Molecular hydrogen promotes retinal vascular regeneration and attenuates neovascularization and neuroglial dysfunction in oxygen-induced retinopathy mice.

BACKGROUND: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice. METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining. RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression. CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.

Timing of general anesthesia for pediatric patients recovering from COVID-19: a prospective cohort study.

OBJECTIVE: To explore the timing of general anesthesia for pediatric patients who have recovered from novel coronavirus infection and summarize anesthesia-related complications. METHODS: We summarized the perioperative management of children under 14 years of age who underwent general anesthesia in our hospital according to national epidemic prevention and control requirements. We compared the incidence of postoperative pulmonary complications within 2 weeks (Group A), 3-4 weeks (Group B), and 5-6 weeks (Group C) after COVID-19 recovery. RESULTS: There were differences among the three groups in terms of decreased blood oxygen saturation (< 94%), secretions, and coughing during the PACU period. The risk of low blood oxygen saturation during PACU decreased as the time of COVID-19 recovery extended in the three groups. Compared to Group A, the risk of low blood oxygen saturation was lower in Group B. The presence of respiratory symptoms and a body temperature above 40℃ increased the risk of decreased blood oxygen saturation. The proportion of children aged 11-14 years and children with high fever experiencing decreased blood oxygen saturation during PACU was higher in Groups A and B. Among the three groups, children with respiratory symptoms and longer illness duration had a higher proportion of decreased blood oxygen saturation during PACU. CONCLUSION: Pediatric patients who have recovered from COVID-19 for more than 2 weeks have a lower risk of postoperative complications after general anesthesia. For children with respiratory system symptoms or high fever, there is a higher risk of transient blood oxygen saturation decrease during PACU. For older children, those with high fever, respiratory system symptoms, or longer illness duration, it is recommended to appropriately extend the time from COVID-19 recovery to surgery.

Intensified perfusion culture (IPC) reduced recombinant protein fragmentation.

Mammalian cells remain the mainstay of biological production host. In industry, cultivating and harvest strategies are sorted in batch mode (e.g., batch, fed-batch, concentrated fed-batch and intensified fed-batch) and continuous mode (e.g., perfusion). To retrieve greater productivity and better product quality, especially for the sensitive products prone to fragmentation, culture modes with various modifications are innovated (e.g., intensified perfusion culture [IPC]). In our study, we demonstrated that the fragmentation of Fc-fusion product (Molecule A) is time-dependent in traditional fed-batch (TFB) culture. The fragmentation proportion increased from 3.8% to 12.4% for Clone A, 0.8% to 1.7% for Clone B and 0.9% to 2.0% for Clone C from Day 10 to Day 14. By applying a novel bioprocess, IPC, which allows continuous feeding of the fresh medium and constant removal of the spent medium without bleeding cells to maintain a defined constant viable cell density, the fragmentation was reduced to 0.3% while the productivity was increased from 2.96 g/L to 15.51 g/L for Clone A. To validate whether the fragmentation reduction is product-sensitive, plasmids carrying the DNA sequences of two other Fc-fusion molecules (Molecule B and Molecule C) were transfected into the host. The results showed consistent fragmentation reducing effect by using IPC. Furthermore, the cultivation scale was expanded to 50 L and 1000 L. A minimum fragmentation level below 0.1% was observed for Molecule C. Our study revealed the capability of IPC in reducing Fc-fusion protein fragmentation and the reproducibility when scaling up while maintaining high productivity.

[Effect of uridine on mitochondrial function].

Uridine is one of the essential nutrients in organisms. To maintain normal cell growth and intracellular metabolism, the uridine must be maintained at certain concentration. Recent studies have shown that uridine can reduce inflammatory response in organisms, participate in glycolysis, and regulate intracellular protein modification, such as glycosylation and acetylation. Furthermore, it can protect cells from hypoxic injury by reducing intracellular oxidative stress, promoting high-energy compounds synthesis. Previous studies have shown that the protective effects of uridine are closely related to its effect on mitochondria. This review summarizes the effect of uridine on mitochondrial function.

