Effect of Guizhi Fulingwan on Ovulation Dysfunction in PCOS-IR Rats by Regulating PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

Objective:To investigate the effect of Guizhi Fulingwan on ovulation dysfunction in rats with polycystic ovary syndrome with insulin resistance （PCOS-IR） induced by letrazole combined with high fat emulsion. Method:A total of 72 female SD rats were randomly divided into control group， model group， metformin group and Guizhi Fulingwan low， medium and high dose groups， with 12 rats in each group. Except for control group， rats were given letrozole 0.001g·kg^-1 combined with high-fat emulsion 15 mL·kg^-1 for 21 consecutive days to establish model of PCOS-IR. Guizhi Fulingwan low， medium and high-dose groups were administrated with Guizhi Fulingwan 0.31， 0.62， 1.24 g·kg^-1 respectively， metformin group was administrated with metformin 0.27 g·kg^-1， control group and model group were administrated with 12 mL·kg^-1 of normal saline daily for 30 days. Hematoxylin-eosin（HE） staining was used to observe ovarian tissue pathology morphology， and enzyme-linked immunoassay method （ELISA） was used to detect serum follicle stimulating hormone （FSH）， luteinizing hormone （LH）， testosterone （T）， fasting insulin （FINS） level，and LH/FSH and insulin resistance index （HOMA-IR） were calculated． Western blot was used to detect the expression levels of autophagy key molecular Atg6 yeast homologue （Beclin-1）， autophagy related gene 5（Atg5）， microtubule associated protein light chain 3 （LC3） Ⅱ proteins in the phosphatidylinositol 3-kinase/protein kinase B/rapamycin target protein （PI3K/Akt/mTOR） signaling pathway and autophagy related indicators in rat ovarian tissue. Beclin-1 and LC3Ⅱ protein expressions were detected by immunohistochemistry （IHC）. Result:Compared with control group， the thickness of follicles and follicular granulosa cells in the ovary of the model group also decreased， and the number of corpus luteum significantly decreased， while the white membrane thickness of the ovary increased， and the number of atresia follicles and cystic dilatation follicles increased significantly. Serum T， LH， LH/FSH， FINS， FINS， HOMA-IR were significantly increased （<italic>P</italic><0.01）. Phosphorylated （p） -PI3K， p-Akt， and p-mTOR proteins in ovarian tissue were all decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The relative expression levels of autophagy-related protein LC3Ⅱ and Beclin-1 were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with model group， the number of follicles in the low， medium and high dose Guizhi Fulingwan group and the metformin group decreased， the number of follicles in atresia and atresia increased， and the follicular granulosa cell layer thickness increased. Serum T， LH， LH/FSH， FINS and HOMA-IR of Guizhi Fulingwan group were significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）， and serum FINS and HOMA-IR of metformin group were significantly decreased （<italic>P</italic><0.01）. The expressions of p-PI3K， p-Akt， and p-mTOR proteins were increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of LC3Ⅱ， Atg5 and Beclin-1 in the medium and high dose groups were significantly decreased （<italic>P</italic><0.01）. Conclusion:Guizhi Fulingwan can activate the PI3K/Akt/mTOR signaling pathway of granular cells， inhibit excessive autophagy of granular cells， improve ovarian function and insulin resistance， and restore ovulation， and the effect is better with high dose.

Effect of Guizhi Fulingwan on Autophagy of Ovarian Granulosa Cells in Mice with Polycystic Ovary Syndrome / 中国实验方剂学杂志

