A fundamental study on postmortem submersion interval estimation by metabolomics analyzing of gastrocnemius muscle from submersed rat models in freshwater.

In forensic practice, determining the postmortem submersion interval (PMSI) and cause-of-death of cadavers in aquatic ecosystems has always been challenging task. Traditional approaches are not yet able to address these issues effectively and adequately. Our previous study proposed novel models to predict the PMSI and cause-of-death based on metabolites of blood from rats immersed in freshwater. However, with the advance of putrefaction, it is hardly to obtain blood samples beyond 3 days postmortem. To further assess the feasibility of PMSI estimation and drowning diagnosis in the later postmortem phase, gastrocnemius, the more degradation-resistant tissue, was collected from drowned rats and postmortem submersion model in freshwater immediately after death, and at 1 day, 3 days, 5 days, 7 days, and 10 days postmortem respectively. Then the samples were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the dynamic changes of the metabolites. A total of 924 metabolites were identified. Similar chronological changes of gastrocnemius metabolites were observed in the drowning and postmortem submersion groups. The difference in metabolic profiles between drowning and postmortem submersion groups was only evident in the initial 1 day postmortem, which was faded as the PMSI extension. Nineteen metabolites representing temporally-dynamic patterns were selected as biomarkers for PMSI estimation. A regression model was built based on these biomarkers with random forest algorithm, which yielded a mean absolute error (± SE) of 5.856 (± 1.296) h on validation samples from an independent experiment. These findings added to our knowledge of chronological changes in muscle metabolites from submerged vertebrate remains during decomposition, which provided a new perspective for PMSI estimation.

Trends in forensic microbiology: From classical methods to deep learning.

Forensic microbiology has been widely used in the diagnosis of causes and manner of death, identification of individuals, detection of crime locations, and estimation of postmortem interval. However, the traditional method, microbial culture, has low efficiency, high consumption, and a low degree of quantitative analysis. With the development of high-throughput sequencing technology, advanced bioinformatics, and fast-evolving artificial intelligence, numerous machine learning models, such as RF, SVM, ANN, DNN, regression, PLS, ANOSIM, and ANOVA, have been established with the advancement of the microbiome and metagenomic studies. Recently, deep learning models, including the convolutional neural network (CNN) model and CNN-derived models, improve the accuracy of forensic prognosis using object detection techniques in microorganism image analysis. This review summarizes the application and development of forensic microbiology, as well as the research progress of machine learning (ML) and deep learning (DL) based on microbial genome sequencing and microbial images, and provided a future outlook on forensic microbiology.

Gut microbiota regulates chronic ethanol exposure-induced depressive-like behavior through hippocampal NLRP3-mediated neuroinflammation.

Chronic ethanol exposure (CEE), which can lead to neuroinflammation, is an increasing risk factor for depression disorder, but the underlying mechanism is not clear. Recent observations have revealed the associations among psychiatric disorders, ethanol exposure and alterations of the gut microbiota. Here, we found that CEE induced depressive-like behavior, which could be alleviated by probiotics and transferred from donor to recipient mice by fecal microbiota transplantation (FMT). Neuroinflammation and the activation of the NLRP3 inflammasome were also observed in recipient mice. The downregulation of NLRP3 in the hippocampus mitigated CEE-induced depressive-like behavior and neuroinflammation but had no significant effect on FMT recipient mice. Moreover, elevated serum inflammatory factors in recipient mice showed a significant mediation effect between the gut microbiota and depressive-like behavior. Together, our study findings indicate that the gut microbiota contributes to both hippocampal NLRP3-mediated neuroinflammation and depressive-like behavior induced by CEE, which may open avenues for potential interventions against CEE-associated psychiatric disorders.

Microbial communities in the liver and brain are informative for postmortem submersion interval estimation in the late phase of decomposition: A study in mouse cadavers recovered from freshwater.

