随着铜绿假单胞菌（铜绿）的耐药性逐年增强，铜绿感染已经成为公共医疗卫生的重点关注问题。线粒体自噬及其介导的线粒体功能障碍在多种细菌感染中已被报道，但线粒体功能障碍在宿主调控铜绿感染中的作用尚不明确。因此，本研究建立铜绿刺激小鼠巨噬细胞感染模型和小鼠急性铜绿感染模型，探讨铜绿是否通过诱导线粒体自噬改变线粒体功能，进而影响宿主免疫炎症反应和细胞毒性，并通过监测生存率和肺组织病理学变化进一步确定线粒体自噬在小鼠铜绿体内感染模型中的作用。结果表明，铜绿引起小鼠腹腔巨噬细胞线粒体功能障碍，并通过线粒体自噬途径清除铜绿刺激引起的活性氧（ROS）累积，从而抑制铜绿引起的促炎性细胞因子分泌并增强细胞毒性。体内实验进一步确认线粒体自噬在铜绿体内感染中的作用。
Mice ; Animals ; Reactive Oxygen Species/metabolism* ; Pseudomonas aeruginosa ; Macrophages/metabolism* ; Mitochondria ; Cytokines/metabolism*

[摘 要] 目的：探讨抑制素β亚基A反义RNA1（INHBA-AS1）对宫颈癌HeLa细胞EMT和鸟氨酸代谢途径的影响及其机制。方法：体外常规培养HeLa细胞，实验分为10组：对照组、阴性对照（NC）组、sh-INHBA-AS1组、PluriSIn 1[硬脂酰辅酶A去饱和酶（stearyl CoA desaturase，SCD）抑制剂]组、NC+PluriSIn 1组、sh-INHBA-AS1+PluriSIn 1组、10058-F4（c-Myc抑制剂）组、NC+10058-F4组、sh-INHBA-AS1+10058-F4组、sh-INHBA-AS1+OE-c-Myc组。平板克隆实验检测各组细胞的增殖能力，FCM检测各组细胞的凋亡情况，Transwell小室实验检测各组细胞的侵袭、迁移能力，qPCR法检测各组细胞中INHBA-AS1、c-Myc、SCD和EMT相关基因（N-cadherin、TGF-β、ZEB1）mRNA的表达，WB法检测各组细胞中c-Myc、SCD、EMT相关（N-cadherin、TGF-β、ZEB1）、S-腺苷-甲硫氨酸脱羧酶（SAMDC）和亚精胺/精胺N1-乙酰转移酶（SSAT）蛋白的表达，ELISA检测各组细胞上清液中鸟氨酸脱羧酶（ODC）的含量。结果：敲减INHBA-AS1表达使HeLa细胞的增殖、侵袭和迁移能力显著降低（均P<0.05）而细胞凋亡率显著升高（P<0.05），qPCR、WB法检测结果显示，敲减INHBA-AS1均可显著抑制HeLa细胞中c-Myc、SCD、N-cadherin、TGF-β、ZEB1和SAMDC的表达（均P<0.05），而促进SSAT的表达（P<0.05），并降低HeLa细胞上清液中ODC的含量（P<0.05）。与c-Myc抑制剂和SCD抑制剂单独处理相比，其联合敲减INHBA-AS1后上述作用更加显著（均P<0.05）；与sh-INHBA-AS1组相比，进一步过表达c-Myc后HeLa细胞的增殖能力显著升高（P<0.05）、SCD和N-cadherin蛋白表达水平显著升高（P<0.05）、细胞上清液中ODC含量显著升高（P<0.05）。结论：INHBA-AS1可通过c-Myc调控SCD的表达，从而影响HeLa细胞鸟氨酸代谢和EMT进程，进而促进HeLa细胞的增殖、侵袭和迁移能力。

