Zhongqiu Lu

The reversal of paraquat-induced mitochondria-mediated apoptosis by cycloartenyl ferulate, the important role of Nrf2 pathway.

In this study, we demonstrate the protective effects of Cycloartenyl ferulate (CF) against Paraquat (PQ)-induced cytotoxicity and elucidate the underlying molecular mechanisms. The results show that, CF could reverse the PQ-induced growth inhibition and release of lactate dehydrogenase in HK-2 human proximal tubular cells. Treatment with PQ induced apoptosis in HK-2 cells, as evidenced by accumulation of sub-G1 cell population, chromatin condensation, DNA fragmentation, and translocation of phosphatidylserine, which were significantly attenuated by co-incubation with CF. Mitochondria-mediated apoptosis pathway contributed importantly to PQ-induced apoptosis, as revealed by the activation of caspase-3/-9, cleavage of PARP, depletion of mitochondrial membrane potential regulated by Bcl-2 family members, and overproduction of reactive oxygen species, which were also effectively blocked by CF. Moreover, treatments of PQ strongly inhibited the expression of Nrf2 and the downstream effectors, HO1 and NQO1. However, co-treatment with CF effectively reversed this action of PQ. Furthermore, silencing of Nrf2 by the siRNA technique significantly blocked the cytoprotective effects of CF against PQ-induced apoptosis, which suggest the important role of Nrf2 signaling pathway an cell apoptosis induced by PQ. Taken together, this study provides a novel strategy for molecular intervention against PQ-induced nephrotoxicity by using phytochemicals.

Resveratrol protects mice from paraquat-induced lung injury: The important role of SIRT1 and NRF2 antioxidant pathways

Sirtuin 1 (SIRT1) acts via the deacetylation of a number of crucial transcription factors and has been implicated in various biological processes, including oxidative stress. Previous studies have indicated that nuclear factor, erythroid 2‑like 2 (NRF2) is an effective target of antioxidant therapy for paraquat (PQ) poisoning. However, the association between SIRT1 and NRF2 and their effects in PQ‑induced oxidative stress remains to be elucidated. The current study demonstrated that PQ exposure upregulated the expression of SIRT1 and NRF2 following 6‑ and 24‑h exposure in the lungs of mice. However, long‑term exposure to PQ significantly decreased the expression of SIRT1 and NRF2. Resveratrol is a SIRT1 activator, and strongly enhanced SIRT1 expression and attenuated the lung injury resulting from PQ exposure in the current study. Additionally, treatment with resveratrol upregulated the expression of NRF2 and glutathione, increased the activity of heme oxygenase‑1, superoxide dismutase and catalase, but depleted the expression of malondialdehyde. The present results demonstrated that resveratrol reduced PQ‑induced oxidative stress and lung injury, potentially through the positive feedback signaling loop between SIRT1 and NRF2.

SIRT1 exerts protective effects against paraquat-induced injury in mouse type II alveolar epithelial cells by deacetylating NRF2 in vitro

Silent information regulator 2-related enzyme 1 (SIRT1), a protein deacetylase, is known to strongly protect cells against oxidative stress-induced injury. The nuclear factor E2-related factor 2 (NRF2)-antioxidant response element (ARE) antioxidant pathway plays important regulatory roles in the antioxidant therapy of paraquat (PQ) poisoning. In the present study, we investigated whether the SIRT1/NRF2/ARE signaling pathway plays an important role in lung injury induced by PQ. For this purpose, mouse type II alveolar epithelial cells (AECs‑II) were exposed to various concentrations of PQ. The overexpression or silencing of SIRT1 was induced by transfecting the cells with a SIRT1 overexpression vector or shRNA targeting SIRT1, respectively. The protein expression levels of SIRT1 and NRF2 were measured by western blot analysis. The superoxide dismutase (SOD) and catalase (CAT) activities, as well as the glutathione (GSH) and malondialdehyde (MDA) levels were measured using respective kits. Heme oxygenase-1 (HO-1) activity was also determined by ELISA. In addition, cell apoptosis was determined by flow cytometry. The protein stability of NRF2 was analyzed using cycloheximide and its acetylation in the cells was also determined. The following findings were obtained: i) SIRT1 overexpression markedly increased NRF2 protein expression; ii) SIRT1 promoted the transcriptional activity of NRF2 and upregulated the expression of the NRF2 downstream genes, SOD, CAT, GSH and HO-1, thus inhibiting the apoptosis of AECs‑II; iii) the inhibition of SIRT1 activity further induced the production of malondialdehyde (MDA), which resulted in increased oxidative damage; iv) SIRT1 promoted the stability of NRF2 by regulating the deacetylation and activation of the NRF2/ARE antioxidant pathway. The findings of this study demonstrate that the protective effects of SIRT1 are associated with the activation of the NRF2/ARE antioxidant pathway in lung injury induced by PQ poisoning.

