ObjectiveTo investigate the possible mechanism affecting liver cirrhosis by splenectomy. MethodsBy subcutaneous administration of 20% carbon tetrachloride(CCl4), liver cirrhosis models were established in splenectomy and nonsplenectomy groups. After HE staining, special staining and immunohistochemical staining, mast cell, Kupffer’s cell and Ito cell were counted under optical microscope. Liver pathological sections and the dynamic changes of these cells in mice were studied respectively in comparison with the normal group.ResultsThe incidence of liver cirrhosis in nonsplenectomy group was significantly higher than that in splenectomy group after the 16th injection of CCl4 (P＜0.05). The count of mast cell was much higher than that in splenectomy group after the 4th and the 8th injection (P＜0.05). Kupffer’s cell and Ito cell significantly increased after the 12th and the 16th injection in nonsplenectomy group compared with splenectomy group (P＜0.05). ConclusionSplenectomy may decline the incidence of hepatic cirrhosis caused by multifactors. In the early stage, splenectomy influences the migration, maturation and accumulation of mast cell. In the middle and late stage, it influences the proliferation of Kupper’s cell and cytokine secretion, thus the Ito cells are activated and proliferation is inhibited, in which extracellular matrix decreases in amount and the degree of hepatic fibrosis is reduced.
【Abstract】Objective To investigate the inhibitory effects of flavonoids quercetin on the occurrence and proliferation of experimental mammary carcinoma. Methods DMBA induced mammary carcinoma was produced in rats. Seventy-nine female Sprague-Dawly rats were divided randomly into four groups: DMBA, DMBA with TAM, DMBA with quercetin and control. Chemicals had been administered to group A, group B, group C and group D respectively for 28 weeks. Samples of breasts were collected for light microscope observation and electromicroscope observation. Their expressions of proliferating cell nuclear antigen (PCNA) and the protein product of H-ras were examined by immunohistochemical staining. Results ①Mammary carcinoma incidence of group A(76.2%) was significantly higher than that of group B(40.9%), group C(45.5%) and group D(0%),P＜0.05, and there was no significant difference between group B and group C (P＞0.05), which indicated that quercetin could inhibit the occurrence of mammary carcinoma. ②Mean mammary tumor diameter of group A (2.37cm) was significantly larger than that of group B(1.82cm) and group C(1.71cm), P＜0.05, and there was no significant difference between group B and group C (P＞0.05), which indicated that quercetin could inhibit the growth of experimental mammary carcinoma. ③Immunohistochemical staining of PCNA showed significant difference between group A and group B, group A and group C (P＜0.05), with no significant difference between group B and group C (P＞0.05), which indicated that quercetin could inhibit the proliferation rate of tumor cells. ④Significant difference between group A and group B, group A and group C (P＜0.05), and no significant difference between group B and group C (P＞0.05), were noticed with immunohistochemical staining of H-ras protein product, which indicated that quercetin could inhibit the activity of Hras protein. Conclusion Quercetin could reduce the mammary carcinoma incidence and its degree of growth, and it may be related with its inhibitory effect on the activity of Hras and the proliferation of tumor cell.
To introduce a rat model of the conversion of acute edematous pancreatitis (AEP) to necrotizing pancreatitis (ANP). One hundred and seven Sprague-Dawley rats were randomized in three experimental groups as follows: sham operation control group and AEP group and ANP group. AEP was induced by pancreatic duct ligation and exocrine stimulation, ANP was induced same as AEP，but with a large dose of dextran-110 (500mg/kg) intravenously. The serum concentration of amylase increased significantly in AEP group and ANP group. Cytosolic free Ca2+ concentration in isolated pancreatic acinar cells increased consistently after induction of ANP. Homorrhage, parenchymal necrosis and calcium deposits in acinar cells were observed in pancreas in ANP group. Ultrastructural examination showed desquamation and necrosis of the endothelium of the pancreatic capillary in ANP group. These results suggest that ischemia may induce the conversion of AEP to ANP via acinar cell Ca2+ overloading. The rat model would seem to be a suitable animal model for studying aggravating mechanism of acute pancreatitis.
