Regulation Effect and Clinical Efficacy of Addition and Subtraction Therapy of Jinkui Shenqiwan Combined with Buzhong Yiqitang on Immune Inflammatory Factors of Patients with Postmenopausal Osteoporosis and Deficiency of Spleen and Kidney / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of addition and subtraction therapy of Jinkui Shenqiwan combined with Buzhong Yiqitang to postmenopausal osteoporosis （PMO） with deficiency of spleen and kidney， and to investigate its regulation effect on immune inflammatory factors. Method:One hundred and sixty patients were randomly divided into observation group and control group， with 80 cases in each group. Both groups got comprehensive western medicine treatment measures. Patients in control group additionally got Zhuanggu Zhitong capsule， 4 capsules/time， 3 times/day. Patients in observation group additionally got addition and subtraction therapy of Jinkui Shenqiwan combined with Buzhong Yiqitang， 1 dose/day. The treatment was continued for 24 weeks. Before and after treatment， lumbar L2-4 bone mineral density （BMD） was detected by Dual energy X-ray absorptiometry （DXA） and lumbar BMD was detected by quantitative CT （QCT）. Scores of traditional Chinese medicine（TCM） syndromes and Chinese osteoporosis-targeted quality of life questionnaire （COQOL） were graded. Levels of Estradiol （E₂）， type Ⅰ procollagen amino terminal pro peptide （PINP）， serum osteocalcin （OC）， osteoprotegerin （OPG）， type Ⅰ collagen cross-linked C-terminal peptide （S-CTX）， tartrate resistant acid phosphatase （TRACP） and urinary pyridinoline （PYD） were detected. Levels of CD4⁺ T cells， CD8⁺ T cells， interleukin-17 （IL-17）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， <italic>γ-</italic>interferon（IFN-<italic>γ</italic>） and interleukin-4 （IL-4） were calculated. The proportion of T helper cell （Th）17 and regulatory T cell （Treg） in CD4⁺ T cells was calculated. Besides， the safety was evaluated. Result:Bone density was detected by DXA in observation group， and its T-value and bone density detected by QCT were all higher than those in control group （<italic>P</italic><0.01）. After treatment， scores of TCM syndrome and COQOL were lower than those in control group （<italic>P</italic><0.01）. Levels of PINP， OC， S-CTX， TRACP and PYD/Cr were all lower than those in control group （<italic>P</italic><0.01）. Levels of OPG， CD8⁺ and Treg were higher than those in control group （<italic>P</italic><0.05）， levels of Th17， Th17/Treg， CD4⁺/CD8⁺， IL-17， TNF-<italic>α</italic> and IFN-<italic>γ </italic>were lower （<italic>P</italic><0.01）， and levels of IL-4 and E₂ were higher than those in control group （<italic>P</italic><0.01）. The clinical efficacy in observation group was better than that in control group （<italic>Z</italic>=2.103， <italic>P</italic><0.05）. Conclusion:On the basis of calcium and vitamin D supplementation， Jinkui Shenqiwan combined with Buzhong Yiqitang can improve levels of E₂ and bone density， reduce clinical symptoms， improve quality of life， regulate bone metabolism index and immune inflammation reaction， with better clinical efficacy and safety.

Effect of glutamate on vascular endothelial growth factor mRNA and protein expressions in hypoxic rat astrocytes in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of glutamate on the expression of vascular endothelial growth factor (VEGF) mRNA and protein in cultured rat astrocytes under hypoxia.</p><p><b>METHODS</b>Cultured rat astrocytes were randomly divided control group, glutamate group, hypoxia group and hypoxia+glutamate group. The cells in the control and glutamate groups were cultured under nomoxic condition (95% air and 5% CO(2)), and those in the other two groups under hypoxic condition (94% N(2), 5% CO(2) and 1% O(2)). The total RNA was extracted from the cells at different time points of hypoxic exposure for real-time FQ-PCR and ELISA to detect the expression of VEGF mRNA and protein in cultured astrocytes, respectively.</p><p><b>RESULTS</b>The expressions of VEGF mRNA and protein underwent no significant changes in the control glutamate groups, but increased obviously in both hypoxia and hypoxia+glutamate groups at 2, 4, 6, 8 and 12 h of hypoxic exposure. At these time points, VEGF expressions at both the mRNA and protein levels were significantly higher in hypoxia+glutamate group than in hypoxia group.</p><p><b>CONCLUSION</b>Glutamate at 1 micromol/L can further increase the expression of VEGF mRNA and protein in astrocytes exposed to hypoxia, which may result from the adaptive changes of glutamate receptors in hypoxic astrocytes.</p>

A simple method for assessment of RNA integrity in laser capture microdissection samples / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples.</p><p><b>METHODS</b>The total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells).</p><p><b>RESULTS</b>When the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples.</p><p><b>CONCLUSION</b>A simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.</p>

Neurons in the corpus callosum of rats: expression of Cav2.2 and their connection / 中南大学学报(医学版)

OBJECTIVE@#To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection.@*METHODS@#Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine.@*RESULTS@#There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum.@*CONCLUSION@#There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.

