Delayed Reaction of Radiation on the Central Nervous System and Bone System in C57BL/6J Mice.

The aim of this study was to provide a suitable mouse model of radiation-induced delayed reaction and identify potential targets for drug development related to the prevention and treatment of radiation injury. C57BL/6J mice were subjected to singular (109 cGy/min, 5 Gy*1) and fractional (109 cGy/min, 5 Gy*2) total body irradiation. The behavior and activity of mice were assessed 60 days after ionizing radiation (IR) exposure. After that, the pathological changes and mechanism of the mouse brain and femoral tissues were observed by HE, Nissl, Trap staining micro-CT scanning and RNA sequencing (RNA-Seq), and Western blot. The results show that singular or fractional IR exposure led to a decrease in spatial memory ability and activity in mice, and the cognitive and motor functions gradually recovered after singular 5 Gy IR in a time-dependent manner, while the fractional 10 Gy IR group could not recover. The decrease in bone density due to the increase in osteoclast number may be relative to the down-regulation of RUNX2, sclerostin, and beta-catenin. Meanwhile, the brain injury caused by IR exposure is mainly linked to the down-regulation of BNDF and Tau. IR exposure leads to memory impairment, reduced activity, and self-recovery, which are associated with time and dose. The mechanism of cognitive and activity damage was mainly related to oxidative stress and apoptosis induced by DNA damage. The damage caused by fractional 10 Gy TBI is relatively stable and can be used as a stable multi-organ injury model for radiation mechanism research and anti-radiation medicine screening.

Ultra-fast screening of free fatty acids in human plasma using ion mobility mass spectrometry.

Free fatty acids are involved in many metabolic regulations in the human body. In this work, an ultra-fast screening method was developed for the analysis of free fatty acids using trapped ion mobility spectrometry coupled with mass spectrometry. Thirty-three free fatty acids possessing different unsaturation degrees and different carbon chain lengths were baseline separated and characterized within milliseconds. Saturated, monounsaturated, and polyunsaturated free fatty acids showed different linearities between collision cross-section values and m/z. The establishment of correlations between structures and collision cross-section values provided additional qualitative information and made it possible to determine free fatty acids which were out of the standards pool but possessed the confirmed linearity. The gas-phase separation made the quantitative analysis reliable and repeatable at a much lower time cost than chromatographic methods. The sensitivity was comparable to and even better than the reported results. The method was validated and applied to profiling free fatty acids in human plasma. Saturated free fatty acids abundance in the fasting state was found to be lower than that in the postprandial state, while unsaturated species abundance was found higher. The method was fast and robust with minimum sample pretreatment, so it was promising in the high-throughput screening of free fatty acids.

[Pharmacology research on PXR as a potential target in screening bioactive components of Chinese material medica].

Pregnane X receptor (PXR) is key transcription factors which mainly regulate the expression of CYP3A genes. At the molecular level, PXR has been revealed the protection mechanism of the body against xenochemicals and a major mode of the drug-drug interactions. Besides playing an important role in drug metabolism and interactions, PXR and its target genes also play an important role in maintaining normal physiological function and homeostasis. Therefore, it is necessary to study the regulation of PXR and its related pharmacological effects of TCM and natural products, and to provide new clues for the new pharmacological pathway.

[Screening active components in compound danshen based on PXR-CYP3A4: an experimental study].

