1.Role of acid-sensing ion channel 1a in global cerebral ischemia-reperfusion injury in rats
Huixian CHENG ; Ming XIA ; Yaomei CUI ; Xianming ZENG ; Yudi ZHOU ; Qiuting ZENG ; Manlin DUAN ; Jianguo XU
Chinese Journal of Anesthesiology 2011;31(10):1260-1263
Objective To investigate the role of acid-sensing ion channel 1a(ASIC1a) in global cerebral ischemia-reperfusion injury in rats.Methods Forty male SD rats weighing 250-300 g were randomly divided into 4 groups (n =10 each): sham operation group (group S),cerebral ischemia-reperfusion group (group I/R),solvent control group (group SC) and group PcTX1 (a ASIC1 a blocker,group P).Global cerebral ischemia-reperfusion was induced by four-vessel occlusion.PcTX1(500 ng/ml)6 μl or solvent 6 μl was injected into the crerbral ventricular at the begining of reperfusion in groups P and SC respectively.The rats were sacrificed at 24 h of reperfusion,and then the hippocampi were removed for determination of Caspase-3,Bcl-2 and Bax protein expression and microscopic examination.Results Compared with group S,the expression of Caspase-3,Bcl-2 and Bax protein was up-regulated in groups I/R,SC and P (P < 0.05).Compared with group I/R,the expression of Caspase-3 and Bax was down-regulated,and the expression of Bcl-2 was up-regulated in group P ( P < 0.05).There was no significant difference in Caspase-3,Bcl-2 and Bax protein expression between groups I/R and SC (P > 0.05).The histopathologic damage was ameliorated in group P as compared with group I/R.Conclusion ASIC1a can induce global cerebral ischemia-reperfusion injury in rats by up-regulating Caspase-3 and Bax expression,and down-regulating Bcl-2 expression and inducing apoptosis.
2.Effects of different doses of PcTx1 on global cerebral ischemia-reperfusion injury in rats
Xianming ZENG ; Yaomei CUI ; Huixian CHENG ; Yunhe ZHU ; Wei GAO ; Qiuting ZENG ; Manlin DUAN ; Jianguo XU
Chinese Journal of Anesthesiology 2012;32(6):708-712
ObjectiveTo explore the effects of different doses of PcTx1,a specific blocker of acid-sensing ion channel 1a,on global cerebral ischemia/repedfusion (I/R) injury in rats,MethodsSixty adult male Sprague Dawley rats (weighing 250-300 g) were randomly divided into 6 grups ( n =10 each):sham operation group (group S),I/R group,different doses of PcTx1 ( 10 ng/ml,group P1 ; 25 ng/ml,group P2 ; 50 ng/ml,group P3 ;and 500 ng/ml,group P4 ) groups.Global cerebral ischemia was induced by the modified procedure of Pulsinelli 4-vessel occlusion.In groups P1,P2,P3 and P4,different doses of PcTx1 ( 10,25,50 and 500 ng/ml),6 μl each,were respectively injected into the lateral cerebral ventricle at the initiation of reperfusion,while equal volume of double distilled water was injected instead in group I/R.Six rats in each group were sacrificed at 24 h of reperfusion,and the brains were immediately removed,Thereafter,the contents of malondialdehyde (MDA),reduced glutathione (GSH) and ritric oxide (NO),the activities of constitutive NO synthase (eNOS) snd inducible NO synthase (iNOS) were detected in hippocampus.Four rats in each group were sacrificed at 72 h of reperfusion,and hematoxylin and eosin staining was used to observe the pathomorphological changes of the hippocampal neurons.ResultsCompared with group S,the other groups showed decreases in the contents of GSH,while increases in the contents of MDA and NO and the activities of cNOS and iNOS ( P < 0.05 or 0.01 ).The contents of GSH increased,while the contents of MDA and NO and the activities of cNOS and iNOS decreased in groups P2,P3 and P4 compared with group I/R ( P < 0.05 or 0.01).Compared with group P1,the contents of GSH increased,the contents of MDA and the activities of cNOS decreased in groups P2,P3 and P4,and the contents of NO and the activities of iNOS decreased in groups P3 and P4 ( P < 0.05 or 0.01 ).Compared with group P2,the activities of iNOS decreased in groups P3 and P4(P < 0.05 or 0.01).The damage to neurons in hippocampal CAI was severe in groups I/R and P1,but it was attenuated in groups P3 and P4.ConclusionPcTx1 25,50 and 500 ng/ml (6 μl)injected into lateral cerebral ventricle can attenuate global cerebral I/R injury in rats,and the dose 50 ng/ml (6 μl) is more suitable.
