1.Effect of propofol preconditioning on endoplasmic reticulum stress induced by hypoxia-reoxygenation in HEPG2 cells
Xiuli YANG ; Weixiang TANG ; Likui WANG ; Yujun SHEN ; Yuanhai LI
Chinese Journal of Anesthesiology 2014;34(4):489-491
Objective To evaluate the effect of propofol preconditioning on endoplasmic reticulum stress induced by hypoxia-reoxygenation (H/R) in HEPG2 cells.Methods HEPG2 cells were randomly divided into 4 groups using a random number table:control group (group C),propofol group (group P),H/R group and H/R + propofol preconditioning group (group PP).In group C,the cells were cultured routinely for 42 h.In group H/R,after being cultured routinely for 6 h,the cells were exposed to 1% O2 + 5% CO2 + 94% N2 for 12 h followed by 12 h reoxygenation.In group PP,the cells were cultured for 6 h in the culture medium containing propofol 10 μmol/L (final concentration),and then H/R was induced.The cell viability was detected by MTT assay.The expression of immunoglobulin heavy chain-binding protein (BIP),C/EBP homologous protein (CHOP) and activated caspase-3 was determined by Western blot.The expression of BIP,CHOP and caspase-3 mRNA was determined by RT-PCR.Results Compared with group C,the cell viability was significantly decreased,and the expression of BIP,CHOP and activated caspase-3 protein and mRNA was up-regnlated in H/R and PP groups,and no significant changes were found in the parameters mentioned above in group P.Compared with group H/R,the cell viability was significantly increased,and the expression of BIP,CHOP and activated caspase-3 protein and mRNA was down-regulated in PP group.Conclusion Propofol preconditioning can promote the cell proliferation and attenuate H/R injury to HEPG2 cells through inhibiting endoplasmic reticulum stress.
2.Identification of common variants within KCNK17 in Chinese Han population.
Zhouping, TANG ; Hu, DING ; Yujun, XU ; Shabei, XU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2010;30(1):13-7
KCNK17 is a member of the acid-sensitive subfamily of tandem pore K(+) channels, which are open at all membrane potentials an red contribute to cellular resting membrane potential. Recent genome-wide study (GWA) has shown that variants within KCNK17 confer genetic susceptibility for increasing ischemic stroke. In an effort to discover additional polymorphism(s), we scrutinized the genetic polymorphisms in the KCNK17. By direct DNA sequencing in 32 individuals, we identified nine sequence variants within the 16 kb of whole KCNK17 gene: one in exon1, one in intron and seven in the promoter region. Haplotypes, their frequencies and linkage disequilibrium coefficients (D'), among polymorphisms were estimated. All the polymorphisms in the 5'-flanking region (SNP2-SNP7) being in complete (or nearly complete) association with each other in the promoter region maybe produce synergistic effect to regulate the expression of KCNK17 gene and then have an influence on the pathogenesis of cerebrovascular diseases. The common haplotypes were observed comprising 88.9% of the total haplotypes in the same block. Bioinformatic analysis predicted several potential transcriptional factors binding sites by SNP -95, -134, -596 and -846. However, these binding sites need to be experimentally verified. The information concerning genetic polymorphisms of KCNK17 gene might provide valuable information for future genetic studies of diseases.
3.The role of transcription factor NF- ?B in vascular endothelial cell impairment induced by high glucose,TNF-? and IL-1?