OBJECTIVE: To screen active components in Compound Danshen (CD) based on pregnane X receptor-cytochrome P450 3A4 (PXR-CYP3A4). METHODS: By using PXR-CYP3A stable transfection human hepatoblastoma G2 (HepG2) cell lines engineering cell strain combined reporter genes technology, active components that induce or inhibit PXR-CYP3A4 paths in CD were screened, and confirmed at the level of enzymic activities. The experiment was divided into the positive control group (RIF 10 micro mol/L), the DMSO group (DMSO 0.1%), each dose of treatment groups (ginsenoside Rc, Rf, Rb2, Rg2, F2, F1, tanshinone I , isoborneol 5, 10, 25, 50, 100, and 200 micro mol/L; each with six duplicates). Cells medium was removed 36, 48, and 60 h after treatment. The activity of CYP3A4 was then determined in the supernant and the fold induction was calculated. RESULTS: Compared with the DMSO group, the fold induction increased when ginsenoside Rc, Rf, Rb2, Rg2, F2, F1, tanshinone I , and isoborneol 50 and 100 micro mol/L was respectively intervened for 36, 48, and 60 h (P <0.05). When cells were treated with isoborneol 200 micro mol/L for 48 and 60 h,the fold induction of ginsenoside Rb2, Rg2, and F1 was significantly higher than that of the RIF group (P <0.05). Enzymic activity results showed that ginsenoside Rc, Rf, Rb2, F2, and F1 could increase the enzyme activity of CYP3A4 at 48 h (P <0.05). CONCLUSION: Ginsenoside Rc, Rf, Rb2, F2, F1, tanshinone I, and isoborneol in DC could induce CYP3A4 enzymes.

Ferroptosis Pathways: Unveiling the Neuroprotective Power of Cistache Deserticola Phenylethanoid Glycosides.

ETHNOPHARMACOLOGICAL RELEVANCE: Cistanche deserticola is a kind of parasitic plant living in the roots of desert trees. It is a rare Chinese medicine, which has the effect of tonifying kidney Yang, benefiting essence and blood and moistening the intestinal tract. Cistache deserticola phenylethanoid glycoside (PGS), an active component found in Cistanche deserticola Ma, have potential kidney tonifying, intellectual enhancing, and neuroprotective effects. Cistanche total glycoside capsule has been marketed to treat vascular dementia disease. AIM OF THE STUDY: To identify the potential renal, intellectual enhancing and neuroprotective effects of PGS and explore the exact targets and mechanisms of PGS. MATERIALS AND METHODS: This study systematically investigated the four types of pathways leading to ferroptosis through transcriptome, metabolome, ultrastructure and molecular biology techniques and explored the molecular mechanism by which multiple PGS targets and pathways synergistically exert neuroprotective effects on hypoxia. RESULTS: PGS alleviated learning and memory dysfunction and pathological injury in mice exposed to hypobaric hypoxia by attenuating hypobaric hypoxia-induced hippocampal histopathological damage, impairing blood‒brain barrier integrity, increasing oxidative stress levels, and increasing the expression of cognitive proteins. PGS reduced the formation of lipid peroxides and improved ferroptosis by upregulating the GPX-4/SCL7A311 axis and downregulating the ACSL4/LPCAT3/LOX axis. PGS also reduced ferroptosis by facilitating cellular Fe2+ efflux and regulating mitochondrial Fe2+ transport and effectively antagonized cell ferroptosis induced by erastin (a ferroptosis inducer). CONCLUSIONS: This study demonstrated the mechanism by which PGS prevents hypobaric hypoxic nerve injury through four types of ferroptosis pathways, achieved neuroprotective effects and alleviated learning and memory dysfunction in hypobaric hypoxia mice. This study provides a theoretical basis for the development and application of PGS.

[Screening of pregnane X receptor activation from ginsenosides].

In order to study effects of ginseng on the metabolism of drug belong to CYP3A4 substrate, screening of pregnane X receptor activation from ginsenosides was performed by reporter assay. Based on PXR-CYP3A stable translation cell lines, 13 ginsenosides were screened for pregnane X receptor activation by reporter assays, and RIF as the positive control. The effect of ginsenosides Rg1 onCYP3A4 mRNA expression was also investigated by RT-PCR. The PXR-CYP3A stable translation cell lines had good response to RIF, and the EC50 is 2.51 micro mol x L(-1). When the condition of final concentration was 10 micromol x L(-1), ginsenoside F2 and protopanaxatriol had moderate inductive effects on PXR. Panaxotriol, Rg2, pseudoginsenoside F11, Rg1, ginsenoside and Rb3 had inhibitory effects on PXR. Ginsenoside Rf1, Rg3, Rh2 and protopanaxdiol had no obvious effects on PXR. Rg1 down-regulated CYP3A4 mRNA expression in a concentration-dependent manner. Activation of pregnane X receptor by ginsenosides may influence the metabolism of drug belong to CYP3A4 substrate, and cause ginseng-drug interactions.

