid 2 like 2 (NFE2L2/Nrf2) and heat shock protein 70 (HSP70) were up-regulated (P < 0.05). Additional administration of recombinant IL-17A protein on the HS model up-regulated the expression level of NF- κB p65 in the liver and lung tissue, additional intraperitoneal injection of IL-17A antibody in mice has a synergistic effect with SK in inhibiting tissue inflammatory response and protecting HS. In summary, SK was proved an effective compound for fulfilling the anti-inflammatory and antioxidative capacity of the HS model by reducing the production and inhibiting the expression of IL-17A.

Endothelial glycocalyx injury is involved in heatstroke-associated coagulopathy and protected by N-acetylcysteine.

Introduction: Damage to endothelial glycocalyx (EGCX) can lead to coagulation disorders in sepsis. Heat stroke (HS) resembles sepsis in many aspects; however, it is unclear whether EGCX injury is involved in its pathophysiology. The purpose of this study was to examine the relationship between the damage of EGCX and the development of coagulation disorders during HS. Methods: We retrospectively collected 159 HS patients and analyzed coagulation characteristics and prognosis of HS patients with or without disseminated intravascular coagulation (DIC). We also replicated a rat HS model and measured coagulation indexes, pulmonary capillary EGCX injury in HS rats. Finally, we evaluated the effect of the antioxidant N-acetylcysteine (NAC) on HS-initiated EGCX injury and coagulation disorders. Results: Clinical data showed that HS patients complicated with DIC had a higher risk of death than HS patients without DIC. In a rat HS model, we found that rats subjected to heat stress developed hypercoagulability and platelet activation at the core body temperature of 43°C, just before the onset of HS. At 24 h of HS, the rats showed a consumptive hypo-coagulation state. The pulmonary capillary EGCX started to shed at 0 h of HS and became more severe at 24 h of HS. Importantly, pretreatment with NAC substantially alleviated EGCX damage and reversed the hypo-coagulation state in HS rats. Mechanically, HS initiated reactive oxidative species (ROS) generation, while ROS could directly cause EGCX damage. Critically, NAC protected against EGCX injury by attenuating ROS production in heat-stressed or hydrogen peroxide (H2O2)-stimulated endothelial cells. Discussion: Our results indicate that the poor prognosis of HS patients correlates with severe coagulation disorders, coagulation abnormalities in HS rats are associated with the damage of EGCX, and NAC improves HS-induced coagulopathy, probably through its protection against EGCX injury by preventing ROS generation.

Antimicrobial peptide cathelicidin LL-37 preserves intestinal barrier and organ function in rats with heat stroke.

Global warming increases the incidence of heat stroke (HS) and HS causes the reduction of visceral blood flow during hyperthermia, leading to intestinal barrier disruption, microbial translocation, systemic inflammation and multiple organ failure. Cathelicidin LL-37 exhibits antimicrobial activities, helps innate immunity within the gut to maintain intestinal homeostasis, and augments intestinal wound healing and barrier function. Thus, we evaluated the effects and possible mechanisms of cathelicidin LL-37 on HS. Wistar rats were placed in a heating-chamber of 42 ̊C to induce HS. Changes in rectal temperature, hemodynamic parameters, and survival rate were measured during the experimental period. Blood samples and ilea were collected to analyze the effects of LL-37 on systemic inflammation, multiple organ dysfunction, and intestinal injury. Furthermore, LS174T and HT-29 cells were used to assess the underlying mechanisms. Our data showed cathelicidin LL-37 ameliorated the damage of intestinal cells induced by HS. Intestinal injury, systemic inflammation, and nitrosative stress (high nitric oxide level) caused by continuous hyperthermia were attenuated in HS rats treated with cathelicidin LL-37, and hence, improved multiple organ dysfunction, coagulopathy, and survival rate. These beneficial effects of cathelicidin LL-37 were attributed to the protection of intestinal goblet cells (by increasing transepithelial resistance, mucin-2 and Nrf2 expression) and the improvement of intestinal barrier function (less cyclooxygenase-2 expression and FITC-dextran translocation). Interestingly, high cathelicidin expression in the ileal samples of inflammatory bowel disease patients was associated with better clinical outcome. These results suggest that cathelicidin LL-37 could prevent heat stress-induced intestinal damage and heat-related illnesses.

