Psoraleae Fructus (PF) is a well-known traditional herbal medicine in China, and it is widely used for osteoporosis, vitiligo, and other diseases in clinical settings. However, liver injury caused by PF and its preparations has been frequently reported in recent years. Our previous studies have demonstrated that PF could cause idiosyncratic drug-induced liver injury (IDILI), but the mechanism underlying its hepatotoxicity remains unclear. This paper reports that bavachin isolated from PF enhances the specific stimuli-induced activation of the NLRP3 inflammasome and leads to hepatotoxicity. Bavachin boosts the secretion of IL-1β and caspase-1 caused by ATP or nigericin but not those induced by poly(I:C), monosodium urate crystal, or intracellular lipopolysaccharide. Bavachin does not affect AIM2 or NLRC4 inflammasome activation. Mechanistically, bavachin specifically increases the production of nigericin-induced mitochondrial reactive oxygen species among the most important upstream events in the activation of the NLRP3 inflammasome. Bavachin increases the levels of aspartate transaminase and alanine aminotransferase in serum and hepatocyte injury accompanied by the secretion of IL-1β via a mouse model of lipopolysaccharide-mediated susceptibility to IDILI. These results suggest that bavachin specifically enhances the ATP- or nigericin-induced activation of the NLRP3 inflammasome. Bavachin also potentially contributes to PF-induced idiosyncratic hepatotoxicity. Moreover, bavachin and PF should be evaded among patients with diseases linked to the ATP- or nigericin-mediated activation of the NLRP3 inflammasome, which may be a dangerous factor for liver injury.
Adenosine Triphosphate ; Animals ; Chemical and Drug Induced Liver Injury/etiology* ; Flavonoids ; Humans ; Inflammasomes ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein ; Nigericin

Objective: To analyze the molecular characteristics of Listeria monocytogenes strains from ready-to eat food in China. Methods: A total of 239 Listeria monocytogenes strains isolated from ready-to-eat food in 2017, all strains underwent whole-genome sequencing (WGS) , and comparisons uncovered population structure derived from lineages, clonal complex, serogroups, antimicrobial susceptibility and virulence, which were inferred in silico from the WGS data. Core genome multilocus sequence typing was used to subtype isolates. Results: All strains were categorized into three different lineages, lineage Ⅱ was the predominant types in food, and IIa was the main serogroups. CC8, CC101 and CC87 were the first three prevalent CCs among 23 detected CCs, accounting for 49.4%. Only 4.6% (11 isolates) of tested strains harbored antibiotic resistance genes, which were mostly trimethoprim genes (7 isolates, 2.9%). All strains were positive for LIPI-1, and only a part of strains harbored LIPI-3 and LIPI-4, accounting for 13.8% (33 isolates) and 14.2% (34 isolates), respectively. ST619 carried both LIPI-3 and LIPI-4. 51.5% (123 isolates) of strains carried SSI-1, and all CC121 strains harbored SSI-2. Different lineages, serogroups and CCs can be separated obviously through cgMLST analysis, and 24 sublineages were highly concordant with CCs. Conclusion Ⅱa was the main serogroups in ready-to-eat food isolates in China; CC8, CC101 and CC87 were the prevalent CCs, and CC87 isolates was hypervirulent isolates, cgMLST method can be adopted for prospective foodborne disease surveillance and outbreaks detection.

Objective To investigate the changes of brain function in moderate and high myopia patients using fractional amplitude of low frequency fluctuation (fALFF),and discuss the correlation between brain function changes and clinical data of patients with myopia.Methods Totally 21 moderate and high myopia patients (myopia group),and 21 healthy volunteers (normal control group) who were matched with myopia patients in age and gender,were selected to take rs-fMRI examination.The difference of fALFF of brain functional activity in patients with myopia and normal controls was compared,and the correlation between the changes of fALFF and clinical data of patients with myopia was analyzed,Results Compared with normal control group,the fALFF values of myopia group in the region of the left inferior frontal gyrus,putamen and right inferior frontal gyrus,putamen and insula were significantly lower (all P ＜ 0.05,AlphaSim corrected).However,in bilateral cingulate gyrus,bilateral anterior cingulate gyrus,left postcentral gyrus,left superior parietal lobule and region,fALFF values were increased (all P ＜ 0.05,AlphaSim corrected).Conclusion Patients with myopia are accompanied by abnormal neuronal activity in many brain areas,which may reflect the dysfunction of language understanding and attention control in myopic patients.

