Objective:To evaluate the effect of tubastatin A (TubA) on pyroptosis during brain injury after cardiac arrest and resuscitation in swine.Methods:Twenty-two conventional male white swine, weighing 34-39 kg, aged 4-6 months, were divided into 3 groups using a random number table: sham operation group (group S, n=6), cardiac arrest-cardiopulmonary resuscitation (CA-CPR) group ( n=8) and CA-CPR+ TubA group ( n=8). The swine model of CA-CPR was established by 9 min of cardiac arrest and 6 min of cardiopulmonary resuscitation in CA-CPR group and CA-CPR+ TubA group. TubA 4.5 mg/kg (in 50 ml of normal saline) was infused over 1 h via the femoral vein starting from 5 min after resuscitation in CA-CPR+ TubA group. Before developing the model and at 1, 2, 4 and 24 h after resuscitation (T 0-4), blood samples were collected from the femoral vein for determination of the concentrations of neuron specific enolase (NSE) and S100β protein in serum (by enzyme-linked immunosorbent assay). Neurological deficit score (NDS) was evaluated at T 4. The animals were then sacrificed, and their brain cortex tissues were harvested to measure the expression of histone deacetylase 6 (HDAC6), caspase-3, cleaved caspase-3, gasdermin E (GSDME) and GSDME N-terminal (N-GSDME) (by Western blot) and contents of high mobility group box 1 (HMGB1), interleukin-1β (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results:Compared with group S, the serum concentrations of NSE and S100β were significantly increased at T 1-4, NDS was increased at T 4, the expression of HDAC6, caspase-3, cleaved caspase-3, GSDME and N-GSDME in brain cortex was up-regulated, and the contents of HMGB1, IL-1β and IL-18 were increased in CA-CPR and CA-CPR+ TubA groups ( P<0.05). Compared with group CA-CPR, the serum concentrations of NSE and S100β were significantly decreased at T 3, 4, NDS was decreased at T 4, the expression of HDAC6, caspase-3, cleaved caspase-3, GSDME and N-GSDME in brain cortex was down-regulated, and the contents of HMGB1, IL-1β and IL-18 were decreased in group CA-CPR+ TubA ( P<0.05). Conclusions:The mechanism by which TubA alleviates brain injury after cardiac arrest and resuscitation may be related to inhibition of pyroptosis in swine.

Objective:To explore the current status of surgery for portal hypertension to grasp current status and future development of surgery in China.Methods:This study is jointly sponsored by China Hepatobiliary & Pancreatic Specialist Alliance & Portal Hypertension Alliance in China (CHESS).Comprehensive surveying is conducted for basic domestic situations of surgery for portal hypertension, including case load, surgical approaches, management of postoperative complications, primary effects, existing confusion and obstacles, liver transplantation(LT), laparoscopic procedures and transjugular intrahepatic portosystemic shunt(TIPS), etc.Results:A total of 8 512 cases of portal hypertension surgery are performed at 378 hospitals nationwide in 2021.Splenectomy plus devascularization predominated(53.0%)and laparoscopy accounted for 76.1%.Primary goal is preventing rebleeding(67.0%) and 72.8% of hospitals used preventive anticoagulants after conventional surgery.And 80.7% of teams believe that the formation of postoperative portal vein thrombosis is a surgical dilemma and 65.3% of hospitals practiced both laparoscopy and TIPS.The major reasons for patients with portal hypertension not receiving LT are due to a lack of qualifications for LT(69.3%)and economic factors(69.0%).Conclusions:Surgery is an integral part of management of portal hypertension in China.However, it is imperative to further standardize the grasp of surgical indications, the handling of surgical operation and the management of postoperative complications.Moreover, prospective, multi-center randomized controlled clinical studies should be performed.