Objective To study the pathology and possible mechanism of experimental hydrochloric acid（HCl） inhalation-indued pulmonary fibrosis in rats.Methods 120 male SD rats were randomly divided into a nomal control group，a bleomycin group，a high dose HCl group，a middle dose HCl group and a low dose HCl group.The bleomycin group was intratracheally injected with bleomycin once to induce pulmonary fibrosis.The three HCl groups were intratracheally injected with HCl once per week.The control group was given saline by the same way.Six rats of each group were randomly sacrificed on day 7，14，28 and 42 respectively.The histological changes of lung tissue were studied by HE and Masson’s trichrome staining.Hydroxyproline level in lung tissue was measured by digestion method.Protein and mRNA expression of transforming growth factor-β1（TGF-β1） were assayed by immunohistochemistry and RT-PCR respectively.Results Alveolitis in three HCl groups was significantl compared to control group，most severe at the second week，then remained at a high level which was equivalent to or exceeded the level of the bleomysin group after 28 days.Pulmonary fibrosis in three HCl groups was also significantly more severe than that in the control group，but milder than that in the bleomysin group.The high-dose and middle-dose HCl groups were not significantly different from the bleomysin group on day 42.There was no difference between three HCl groups in the earlier period，but the high-dose HCl group has a significantly difference from low-dose group on day 42.The content of hydroxyproline in high-dose and middle-dose HCl groups was also significantly higher than that in the control group.On day 42 hydroxyproline content in high-dose HCl dose rather middle –or low dose group was similiar with the level of bleomysin group.Content of TGF-β1 mRNA in three HCl groups was comparable to the level of bleomysin group on day 28 and exceeded on day 42.The expression of TGF-β1 in three HCl groups was not significantly different from the bleomysin group on day 42.Conclusion Experimental acid aspiration might contribute to pulmonary fibrosis in rats.Acid induced alveolar epithelial cell damage，abnormal proliferation and repair and fibrosis could be involved..
Objective To observe the effect of intravitreal injection of mouse nerve growth factor (NGF) on interphotoreceptor retinoid-binding protein (IRBP) in the vitreous of diabetic rats at early stages. Methods Ninety-six male Sprague Dawley (SD) rats were divided into control group (group A, 24 rats) and experimental group (72 rats). The rats in experimental group were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into positive control group (group B), normal saline group (group C) and NGF group (group D), 24 rats in each group. The rats in the group A and B were not intervened. The rats were received intravitreal injection with 4mu;l normal saline (group C) or 4 mu;l (0.5 mu;g/mu;l) NGF (group D). At 2, 4, 6 and 8 weeks after injection, IRBP levels were detected by enzymelinked immunosorbent assay (ELISA); hematoxylin-eosin (HE) staining and light microscope were used to observe the morphological changes of the retina; transmission electron microscope was used to observe the retinal ultrastructure.Results At 2 weeks after injection, there was no significant difference in IRBP expression between group A,B,C and D (F=2.833，P=0.052). At 4, 6, 8 weeks after injection, the differences of IRBP expression between group A, B, C and D were significant (F=22.252, 108.459, 105.726; P=0.000). At different time points after injection, there was no significant difference in IRBP expression of group A (F=1.462, P=0.241), but there were significant differences in IRBP expression of group B, C and D (F=150.98, 63.519, 64.604; P=0.000). Light microscope found that the retinal structure was clear in group A and in group B, C, D at 2, 4 weeks after injection; the retinal thickness were thinner in group B, C, D at 8 weeks after injection. Transmission electron microscope displayed that the structure of rod outer segments was clear in group A and in group B, C, D at 2 weeks after injection; partly unclear structure of rod outer segments and slightly enlarged gap were observed in group B, C, D at 4, 8 weeks after injection. Conclusion Intravitreal injection with NGF can stabilize the IRBP expression in the vitreous of diabetic rats at early stages effectively.
