Break the domestic public hospital clinical research evaluation system under the background of"five only" / 现代医院

Objective The purpose of this article is to summarize and review the current status of the construction of clinical research evaluation systems in domestic public hospitals,identify existing problems in the evaluation system,and propose development strategies and suggestions.Methods Retrieved relevant articles,dissertations and policies from the past five years(2018-2022),screened the titles,viewed the full texts of 52 selected papers and their references,and summarized them.Results The"five-only"indicators have long been an important indicator for evaluating clinical research in public hospitals,but in today's scientific research environment and policy environment,the"five-only"evaluation system has revealed its utilitarian draw-backs and gradually evolved into a hindrance to scientific research.It is urgent to break through the"five-only"orientation and establish a clinical research evaluation system oriented towards"transforming and applying transformation of scientific research achievements".Conclusion The evaluation system for clinical research should break the previous"five-only"evaluation model based on quantity-oriented scientific research evaluation.We can draw on the framework of the research output,influence,and environment indicators in the UK's REF Excellence Framework model,combine the American APT system and the Chinese STEM indicator dimensions,explore multi-outcome evaluation,integrate developmental indicators,and continuously improve the indica-tor system and application methods in practice to promote the development of clinical research in public hospitals.

Association between preoperative serum β 2-microglobulin concentrations and postoperative delirium in elderly patients / 中华麻醉学杂志

Objective:To evaluate the association between preoperative serum β 2-microglobulin (β 2MG) concentrations and postoperative delirium (POD) in elderly patients. Methods:The study selected patients who underwent knee or hip arthroplasty under spinal-epidural anesthesia on an elective basis at Qingdao Municipal Hospital from May 2021 to November 2022. The patients were divided into a POD group and a non-POD group based on the occurrence of POD. The study was conducted as part of the Perioperative Neurocognitive Impairment and Biomarkers Lifestyle Cohort, which was a nested case-control study. The study collected baseline data from two groups of patients and analyzed the differences between them. Logistic regression was used to identify the risk factors for POD. The stability of the regression model was tested using sensitivity analysis. The mediation model was used to examine whether cerebrospinal fluid (CSF) biomarkers mediated the relationship between β 2MG and POD. The receiver operating characteristic curve was drawn and the area under the curve was calculated to evaluate the accuracy of preoperative β 2MG concentrations and CSF biomarker concentration in predicting POD. Results:There were 57 cases in POD group and 449 cases in non-POD group. The results of logistic regression analysis showed that the increased β 2MG and CSF total tau protein (t-tau) concentrations were risk factors for POD, and the increased CSF β-amyloid 42 concentration was a protective factor for POD after adjustment for multiple confounders such as age, gender, education, Mini-Mental State Examination, history of hypertension and infusion volume ( P<0.05). The results of mediation analysis showed that the serum β 2MG′s effect on POD was partly mediated by t-tau (18.1%). The results of the receiver operating characteristic curve showed that the area under the curve of the β 2MG concentration combined with the CSF biomarker concentration was 0.742. Conclusions:Elevated preoperative serum β 2MG concentration is a risk factor for POD in elderly patients, and the relationship may be partly mediated by CSF t-tau.

Relationship between preoperative serum bilirubin concentration and postoperative delirium in patients undergoing knee or hip replacement / 中华麻醉学杂志

Objective:To evaluate the relationship between preoperative serum bilirubin concentrations and postoperative delirium (POD) in the patients undergoing knee or hip replacement.Methods:Medical records from 413 patients undergoing knee or hip arthroplasty were selected from August 2020 to October 2023 at Qingdao Municipal Hospital using a nested case-control design based on the PNDABLE study cohort. The patients were divided into POD group ( n=77) and non-POD group ( n=336) according to whether POD occurred. Univariate analysis was used to analyze the influencing factors. Logistic regression was used to identify the risk factors for POD. The significance of mediation effect was tested. The receiver operating characteristic curve was drawn to evaluate the accuracy of risk factors in predicting POD. Results:There were significant differences in age, education time, ratio of diabetes history, Memorial Delirium Assessment Scale score, total bilirubin concentration, direct bilirubin concentration, indirect bilirubin concentration, Aβ 42 concentration, p-tau concentration, t-tau concentration, Aβ 42/p-tau ratio and Aβ 42/t-tau ratio between POD group and non-POD group ( P<0.05). The results of logistic regression analysis showed that preoperative serum total bilirubin, direct bilirubin and indirect bilirubin were risk factors for POD ( P<0.05). The results of mediation effects showed that the concentration of total tau protein in CSF partly mediated the relationship between high serum total bilirubin, direct bilirubin and indirect bilirubin concentrations and POD. The area under the receiver operating characteristic curve of total bilirubin, direct bilirubin and indirect bilirubin combined with CSF biomarker concentrations in predicting POD was 0.83 ( P<0.001). Conclusions:Preoperative elevated concentrations of total bilirubin, direct bilirubin and indirect bilirubin are risk factors for POD in the patients undergoing knee or hip replacement. CSF t-tau concentration has a partly mediating role in the association between serum total bilirubin, direct bilirubin and indirect bilirubin concentrations and the development of POD.

