1.Inhibitory effect of rapamycin on connective tissue growth factor-stimulated cell proliferation and fibronectin secretion in myofibroblasts
Xuxia GAO ; Haichang HUANG ; Xiaomei LI
Chinese Journal of Nephrology 2009;25(9):678-682
Objective To investigate the inhibitory effect and associated mechanism of rapamycin on proliferation and extracellular matrix (ECM) secretion in myofibroblasts stimulated by connective tissue growth factor (CTGF). Methods Primary cultivated myofibroblasts were divided into 6 groups: control, CTGF (100 μg/L), rapamycin 20 μg/L+CTGF 100 μg/L, rapamycin 40 μg/L +CTGF 100 μg/L, rapamycin 20 μg/L, and rapamycin 40 μg/L alone. 5'-bromodeoxyuridine (BrdU) incorporation assay was used to detect the myofibroblast proliferation.Western blot was used to analysis the secretory FN protein in the supernatant medium of cultured myofibroblasts and the ERK1/2 phosphorylation in myofibroblasts. Results CTGF (100 μg/L)incubation significantly increased the number of Brdu positive myofibroblasts(P<0.01) and the level of FN protein secretory (P<0.05) in cell supernatant medium compared with control group,respectively. The number of Brdu positive myofibroblasts markedly decreased by 62% and 70% (P <0.05) in rapamycin 20 μg/L+CTGF 100 μg/L and rapamycin 40 μg/L+CTGF 100 μg/L groups, respectively. The FN protein levels in supernatant were decreased by 15% and 44% compared with CTGF 100 μg/L group, respectively; but the difference of FN protein levels was significant only in rapamycin 40 μg/L group (P<0.05). CTGF could activate ERK1/2 at 10 minutes; but as myofibroblasts were pretreated with rapamycin 40 μg/L for 30 min, it abolished CTGF-induced ERK1/2 phosphoralation. PD98059, the specific inhibitor of ERK1/2, could block the effect of CTGF-induced proliferation (7%±5% vs 85%±7%, P<0.01) and FN secretion (1.0±0.1 vs 1.6±0.3, P<0.05). Conclusions Rapamycin partially suppresses the proliferation and ECM secretion of myofibroblasts induced by CTGF. Its effect may be through inhibiting CTGF-induced activation of ERKI/2 signaling pathway.
2.Pravastatin improves level of antithrombin Ⅲ in rats combined nephrotic syndrome with hypercoagulability
Xuxia GAO ; Daoyou ZHANG ; Jianguo SONG ; Hui ZHANG
Chinese Journal of Clinical Pharmacology and Therapeutics 2006;11(7):801-805
AIM: To investigate the anticoagulant effect of pravastatin and low molecular weight heparin (LMWH), as well as their combination, over time, in a rat model of experimental nephrotic syndrome. METHODS: Healthy SD male rats were chosen randomly to perform nephrotic syndrome models by single injection of adriamycin via tail vein, the matched normal control rats received single injection of equivalent 0.9% sodium chloride instead. After 14 days, the models were set up and randomized into model control group, pravastatin group (pravastatin 2 mg·kg-1·d-1 gavage once a day), LMWH group(LMWH 200 U·kg-1·d-1 intraperitoneal injection once a day) and combined treatment group(pravastatin 2 mg·kg-1·d-1 gavage+ LMWH 200 μ·kg-1·d-1 intraperitoneal injection), then all rats underwent measuring proteinuria every two weeks and fibrinogen, antithrombinⅢ(ATⅢ), D-dimer, platelet count, serum total protein and serum albumin after 4 weeks of treatment. RESULTS: The concentration of fibrinogen and D-dimer in model group was higher significantly than that in control group, and the level of ATⅢ was lower remarkably than that in control group, but platelet count had no obvious change; Compared with model group, pravastatin could increase the level of ATⅢ and decrease the concentration of D-dimer, but the concentration of fibrinogen and platelet count did not change obviously; LMWH and combined treatment could also decrease level of D-dimer, but had no great effects on ATⅢ, fibrinogen and platelet count; all treatment group had no obvious change of serum total protein and serum albumin. CONCLUSION: Adriamycin-induced nephrotic syndrome rat models have hypercoagulability and pravastatin can increase the level of ATⅢ.
