The antioxidant effect of preischemic dexmedetomidine in a rat model: increased expression of Nrf2/HO-1 via the PKC pathway

Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.

Mechanism of berberine reverses drug resistance of K562/A02 cells by down-regulating protein kinase C-alpha / 国际中医中药杂志

Objective:To observe the effect of berberine on leukemia drug-resistant cell strain K562/A02 to Adriamycin resistance and protein kinase C-alpha (PRKCA) and explore its possible mechanism.Methods:The leukemia K562 cells of human chronic myeloid and Adriamycin resistant strain K562/A02 were cultured in vitro with 2.5-50.0 μmol/L doxorubicin to treat thoese cells and drug resistance of K562 and K562/A02 to Adriamycin was detected, the 50% inhibitory concentration (IC 50) of the drug was calculatedthe resistance of K562 and K562/A02 to doxorubicin was detectd , and, K562/A02 cells were treated with doxorubicin solution at a final concentration of 5 μmol/L, and K562/A02 cells were divided into control group, inhibitor group (50 μmol/L PRKCA inhibitor), low dose berberine group, medium dose berberine group and high dose berberine group. Cell counting (CCK-8) method was used to detect the inhibition rate of cell proliferation, the apoptosis was detected by flow cytometry, real-time fluorescent quantitative PCR assay detects PRKCA, MRP, multidrug resistance related genes (MDR1) levels, and the protein expressions of protein kinase C-α (PRKCA), multidrug resistance related protein (MRP), P-glycoprotein (P-gp) were detected by Western blotting. Results:The IC 50 concentration of K562/A02 to Adriamycin was significantly higher than K562. Compared with the control group, the inhibition rate of cell proliferation and the apoptosis rate in the inhibitor group, low-dose berberine group, medium-dose berberine group, and high-dose berberine group were significantly increased ( P<0.05), the expression of PRKCA mRNA (0.45±0.08, 0.92±0.10, 0.57±0.05, 0.35±0.04 vs. 1.00±0.12), MDR1 gene (0.73±0.08, 0.87±0.09, 0.65±0.07, 0.41±0.05 vs. 1.00±0.11) and PRKCA (0.59±0.09, 0.78±0.12, 0.61±0.11, 0.42±0.07 vs. 0.96±0.14), MRP (0.62±0.08, 0.79±0.13, 0.62±0.10, 0.41±0.06 vs. 0.98±0.14), P-gp (0.55±0.08, 0.75±0.12, 0.59±0.09, 0.35±0.06 vs. 0.92±0.15) were significantly reduced ( P<0.05), and berberine was dose-dependent ( P<0.05); Overexpression of PRKCA can inhibit the effect of berberine on reversing the drug resistance of K562/A02 cells. Conclusion:Berberine may reverse the drug resistance of K562/A02 to Adriamycin by down-regulating PRKCA.

Effects of dexmedetomidine on alveolar epithelial barrier function in rats with VILI and the role of PKC / 中华麻醉学杂志