Construction and identification of a plasmid with EGFP reporter gene for enhancer function analysis / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a plasmid vector with EGFP reporter gene for functional analysis of enhancers.</p><p><b>METHODS</b>EGFP DNA was amplified by PCR from plasmid pEGFP-N1 DNA and subcloned into plasmid PGL3-promoter backbone without luc(+) gene to construct the enhancer-identifying vector pEGFP-enhancer. Different copies of hypoxia response element (HRE) sequence were synthetized and subcloned into the multiple cloning site of the plasmid pEGFP-enhancer. Using Lipofectamine 2000, the recombined pEGFP-HRE and pEGFP-5HRE plasmids were transfected into the Hela cells respectively. After hypoxic or normoxic cell culture, EGFP expression in the cells was detected by flow cytometry and fluorescence microscopy.</p><p><b>RESULTS</b>After hypoxic exposure, the fluorescence intensity of EGFP in the Hela cells transfected with the plasmid increased with the enhancer HRE copies, while the fluorescence intensity underwent no significant changes after normoxic cell culture.</p><p><b>CONCLUSION</b>we have successfully constructed the enhancer expression vector plasmid pEGFP-enhancer, which can identify the activity of the enhancers through EGFP expression.</p>

Effect of ligustrazine on nNOS expression and neuranagenesis in adult rats after cerebral ischemia-reperfusion injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of ligustrazine on cell proliferation in the subventricular zone (SVZ) and dentate gyrus (DG) and nNOS expression in rat brain after cerebral ischemia-reperfusion injury.</p><p><b>METHODS</b>Male SD rats were randomly divided into normal control group, sham operation group, model group and ligustrazine treatment group. The latter two groups were further divided into 5 subgroups for observation at 1, 3, 7, 14 and 21 days after reperfusion following a 2-hour middle cerebral artery occlusion (MCAO). The cells in S phase were labeled with BrdU, and immunohistochemistry was employed to detect BrdU- and nNOS-positive cells. The numbers of BrdU-positive cells in the SVZ and DG were measured. The expression of nNOS was detected by Western blotting.</p><p><b>RESULTS</b>nNOS expression increased significantly in the model group as compared to the sham operation group (P<0.05), and ligustrazine treatment significantly lowered the expression level in comparison with the model group (P<0.05). Compared with the model group, a significant increase in BrdU-positive cells occurred in the SVZ of rats 1 and 3 days after igustrazine treatment (P<0.05), along with an increase of DG BrdU-positive cells.</p><p><b>CONCLUSION</b>Ligustrazine significantly restrains ischemia-reperfusion injury-induced nNOS activity enhancement and promotes cell proliferation in the SVZ and DG of adult rats after ischemia-reperfusion injury.</p>

Effect of ligustrazine on cell proliferation in subventricular zone in rat brain with focal cerebral ischemia-reperfusion injury / 中南大学学报(医学版)

OBJECTIVE@#To observe the effect of ligustrazine on cell proliferation in subventricular zone (SVZ) in rat brain with focal cerebral ischemia reperfusion injury.@*METHODS@#Male SD rats were randomly divided into a normal group,a sham operation group,a ligustrazine treatment group, and a control group. The ligustrazine treatment group and the control group were further divided into 5 subgroups: 1d, 3d, 7d, 14d, and 21d reperfusion after 2h middle cerebral artery occlusion (MCAO). The focal cerebral ischemia-reperfusion model was made by MCAO. S phase cells were labelled with BrdU. Immunohistochemistry method was conducted to detect the BrdU positive cells. The total number of BrdU positive cells in the SVZ was measured. The expression of neuro nitric oxide synthase (nNOS) was detected with Western blot method.@*RESULTS@#There was a significant increase of BrdU positive cells in SVZ of ligustrazine treatment in the 1d and 3d group compared with that of the control group (P<0.01). The total number of BrdU positive cells reached a peak in 7d group and declined afterwards. Cells proliferated also in SVZ on the contralateral side, and peaked at 7d. The nNOS expression of ligustrazine administration after the focal cerebral ischemia-reperfusion decreased at 1d and 3d after the reperfusion compared with that of the control group (P<0.05), and increased at 7d, but with no significant difference compared with that of the control group.@*CONCLUSION@#Ligustrazine may promote the cell proliferation in SVZ of adult rats with ischemia-reperfusion injury by decreasing the nNOS expression.