Aim To find out the relationship between muscarinic receptor and reactive oxygen species (ROS) and the probable differences between the four muscarinic receptor subtypes. Methods We transfected the plasmid encoding muscarinic receptor (including subtypes: M_1, M_2, M_3 and M_4) into PC12 cells. Then PC12 cells were exposed to hydrogen peroxide (H_2O_2), carbachol and other inhibitors such as atropine, LY294002 and PD98059. Results The results showed that activation of muscarinic receptor by carbachol protected PC12-M_1, PC12-M_2,PC12-M_3 and PC12-M_4 cells from apoptosis induced by H_2O_2. There was no statistical difference in the protective effect between these four muscarinic receptor subtypes. By using the inhibitors, we found that atropine and LY294002 blocked the protective effect of activation of muscarinic receptor on apoptosis induced by H_2O_2. Conclusion Activation of muscarinic receptor retarded the apoptosis induced by H_2O_2. There was no difference between the four muscarinic receptor subtypes. The protective effect was mainly mediated by the activation of muscarinic receptor and phosphatidylinositol-3 kinase (PI3K).

Objective:To investigate the value of chest CT quantitative index in clinical classification and lung injury severity evaluation of COVID-19.Methods:The current study retrospectively analyzed the clinical and CT data of 438 patients with COVID-19 between January 2020 and March 2020 in Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology. The clinical types included common type ( n=146), severe type ( n=247) and critical type ( n=45). The chest CT indexes of all patients were quantitatively analyzed by artificial intelligence (AI) deep learning, including whole lung volume, CT lung opacification, ground glass opacification volume (GGO volume; CT value<-300 HU), solid opacification volume (SO volume; CT value ≥-300 HU) and the ratio of volume to the whole lung volume, the ratio of SO volume to GGO volume (SO volume/GGO volume). Kruskal-Wallis test was used to conduct statistical analysis of the differences in quantitative parameters among clinical types, and multiple ordered logistic regression was used to analyze the correlation between quantitative parameters and clinical types. Results:Among the 438 patients diagnosed with COVID-19, severe and critical patients were older ( P<0.05), and most of the critical patients were male ( P<0.05). The main clinical manifestations of all clinical types were fever, followed by cough, fatigue, chest tightness, dyspnea, gastrointestinal symptoms and so on. GGO volume was the main CT manifestation of all the three clinical subtypes. The whole-lung opacification volume, GGO volume, SO volume and their proportions in whole-lung volume significantly increased from common, severe to critical types (all P<0.05). SO volume/GGO volume increased with the severity of clinical type [common type 0.12 (0.03, 0.34), severe type 0.29 (0.11, 0.59), critical type 0.61 (0.39, 0.97)]. Multiple ordered logistic regression analysis showed that whole-lung opacification volume (OR=1.009), SO volume/GGO volume (OR=1.866), GGO volume (OR=1.008) and SO volume (OR=1.016) had a significant positive effect on the severity of clinical typing ( P<0.01). Conclusion:Quantitative indicators of chest CT based on deep learning algorithm (SO volume, GGO volume, SO volume/GGO volume) are closely related to the clinical severity of COVID-19.

AIM To investigate the protection of plicamycin on apoptosis in cerebellar granule neurons (CGN) of rat. METHODS TUNEL, Hoechst 33258 staining, agarose gel electrophoresis and fluorescein diacetate staining were used to detect morphological and biochemical characteristics of apoptosis in primary rat CGN. RESULTS Being pre-incubated with plicamycin for 1 h and lasting for 24 h, rat CGN apoptosis induced by low potassium basal modified Eagle′s medium for 24 h was inhibited in a plicamycin concentration-dependent manner. This effective concentrations of plicamycin were from 50 to 200 nmol·L-1, and the maximum inhibitory rate of plicamycin on CGN apoptosis was near 80% at 200 nmol·L-1. CONCLUSIONPlicamycin inhibits rat CGN apoptosis induced by low potassium.

AIM: To analyze the subcellular localization of Arnt2 in rat cerebellar granule neurons (CGNs). METHODS: Based on the amino acids sequence of Arnt2 (LOCUS:NP_036913), the subcellular localization of Arnt2 in eukaryotic cells and the nuclear export signals (NES) of Arnt2 were predicted in CBS bioinformatics database. The subcellular localization of Arnt2 in rat cerebellar granule neurons was detected by the method of laser scanning confocal microscopy (LSM) analysis. RESULTS: It was predicted that Arnt2 located in nuclei of eukaryotic cells with the most probability, while located in cytoplasmic mitochondria with a slight possibility. A nuclear export signal was found in Arnt2 amino acids sequence, it was identified to be the leucine of No.143 that located in N-terminal of Arnt2 amino acids sequence. Finally, the result of LSM analysis shows nuclear localization of Arnt2 in rat CGNs. CONCLUSION: Arnt2 is located in nuclei of normal rat CGNs, it suggests that Arnt2 has the tendency to translocate into mitochondria after induced by some of inducible factors, for both the possibility of mitochondria localization and NES exist in Arnt2 amino acids sequence.

