The involvement of oxidative stress and the TLR4/NF-κB/NLRP3 pathway in acute lung injury induced by high-altitude hypoxia.

OBJECTIVE: This study investigated the effect of oxidative stress and the TLR4/NF-κB/NLRP3 pathway on the pathogenesis of acute lung injury (ALI) induced by high-altitude hypoxia. METHODS: Rats were placed in an animal hyperbaric oxygen chamber to establish a rat model of ALI induced by high-altitude hypoxia after treatment with N-acetylcysteine (NAC; a reactive oxygen species [ROS] inhibitor) or/and MCC950 (an NLPR3 inflammasome inhibitor). After modeling, the wet-to-dry weight ratio (W/D) of rat lung tissues was calculated. In lung tissues, ROS levels were detected with immunofluorescence, the enzyme activity was tested with the kit, and the expression of TLR4/NF-κB/NLRP3 pathway-related genes and proteins was measured with western blotting and qRT-PCR. The levels of inflammatory factors in the serum were quantified with ELISA. RESULTS: After modeling, rats showed significantly increased W/D, ROS levels, and Malondialdehyde (MDA) concentrations and markedly diminished Superoxide dismutase (SOD) and Glutathione (GSH) concentrations in lung tissues (all P < 0.01), accompanied by substantially enhanced serum levels of TNF-α, IL-6, and IL-1ß, significantly reduced serum levels of IL-10, and remarkably augmented TLR4, NLRP3, p-NF-κB p65, NF-κB p65 mRNA, and Caspase-1 expression in lung tissues (all P < 0.01). Furthermore, treatment with NAC or MCC950 alone or in combination prominently lowered the W/D of lung tissues (P < 0.01), serum levels of TNF-α (P < 0.05), IL-6 (P < 0.05), and IL-1ß (P < 0.01), and NF-κB p65 expression and phosphorylation (P < 0.05, P < 0.01) while significantly increasing SOD and GSH concentrations (P < 0.05, P < 0.01) and serum levels of IL-10 (P < 0.01) in modeled rats. Meanwhile, treatment of NAC alone or combined with MCC950 significantly reduced MDA concentration and ROS levels (P < 0.05, P < 0.01) in modeled rats, and treatment of MCC950 alone or combined with NAC considerably declined TLR4, NLRP3, and Caspase-1 expression in modeled rats (P < 0.05, P < 0.01). CONCLUSION: Inhibition of oxidative stress and the TLR4/NF-κB/NLRP3 pathway can ameliorate ALI in rats exposed to high-altitude hypoxia.

Huangqi Baihe Granules alleviate hypobaric hypoxia-induced acute lung injury in rats by suppressing oxidative stress and the TLR4/NF-κB/NLRP3 inflammatory pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangqi Baihe Granules (HQBHG) are a modified formulation based on the traditional recipe "Huangqi Baihe porridge" and the Dunhuang medical prescription "Cistanche Cistanche Soup." The Herbal medicine moistens the lungs and tones the kidneys in addition to replenishing Qi and feeding Yin, making it an ideal choice for enhancing adaptability to high-altitude hypoxic environments. AIM OF THE STUDY: The purpose of this study was to examine a potential molecular mechanism for the treatment and prevention of hypoxic acute lung injury (ALI) in rats using Huangqi Baihe Granules. MATERIALS AND METHODS: The HCP-III laboratory animal low-pressure simulation chamber was utilized to simulate high-altitude environmental exposure and establish an ALI model in rats. The severity of lung damage was evaluated using a battery of tests that included spirometry, a wet/dry lung ratio, H&E staining, and transmission electron microscopy. Using immunofluorescence, the amount of reactive oxygen species (ROS) in lung tissue was determined. Superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and myeloperoxidase (MPO) levels in lung tissue were determined using this kit. Serum levels of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta), and antiinflammatory cytokines like interleukin-10 (IL-10) were measured using an enzyme-linked immunosorbent assay kit. Gene expression changes in lung tissue were identified using transcriptomics, and the relative expression of proteins and mRNA involved in the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB p65)/Nod-like receptor protein 3 (NLRP3) pathway were determined using western blotting and quantitative real-time PCR. RESULTS: HQBHG was shown to enhance lung function considerably, decrease the wet/dry ratio of the lungs, attenuate lung tissue damage, suppress ROS and MDA formation, and increase SOD activity and GSH expression. The research also demonstrated that HQBHG inhibited the activation of the TLR4/NF-κB p65/NLPR3 signaling pathway in lung tissue, reducing the release of downstream pro-inflammatory cytokines. CONCLUSIONS: HQBHG exhibits potential therapeutic effects against ALI induced by altitude hypoxia through suppressing oxidative stress and inflammatory response. This suggests it may be a novel drug for treating and preventing ALI.

