Role of PKC and MMPs in the pathogenesis of the rat experimental atherosclerosis and the action of Danshen injection

AIM: To study the effects of protein kinase C on the expression of MMPs that may play an important role in the formation of atherosclerosis and the possible mechanism of Danshen injection to treat atherosclerosis. METHODS: 50 Sprgue-Dawley (SD) rats were divided into three groups randomly: control group (group C), model group (group M) and Denshen treatment group (group D). The serum was collected to measure the level of cholesterol, triglyceride, low density lipoprotein cholesterol（LDL-ch）. The expression of PKC and MMPs were measured by immunohistochemistry. Light microscope and electron microscope were also used. RESULTS: ① The cholesterol, triglyceride, LDL-ch concentrations in group M and group D were significantly higher than those in group C (P<0.01), cholesterol, triglycerideand LDL-ch contents were lower in group D than group M (P<0.01). ② The expression of PKC, MMP-2, MMP-9 in group M were significantly higher than those in group C (P<0.01), and were significantly lower in group D than those in group M (P<0.01). The expression of PKC was correlated with MMP-2 and MMP-9. ③ Light microscope showed that there were plaques in intima and serious calcification, necrosis, obviously irregular thickness in media of group M, but slightly in group D. ④ Electron microscope showed that smooth muscle cells of group M were necrosed , collage grew abundantly and alined disorderly, most of the endothelial cells exfoliated, but slightly in group D. CONCLUSIONS: ① MMPs play an important role in the generation and development of atherosclerosis. ② The activation of PKC may take part in the formation of atherosclerosis and it may be the upstream mechanism of the expression of MMPs. ③ The reduced expression of MMPs and PKC is a part of mechanism for Danshen to prevent and treat atherosclerosis.     

Apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol

AIM: To investigate the molecular mechanism of the apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol. METHODS: Human primary gastric cancer cells were planted into nude mice to establish the cancer model. Resveratrol at different doses were injected near the carcinoma on the nude mice. After treatment, transmission electron microscope and TUNEL staining method were used to detect the apoptosis of implanted tumor cells. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-related genes bcl-2 and bax in implanted tumor. RESULTS: Resveratrol significantly inhibited carcinoma growth when it was injected near the carcinoma. The apoptotic cells in implanted tumor induced by resveratrol were detected by transmission electron microscope and TUNEL staining, immunohistochemical staining and RT-PCR showed resveratrol inhibited bcl-2 expression and increased bax expression in human primary gastric cancer cells. CONCLUSION: Resveratrol inhibits implanted tumor of human primary gastric cancer cells in nude mice through inducing apoptosis. This apoptosis may be mediated by down-regulation of bcl-2 expression and up-regulation of bax expression.     

Effects of Ginkgolide B on glutamate-induced apoptosis and calcium overload in cultured rat cortical neurons

AIM: To observe the effects of ginkgolide B on the neuron apoptosis induced by glutamate and explore whether this effects are related to the changes of calcium in neurons. METHODS: Primary neuron culture was prepared according to a previously reported procedure with slight modification. Neuron damage was induced by 0.8 mmol/L glutamate. The cell survival rate was examined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. The neuron morphological changes, Hoechst 33258 unclear-staining analysis and agarose gel electrophoresis of DNA were measured as the indexes of cell apoptosis. Intracellular free Ca2+ concentration in neurons was measured by using the fluorescent Ca2+ indicator Fura-2/AM. RESULTS: The cells, exposed to glutamate (0.8 mmol/L), showed characteristic change of apoptosis and calcium overload, which were relieved by the treatment of ginkgolide B (10-250 μmol/L), with survival increasing and cell morphology restoring and DNA fragment decreasing. CONCLUSIONS: Ginkgolide B prevents the neurons from glutamate neurotoxicity by inhibiting glutamate-induced apoptosis. The potential mechanism of its action may be related to the competitive PAF receptor binding of ginkgolide B and decreasing the intracellular calcium concentration in neurons.     

