ObjectiveA middle cerebral artery occlusion (MCAO) mouse model is established by electrocoagulation of the middle cerebral artery. The study examines the mechanism by which exosomes (EXO) derived from human amniotic mesenchymal stem cells (hAMSCs) improve ischemic stroke and regulate neural ferroptosis-related injury. MethodsThirty-two SPF-grade male C57BL/6J mice aged 6 - 8 weeks were randomly divided into four groups (n=8 per group): sham group (Sham), model group (MCAO), MCAO plus normal saline group (MCAO+NaCl), and MCAO plus exosome group (MCAO+EXO). The mouse MCAO model was established by electrocoagulation of the middle cerebral artery. Mice in the Sham group underwent exposure of the middle cerebral artery without electrocoagulation. Twenty-four hours before MCAO induction, mice in the MCAO+EXO group received a tail vein injection of 100 μL of exosomes derived from the culture supernatant of hAMSCs at a concentration of 9.5×10¹¹ particles/mL. Mice in the MCAO+NaCl group were injected with an equal volume of normal saline via the tail vein. Twenty-four hours after model establishment, neurological deficits were evaluated using the Longa neurological deficit scoring system. Cerebral infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Hematoxylin and eosin (HE) staining was performed to evaluate morphological changes of neurons in the ischemic brain regions. The contents of ferrous iron (Fe²⁺), malondialdehyde (MDA), total glutathione (total GSH), oxidized glutathione (GSSG), and reduced glutathione (GSH) in the infarct core and peri-infarct regions were determined using microcolorimetric assays to evaluate differences among groups. The mRNA expression levels of ferroptosis-related factors, including nuclear factor erythroid 2-related factor 2 (NRF2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in the infarct core and peri-infarct regions were measured by real-time quantitative PCR. Protein expression levels of NRF2, SLC7A11, and GPX4 in the infarct and peri-infarct regions of each group were analyzed by Western blotting. ResultsCompared with the MCAO group, the Longa neurological deficit score was significantly reduced in the MCAO+EXO group (P<0.01). Prominent cerebral infarction was observed in the MCAO group, whereas the infarct volume ratio was markedly decreased in the MCAO+EXO group compared with the MCAO group (P<0.001). Histopathological analysis revealed that mice in the MCAO group exhibited obvious neuronal damage, including cytoplasmic vacuolar degeneration, nuclear pyknosis and fragmentation, unclear nuclear structure, and disorganized neuronal arrangement, compared with the Sham group. In contrast, neurons in the MCAO+EXO group showed relatively preserved morphology, with intact cellular structures and large, regular nuclei located centrally within the cells. Biochemical analysis demonstrated that Fe²⁺ and MDA levels in the infarct core and peri-infarct regions were significantly increased in the MCAO group compared with the Sham group (P<0.001). These levels were significantly reduced in the MCAO+EXO group compared with the MCAO group (P<0.01). In addition, total glutathione (total GSH), oxidized glutathione (GSSG), and reduced glutathione (GSH) levels were markedly decreased in the MCAO group relative to the Sham group (P<0.01). Compared with the MCAO group, the MCAO+EXO group exhibited significantly increased levels of total GSH and GSH (P<0.001), while no significant change was observed in GSSG levels (P>0.05). Furthermore, both mRNA and protein expression levels of nuclear factor erythroid 2-related factor 2 (NRF2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were significantly downregulated in the MCAO group compared with the Sham group (P<0.01, P<0.001). In contrast, both mRNA and protein expression levels of NRF2, SLC7A11, and GPX4 were significantly upregulated in the MCAO+EXO group compared with the MCAO group (P<0.05). ConclusionIn the mouse MCAO model, tail vein injection of exosomes derived from hAMSCs can improve motor function, reduce infarct area, protect neuronal cell morphology, and reduce the degree of nerve injury. Exosomes may exert a protective effect by activating the NRF2/SLC7A11/GPX4 pathway and reducing ferroptosis in neuronal cells of MCAO model mice.