Yujun XIAO ; Ailong HUANG ; Chunlin LI ; Huacong DENG ; Mingque XIANG ; Ni TANG
Chinese Journal of Endocrinology and Metabolism 1986;0(04):-
Objective To investigate the role of NF-?B in high glucose (HG) and cytokines (TNF-? and IL-1?)-induced impairment of ECV-304 cells (vascular endothelial cell line). Methods Recombinant adenovirus containing NF-?B supper-repressor I?B?M with mutant I?B? was constructed. Western blot, electrophoretic mobility shift assay (EMSA) and thiazolyl blue viability assay were applied in this study. Results TNF-?-induced I?B? degradation and NF-?B activation (P
4.Effect of HMGB1 and expression of TLR-4 in renal injury of SLE
Shuxia LIU ; Huifang GUO ; Yujun ZHANG ; Qingjuan LIU ; Lijuan TANG ; Huijun DUAN
Chinese Journal of Immunology 2001;0(10):-
Objective:To investigate the effect of HMGB1 and TLR-4 in the renal injury of SLE.Methods:The level of HMGB1 in serum from 16 patients suffering from SLE without kidney damage,18 patient with lupus nephritis (LN),and 12 healthy individuals were measured by ELISA.The fresh PBMCs were isolated and the total RNA was extracted.Then the mRNA expression of HMGB1 was amplified by RT-PCR.Flow cytometric analysis(FCM) was performed to study cell surface markers and the expression of TLR-4.Results:RT-PCR and ELISA results showed that the expressions of mRNA and protein were higher in patients with LN than in SLE without kindey damage and healthy people.The expression of TLR-4 in CD14+ monocytes of patients with LN was higher either,while there were no significant changes in CD3+ T cells among LN,SLE and healthy control.Conclusion:PBMCs in patients with LN could synthesize and secrete HMGB1 initiatively,which are correlated with serum HMGB1 level.HMGB1 might play a role in autoimmunity of lupus nephritis partly by activation of monocytes through its binding to TLR-4.
5.The immunoregulatory effects of bone marrow-derived mesenchymal stem cells combined with leflunomide on mice T-lymphocytes
Yingying QIU ; Jing LI ; Yujun YIN ; Yu TANG ; Haiyan YOU ; Wei ZHU ; Zhijun JIAO
Chinese Journal of Rheumatology 2008;12(7):469-472
Objective To investigate the immunoregulatory effects of bone marrow-derived mesenehy-real stem ceils (BMSCs) combined with leflunomide (LEF) on mice T-lymphocytes in vitro. Methods BMSCs from BALB/c mice were isolated and expanded. The purity of BMSCs was identified by flow cytometry (FCM). The BALB/c mice's spleen lymphocytes were isolated by using EZ-Sep<'TM> Mouse 1X. Under ConA stimulation, spleen lymphocytes were pretreated with LEF, then washed and co-cuhured with BMSCs. We set up four groups to investigate in this study: group A, spleen lymphocytes alone; group B, spleen lymphocytes with BM- SCs; group C, LEF-pretreated spleen lymphocytes alone and the group D, LEF-pretreated spleen lymphocytes with BMSCs. T-lymphocytes proliferation was assessed by MTT. FCM was used to analysis T-lymphocytes apoptosis and surface markers of CD69 and CD28. The mRNA expression of interleukin (IL)-2, IL-10 were detected by real-time RT-PCR. Results In vitro, the T-lymphocytes'values of A570 nm were significantly lower in group B and group C, compared with group A (group B vs group C vs group A, 0.578±0.042 vs 0.502± 0.040 vs 0.778±0.035, P<0.01), while the value of A<,570 nm>in group D was 0.218±0.033, which was also obviously lower than that in group B and group C (P<0.01). There were no suppressing effects on T-lympho-cytes'activation and expression of IL-2 had been observed. The proportion of apoptotic T-lymphocytes in group B and group D were (2.29±0.32)% and (4.22±0.98)%, which was significandy lower than that in group A (8.08±1.20) (P<0.01). The expression of IL-10 in group B and C were also lower than that in group A (group B vs group C vs group A, 0.098±0.039 vs 0.054±0.022 vs 1.000, P<0.01 ). Either, the expression of IL-10 in group D was 0.023±0.015, which was obviously lower than that in group B and group C (P<0.01). Conclusion BMSCs combined with LEF show synergistic immunoregulatary effects on mice's T-lymphoeytes.
6.Relationship between ABCA3 gene polymorphisms and susceptibility with neonatal respiratory distress syndrome
Wenjun TIAN ; Xiuqi CHEN ; Qiufen WEI ; Shangying TANG ; Huijuan QIN ; Yujun CHEN
Chinese Pediatric Emergency Medicine 2015;22(3):161-164
Objective To investigate ABCA3 gene polymorphism and its relationship with neonatal respiratory distress syndrome (NRDS) in the Guangxi Zhuang Autonomous Region of China by genotyping and haplotype analysis.Methods Using a tagging single nucleotide polymorphism (tSNP) strategy and TaqMan (r) real-time PCR,we genotyped 4 tSNPs (rs4787273,rs 1 50929,rs 11867129,and rs 17135889) and one additional coding SNP(rs13332514) of the ABCA3 gene in preterm infants with NRDS(NRDS group,n =45) and without NRDS (non-NRDS group,n =45) and subsequently predicted the haplotypes.The minor allele frequency and the haplotype 'distribution were compared between the two groups.Results The minor allele A(0.14 vs.0.05,P =0.046) and genotype AG (0.289 vs.0.111,P =0.035) frequency of SNP rs17135889 in NRDS group were significantly higher than those in non-NRDS group.Totally 6 haplotypes occurred at a frequency ≥0.01,among which,the haplotype TGGAG,depended on rs17135889,was significantly higher in NRDS group than that in non-NRDS group (0.061 vs.0.000,P =0.014).Conclusion The results suggested that SNP rs17135889 of ABCA3 gene might be related to NRDS in preterm population of the Guangxi Zhuang Autonomous Region.Allele A contributes to NRDS susceptibility in preterm infants.
