PURPOSE:To verify existance of a-,~-,and 3'-protein kinase C(PKC)subspecies and their localization in rabbit retina. METHODS: Using an immunohistoehemical technique with mono- elonal antibodies against PKC isozymes- I (a),-I[ (13),and -~[ (Y) to characterize the distribution of PKC in rabbit retina. RESULTS:There is a positive immunostaining for a-,13-,and ~-PKC in rabbit retina. The immunoreactivity of a-PKC was observed mainly in the bipolar cells of inner nuclear layer and the outer segments of photorecptors. The positive immunostaining of 13-PKC could be seen in the ganglion cells,inner plexiform layer,inner nuclear layer,and the outer segments of photoreceptors. A diffuse and weak staining of Y-PKC is recognized in the ganglion cell layer,inner plexifrom layer,inner nuclear layer, and the outer segments of photoreceptors. CONCLUSION:The protein kinase C sub- speeies-a,-~,and-'Y are present in retina which is a part of the central nervous system
ObjectiveTo review the recent progress of the molecular targeted therapy for thyroid cancer. MethodsThe literatures of molecular etiology for thyroid cancer, mechanism and evaluation of targeted therapy via Medline and CHKD database were reviewed. ResultsSo far, four molecular targeted drugs (Sorafenib, Lenvatinib, Vandetanib, and Cabozantinib) have been approved for treatment of advanced thyroid cancer by FDA. They can mainly improve the patient's progression-free survival. Besides, several new molecular targeted drugs have accomplishedⅠphase or Ⅱ phase clinical trials. These drugs may be new options for treatment of advanced thyroid cancer in the future. ConclusionsMolecular targeted drugs have been the main therapeutic method for advanced thyroid cancer. However, we should invent more effective new drugs and investigate the drug combination to improve the therapeutic effect.
Diabetic retinopathy (DR), a neurovascular complication of diabetes, presents a multifaceted pathogenesis that encompasses numerous biological processes and molecular mechanisms. Endoplasmic reticulum stress (ERS) plays a critical role in the maintenance of cellular homeostasis, and diabetic neuro-microangiopathy is driven by high glucose, which activated ERS through the promotion of protein misfolding, oxidative stress, and disturbances in calcium homeostasis. ERS activates the unfolded protein response, thereby influencing the onset and progression of DR through modulating mitochondria-associated endoplasmic reticulum membranes, autophagy, apoptosis, microvascular function, oxidative stress, and inflammation pathways. Currently, the principal interventions against ERS comprise the modulation of the ERS signalling axis and its interactions with associated pathological processes such as autophagy, oxidative stress, and inflammation, through pharmacological and molecular mechanisms. These interventions are directly or indirectly shown to inhibit persistent ERS and are demonstrated to ameliorate DR. With the in-depth study of ERS and the research and development of various drugs for ERS, it is expected to bring novel insights and strategies for DR management in the future.
Objective To investigate the alteration of protein kinase C (PKC) and endothelin system in early diabetic rats, and the effect of specific PKC inhibitor on the expression of retinal endothelin-1 (ET-1). Methods The rats model with streptozotocin(STZ)-induced diabetes were set up. The expression of retinal PKC was detected by enzyme-linked immunoabsorbent assay (ELISA). The expression of retinal ET-1, ET-3, ET-A and ET-B receptor mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The alteration of retinal ET-1 mRNA after intravitreal injection of PKC inhibitor GF109203X in diabetic rats was also observed. Results The activities of membranous PKC were significantly increased in 2-week diabetic rats compared with that in normal rats（t=3.296 , P=0.008）, while activities of cytosolic PKC were unchangeable（t=0.138, P=0.894）. The expression of retinal ET-1 mRNA was significantly increased（P=0.008）, while no change was found in expression of ET-3, ET-A and ET-B mRNA（P=0.918，P=0.889，P=0.500）. After intravitreal in jection of 10-5、10-6、10-7 mol/L PKC inhibitor GF109203X in diabetic rats, the expression of retinal ET-1 mRNA was decreased in a dose-dependent manner compared with the control rats. Conclusion Activation of PKC and increased expression of ET-1 could be found in the retina of early diabetic rats, and PKC inhibitor could inhibit the expression of retinal ET-1. (Chin J Ocul Fundus Dis，2004，20：168-171)
