AIM:To investigate whether cigarette smoke(CS)promotes the expression of endoplasmic reticu-lum-associated apoptosis protein CCAAT/enhancer-binding protein homologous protein(CHOP)in rat lung tissues. METHODS:Adult male Wistar rats(n=40)were randomly divided into 4 groups with 10 rats in each group: control group,CS-2 group(exposed to CS for 2 months),CS-4 group(exposed to CS for 4 months)and ex-smoking(Ex-S)group (exposed to CS for 4 months and then quit smoking for 1 month).The percentage of forced expiratory volume in 0.3 second to forced vital capacity(FEV0.3/FVC)and peak expiratory flow(PEF)were measured.TUNEL assay was used to detect the apoptotic cells.In situ hybridization and RT-PCR were used to determine the mRNA expression of CHOP.The methods of immunohistochemistry and Western blot were used to determine the protein expression of CHOP.Western blot was also used to determine the protein levels of protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK,eukaryotic initiation factor(eIF)2αand p-eIF2α.RESULTS:The pulmonary function greatly decreased in the rats exposed to CS for 2 months in comparison with control group(P<0.05),markedly decreased in the rats exposed to CS for 4 months as com-pared with the rats after exposure to CS for 2 months(P<0.05),and was improved little in ex-smoking rats(P>0.05). The structural destruction of the lung was observed in the rats exposed to CS for 2 months,and more obvious changes were found in the rats exposed to CS for 4 months.However,the structural destruction of the lung remained obvious in ex-smok-ing rats.The apoptotic cells were markedly increased in the rats exposed to CS for 2 months and were even more in the rats exposed to CS for 4 months.The apoptotic cells were alveolar epithelial cell I(ACE I),ACE II,vascular endothelial cells and bronchial epithelial cells.The protein levels of p-PERK,p-eIF2αand CHOP were remarkably increased in the rats af-ter exposure to CS for 2 months compared with the control rats(P<0.05),significantly elevated in the rats exposed to CS for 4 months compared with the rats exposed to CS for 2 months(P<0.05),and slightly decreased in ex-smoking rats in comparison with the rats after exposure to CS for 4 months(P>0.05).The total protein levels of PERK and eIF2αdid not change between the control rats and those exposed to CS.CONCLUSION: CS promotes the development of chronic ob-structive pulmonary disease(COPD)by inducing the expression of endoplasmic reticulum-associated apoptosis protein CHOP via PERK/eIF2α/CHOP signaling pathway.

OBJECTIVETo study the expression of lung Krüppel-like transcription factor (KLF2/LKLF) in lung tissues of rats with chronic obstructive pulmonary disease (COPD) and the relationship between KLF2 and NF-E2-related factor 2 (Nrf2), and make further explore the effects of KLF2 on the expression of gamma-glutamylcysteine synthetase (gamma-GCS).

METHODSTwenty-two male SD rats were randomly divided into a COPD group (n = 10) and a normal control group (n = 11). The rat model of COPD established by cigarette smoking and intratracheal instillation of lipopolysaccharide (LPS), and lung tissues were obtained. The expressions of KLF2, Nrf2, gamma-GCS mRNA and protein in lung tissues were measured by immunohistochemistry (IHC), Western blot, in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR). To explore the relationship between KLF2 and Nrf2 protein,we utilize the method of co-immunoprecipitation (CO-IP).

RESULTSIHC and Western blot showed that protein expressions of KLF2, Nrf2, gamma-GCS were higher in the lung tissues from rats with COPD than those in the control groups (all P < 0.05). The levels of KLF2, gamma-GCS mRNA were markedly increased in the COPD group (all P < 0.01) while Nrf2 mRNA expression in COPD group had no significant difference with that in control group ( P > 0.05). CO-IP result showed that KLF2 were obviously present in immunoprecipitates of Nrf2 (P < 0.01) . Linear correlation analysis showed that the level of KLF2 protein was positively correlated with the level of Nrf2 protein (P < 0.05), and KLF2, Nrf2 proteins were positively correlated with gamma-GCS mRNA and protein (all P < 0.05).

