Association between bile acids and nonalcoholic fatty liver disease / 临床肝胆病杂志

With the rapid increase in the prevalence rate of nonalcoholic fatty liver disease (NAFLD), new treatment methods are needed to prevent disease progression to liver fibrosis, liver cirrhosis, and liver cancer. Although great efforts have been made to clarify the pathological mechanisms of NAFLD disease progression, there are still no effective treatment methods at present. Bile acids (BAs) regulate systemic metabolism by activating nuclear receptors and G protein-coupled receptors and have been identified as important signaling molecules involved in lipid, glucose, and energy metabolism. Dysregulation of BA homeostasis is associated with the severity of NAFLD. This article summarizes the important ligands in BA metabolism and their role in the progression of NAFLD, in order to provide a basis for the treatment of NAFLD by targeting BA messengers.

Role of glucose and lipid metabolism mediated by the bile acid receptor Takeda G protein-coupled receptor 5 in nonalcoholic fatty liver disease / 临床肝胆病杂志

Nonalcoholic fatty liver disease (NAFLD) has gradually become a prominent cause affecting human liver health, and the development and progression of NAFLD are associated with metabolic dysfunction, with glucose and lipid metabolism disorder as the key link in this process. Takeda G protein-coupled receptor 5 (TGR5) is one of the main receptors of bile acid and is extensively expressed in the body, and glucose and lipid metabolism mediated by TGR5 plays an important role in the human body. This article summarizes the role and mechanism of TGR5 in glucose and lipid metabolism and the research findings of the treatment of NAFLD based on TGR5, in order to provide a reference for basic and clinical research.

Research advances in the bile acid membrane receptor TGR5 in biliary tract diseases / 临床肝胆病杂志

TGR5 is a bile acid-activated G protein-coupled receptor and plays an important role in the physiological and pathological processes of the biliary system. This article describes the normal expression of TGR5 in the liver and bile duct under normal physiological conditions and its functions including the regulation of bile acid secretion and metabolism and cytoprotection. This article also summarizes the changes in the expression and function of TGR5 under pathophysiological conditions and the mechanism of TGR5 in affecting the development and progression of biliary tract diseases through inflammatory response and cell proliferation and apoptosis. TGR5 may be a potential target for the treatment of biliary tract diseases in the future.

Role of GPR30 in reduction of ketamine-induced long-term cognitive dysfunction by 17β estradiol in neonatal rats / 中华麻醉学杂志

Objective:To evaluate the role of G protein-coupled receptor 30 (GPR30) in reduction of ketamine-induced long-term cognitive dysfunction by 17β estradiol in neonatal rats.Methods:Thirty healthy male Sprague-Dawley rats, aged 7 days, weighing 11-18 g, were divided into 5 groups ( n=6 each) using a random number table method: control group (group C), ketamine group (group K), 17β estradiol plus ketamine group (group KE), GPR30 agonist G1 plus ketamine group (group G1K) and GPR30 inhibitor G15 plus 17β estradiol plus ketamine group (group G15EK). Ketamine 75 mg/kg was intraperitoneally injected in group K. In group EK, 17β estradiol 600 μg/kg was subcutaneously injected, and ketamine 75 mg/kg was intraperitoneally injected.In group G1K, G1 200 μg/kg was subcutaneously injected, and ketamine 75 mg/kg was intraperitoneally injected.In group G15EK, G15 300 μg/kg and 17β estradiol 600 μg/kg were subcutaneously injected, and ketamine 75 mg/kg was intraperitoneally injected.The equal volume of normal saline was intraperitoneally given in group C. The injection was performed every 24 h for 3 consecutive days.All the rats were allowed to grow up till postnatal day 60, and then Morris water maze test was performed to evaluate their spatial learning and memory function.The rats were sacrificed after the end of Morris water maze test, and hippocampi were removed for determination of contents of acetyl cholinesterase (AChE) and acetylcholine (ACh) by enzyme-linked immunosorbent assay. Results:Compared with group C, the escape latency was significantly prolonged on the 3-5 training days, the frequency of crossing the platform and percentage of time of staying at the target quadrant were decreased, the content of AChE was increased, and the content of ACh was decreased in group K ( P<0.05). Compared with group K, the escape latency was significantly shortened on the 3-5 training days, the frequency of crossing the platform and percentage of time of staying at the target quadrant were increased, the content of AChE was decreased, and the content of ACh was increased in EK and G1K groups ( P<0.05). Compared with EK and G1K groups, the escape latency was significantly prolonged on the 3-5 training days, the frequency of crossing the platform and percentage of time of staying at the target quadrant were decreased, the content of AChE was increased, and the content of ACh was decreased in group G15EK ( P<0.05). Conclusion:GPR30 is involved in reduction of ketamine-induced long-term cognitive dysfunction by 17β estradiol, which is related to regulating the contents of AChE and ACh in hippocampi of neonatal rats.

