Protection of Icariin Against Hydrogen Peroxide-Induced MC3T3-E1 Cell Oxidative Damage.

OBJECTIVE: The aim of the present study was to evaluate the potential protective mechanism of icariin against oxidative damage caused by hydrogen peroxide in MC3T3-E1 cells. METHODS: MC3T3-E1 cells were treated with different concentrations of icariin to explore the optimal dose of icariin. MC3T3-E1 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2 O2 ; 0, 0.1, 0.2, 0.5, 1, and 2 mM) for 24 h to induce oxidative damage and cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Then, cells were divided into five groups: control, H2 O2 (0.2 mM), icariin (0.1 µM) and H2 O2 (0.2 mM), + icariin (0.1 µM). Cell viability was detected by CCK-8 assay. In addition, the content of glutathione and superoxide dismutase and the activity level of malondialdehyde in these treatment groups were determined. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were also performed to measure the early and late osteogenesis, respectively. Protein expression of ß-catenin and cyclin D1 was measured by western blot assay. Then, we used an antagonist of Wnt/ß-catenin signaling pathway (DKK-1) and western blot analysis to further explore potential mechanism. RESULTS: After 24 h of exposure to 0.2 mM H2 O2 , the viability of MC3T3-E1 cells was significantly decreased compared to that of the control cells. We first found that icariin can promote cell proliferation of MC3T3-E1 cells in a dose-dependent manner, with the dosage 0.1 µM showing the best pro-proliferative effect. Furthermore, icariin could promote the protein expression of OSX and RUNX2. The results showed that icariin can reverse the inhibitory osteogenic effects of MC3T3-E1 caused by H2 O2 . In addition, icariin could increase the Wnt-signaling related proteins. The results showed that MC3T3-E1 cells in the H2 O2 (0.2 mM) + icariin (0.1 µM) + Wnt-signaling antagonist (DKK-1) group had weaker ALP and ARS staining compared with that observed in the control and H2 O2 (0.2 mM) + icariin (0.1 µM) groups. The ALP activity and calcium content were decreased in the 0.2 mM H2 O2 + 0.1 µM icariin + DKK-1 group compared to that observed in the 0.2 mM H2 O2 + 0.1 µM icariin group. CONCLUSION: The results showed that icariin can increase the viability of MC3T3-E1 cells, reverse the oxidative stress induced by H2 O2 and protect MC3T3-E1 cells against H2 O2 -induced inhibition of osteogenic differentiation, which may occur through the Wnt/ß-catenin signaling pathway.

Serologic Response to SARS-CoV-2 in COVID-19 Patients with Different Severity.

The immense patient number caused by coronavirus disease 2019 (COVID-19) global pandemic brings the urge for more knowledge about its immunological features, including the profile of basic immune parameters. In this study, eighty-eight reported COVID-19 patients in Wuhan were recruited from January to February, 2020, including 32 severe/critical cases and 56 mild/moderate cases. Their mean age was 56.43 years (range 17-83) and gender ratio (male/female) was 43:45. We tested SARS-CoV-2 RNA with commercial kits, investigated the level of serologic IgM and IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using magnetic particle chemiluminescence immunoassays, and compared the results of serologic tests and nucleic acid test (NAT). Among 88 patients, 95.45% were confirmed as positive by the combination of NAT and antibody test, which was significantly higher (P < 0.001) than by single nucleic acid test (73.86%) or serologic test (65.91%). Then the correlation between temporal profile and the level of antibody response was analyzed. It showed that seroconversion started on day 5 after disease onset and IgG level was rose earlier than IgM. Comparison between patients with different disease severity suggested early seroconversion and high antibody titer were linked with less severe clinical symptoms. These results supported the combination of serologic testing and NAT in routine COVID-19 diagnosis and provided evidence on the temporal profile of antibody response in patients with different disease severity.

The Cellular and Molecular Mechanisms Underlying Silver Nanoparticle/Chitosan Oligosaccharide/Poly(vinyl alcohol) Nanofiber-Mediated Wound Healing.

