Methamphetamine reduces expressions of tight junction proteins, rearranges F-actin cytoskeleton and increases the blood brain barrier permeability via the RhoA/ROCK-dependent pathway.

Methamphetamine (METH) is a psychostimulant with severe neurotoxicity, which is related to an increase of blood-brain barrier (BBB) permeability. However, the exact mechanisms have not been fully illuminated. In the present study, male Sprague Dawley rats were treated with METH or saline with 8 injections (i.p.) at 12-h intervals and sacrificed 24 h after the last METH injection. To evaluate BBB permeability, 6 rats were administered with Evans blue (EB) by tail vein injection 1 h prior to sacrifice. EB levels significantly increased in both left and right frontal lobes in METH-treated rats, suggesting increase of BBB permeability, which was proved by the rearrangement of F-actin cytoskeleton and decreased expressions of tight junction (TJ) proteins in hippocampus. Over-expressions of RhoA, ROCK, myosin light chain (MLC), cofilin, phosphorylation (p)-MLC, p-cofilin and matrix metalloproteinase (MMP)-9 were observed, indicating activated RhoA/ROCK pathway. Rat brain microvascular endothelial cells (RBMECs) were isolated and treated with inhibitors of RhoA and ROCK followed by METH. Pretreatments of the inhibitors significantly decreased expressions of RhoA, ROCK, MLC, cofilin, p-MLC and p-cofilin, increased expressions of TJ proteins, suppressed F-actin cytoskeleton rearrangement and reduced the permeability of RBMECs. These results suggested that METH increased BBB permeability through activating the RhoA/ROCK pathway, which resulted in F-actin cytoskeleton rearrangement and down-regulation of TJ proteins.

Diagnostic role of serum tryptase in anaphylactic deaths in forensic medicine: a systematic review and meta-analysis.

Postmortem diagnosis of sudden death due to anaphylaxis can be very difficult due to the non-specific pathological findings in forensic practice. Postmortem serum tryptase has been used as an indicator of possible ante-mortem anaphylaxis. Though many previous studies have been conducted to explore the diagnostic significance of serum tryptase for lethal anaphylaxis, inconsistent results were documented. In this study, we made a retrospective study and presented a systematic review and meta-analysis that aims to summarize the diagnostic significance of postmortem serum tryptase in the deceased with and without anaphylactic shock and to calculate a cutoff value for future reference in the identification of deaths due to anaphylactic shock. A complete literature search in the PubMed, Cochrane Library, CNKI and Embase databases (published prior to March 1st, 2017) was performed. The quality of the eligible literature was evaluated according to the Newcastle-Ottawa Quality Assessment Scale (NOS), and the relevant data was extracted. The procedure of meta-analysis was performed by RevMan 5.3 software. Subgroup analysis was performed according to different causes of death. A total of nine studies with 296 patients were identified. The NOS of each included study was equal to 7. The results indicated that high concentrations of tryptase were significantly associated with anaphylactic shock when compared to the other causes of death. The weighted mean difference (WMD) was 29.53 (95% CI = 7.58-51.47, p = 0.008). Similar results were detected in the subgroup analysis when compared to deaths due to cardiovascular disease (CVD). However, no obvious elevation of tryptase in decedents with CVD compared to the other cause of death was observed (WMD = 4.42, 95% CI = -0.94-9.79). We concluded that high serum tryptase is a promising diagnostic biomarker for deaths due to anaphylactic shock, especially when it is higher than 30.4 µg/L.

CXCL1 and CXCR2 as potential markers for vital reactions in skin contusions.

Detection of the vitality of wounds is one of the most important issues in forensic practice. This study investigated mRNA and protein levels of CXCL1 and CXCR2 in skin wounds in mice and humans. Western blot analysis of CXCL1 and CXCR2 protein levels showed no difference between wounded and intact skin. However, mRNA levels demonstrated higher expression of CXCL1 and CXCR2 in contused mouse and human skin, compared with intact skin. At postmortem there were no remarkable changes in CXCL1 and CXCR2 mRNA levels in contused mouse skin. Increased mRNA expression was observed in contused mouse skin up to 96 h and 72 h after death for CXCL1 and CXCR2 respectively. In human samples of wounded skin, increased CXCL1 mRNA levels were detected up to 48 h after autopsy in all 5 cases, while increased CXCR2 mRNA levels were observed 48 h after autopsy in 4 of 5 cases. These findings suggest that the levels of CXCL1 and CXCR2 mRNA present in contused skin can be used as potential markers for a vital reaction in forensic practice.

Gut Microbiota Participates in Polystyrene Microplastics-Induced Hepatic Injuries by Modulating the Gut-Liver Axis.

