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ABSTRACT: Objective: The objective of this study is to evaluate the effect and mechanism of aging on iodinated-contrast-media-induced nephropathy in male rats. Methods: Twenty-four healthy male rats were initially divided into 12-month-old and 24-month-old age groups (adult and older age groups, respectively; n = 12/group); subsequently, each age group was randomly divided into saline control (NS) and contrast media (CM) groups (n = 6/group). CM (76% diatrizoate, 10 mL/kg b.w.) was given through the caudal vein. Urinary creatinine (Ucr) and serum creatinine (Scr) were detected by an automatic biochemical analyzer. The activities of renal malondialdehyde (MDA), superoxide dismutase (SOD), angiotensin-converting enzyme (ACE), angiotensin II (Ang II), and reduced form of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) were determined by spectrophotometric assays with commercially available kits according to the manufacturers' protocols. Renal histological changes were observed by hematoxylin and eosin staining and scored semiquantitatively. Results: In diatrizoate-injected aged rats, Scr, the activities of ACE, Ang II, MDA, and NADPH oxidase in renal tissues were significantly increased (p < 0.01). The histologic scores were higher in the aged animals with CM treatment than those of control or adult rats (p < 0.01). There was an increasing trend but no significant statistical difference in renal ACE, Ang II, MDA, and NADPH oxidase or histologic scores in adult CM-injected rats compared with control animals (p > 0.05). Conclusions: Older age is an aggravating factor of iodinated-contrast-media-induced nephropathy in male rats. Oxidative stress and the renin-angiotensin system (RAS) may play an important role in nephrotoxicity induced by iodinated contrast media, especially in aged male rats.
Renal Failure 11/2012; · 0.82 Impact Factor
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ABSTRACT: Objective: To elucidate the relationship of oxidative stress and specificity protein 1 (Sp1) in the process of epithelial-to-mesenchymal transdifferentiation (EMT) and also to investigate the molecular mechanism of protective effect of probucol on the pathogenesis of diabetic kidney disease (DKD). Methods: Thirty male Sprague-Dawley (SD) rats were randomly divided into control group, diabetic group, and diabetic group under probucol therapy (n = 10 per group). The biochemical indicators including 24-h urinary total protein (24-h UTP) excretion, blood glucose (BG), lipids [triglycerides (TGs), total cholesterol (TC)], serum creatinine (Scr), creatinine clearance rate (Ccr), kidney tissue malondialdehyde (MDA) level, and glutathione peroxidase (GSH-Px) activity were assessed in all groups. The renal pathological changes were evaluated by hematoxylin and eosin (HE) and Masson staining. The protein expression of Sp1, α-smooth muscle actin (α-SMA), and E-cadherin was also measured and analyzed by immunohistochemistry and Western blotting. Results: Compared with the control group, the BG, TC, Scr, 24-h UTP, and MDA level of renal tissue increased significantly and the Ccr reduced in the rats of diabetic group (all p < 0.01). The pathological scores and the expression of Sp1 and α-SMA in renal tissue were up-regulated (p < 0.01) and the expression of E-cadherin was down-regulated significantly in the diabetic animals (p < 0.01). In the diabetic animals treated with probucol, the renal injuries were alleviated (p < 0.01). Conclusions: Oxidative stress may play an important role in the EMT process of tubular epithelial cells. Probucol could ameliorate renal disease progression in this model of diabetic nephropathy, which might be due to an antioxidant action, down-regulation of Sp1 protein expression, and inhibition of renal tubular EMT.