Introduction: Bodies recovered from water, especially in the late phase of decomposition, pose difficulties to the investigating authorities. Various methods have been proposed for postmortem submersion interval (PMSI) estimation and drowning identification, but some limitations remain. Many recent studies have proved the value of microbiota succession in viscera for postmortem interval estimation. Nevertheless, the visceral microbiota succession and its application for PMSI estimation and drowning identification require further investigation. Methods: In the current study, mouse drowning and CO2 asphyxia models were developed, and cadavers were immersed in freshwater for 0 to 14 days. Microbial communities in the liver and brain were characterized via 16S rDNA high-throughput sequencing. Results: Only livers and brains collected from 5 to 14 days postmortem were qualified for sequencing. There was significant variation between microbiota from liver and brain. Differences in microbiota between the cadavers of mice that had drowned and those only subjected to postmortem submersion decreased over the PMSI. Significant successions in microbial communities were observed among the different subgroups within the late phase of the PMSI in livers and brains. Eighteen taxa in the liver which were mainly related to Clostridium_sensu_stricto and Aeromonas, and 26 taxa in the brain which were mainly belonged to Clostridium_sensu_stricto, Acetobacteroides, and Limnochorda, were selected as potential biomarkers for PMSI estimation based on a random forest algorithm. The PMSI estimation models established yielded accurate prediction results with mean absolute errors ± the standard error of 1.282 ± 0.189 d for the liver and 0.989 ± 0.237 d for the brain. Conclusions: The present study provides novel information on visceral postmortem microbiota succession in corpses submerged in freshwater which sheds new light on PMSI estimation based on the liver and brain in forensic practice.

Postmortem submersion interval estimation of cadavers recovered from freshwater based on gut microbial community succession.

Microbial community succession during decomposition has been proven to be a useful tool for postmortem interval (PMI) estimation. Numerous studies have shown that the intestinal microbial community presented chronological changes after death and was stable in terrestrial corpses with different causes of death. However, the postmortem pattern of intestinal microbial community succession in cadavers retrieved from water remains unclear. For immersed corpses, the postmortem submersion interval (PMSI) is a useful indicator of PMI. To provide reliable estimates of PMSI in forensic investigations, we investigated the gut microbial community succession of corpses submersed in freshwater and explored its potential application in forensic investigation. In this study, the intestinal microbial community of mouse submersed in freshwater that died of drowning or CO2 asphyxia (i.e., postmortem submersion) were characterized by 16S rDNA amplification and high-throughput sequencing, followed by bioinformatic analyses. The results demonstrated that the chronological changes in intestinal bacterial communities were not different between the drowning and postmortem submersion groups. α-diversity decreased significantly within 14 days of decomposition in both groups, and the ß-diversity bacterial community structure ordinated chronologically, inferring the functional pathway and phenotype. To estimate PMSI, a regression model was established by random forest (RF) algorithm based on the succession of postmortem microbiota. Furthermore, 15 genera, including Proteus, Enterococcus, and others, were selected as candidate biomarkers to set up a concise predicted model, which provided a prediction of PMSI [MAE (± SE) = 0.818 (± 0.165) d]. Overall, our present study provides evidence that intestinal microbial community succession would be a valuable marker to estimate the PMSI of corpses submerged in an aquatic habitat.

Advances in artificial intelligence-based microbiome for PMI estimation.

Postmortem interval (PMI) estimation has always been a major challenge in forensic science. Conventional methods for predicting PMI are based on postmortem phenomena, metabolite or biochemical changes, and insect succession. Because postmortem microbial succession follows a certain temporal regularity, the microbiome has been shown to be a potentially effective tool for PMI estimation in the last decade. Recently, artificial intelligence (AI) technologies shed new lights on forensic medicine through analyzing big data, establishing prediction models, assisting in decision-making, etc. With the application of next-generation sequencing (NGS) and AI techniques, it is possible for forensic practitioners to improve the dataset of microbial communities and obtain detailed information on the inventory of specific ecosystems, quantifications of community diversity, descriptions of their ecological function, and even their application in legal medicine. This review describes the postmortem succession of the microbiome in cadavers and their surroundings, and summarizes the application, advantages, problems, and future strategies of AI-based microbiome analysis for PMI estimation.

Spray dried inhalable ivacaftor co-amorphous microparticle formulations with leucine achieved enhanced in vitro dissolution and superior aerosol performance.

The present study aimed to develop inhalable powder formulations with both dissolution enhancement and superior aerodynamic properties for potential pulmonary delivery of a poorly water-soluble drug, ivacaftor (IVA). The IVA-leucine (LEU) microparticle formulations were produced by spray drying and the physicochemical, aerosolization and cytotoxicity properties were characterized. Co-amorphous microparticle formulation was formed at the IVA: LEU 3:1 M ratio with hydrogen bond interactions as indicated by Fourier transform infrared spectroscopy (FTIR) results. Dissolution rate of the co-spray dried formulations was significantly improved as compared with the IVA alone or physical mixtures. The co-spray dried formulations exhibited > 80% fine particle fraction (FPF) and > 95% emitted dose percentage (ED) values respectively, with superior physical and aerosolization stability under 40℃ at 75% RH for 30 days. The laser scanning confocal microscopy results demonstrated that more IVA was uptake by Calu-3 cell lines for the co-spray dried formulation. In summary, our results demonstrated that co-spray drying IVA with LEU could achieve enhanced in vitro release and superior aerodynamic properties for pulmonary delivery of IVA.