Objective:To evaluate the effects of dexmedetomidine on alveolar epithelial barrier function in rats with ventilator-induced lung injury (VILI), and the role of protein kinase C (PKC).Methods:One hundred clean-grade male Sprague-Dawley rats, weighing 270-320 g, aged 4-5 months, were divided into 5 groups ( n=20 each) using a random number table method: control group (group C), VILI group (group V), PKC inhibitor group (group B), dexmedetomidine group (group D), and dexmedetomidine plus PKC agonist group (DP group). The VILI model was developed by mechanical ventilation with a tidal volume of 40 ml/kg for 4 h in anesthetized animals.Group C breathed air autonomously for 4 h without mechanical ventilation.Group V was mechanically ventilated for 4 h. In group B, bisindolvlmaleimide I 0.12 mg/kg was injected intramuscularly 1 h before mechanical ventilation.In D and DP groups, dxmedetomidine 5.0 μg/kg was injected intravenously at 20 min before mechanical ventilation, and dexmedetomidine was intravenously infused at the rate of 5.0 μg·kg -1·h -1 during mechanical ventilation.In group DP, PKC agonist phorbol-12-myristic acid-13-acetate 15 μg/kg was intraperitoneally injected at 30 min before mechanical ventilation.At 4 h of mechanical ventilation, oxygenation index (OI), lung permeability index (LPI) and wet/dry lung weight (W/D) ratio were measured, the pathological changes of lung tissues were observed, and lung injury was assessed and scored.The expression of PKC, occludin and ZO-1 protein was detected by Western blot, and the expression of PKC mRNA, occludin mRNA and ZO-1 mRNA was determined by real-time polymerase chain reaction. Results:Compared with group C, OI was significantly decreased, LPI, W/D ratio and lung injury score were increased, the expression of PKC protein and mRNA was up-regulated, and the expression of occludin and ZO-1 protein and mRNA was down-regulated in V and DP groups ( P<0.05), and no significant change was found in the parameters mentioned above in B and D groups ( P>0.05). Compared with group V, OI was significantly increased, LPI, W/D ratio and lung injury score were decreased, the expression of PKC protein and mRNA was down-regulated, and the expression of occludin and ZO-1 protein and mRNA was up-regulated in B, D and DP groups ( P<0.05). Compared with group D, OI was significantly decreased, LPI, W/D ratio and lung injury score were increased, the expression of PKC protein and mRNA was up-regulated, and the expression of occludin and ZO-1 protein and mRNA was down-regulated in group DP ( P<0.05). Conclusions:Dexmedetomidine can reduce the damage to alveolar epithelial barrier function in rats with VILI, and the mechanism is related to inhibition of PKC activation and up-regulation of the expression of occludin and ZO-1.

Bitter gourd extract improves glucose homeostasis and lipid profile via enhancing insulin signaling in the liver and skeletal muscles of diabetic rats

Objective: To investigate the modulatory effects of bitter gourd extract on the insulin signaling pathway in the liver and skeletal muscle tissues of diabetic rats. Methods: The ethanolic extract of bitter gourd was prepared and its contents of total polyphenols and flavonoids were assayed. A neonatal streptozotocin-induced diabetic rat model was established and the diabetic rats were assigned into different groups and were treated with different doses of bitter gourd extract (100, 200, 400, or 600 mg/kg) or with glibenclamide (0.1 mg/kg) for 30 d. Fasting blood glucose, insulin, and lipid profile were evaluated and the insulin signaling pathway in the liver and skeletal muscle of rats was investigated. The correlations between homeostasis model assessment (HOMA) and the components of insulin signaling pathway were also evaluated. Results: Different doses of bitter gourd extract significantly ameliorated fasting blood glucose level and HOMA index for insulin resistance. Moreover, bitter gourd extract increased serum insulin and improved disrupted serum lipid profile. The levels of insulin receptor substrate-1 (IRS-1), p-insulin receptor β (p-IR-β), protein kinase C (PKC), GLUT2, and GLUT4 were improved by treatment with bitter gourd extract. The best results were obtained with 400 mg/kg dose of the extract, the effect of which was equivalent to that of glibenclamide. HOMA in the bitter gourd treated rats was negatively correlated with p-IR-β, IRS-1 and PKC in hepatic and skeletal muscle. HOMA was also negatively correlated with skeletal muscle GLUT4. Conclusions: Bitter gourd extract improves glucose homeostasis and lipid profile in diabetic rats via enhancement of insulin secretion and sensitivity. Therefore, bitter gourd can be used as a potential pharmacological agent for the treatment of type 2 diabetes mellitus.