Clearance rate and BP-ANN model in paraquat poisoned patients treated with hemoperfusion.

In order to investigate the effect of hemoperfusion (HP) on the clearance rate of paraquat (PQ) and develop a clearance model, 41 PQ-poisoned patients who acquired acute PQ intoxication received HP treatment. PQ concentrations were determined by high performance liquid chromatography (HPLC). According to initial PQ concentration, study subjects were divided into two groups: Low-PQ group (0.05–1.0u2009μg/mL) and High-PQ group (1.0–10u2009μg/mL). After initial HP treatment, PQ concentrations decreased in both groups. However, in the High-PQ group, PQ levels remained in excess of 0.05u2009μg/mL and increased when the second HP treatment was initiated. Based on the PQ concentrations before and after HP treatment, the mean clearance rate of PQ calculated was 73u2009±u200915%. We also established a backpropagation artificial neural network (BP-ANN) model, which set PQ concentrations before HP treatment as input data and after HP treatment as output data. When it is used to predict PQ concentration after HP treatment, high prediction accuracy (R = 0.9977) can be obtained in this model. In conclusion, HP is an effective way to clear PQ from the blood, and the PQ concentration after HP treatment can be predicted by BP-ANN model.

Temporizing surgical management improves outcome in patients with Vibrio necrotizing fasciitis complicated with septic shock on admission

BACKGROUNDnNecrotizing fasciitis (NF) caused by Vibrio infection is one of the most fatal diseases, resulting in high morbidity and mortality. Early diagnosis and effective surgical intervention are the mainstays for better outcomes for affected patients. Currently, standard surgical management calls for prompt and aggressive debridement and amputation. However, due to its rapid progression and deterioration, 50-60% of Vibrio NF cases present with septic shock and multiple organ dysfunction on admission. These patients, who usually have many surgical contraindications, are unable to tolerate a prolonged aggressive surgical debridement. Therefore, determining the optimal surgical intervention for these particularly severe patients remains a formidable problem in emergency medicine.nnnMETHODSnA retrospective study was conducted on patients who underwent surgery for Vibrio NF and septic shock on admission to the emergency room from April 2001 to October 2012. These patients received the same treatment protocol, with the exception of the initial surgical intervention strategy. Nineteen patients were treated with a temporizing strategy, which called for simple incisions and drainage under regional anesthesia, followed by complete debridement 24h later. Another fifteen patients underwent aggressive surgical debridement during the first operative procedure. Basic demographics, laboratory results on admission, clinical course and outcomes were compared to assess the efficacy and safety of two initial surgical treatment methods: the temporizing strategy and the aggressive strategy.nnnRESULTSnThirty-four patients were included in this study, and the average age was 51.65 years. Chronic liver disease was the most prevalent preexisting condition (50.00%) and the lower limbs were most commonly involved in infection (76.47%). In this patient population, 19 cases underwent surgery with a temporizing therapeutic strategy, while the remaining 15 cases were treated with an aggressive surgical strategy. There were no differences between the two groups with respect to demographics, severity of illness and laboratory data. Compared with those treated with the aggressive strategy, patients treated with the temporizing strategy had shorter operation time (40.79 ± 16.61 vs. 102.00 ± 18.97 min, p<0.001), less bleeding (120.53 ± 67.20 vs. 417.33 ± 134.72 mL, p<0.001), a reduced amount of intraoperatively administrated fluid (3144.70 ± 554.71 vs. 1637.40 ± 302.11 mL, p<0.001), decreased maximum dose of dopamine (15.73 ± 5.64 vs. 10.47 ± 5.61 μg/kg/min, p=0.011) and noradrenaline (20.13 ± 7.50 vs. 13.37 ± 6.18 μg/kg/min, p=0.007), lower arterial lactate values at the end of surgery (5.56±1.99 vs. 8.66 ± 3.25 mmol/L, p=0.004), and, most importantly, lower mortality (26.32% vs. 60.00%, p=0.048). All other treatment conditions, such as duration of vasopressor therapy, number of debridement procedures, rate of amputation, ICU length of stay and hospital length of stay, were the same for both groups.nnnCONCLUSIONnThe temporizing strategy, with early initiation of simple incisions and drainage under regional anesthesia followed by complete debridement 24h later, is more feasible and effective for patients with Vibrio NF complicated with septic shock, as compared with the aggressive surgical debridement strategy.