The trend, problems discovered, and enlightenment to hospital management of medical insurance fund unannounced inspection / 中华医院管理杂志

The rational use of medical insurance fund(MIF) plays an important role in promoting the high-quality development of public hospitals, and the supervision of MIF is in a trend of under the rule of law, normalization, professionalization and standardization, and unannounced inspection will become the norm. The authors systematically analyzed three main trends of MIF unannounced inspections, namely, gradually increasing intensity, constantly innovating methods, and increasingly serious consequences. The problems exposed in unannounced inspections were sorted out from five dimensions: form of results, severity, scope of attribution, subjective intention, and regulatory screening ideas. The enlightenment of MIF unannounced inspections to hospital management was explored from four aspects: compliance awareness, organizational system, fine management, and daily supervision. It was proposed that public hospitals should transform their roles and positions, improve the working mechanism of departmental collaboration, and achieve fine management in policy understanding, system formulation, process design, information support, data governance, regulatory implementation, personnel training, and performance matching. At the same time, internal simulated unannounced inspections in hospitals should be regarded as a routine work.

Clinical characteristics and prognosis of 28 cases of infant acute lymphoblastic leukemia / 中华儿科杂志

Objective: To analyze the clinical characteristics and prognosis of patients with infant acute lymphoblastic leukemia (IALL). Methods: A retrospective cohort study.Clinical data, treatment and prognosis of 28 cases of IALL who have been treated at Beijing Children's Hospital, Capital Medical University and Baoding Children's Hospital from October 2013 to May 2023 were analyzed retrospectively. Based on the results of fluorescence in situ hybridization (FISH), all patients were divided into KMT2A gene rearrangement (KMT2A-R) positive group and KMT2A-R negative group. The prognosis of two groups were compared. Kaplan-Meier method and Log-Rank test were used to analyze the survival of the patients. Results: Among 28 cases of IALL, there were 10 males and 18 females, with the onset age of 10.9 (9.4,11.8) months. In terms of immune classification, 25 cases were B-ALL (89%), while the remaining 3 cases were T-ALL (11%). Most infant B-ALL showed pro-B lymphocyte phenotype (16/25,64%). A total of 22 cases (79%) obtained chromosome karyotype results, of which 7 were normal karyotypes, no complex karyotypes and 15 were abnormal karyotypes were found. Among abnormal karyotypes, there were 4 cases of t (9; 11), 2 cases of t (4; 11), 2 cases of t (11; 19), 1 case of t (1; 11) and 6 cases of other abnormal karyotypes. A total of 19 cases (68%) were positive for KMT2A-R detected by FISH. The KMT2A fusion gene was detected by real-time PCR in 16 cases (57%). A total of 24 patients completed standardized induction chemotherapy and were able to undergo efficacy evaluation, 23 cases (96%) achieved complete remission through induction chemotherapy, 4 cases (17%) died of relapse. The 5-year event free survival rate (EFS) was (46±13)%, and the 5-year overall survival rate (OS) was (73±10)%.The survival time was 31.3 (3.3, 62.5) months. There was no significant statistical difference in 5-year EFS ((46±14)% vs. (61±18)%) and 5-year OS ((64±13)% vs. (86±13)%) between the KMT2A-R positive group (15 cases) and the KMT2A-R negative group (9 cases) (χ2=1.88, 1.47, P=0.170, 0.224). Conclusions: Most IALL patients were accompanied by KMT2A-R. They had poor tolerance to traditional chemotherapy, the relapse rate during treatment was high and the prognosis was poor.