Objective To silence the beclin1 gene expression by using RNA interference technology in AR42J rat pancreatic acinar cells,and build a stable AR42J line silencing beclin1.Methods Three kinds of shRNA targeting rat beclin1 mRNA and negative control shRNA were designed and synthesized,and were inserted into the plasmids GV112,respectively.The recombinant plasmids were named as p-sh-Beclin1-1,psh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC.Lipo3000 was used to transfect the recombinant plasmid into AR42J cells,the expression of beclin1 mRNA were detected by RT-PCR to screen for the most efficient silencing plasmid,and then it was packaged into lentiviral (LV).AR42J cells were infected with LV and screened by puromycin.Beclin1 mRNA and protein expression was determined by RT-PCR and Western blot.Results The recombinant plasmid was confirmed by agarose gel electrophoresis and sequencing showed that shRNA sequences were in line with expectations.The beclin1 mRNA inhibition rates of AR42J cells after p-sh-Beclin1-1,p-sh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC transfection were (17.8 ± 4.0) ％,(30.6 ± 2.8) ％,(45.8 ± 7.7) ％,(7.0 ± 11.8) ％,respectively.The inhibition rates of three p-sh-Beclin1 transfection cells were significantly higher than that in non-transfection cells,and the difference was statistically significant (P ＜ 0.05).While the inhibition rate of p-shRNA-NC transfection cells was not significantly different from that of non-transfection cells,p-sh-Beclin 1-3 with highest rate of inhibition was packaged by LV,and infected AR42J cells,then puromycin was applied to screen,inhibition rate of beclin mRNA expression in LV infection cells was (86.1 ± 1.2) ％,and the protein expression inhibition rate was (87.9 ± 2.8) ％,and the difference between infection and non-infection groups was statistically significant (P ＜ 0.05).Conclusions The stable AR42J line silencing beclin1 is successfully established,which can provide a new cell model for future research of the role of beclin1 in the pathogenesis of acute pancreatitis.

Objective To explore the expression of CD44v8 protein in human bladder and urothelial carcinoma ,as well as the value in diagnosis of human bladder and urothelial carcinoma .Methods RT‐PCR was used to analysis the expression of CD44v8 protein in 75 patients with bladder and urothelial carcinoma in the pathological stage and clinical stage and 20 subjects of normal bladder mucosa were collected as control .Results CD44v8 protein was negative in all normal bladder and urothelial mucosa ,the copy number was less than 1 × 102 copy/mL ;26 cases of bladder and urothelial carcinoma was positive ,and the positive rate of CD44v8 was 34 .7% ,and Ct values were less than 35 and copy number was greater than 1 × 104 copy/mL .Positive rate was correla‐ted with high pathological grades and TNM stages ,but no significant difference was observed in recurrence of tumor .Conclusion CD44v8 could be useful indicator for the assessment of pathological grades and TNM stages of bladder and urothelial carcinoma .

Objective To establish a stable AR42J cell line expressing EGFP LC3.Methods The EGFP LC3 overexpressed Lentivirus was constructed and transfected into pancreatic acinar cells (AR42J) of rats.The rats with Lentiviral EGFP transfection were treated as negative control.The transfection efficiency was detected by inverted fluorescence microscope and flow cytometry.The EGFP LC3 protein expression in the stable cell lines were analyzed by Western blot.The cells were treated with thapsigargin to establish endoplasmic reticulum stress model,and the LC3,PERK protein expressions were detected by Western blot.Results The transfection efficiency of Lentiviral EGFP LC3 of AR42J cell was ＞ 85％,which could achieve stable passage.The expression of LC3 mRNA of AR42J cells transfected with Lentiviral EGFP LC3 was 9.14 ±0.32 folds higher than that of negative control,which had no expression of LC3 protein,only EGFP expression.However,compared with non-transfection group,the LC3 mRNA expression in EGFP group was not significantly different.Conclusions A pancreatic acinar cell line (AR42J) of rat stably expressing EGFP LC3 protein is successfully constructed.And it may provide a new model for further research of the relationship between acute pancreatitis and autophagy.