Biological effects of exposure to 2650 MHz electromagnetic radiation on the behavior, learning, and memory of mice.

BACKGROUND: With the development of communication technology, the public is paying increasing attention to whether electromagnetic radiation is harmful to health. Mobile phone communication has entered the 5G era, and there are almost no reports on electromagnetic radiation at 2650 MHz. Therefore, it is necessary to evaluate the risk of adverse effects of 5G mobile phone EMR exposure on the human brain. METHODS: Male animals were continuously exposed to 2650 MHz-EMR for 28 days with a whole-body averaged specific absorption rate (WBSAR) of 2.06 W/kg for 4 h per day. Mouse behavior was assessed using the open-field test (OFT), elevated-plus maze (EPM), and tail suspension test (TST). The Morris water maze (MWM), HE staining, and TUNEL staining were used to evaluate the spatial memory ability and pathological morphology of hippocampal dentate gyrus cells. Additionally, the expression levels of brain-derived neurotrophic factor (BDNF), aminobutyric acid (GABA), and glucocorticoid (GR) in the hippocampus were detected by western blotting and immunohistochemistry, while the corticosterone (CORT) level in serum was detected by ELISA. RESULTS: In the OFT, the total distance traveled, central distance traveled, and residence time significantly decreased in the EMR exposure group (p < .05). In EPM, the percentage of the number of times to open the arm and the percentage of time to open the arm significantly decreased in the EMR exposure group. However, in the TST, the two groups had no significant difference in the 4-min immobility time. In the MWM, the escape latency of the EMR exposure group was shorter than that of the control group, with no significant difference. Furthermore, CORT levels in serum were significantly increased in the EMR exposure group (p < .05), while the expression of BDNF and GR proteins in the hippocampus was reduced (p < .05), but there was no significant difference in GABA expression. CONCLUSIONS: Our results indicate that exposure to 2650 MHz-EMR (WBSAR: 2.06 W/kg, 28 days, 4 h per day) had no significant effect on the spatial memory ability of mice (in comparison to little effect). The exposure may be associated with anxiety-like behavior in mice but not related to depression-like behavior in mice.

Time-course effects and mechanisms of hypobaric hypoxia on nervous system in mice.

OBJECTIVE: The aim of this study was to investigate the effect of time course on neurological impairment after acute hypobaric hypoxia exposure in mice and clarify the mechanism of acclimatization, so as to provide a suitable mice model and identify potential target against hypobaric hypoxia for further drug research. METHOD: Male C57BL/6J mice were exposed to hypobaric hypoxia at a simulated altitude of 7000 m for 1, 3, and 7 days (1HH, 3HH and 7HH respectively). The behavior of the mice was evaluated by novel object recognition (NOR) and morris water maze test (MWM), then, the pathological changes of mice brain tissues were observed by H&E and Nissl staining. In addition, RNA sequencing (RNA-Seq) was performed to characterize the transcriptome signatures, and enzyme-linked immunosorbent assay (ELISA), Real-time polymerase chain reaction (RT-PCR), and western blot (WB) were used to verify the mechanisms of neurological impairment induced by hypobaric hypoxia. RESULT: The hypobaric hypoxia condition resulted in impaired learning and memory, decreased new object cognitive index, and increased escape latency to the hidden platform in mice, with significant changes seen in the 1HH and 3HH groups. Bioinformatic analysis of RNA-seq results of hippocampal tissue showed that 739 differentially expressed genes (DEGs) appeared in the 1HH group, 452 in the 3HH group, and 183 in the 7HH group compared to the control group. There were 60 key genes overlapping in three groups which represented persistent changes and closely related biological functions and regulatory mechanisms in hypobaric hypoxia-induced brain injuries. DEGs enrichment analysis showed that hypobaric hypoxia-induced brain injuries were associated with oxidative stress, inflammatory responses, and synaptic plasticity. ELISA and WB results confirmed that these responses occurred in all hypobaric hypoxic groups while attenuated in the 7HH group. VEGF-A-Notch signaling pathway was enriched by DEGs in hypobaric hypoxia groups and was validated by RT-PCR and WB. CONCLUSION: The nervous system of mice exposed to hypobaric hypoxia exhibited stress followed by gradual habituation and thus acclimatization over time, which was reflected in the biological mechanism involving inflammation, oxidative stress, and synaptic plasticity, and accompanied by activation of the VEGF-A-Notch pathway.