Objective To observe the retinal toxicity of repeated intravitreal injection with bevacizumab (Avastin) in diabetic rats. Methods Forty male Sprague Dawley (SD) rats were randomly divided into normal group (Group A, 10 rats) and diabetes mellitus group (30 rats). The rats in diabetes mellitus group were induced with streptozotocin injection for diabetic retinopathy model. And then randomly divided into diabetic retinopathy (DR) group (Group B,10 rats), the rats were not intervened; the left eyes of the other 20 rats were intravitreal injected with bevacizumab 3 mu;l (25 mg/ml) for 3 times as experimental group (Group C) ; the right eyes of the 20 rats were not intervened as experimental control group (Group D). 20 days after last intravitreal injection, retinal function was measured by Flicker Electroretinogram (F-ERG);retinal vascular pattern was determined by fluorescence microscopy of ethidium bromide (EB) stained retinal flat mounts; retinal morphological changes were determined by light microscope on hematoxylin-eosin (HE) stained sections;Thy-1 and VEGF expression was measured by immunohistochemistry staining. Results F-ERG showed that, the differences of a-and b-waves, the b-wave amplitude and the Ops-wave amplitude in the implicit time between group A, B, C and D were significant (F=33.165, 36.162,19.955, 23.243; P＝0.000); the differences of a-wave amplitude between group A,B,C and D was not significant (F=0.097,P＝0.961). Retinal blood vessel pattern was normal in Group A; retinal vascular vessels were tortuous and irregularly expanded in Group B; retinal vascular vessels of Group C were regular and thinner than Group A; microaneurysm were showed in Group D. Light microscope displayed that the layers of the rat retina of Group A were regular, the retinal architectures of Group B were irregular, the retinal layers were regular in Group C, the retinal layers were irregular in Group D. Immunohistochemistry staining discovered that Thy-1 and VEGF were mainly expressed in ganglion cell layer（GCL）. Conclusion Repeated intravitreal injection of bevacizumab is toxic to retina of diabetes mellitus rats.
Objective To observe the inhibition effect of the hypoxia inducible factor-1alpha; (HIF-1alpha;) specific siRNA on the expression of vascular endothelial growth factor (VEGF) mRNA in retinal tissues in diabetic rat. Methods This is a randomized controlled study. HIF-1alpha; specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector. Fifty-four healthy Sprague Dawley (SD) rats were divided into control group (15 rats) and experimental group (39 rats). The experimental rats were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into diabetic retinopathy (DR) group (15 rats), vector group (12 rats) and gene therapy group (12 rats). LipofectamineTM2000 mixed with pSilencer2.1-U6neo plasmid or HiF-1alpha; siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively. Nothing was transfected into DR and control group. The expression of VEGF mRNA in retinas was measured by real-time RT-PCR. The inhibition efficiency of VEGFmRNA was calculated at 24, 48, 72 hours and 1 week after injection respectively. Significant differences between groups were evaluated by oneway analysis and LSD-t analysis. Results HIF-1alpha; siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group, increased obviously in the DR and vector group, decreased in the gene therapy group. There was no statistically significant between DR and vector group (t=0.669，0.142,0.151，0.025；P=0.514，0.889，0.882，0.980). The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group (t=8.768, 13.695, 11.285, 8.253;P=0.000). The inhibition efficiency of VEGFmRNA was 32.76%, 43.60%, 47.70%, 50.86% at 24, 48, 72 hours and 1 week after injection. Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1alpha; siRNA recombinant plasmid.