Effect of intravenous induction with different doses of Esketamine combined with propofol and sufentanil on intraocular pressure among pediatric strabismus surgery: a randomized clinical trial.

BACKGROUND: It is well-established that maintaining stable intraocular pressure (IOP) within the normal range during ophthalmic surgery is important. Esketamine is a commonly used drug in pediatric general anesthesia due to its good analgesic and sedative effects. However, its application in ophthalmic surgery is limited because it can increase IOP. The effect of esketamine combined with other common anesthetics on IOP has been underinvestigated. This study aimed to investigate the effect of different doses of esketamine combined with propofol and sufentanil on IOP during intravenous induction of general anesthesia for pediatric strabismus surgery. METHODS: A total of 181 children with strabismus undergoing unilateral eye surgery under general anesthesia were recruited. Intravenous induction included the use of sufentanil 0.1 µg/kg, propofol 3 mg/kg, and esketamine. Base on the dosage of esketamine, the patients were randomly allocated into three groups: esketamine low (EL) group with 0.25 mg/kg (n = 62), esketamine high (EH) group with 0.5 mg/kg (n = 60), and normal saline (NS) group (n = 59). Hemodynamic parameters, respiratory parameters, and IOP of the non-surgical eye were recorded and compared among the three groups at different time points: before induction (T0), 1 min after induction but before laryngeal mask insertion (T1), immediately after laryngeal mask insertion (T2), and 2 min after laryngeal mask insertion (T3). RESULTS: There were no significant differences in age, gender, body mass index (BMI), and respiratory parameters among the three groups at T0. The IOP at T1, T2, and T3 was lower than that at T0 in all three groups. The EH group (12.6 ± 1.6 mmHg) had a significantly higher IOP than the EL group (12.0 ± 1.6 mmHg) and the NS group (11.6 ± 1.7 mmHg) at T1. However, no difference was found between the EL and NS groups at any time point. Systolic blood pressure (SBP) and heart rate (HR) at T1, T2, and T3 were lower than at baseline, and SBP and HR were higher at T2 than at T1. Additionally, the EH group had a significantly higher HR at T1 than the other two groups. There was no significant difference in diastolic blood pressure (DBP) among the three groups at any time point. CONCLUSION: Propofol combined with sufentanil significantly decreased IOP during the induction of general anesthesia. Although a dose of 0.5 mg/kg esketamine elevated IOP compared to the low-dose and control groups after induction, the IOP remained lower than baseline. 0.25 mg/kg esketamine combined with propofol and sufentanil had little effect on IOP. Therefore, we advocate that a maximum dose of 0.5 mg/kg esketamine combined with propofol and sufentanil will not elevate IOP compared to baseline in pediatric strabismus surgery. TRIAL REGISTRATION: The registration number is ChiCTR2200066586 at Chictr.org.cn. Registry on 09/12/2022.

High Concentration Hydrogen Mitigates Sepsis-Induced Acute Lung Injury in Mice by Alleviating Mitochondrial Fission and Dysfunction.

Background: Multiple organ failure (MOF) is the main cause of early death in septic shock. Lungs are among the organs that are affected in MOF, resulting in acute lung injury. A large number of inflammatory factors and stress injury in sepsis can lead to alterations in mitochondrial dynamics. Numerous studies have confirmed that hydrogen can alleviate sepsis in the animal model. The purpose of this experiment was to explore the therapeutic effect of high concentration (67%) hydrogen on acute lung injury in septic mice and its mechanism. Methods: The moderate and severe septic models were prepared by cecal ligation and puncture. Hydrogen with different concentrations was inhaled for one hour at 1 h and 6 h after the corresponding surgery. The arterial blood gas of mice during hydrogen inhalation was monitored in real time, and the 7-day survival rate of mice with sepsis was recorded. The pathological changes of lung tissues and functions of livers and kidneys were measured. The changes of oxidation products, antioxidant enzymes and pro-inflammatory cytokines in lungs and serums were detected. Mitochondrial function was measured. Results: The inhalation of 2% or 67% hydrogen improves the 7-day survival rate and reduces acute lung injury as well as liver and kidney injury in sepsis. The therapeutic effect of 67% hydrogen inhalation on sepsis was related to increasing antioxidant enzyme activity, reducing oxidation products and pro-inflammatory cytokines in lungs and serums. Compared with the Sham group, mitochondrial dysfunction was alleviated in hydrogen groups. Conclusions: Hydrogen inhalation by high or low concentration can both significantly improve sepsis; however, a high concentration demonstrates a better protective effect. High concentration hydrogen inhalation can significantly improve the mitochondrial dynamic balance and reduce the lung injury in septic mice.