CONCLUSIONThe expression of KLF2 is significantly up-regulated in COPD, which maybe up-regulate gamma-GCS mRNA expression by increasing Nrf2 expression and nuclear translocation against oxidative stress.

Animals ; Dipeptides ; metabolism ; Kruppel-Like Transcription Factors ; metabolism ; Lung ; pathology ; physiopathology ; Male ; NF-E2-Related Factor 2 ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; pathology ; physiopathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification. METHODS: The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR. RESULTS: The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells. CONCLUSION The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.
Animals ; Cloning, Molecular ; Cricetinae ; Dependovirus ; genetics ; metabolism ; Epithelium, Corneal ; cytology ; metabolism ; Genetic Vectors ; Herpesvirus 1, Human ; enzymology ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection

Objective To investigate the effects of bradykinin (BK) on the proliferation of rabbit corneal endothehal ceils (RCECs) and the expression of tight junction-related proteins zonula occludens-1 (ZO-1) and zonula occludens-1-associated nucleic-acid-binding protein (ZONAB),and to explore the underlying mechanisms of BK on cell proliferation in corneal endothelium.Methods RCECs at logarithmic growth phase were treated with different concentrations of BK (0.01,0.1,1,10 μmol · L-1) BK group,with the controls left untreated.Morphological changes of cells in each group were examined under phase-contrast microscope,and MTT assays were used to detect cell proliferation at 24 h,48 h,72 h,96 h after BK treatment.And,at 72 h,the expression levels of ZO-1 and ZONAB protein were determined by Western blot.Results After 72 h of treatment,the cells in each group were fused into pieces and closely linked into a monolayer;but after 96 h,the growth of the cells was restricted,with the intercellular space become larger and the cells exfoliated.Compared with the control group,BK induced a significant increase of absorbance value and cell viability,and the differences were statistically significant (all P ＜ 0.001),and the promoting effects showed a concentration-dependent manner,and 1 μmol · L-1 BK demonstrated the strongest regulative effect (P ＜ 0.001).Western blot results showed that BK upregulated the expression of ZO-1 and ZONAB protein in a concentration-dependent manner.Conclusion BK can stimulate the proliferation of RCECs in a time-and concentration-dependent manner,and the mechanisms are probably associated with ZO-1/ZONAB-mediated signaling pathway.

To reveal the epidemiological and pathogenic characteristics of Hand-foot-and-mouth disease (HFMD) in Hainan province in 2010, epidemiology data of HFMD reporting cases were analyzed, clinical specimens from 1346 HFMD cases were collected for enterovirus (EV) detection. Viral isolation was performed for EV nucleic acid positive samples. Complete VP1 encoding region of EV71 were sequenced and analyzed with Sequencher (version 5.0) and MEGA software (version 5.0). The epidemiology data showed that all 18 prefectures in Hainan had reporting cases during 2010, with higher incidence in the northeast; and the children less than 4 years old accounted for the majority of the suffered; the epidemic reached peak during September to October, which was different from other Provinces in China. The laboratory results indicated that EV71 and CA16 were identified as the major causative pathogens in Hainan in 2010, however, EV71 infection was absolutely dominant among severe and fatal cases. In addition, some HFMD cases were identified associated with other serotypes of EV infections. Molecular epidemiological analysis showed that all the EV71 strains belonged to C4a evolutionary branch, which is the dominant evolutionary branch in China in recent years, and at least three transmission chains existed. This study has an important information in clarifying the characteristics of epidemics and transmission of HFMD in Hainan, and to provide the guidance for HFMD prevention and control in the future.
Capsid Proteins ; genetics ; Child, Preschool ; China ; epidemiology ; Disease Outbreaks ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; Phylogeny ; Seasons