Relationship among subjective well-being,depression-anxiety-stress and dispositional mindfulness in professional athletes / 中国心理卫生杂志

Objective:To explore the relationship among subjective well-being and depression-anxiety-stress and dispositional mindfulness in professional athletes,and the role of cognitive fusion and experiential avoidance and age in the relationship.Methods:Totally 423 professional athletes were selected and assessed with the Satisfac-tion with Life Scale(SWLS),Positive Affect Scale(PAS),Depression-Anxiety-Stress Scale(DASS-21),Five Fac-et Mindfulness Questionnaire(FFMQ),Cognitive Fusion Questionnaire(CFQ),and Acceptance and Action Ques-tionnaire-Ⅱ(AAQ-Ⅱ).Results:After controlling for gender,the FFMQ scores were positively correlated with SWB scores(β=0.35),and negatively correlated with DASS-21 scores(β=-0.40).The scores of CFQ and AAQ-Ⅱplayed a sequential mediating role in the relationship between FFMQ scores and the scores of SWB and DASS-21(β=0.04,-0.12).Age moderated the relationship between the scores of FFMQ and SWB(β=-0.01).Conclu-sion:The subjective well-being and depression-anxiety-stress may be related to dispositional mindfulness in profes-sional athletes,and age could moderate the relationship between subjective well-being and dispositional mindful-ness.

Clinical features and management analysis of 11 cases of laryngocele / 中华耳鼻咽喉头颈外科杂志

Objective: To summarize clinical features and our experience of the diagnosis and treatment of laryngocele. Methods: Clinical data of 11 laryngocele patients in department of Otorhinolaryngology Head and Neck Surgery of the Second Affiliated Hospital of Shanxi Medical University from January 2012 to December 2021 were retrospectively reviewed, including 9 men and 2 women, aged from 12 to 75 years, with median age of 56 years. Electronic laryngoscope was performed in 10 of all patients, laryngeal CT in 10 and cervical color ultrasound in 5 before operation.All the operations were performed under general anesthesia, and the external cervical approach was used for external and combined laryngocele. The internal laryngocele was resected by low temperature plasma through transoral endoscopy. Patients were followed up regularly after operation to evaluate the effect. Clinical feature, types of lesions, imaging findings, surgical approaches and follow-up results were analyzed through descriptive statistical method. Results: Eleven laryngocele patients were divided into mixed type (n=6), internal type (n=4) and external type (n=1).Nine patients presented with hoarseness or dysphonia, 7 with cervical mass and 1 with airway obstruction. Surgical resections were done through external cervical approach (n=7)or transoral endoscopic approach (n=4). All the operations were successful and no complication occurred. All cases were followed up from 17 to 110 months. No recurrence was encountered. Conclusions: Laryngocele is a rare lesion with atypical clinical presentation. Preoperative imaging including CT scan and electronic laryngoscope is essential to evaluate the location, and extent of the lesion, and to make the surgical plan.Complete surgical excision is required. Surgical resection is the only effective method for the treatment of laryngocele.

Clinical and ASS1 gene variant analysis of three Chinese pedigrees affected with Citrullinemia type I / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical and genetic characteristics of three Chinese pedigrees affected with Citrullinemia type I (CTLN1).@*METHODS@#Three children diagnosed at the Children's Hospital Affiliated to Shandong University from 2017 to 2020 were selected as the study subjects. Genomic DNA was extracted from peripheral blood samples of the probands and their parents. Next generation sequencing (NGS) was carried out to detect pathological variants of the probands. Sanger sequencing was used for validating the candidate variant among the pedigrees.@*RESULTS@#The probands have respectively carried compound heterozygous variants of c.207_209delGGA and c.1168G>A, c.349G>A and c.364-1G>A, c.470G>A and c.970G>A of the ASS1 gene, which were respectively inherited from their parents.@*CONCLUSION@#The newly discovered c.207_209delGGA and c.364-1G>A variants have enriched the mutational spectrum of the ASS1 gene. And the mutation spectrum of Chinese CTLN1 patients is heterogeneous.

Analysis of clinical features and ATRX gene variants in a Chinese pedigree affected with X-linked alpha thalassemia mental retardation (ATR-X) syndrome / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical characteristics and genetic basis of two brothers featuring X-linked alpha thalassemia mental retardation (ATR-X) syndrome.@*METHODS@#An infant who had presented at the Qilu Children's Hospital in 2020 for unstable upright head and inability to roll over and his family were selected as the study subjects. The clinical features of the child and one of his brothers were summarized, and their genomic DNA was subjected to targeted capture and next generation sequencing (NGS).@*RESULTS@#The brothers had presented with mental retardation and facial dysmorphisms. NGS revealed that they had both harbored a hemizygous c.5275C>A variant of the ATRX gene located on the X chromosome, which was inherited from their mother.@*CONCLUSION@#The siblings were diagnosed with ATR-X syndrome. The discovery of the c.5275C>A variant has enriched the mutational spectrum of the ATRX gene.