Objective:To study the crosstalk between the activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) - X-box binding protein 1 (XBP1) pathway in oxygen-glucose deprivation/reoxygenation (OGD/R)-injured mouse hippocampal neuronal cell line HT22.Methods:The OGD/R-injured HT22 cell model was used to observe the changes of the indicators of endoplasmic reticulum stress (ERS), cell viability, and apoptosis at different OGD/R time points (0, 3, 6, 12, and 24 hours). HT22 cells in the logarithmic growth phase were randomized into blank control group, control+ATF6 activator (AA147) group, control+IRE1 inhibitor (4μ8c) group, OGD/R model group, OGD/R+AA147 group and OGD/R+4μ8c group (10 μmol/L AA147 or 16 μmol/L 4μ8c was given during the whole process in the AA147 group and 4μ8c group). Western blotting was used to detect the expression of ERS-related proteins [glucose-regulated protein 78 (GRP78), phosphorylated-inositol-requiring enzyme 1 (p-IRE1), and phosphorylated-eukaryotic translation initiation factor-2α (p-eIF2α)], and apoptosis-related proteins (Bcl-2, Bax, caspase-3, and cleaved caspase-3). The mRNA of ERS-related genes, and ATF6 [homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (Herpud1), protein disulfide isomerase associated 4 (Pdia4) and Sel-1 suppressor of lin-12-like (Sel1L)] and spliced XBP1 [XBP1s, include DnaJ heat shock protein family member B9 (Erdj4), Sec24 related gene family, member D (Sec24d) and signal sequence receptor, gamma (Ssr3)] induced transcriptional response-related genes were measured by real-time quantitative polymerase chain reaction (RT-qPCR). A cell counting kit-8 (CCK-8) assay was used to detect the viability of HT22 cells. Immunofluorescence was utilized to test the expression of cleaved caspase-3.Results:Compared with the blank control group, the expression of ERS-related proteins p-IRE1 and p-eIF2α were significantly increased at 12 hours and 3 hours following OGD/R, respectively (p-IRE1/β-actin: 2.09±0.10 vs. 1.00±0.00, p-eIF2α/β-actin: 1.39±0.11 vs. 1.00±0.00, both P < 0.01). The mRNA expressions of ERS-related genes [ATF6, XBP1s, unspliced XBP1 (XBP1u), activating transcription factor 4 (ATF4), CCAAT/EBP homologous protein (CHOP)] were also upregulated in different OGD/R timepoint in HT22 cells, which indicated ERS was activated in OGD/R-stimulated HT22 cells. Compared with the OGD/R model group, the expression of protein p-IRE1 was not changed, but the mRNA of XBP1s and XBP1u were obviously downregulated in the OGD/R+AA147 group [XBP1s (2 -ΔΔCt): 0.76 (0.71, 0.92) vs. 1.13 (1.03, 1.29), XBP1u (2 -ΔΔCt): 0.29±0.05 vs. 0.52±0.04, both P < 0.01], whereas the expressions of XBP1s-induced transcriptional response downstream genes did not change significantly. Compared with the OGD/R model group, the protein of short-form ATF6 (sATF6) and GRP78 were not changed after administration of 4μ8c, neither was the mRNA expression of ATF6-induced transcriptional response-related genes. These results showed that the mRNA expression of XBP1s and XBP1u were inhibited by AA147-induced activation of ATF6, but no crosstalk was observed between the transcriptional response induced by ATF6 and XBP1s. Compared with the blank control group, the cell viability decreased significantly at OGD/R 3 hours [(44.64±5.12) % vs. (99.13±5.76) %, P < 0.01], the ratios of apoptosis-related proteins Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly increased at OGD/R 3 hours and OGD 0 hour, respectively (Bax/Bcl-2: 6.15±1.65 vs. 1.00±0.00, cleaved caspase-3/caspase-3: 17.48±2.75 vs. 1.00±0.00, both P < 0.01), which indicated that apoptosis was activated in OGD/R-treated HT22 cells. Compared with the OGD/R model group, the cell viability decreased significantly [(36.52±17.78)% vs. (69.90±9.43)%, P < 0.01], and the ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly upregulated in the OGD/R+AA147 group in HT22 cells (Bax/Bcl-2: 2.06±0.31 vs. 1.10±0.25, cleaved caspase-3/caspase-3: 3.35±0.59 vs. 0.55±0.09, both P < 0.01). Conclusion:Under our experimental conditions, no obvious crosstalk between the transcriptional response induced by ATF6 and XBP1s was observed, while ATF6 activation induced by AA147 suppressed mRNA expression of XBP1s and XBP1u and promoted cell death in OGD/R-treated HT22 cells.

