Objective To investigate the mechanism by which Nicotinamide adenine dinucleotide(NADH)regu-lates anti-tuberculosis drug-induced liver injury and apoptosis in mice through SIRT1/Nrf2 pathway.Methods Twenty-four six-week-old SPF male mice were randomly divided into four groups according to body weight,ADLI group[90 mg/(kg·d)Isoniazid,135 mg/(kg·d)Rifampicin,315 mg/(kg·d)Pyrazinamide were given by gavage],control group[thesame volume of saline was given by gavage as antituberculosis drug-induced liver injury(ADLI)group],NADH group(30 mg/kg NADH wasgiven by gavage on the basis of control group)and NADH intervention group(30 mg/kg NADH wasgiven by gavage on the basis of ADLI group),with sixmice in each group.They were gavaged continuously for seven days,and their seruand liver tissues were collected.The mRNA and protein expression of silence information regulator 1(SIRT1),nuclear factor erythroid 2-related factor 2(Nrf2)in SIRT1/Nrf2 pathway,apoptosis indicators B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)and caspase-3 were detected by qRT-PCR and Western blot,respectively.HE staining was performed to observe the morphology of liver tissue.The liver was weighedandthe liver index was obtained by dividingweight by body weight.The levels of glutamate aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH),which are indicators of liver injury,were detected by microplate method.Results Compared with control group,the protein and mRNA expression of SIRT1,Nrf2decreased significantly in ADLI group.Liver tissue struc-ture wasdisturbed,hepatocytes were obviously swollen,and their boundary was unclear.The weight of mice de-creased,but liver index increased.The mRNA and protein expression level of anti-apoptotic factor Bcl-2 decreased,while that of Bax and caspase-3 was raised.The level of ALT,AST and LDH were also elevated.The differences a-bove were statistically significant(P＜0.05).Compared with ADLI group,the protein and mRNA expression of SIRT1,Nrf2 were higher after NADH intervention.Liver tissue structure became clear,and hepatocytes were po-lygonal.The protein and mRNA expression of anti-apoptosis factor Bcl-2 was elevated and while that of Bax and caspase-3 was lower.The weight of mice increased and liver index decreased.The expression of ALT,AST and LDH decreased.The differences above were statistically significant(P＜0.05).Conclusion NADH may allevi-ate anti-tuberculosis drug-induced liver injury and apoptosis in mice by regulating SIRT1/Nrf2 pathway.

Objective: To isolate and identify Prevotella nigrescens (P.nigrescens) in gingival crevicular fluid of patients with chronic periodontitis and to analyze its tumor-promoting role in esophageal squamous cell carcinoma (ESCC). Methods: Samples of gingival crevicular fluid were collected from patients with chronic periodontitis and cultured on GAM agar medium under anaerobic conditions. Black colonies on GAM agar plates were picked for subculture, and then the bacteria were isolated and purified. Bacterial identification was conducted using Gram staining and 16S rDNA sequencing analysis. The isolated bacterial species was used to infect ESCC NE6-T cells and the changes in biological characteristics of the infected cells were evaluated. Results: Multiple bacterial colonies were observed after anaerobic culturing of gingival crevicular fluid samples for 120 h on GAM agar plates. A single bacterial colony with pure black and smooth appearance was obtained from grey black bacterial colonies with streak method. It was a Gram-negative bacterium with bead-like shape. It showed 99.78% 16S rDNA sequence identity with P. nigrescens F0103 and was named P. nigrescens LY01. P. nigrescens LY01 infection promoted the in vitro proliferation, migration and invasion of NE6-T cells and the growth of subcutaneous xenograft in nude mice. In addition, it could also induce the upregulation of Ki67 and activation of p-STAT3. Conclusions P. nigrescens inhabiting in gingival sulcus might promote the progression of ESCC in human with chronic periodontitis.

In this study,we developed a novel on-line solid phase extraction (SPE)-ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS)-based analytical method for simulta-neously quantifying 12 illicit drugs and metabolites (methamphetamine,amphetamine,morphine,co-deine,6-monoacetylmorphine,benzoylecgonine,3,4-methylenedioxymethamphetamine,3,4-methylenedioxyamphetamine,cocaine,ketamine,norketamine,and methcathinone) and cotinine(COT) in wastewater samples.The analysis was performed by loading 2 mL of the sample onto an Oasis hydrophilic-lipophilic balance cartridge and using a cleanup step (5％ methanol) to eliminate interference with a total run time of 13 min.The isotope-labeled internal standard method was used to quantify the target substances and correct for unavoidable losses and matrix effects during the on-line SPE process.Typical analytical characteristics used for method validation were sensitivity,linearity,precision,repeatability,recovery,and matrix effects.The limit of detection (LOD) and limit of quantification (LOQ)of each target were set at 0.20 ng/L and 0.50 ng/L,respectively.The linearity was between 0.5 ng/L and 250 ng/L,except for that of COT.The intra-and inter-day precisions were ＜10.45％ and 25.64％,respec-tively,and the relative recovery ranged from 83.74％ to 162.26％.The method was used to analyze various wastewater samples from 33 cities in China,and the results were compared with the experimental re-suits of identical samples analyzed using off-line SPE.The difference rate was between 19.91％and-20.44％,and the error range could be considered acceptable.These findings showed that on-line SPE is a suitable alternative to off-line SPE for the analysis of illicit drugs in samples.

OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*