Immunosupressive mechanism of cornus officinalis glycosides on the corneal allograft rejection in rats

AIM: To investigate the influence of the cornus officinalis glycosides (COG) on immunological function of corneal transplantation model of rats, and to clarify the immunosuppressive mechanism of COG through observing the activation of lymphocytes in blood. METHODS: Wister rats were used as recipients and SD rats were used as corneal graft donors, then the corneal allografts transplantation model on the closed colony rats were set up. Splenocytes proliferation and mixed lymphocyte reaction of Wister rats activated by ConA were observed. The phenotype change of CD4, CD8, CD25 in blood in different time postoperatively were observed by the di-sign flow cytometry, and the rate of CD4/CD8 was calculated. RESULTS: 1. The COG suppressed the proliferation of T lymphocytes and one-way mixed lymphocyte reaction on the corneal allografting. 2. The phenotype change of lymphocytes in boold was as follows: there was no significant difference between the different time of the CD4, CD8 expression and the CD4/CD8 rate in blood of the control group. The CD4 positive cells expressed CD25 postoperatively increased obviously. The CD4/CD8 rate of medicine group had the tendency to decrease. The CD4 positive cells expressed CD25 postoperation in the medicine group were less than that in the control group obviously. CONCLUSION: The suppression of the T lymphocyte proliferation, mixed lymphocyte reaction, CD molecule expressed by the activated T lymphocytes and the IL-2 receptor expression may be the main immunosuppressive mechanisms of Cornus officinalis glycosides on the cell-mediated immunity.     

Effect of glycine on body weight and lipid metabolism in mice

AIM: To study the effect of glycine on body weight and lipid metabolism in mice. METHODS: Mice were randomly divided into four groups, control, high-fat diet group (HF), glycine group (Gly), and glycine treatment group (Gly+HF). The mice were fed with normal diet and high-fat diet for 12 days, respectively, and then were treated with glycine solution by intraperitoneal injection. Finally, the serum was collected to measure serum lipid, epididymal fat-pads, pararenal fat-pads and abdominal fat-pads were taken out to weight. Moreover, pathologic changes in liver were observed. RESULTS: The body weight of HF group was obviously lower than that in control; the body weight and adipose tissue content of Gly group were obviously lower than those in control; the body weight and adipose tissue content of Gly+HF group were obviously lower than those in HF group; the levels of TC and TG in Gly group were obviously lower than those in control; there was no statistical difference in serum lipid between Gly+HF and HF groups. In addition, marked hepatic steatosis in HF group, and slight hepatic steatosis in Gly+HF group were found. CONCLUSION: Gly can decrease the body weight and adipose tissue content and serum lipid in normal diet and high-fat diet mice, and it also can prevent hepatic steatosis.     

Study on animal intracorneal implantation model with xenogeneic swine corneal stroma

AIM: To study the immunogenicity and biocompatibility of xenogeneic swine corneal stroma as biological carrier for cornea reconstruction and to reconstruct corneal endothelial tissue with this carrier in vitro. METHODS: (1) The lymphocytes from the peripheral blood of F344 rats were immunologically labeled by anti-rat CD25-FITC and anti-rat CD4/CD8-PE, then determined by flow cytometry (FCM) at 12th， 90th day after intracorneal implantation with fresh and dehydrated swine corneal stroma. (2) The fresh and dehydrated grafts made of swine corneal stroma were implanted intralamellarly in corneas of New Zealand rabbits. Clinical examinations were performed monthly and histological examinations were made at 14th， 30th， 60th， 120th and 240th day. (3) The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology of reconstructed tissue was tested by microscope. RESULTS: (1) Compared to isograft group and negative control, the expression CD4＋CD25＋， CD8＋CD25＋ and CD4＋/CD8＋ of xenograft rat group implantation with swine corneal stroma did not appear significantly different in statistics (P>0.05). (2) In the total 12 rabbits, all the cornea grafts survived without rejection reaction， corneal haze or corneal neovascularization. The fresh grafts got transparent after 2 months, and the dehydrated grafts got transparent after 6 months. Histological study demonstrated both fresh and dehydrated stroma grafts had fused with rabbits'corneal stroma very well without lymphocytes infiltrating. (3) As shown in histological observations, the reconstructed cat corneal endothelial tissue was similar to the nature tissue. Cultured cat endothelial cells connected tightly to each other and attached to the Descemet's membrane closely. CONCLUSION: Swine corneal stroma has low immunogenicity and satisfying biocompatibility,it is an ideal biological carrier for cornea reconstruction in vitro.     