BACKGROUND: The study aims to correlate osteogenesis with autophagy during the mineralization induction of MC3T3-e1 through exploring the expression of runt-related transcription factor 2 (RUNX2)/lysosomal-associated transmembrane protein 5 (LAMPT5). METHODS: The induction of mineralization in MC3T3-e1 was followed by detecting the expressions of osteogenesisrelated indexes such as RUNX2, alkaline phosphatase (ALP), osteocalcin (OCN), and LAPTM5 using RT-qPCR and Western blot from 0 to 14 days. Transmission electron microscope was utilised in visualizing the alterations of autophagosomes, which was followed by immunofluorescence detecting the subcellular localization of autophagy-related index sequestosome 1 (P62) and microtubule-associated protein 1 light 3 (LC3) protein and scrutinising the expression of P62 mRNA and P62 and LC3 proteins. RESULTS: Induction of MC3T3-e1 mineralization demonstrated an increased expression of osteogenesis-related indicators such as RUNX2, ALP, OCN, and LAPTM5 (p < 0.05), as evident from the results of RT-qPCR and Western blot. Meanwhile, the expression of autophagosomes increased one day after mineralization induction and then experienced a gradual decline, and enhanced expression of LC3 protein was noted on days 1–2 of mineralization induction but was then followed by a corresponding reduce. In contrast, a continuous increase was reported in the expression of P62 mRNA and protein, respectively (p < 0.05). Up- and down-regulating RUNX2/LAPTM5 expression alone confirmed the aforementioned results. CONCLUSION It was therefore proposed that RUNX2 may be responsible for an early increase and then a gradual decrease in LAPTM5-mediated autophagy through the regulation of its high expression. Meanwhile, increased LAPTM5 expression in osteogenic mineralization presumed that RUNX2/LAPTM5 promoted autophagy and osteogenic expression, which may play a bridging role in the regulation of autophagy and osteogenesis.

Objective:To compare the severity of brain injury between asphyxia and electrical stimulation induced cardiac arrest in rats.Methods:Forty-two healthy male Sprague-Dawley (SD) rats were randomized into sham group ( n = 6), asphyxia group ( n = 18) and electrical stimulation group ( n = 18). Rats in each group were given invasive mechanical ventilation and femoral blood vessels catheterization for monitoring blood pressure and fluid infusion. In the asphyxia group, the tracheal tube was clamped to induce cardiac arrest, and in the electrical stimulation group, the esophageal electrical stimulation was used to induce cardiac arrest, and cardiopulmonary resuscitation (CPR) was performed 4 minutes after cardiac arrest. In the sham group, only tracheal intubation and femoral artery intubation were performed after anesthesia, but cardiac arrest was not induced. Animals were allowed to survive until 72 hours after resuscitation, and survival analysis was performed using Kaplan-Meier curves. At 24 hours and 72 hours after resuscitation, the neurological deficit score (NDS) was measured. The vena cava blood was collected, and the brain injury associated serum biomarkers, neuron-specific enolase (NSE) and S100B, were detected by enzyme-linked immunosorbent assay (ELISA). The brain tissues were then harvested to perform hematoxylin-eosin (HE) staining for observing pathological changes in the hippocampal CA1 area with light microscopy. Results:Cardiac arrest was successfully induced in both the asphyxia group and the electrical stimulation group, 94.4% (17/18) and 88.9% (16/18) animals were resuscitated successfully in the two groups respectively. Kaplan-Meier curves analysis showed that 72-hour cumulative survival rate was similar in the asphyxia group and the electrical stimulation group (Log-Rank test: χ2 = 0.040, P = 0.841). Both asphyxia group and electrical stimulation group had higher NDS score than sham group at 24 hours after resuscitation (37.50±4.26, 32.17±4.02 vs. 8.33±2.33, both P < 0.01). NDS score showed a downwards trend at 72 hours after resuscitation in both model groups, and the decline was more significant in the electrical stimulation group, which was