Objective To construct an in vitro"metabolic memory"cell model of HT-22 mouse hippocampal neurons induced by high glucose,and to investigate the effect of"metabolic memory"on apoptosis and histone acetylation in HT-22 cells.Methods HT-22 cells were cultured in high glucose medium(glucose concentration was 55 mmol/L)and conventional glucose medium(glucose concentration was 25 mmol/L),and cells were divided into the control group(NG 4,6 and 8 groups,25 mmol/L glucose was cultured for 4,6 and 8 days,respectively),the high glucose group(HG 4,6 and 8 groups,respectively)and the metabolic memory group(HG2NG2,HG2NG4,HG2NG6,HG4NG2 and HG4NG4 groups,high glucose culture for 2 days to 25 mmol/L glucose culture for 2,4 or 6 days,high glucose culture for 4 days to 25 mmol/L glucose culture for 2 or 4 days).Cell viability was detected by CCK-8 method.The release of lactate dehydrogenase(LDH)in cell culture supernatant was detected,and the optimal time to establish a"metabolic memory"model was selected.Subsequently,cells were divided into the NG4 group,the NG8 group,the HG4 group,the HG4NG4 group and the HG8 group,and the cell morphology of each group was observed by optical microscope.The apoptosis rate was detected by flow cytometry.The activities of deacetylase(HDAC)and histone acetyltransferase(HAT)were detected by enzyme-linked immunosorbent assay(ELISA).Western blot assay was used to detect expression levels of histone deacetylase 4(HDAC4),B lymphocyte tumor 2(Bcl-2),Bcl-2 related X protein(Bax)and Caspase-3 protein.Results The HG4NG4 group was the ideal cell model with high glucose metabolic memory.Cells of the NG4 group and the NG8 group were interwoven into a dense network,growing well,with spindle shaped cells and distinct synaptic structures.However,in the HG4 group and the HG8 group,the cell body became round,synaptic structure disappeared and growth was inhibited.In the HG4NG4 group,the number of cells increased but their morphology was damaged.Results of flow cytometry showed that compared with the NG8 group,the apoptosis rates were significantly increased in the HG8 group and the HG4NG4 group(P＜0.05).ELISA results showed that compared with the NG8 group,the expression levels of HDAC4,Bax,and Caspase-3 proteins increased in the HG8 group and the HG4NG4 group,while the expression level of Bcl-2 protein significantly decreased(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels of HAT and HDAC in the HG4NG4 group.Western blot reslts showed that compared with the NG8 group,the levels of HDAC4,Bax and Caspase-3 protein increased in the HG8 group and the HG4NG4 group(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels in the HG4NG4 group.Conclusion HT-22 mouse hippocampal neurons cultured with 55mmol/L high glucose for 4 days,and then cultured with 25 mmol/L glucose for 4 days are the ideal"metabolic memory"cell model.The mechanism may be related to the increased activity of HDAC,HAT and HDAC4 expression in the hyperglycemic model.

OBJECTIVE To investigate the pharmacodynamic effects of the Citri Grandis Exocarpium extract on anorexia model immature rats.METHODS Ten immature rats were randomly selected as the control group and given regular diet， while anorexia model was induced in the remaining 50 immature rats using the etiological simulation method. The immature model rats were divided into the model group， Jianwei xiaoshi tablet group （0.65 g/kg）， Citri Grandis Exocarpium extract low-dose， medium- dose and high-dose groups （0.5， 1.0， 2.0 g/kg）. After 14 d of continuous intragastric administration， the body mass and food intake of rats in each group were observed， and the gastric acid concentration， pepsinase activity， serum levels of gastrin （GAS）， motilin （MTL） and gastric inhibitory peptide （GIP） were measured. The histopathological changes in the gastric tissue were observed.RESULTS Compared with control group， the food intake， gastric acid concentration， pepsin activity， the levels of GAS and MTL were significantly decreased and GIP level was significantly increased in the immature rats of model group （P＜0.05）； gastric histopathological examination showed significant gastric mucosal congestion and loosening and detachment of epithelial cells. Compared with model group， all administration groups showed different degrees of improvement in the above indexes and decreased gastric histopathological damage after drug intervention.CONCLUSIONS Citri Grandis Exocarpium extract improves gastric motility and protects gastric mucosa in the immature rats by regulating the concentration of gastric acid， pepsin activity， the levels of GAS， MTL and GIP， thus improving anorexia in immature rats.

