Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis

AIM:To investigate the location and expression of TIMP-1and TIMP-2 in the liver of normal and experimental hepaticfibrosis in rats.METHODS:The rat models of experimental immunityhepatic fibrosis (n=20) were prepared by the means ofimmunologic attacking with human serum albumin (HSA),and normal rats (n=10) served as control group.Bothimmunohistochemistry and in situ hybridization methodswere respectively used to detect the TIMP-1 and TIMP-2mRNA and related antigens in liver.The liver tissue wasdetected to find out the gene expression of TIMP-1 andTIMP-2 with RT-PCR.RESULTS:The TIMP-1 and TIMP-2 related antigens in liversof experimental group were expressed in myofibroblasts andfibroblasts (TIMP-1:482±65 vs 60±20;TIMP-2:336±48 vs50±19,P<0.001).This was the most obvious in portal areaand fibrous septum.The positive signals were located incytoplasm,not in nucleus.Such distribution and location wereconfirmed by situ hybridization (TIMP-1/β-acton:1.86±0.47vs 0.36±0.08;TIMP-2/β-actin:1.06±0.22 vs 0.36±0.08,P<0.001).The expression of TIMP-1 and TIMP-2 was seenin the liver of normal rats,but the expression level was verylow.However,the expression of TIMP-1 and TIMP-2 in theliver of experimental group was obviously high.CONCLUSION:In the process of hepatic fibrosis,fibroblastsand myofibroblasts are the major cells that express TIMPs.The more serious the hepatic fibrosis is in the injured liver,the higher the level of TIMP-1 and TIMP-2 gene expression.     

Expression of glutathione S-transferase placental mRNA in hepatic preneoplastic lesions in rats

ＥｘｐｒｅｓｓｉｏｎｏｆｇｌｕｔａｔｈｉｏｎｅＳｔｒａｎｓｆｅｒａｓｅｐｌａｃｅｎｔａｌｍＲＮＡｉｎｈｅｐａｔｉｃｐｒｅｎｅｏｐｌａｓｔｉｃｌｅｓｉｏｎｓｉｎｒａｔｓＺＨＵＨｕａｎＺｈａｎｇ，ＺＨＡＮＧＸｉｎｇＬｉａｎｄＣＨＥＮＹｉＳｈｅｎ...     

Expression and significance of HIF-1α and VEGF in rats with diabetic retinopathy

Objective:To investigate the expression of hypoxia inducible iaclor-1α(HIF-1α)and vascular endothelial growth factor(VECF)in diabelic retinopathy(DR)rats and its effect on the DR occurrence and development.Methods:A total of 120 SD rats were randomly divided into trial group and control group with 60 in each.STZ.i.p.was used in the trial group to establish the DM model,citrate buffer salt of same amount was used up.to the control group.1,3 and 6 months after injection,respective 20 rats were sacrificed in each group to observe expression of HIF-1αand VEGF in the rat retina tissue at different lime points.Results:Expression of HIF-1αand VEGF were negative in the control group;expression of HIF-1αand VKGF protein in retinal tissue were weak after 1 month of DR mold formation.It showed progressive enhancement along with the progression in different organizations,differences between groups were significant(P0.05).Conclusions:Expressions of HIF-1αand VF.GF were;correlated with disease progression in early diabelic relinopathy.Retinal oxygen can induce over-expression of HIF-1αand VEGF.It shows that HIF-1αand VEGF play an important role in the pathogenesis of DR.     

Expression of Bcl-2 and NF-κB in brain tissue after acute renal ischemia-reperfusion in rats

Objective:To investigate the effect of acute renal ischemia reperfusion on brain tissue.Methods:Fourty eight rats were randomly divided into four groups(n=12):sham operation group,30 min ischemia 60 min reperfusion group,60 min ischemia 60 min reperfusion group,and120 min ischemia 60 min reperfusion group.The brain tissues were taken after the experiment.TUNEL assay was used to detect the brain cell apoptosis,and western blot was used to detect the expression of apoptosis-related proteins and inflammatory factors.Results:Renal ischemiareperiusion induced apoptosis of brain tissues,and the apoptosis increased with prolongation of ischemia time.The detection at the molecular level showed decreased Bcl-2 expression,increased Bax expression,upreguiated expression of NF- κB and its downstream factor COX-2/PGE2.Conclusions:Acute renal ischemia-reperfusion can cause brain tissue damage,manifested as induced brain tissues apoptosis and inflammation activation.     

Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-pi and a-smooth muscle actin in rats with hepatic fibrosis

AIM: To investigate the effect of interferon-a (IFN-α) on preventing or reversing hepatic fibrosis in rat experimental model induced by CCI4. METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls, n = 18), group B (fibrotic model controls, n = 22), group C (IFN-α prevention, n = 22) initially treated with intramuscular injection of IFN-a in saline daily at the doses of 1× 105 U for 6 wk, group D (IFN-a treatment, n = 24) treated with intra-muscular injection of IFN-a in saline daily at the doses of 1×105 U for 6 wk after the first 6 wk, group E (0.9% sodium chloride treatment control, n = 24) treated with intra-muscular injection of 0.01 mL/kg daily for 6 wk after the first 6 wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemical and electron microscopic studies for the expressions of transforming growth factor-pi (TGF- μ41) and α-smooth muscle actin (α-SMA). RESULTS: The expressions of TGF-pl, the number of activated hepatic stellate cells and a-SMA in hepatic tissue of group C were significantly less than those of group B (P<0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P<0.01). CONCLUSION: IFN-a can inhibit the production of TGF-pl, decrease HSC activation and stimulate its apoptosis.     

Effect of L-arginine on calcium in hepatic mitochondrion in rats with obstructive jaundice

BACKGROUND: There is much debate over the regulation of mitochondrial calcium overload and reducing the impairment of energy metabolism in hepatic cells. It has not been reported whether L-arginine (L-Arg) can affect hepatic mitochondrial calcium overload. This study was undertaken to investigate the protective effect of L-Arg on Ca2+ handling of hepatic mitochondrion in rats with obstructive jaundice and to clarify its possible mechanism. METHODS: Seventy-two male SD rats were randomly divided into 3 groups: sham operation+normal saline group (SO group), common bile duct ligation+normal saline group (BDL group), and common bile duct ligation+ L-Arg group (L-Arg group). The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and Ca2+ in rat hepatic mitochondrion were examined at the 7th, 14th and 21st day after operation. RESULTS: The Ca2+ and MDA levels of hepatic mitochondrion increased significantly but their SOD content decreased markedly at each time point in the BDL group. Except at the 21st day, the Ca2+ and MDA, contents of hepatic mitochondrion were significantly lower, and SOD concentrations were higher in the L-Arg group than those in the BDL group at the 7th and 14th day (P<0.01). CONCLUSION: L-Arg has a protective effect on mitochondrion in the early and mid stages of obstructive jaundice.     

Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-β1 and α-smooth muscle actin in rats with hepatic fibrosis

AIM: To investigate the effect of interferon-α(IFN-α) on preventing or reversing hepatic fibrosis in rat experimental model induced by CCl4.METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls,n=18), group B (fibrotic model controls, n=22), group C (IFN-α prevention, n=22) initially treated with intra-muscular injection of IFN-α in saline daily at the doses of 1&#215;105U for 6wk, group D (IFN-α treatment, n=24) treated with intra-muscular injection of IFN-α in saline daily at the doses of 1&#215;105U for 6wk after the first 6wk, group E (0.9% sodium chloride treatment control, n=24) treated with intra-muscular injection of 0.01mL/kg daily for 6wk after the first 6wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemical and electron microscopic studies for the expressions of transforming growth factor-β1(TGF-β1) and α-smooth muscle actin (α-SMA).RESULTS: The expressions of TGF-β1, the number of activated hepatic stellate cells and α-SMA in hepatic tissue of group C were significantly less than those of group B(P&lt;0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P&lt;0.01).CONCLUSION: IFN-α can inhibit the production of TGF-β1, decrease HSC activation and stimulate its apoptosis.     