Protective effect of San Huang Pill and its bioactive compounds against ulcerative colitis in Drosophila via modulation of JAK/STAT, apoptosis, Toll, and Nrf2/Keap1 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: San Huang Pill (SHP) is a prescription in Dunhuang Ancient Medical Prescription, which has the efficacy of heat-clearing and dampness-drying, and is a traditional formula for the treatment of gastrointestinal diseases. However, its efficacy and mechanism in treating ulcerative colitis (UC) are still unclear. AIM OF THE STUDY: To investigate the protective effects of SHP and its bioactive compounds against Dextran Sulfate Sodium (DSS)-induced intestinal damage using the Drosophila melanogaster model, and to detect the molecular mechanism of SHP in the treatment of UC. METHODS: Survival rate, locomotion, feeding, and excretion were used to explore the anti-inflammatory effects of SHP. The pharmacotoxicity of SHP was measured using developmental analysis. Intestinal integrity, intestinal length, intestinal acid-base homeostasis, and Tepan blue assay were used to analyze the protective effect of SHP against DSS-induced intestinal damage. The molecular mechanism of SHP was detected using DHE staining, immunofluorescence, real-time PCR, 16 S rRNA gene sequencing, and network pharmacology analysis. Survival rate, intestinal length, and integrity analysis were used to detect the protective effect of bioactive compounds of SHP against intestinal damage. RESULTS: SHP supplementation significantly increased the survival rate, restored locomotion, increased metabolic rate, maintained intestinal morphological integrity and intestinal homeostasis, protected intestinal epithelial cells, and alleviated intestinal oxidative damage in adult flies under DSS stimulation. Besides, administration of SHP had no toxic effect on flies. Moreover, SHP supplementation remarkably decreased the expression levels of genes related to JAK/STAT, apoptosis, and Toll signaling pathways, increased the gene expressions of the Nrf2/Keap1 pathway, and also reduced the relative abundance of harmful bacteria in DSS-treated flies. Additionally, the ingredients in SHP (palmatine, berberine, baicalein, wogonin, rhein, and aloeemodin) had protection against DSS-induced intestinal injury, such as prolonging survival rate, increasing intestinal length, and maintaining intestinal barrier integrity. CONCLUSION: SHP had a strong anti-inflammatory function, and remarkably alleviated DSS-induced intestinal morphological damage and intestinal homeostatic imbalance in adult flies by regulating JAK/STAT, apoptosis, Toll and Nrf2/Keap1 signaling pathways, and also gut microbial homeostasis. This suggests that SHP may be a potential complementary and alternative medicine herb therapy for UC, which provides a basis for modern pharmacodynamic evaluation of other prescriptions in Dunhuang ancient medical prescription.

Angelica sinensis polysaccharide extends lifespan and ameliorates aging-related diseases via insulin and TOR signaling pathways, and antioxidant ability in Drosophila.

Angelica sinensis polysaccharide (ASP) is one of the principal active components of Angelica sinensis (AS) that is widely used in natural medicine and has various pharmacological activities, including antioxidant, anti-inflammatory, and enhancing immunity. However, its pharmacological role of anti-aging needs to be clarified. Here, we detected the beneficial effect and mechanism of ASP on healthy aging and aging-related diseases using the Drosophila melanogaster model. The results showed that oral administration of ASP remarkably extended lifespan, increased reproduction, improved climbing ability, and increased resistance to starvation and oxidative stress in aged flies, mainly via inhibiting insulin signaling (IIS) and TOR signaling and boosting antioxidant ability. Further, ASP supplementation protected against aging-induced intestinal homeostasis imbalance via inhibiting intestinal stem cells (ISCs) hyperproliferation and oxidative damage, improved sleep disorders via rescuing sleep rhythm in aged flies, and had a neuroprotective effect on Aß42 transgenic flies. Taken together, our findings shed light on the possibility that ASP could increase lifespan, improve healthy aging, and ultimately reduce the incidence of age-related illnesses. It holds promise as a candidate for anti-aging intervention and treatment for aging-associated disorders.