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture.

Chinese hamster ovary (CHO) cells represent a group of predominantly used mammalian hosts for producing recombinant therapeutic proteins. Known for their rapid proliferation rates, CHO cells undergo aerobic glycolysis that is characterized by fast glucose consumption, that ultimately gives rise to a group of small-molecule organic acids. However, only the function of lactate has been extensively studied in CHO cell culture. In this study, we observed the accumulation of acetate from the late exponential phase to harvest day, potentially contributing to the pH decline in late culture stage regardless of lactate consumption. In addition, we evaluated the acidification of the fresh media and the cell culture suspension, and the data revealed that acetate presented a lower acidification capacity compared to lactate and exhibited limited inhibitory effect on cells with less than 20 mM supplemented in the media. This study also explored the ways to control acetate accumulation in CHO cell culture by manipulating the process parameters such as temperature, glucose, and pH control. The positive correlation between the specific glucose consumption rate and acetate generation rate provides evidence of the endogenous acetate generation from overflow metabolism. Reducing these parameters (temperature, glucose consumption) and HCl-controlled low pH ultimately suppress acetate build-up. In addition, the specific acetate generation rate and relevant glucose consumption rate are found to be a metabolic trait associated with specific cell lines. Taken together, the results presented in these experiments provide a means to advance industrial CHO cell culture process control and development.

Two new photoactive metal-organic compounds for degradation of methylene blue and treatment in ophthalmic local anesthesia.

Two new metal-organic compounds, namely [Cu(CrO4)(4,4'-bipy)(H2O)]·n(H2O) (1) together with [Mn(Cr2O7)(bpp)2]n (2) (4,4'-bipy is 4,4'-bipyridine and bpp is 1,3-bis(4-pyridyl)propane), were hydrothermally generated, which were characterized structurally through a series of characterization techniques. Moreover, compounds 1 and 2 have 2.95 eV and 3.02 eV of narrow optical band gap values, and possess outstanding photocatalytic effects for the methylene blue degradation under irradiation of visible light. The application of above compounds in the ophthalmic local anesthesia was examined and the specific mechanism was tested. First of all, the acetylcholine content in the synaptic cleft was measured with enzyme linked immunosorbent assay (ELISA) assay after treated with the CPs. The acetylcholine receptor relative expression on nerve cells was subsequently measured via real time reverse transcription-polymerase chain reaction (RT-PCR) under the treatment of compounds. In the end, the complexes' toxicity was evaluated by Cell Counting Kit-8 (CCK-8) detection.

Effects of preoperative nasal spray esketamine on separation anxiety and emergence agitation in pediatric strabismus surgery: A randomized clinical trial.

OBJECTIVE: To investigate the effects of preoperative nasal spray esketamine on separation anxiety and postoperative emergence agitation in pediatric strabismus surgery. METHOD: Ninety children aged 3 to 6 years who underwent elective strabismus surgery were randomly divided into 3 groups that received 0.5 mg/kg (group S1), 1 mg/kg of esketamine (group S2), and the same volume of normal saline (group C) by nasal spray 10 minutes before surgery. The observation indicators of this test include the Ramsay sedation score, separation anxiety score, mask induction score, and the incidences of postoperative emergence agitation. Patient's heart rate, blood oxygen, post anesthesia care unit stay time, and any adverse events were recorded. RESULTS: The Ramsay sedation score was significantly lower in group C than those in groups S1 and S2 (P < .001). The separation anxiety scores and the mask induction scores were significantly higher in group C than those in groups S1 and S2 (P < .001). The incidences of emergence agitation in groups S1 and S2 were significantly lower than that in C group (P < .001). No obvious clinical complication was observed. CONCLUSION: Preoperative nasal spray esketamine reduced the preoperative separation anxiety and decrease emergence agitation in pediatric strabismus surgery.