Single-cell transcriptomics profiling the compatibility mechanism of As2O3-indigo naturalis formula based on bone marrow stroma cells.

Compound realgar natural indigo tablet is the only oral arsenic agent widely used in acute promyelocytic leukemia (APL) treatment. However, as a therapeutic drug for diseases of the blood system, the scientific knowledge of As2O3-indigo naturalis formula compatibility has not been studied in bone marrow stromal cells (BMSCs). We chose arsenic trioxide (As2O3: A), tanshinone IIA (T) and indirubin (I) as representative active compounds of realgar, indigo naturalis, and Salvia miltiorrhiza, respectively, to evaluated the pharmaceutical mechanism and the compatibility of ATI (drug combination) using single-cell RNA sequencing (scRNA-seq). The overlapped genes associated with both disease and drug were selected in BMSCs for in-depth analysis. Results show that joint applications of ATI had the strongest therapeutic efficacy in a murine APL model. Lepr-MSCs, OLCs and BMECs were the sensitive cell groups targeted by ATI in the murine APL model. ATI could regulate the related genes of osteogenic differentiation, adipogenic differentiation, and endothelial cell migration in bone marrow mesenchymal lineage cells in murine APL model and improve normal hematopoiesis-related gene expression and poor prognosis of Lepr-MSCs, OLCs and BMECs in mice with leukemia according to scRNA-seq data. The strongest regulatory effects were found in the joint applications of ATI. ATI combination had the potential mechanism to maintain the stability of the hematopoietic microenvironment and promote hematopoiesis to assist in the treatment of APL. This study illustrated the potential mechanism of ATI in regulating BMSCs from the overall perspective of the hematopoietic microenvironment, and broadened the scientific understanding of ATI compatibility in BMSCs.

Pinelliae rhizoma alleviated acute lung injury induced by lipopolysaccharide via suppressing endoplasmic reticulum stress-mediated NLRP3 inflammasome.

Pinelliae rhizoma (PR), one kind of commonly-used Chinese herbs, is generally prescribed to treat various respiratory diseases, including acute lung injury (ALI). However, the accurate bioactive ingredients of PR and the underlying pharmacological mechanism have both not been fully elucidated. Therefore, this study aimed to identify the bioactive ingredients that could alleviate lipopolysaccharide (LPS)-induced ALI and explore the possible mechanism involved. Our results confirmed that LPS infection indeed caused acute inflammatory damage in mice lung, accompanying with the enhancement of IL-1ß contents and the activation of the NLRP3 inflammasome in lung tissue and macrophagocyte, all of which were remarkably ameliorated by PR treatment. Next, mechanistically, LPS was found to trigger endoplasmic reticulum (ER) stress and downstream cellular calcium ions (Ca2+) release via activating Bip/ATF4/CHOP signaling pathway. Like PR, 4-PBA (a specific inhibitor of ER stress) not only obviously reversed Bip/ATF4/CHOP-mediated ER stress, but also significantly attenuated LPS-induced activation of the NLRP3 inflammasome. Furthermore, the bioactive ingredients of PR, which generated the anti-inflammatory effects, were screened by metabolomics and network pharmacology. In vitro experiments showed that chrysin, dihydrocapsaicin, and 7,8-dihydroxyflavone (7,8-DHF) notably suppressed LPS-induced ER stress and following NLRP3 inflammasome activation. In conclusion, our findings suggested that PR alleviated LPS-induced ALI by inhibiting ER stress-mediated NLRP3 inflammasome activation, which is mainly relevant with these three bioactive ingredients. This study provided a theoretical basis for the clinical application of PR to treat ALI, and these bioactive ingredients of PR would be promising therapeutic drugs for the treatment of ALI.

microRNA arm-imbalance in part from complementary targets mediated decay promotes gastric cancer progression.