Objective:To investigate the effect of Guizhi Fulingwan on autophagy of ovarian granulosa cells in mice with polycystic ovary syndrome （PCOS）. Method:Twenty SD mice were randomized into a normal group （<italic>n</italic>=10） and a PCOS model group （<italic>n</italic>=10）， followed by PCOS modeling and <italic>in vitro</italic> culture of extracted ovarian granulosa cells. The ovarian granulosa cells of normal mice were classified into the control group and treated with 10% blank serum while those of PCOS mice into the experimental groups and with 10% Guizhi Fulingwan-containing serum at different concentrations （17.6， 35.1， 70.2 mg·kg^-1） and 10% metformin-containing serum （25 mg·kg^-1）， respectively， for 72 h. During the modeling， the changes in mouse body weight were measured. After modeling， the ovarian morphology was observed by microscopy， and the fasting blood glucose （FBG） was measured by Roche glucometer. Following the detection of fasting insulin （FI） and testosterone （T） levels by radioimmunoassay， the proliferation of ovarian granulosa cells was determined using cell counting kit-8 （CCK-8） to figure out the maximal dose of drug-containing serum that did not obviously affect the cell viability for subsequent assay. The autophagy of ovarian granulosa cells was examined by flow cytometry， and the protein expression levels of intracellular microtubule-associated protein 1 light chain 3Ⅰ （LC3Ⅰ）， LC3Ⅱ， Beclin1， and p62 were assayed by Western blott. Result:Compared with the blank group， the model group showed increased body weight and elevated FI， FBG， and T levels （<italic>P</italic><0.05，<italic>P</italic><0.01）， indicating the successful modeling of PCOS mice. Flow cytometric assay proved that the incubation with 10% Guizhi Fulingwan serum-containing medium resulted in a decline of autophagy （<italic>P</italic><0.05）. As demonstrated by Western blot assay results， the protein expression levels of Beclin1 and LC3 Ⅱ/Ⅰ in the model group increased significantly as compared with those of the blank group， whereas the expression level of p62 decreased significantly （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the model group， the medium- and high-dose Guizhi Fulingwan groups exhibited significantly down-regulated Beclin1 and LC3 Ⅱ/Ⅰ levels but remarkably up-regulated p62 （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Guizhi Fulingwan inhibits the autophagy of ovarian granulosa cells by down-regulating the protein expression levels of Beclin1 and LC3 Ⅱ/Ⅰ.

Morphological observation of cellular uptake of beta amyloid protein in cultured human neuroblastoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the cellular uptake of beta amyloid protein (Abeta) by cultured human neuroblastoma (SH-SY5Y) cells and the location of Abeta in the subcellular structures.</p><p><b>METHODS</b>The time course of cellular uptake of Abeta1-42-fluo in the SH-SY5Y cells was observed directly under laser scanning confocal microscope (LSCM). Image analysis was conducted to compare the differences of cellular Abeta uptake after treatment of the cells with different concentrations of extracellular Abeta for 24 h. Multiple immunofluorescence staining was employed to identify the location of Abeta in the subcellular structures.</p><p><b>RESULTS</b>SH-SY5Y cells showed Abeta internalization after incubation with Abeta1-42-fluo (200 nmol/L) for 1 h, and the quantity of Abeta uptake was time-dependent. A higher concentration of extracellular Abeta1-42-fluo resulted in increased Abeta uptake, which differed significantly between the 3 groups with treatment at different concentrations (P<0.01 or 0.05). Immunofluorescence staining revealed a co-localization of part of the Abeta and Lamp-1 (a lysosome marker) in the cytosome.</p><p><b>CONCLUSION</b>SH-SY5Y cells can clear Abeta through a time- and dose-dependent cellular uptake mechanism. Part of the Abeta uptaken in the cytoplasm is located in the lysosome .</p>

Vasoactive intestinal peptide enhances angiogenesis after focal cerebral ischemia / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of vasoactive intestinal peptide (VIP) on angiogenesis after focal cerebral ischemia.</p><p><b>METHODS</b>Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 120 min in adult SD rats with intracerebroventricular VIP administration at the beginning of reperfusion. Immunohistochemistry was performed to assay BrdU immunoreactive endothelial cells, expressions of VEGF, flt-1 and flk-1 in the ischemic zone, and the protein expressions of vascular endothelial growth factor (VEGF) in the brain was measured using Western blotting.</p><p><b>RESULTS</b>Immunohistochemical staining revealed significantly increased BrdU immunoreactive endothelial cells on the margins of the ischemic lesion in rats treated with VIP as compared with that in the control rats (P<0.05). VIP significantly increased the number of VEGF immunoreactive cells and flt-1- and flk-1-positive endothelial cells in comparison with the control group (P<0.01). Western blotting showed that VIP treatment resulted in significantly increased VEGF protein level in the ipsilateral hemisphere (P<0.05).</p><p><b>CONCLUSIONS</b>VIP enhances angiogenesis in the ischemic brain by increasing the expressions of VEGF in the brain tissue and its receptors flt-1 and flk-1 in the endothelial cells.</p>

Survival and prognostic factors in resected satellite-nodule T4 non-small cell lung cancer / 中华外科杂志

