ObjectiveTo study the changes of levels of α subunits of stimulatory (Gsα) and inhibitory guanine nucleotide binding protein (Giα) in newborn guinea pig (0 2 days old) myocardium undergoing global ischemic reperfusion, and influences on the changes by St.Thomas Ⅱ and cold blood cardioplegic solution.MethodsThirty newborn guinea pigs were randomly assigned to three groups. GroupⅠ ( n = 10): the newborn hearts suffered by hypothermic global ischemia; group Ⅱ( n =10): the newborn hearts arrested by St. Thomas Ⅱ , and group Ⅲ ( n = 10): the newborn hearts arrested by cold blood cardioplegic solution. Levels of Gsα and Giα were investigated with Western blot analysis.ResultsNo differences of levels of Gsα and Giα were found in three groups before ischemia ( P gt;0.05). The level of Gsα after ischemia was significantly decreased than before ischemia in groupⅠand group Ⅱ ( P lt; 0 01), whereas no pronounced changes in group Ⅲ ( P gt;0.05) were noted after ischemia. The level of Gsα in group Ⅲ was not significantly changed after reperfusion compared with before ischemia( P gt;0 05), and it was much higher than those in groupⅠand group Ⅱ ( P lt; 0 01). Level of Giα was found not markedly changed in group Ⅲ after reperfusion compared with that before ischemia, but was notable higher in groupⅠand group Ⅱ( P lt;0.01). ConclusionsSignificant decrease of level of Gsα, whereas marked increase of level of Giα are found in myocardium of newborn guinea pig undergoing hypothermic (20℃) ischemic reperfusion. No impact of St. Thomas Ⅱ on these changes is verified, but recovery to the level of Gsα and Giα before ischemia is achieved by cold blood cardioplegic solution after ischemia and reperfusion. Unbalance between Gsα and Giα is the one of the mechanisms of ischemic reperfusion injury for immature myocardium.
Objective To study the function and mechanism of G protein coupled receptor kinase interacting protein 1(GIT1) RNA hairpin (GIT1-RNAh) in osteoblast migration. Methods The sixth passage osteoblasts were divided into 2 groups and were infected by GIT1-RNAh (experimental group) and green fluoresence protein RNA hairpin (GFP-RNAh) (control group) adenovirus for 12 hours respectively. Each group was further classfied into two groups according to with or without platelet-drived growth factor (PDGF) stimulation. The GIT1 expression and Paxillindistribution was analyzed by immunofluorescence staining. Paxillin phosphorylation was detected by Western Blot. The localization of Paxillin was determined by co-immunofluorescence staining after transfection with cyanine fluorescence protein tagged GIT1RNAh (CFP-GIT1-RNAh)(experimental group) and GFP-RNAh (CFP-GFP-RNAh)(control group). The role of GIT1-RNAh (experimental group) and GFP-RNAh (control group) adenovirus in osteoblasts migration was determined by wound healing assay. Results Immunofluorescence staining results showed that the GIT1-RNAh significantly inhibited endogenous GIT1 expression, interfered Paxillin distribution.Western Blot results showed that Paxillin phosporylation was obviously inhibited in osteoblasts infected with GIT1-RNAh adenovirus (P＜0.05). The wound healing assay results howed that GIT1-RNAh adenovirus significantly inhibited osteoblast migration induced by PDGF. Conclusion GIT1-RNAh inhibits osteoblasts migration by interfering paxillin distribution and decrease Paxillin phosphorylation.
The mechanisms of general anesthesia, which was introduced about 170 years ago, remain poorly understood. Even less well understood are the effects of general anesthesia on the human body. Recently we identified 18 G-protein coupled receptor (GPCR) genes of Daphnia pulex, an invertebrate model organism. Phylogenetic analysis identified these genes to be the homologs of the human γ-aminobutyric acid, type B (GABAB) receptor, metabotropic glutamate receptors (mGluR), adrenergic receptor, serotonin (5-HT) receptor, dopamine receptor and muscarinic acetylcholine receptor (mAChR). Using reverse transcription and quantitative PCR techniques, we systematically measured the effects of propofol, etomidate and ethanol on these 18 GPCR mRNA expressions in Daphnia pulex.
