OBJECTIVE: To explore the therapeutic effects and underlying mechanisms of Dendrobium officinale (Tiepi Shihu) extract (DOE) on insomnia. METHODS: Forty-two male Sprague-Dawley rats were randomly divided into 6 groups (n=7 per group): normal control, model control, melatonin (MT, 40 mg/kg), and 3-dose DOE (0.25, 0.50, and 1.00 g/kg) groups. Rats were raised in a strong-light (10,000 LUX) and -noise (>80 db) environment (12 h/d) for 16 weeks to induce insomnia, and from week 10 to week 16, MT and DOE were correspondingly administered to rats. The behavior tests including sodium pentobarbital-induced sleep experiment, sucrose preference test, and autonomous activity test were used to evaluate changes in sleep and emotions of rats. The metabolic-related indicators such as blood pressure, blood viscosity, blood glucose, and uric acid in rats were measured. The pathological changes in the cornu ammonis 1 (CA1) region of rat brain were evaluated using hematoxylin and eosin staining and Nissl staining. Additionally, the sleep-related factors gamma-aminobutyric acid (GABA), glutamate (GA), 5-hydroxytryptamine (5-HT), and interleukin-6 (IL-6) were measured using enzyme linked immunosorbent assay. Finally, we screened potential sleep-improving receptors of DOE using polymerase chain reaction (PCR) array and validated the results with quantitative PCR and immunohistochemistry. RESULTS: DOE significantly improved rats' sleep and mood, increased the sodium pentobarbital-induced sleep time and sucrose preference index, and reduced autonomic activity times (P<0.05 or P<0.01). DOE also had a good effect on metabolic abnormalities, significantly reducing triglyceride, blood glucose, blood pressure, and blood viscosity indicators (P<0.05 or P<0.01). DOE significantly increased the GABA content in hippocampus and reduced the GA/GABA ratio and IL-6 level (P<0.05 or P<0.01). In addition, DOE improved the pathological changes such as the disorder of cell arrangement in the hippocampus and the decrease of Nissel bodies. Seven differential genes were screened by PCR array, and the GABA_A receptors (Gabra5, Gabra6, Gabrq) were selected for verification. The results showed that DOE could up-regulate their expressions (P<0.05 or P<0.01). CONCLUSION DOE demonstrated remarkable potential for improving insomnia, which may be through regulating GABA_A receptors expressions and GA/GABA ratio.
Animals ; Dendrobium/chemistry* ; Rats, Sprague-Dawley ; Male ; Sleep Initiation and Maintenance Disorders/blood* ; Plant Extracts/therapeutic use* ; Receptors, GABA-A/metabolism* ; Noise/adverse effects* ; Light/adverse effects* ; gamma-Aminobutyric Acid/metabolism* ; Sleep/drug effects* ; Rats ; Receptors, GABA/metabolism*

Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the "bubble" structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to pyruvate kinase M2 (PKM2)-dependent conventional caspase-3/gasdermin E (GSDME) cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knockdown significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-programmed death-1 (anti-PD-1). In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.
Pyroptosis/immunology* ; Humans ; Endoplasmic Reticulum/immunology* ; Animals ; Nogo Proteins/antagonists & inhibitors* ; Mice ; Cell Line, Tumor ; Xanthones/pharmacology* ; Neoplasms/pathology* ; Mice, Nude