Effects of ICI 182, 780, an ERα and ERß antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells / Efeitos do antagonista seletivo do REα e do REß ICI 182, 780 e do agonista de GPER G-1 no processo de autofagia em células de câncer de mama

ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.

Mas-related G protein-coupled receptor X2: a novel receptor for non-IgE-dependent activation of mast cells / 中华皮肤科杂志

Mast cells (MC),a major participant in inflammation and allergy,can initiate an early defense response to foreign invaders.Besides expressing high-affinity IgE receptor,MC also expresse a large number of G protein-coupled receptors (GPCRs).Various studies have shown that cationic micromolecules represented by substance P and many peptidergic drugs can induce MC degranulation through a novel receptor,Mas-related G protein-coupled receptor X2 (MrgprX2).MrgprX2 plays an important role in allergy,itching and pseudo-allergic drug reactions.It will help explain some clinical difficulties which can not be explained by classical IgE-dependent activation of MC,and provides potential therapeutic targets for MC-mediated diseases.

Understanding Bile Acid Signaling in Diabetes: From Pathophysiology to Therapeutic Targets

Diabetes and obesity have reached an epidemic status worldwide. Diabetes increases the risk for cardiovascular disease and non-alcoholic fatty liver disease. Primary bile acids are synthesized in hepatocytes and are transformed to secondary bile acids in the intestine by gut bacteria. Bile acids are nutrient sensors and metabolic integrators that regulate lipid, glucose, and energy homeostasis by activating nuclear farnesoid X receptor and membrane Takeda G protein-coupled receptor 5. Bile acids control gut bacteria overgrowth, species population, and protect the integrity of the intestinal barrier. Gut bacteria, in turn, control circulating bile acid composition and pool size. Dysregulation of bile acid homeostasis and dysbiosis causes diabetes and obesity. Targeting bile acid signaling and the gut microbiome have therapeutic potential for treating diabetes, obesity, and non-alcoholic fatty liver disease.

Role of GPR30 in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in hippocampus of newborn rats: the relationship with p-ERK1/2 / 中华麻醉学杂志

Objective@#To evaluate the role of G protein-coupled receptor 30 (GPR30) in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats and the relationship with phosphorylated extracellular signal-regulated kinase 1/2 (p-ERKl/2).@*Methods@#Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 7 days, weighing 11-18 g, were divided into 4 groups (n=6 each) using a random number table method: control group (group C), ketamine group (group K), 17β estradiol plus ketamine group (group EK), and GPR30 inhibitor G15 plus 17β estradiol plus ketamine group (group G15EK). Ketamine 75 mg/kg (diluted to 0.1 ml in normal saline) was intraperitoneally injected every 24 h for 3 consecutive days in group K. In group EK, 17β estradiol 600 μg/kg was subcutaneously injected and ketamine 75 mg/kg was intraperitoneally injected every 24 h for 3 consecutive days.G15 300 μg/kg and 17β estradiol 600 μg/kg were subcutaneously injected and ketamine 75 mg/kg was intraperitoneally injected every 24 h for 3 consecutive days in group G15EK.The equal volume of normal saline 0.1 ml was intraperitoneally injected instead in group C. The animals were sacrificed at 24 h after the last injection for determination of the expression of cleaved caspase-3, ERK1/2 and phosphorylated ERK1/2(p-ERK1/2) (by Western blot).@*Results@#There was no significant difference in the expression of ERK1/2 in hippocampus among the four groups (P>0.05). Compared with group C, the expression of cleaved caspase-3 was significantly up-regulated, and the expression of p-ERK1/2 was down-regulated in K and G15EK groups (P<0.05). Compared with group K, the expression of cleaved caspase-3 was significantly down-regulated, and the expression of p-ERK1/2 was up-regulated in group EK (P<0.05). Compared with group EK, the expression of cleaved caspase-3 was significantly up-regulated, and the expression of p-ERK 1/2 was down-regulated in group G15EK (P<0.05).@*Conclusion@#GPR30 is involved in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats, which is related to up-regulating the expression of p-ERKl/2.