Sepsis is a complex syndrome caused by multiple pathogens and involves multiple organ failure, particularly spleen dysfunction. In 2017, the worldwide incidence was 48.9 million sepsis cases and 11 million sepsis-related deaths were reported (Rudd et al., 2020). Inflammation, oxidative stress, and apoptosis are the most common pathologies seen in sepsis. Liensinine (LIE) is a bisbenzylisoquinoline-type alkaloid extracted from the seed embryo of Nelumbo nucifera. Lotus seed hearts have high content of LIE which mainly has antihypertensive and antiarrhythmic pharmacological effects. It can exert anti-carcinogenic activity by regulating cell, inflammation, and apoptosis signaling pathways (Manogaran et al., 2019). However, its protective effect from sepsis-induced spleen damage is unknown. In this research, we established a mouse sepsis model induced by lipopolysaccharide (LPS) and investigated the protective effects of LIE on sepsis spleen injury in terms of inflammatory response, oxidative stress, and apoptosis.
Mice ; Animals ; Lipopolysaccharides/pharmacology* ; Spleen ; Inflammation ; Apoptosis ; Sepsis ; Oxidative Stress

Alcoholic liver disease(ALD)is the most frequent liver disease worldwide,resulting in severe harm to personal health and posing a serious burden to public health.Based on the reported antioxidant and anti-inflammatory capacities of scutellarin(SCU),this study investigated its protective role in male BALB/c mice with acute alcoholic liver injury after oral administration(10,25,and 50 mg/kg).The results indicated that SCU could lessen serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels and improve the histopathological changes in acute alcoholic liver;it reduced alcohol-induced malondialdehyde(MDA)content and increased glutathione peroxidase(GSH-Px),catalase(CAT),and superoxide dismutase(SOD)activity.Furthermore,SCU decreased tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and IL-1β messenger RNA(mRNA)expression levels,weakened inducible nitric oxide synthase(iNOS)activity,and inhibited nucleotide-binding oligomerization domain(NOD)-like receptor protein 3(NLRP3)inflammasome activation.Mechanistically,SCU suppressed cytochrome P450 family 2 subfamily E member 1(CYP2E1)upregulation triggered by alcohol,increased the expression of oxidative stress-related nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)pathways,and suppressed the inflammation-related degradation of inhibitor of nuclear factor-κB(NF-κB)-α(IκBα)as well as activation of NF-κB by mediating the protein kinase B(AKT)and p38 mitogen-activated protein kinase(MAPK)pathways.These findings demonstrate that SCU protects against acute alcoholic liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and suppressing inflammation by regulating the AKT,p38 MAPK/NF-κB pathways.

Drinking culture has high significance in both China and the world, whether in the entertainment sector or in social occasions; according to the World Health Organization's 2018 Global Alcohol and Health Report, about 3 million people died from excessive drinking in 2016, accounting for 5.3% of the total global deaths that year. Oxidative stress and inflammation are the most common pathological phenomena caused by alcohol abuse (Snyder et al., 2017). Scutellarin, a kind of flavonoid, is one of the main active ingredients extracted from breviscapine. It exerts anti-inflammatory, antioxidant, and vasodilation effects, and has been used to treat cardiovascular diseases and alcoholic liver injury. Although scutellarin can effectively alleviate multi-target organ injury induced by different forms of stimulation, its protective effect on alcoholic brain injury has not been well-defined. Therefore, the present study established an acute alcohol mice brain injury model to explore the effect of scutellarin on acute alcoholic brain injury. The study was carried out based on the targets of oxidative stress and inflammation, which is of great significance for the targeted therapy of clinical alcohol diseases.
Animals ; Apigenin/therapeutic use* ; Brain Injuries/drug therapy* ; Glucuronates/therapeutic use* ; Humans ; Mice ; Oxidative Stress

