Hyperbaric Oxygen Treatment Improves Hearing Level Attenuating TLR4/NF-κB Mediated Inflammation in Sudden Sensorineural Hearing Loss Patients / 生物医学与环境科学（英文）

Objective@#Hyperbaric oxygen treatment (HBOT) has demonstrated efficacy in improving hearing levels of patients with idiopathic sudden sensorineural hearing loss (ISSHL); however, the underlying mechanisms are not well understood. HBOT alleviates the inflammatory response, which is mediated by Toll-like receptor (TLR) 4 and nuclear factor (NF)-κB. In this study we investigated whether HBOT attenuates inflammation in ISHHL patients alteration of TLR4 and NF-κB expression.@*Methods@#ISHHL patients ( = 120) and healthy control subjects ( = 20) were enrolled in this study. Patients were randomly divided into medicine group treated with medicine only ( = 60) and HBO group receiving both HBOT and medicine ( = 60). Audiometric testing was performed pre- and post-treatment. TLR4, NF-кB, and TNF-α expression in peripheral blood of ISSHL patients and healthy control subjects was assessed by ELISA before and after treatment.@*Results@#TLR4, NF-κB, and TNF-α levels were upregulated in ISSHL patients relative to healthy control subjects; the levels were decreased following treatment and were lower in the HBO group than that in the medicine group post-treatment ( < 0.05 and < 0.01).@*Conclusion@#HBOT alleviates hearing loss in ISSHL patients by suppressing the inflammatory response induced by TLR4 and NF-κB signaling.

The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels / 中华心血管病杂志

Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.

Hyperbaric oxygen improves functional recovery of rats after spinal cord injury via activating stromal cell-derived factor-1/CXC chemokine receptor 4 axis and promoting brain-derived neurothrophic factor expression / 中华医学杂志(英文版)

BACKGROUND@#Spinal cord injury (SCI) is a worldwide medical concern. This study aimed to elucidate the mechanism underlying the protective effect of hyperbaric oxygen (HBO) against SCI-induced neurologic defects in rats via exploring the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis and expression of brain-derived neurotrophic factor (BDNF).@*METHODS@#An acute SCI rat model was established in Sprague-Dawley rats using the Allen method. Sixty rats were divided into four groups (n = 15 in each group): sham-operated, SCI, SCI treated with HBO (SCI + HBO), and SCI treated with both HBO and AMD3100 (an antagonist of CXCR4; SCI + HBO + AMD) groups. The rats were treated with HBO twice a day for 3 days and thereafter once a day after the surgery for up to 28 days. Following the surgery, neurologic assessments were performed with the Basso-Bettie-Bresnahan (BBB) scoring system on postoperative day (POD) 7, 14, 21, and 28. Spinal cord tissues were harvested to assess the expression of SDF-1, CXCR4, and BDNF at mRNA and protein levels, using quantitative real-time polymerase chain reaction, Western blot analysis, and histopathologic analysis.@*RESULTS@#HBO treatment recovered SCI-induced descent of BBB scores on POD 14, (1.25 ± 0.75 vs. 1.03 ± 0.66, P < 0.05), 21 (5.27 ± 0.89 vs. 2.56 ± 1.24, P < 0.05), and 28 (11.35 ± 0.56 vs. 4.23 ± 1.20, P < 0.05) compared with the SCI group. Significant differences were found in the mRNA levels of SDF-1 (mRNA: day 21, SCI + HBO vs. SCI + HBO + AMD, 2.89 ± 1.60 vs. 1.56 ± 0.98, P < 0.05), CXCR4 (mRNA: day 7, SCI + HBO vs. SCI, 2.99 ± 1.60 vs.1.31 ± 0.98, P < 0.05; day 14, SCI + HBO vs. SCI + HBO + AMD, 4.18 ± 1.60 vs. 0.80 ± 0.34, P < 0.05; day 21, SCI + HBO vs. SCI, 2.10 ± 1.01 vs.1.15 ± 0.03, P < 0.05), and BDNF (mRNA: day 7, SCI + HBO vs. SCI, 3.04 ± 0.41 vs. 2.75 ± 0.31, P < 0.05; day 14, SCI + HBO vs. SCI, 3.88 ± 1.59 vs. 1.11 ± 0.40, P < 0.05), indicating the involvement of SDF-1/CXCR4 axis in the protective effect of HBO.@*CONCLUSIONS@#HBO might promote the recovery of neurologic function after SCI in rats via activating the SDF-1/CXCR4 axis and promoting BDNF expression.

Simultaneous determination of six components in water extract and alcohol precipitation liquid in Astragali Radix by HPLC-UV-ELSD / 中国中药杂志

A HPLC-UV-ELSD method was established for simultaneous determination of six components in two intermediates of Shenqi Fuzheng injection (SFI) and the feasibility of establishing quantitative analysis of multi-components by single marker (QAMS) methods on different detectors was further explored. Calycosin-7-O-β-D-glucoside and astragloside Ⅳ were selected as internal reference substances for respectively flavonoids and saponins, and relative correlation factors (RCF) of formononetin-7-O-β-D-glucoside, 9, 10-dimethoxypterocarpan-3-O-β-D-glucopyranoside, 2'-dihydroxy-3', 4'-dimethoxyisoflavan-7-O-β-D-glucopyranoside and astragloside Ⅱ were calculated. Eventually, quantitative results of the 14 samples were compared between QAMS and external standard method. The sample concentrations calculated by QAMS were similar with concentrations calculated by external standard method, and the absolute values of relative deviations were generally less than 5% according to the UV detection of flavonoids. On the basis of ELSD detection for saponins, however, the absolute values of relative deviation of the two methods ranged from 0.48% to 23.17%. The QAMS method built on ultraviolet (UV) detectors was stable and can be used as a substitute method to reduce the consumption of standard compounds; meanwhile, the accuracy of QAMS method built on evaporative light scattering detector (ELSD) was inferior to that of external standard method, and the working principle of ELSD and feasible concentration range remain to be further studied.