Role of GPR30 in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in hippocampus of newborn rats: the relationship with p-ERK1/2 / 中华麻醉学杂志

Objective To evaluate the role of G protein-coupled receptor 30 (GPR30) in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats and the relationship with phosphorylated extracellular signal-regulated kinase 1/2 (p-ERKl/2).Methods Twentyfour clean-grade healthy male Sprague-Dawley rats,aged 7 days,weighing 11-18 g,were divided into 4 groups (n =6 each) using a random number table method:control group (group C),ketamine group (group K),17β estradiol plus ketamine group (group EK),and GPR30 inhibitor G15 plus 17β estradiol plus ketamine group (group G15EK).Ketamine 75 mg/kg (diluted to 0.1 ml in normal saline) was intraperitoneally injected every 24 h for 3 consecutive days in group K.In group EK,17β estradiol 600 μg/kg was subcutaneously injected and ketamine 75 mg/kg was intraperitoneally injected every 24 h for 3 consecutive days.G15 300 μg/kg and 17β estradiol 600 μg/kg were subcutaneously injected and ketamine 75 mg/kgwas intraperitoneally injected every 24 h for 3 consecutive days in group G15EK.The equal volume of normal saline 0.1 ml was intraperitoneally injected instead in group C.The animals were sacrificed at 24 h after the last injection for determination of the expression of cleaved caspase-3,ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) (by Western blot).Results There was no significant difference in the expression of ERK1/2 in hippocampus among the four groups (P＞0.05).Compared with group C,the expression of cleaved caspase-3 was significantly up-regulated,and the expression of p-ERK1/2 was down-regulated in K and G15EK groups (P＜0.05).Compared with group K,the expression of cleaved caspase-3 was significantly down-regulated,and the expression of p-ERK1/2 was up-regulated in group EK (P＜0.05).Compared with group EK,the expression of cleaved caspase-3 was significantly up-regulated,and the expression of p-ERK 1/2 was down-regulated in group G15EK (P＜0.05).Conclusion GPR30 is involved in 17β estradiol-induced inhibition of ketamine-caused neuroapoptosis in the hippocampus of newborn rats,which is related to up-regulating the expression of p-ERKl/2.

Effect of levocetirizine hydrochloride on the growth of human dermal papilla cells: a preliminary study / 中华皮肤科杂志

Objective To preliminarily evaluate the effect of levocetirizine hydrochloride at different concentrations on the growth of in vitro cultured human dermal papilla cells,and to explore its mechanism.Methods Human dermal papilla cells were divided into several groups to be cultured with Dulbecco's modified eagle medium (DMEM) containing 0 (control group),1,10,100,1 000,10 000 μg/L levocetirizine hydrochloride respectively for 48 hours.Immunofluorescence staining was performed to observe the growth of the dermal papilla cells,and methyl thiazolyl tetrazolium (MTT) assay to evaluate the proliferative activity of the dermal papilla cells.Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expression of cyclooxygenase 2 (COX-2),prostaglandin D2 synthase (PTGDS),prostaglandin E2 (PGE2),prostaglandin F2alpha (PGF2α),G protein-coupled receptor 44 (GPR44),protein kinase B (AKT) and glycogen synthase kinase 3β (GSK3β),and Western blot analysis to determine the protein expression of PTGDS.After 24-hour culture with DMEM containing levocetirizine hydrochloride at different concentrations,enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of prostaglandin D2 (PGD2) and PGD2R receptor in the culture supernatant of the human dermal papilla cells.Statistical analysis was carried out with SPSS 17.0 software using one-way analysis of variance for the comparison of the above indices among the groups,and least significant difference (LSD)-t test for multiple comparisons.Results Immunofluorescence staining showed that human dermal papilla cells grew well and reached over 90％ confluence in the 100 μg/L levocetirizine hydrochloride group.MTT assay revealed that there were significant differences in the proliferation rate among all the groups (F =42.22,P ＜ 0.05),and the proliferation rate was significantly higher in the 100 μg/L levocetirizine hydrochloride group (115.80％ ± 5.10％) than in the control group (100％,t =28.26,P ＜ 0.05).The mRNA expression(2-△△Ct) of COX-2,PGF2a,PTGDS,GPR44 and AKT all significantly differed among these groups (F =1.97,3.66,2.17,2.66 and 7.32 respectively,all P ＜ 0.05),while no significant difference in the mRNA expression of PGE2 and GSK3β was observed among these groups (F =0.87 and 1.19 respectively,both P ＞ 0.05).The 100 μg/L levocetirizine hydrochloride group showed significantly decreased mRNA expression of COX-2,PTGDS and GPR44 (0.84± 0.08,0.81±0.10 and 0.85 ± 0.09 respectively) compared with the control group (t =1.97,2.17 and 2.66 respectively,all P ＜ 0.05),but significantly increased mRNA expression of PGF2α and AKT (1.96 ± 0.25 and 1.74 ± 0.32 respectively) compared with the control group (t =3.66,7.32 respectively,both P ＜ 0.05).Moreover,the protein expression of PTGDS,PGD2 and PGD2R significantly differed among these groups (all P ＜ 0.05),and was significantly lower in the 100 μg/L levocetirizine hydrochloride group than in the control group (P ＜ 0.05).Conclusion Levocetirizine hydrochloride can promote the in vitro growth of human dermal papilla cells,likely by inhibiting the PGD2-GPR44 pathway and activating the AKT signal pathway.