ObjectiveTo verify the expression change of insulin-like growth factor-Ⅰ (IGF-Ⅰ) protein and its mRNA before and after Roux-en-Y gastric bypass surgery (RYGB) in obese rats, and to investigate the relationship between the expression of IGF-Ⅰ and proliferation/apoptosis of adipose cells. Methods① Seventy male SD rats were raised at the SPF level circumstance and were randomly divided into control group (NC group, 10 rats) and high fat diet group (60 rats). Rats of high fat diet group were given specific high fat formula diet, rats of NC group were given particular formula diet. After 6 weeks, the body weights of the rats in high fat diet group were measured, and the 20 rats of top weight were selected. The 20 obese rats were randomly divided into 2 groups:gastric bypass (GB) group (n=10) and sham-operation group (SO group, n=10). RYGB were administered to the rats of GB group, and for rats of SO group, sham operations were performed. Rats of NC group did not receive any surgery. Inguinal adipose tissues[represented the subcutaneous adipose tissue (SAT)] and epididymal adipose tissues[on behalf of visceral adipose tissue (VAT)] were taken during operation in rats of GB group and SO group respectively (0.5 g), and 12 weeks after operation in all rats of three groups. The expressions of IGF-Ⅰ protein and its mRNA in adipose tissue were detected by Western blot and real-time fluorescence quantitative PCR. ② Transfection experiment. SAT cells were divided into blank control group (BC group, without transfection), IGF-Ⅰ(+) group (gene overexpression group), IGF-Ⅰ(+) empty vector group, IGF-Ⅰ(-) group (gene silencing group), and IGF-Ⅰ(-) empty vector group. Cells were transfected with corresponding vectors with 3 duplicated holes of each group. Cell viability and apoptosis assays were carried out in 48 hours after transfection. Expressions of protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), phosphoinositide 3-kinase (PI3K), and phosphorylated phosphoinositide 3-kinase (p-PI3K) were detected by Western blot meanwhile. ③ Wortmannin experiment. SAT cells were divided into Wortmannin (+) IGF-Ⅰ(+) group, Wortmannin (+) IGF-Ⅰ(-) group, Wortmannin (-) IGF-Ⅰ(+) group, and Wortmannin (-) IGF-Ⅰ(-) group, which were transfected with corresponding vectors for 24 hours, then adding Wortmannin (0.1 mmol/L). After 24 hours, the expression levels of AKT, p-AKT, p-PI3K, PI3K, and GAPDH were detected by Western blot. Results① PCR results showed that, in SAT, compared with preoperative GB group, the expression levels of IGF-Ⅰ mRNA and its protein in postoperative GB group were both lower (P < 0.01). However, the expression levels of IGF-Ⅰ mRNA and its protein between preoperative SO group and postoperative SO group showed no significant difference (P > 0.05). In VAT, the expression levels of IGF-Ⅰ mRNA and its protein in 5 groups showed no significant difference (P > 0.05). ② The MTT results showed that, IGF-Ⅰ(+) group harbored stronger proliferation abilities compared with its negative control group (P=0.04), whereas IGF-Ⅰ(-) group had lower abilities compared with its negative control group (P=0.04). The results of flow cytometry assay showed that, the apoptosis rate of IGF-Ⅰ(+) group was lower (P=0.04) than that of the corresponding negative control group, and it was higher in IGF-Ⅰ(-) group than that of the corresponding negative control group (P=0.04). ③ Compared with IGF-Ⅰ(+) empty vector group, p-PI3K/PI3K ratio (P=0.03) and p-AKT/AKT (P=0.04) ratio of IGF-Ⅰ(+) group were increased; compared with IGF-Ⅰ(-) empty vector group, p-PI3K/PI3K ratio (P=0.04) and p-AKT/AKT ratio (P=0.04) of IGF-Ⅰ(-) group were decreased. The p-AKT/AKT ratio of Wortmannin (-) IGF-Ⅰ(+) group was higher (P < 0.05) than that of Wortmannin (+) IGF-Ⅰ(+) group; the p-AKT/AKT ratio of Wortmannin (-) IGF-Ⅰ(-) group was lower than that of Wortmannin (-) IGF-Ⅰ(+) group (P < 0.05). ConclusionsIGF-Ⅰ is involved in the accumulation of subcutaneous fat in rats. RYGB can significantly reduce the expression levels of IGF-Ⅰ mRNA and its protein in subcutaneous fat of rats, so as to achieve the effect of weight loss.