3.A New Canal Instrument Sterilizer:Research and Developevment
Yuehua GAO ; Xuxia PENG ; Jing ZHOU ; Jiechun CHEN
Chinese Journal of Nosocomiology 2006;0(02):-
OBJECTIVE To make a new sterilizer to disinfect the instruments used for the root pulp treatment.METHODS After studying the SL sterilizer used in Japan,a new sterilizer was made.Studied how long the sterilizer could be used after putting through the electric.The effect of it was compared with the Eurouda sterilizer E6-18/24 by the method of culturing the bacteria.RESULTS 208.8 seconds after putting through the electric,the sterilizer could be used to sterilize the apparatus.And the effect of the new sterilizer was the same as the Eurouda sterilizer E6-18/24.CONCLUSIONS The effect of the sterilizer is testified,and the sterilizer could be used for the root pulp treatment instruments.
4.Study on the immune effect of heparin-binding hemagglutinin adhesin
Lihui NIE ; Zhaogang SUN ; Xuxia ZHANG ; Yi LIU ; Mengqiu GAO ; Chuanyou LI
Chinese Journal of Microbiology and Immunology 2009;29(9):792-795
Objective To investigate the immune protection of heparin-hinding hemagglutinin ad-hesin(HBHA) and to estimate its potential diagnostic value. Methods Native HBHA were used to stimu-late peripheral blood mononuelear cells (PBMCs) from different infected-cases including PPD negative healthy control, PPD positive latent tuberculosis(LTB) infection, pulmonary tuberculosis, and the IFN-γ/in the supernatant of culture was detected. Meanwhile, HBHA specific IgG antibody in the sera was detected by ELISA. Results The middle level of HBHA specific IFN-γ of the three groups were 49.5 pg/ml, 781.9 pg/ml and 341.8 pg/ml, respectively. IFN-γ of latent tuberculosis group was much higher than that of the control, and slightly higher than that of the patients with pulmonary tuberculosis. And the absorbency of the IgG antibody to HBHA in the three groups was 0.212±0.066, 0.224 ± 0.076 and 0.285±0.078. lgG an-tibody in the patients with pulmonary tuberculosis is higher than that of the healthy, including the control and the latent tuberculosis infection. Conclusion HBHA has good immunogenieity, and it can stimulate the LTB to release high level IFN-γ, suggests that the LTB doesn't develop active tuberculosis may rely on its protection. HBHA specific. IFN-γ release may identify 1,333 from the healthy. Anti-HBHA antibody plays an auxiliary role in the diagnosis of pulmonary tuberculosis.
5.Evaluation of applying γ-H2AX as a radiation biodosimetry with an animal model
Jing WANG ; Yaping ZHANG ; Defang DING ; Yun GAO ; Xuxia ZHANG ; Junxiang ZHANG ; Honghong CHEN
Chinese Journal of Radiological Medicine and Protection 2015;35(5):329-333
Objective To evaluate the potential feasibility of γ-H2AX foci as a biodosimetry after exposure to ionizing radiation by comparing DNA double-strand break repair kinetics in rat blood lymphocytes with that in human lymphocytes.Methods Peripheral blood lymphocytes separated from Sprague-Dawley(SD) male rats and healthy adults were exposed to γ-rays,and some rats were also subjected to total body irradiation.The inductions of DNA repair-related foci of γ-H2AX,pATM (S1981) and pDNA-PKcs (T2609) were detected with immunofluorescence staining technique at different time points post-irradiation,and the status of their co-localization was analyzed.Results The induction kinetics of γ-H2AX foci in rat lymphocytes was similar to that observed in human lymphocytes.The frequencies of γ-H2AX foci peaked at 30 min after γ-ray irradiation (trst =62.64,th =28.52,P < 0.05),then decreased rapidly after 6 h post-irradiation (trat =45.96,th =14.80,P <0.05),and the residual foci number remained only about 3%-8% of its maximal value at 24 h post-irradiation.At 30 min after γ-ray irradiation,the frequencies of pATM (S1981) and pDNA-PKcs (T2609) foci in rat and human lymphocytes significantly higher than those of nonirradiated control (trat =21.05,25.80,th =11.07,29.52,P < 0.05),and the frequencies of co-localization of pATM (S1981) or pDNA-PKcs (T2609) foci with γ-H2AX foci also markedly increased by 26%-32% in irradiated lymphocytes of rat and human (trat =5.34,9.14,thuman =18.32,51.28,P <0.05).Moreover,γ-H2AX foci incidence in rat lymphocytes in vitro was consistent with that induced by total body irradiation of rat.The number of γ-H2AX foci in irradiated rat lymphocytes increased with irradiation dose in a linear dose-dependent manner,its slope was similar to that of irradiated human lymphocytes reported by other laboratory.Conclusions Rat is a useful animal model to evaluate radiation biodosimetry with γ-H2AX foci in lymphocytes.The co-activation of ATM and DNA-PK plays an important role in DSB repair in the irradiated lymphocytes of rat and human.