High concentration of hydrogen gas alleviates Lipopolysaccharide-induced lung injury via activating Nrf2 signaling pathway in mice.

BACKGROUND AND AIMS: The lung is the first organ to fail in sepsis. Our previous studies have proven that 2% molecular hydrogen (H2) inhalation remain a protective effect on a septic animal model via its anti-inflammatory and anti-apoptosis properties. This current research aims to observe the therapeutic effect of high concentration hydrogen (67%, HCH) on lipopolysaccharide (LPS) induced acute lung injury (ALI), and further investgate the role of Nrf2 signaling pathway. METHODS: ALI model was induced by LPS areosol inhalation. HCH were treated for 1 h at 1 and 6 h after modelling. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 4 and 24 h after the exposure of LPS. The histological scores, wet/dry weight ratios, myeloperoxidase (MPO) activity, protein content and cytokine levels in BALF, apoptosis condition of lung cells, expression of Nrf2 and NF-κB were assessed in both wild type and Nrf2-knockout mice. RESULTS: HCH Inhalation significantly alleviated LPS-induced pathological alterations of lung, and reduced the protein concentration, the wet/dry weight ratio, and the MPO activity of lung tissue. HCH Inhalation improved LPS-induced increasement in caspase-3 activity and the number of TUNEL-positive cells. HCH inhalation attenuated the LPS induced increased total cell content and polymorphonuclear granulocyte content, and pro-inflammatory cytokines, Nrf2 and NF-κB expression. HCH could not produce protective effct in Nrf2-knockout mice. CONCLUSION: HCH can effectively alleviate LPS-induced ALI, which may be related to activation of Nrf2 signaling pathway and inhibition of inflammatory response and cell apoptosis mediated by NF-κB.

MicroRNA-1297 suppressed the Akt/GSK3ß signaling pathway and stimulated neural apoptosis in an in vivo sevoflurane exposure model.

OBJECTIVE: Common inhalation anesthetics used for clinical anesthesia (such as sevoflurane) may induce nerve cell apoptosis during central nervous system development. Furthermore, anesthetics can produce cognitive impairments, such as learning and memory impairments, that continue into adulthood. However, the precise mechanism remains largely undefined. We aimed to determine the function of microRNA-1297 (miR-1297) in sevoflurane-induced neurotoxicity. METHODS: Reverse transcription-polymerase chain reaction assays were used to analyze miR-1297 expression in sevoflurane-exposed mice. MTT and lactate dehydrogenase (LDH) assays were used to measure cell growth, and neuronal apoptosis was analyzed using flow cytometry. Western blot analyses were used to measure PTEN, PI3K, Akt, and GSK3ß protein expression. RESULTS: In sevoflurane-exposed mice, miR-1297 expression was up-regulated compared with the control group. MiR-1297 up-regulation led to neuronal apoptosis, inhibition of cell proliferation, and increased LDH activity in the in vitro model of sevoflurane exposure. MiR-1297 up-regulation also suppressed the Akt/GSK3ß signaling pathway and induced PTEN protein expression in the in vitro model. PTEN inhibition (VO-Ohpic trihydrate) reduced PTEN protein expression and decreased the effects of miR-1297 down-regulation on neuronal apoptosis in the in vitro model. CONCLUSION: Collectively, the results indicated that miR-1297 stimulates sevoflurane-induced neurotoxicity via the Akt/GSK3ß signaling pathway by regulating PTEN expression.

The impact of topical lidocaine and timing of LMA removal on the incidence of airway events during the recovery period in children: a randomized controlled trial.