OBJECTIVE: To investigate the protective effect and potential mechanism of tubastatin A (TubA), a specific inhibitor of histone deacetylase 6 (HDAC6), on renal and intestinal injuries after cardiopulmonary resuscitation (CPR) in swine. METHODS: Twenty-five healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 10) and TubA intervention group (n = 9) using a random number table. The porcine model of CPR was reproduced by 9-minute cardiac arrest induced by electrical stimulation via right ventricle followed by 6-minute CPR. The animals in the Sham group only underwent the regular operation including endotracheal intubation, catheterization, and anesthetic monitoring. At 5 minutes after successful resuscitation, a dose of 4.5 mg/kg of TubA was infused via the femoral vein within 1 hour in the TubA intervention group. The same volume of normal saline was infused in the Sham and CPR model groups. Venous samples were collected before modeling and 1, 2, 4, 24 hours after resuscitation, and the levels of serum creatinine (SCr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (I-FABP) and diamine oxidase (DAO) in serum were determined by enzyme-linked immunoadsordent assay (ELISA). At 24 hours after resuscitation, the upper pole of left kidney and terminal ileum were harvested to detect cell apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) were detected by Western blotting. RESULTS: After resuscitation, renal dysfunction and intestinal mucous injury were observed in the CPR model and TubA intervention groups when compared with the Sham group, which was indicated by significantly increased levels of SCr, BUN, I-FABP and DAO in serum. However, the serum levels of SCr and DAO starting 1 hour after resuscitation, the serum levels of BUN starting 2 hours after resuscitation, and the serum levels of I-FABP starting 4 hours after resuscitation were significantly decreased in the TubA intervention group when compared with the CPR model group [1-hour SCr (μmol/L): 87±6 vs. 122±7, 1-hour DAO (kU/L): 8.1±1.2 vs. 10.3±0.8, 2-hour BUN (mmol/L): 12.3±1.2 vs. 14.7±1.3, 4-hour I-FABP (ng/L): 661±39 vs. 751±38, all P < 0.05]. The detection of tissue samples indicated that cell apoptosis and necroptosis in the kidney and intestine at 24 hours after resuscitation were significantly greater in the CPR model and TubA intervention groups when compared with the Sham group, which were indicated by significantly increased apoptotic index and markedly elevated expression levels of RIP3 and MLKL. Nevertheless, compared with the CPR model group, renal and intestinal apoptotic indexes at 24 hours after resuscitation in the TubA intervention group were significantly decreased [renal apoptosis index: (21.4±4.6)% vs. (55.2±9.5)%, intestinal apoptosis index: (21.3±4.5)% vs. (50.9±7.0)%, both P < 0.05], and the expression levels of RIP3 and MLKL were significantly reduced [renal tissue: RIP3 protein (RIP3/GAPDH) was 1.11±0.07 vs. 1.39±0.17, MLKL protein (MLKL/GAPDH) was 1.20±0.14 vs. 1.51±0.26; intestinal tissue: RIP3 protein (RIP3/GAPDH) was 1.24±0.18 vs. 1.69±0.28, MLKL protein (MLKL/GAPDH) was 1.38±0.15 vs. 1.80±0.26, all P < 0.05]. CONCLUSIONS TubA has the protective effect on alleviating post-resuscitation renal dysfunction and intestinal mucous injury, and its mechanism may be related to inhibition of cell apoptosis and necroptosis.
Male ; Animals ; Swine ; Abdominal Injuries ; Apoptosis ; Cardiopulmonary Resuscitation ; Kidney Diseases