significantly different as compared with asphyxia group (14.00±2.89 vs. 26.33±4.84, P < 0.05). ELISA results showed that the levels of serum NSE at 24 hours after resuscitation in the asphyxia and electrical stimulation groups were significantly higher than those in the sham group (μg/L: 1.02±0.07, 1.02±0.02 vs. 0.87±0.02, both P < 0.05). NSE kept increasing at 72 hours after resuscitation in the asphyxia group, which showed significant difference as compared with sham group (μg/L: 1.03±0.05 vs. 0.87±0.02, P < 0.01). But it had almost recovered to the normal level in the electrical stimulation group without significant difference as compared with sham group (μg/L: 0.96±0.04 vs. 0.87±0.02, P > 0.05). There was no significant difference in S100B level at different time points after resuscitation among three groups. It was displayed under light microscope that there was no significant neuronal damage in the hippocampal CA1 area in the two model groups at 24 hours after resuscitation as compared with the sham group. At 72 hours, there were certain damages in the hippocampal CA1 area in both model groups, which were more obvious in the asphyxia group. Conclusions:Both cardiac arrest models induced by asphyxia and electrical stimulation show a certain degree of brain injuries after resuscitation. Brain injuries are more severe in asphyxia-induced cardiac arrest compared with trans-esophageal electrical stimulation method.

With the popularization of cardiopulmonary resuscitation (CPR) technology, the success rate of restoration of spontaneous circulation (ROSC) is gradually improved, and the survival rate and neurological outcome of patients with cardiac arrest are improved. Currently, therapeutic methods for cerebral resuscitation after cardiac arrest are limited. In addition to mild hypothermia for clinical application, the majority of drugs remain in the animal experimental stage. Finding effective brain protection drugs has become a hot spot in the field of brain resuscitation research. This article will review the pharmaceutical progress of research for cerebral resuscitation after cardiac arrest, so that we can study the brain protection mechanism of these drugs better and more targeted.
Cerebrovascular Circulation/drug effects* ; Heart Arrest/drug therapy* ; Humans ; Pharmaceutical Research/trends* ; Resuscitation/methods*

Objective To investigate the spectrum of CYP21A2 gene mutation and the correlation between genotype and phenotype in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas.Methods Genomic DNA was extracted from the peripheral blood samples of the proband.Locus-specific PCR,direct sequencing of PCR amplification products,and multiplex ligation-dependent probe amplification were applied to detect pathogenic gene CYP21A2 and the relationship between genotypes and phenotypes was analyzed.Results (1) Of 35 patients with 21-hydroxylase deficiency,25 were classified as salt-wasting phenotype and 10 were simple virilizing phenotype.(2) 69 mutant alleles were detected in a total of 70 alleles in 35 patients.Only one mutant allele was detected in one patient.Two mutant alleles were detected in all other patients,with the mutation detection rate 98.6％.(3) A total of 6 types of mutations were detected,of which c.293-13C/A＞G (I2G) was the most common,accounting for 57.1％ (40/70),followed by 18.6％ (13/70) for large gene deletion or conversion,and 14.3％ (10/70) for p.I173N.In addition,a novel mutation,c.949C＞T (p.R317X),which has not been reported previously,was detected as a pathogenic mutation.(4) Correlation analysis of genotype and phenotype in 35 children showed that the phenotype predicted by genotype was consistent with the actual salt-wasting phenotype in 31 children,and those in three children were inconsistent with the actual clinical phenotype.Conclusion The mutation characteristics of CYP21A2 gene in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas are slightly different from those reported in other regions in China.A mutation c.949C＞T has not been reported,which enriches the mutation spectrum of CYP21A2 gene and provide the foundation for genetic counseling and prenatal diagnosis.