BACKGROUND: The timing of laryngeal mask airway (LMA) removal remains undefined. This study aimed to assess the optimal timing for LMA removal and whether topical anesthesia with lidocaine could reduce airway adverse events. METHODS: This randomized controlled trial assessed one-to-six-year-old children with ASA I-II scheduled for squint correction surgery under general anesthesia. The children were randomized into the LA (lidocaine cream smeared to the cuff of the LMA before insertion, with mask removal in the awake state), LD (lidocaine application and LMA removal under deep anesthesia), NLA (hydrosoluble lubricant application and LMA removal in the awake state) and NLD (hydrosoluble lubricant application and LMA removal in deep anesthesia) groups. The primary endpoint was a composite of irritating cough, laryngeal spasm, SpO2 < 96%, and glossocoma in the recovery period in the PACU. The secondary endpoints included the incidence of pharyngalgia and hoarseness within 24 h after the operation, duration of PACU stay, and incidence of agitation in the recovery period. The assessor was unblinded. RESULTS: Each group included 98 children. The overall incidence of adverse airway events was significantly lower in the LA group (9.4%) compared with the LD (23.7%), NLA (32.6%), and NLD (28.7%) groups (P=0.001). Cough and laryngeal spasm rates were significantly higher in the NLA group (20.0 and 9.5%, respectively) than the LA (5.2 and 0%, respectively), LD (4.1 and 1.0%, respectively), and NLD (9.6 and 2.1%, respectively) groups (P=0.001). Glossocoma incidence was significantly lower in the LA and NLA groups (0%) than in the LD (19.6%) and NLD (20.2%) groups (P< 0.001). At 24 h post-operation, pharyngalgia incidence was significantly higher in the NLA group (15.8%) than the LA (3.1%), LD (1.0%), and NLD (3.2%) groups (P< 0.001). CONCLUSIONS: LMA removal in the awake state after topical lidocaine anesthesia reduces the incidence of postoperative airway events. TRIAL REGISTRATION: ChiCTR, ChiCTR-IPR-17012347 . Registered August 12, 2017.

Establishment and optimization of a high-throughput mimic perfusion model in ambr® 15.

OBJECTIVES: To establish an automated high-throughput mimic perfusion scale-down model (SDM) in ambr® 15 system. RESULTS: An optimized SDM for mimic perfusion was developed in ambr® 15 system. Cell retention in ambr® 15 was realized by sedimentation and supernatant removal with a retention rate > 95%. Although the SDM couldn't reach the viable cell density (VCD) at a bench scale bioreactor (BR), it maintained VCD at approximately 30 × 106 cells/mL with a cell bleeding rate estimated theoretically and predicted the cell specific perfusion rate (CSPR). A base-feeding strategy was developed to alleviate the pH drop during sedimentation which would adversely have an impact on cell growth, and showed an apparent cell viability improvement from 79.6% (control) to 90.1% on Day 18. The optimized SDM for mimic perfusion was employed for media screening in two cell lines. CONCLUSIONS: A small-scale high-throughput perfusion model in ambr® 15 was developed, optimized to improve cell viability, and as a result, utilized for media screening in two cell lines.

Flumazenil reduces respiratory complications during anesthesia emergence in children with preoperative upper respiratory tract infections.

Despite its benignity, upper respiratory infections (URIs) 1 increase the risk of postoperative respiratory complications during the perioperative and postoperative periods. Flumazenil could improve the symptoms of respiratory obstruction.To evaluate the effect of flumazenil on the occurrence of respiratory complications during anesthesia emergence of children with preoperative URI.This was a prospective study of 164 consecutive pediatric patients who underwent strabismus surgery under general anesthesia at the Tianjin Eye Hospital between August 2016 and April 2017. The patients were grouped as: normal airway (N), recent mild URI (I), normal airway and flumazenil (NF), and recent mild URI and flumazenil (group IF) (n = 41/group). An initial dose of flumazenil (0.1 mg) was administrated intravenously to groups IF and NF. The intraoperative and postoperative respiratory complications were recorded by one anesthesiologist unaware of the grouping.All patients underwent surgery uneventfully. The incidence of postoperative respiratory complications in post-anesthesia care unit (PACU) was higher in group I compared with the other 3 groups (IF: 17%; I: 41%; NF: 5%; N: 10%; P = .0147). During the PACU period, significant differences among groups were seen for cough (IF: 15%; I: 20%; NF: 2%; N: 0%; P = .004), secretion (IF: 17%; I: 29%; NF: 5%; N: 7%; P = .007), low oxygen saturation (IF: 12%; I: 32%; NF: 2%; N: 7%; P = .001), and glossocoma (IF: 15%; I: 34%; NF: 10%; N: 32%; P = .015).Respiratory complications during anesthesia emergence were higher in patients with recent preoperative URI compared to patients with healthy airways. Postoperative flumazenil could reduce the incidence of glossocoma.