Objective To investigate the changes and significance of autophagy in rats with experimental acute necrosis pancreatitis (ANP).Methods According to method of random number,18 rats were randomly divided into control group,ANP group,ANP+rapamycin (RAP) group.The ANP rat model was established by intraperitoneal injection of 20％ L-arginine.The rats of ANP+RAP group were intraperitoneal injected with RAP 1.2 mg/kg at 30 minutes before modeling.The rats of control group were intraperitoneal injected with 0.9％ NaCl solution.The blood was drawed from the hearts nine hours after modeling for subsequent experiments.Serum levels of trypsinogen activation peptide (TAP),interleukin (IL-1),IL-6 and tumor necrosis factor (TNF) α were measured with enzyme-linked immunosorbent assay.The pancreatic tissues were pathologically scored.Autophagy-related structures in rat pancreatic acinar cells were observed by transmition electron microscopy.The expression of autophagy marker microtuble assciated protein 1 light chain 3 (LC3)-Ⅱ and Beclin-1 at mRNA and protein level were measured by quantitative real-time polymerase chain reaction (qRT-PCR),Western bloting and immunohistochemistry.The single factor analysis of variance was used for mean comparison among groups.Results A rat model of ANP was successfully established.Histopathological score of pancreas acinar cell necrosis of ANP+RAP group (2.19±1.38) was higher than that of ANP group (0.97±0.68),and the difference was statistically significant(F=33.75,P＜0.05).The results of Western blotting indicated that the protein expression of LC3-Ⅱ and Beclin-1 in ANP group (35.25±2.68 and 49.40±5.28)were higher than those in control group (1.54±0.16 and 0.78±0.06),furthermore the expressions in ANP+RAP group(123.53±3.21 and 76.41±3.80) were higher than those in ANP group,and the differences were statistically significant(F=2 045.54,326.87,both P＜0.01).Immunohistochemistry results also indicated that the LC3Ⅱ and Beclin-1 expression at protein level of ANP+RAP group (7 570.63±4 357.67 and 3 418.09±2 035.78) were higher than those of ANP group (1 926.53±1 414.44 and 536.11±403.10),and the differences were statistically significant (F=39.83,41.58,both P＜0.01).The expression of Beclin-1 at mRNA level of ANP group (107.12±29.10) was statistically higher than that of control group(7.01 ±3.39),and the difference was statistically significant (F=3.61,P＜0.05),but the expression of ANP+RAP group (97.63 ± 65.38)was no significant difference compared with ANP group.However,the expression of LC3-Ⅱ at mRNA level of ANP+ RAP group (4.37 ± 1.67) was statistically higher than that of ANP group (1.76 ± 1.59),and the difference was statistically significant(F=16.10,P＜0.05),but the expression of ANP group was no significant difference compared with control group (1.51 ±0.95).The result of electron microscopy showed that autophagy related structures increased in ANP group compared with that of control group,which of ANP+RAP group was more.The serum levels of TAP,IL-1 and IL-6 of ANP + RAP group were (36.47 ± 1.71) pmol/L,(122.88± 26.67) pg/mL and (107.39±13.95) pg/mL,which were all higher than those of ANP group ((25.63 ± 6.05) pmol/L,(98.06 ±9.29) pg/mL and (86.16± 7.20) pg/mL),and the differences were statistically significant (F=116.71,50.45,79.67; all P＜0.01).There was no significant difference in TNFα between ANP+ RAP group ((140.80±60.82) pg/mL) and ANP group ((105.23±6.95) pg/mL,F=14.76,P＞0.05).Conclusions Autophagy increased in rats with ANP.Promoting autophagy could significantly activate trypsinogen,aggravate pancreatic injury and increase inflammation reaction,which indicated that autophagy might involve in the pathogenesis of ANP through trypsinogen activation.