Role of GSTM1 expression in mild hypothermia-induced mitigation of cerebral ischemia-reperfusion injury: relationship with microglial polarization / 中华麻醉学杂志

Objective:To evaluate the role of glutathione S-transferase μ1 (GSTM1) expression in mild hypothermia-induced mitigation of cerebral ischemia-reperfusion (I/R) injury and the relationship with microglial polarization.Methods:Eighty clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (S group), cerebral I/R group (I/R group), mild hypothermia group (H group), and GSTM1 inhibitor + mild hypothermia group (IH group). The rat model of cerebral I/R injury was prepared using the filament occlusion method. The filament was removed to restore blood flow after the left middle cerebral artery was blocked for 2 h, and the rats′ brain and rectal temperature were maintained at 36-37 ℃ during the period. The vessels were only isolated and ligated without occlusion in S group. In H group, the entire body was wiped with 75% ethanol immediately after removing the filament, and the brain and rectal temperatures were maintained at 32-33 ℃ for 3 h, and the other procedures were the same as those previously described in I/R group. In IH group, GSTM1 inhibitor itaconic acid 8.6 mg/kg was intraperitoneally injected at 24 and 1 h before developing the model, and the other procedures were the same as those previously described in H group. Neurological deficits were evaluated using a modified neurological severity score (mNSS) at 24 h of reperfusion, and then the animals were sacrificed and the brains were removed for observation of cerebral infarction (by TTC staining) and for determination of the expression of GSTM1, M1-type microglial marker inducible nitric oxide synthase (iNOS), and M2-type microglial marker arginase-1 (Arg-1) (by Western blot), expression of GSTM1, iNOS and Arg-1 mRNA (quantitative real-time polymerase chain reaction) and contents of interleukin-6 (IL-6), IL-10, tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) (by enzyme-linked immunosorbent assay). Results:Compared with S group, the mNSS and percentage of cerebral infarct size were significantly increased, and the expression of iNOS and Arg-1 protein and mRNA was up-regulated, the expression of GSTM1 and mRNA was down-regulated, and the contents of IL-6, TNF-α, IL-10 and TGF-β were increased in the other three groups ( P<0.05). Compared with I/R group and IH group, the mNSS and percentage of cerebral infarct size were significantly decreased, and the expression of iNOS protein and mRNA was down-regulated, the expression of Arg-1 protein and mRNA and GSTM1 was up-regulated, the contents of TNF-α and IL-6 were decreased, and the contents of TGF-β and IL-10 were increased in H group ( P<0.05). Conclusions:Up-regulated expression of GSTM1 is involved in mild hypothermia-induced mitigation of cerebral I/R injury, which is associated with inhibition of microglial polarization toward the M1 phenotype and promotion of polarization toward the M2 phenotype.

Advances of microglia and their exosomes in central neurodegenerative diseases / 中华老年医学杂志

Microglia are widely present in the central nervous system and participate in various pathophysiological processes.They play an important role in degenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.In recent years, the study of exosomes produced by microglia activation involved in the pathophysiological processes of various diseases has attracted extensive attention, but the role of exosomes has not been fully clarified.This article reviewed the characteristics and functions of microglia, the characteristics and functions of microglia-derived exosomes and their roles in central neurodegenerative diseases.

Effect of selective cerebral mild hypothermia on SUMO2/3 modification of Drp1 in a rat model of cerebral ischemia-reperfusion / 中华麻醉学杂志

