Establishment of a screening cell model for cannabinoid type 1 receptor agonists / 药学学报

Cannabinoid receptors are one of the most expressed G protein-coupled receptors in the central nervous system, which are potential drug targets for inflammation, pain and drug abuse. Cannabinoid receptors are composed of type 1 receptor (CB1R), type 2 receptor (CB2R) and other receptors, of which CB1R plays a vital role in regulating central memory, cognition, and motor function. Therefore, screening CB1R agonists has potential value in treating nervous system diseases. In this study, the intracellular loop 3 (ICL3) domain of CB1R was replaced with a circular-permutated enhanced green fluorescent protein (cpEGFP). After infecting HEK 293T cells with lentivirus particles, we obtained a stable cell line that was overexpressed human CB1R-cpEGFP after puromycin selection. The interaction between receptor agonists and CB1R led to the change of receptor conformation, resulting in de-protonation of the EGFP, and enhancing the fluorescence intensity. Therefore, active CB1R compounds could be verified by measuring the fluorescence intensity. Using CB1R agonist arachidonyl-2′-chloroethylamide (ACEA) as a positive control to evaluate the reliability of this model, studies have shown that ACEA could induce receptor activation and increase fluorescence intensity, while antagonist rimonabant inhibited receptor activation with unchanged fluorescence intensity. In conclusion, this study successfully constructed a fluorescent probe screening model for CB1R agonists.

The screening model for dopamine receptor agonists by a dopamine sensor / 药学学报

Parkinson's disease (PD) is a degenerative disease of the central nervous system due to the loss or death of dopaminergic neurons in the substantia nigra. Clinically, levodopa is the most effective and commonly used drug for PD treatment. However, long-term levodopa therapy is prone to motor complications and other side effects caused by excessive peripheral dopamine production, which has become an urgent problem to be solved in PD treatment. Dopamine receptor (DR) agonists are similar to dopamine. They can directly stimulate postsynaptic dopamine receptors, produce the same effect as dopamine, delay the application of levodopa as much as possible, and reduce complications caused by long-term use of levodopa. Therefore, screening effective dopamine receptor agonists has become a key issue in the study and treatment of PD. In order to establish a rapid, stable and reliable method for dopamine receptor agonist screening, this study used the human dopamine receptor 2 (DRD2) gene fused with a circular permuted EGFP (cpEGFP) to construct a recombinant gene, packaged with lentiviral vector, and the vector replaced the parted inner transmembrane domain of the third intracellular loop (ICL3) of genetically-encoded GPCR-activation based (GRAB) sensors. The fluorescence of GPCR-fused cpEGFP is regulated by conformational changes mediated by the interaction of dopamine receptor agonists with GPCRs without altering GPCR activity. The HEK293T cells were infected with viral vector, screened by puromycin to select highly expressed cells. Dopamine receptor agonists (including dopamine, bromocriptine mesylate, cabergoline, pramipexole) were used as positive drugs to explore the best screening and detection conditions, establishing a stable model to evaluate the dopamine receptor agonist. The results showed that the optimal filter for the dopamine receptor agonist in this study was the cell seeding count of 7×104, and the effective concentration of the positive drug was 1-100 µmol·L-1. In addition, pretreated with 10 µmol·L-1 dopamine receptor antagonists (including chlorprothixol hydrochloride, domperidone, and sulpiride), the positive fluorescence signal of overexpressed DRD2-cpEGFP HEK293T cells could not be detected when exposed to 10 µmol·L-1 dopamine receptor agonists, which proved that dopamine receptor antagonists could block the activity of dopamine receptor agonists, so they cannot activate dopamine receptor allosteric, indicating that the model has good specificity and can also be used for the screening and detection of new dopamine receptor antagonists. In summary, the study constructs a stable dopamine sensor detection system, which can effectively screen potential dopamine receptor agonists. The operation procedures are simple and rapid. And it can be used for a large-scale screening providing a fundamental methodology for drug development and PD treatment targeted on DRD2.