Background:Damage of intestinal mucosal barrier is a key factor in the development and progress of acute pancreatitis(AP),and is closely related with the prognosis of the disease. Aims:To investigate the protective effect and possible mechanism of mucoprotective agent teprenone on intestinal mucosal barrier in rats with experimental AP. Methods:Forty-five adult male Sprague-Dawley rats were randomly divided into normal control group(n = 5),AP model group(n = 20)and teprenone treated group(n = 20). AP model was established by subcutaneous injection of cerulein at abdominal wall. Rats in treated group were intervened with teprenone intragastrically before and after model establishment. ELISA was used for measurement of serum interleukin-1(IL-1),IL-6,tumor necrosis factor-α(TNF-α)and amylase;histopathological and ultrastructural changes of small intestinal mucosa were observed by light microscope and transmission electron microscope;Western blotting was used to detect the expressions of tight junction protein occludin and ZO-1. Results:Serum levels of IL-1,IL-6,TNF-α and amylase in AP model group were significantly higher than those in normal control group(P < 0. 05),accompanied by necrosis and exfoliation of small intestinal villus,widening of intercellular tight junctions and downregulation of occludin and ZO-1 expression. While in teprenone treated group,serum levels of proinflammatory cytokines and amylase were significantly decreased as compared with AP model group(P < 0. 05),the villus of small intestine remained intact,and dense tight junctions were observed. Expressions of occludin and ZO-1 in teprenone treated group were upregulated. Conclusions:Teprenone may protect against intestinal mucosal barrier injury in AP model rats by upregulating tight junction protein expression.

Objective To observe the changes of zymophagy during experimental acute pancreatitis (AP) induced by caerulein.Methods Pancreatic acinar cell line AR42J cells were cultured in 6-well plates till 90％ confluent and then divided into AP group and control group.Caerulein (1 × 10-8 mol/L) was added into AP group to establish AP cell model,and 1640 cell culture medium was added into control group.After caerulein treated for one,four,six,eight,12 and 24 hours,cells and cell culture supernatant were collected.The levels of cytokine interleukin (IL)-1,tumor necrosis factor (TNF)α,trypsinogen activation (TAP) and amylase were measured with enzyme-linked immunosorbent assay (ELISA) method.The expression of LC3 and Beclin1 at mRNA of each group were detected by reverse transcription-polymerase chain reaction (RT-PCR).The LC3B protein level of each group were detected by Western blotting.The changes of autophagosome and zymophagosome were observed by transmission electron microscopy.The difference between AP group and control group was analyzed by analysis of variance.Results The level of IL-1,TNFα,amylase and TAP in cell culture supernatant of control group was (18.83±7.10) pg/mL,(14.20±3.79) pg/mL,(10.03±2.85) U/L and (39.48±8.62) pg/mL,respectively.Those of AP group significantly increased at first hour ((62.13±11.25) pg/mL,F=3.32,P＜0.01 ; (30.98±7.11) pg/mL,F=3.05,P＜0.05; (25.06±6.82) U/L,F=2.90,P＜0.05 and (128.51± 18.30) pg/mL),F=2.62,P＜0.01,at fourth or sixth hour reached peak (IL-1 at fourth hour:(71.96± 15.82) pg/mL,F=7.25,P＜0.01;TNFα at sixth hour:(39.92±8.94) pg/mL,F=4.93,P＜0.05; amylase at fourth hour:(28.83 ± 8.31) U/L,F=2.06,P＜0.05; TAP at fourth hour:(146.29± 29.36) pg/mL,F=0.14,P＜0.01) and then gradually decreased.At fourth and sixth hour,the expression of LC3 at mRNA level in AP group was 3.18±0.82,1.71±0.14,respectively,while the expression of Beclin-1 rnRNA at first,fourth hour was 2.44±0.34 and 4.13±0.30,all of them were significantly increased compared with those of control group (0.21±0.04 and 0.30±0.08,LC3 mRNA F=0.79、0.06; Beclin mRNA F=2.31、0.36,all P＜ 0.05).There were no significant differences at other time points.The numbers of autophagosome and zymophagosome of AP group were significantly higher than those of control group under transmission electron microscopy.Conclusion Zymophagy occurred during AP cell model induced by caerulein,which suggested that zymophagy might involve in the mechanism of AP.