Comparison of hematological traits and oxygenation properties of hemoglobins from highland and lowland Asiatic toad (Bufo gargarizans).

The Asiatic toad (Bufo gargarizans) belonging to the family of Bufonidae (Anura: Amphibia) is successfully residing on the Qinghai-Tibetan Plateau (QTP). To investigate whether the oxygen delivery undergoes adaptive adjustments to high-altitude environments in Asian toads inhabiting the QTP (Zoige County, 3446 m), choosing low-altitude populations (Chengdu City, 500 m) as control, we measured hematological traits, O2 affinities of whole blood, Hb-O2 affinities of purified Hbs, their sensitivities to temperature, and allosteric effectors (H+, Cl- and ATP). Our results showed that high-altitude Asiatic toads possessed significantly increased hemoglobin concentration, hematocrit, and red blood cell count, but significantly decreased erythrocyte volume compared with low-altitude toads. The whole blood and purified Hbs of high-altitude Asiatic toads both exhibited significantly higher O2 affinities compared with low-altitude toads. Substantially increased intrinsic Hb-O2 affinities of high-altitude Asiatic toads Hbs are likely to be the main reason for its elevated Hb-O2 affinities given the anionic cofactor sensitivities of high- and low-altitude toads were similar. The Hbs of high-altitude toads were also characterized by distinctly strong Bohr effects at the low temperature and low-temperature sensitivities. The adaptive adjustments of hematological traits could enhance the blood-O2 carrying capacity of high-altitude Asiatic toads. The increased Hb-O2 affinities could safeguard the pulmonary O2 uploading under hypoxia. The strong Bohr effects at the low temperature could help the release of O2 in metabolic tissues and cold limbs, while low-temperature sensitivity could minimize the effect of temperature fluctuation on the Hb-O2 affinity.

Freeze tolerance and the underlying metabolite responses in the Xizang plateau frog, Nanorana parkeri.

The frog Nanorana parkeri (Dicroglossidae) is endemic to the Tibetan Plateau, and overwinters shallow pond within damp caves for up to 6 months. Herein, we investigate the freeze tolerance of this species and profile changes in liver and skeletal muscle metabolite levels using an untargeted LC-MS-based metabolomic approach to investigate molecular mechanisms that may contribute to freezing survival. We found that three of seven specimens of N. parkeri could survive after being frozen for 12 h at - 2.0 °C with 39.91% ± 5.4% (n = 7) of total body water converted to ice. Freezing exposure induced partial dehydration of the muscle, which contributed to decreasing the amount of freezable water within the muscle and could be protective for the myocytes themselves. A comparative metabolomic analysis showed that freezing elicited significant responses, and a total of 33 and 36 differentially expressed metabolites were identified in the liver and muscle, respectively. These metabolites mainly participate in alanine, aspartic acid and glutamic acid metabolism, arginine and proline metabolism, and D-glutamine and D-glutamate metabolism. After freezing exposure, the contents of ornithine, melezitose, and maltotriose rose significantly; these may act as cryoprotectants. Additionally, the content of 8-hydroxy-2-deoxyguanine, 7-Ketocholesterol and hypoxanthine showed a marked increase, suggesting that freezing induced oxidative stress in the frogs. In summary, N. parkeri can tolerate a brief and partial freezing of their body, which was accompanied by substantial changes in metabolomic profiles after freezing exposure.

Metabolic characteristics of overwintering by the high-altitude dwelling Xizang plateau frog, Nanorana parkeri.

