1.Detection of streptomycin-resistance associated rpsL and rrs gene mutations in Mycobacterium tuberculosis by PCR-single-strand-conformational polymorphism
Zhaodong LI ; Hui WEI ; Dapeng FAN ; Peng DU ; Aihua SUN
Chinese Journal of Clinical Infectious Diseases 2011;04(5):275-277
ObjectiveTo establish a novel rapid detection method based on PCR-single-strand-conformational polymorphism (PCR-SSCP) to determine mutation of streptomycin-resistance associated rpsL and rrs genes in isolates of Mycobacterium tuberculosis (MTB).MethodsStreptomycin-resistance of 112 MTB isolates was detected using the routine drug susceptibility test,and a special PCR-SSCP assay was established.The mutations of rpsL and rrs genes in streptomycin-resistant MTB isolates were detected by PCR-SSCP and PCR direct sequencing (PCR-DS) ; the results from two techniques were compared.Results All isolates had both rpsL and rrs genes.Fifty-two isolates (46.4%) were streptomycin susceptible,in which only 1 isolate showed abnormal PCR-SSCP fragments from rrs gene,and the specificity of PCR-SSCP was 98.1% (51/52).Sixty isolates (53.6%) were streptomycin-resistant,in which 46 (76.6%) and 11 ( 18.3% ) isolates presented the abnormal PCR-SSCP fragments of rpsL and rrs gene,respectively.One streptomycin-resistant isolate showed abnormal PCR-SSCP fragments from both rpsL and rrs genes.The sensitivity of PCR-SSCP was 93.3% (56/60).ConclusionThe PCR-SSCP that established in this study is a specific and sensitive method for rapid detection of the streptomycin-resistance associated mutations in rpsL and rrs genes of MTB.
2.Expression and significance of angiopoietin-2 and Tie-2 receptors in a rat model of acute lung injury
Zhijiang QI ; Xiaozhi WANG ; Zhaodong HAN ; Bojiang WANG ; Ting SUN
Chinese Journal of Pathophysiology 2010;26(2):314-317
AIM: To explore the expressions and significance of angiopoietin-2 (Ang-2) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie-2) receptors in a rat model of acute lung injury (ALI). METHODS: Wistar rats (n=42) were divided into control group (n=12) and cecal ligation and puncture (CLP) group (n=30). Control group underwent sham operation, and CLP group underwent cecal ligation and puncture to make the model of ALI. 12 h after sham operation or CLP, 6 rats in each group were killed, and arterial blood gas analysis and lung coefficient were tested. The expressions of Ang-2 and Tie-2 receptors in lung tissue were observed by immunohistochemical method. Blood samples of the rest rats were collected from vena caudalis, and Ang-2 levels were measured by enzyme linked immunosorbent assay (ELISA). The mortality rate in each group within 36 h was compared. The lung architecture was observed under microscope. RESULTS: The lung architecture in control group was clear and intact. Alveolar septum was thicker, blood capillary was congested, and neutrophils and macrophages were infiltrated in the lung tissue in CLP group. Tie-2 receptors were expressed in bronchial epithelial cells, smooth muscle cells and endothelial cells in control group. Besides the similar expression as control group, high expression of Tie-2 on neutrophils and macrophages in CLP group was observed. In the adhesion location of Tie-2 receptors positive inflammatory cells, there was stronger staining in endothelial cells. Ang-2 was expressed in smooth muscle cells, bronchial epithelial cells and endothelial cells in control group. The Ang-2 level in CLP group were higher than that in control group [(8.14±1.74) μg/L vs (4.63±0.49) μg/L, P<0.01], and the Ang-2 level of dead rats was higher than that of survival rats within 36 h in CLP group [(8.95±1.61)μg/L vs (6.80±0.96)μg/L, P<0.01]. Oxygen partial pressure in control group was lower (P<0.01) and lung coefficient was higher (P<0.01) than that in CLP group. CONCLUSION: Ang-2 and Tie-2 receptors may participate in the pathophysiology of ALI, and Ang-2 level is correlated with mortality.