Objective:To investigate the role of cholinergic anti-inflammatory pathway in the regulation of peptide transporter 1 (PepT1) expression in small intestinal epithelium of septic rats by Ghrelin.Methods:One hundred adult male Sprague-Dawley (SD) rats were randomly divided into sham operation group, sepsis group, sepsis+vagotomy group, sepsis+Ghrelin group, and sepsis+vagotomy+Ghrelin group, with 20 rats in each group. In the sham operation group, the cecum was separated after laparotomy, without ligation and perforation. In the sepsis group, the rats received cecal ligation puncture (CLP). In the sepsis+vagotomy group, the rats received CLP and vagotomy after laparotomy. In the sepsis+Ghrelin group, 100 μmol/L Ghrelin was intravenously injected after CLP immediately. The rats in the sepsis+vagotomy+Ghrelin group received CLP and vagotomy at the same time, then the Ghrelin was intravenously injected immediately with the same dose as the sepsis+Ghrelin group. Ten rats in each group were taken to observe their survival within 7 days. The remaining 10 rats were sacrificed 20 hours after the operation to obtain venous blood and small intestinal tissue. The condition of the abdominal intestine was observed. The injury of intestinal epithelial cells was observed with transmission electron microscopy. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in serum and small intestinal tissue were detected by enzyme-linked immunosorbent assay (ELISA). The brush border membrane vesicle (BBMV) was prepared, the levels of mRNA and protein expression of PepT1 in the small intestinal epithelium were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting.Results:All rats in the sham operation group survived at 7 days after operation. The 7-day cumulative survival rate of rats in the sepsis group was significantly lower than that in the sham operation group (20% vs. 100%, P < 0.05). The cumulative survival rate of rats after Ghrelin intervention was improved (compared with sepsis group: 40% vs. 20%, P < 0.05), but the protective effect of Ghrelin was weakened after vagotomy (compared with sepsis+Ghrelin group: 10% vs. 40%, P < 0.05). Compared with the sham operation group, in the sepsis group, the small intestine and cecum were dull red, the intestinal tubules were swollen and filled with gas, the intestinal epithelial cells were seriously injured under transmission electron microscopy, the levels of TNF-α and IL-1β in serum and small intestinal were significantly increased, and the expression levels of PepT1 mRNA and protein in the small intestinal epithelium were significantly decreased. It indicated that the sepsis rat model was successfully prepared. After vagotomy, the intestinal swelling and gas accumulation became worse in septic rats, leading to the death of all rats. Compared with the sepsis group, the abdominal situation in the sepsis+Ghrelin group was improved, the injury of intestinal epithelial cells was alleviated, the serum and small intestinal TNF-α and IL-1β were significantly decreased [serum TNF-α (ng/L): 253.27±23.32 vs. 287.90±19.48, small intestinal TNF-α (ng/L): 95.27±11.47 vs. 153.89±18.15, serum IL-1β (ng/L): 39.16±4.47 vs. 54.26±7.27, small intestinal IL-1β (ng/L): 28.47±4.13 vs. 42.26±2.59, all P < 0.05], and the expressions of PepT1 mRNA and protein in the small intestinal epithelium were significantly increased [PepT1 mRNA (2 -ΔΔCt): 0.66±0.05 vs. 0.53±0.06, PepT1 protein (PepT1/GAPDH): 0.80±0.04 vs. 0.60±0.05, both P < 0.05]. Compared with the sepsis+Ghrelin group, after vagotomy in the sepsis+vagotomy+Ghrelin group, the effect of Ghrelin on reducing the release of inflammatory factors in sepsis rats was significantly reduced [serum TNF-α (ng/L): 276.58±19.88 vs. 253.27±23.32, small intestinal TNF-α (ng/L): 144.28±12.99 vs. 95.27±11.47, serum IL-1β (ng/L): 48.15±3.21 vs. 39.16±4.47, small intestinal IL-1β (ng/L): 38.75±4.49 vs. 28.47±4.13, all P < 0.05], the up-regulated effect on the expression of PepT1 in small intestinal epithelium was lost [PepT1 mRNA (2 -ΔΔCt): 0.58±0.03 vs. 0.66±0.05, PepT1 protein (PepT1/GAPDH): 0.70±0.02 vs. 0.80±0.04, both P < 0.05], and the injury of small intestinal epithelial cells was worse. Conclusion:Ghrelin plays a protective role in sepsis by promoting cholinergic neurons to inhibit the release of inflammatory factors, thereby promoting the transcription and translation of PepT1.