6.Role of GSK-3β/β-catenin signaling pathway in human renal proximal tubular epithelial cell injury induced by depleted uranium
Qiang LI ; Yizhong BAO ; Xuxia ZHANG ; Yun GAO ; Defang DING ; Xiangyi REN ; Honghong CHEN
Chinese Journal of Radiological Medicine and Protection 2017;37(3):171-176
Objective To investigate the effects of glycogen synthase kinase-3β (GSK-3β) and β-catenin signaling on the human renal proximal tubular epithelial HK-2 cell injury induced by depleted uranium(DU),and provide a new enlightenment for the development of DU antidotes.Methods H K-2 cells were exposed to different concentrations of DU for 3-24 h,then the protein expressions of kidney injury molecule 1 (KIM-1),neutrophil gelatinase-associated lipocalin (NGAL) and nuclear β-catenin were detected by immunofluorescence staining.The protein expressions of p-GSK-3 β(S9),GSK-3β and cmyc were detected by Western blot assay.HK-2 cells were transiently transfected by GSK-3β (KD) plasmid or treated by TDZD-8 to inhibit the activity of GSK-3β specifically.Other HK-2 cells were transiently transfected by β-catenin plasmid to overexpress the β-catenin protein.Results The percentages of KIM-1 and NGAL-positive cells increased with DU exposure time and concentrations from 300 and 600 μmol/L,and they were significantly higher than those of the blank control at 6-24 h of DU exposure (KIM-1-positive cells:t =11.06,18.97,30.49,P <0.05;t =6.79,16.02,85.45,P < 0.05;NGAL-positive cells:t =11.78,11.37,34.29,P <0.05;t =7.34,21.63,36.84,P <0.05).In contrast,the ratio of p-GSK-3β (S9) to GSK-3β and percentage of nuclear β-catenin-positive cells were significantly higher than that of the blank control at 3-24 h of DU exposure (p-GSK-3β(S9)/GSK-3β:t =3.95,4.69,5.40,3.34,P < 0.05;nuclear β-catenin-positive cells:t =4.61,6.52,36.64,14.93,P < 0.05) with a maximum response at 9 h of DU exposure accompanied with corresponding increase of protein level of c-myc,a downstream target gene of β-catenin.Transient transfection of HK-2 cells with GSK-3β (KD) plasmid significantly inhibited the activity of GSK-3β (t =8.07,P < 0.05) and reduced the DU-increased percentage of KIM-1-positive cells (t =24.77,P < 0.05).Treatment cells with TDZD-8 inhibited the activity of GSK-3β and enhanced the percentage of nuclear β-catenin-positive cells,and it also significantly reduced the percentage of KIM-1-positive cells in HK-2 cells exposed to DU (t =6.25,6.73,P < 0.05).Moreover,overexpression of β-catenin significantly reduced DU-induced cell injury (t =7.48,P < 0.05).Conclusions GSK-3β/β-catenin signaling plays a key role in regulating the DU-induced cytotoxicity of HK-2 cells.Inhibition of GSK-3β activity and overexpression of β-catenin can protect the HK-2 cells from DU-induced damage.
7.Earlier renal fibrosis aggravates acute kidney injury induced by ischemia reperfusion in mice
Xuxia GAO ; Liyuan HAN ; Liping MA ; Yixin WANG
Chinese Journal of Nephrology 2018;34(12):924-929
Objective To investigate the influence of earlier renal fibrosis on ischemia and reperfusion induced acute kidney injury. Methods Male C57BL/6 mice at eight to twelve weeks old age were divided into 4 groups randomly: (1)Sham (n=3); (2)Unilateral ureter obstruction (UUO, n=6):UUO for 3 days (UUO3d, n=3) and UUO for 5 days (UUO5d, n=3);(3)Ischemia and reperfusion (IR, n=7): bilateral kidney ischemia for 40 minutes followed by 24 hours of reperfusion; (4)UUO for 3 days plus IR (UUO3d+IR, n=6): bilateral kidney ischemia after UUO 2 days for 40 minutes followed by 24 hours of reperfusion, and the real time for UUO was 3 days. Pathologic analysis for acute or chronic injury was performed on paraffin embedded kidney sections with hematoxylin and eosin (HE) or Masson staining. Apoptosis was detected by immunohistochemistry(IHC) and Western blotting with anti-caspase-3 antibody, and proliferation was observed by IHC with anti-ki67 antibody. Results On kidney sections with HE or Masson staining, it showed that the chronic kidney lesions and fibrosis got more severe as time of UUO prolonged from 3 days to 5 days; the area of matrix deposition increased in UUO5d and UUO3d mice significantly compared to Sham mice (P<0.05) and was smaller in UUO3d mice compared with UUO5d mice obviously (P<0.05). Acute kidney injury could be observed in UUO3d+IR mice, such as massive inflammatory cells infiltration, tubules dilation, brush border disappearance, tubular epithelial cells vacuolar degeneration, necrosis, casting formation, coexisting with chronic lesions: thinner cortex, broadened interstitial space, and increased blue stained matrix. Acute kidney injury score in UUO3d+IR mice was higher than that in IR mice significantly (P<0.05), and serum creatinine level increased significantly in UUO3d+IR mice compared to Sham mice (P<0.05). Caspase-3 expression increased and ki67 positive tubular cells decreased in UUO3d+IR mice than those in IR mice obviously (P<0.05). Conclusion Earlier renal fibrosis aggravates acute kidney injury induced by ischemia reperfusion in mice through increasing apoptosis and decreasing proliferation of tubular epithelial cells.