LGR5 and ALDH1A1 served as prognostic and predictive markers for non-small cell lung cancer / 中华核医学与分子影像杂志

Objective To investigate the value of leucine-rich repeat-containing G protein coupled receptor 5 (LGR5) and aldehyde dehydrogenase 1A1 (ALDH1A1) in progression and prognosis of non small-cell lung cancer (NSCLC),in order to find new targets for NSCLC-targeted imaging and radiation therapy.Methods Fresh tissues (n =24) and paraffin embedding tissues (n =109) of patients with NSCLC were collected from the First Affiliated Hospital of Soochow University between November 2009 and March 2012.Quantitative real time-PCR was used for the investigation of expression of LGR5 and ALDH1A1 mRNA in 24 NSCLC patients.Immunohistochemistry (IHC) was used for detecting LGR5 and ALDH1A1 expressions in NSCLC tissues and adjacent normal tissues.Data were analyzed by Mann-Whitney u test,x2 test,Pearson correlation analysis,Kaplan-Meier method,Cox proportional hazards regression model.Results Compared with adjacent normal tissues,LGR5 and ALDH1A1 mRNA were frequently increased in NSCLC tissues (u values:150,74,both P＜0.01),and the expression of LGR5 and ALDH1A1 mRNA was significantly correlated (r=0.416,P＜0.05).Positive LGR5 and ALDH1A1 expression was defined in 28.44％ (31/109) and 41.28％ (45/109) of the NSCLC tumors,respectively.Further analysis indicated that 24 of the LGR5 positive samples (77.42％,24/31) expressed ALDH1A1 (r=0.388,P＜0.01).LGR5 and ALDH1A1 ex pressions in NSCLC with higher TNM stage were significantly higher than those in NSCLC with lower TNM stage (x2 values:4.64,5.24,both P＜0.05).Coexpression of LGR5 and ALDH1A1 in NSCLC with lymph node metastasis was higher than that in NSCLC without lymph node metastasis (x2=4.12,P＜0.05).High expression of LGR5 or ALDH1A1 was related to poor prognosis (x2 values:6.24,4.18,both P＜0.05),and NSCLC patients with coexpression of LGR5 and ALDH1A1 had a poorer prognosis than the others (x2 =10.63,P＜0.01).Both of them were independent risk factors of a poorer prognosis (corrected hazard ratio (95％ CI):2.361(1.106-5.037),2.306(1.101-4.830);both P＜0.05).Conclusions The expressions of LGR5 and ALDH1A1 are closely associated with the tumorigenesis,metastasis and poor prognosis of NSCLC.LGR5 and ALDH1A1 might be new targets for NSCLC-targeted tumor imaging and radiation therapy.