Objective To study the effect of human CCR4-NOT transcription complex subunit 7 (CNOT7) gene knockdown on the immune microenvironment of HepG2 cells and explore its significance. Methods We designed a cell transfection protocol and performed the experiment with three groups:CNOT7-targeted knockdown group, control group, and CNOT7 overexpression group. The transfection efficiency was assessed using inverted fluorescence microscopy, and the expression level of CNOT7, transforming growth factor-β1 (TGF-β1), and nuclear factor-kappa B (NF-κB) p65 proteins was determined by Western blotting. The concentration of TGF-β1 secreted in the cell culture supernatant was measured by ELISA. The sensitivity of tumor cells to the killing function of natural killer (NK) cells was detected by flow cytometry. Results Compared with the control group, the expression level of TGF-β1 and NF-κB p65 proteins was significantly decreased in the CNOT7-targeted knockdown group, and the TGF-β1 concentration in the culture supernatant was also significantly reduced. However, in the CNOT7 overexpression group, the expression level of the two proteins and TGF-β1 concentration were significantly increased. NK cells were co-cultured with tumor cells, and the apoptosis rate of HepG2 cells transfected with CNOT7-specific shRNA was significantly increased. However, in the CNOT7 overexpression group, the apoptosis rate was significantly decreased. Conclusion CNOT7 forms the immune microenvironment of hepatocellular carcinoma. Targeted knockdown of CNOT7 can reduce TGF-β1 secretion and enhance the killing function of NK cells toward HepG2 cells.

Objective To analyze tumor immune microenvironment and related mechanisms in liver cancer.Methods We included 10 cases of hepatocellular carcinoma,hepatitis B patients and healthy volunteers from January 2015 to December 2017 in Shanxi Grand Hospital.We first detected the peripheral and local GM-CSF level in each group,detected myeloid-derived suppressor cells (MDSCs) GM-CSF and pathway-related protein expression.from liver cancer,tumor margin and normal liver tissue through flow cytometry and immunohistochemistry,Finally,we transfected the CCR4-NOT transcriptional complex subunit 7 (CNOT7) recombinant plasmid in the hepatoma cell line,and then detected the related protein expression.Results There was no significant difference for peripheral blood GM-CSF level between liver cancer group,hepatitis group and control group (P＞0.05).The level of local GM-CSF was (32.2±8.9) ng/L,which was higher than that of hepatocellular carcinoma (9.7±2.7) ng/L and normal liver tissue (11.6±2.9) ng/L.The difference was statistically significant (P＜0.05).The proportion of MDSCs at the edge of the tumor was (9.9 ±3.6) ％,which was higher than that of liver cancer (4.0± 1.5) ％ and normal liver tissue (6.3±2.3) ％,and the difference was statistically significant (P＜0.05).Immunohistochemistrydata was consistent with previous data.Compared with normal liver tissue,CNOT7 and STAT3 were highly expressed in liver cancer tissues,while STAT1 was lowly expressed.HepG2 human hepatoma cells were selected for transfection.Compared with the empty plasmid group,CNOT7 expression was decreased in the knocking out group at the same time STAT1 expression was increased,STAT3 and GM-CSF expression was decreased.Conclusion In hepatocellular carcinoma,the secretion of GM-CSF increased and the number of MDSCs increased.Knocking out CNOT7 reduced GM-CSF secretion and activate the JAK/STAT signaling pathway.

Objective To investigate the protective effects of a Yiqi Huatan Quyu formulation on liver injury in chronic intermittent hypoxia rats.Methods Thirty healthy male Sprague-Dawley rats of SPF grade were randomly divided into 3 groups:Group A(normoxia and normal saline gavage),Group B(chronic intermittent hypoxia and normal saline gavage)and Group C(chronic intermittent hypoxia and Yiqi Huatan Quyu formulation gavage)(n =10 in each group).After 8 weeks of treatment,the liver tissue was extracted for protein and RNA.Western blot was used to test the protein levels of B-cellymphoma-2 associated X protein (BAX) and B-cellymphoma-2 (BCL-2),and Realtime PCR was used to test gene expression of BAX and BCL-2.Transferase-mediated dUTP nick end-labeling(TUNEL)was used to assay the apoptotic rate of liver cells.Results The expression of the BAX protein and BAX gene was higher and the expression of the BCL-2 protein and BCL-2 gene was lower in Group B than in Group A(t =13.490 and 16.480,P =0.005 and 0.002;t =5.142 and 9.776,P =0.036 and 0.001,respectively).The levels of the BAX protein and BAX gene were lower and the levels of the BCL-2 protein and BCL-2 gene were higher in Group C than in Group B(t =6.088 and 15.240,P =0.026 and 0.005;t =5.296 and 16.380,P =0.034 and 0.004,respectively).The apoptosis rate of hepatocytes was higher in Group B than in Group A(t =7.698,P =0.002),and was lower in Group C than in Group B(t=4.345,P =0.012).Conclusions The Yiqi Huatan Quyu formulation may alleviate the apoptosis of liver cells in chronic intermittent hypoxia rats by upregulating BCL-2 and down-regulating BAX levels,thus exerting protective effects on the liver.