Objective To observe the effect of pigment epithelium-derived factor (PEDF）on glutamate metabolism in diabetic rat retina. Methods 78 Sprague-Dawley rats were randomly divided into the model group, model control group, PEDF intervention group and intervention control group. There were some dead and euglycemia rats at the end of experiment, so only 12 rats in each group were included in the statistical analysis. The diabetic retinopathy rat model of the model, PEDF intervention and intervention control group were induced with streptozotocin injection. The rats in the model group were not intervened. The monthly-age matched normal rats of model group were in the model control group. The left eyes of rats were received intravitreal injection with 5 mu;l (0.1 mu;g/mu;l) PEDF (PEDF intervention group) or 5 mu;l phosphate buffer solution (intervention control group). The expressions of L-glutamate/L-aspartate transporter in retina were analyzed by western blot and real time RT-PCR techniques and glutamate content in retina was analyzed by high-pressure liquid chromatography (HPLC). Cultured rat Muuml;ller cells were divided into the control,experimental, PEDF intervention and intervention control group, GLAST expressions were detected by fluorescence immunofluorescence and real-time RT-PCR techniques. The glutamate up-take activity of Muuml;ller cells was determined by intracellular [3H] labeled D, L-glutamate concentration with scintillation counting. Results Western blot and real-time RT-PCR showed that GLAST expression decreased (real-time RT-PCR：t=8.86,Plt;0.01；Western blot：t=3.42,P＜0.05), glutamate content increased（t=4.01,P＜0.05）in model group compared with the model control group; GLAST expression increased (real-time RT-PCR：t=3.56,P＜0.05；Western blot：t=3.52,P＜0.05), glutamate content decreased（t=4.36,P＜0.05）in the PEDF intervention group compared with the intervention control group. Real-time RT-PCR and fluorescence immunofluorescence showed that high glucose down-regulate GLAST expressions in Muuml;ller cells (rea-time RT-PCR：t=3.48,P＜0.05；fluorescence immunofluorescence：t=4.72,P＜0.05 ) and impair glutamate uptake activity of Muuml;ller cells (t=3.81, Plt;0.05). Under high glucose conditions, PEDF up-regulated GLAST expression significantly (real-time RT-PCR：t=6.82,P＜0.01；fluorescence immunofluorescence：t=3.72,P＜0.05) and ameliorated the glutamate up-take activity of Muuml;ller cells（t=4.14, Plt;0.05). Conclusions In diabetic rats, PEDF may improve the activity of GLAST in Muuml;ller cells, thus ameliorate retinal glutamate metabolism and inhibit death of retinal ganglion cells.
Objective To observe the effect of diabetic retinopathy on endothelial progenitor cells (EPCs) from peripheral blood. Methods Sixty male Wistar rats were divided into control group and diabetes group. The rats in diabetes group were induced with streptozotocin (STZ) injection for diabetic retinopathy model. Flow cytometry was used to identify and count the number of EPCs from peripheral blood at 1 week, 1, 3 and 6 months after injection. All eyeballs were examined by hematoxylin and eosin (HE) staining, periodic acidSchiff's (PAS) staining of trypsin-digested retinal vessels flat preparation and transmission electron microscope. EPCs count, and the relationship between DR morphological changes and EPCs count were compared and analyzed. Results The quantity of EPCs from peripheral blood at 1 week, 1, 3 and 6 months after STZ injection were 25plusmn;7, 28plusmn;8, 39plusmn;7, 43plusmn;7 cells per 200 000 monocytes respectively, which decreased compared with the control group 45plusmn;4 cells per 200 000 monocytes (F=8.933，Plt;0.01). The quantity of EPCs was gradually increased at 1 week, 1, 3 and 6 months after STZ injection, accompanied with responsive pathological changes of retinal structure and vessels. The thickness of retina at 1 week and 1 month after injection were reduced slightly. The number of retinal ganglion cells reduced, with the time passing by. Endothelial cells were edema, mitochondrial was swollen, capillary basement membrane was thicken, lumen was significant stenosis, lumen occlusion and retinal artery aneurysm were observed at 6 months after STZ injection. Conclusion The number of EPCs increases gradually throughout the development of DR.