Analysis of B-cell clonality in the hepatic tissue of patients with Sjögren's syndrome

OBJECTIVE: We investigated the incidence of B-cell clonality in the minor salivary gland and liver (extra-glandular lesion) of patients with Sj?gren's syndrome (SS). We also compared B-cell clonality in the minor salivary gland and liver in the same individuals, and compared its incidence among patients with various liver diseases, such as primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH). METHODS: A minor salivary gland biopsy was performed on 35 patients with SS (30 patients with primary SS, and five patients with secondary SS). A liver biopsy was performed on nine patients with SS associated with bile duct lesions, two patients with PBC, one patient with AIH, one patient with drug-induced liver dysfunction, and three patients with viral hepatitis. DNA was extracted from each tissue sample and then subjected to Polymerase Chain Reaction (PCR). B-cell clonality was analysed by assessing the rearrangement of the immunoglobulin heavy chain (IgH) gene by PCR. RESULTS: B-cell clonality was confirmed in the minor salivary gland biopsy sample in 23 of the 35 patients (65.7%), and in the liver biopsy sample (non-exocrine organ involvement) in seven of the nine patients (77.8%). The presence or absence of B-cell clonality was investigated in both the minor salivary gland and liver in seven patients, but B-cell clonality was confirmed in both tissues in only one patient, and the pattern of clonality in the minor salivary gland differed from that in the liver. B-cell clonality was detected in the liver of the PBC and AIH patients. CONCLUSION: B-cell clonality is a phenomenon that is observed frequently in SS lesions in the salivary glands and liver. The appearance of B-cell clonality was shown to be attributable to antigen-driven clonal expansion.     

Expression of p53, p16 and COX-2 in pancreatic cancer with tissue microarray

BACKGROUND: Pancreatic cancer development and progression is driven by the accumulation of genetic changes. In this study we constructed tissue microarray containing specimens from pancreatic cancer, adjacent non-cancer tissue and normal tissue to survey the expression of p53, p16 and cyclooxyganase-2 (COX-2). METHODS: Tissue microarray containing 337 specimens from different stages of pancreatic cancer, adjacent noncancer tissue and normal tissues was constructed, and the expression of p53, p16 and COX-2 was assayed by immunohistochemistry to consecutive formalin-fixed tissue microarray sections. RESULTS: The expression of p53, p16 and COX-2 was significantly higher in tumorous tissues than in non-tumorons ones. A significant relationship was observed between p53 and COX-2, or p16 and COX-2. But no obvious correlation was seen between p53 and p16 expressions. Logistic regression analysis showed p53 and COX-2 as dependent predictors in pancreatic carcinogenesis, and a reciprocal relationship to neoplastic progression between p53 and COX-2. CONCLUSION: Combination analysis of p53 and COX-2 may be useful in predicting pancreatic carcinogenesis.     

Therapeutic effects and molecular mechanisms of anti-fibrosis herbs and selenium on rats with hepatic fibrosis

AIM:To study the therapeutic effects of anti-fibrosis herbsand selenium on hepatic fibrosis induced by carbontetrachloride (CCl_4) in rats and the underlining molecularmechanisms.METHODS:Fifty-three Wistar rats were randomly dividedinto:normal control group,model control group,colchicinegroup,anti-fibrosis herbs group (AF group) and anti-fibrosisherbs plus selenium group (AS group).The last four groupswere administered with CCl_4 at the beginning of experimentto induce hepatic fibrosis.Then colchicine,anti-fibrosis herbsand selenium were used to treat them.The normal controlgroup and the model control group were given normal salineat the same time.At the end of the 6~(th) week,rats in eachgroup were sacrificed.Blood and tissue specimens weretaken.Serum indicators (ALT,AST,HA,LN) were determinedand histopathological changes were graded.Lymphocyte CD_4and CD_8 were examined by flow cytometry.Expression ofTGF-β_1 and NF-κB was detected by immunohistochemistryand expression of TGF-β_1 mRNA was detected by semi-quantified RT-PCR.RESULTS:Histological grading showed much a smallerdegree of hepatic fibrogenesis in AS group and AF groupthan that in colchicine group and model control group.Theserum content of ALT,AST,HA and LN in AF group and ASgroup were significantly lower than that in colchicine group(ALT:65.8±26.5,67.3±18.4 and 96.2±20.9 in AF,AS andcolchicine groups respectively;AST:150.8±34.0,154.6±27.3and 215.8±24.6 respectively;HA:228±83,216±58 and416±135 respectively;LN:85.9±15.0,80.6±18.6 and106.3±14.2 respectively) (P<0.05).The level of CD_4 andCD_4/CD_8 ratio in AF group and AS group was significantly higherthat those in cochicine group (CD_4:50.8±3.8,52.6±3.4 and40.2±2.1 in AF,AS and colchicine groups respectively;CD_4/CD_8 ratio:1.45,1.46 and 1.26,respectively (P<0.05).The expression level of NF-κB and TGF-β_1 in the liver tissuesof AF and AS treatment groups was markedly decreasedcompared with that in cochicine group,and TGF-β_1 mRNAwas also markedly decreased (1.07±0.31 and 0.98±0.14 vs2.34±0.43,P<0.05).CONCLUSION:Anti-fibrosis herbs and selenium havebeneficial effects on hepatic fibrosis in rats by enhancingimmunity and inhibiting NF-κB and TGF-β_1 expressions.     