Objective:To evaluate the effect of selective cerebral mild hypothermia on small ubiquitin-like modifier 2/3 (SUMO2/3) modification of dynamin-related protein 1 (Drp1) in a rat model of cerebral ischemia-reperfusion (I/R).Methods:Sixty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 240-260 g, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (S group), cerebral I/R group (I/R group), selective cerebral mild hypothermia group (HT group) and normal temperature group (NT group). The operation was performed under the monitoring of cerebral temperature and rectal temperature.Only the cervical blood vessels were exposed in S group, while focal cerebral I/R was induced by 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion in anesthetized animals in the other three groups.In HT group and NT group, 4 and 37 ℃ normal saline was perfused through the left internal carotid artery at a rate of 80 ml·kg -1·h -1 for 15 min, respectively. Modified neurological severity score (mNSS) was assessed at 24 h of reperfusion. Then the rats were sacrificed under deep anesthesia, brains were removed, brain tissues were obtained for determination of the percentage of cerebral infarct size (by TTC staining), and the ischemic penumbra tissues in the cerebral cortex were removed for examination of the ultra-structural changes of mitochondria (with a transmission electron microscope) and for determination of the SUMO2/3 modification of Drp1 (by CO-IP), expression of total Drp1 (T-Drp1) and total cytochrome c (T-Cytc) (by Western blot), and expression of mitochondrial outer membrane Drp1 (M-Drp1) and cytoplasmic Cytc (C-Cytc) (by Western blot) after isolation of mitochondria and cytoplasm. Results:Compared with S group, the mNSS and percentage of cerebral infarct size were significantly increased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was up-regulated, and SUMO2/3 modification of Drp1 in ischemic penumbra area was increased ( P<0.05), the fragmentation of mitochondria was aggravated, and cristae rupture and vacuolation were obvious in the other three groups. Compared with I/R group, the mNSS and percentage of cerebral infarct size were significantly decreased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was down-regulated, SUMO2/3 modification of Drp1 was increased ( P<0.05), the fragmentation of mitochondria was significantly attenuated, and cristae rupture and vacuolation were weakened in HT group. There were no significant differences in these detection parameters between NT group and I/R group ( P>0.05). Conclusions:The mechanism by which selective cerebral mild hypothermia alleviates the cerebral I/R injury is related to increased SUMO2/3 modification of Drp1, decreased binding of Drp1 to mitochondrial outer membrane, and reduced mitochondrial excessive fission in rats.

Progress of single-cell sequencing in lymphoma / 白血病·淋巴瘤

Single-cell sequencing (SCS) sequences the genetic information of a single cell to better understand the differences amongst cells and reveal the unique changes of each cell type. The specific analysis of cell subsets at the single-cell level can accurately evaluate tumor cells and microenvironment cells to reveal the complexity of molecular components and the difference from the corresponding components in non-malignant tissues. Lymphoma is highly heterogeneous, some have unknown pathological types, etiology and poor prognosis. SCS is helpful to clarify the molecular mechanisms of lymphomagenesis and pathological staging, and guide clinical practice. This article reviews SCS and its application in lymphoma.

Polydatin improves intestinal barrier injury after traumatic brain injury in rats by reducing oxidative stress and inflammatory response via activating SIRT1-mediated deacetylation of SOD2 and HMGB1 / 南方医科大学学报

OBJECTIVE@#To investigate the protective effect against intestinal mucosal injury in rats following traumatic brain injury (TBI) and explore the underlying mechanism.@*METHODS@#SD rat models of TBI were established by fluid percussion injury (FPI), and the specimens were collected at 12, 24, 48, and 72 h after TBI. Another 15 rats were randomly divided into shamoperated group (n=5), TBI with saline treatment (TBI+NS) group (n=5), and TBI with PD treatment (TBI+PD) group (treated with 30 mg/kg PD after TBI; n=5). Body weight gain and fecal water content of the rats were recorded, and after the treatments, the histopathology of the jejunum was observed, and the levels of D-lactic acid (D-LAC), diamine oxidase (DAO), ZO-1, claudin-5, and reactive oxygen species (ROS) were detected. Lipid peroxide (LPO) and superoxide dismutase (SOD) 2 content, jejunal pro-inflammatory factors (IL-6, IL-1β, and TNF- α), Sirt1 activity, SOD2 and HMGB1 acetylation level were also determined after the treatments.@*RESULTS@#The rats showed significantly decreased body weight and fecal water content and progressively increased serum levels of D-LAC and DAO after TBI (P < 0.05) with obvious jejunal injury, significantly decreased expression levels of ZO-1 and claudin-5, lowered SOD2 and Sirt1 activity (P < 0.05), increased expression levels of LPO, ROS, and pro-inflammatory cytokines, and enhanced SOD2 and HMGB1 acetylation levels (P < 0.05). Compared with TBI+NS group, the rats in TBI+PD group showed obvious body weight regain, increased fecal water content, reduced jejunal pathologies, decreased D-LAC and DAO levels (P < 0.05), increased ZO-1, claudin-5, SOD2 expression levels and Sirt1 activity, and significantly decreased ROS, LPO, pro-inflammatory cytokines, and acetylation levels of SOD2 and HMGB1 (P < 0.05).@*CONCLUSION@#PD alleviates oxidative stress and inflammatory response by activating Sirt1-mediated deacetylation of SOD2 and HMGB1 to improve intestinal mucosal injury in TBI rats.

m7G-lncRNAs are potential biomarkers for prognosis and tumor microenvironment in patients with colon cancer / 南方医科大学学报