Dietary pollution by polystyrene microplastics (MPs) can cause hepatic injuries and microbial dysbiosis. Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, exerts beneficial effects on the liver by modulating the gut microbiota. However, the role of microbiota in MPs-induced hepatic injuries and the protective effect of EGCG have not been clarified. Here, 5 µm MPs were orally administered to mice to induce hepatic injuries. Subsequently, antibiotic cocktail (ABX) and fecal microbial transplant (FMT) experiments were performed to investigate the underlying microbial mechanisms. Additionally, EGCG was orally administered to mice to explore its protection against MPs-induced hepatic injuries. Our results showed that MPs activated systemic and hepatic inflammation, promoted fibrosis, and altered the liver metabolome; meanwhile, MPs damaged the gut homeostasis by disturbing the gut microbiome, promoting colonic inflammation, and impairing the intestinal barrier. Notably, MPs reduced the abundance of the probiotics Akkermansia, Mucispirillum, and Faecalibaculum while increasing the pathogenic Tuzzerella. Interestingly, the elimination of gut microbiota mitigated MPs-induced colonic inflammation and intestinal barrier impairment. Moreover, ABX ameliorated MPs-induced systemic and hepatic inflammation but not fibrosis. Correspondingly, microbiota from MPs-administered mice induced colonic, systemic, and hepatic inflammation, while their profibrosis effect on the liver was not observed. Finally, EGCG elevated the abundance of probiotics and effectively repressed MPs-induced colonic inflammation. MPs-induced systemic and hepatic inflammation, fibrosis, and remodeling of the liver metabolome were also attenuated by EGCG. These findings illustrated that gut microbiota contributed to MPs-induced colonic and hepatic injuries, while EGCG could serve as a potential prevention strategy for these adverse consequences.

Obeticholic acid protects against methamphetamine-induced anxiety-like behavior by ameliorating microbiota-mediated intestinal barrier impairment.

Methamphetamine (Meth) abuse can cause severe anxiety disorder and interfere with gut homeostasis. Obeticholic acid (OCA) has emerged as a protective agent against diet-related anxiety that improves gut homeostasis. The potential for OCA to ameliorate Meth-induced anxiety, and the microbial mechanisms involved, remain obscure. Here, C57/BL6 mice were intraperitoneally injected with Meth (15 mg/kg) to induce anxiety-like behavior. 16 S rRNA sequence analysis and fecal microbiome transplantation (FMT) were used to profile the gut microbiome and evaluate its effects, respectively. Orally administered OCA was investigated for protection against Meth-induced anxiety. Results indicated that Meth mediated anxiety-like behavior, aroused hippocampal neuroinflammation through activation of the TLR4/MyD88/NF-κB pathway, weakened intestinal barrier and disturbed the gut microbiome. Specifically, abundance of anxiety-related Rikenella was increased. FMT from Meth-administrated mice also weakened intestinal barrier and elevated serum LPS, inducing hippocampal neuroinflammation and anxiety-like behavior in recipient mice. Finally, OCA pretreatment ameliorated Meth-induced impairment of gut homeostasis by reshaping the microbial composition and improving the intestinal barrier. Meth-induced anxiety-like behavior and hippocampal neuroinflammation were also ameliorated by OCA pretreatment. These preliminary findings reveal the crucial role of gut microbiota in Meth-induced anxiety-like behavior and neuroinflammation, highlighting OCA as a potential candidate for the prevention of Meth-induced anxiety.

Transcutaneous auricular vagal nerve stimulation releases extrapineal melatonin and reduces thermal hypersensitivity in Zucker diabetic fatty rats.

Type 2 diabetes (T2D) is the most common comorbidity of COVID-19, and both are related to the lack of circulating melatonin. In addition, chronic pain is a common sequela of both COVID-19 and T2D. Using a neuropathic pain model produced by sciatic nerve chronic constriction injury in Zucker diabetic fatty rats, a verified preclinical genetic T2D neuropathy animal model, this study aimed to show that transcutaneous auricular vagal nerve stimulation (taVNS) could elevate plasma melatonin concentration, upregulate the expression of melatonin receptors (MTRs) in the amygdala, and relieve peripheral neuropathic pain. Furthermore, taVNS would restore melatonin levels and relieve pain even in pinealectomized rats. On the contrary, intraperitoneally injected luzindole, a melatonin receptor antagonist, would attenuate the antinociceptive effects of taVNS. In conclusion, the mechanism of the therapeutic effect of taVNS on chronic pain involves the release of extrapineal melatonin and the positive regulation of the expression of central MTRs. This beneficial efficacy should be considered during COVID-19 rehabilitation in individuals with diabetes.

Antibiotics Attenuate Methamphetamine-Induced Hepatotoxicity by Regulating Oxidative Stress and TLR4/MyD88/Traf6 Axis.