Strand-selection is the final step of microRNA biogenesis in which functional mature miRNAs are generated from one or both arms of precursor. The preference of strand-selection is diverse during development and tissue formation, however, its pathological effect is still unknown. Here we find that two miRNA arms from the same precursor, miR-574-5p and miR-574-3p, are inversely expressed and play exactly opposite roles in gastric cancer progression. Higher-5p with lower-3p expression pattern is significantly correlated with higher TNM stages and poor prognosis of gastric cancer patients. The increase of miR-574-5p/-3p ratio, named miR-574 arm-imbalance is partially due to the dynamic expression of their highly complementary targets in gastric carcinogenesis, moreover, the arm-imbalance of miR-574 is in turn involved and further promotes gastric cancer progression. Our results indicate that miR-574 arm-imbalance contribute to gastric cancer progression and re-modification of the miR-574-targets homeostasis may represent a promising strategy for gastric cancer therapy.

Tanshinone IIA ameliorates dextran sulfate sodium-induced inflammatory bowel disease via the pregnane X receptor.

Tanshinone IIA (Tan IIA) (C19H18O3) is one of the major active lipophilic components in a conventional Chinese medicine called danshen, and it has long been used in the People's Republic of China and other neighboring countries to treat patients suffering from inflammatory bowel disease (IBD). Previous experiments by many teams determined which mechanism of Tan IIA is relevant to the treatment of IBD associated with inflammation and the pregnane X receptor (PXR). The current study demonstrated that Tan IIA is an efficacious PXR agonist and its ability to induce CYP3A4 mRNA and protein expression was mediated by the transactivation of PXR, a known target of abrogating inflammation in IBD. Clinical symptoms in mice and histological assessment data suggested that administration of Tan IIA in mice demonstrated significant protection and showed that in DSS-induced IBD it acts in a concentration-dependent manner. PXR-silenced mice treated with Tan IIA demonstrated low protection against DSS-induced mouse IBD and exacerbated the severity of IBD compared with wild-type mice; PXR-silenced mice demonstrated the necessity for PXR in Tan IIA-mediated upregulation of xenobiotic metabolism genes. The IBD treatment effects of Tan IIA are partially due to PXR-mediated upregulation of xenobiotic metabolism and downregulation of inflammatory mediators. The novel findings reported here may contribute to the effective utilization of Tan IIA and its derivatives as a PXR ligand in the treatment of human IBD. This suggests that Tan IIA may have considerable clinical utility.

The protective effect of piperine on dextran sulfate sodium induced inflammatory bowel disease and its relation with pregnane X receptor activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Inflammatory bowel disease (IBD) is associated with chronic inflammation of the intestinal tract. Piperine (1-peperoylpiperidine), the primary lipophilic component in black pepper (Piper nigrum) and long pepper (Piper longum), has been reported to be effective for anti-inflammatory. Rencently, several ethnopharmacological purity compounds, such as baicalin and artemisinin, are reported to have potentially therapeutic role in treating IBD. In the present study, the effects of piperine on pregnane X receptor (PXR)-mediated CYP3A expression and its therapeutic role in IBD were investigated. MATERIALS AND METHODS: LS174T cells and C57BL/6J mice were treated by the piperine. Gene expressions were analyzed by real-time PCR, Western blot analysis, transient transfections assay and histological analysis. RESULTS: Data indicated that treatment of LS174T cells with piperine markedly increased both CYP3A4 and PXR mRNA and protein. Transient transfection experiments indicated that transcriptional activation of the CYP3A4 gene via piperine was PXR-dependent. Data show that pre-administration of piperine decreased clinical hallmarks of colitis in DSS-treated PXR mice as measured by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. Inflammatory mediators (CCR2, ICAM-1, IL-1ß, IL-6, IL-10, iNOS, MCP-1, and TNFα) after DSS treatment were significantly decreased in mice pretreated with piperine but corresponding conditions did not occur in mice with down-regulation of PXR by small interfering RNA (siRNA). CONCLUSION: Piperine is a potential agonist of PXR and an inducer of PXR, which may induce CYP3A4 gene expression at the mRNA and protein levels. These results establish that piperine may contribute to prevention or reduction of colonic inflammation.