OBJECTIVE@#To assess the value of m7G-lncRNAs in predicting the prognosis and microenvironment of colorectal cancer (CRC).@*METHODS@#We screened m7G-lncRNAs from TCGA to construct an m7G-lncRNAs risk model using multivariate Cox analysis, which was validated using ROC and C-index curves. Calibration and nomogram were used to predict the prognosis of CRC patients. Point-bar charts and K-M survival curves were used to assess the correlation of risk scores with the patients' clinical staging and prognosis. CIBERSORT and ESTIMATE were used to explore the association between the tumor microenvironment and immune cell infiltration in patients in high and low risk groups and the correlation of risk scores with microsatellite instability, stem cell index and immune checkpoint expression. A protein-protein interaction network was constructed, and the key targets regulated by m7G-lncRNAs were identified and validated in paired samples of CRC and adjacent tissues by immunoblotting.@*RESULTS@#We identified a total of 1722 m7G-lncRNAs from TCGA database, from which 12 lncRNAs were screened to construct the risk model. The AUCs of the risk model for predicting survival outcomes at 1, 3 and 5 years were 0.727, 0.747 and 0.794, respectively. The AUC of the nomogram for predicting prognosis was 0.794, and the predicted results were consistent with actual survival outcomes of the patients. The patients in the high-risk group showed more advanced tumor stages and a greater likelihood of high microsatellite instability than those in the low-risk group (P < 0.05). The tumor stemness index was negatively correlated with the risk score (r=-0.19; P=7.3e-05). Patients in the high-risk group had higher stromal cell scores (P=0.0028) and higher total scores (P=0.007) with lowered expressions of activated mast cells (r=-0.11; P=0.045) and resting CD4+ T cells (r=-0.14; P=0.01) and increased expressions of most immune checkpoints (P < 0.05). ATXN2 (P= 0.006) and G3BP1 (P=0.007) were identified as the key targets regulated by m7G-lncRNAs, and their expressions were both higher in CRC than in adjacent tissues.@*CONCLUSION@#The risk model based on 12 m7G-lncRNAs has important prognostic value for CRC and can reflect the microenvironment and the efficacy of immunotherapy in the patients.

Optimization of the extraction technology of modified Tabusen- 2 and its inhibitory effects on osteoclast differen- tiation / 中国药房

OBJECTIVE To optimize the extraction technology of modified Tabusen- 2（MT-2），and to investigate inhibitory effects of the extract obtained by the optimal technology on osteoclast differentiation. METHODS The index components of MT- 2 process optimization were selected by using network pharmacology. Based on single factor tests ，the extraction technology of MT- 2 was optimized by Box-Behnken design-response surface methodology according to the comprehensive score of contents of above index components ，and then validated. RAW 264.7 cells were induced by receptor activator of nuclear factor-κB ligand（100 ng/mL） to prepare osteoclast differentiation model. Inhibitory effects of MT- 2 extract（18.6，37.2，74.4 ng/mL）obtained by the optimal technology on osteoclast differentiation were investigated. RESULTS The index components screened by network pharmacology included chlorogenic acid ，terpineol diglucoside ，isochlorogenic acid A ，1，5-dicaffeoylquinic acid ，hydroxysafflower yellow A ， ginsenoside Rg 1 and ginsenoside Rb 1. The optimal extraction technology of MT- 2 was ethanol volume fraction of 60％ ，the solid-liquid ratio of 1 ∶ 14（g/mL），extraction time of 94 min and extraction times of twice. The average comprehensive score obtained by the three validation experiments was 95.50，and the relative error with the predicted value （95.75）was －0.26％. Compared with osteoclastic differentiation model cells ，the cells treated with MT- 2 extract prepared by the optimal technology were mostly mononuclear round cells ，and the number of osteoclasts decreased significantly （P＜0.05），its inhibitory effects tended to strengthen with the increase of drug concentration. CONCLUSIONS The optimal extraction technology of MT- 2 is stable and feasible. Obtained extract can inhibit osteoclast differentiation.