Objective:To investigate the role of LncRNA-TUG1 in the injury of intestinal epithelial cells induced by lipopolysaccharide (LPS).Methods:LPS was used to treat HIEC-6 human intestinal epithelial cells for 24 h to construct a sepsis injury model. Whole transcriptome RNA sequencing was used to analyze the expression changes of mRNA, microRNA and lncRNA in HIEC-6 cells after LPS treatment. Real-time fluorescence quantitative (qRT-PCR) and Western blot was performed to detect the expression changes of lncRNA-TUG1, microRNA-132-3p (miR-132-3p), SIRT1 mRNA and SIRT1 protein in HIEC-6 cells after LPS treatment. The expression levels of LncRNA-TUG1, miR-132-3p and SIRT1 were artificially changed by in vitro transfection. qRT-PCR and Western blot were used to confirm the regulatory effect of lncRNA-TUG1 on microRNA-132-3p and SIRT1. CCK-8 and flow cytometry were used to analyze the effects of LncRNA-TUG1, miR-132-3p and SIRT1 on the proliferation and apoptosis of HIEC-6 cells. The dual luciferase report analysis was used to verify the targeting relationship between LncRNA-TUG1, miR-132-3p and SIRT1. Statistical analysis was performed using SPSS 17.0, and differences between the two groups were compared using independent sample t test. Results:RNA sequencing results showed that the expressions of lncRNA-TUG1 and SIRT1 were decreased in HIEC-6 cells after LPS treatment ( t=3.26, P<0.05 and t=2.55, P<0.05), but the expression of miR-132-3p was increased ( t=4.12, P<0.05). In vitro cell experiments, the expression of lncRNA-TUG1 and SIRT1 were decreased in HIEC-6 cells treated with LPS ( t=5.69, P<0.05 and t=5.712, P<0.05), while the expression of miR-132-3p was increased ( t=3.88, P<0.05). Overexpression of lncRNA-TUG1 increased the proliferation rate ( t=6.55, P<0.05) and decreased the apoptosis rate ( t=3.94, P<0.05) of LPS-treated cells. Upregulation of lncRNA-TUG1 decreased the expression of miR-132-3p ( t=4.66, P<0.05), and increased the mRNA and protein levels of SIRT1 ( t=3.91, P<0.05). Transfection of miR-132-3P mimic could inhibit the mRNA ( t=4.08, P<0.05) and protein levels of SIRT1. In LPS-treated cells, the cells co-transfected with miR-132-3pmimic and siRNA-SIRT1 had a lower proliferation rate ( t=4.55, P<0.05 and t=5.67, P<0.05) and a higher apoptosis rate ( t=3.90, P<0.05 and t=4.22, P<0.05) than those transfected with only pcDNA3.1-lncRNA-TUG. Conclusions:lncRNA-TUG1 may act as a ceRNA to regulate miR-132-3p/SIRT1, therefore alleviating HIEC-6 cell injury caused by LPS. Intervention of lncRNA-TUG1/miR-132-3p/SIRT1 regulatory pathway may become a potential strategy to prevent sepsis-induced intestinal mucosal damage.

objective:To investigate the tolerability of early enteral nutrition (EN), and to further explore the association of early EN with clinical outcome in critically ill patients with hemodynamic instability.Methods:The adult patients from Zhejiang Provincial People’s Hospital with an expected admission to ICU for at least 24 h were consecutively recruited from May 2014 to May 2016, and all clinical, laboratory, and survival data were prospectively collected. The AGI grade was daily assessed on the first week of ICU admission. Enteral nutrition (EN) started after 6 h of hemodynamic stability (MAP ≥ 65 mmHg) when the patients took vasoactive medication. The patients were divided into three groups based on the timing of EN initiation: early EN group (EN initiation within 48 h of ICU admission), late EN group (EN initiation at more than 48 h of ICU admission), and no initiation of enteral feeding within 7 days of ICU admission.Results:Of 201 patients enrolled, the mean age was 65.3 ± 16.4 years, APACHE II score was 22.4 ± 6.85, and 191 patients (95.0%) took mechanical ventilation. There were no differences in high gastric residual volume, diarrhea, and gastrointestinal (GI) bleeding between the early EN group and late EN group ( P>0.05). Whereas, patients in the no initiation of EN within 7 days of ICU admission had a lower prevalence of gastric residual volume (16.7% vs. 33.3%, P=0.05), but higher prevalence of GI bleeding (47.2% vs. 26.1%, P=0.02). Compared with those in the late EN group and in no initiation of EN within 7 days of ICU admission, patients in the early EN group had lower 28- (30.4% vs. 47.9% vs. 55.6%, P=0.01) and 60-day mortality rates (38.0% vs. 53.4% vs. 63.9%, P=0.017). Multivariate Cox regression analysis showed that the timing of EN initiation on the admission to ICU (early EN vs. late EN, χ 2≥5.83, P<0.05; early EN vs. no initiation of EN, χ 2≥7.90, P<0.01), serum creatinine ( χ 2=5.06, P<0.05), plasma albumin ( χ 2≥6.41, P<0.01), AGI grade ( χ 2≥8.15, P<0.01), and APACHE II score ( χ 2≥9.62, P<0.01) were independent predictors for 28- and 60-day mortality. Conclusions:Early EN on admission to ICU could be tolerated, and is significantly associated with lower risk of 28- and 60-day mortality in critically ill patients with vasoactive medication to maintain hemodynamic stability.