Improvement in heat stress-induced multiple organ dysfunction and intestinal damage through protection of intestinal goblet cells from prostaglandin E1 analogue misoprostol.

AIMS: Heat stroke is a life-threatening disorder triggered by thermoregulatory failure. Hyperthermia-induced splanchnic hypoperfusion has been reported to induce intestinal barrier dysfunction and systemic immune response that ultimately cause multiple-organ failure and death. Intestinal goblet cells contribute greatly to the formation of mucus barrier, which hinders translocation of gut microorganisms. Studies have reported that misoprostol can not only alleviate ischemic injury but also protect GI mucosal layer. Therefore, we evaluated the effects of misoprostol on intestinal goblet cells after heat stress and on multiple-organ dysfunction in heat stroke rats. MAIN METHODS: Heat stress was established in the heating chamber and followed by misoprostol treatment. Changes in hemodynamics, organ function indices, inflammation, oxidative stress, and survival rate were analyzed. Furthermore, ilea and LS174T cells were used to examine intestinal functions. KEY FINDINGS: Heat stress caused dysfunction of intestinal goblet cells and damage to ilea by increasing oxidative stress and apoptosis. Increased nitrosative stress and inflammation accompanied by hypotension, hypoperfusion, tachycardia, multiple-organ dysfunction, and death were observed in the heat stroke rat model. Treatment of LS174T cells with misoprostol not only decreased oxidative stress and apoptosis but also reduced cytotoxicity caused by heat stress. Moreover, misoprostol prevented disruption of the enteric barrier, multiple-organ injury, and death in rats with heat stroke. SIGNIFICANCE: This study indicates that misoprostol could alleviate intestinal damage and organ injury caused by heat stress and be a potential therapy for heat-related illnesses.

Anti-Inflammatory and Anti-Oxidative Effects of Isorhamnetin for Protection Against Lung Injury in a Rat Model of Heatstroke in a Dry-Heat Environment.

BACKGROUND Isorhamnetin is a natural flavonoid compound with anti-inflammatory and antioxidant properties. However, its roles in alleviating lung injury associated with heatstroke remain unclear. Therefore, this study aimed to evaluate the protective effects of different isorhamnetin doses on lung injury in heatstroke rat models exposed to a dry-heat environment. MATERIAL AND METHODS Fifty Sprague-Dawley rats were randomly divided into 5 groups: normal control (0.9% saline), heatstroke (0.5% CMCNa), and isorhamnetin (25, 50, and 100 mg/kg) groups; treatments were administered by gavage daily for 7 days. All rats, except those in the control group, were exposed to a dry-heat environment (41±1°C, 10±2% relative humidity) for 150 min to induce heatstroke. Pathological changes, ultrastructure, edema, inflammation, and oxidative stress in the lungs were assessed. RESULTS Compared with the heatstroke group, rats treated with 100 mg/kg isorhamnetin showed amelioration of histopathological and ultrastructural changes in the lungs; decreased lung injury scores (P<0.05) and wet/dry weight ratios (P<0.01); lower levels of phospho-nuclear factor-kappaB (P<0.05), high-mobility group box 1 (P<0.01), tumor necrosis factor-alpha (P<0.01), interleukin (IL)-1ß (P<0.01), and IL-6 (P<0.01); lower malondialdehyde contents (P<0.01); and higher superoxide dismutase (P<0.01) and catalase activities (P<0.05). CONCLUSIONS In a dry-heat environment, isorhamnetin protected against lung injury in heatstroke rat models via anti-inflammatory and anti-oxidative mechanisms.