Renal Failure 09/2012; 34(10):1244-51. · 0.82 Impact Factor
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Ying Li,
Qiong Chen,
Fu-You Liu,
You-Ming Peng,
Shuai Wang,
Ji Li,
Jun Li, Shao-Bin Duan,
Lin Sun,
Guang-Hui Ling,
Jun-Hui Luo
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ABSTRACT: Norcantharidin (NCTD) was shown in our previous studies to attenuate renal tubulointerstitial fibrosis in rat models with diabetic nephropathy (DN). The aim of this study was to determine the effects of NCTD on the expression of extracellular matrix (ECM) and TGF-β1 in HK-2 cells stimulated by high glucose and on calcineurin (CaN)/NFAT pathway. Whether or not the antifibrotic effect of NCTD on renal interstitium was dependent on its inhibition of CaN pathway was also investigated. Experimental concentrations of NCTD were verified by cytotoxic test and MTT assay. HK-2 cells were transfected with CaN small interference RNA (siRNA). The mRNA and protein expressions of FN, ColIV, TGF-β1, and CaN in HK-2 cells were detected by real-time PCR and western blot. The CaN/NFAT pathway was examined by indirect immunofluorescence and western blot. Our study revealed that NCTD concentrations over 5 mg/l had overt cytotoxicity on HK-2 cells. Meanwhile, both 2.5 and 5 mg/l NCTD inhibited HK-2 cell proliferation (P < 0.05). NCTD inhibited the upregulation of FN, ColIV, and TGF-β1 of HK-2 cells stimulated by high glucose (P < 0.05), and also significantly downregulated the expression of CaN mRNA and protein in HK-2 cells (P < 0.05). In addition, not only was the nuclear translocation of NFATc inhibited, but its protein level in the nucleus was also reduced. Following CaN siRNA transfection, CaN mRNA and protein expression were significantly decreased. In contrast, the protein levels of FN, ColIV, and TGF-β1 increased in HK-2 cells stimulated by high glucose (P < 0.05). However, NCTD treatment downregulated their expression. These results indicated that NCTD could decrease the expression of ECM and TGF-β1 in HK-2 cells stimulated by high glucose, downregulate CaN expression, and block the CaN/NFAT signaling pathway. However, the effect of NCTD on inhibition of the expression of ECM and TGF-β1 was not associated with its inhibition of the CaN/NFAT pathway.
Laboratory Investigation 09/2011; 91(12):1706-16. · 3.64 Impact Factor
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ABSTRACT: There is rapidly accumulating evidence that use of oxidized hair dye causes various forms of nephrotoxic injury. However, the regulation and implication of the nephrotoxic injury resulting from p-aminophenol (PAP) and p-paraphenylenediamine (PPD), the main components of oxidized hair dye, remain unknown.
To clarify the effect of PAP and PPD, we analyzed the proliferation, lactate dehydrogenase (LDH) levels, apoptosis and subsequent mRNA levels of caspase-3 in HK-2 cells stimulated with different concentrations of PAP or PPD. Cell proliferation was assessed by MTT assay. The production of LDH was determined by Hitachi 7170 biochemical analyzer. The apoptosis of cell was analyzed using flow cytometry and scanning electron microscopy. The mRNA levels of caspase-3 were quantitatively measured by real-time PCR.
The proliferation of HK-2 cells was significantly inhibited by PAP, PPD and each mixed with hydrogen peroxide (H2O2). The level of apoptosis of HK-2 cells, the mRNA levels of caspase-3 and LDH production by PAP or PPD stimulation was significantly higher than controls or after H2O2. A typical apoptotic morphological change was observed under electron microscopy in response to PAP or PPD.
It appears that caspase-3 may play a key role in the nephrotoxic injury resulting from PAP, PPD or its oxidized form.
Journal of nephrology 07/2011; 25(4):481-9. · 1.65 Impact Factor
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ABSTRACT: The present study aims to observe the effects of NCTD (norcantharidin) on proliferation and FN (fibronectin) expression in human renal proximal tubular epithelial cell line (HK-2) induced by albumin in vitro. HK-2 cells were divided into control group, albumin group and different concentration of NCTD intervention groups. Proliferation of HK-2 cells was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], FN protein in culture media of HK-2 cells was examined by ELISA, and FN mRNA was analysed by RT-PCR (reverse transcription-PCR). We chose less than 5.0 mg/l of NCTD as the experimental concentration for the cytotoxicity test. MTT score was higher in the albumin group than in the control group (P<0.05). As compared with that of the albumin group, MTT score and FN protein concentration decreased, FN mRNA significantly down-regulated in NCTD intervention groups respectively (P<0.05). Our study showed that NCTD could inhibit the albumin-induced cell proliferation and FN expression in HK-2 cells, which might further prove the anti-fibrotic role of NCTD in proteinuria-associated tubulointerstitial damage.