Ras homolog family (Rho)/ Rho-associated coiled-coil kinase (ROCK) signaling pathway widely exists in human and mammal cells, which is closely related to inhibition of repair after optic nerve damage. The expression level of Rho/ROCK signaling pathway-related proteins is up-regulated in glaucoma, and related with the death of retinal ganglionic cell (RGC) and the axon activity. ROCK inhibitors can protect the surviving RGC and promote axon extension with a dose-dependent manner. ROCK inhibitors also can inhibit glial scar formation, lower intraocular pressure and inhibit inflammatory response to some degrees. Rho/ROCK signaling pathway correlates with the optic nerve disease progression, and ROCK inhibitors hope to become a new therapeutic drug.
ObjectiveTo detect the expressions of takeda G protein-coupled receptor 5 (TGR5) and mortalin protein 75 in the tissues of patients with intrahepatic cholangiocarcinoma (ICC), and to explore their relationship with prognosis.MethodsA total of 94 ICC patients who were admitted to Anyang District Hospital and received surgical treatment from March 2015 to March 2018 were selected as the research objects. The expressions of TGR5 and mortalin protein 75 in ICC cancer tissues and adjacent tissues were detected by immunohistochemistry and Western blot (WB). The relationship between the expressions of TGR5 and mortalin protein 75 in ICC cancer tissues and clinicopathological parameters and prognosis was analyzed. Multivariate Cox proportional hazards regression was used to analyze the risk factors of poor prognosis in patients with ICC. ROC curve was used to analyze the diagnostic value of TGR5 and mortalin protein 75 for poor prognosis in patients with ICC.ResultsImmunohistochemical results showed that the positive expression rates of TGR5 and mortalin protein 75 in cancer tissues were significantly higher than those in adjacenttissues (P<0.05). WB results showed that the protein expression levels of TGR5 and mortalin protein 75 in cancer tissues were significantly higher than those in adjacent tissues (P<0.05). The expression of TGR5 protein in cancer tissues of ICC patients was correlated with gender, tumor diameter, degree of differentiation, TNM staging, satellite focus, and liver cirrhosis (P<0.05). The expression of mortalin protein 75 was correlated with tumor diameter, TNM staging, nerve involvement, satellite focus, and liver cirrhosis (P<0.05). There were significant differences in gender, tumor diameter, TNM staging, microvascular invasion, satellite focus, liver cirrhosis, and the expressions of TGR5 and mortalin protein 75 between the poor prognosis group and the good prognosis group (P<0.05). The cumulative 3-year overall survival rate of TGR5 positive patients (32.00%) was significantly lower than that of TGR5 negative patients (63.16%), χ2=6.228, P=0.013; the cumulative 3-year overall survival rate of mortalin protein 75 positive patients (32.91%) was significantly lower than that of mortalin protein 75 negative patients (66.67%), χ2=6.079, P=0.014. Multivariate Cox proportional hazards regression analysis showed that the positive expression of TGR5 and mortalin protein 75, TNM Ⅲ+Ⅳphase, satellite focus, and cirrhosis were risk factors for poor prognosis in ICC patients (P<0.05). ROC results showed that when the expression level of TGR5 was 0.932 as the cut-off value, its AUC in the diagnosis of poor prognosis of ICC patients was 0.783, the sensitivity was 72.4%, the specificity was 72.2%; when the expression level of mortalin protein 75 was 0.756 as the cut-off value, its AUC in the diagnosis of poor prognosis of ICC patients was 0.805, the sensitivity was 84.4%, the specificity was 63.9%; the AUC of combined diagnosis of TGR5 and mortalin protein 75 was 0.884, the sensitivity was 79.3%, the specificity was 83.3%.ConclusionsThe high expressions of TGR5 and mortalin protein 75 in cancer tissues of ICC patients are associated with poor prognosis, and they are risk factors for poor prognosis. The combined detection of TGR5 and mortalin protein 75 has a certain value in predicting poor prognosis, and can be used as potential biological indicators.