Bovine Lactoferrin Alleviates Pulmonary Lipid Peroxidation and Inflammatory Damage in Heat Stroke Rats.

Lung injury occurring in the early stage of heat stroke (HS) leads to hypoxia and further aggravation of other organic damage. Lactoferrin (LF) is an iron binding protein with anti-inflammatory and antioxidant effects. This study focuses on the protection of preadministration of bovine lactoferrin (BLF) against lung injury in rats with HS. Sixty-four Sprague-Dawley male rats were divided into four groups randomly: control (CON)+phosphate-buffered saline (PBS) (n = 16), HS+PBS (n = 16), HS+low-dose BLF (LBLF) (n = 16), and HS+high-dose BLF (HBLF) (n = 16). CON+PBS and HS+PBS were preadministered 10 mL/kg PBS for 1 week. HS+LBLF and HS+HBLF were preadministered 100 and 200 mg/kg BLF for 1 week, respectively. The HS onset time and the survival rate were recorded, and bronchoalveolar lavage fluid was obtained to measure protein concentration. Lung was obtained for pathological analysis and wet/dry weight ratio measurement; later, the content of malondialdehyde (MDA), activity of myeloperoxidase (MPO), and superoxide dismutase (SOD) were measured in lung tissue homogenate. The results indicated that BLF preadministration could delay the HS onset time, enhance the survival rate, the levels of serum inflammatory cytokine and MDA content in HS+LBLF and HS+HBLF showed significant reduction compared with HS+PBS, while a significant elevation of SOD activity and reduction of MPO activity in HS+HBLF. Our results demonstrate that BLF preadministration could relieve lung injury in HS rats by enhancing thermal endurance, and alleviating serum inflammatory response and pulmonary oxidative stress damage.

[Hydroxysafflor yellow A attenuates heat stroke-induced acute lung injury in mice by inhibiting necroptosis].

OBJECTIVE: To investigate the protective effect of hydroxysafflor yellow A (HSYA) against heat stroke (HS)-induced acute lung injury and its possible mechanism. METHODS: The optimal dose of HSYA pretreatment via intraperitoneal injection prior to HS was determined in a mice by observing heat tolerance of the mice. C57BL/6J mice were pretreated with HSYA at the optimal dose or with Nec-1 (a RIP1 activation inhibitor) before HS, and the changes in core body temperature and survival of the mice were observed during the 72-h recovery period. At different stages of recovery, lung tissues, bronchoalveolar lavage fluid and blood samples were collected from the mice for assessing lung tissue pathology, wet-to-dry weight ratio and water content of the lungs; leukocyte and neutrophil counts, total protein levels and HMGB1 level in the bronchoalveolar lavage fluid (BLF) were also detected. Serum levels of TNF-α, IL-6 and HMGB1 were detected with ELISA, and the expression levels of RIP1, RIP3, MLKL-s358, MLKL and MLKL-s358 proteins in the lung tissues were detected using Western blotting. RESULTS: HSYA pretreatment at the moderate and high doses significantly improved heat tolerance of the mice with comparable effects. At the optimal dose of 2.25 mg/kg, HSYA pretreatment significantly increased heat tolerance of the mice (P<0.05), showing a similar effect with Nec-1 pretreatment. Pretreatment with HSYA and Nec-1 both significantly increased survival rate of the mice (P<0.05), lowered histopathological score and water content of the lungs, and reduced the levels of TNF-α, IL-6 and HMGB1 (P<0.05), leukocyte and neutrophil counts, and total protein and HMGB1 levels in the BLF (P<0.05). The mice during recovery from HS showed significantly increased RIP1 expression and MLKL-s358 phosphorylation level in the lung tissue (P<0.05), which were obviously lowered by HSYA pretreatment of the mice. CONCLUSION: Severe HS results in necroptosis in the lung tissue of mice, which can be alleviated by HSYA pretreatment.

CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) Deficiency Attenuates Heatstroke-Induced Intestinal Injury.

The intestine is one of the main target organs involved in the pathological process of heatstroke. CCAAT/enhancer-binding protein homologous protein (CHOP) is involved in endoplasmic reticulum (ER) stress-induced apoptosis. This study aimed to explore the role of CHOP in heatstroke-induced intestinal injury and potential therapy. An in vitro heat stress (HS) model using Caco-2 cells was employed. We observed the role of CHOP in apoptosis-mediated intestinal epithelial cell injury secondary to HS by evaluating cell viability, lactate dehydrogenase release, apoptosis levels, and GRP78, PERK, ATF4, CHOP, Bcl-2, and BAX mRNA and protein expression. To further study the role of CHOP in HS-induced intestinal barrier dysfunction, we assessed transepithelial electrical resistance, paracellular tracer flux, ultrastructure of tight junctions, and protein expression of ZO-1 and occludin. Male wild-type mice and CHOP knockout mice were used for in vivo experiments. We evaluated serum d-lactate and diamine oxidase levels, histopathological changes, intestinal ultrastructure, and ZO-1 and occludin protein expression. HS activated the PERK-CHOP pathway and promoted apoptosis by upregulating BAX and downregulating Bcl-2; these effects were prevented by CHOP silencing. Intestinal epithelial barrier function was disrupted by HS in vitro and in vivo. CHOP silencing prevented intestinal barrier dysfunction in Caco-2 cells, whereas CHOP knockout mice exhibited decreased intestinal mucosal injury. The ER stress inhibitor 4-phenylbutyrate (4-PBA) prevented HS-induced intestinal injury in vitro and in vivo. This study indicated that CHOP deficiency attenuates heatstroke-induced intestinal injury and may contribute to the identification of a novel therapy against heatstroke associated with the ER stress pathway.

Pharmacological Activation Of Aldehyde Dehydrogenase 2 Protects Against Heatstroke-Induced Acute Lung Injury by Modulating Oxidative Stress and Endothelial Dysfunction.

Heatstroke (HS) can cause acute lung injury (ALI). Heat stress induces inflammation and apoptosis via reactive oxygen species (ROS) and endogenous reactive aldehydes. Endothelial dysfunction also plays a crucial role in HS-induced ALI. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that detoxifies aldehydes such as 4-hydroxy-2-nonenal (4-HNE) protein adducts. A single point mutation in ALDH2 at E487K (ALDH2*2) intrinsically lowers the activity of ALDH2. Alda-1, an ALDH2 activator, attenuates the formation of 4-HNE protein adducts and ROS in several disease models. We hypothesized that ALDH2 can protect against heat stress-induced vascular inflammation and the accumulation of ROS and toxic aldehydes. Homozygous ALDH2*2 knock-in (KI) mice on a C57BL/6J background and C57BL/6J mice were used for the animal experiments. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro experiment. The mice were directly subjected to whole-body heating (WBH, 42°C) for 1 h at 80% relative humidity. Alda-1 (16 mg/kg) was administered intraperitoneally prior to WBH. The severity of ALI was assessed by analyzing the protein levels and cell counts in the bronchoalveolar lavage fluid, the wet/dry ratio and histology. ALDH2*2 KI mice were susceptible to HS-induced ALI in vivo. Silencing ALDH2 induced 4-HNE and ROS accumulation in HUVECs subjected to heat stress. Alda-1 attenuated the heat stress-induced activation of inflammatory pathways, senescence and apoptosis in HUVECs. The lung homogenates of mice pretreated with Alda-1 exhibited significantly elevated ALDH2 activity and decreased ROS accumulation after WBH. Alda-1 significantly decreased the WBH-induced accumulation of 4-HNE and p65 and p38 activation. Here, we demonstrated the crucial roles of ALDH2 in protecting against heat stress-induced ROS production and vascular inflammation and preserving the viability of ECs. The activation of ALDH2 by Alda-1 attenuates WBH-induced ALI in vivo.