3.The relevance of Mycobacterium tuberculosis isoniazid-resistance and mutations in two different regions of the katG gene
Zhaodong LI ; Hui WEI ; Yan ZHANG ; Yu CHEN ; Peng DU ; Aihua SUN
Chinese Journal of Laboratory Medicine 2011;34(2):125-129
Objective To analyze and compare the mutations in two different regions of the katG gene and study the relevance of Mycobacterium tuberculosis isoniazid-resistance and mutations in two different regions of the katG gene. Methods Fifty-three INH-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from Zhejiang province were analyzed. PCR was used to amplify two regions of the katG gene (GenBank accession no. U06258) region 1 (from codon 1 to codon 150) and region 2 ( from codon 227 to codon 470) which were then sequenced in order to identify mutations. Results Three strains resistant to INH did not contain mutations in either region. Fourteen strains carried mutations in region 1. Among them 5 strains barbered deletions, and showed high-level resistance to isoniazid. Five strains had mutations only in region 1. Region 2 carried multiple point mutations, especially at codon 315, and there were S315 N ( AGC→AAC ) substitution in 18 of those cases. The frequency of mutations in the katG S315 of high-level INH-resistance isolates ( 84. 4%, 27/32) was significantly higher than those of low-level INH-resistance isolates( 15.6%, 5/32 ), there was statistically significant difference (x2 = 30. 25, P < 0. 01 ).katG S315 mutations in high-level INH-resistance frequency (84. 4%, 27/32) was significantly higher than the other mutations of katG gene of high-level INH-resistance frequency (27. 7%, 5/18 ), there was significant difference (x2 = 16.02, P < 0. 01 ). The analysis of region 2 allowed INH resistance to be diagnosed in 84. 9% of the strains. Five strains had mutations only in region 1 ,which allowed the proportion of INH-resistant strains identified to be increased to 94. 3%. Conclusions The number of mutations at codon 315 was high. Mutation type and location closely related with drug resistance and the analysis of region 1 resulted in a 9. 4% increase in the rate at which mutations were identified.
4.Protein expression changes of mitochondrial apoptotic pathway during the reverse effects of lipid emulsion on bupivacaine cardiotoxicity
Jing TANG ; Xiying YANG ; Zhaodong JUAN ; Haoyun ZHANG ; Lina SUN ; Zheng ZHU
The Journal of Clinical Anesthesiology 2017;33(6):602-604
Objective To detect the protein expression changes of mitochondrial apoptotic pathway during the reverse effects of lipid emulsion on bupivacaine cardiotoxicity, so as to investigate the probable mechanism concerning the reverse effect of lipid emulsion on bupivacaine cardiotoxicity.Methods The ventricular muscles of 15 healthy SD neonatal mice (1-3 d) were chosen to conduct primary culture in vitro.And the cardiomyocytes were cultivated in a medium containing bupivacaine for 24 hours to establish its bupivacaine poisoning model.The cultured cardiomyocytes were divided into three groups: control group (group C);bupivacaine group (group B);and bupivacaine+lipid emulsion group (group BL).Flow cytometry was applied to examine the apoptosis of cardiomyocytes, and the Western blot was employed to detect the protein expression variation of cytochrome C (Cyto-C) and cleaved casepase-3.Results Compared with group C, the apoptosis rate was remarkably increased in both group B and group BL and that of the group B was dramatically higher than that of the group BL, with a statistical significance (P<0.05).Compared with group C, the protein expression levels of both Cyto-C and cleaved casepase-3 were significantly increased in groups B and BL (P<0.05), and the protein expression levels of both Cyto-C and cleaved casepase-3 in group B were significantly higher than those in group BL (P<0.05).Conclusion Lipid emulsion can regulate apoptosis through inhibiting the release of mitochondrial Cyto-C and reducing casepase-3 activation, thus it protects cardiomyocytes.