Objective To investigate the appropriate age of primary and secondary school students for cardiopulmonary resuscitation (CPR) training.Methods A total of 437 students aged 9-15 years at 3 to 6 grade in the primary schools or 1 to 2 grade in the secondary schools were selected from 2 Yuyao primary and secondary schools by stratified random sampling between March 2017 and January 2018.The numbers of students with the age of 9,10,11,12,13,14 and 15 y were 61,62,66,64,63,63 and 58,respectively.All students received chest compression training provided by Yuyao emergency department People's Hospital according to the 2015 Cardiopulmonary Resuscitation Guidelines.The training included 30 min theoretic teaching and 6 min practice in the simulator.The quality of chest compression performed by students was assessed;the depth,rate,position and retention of chest compression were recorded.Results The mean depth of chest compression in the students aged 9-15 years was 3.8,4.1,4.6,5.1,5.2,5.6 and 5.6 cm,respectively;the accuracy rate was 24.6％(14/61),25.8％ (16/62),50.2％ (33/66),70.5％ (45/64),79.4％(50/63),88.9％(56/63) and 91.4(53/58),respectively.Compared with the students aged 9-11 years,the mean depth of chest compression was significantly increased and accuracy rate was significantly improved in the students aged 12-15 years (Compared with 9-y students,t=-8.936,-9.502,-10.640 and-11.370;x2=35.019,47.599,63.013 and 65.671;compared with 10-y students,t=-6.927,-8.179,-10.70 and-11.047;x2=24.977,35.967,50.916 and 52.727;compared with 1 1-y students,t=-3.095,-4.177,-6.785 and-6.995;x2=5.586,12.114,22.786 and 24.870;all P＜0.05).The mean rate of chest compression was 110-116/min and its accuracy rate was 86.4％-95.2％;the accuracy rate of chest compression position was 90.9％-96.8％ in all students,there were no significant differences among the 7 groups.The mean retention rate of chest compression in the 7 groups was 81.3％(122/150),67.3％(101/150),64.7％ (94/150),48.0％(72/150),48.7％(73/150),33.3％(50/150) and 27.3％(41/150),respectively.Compared with the students aged 9-11 years,the mean retention rate of chest compression was significantly decreased in the students aged 12-15 years (compared with the 9-y students,x2=36.472,35.179,70.64 and 119.92;compared with 10-y students,x2=11.483,10.728,34.682 and 72.150;compared with 11-y students,x2=6.528,5.927,25.855 and 59.11;all P＜0.05).Correlation analysis showed that the depth (r=0.96,0.89,0.91 and 0.86;P＜0.01) and retention rate (r=-0.99,-0.90,-0.93 and-0.86;all P＜0.01) of chest compression were significantly associated with the age,body weight,height and body mass index of students.Conclusion The students with an age of 12 years or more are able to effectively perform chest compression;thus,12 years and above might be the appropriate age for CPR training.