Ageing related thyroid deficiency increases brain-targeted transport of liver-derived ApoE4-laden exosomes leading to cognitive impairment.

Alzheimer's disease (AD) is the prevalent cause of dementia in the ageing world population. Apolipoprotein E4 (ApoE4) allele is the key genetic risk factor for AD, although the mechanisms linking ApoE4 with neurocognitive impairments and aberrant metabolism remains to be fully characterised. We discovered a significant increase in the ApoE4 content of serum exosomes in old healthy subjects and AD patients carrying ApoE4 allele as compared with healthy adults. Elevated exosomal ApoE4 demonstrated significant inverse correlation with serum level of thyroid hormones and cognitive function. We analysed effects of ApoE4-containing peripheral exosomes on neural cells and neurological outputs in aged or thyroidectomised young mice. Ageing-associated hypothyroidism as well as acute thyroidectomy augmented transport of liver-derived ApoE4 reach exosomes into the brain, where ApoE4 activated nucleotide-binding oligomerisation domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome by increasing cholesterol level in neural cells. This, in turn, affected cognition, locomotion and mood. Our study reveals pathological potential of exosomes-mediated relocation of ApoE4 from the periphery to the brain, this process can represent potential therapeutic target.

The neuroprotective mechanism of lithium after ischaemic stroke.

Stroke causes degeneration and death of neurones leading to the loss of motor function and frequent occurrence of cognitive impairment and depression. Lithium (Li+), the archetypal mood stabiliser, is neuroprotective in animal models of stroke, albeit underlying mechanisms remain unknown. We discover that Li+ inhibits activation of nucleotide-binding oligomerisation domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes in the middle cerebral artery occlusion (MCAO) stroke model in mice. This action of Li+ is mediated by two signalling pathways of AKT/GSK3ß/ß-catenin and AKT/FoxO3a/ß-catenin which converge in suppressing the production of reactive oxygen species (ROS). Using immunocytochemstry, MRI imaging, and cell sorting with subsequent mRNA and protein quantification, we demonstrate that Li+ decreases the infarct volume, improves motor function, and alleviates associated cognitive and depressive impairments. In conclusion, this study reveals molecular mechanisms of Li+ neuroprotection during brain ischaemia, thus providing the theoretical background to extend clinical applications of Li+ for treatment of ischemic stroke.

Changes on proteomic and metabolomic profile in serum of mice induced by chronic exposure to tramadol.

Tramadol is an opioid used as an analgesic for treating moderate or severe pain. The long-term use of tramadol can induce several adverse effects. The toxicological mechanism of tramadol abuse is unclear. Limited literature available indicates the change of proteomic profile after chronic exposure to tramadol. In this study, we analyzed the proteomic and metabolomic profile by TMT-labeled quantitative proteomics and untargeted metabolomics between the tramadol and the control group. Proteomic analysis revealed 31 differential expressed serum proteins (9 increased and 22 decreased) in tramadol-treated mice (oral, 50 mg/kg, 5 weeks) as compared with the control ones. Bioinformatics analysis showed that the dysregulated proteins mainly included: enzyme inhibitor-associated proteins (i.e. apolipoprotein C-III (Apoc-III), alpha-1-antitrypsin 1-2 (Serpina 1b), apolipoprotein C-II (Apoc-II), plasma protease C1 inhibitor, inter-alpha-trypsin inhibitor heavy chain H3 (itih3)); mitochondria-related proteins (i.e. 14-3-3 protein zeta/delta (YWHAZ)); cytoskeleton proteins (i.e. tubulin alpha-4A chain (TUBA4A), vinculin (Vcl)). And we found that the differential expressed proteins mainly involved in the pathway of the protein digestion and absorption. Metabolomics analysis revealed that differential expressed metabolites mainly involved in protein ingestion and absorption, fatty acid biosynthesis, steroid hormone biosynthesis and bile secretion. Our overall findings revealed that chronic exposure to tramadol changed the proteomic and metabolomic profile of mice. Moreover, integrated proteomic and metabolomic revealed that the protein digestion and absorption is the common enrichment KEGG pathway. Thus, the combination of proteomics and metabolomics opens new avenues for the research of the molecular mechanisms of tramadol toxicity.