Wound healing is a complex pathophysiological process that occurs frequently in everyday pathology and remains a challenge during the treatment of trauma. Previously, we prepared silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) (PVA/COS-AgNP) nanofibers via an electrospinning technique. These nanofibers promoted the proliferation of human skin fibroblasts (HSFs) and the expression of transforming growth factor TGF-ß1 in the early stage of wound repair, although the specific mechanisms remain unclear. Therefore, considering that TGF-ß1 has emerged as a major modulator of wound healing, the objective of this study was to further understand whether the molecular mechanisms responsible for PVA/COS-AgNP nanofiber-mediated wound healing include the TGF-ß1/Smad signal transduction pathway. In this study, we used human skin fibroblasts (HSFs) to investigate the molecular and cellular mechanisms underlying PVA/COSAgNP nanofiber-mediated wound healing. Cell adhesion and proliferation experiments, immunofluorescence staining, hydroxyproline content measurements, flow cytometry, quantitative real-time PCR (qRT-PCR), and western blotting (WB) were used to analyze the wound healing mechanisms of human skin fibroblasts treated with various concentrations of PVA/COS-AgNP nanofibers and the combined application of silver nanofibers and SB431542 (an inhibitor of the TGF-ß1 receptor kinase). Our study showed that PVA/COS-AgNP nanofibers markedly promoted fibroblast proliferation, collagen synthesis, and cell adherence. We also found that treating fibroblasts with PVA/COS-AgNP nanofibers stimulated cell cycle progression from G1 into the S and G2 phases, reducing the proportion of cells in the G0/G1 phase and inducing S and G2/M arrest. Importantly, the cell factors associated with the TGF-ß1/Smad signal transduction pathway, such as TGF-ß1, TGFßRI, TGFßRII, pSmad2, pSmad3, collagen I, collagen III, and fibronectin were also up-regulated. Moreover, this enhancing effect was markedly inhibited by the TGFßRI receptor inhibitor, SB431542. Therefore, the PVA/COS-AgNP nanofibers used to accelerate wound healing do so by activating the TGF-ß1/Smad signal transduction pathway.

Alu RNA accumulation in hyperglycemia augments oxidative stress and impairs eNOS and SOD2 expression in endothelial cells.

Endothelial dysfunction resulting from oxidative stress and inflammation plays a dominant role in hyperglycemia-induced vasculopathy. While double-stranded RNA (dsRNA) accumulates in redox and inflammatory conditions, its precise role in hyperglycemia-associated endothelial dysfunction remains unclear. This study aimed to investigate whether and how endogenous dsRNA contributes to endothelial dysfunction via oxidative stress. We used a dsRNA-specific antibody J2 to detect and immunoprecipitate cellular dsRNA. Acquired dsRNA was recognized by cDNA library construction and DNA sequencing. Quantitative PCR, ELISA and immunoassays were performed to identify changes induced by acquired dsRNA in primary human umbilical vein endothelial cells (HUVEC). Our data showed that endogenous dsRNA homologous to Alu Sc subfamily accumulated in hyperglycemic HUVEC. Comparing Alu-transfected HUVEC with high-glucose treated HUVEC, we found that Alu RNA elicited the production of reactive oxygen species (ROS) and up-regulated interleukin-1ß (IL-1ß) expression and secretion in a similar manner as high-glucose treatment. Moreover, Alu RNA impeded the expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase 2 (SOD2), increased ROS production and activated nuclear factor NFκB by chemically scavenging ROS and inactivation of NFκB. The repressed expression of eNOS and SOD2 resulted from Alu RNA-mediated negative regulatory mechanisms. Our study uncovered endogenous Alu RNA accumulation in hyperglycemic endothelial cells that provoked endothelial oxidative stress and dysfunction by suppressing SOD2 and eNOS expression at both transcription and translation levels via NFκB signaling pathway. These findings suggest a novel regulatory mechanism that involves endogenous dsRNA in endothelial oxidative stress and dysfunction.

[Comparative study on the treatment of acromioclavicular joint dislocation: coracoclavicular ligament reconstruction combined with hook plate fixation or suture-anchor fixation].