Objective To compare the differences in cardiac functions and myocardial injury between asphyxia and trans-oesophageal pacing induced rat cardiac arrest models.Methods Healthy adult male Sprague-Dawley (SD) rats were randomly divided into sham group,asphyxia group and electrical stimulation group by random number table.The rats in the latter two groups were randomly divided into two subgroups (24 hours and 72 hours)according to the sampling time after successful resuscitation,with 6 rats in each group.All rats were mechanically ventilated for 20 minutes,in electrical stimulation group,cardiac arrest was induced by trans-oesophageal cardiac pacing for about 3 minutes (intensity 30 V,frequency 50 Hz,pulse duration 2 ms),and in asphyxia group,cardiac arrest was induced by clipping trachea for about 3 minutes.Cardiopulmonary resuscitation (CPR) was initiated 4 minutes after cardiac arrest.Echocardiographic examination was performed at 2 hours after return of spontaneous circulation (ROSC) with cardiac color ultrasound apparatus.Cardiac tissues were harvested at 24 hours and 72 hours after ROSC,hematoxylin-eosin (HE) staining was performed,and myocardial damage was observed under light microscope.The levels of cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP) in serum were determined by enzyme-linked immunosorbent assay (ELISA).Results There was no significant difference in ROSC rate between the asphyxia group and electrical stimulation group [94.4％ (17/18) vs.88.9％ (16/18),P ＞ 0.05].The heart rate (HR),mean arterial pressure (MAP),left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) at 2 hours after ROSC in asphyxia group and electrical stimulation group were significantly lower than those in sham group [HR (bpm):401.50± 19.76,370.67± 18.63 vs.430.17± 18.38,MAP (mmHg,1 mmHg =0.133 kPa):107.17± 12.92,92.50±9.35 vs.125.67±5.72,LVEF:0.60±0.02,0.54±0.03 vs.0.63±0.01,LVFS:(48.40±2.52)％,(40.33±3.32)％ vs.(55.47 ± 2.38)％,all P ＜ 0.05],and the decrease in electrical stimulation group was more significant (all P ＜ 0.05).Compared with sham group,the levels of cTnI and BNP in serum of electrical stimulation group were significantly increased at 24 hours after ROSC [cTnI (ng/L):51.57±13.04 vs.38.23±5.57,BNP (ng/L):1 919.61±823.22 vs.977.47 ±445.18,both P ＜ 0.05],but there was no significant difference in cTnI or BNP of serum between asphyxia group and sham group [cTnI (ng/L):46.84 ± 11.04 vs.38.23 ± 5.57,BNP (ng/L):1 144.13±390.05 vs.977.47 ± 445.18,both P ＞ 0.05].There was no significant difference in cTnI or BNP of serum at 72 hours after ROSC among all the groups.The results of HE stain showed that the pathological injury of myocardium in electrical stimulation group was more serious than that in asphyxia group,characterized by more severe myocardial edema and partial myocardial cell lysis.Conclusion The cardiac function after cardiac arrest-CPR was decreased in both asphyxia group and electrical stimulation group,but electrical stimulation had a heavier cardiac function injury than asphyxia.

Objective: To investigate the influencing factors of the dose coverage of unplanned internal mammary lymph node (IMN) irradiation in patients receiving chemotherapy after mastectomy. Methods: Clinical data of 138 patients receiving radiotherapy in the upper and lower lymph node drainage area of the thoracic wall and clavicle [three-dimensional conformal radiotherapy (3DCRT), field-in-field forward intensity-modulated radiotherapy (F-IMRT) or inverse IMRT (I-IMRT)] were retrospectively analyzed. The IMN was delineated according to the Radiation Therapy Oncology Group (RTOG) criteria. The unplanned irradiation dose of the IMN was obtained. The correlation between the IMN irradiation dose, clinical characteristics and specific parameters of radiotherapy during the unplanned irradiation was statistically analysed. Results: The mean dose of unplanned IMN irradiation was 32.85 Gy (range: 2.76-50.93 Gy). In total, 7.3% of breast cancer patients obtained the therapeutic dose of≥ 45 Gy. Body weight, body mass index (BMI), body surface area (BSA) and thoracic transverse diameter (D_T) were lower, whereas the planning target volume of IMN (VIMN) included in the chest wall PTV (IMNin) and the ratio of IMNin to V_IMN were higher compared with those of their counterparts with insufficient therapeutic dose. Multivariate regression analysis demonstrated that body weight, thoracic anteroposterior diameter (D_AP), D_T, R_IMNin and PTV volume were the influencing factors of the dose coverage of unplanned IMN irradiation (P=0.000, 0.000, 0.001, 0.000 and 0.034). Conclusions For patients receiving chemotherapy after mastectomy, the dose coverage significantly varies when the IMN is the unplanned target. Partial patients achieve the therapeutic dose. The dose coverage of unplanned IMN irradiation is influenced by physical characteristics, anatomical features and technical parameters of radiotherapy, which should be emphasized during the study design and result analysis.