Objective To study the regulation of dendritic cells by recombinant glycated polylysine-coupled MIP-3α-FL double-gene targeting expression vector in liver cancer immune microenvironment.Methods H22 hepatocarcinoma cells were transfected with recombinant plasmid of MIP-3α-FL (shMIP-3α-FL) and injected into hepatoma model mice.The survival time,tumor size were compared.Flow cytometry was used to measure the number and phenotype of tumor infiltrating DCs.Results Western blot and ELISA demonstrated that the secretion of MIP-3α and FL in H22 cells was significantly increased after transfection with MIP-3α-FL.The survival time of the mice in the experimental group was significantly prolonged,the tumor size decreased.Flow cytometry showed that the number of tumor-infiltrating DCs in the experimental group was significantly higher than that in the control group;the expression of CD80 and CD86 in the infiltrating dendritic cells (TIDCs) was significantly higher than that of the control group.Conclusions The co-action of MIP-3α and FL can significantly promote DC accumulation,maturation,and conjugate glycosylated polylysine carriers increase the precision of targeting and enhance the antigenpresentation of the DCs.

Objective To study the action of CNOT 7 (CCR4-NOT transcription complex subunit 7 human)gene and its mechanisms in the process of Vγ 9Vδ2T cell immunologic tolerance of HepG2 cells (Hepatoblastoma G2 Cell Line).Methods The shCNOT 7 (Recombinant plasmid of CNOT 7) and control vector shRNA were transfected into HepG2 cells.Vγ9Vδ2T cytokine stimulated each group before and after cell transfection,Cell apoptosis was detected by flow cytometry (FCM),CNOT 7 protein and STAT1,STAT3 expression level was detected by Western blot.CNOT 7,STAT1 and STAT3 protein expression levels of HepG2 liver cancer cell lines and L02 normal liver cell line was assayed by Western blot.Results When stimulated by Vγ9Vδ2T cytokine,the apoptosis rate of gene-knockdown group significantly improved from (7.55％ ±2.63％) to (20.59％ ±3.12％).Compared with L02 cells,the CNOT7 protein expression of HepG2 cells increased (F =28.76,P ＜ 0.01),STAT3 protein expression increased (F =110.29,P ＜ 0.01),while STAT1 protein expression was down-regulated (F =35.67,P ＜ 0.01).CNOT 7 knockout could induce HepG2 cells STAT1 expression (t =6.69,P ＜ 0.05).Conclusions CNOT 7 gene could induce HepG2 cells Vγ 9Vδ2T cellular immune tolerance.CNOT 7 knockout could reverse the Vγ 9Vδ2T cell immunologic tolerance of HCC.

OBJECTIVE To observe the efficacy of specific immunotherapy (SIT) with Alutard and NHD in children with allergic rhinitis due to different level of skin prick test (SPT) with dermatophagoides farinae (DF) and dermatophagoides pteronyssinus (DP). METHODS A total of 178 children with persistent allergic rhinitis were included in this study. Their age ranged from 6 to 12 years. They were divided into 3 groups according to level of SPT. Group 1: The level of skin index (SI) of DF is greater than that of DP, Group 2: The level of SI of DF is equal to that of DP and Group 3: The level of SI of DF is less than that of DP. The children in each group were randomly divided into 2 subgroups: Alutard group and NHD group. The children were given SIT with Alutard or NHD for one year. Symptom and medication scores were recorded and analyzed. RESULTS After receiving therapy for 3 months and 6 months, symptom and medication scores of the Group 1 and 2 in the NHD group were lower than those in the Alutard group (P<0.05); Symptoms and medication scores of the Group 3 in the Alutard group were lower than those in the NHD group (P<0.05). After receiving therapy for 9 and 12 months, the symptom and medication scores of the NHD and Alutard group in all the three groups showed no statistical difference (P>0.05). CONCLUSION The efficacy of SIT with Alutard and NHD is different in children with allergic rhinitis with different levels of SPT due to DF and DP after 3 and 6 months, but is similar after 9 and 12 months. SIT with Alutard and NHD is effective in treating children with allergic rhinitis.