ObjectiveTo explore the mechanism by which Qidan Yifei Tongqiao granules （QDYF） alleviate nasal mucosal remodeling in allergic rhinitis （AR） via the interleukin-33 （IL-33）/growth stimulation expressed gene 2 （ST2）/interleukin-1 receptor accessory protein （IL-1RAP） signaling pathway from the perspective of Qi-replenishing and blood-activating therapy. MethodsFirst， according to the previous network pharmacology results， this study predicted the potential mechanisms of QDYF in treating AR by screening key pathways， components， and targets. Molecular docking was performed via AutoDock and PyMOL 2.5.5. Subsequently， a rat model of ovalbumin （OVA）-induced AR was used for validation through in vivo experiments. Forty-eight rats were assigned into 6 groups： Control， model， low-dose QDYF （QDYF-L， 4.04 g·kg^-1）， medium-dose QDYF （QDYF-M， 8.08 g·kg^-1）， high-dose QDYF （QDYF-H， 16.16 g·kg^-1）， and loratadine （0.9 mg·kg^-1）. After 14 days of intervention， behavioral scores of the rats were observed. The morphological changes of nasal mucosa tissue were observed by hematoxylin-eosin （HE） staining. Masson staining was used to observe collagen fiber deposition in the nasal mucosal tissue and to calculate the collagen volume fraction （CVF）. The expression of E-cadherin （E-cad） in the nasal mucosa tissue was detected by immunofluorescence. The serum levels of helper T cell 2 （Th2） cytokines interleukin-4 （IL-4）， interleukin-5 （IL-5）， and interleukin-13 （IL-13） as well as helper T cell 1 （Th1） cytokines interleukin-2 （IL-2） and interferon-γ （INF-γ） were quantified by enzyme-linked immunosorbent assay （ELISA）. The protein levels of transforming growth factor-beta 1 （TGF-β₁）， IL-33， ST2， and IL-1RAP in the nasal mucosa tissue were determined by Western blot. ResultsIL-33， ST2， and IL-1RAP had strong binding ability with the main active ingredients—wogonin， 7-methoxy-2-methylisoflavone， formononetin， naringenin， stigmasterol， and beta-sitosterol of QDYF， with the binding energy < -4.25 kcal⋅mol^-1（1 cal≈4.184 J）. The results of in vivo experiments showed that compared with the control group， the model group exhibited increased behavioral scores （P<0.05）， aggravated pathological damage of nasal mucosa， increased collagen fiber deposition and CVF （P<0.05）， elevated serum levels of IL-4， IL-5， and IL-13， up-regulated protein levels of TGF-β₁， IL-33， ST2， and IL-1RAP in the nasal mucosa （P<0.05）， down-regulated expression of E-cad， and declined serum levels of IL-2， IFN-γ， and IFN-γ/IL-4 ratio （P<0.05）. Compared with the model group， the QDYF groups and loratadine group showed reduced behavioral scores （P<0.05）， alleviated pathological damage of nasal mucosa， reduced collagen fiber deposition and CVF （P<0.05）， and up-regulated E-cad expression （P<0.05）. Compared with the model group， the QDYF-H group and the loratadine group showed raised levels of INF-γ and IFN-γ/IL-4 ratio （P<0.05）， declined serum levels of IL-4， IL-5， and IL-13， and down-regulated protein levels of TGF-β₁， IL-33， ST2， and IL-1RAP in the nasal mucosa （P<0.05）. In addition， the QDYF-H group exhibited an elevated serum IL-2 level （P<0.05）. The QDYF-M group showed down-regulated protein levels of TGF-β₁， IL-33 and IL-1RAP in the nasal mucosa （P<0.05）. The QDYF-L group demonstrated a down-regulated protein level of ST2 in the nasal mucosa （P<0.05）. ConclusionQDYF may regulate the Th1/Th2 balance through the IL-33/ST2/IL-1RAP signaling pathway， thereby ameliorating nasal mucosal tissue remodeling and alleviating AR.

