[Effect of Huoxue injection on oxidized low-density lipoprotein induced activation of human umbilical vein endothelial cells].

OBJECTIVE: To observe effect of Huoxue injection on the expression of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), the adherence of monocytes to endothelial cells, and the regulation role of nuclear factor kappa B (NF-kappaB) in cultured human umbilical vein endothelial cells (HUVEC) injury induced by the oxidized low-density lipoprotein (ox-LDL). METHOD: The ox-LDL (100 mg x L(-1)) was added to the cultured HUVEC to prepare the injury model of HUVEC. The adhesive percentage between HUVEC treated with ox-LDL and monocytes was determined by protein quantification. Expression of mRNA and protein of ICAM-1 and VCAM-1 were determined by RT-PCR and flow cytometry respectively. The percentage of positive cells and the ratio of nuclei and cytoplasm of NF-kappaB p65 staining in HUVEC the were examined by cell immunochemistry. RESULT: Treatment of HUVEC with ox-LDL for 12, 24 hours significantly increased adhesion of monocytes to HUVEC and enhanced the expressions of mRNA and protein of ICAM-1 and VCAM-1. The percentage of positive cells and the ratio of nuclei and cytoplasm of NF-kappaB p65 staining in HUVEC were significantly increased after treatment with ox-LDL for 24 hours. Huo Xue Injection could significantly inhibit the adhesion between monocyte and HUVEC, the expression of mRNA and protein of ICAM-1 and VCAM-1, and declined the percentage of positive cells and the ratio of nuclei and cytoplasm of NF-kappaB p65 staining in HUVEC. The effects were strengthened with increasing the deal of Huoxue injection. CONCLUSION: Huoxue injection has an inhibitory effect on the adherence of monocytes to HUVEC, probably by way of down-regulating the expression of mRNA and protein of ICAM-1 and VCAM-1 in HUVEC. The mechanism is probably associated with inhibiting the activation of NF-kappaB p65 of HUVEC. The effects of Huoxue injection can bring about the protective effect to endothelial cells injury induced by ox-LDL.

[Effect of effective components of Huanglian Jiedu decocting on NF-kappaB in cultured rat cerebral microvascular endothelial cells injury induced by hypoxia and reoxygenation].

OBJECTIVE: To investigate the protective mechanism of geniposide, baicalin and berberine on hypoxia and reoxygenation injury in cultured rat cerebral microvascular endothelial cells. METHOD: To establish a model of hypoxia four hours and reoxygenation twelve hours injury in rat cerebral microvascular endothelial cells in vitro. The injured cells were treated with geniposide (0. 128, 0.064, 0.032 micromol mL(-1), baicalin (0.028, 0.014, 0.007 micromol mL(- 1)) and berberine (0.024, 0.012, 0.006 micromol mL(-1)). The expression of p65 subunit of NF-kappaB was detected by immunocytochemical assay and techniques of image quantitative analysis. The protein expression of NF-kappaB was calculated with the mean optical density and mean area. The nuclear translocation of NF-kappaB was calculated with the percentage of positive cells and ratios of light transmittance of cytoplasm and cell nucleus. RESULT: Compared with the normal group, both the protein expression and the nuclear translocation of NF-kappaB of model group were significant increased (P <0.01). Compared with the model group, the mean optical density of all treated groups was decreased ,but these was no significant difference between them. As compared with model group, the mean area of all treated groups was significant decreased (P < 0.01). The percentage of nuclear translocation of all treated groups is not only lower than that of the model group but higher than that of the normal group (P <0.01). Compared with the model group, the ratios of light transmittance of cytoplasm and cell nucleus of all treated groups was significantly elevated (P <0.01). CONCLUSION: The results suggesed that geniposide, baicalin and berberine could protect hypoxia/reoxygenation injuried rat cerebral microvascular endothelial cells injury. One of the mechanism may lie in inhibiting both the protein expression and the nuclear translocation of NF-kappaB.

[Effect of effective components of huanglian jiedu decoction on hypoxia, reoxygenation injury and the expression of VCAM in cultured rat cerebral microvascular endothelial cells].