Anaplastic lymphoma kinase-translocation renal cell carcinoma: clinical and pathological analysis / 中华病理学杂志

Objective: To investigate the clinicopathological features, molecular characteristics, differential diagnosis and prognosis of anaplastic lymphoma kinase (ALK)-translocation renal cell carcinoma. Methods: Two cases of ALK-translocation renal cell carcinoma diagnosed from January 2011 to December 2020 were retrospectively analyzed to characterize their morphological features, immunohistochemical expression and prognosis. Multiple molecular studies including fluorescence in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR), and next-generation sequencing were performed to characterize the genetic alterations. Results: Two patients included one male and one female, with 59 and 57 years old, respectively. Morphologically, case 1 resembled collecting duct carcinoma or renal medullary carcinoma, which demonstrated tubular, microcapsule and reticular structures, with a remarkable myxoid background and lymphocytes infiltration; case 2 resembled Xp11.2 translocation renal cell carcinoma or type 2 papillary renal cell carcinoma, which demonstrated tubular papillary and focal solid structures, with flocculent cytoplasm and many foamy histiocytes, but without myxoid background and lymphocytes infiltration. Immunohistochemistry showed strongly positive expression of ALK. CK7, E-cadherin, vimentin, PAX8 and CD10 showed various degrees of expression, and other antibodies were nonreactive. A variety of molecular assays showed definite ALK gene translocation, with rare VCL-ALK gene fusion (VCL exon and 16-ALK exon 20) in case 1, and EML4-ALK gene fusion (EML4 exon and 2-ALK exon 20) in case 2. Conclusions: ALK-translocation renal cell carcinoma is rare with various morphological features, and is easy to miss and misdiagnose. The characteristic ALK expression and molecular detection of ALK translocation are helpful for diagnosing this type of renal cell carcinoma.

Papillary renal neoplasm with reverse polarity: a clinicopathological analysis / 中华病理学杂志

Objective: To study the clinical pathological characteristics, immunophenotype, molecular changes and prognosis of the papillary renal neoplasm with reverse polarity (PRNRP). Methods: Nine cases of PRNRP, diagnosed from 2013 to 2019, were retrieved from the Department of Pathology of Nanjing Jinling Hospital, Nanjing University School of Medicine. Histomorphology, immunophenotype and molecular genetics were analyzed with review of the literatures. Results: There were five male and four female patients, aged from 49 to 70 years, with an average age of 60.1 years. During a mean follow-up of 29 months, one patient died for other cause, and the others survived without disease. Microscopically, the tumor cells arranged in papillary structure with a fibrovascular core, the surface of which was covered with a single layer of cuboidal or columnar cells. The most prominent feature was that the tumor nuclei located at the top of the cytoplasm far from the basement membrane, and they were monotonous in size and arranged neatly with no or few nucleoli. Immunohistochemically, all nine cases of PRNRP showed diffuse positive expression of CK7 and E-cadherin, various degrees of P504s expression, and no expression of CD10 and CD117, with a Ki-67 index of 1%-3%. Unlike other papillary renal cell carcinoma, the nine cases of PRNRP all showed characteristic positive expression of GATA3. The fluorescence in situ hybridization assay showed that the majority of PRNRPs (8/9) did not have triploids on chromosomes 7 and 17. The sequencing of the KRAS gene confirmed the presence of a nonsense KRAS mutation in 8 of the 9 cases. Conclusions: PRNRP is a subtype of papillary renal cell carcinoma with characteristic morphological, immunophenotypic and molecular features, and indolent behaviors. More data are needed to define PRNRP as "carcinoma", and a definitive diagnosis of PRNRP is of great significance for proper treatment choice and accurate prognostication.

Succinate dehydrogenase-deficient renal cell carcinoma:a clinicopathological, ultrastructural and molecular analysis / 中华病理学杂志

Objective: To investigate the clinicopathological features, immunophenotype, ultrastructure, genetic alterations and prognosis of succinate dehydrogenase-deficient renal cell carcinoma (SDH RCC). Methods: A total of 11 SDH RCCs, diagnosed from 2010 to 2019, were selected from the Department of Pathology of Nanjing Jingling Hospital, Nanjing University School of Medicine for clinicopathologic, immunohistochemical (IHC), ultrastructural investigation and follow-up. The molecular features of seven cases were analyzed by the panel-targeted DNA next generation sequencing (NGS). Results: There were seven males and four females, with ages ranging from 24 to 62 years (mean 41.4 years, median 41 years). Microscopically, SDH RCC was mainly composed of solid and tubular structures with local cystic change. Four cases showed nested or trabecular structure distributed in a loose hypocellular connective tissue or around scar, similar to oncocytoma. The neoplastic cells demonstrated flocculent eosinophilic cytoplasm with typical intracytoplasmic vacuoles. Immunohistochemically, eight cases were negative for SDHB; three cases showed focal and weak expression, whereas normal renal tubular and vascular endothelial cells demonstrated strong cytoplasmic staining. NGS of DNA targeted-panel detected pathogenic mutations of SDHB gene in seven cases (including three cases with equivocal IHC expression of SDHB), without any mutations in other SDH related genes. There were four cases of SDHB missense mutation, one case of frameshift mutation, one case of splicing mutation, and one case of acquired stop codon mutation. Conclusions: SDH RCC is a distinct variant of RCCs with genetic tendency or with hereditary cancer syndrome. NGS is recommended to detect the related gene mutations for a definitive diagnosis. The patients should be closely followed up.