OBJECTIVE: To investigate the clinical outcomes of acromioclavicula (AC) joint dislocation treated with coracoclavicular (CC) ligament reconstruction and hook plate fixation/suture-anchor fixation. METHODS: There were 105 patients with Rockwood type III or severer AC joint dislocations were randomly divided into two groups from February 2007 to April 2010. They were treated with CC ligament reconstruction using double bundle of Palmaris longus (hook plate fixation group, 54 cases), and subsequently fixed with hook plates or suture-anchors (suture-anchors group, 51 cases). Patients were followed up, and the AC distance and CC distance were measured on the postoperative X-ray films, and the outcomes were assessed according to Karlsson criteria and Constant-Murley shoulder score. Ranked data was analyzed with the use of χ(2) test and measurement data with two sample t test. RESULTS: Eighty-nine patients were followed up for 24-42 months, average 30 months. There were 46 cases in hook plate fixation group and 43 cases in suture-anchor fixation group, without significant difference in age, gender, injured side and Rockwood classification between both groups. Between the two groups, no statistical difference was detected in the AC and CC distance measured within 6 months after operation (P > 0.05). The AC and CC distances of hook plate fixation group measured in 24 months postoperatively were larger than those in suture-anchor fixation group, respectively (F = 1.904 and 1.854, P < 0.05). In hook plate fixation group, the AC and CC distances measured in 24 months postoperatively were larger than those measured in 6 month postoperatively, respectively (F = 1.863 and 1.842, P < 0.05). According to Constant-Murley shoulder score, the average score was 88.5 for hook plate fixation group and 92.7 for suture-anchor fixation group (F = 0.475, P = 0.017). According to Karlsson criteria, the excellent and good rate of the functional recovery was 95.4% in suture-anchor fixation group, better than hook plate fixation group (χ(2) = 4.564, P = 0.033). CONCLUSIONS: The clinical outcomes of AC joint dislocation treated with CC ligament reconstruction and suture-anchor fixation are better than those treated with CC ligament reconstruction and hook plate fixation. The AC and CC distances increase after the removal of hook plate, which may be associated with poor functional recovery.

[Effect of intravenous patient-controlled intravenous analgesia with small dose of ketamine during shock stage on cytokine balance in patients with severe burn].

OBJECTIVE: To investigate the influences of patient-controlled intravenous analgesia (PCIA) with small dose of ketamine solely or combined with fentanyl during shock stage on cytokine balance in patients with severe burn. METHODS: Forty-five patients with severe burn and hospitalized within 24 hours after injury were randomly divided into three groups (n=15 in each group): conventional analgesia therapy group (group CAT), patient controlled intravenous ketamine analgesia group (group PCIKA, intravenous small dose ketamine), and patient controlled intravenous ketamine and fentanyl analgesia group (group PCIKFA, intravenous small dose ketamine combined with fentanyl). In group CAT, patients received intramuscular injection of pethidine 50 mg and phenergan 25 mg when patients complained of pain. In group PCIKA, patients received intravenous infusion with a mixture of ketamine 20 g/L + droperidol 50 mg/L, and in group PCIKFA with a mixture of ketamine 10 g/L + fentanyl 5 mg/L + droperidol 50 mg/L. In two groups of PCIA, patients received loading dose of 1 ml, with background infusion of 1.5 ml/h. Pain scores and side effects of analgesics were determined or observed at the time points of before analgesia and 1, 8, 24, 48 hours after analgesia, and serum cytokines [including interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-alpha)] were measured at the same time points. RESULTS: Pain scores in PCIFA and PCIKFA groups were significantly lower than that of before analgesia and group CAT (all P<0.01). There were no significant differences in heart rate (HR) and mean artery pressure (MAP) in three groups. No significant difference in side effects was found in three groups. The serum levels of cytokines were significantly lower in two PCIA groups than those of group CAT (all P<0.01). CONCLUSION: Patient-controlled intravenous analgesia with small doses of ketamine or combined with fentanyl during shock stage in patients with severe burn give efficient and safe pain relief, and cytokine balance.