Objective:To investigate the distribution of iron deposition in the substantia nigral (SN) subregions on quantitative susceptibility mapping (QSM) and the change of swallow tail sign (STS) in patients with relapsing-remitting multiple sclerosis (RRMS) of different disease stages.Methods:The clinical and imaging data of 53 patients with RRMS (case group) diagnosed at the First Hospital of Chongqing Medical University from November 2019 to December 2021 were retrospectively analyzed. The case group was divided into 0-5 years subgroup, 6-10 years subgroup, and >10 years subgroup according to the disease duration; another 37 age-and gender-matched healthy volunteers were recruited as the control group during the same period. All subjects underwent MRI and QSM reconstruction. First, the SN was divided into four subregions: rostral anterior-SN (aSNr), rostral posterior-SN (pSNr), caudal anterior-SN (aSNc), and caudal posterior-SN (pSNc) on the QSM, and the quantitative susceptibility value (QSV) of each subregion was measured, and then the STS of the SN was observed and scored on the susceptibility weighted imaging (SWI) generated by post-processing. ANOVA was used to compare the differences in the QSV of each subregion of SN among the groups, and the probability of abnormal STS was compared using the χ 2 test. Spearman′s test was used to analyze the correlation between the QSV of each subregion of SN and the STS score. Results:The differences in QSV of aSNr, pSNr, aSNc, and pSNc were statistically significant among the 0-5 years subgroup, 6-10 years subgroup,>10 years subgroup of RRMS patients and the control group ( P<0.05). The QSV of aSNr, pSNr, and aSNc in 0-5 years subgroup was higher than those in the control group ( P was 0.039, 0.008, 0.039, respectively). The QSV of aSNr, aSNc, and pSNc in the 6-10 years subgroup were higher than those in the 0-5 years subgroup ( P was <0.001, 0.020, 0.015, respectively). The QSV of the aSNc, pSNc in >10 years subgroup were lower than those in the 6-10 years subgroup ( P=0.037, 0.006). The QSV of aSNr, pSNr in >10 years subgroup were higher than those in the control group ( P was <0.001, 0.001). There were 7 cases of abnormal STS in the 0-5 years subgroup, 11 cases in the 6-10 years subgroup, 12 cases in >10 years subgroup, and 9 cases in the control subgroup, and there was a statistically significant difference in the probability of abnormal STS among the subgroups of the RRMS patients and the control subgroup (χ 2=16.20, P=0.011). Both the scores of STS in the 6-10 years subgroup and >10 years group were positively correlated with the QSV in pSNc ( r s=0.65, P=0.006; r s=0.48, P=0.045). Conclusions:In RRMS patients, SN iron deposition is concentrated on aSNr, pSNr, and aSNc in the 0-5 years subgroup and on aSNr, aSNc and pSNc in the 6-10 years subgroup. The QSVs of all SN subregions have a downward trend in >10 years subgroup compared with that in the 6-10 years subgroup. Both the QSVs of the pSNc in the 6-10 years group and >10 years group are positively related to STS scores. These help explore the potential progression pattern of SN iron deposition in RRMS patients and the cause of abnormal STS in RRMS patients.

Objective The basic biological, echocardiography and gene sequencing parameters of mice overexpressing Slit2 gene (Slit2-Tg mice) were collected and evaluated, and to provide a reference for the application of Slit2-Tg mice in biomedical research. Methods Slit2-Tg and C57BL/6 J mice were inbred. The genotypes of the mice were determined by a PCR assay. The blood samples were collected for blood routine and biochemical tests. The tissues of main organs were collected for protein expression and pathological analysis. Echocardiography and transcriptome sequencing was carried out for analyzing the heart function and gene expression, respectively. Results The litter size was significantly higher in the Slit2-Tg mice than in C57BL/6 J mice. Human Slit2 gene and protein expressions were detected in the main organs of Slit2-Tg mice. Organ coefficient of spleen was significantly increased in Slit2-Tg mice, but the tissue structure appeared normal. There were significant changes in the counts of erythrocytes, platelets, eosinophils, and biochemistry of glucose, globulin, urea nitrogen, triglycerides, HDL, and atherosclerosis index. Echocardiography showed no significant differences in the morphology and function of the Slit2-Tg hearts except in the left ventricular anterior wall thickness at the end-diastolic state. Compared with the C57BL/6 J mice, 535 genes out of 17513 genes in the Slit2-Tg hearts were increased or decreased, mainly involving 15 biological process or signal transduction pathways. Conclusions This study has collected the biological parameters of Slit2-Tg mice and suggests that this model animal is suitable for the studies of cardiovascular diseases.