Role of exosomes in neuronal injury induced by M1 microglia / 中华麻醉学杂志

Objective:To evaluate the role of exosomes in neuronal injury induced by M1 microglia.Methods:Liposolysaccharide 100 ng/ml and interferon-γ (IFN-γ)20 ng/ml were added to well-growing BV2 microglia to induce the polarization of microglia into M1 phenotype.Cell supernatant of M1 microglia was collected and M1 microglia exosomes (M1-exo) were extracted with exosome kit.The well-growing N2a cells were divided into 4 groups ( n=24 each) using a random number table method: control group (group C), M1 microglia group (group M), exosome group (group E), and exosome inhibitor+ M1 microglia group (group G+ M). The cells in group C were conventionally cultured, the cells in group M were cultured with the supernatant of M1 microglia for 24 h, and the cells in group E were cultured with M1 microglia-derived exosomes for 24 h. In G+ M group, exosome inhibitor GW4869 was added, M1 microglia were incubated for 24 h, then the supernatant was collected and added to N2a cells, and the cells were incubated for 24 h. Cell viability of N2a cells was measured by the cell counting kit 8 assay, cell apoptosis rate was determined by flow cytometry.The expression of apoptosis-related genes Bcl-2 and Bax mRNA was detected by quantitative real-time-polymerase chain reaction, and the expression of apoptosis-related genes Bcl-2 and Bax protein was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the apoptosis rate was increased, the expression of Bcl-2 protein and mRNA was down-regulated, and the expression of Bax protein and mRNA was up-regulated in the other three groups ( P<0.05). Compared with group M, the cell viability was significantly increased, the apoptosis rate was decreased, the expression of Bcl-2 protein and mRNA was up-regulated, and the expression of Bax protein and mRNA was down-regulated in group G+ M ( P<0.05). There was no significant difference in the above indexes between group E and group M ( P>0.05). Conclusions:M1 microglia can mediate neuronal injury via exosomes.

Role of miR-205-3p in oncosis in astrocytes subjected to oxygen-glucose deprivation and restoration: relationship with AQP4 / 中华麻醉学杂志

Objective:To evaluate the role of miR-205-3p in oncosis in astrocytes subjected to oxygen-glucose deprivation and restoration (OGD/R) and the relationship with aquaporin4 (AQP4).Methods:Primary astrocytes were cultured in vitro to the logarithmic growth phase and divided into 5 groups ( n=16 each) using a random number table method: control group (C group), OGD/R group (O group), OGD/R+ miR-205-3p mimic group (M group), OGD/R+ miR-205-3p inhibitor group (I group), and OGD/R+ negative control group (NC group). Cells were cultured routinely in C group.Cells were subjected to 4 h of oxygen-glucose deprivation in a 37℃ anaerobic incubator (containing 94% N 2, 1% O 2 and 5% CO 2) followed by restoration of O 2-glucose supply for 24 h in O group.Cells in M, I and NC groups were transfected with miR-205-3p mimic, miR-205-3p inhibitor and miR-205-3p negative control for 48 h, respectively, and then cells were subjected to 4 h of oxygen-glucose deprivation followed by restoration of O 2-glucose supply for 24 h. The cell viability was evaluated by CCK-8 assay, the cell injury and oncosis were analyzed by flow cytometry, the expression of AQP4 mRNA was detected by quantitative reverse transcription-polymerase chain reaction, and the expression of AQP4 and porimin was detected by Western blot. Results:Compared with C group, the expression of miR-205-3p was significantly down-regulated, the cell viability was decreased, the rates of cell injury and oncosis were increased, and the expression of AQP4 protein and mRNA and porimin was up-regulated in O group ( P<0.05). Compared with O group, the expression of miR-205-3p was significantly up-regulated, the cell viability was increased, the rates of cell injury and oncosis were decreased, and the expression of AQP4 protein and mRNA and porimin was down-regulated in M group, the expression of miR-205-3p was significantly down-regulated, the cell viability was decreased, the rates of cell injury and oncosis were increased, and the expression of AQP4 protein and mRNA and porimin was up-regulated in I group ( P<0.05), and no significant changes were found in NC group( P>0.05). Conclusions:miR-205-3p is involved in oncosis in astrocytes subjected to OGD/R, which is associated with regulation of AQP4 expression.

Role of miR-20a-5p in M1 microglia aggravating oxygen-glucose deprivation and restoration-induced injury to neurons: relationship with MFN2 / 中华麻醉学杂志