Correlation between expression of peroxisome proliferator-activated receptor γ and oxidative stress in brains of rats with chronic fluorosis

正Objective To detect the expression of peroxisome proliferator-activated receptorγ(PPARγ) in the brains of rats with chronic fluorosis and elucidate the relationship between PPARγand oxidative stress in chronic fluorosis.Methods According to body weight (100-120 g),sixty healthy SD rats were divided into control group(less than 0. 5 mg/L fluoride in drinking water), low fluoride group (5. 0 mg/L fluoride in drinking water,prepared by NaF), and high fluoride group (50. 0 mg/L fluoride in drinking water) via the random number table     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model Ⅰ, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO) activities assayed. NF-κB p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-α and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25±1.39 and 6.24±1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69±0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model II group were higher than that of normal control (9.7±1.96 vs1.7±0.15, 84.09±14.52 vs 16.03±6.21, 77.69±8.09 vs 13.41±4.91 P<0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r= 0.8235, 0.8780, 0.8572, 0.9152, 0.8247; P<0.05). CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model Ⅰ, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO)activities assayed. NF-~B p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-α (and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25&#177;1.39 and 6.24&#177;1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69+0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model Ⅱ group were higher than that of normal control(9.7&#177;1.96 vs 1.7&#177;0.15, 84.09&#177;14.52 vs 16.03&#177;6.21,77.69&#177;8.09 vs 13.41&#177;4.91 P&lt;0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152,0.8247; P&lt;0.05). CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Expression of TNF-α and VEGF in the esophagus of portal hypertensive rats

AIM: To investigate the expression of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF) in the development of esophageal varices in portal hypertensive rats. METHODS: Thirty male Sprague-Dawley (SD) rats in the model group in which a two-stage ligation of portal vein plus ligation of the left adrenal vein was performed, were divided into three subgroups (M7, M14, and M21) in which the rats were kiued on the seventh day, the 14^th d and the 21 d after the complete portal ligation. Thirty male SD rats, which underwent the sham operation in the control group, were also separated into three subgroups (C7, C14 and C21) corresponding to the models. The expression of TNF-α and VEGF in the esophagus of all the six subgroups of rats were measured with immunohistochemical SP technique. RESULTS: The portal pressure in the three model subgroups was significantly higher than that in the corresponding control subgroups (23.82&#177;1.83 vs 11.61&#177;0.86 cmH2O, 20.90&#177;3.27 vs 11.43&#177;1.55 cmH2O and 20.68&#177;2.27 vs 11.87&#177;0.79 cmH2O respectively, P&lt;0.01), as well as the number (9.3&#177;1.6 vs 5.1&#177;0.8, 11.1&#177;0.8 vs 5.4&#177;1.3 and 11.7&#177;1.5 vs 5.2&#177;1.1 respectively, P&lt;0.01) and the total vascular area (78 972.6&#177;3 527.8 vs 12 993.5&#177;4 994.8 um^2, 107 207.5&#177;4 6461.4 vs 11 862.6&#177;5 423.2 um^2 and 110 241.4&#177;49 262.2 vs 11 973.7&#177;3 968.5 um^2 respectively, P&lt;0.01) of submucosal veins in esophagus. Compared to the corresponding controls, the expression of TNF-α and VEGF in M21 was significantly higher (2.23&#177;0.30 vs 1.13&#177;0.28 and 1.65&#177;0.38 vs 0.56&#177;0.30 for TNF-α and VEGF respectively, P &lt;0.01), whereas there was no difference in M7 (1.14&#177;0.38 vs 1.06&#177;0.27 and 0.67&#177;0.35 vs 0.50&#177;0.24 for TNF-α and VEGF respectively, P&gt;0.05) and M14 (1.20&#177;0.25 vs 1.04&#177;0.26 and 0.65&#177;0.18 vs 0.53&#177;0.25 for TNF-α and VEGF respectively, P&gt;0.05). And the expression of TNF-α and VEGF in M21 was significantly higher than that in M7 (2.23&#177;0.30 vs 1.14&#177;0.38 and 1.65&#177;0.38 vs 0.67&#177;0.35 for TNF-α and VEGF respectively, P&lt;0.01) and M14 (2.23&#177;0.30 vs 1.20&#177;0.25 and 1.65&#177;0.38 vs 0.65&#177;0.18 for TNF-α and VEGF respectively, P&lt;0.01), but there was no difference between M7 and M14 (1.14&#177;0.38 vs 1.20&#177;0.25 and 0.67&#177;0.35 vs 0.65&#177;0.18 for TNF-α and VEGF respectively, P &gt;0.05). CONCLUSION: In the development of esophageal varices in portal hypertensive rats, increased TNF-α and VEGF may be not an early event, and probably play a role in weakening the esophageal wall and the rupture of esophageal varices.     