8.Mitochondrial diabetes mellitus:a case report
Jinxin ZHANG ; Xuxia GAO ; Liping MA
Chinese Journal of Diabetes 2024;32(1):59-61
Mitochondrial diabetes mellitus(MDM)is a type of diabetes caused by mitochondrial gene mutations resulting in progressive secretory function defects of pancreatic islet β cells.MDM is a rare single-gene genetic disease,accounting for about 1%of all diabetes mellitus.We reported a case of MDM patient and their family.
9.Analysis of SLC25A13 gene mutations in five infants with neonatal intrahepatic cholestasis caused by citrin deficiency.
Junjie XU ; Min GAO ; Yuqiang LYU ; Yunping TANG ; Xuxia WEI ; Lu YANG ; Kaihui ZHANG ; Yi LIU ; Zhongtao GAI
Chinese Journal of Medical Genetics 2018;35(1):34-38
OBJECTIVE To identify potential mutations in five infants with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). METHODS The SLC25A13 gene was analyzed by next-generation sequencing. Suspected mutations were confirmed by PCR and Sanger sequencing in the probands and their parents. Impact of novel mutations was predicted with PolyPhen-2 software. RESULTS All neonates have harbored mutations of the SLC25A13 gene. Eight mutations were discovered, which included two novel mutations (c.1357A>G and c.1663dup23). All parents were found to be carriers of the mutations. CONCLUSION Mutations of the SLC25A13 gene probably underlie the NICCD among the five patients, among which 851del4 and 1638-1660dup were the most common ones. This has enriched the spectrum of SLC25A13 mutation in association with NICCD.
10.Analysis of a child with Johanson-Blizzard syndrome due to novel compound heterozygous variants of UBR1 gene.
Xiaoli FU ; Li ZHANG ; Xuxia WEI ; Yuqiang LYU ; Lu YANG ; Min GAO ; Zhongtao GAI ; Yi LIU
Chinese Journal of Medical Genetics 2022;39(12):1379-1384
OBJECTIVE:
To analyze the clinical and genetic characteristics of a boy featuring unexplained developmental delay, malnutrition and distinct facial appearance.
METHODS:
Physical examination was carried out for the child. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA and trio-whole exome sequencing. Candidate variants were verified by Sanger sequencing.
RESULTS:
The patient had facial dysmorphism including nasal alae aplasia, scalp defect and teeth deformities, in addition with recurrent diarrhea due to pancreatic exocrine insufficiency. DNA sequencing revealed that he has harbored compound heterozygous variants of the UBR1 gene, namely c.3167C>G (p.S1056X) and c.1911+14C>G, which were inherited from his father and mother, respectively. Database search has suggested the c.3167C>G to be a novel nonsense variant and c.1911+14C>G a known splicing variant. Based on the guidelines of the American College of Medical Genetics and Genomics, the two variants were predicted to be pathogenic and likely pathogenic, respectively.
CONCLUSION
The child was diagnosed with Johanson-Blizzard syndrome due to the compound heterozygous variants of the UBR1 gene. Above finding has enriched the mutational spectrum of the UBR1 gene and provided a basis for genetic counseling for this family.
Child
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Humans
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Male
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Ectodermal Dysplasia/genetics*
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Pancreatic Diseases/genetics*
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Ubiquitin-Protein Ligases/genetics*