3.Effect of hydrogen-rich saline on expression of miR-210 and miR-21 in hippocampus during global cerebral ischemia-reperfusion in rats
Qiuting ZENG ; Wei GAO ; Yu ZOU ; Ying ZHOU ; Xuejun SUN ; Manlin DUAN ; Jianguo XU
Chinese Journal of Anesthesiology 2013;(2):239-241
Objective To evaluate the effects of hydrogen-rich saline on the expression of miR-210 and miR-21 in hippocampus during global cerebral ischemia-reperfusion (I/R) in rats.Methods Seventy-two healthy male Sprague-Dawley rats,aged 9-10 weeks,weighing 250-300 g,were randomly divided into 3 groups (n =24 each):sham operation group (group S),group I/R,and hydrogen-rich saline group (group H).Global cerebral I/R was produced by 4-vessel occlusion method.In group H,0.6 mmol/L hydrogen-rich saline 5 ml/kg was injected intraperitoneally at 0 and 6 h of reperfusion,while the equal volume of normal saline was injected instead of hydrogen-rich saline in the other two groups.Rats were sacrificed at 24 and 72 h of reperfusion,and then the bilateral hippocampi were removed for detection of the expression of miR-210 and miR-21 using RT-PCR.The global brain tissues were also obtained and stained with HE for examination of the changes in pyramidal cells in the CA1 region of hippocampus.Results Compared with group S,the expression of miR-210 and miR-21 was significantly up-regulated,and the number of pyramidal cells was decreased in group I/R (P < 0.05).Compared with group I/R,the expression of miR-210 and miR-21 was significantly down-regulated,and the number of pyramidal cells was increased in group H (P < 0.05).The pathological changes were significantly ameliorated in group H.Conciusion The mechanism by which hydrogen-rich saline attenuates global cerebral I/R injury is related to downregulation of the expression of miR-210 and miR-21 in rat hippocampus.
4.Effect of hydrogen-rich saline on regulatory T cells in peripheral blood during global cerebral ischemia-reperfusion in rats
Qian LI ; Qiuting ZENG ; Pan YU ; Changsong ZHU ; Tao LIU ; Xingdong CHEN ; Manlin DUAN ; Jianguo XU
Chinese Journal of Anesthesiology 2014;(3):370-372
Objective To evaluate the effect of hydrogen-rich saline on the regulatory T cells (Tregs ) in the peripheral blood during global cerebral ischemia-reperfusion (I/R ) in rats .Methods Seventy-seven male Sprague-Dawley rats ,aged 2-3 months ,weighing 260-300 g ,were randomly divided into 3 groups using a random number table:sham operation group (group S , n=11) ,group I/R (n=33) ,and hydrogen-rich saline group (group H , n=33 ) .Global cerebral I/R was produced by 4-vessel occlusion method .The bilateral carotid arteries were blocked for 15 min followed by reperfusion in I/R and H groups .In group H ,0.6 mmol/L hydrogen-rich saline 5 ml/kg was injected intraperitoneally at 0 and 6 h of reperfusion ,while the equal volume of normal saline was injected instead in the other two groups .Before ischemia (T0 ) in group S and at 6 ,24 and 72 h of reperfusion (T1-3 ) in I/R and H groups ,7 rats were chosen ,the blood samples from the peripheral vein were collected for determination of the number of Tregs . Then the animals were sacrificed and the spleen was removed for measurement of transforming growth factor-β1 (TGF-β1) content .The left 4 rats of each group were sacrificed at T0 and T1-3 and the brains were obtained for examination of the pyramidal cell morphology in the hippocampal CA 1 region and for determination of the number of pyramidal cells in brain tissues .Results Compared with group S , the number of pyramidal cells in the hippocampal CA1 region ,the number of Tregs in the peripheral blood and content of TGF-β1 in the spleen were significantly decreased at T1-3 in group I/R ( P<0.05) .Compared with group I/R ,the number of pyramidal cells in the hippocampal CA 1 region and the number of Tregs in the peripheral blood at T2-3 ,and the content of TGF-β1 in the spleen at T1-3 were significantly increased ( P<0.05) ,and the pathological changes of pyramidal cells were attenuated in group H .Conclusion The mechanism by which hydrogen-rich saline attenuates global cerebral I/R injury may be related to the increased number of Tregs in peripheral blood and promoted secretion of TGF-β1 in rats .
