AIMTo investigate the effects of sulfated polymannuroguluronate (SPMG), a novel candidate anti-AIDS drug in Phase II clinical trial, on Tat-induced release of proinflammatory cytokines (i.e., TNFalpha, IL-1beta and IL-6) and its related mechanism.

METHODSThe effects of SPMG on Tat induced TNFalpha (4 h), IL-1beta and IL-6 (6 h) secretion in THP-1 cells were measured by ELISA. Western blotting analysis was used to study the effects of SPMG on Tat induced PKCzeta, PKCtheta and PKCsigma phosphorylation.

RESULTSSPMG (50 to 100 microg x mL(-1)) markedly suppressed TNFalpha, IL-1beta and IL-6 secretion in Tat activated THP-1 cells. In THP-1 cells the phosphorylation levels of PKCzeta, PKCtheta and PKCsigma significantly increased following Tat stimulation, and only PKCsigma phosphorylation levels was inhibited by SPMG (50 to 100 microg x mL(-1)).

CONCLUSIONSPMG suppresses the secretion of proinflammatory cytokines in THP-1 cells may be by inhibiting PKCsigma activation.

Cell Line, Tumor ; Gene Products, tat ; pharmacology ; Humans ; Interleukin-1beta ; secretion ; Interleukin-6 ; secretion ; Isoenzymes ; metabolism ; Phosphorylation ; Polysaccharides ; pharmacology ; Protein Kinase C ; metabolism ; Protein Kinase C-delta ; metabolism ; Protein Kinase C-theta ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVE: To predict the targets and pathways in the therapeutic mechanism of Guizhi Gancao Decoction (GZGCD) against heart failure (HF) based on network pharmacology. METHODS: The chemical components of GZGCD were analyzed using the databases including TCMSP, TCMID and TCM@Taiwan, and the potential targets of GZGCD were predicted using the SwissTargetPrediction database. The targets of HF were obtained using the databases including DisGeNET, Drugbank and TTD. The intersection targets of GZGCD and HF were identified using VENNY. Uniport database was used to convert the information, and the components-targets-disease network was constructed using Cytoscape software. The Bisogene plug-in, Merge plug-in, and CytoNCA plug-in in Cytoscape software were used for protein-protein interaction (PPI) analysis to acquire the core targets. Metascape database was used for GO and KEGG analysis. The results of network pharmacology analysis were verified with Western blot analysis. Three factors (PKCα, ERK1/2 and BCL2) were screened according to the degree value of network pharmacology results and the degree of correlation with heart failure process. The pentobarbtal sodium was dissolvein H9C2 cells treated with serum-free high glucose medium to simulate the ischemic anoxic environment of heart failure. The total proteins of myocardial cells were extracted. The protein contents of PKCα, ERK1/2 and BCL2 were determined. RESULTS: We identified a total of 190 intersection targets between GZGCD and HF using Venny database, involving mainly the circulatory system process, cellular response to nitrogen compounds, cation homeostasis, and regulation of the MAPK cascade. These potential targets were also involved in 38 pathways, including the regulatory pathways in cancer, calcium signal pathway, cGMP-PKG signal pathway, and cAMP signal pathway. Western blot analysis showed that in an in vitro H9C2 cell model of HF, treatment with GZGCD downregulated PKCα and ERK1/2 expressions and upregulated BCL2 expression. CONCLUSION The therapeutic mechanism of GZGCD for HF involves multiple targets including PRKCA, PRKCB, MAPK1, MAPK3, and MAPK8 and multiple pathways including the regulatory pathway in cancer and the calcium signaling pathway.
Humans ; Protein Kinase C-alpha ; Network Pharmacology ; Heart Failure/drug therapy* ; Proto-Oncogene Proteins c-bcl-2

OBJECTIVETo study the target killing effect of soluble Fas(sFas) coupled with protein kinase C(PKC) inhibitor on colorectal carcinoma cells.

METHODSThe extracellular region of Fas protein was cloned and amplified by RT-PCR, and the expressing vector pGEX-4T-1-sFas was constructed. The sFas protein was purified by GST fusion protein purification system and coupled with Calphostin C(one kind of PKC inhibitor). The killing effect of soluble Fas coupled with PKC inhibitor on FasL-positive colorectal carcinoma cells was detected.

RESULTSAfter amplifying and cloning, the extracellular region of Fas protein, a 571 bp fragment, was proved by limited enzyme cutting and DNA sequencing. The expressed and purified protein was identified by Western Blot after transformed into E. coli BL21. The coupled sFas-Calphostin C showed suppressant activity on PKC kinase by the PKC kinase activity assay kit. The growth suppression rate of FasL-positive colorectal carcinoma HR-8348 cells treated with sFas-Calphostin C was significantly higher than that of FasL-negative cells, but the killing effect of sFas-Calphostin C on normal human monocyte was not obvious. Compared with 5-Fu alone, the growth suppression rate of FasL-positive colorectal carcinoma HR-8348 cells was significantly raised by sFas-Calphostin C combined with 5-Fu.