Cell Biology International 06/2011; 35(12):1239-41. · 1.48 Impact Factor
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ABSTRACT: To investigate the effects of norcantharidin (NCTD) on tubulointerstitial fibrosis of diabetic nephropathy (DN) in streptozotocin-induced rat model.
Sprague-Dawley rats were randomly divided into control group, model group, low-dose NCTD (0.05 mg/kg/day) group, and high-dose NCTD (0.1 mg/kg/day) group. The model group was induced by injection intraperitoneally with 30 mg/kg streptozotocin in 0.1 mol/L sodium citrate solution (pH 4.5), after high-calorie foods were given for 2 months. NCTD was administered daily after the DN rat model was built. Rats were sacrificed at the end of the third and the eighth week; renal fibrosis and the expression of FN, collagen IV, TGF-β1, and calcineurin (CaN) were detected by Masson and immunohistochemistry staining, respectively.
Tubulointerstitial fibrosis was observed in DN rats, this kind of pathological changes was ameliorated in NCTD treatment group (p < 0.05). The expressions of FN, collagen IV, and TGF-β1 protein increased in the tubulointerstitial field of DN rats compared with the rats in control group. NCTD treatment could dose-dependently decrease their expression and reverse the fibrotic degree (p < 0.05). Meanwhile, the expression of CaN was detected in tubular fields of normal kidney and increased in the tubulointerstitial field in DN rats. However, NCTD downregulated its expression in a dose-dependent manner (p < 0.05).
NCTD could downregulate FN, collagen IV, and TGF-β1 expression in tubulointerstitial fields and attenuate tubulointerstitial fibrosis in the early stage of DN rats. NCTD also alleviated the expression of CaN in tubules in DN. The relationship between the role of NCTD's anti-tubulointerstitial fibrosis and its inhibition to CaN expression remains to be further elucidated.
Renal Failure 01/2011; 33(2):233-41. · 0.82 Impact Factor
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ABSTRACT: Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor-beta1 (TGF-beta1) inducing fibrosis, has recently been implicated in peritoneal fibrosis. Extracellular matrix (ECM) accumulation and angiogenesis are characteristic changes in peritoneal fibrosis. In this study we investigated the effect of CTGF knockdown via interference RNA (RNAi) on ECM production and vascular endothelial growth factor (VEGF) expression induced by TGF-beta1 in human peritoneal mesothelial cells (HPMCs). Four CTGF short hairpin RNA (shRNA) expression constructs were generated using the pRetroSuper vector, and infectious retroviral particles were prepared to infect HPMCs. Expression levels of CTGF, fibronectin(FN), collagen 1 (col 1), laminin, and VEGF mRNA and protein were measured by semi-quantitative reverse transcription polymerase chain reaction and western blot assay. CTGF expression was increased after stimulation with TGF-beta1, but inhibited using each of the four independent CTGF shRNA constructs (P < 0.01). Moreover, expression of ECM proteins (FN, col 1, and laminin) and VEGF were upregulated after incubation with TGF-beta1, but elevated levels of ECM and VEGF induced by TGF-beta1 were significantly inhibited by RNAi (P < 0.01), but not by the empty retroviral vector (P > 0.05). From these results, we concluded that retrovirus-mediated CTGF shRNA can effectively inhibit ECM production and VEGF expression induced by TGF-beta1 in HPMCs. This study suggests that downregulation of CTGF may represent a potential therapeutic approach for peritoneal fibrosis through decreasing ECM accumulation and angiogenesis.