Objective:To evaluate the clinical application value of endoscopic ultrasonography (EUS) in the etiological diagnosis of patients initially diagnosed with idiopathic acute pancreatitis (IAP).Methods:Clinical data of 128 patients who underwent further EUS and magnetic resonance cholangiopancreatography (MRCP) after initial diagnosis of IAP at the Gastrointestinal Endoscopy Center of the First Affiliated Hospital of Naval Medical University between January 2015 and February 2022 were collected and divided into a single-episode group (single-episode group, 51 cases) and a multiple-episode group (recurrent group, 77 cases) based on the number of AP episodes. The data and the diagnosis of the etiology of IAP in the two groups by EUS were analyzed and compared with the etiological diagnosis results of MRCP.Results:The differences on basic information such as gender, age, history of smoking, history of alcohol consumption, family history of pancreatic disease, history of cholecystectomy, abnormality of liver function, and severity of pancreatitis between the single-episode group and recurrent group of IAP patients were not statistically significant. The etiology was clarified in 79 (62%) IAP patients after EUS examination, of which 55 (43%) cases had biliary disease (gallstones, microlithiasis, biliary sludge) and 24 (19%) cases had pancreatic disease (chronic pancreatitis, pancreatic divisum, pancreatic interstitial or pancreatic ductal changes). The percentage of patients with biliary disease as the cause of IAP was significantly higher in the single-episode group than in the recurrent group (59% vs 32%), while the percentage of patients with pancreatic disease as the cause of IAP was higher in the recurrent group than in the single-episode group (25% vs 10%), with statistically significant differences ( P values=0.004 and 0.035, respectively). The performance of EUS in diagnosing the etiology of IAP was significantly higher than that of MRCP (62% vs 19%, P=0.032), where EUS was more accurate in detecting biliary microlithiasis or biliary sludge (43% vs 9%, P<0.01). EUS was also superior to MRCP in identifying subtle changes in chronic pancreatitis lesions (small pancreatic nodules, patchy hyperechogenicity, etc.) and intraductal papillary mucinous neoplasms(17% vs 7%, P<0.05), but was inferior to MRCP in identifying pancreatic divisum (2 cases vs 4 cases). Conclusions:In view of high diagnostic accuracy and safety of EUS in diagnosing biliary diseases, and based on the fact that most IAPs in China are due to biliary diseases, EUS based management strategy can be considered to be a reasonable approach for evaluation of IAP patients. The MRCP can be used as a supplement to the EUS to identify a controversial etiology.

Objective:To evaluate diagnostic value of contrast-enhanced harmonic endoscopic ultrasonography (CEH-EUS) for pancreatic cystic lesions.Methods:Endoscopic and clinical follow-up data of patients with pancreatic cystic lesions diagnosed by EUS in Department of Gastroenterology of the First Affiliated Hospital of Naval Medical University with CEH-EUS video from March 2013 to April 2020 was retrospectively analyzed.Results:A total of 36 patients were included. There were 16 cases of serous cystadenomas (SCA), 10 cases of mucinous cystic neoplasm (MCN), 5 cases of intraductal papillary mucinous neoplasms (IPMN, 3 with complex type, 2 with main pancreatic duct type) and 5 cases of pancreatic pseudocyst (PPC). 87.5%(14/16) of SCA and 86.7%(13/15) of MCN+ IPMN had hyperenhanced cystic wall with obvious peak and similar washout as surrounding tissue, whereas only 20%(1/5) PPC had hyperenhanced cystic wall. The hyperenhancing effect of PPC was significantly lower than that of SCA and MCN+ IPMN ( P=0.0035 and P=0.0048, respectively ). Mural nodules were detected in 17 cases of pancreatic cystic lesions by EUS, of which 3 cases had hyperenhanced mural modules and 14 cases had hypoenhanced mural nodules by CEH-EUS. Patients showing hyperenhanced mural modules were all finally diagnosed as pancreatic malignancy (1 IPMN, 2 MCN), and the accuracy was 100%. Conclusions:CEH-EUS can have a obvious advantage of differentiating pseudocyst and other pancreatic cystic lesions, while not very useful for differentiating SCA and MCN. Pancreatic cystic lesions showing hyperenhanced mural nodules under CEH-EUS may imply malignancy potential.