The Xizang plateau frog, Nanorana parkeri, has the highest altitudinal distribution of all frogs in the world and survives the cold of winter without feeding by entering into a hibernating state. However, little attention has been paid to its physiological and biochemical characteristics that support overwintering underwater in small ponds. Here, we measured metabolic rate and heart rate, and collected liver and muscle samples from N. parkeri in summer and winter for analysis of mitochondrial respiration rate, and activities and relative mRNA transcript expression of metabolic enzymes. Compared with summer-collected frogs, both resting metabolic rate and heart rate were significantly reduced in winter-collected frogs. Both state 3 and state 4 respiration of liver mitochondria were also significantly reduced in winter but muscle mitochondria showed a decline only in state 3 respiration in winter. The activities and corresponding mRNA expression of cytochrome c oxidase showed a marked decline in winter, whereas the activities and corresponding mRNA expression of lactate dehydrogenase increased in winter-collected frogs, compared to summer. The thermal sensitivity (Q10 values) for state 3 respiration rate by liver mitochondria, and activities of lactate dehydrogenase, and cytochrome c oxidase all increased in winter-collected frogs, compared with summer frogs, suggesting that overwintering frogs were more sensitive to changes in external temperature. Enzyme changes mainly result from lower overall quantities of these enzymes as well as post-translational modifications. We conclude that overwintering N. parkeri exhibit a seasonal, temperature-independent suppression of metabolism that is mediated at multiple levels: physiological, mitochondrial, gene expression and enzyme activity levels.

Suppressed LPS-mediated TLR4 signaling in the plateau zokor (Eospalax baileyi) compared to the bamboo rat (Rhizomys pruinosus) and rat (Rattus norvegicus).

Ecological immunology involves the study of the immune function of wildlife, which is seldom compared with that of model animals. Here, we evaluated and compared the level of the innate immune response in the plateau zokor (Eospalax baileyi), an indigenous underground rodent from the Tibetan Plateau, with that in the bamboo rat (Rhizomys pruinosus) and Sprague-Dawley (SD) rat (Rattus norvegicus). The spleen was observed by ordinary light and transmission electron microscopy, and the spleen index was calculated. After liposaccharide (LPS) challenge, the expression of Toll-like receptor 2 (TLR2), TLR4, and hypoxia-inducible factor 1α (HIF-1α) in the spleen was detected by Western blot analysis and immunofluorescence. The expression of nuclear factor-κB1 (NF-κB1) and mitogen-activated protein kinase 14 (MAPK14) in the spleen was detected by real-time quantitative polymerase chain reaction, and the levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-ß (IFN-ß) in the spleen were detected by enzyme-linked immunoassay. The spleen index of the plateau zokor was lower than that of the bamboo rat and SD rat. The expression of TLR4, NF-κB1, and MAPK14 and the levels of IL-6 and TNF-α in the spleen of the plateau zokor were lower than those of the bamboo rat and SD rat, while the expression of TLR2 and HIF-1α and the level of IFN-ß were higher than those of the bamboo rat and SD rat. We speculate that suppression of the TLR4 signaling pathway in the plateau zokor is an adaptation to hypoxic tunnels that decreases antigenic risk and maintains immune homeostasis. Moreover, the spleen of the plateau zokor is reduced in size, reducing the innate immunity investment in the spleen. We also noted that high levels of HIF-1α in the spleen of the plateau zokor suppressed crosstalk between HIF-1α and TLR4, promoting the innate immune response.

The levels of oxidative stress and antioxidant capacity in hibernating Nanorana parkeri.

The effect of hibernation on oxidative stress and antioxidant defense was assessed in the frog Nanorana parkeri which inhabits the southern Tibetan Plateau. We compared the indices of oxidative stress (GSSG/GSH), the degree of oxidative damage (content of carbonyl proteins and lipid peroxide products) and the activities of antioxidant enzymes (SOD, CAT, GPx, GST and GR) in liver, brain, heart and muscle of N. parkeri sampled during summer and winter. Obtained results showed that hibernation induced a significant decrease in the level of GSH in heart, liver, and muscle, while the ratio of GSSG/GSH markedly increased in all tissues except for muscle. Regarding oxidative damage, significant increases in TBARS were observed in all tissues of N. parkeri in the midst of hibernation, and the lipid peroxides level also clearly elevated in these tissues except the liver. In liver and brain, the level of carbonyl proteins was significantly higher in winter relative to summer. Additionally, the activity of antioxidant enzymes obviously reduced in the liver of hibernating N. parkeri. The total antioxidant capacity was also significantly lower in all tissues during winter than summer. In conclusion, hibernation in N. parkeri induced oxidative stress which was supported by oxidative damage to lipids and proteins with suppression of antioxidant defense.