Therapeutic apheresis and dialysis: official peer-reviewed journal of the International Society for Apheresis, the Japanese Society for Apheresis, the Japanese Society for Dialysis Therapy 02/2010; 14(1):27-34. · 1.39 Impact Factor
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ABSTRACT: Norcantharidin (NCTD), the demethylated analog of cantharidin isolated from Mylabris, is an anticancer drug routinely used against various human cancers in China. The aims of this study are to learn if NCTD has a protective action against severe proteinuria and consequent interstitial inflammation and fibrosis, and if the inhibition of nuclear factor-kappaB (NF-kappaB) and connective tissue growth factor (CTGF) by NCTD might be involved. Male Sprague-Dawley rats with protein overload nephropathy induced by intraperitoneally injected bovine serum albumin were used as a model. The histopathological examination of kidney tissue in the 9th week by light microscopy and scanning electron microscopy revealed that inflammatory cells had extensively infiltrated into the tubulointerstitial areas with interstitial fibrosis. The administration of NCTD at 0.1 mg/kg/day to the bovine-serum-albumin-injected animal models effectively reduced the proteinuria, and prevented the proteinuria-induced interstitial inflammation and fibrosis. Expressions of the NF-kappaB p65 subunit and CTGF, detected by immunohistochemistry, Western blotting and reverse-transcription polymerase chain reaction, were upregulated in protein overload nephropathy and were attenuated by NCTD. Inhibition of the expressions of the NF-kappaB p65 subunit and CTGF was one beneficial effect of NCTD. These results suggest that in addition to the antiproteinuric action of NCTD, due to its anti-inflammatory and antifibrotic effects as shown in the present study, it may become a therapeutic agent for proteinuria and its associated chronic inflammatory and fibrotic nephropathy.
American Journal of Nephrology 02/2008; 28(3):465-77. · 2.54 Impact Factor
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ABSTRACT: To explore the protective effect of amlodipine on the cytotoxicity induced by contrast media (meglumine diatrizoate) in human kidney cells (HKC).
An HKC line was used. The experiment was divided into 4 groups: a model group (diatrizoate 111g/L), a prevention group (diatrizoate 111g/L+amlodipine 10(-5)mol/L), an amlodipine control group (amlodipine 10(-5)mol/L), and a culture medium control group (simple none blood serum DMEM-F12 medium). Cytotoxicity induced by meglumine diatrizoate was analysed by methyl thiazolyl tetrazolium (MTT) test, lactate dehydrogenase (LDH) assay, Hochest33258 fluorescence stained cytospins, and flow cytometric DNA analysis. The protein expression of Bax was determined by Western blot, and caspase-3 activity was examined by fluorometric method.
In the prevention group, the cell viability increased significantly (P<0.05), LDH levels decreased (P<0.05), and the apotosis was lower than that of the model group (P<0.05) .Bax protein expression and caspase 3 activity decreased (P<0.05).
Amlodipine can inhibit the HKC apoptosis and protect the renal tuble cell from injury induced by meglumine diatrizoate.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 10/2007; 32(5):806-11.
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ABSTRACT: To compare the nephrotoxicity of high- and low-osmolar contrast media (HOCM and LOCM), and to determine the protective role of forsinopril or telmisartan and its possible mechanism.
Forty eight healthy SD rats were randomly divided into 6 groups: a normal control group, a glycerol control group, a low-osmolar contrast media (LOCM) group, a high-osmolar contrast media (HOCM) group, a forsinopril group, and a telmisartan group. Glycerine for inducing kidney damage was given to all rats except the normal control group. Twenty-four hours after the injection of glycerine, the mixed forsinopril suspension (10mg/kg) or telmisartan (5mg/kg) was poured into the stomach in the preventive group. Serum creatinine (SCr) and plasm angiotensin II (AngII) levels were detected by an automatical biochemical analyzer and radioimmunoassay; caspase-3 activity and claudin-1 expression of the renal tissue were detected by fluorometric method and immunohistochemical method. The renal injury was assessed by hematoxylin and eosin (HE) staining and terminal deoxynucleotide mediated nick and labeling (TUNEL) staining, respectively.