Objective To evaluate the supercutaneous fixation with locking plate for treatment of open metacarpal fractures.Methods From March 2015 to November 2016,16 open metacarpal fractures were treated with supercutaneous locking plate after emergency debridement at Department of Hand Surgery,The Second Affiliated Hospital to Wenzhou Medical University.They were 11 men and 5 women,from 20 to 67 years of age (average,37.5 years).There were 2 cases of the 1st metacarpal fracture,6 ones of the 2ed metacarpal fracture,one of the 3rd metacarpal fracture,2 ones of the 4th metacarpal fracture and 5 ones of the 5th metacarpal fracture.All fractures were open injury.By the Gustilo-Anderson classification,there were 9 cases of type Ⅱ and 7 ones of type Ⅲ A.Functional rehabilitation was encouraged immediately after operation.The outcomes were evaluated at the 4th and 24th weeks postoperatively according to the Total Angle of Motion (TAM) for digital joints recommended by the Hand Surgery Society,Chinese Medical Association.Results The mean operation time was 46.8 minutes (from 35 to 108 minutes).All cases were followed up for an average of 9.8 months (from 7 to 25 months).All fractures healed without breakage or loosening of the supercutaneous locking plate after an average of 6.2 weeks(from 4.3 to 7.8 weeks).Nail tract infection occurred in 3 cases.According to the TAM at the 4th week postoperatively,5 cases were rated as excellent,8 as good,2 as fair and one as poor;according to the TAM at the 24th week postoperatively,11 cases were rated as excellent,4 as good and one as fair.Conclusion For open metacarpal fractures with severe contamination or soft tissue injury,supercutaneous locking plate fixation can achieve fine clinical outcomes.

Objective:To evaluate the clinical application value of gastrointestinal contrast-enhanced ultrasound combined gas injection method in verifying the location of nasointestinal tube in critically ill patients.Methods:Data of 60 critically ill patients who had the indications of indwelling nasointestinal tube were collected from September 1,2015 to September 1,2016 in the Intensive Care Unit of Zhejiang Provincial People(s) Hospital.The position of nasointestinal tube in patients who underwent bedside blind insertion would be confirmed routinely through gas injection auscultation method.After tube was inserted,its route was scanned by ultrasound with gas perfusion assistance.Afterwards,rapid gas perfusion was used until suspicious tube end position was determined.Furthermore,oral ultrasound contrast agent was injected into the tube if instantaneous strong echo of gas was observed in localized lumen,and contrast agent filling meant the placement being successful.Two methods of position confirmation of nasointestinal tube in critically ill patients included gastrointestinal contrast enhanced ultrasound combined gas injection and gas injection auscultation only,and the effect of the two methods was compared and confirmed by chest and abdominal X ray examinations to verify the location of nasointestinal tube below pylorus.Results:A total of 60 patients were included in this study,58 patients(96.7％)in gastrointestinal contrast enhanced ultrasound combined gas injection group were successfully positioned.Among them,the placements of tube in 56 cases were below pylorus,while 2 cases were above pylorus.The sensitivity,specificity,positive predictive value,negative predictive value and accuracy of location of gastrointestinal contrast enhanced ultrasound combined gas injection method were 96.6％,100％,100％,50％,96.7％ and of gas injection auscultation method were 74.1％,50％,97.7％,6.3％ and 73.3％.The differences of the sensitivity,specificity,negative predictive value and accuracy between the two methods were statistically significant (P ＜ 0.05).Conclusion:Gastrointestinal contrastenhanced ultrasound combined gas injection method is a safe,simple and convenient method with high sen-sitivity,specificity,negative predictive value and accuracy in confirming the location of the nasointestinal tube.