Incidence, risk factors and impact on outcomes of secondary infection in patients with septic shock: an 8-year retrospective study

Secondary infection in septic patients has received widespread attention, although clinical data are still lacking. The present study was performed on 476 patients with septic shock. Time trends for mortality were analyzed using Spearman’s rank correlation test. Risk factors for secondary infection were investigated by binary logistic regression. The extended Cox model with time-varying covariates and hazard ratios (HR) was performed to determine the impact of secondary infection on mortality. Differences in hospital length of stay (LOS) between patients with and without secondary infection were calculated using a multistate model. Thirty-nine percent of septic shock patients who survived the early phase of the disease developed secondary infection. There was a statistically significant increased odds ratio for secondary infection in older patients and patients with a longer LOS in the intensive care unit (ICU), a higher Sequential Organ Failure Assessment (SOFA) score, and endotracheal intubation. Secondary infection significantly reduced the rate of discharge (HR 5.607; CI95 3.612–8.704; Pu2009<u20090.001) and was associated with an increased hospital LOS of 5.46 days. The present findings represent a direct description of secondary infection in septic shock patients and highlight the influence of this condition on septic shock outcomes.

Cycloartenyl Ferulate Inhibits Paraquat-Induced Apoptosis in HK-2 Cells With the Involvement of ABCC1

Nephrotoxicity induced by chemicals such as paraquat (PQ) is a common clinical phenomenon; therefore, searching for drugs with renal protective effect is of a great practical significance. Our previous investigation found that cycloartenyl ferulate (CF) can antagonize the cytotoxic effect of PQ, and recent studies also revealed a variety of bioactivities of CF. However, specific molecular mechanisms underlying the protective effect of CF have not been explored yet. HPLC detection of PQ content indicated that CF reduced PQ accumulation in HK‐2 cells and thereby improved cell survival. Western blot results showed that both PQ and CF did not affect the expression of ABCB1; however, while PQ suppressed the expression of ABCC1, CF upregulated ABCC1 expression and thereby reversed the inhibitory effect of PQ on ABCC1 expression. Meanwhile, HK‐2 cells did not express ABCG2. When the expression of ABCC1 was knocked down with siRNA, the inhibitory effect of CF on intracellular PQ accumulation was blocked. Further flow cytometric analysis showed that while PQ significantly induced the appearance of sub‐G1 apoptotic peak in cells, CF evidently inhibited apoptosis. TUNEL‐DAPI double‐staining also detected that PQ significantly induced the occurrence of DNA fragmentation in cells, whereas CF effectively inhibited the effect of PQ. Further results showed that ABCC1 siRNA effectively abolished the protective effect of CF on PQ‐induced apoptosis. Taken together, these data demonstrated that in HK‐2 cells, CF could antagonize PQ‐induced toxicity with the involvement of regulatiion of ABCC1 protein expression, which provides a new strategy for treatments of nephrotoxicity. J. Cell. Biochem. 117: 872–880, 2016.

An Effective Machine Learning Approach for Prognosis of Paraquat Poisoning Patients Using Blood Routine Indexes

The early identification of toxic paraquat (PQ) poisoning in patients is critical to ensure timely and accurate prognosis. Although plasma PQ concentration has been reported as a clinical indicator of PQ poisoning, it is not commonly applied in practice due to the inconvenient necessary instruments and operation. In this study, we explored the use of blood routine indexes to identify the degree of PQ toxicity and/or diagnose PQ poisoning in patients via machine learning approach. Specifically, we developed a method based on support vector machine combined with the feature selection technique to accurately predict PQ poisoning risk status, then tested the method on 79 (42 male and 37 female; 41 living and 38 deceased) patients. The detection method was rigorously evaluated against a real‐world data set to determine its accuracy, sensitivity and specificity. Feature selection was also applied to identify the factors correlated with risk status, and the results showed that there are significant differences in blood routine indexes between dead and living PQ‐poisoned individuals (p‐value < 0.01). Feature selection also showed that the most important correlated indexes are white blood cell and neutrophils. In conclusion, the toxicity or prognosis of PQ poisoning can be preliminarily ascertained by blood routine testing without PQ concentration data, representing an additional tool and innovative approach to assess the prognosis of PQ poisoning.