ObjectiveThe purpose of this study was to explore the expression of Corticotropin releasing hormone (CRH), Corticotropin releasing hormone receptor 1 (CRHR1), Protein kinase C (PKC) in epileptogenic zone of Infantile spasm (IS).MethodsCollected 17 cases of tissues of IS patients from operation and 6 cases of normal brain tissues from clinical autopsy during June 2011 to June 2014. Westen blot was used to detected the protein expression of CRH, CRHR1, PKC. PCR was used to exam the mRNA expression of CRH, CRHR1, PKC. Immunohistochemistry and fluorescenceimmuno assay were used to detect the expression of CRH, CRHR1, PKC.ResultsThe mRNA expression of CRH and CRHR1 in IS group are higher than control group, and the protein expression of CRH and CRHR1 in IS group are higher than control group. CRH are slightly expressed in the controls, medium and strong expressed in IS, CRH and NF200 both expressed in IS; CRH is negative in GFAP positive astrocyte; CRH is negative in HLA positive microglial cell. CRHR1 are slightly and medium expressed in the controls, medium and strong expressed in IS, CRHR1 and NF200 both expressed in IS; CRHR1 and GFAP are both positive in astrocyte; CRHR1 and HLA are both positive in microglial cell. PKC are slightly and medium expressed in the controls, medium and strong expressed in IS, PKC and NF200 both expressed in IS; PKC and GFAP are both positive in astrocyte; PKC and HLA are both positive in microglial cell. Spearman analysis showed positive correlation between the expression of CRH, CRHR1, PKC with epileptic spasm in IS patients, as well as positive correlation between PKC with CRHR1.ConclusionsOver expression of CRH, CRHR1, PKC with epileptic spasm in IS patients were positive related with epileptic seizure in IS patients, indicated that CRH signal pathway is related with IS pathogenesis.
Objective To investigate the effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial (RPE) cells and the signal transduction pathway. Methods Reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of syndecan-1 mRNA and protein in normal RPE cells. The expression of syndecan-1 in RPE cells stimulated by different cytokines was detected and quantitatively analyzed by image process of immunofluorescence. The stimulation included 7 and 35 ng/ml tumor necrosis factor (TNF)-alpha; for 24 hours, 1 and 6 mu;g/ml lipopolysaccharide (LPS) for 11 hours, 7 ng/ml TNF-alpha; for 0 to 24 hours (once per 2 hours, and 13 times in total), and 30% supernatant of monocyte/macrophage strain (THP-1 cells) for 3, 14 and 43 hours. The effect of 30% supernatant of THP-1 cells was assayed after pretreated by PD098059［the specific inhibitor of extracellular signal regulated kinase(ERK) 1/2］for 2 hours. After exposed to 30% supernatant of THP-1 cells for 3 hours and treated by 0.25% trypsin for 5 minutes, RPE cells attaching was evaluated by methyl thiazolyl tetrazolium assay. Results In normal human RPE cells, expressions of syndecan-1 mRNA and protein were detected, and b syndecan-1 positive yellowish green fluorescence was found in the cell membrane and cytoplasm while light green fluorescence was in the nucleus. As the concentration and stimulated time of TNF-alpha; or LPS increased, the fluorescence intensity decreased（Plt;0.01）, and after exposed to 30% supernatant of THP-1 cells, weaker fluorescence intensity was detected (Plt;0.001). Pretreatment with 50 mu;mol/L PD098059 for 2 hours partly inhibited the effect of THP-1 cells supernatant. After exposed to 30% supernatant of THP-1 cells for 3 hours, the number of attached cells decreased compared with the controls（Plt;0.05）. Conclusions TNF-alpha; and LPS down-regulate the expression of syndecan-1 in cultured human RPE cells. The supernatant of THP-1 cells down-regulates the expression of syndecan-1 and lessens the cells attaching, which is at least mediated by ERK 1/2 pathway. (Chin J Ocul Fundus Dis, 2006, 22: 113-116)