An LC-MS/MS method for simultaneous analysis of the cystic fibrosis therapeutic drugs colistin, ivacaftor and ciprofloxacin.

Inhaled antibiotics such as colistin and ciprofloxacin are increasingly used to treat bacterial lung infections in cystic fibrosis patients. In this study, we established and validated a new HPLC-MS/MS method that could simultaneously detect drug concentrations of ciprofloxacin, colistin and ivacaftor in rat plasma, human epithelial cell lysate, cell culture medium, and drug transport media. An aliquot of 200 µL drug-containing rat plasma or cell culture medium was treated with 600 µL of extraction solution (acetonitrile containing 0.1% formic acid and 0.2% trifluoroacetic acid (TFA)). The addition of 0.2% TFA helped to break the drug-protein bonds. Moreover, the addition of 0.1% formic acid to the transport medium and cell lysate samples could significantly improve the response and reproducibility. After vortexing and centrifuging, the sample components were analyzed by HPLC-MS/MS. The multiple reaction monitoring mode was used to detect the following transitions: 585.5-101.1 (colistin A), 578.5-101.1 (colistin B), 393.2-337.2 (ivacaftor), 332.2-314.2 (ciprofloxacin), 602.3-101.1 (polymyxin B1 as internal standard (IS)) and 595.4-101.1 (polymyxin B2 as IS). The running time of a single sample was only 6 min, making this a time-efficient method. Linear correlations were found for colistin A at 0.029-5.82 µg/mL, colistin B at 0.016-3.14 µg/mL, ivacaftor at 0.05-10.0 µg/mL, and ciprofloxacin at 0.043-8.58 µg/mL. Accuracy, precision, and stability of the method were within the acceptable range. This method would be highly useful for research on cytotoxicity, animal pharmacokinetics, and in vitro drug delivery.

Hepatotoxicity of nutmeg: A pilot study based on metabolomics.

Incidences of abuse and poisoning have been reported for nutmeg, a household spice made from grinding the seed of Myristica fragrans, owing to its hallucinogenic properties. However, there have been no reports on nutmeg hepatotoxicity in relation to dose and duration of exposure. To investigate the hepatotoxicity of different nutmeg exposure durations and doses, male mice were administered daily with normal saline, 1.0 g/kg nutmeg, or 4.0 g/kg nutmeg by intragastrical gavage for either 7 or 14 days (for a total of six treatment groups, n = 6). Body weight of each mouse was monitored daily. Histological analysis of liver tissues was performed using hematoxylin and eosin (H&E) staining to investigate the morphological changes in hepatocytes. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined using enzyme-linked immunosorbent assay (ELISA) to investigate liver function. Metabolomics and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed between treatment groups for identifying differential metabolites. Mice in the nutmeg exposure groups exhibited slow growth trends, hepatocyte damage, and significantly elevated serum AST and ALT levels associated with nutmeg dose and exposure duration. Metabolomics and KEGG enrichment pathway analyses also revealed differential levels of some metabolites related to liver function upon nutmeg exposure. Therefore, the present study reasonably speculates that nutmeg exposure may cause liver damage and affect liver function depending on the dose and duration.

Toxicology of tramadol following chronic exposure based on metabolomics of the cerebrum in mice.

Tramadol is an opioid used as an analgesic for treating moderate or severe pain. The long-term use of tramadol can induce several adverse effects. The toxicological mechanism of tramadol abuse is unclear. Metabolomics is a very useful method for investigating the toxicology of drug abuse. We investigated the impact of chronic tramadol administration on the cerebrum of mice, focusing on the metabolites after tramadol administration. The mice received 20 or 50 mg/kg body weight tramadol dissolved in physiological saline daily for 5 weeks via oral gavage. Compared with the control group, the low dose tramadol group showed seven potential biomarkers, including gamma-hydroxybutyric acid, succinate semialdehyde, and methylmalonic acid, which were either up- or down-regulated. Compared with the control group, the high dose tramadol group showed ten potential biomarkers, including gamma-hydroxybutyric acid, glutamine, and O-phosphorylethanolamine, which were either up- or down-regulated. The up-regulated gamma-hydroxybutyric acid and the down-regulated succinate semialdehyde revealed that the neurotransmitter system was disrupted after tramadol abuse. Compared with the low dose tramadol group, there were twenty-nine potential biomarkers in the high dose tramadol group, mainly related to the pentose phosphate pathway and glycerophospholipid metabolism. In conclusion, metabolomics in the tramadol abuse group demonstrated that long-term tramadol abuse can result in oxidative damage, inflammation, and disruption of the GABA neurotransmitter system, which will help to elucidate the toxicology of tramadol abuse.