OBJECTIVE: To investigate the mechanism of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC) in alleviating brain injury after resuscitation in swine with cardiac arrest (CA). METHODS: Twenty-nine healthy male large white swine were randomly divided into Sham group (n = 9), cardiopulmonary resuscitation (CPR) group (n = 10) and hESC-MSC group (n = 10). The Sham group only completed animal preparation. In CPR group and hESC-MSC group, the swine model of CA-CPR was established by inducing ventricular fibrillation for 10 minutes with electrical stimulation and CPR for 6 minutes. At 5 minutes after successful resuscitation, hESC-MSC 2.5×10⁶/kg was injected via intravenous micropump within 1 hour in hESC-MSC group. Venous blood samples were collected before resuscitation and at 4, 8, 24, 48 and 72 hours of resuscitation. The levels of neuron specific enolase (NSE) and S100B protein (S100B) were detected by enzyme linked immunosorbent assay (ELISA). At 24, 48 and 72 hours of resuscitation, neurological deficit score (NDS) and cerebral performance category (CPC) were used to evaluate the neurological function of the animals. Three animals from each group were randomly selected and euthanized at 24, 48, and 72 hours of resuscitation, and the hippocampus tissues were quickly obtained. Immunofluorescence staining was used to detect the distribution of hESC-MSC in hippocampus. Immunohistochemical staining was used to detect the activation of astrocytes and microglia and the survival of neurons in the hippocampus. The degree of apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: The serum NSE and S100B levels of brain injury markers in CPR group and hESC-MSC group were significantly higher than those in Sham group at 24 hours of resuscitation, and then gradually increased. The levels of NSE and S100B in serum at each time of resuscitation in hESC-MSC group were significantly lower than those in CPR group [NSE (μg/L): 20.69±3.62 vs. 28.95±3.48 at 4 hours, 27.04±5.56 vs. 48.59±9.22 at 72 hours; S100B (μg/L): 2.29±0.39 vs. 3.60±0.73 at 4 hours, 2.38±0.15 vs. 3.92±0.50 at 72 hours, all P < 0.05]. In terms of neurological function, compared with the Sham group, the NDS score and CPC score in the CPR group and hESC-MSC group increased significantly at 24 hours of resuscitation, and then gradually decreased. The NDS and CPC scores of hESC-MSC group were significantly lower than those of CPR group at 24 hours of resuscitation (NDS: 111.67±20.21 vs. 170.00±21.79, CPC: 2.33±0.29 vs. 3.00±0.00, both P < 0.05). The expression of hESC-MSC positive markers CD73, CD90 and CD105 in the hippocampus of hESC-MSC group at 24, 48 and 72 hours of resuscitation was observed under fluorescence microscope, indicating that hESC-MSC could homing to the damaged hippocampus. In addition, compared with Sham group, the proportion of astrocytes, microglia and apoptotic index in hippocampus of CPR group were significantly increased, and the proportion of neurons was significantly decreased at 24, 48 and 72 hours of resuscitation. Compared with CPR group, the proportion of astrocytes, microglia and apoptotic index in hippocampus of hESC-MSC group decreased and the proportion of neurons increased significantly at 24 hours of resuscitation [proportion of astrocytes: (14.33±1.00)% vs. (30.78±2.69)%, proportion of microglia: (12.00±0.88)% vs. (27.89±5.68)%, apoptotic index: (12.89±3.86)% vs. (52.33±7.77)%, proportion of neurons: (39.44±3.72)% vs. (28.33±1.53)%, all P < 0.05]. CONCLUSIONS Application of hESC-MSC at the early stage of resuscitation can reduce the brain injury and neurological dysfunction after resuscitation in swine with CA. The mechanism may be related to the inhibition of immune cell activation, reduction of cell apoptosis and promotion of neuronal survival.
Animals ; Heart Arrest/therapy* ; Cardiopulmonary Resuscitation ; Swine ; Humans ; Male ; Human Embryonic Stem Cells/cytology* ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/cytology* ; Phosphopyruvate Hydratase/blood* ; Brain Injuries/therapy* ; S100 Calcium Binding Protein beta Subunit ; Apoptosis ; Disease Models, Animal