Cryopreservation is the primary technique for in vitro preservation of allogeneic tissue. However, its success is often hindered by factors such as low temperature, ischemia, and hypoxia. This study investigated the potential of Sini Decoction, known for its antioxidant and anti-apoptotic properties, to reduce cryopreservation-induced injury in rats' sciatic nerves. Sini Decoction was prepared according to the Chinese Pharmacopoeia, and its cytotoxicity on Rsc96 cells was assessed by using the CCK-8 method. Sini Decoction at concentrations of 4, 8, and 16 mg·mL~(-1), termed as low-(SL), medium-(SM), and high-(SH) doses group, was used for cryopreservation of rats' sciatic nerves. A normal control(NC) group and a fresh nerve control(fresh) group were set. Flow cytometry and TUNEL staining were used to detect the apoptosis of neural tissue cells after cryopreservation. Western blot was used to detect the expression of apoptosis-related proteins(Bcl-2, Bax, caspase-3, and caspase-8) and nerve regeneration proteins(NGF and BDNF) in vitro after cryopreservation. Oxidative damage of neural tissue after cryopreservation was evaluated by measuring levels of GSH, SOD, MDA, ROS, and ATP. Cryopreserved nerves were then used for allogeneic transplantation. One week after transplantation, CD4~+ and CD8~+ fluorescent double staining assessed inflammatory cell invasion in the transplanted nerve segment, and ELISA evaluated the expression of serum inflammatory factors(IL-1, IFN-γ, and TNF-α) in recipients. Twenty weeks after transplantation, electrophysiology and NF200 neurofilament staining were used to evaluate nerve regeneration. RESULTS:: showed that Sini Decoction at concentrations of below 32 mg·mL~(-1) exhibited no cytotoxicity to Rsc96 cells. During in vitro nerve cryopreservation, Sini Decoction significantly reduced cell apoptosis, ROS, and MDA production compared to the NC group. In the SH group, the protein expression of NGF and BDNF in vitro, as well as ATP, SOD, and GSH production, were significantly increased. In the rejection reaction one week after transplantation, compared to the fresh nerve transplantation group, the SL and SM groups showed reduced CD4~+ and CD8~+ T cell invasion in the transplanted nerve segment and down-regulated IL-1, IFN-γ, and TNF-α expression in recipient serum. Twenty weeks after transplantation, the electrophysiological test results of CMAP, NCV, and NF200 neurofilament protein fluorescent staining in the SM and SH groups were superior to those in the NC and fresh groups. These findings indicate that Sini Decoction offers protective benefits in the cryopreservation of rats' sciatic nerves and holds significant potential for the in vitro preservation of tissue and organs.
Animals ; Apoptosis/drug effects* ; Rats ; Oxidative Stress/drug effects* ; Sciatic Nerve/cytology* ; Cryopreservation ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Rats, Sprague-Dawley ; Protective Agents/pharmacology*

This study aims to explore the pharmacodynamic material basis of Jinbei Oral Liquid(JBOL) against idiopathic pulmonary fibrosis(IPF) based on serum pharmacochemistry and network pharmacology. The ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(UHPLC-Q-TOF-MS/MS) technology was employed to analyze and identify the components absorbed into rat blood after oral administration of JBOL. Combined with network pharmacology, the study explored the pharmacodynamic material basis and potential mechanism of JBOL against IPF through protein-protein interaction(PPI) network construction, "component-target-pathway" analysis, Gene Ontology(GO) functional enrichment, and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. First, a total of 114 compounds were rapidly identified in JBOL extract according to the exact relative molecular mass, fragment ions, and other information of the compounds with the use of reference substances and a self-built compound database. Second, on this basis, 70 prototype components in blood were recognized by comparing blank serum with drug-containing serum samples, including 28 flavonoids, 25 organic acids, 4 saponins, 4 alkaloids, and 9 others. Finally, using these components absorbed into blood as candidates, the study obtained 212 potential targets of JBOL against IPF. The anti-IPF mechanism might involve the action of active ingredients such as glycyrrhetinic acid, cryptotanshinone, salvianolic acid B, and forsythoside A on core targets like AKT1, TNF, and ALB and thereby the regulation of multiple signaling pathways including PI3K/AKT, HIF-1, and TNF. In conclusion, JBOL exerts the anti-IPF effect through multiple components, targets, and pathways. The results would provide a reference for further study on pharmacodynamic material basis and pharmacological mechanism of JBOL.
Drugs, Chinese Herbal/pharmacokinetics* ; Animals ; Tandem Mass Spectrometry ; Network Pharmacology ; Rats ; Chromatography, High Pressure Liquid ; Rats, Sprague-Dawley ; Male ; Idiopathic Pulmonary Fibrosis/metabolism* ; Humans ; Administration, Oral ; Protein Interaction Maps/drug effects* ; Signal Transduction/drug effects*