Optimization of inhalable liposomal powder formulations and evaluation of their in vitro drug delivery behavior in Calu-3 human lung epithelial cells.

Inhalation therapy has advantages for the treatment of multidrug resistant bacterial lung infections with high drug concentrations at the infection sites in the airways and reduced systemic exposure. We have developed liposomal formulations for pulmonary delivery of synergistic ciprofloxacin (Cipro) and colistin (Col) as the potential candidate for treatment of lung infections caused by multidrug resistant Gram-negative bacteria. This study aims to: (1) further optimize the powder formulation by adding drying stabilizers (polyvinyl pyrrolidone or poloxamer) to protect the liposomes during spray-freeze-drying; (2) evaluate the transport and cellular uptake of drugs in a human lung epithelial Calu-3 cell model. The liposomal powder formulations were produced using the ultrasonic spray-freeze-drying technique. The optimal formulation (F5) used mannitol (8% w/v) and sucrose (2% w/v) as the internal lyoprotectants. Adding external lyoprotectants/aerosolization enhancers (i.e. 8% w/v mannitol, 2% w/v sucrose and 1%, w/w PVP 10) produced the superior rehydrated EE values of ciprofloxacin and colistin (50.2 ± 0.9% for Cipro and 37.8 ± 1.2% for Col) as well as satisfactory aerosol performance (FPF: 34.2 ± 0.8% for Cipro and 33.6 ± 0.9% for Col). The cytotoxicity study indicated that F5 with the colistin concentration at 50 µg/mL and ciprofloxacin at 200 µg/mL was not cytotoxic to human lung epithelial Calu-3 cells. The intracellular uptake of ciprofloxacin was concentration-dependent in Calu-3 cells and the uptake of A-B was more than that of B-A for all samples (p < 0.05). This study demonstrates that co-delivery of ciprofloxacin and colistin in a single liposome can lower the transport capability of both drugs across the Calu-3 cell monolayer and their accumulation in the cells. These findings indicate that co-loaded liposomal powder of ciprofloxacin and colistin is a promising potential treatment for respiratory infections caused by multidrug resistant Gram-negative bacteria.

Toxicology of paraquat and pharmacology of the protective effect of 5-hydroxy-1-methylhydantoin on lung injury caused by paraquat based on metabolomics.

Paraquat (PQ) is a non-selective herbicide and is exceedingly toxic to humans. The mechanism of PQ toxicity is very complex and has not been clearly defined. There is no specific antidote for PQ poisoning. 5-hydroxy-1-methylhydantoin (HMH) is an intrinsic antioxidant and can protect against renal damage caused by PQ. The mechanism of PQ toxicology and the possible effects of HMH on PQ-induced lung injury were determined in this study. It was found that PQ decreased superoxide dismutase (SOD) activity and elevated the level of malondialdehyde (MDA), while HMH elevated SOD activity and decreased the level of MDA. Based on metabolomics, the citrate cycle, glutathione metabolism, taurine and hypotaurine metabolism, regulation of lipolysis in adipocytes, inflammatory mediator regulation of TRP channels, purine and pyrimidine metabolism, aldosterone synthesis and secretion, and phenylalanine metabolism were changed in the PQ group. Compared with the PQ group, the levels of N-acetyl-l-aspartic acid, L-glutamic acid, L-aspartic acid, mesaconic acid, adenosine 5' monophosphate, methylmalonic acid, cytidine, phosphonoacetic acid, hypotaurine, glutathione (reduced) and cysteinylglycine increased, while the levels of corticosterone, xanthine, citric acid, prostaglandin G2, 4-pyridoxic acid and succinyl proline decreased in the HMH group. These metabolites revealed that HMH can alleviate inflammation caused by PQ and elevate the activity of intrinsic antioxidants. In conclusion, our results revealed PQ toxicology and the pharmacology underlying the protective effect of HMH on lung injury due to PQ. Toxicity caused by PQ results in lipid peroxidation and an increase in reactive oxygen species (ROS), nitric oxide (NO), damage to the biliary system, gastrointestinal system and nervous system, in addition to lungs, kidneys, and the liver. HMH is a good antioxidant and protects against lung injury caused by PQ. In summary, HMH efficiently reduced PQ-induced lung injury in mice.