Effect of electroacupuncture at "Zusanli" (ST 36) on duodenal mast cells, NGF and NTRK1 in rats with functional dyspepsia / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) at "Zusanli" (ST 36) on duodenal mast cells, nerve growth factor (NGF) and neurotrophic tyrosine kinase receptor type 1 (NTRK1), and to explore the mechanism of electroacupuncture at Zusanli (ST 36) on functional dyspepsia (FD).@*METHODS@#Sixty SPF-grade 10-day-old SD rats were randomly divided into a normal group, a model group, a ketotifen group and an EA group, 15 rats in each group. The FD model was prepared by iodoacetamide combined with rat tail clamping method in the model group, the ketotifen group and the EA group. The rats in the ketotifen group were injected intraperitoneally with ketotifen (1 mg•kg-1•d-1) for 7 days; the rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36), with disperse-dense wave, frequency of 2 Hz/50 Hz and intensity of 0.5 mA, 20 min each time, once a day for 14 days. The gastric emptying rate and small intestinal propulsion rate in each group were observed; the morphology of duodenal mucosa was observed by HE staining; the toluidine blue staining was used to observe the number and degranulation of mast cells in duodenal mucosa; the protein and mRNA expressions of NGF, NTRK1 in duodenum were detected by Western blot and real-time PCR; the level of interleukin-1β (IL-1β) in duodenum was measured by ELISA.@*RESULTS@#Compared with the normal group, the gastric emptying rate and small intestinal propulsion rate in the model group were decreased (P<0.01); compared with the model group, the gastric emptying rate and small intestinal propulsion rate in the ketotifen group and the EA group were increased (P<0.01); the small intestinal propulsion rate in the EA group was higher than that in the ketotifen group (P<0.01). In the model group, local defects in duodenal mucosa were observed with a small amount of inflammatory cell infiltration; no obvious abnormality was found in duodenal mucosa of the other groups. Compared with the normal group, the mast cells of duodenal mucosa in the model group were increased significantly with significant degranulation; compared with the model group, the mast cells of duodenal mucosa in the ketotifen group and the EA group were decreased significantly, and the degranulation was not obvious. Compared with the normal group, the protein and mRNA expressions of NGF, NTRK1 as well as the level of IL-1β in duodenum in the model group were increased (P<0.01); compared with the model group, the protein and mRNA expressions of NGF, NTRK1 as well as the levels of IL-1β in duodenum in the ketotifen group and the EA group were decreased (P<0.01, P<0.05); compared with the ketotifen group, the mRNA expression of NGF, as well as the protein and mRNA expressions of NTRK1 in duodenum in the EA group were decreased (P<0.05, P<0.01).@*CONCLUSION@#EA at "Zusanli" (ST 36) could inhibit the activation of duodenal mast cells and regulate the expressions of NGF and its receptor to improve the low-grade inflammatory response of duodenum, resulting in treatment effect on FD.

The distribution of sleep duration in mid－pregnancy and its association with prehypertension / 中华疾病控制杂志

@#Objective To study the distribution of sleep duration in mid－pregnancy women and examine its association with prehypertension ( PHT) ． Methods In the baseline survey of a prospective cohort study，943 women in mid－pregnancy were recruited in Guangzhou，China in 2017－2018． A standardized questionnaire was used to assess demographic characteristics，sleep duration and other lifestyles． We obtained maternal blood pressure values，weights，heights，and medical histories from medical records． Multivariate logistic regression was conducted to examine the association between sleep duration and PHT． Ｒesults The average daily sleep duration of women in mid －pregnancy was ( 10. 41 ± 1. 67 ) hours，and it was negatively related to age and educational level． Overall，98. 33% of pregnant women had a daily sleep duration ≥ 7 h and the distribution was related to passive smoking． The average night time sleep duration was ( 9. 48±1. 21 ) hours，and it was negatively related to age and educational level． The daytime sleep duration was ( 0. 93 ± 0. 69 ) hours，and it was positively associated with physical activity． The average bedtime was( 22 ∶ 42 ± 1．24) ，and it was positively associated with passive smoking． The prevalence of PHT was 9. 61%． We did not observe any significant association between sleep duration and PHT． Conclusions The mid－pregnancy women in Guangzhou had relatively long sleep duration， and it differed by maternal age，educational level，physical activity，and passive smoking． There was no significant association between sleep duration and PHT．

