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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.
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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.
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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.
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Objective To investigate the prevalence of feeding intolerance (FI),and to explore the FI within 7 days of ICU admission in association with clinical outcome in critically ill patients.Methods The adult patients from 14 general ICUs in Zhejiang Province with an expected admission to ICU for at least 24h were recruited from March 2014 to August 2014,and all clinical,laboratory,and survival data were prospectively collected.The AGI (acute gastrointestinal injury) grade was daily assessed based on gastrointestinal (GI) symptoms,feeding details and organ dysfunction within the first week of ICU stay.The intra-abdominal pressures (IAP) was measured using AbViser device.Results Of 550 patients enrolled,418 were assessed in GI symptoms and feeding details within 7 days of ICU stay.The mean age and SOFA score were (65.1 ± 18.3) years and (8.96 ±4.10),respectively.Of them,355 patients (84.9％) were under mechanical ventilation support,and 37 (8.85％) received renal replacement therapy.The mean length of time for enteral feeding was (30.8 ±26.2) h,and the prevalence of FI on the 3rd and 7th day of ICU stay accounted for 39.2％ and 25.4％,respectively.Compared to those with FI within 7 days of ICU stay,the patients without FI had higher rate of successively weaning from mechanical ventilation (21.3％ vs.5.7％,P =0.003) and higher rate of withdrawal of vasoactive medication (45.5％ vs.20.0％,P =0.037),as well as lower mortality rate of 28-day (24.4％ vs.38.7％,P =0.004) and 60-day (29.6％ vs.44.3％,P =0.005).In multivariate Cox regression model with adjustment for age,sex,participant center,serum creatinine and lactate,AGI grade on the first day of ICU stay,and comorbidities,the FI within 7 days of ICU stay (x2 ≥ 7.24,P ＜ 0.01) remained to be independent predictors for 60-day mortality.After further adjusted for SOFA score,the FI within 7 days of ICU stay (HR =1.71,95％ CI:1.18-2.49;P =0.006) and AGI grade on the first day of ICU stay (HR =1.33,95 ％ CI:1.07-1.65;P =0.009) could provide independent prognostic values of 60-day mortality.Conclusions There is high rate of FI occurred within 7 days of ICU stay,and is significantly associated with worse outcome.In addition,this study also provides evidence to further support that measurement of gastrointestinal dysfunction could increase value of SOFA score in outcome prediction for the risk of 60-day mortality.
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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.
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Objective To develop a method for determination of ultra-trace 2-chlorovinylarsonic acid(CVAOA)in human urine specific to lewisite exposure. Methods The conditions for dispersive liquid-phase microextraction(DLPME)were optimized by orthogonal experiments with results as follows:a mixture of 250μl methanol(dispersive solvent),250μl ethyl acetate(extraction sol?vent),and 3,4-dimercaptotoluene(DMT)(derivatization reagent,using 1∶100 mole ratio of CVAOA to DMT)was injected into 1 ml urine when pH was adjusted to 1. In the next 60 min,the CVAOA was derivated and the CVAOA-DMT derivative was extracted simul?taneously at 90℃. The derivative was then analyzed by gas chromatography/tandem mass spectrometry-select reaction monitoring〔GC/MS/MS(SRM)〕. Results The linear calibration extended from 50 pg/ml to 1μg/ml(r2=0.9999)with the relative standard deviations (RSD)less than 10%. The limit of detection was 18 pg/ml and the limit of quantitation was 56 pg/ml,which was much lower than that of the reported DLPME methods. When detecting human urine samples with low,middle and high spiked concentrations(0.5,5 and 50 ng/ml)of lewisite,the analysis accuracies ranged from 98.2%to 104%and the RSD ranged from 6.9%to 8.9%. Conclusion The method developed in this study has high specificity and sensitivity as well as good precision and accuracy. It is s imple and can be readi?ly applied to the exposed sample analysis.
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Objective To investigate the impact of early initiation of continuous renal replacement therapy (CRRT) based on Kidney Disease: Improving Global Outcomes (KDIGO) classification on the prognosis of critically ill patients with acute kidney injury (AKI). Methods A retrospective analysis of clinical data of patients diagnosed as AKI in Department of Critical Care Medicine of Zhejiang Provincial People's Hospital from January 2011 to January 2015 was conducted. All patients included should be 18 years old or older, having stayed in intensive care unit (ICU) for more than 48 hours, and received CRRT. All subjects were divided into three groups according to their renal function before CRRT according to the KDIGO-AKI guideline: AKI-stage 1 group, AKI-stage 2 group and AKI-stage 3 group. The general condition, original disease, severity of disease, duration of mechanical ventilation, the length of ICU or hospital stay, 28-day survival rate and in-hospital mortality rate were compared among these three groups. Additionally, risk factors for the 28-day survival rate and hospital mortality of critically ill patients with AKI were screened by logistic regression analysis. Results A total of 258 critically ill patients with AKI were enrolled, with 64 cases in AKI-stage 1 group, 62 cases in AKI-stage 2 group, and 132 cases in AKI-stage 3 group. 116 patients survived with 28-day survival rate of 44.96%. 154 patients died with hospital mortality 59.69%. The precipitating factors of AKI in all three groups (stage 1, stage 2, and stage 3) were similar, with sepsis, heart failure and poisoning (drugs or poison) being the main triggers for AKI, accounting for 35.66%, 19.38% and 13.18%, respectively. There were significant differences in the rate of vasoactive agent usage (31.25%, 41.94%, 50.00%, χ2 = 6.241, P = 0.044), acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score (20.87±7.37, 17.19±7.02, 22.58±7.95, F = 5.292, P = 0.006) and sequential organ failure assessment (SOFA) score (8.41±3.46, 6.22±2.43, 9.58±3.71, F = 10.328, P = 0.000), while there was no significant difference in gender, age, primary disease, time from ICU admission to the beginning of CRRT, mean arterial pressure (MAP), lactate level or 24-hour lactate clearance rate (LCR), mechanical ventilation time, the length of ICU or hospital stay, 28-day survival rate or hospital mortality among these three groups (all P > 0.05). According to the logistic regression analysis, time from ICU admission to start of CRRT and lactate level were the independent risk factors for 28-day survival rate or hospital mortality of critically ill patients with AKI [odds ratio (OR) for 28-day survival rate was 0.850 and 0.774, 95% confidence interval (95%CI) was 0.752-0.960 and 0.638-0.940, P value was 0.009 and 0.010, respectively; OR for hospital mortality was 0.884 and 0.756, 95%CI was 0.781-1.000 and 0.610-0.939, P value was 0.049 and 0.011, respectively]. Conclusion Early initiation of CRRT based on KDIGO-AKI classification could not improve the prognosis of critically ill patients with AKI, the optimal timing of RRT for such patients remains to be further explored.