Objective:To evaluate the role of miR-20a-5p in M1 microglia aggravating oxygen-glucose deprivation and restoration (OGD/R)-induced injury to neurons and the relationship with mitofusin2 (MFN2).Methods:The well-growing BV2 microglia (M0 type) were polarized into M1 phenotype by lipopolysaccharide (100 ng/ml) and IFN-γ (20 ng/ml) and identified by quantitative real-time polymerase chain reaction and immunofluorescence.The well-growing N2a cells were divided into 6 groups ( n=6 each) by the random number table method: control group (group C), OGD/R group, M0 microglia co-culture group (group M0), M1 microglia co-culture group (group M1), miR-20a-5p inhibitor transfection group (group I) and negative control group (group NC). The cells were routinely cultured in group C, and the cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply to develop the model of OGD/R injury in group OGD/R.The cells were subjected to OGD for 3 h and were co-cultured with M0 microglia for 24 h during restoration of oxygen-glucose supply in group M0.The cells were subjected to OGD for 3 h and were co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply in group M1.In group I and group NC, cells were transfected with miR-20a-5p inhibitor and negative control miRNA into M1 microglia, respectively, and N2a cells were subjected to OGD for 3 h and co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply.The cell viability was determined by cell counting kit-8 assay, amount of lactate dehydrogenase (LDH) released was determined, the expression of miR-20a-5p and MFN2 mRNA was detected by quantitative real-time polymerase chain reaction, and MFN2 expression was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the amount of LDH released was increased, and the expression of MFN2 protein and mRNA was down-regulated in the other five groups, miR-20a-5p expression was significantly up-regulated in OGD/R, M0 and M1 groups, and miR-20a-5p expression was significantly down-regulated in group I ( P<0.05). There were no significant differences in the cell viability, amount of LDH released, and expression of miR-20a-5p, MFN2 protein and mRNA between group OGD/R and group M0 ( P>0.05). Compared with group OGD/R and group M0, the cell viability was significantly decreased, the amount of LDH released was increased, and the expression of MFN2 protein and mRNA was down-regulated, and miR-20a-5p expression was up-regulated in group M1 ( P<0.05). Compared with group M1, the cell viability was significantly increased, the amount of LDH released was decreased, the expression of MFN2 protein and mRNA was up-regulated, and miR-20a-5p expression was down-regulated in group I ( P<0.05). Conclusions:The mechanism by which M1 microglia aggravates OGD/R-induced damage to N2a cells may be related to the up-regulation of miR-20a-5p expression in M1 microglia and the inhibition of MFN2 expression in N2a cells.

Role of exosomes in M2 microglia-induced reduction of oxygen-glucose deprivation and restoration injury to astrocytes / 中华麻醉学杂志

Objective:To evaluate the role of exosomes in M2 microglia-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury to astrocytes.Methods:The primary astrocytes were cultured in vitro to the logarithmic growth phase and divided into 5 groups ( n=14 each) using a random number table method: control group (group C), OGD/R group (group O), OGD/R+ M2 microglia group (O+ M2 group), OGD/R+ M2 microglia+ GW4869 group (O+ M2+ G group) and OGD/R+ M2 microglia-derived exosome group (O+ M2-E group). Cells in group C were cultured routinely.Cells in group O were subjected to 4 h of oxygen-glucose deprivation (OGD) and 24 h of restoration of O 2-glucose supply.In group O+ M2, cells were subjected to 4 h of OGD, and the supernatant of M2 microglia 2 ml was added to the medium during restoration of O 2-glucose supply, and the cells were cultured for 24 h. In group O+ M2+ G, cells were subjected to 4 h of OGD, and the supernatant of M2 microglia 2 ml treated with the exosome inhibitor GW4869 10 μmol/L was added to the medium during restoration of O 2-glucose supply, and the cells were cultured for 24 h. In group O+ M2-E, cells were subjected to 4 h of OGD, and the M2 microglia-derived exosome 10 μg/ml was added to the medium during restoration of O 2-glucose supply, and the cells were cultured for 24 h. The morphological changes of cells were observed with a light microscope, the cell viability was detected by CCK-8 assay, the expression of aquaporin 4 (AQP4) mRNA was detected by quantitative real-time polymerase chain reaction, and the expression of AQP4 and porimin was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the expression of AQP4 protein and mRNA and porimin was up-regulated ( P<0.05), and cell swelling occurred in the other four groups.Compared with group O, the cell viability was significantly increased, and the expression of AQP4 protein and mRNA and porimin was down-regulated in O+ M2 and O+ M2-E groups ( P<0.05), and no significant change was found in the parameters mentioned above ( P>0.05), and the cell viability was significantly attenuated in group O+ M2+ G.Compared with group O+ M2, the cell viability was significantly decreased, and the expression of AQP4 protein and mRNA and porimin was up-regulated in group O+ M2+ G ( P<0.05), and no significant change was found in the parameters mentioned above ( P>0.05), and the degree of cell swelling was increased in group O+ M2-E. Conclusions:M2 microglia can mitigate OGD/R injury to astrocytes through exosomes.