Objective:To investigate the effect of lenalidomide (LEN) combined with temozolomide (TMZ) on proliferation, invasion, drug resistance and O6-methylguanine-DNA methyltransferase ( MGMT) gene epigenetic modification of TMZ-resistant human glioblastoma cell line U251/TR. Methods:A TMZ-resistant human glioma cell line, U251/TR, was successfully established by stepwise exposure of U251 parental cells to TMZ. U251/TR cells were divided into dimethyl sulfoxide (DMSO) group, LEN group, TMZ group and LEN+TMZ group (DMSO group: without any drug intervention; LEN group, TMZ group, and LEN+TMZ group were pretreated with 100 μmol/L LEN, 200 μmol/L TMZ, 100 μmol/L LEN+200 μmol/L TMZ, respectively; the drugs were administered once every 24 h). The proliferation rate of these cells in each group was detected by sulfonylrhodamine B colorimetric assay at different time points (24, 48, 72, and 96 h after treatment). At 96 h after treatment, the invasion and migration abilities of cells in each group were detected by Transwell assay; the proliferation cycle of cells in each group was detected by flow cytometry; Western blotting, immunohistochemical staining and immunofluorescence staining were used to detect the MGMT protein expression, and the MGMT mRNA expression in cells of each group was detected by reverse transcription-PCR; methylation specific PCR was used to detect the MGMT gene promoter methylation in each group of cells. Results:The cell proliferation rate of LEN+TMZ group was significantly decreased as compared with TMZ, LEN, and DMSO groups at 24, 48, 72 and 96 h after treatment ( P<0.05). At 96 h after treatment, LEN+TMZ group had significantly decreased number of transmembrane cells, and significantly increased ratio of cells at G0/G1 phase as compared with the other 3 groups ( P<0.05); the MGMT protein and mRNA expression levels in TMZ group and LEN+TMZ group were significantly lower than those in LEN group and DMSO group ( P<0.05); and the number of cells with strong or moderate MGMT expression in TMZ group and LEN+TMZ group was obviously less than that in LEN group and DMSO group, and the MGMT fluorescence intensity in TMZ group and LEN+TMZ group (+) was obviously lower than that in LEN group (+++) and DMSO group (+++). The MGMT gene promoter was unmethylated in all groups. Conclusion:LEN alone does not obviously inhibit the proliferation and invasion of U251/TR cells; but LEN combined with TMZ could inhibit the proliferation and invasion of U251/TR cells and co-reverse the drug resistance of U251/TR cells, whose mechanism is not related to the changes of MGMT gene promoter methylation.