CONCLUSIONThe recombinant of soluble Fas and PKC inhibitor shows target killing effect on colorectal carcinoma cells.

Colorectal Neoplasms ; therapy ; Fas Ligand Protein ; pharmacology ; Humans ; Naphthalenes ; pharmacology ; Protein Kinase C ; antagonists & inhibitors ; Recombinant Proteins ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

OBJECTIVETo determine effects of activating protein kinase C (PKC) on ventricular action potential duration restitution (APDR) and Burst stimulus induced arrhythmia in Langendorff-perfused rabbit hearts.

METHODSMale rabbits were equally divided into three groups randomly: control group (Tyrode's solution perfusion), PKC agonist phorbol-12-myristate-13-acetate (PMA, 100 nmol/L) group and PKC inhibitor bisindolylmaleimide (BIM, 500 nmol/L) group. Thirty minutes after perfusion, the monophasic action potential (MAP) and effective refractory period (ERP) were determined in right basal ventricle (RB), right apex (RA), left basal ventricle (LB) and left apex (LA) of all the animals, and APDR curve was drawn. Burst stimulus method was used to induce ventricular arrhythmia in perfused rabbit hearts; Real-time PCR was used to detect the mRNA expression of PKC in four different areas of ventricle.

RESULTSCompared with the control group, the ERP, 90% of monophasic action potential duration (MAPD(90)) and ERP/MAPD(90) were significantly shortened (all P < 0.01), the max slopes (S(max)) of APDR curve were significantly steeper (RB: 1.22 ± 0.23 vs. 0.65 ± 0.19; RA: 2.99 ± 0.29 vs. 1.02 ± 0.18; LB: 1.84 ± 0.21 vs. 0.85 ± 0.12; LA: 4.02 ± 0.32 vs.1.12 ± 0.23, all P < 0.01) and the incidences of ventricular arrhythmia were significantly increased in the PMA group. All parameters were similar between the BIM group and the control group (all P > 0.05).

CONCLUSIONActivating PKC could enhance the max slopes of APDR curve at various ventricular areas and subsequently increase arrhythmia susceptibility in Langendorff-perfused rabbit hearts.

Action Potentials ; Animals ; Arrhythmias, Cardiac ; physiopathology ; Heart ; drug effects ; physiopathology ; Male ; Protein Kinase C ; pharmacology ; Rabbits

OBJECTIVETo observe the role of PKC-potentiated inhibitory protein for protein phosphatase 1 of 17 x 10(3) (CPI-17) in vascular calcium sensitivity regulated by protein kinase Calpha (PKCalpha) and Cepsilon (PKCepsilon) in rats with hemorrhagic shock (HS).

METHODSEight Wistar rats were used to reproduce 2 h HS model. Superior mesenteric artery (SMA) rings from HS rats were randomly divided into 2 h shock group (without treatment), PKCalpha agonist group (with addition of thymelea toxin into the nutrient solution), CPI-17 antibody + PKCalpha agonist group [incubation with thymelea toxin and CPI-17 antibody (1:800)], PKCepsilon agonist group (with addition of carbachol into the nutrient solution), and CPI-17 antibody + PKCepsilon agonist group [incubation with carbachol and CPI-17 antibody (1:800)]. SMA rings from another eight normal rats were used as normal control group. Calcium sensitivity indices (Emax, pD2) of SMA rings were measured by isolated organ perfusion system. Hypoxic VSMCs in primary culture were randomly divided into 2 h hypoxia group, PKCalpha agonist group (with above-mentioned treatment), PKCepsilon agonist group (with above-mentioned treatment), normal VSMCs were used as normal control group. Protein expression and phosphorylation of CPI-17 were measured via Western blot.

RESULTSEmax and pD2 in all the experimental groups were lower than those in normal control group (P < 0.01). Emax in PKCalpha agonist group and PKCepsilon agonist group was increased (5.8 +/- 0.8, 5.8 +/- 0.9 mN, respectively) as compared with that of 2 h shock group (4.1 +/- 0.6 mN, P < 0.01). Protein expression and phosphorylation of CPI-17 in VSMC were significantly decreased in 2 h hypoxia group, compared with those in normal control group (P < 0.05), and those in PKCalpha agonist and PKC agonist groups (P < 0.05 or P < 0.01).

CONCLUSIONSPKCalpha and PKCepsilon may regulate vascular calcium sensitivity through change in protein expression and activity of CPI-17 in HS rats.

Animals ; Calcium ; blood ; pharmacology ; Female ; Male ; Muscle Proteins ; metabolism ; Phosphoproteins ; metabolism ; Phosphorylation ; Protein Kinase C-alpha ; metabolism ; Protein Kinase C-epsilon ; metabolism ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; metabolism

AIMTo investigate the effect of ciliary neurotrophic factor (CNTF) on the nuclear translocation of protein kinase C (PKC) following NMDA administration in the primary cultured hippocampal neurons.

METHODS(1) PKCGAMMA or PKCepsilon- immunocytochemistry staining method was used after treating neurons with NMDA or CNTF. (2) The gray of the nucleus of the PKC-positive neurons were measured under the image pattern analysis system.