Role of lipopolysaccharide in the development of hepatopulmonary syndrome

AIM: The goal of the present study was to evaluate the possibility about enterogenous endotoxemia in pathogenesis of hepatopulmonary syndrome. METHODS: The rat model of cirrhosis was prepared with compound factors. A small dose of lipopolysaccharide (LPS) was administered intraperitoneally once to aggravate endotoxemia of animal with cirrhosis. The normal rats injected with LPS or injected with LPS combined with glycine (LPS antagonist) were designed as controls. RESULTS: Hepatopulmonary syndrome of rats with cirrhosis had occurred in the end of eighth weeks. Pulmonary pathological changes of cirrhosis rats were exacerbated after administration of a given dose of LPS. Glycine sharply antagonized the biological effect of LPS in vivo and in vitro, inhibited the production of TNF-α by LPS and alleviated various pathological changes of hepatopulmonary syndrome. CONCLUSIONS: Enterogenous endotoxemia in cirrhosis rats might be an important mechanism in the development of hepatopulmonary syndrome. Endotoxin and its mediating effect by way of cytokines (TNF-α) may play a role in the pathogenesis of hepatopulmonary syndrome.     

Deoxyribozyme inhibits the proliferation of human umbilical artery smooth muscle cells

AIM: To observe the inhibition effects of 10-23 deoxyribozyme (DNAzyme) specific to human proliferating cell nuclear antigen (PCNA) gene on the proliferation of human umbilical artery smooth muscle cells (HUASMC) in vitro. METHODS: Using lipofectamine (Lip)-mediated method, the DNAzyme specific to PCNA was introduced into HUASMC. ［3H］-TdR incorporation was determined. The cell proliferation was examined by MTT assay and cell cycle was detected by flow cytometry. RESULTS: ［3H］-TdR incorporation in 1.0 μmol/L DNAzyme group was lower than that in control group (P<0.05) at 2 days after transfection. Compared with control group, the A value of MTT cholorimetric analysis decreased in 1.0 μmol/L DNAzyme group and antisense oligodeoxynucleotide (ASODN) group, at 2, 3 and 5 days after transfection significantly (P<0.01). The decrease in A value showed a dose-dependent manner within the experimental range at 5 days. After 2 days, the percentage of quiescence cells (G0/G1) was 73.8%, 54.7% and 41.1% in DNAzyme, ASODN and control groups, respectively. CONCLUSION: The DNAzyme specific to PCNA suppresses the proliferation of HUASMC effectively in vitro.     

Relation between the time-spatiotemporal expression of caspase-9 and photoreceptor apoptosis as well as electroretinography in RCS rats

AIM: To investigate the relation between the time- spatiotemporal expression of caspase-9 and photoreceptor apoptosis as well as electroretinography (ERG) in Royal College of Surgeons (RCS) rats. METHODS: Twelve non-pigmented degenerative RCS rats at age of 15, 20, 25, 30 days were sacrificed after ERG examination. The caspase-9 expression was analyzed with immunohistochemical staining, fluoro-metric activity assay and Western blotting. Apoptotic retinal neurons were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. RESULTS: The latency and amplitude of b-wave were (58.60±3.42) ms and (109.07±14.50) mV, (56.45±1.08) ms and (109.00±7.35) mV, respectively, in 15 and 20 days old degenerative RCS rats. The ERG resembled flat form in the degenerative RCS rats at age of 25 and 30 days. TUNEL-positive cells were located only in the outer nuclear layer, mostly prevailed in 25-day-old group. Caspase-9 protein expressed in the rat retina started to be found immunohistochemically in 25-day-old and was most predominant in the inner segment. Fluorometric activity assay showed that caspase-9 activity reached its peak, (10.98±0.13) nmol·g-1·min-1 at 20th day. Western blot assay revealed the small cleavage fragmentation at 20th day. No cleavage fragmentation was revealed in the 15-day-old degenerative RCS rats and all other control rats. CONCLUSION: The activation and time-spatiotemporal expression of caspase-9 are coincidence with photoreceptor apoptosis as well as the loss of vision function in RCS degenerative rats, which implicates the important role of caspase-9 in photoreceptor apoptosis.