Tanshinone IIA exerts protective effects in a LCA-induced cholestatic liver model associated with participation of pregnane X receptor.

Tanshinone IIA (Tan IIA) is one of the main natural active ingredients purified from Salvia miltiorrhiza radix, which has long been used in clinical practice in China to treat diseases including liver fibrosis, Alzheimer׳s disease, and cardiovascular diseases. Tan IIA has hepatoprotective properties, and is an efficacious PXR agonist. Our study was designed to observe the function and mechanism of the hepatoprotective properties of Tan IIA. HepG2 cells were used to investigate the vitrol effects of Tan IIA on PXR and CYP3A4. Gut-formed LCA is hepatotoxic, and has been implicated in the pathogenesis of cholestatic diseases. To further investigate the hepatoprotective mechanisms of Tan IIA against LCA-induced cholestasis in vivo, we choose the normal mice and siRNA-treated mice. The in vitro study demonstrated that the effect of Tan IIA on CYP3A4 was mediated by transactivation of PXR in a dose- and time-dependent manner. The in vivo experiments using PXR siRNA revealed that Tan IIA could protect against LCA-induced hepatotoxicity and cholestasis in a dose-dependent manner. These effects were partially caused by the upregulation of PXR, as well as Cyp3a11, Cyp3a13, and Mdr1, which are the enzymes responsible for LCA metabolism. This is the first report showing that the hepatoprotective effects of Tan IIA are partly mediated by PXR.

The Correlation between Chemical Composition, as Determined by UPLC-TOF-MS, and Acute Toxicity of Veratrum nigrum L. and Radix paeoniae alba.

The eighteen incompatible medicaments is an important theory in traditional Chinese medicine. The theory suggests that drugs in the eighteen incompatible medicaments can be toxic when used together. Veratrum nigrum L. and Radix paeoniae alba belong to the eighteen incompatible medicaments and have been prohibited for thousands of years. This study offers preliminary insight into the mechanism and chemical constituents responsible for the incompatibility and toxicity of these two agents. Specifically, we performed toxicology studies to identify and quantify the constituent substances of the two agents. Experiments revealed that acute toxicity increases when the dose of V. nigrum L. is higher than, or equal to, RPA. UPLC-TOF-MS analysis showed that, although the volumes of V. nigrum L. were the same, the content of some veratrum alkaloids changed significantly and had a trend toward a highly positive correlation (|r| ≥ 0.8) with toxicity. This suggests that the increased toxicity of the V. nigrum L. and RPA combination was due mainly to increased content of the special veratrum alkaloids. The cytotoxicity of veratridine in SH-SY5Y cells was decreased with increasing paeoniflorin concentrations. This study provides insight into the mechanism behind the incompatibility theory of TCM.

Artemisinin protects against dextran sulfate-sodium-induced inflammatory bowel disease, which is associated with activation of the pregnane X receptor.

Artemisinin has been used to treat malaria for centuries in the context of traditional Chinese medicine. In the present study, the effects of artemisinin on pregnane X receptor (PXR)-mediated CYP3A expression and its therapeutic role in inflammatory bowel disease were investigated. LS174T cells exposed to artemisinin at various concentrations and for different periods of time were examined with respect to the specific induction of CYP3A4 and PXR mRNA expression. Transient transfection experiments showed transcriptional activation of the CYP3A4 gene through artemisinin to be PXR-dependent. An electrophoretic-mobility shift assay (EMSA) showed that artemisinin activates the DNA-binding capacity of the PXR for the CYP3A4 element. These results indicate that the induction of CYP3A4 by artemisinin is mediated through the activation of PXR. Using animal models, it was demonstrated that artemisinin abrogates dextran sulfate sodium (DDS)-induced intestinal inflammation. Preadministration of artemisinin ameliorated the clinical hallmarks of colitis in DSS-treated mice as determined by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. Artemisinin was found to prevent or reduce the severity of colonic inflammation by inducing CYP3A expression by activation of PXR.