RESULTS(1) After NMDA administration of different concentration and time, Nucleus appear PKCgamma and PKCepsilon activities, especially the 100 micromol/L NMDA 30 min group. (2) The gray of nucleus in CNTF + 500 micromol/L NMDA group is similar to control group.

CONCLUSIONNMDA can induce nuclear translocation of PKC in the primary cultured hippocampal neurons, and CNTF can inhibit the translocation. It suggests that the inhibition of PKC translocation induced by NMDA is one of the important reasons for the neuro-protective effects of CNTF.

Animals ; Cells, Cultured ; Ciliary Neurotrophic Factor ; pharmacology ; Hippocampus ; cytology ; N-Methylaspartate ; pharmacology ; Neurons ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Protein Transport ; Rats ; Rats, Sprague-Dawley

OBJECTIVESerum pregnancy-associated plasma protein A (PAPP-A) is increased in acute coronary syndrome patients and related to prognosis. We investigated the effects of C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-alpha) on PAPP-A mRNA expression in monocytes.

METHODSMonocytes were isolated by Ficoll density gradient centrifugation from blood of healthy volunteers. The PAPP-A expressions at mRNA level post CRP or rhTNF-alpha stimulation were measured by RT-PCR.

RESULTSPAPP-A mRNA expression in peripheral blood monocytes increased 2 hours (0.2128 +/- 0.0136) and peaked 24 hours (0.6837 +/- 0.1360) after CRP (20 mg/L) stimulation compared with control group (0.1842 +/- 0.0101). PAPP-A mRNA expression increased rapidly, peaked 2 hours (1.2546 +/- 0.0866) and remained elevated up to 24 hours (0.8203 +/- 0.0413) after rhTNF-alpha (100 ng/ml) stimulation. The effects of CRP and TNF-alpha were dose-dependent. PAPP-A mRNA expression of monocytes were 0.2544 +/- 0.0611, 0.4177 +/- 0.1200, 0.5828 +/- 0.0152, 0.6837 +/- 0.1360 after stimulated with CRP (1, 5, 10, 20 mg/L), and 0.2424 +/- 0.1378, 0.3335 +/- 0.0196, 0.5742 +/- 0.0131, 0.6913 +/- 0.0219 and 0.8203 +/- 0.0413 after stimulated with rhTNF-alpha (5, 10, 25, 50 and 100 ng/ml). Actinomycin D, the DNA-directed RNA polymerase inhibitor, completely blocked CRP and TNF-alpha induced PAPP-A expression.

CONCLUSIONSPAPP-A mRNA expression could be stimulated by CRP and TNF-alpha in human peripheral blood monocytes which might be responsible for the increased serum PAPP-A level in patients with acute coronary syndromes.

C-Reactive Protein ; adverse effects ; pharmacology ; Cells, Cultured ; Humans ; Monocytes ; drug effects ; metabolism ; Pregnancy-Associated Plasma Protein-A ; metabolism ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

The previous study indicated that aquaporin 4 (AQP4) deficiency attenuated opioid physical dependence. However, the underlying mechanism remains unknown. In the present study, the effects of AQP4 deficiency on the expression of three factors, protein kinase C (PKC) α, PKCγ and c-Fos in the spinal cord, which are known to be concerned with spinal neuronal sensitization and opiate dependence, were investigated in AQP4 knockout mice using Western blotting analysis. It was observed that AQP4 deficiency reduced the score of naloxone-precipitated abstinent jumping after repeated morphine administration compared with wild-type (P < 0.001). Meanwhile, the protein levels of PKCα and c-Fos in the spinal cord of AQP4 knockout mice were significantly higher than those in the wild-type mice; while the expression of PKCγ was decreased remarkably by AQP4 knockout during the withdrawal (P < 0.01). These data suggest that AQP4 deficiency-attenuated morphine withdrawal responses may be partially attributed to the changes in the spinal expression of PKCα, PKCγ or c-Fos.
Analgesics, Opioid ; pharmacology ; Animals ; Aquaporin 4 ; deficiency ; genetics ; Mice ; Mice, Knockout ; Morphine ; pharmacology ; Naloxone ; pharmacology ; Protein Kinase C ; metabolism ; Protein Kinase C-alpha ; metabolism ; Spinal Cord ; metabolism ; Substance Withdrawal Syndrome ; metabolism

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
A549 Cells ; Adenosine Diphosphate Ribose/pharmacology* ; Apoptosis ; Apoptosis Regulatory Proteins ; Arsenic ; Arsenites ; Cacodylic Acid/pharmacology* ; Caspase 3 ; Caspases/pharmacology* ; Cytochromes c/pharmacology* ; Humans ; Mitochondrial Proteins/pharmacology* ; Poisons ; Propidium/pharmacology* ; RNA, Messenger ; Sincalide/pharmacology* ; Sodium Compounds ; bcl-Associated Death Protein/metabolism*

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estrogen Antagonists ; pharmacology ; Glioma ; pathology ; Humans ; Protein Kinase C ; antagonists & inhibitors ; Tamoxifen ; pharmacology