OBJECTIVE: To investigate the protective mechanism of geniposide, baicalin and berberine on hypoxia and reoxygenation injury in cultured rat cerebral microvascular endothelial cells. METHOD: A model of four hours hypoxia and twelve hours reoxygenation injury in rat cerebral microvascular endothelial cells in vitro was established. The injured cells were treated with geniposide (0.128, 0.064, 0.032 mmol x L(-1)), baicalin (0.028, 0.014, 0.007 mmol L(-1)) and berberine (0.024, 0.012, 0.006 mmol L(-1)), respectively. The immunocytochemical method and techniques of image quantitative analysis were used to detect the mean optical density and mean area in order to match the protein expression of VCAM-1. The method of RT-PCR was adopted to observe and match the mRNA expression of VCAM-1. RESULT: As compared with the normal group, the mean optical density, the mean area and the mRNA expression of VCAM-1 of model group were significant increased (P < 0.01, P < 0.01, P < 0.01). As compared with the model group, both the mean optical density and the mean area of all treated groups were decreased, and there was significant difference between them (P < 0.01, P < 0.01). As compared with normal group, the mean optical density of baicalin (0.007 mmol x L(-1)) and berberine (0.012, 0.006 mmol x L(-1)) were significant decreased (P < 0.05, P < 0.01, P < 0.01), but there was no significant difference between the other groups and the normal group. As compared with normal group, the mean area of baicalin (0.0014 mmol x L(-1)) was significant decreased (P < 0.05), but there was significant difference between the other groups and the normal group. The mRNA expression of all treated groups was not only lower than that of the model group but also higher than that of the normal group (P < 0.05, P < 0.05). CONCLUSION: The results suggest that geniposide, baicalin and berberine, which are effective compositions of huanglian jiedu decoting, can protect hypoxia-reoxygenation injuried rat cerebral microvascular endothelial cells. One of the protected mechanisms is that they can inhibit the expression of VCAM-1.

Effects of Radix notoginseng extracts drug-containing serum on expressions of bcl-2, Bax and p21WAF1 proteins in MNNG transformed GES-1 cells.

OBJECTIVE: To investigate the effects of Radix notoginseng extracts drug-containing serum on the expressions of apoptosis-regulating proteins including Bax, bcl-2 and p21WAF1 in precancerous gastric cells. METHODS: The N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) transformed eternalized human gastric mucosa epithelium GES-1 cell line (MC cell) was used in vitro as a model of gastric precancerous lesion. The medicated canine serum was prepared by feeding to the adult Beagle dog with Radix notoginseng extracts and obtaining the serum after 2-hour medication. MC cells were cultured with medicated canine serum (medicated serum group) or non-medicated canine serum (normal control group) for 72 hours. Expressions of Bax, bcl-2 and p21WAF1 proteins were detected by immunocytochemical assay and the average optical density of the cells was determined by an image analysis system. RESULTS: Compared with those of the normal control group, Bax and p21WAF1 expressions in medicated serum group were significantly enhanced (P<0.01), while the expression of bcl-2 was significantly reduced (P<0.01). CONCLUSION: Radix notoginseng extracts may inhibit the proliferation and promote the apoptosis of precancerous gastric cells through altering expressions of the bcl-2, Bax and p21WAF1 genes.

[Protective action of effective components of Huanglian Jiedu decoction on hypoxia and reoxygenation injury in cultured rat cerebral microvascular endothelial cells].

OBJECTIVE: To investigate the protective effect of geniposide, baicalin and berberine for the rat cerebral microvascular endothelial cell. METHOD: The model of hypoxia and reoxygenation injury in rat cerebral microvascular endothelial cells in vitro was established. Both normal and model cells were treated with geniposide (1.024, 0.512, 0.256, 0.128, 0.064, 0.032, 0.016, 0.008 micromol x mL(-1)), baicalin (0.224, 0.112, 0.056, 0.028, 0.014, 0.007, 0.003 micromol x mL(-1)) and berberine (0.192, 0.096, 0.048, 0.024, 0.012, 0.006, 0.003 micromol x mL(-1)). Cell activity was measured by methyl thiazolyl tetrazolium (MTT) test. RESULT: After hypoxia/hypoglycemia cultures for 4 hour and reoxygenation for 12 hour, geniposide (0.128, 0.064, 0.032 micromol x mL(-1)), baicalin (0.028, 0.014, 0.007 micromol x mL(-1)) and berberine (0.024, 0.012, 0.006 micromol x microL(-1) could protect the injuried cerebral microvascular endothelial cells. CONCLUSION: Appropriate concentration of geniposide, baicalin and berberine, which are effective components of Huanglian Jiedu decoction, could protect the injuried cerebral microvascular endothelial cells.