As the world's second largest vector of pathogens,ticks can spread a variety of pathogens by sucking the host's blood.Ticks not only threaten human life and health,but also cause great economic losses in animal husbandry.Artificial breeding of ticks can provide a stable environment for the growth and reproduction of ticks,thereby generating sufficient exper-imental materials for understanding ticks'biological characteristics,studying tick-borne pathogens,and developing anti-tick drugs and vaccines.Current methods of breeding ticks in the laboratory can be roughly divided into two categories:breeding methods using host animals or artificial membranes.The selection of breeding method must be comprehensively considered,ac-cording to tick types,blood-sucking habits,living environments,and other aspects.The development processes of the two methods,and their respective advantages and disadvantages,are described and discussed,to assist laboratories in artificial breeding of ticks.

Arsenic is a semi-metal,and lipid-soluble arsenic compounds are one of the widespread forms in the environment and food chain,but there is a lack of standards for lipid-soluble arsenic compounds,which is one of the bottlenecks in the current analytical detection and toxicological studies of organic arsenic.In this study,four saturated arsenic-containing hydrocarbons,AsHC 318,AsHC 332,AsHC 346,and AsHC 374(The number is relative molecular mass),were successfully synthesized in three steps by using dimethylarsinic acid,potassium iodide,sodium hydroxide,and four brominated alkanes(1-Bromotetradecane,1-bromopentadecane,1-bromohexadecane,and 1-bromooctadecane)as raw materials.The structures of these four saturated arsenic-containing hydrocarbons were characterized by proton nuclear magnetic resonance(1H NMR)spectroscopy,13C nuclear magnetic resonance(13C NMR)spectroscopy,and high-resolution mass spectrometry(HR-MS).The yields of the method were 8%-10%,and the synthesized compounds could be used in subsequent toxicity evaluation experiments to assess the toxic effects and mechanisms of action of arsenic-containing hydrocarbons.This study provided an effective method for synthesis of arsenic-containing hydrocarbons,enriching the synthesis methods of arsenic-containing hydrocarbons,and provided raw materials for the subsequent toxicological studies of arsenic-containing hydrocarbons.

As the world's second largest vector of pathogens,ticks can spread a variety of pathogens by sucking the host's blood.Ticks not only threaten human life and health,but also cause great economic losses in animal husbandry.Artificial breeding of ticks can provide a stable environment for the growth and reproduction of ticks,thereby generating sufficient exper-imental materials for understanding ticks'biological characteristics,studying tick-borne pathogens,and developing anti-tick drugs and vaccines.Current methods of breeding ticks in the laboratory can be roughly divided into two categories:breeding methods using host animals or artificial membranes.The selection of breeding method must be comprehensively considered,ac-cording to tick types,blood-sucking habits,living environments,and other aspects.The development processes of the two methods,and their respective advantages and disadvantages,are described and discussed,to assist laboratories in artificial breeding of ticks.

Objective To investigate the effect of the combination of Hyssop and White Peony on the nuclear factor E2-related factor-2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway induced by the combination of NG-nitro-L-arginine methyl ester,hydrochloride(L-NAME)and Aconite in mice with hypertensive kidney injury and its therapeutic mechanism.Methods A total of 80 KM mice were randomly divided into blank group,model group and experimental A,B,C,D,E and F groups,with 10 mice per group.Except for the blank group,the remaining 7 groups were given aconite decoction+L-NAME by gavage to prepare a hypertensive kidney injury model.Experimental A,B,C,D,E,and F groups were given 6.83 g·kg-1·d-1 of different proportions of Hyssop-White Peony solution in different proportions with 5∶0,4∶1,3∶2,2∶3,1∶4,0∶5,respectively.Blank and model groups were given an equal volume of distilled water by gavage.All 8 groups of mice were intervened for 6 weeks.The blood pressure changes of mice in each group were compared;the levels of blood urea nitrogen(BUN)in serum and superoxide dismutase(SOD)in kidney tissue were detected by kit;and the expression of Nrf2 and HO-1 in kidney tissue was detected by immunohistochemistry.Results The systolic blood pressure levels of blank group,model group and experimental C,D groups were(99.56±4.13),(140.11±7.33),(106.23±8.41)and(105.97±6.43)mmHg;the blood urea nitrogen contents were(172.00±15.90),(352.64±17.23),(201.76±12.14)and(192.72±12.77)μg·mL-1;BUN contents were(232.14±8.58),(165.44±6.17),(205.06±5.34)and(182.94±9.91)μg·mL-1;the expression levels(optical density)of Nrf2 were 2.06±0.14,2.25±0.22,2.63±0.22 and 2.83±0.21;the expression levels(optical density)of HO-1 were 2.18±0.12,2.25±0.10,2.64±0.16 and 2.65±0.28,respectively.The above indexes in experimental C and D groups were statistically significant compared with those of the model group(all P＜0.01).Conclusion Hyssop and White Peony can enhance the ability of L-NAME and Aconite to resist oxidative stress in mice with hypertensive kidney injury induced by the combination of Nrf2/HO-1 pathway,and inhibit the process of apoptosis of kidney cells caused by kidney injury caused by hypertension,thereby exerting its protective effect on the kidney.