Combination therapy of molecular hydrogen and hyperoxia improves survival rate and organ damage in a zymosan-induced generalized inflammation model.

Multiple organ dysfunction syndrome (MODS) is a leading cause of mortality in critically ill patients. Hyperoxia treatment may be beneficial to critically ill patients. However, the clinical use of hyperoxia is hindered as it may exacerbate organ injury by increasing reactive oxygen species (ROS). Hydrogen gas (H2) exerts a therapeutic antioxidative effect by selectively reducing ROS. Combination therapy of H2 and hyperoxia has previously been shown to significantly improve survival rate and organ damage extent in mice with polymicrobial sepsis. The aim of the present study was to investigate whether combination therapy with H2 and hyperoxia could improve survival rate and organ damage in a zymosan (ZY)-induced generalized inflammation model. The results showed that the inhalation of H2 (2%) or hyperoxia (98%) alone improved the 14-day survival rate of ZY-challenged mice from 20 to 70 or 60%, respectively. However, combination therapy with H2 and hyperoxia could increase the 14-day survival rate of ZY-challenged mice to 100%. Furthermore, ZY-challenged mice showed significant multiple organ damage characterized by increased serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen and creatinine, as well as lung, liver and kidney histopathological scores at 24 h after ZY injection. These symptoms where attenuated by H2 or hyperoxia alone; however, combination therapy with H2 and hyperoxia had a more marked beneficial effect against lung, liver and kidney damage in ZY-challenged mice. In addition, the beneficial effects of this combination therapy on ZY-induced organ damage were associated with decreased serum levels of the oxidative product 8-iso-prostaglandin F2α, increased activity of superoxide dismutase and reduced levels of the proinflammatory cytokines high-mobility group box 1 and tumor necrosis factor-α. In conclusion, combination therapy with H2 and hyperoxia provides enhanced therapeutic efficacy against multiple organ damage in a ZY-induced generalized inflammation model, suggesting the potential applicability of H2 and hyperoxia in the therapy of conditions associated with inflammation-related MODS.

HYDROGEN-RICH MEDIUM AMELIORATES LIPOPOLYSACCHARIDE-INDUCED BARRIER DYSFUNCTION VIA RHOA-MDIA1 SIGNALING IN CACO-2 CELLS.

Gastrointestinal barrier dysfunction is associated with the severity and prognosis of sepsis. Hydrogen gas (H2) can ameliorate multiple organ damage in septic animals. Ras homolog gene family member A (RhoA) and mammalian diaphanous-related formin 1 (mDia1) are important to regulate tight junction (TJ) and adherens junction (AJ), both of which determine the integrity of the intestinal barrier. This study was aimed to investigate whether H2 could modulate lipopolysaccharide (LPS)-stimulated dysfunction of the intestinal barrier and whether RhoA-mDia1 signaling is involved. Caco-2 cells were exposed to different concentrations of LPS (1 µg/mL-1 mg/mL). The permeability of the intestinal barrier was evaluated by transepithelial resistance (TER) and fluorescein-isothiocyanate-dextran flux. Expression and distribution of occludin and E-cadherin were analyzed by Western blot and immunofluorescence. RhoA activity was measured by G-Lisa assay, and mDia1 expression was assessed by Western blot. LPS (100 µg/mL) decreased TER and increased fluorescein-isothiocyanate-dextran flux, which were alleviated by H2-rich medium. Also, H2 down-regulated LPS-induced oxidative stress. Moreover, H2 improved the down-regulated expression and redistribution of occludin and E-cadherin caused by LPS. Additionally, H2 alleviated LPS-caused RhoA activation, and the beneficial effects of H2 on barrier were counteracted by RhoA agonist CN03. Rho inhibitor C3 exoenzyme mitigated LPS-induced barrier breakdown. Furthermore, H2-rich medium increased mDia1 expression, and mDia1 knockdown abolished protections of H2 on barrier permeability. mDia1 knockdown eliminated H2-induced benefits for occludin and E-cadherin. These findings suggest that H2 improves LPS-induced hyperpermeability of the intestinal barrier and disruptions of TJ and AJ by moderating RhoA-mDia1 signaling.