Chinese medicine Yu-Ping-Feng-San attenuates allergic inflammation by regulating epithelial derived pro-allergic cytokines / 中国天然药物

This study aimed to investigate the mechanisms of Yu-Ping-Feng-San (YPFS) on attenuating allergic inflammation in the initial stage of atopic dermatitis (AD). AD mouse model was established with fluorescein isothiocyanate (FITC) sensitization and elicitation. Epithelial barrier structure was observed with transmission electron microscope. The populations of dendritic cells (DCs) and group 2 innate lymphoid cells (ILC2s) were detected by flow cytometry. Human immortalized keratinocyte (HaCaT) cells were stimulated with Poly(I:C)/TNF-α in vitro to assessthymic stromal lymphopoietin (TSLP), interleukin (IL)-33 and nuclear factor-κB (NF-κB) levels or expressions by immunofluorescence, enzyme linked immunosorbent assay (ELISA) and western blot. In the initial stage of AD, ear swelling and infiltration of inflammatory cells in ear tissues were markedly attenuated with YPFS treatments. The damaged structures of ear epithelium and the increased levels of Th2-cytokines induced by FITC were significantly rescued in YPFS-treated mice. The production of pro-allergic cytokines, TSLP and IL-33, as well as the cell populations of their target cells DCs and ILC2s were decreased in AD model, respectively. Likewise, the levels of TSLP and IL-33 in Poly(I:C)/TNF-α-stimulated HaCaT cells showed the same results. Lower levels of p-NF-κB were detected with YPFS treatment, and the expressions of TSLP and IL-33 could be further decreased with inhibiting of NF-κB. Therefore, YPFS attenuates allergic inflammation in the initial stage of AD probably through regulating NF-κB-TSLP/IL-33 pathway, which may provide a novel effective target for the prevention and treatment of allergic diseases.

Comparative study of differentiation potential of mesenchymal stem cells derived from orofacial system into vascular endothelial cells / 北京大学学报(医学版)

OBJECTIVE@#To compare the proliferation and capacity of differentiation to vascular endothelial cells and angiogenesis induction among stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC) and human bone marrow mesenchymal stem cells (BMSC) from orofacial bone.@*METHODS@#SHED and DPSC were isolated from pulp tissue of the patients. BMSC were isolated from orthognathic or alveolar surgical sites. The surface markers of the cells were detected by flowcytometry. Cell counting kit-8 (CCK-8) assays were conducted to detect the proliferation ability of the cells. The cells were induced into endothelial cells with conditional medium and then the induced cells were cultured in Matrigel medium. The expression of angiogenesis-related genes such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand Factor (vWF) were quantified by real-time PCR. The cells were cultured in chick embryo chorioallantoic membrane (CAM) and the vessels were counted after 5 days.@*RESULTS@#The cell surface markers CD73, CD90, CD105 and CD146 of all the stem cells were positive, CD34 and CD45 were negative. The CD146 positive rate of SHED and DPSC was higher than that of BMSC. SHED had a higher proliferation rate than DPSC and BMSC. After angiogenic induction for 14 d, 3 kinds of cells emanated pseudopodia formed grid structure long vasculature in Matrigel media. The total length of tube formation of induced BMSC (7 759.7 μm) and SHED (7 734.3 μm) was higher than DPSC (5 541.0 μm). The meshes number of induced SHED (70.7) was higher than DPSC (60) and BMSC (53.7) in Matrigel medium. The expression of CD31, VEGFR2 and vWF genes of SHED were higher than those of BMSC and DPSC. VEGFR1 gene expression of BMSC was higher than that of the other groups, and SHED was higher than DPSC. The expression of VEGF showed no difference among the cells. No deference was showed between the effect of the stem cells and negative control on new formed vessels in CAM. The total length of vessels of SHED (30.4 mm) was higher than that of the negative control (20.9 mm) and BMSC (28.0 mm).@*CONCLUSION@#SHED, DPSC and BMSC can differentiate into vascular endothelial cells. SHED showed a stronger angiogenesis differentiation and proliferation potential compared with DPSC and BMSC.