Changes in expression of spinal endothelin-1 and its receptors in a mouse model of bone cancer pain / 中华麻醉学杂志

Objective To evaluate the changes in the expression of spinal endothelin-1 (ET-1) and its receptors in a mouse model of bone cancer pain (BCP).Methods Ninety-six healthy male SPF C3H/HeN mice,aged 4-6 weeks,weighing 20-25 g,were divided into 2 groups (n=48 each) using a random number table:sham operation group (group S) and BCP group.BCP was produced by injecting α-MEM 20 μl containing 1×104 cells/μ1 NCTC 2472 osteosarcoma cells into the distal medullary cavity of the right femur bone.In group S,t-MEM 20 μl was injected into the distal medullary cavity of the right femur bone.Mechanical paw withdrawal threshold (MWT) and the number of spontaneous flinches (NSF) were measured on 1 day before inoculation (T0) and 4,7,10,14 and 21 days after inoculation (T1-5).Twelve mice of each group were randomly sacrificed at T0,2,4,5,and the lumbar enlargement segments of the spinal cord were harvested to detect the expression of ET-1,endothelin type A receptor and endothelin type B receptor protein and mRNA (using Western blot or real-time polymerase chain reaction).Results The MWT was significantly lower and the NSF was higher at T1 in group S and at T1-5 in group BCP than at T0 (P＜0.05).Compared with group S,the MWT was significantly decreased and the NSF was increased at T2-s,and the expression of ET-1,endothelin type A receptor and endothelin type B receptor protein and mRNA was down-regulated at T2,4,5 in group BCP (P＜0.05).Conclusion The pathophysiological process of BCP is associated with down-regulating the expression of spinal ET-1 and its receptors in mice.

Research advances in the pathogenesis of bile acid-related non-alcoholic fatty liver disease and related pharmacotherapy / 临床肝胆病杂志

Non-alcoholic fatty liver disease (NAFLD) has become a new health issue in the world due co its increasing incidence rate,and in particular,nonalcoholic steatohepatitis is progressive and has poor prognosis.Therefore,there is an argent need to search for the methods for the prevention of disease progression and treatment.Bile acid,as an important metabolite and signal molecule,can adjust the metabolism of lipids and carbohydrates and energy balance inside and outside the liver.Bile acid interacts with is receptors,such as the farnesoid X receptor and Takeda G-protein coupled receptor 5,bile acid transporter,and gut microbiota and is involved in the pathogenesis of NAFLD and nonalcoholic steatohepatitis at different levels.This article summarizes the research advances in the pathogenesis of bile acid-related NAFLD and related pharmacotherapy.

The study of 5-Hydroxytryptamine 2B receptor-nanodisc self-assembling and its ligand binding activity / 中国医师杂志

Objective To explore the application of nanodisc in functional and drug discovery research of G protein-coupled receptor (GPCR).Methods The purified recombinant 5-Hydroxytryptamine 2B receptor (5-HT2BR) was reconstituted into nanodisc complex.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclution chromatography were performed to evaluate the reconstitution reaction,followed by the use of surface plasmon resonance to validate the ligand-binding activity of 5-HT2BR after reconstitution.Results 5-HT2B R was effectively self-assembled into nanodisc while maintained its binding activity toward the antagonist SB204741.Conclusions The presented study provided potential application of 5-HT2B R-nanodisc for the development of subtype-selective drugs against 5-HT2B R and the fundamental of utilizing nanodisc for GPCR structural and functional studies as well as drug discovery.

Relationship between TDAG8 and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration-induced apoptosis in rat neurons / 中华麻醉学杂志