Objective:To explore the protective efficacy of sulforaphane (SFN) in alleviating intestinal mucosal injury after resuscitation in pigs and its possible mechanism.Methods:This experiment was performed in the laboratory animal center, Zhejiang university. Using a random number table, twenty-four domestic healthy male white pigs were randomly divided into the Sham group, cardiopulmonary resuscitation (CPR) group, and SFN group, in which the Sham group had 6 pigs, and the other two groups had 9 pigs, respectively. The experimental parameters of 10 min of cardiac arrest and 6 min of CPR were chosen to establish the porcine model of CPR in the CPR and SFN groups. At 5 min after resuscitation, a dose of 2 mg/kg of SFN was infused via the femoral vein within 10 min in the SFN group. At 1 h, 2 h, 4 h, and 24 h after resuscitation, vein samples were collected, and then the levels of intestinal fatty acid binding protein (IFABP) and diamine oxidase (DAO) in serum were measured by ELISA. Subsequently, 6 pigs were chosen to be euthanized in each group, and then tissue samples were harvested from distal ileum to measure the level of cell apoptosis by TUNEL, the activities of superoxide dismutase (SOD) and catalase (CAT) and the contents of glutathione (GSH) and malondialdehyde (MDA) by biochemical method, the contents of 4-hydroxy-2-nonenal (4-HNE) by ELISA, the fluorescence intensity of reactive oxygen species (ROS) by immunofluorescence staining, and the expression levels of zonula occluden-1 (ZO-1), occludin, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) by Western blot. Continuous variables were compared with one way analysis of variance among the three groups, and Bonferroni test was used for further pairwise comparison.Results:During the observation period after resuscitation, the serum levels of biomarkers of intestinal mucosal injury including IFABP and DAO were significantly higher in the CPR and SFN groups than in the Sham group (all P<0.05). However, the serum levels of IFABP at 2 h, 4 h, and 24 h after resuscitation and the serum levels of DAO at 1 h, 2 h, 4 h, and 24 h after resuscitation were significantly lower in the SFN group than in the CPR group (all P<0.05). At 24 h after resuscitation, apoptotic index was significantly increased, SOD and CAT activities and GSH contents were significantly decreased, MDA and 4-HNE contents and ROS production were significantly increased, ZO-1 and occludin expression were significantly down-regulated, and Nrf2 and HO-1 expression were significantly up-regulated in the CPR and SFN groups when compared with the Sham group (all P<0.05). However, apoptotic index was significantly decreased, SOD and CAT activities and GSH contents were significantly increased, MDA and 4-HNE contents and ROS production were significantly decreased, and ZO-1, occludin, Nrf2, and HO-1 expression were significantly up-regulated in the SFN group when compared to the CPR group (all P<0.05). Conclusion:SFN could effectively protect against intestinal mucosal injury after resuscitation in pigs, and its mechanism was possibly related to the inhibition of oxidative stress and cell apoptosis via the activation of Nrf2/HO-1 pathway.

Objective:To establish pig cardiac arrest resuscitation model, and explore the effect of automatic compression synchronous ventilation (ACSV) on cardiopulmonary resuscitation in pigs.Methods:Twelve male white pigs with body weight of (38±3) kg were divided into ACSV group and intermittent positive pressure ventilation (IPPV) group with 6 pigs in each group by random number table method. A porcine cardiac arrest and resuscitation model was prepared with ventricular fibrillation induced by alternating current release via right ventricular electrode for 6 min and compression for 8 min. Mechanical chest external compression depth 5 cm, frequency 100 times/min. The tidal volume of ACSV group was 3 mL/kg and the frequency was 100 times/min. In the IPPV group, the tidal volume was 7 mL/kg and the frequency was 10 times/min. Arterial blood was drawn before resuscitation and at 1, 4 and 7min during resuscitation for blood gas analysis. Coronary perfusion pressure (CPP), end-respiratory carbon dioxide (ETCO 2) and carotid blood flow (CBF) were monitored during resuscitation. Stroke volume (SV) and global ejection fraction (GEF) were recorded by pressure monitoring catheter before and 1, 2 and 4 h after resuscitation. Venous blood samples were collected at each time point and 24 h after resuscitation to detect cardiac troponin I (cTnI), neuron specific enolase (NSE), alamine aminotransferase (ALT), creatinine (Cr), and intestinal fatty acid binding protein (IFABP). Results:(1) During resuscitation, CPP, ETCO 2 and CBF in ACSV group were slightly higher than those in IPPV group, but the differences between groups were not statistically significant. (2) There was no significant difference in pH, PaCO 2, HCO 3- and lactic acid between the two groups during resuscitation. The PaO 2 in ACSV group was higher than that in IPPV group, and the difference was statistically significant at 4 and 7 min. (3) The success rate of resuscitation in both groups was 83.3%, and there was no significant difference in SV and GEF before and after resuscitation. (4) After resuscitation, cTnI, NSE, ALT, Cr, iFABP and other indexes in ACSV group were lower than those in IPPV group, and there were statistically significant differences in cTnI at 24 h after resuscitation, ALT at 2 h and 24 h after resuscitation, and IFABP at 4 h and 24 h after resuscitation (all P<0.05). Conclusions:This study preliminarily suggested that the novel ACSV could significantly improve the oxygen supply level during cardiopulmonary resuscitation in pigs, while keeping the compression efficiency unchanged, avoiding hyperventilation, and reducing multiple organ damage after resuscitation, which is worthy of further study.