AIM: To Screen and identify differentially expressed genes that involved in apoptosis model in rat cerebellar granule neurons (CGNs).METHODS: The rat cerebellar granule neurons were isolated and primarily cultured. Fluorescent differential display RT-PCR (FDD RT-PCR) was performed to screen differentially expressed ESTs in the apoptosis model of primarily cultured rat CGNs. ESTs were subcloned into pGEM-T EasyTM vector and then sequenced. Alignment assay in non-redunant database was applied for encoding information. Reverse Northern blotting was used to appraise the results from DDRT-PCR.RESULTS: 164 pieces of differentially expressed ESTs were obtained by FDDRT-PCR. 17 of them were subcloned and sequenced. 5 ESTs of 17 were confirmed to be positive results by reverse Northern blotting. CONCLUSION: DD-PCR is a rapid, simple-operation and sensitive method for screening differentially expressed genes, which would contribute to the molecular mechanisms of apoptosis/survive of CGNs.

BACKGROUND:Most bone marrow mesenchymal stem cel s are infused intravenously and have very low efficiency of homing to the bone marrow. However, cel infusion via the femoral approach is little reported. OBJECTIVE:To explore the distribution of luciferase gene modified red fluorescent protein transgenic bone marrow mesenchymal stem cel s in vivo through different infusion routes. METHODS:Luciferase gene modified bone marrow mesenchymal stem cel s at different gradients (5×106, 1×106, 1×105, 1×104) were seeded or injected into the in vitro pore plate or free femurs to observe the fluorescence imaging and select the best concentration of cel s. Luciferase gene modified bone marrow mesenchymal stem cel s at the best cel concentration were injected into the mice via the femur and the tail vein, respectively. The distribution of fluorescence and cel number in the mice were explored by using bioluminescence, pathological examination, flow cytometry and quantitative PCR. RESULTS AND CONCLUSION:Ex vivo fluorescence intensity of luciferase gene modified bone marrow mesenchymal stem cel s was positively correlated with the cel concentration;fluorescent cel s in vivo appeared in the femur first and then quickly spread to the lungs in the femur group, while fluorescent cel s in the tail vein group spread to the lungs quickly after cel infusion. Fluorescent cel s could be seen in the spleen, liver and other organs 24 hours later in the two groups. The distribution and migration of cel s in mice could be observed successful y by bioluminescence;5 minutes after cel infusion, the lungs of mice in the two groups began to emit fluorescence that could spread to the liver, spleen and other tissues 24 hours later, and the fluorescence intensity reached its peak after 15 minutes. The distribution of bone marrow mesenchymal stem cel s in mice had no significant difference between the femur group and the tail vein group. To conclude, cel injection through the bone marrow cavity and tail vein fails to promote the homing of bone marrow mesenchymal stem cel s to the bone marrow.

AIM: To clone the full-length of 75A EST. METHODS: After the extraction of total RNA from primary cultured rat cerebellar granule cell of 7DIV in the medium containing 25 mmol/L KCl, T_4 DNA ligase-mediated 5' RACE was used to retrieve 5' unknown sequence of 75A EST, and the first round 5' RACE PCR product was subcloned into pGEM-T easy vector for sequence and homogeneous analysis. RESULTS: The first round of 5' RACE produce a 2.5 kb band, and 75A EST was identified to be partial sequence of Neuron-derived orphan receptor (Nor1) gene. After two more rounds RACE, we firstly cloned the full-length of Nor-1 cDNA. CONCLUSION: T_4 DNA ligase mediated 5' RACE is an efficient method to retrieve information about the 5' termini of mRNAs, and lay a foundation for further study which role Nor1 play in the cerebellar granule cell differentiation or survive.

【Objective】To investigate the molecular mechanism of dopamine (DA)-induced apoptosis in cultured cerebellar granule neurons (CGNs) and the effect of muscarinic cholinergic receptor (mAChR) agonist carbachol on it.【Methods】The apoptosis of neurons was measured by phase-contrast microscopy,Hoechst 33258 nucleus staining and DNA fragmentation agarose gel electrophoresis.The neuronal viability was measured by fluorescein diacetate (FDA) staining.The activation of extracellular signal-regulated protein kinase (ERK) was determined by Western blot.【Results】Dopamine increases the phosphorylation of ERK and induces apoptosis in CGNs,which is blocked by both carbachol and PD 98059.The protective effect and the inhibiting ERK phosphorylation of carbachol were blocked by atropine.【Conclusion】DA-induced apoptosis in CGNs may be mediated by activation of ERK.Carbachol protects CGNs from DA-induced apoptosis by activating mAChR and subsequent inhibition of activation of ERK.