Prognosis and survival analysis of paraquat poisoned patients based on improved HPLC-UV method.

UNLABELLEDnParaquat (PQ) has caused deaths of numerous people around the world. In order to assess the lethal plasma concentration, the patients who acquired acute PQ intoxication were analyzed by plasma concentration monitoring. The plasma PQ concentrations were determined by high performance liquid chromatography (HPLC) which used 5-bromopyrimidine as internal standard and trichloroacetic acid-methanol (1:9) as protein precipitant. The liver, kidney and coagulation function were determined by automatic biochemical analyzer. According to plasma PQ concentration, 90 patients were divided into four groups: trace PQ group (<50ng/mL), low PQ group (<1000ng/mL), medium PQ group (1000-5000ng/mL) and high PQ group (>5000ng/mL). The clinical data from the four groups was statistically analyzed. The results showed the developed HPLC methods exhibited a high degree of accuracy and good linearity within 50-25000ng/mL (R=0.9998). The Spearmans correlation analysis showed PQ concentration had a strong relationship to total bilirubin, direct bilirubin, aspartic transaminase, urea nitrogen, prothrombin time, prothrombin activity, and international normalized ratio (P<0.01). The cured or survival PQ poisoned patients among the trace PQ group, the low PQ group, the medium PQ group, and the high PQ group were 19/19 (100%), 19/21 (90.47%), 11/25 (44.0%), and 0/25 (0%) respectively. The mean hospital days were (10.37±8.04), (18.76±12.06), (16.76±14.44), and (4.04±5.41) days respectively. The Cox regression analysis indicated that plasma PQ concentration was highly related to prognosis (P<0.05). In conclusion, no patient presenting with a PQ concentration over 5000ng/mL survived. The plasma PQ level is related to liver, kidney and coagulation function, which can be used as an important clinical index to judge the prognosis of PQ poisoned patients.nnnCHEMICAL COMPOUNDSnParaquat (PubChem CID: 15938), 5-bromopyrimidine (PubChem CID: 78344), acetonitrile (PubChem CID: 6342), sodium dihydrogen phosphate (PubChem CID: 23672064), sodium heptanesulfonate (PubChem CID: 23672332), methylprednisolone (PubChem CID: 6741), cyclophosphamide (PubChem CID: 2907).

Mifepristone-inducible recombinant adenovirus attenuates paraquat-induced lung injury in rats

Objective: To investigate the effects of overexpression of nuclear factor E2-related factor-2 (NRF2) on lung injury in rats exposed to paraquat (PQ) poisoning. Methods: A mifepristone (RU486)-inducible recombinant adenoviral vector carrying the human NRF2 gene (Ad-RUNRF2) was constructed and transfected via airway into the rats 7 days before the administration of RU486. Rats were orally challenged with PQ at 20 mg/kg 24 h after the injection of RU486. On days 0.5, 3 and 21 after PQ poisoning, the expressions of NRF2 and cytokines related to inflammation and oxidation in lung tissue were examined. Results: RU486 remarkably enhanced NRF2 mRNA and NRF2 protein levels in Ad-RUNRF2-transfected rats in a dose-dependent manner (p < 0.01). PQ stimulated compensatory overexpression of NRF2, heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO-1) in lungs on days 0.5 and 3 after exposure (p < 0.05), but depleted the expression of catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione (GSH), with an increased malondialdehyde (MDA) (p < 0.05). However, pretreatment with Ad-RUNRF2 and RU486 strongly enhanced the expression levels of NRF2, HO-1, NQO-1, CAT and GSH-Px in the lungs of PQ intoxicated rats, with increased GSH and decreased MDA (p < 0.05). Pretreatment with Ad-RUNRF2 and RU486 also strongly suppressed the PQ-induced activation of nuclear factor κB (NF-κB) and decreased the levels of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). In addition, Ad-RUNRF2 and RU486 induction significantly reduced PQ-induced pathological changes in lungs and attenuated lung oedema and protein leakage caused by PQ (p < 0.05). Conclusion: RU486-induced overexpression of NRF2 in lungs transfected with Ad-RUNRF2 can ameliorate PQ-induced lung injury by the activation of the NRF2-antioxidant response element (ARE) pathway.