Bioinformatic and Expression Analysis of Ventricular Septal Defect-associated Long Non-coding RNA TUC40- / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the potential role of TUC40- in human and mouse embryonic heart development.</p><p><b>METHODS</b>Bioinformatics databases including NCBI,UCSC,and Uniprot and software including Clustal,DNAMAN,and MEGA 6 were used to collect information of TUC40- and uc.40-. The expression profile at key time points of heart development was investigated by strand-specific quantitative real time polymerase chain reaction.</p><p><b>RESULTS</b>Uc.40- was conservative in sequence, genomic location, and transcription factor binding sites across human and mouse. Pbx1/TUC40- showed negative trend during embryonic mouse heart maturation.</p><p><b>CONCLUSIONS</b>Various levels of conservation of uc.40- suggests similar functions of TUC40- in these two species. TUC40- may play its roles in human and mouse embryonic heart development by regulating Pbx1.</p>

Hyperbaric oxygen intervention on expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in spinal cord injury models in rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hyperbaric oxygen (HBO) intervention is a main therapeutic method and the curative effect has been certified for spinal cord injury (SCI), but the mechanisms of the neuroprotective effect of HBO on SCI remain elusive. This study aimed to observe the change in expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) after SCI at different time points and to investigate the neuroprotective mechanism of HBO on SCI in rats.</p><p><b>METHODS</b>A total of 160 adult Sprague-Dawley rats, weighing between 250 and 300 g, were randomly assigned to four experimental groups (n = 40 per group). SCI group: SCI was created with a special NYU impactor of Allen's by a 25 gramcentimeter impacting energy on T10 of the spinal cord. SCI+HBO group: HBO therapy after SCI model was established. Sham operation (SH) group: only laminectomy of T10 and no impact on the spinal cord was done. SH+HBO group: HBO therapy after sham operation. The hindlimb functional recovery was evaluated using Basso, Beattie, and Bresnahan (BBB) score and the expressions of HIF-1α and VEGF were observed with fluorescent quantitation PCR and Western blotting method of six rats picked randomly from each group at different time points of 1, 3, 7, and 14 days after operation.</p><p><b>RESULTS</b>Rats in the SCI group and SCI+HBO group were paralyzed completely after operation with BBB 0-1 score. Rats in the SH group and SH+HBO group could walk after sham operation with BBB 20-21 score. The BBB score of rats in the SCI+HBO group (4.67±1.97 and 10.83±2.23) was higher than that in the SCI group (1.83±0.75 and 6.67±2.16) at 7 and 14 days time points obviously (P < 0.05). The expressions of HIF-1a and VEGF in the SCI group and SCI+HBO group were higher than in the SH group and SH+HBO group at any time point obviously (P < 0.05), while the SCI+HBO group presented the least expression of HIF-1α mRNA and protein (3.82±0.41 and 0.59±0.06; 2.26±0.41 and 0.37±0.05; 1.58±0.26 and 0.29±0.05) than that in the SCI group (6.36±0.58 and 0.76±0.07; 3.55±0.47 and 0.51±0.07; 2.27±0.39 and 0.40±0.06) respectively at 3, 7, and 14 days time points (P < 0.05) with significant difference and more expression of VEGF mRNA and protein (5.83±0.77 and 0.72±0.06; 4.59±0.51 and 0.63±0.06) than that in the SCI group (3.06±0.30 and 0.48±0.07; 2.25±0.24 and 0.39±0.09) respectively at 7 and 14 days time points (P < 0.05) with significant difference.</p><p><b>CONCLUSIONS</b>HBO could improve the hind limb functional recovery after SCI in rats. The elevation and duration of the expression of VEGF and the reduction of expression of HIF-1α by HBO intervention may be inversely related in the repair of damaged spinal cord and neuroprotective effect.</p>

Effects of hyperbaric oxygen preconditioning on ischemia-reperfusion inflammation and skin flap survival / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hyperbaric oxygen preconditioning (HBO) is a new method of ischemia preconditioning. In this study, we examined its effects on skin flap survival and the mechanisms involved.</p><p><b>METHODS</b>Thirty-six rats were divided into three groups: HBO preconditioning, control, and sham groups. An extended epigastric adipocutaneous flap based on the right superficial epigastric artery and vein was raised. A 3-hour period of flap ischemia was induced by clamping the pedicle vessels with a microvascular clamp. At the end of ischemia induction, the clamp was removed and the flap was resutured. Rats in the HBO preconditioning group were treated with HBO four times before surgery. Microcirculation in the skin flap was measured on postoperative days 1, 3 and 5. The size of the flap was measured on postoperative day 5, before the animals were sacrificed. Samples of the skin flap were prepared and stained with hematoxylin and eosin. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the flap samples were measured.</p><p><b>RESULTS</b>Surviving flap size was significantly higher in the HBO preconditioning group compared with controls, with a reduced inflammatory response and increased perfusion. IL-1, TNF-α, and IL-6 levels in the HBO preconditioning group were lower than in controls.</p><p><b>CONCLUSIONS</b>HBO preconditioning improved flap survival in this ischemia-reperfusion rat model. The mechanisms responsible for this effect may relate to attenuation of the inflammatory response and increased flap perfusion following HBO preconditioning.</p>