Objective:To investigate the correlation between serum vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and coagulation index and prognosis of differentiated thyroid cancer (TC) .Methods:136 TC patients admitted in Department of Head and Neck Surgery Shanxi Cancer Hospital from Jan. 2021 to Dec. 2022 were selected as TC group, and 80 patients with confirmed thyroid benign nodule during the same period were selected as control group. VEGF, vWF and coagulation indexes were compared between the two groups. The tumor free survival was calculated by Kaplan-Meier method.Results:Compared with the control group, the levels of VEGF, vWF, TT, Fbg in TC group were higher, and the levels of PT, APTT were lower ( P < 0.001). Compared with patients < 55 years old, the levels of VEGF, vWF, TT and Fbg were higher in patients ≥55 years old, and the levels of PT and APTT were lower ( P < 0.001). Compared with papillary carcinoma, the levels of VEGF, vWF, TT and Fbg were higher in follicular carcinoma patients, and the levels of PT and APTT were lower ( P < 0.001). The levels of VEGF, vWF, TT and Fbg were higher and the levels of PT and APTT were lower in patients with TNM stage III to IV than in patients with TNM stage I to II ( P < 0.001). Compared with patients without lymph node metastasis, VEGF, vWF, TT, Fbg levels were higher in patients with lymph node metastasis, and PT, APTT levels were lower ( P < 0.001). Compared with patients with tumor size < 2 cm, VEGF and vWF levels were higher in patients with tumor size ≥ 2 cm ( P < 0.001). Survival curve analysis showed that the tumor free survival of high VEGF, high vWF, high TT and high Fbg groups was shorter than that of low VEGF, low vWF, low TT and low Fbg groups (Log-rank=9.149, 8.856, 4.683, 5.867, P < 0.001) . Conclusion:VEGF, vWF and coagulation indexes in TC patients were correlated with clinicopathological features, and were helpful to predict postoperative tumor-free survival.

Objective:To investigate the correlation between serum vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and coagulation index and prognosis of differentiated thyroid cancer (TC) .Methods:136 TC patients admitted in Department of Head and Neck Surgery Shanxi Cancer Hospital from Jan. 2021 to Dec. 2022 were selected as TC group, and 80 patients with confirmed thyroid benign nodule during the same period were selected as control group. VEGF, vWF and coagulation indexes were compared between the two groups. The tumor free survival was calculated by Kaplan-Meier method.Results:Compared with the control group, the levels of VEGF, vWF, TT, Fbg in TC group were higher, and the levels of PT, APTT were lower ( P < 0.001). Compared with patients < 55 years old, the levels of VEGF, vWF, TT and Fbg were higher in patients ≥55 years old, and the levels of PT and APTT were lower ( P < 0.001). Compared with papillary carcinoma, the levels of VEGF, vWF, TT and Fbg were higher in follicular carcinoma patients, and the levels of PT and APTT were lower ( P < 0.001). The levels of VEGF, vWF, TT and Fbg were higher and the levels of PT and APTT were lower in patients with TNM stage III to IV than in patients with TNM stage I to II ( P < 0.001). Compared with patients without lymph node metastasis, VEGF, vWF, TT, Fbg levels were higher in patients with lymph node metastasis, and PT, APTT levels were lower ( P < 0.001). Compared with patients with tumor size < 2 cm, VEGF and vWF levels were higher in patients with tumor size ≥ 2 cm ( P < 0.001). Survival curve analysis showed that the tumor free survival of high VEGF, high vWF, high TT and high Fbg groups was shorter than that of low VEGF, low vWF, low TT and low Fbg groups (Log-rank=9.149, 8.856, 4.683, 5.867, P < 0.001) . Conclusion:VEGF, vWF and coagulation indexes in TC patients were correlated with clinicopathological features, and were helpful to predict postoperative tumor-free survival.