Comparison of the properties of CD146 positive and CD146 negative subpopulations of stem cells from human exfoliated deciduous teeth / 北京大学学报(医学版)

OBJECTIVE@#Stem cells from human exfoliated teeth (SHED) were sorted by magnetically activated cell sorting (MACS) technique to obtain the CD146 positive and negative cell subpopulation. Then the biological characteristics of these subpopulations were compared to explore their specific application potential in tissue engineering.@*METHODS@#In this study, freshly extracted deciduous teeth without any caries or dental pulp disease were obtained. SHED was isolated using enzyme digestion method and then sorted by MACS, CD146 positive cells and CD146 negative cells were obtained after cell sorting. The biological characteristics of the unsorted mixed cells, CD146 positive subpopulation and CD146 negative subpopulation were compared. The proliferation ability was detected through cell counting kit-8 (CCK-8) and colony-forming unit (CFU). After osteogenic induction, alizarin red staining was performed and the gene expression of osteogenic related markers was detected by quantitative real-time polymerase chain reaction(qPCR). After adipogenic induction, oil-red O staining was performed and the gene expression of adipogenic related markers was detected. After neurogenic differentiation induction, the expression of neural markers was detected by immunofluorescence and the gene expression of neural markers was detected by qPCR.@*RESULTS@#SHED of the fifth passage was sorted by MACS. And the CD146 positive cell subpopulation and CD146 negative cell subpopulation were obtained. CCK8 assay showed that the proliferative tendency of the three cell groups was consistent, but the proliferation potential of CD146 positive and negative cell subpopulations was significantly lower than that of the unsorted cells. The colony forming rates of the unsorted mixed cell group, CD146 positive and negative populations were 28.6%±3%,17.1%±2.3% and 27.5%±2.5%, respectively. After 21 days of osteogenic induction, alizarin red staining and qPCR showed that the CD146 positive cell population had more mineralized nodule formation and expressed higher level of osteogenic related genes compared with the other two groups. After 21 days of adipogenic induction, oil red O staining and qPCR results showed that the CD146 negative subpopulation produced more lipid droplets and the expression of lipid related genes increased more significantly. After 14 days of neural induction, cell immunofluorescence and qPCR results showed that the unsorted mixed cell group and CD146 positive subpopulation expressed glial cell marker, and the expressions of neural precursor cells and neuronal marker increased significantly in negative subpopulation.@*CONCLUSION@#The unsorted mixed cells showed better proliferative potential than CD146 positive and negative subpopulations. The CD146 positive subpopulation was most potent in osteogenic differentiation; it was more suitable for bone tissue engineering. The CD146 negative cells had stronger adipogenic differentiation potential than the other two cell groups; different subpopulations differed in neural differentiation.

Lipoic Acid Combined with Epalrestat versus Lipoic Acid in Treating Diabetic Peripheral Neuropathy:A Meta-analysis / 中国医学科学院学报

Objective To compare the clinical effectiveness of lipoic acid combined with epalrestat versus lipoic acid in treating diabetic peripheral neuropathy(DPN). Methods Randomized controlled trials(RCTs) and clinical controlled trials on lipoic acid versus epalrestat for DPN before February 2016 were searched through five databases:CNKI,CBM,VIP,Wanfang,and PubMed. The quality of the included trials were assessed using Cochrane software and Jadad scores. Data were analyzed with Review Manager 5.3 software. Results Nine studies were included in the analysis. Meta analysis showed that the lipoic aid monotherapy was significantly inferior to lipoic acid-epalerestat combination therapy [RR=0.58,95%Cl(0.47,0.71),P<0.00001]. Inferiority of the lipoic acid monotherapy was also shown in nerve conduction velocity with WMDs of-4.94 [95%Cl(-7.41,-2.46),P<0.0001] for median motor nerve conduction velocity(MNCV),-5.08 [95%Cl(-7.68,-2.49),P=0.0001] for peroneal MNCV,-4.24 [95%Cl(-6.20,-2.29),P<0.0001] for median sensory nerve conduction velocity(SNCV),and-3.66 [95%Cl(-5.02,-2.31),P<0.00001] for peroneal SNCV. Sensitivity analysis showed that the results were robust. However,the included trials were limited by simple design,few subjective indicators,and short follow-up time. Conclusions Lipoic acid combined with epalrestat is better than lipoic acid alone in the treatment of DPN,as well as the MNCV and SNCV of median or peroneal nerve. Due to the low quality of the included studies,high-quality RCTs are warranted to validate the results.