7.Proliferation of renal cells involved by NF-?B/COX-2 signal pathway in diabetic nephropthy
Shuxia LIU ; Yujun ZHANG ; Jingkun ZHANG ; Qingjuan LIU ; Lijuan TANG ; Huijun DUAN
Basic & Clinical Medicine 2006;0(09):-
Objective To investigate the correlation between NF-?B/COX-2 signal pathway and cell proliferation in diabetic nephropathy. Methods Uninephrectomized STZ-induced male Wister rats were used as animal model. Using immunohistochemistry to detect NF-?B and COX-2 protein expressions in diabetic kidneys at the 16th week. HKC were cultured separately in normal or high glucose medium for 24,48,72 h.The expression of NF-?B and COX-2 protein was detected by flow cytometry and the expression of PCNA was detected by immunocytochemical staining. Results 1 Volum of glomeruli, mesangial matrix, thickness of glomerular and tubular basement membrane increased in diabete group; 2 COX-2 were expressed in cytoplasm of tubules and glomeruli by immunohistochemistry. Compared with control group, the expression of COX-2 was higher; activated NF-?B expressed in nucli of both tubules and glomeruli, There was light stainings for in control group, while enhanced stainings were observed in DM, there was a positive correlation between NF-?B and COX-2.3 Compared with those in HKC cultured in the medium with normal level glucose, the stainings were strengthened for PCNA in HKC exposed to high glucose from 24 h. 4 By FCM, the expression of NF-?B and COX-2 in HKC cultured in high glucose medium was higherthan that in normal glucose medium; the expression of NF-?B and PCNA was positively correlated with the expression of COX-2. Conclusion Activating NF-?B and elevating the expression of COX-2 play an important role in regulating cell proliferation, which may be one of the injury mechanisms of the renal cells during diabetic nephropathy.
8.Immunoregulatory effects of mesenchymal stem cells on T-lymphocytes of lupus nephritis
Yingying QIU ; Jing LI ; Jianqiang HE ; Yujun YIN ; Yu TANG ; Haiyan YOU
Chinese Journal of Rheumatology 2009;13(6):-
Objective To investigate the immunoregulatory effects of mesenchymal stem cells (MSCs) on the peripheral T-lymphocytes of lupus nephritis in vitro. Methods MSCs were isolated and expanded from human bone marrow cells. The purity of MSCs was identified by flow cytometry (FCM). The MSCs (4×104, 1×104, 2×103) were added into wells containing peripheral blood lymphocytes (2×105) from lupus nephritis in the presence of phytohemagglutinin [PHA). Samples were divided into the following groups: group A:T-lymphocytes alone; group B: MSCsl with T-lymphocytes(MSCsl:T=1:5); group C: MSCs2 with T-lymphocytes (MSCs2:T=1:20); group D: MSCs3 with T-lymphocytes (MSCs3:T=1:100). The proliferation of T-lymphocytes was assessed by MTT. FCM was used to analyze the apoptosis of T-lymphocytes and surface markers of CD28 and CD152. The gene expression of interferon γ (IFN-γ), interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1) were detected by real-time RT-PCR. Results In vitro, with the presence of MSCs, peripheral blood T-lymphocytes from lupus nephritis were statistically significantly decr-eased in their proliferative activities , apoptosis and CD28 expression in a dose-dependent manner. No inhibitory effects on CD152 expression of T-lymphocytes had been observed . MSCs promoted the gene expression of TGF-β1 and inhibited the gene expression of IL-10, IFN-γ. Conclusion MSCs can inhibit the proliferative activities, apoptosis and CD28 expression of peripheral blood T-lymphocytes of lupus nephritis, increase gene expression of TGF-β1 and lower the gene expression of IL-10, IFN-γ, which may play an important role in it's immunosuppressive effects on lupus nephritis.