NLRP3 ablation enhances tolerance in heat stroke pathology by inhibiting IL-1ß-mediated neuroinflammation.

BACKGROUND: Patients with prior illness are more vulnerable to heat stroke-induced injury, but the underlying mechanism is unknown. Recent studies suggested that NLRP3 inflammasome played an important role in the pathophysiology of heat stroke. METHODS: In this study, we used a classic animal heat stroke model. Prior infection was mimicked by using lipopolysaccharide (LPS) or lipoteichoic acid (LTA) injection before heat stroke (LPS/LTA 1 mg/kg). Mice survival analysis curve and core temperature (TC) elevation curve were produced. NLRP3 inflammasome activation was measured by using real-time PCR and Western blot. Mice hypothalamus was dissected and neuroinflammation level was measured. To further demonstrate the role of NLRP3 inflammasome, Nlrp3 knockout mice were used. In addition, IL-1ß neutralizing antibody was injected to test potential therapeutic effect on heat stroke. RESULTS: Prior infection simulated by LPS/LTA injection resulted in latent inflammation status presented by high levels of cytokines in peripheral serum. However, LPS/LTA failed to cause any change in animal survival rate or body temperature. In the absence of LPS/LTA, heat treatment induced heat stroke and animal death without significant systemic or neuroinflammation. Despite a decreased level of IL-1ß in hypothalamus, Nlrp3 knockout mice demonstrated no survival advantage under mere heat exposure. In animals with prior infection, their heat tolerance was severely impaired and NLRP3 inflammasome induced neuroinflammation was detected. The use of Nlrp3 knockout mice enhanced heat tolerance and alleviated heat stroke-induced death by reducing mice hypothalamus IL-1ß production with prior infection condition. Furthermore, IL-1ß neutralizing antibody injection significantly extended endotoxemic mice survival under heat stroke. CONCLUSIONS: Based on the above results, NLRP3/IL-1ß induced neuroinflammation might be an important mechanistic factor in heat stroke pathology, especially with prior infection. IL-1ß may serve as a biomarker for heat stroke severity and potential therapeutic method.

Ethyl pyruvate ameliorates heat stroke-induced multiple organ dysfunction and inflammatory responses by induction of stress proteins and activation of autophagy in rats.

Objective: Heat stroke (HS) elicits the systemic inflammatory responses that result in multiple organ dysfunction (MOD). Heat shock response and autophagy are activated during heat stress for removal of damaged organelles and proteins, emerging as a major regulator of cellular homeostasis. Ethyl pyruvate (EP) is a derivative of pyruvic acid and possesses antioxidant and anti-inflammatory effects. This study aims to investigate the effects of EP on MOD in HS rats and explore the possible mechanisms.Method: Anesthetized rats were placed in a heating chamber (42 °C) to elevate the core body temperature attaining to 42.9 °C. Rats were then moved to room temperature and monitored for 6 h. EP (60 mg/kg, i.v.) was administered 30 min prior to heat exposure.Results: Results showed that EP significantly reduced HS-induced increases in plasma levels of LDH, CPK, GPT and CK-MB, reversed the decrease of platelet counts, and alleviated intestinal mucosal and pulmonary damage. Moreover, EP reduced pro-inflammatory protein, including TNF-α, IL-6, IL-1ß, HMGB1 and iNOS, and induced stress proteins, heme oxygenase-1 (HO-1), heat shock protein (HSP) 70 and HSP90 in the liver of HS rats. The levels of HS-activated autophagy-regulatory proteins were affected by EP, in which the phosphorylated mTOR and AKT were reduced, and the phosphorylated AMPK increased, accompanied with upregulation in ULK1, Atg7, Atg12 and LC3II, and downregulation of p62.Conclusion: In conclusion, EP ameliorated HS-induced inflammatory responses and MOD, and the underlying mechanism is associated with the induction of the stress proteins HO-1 and HSP70 as well as restorage of autophagy.