5.The influence of M2 macrophages on tumor-promoting effect of gastric cancer-derived mesenchymal stem cells in gastric cancer microenvironment
Zhaodong SUN ; Ting ZHANG ; Juan HUO ; Wei LI
Chongqing Medicine 2018;47(16):2126-2130
Objective To investigate the effect of M2 macrophages resident in gastric cancer microenvironment on the tumor-promoting effect of human gastric cancer-derived mesenchymal stem cells (GC-MSCs).Methods Macrophages in BALB/c mice were depleted by using clodronate liposomes.The tumor volumes and weights in nude mice co-injected with GC-MSCs and BGC-823 with and without macrophage depletion were recorded.Tumor tissues of nude mice and gastric cancer patients were collected,and M2 macrophage-associated genes and proteins were detected by RT-PCR and western blot.Furthermore,the regulating effect of GC-MSCs on macrophage polarization to M2-subtype was validated in the co-culture experiment in vitro.Results Tumor growth in GC-MSCs co-injected mice was significantly inhibited by macrophage depletion (P=0.009).Results of RT-PCR and western blot showed that the transcription and expression of M2 macrophage-associated proteins were significantly higher in tumor tissues from GC-MSCs co-injected mice than those in the control group.Moreover,the transcription and expression levels of M2 macrophage-associated proteins were also high-er in gastric cancer tissues than those in the corresponding adjacent normal tissues.After co-culture with GC-MSCs directly,the expressions of M2 macrophage-associated proteins were significantly up-regulated in THP-1-derived macrophages.Conclusion M2 macrophages in gastric cancer microenvironment might play a critical role in the tumor-promoting effect of GC-MSCs.
6.Effect of sevoflurane preconditioning on expression of metabotropic glutamate receptor typeⅡ dur-ing focal cerebral ischemia-reperfusion in rats
Zheng ZHU ; Meiyan SUN ; Rui ZHANG ; Danyang MA ; Xiaoli ZHUANG ; Yan LI ; Du ZHENG ; Zhaodong JUAN ; Xiaoyong ZHAO
Chinese Journal of Anesthesiology 2018;38(7):882-885
Objective To evaluate the effect of sevoflurane preconditioning on the expression of metabotropic glutamate receptor type Ⅱ( mGluRⅡ) during focal cerebral ischemia-reperfusion ( I∕R) in rats. Methods Forty-eight clean-grade healthy male Sprague-Dawley rats were divided into 3 groups (n=16 each) using a random number table method: sham operation group ( group S), cerebral I∕R group (group I∕R) and sevoflurane preconditioning group (group Sev). Rats were anesthetized with 10% chloral hydrate 3 ml∕kg. Focal cerebral I∕R was produced by occlusion of the right middle cerebral artery for 2 h fol-lowed by 24 h reperfusion. In group Sev, 2. 7% sevoflurane was inhaled for 1 h and 24 h later focal cerebral I∕R was produced. At 24 h after reperfusion, neurological deficit was scored, the cerebral infarct size was determined by TTC staining, the cell apoptosis in ischemic penumbra was observed by TUNEL, IκB-α ex-pression was detected by Western blot, and mGluRⅡexpression was determined by immunofluorescent stai-ning. The apoptosis rate was calculated. Results Compared with group S, the neurological deficit score, cerebral infarct size and apoptosis rate were significantly increased, the expression of mGluRⅡwas up-regu-lated, and the expression of IκB-α was down-regulated in I∕R and Sev groups ( P<0. 05). Compared with group I∕R, the neurological deficit score, cerebral infarct size and apoptosis rate were significantly de-creased, the expression of mGluRⅡwas down-regulated, and the expression of IκB-α was up-regulated in group Sev (P<0. 05). Conclusion Sevoflurane preconditioning reduces focal cerebral I∕R injury through inhibiting the expression of mGluRⅡ in rats.