[Protect effects of Qingkailing injection on mitochondrion membrane potential during injury induced by hypoxia-hypoglycemia and reoxygenation in cultured rat hippocampal neurons].

OBJECTIVE: To investigate the protect effects of Qingkailing injection on mitochondrion membrane potential (MMP) during injury induced by hypoxia-hypoglycemia and reoxygenation in cultured rat hippocampal neurons. METHOD: Mitochondrion activity was measured by methyl thiazolyl tetrazolium (MTT) test. MMP and apoptosis were measured by flow cytometry. intracellular free calcium concentration ([Ca2+]i) was measured with confocal laser scanning microscopy. RESULT: Hypoxia-hypoglycemia cultures for 5 hour and reoxygenation for 3 hour induced intracellular[Ca2+]i and apoptosis rate significantly increased. The effects were increased with the extending time of reoxygenation. MMP and mitochondrion activity declined significantly after 3 hour reoxygenation. The effects were declined with the extending time of reoxygenation. Qingkailing injection might have significantly decrease intracellular [Ca2+]i and apoptosis rate, increase MMP and mitochondrion activity. CONCLUSION: Qingkailing Injection might have significantly inhibit the decline in MMP induced by hypoxia-hypoglycemia and reoxygenation, and had effects of stable it and anti-neuronal apoptosis. The effects might be related to inhibit overload of intracellular free calcium.

[Effects of Astragalus membranaceus and Panax notoginseng on the transformation of bone marrow stem cells and proliferation of EPC in vitro].

OBJECTIVE: To investigate the effect and the possible mechanism underlying the promotional effect of Astragalus membranaceus and Panax notoginseng on the transformation of bone narrow stem cells and proliferation of EPC. METHOD: The marrow blood was collected in the patients with ischemia of lower limbs and BM-MNCs were separated and proliferated under different conditions. A. morphologic observation was performed and the ratio of CD34+ cells was measured. RESULT: The shuttle shaped cells lined up as bunches with several round cells scattered. The ratio of CD34+ cells was significantly increased in groups treated with medium (P < 0.01) and lower (P < 0.05) dosages of A. membranaceus and medium (P < 0.01) and high dosages (P < 0.01) of P. notoginseng respectively as compared with control group. CONCLUSION: A. membranaceus and P. notoginseng can promote the transformation and proliferation of EPC.

[The protective effect of cholic acid on an in vitro injury model of ischemia-reperfusion in cerebral microvascular endothelial cells].

OBJECTIVE: To establish an in vitro injury model of ischemia-reperfusion in cerebral microvascular endothelial cells of rats and observe the protective effect of cholic acid. METHOD: Cultured rat microvascular endothelial cells were subjected to the oxygen-glucose deprivation (OGD) (Krebs solution) and recovery of oxygen-glucose, which simulated in vitro ischemia and reperfusion injury, and treated with cholic acid. The A value was measured with MIT chromatometry. RESULT: Cultured cells were impaired after OGD for 4 hours and recovery of oxygen-glucose for 12 hours, the A value of the cells treated with cholic acid was significantly higher than that of the cells without treatment (P < 0.01). CONCLUSION: Cholic acid could obviously protect rat cerebral microvascular endothelial cells from injury induced by an in vitro ischemia-reperfusion.

[Apoptosis-promoting effect of Panax notoginseng extracts on MNNG-transformed GES-1 cells].