ISSR analysis on genetic diversity of traditional medicinal plant Stemona tuberosa / 中草药

Objective To analyze the relationship and genetic diversity of Stemona tuberosa in different populations. Methods ISSR molecular technique was be used to decipher the genetic diversity of S. tuberosa in South of the Yangtze River, The genetic relationships among different populations were analyzed based on software POPGEN32 and the DNA molecular dendrogram was structured according to the software NTSYSpc-2.10E. Results Seventy-four obvious bands of different S. tuberosa populations were amplified with seven suitable ISSR primers, 74 of them were polymorphic sites, and the percentage of polymorphism bands reached to 100%; Effective number of alleles (Ne) is 1.524 4, the average Nei's genetic diversity (He) index is 0.314 6, the average Shannon's information index is 0.478 6, the genetic distance among populations is 0-0.950 2, and the average distance is 0.416 3. Conclusion The study revealed that a relative high genetic diversity occurred in different populations of S. tuberosa, the genetic variation is related to geographical distance in different populations of S. tuberosa. The study will throw light on how to introduce and cultivate traditional Chinese medicinal plant, how to screen peculiar pyrrolidine alkaloids from S. tuberosa, and how to build to scientific standards and theoretical basis for traditional geo-authentic crude drug in China.

Effect of Modified Baizhu Powder on Gastrointestinal Functions in Mouse Models with Stomach-cold Functional Dyspepsia / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effect of modified Baizhu (Rhizoma Atractylodis Macrocephalae) powder on the gastrointestinal function in mouse models with stomach-cold functional dyspepsia. Meanwhile,the mouse models were administered with Shihu (dendrobium), a traditional Chinese drug with cold nature and flavour, to explore the way via which it exert its effect on specific symptoms. Methods: Mouse models with stomach-cold functional dyspepsia were established by ice water and ice NaOH. The effects of modified Baizhu powder and dendrobium on mice were observed in terms of water intake, weight change,small intestine propulsion rate, intestinal absorption function, and effects on ghrelin and motilin.</p><p><b>RESULTS</b>The modified Baizhu powder effectively increased food intake, water intake, body weight (P<0.05) and swimming time (P<0.01), increased the small intestine propulsion rate and serum D-xylose content (P<0.05), and up-regulated ghrelin (P<0.05). Also, it showed a trend to down-regulate the motilin, although the change was not statistically significant (P>0.05). In contrast,the use of Shihu aggravated symptoms in the mouse models. Conclusion: The changes in ghrelin and motilin levels may be the neuro-endocrine mechanisms via which the modified Baizhu powder and Shihu exert their effects on mouse models.</p>

Inhibitory effect of E2F-1-silencing lentivirus vector on chemoresistance of subcutaneous human gastric cancer in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effects of E2F-1-silencing lentivirus vector on the growth and chemoresistance of subcutaneous human gastric cancer in nude mice.</p><p><b>METHODS</b>Thirty-six nude mice were inoculated subcutaneously with chemoresistant SGC-7901/DDP cells to establish subcutaneous tumor models of gastric carcinoma. The mice were randomly divided into E2F-1/RNAi-LV group, LV-scrRNAi group and PBS group (n = 12). E2F-1/RNAi-LV, LV-scrRNAi or PBS (0.1 ml per time) was injected into the mice, respectively, every two days. The nude mice received an intraperitoneal injection of cisplatin (25 mg/kg) every two days. The tumor volume was measured and histopathological changes of the tumors were observed by HE staining. The expressions of E2F-1, c-Myc, survivin, MDR1 and MRP were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Apoptosis in tumor xenografts was determined by in situ TUNEL labeling technique.</p><p><b>RESULTS</b>The mean tumor growth rate of the E2F-1/RNAi-LV group was significantly slower than that of the LV-scrRNAi and control groups (P < 0.05). The tumor volume of the E2F-1/RNAi-LV group was (745.13 ± 154.42)mm(3), significantly lower than that of the LV-scrRNAi and PBS groups (P < 0.05). Compared with that in the LV-scrRNAi and PBS groups, the expressions of mRNA and protein of E2F-1, c-Myc, survivin, MDR1 and MRP were significantly decreased in the E2F-1/RNAi-LV group (P < 0.05). The apoptotic rate in the E2F-1/RNAi-LV treatment group was (27.5 ± 9.7)%, significantly higher than (7.0 ± 1.1)% in the LV-scrRNAi group and (7.3 ± 1.2)% in the PBS group (P < 0.05).</p><p><b>CONCLUSION</b>Intra-tumoral injection of E2F-1/RNAi-LV shows significantly inhibitory effect on the tumor growth and chemoresistance of subcutaneous human gastric cancer in nude mice.</p>