In diatrizoate-injected rats, SCr and AngII levels were increased (P<0.05). Expression of claudin-1 protein and caspase-3 activity in the renal tissue was upregulated. The histologic changes and percentage of apoptotic cells were milder in the LOCM rats than those in the HOCM rats. In the group pretreated with forsinopril or telmisartan, no increase in the levels of SCr and AngII was discovered. The expression of claudin-1 protein and caspase-3 activity was significantly lower than that in the HOCM group. The renal injuries induced by diatrizoate were alleviated.
Both HOCM and LOCM could cause cellular apoptosis in the kidney.LOCM was less toxic to rat kidney than HOCM. Nephrotoxicity induced by HOCM might be related to caspase-3, claudin-1 and AngII. Forsinopril or telmisartan may protect the renal tissue from nephrotoxicity induced by diatrizoate.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 10/2007; 32(5):812-8.
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Fu-you Liu,
Li Xiao,
You-ming Peng, Shao-bin Duan,
Hong Liu,
Ying-hong Liu,
Gui-hui Ling,
Fang Yuan,
Jun-xiang Chen,
Xiao Fu,
Jian-lian Zhu
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ABSTRACT: The peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor beta1 (TGF-beta1) plays a key role in regulating tissue repair and remodelling after injury. Connective tissue growth factor (CTGF), a downstream mediator of TGF-beta1 inducing fibrosis, has been implicated in peritoneal fibrosis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis that can hasten peritoneal fibrosis. In this study, we investigated the effect of small interfering RNA (siRNA) of CTGF by pRETRO-SUPER (PRS) retrovirus vector on the expression of CTGF and VEGF in human peritoneal mesothelial cells.
Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect human peritoneal mesothelial cell (HPMC). The cells were divided into seven groups: low glucose DMEM, low glucose DMEM + TGF-beta1 5 ng/ml, low glucose DMEM + TGF-beta1 5 ng/ml + PRS-CTGF-siRNA(1-4) and low glucose DMEM + TGF-beta1 5 ng/ml + PRS. The expression of CTGF and VEGF were measured by semiquantitative RT-PCR and Western blot.
Low levels of CTGF and VEGF were detected in confluent HPMCs. Following stimulation with TGF-beta1, the levels of CTGF and VEGF were significantly upregulated (P < 0.01). Introduction of PRS-CTGF-siRNA(1-4) resulted in the significant reduction of CTGF mRNA and protein, and VEGF mRNA (P < 0.01), especially in groups PRS-CTGF-siRNA1 and PRS-CTGF-siRNA4. The introduction of PRS void vector did not have these effects (P > 0.05).
The expression of CTGF siRNA mediated by PRS retrovirus vector can effectively reduce the level of CTGF and VEGF induced by TGF-beta1 in cultured HPMCs. This study may provide potential therapeutic strategies to prevent the peritoneal fibrosis.
Chinese medical journal 02/2007; 120(3):231-6. · 0.86 Impact Factor
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ABSTRACT: Tubulointerstitial damage (TID) is an important mediator in the progression of chronic proteinuric nephropathies. Our aim in this study was to evaluate the relationship between several clinical predictors and TID in adult-onset primary nephrotic syndrome in China.
One hundred ninety-five adult inpatients who were diagnosed with primary nephrotic syndrome based on clinical presentation and biopsy results were enrolled in this study from March 2003 to September 2005. The degree of TID was graded by a semiquantitative method including <2 score and >or=2 score.
In all patients, the rate of glomerulosclerosis was correlated with the severity of TID. Serum creatinine and uric acid (r = 0.183, p = 0.012 and r = 0.377, p = 0.00001, respectively) but not serum lipid or total 24-h urinary protein were related with TID. In 64 patients, urinary excretion of IgG (r = 0.443, p = 0.00001) but not of albumin, transferrin, retinal-binding protein, or alpha1-microglobulin were significantly associated with the extent of TID. Proteinuria selectivity index based upon IgG also correlated significantly with the extent of TID (p = 0.0001) (score 0-1 vs. score >or=2).
These results showed that serum creatinine and uric acid, the excretion of urinary IgG and proteinuria selectivity index based upon IgG, were highly correlated with the severity of TID in adult-onset primary nephrotic syndrome. These clinical parameters might be useful for predicting the development and progression of proteinuric nephropathy as independent risk factors.