Plant-produced human recombinant erythropoietic growth factors support erythroid differentiation in vitro.

Clinically available red blood cells (RBCs) for transfusions are at high demand, but in vitro generation of RBCs from hematopoietic stem cells requires significant quantities of growth factors. Here, we describe the production of four human growth factors: erythropoietin (EPO), stem cell factor (SCF), interleukin 3 (IL-3), and insulin-like growth factor-1 (IGF-1), either as non-fused proteins or as fusions with a carrier molecule (lichenase), in plants, using a Tobacco mosaic virus vector-based transient expression system. All growth factors were purified and their identity was confirmed by western blotting and peptide mapping. The potency of these plant-produced cytokines was assessed using TF1 cell (responsive to EPO, IL-3 and SCF) or MCF-7 cell (responsive to IGF-1) proliferation assays. The biological activity estimated here for the cytokines produced in plants was slightly lower or within the range cited in commercial sources and published literature. By comparing EC50 values of plant-produced cytokines with standards, we have demonstrated that all four plant-produced growth factors stimulated the expansion of umbilical cord blood-derived CD34+ cells and their differentiation toward erythropoietic precursors with the same potency as commercially available growth factors. To the best of our knowledge, this is the first report on the generation of all key bioactive cytokines required for the erythroid development in a cost-effective manner using a plant-based expression system.

[Comparison on ingredients between cultured cell and cultivated Saffron pistils].

OBJECTIVE: To compare the differences of active ingredients between tissue cultured cells and cultivated saffron pistils. METHOD: The experiment was carried out by the Fourier transform infrared spectroscopy (FTIR) spectra and high performance liquid chromatography (HPLC). RESULT: The data indicated that the species and contents active ingredients in saffron pistils from different places were different. The species of active ingredients in tissue cultured cells are less than those in cultivated saffron pistils. However, the quantity of crocin A, which showed good anticancer effect, is 2-3 times more than that in cultivated saffron pistils. CONCLUSION: The active ingredients of the tissue cultured cells are similar to those of saffron pistils, but their contents are different. Therefore, the tissue cultured cells can only be the part-substitutes of cultivated saffron pistils.

[Analysis of contents of berberine in Coptis chinensis of Lichuan].

OBJECTIVE: To determine the contents of berberine in Coptis chinensis of Lichuan and establish its best cultivation scheme. METHOD: We used high performance liquid chromatography (HPLC) to measure the contents of berberine at different altitude, growth age, and leaves. RESULTS: Analytic data showed that the growth age and firry woods shading did not affect the contents of berberine in rhizome of Coptis chinensis. Low altitude was more suitable for Coptis chinensis to synthesize berberine. The contents of berberine in rhizome of Coptis chinensis with floral leaf were higher than those with lusterless and lustrous leaves, but no significant difference. CONCLUSION: The synthesis of berberine is closely correlated with shading conditions. It is recommended that the shading ratio should be reduced or the sheds removed in the middle growth age (2-3 years), and then the herb should be reshaped at the last year to enhance the synthesis of berberine, so as to obtain high-quality Coptis chinensis in the harvest.