Objective:To investigate the relationship between the virulence and the carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection,and to identify carbapenem-resistant and hypervirulent Klebsiella pneumoniae(CR-HVKP)strains.Methods:A total of 192 Klebsiella pneumoniae strains were isolated from blood culture of patients with bloodstream infections from 2016 to 2019,of which 96 isolates were carbapenem-resistant Klebsiella pneumoniae(CRKP)and 96 were carbapenem-sensitive Klebsiella pneumoniae(CSKP).The drug susceptibility was detected by VITEK-2 automatic microbial analyzer;carbapenemase genes,virulence genes and capsule typing were detected by polymerase chain reaction;the high viscosity phenotype of strains was detected by string test,and the genome characteristics of CR-HVKP were detected by whole genome sequencing.Serum killing and biofilm formation test were used to further verify the virulence of CR-HVKP.Results:There were significant differences in drug resistance to common antibiotics,except for minocycline between CSKP and CRKP isolates(all P<0.05).92 out of 96 CRKP isolates carried carbapenemase genes,mainly blaKPC-2.The string tests were positive in 4 isolates of CRKP and 36 isolates of CSKP(P<0.05).The detection rates of virulence genes Kfu,aerobictin,iutA,ybtS,rmpA,magA,allS,and capsule antigen K1 and K2 in CSKP group were significantly higher than those in CRKP group(all P<0.05).One HVKP strain was detected in the CRKP group(CR-HVKP)and 36 HVKP was detected in the CSKP group(P<0.05).The CR-HVKP strain belonged to the MLST412,serotype K57,expressed iutA,entB,mrkD,fimH,and rmpA virulence genes,and showed strong biofilm formation and significantly increased serum resistance.Whole genome sequencing results showed that this CR-HVKP isolate carried blaSHV-145,blaTEM-1,blaCTX-M-3,fosA6,oqxA5,oqxB26,and aac(3)-Ⅱd resistance genes,accompanied by abnormalities in outer membrane protein K(OmpK)35 and OmpK36.Conclusions:The drug resistance of CRKP is significantly higher than that of CSKP,while CRKP carrying fewer virulence genes in both number and types compared to CSKP.A new MLST type of carbapenem-resistant and hypervirulent Klebsiella pneumoniae strain has been detected,which requires clinical awareness and epidemiological monitoring.

Amorphous solid dispersion (ASD) is one of the most effective formulation approaches to enhance the water solubility and oral bioavailability of poorly water-soluble drugs. However, maintenance of physical stability of amorphous drug is one of the main challenges in the development of ASD. Crystallization is a process of nucleation and crystal growth. The nucleation is the key factor that influences the physical stability of the ASD. However, a theoretical framework to describe the way to inhibit the nucleation of amorphous drug is not yet available. We reviewed the methods and theories of nucleation for amorphous drug. Meanwhile, we also summarized the research progress on the mechanism of additives influence on nucleation and environmental factors on nucleation. This review aims to enhance the better understanding mechanism of nucleation of amorphous drug and controlling over the crystal nucleation during the ASD formulation development.