ObjectiveTo observe the effect of G protein alpha inhibitory subunit (Gαi) 1 and Gαi3 on signal transduction and angiogenesis induced by Netrin-1 (NTN1) and explore the possible mechanisms. MethodsTwenty male C57BL/6J mice aged 6 to 8 weeks were randomly assigned to a control group and a diabetic group, with 10 mice in each group. Diabete group mice were induced by streptozotocin to establish diabetes model. 12 weeks after modeling, quantitative real-time polymerase chain reaction and Western blot were performed to detect the expression of Ntn1, Gαi1 and Gαi3 in diabetic retinas. Additionally, 35 male C57BL/6J mice aged 2 weeks were randomly stratified into three groups: a control group, an intravitreal injection of NTN1 group (NTN1 group), and a retinal endothelial cell-specific Gαi1/Gαi3 knockdown coupled with intravitreal NTN1 injection group (Gαi1/Gαi3 eKD+NTN1 group), with 15 mice in each of the normal control and NTN1 groups, and 5 mice in the Gαi1/Gαi3 eKD+NTN1 group. Isolectin B4 staining was performed to observe retinal neovascularization. In vitro, human umbilical vein endothelial cells (HUVEC) were divided into four groups: negative control lentiviral transfection group (shC group), negative control lentiviral transfection+NTN1 treatment group (shC+NTN1 group), Gαi1/Gαi3 knockdown group (shGαi1/Gαi3 group), and Gαi1/Gαi3 knockdown+NTN1 treatment group (shGαi1/Gαi3+NTN1 group). The effects of NTN1, Gαi1, and Gαi3 on HUVEC proliferation were assessed using the EdU assay. Transwell assays were conducted to determine the effects on HUVEC migration, and Matrigel assays were used to evaluate the effects on HUVEC tube formation. Protein kinase B (Akt), phosphorylated Akt (p-Akt), ribosomal protein S6 kinase (S6K), phosphorylated S6K (p-S6K), extracellular regulatory protein kinase (Erk1/2), phosphorylated Erk1/2 (p-Erk1/2) protein expression on HUVEC were detected by Western blot. ResultsCompared with the control group, the relative expression levels of Ntn1, Gαi1, and Gαi3 mRNA and protein in the diabetic group retina were significantly increased, with statistically significant differences (t=11.800, 9.298, 10.620, 7.503, 3.432, 8.037; P＜0.000 1). Compared with the shC group, the relative expression levels of Gαi1 and Gαi3 mRNA and protein in the shGαi1/Gαi3 group in HUVEC were significantly reduced, showing statistically significant differences (t=16.310, 16.300, 13.600, 9.068; P＜0.000 1). HUVEC proliferation rate, migration number and lumen formation number: compared with shC group, shC+NTN1 group significantly increased, while shGαi1/Gαi3 group and shGαi1/Gαi3+NTN1 group significantly decreased, and the differences were statistically significant (F=62.750, 49.830, 54.900; P＜0.000 1). Compared with the control group, the relative expression levels of Gαi1 and Gαi3 mRNA and protein in retina were significantly decreased in the Gαi1/Gαi3 eKD group, showing statistically significant differences (t=10.920, 13.460, 9.219, 10.500; P＜0.000 1). Retinal neovasculogenesis area: compared with the normal control group, the area of retinal neovasculogenesis increased significantly in the NTN1 group, but decreased significantly in the Gαi1/Gαi3 eKD+NTN1 group, with statistical significance (F=24.010, P＜0.000 1). The protein expression of p-Akt relative to Akt, p-S6K relative to S6K, and p-Erk1/2 relative to Erk1/2: compared with shC group, the protein expression of shC+NTN1 group was significantly increased, while that of shGαi1/Gαi3 group and shGαi1/Gαi3+NTN1 group was significantly decreased, with statistical significance (F=78.610, 144.400, 77.010; P＜0.000 1). ConclusionsNTN1 induces Gαi1/Gαi3 to mediate activation of downstream Akt-mammalian target proteins of rapamycin and Erk1/2, thereby promoting angiogenesis in vivo and in vitro environments. Knocking down Gαi1/Gαi3 significantly reduces the NTN1-induced angiogenesis effect.