Methamphetamine (METH) is a major psychostimulant drug of abuse worldwide, and its neurotoxicity has been studied extensively. In addition to neurotoxicity, METH can also induce hepatotoxicity. The underlying mechanism of intestinal microorganisms in METH-induced hepatotoxicity remains unclear. In this study, mice have received antibiotics intragastrically or PBS once each day for 1 week, followed by METH or saline. The antibiotics attenuated METH-induced hepatotoxicity as evidenced by histopathological observation and biochemical analysis; furthermore, they alleviated METH-induced oxidative stress. The effect of antibiotics on METH-induced hepatotoxicity was investigated using RNA-sequencing (RNA-seq). The RNA-seq results demonstrated that antibiotics could regulate 580 differentially expressed genes (DEGs), of which 319 were upregulated after METH treatment and then downregulated with antibiotic pretreatment and 237 were first downregulated after METH administration and then upregulated after antibiotic pretreatment, in addition to 11 upregulated and 13 downregulated ones simultaneously in METH and antibiotic-pretreated groups. RNA-seq analyses revealed that TLR4 is one of the hub genes. Western blot analysis indicated that antibiotics inhibited the increase of TLR4, MyD88 and Traf6 induced by METH. This research suggests that antibiotics may play an important role in preventing METH-induced liver injury by regulating oxidative stress and TLR4/MyD88/Traf6 axis, though further investigation is required.

Lactulose attenuates METH-induced neurotoxicity by alleviating the impaired autophagy, stabilizing the perturbed antioxidant system and suppressing apoptosis in rat striatum.

Methamphetamine (METH) is a widely abused psychostimulant. Lactulose is a non-absorbable sugar, which effectively decreases METH-induced neurotoxicity in rat. However, the exact mechanisms need further investigation. In this study, 5-week-old male Sprague Dawley rats received METH (15 mg/kg, 8 intraperitoneal injections, 12-h interval) or saline and received lactulose (5.3 g/kg, oral gavage, 12-h interval) or vehicle 2 days prior to the METH administration. Compared to the control group, in the METH alone group, cytoplasmic vacuolar degeneration in hepatocytes, higher levels of alanine transaminase, aspartate transaminase and ammonia, overproduction of reactive oxygen species (ROS) and increase of superoxide dismutase activity in the blood were observed. Moreover, in rat striatum, expressions of nuclear factor erythroid 2-relatted factor-2 (Nrf2) and heme oxygenase-1 were suppressed in the nucleus, although over-expression of Nrf2 were observed in cytoplasm. Over-expressions of BECN1 and LC3-II indicated initiation of autophagy, while overproduction of p62 might suggest deficient autophagic vesicle turnover and impaired autophagy. Furthermore, accumulation of p62 cloud interact with Keap1 and then aggravate cytoplasmic accumulation of Nrf2. Consistently, over-expressions of cleaved caspase 3 and poly(ADP-ribose) polymerase-1 suggested the activation of apoptosis. The pretreatment with lactulose significantly decreased rat hepatic injury, suppressed hyperammonemia and ROS generation, alleviated the impaired autophagy in striatum, rescued the antioxidant system and repressed apoptosis. Taken together, with decreased blood ammonia, lactulose pretreatment reduced METH-induced neurotoxicity through alleviating the impaired autophagy, stabilizing the perturbed antioxidant system and suppressing apoptosis in rat striatum.

IL-6 and IL-20 as potential markers for vitality of skin contusion.

The detection of vitality of wounds is very important in forensic practice. This study is performed using quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) in both mouse and human skin wounds for the application of IL-6 and IL-20 in order to differentiate intravital wounds from postmortem wounds. RT-qPCR analysis of contused mouse skin showed that increased IL-6 and IL-20 mRNA levels were found in comparison to intact skin tissues. The increased mRNA expressions of IL-6 and IL-20 were observed until 72 h after death in contused mouse skin, whereas there were no marked changes in these two cytokines in the postmortem contusion group. The alterations of IL-6 and IL-20 can also be detected in human skin wound samples. These finding suggest that mRNA levels of IL-6 and IL-20 might be used as potential markers for vital reaction.

RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study.

Detection of the vitality of wounds is essential in forensic practice. The present study used Illumina RNA-seq technology to determine gene expression profiles in contused mouse skin. In obtained high quality sequencing reads, the reads were mapped onto a reference transcriptome (Mus_musculus.GRCm38.83). The results revealed that there were 659 up-regulated and 996 down-regulated differentially expressed genes (DEGs) in contused mouse skin. The DEGs were further analyzed using the Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes databases. Genes from different functional categories and signalling pathways were enriched, including the immune system process, immune response, defense response, cytokine-cytokine receptor interaction, complement and coagulation cascades and chemokine signalling pathway. Expression patterns of 11 DEGs were verified by RT-qPCR in mice skins. In addition, alterations of five DEGs were also analyzed in postmortem human wound samples. The results were in concordance with the results of RNA-seq. These findings suggest that RNA-seq is a powerful tool to reveal DEGs as potential markers for vital reaction in terms of forensic practices.