Objective To investigate the effect of hypericin on the activity of protein kinase C (PKC) in cultured human retinal pigment epithelium (RPE) cells in vitro.Methods RPE cells were cultured in standard medium with 10% serum concentrations containing 0.5 to 5.0 μmol/L hypericin with or without preincubation of phorbol 12-myristate 13-acetate (PMA). The activities of cytosolic PKC (c-PKC) and membranous PKC (m-PKC) were assayed by PKC kit. Results The original activities of c-PKC and m-PKC of RPE cells were (35.34±4.10) pmol·min-1·mg-1and (62.52±8.80) pmol·min-1·mg-1.The activity of c-PKC in RPE cells with PMA preincubation decreased rapidly in 5 minutes, with a subsequent slow decrease after 20 minutes and a decrease to 18% of the activity of c-PKC in RPE cells without PMA preinubation after 60 minutes. While the activity of m-PKC in RPE cells with PMA preincubation increased gradually after 5 minutes and reduced after reached the peak at 40 minutes, and then returned to baseline after 60 minutes, eventually decreased below 30% of the control group. When RPE cells were cultured with PMA for 48 hours, the activities of c-PKC and m-PKC were hardly detectable, while RPE cells were cultured with both PMA and hypericin, hypericin could counteract most of down-regulation by PMA. Conclusion Hypericin may inhibit the translocation of PKC in RPE cells,change the activity of PKC, promote the apoptosis of RPE cells likely,and then prevent proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis，2003，19：55-58)
PURPOSE:To evaluate the activitv of protein kinase C(PKC) in response to retinal photochemical insult in rat. Furthermore, to investigate the effect of dexamethasone(DXM ) on PKC activity. METHODS :The experiments were performed on 48 SI') rats whieh were separated into two groups,control and treated groups,and the latter received daily intraperitoneal injections of DXM (1 mg/kg)for 5 consecutive days,starting 3 days before light exposure. The animals were continually exposed to green fluorescent light (510nm~560nm) with an illuminance level of (1 900plusmn;106.9)lx for 24 hrs.The retinal enzyme activity of PKC was tested at 6 hrs,1 day,3 days,7 days,and 14 days after light exposure respectively. RESULTS:In animal models,PKC activity showed a transient increase in both groups at 6 hrs after light exposure and then decrease persistently there alter. The activity of PKC was unresponsive to DXM intervention. CONCLUSIONS :These results suggested that the persistent lower PKC activity might result in disturbance of retinal function in rat retinal photochemical injury. （Chin J Ocul Fundus Dis,1997,13: 78-80）
Abstract： Objective To study the changes of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) expression of isolated rat hearts after diazoxide preconditioning (DPC), and to explore the possible mechanism of cAMP signaling pathway in myocardial protection by DPC. Methods Isolated working heart Langendorff perfusion models of 40 Wistar rats were set up and were divided randomly into four groups. For the ischemia reperfusion injury(I/R) group (n=10), 30 min of equilibrium perfusion was followed by a 60 min reperfusion of KrebsHenseleit (K-H) fluid. The DPC group (n=10) had a 10 min equilibrium perfusion and two cycles of 5 min of 100 μmol/L diazoxide perfusion followed by a 5 min diazoxidefree period before the 30 min ischemia and the 60 min reperfusion of K-H fluid. The blank control group (control group, n=10) and the Dimethyl Sulphoxide(DMSO) group (n=10) were perfused with the same treatment as in the DPC group except that diazoxide was replaced by natriichloridum and DMSO respectively. The activity of creatine kinase (CK) in coronary outflow, the activity of malonyldialdehyde (MDA) and superoxide dismutase (SOD) in myocardium were detected. And the scope of myocardial infarction and the concentrations of myocardial cAMP and PKA were also assessed. Results Compared with the I/R group, the level of MDA for the DPC group decreased significantly (8.28±2.04 nmol/mg vs. 15.52±2.18 nmol/mg, q=11.761,Plt;0.05), the level of SOD increased significantly (621.39±86.23 U/mg vs. 477.48±65.20 U/mg, q=5.598,Plt;0.05). After a 30 min reperfusion, compared with the I/R group, the content of CK decreased significantly (82.55±10.08 U/L vs. 101.64±19.24 U/L, q=5.598, Plt;0.05) and the infarct size reduced significantly (5.63%±9.23% vs.17.58%±5.76%, q=6.176,Plt;0.05) in the DPC group. The cAMP concentration in the DPC group was much higher than that in the I/R group (0.64±0.07 pmol/g vs. 0.34±0.05 pmol/g, q=14.738,Plt;0.05), and PKA concentration was also much higher than that in the I/R group ［17.13±1.57 pmol/(L·min·mg) vs. 12.85±2.01 pmol/(L·min·mg), Plt;0.05］. However, there were no significant differences between the I/R group, DMSO group and the control group in the above indexs (Pgt;0.05). Conclusion DPC significantly improves the releasing of cAMP and PKA, decreases oxygen free radicals, and relieves myocardial ischemia reperfusion injury. The cAMP signaling pathway may be involved in triggering the process of myocardial protection mechanisms of DPC.