Objective:To investigate the etiology of necrotizing pneumonia (NP) in children and the clinical characteristics of NP caused by different pathogens in China.Methods:A retrospective, case-control study was performed in children with NP who were admitted to 13 hospitals in China from January 2008 to December 2019. The demographic and clinical information, laboratory data, etiological and radiological findings were analyzed. The data were divided into three groups based on the following years: 2008-2011, 2012-2015 and 2016-2019, and the distribution characteristics of the pathogens in different period were compared. Meanwhile, the pathogens of pediatric NP in the southern and northern China were compared. And the clinical characteristics of the Mycoplasma pneumoniae (MP) NP and the bacterial NP were also compared. T-test or Mann-Whitney nonparametric test was used for comparison of numerical variables, and χ 2 test was used for categorical variables. Results:A total of 494 children with NP were enrolled, the median ages were 4.7 (0.1-15.3) years, including 272 boys and 222 girls. Among these patients, pathogens were identified in 347 cases and the pathogen was unclear in the remaining 147 cases. The main pathogens were MP (238 cases), Streptococcus pneumoniae (SP) (61 cases), Staphylococcus aureus (SA) (51 cases), Pseudomonas aeruginosa (13 cases), Haemophilus influenzae (10 cases), adenovirus (10 cases), and influenza virus A (7 cases), respectively. MP was the most common pathogen in all three periods and the proportion increased yearly. The proportion of MP in 2016-2019 was significantly higher than that in 2012-2015 (52.1% (197/378) vs. 36.8% (32/87), χ 2= 6.654, P=0.010), while there was no significant difference in the proportion of MP in 2012-2015 and that in 2008-2011 (36.8% (32/87) vs. 31.0% (9/29), χ2=0.314, P=0.575).Regarding the regional distribution, 342 cases were in the southern China and 152 in the northern China. Also, MP was the most common pathogen in both regions, but the proportion of MP was higher and the proportion of SP was lower in the north than those in the south (60.5% (92/152) vs. 42.7% (146/342), χ 2=13.409, P<0.010; 7.9% (12/152) vs. 14.3% (49/342), χ 2= 4.023, P=0.045). Comparing the clinical characteristics of different pathogens, we found that fever and cough were the common symptoms in both single MP and single bacterial groups, but chest pain was more common (17.0% (34/200) vs. 6.1% (6/98), χ 2=6.697, P=0.010) while shortness of breath and wheezing were less common in MP group (16.0% (32/200) vs. 60.2% (59/98), χ 2=60.688, P<0.01; 4.5% (9/200) vs. 21.4% (21/98), χ 2=20.819, P<0.01, respectively). The white blood cell count, C-reactive protein and procalcitonin in the bacterial group were significantly higher than those in the MP group (14.7 (1.0-67.1)×10 9/L vs. 10.5 (2.5-32.2)×10 9/L, 122.5 (0.5-277.3) mg/L vs. 51.4 (0.5-200.0) g/L, 2.13 (0.05-100.00) μg/L vs. 0.24 (0.01-18.85) μg/L, Z=-3.719, -5.901 and -7.765, all P<0.01). Conclusions:The prevalence of pediatric NP in China shows an increasing trend during the past years. MP, SP and SA are the main pathogens of NP, and the most common clinical symptoms are fever and cough. The WBC count, C-reactive protein and procalcitonin in bacterial NP are significantly higher than those caused by MP.

Objective To study the effect of hyperbaric oxygen in the treatment of severe traumatic brain injury.Methods One hundred and ninety-four patients with severe traumatic brain injury were selected.The patients were divided into control group and observation group according to admission date (even number or odd number) with 97 cases each.The control group received only neurosurgical treatment, and the observation group received hyperbaric oxygen combined with neurosurgical treatment.The therapeutic effects of the 2 groups were compared.Results The waking time in observation group was significantly shorter than that in control group: (50.19 ± 5.23) h vs.(191.52 ± 9.67)h,the Glasgow coma scale(GCS)score and functional independence measure(FIM)score after treatment were significantly higher than those in control group:(12.21 ± 1.41)scores vs.(8.54 ± 1.38) scores and (85.91 ± 10.24) scores vs.(67.63 ± 9.76) scores, and there were statistical differences (P<0.05).The good recovery rate in observation group was significantly higher than that in control group:43.30%(42/97)vs.17.53%(17/97),the rate of plant survival and death was significantly lower than that in control group:10.31%(10/97)vs.31.96%(31/97),and there was statistical difference(P<0.05).The observation group did not appear hyperbaric oxygen treatment related adverse reactions.The incidences of lung infection,upper gastrointestinal bleeding and renal function impairment in the observation group were significantly lower than those in control group:15.46%(15/97)vs.38.14%(37/97),5.15%(5/97)vs.16.49% (16/97) and 6.19% (6/97) vs.16.49% (16/97), and there were statistical differences (P<0.05).Conclusions Compared with only neurosurgical treatment in patients with severe traumatic brain injury,hyperbaric oxygen combined with neurosurgical treatment has more advantages in shortening the waking time,improving self-care ability and prognosis,and reducing complications.