Cytotoxic constituents from the leaves of Broussonetia papyrifera / 中国天然药物

AIM@#To investigate the chemical constituents from the leaves of Broussonetia papyrifera.@*METHODS@#The chemical constituents were isolated and purified by macroporous adsorptive resin D101, silica gel, and ODS column chromatography and preparative HPLC. Their structures were elucidated on the basis of 1D and 2D NMR analyses. In addition, their cytotoxic activity against human hepatoma carcinoma cells (HepG-2) were evaluated by the MTT method. Furthermore, RP-HPLC and colorimetric methods were used for the analysis of cosmosiin and total flavonoids.@*RESULTS@#A new lignan, together with five known compounds were obtained, and their structures were characterized as (+)-pinoresinol-4'-O-β-D-glucopyranosyl-4″-O-β-D-apiofuranoside (1), cosmosiin (2), luteolin-7-O-β-D-glucopyranoside (3), liriodendrin (4), 3, 5, 4'-trihydroxy-bibenzyl-3-O-β-D-glucoside (5), and apigenin-6-C-β-D-glucopyranside (6). Furthermore, RP-HPLC and colorimetric methods were established for the analysis of cosmosiin and total flavonoids.@*CONCLUSION@#Compound 1 was a new lignan, and compounds 5 and 6 were isolated for the first time from the title plant. Compounds 1, 4 and 6 showed definite activities against HepG-2, while the other compounds didn't show inhibitory effects. The optimal harvest time of B. papyrifera (L.) Vent. is September.

Effects of 5-FU combined compound ginseng and astragalus on biological behavior of human gastric cancer MGC-803 cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the in vitro effects of 5-fluorouracil (5-FU) combined Compound Ginseng and Astragalus (CGA) on the biological behaviors such as the proliferation, the cloning, apoptosis and migration of human gastric cancer MGC-803 cells.</p><p><b>METHODS</b>The cell proliferation inhibition rate was detected by MTT assay, and the median effective concentrations of different drugs used alone/combination were calculated. The cell cycle and apoptosis were observed by flow cytometry. The formation of the cell colony was detected by Giemsa staining. The drugs' inhibition on cell migration was detected by cell scratch experiment.</p><p><b>RESULTS</b>CGA and 5-FU both could inhibit the growth of the human gastric cancer cell line MGC-803 cells, and their effects were enhanced along with increased drug concentrations. Compared with CGA or 5-FU alone, CGA +5-FU got higher cell growth inhibition rate (P<0.05), and the effects were enhanced along with increased concentrations. CGA and 5-FU both could induce the apoptosis of MGC-803 cells, inhibit the formation of cell cloning, block cells at G0/G1 phase, and inhibit the cell migration. Compared with CGA or 5-FU alone, CGA + 5-FU got higher apoptosis rate of MGC- 803 cells, and more cells blocked at G0/G1 phase (P<0.05). Besides, the MGC-803 cells were inhibited at G0/G1 phase. Compared with CGA or 5-FU alone, CGA +5-FU obviously lowered formation of cell cloning and area of cell migration (P<0.05). The median effective concentration of CGA +5-FU was less than the sum of the median effective concentration of CGA and 5-FU.</p><p><b>CONCLUSION</b>Compared with CGA or 5-FU alone, CGA +5-FU could better inhibit the cell growth of human gastric cancer MGC- 803 cells, suppress the formation of cell cloning, induce cell apoptosis, block the cell cycle at G0/G1 phase, and inhibit the cell migration.</p>

Lentivirus-mediated RNA interference targeting E2F-1 inhibits human gastric cancer MGC-803 cell growth in vivo

The E2F-1 transcription factor is post-translationally modified and stabilized in response to various forms of DNA damage to regulate the expression of cell-cycle and pro-apoptotic genes. The sustained overexpression of E2F-1 is a characteristic feature of gastric cancer. In this study, we investigated the role of short hairpin RNA (shRNA) targeting E2F-1 gene on human gastric cancer MGC-803 cell growth in vivo, and preliminarily revealed the mechanism. Thus, we constructed recombinant pGCSIL-GFP-shRNA-E2F-1 lentiviral vector to knock down E2F-1 expression in human gastric cancer MGC-803 cells in vivo, and studied the effect of E2F-1 shRNA on growth of MGC-803 tumor and evaluated its treatment efficacy. Our data demonstrated that in a mouse model of established gastric cancer, intratumor injection of lentiviral shRNA targeting E2F-1 definitely decreased the endogenous E2F-1 mRNA and protein expression in MGC-803 tumor, and inhibited tumor growth and promoted tumor cells apoptosis. Moreover, we found that E2F-1 shRNA increased the expression of phosphatase and tensin homolog (PTEN), activated caspase-3 and caspase-9, and suppressed nuclear factor (NF)-kappaB expression in tumor tissue as determined by reverse transcription (RT)-PCR and western blotting. In summary, shRNA targeting of E2F-1 can effectively inhibits human gastric cancer MGC-803 cell growth in vivo and may be a potential therapeutic strategy for gastric cancer.