Objective To investigate the relationship between T?cell death?associated gene 8 ( TD?AG8) and endogenous neuron?protective mechanism against oxygen?glucose deprivation and restoration ( OGD∕R)?induced apoptosis in rat neurons. Methods The primary cortical neurons obtained from fetal rats were seeded in 6?well plates at a density of 1×105 cells∕ml and divided into 5 groups using a random number table: control group ( group C, n=24 ) , group OGD∕R ( n=48 ) , TDAG8 agonist BTB09089 group (group BTB, n=24), TDAG8?siRNA group ( group siRNA, n=24), and blank vehicle group ( group V, n=24) . The medium was replaced with glucose?and serum?free Locke′s buffer, and the neu?rons were exposed to 95% N2?5% CO2 in an air?tight incubator at 37℃ for 60 min followed by routine cul?ture to establish the model of OGD∕R. In BTB, siRNA and V groups, 20 μmol∕L TDAG8 agonist BTB09089, 200 pmol∕L TDAG8?siRNA, and 6 μl∕200 μl transfection reagent were added, respectively, at 24 h before oxygen?glucose restoration. At 6 h of oxygen?glucose restoration, the neuronal viability and a?mount of lactic dehydrogenase ( LDH) released were measured, and the expression of TDAG8 and caspase?3 mRNA in neurons was detected by fluorescent quantitative real?time polymerase chain reaction. In group OGD∕R, the expression of TDAG8 and caspase?3 was measured by Western blot at 0, 3, 6, 12 and 24 h of oxygen?glucose restoration. In C, OGD∕R, BTB, siRNA and V groups, the expression of TDAG8, caspase?3 and p?Akt was detected at 6 h of oxygen?glucose restoration. Results In group OGD∕R, the ex?pression of TDAG8 was gradually up?regulated after oxygen?glucose restoration, and the expression of caspase?3 peaked at 6 h of oxygen?glucose restoration. Compared with group C, the neuronal viability was significantly decreased, the amount of LDH released was significantly increased, and the expression of TD?AG8 and caspase?3 protein and mRNA and p?Akt was significantly up?regulated in OGD∕R, V and siRNA groups ( P0?05) . Conclusion TDAG8 is partially involved in the endogenous neuron?protective mechanism against OGD∕R?induced apoptosis in rat neurons, which may be related to activation of Akt signaling pathway.

Role of spinal neuronal Mas-related gene receptor C in maintenance of bone cancer pain in mice / 中华麻醉学杂志

Objective To evaluate the role of spinal neuronal Mas-related gene receptor C (MrgC) in the maintenance of bone cancer pain (BCP) in mice.Methods A total of 132 SPF male C3H/HeJ mice, aged 8-10 weeks, weighing 18-22 g, were randomly divided into 4 groups (n=33 each) using a random number table: sham operation group (S group) , BCP group, bovine adrenal medulla peptide 8-22 (BAM8-22, a highly selective MrgC agonist) group (group BAM), and MrgC antibody group (group MA).BCP was produced by injecting α-MEM 20 μl containing 2×105NCTC2472 cells into the distal medullary cavity of right femur bone.While α-MEM 20 μl was injected only in group S.The artificial cerebrospinal fluid 5 μl was injected intrathecally in S and BCP groups, and BAM 8-22 8 nmol/5 μl and MrgC antibody 5 μl were injected intrathecally in BAM and MA groups, respectively, once a day for 7 consecutive days starting from the day 14 after inoculation of the tumor cells.At 1 day before inoculation (T0), before administration (T1) , and at 14, 16 19 and 21 days after inoculation (T2-5, at 0.5 h before the initial administration and 2 h after each administration) , the number of spontaneous flinches (NSF) and mechanical paw withdrawal threshold (MWT) were measured.Five animals selected from each group at each time point were sacrificed, and the lumbar enlargement segments of the spinal cord were removed for determination of MrgC expression in the spinal neurons (by immunofluorescence).Results Compared with group S, NSF was significantly increased, MWT was decreased, and the expression of MrgC was up-regulated at T1-5 in BCP, BAM and MA groups.Compared with group BCP, NSF was significantly decreased, MWT was increased, and the expression of MrgC was up-regulated at T2-5 in group BAM, and NSF was significantly increased, MWT was decreased, and the expression of MrgC was down-regulated at T2-5 in group MA.Conclusion Spinal neuronal MrgC is involved in the maintenance of BCP in mice.

Stem cell marker LGR5：a new target for cancer therapy / 中国组织工程研究

BACKGROUND:Recent studies have shown that cancer stem cels play a key role in the development of tumors, therefore, the research about cancer stem cels’ markers can deepen the understanding of the development and clinical diagnosis of the tumor. OBJECTIVE:To review the research progression of stem cel marker LGR5. METHODS: The first author retrieved PubMed database and Wanfang database for papers regarding LGR5 and stem cel/cancer stem cel published between January 1998 and December 2014 using the key Words “LGR5, stem cel, cancer stem cel” in English and Chinese, respectively. A total of 178 papers were initialy retrieved. After 123 papers with independent objective and out-of-date contents were excluded, 55 papers were suitable for final analysis. RESULTS AND CONCLUSION: LGR5 is the surface marker of intestinal, stomach, hair folicle stem cels, which has a great relationship with occurrence, development and prognosis of colorectal cancer, stomach cancer, lung cancer, ovarian cancer, liver cancer, basal cel carcinoma. As a candidate marker of cancer stem cels, LGR5 can be the new treatment target. Studies have shown that R-spondins (RSPOs) is a high affinity ligand of LGR5. They participate in the Wnt signaling pathway, regulating cel proliferation and differentiation.