5.Role of mitochondrial permeability transition pore of hippocampai neurons in process of hydrogen-rich saline attenuating global cerebral ischemia-reperfusion injury in rats
Yaomei CUI ; Ming XIA ; Huixian CHENG ; Xianming ZENG ; Jian ZONG ; Kangli HUI ; Xuejun SUN ; Manlin DUAN ; Jianguo XU
Chinese Journal of Anesthesiology 2011;31(9):1139-1142
Objective To investigate the role of mitochondrial permeability transition pore (mPTP) of hippocampal neurons in process of hydrogen-rich saline attenuating global cerebral ischemia-reperfusion (I/R) injury in rats.Methods Seventy-two male Sprague Dawley rats,weighing 250-300 g,were randomly divided into six groups ( n =12 each):sham operation group (group S),cerebral ischemia-reperfusion group (group IR),normal saline group (group NS),hydrogen-rich saline group (group H),atractyloside group (group A) and hydrogen-rich saline + atractyloside group (group HA).Global cerebral I/R injury was produced by four-vessel occlusion method.Bilateral vertebral arteries were cauterized.Then bilateral common carotid arteries were occluded for 15min and followed by reperfusion.In groups H and HA,hydrogen-rich saline 5 ml/kg was injected intraperitoneally immediately after reperfusion,while equal volume of normal saline was injected in the other four groups.The rats in groups A and HA received intracerebroventricular injection of atractyloside 15 μl 10 min before reperfusion,while groups NS and H received intracerebroventricular injection of equal volume of normal saline.After the neurological behavior was evaluated at 24 h of reperfusion,8 rats in each group were sacrificed and the hippocampi were immediately isolated and homogenized followed by density gradient centrifugation.The opening degree of mPTP was assayed with spectrophotometry and the mitochondrial membrane potential (MMP) was detected with Rhodamine 123 method.Four rats in each group were killed at 72 h of reperfusion and the brains were removed for microscopic examination of the area CA1 of the hippocampus and determination of the number of normal pyramidal neurons.Results Compared with group S,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in the other five groups ( P < 0.05).The neurological behavior was better,MMP was increased and mPTP opening degree was decreased in groups H and HA as compared with group IR ( P < 0.05).Compared with group H,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in group HA ( P < 0.05).Compared with group IR,the number of normal pyramidal neurons at 72 h of reperfusion in the CA1 region of the hippocampus was higher in group HA ( P <0.05).The injury of the CA1 region of the hippocampus at 72 h of reperfusion was attenuated in group H as compared with groups IR,NS,A and HA.Conclusion Hydrogen-rich saline can attenuate global cerebral I/R injury throngh inhibiting the mPTP opening and reducing the dissipation of MMP,thus maintaining the mitochondrial function.