Modulation of microglial phenotypes by dexmedetomidine through TREM2 reduces neuroinflammation in heatstroke.

No FDA approved pharmacological therapy is available to reduce neuroinflammation following heatstroke. Previous studies have indicated that dexmedetomidine (DEX) could protect against inflammation and brain injury in various inflammation-associated diseases. However, no one has tested whether DEX has neuro-protective effects in heatstroke. In this study, we focused on microglial phenotypic modulation to investigate the mechanisms underlying the anti-inflammatory effects of DEX in vivo and in vitro. We found that DEX treatment reduced the expression of CD68, iNOS, TNF-α, and IL-1ß, and increased the expression of CD206, Arg1, IL-10 and TGF-ß in microglia, ameliorating heatstroke induced neuroinflammation and brain injury in mice. TREM2, whose neuro-protective function has been validated by genetic studies in Alzheimer's disease and Nasu-Hakola disease, was significantly promoted by DEX in the microglia. TREM2 esiRNA reversed the DEX-induced activation of PI3K/Akt signalling. Overall these findings indicated that DEX may serve, as a potential therapeutic approach to ameliorate heatstroke induced neuroinflammation and brain injury via TREM2 by activating PI3K/Akt signalling.

Protective effect of L-carnitine on myocardial injury in rats with heatstroke.

PURPOSE: To investigate the protective effect of L-carnitine on myocardial injury in rats with heatstroke. METHODS: orty-eight rats were randomly divided into control, heatstroke and 25, 50 and 100 mg/kg L-carnitine groups. The last three groups were treated with 25, 50 and 100 mg/kg L-carnitine, respectively, for seven successive days. Then, except for the control group, the other four groups were transferred into the environment with ambient temperature of (39.5 ± 0.4 °C) and relative humidity of (13.5 ± 2.1%) for 2 h. The core temperature (Tc), mean arterial pressure (MAP), heart rate (HR) and serum and myocardial indexes were detected. RESULTS: Compared with the heatstroke group, in the 100 mg/kg L-carnitine group, the Tc was significantly decreased, the MAP and HR were significantly increased, the serum creatine kinase, lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, tumor necrosis factor α and interleukin 1ß levels were significantly decreased, the myocardial superoxide dismutase and glutathione peroxidase levels were significantly increased, the myocardial malondialdehyde level was significantly decreased and the cardiomyocyte apoptosis index and myocardial caspase-3 protein expression level were remarkably decreased (p < 0.05). CONCLUSIONS: The L-carnitine pretreatment can alleviate the myocardial injury in heatstroke rats through reducing the inflammatory response, oxidative stress and cardiomyocyte apoptosis.

The efficacy of steroids in reducing morbidity and mortality from extreme hyperthermia and heatstroke-A systematic review.

Severe hyperthermia from classical or exertional heatstroke, or from drug ingestion or other noninfective pyrogens, is associated with a high mortality and morbidity. A systemic pro-inflammatory response occurs during heatstroke, characterized by elevated cytokines with endotoxemia from elevated lipopolysaccharide (LPS) levels. Corticosteroids reduce LPS and cytokine levels, suggesting that they may improve outcome. A systematic review searching Embase, MEDLINE, and PubMed from the earliest date available until September 2019 was conducted, according to the PRISMA guidelines, with five papers identified. In four studies, systemic steroids administered before or at the onset of heat stress improved mortality or reduced organ dysfunction. Survival time was greatest when steroid administration preceded heat stress. In one study, a nonsignificant increase in mortality was seen. A dose response was observed, with higher doses extending survival time. Animal studies suggest that steroids improve mortality and/or organ dysfunction after an episode of heat stress or extreme hyperthermia.