Expression of TNF-α and VEGF in the esophagus of portal hypertensive rats

AIM: To investigate the expression of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF) in the development of esophageal varices in portal hypertensive rats. METHODS: Thirty male Sprague-Dawley (SD) rats in the model group in which a two-stage ligation of portal vein plus ligation of the left adrenal vein was performed, were divided into three subgroups (M7, M14, and M21) in which the rats were kiued on the seventh day, the 14th d and the 21 d after the complete portal ligation. Thirty male SD rats, which underwent the sham operation in the control group, were also separated into three subgroups (C7, C14 and C21) corresponding to the models. The expression of TNF-α and VEGF in the esophagus of all the six subgroups of rats were measured with immunohistochemical SP technique. RESULTS: The portal pressure in the three model subgroups was significantly higher than that in the corresponding control subgroups (23.82±1.83 vs 11.61±0.86 cmH2O, 20.90±3.27 vs11.43±1.55 cmH2O and 20.68±2.27 vs 11.87±0.79 cmH2O respectively, vs P<0.01), as well as the number (9.3±1.6 vs 5.1?.8, 11.1±0.8 vs 5.4±1.3 and 11.7±1.5 vs 5.2?.1 respectively, P<0.01) and the total vascular area (78 972.6±3 527.8 vs 12 993.5±4 994.8 μm2, 107 207.5±4 6461.4 vs 11 862.6±5 423.2 μm2 and 110 241.4±49 262.2 vs 11 973.7±3 968.5 μm2 respectively, P<0.01) of submucosal veins in esophagus. Compared to the corresponding controls, the expression of TNP-α and VEGF in M21 was significantly higher (2.23±0.30 vs 1.13±0.28 and 1.65±0.38 vs 0.56±0.30 for TNF-α and VEGF respectively, P <0.01), whereas there was no difference in M7(1.14±0.38 vs 1.06±0.27 and 0.67±0.35 vs 0.50±0.24 for TNPa and VEGF respectively, P>0.05) and M14 (1.20±0.25 vs 1.04±0.26 and 0.65±0.18 vs 0.53±0.25 for TNF-α and VEGF respectively, P>0.05). And the expression of TNF-α and VEGF in M21 was significantly higher than that in M7 (2.23±0.30 vs1.14±0.38 and 1.65±0.38 vs 0.67±0.35 for TNF-α and VEGF respectively, P<0.01) and M14(2.23±0.30 vs 1.20±0.25 and 1.65±±0.38 vs 0.65±0.18 for TNF-a and VEGF respectively, P<0.01), but there was no difference between M7and M14(1.14±0.38 vs1.20±0.25 and 0.67±0.35 vs 0.65±0.18 for TNF-α and VEGF respectively, P>0.05). CONCLUSION: In the development of esophageal varices in portal hypertensive rats, increased TNF-α and VEGF may be not an early event, and probably play a role in weakening the esophageal wall and the rupture of esophageal varices.