6.A study on gene mutation of coagulation factor Ⅺ protein secretion disorder and its mechanism
Shuting JIANG ; Yuan CHEN ; Meina LIU ; Manlin ZENG ; Kaiqi JIA ; Lihong YANG ; Yanhui JIN ; Mingshan WANG
Chinese Journal of Laboratory Medicine 2022;45(5):488-493
Objective:To investigate the molecular pathogenesis of a newly discovered gene mutation in a family with hereditary coagulation factor Ⅺ(FⅪ) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in September 2021 due to "calculus of intrahepatic duct". The patient had no symptoms of spontaneous bleeding.The clinical data and blood samples of the proband and her family members (10 persons in 3 generations) were collected.The activated partial thromboplastin time (APTT) and FⅪ activity (FⅪ:C) were performed by the one-stage clotting assay. FⅪ antigen (FⅪ:Ag) were detected by enzyme linked immunosorbent assay (ELISA). Genomic DNA extracted from peripheral blood cells of subjects was used as template to analyze F11 gene mutation by DNA direct sequencing. Bioinformatics software was used to analyze the effects of mutations on protein structure and function. Wild-type and mutant FⅪ protein expression vectors were constructed and transient transfected into HEK293T cells. The total RNA was extracted from positive transfected cells and then reversely transcribed into cDNA. The mRNA expression level of F11 gene in transfected cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The content of FⅪ:Ag and the expression of FⅪ protein in transfected cell lysates and culture supernatant were detected by ELISA and western blot.Results:The APTT of the proband was significantly prolonged to 107.9s (reference range 29.0-43.0s), while FⅪ:C and FⅪ:Ag were significantly decreased to 2% (reference range 84%-122%) and 5% (reference range 76%-127%), respectively. Gene sequencing analysis indicated that the proband had c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation in exon 6 and 13 of the F11 gene, respectively. Bioinformatics analysis showed that the amino acids at site 161 of FⅪ protein were threonine (Thr) in the matrix composed of five different species, indicating that Thr161 site was highly conserved among homologous genes in different species. p.Thr161Met heterozygous mutation affected the stability of local intermolecular structure of FⅪ protein. In vitro expression experiments of p.Thr161Met mutation showed that FⅪ protein had a normal synthesis in the cells but secretion dysfunction.Conclusions:c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation were mainly responsible for the decrease of FⅪ in this family. p.Thr161Met mutation was first reported in the world and did not affect the normal synthesis of FⅪ protein, but caused secretion dysfunction.
7.Antithrombin deficiency caused by SERPINC1 gene mutations predispose to venous thrombotic events
Haixiao XIE ; Yuan CHEN ; Qiyu XU ; Xingxing ZHOU ; Kaiqi JIA ; Manlin ZENG ; Lihong YANG ; Mingshan WANG
Chinese Journal of Laboratory Medicine 2023;46(2):203-208
Objective:To analyze 12 antithrombins (AT) gene mutations that cause AT deficiency and discuss the relationship between the SERPINC1 gene. mutations and venous thrombotic events.Methods:This study belongs to case series of observational studies. Collected the clinical data of 12 AT deficiency cases in the First Affiliated Hospital of Wenzhou Medical University from April 2014 to April 2021 and collected the blood samples before treatment. The AT activity (AT: A) and AT antigen (AT: Ag) was detected by chromogenic substrate and immunoturbidimetry, respectively. The 7 exons and flanking sequences of the SERPINC1 gene were sequenced directly by PCR, the suspected mutations were validated by reverse sequencing. Analyzed the correlation between the SERPINC1 gene. mutations and venous thrombotic events and figured out the proportion.Results:The AT: A of the 12 patients all decreased significantly, ranging from 30% to 66%, and the AT: Ag of the 7 patients decreased accordingly, showing type Ⅰ AT deficiency, and the AT: Ag of the other 5 patients were normal, presented type Ⅱ AT deficiency. 12 mutations were found including 6 heterozygous mutations which were discovered for the first time: c.456_458delCTT(p.phe121del), c.318_319insT(p.Asn75stop), c.922G>T(p.Gly276Cys), c.938T>C (p.Met281Thr), c.1346T>A(p.Leu417Gln)and c.851T>C(p.Met252Thr). All 12 patients had venous thrombosis, and 3 cases including 2 compound heterozygotes and 1 single heterozygote all suffered from deep venous thrombosis (DVT) when they were younger without obvious triggers. The other 9 patients all combined with the other thrombotic factors including old age, hypertensive, smoking, pregnancy, and prolonged immobilization.Conclusion:Patients with AT deficiency caused by SERPINC1 gene defects are prone to venous thrombosis, especially combined with other thrombotic factors.