Objective:To evaluate the safety and efficacy of a novel domestic extracorporeal membrane oxygenation (ECMO) mainframe in a porcine model, and to provide the basis for further clinical application.Methods:Five domestic healthy male white pigs, weighing (51±4) kg, were selected. The ECMO system was established by using a novel ECMO mainframe with imported membrane oxygenator and pipeline, and continued to run for 72 hours. ECMO parameters are as follows: veno-arterial ECMO, centrifugal pump speed 3 000-3 500 r/min, continuous infusion of heparin anticoagulation to maintain the activate clotting time (ACT) of 140-200 s. Real-time monitoring of speed, flow, pressure before pump, pressure after pump, pressure after membrane and other equipment parameters, and the equipment performance was scored. The changes of hemodynamics, blood lactic acid and blood routine were monitored dynamically. Repeated measures analysis of variance was used to compare different time points within the group. At the end of the experiment, the thrombosis in the pump head and oxygenator was observed. The animals were sacrificed to obtain the tissue samples of the main organs for gross observation and pathological injury evaluation.Results:All animals successfully ran the ECMO system for 72 hours. (1) The centrifugal pump speed should be maintained at 3 029-3 483 r/min, the flow rate was maintained at 2.24-2.60 L/min, The pressure before the pump between minus 107.57 and minus 31.86 mmHg, the pressure after the pump was 197.50-282.43 mmHg, and the pressure after the membrane was 178.71-261.5 mmHg, all were in the normal range, and there was no significant difference between different time points (all P>0.05). The performance scores of the mainframe were all 4 points or above, indicating that the use requirements were met. (2) The heart rate of the animals was 50-80 beats /min, the mean arterial pressure was 85-115 mmHg, and the lactic acid was 0.996-2.25 mmol/L, all within the normal range, and there was no significant difference between different time points (all P>0.05). The free hemoglobin was 8.98-16.39 mg/L, and the hemoglobin was 6.58-7.52 g/L, both within a reasonable range, and there was no significant difference between different time points (all P>0.05). The platelet count was 69.6-231.6×10 9/L, and showed a continuous downward trend ( P<0.05). ACT was maintained at 135-169 s, which was within the target range, and there was no significant difference between different time points ( P<0.05). (3) At the end of the experiment, there was no obvious thrombosis in the pump head and oxygenator, no obvious thrombosis or infarction in the heart, brain, liver, lung and kidney, and no obvious hemorrhage or necrosis under the microscope. Conclusions:The ECMO established by the novel domestic ECMO mainframe combined with imported membrane oxygenator and pipeline ran smoothly for 72 hours, achieving the target of effect and safety.