Objective:This study is conducted to analyze the impact of county medical alliance on patient flows under the NRCMS in Dingyuan county of Anhui province. Methods: data on patient-flow were collected through the NRCMS information platform and related policy documents for 2015-2016, and interviews were conducted to study the main reform practices in the county medical alliance. Results: The patient flow under the NRCMS varied in the way of 1) the total number of inpatients seeking treatment outside of the county decreased by 3.31%;2) the treat-ment volume of county-level hospitals and township health centers were increasing,and the latter one is growing faster than the former,meanwhile,the treatment volume of the village clinics declined,and 3) the hospitalization expenses per inpatient for patients who seek treatment outside the county are much higher than the expenses incurred within the county. Conclusions:the development of county-level medical alliance has influence on patients with common disea-ses who used to seek treatment by helping to reduce the number of inpatients outside the county and reduce the pres-sure of the new rural cooperative fund. However, significant problems still exist such as shortage of talented practi-tioners,and lagging development of information technology system.

OBJECTIVE: To investigate the expression of STAT3 gene in patients with acute myeloid leukemia and its correlation with clinical characteristics. METHODS: The real-time quantitative RT-PCR was used to detect the level of STAT3 mRNA in bone marrow samples from 38 newly diagnosed patients with acute myeloid leukemia(AML), and its relevance with clinical characteristics and prognosis were statistically analyzed. Western blot was employed to detect the STAT3 protein level in AML patients. The bone marrow cells from 15 healthy subjects were used as control. RESULTS: At the mRNA level, the expression level of STAT3 in the AML group was significantly higher than that in control group (P<0.05). The level of STAT3 in AML group correlated positively with the risk factors of patients (P<0.01，r=0.592）. The STAT3 expression level in the high-risk group was statistically higher than that in the standard-risk group and the control group (P<0.01，P<0.01). Furthermor, there was no statistical difference between the sub-groups of AML (P>0.05). The median survival time of patients in STAT3 low expression group was logner than that in high expression group, but the difference was not statistically significant (P>0.005). The level of STAT3 protein in AML patients was significantly higher than that in control group (P<0.05）. CONCLUSION The STAT3 gene is highly expressed in AML patients, which may be used as a predictor for high-risk of AML.
Bone Marrow ; Humans ; Leukemia, Myeloid, Acute ; Prognosis ; RNA, Messenger ; STAT3 Transcription Factor ; genetics

Consistency in quality of traditional Chinese medicine granules is an important factor to ensure reproducible clinical efficacy. In this study rhubarb dispensing granules were utilized to construct an efficacious near-infrared spectroscopy (eNIRS) assay by combining NIRS and biopotency. A NIR method for assaying rhubarb dispensing particles was established, and information on different batches was collected. The diarrhea-inducing biopotency of rhubarb dispensing granules was determined based on a constipation model induced by diphenoxylate in mice. The animal protocol was approved by the Animal Ethic Committee​ of 302 Hospital of Chinese PLA People's Liberation Army (ID: IACUC-2019-0010). Ten anthraquinones were determined in rhubarb dispensing granules by UPLC. The correlation between NIR and biopotency was analyzed and five characteristic bands that correlated highly with bioactivity were identified, including 4 011-4 390, 4 859-5 461, 7 012-7 493, 10 992-11 312 and 11 871-12 489 cm^-1. There were some differences in the main bands of different chemical constituents. In summary, five active bands based on NIRS were identified and found to be able to achieve rapid on-line detection of rhubarb dispensing granule quality. This research model may also provide reference for quality control of other Chinese medicine dispensing granules.