<p><b>OBJECTIVE</b>To study the survival and prognostic implication in surgically resected satellite-nodule T4 (T4 satellite) non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>From January 1995 to March 2005, the complete resection was performed to 42 patients with NSCLC who were postoperatively identified as pathologic-stage T4 satellite. Survival and associations between clinicopathological parameters and prognosis were analyzed. Thirty-two patients with pathologic stage local-invasion T4 (T4 invasion) NSCLC who underwent resection at the same time were also analyzed.</p><p><b>RESULTS</b>The 1-, 3- and 5-year survival was 76.2%, 57.1% and 46.0% for patients with T4 satellite, while 62.3%, 31.5% and 20.0% for patients with T4 invasion. There was a significant higher survival in T4 satellite group when compared to that in T4 invasion group (P < 0.05). Furthermore, patients with T4 satellite N0M0 got a better survival than those with T4 satellite N1-2M0, T4 invasion N0M0 and T4 invasion N1 -2M0 (P < 0.05). For patients with T4 satellite, univariate analysis showed that histology, main tumor size, lymph node status and adjuvant chemotherapy were linked with survival, while main tumor size, lymph node status and adjuvant chemotherapy served as the independent prognostic factors with multivariate analysis.</p><p><b>CONCLUSIONS</b>Patients with completely resected T4 satellite NSCLC have a better prognosis than those with T4 invasion. Main tumor size over 3 cm, lymph node metastasis or no adjuvant chemotherapy means an unfavorable prognosis.</p>

Internalization of beta amyloid protein in primary cultured cortical neurons in mouse / 中南大学学报(医学版)

OBJECTIVE@#To observe the internalization of beta-amyloid protein (Abeta) in primary cultured neurons and effect of astrocyte on it.@*METHODS@#The purified cortical neurons of mouse were cultured for 14 d, and were divided into a control group and an Abeta group. Each group was further divided into 3 subgroups. The neurons and 3 different concentration fluorescein or Abeta1-42-fluo were co-incubated for 24 h. The internalization of Abeta and the location of Abeta in subcellular structure were examined by the laser scanning confocal microscope combined with the image analysis method directly or after immunofluorescence staining. Neurons and astrocytes were co-cultured for 14 d. The cultured neurons and astrocytes were divided into a control group and a Abeta group. The cultures were treated with 200 nmol/L fluorescein or 200 nmol/L Abeta1-42-fluo for 24 h respectively. The effect of astrocyte on the internalization was analyzed by the above method.@*RESULTS@#There were no fluorescent granules within neurons in every fluorescein group. The purified cortical neurons could internalize 100 nmol/L, or 200 nmol/L Abeta1-42-fluo in 24 h. The fluorescent granules of Abeta1-42 distributed within perikaryon and processes. The internalization was related to the concentration of Abeta. The part of Abeta was located in the lysosome of neurons indicated by immunofluorescence staining. Compared with the purified neurons, the neurons co-cultured with the astrocytes internalized Abeta increased in the internalization of Abeta. There was significant difference between the purified neurons and the co-cultured neurons with astrocytes (P<0.05).@*CONCLUSION@#Neurons could internalize the proper concentration of Abeta. Astrocyte might facilitate the internalization of Abeta in neurons.

Neuroprotective effects of vasoactive intestinal peptide against rat cerebral ischemia / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the neuroprotective effect of vasoactive intestinal peptide (VIP) in rat ischemic brain injury.</p><p><b>METHODS</b>VIP was administered via intracerebroventricular injection in SD rats prior to focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery. The infarct volume was assessed with TTC staining, and immunohistochemistry was performed to analyze the S100beta expression in the cerebral tissue, with the serum concentrations of S100beta detected by double-antibody sandwich enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>After VIP injection, the relative infarct volume in the rats with cerebral ischemia was significantly reduced by 32.3% as compared with the volume in the control group on day 1 (P<0.05), and the number of S100beta-positive cells was significantly decreased in the cerebral tissue (P<0.05). The injection also resulted in significantly decreased serum S100beta concentrations in the rats (P<0.05).</p><p><b>CONCLUSION</b>VIP injection can reduce the infarct volume in rats with focal cerebral ischemia, suggesting the neuroprotective effect of VIP in brain ischemia possibly by reducing S100beta overexpression.</p>