The effects and mechanisms of G protein-coupled receptor 91 on blood-retinal barrier in diabetic rats / 中华眼底病杂志

Objective To investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats.Methods A lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed.Healthy male Sprague-Dawley (SD) rats were selected in this study.The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A,n=15),the rats received injections of an equal volume of 0.1％ citrate buffer;(2) streptozocin (STZ) group (Group B,n=15),the rats received injections of STZ;(3) LV.shScrambled group (Group C,n=15),diabetic rats received an intravitreal injection of 1 μl 1 × 10s TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes;(4) LV.shGPRg1 group (Group D,n=15),diabetic rats received an intravitreal injection of 1 μl 1 × 108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles.At 12 weeks after intravitreal injection,immunohistochemistry and Western blot were used to assess the expression of GPRg1,p-extracellular signal-regulated kinase(ERK)1/2,t-ERK1/2,p-Jun N-terminal kinase (JNK),t-JNK,p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK.Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel.Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF.Results Immunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer.Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31,P＜0.01).HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina,while the telangiectatic vessels attenuated in Group D.It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)％ compared with Group C and there was significant difference (F =30.35,P＜0.05).The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15,P＜0.05).The results of Western blot indicated that compared with Group A,the expressions of p-ERK1/2,p-JNK and p-p38 MAPK were significantly upregulated (q=6.38,2.94,3.45;P＜0.05).Meanwhile,the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50,P＜0.05).Conclusions The intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats.GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.

Influence of pertussis toxin on GPER-mediated activation of phosphatidylinositol 3-kinase/protein kinase R signaling induced by 17β-estradiol in endometrial carcinoma cells / 中华妇产科杂志

Objective To investigate the influence of pertussis toxin(PTX)on G protein-coupled estrogen receptor(GPER)-mediated activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt)signaling activated by 17 β-estradiol(17β-E2)in endometrial carcinoma cells.Methods Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells.Changes of levels of GPER,ERα and ERβ protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations(0,0.1,0.5,1.0 μg/ml),and then co-stimulated with with 1 × 10-6 mol/L 17β-E2 respectively at different time (Ishikawa 30 minutes,HEC-1A 15 minutes).Results(1)Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell.(2)After co-treated with PTX at different concentrations(0,O.1,0.5,1.0 μg/ml)and 10-6 mol/L 17β-E2,in Ishikawa cell,the ratio of pAkt/Akt was 0.74 ±0.54,0.34 ±0.06,0.18 ±0.03,0.07 ±0.15,the gray values of GPER was 0.872 ± 0.490,0.395 ± 0.054,0.145 ± 0.014,0.034 ± 0.008,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which was most obviously when the concentration was 1.0 μg/ml(F =63.729,P =0.0001;F =160.284,P =0.0001);ERα and ERβ protein had no significant change among different groups(P ＞0.05).In HEC-1A cell,the ratio of pAkt/Akt was 0.73 ±0.09,0.26 ±0.14,0.11 ±0.03,0,the Gray values of GPER is 0.927 ±0.134,0.485 ± 0.022,0.194 ± 0.004,0,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which were also completely inhibited when the concentration was 1 μg/ml(F =1039.321,P =0.0001;F =109.646,P =0.0001),ERα protein had no significant differences(P ＞ 0.05)among different groups.ERβ was negatively expressed.Conclusion The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.

GPRC5A and lung cancer / 国际肿瘤学杂志

The G protein-coupled receptor family C,member 5,group A (GPRC5A) gene is known as retinoic acid-induced gene,which is mainly distributed in lung tissue.The expression of GPRC5A in lung cancer is significantly decreased compared with normal lung.GPRC5A leads to lung cancer through knockout mice,which is proven to be a suppressor gene of lung cancer.GPRC5A may be a novel biomarker for the diagnosis of lung cancer and a new target for the treatment of lung cancer.