Objective To investigate the clinical effect of minimally invasive extraction of anterior tooth residual root after root separation.Methods A total of 400 patients receivinganterior tooth residual root extraction were collected in the clinic of oral and maxillofacial surgery department between January 2015 and December 2016.The patients were divided into a control group and a study group according to their sequence to see the doctor,with an odd for the study group and an even for the control group.In the study group,residual roots were separated mesiodistally by high speed turbine before using minimally invasive extraction tool;while in the control group residual roots were extracted only using minimally invasive extraction tool.The surgical duration,postoperative damage rate of the lip side plate,degree of pain and patient satisfaction in the two groups were analyzed.Results The surgical duration was shorter in the study group compared with the control group (P ＜ 0.05).The postoperative damage rate of the lip side plate and the degree of pain were lower,while patient satisfaction was higher in the study group than in the control group (P ＜ 0.05).Conclusions The postoperative damage rate of the lip side plate is significantly lower in minimally invasive extraction of anterior tooth residual root after root separation.Smaller trauma is conducive to the implant afterwards.Root separation in minimally invasive extraction of anterior tooth residual root is valuable for clinical application.

Objective: To evaluated the unplanned coverage dose to the internal mammary chain (IMC) in patient treated with postmastectomy radiotherapy (PMRT). Methods: One hundred and thirty eight patients with breast cancer receiving radiotherapy (RT) in our hospital were retrospectively analyzed. Patients were divided into three groups: three-dimensional conformal radiotherapy (3D-CRT) group, forward intensity-modulated radiotherapy (F-IMRT) group and inverse IMRT (I-IMRT) group. The IMC were contoured according to Radiation Therapy Oncology Group (RTOG) consensus, and were not include into the planning target volume (PTV). The incidental irradiation dose to IMC among the three groups and the first three intercostal spaces IMC (ICS-IMC 1-3) were all compared, and explored the relationship between the mean doses (Dmean) of IMC and the OARs (ipsilateral lung and heart). Results: The dose delivered to IMC showed no difference in CRT, F-IMRT and I-IMRT(33.80 Gy, 29.65 Gy and 32.95 Gy). And 10.42%, 2.04%, and 9.76% patients achieved ≥45 Gy when treated with CRT, F-IMRT and I-IMRT. For the IMC dose in the first three intercostal spaces (ICS1-3), there was no difference to the three treatment plannings. The Dmean, V₂₀, V₃₀, V₄₀ and V₅₀ of the ICS-IMC2 and ICS-IMC3 were all obviously superior than ICS-IMC1 for all these three plannings. Moderate positive correlation was founded between Dmean for IMC and Dmean for heart for left breast cancer patients underwent CRT (r=0.338, P=0.01). Whereas for F-IMRT and I-IMRT groups, positive correlation were founded between Dmean for IMC and Dmean and V₂₀ for ipsilateral lung for all patients (F-IMRT: r=0.366, P=0.010; r=0.318, P=0.026; I-IMRT: r=0.427, P=0.005; r=0.411, P=0.008). Conclusions In 3D-CRT, F-IMRT and I-IMRT planning methods, partial patients get IMC irradiated doses that could achieve therapeutic doses. Compared with 3D-CRT, F-IMRT and I-IMRT further reduced the dose of irradiated organs. However, there is no difference in the dose coverage of IMC for the three planned approaches when the IMC made an unplanned target.