OBJECTIVE: To study the apoptosis-promoting effect of the serum from Panax notoginseng extracts-fed dog on precancerous gastric cells by means of flow cytometry. METHODS: In the experiment, we adopted eternalized human gastric mucosa epithelium GES-1 cells transformed by N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (MC cells) as the model of precancerous lesions for study in vitro. We took the serum of a dog before and at two different points of time (2 and 6 hours) after feeding the dog with Panax notoginseng extracts for experiment. The MC cells were cultured in mediums with different concentrations of the medicated serum at 2- or 6-hour point of time for 72 hours. By means of flow cytometry, we examined the apoptosis-promoting effects of the serums on the MC cells. RESULTS: The medicated serums at these 2 points of time had significant effects in promoting MC cell apoptosis. The proportions of apoptotic cells in culture mediums with medicated serums had a significant increase as compared with those in culture mediums with non-medicated serums (serum obtained before administration of extracts to the dog) under the same conditions (P<0.05). The number of MC cells in G(0)/G(1)phase was decreased (P<0.05) and that in G(2)/M phase increased (P<0.05), while no consistent changes were observed in S phase. CONCLUSION: The medicated serums obtained at the two different points of time have significant apoptosis-promoting effects on MC cells. They decrease the number of MC cells in G(0)/G(1) phase and increase the number of MC cells in G(2)/M phase. This is probably responsible for the effects of Panax notoginseng extracts in inhibiting the proliferation of MC cells and promoting its apoptosis.

[Protective effects of salidroside on injury induced by hypoxia/hypoglycemia in cultured neurons].

OBJECTIVE: To study the protective effects of salidroside on injury induced by hypoxia/hypoglycemia in cultured SH-SY5Y cell. METHOD: Apoptosis and intracellular free calcium concentration ([Ca2+]i) were measured by flow cytometry, morphological changes and neuronal necrosis were observed with fluorescence microscope, and the lactic dehydrogenate (LDH) release was measured by LDH kits. RESULT: Hypoxia/hypoglycemia cultures for 2 hours induced neuronal apoptosis and necrosis. They were 18.59% (P < 0.01) and 4.94% (P < 0.01) respectively. There were morphological changes including chromatin condensation, nuclear fragmentation and formed apoptotic bodies after exposed to hypoxia/hypoglycemia for 2, 4, 6, 12 hours. After 2 hours of hypoxia/hypoglycemia, neuronal [Ca2+]i and the release of LDH were significantly increased. They were 8.46 nmol/L (P < 0.01) and 16.59% (P < 0.01) respectively. The effects were enhanced with the extending time of hypoxia/hypoglycemia. Salidroside might have significantly decreased the percentage of neuronal apoptosis and necrosis, reduced neuronal [Ca2+]i and the release of LDH. The effects of salidroside were strengthened with the increasing of Salidroside dosage. CONCLUSION: Salidroside has effect of anti-neuronal apoptosis. This effect might be related to its function of decreasing intracellular free calcium concentration.

[Inhibiting effects of Panax notoginseng extracts on proliferation of GES-1 cells and MNNG-transformed GES-1 cells].

OBJECTIVE: Through cell cultivation, we studied the inhibiting effects of the serum of the dog fed with Panax notoginseng extracts on precancerous gastric cells, trying to find the best time points or periods when the extracts' function was the strongest after administration of the extracts to the dog. METHODS: The experiments adopted eternalized human gastric mucosa epithelium GES-1 cells and MC cells gained from GES-1 cells transformed by N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) as the model of precancerous lesions for study in vitro. We took the serum of a dog before and at different points of time after feeding the dog with Panax notoginseng extracts for experiment. By means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, we examined the inhibiting effects of the serum after culturing the GES-1 and MC cells for 72 hours with different concentration (8%, 4%, 2%) of medicated serum obtained from the dog at different points of time, so as to find that, at which points of time the medicated serum obtained, it would be the most effective. RESULTS: The results showed that the GES-1 and MC cells inhibition rates of medicated serum from the points of 2-hour and 6-hour were the highest, and the culture medium containing 8% of medicated serum from these two points had prominent inhibiting effects on both kinds of cells. The GES-1 cells inhibition rate in culture medium containing 8% of medicated serum from the point of 2-hour was 70.8% (P<0.01) and that of the MC cells was 45.3% (P<0.01). The GES-1 cells inhibition rate in culture medium containing 8% of medicated serum from the point of 6-hour was 88.5%(P<0.01) and that of the MC cells was 42.4% (P<0.01). CONCLUSION: The points of time with the strongest inhibiting effects are 2 hours and 6 hours after being fed with Panax notoginseng extracts. At these two points, the serum is most effective in inhibiting the proliferation of GES-1 and MC cells.