Objective To investigate the effects of sugammadex on postoperative pulmonary com-plications(PPCs)and postoperative recovery after thoracoscopic lung resection surgery.Methods A total of 263 patients scheduled for thoracoscopic lung resection surgery between November 2021 and July 2023,112 males and 151 females,aged 18-64 years,BMI 18.5-28.0 kg/m2,ASA physical status Ⅰ-Ⅲ,were randomly divided into three groups:the sugammadex group(group S,n=88),the neostigmine group(group N,n=87),and the control group(group C,n=88).The patient was sent to postanesthesia care unit(PACU)after operation,when the train of four(TOF)count reached 2,group S was given sugamma-dex 2 mg/kg,group N was given neostigmine 0.04 mg/kg+atropine 0.02 mg/kg,and group C was given equal volume of normal saline.The incidence of PPCs from the end of the surgery to the time of discharge was recorded.The time from the end of surgery to extubation,the time from drug administration to recovery of the train of four ratio(TOFr)to 0.9,the TOFr immediately after extubation,the length of stay in PACU,hypoxemia after extubation(SpO2＜90％)were recorded,and the incidence rate of postoperative residual neuromuscular block(PRNB)was calculated.The time of first getting out of the bed for activity,the number of total and effective compressions by the analgesia pump within 48 hours after surgery,the inci-dence of rescue analgesia,the clinical pulmonary infection score(CPIS),the numbers of postoperative nau-sea and vomiting(PONV),total drainage of the chest tube,duration of the chest tube insertion,and the length of postoperative hospital stay were recorded.Results Compared with group C,the incidence of PPCs,PRNB and hypoxemia after extubation were significantly decreased,time from the end of surgery to extubation,time from drug administration to recovery of TOFr to 0.9,the length of stay in PACU,and the first postoperatively out of bed activity time were significantly shortened,the TOFr immediately after extuba-tion was significantly increased,and CPIS was significantly decreased in group S(P＜0.05);the time from the end of surgery to extubation,time from drug administration to recovery of TOFr to 0.9,the length of stay in PACU were significantly shortened,the TOFr immediately after extubation was significantly in-creased,PRNB after extubation were significantly decreased in group N(P＜0.05).Compared with group N,the incidence of PRNB after extubation were significantly decreased,the time from the end of surgery to extubation,the time from drug administration to recovery of TOFr to 0.9,the length of stay in PACU,and the first postoperatively out of bed activity time were significantly shortened,the TOFr immediately after ex-tubation was significantly increased in group S(P＜0.05).There was no significant difference in other in-dexes between the three groups.Conclusion Sugammadex can rapidly antagonize the residual muscle re-laxation,decrease the rate of PPCs and PRNB,and promote rapid recovery of patients after thoracoscopic lung resection surgery.

Objective:To investigate the feasibility, safety and effectiveness of colonic interposition with vascular anastomosis in reconstructing the entire esophagus and hypopharynx after resection of hypopharyngeal cancer with esophageal cancer.Methods:We conducted a retrospective analysis of 4 male patients with simultaneous multiple primary cancers of the hypopharynx and esophagus, aged 47 to 58, treated in the Department of Head and Neck Surgery at the Hunan Cancer Hospital from February to August 2019. All cases underwent total hypopharyngectomy and total esophagectomy, of whom, three cases presented with total laryngectomy and one case with larynx preservation. Colonic interposition was performed using the left colic artery as a pedicle, with an average colonic length of 48.5 cm. The colon was elevated through the esophageal bed to the neck, and the branch of the colonic mesenteric artery was anastomosed to one of the neck arteries, including the inferior thyroid artery in one case, the transverse cervical artery in two cases, and the superior thyroid artery in one case, and all venous anastomoses were performed with the internal jugular veins.Results:The postoperative neck and abdominal wounds healed well without anastomotic leakage, and all patients were able to resume a regular oral diet within 21-30 days postoperatively. During the follow-up of 48-52 months, two cases died due to tumor recurrence, while the remaining two cases were disease-free survivals.Conclusion:Colonic interposition with vascular anastomosis is a safe and reliable reconstruction method suitable for repairing long-segment upper digestive tract defects after resection of hypopharyngeal cancer with esophageal cancer.