The effects of chronic hypoxic hypercapnia on the TLR4 and NFkappaB in hippocampus neuron in rats / 中国应用生理学杂志

<p><b>AIM</b>To study the expression and effect of TLR4 and NFkappaB protein in hippocampus neuron in rats exposed to chronic hypoxic hypercapnia.</p><p><b>METHODS</b>The disorder of learning-memory in pulmonary hypertension rat model was reproduced by chronic hypoxic hypercapnia. Thirty rats were randomly divided into three groups: normal control group, hypoxic hypercapnia 2-week and 4-week group. The number of apoptosis neurons in hippocampus CA1/3 was counted by TUNEL method. Activity of TLR4 and NFkappaB in hippocampus CA1/3 was detected by using SP immunocytochemical technique.</p><p><b>RESULTS</b>The expression of TLR4 protein in hippocampus CA1/3 in group 2HH( CA1: 0.1275 +/- 0.0242, CA3: 0.1156 +/- 0.0376) and 4HH (CA1: 0.1522 +/- 0.0187, CA3: 0.1427 +/- 0.0453) were significantly higher than those in the NC group (P < 0.05, P < 0.01). The positive expression of NFkappaB were showed in cell nucleus in group 2HH (CA1: 0.1326 +/- 0.0324, CA3: 0.1301 +/- 0.0112) and group 4HH (CA1: 0.1612 +/- 0.0428, CA3: 0.1578 +/- 0.0365), and significantly higher than those in the NC group (P < 0.05, P < 0.01). The apoptosis of neural cells in hippocampus CA1/3 gradually increased with the time of exposure, and reached peak at 4 weeks (P < 0.01 vs NC group).</p><p><b>CONCLUSION</b>The activation of TLR4 and NFkappaB may play an important role in the apoptosis of hippocampus neural cells in rat exposed to chronic hypoxic hypercapnia.</p>

Effects of ginkgolide B against damage of cultured hippocampal neurons caused by glutamate / 中国应用生理学杂志

<p><b>AIM</b>To investigate protective effects of ginkgolide B (GB) in different administration modes on glutamate-induced neuronal damage.</p><p><b>METHODS</b>Essential GB were obtained by supercritical CO2 fluid extraction. Glutamate excitotoxicity were examined in primary cultures from neonatal Wistar rat, by using of Trypan blue dye staining, testing the lactate dehydrogenase leakage from cultured neurons and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method. The protective effects of GB in different administration modes (pre-treatment and post-treatment) were adopted and compared with the NMDA receptor uncompetitive antagonist-MK-801 in acute-treatment.</p><p><b>RESULTS</b>Treatment with GB in two administration modes both could increase ratio of surviving neuron, decrease LDH efflux and reduce ratio of neuron apoptosis in different degree, depended on dose in certain range. The protective effect of pre-treatment was superior to post-treatment, but inferior to MK-801.</p><p><b>CONCLUSION</b>GB can protect neurons against glutamate damage, and preventive using has more efficiency. The potential mechanism of its neural protection may be not only related to PAF receptor. If the predominant protection effect of GB in pretreatment is considered, precautionary intervention to high-risk population could have more value.</p>

Dynamic and long-lasting expression of thrombin receptor-1 after intracerebral hemorrhage in rats / 中国应用生理学杂志

<p><b>AIM</b>To study the dynamic and long-lasting expression of thrombin receptor after acute intracerebral hemorrhage (ICH) in rats.</p><p><b>METHODS</b>36 rats were randomly divided into 6 groups (n = 6): Normal group and ICH model groups at 6 hours, 24 hours, 3 days, 7 days and 14 days. ICH models were produced with the induction of collagenase type VII-S. Immunohistochemical method was used to detect PAR-1 protein and RT-PCR technique was used to detect PAR-1mRNA in brain tissue around the haematoma in different groups.</p><p><b>RESULTS</b>PAR-1 protein and mRNA were mild positive in normal group. In model groups, intensity of PAR-1 expression started to enhance at 6 hours, and enhanced more at 24 hours. PAR-1 expression reached the peak at 3 days and began to descend. At 7 days the descent was obvious and there was further descent at 14 days. At each time point, the PAR-1 protein positive cell number and PAR-1mRNA absorbance ratio in ICH model groups were significantly higher than those in normal group (P < 0.05 or P < 0.01). In addition, PAR-1 proteins were obviously expressed in vivo in brain capillary endothelial cell.</p><p><b>CONCLUSION</b>Functional PAR-1 exists in brain capillary endothelial cells. Activation of PAR-1 after ICH due to the stimulation of thrombin is not only the initiating agent of cerebral edema after ICH, but also participates the development of cerebral edema.</p>