9.Effect of propofol pretreatment on endoplasmic reticulum stress-mediated apoptosis during focal cerebral ischemia-reperfusion in rats
Weixiang TANG ; Likui WANG ; Yiqiao WANG ; Xiuli YANG ; Zhixin GAO ; Yujun SHEN ; Yuanhai LI
Chinese Journal of Anesthesiology 2012;(12):1488-1490
Objective To evaluate the effect of propofol on the endoplasmic reticulum stress-mediated apoptosis during focal cerebral ischemia-reperfusion (I/R) in rats.Methods Eighty adult male Sprague-Dawley rats,weighing 240-280 g,were randomly divided into 4 groups (n =20 each):shame operation group (group S) ; focal cerebral I/R group (group FCIR); propofol pretreatment group (group P); intralipid pretreatment group (group Ⅰ).Focal cerebral I/R was induced by 4 h middle cerebral artery occlusion followed by reperfusion.Propofol was infused at a rate of 12 mg· kg-1 · h-1 starting from 30 min before ischemia until 15 min of ischemia in group P,while intralipid was given instead of propofol in group I.Neurological deficit scores (NDSs) were measured at 6 h of reperfusion in 10 rats chosen from each group and the rats were then sacrificed.Their left brains were removed for determination of brain water content.The left 10 rats were sacrificed and their brains were immediately removed for determination of the expression of C/EBP homologous protein (CHOP),Bcl-2,and activated caspase-3 in the left hippocampi by Western blot.Results Compared with group S,NDSs and brain water content were significantly increased,the expression of CHOP and activated caspase-3 was up-regulated,and the expression of Bcl-2 was down-regulated in group FCIR,NDSs was increased in group P (P < 0.05).Compared with group FCIR,NDSs and brain water content were significantly decreased,the expression of CHOP and activated caspase-3 was down-regulated,and the expression of Bcl-2 was up-regulated in group P,and no significant change was found in each parameter in group Ⅰ (P > 0.05).Conclusion Propofol can reduce focal cerebral I/R injury through inhibition of the endoplasmic reticulum stress-mediated apoptosis in rats.
10.The effect of HMGB1 on the renal injure of systemic lupus erythematosus mediated in part via the TLR4 pathway
Shuxia LIU ; Jun HAO ; Huifang GUO ; Yujun ZHANG ; Qingjuan LIU ; Lijuan TANG ; Ning CHEN ; Haifiang WU ; Huijun DUAN
Chinese Journal of Microbiology and Immunology 2008;28(12):1079-1083
Objective To investigate the relationship between the effect of high mobility group protein box 1 (HMGBI) on the renal injure of systemic lupus erythematosus (SLE) and the expression of Toll-like receptor 4(TLR4). Methods The level of HMGB1, MMP-2 and TIMP-2 in serum from 16 pa-tients with SLE, 18 patients with lupus nephritis(LN) and 12 healthy people were measured by ELISA. The fresh peripheral blood mononuelear cell (PBMC) were isolated and the total RNA was extracted. Then the mRNA expression of HMGB1 was amplified by RT-PCR. Flow cytometry analysis was performed to study cell surface markers and the expression of TLR4. Results RT-PCR and ELISA results showed that the expres-sions of mRNA and level of HMGB1 protein in serum were higher in patients with LN than those in SLE and healthy people. The expression of TLR4 in CD14+ monecytes of patients with LN was higher than that with SLE and healthy people, while there were no significance in CD3+ T cells among LN, SLE and healthy peo-ple. The expressions of MMP-2 and TIMP-2 in serum of LN was lower than that in SLE and healthy people, at the same time the ratio of MMP-2/TIMP-2 decreased in LN group. HMGB1 mRNA and CD14+/TLR4+ was negatively correlated with the ratio of MMP-2/TIMP-2, and the level of HMGB1 in serum was positively correlated with proteinuria, while negatively correlated with the ratio of MMP-2/TIMP-2 in LN. Conclusion HMGB1 is one of the important cytokine in the pathogenesis of lupus nephritis. HMGBI might play a role in proteinuria of lupus nephritis in part via TLR4 pathway to activate monocytes and decrease the expression of MMP-2/TIMP-2.