7.Effects of trioxygen preconditioning on cerebral ischemia/reperfusion injury and glutamate receptor in rats
Lin ZHANG ; Yunqi LI ; Yangyang LIU ; Xiaotong SUN ; Zhaodong JUAN ; Rui ZHANG ; Li'na SUN
Chinese Critical Care Medicine 2022;34(3):280-283
Objective:To study the effects of trioxygen pretreatment on cerebral ischemia/reperfusion (I/R) injury in rats.Methods:A total of 24 clean grade male Sprague-Dawley (SD) rats were randomly divided into Sham group, brain I/R group (I/R group) and Ozone pretreatment group (Ozone group), with 8 rats in each group. The animals were routinely fed, and the operation was performed 5 days after the intervention of Ozone group by intraperitoneal injection of trioxygen water (concentration 80 mg/L, 0.01 mL/g), and the Sham group and I/R group were injected with equal volume normal saline. The Sham group only separated the arteries without ligation, and the I/R group and Ozone group established the rat cerebral I/R model. Neurological deficit score (NDS) was performed 2 hours after ischemia and modified neurological deficit score (mNSS) was performed 24 hours after reperfusion. Brain tissue was collected after anesthesia. Cerebral infarction was observed by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and the percentage of cerebral infarction volume was calculated. Protein expression of metabolic glutamate receptor 5 (mGluR5) and ionic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA2 in cerebral ischemic penumbra was determined by Western blotting.Results:Compared with the Sham group, NDS score, mNSS score and percentage of cerebral infarction volume in I/R group were increased [NDS score: 2.63±0.52 vs. 0, mNSS score: 9.63±1.19 vs. 1.13±0.64, cerebral infarction volume: (41.25±2.93)% vs. 0%, all P < 0.05], and expressions of mGluR5 and GluA2 in penumbra area of cerebral ischemia were decreased [mGluR5 protein (mGluR5/β-actin): 0.44±0.14 vs. 1.00±0.10, GluA2 protein (GluA2/β-actin): 0.23±0.08 vs. 1.00±0.25, both P < 0.05]. Compared with the I/R group, mNSS score and percentage of cerebral infarction volume in the Ozone group were decreased [mNSS score: 7.00±1.20 vs. 9.63±1.19, cerebral infarction volume: (27.23±6.21)% vs. (41.25±2.93)%, both P < 0.05], and mGluR5 and GluA2 expressions in the penumbra of cerebral ischemia were up-regulated [mGluR5 protein (mGluR5/β-actin): 0.81±0.10 vs. 0.44±0.14, GluA2 protein (GluA2/β-actin): 0.76±0.13 vs. 0.23±0.08, both P < 0.05]. Conclusion:Trioxygen preconditioning can alleviate cerebral I/R injury in rats, and its mechanism may be related to the upregulation of GluR5 and GluA2 in the ischemic penumbra.
8.Effect of clemastine fumarate on TLR4/PI3K/Akt signaling pathway during hypoxia-reoxygenation in rat cardiomyocytes
Ru YAN ; Feng YUE ; Yongxin LIU ; Xiaoxiao YUAN ; Meiyan SUN ; Rui ZHANG ; Zhaodong JUAN ; Yaru HUANG ; Jizhe SHEN
Chinese Journal of Anesthesiology 2019;39(5):610-612
Objective To evaluate the effect of clemastine fumarate on Toll-like receptor 4/phosphatidylinositol-3-kinase/serine-threonine kinase (TLR4/PI3K/Akt) signaling pathway during hypoxia-reoxygenation (H/R) in rat cardiomyocytes.Methods H9C2 cells of rats cultured in vitro were seeded in culture wells or dishes at a density of 1×105 cells/ml and divided into 3 groups (n=11 each) by using a random number table method:control group (group C),H/R group and clemastine fumarate group (CF group).Cardiomyocytes were exposed to 5% CO2-95% N2in a low-glucose DMEM medium at 37℃ for 4 h followed by 4 h reoxygenation.At 4 h of reoxygenation,the cell viability was detected by CCK-8 assay,the ultrastructure was observed with a transmission electron microscope,the expression of TLR4,PI3K,phosphorylated Akt (p-Akt) and caspase-3 was detected by Western blot,and the expression of TLR4,PI3K and caspase-3 was detected by immunofluorescence.Results Compared with group C,the cell viability was significantly decreased,the expression of TLR4 and caspase-3 was up-regulated,and the expression of PI3K and p-Akt was down-regulated in group H/R (P<0.05).Compared with group H/R,the cell viability was significantly increased,the expression of TLR4 and caspase-3 was down-regulated,the expression of PI3K and p-Akt was up-regulated (P<0.05),and the mitochondrial damage was significantly attenuated in group CF.Conclusion The mechanism by which clemastine fumarate alleviates H/R injury to rat cardiomyocytes may be related to inhibiting TLR4 expression and activating PI3K/Akt signaling pathway.