8.Analysis of two propositas with inherited hypodysfibrinogenemia
Yuan CHEN ; Kaiqi JIA ; Anqing ZOU ; Manlin ZENG ; Lihong YANG ; Jianrong YANG ; Xiaolong LI ; Yanhui JIN ; Mingshan WANG
Chinese Journal of Laboratory Medicine 2022;45(12):1207-1213
Objective:To analyze the phenotype and genotype of two propositi with inherited hypodysfibrinogenaemia caused by compound heterozygous mutations, and investigate the molecular mechanism.Metheds:Two propositi and their family members(7 person in 3 generations and 10 person in 3 generations,respectively) were investigated. The activity of plasma fibrinogen (Fg:C) and thrombin time (TT) were analyzed by coagulation method, the antigen of plasma fibrinogen (Fg:Ag) was detected by immunoturbidimetry. All of the exons and flanking sequences of FGA,FGB,FGG of two propositi were amplified by PCR, followed by direct sequencing. The ClustalX-2, 1-win software was used to analyze the conservatism of mutated gene locus. PROVEAN and Mutation Taster were applied to analyze the pathogenicity of mutated amino acid. The changes of the protein spatial structure and intermolecular interaction were analyzed by Pymol.Results:Fg:C and Fg:Ag of proposita A and B were both significantly decreased (0.74 and 0.78 g/L, 0.96 and 0.94 g/L, respectively). Gene analysis revealed that proposita A and B both carried a heterozygous mutation c.2185G>A(p.AαGlu710Lys) in exon 6 of FGA. Furthermore, proposita A also carried a heterozygous mutation c.701G>T(p.γTrp208Leu) in exon 7 of FGG, and proposita B carried a heterozygous mutation c.1015A>C(p.γSer313Arg) in exon 8 of FGG. Phylogenetic analysis suggested that p.AαGlu710,p.γTrp208 and p.γSer313 were highly conserved among homologous species. All variants were predicted to be deleterious by two online bioinformatic softwares. The protein model analysis indicated that protein spatial structure and intermolecular hydrogen bonds were changed by these variants, which destroyed the stability of Fg.Conclusion:The compound heterozygous mutations of p.AαGlu710Lys and p.γTrp208Leu,p.AαGlu710Lys and p.γSer313Arg might account for the hypodysfibrinogenemia in two propositi.
9.Genetic analysis of two Chinese pedigrees affected with Hereditary hypofibrinemia due to missense variants.
Xiaoyong ZHENG ; Yi CHEN ; Mengzhen WEN ; Yanhui JIN ; Manlin ZENG ; Kaiqi JIA ; Yuan CHEN ; Mingshan WANG ; Lihong YANG
Chinese Journal of Medical Genetics 2023;40(3):276-281
OBJECTIVE:
To retrospectively analyze the clinical phenotypes and genetic variants in two Chinese pedigrees affected with Hereditary hypofibrinemia (IFD) and explore their molecular pathogenesis.
METHODS:
Two probands and their pedigree members were admitted to the First Affiliated Hospital of Wenzhou Medical University on March 30, 2021 and May 27, 2021, respectively. Clinical phenotypes of the probands were collected, and blood clotting indexes of the probands and their pedigree members were determined. Variants of the FGA, FGB and FGG genes were analyzed by Sanger sequencing, and candidate variants were verified by sequence comparison. Bioinformatic software was used to analyze the conservation of the amino acids and pathogenicity of the proteins. Alteration in protein structure and intermolecular force before and after the variant was analyzed by simulating the protein model.
RESULTS:
Proband 1, a 18-year-old male, had significantly low plasma fibrinogen activity (Fg:C) and plasma fibrinogen antigen (Fg:Ag), respectively at 0.80 g/L and 1.00 g/L. Proband 2, a 43-year-old male, had slightly low Fg:C and Fg:Ag at 1.35 g/L and 1.30 g/L, respectively. The Fg:C and Fg:Ag of proband 1's father, proband 2's father and son were also below the normal level. Genetic testing showed that proband 1 had harbored a heterozygous missense variant of c.688T>G (p.Phe230Val) in exon 7 of the FGG gene, which was inherited from his father. Proband 2, his father and son all had harbored a heterozygous variant of c.2516A>C (p.Asn839Thr) in exon 6 of the FGA gene. Homology analysis showed that the Phe230 and Asn839 residues were highly conserved among homologous species. Bioinformatic analysis predicted that both p.Phe230Val and p.Asn839Thr were pathogenic variants.
CONCLUSION
Analysis of protein simulation model showed that the p.Asn839Thr variant has changed the hydrogen bo`nd between the amino acids, thus affecting the stability of the protein structure. The heterozygous missense variants of p.Phe230Val and p.Asn839Thr probably underlay the IFD in the two pedigrees.
Humans
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Male
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Amino Acids
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East Asian People
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Exons
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Pedigree
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Retrospective Studies
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Afibrinogenemia/genetics*
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Mutation, Missense
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Fibrinogen/genetics*