Archives of Medical Research 11/2006; 37(8):981-6. · 1.88 Impact Factor
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Chinese medical journal 10/2005; 118(18):1552-6. · 0.86 Impact Factor
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ABSTRACT: To explore the effects of exogenous transforming growth factor-beta 1 (TGFbeta1) on peripheral nerve regeneration after the peripheral nerve injury and if TGFbeta1 regulates the expression of basic fibroblast growth factor (bFGF) in the anterior horn motoneurons of spinal cord during regeneration.
Forty-eight rats were crushed on the right sciatic nerve and then randomly divided into 2 groups: TGFbeta1 group and NS group. In TGFbeta1 group, TGFbeta1 50 microL (0.1 microg/mL) was injected into the proximal nerve near to the crushed nerve and after the operation the injured leg was injected with equal TGFbeta1 whereas the NS was replaced in the NS group. The rats of each group survived for 3, 7, 14 and 21 days after the lesion. The bFGF expression in the anterior horn motoneurons of spinal cord was detected by immunohistochemistry (IHC). Semi-thin section and Fast Blue retrograde tracing were also performed with the rats surviving for 21 days to observe the regeneration of distal end in the injured right sciatic nerve.
The number of bFGF immunoreactive positive motoneurons in TGFbeta1 group was obviously higher than that of the NS group (P < 0.05). In the distal sciatic nerve of the rats treated with TGFbeta1, the number and diameter of regenerating myelinated axons and the thickness of myelinated sheath were more than those of the NS group (P < 0.05). The number of motoneurons in spinal cord and neurons in dorsol root ganglia (DRG) labelled with Fast Blue in the NS group was obviously lower than in the TGFbeta1 group (P < 0.01).
Exogenous TGFbeta1 plays an important role in promoting the peripheral nerve regeneration; TGFbeta1 up-regulates the bFGF expression in the anterior horn motoneurons of spinal cord during the peripheral nerve regeneration.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 08/2005; 30(4):447-51.
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ABSTRACT: To determine the effect of transforming growth factor beta1 (TGFbeta1) antisense RNA on the synthesis and secretion of extracellular matrix in human peritoneal mesothelial cells (HPMC).
The recombinant human TGFbeta1 antisense eukaryotic expression vector was transfected into HPMC. Fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) were detected by ELISA method. The expression of FN, collagen I (col I) and PAI-1 mRNA were detected by RT-PCR method.
The mRNA of FN, Col I,nd PAI-1 in HPMC were repressed by TGFbeta1 antisense RNA 24 hours after the transfection in comparison with the control group, which decreased 17%, 26%, and 9.6%, respectively. The protein of FN and PAI-1 were inhibited 48 hours after the transfection (P < 0.05).
Recombinant human TGFbeta1 antisense eukaryotic expression vector can inhibit the synthesis and secretion of extracellular matrix in HPMC which may be effective in gene therapy for peritoneal fibrosis.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 11/2004; 29(5):548-51.
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ABSTRACT: To establish a culture model of peritoneal mesothelial cells in vitro separated by infusing the digestive fluid in the rat abdominal cativity.
Peritoneal mesothelial cells (PMC) were digested with 0.125% trypsin-0.01% EDTA Na2 in the abdominal cavity. We collected coelio-perfusate and cultured it. The isolated cells were identified by the phase contrast microscope, electron microscope and immunohistochemical analysis.
The confluent cells showed a uniform cobblestone-like appearance under the phase contrast microscope; numerous surface microvilli could be found under the electron microscope and the purity of the cells was over 95%. Immunohistochemical studies revealed a positive staining for cytokeratin and vimentin, but a negative staining for Factor VIII associated antigen and CD45.
The model of rat peritoneal mesothelial cells has been established successfully and it will provide an experimental basis of studying the fibrosis in peritoneal dialysis.
Hunan yi ke da xue xue bao = Hunan yike daxue xuebao = Bulletin of Hunan Medical University 01/2003; 27(6):542-4.