Objective To explore the curative effect of deep-brain magnetic stimulation (DMS) on learned helplessness behavior in the chronic restrained stress (CRS) rat model. Methods Twenty-nine Sprague-Dawley rats were randomly divided into control group (n=8) and CRS group (n=21). CRS group was exerted chronic restrained stress, while the control group did not receive any stress, for three weeks. Then learned helplessness behavior was tested using Forced Swimming Test (FST) and the hopeless rats of the CRS group were divided ran-domly into sham group (n=6), DMS group (n=8) and citalopram group (n=7), that received corresponding treatment respectively. They were evaluated with FST again after one-week treatment. Results The immobile time in FST was longer in CRS group than in the control group after three-week stress (F=11.260, P=0.002). After one-week treatment, no significant improvement was found in the citalopram group (F=1.565, P=0.235), however, the immobile time in DMS group decreased (F=6.277, P=0.025), and was shorter than that in the sham group (F=5.560, P=0.036). Conclusion CRS could result in learned helplessness behavior, which could be alleviated with one-week DMS.

Objective To study the effect of mitogen-activated protein kinas/extracellular signalregulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA).Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic synovectomy of RA patients with active disease activity [disease activity score(DAS)28 ≥3.2].The RA-FLS were primarily cultured by the explanted culture,and then were treated with Shh agonist purmorphamine,inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126,respectively.Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2),which was the critical protein of MAPK/ERK signaling.The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8),and the cell proliferation rate was detected using a flow cytometry.Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis.Results Compared with the control group,purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L,and the peak activations of p-ERK1/2 took place at 15 min (P＜0.01).Cyclopamine and U0126 decreased the expression ofp-ERK1/2 protein (P＜0.01).After the RA-FLS treated with purmorphmine(1 μmol/L)for 48 hours,the cell proliferation activity was (114±4)％ and the percentage of S phase cells was (8.39±0.60)％,which was significantly higher than those of the control group (100±0)％ (P＜0.01) and (3.29±0.69)％ (P＜0.01).After treated with cyclopamine (10 μmol/L) for 48 hours,the cell proliferation activity of RA-FLS was (89±1)％ (P＜0.05) and the percentage of S phase cells was (1.53±0.22)％ (P＜0.05).When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L),the cell proliferative activity was (89±2)％ (P＜0.05) and the percentage of S phase cells was(1.07±0.25)％(P＜ 0.05).Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling,which in turn contributes to hyperplasia of synovium and ultimately leading to RA.

AIM:To determine the effects and mechanisms of interleukin-22 (IL-22) on the fibroblast-like sy-noviocytes ( FLSs) from rheumatoid arthritis ( RA) patients.METHODS:RA-FLSs were cultured by tissue culture meth-od.RA-FLSs were incubated with different concentrations of IL-22 (0,1,10,100μg/L) for 24 h, 48 h and 72 h.The cell viability was examined by CCK-8 assay.IL-22 at concentration of 10 μg/L was used to stimulate RA-FLSs for 24 h, and the change of cell cycle distribution was identified by flow cytometry.The effects of IL-22 at concentrations of 0, 1, 10, 100μg/L and/or STA-21 (a STAT3 inhibitor at concentrations of 0, 25, 50μmol/L) on the protein levels of Bcl-2 and p-STAT3 in the RA-FLSs were determined by Western blot.RESULTS:Compared with control group, stimulation of rhIL-22 at different concentrations for 24 h, 48 h and 72 h, the cells viabilityof RA-FLSs were obviously increased ( P<0.05 ) . After co-cultured with 10 μg/L rhIL-22 for 24 h, the percentages of RA-FLSs were obviously increased in the G2/M+S phase and decreased in the G0/G1 phase.At the same time, rhIL-22 increased, but STA-21 decreased the protein levels of Bcl-2 but p-STAT3 in the RA-FLSs obviously (P<0.05).Treatment with STAT3 inhibitor STA-21 reversed the effect of IL-22-induced Bcl-2 upregulation in the RA-FLSs ( P<0.01 ) .CONCLUSION: STAT3 is critical in the process of IL-22-induced Bcl-2 upregulation in RA-FLSs, indicating that IL-22 may play a role in the apoptosis of RA-FLSs.