Orthodontic tooth movement elicits alveolar bone remodeling and orofacial pain that is manifested by tooth mechanical hyperalgesia. Nerve growth factor (NGF) is upregulated in periodontium and may modulate tooth mechanical hyperalgesia. The objectives were to examine the role of NGF in tooth mechanical hyperalgesia and to elucidate the underlying mechanisms. Tooth mechanical hyperalgesia was induced by ligating closed coil springs between incisors and molars in Sprague-Dawley rats. Retrograde labeling was performed by periodontal administration of fluor-conjugated NGF and the detection of fluorescence in trigeminal ganglia (TG). Lentivirus vectors carrying NGF shRNA were employed to knockdown the expression of NGF in TG. The administration of agonists, antagonists, and virus vectors into TG and periodontium was conducted. Tooth mechanical hyperalgesia was examined through the threshold of biting withdrawal. Our results revealed that tooth movement elicited tooth mechanical hyperalgesia that could be alleviated by NGF neutralizing antibody and that NGF was upregulated in periodontium (mainly in periodontal fibroblasts) and TG. Retrograde labeling revealed that periodontal NGF was retrogradely transported to TG after day 1. Acid-sensing ion channel 3 (ASIC3) and NGF were co-expressed in trigeminal neurons and the percentage of co-expression was significantly higher following tooth movement. The administration of NGF and NGF neutralizing antibody into TG could upregulate and downregulate the expression of ASIC3 in TG, respectively. NGF aggravated tooth mechanical hyperalgesia that could be alleviated by ASIC3 antagonist (APETx2). Moreover, NGF neutralizing antibody mitigated tooth mechanical hyperalgesia that could be recapitulated by ASIC3 agonist (GMQ). NGF-based gene therapy abolished tooth mechanical hyperalgesia and downregulated ASIC3 expression. Taken together, in response to force stimuli, periodontal fibroblasts upregulated the expressions of NGF that was retrogradely transported to TG, where NGF elicited tooth mechanical hyperalgesia through upregulating ASIC3. NGF-based gene therapy is a viable method in alleviating tooth-movement-induced mechanical hyperalgesia.

OBJECTIVETo explore the impact of neutrophil gelatinase-associated lipocalin (NGAL) knockdown by NGAL siRNA encapsulated with urocanic acid-modified chitosan nanoparticles (UAC) on the proliferation, migration and apoptosis of human colon cancer cells.

METHODSNGAL siRNA was encapsulated by UAC and chitosan (CTS) respectively, and then was transfected into human colon cancer cell lines HT29. The NGAL mRNA was detected by real-time quantitative PCR (RT-QPCR). Relationships of NGAL gene silencing with the proliferation, migration and apoptosis of HT29 cell were analyzed.

RESULTSUnder the fluorescence microscope, the transfection efficiency of siRNA in UAC group was (37.52±7.17)%, which was significantly higher than (11.32±3.39)% in CTS group (t=6.102, P=0.005). Forty-eight hours after transfection, RT-QPCR examination showed that the level of NGAL mRNA expression was 0.350 in UAC group and 0.529 in CTS group with significant difference (t=-3.743, P=0.02), meanwhile both levels were significantly lower as compared to control group(F=163.538, P<0.001). Proliferation analysis revealed that after silencing NGAL gene, proliferation rate of UAC group and CTS group was slightly lower than control group, and no significant differences were found (F=9.520, P=0.438). However, migration assay demonstrated that the 24-hour migration rate of UAC group and CTS group was significantly lower than that of control group (F=6.756, P=0.029), meanwhile the migration rate of UAC group was slightly lower than that of CTS group [(77.90±7.14)% vs. (87.67±3.98)%, t=-1.704, P=0.164]. Apoptosis detection revealed that the apoptosis rate in UAC group was significantly higher than that in CTS group and the control group 2 days after transfection [(15.800±1.054)% vs. (12.900±0.656)%, (11.933±1.914)%, F=7.004, P=0.027].

CONCLUSIONSThe encapsulated ability and transfection efficiency of chitosan modified by urocanic acid elevate significantly. Silencing NGAL gene by UAC carrier can down-regulate the expression of NGAL mRNA in HT29 colon cell line, inhibit their migration and facilitate their apoptosis.

Objective To explore the effect of NGAL knockdown by NGAL siRNA encapsulated with urocanic acid-modified chitosan nanoparticles (UAC).MethodsNGAL siRNA encapsulated by UAC and chitosan (CTS) respectively, which were then used to transfect human colon cancer cell lines HT29.The expression level of NGAL protein were detected by Enzyme Linked Immunosorbent Assay(ELISA).ResultsThe ELISA study revealed that the expression level of NGAL protein in UAC group(average 0.583μg/L) was significantly lower than in CTS group (average 0.772μg/L) and control group(average 1.071μg/L) (P<0.05).ConclusionThe NGAL expression of mRNA and protein in HT29 cells could be down-regulated by siRNA encapsulated by UAC.