[Exploration about the protection mechanism of 5-hydroxy-1-methylhydantoin on paraquat poisoning model].

OBJECTIVE: To investigate the effects of 5-hydroxy-1-methylhydantoin (HMH) on kidney injury induced by paraquat (PQ). METHODS: Fifteen SPF healthy Kunming mice were randomly divided into normal saline (NS) control group, PQ poisoning model group and HMH intervention group, with 5 mice in each group. PQ poisoning model was challenged by one-time gavage of 30 mg/kg PQ solution. The NS group received the same amount of NS by gavage. The HMH group was given 100 mg/kg of HMH immediately after the model was made and continued to be gavaged. Mice in each group were sacrificed 1 day after HMH gavage and heart blood and renal tissue were harvested for examination. The morphological changes of renal tissue were observed under light microscope by hematoxylin-eosin (HE) staining. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in renal tissue were detected according to the instructions of the kit. The expression of heme oxygenase-1 (HO-1) and interleukin-1ß (IL-1ß) in renal tissues were detected by Western Blot. The serum metabolites were detected by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), the overall distribution of each sample was observed by principal component analysis (PCA), the accuracy of the model was evaluated by multidimensional analysis orthogonal partial least squares-discriminant analysis (OPLS-DA), and the difference metabolites were screened by variable importance in the projection (VIP) value > 1. RESULTS: Light microscopic observation showed that: glomerular structure in NS group was clear, there was no hyperemia and inflammatory cell infiltration in renal interstitium and blood vessels. In PQ group, some glomeruli atrophy and necrosis, capillary congestion in glomeruli, infiltration of inflammatory cells around glomeruli, swelling of renal tubular epithelial cells, slight stenosis of lumen, and occasional necrosis and exfoliation of epithelial cells occurred. The degree of kidney injury in HMH group was significantly less than that in PQ group. Compared with the NS group, the content of MDA in the PQ group was significantly increased (nmol/g: 6.70±0.84 vs. 2.70±0.43, P < 0.01) and the activity of SOD was significantly decreased (kU/L: 33.30±4.66 vs. 50.20±3.23, P < 0.05), the protein expression of HO-1 and IL-1ß were significantly increased (HO-1/ß-actin: 1.11±0.12 vs. 0.61±0.13, IL-1ß/ß-actin: 0.93±0.13 vs. 0.32±0.06, both P < 0.05). Compared with the PQ group, the content of MDA in the HMH group was significantly decreased (nmol/g: 5.10±0.93 vs. 6.70±0.84, P < 0.05) and the activity of SOD was significantly increased (kU/L: 61.00±9.02 vs. 33.30±4.66, P < 0.05), the protein expression of HO-1 was significantly decreased (HO-1/ß-actin: 0.77±0.07 vs. 1.11±0.12, P < 0.05), however, there was no significant difference in the protein expression of IL-1ß (IL-1ß/ß-actin: 0.87±0.13 vs. 0.93±0.13, P > 0.05). Metabolite detection results showed that: compared with NS group, the levels of creatinine, glycine, succinic acid, fumaric acid and citric acid were significantly increased in the PQ group (VIP value was 1.50, 1.58, 1.64, 1.74 and 1.95 respectively, all P < 0.05), while the levels of palmitic acid,α-tocopherol and 6-phosphogluconic acid were significantly decreased (VIP value was 1.10, 1.55 and 1.56 respectively, all P < 0.05). Compared with the PQ group, the levels of creatinine and citric acid were significantly decreased in the HMH group (VIP value was 1.50 and 1.86, both P < 0.05), while trans-4-hydroxy-proline, D-glyceric acid, 2, 6-fructose phosphate, 6-phosphate gluconic acid and aminomalonic acid were significantly increased (VIP value was 1.36, 1.55, 1.63, 1.68 and 1.76 respectively, all P < 0.05). CONCLUSIONS: HMH protects kidney injury caused by PQ poisoning by correcting tricarboxylic acids cycle disturbance, lipid peroxidation and energy metabolism disturbance, and its mechanism is related to the regulation of HO-1 protein expression through Nrf2 pathway.