Effects of Wp-PKC on gonadal development of Whitmania pigra / 中国中药杂志

Protein kinase C(PKC) is a kind of kinase which is widely involved in cell proliferation and development. PKC(Wp-PKC) in Whitmania pigra body belongs to classic PKC. In order to investigate the effect of Wp-PKC on the development of Wh. pigra germ cells, 17β-estradiol(17β-E2)(100 ng·mL~(-1)) and methyltestosterone(MT)(150 μg·L~(-1)), 150 μg·L~(-1)(MT)+0.5 mg·L~(-1) PKC, 0.5 mg·L~(-1) PKC inhibitor were added to Wh. pigra culture water, and no addition group(control group) was added, and the effects on the development of Wh. pigra germ cells and the expression of Wp-PKC were observed. The results showed that: Wp-PKC in male gonads was always higher than that in female gonads; MT promoted the development of male gonads in Wh. pigra, while the expression of Wp-PKC was significantly higher than that in the control; 17β-E2 promoted the development of female gonads in Wh. pigra and Wp-PKC expression significantly lower than that of the control; while the development of the female and male gonads in the PKC inhibitor group was inhibited, the expression of Wp-PKC was significantly lower than that of the control. In summary, Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads.

A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium / 中国天然药物

Cudrania tricuspidata Bureau (CTB), a species of the Moraceae plant, has been used as a bruise recovery treatment. This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease (IBD). We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C. CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB, which are responsible for the expression of cell-cycle-related proteins (CDK2, CDK4, cyclin D1 and cyclin E) during its promotion of cell proliferation. Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4% (W/V) for seven days. We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score, which was composed of body weight change, diarrhea, and hematochezia in ICR mice treated with dextran sulfate sodium. Hence, we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC, JNK and NF-κB in colon epithelial cells.

Cloning and expression analysis of protein kinase C gene from Whitmania pigra / 中国中药杂志

Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

Expression and localization of diacylglycerol kinase zeta and protein kinase C beta II in mouse back skin with different coat colors / 中国组织工程研究

BACKGROUND: In the skin and hair pigmentation mechanism, diacylglycerol kinase ζ (DGKζ) has not been reported to participate in pigmentation. OBJECTIVE: To investigate the expression and localization of DGKζ and protein kinase C βII (PKCβII) in the mouse back skin with different coat colors and their correlation with the formation of coat color. METHODS: The back skin of 2-month-old mice with different coat colors (white, gray, and black, 6 mice for each color) was taken to detect DGKζ and PKCβII mRNAs by real-time PCR, DGKζ, PKCβII and p-PKCβII proteins by western blot, and DGKζ and p-PKCβII expression by immunohistochemistry. The study protocol was approved by the Animal Ethics Committee of the Animal Experimental Center of Shanxi Medical University. RESULTS AND CONCLUSION: DGKζ and PKCβII mRNAs were both expressed in the back skin of mice with three kinds of coat colors, with its relative expression amount being: gray group > white group; black group > white group (P white group; black group > white group (P < 0.05). Immunohistochemistry staining showed that DGKζ and p-PKCβII were positively expressed in the outer root sheath of hair follicle, hair matrix cells and melanocytes in the hair matrix in the back skin of gray and black mice, while were negative in the hair follicles of white mice. According to the expression and localization of DGKζ, PKCβII in white, gray and black mouse back skin, we can speculate that DGKζ and PKCβII may participate in the formation of the coat color.

Regulatory effect of metabolic glutamate receptor 1 on maltolate aluminum-induced synaptic plasticity in rats / 中国职业医学

OBJECTIVE: To observe the effect of maltolate aluminum on synaptic plasticity in the hippocampus of rats and to explore the regulatory effect and mechanism of metabotropic glutamate receptor 1(mGluR1). METHODS: Specific pathogen free healthy adult male SD rats were randomly divided into control group, aluminum group, aluminum agonist group and aluminum antagonist group, 8 rats in each group. The rats in the control group received no treatment; the rats in aluminum group were injected with 5 μL 10 mmol/L maltolate aluminum solution into the lateral ventricle; the rats in aluminum agonists and aluminum antagonist group were injected with 3 μL 10 mmol/L maltolate aluminum solution plus 2 μL 0.1 μmol/L mGluR1 agonist or 2 μL 0.2 μmol/L mGluR1 antagonists into the lateral ventricle, respectively.Maltolate aluminum solution was injected every 2 days and continued for 10 days. After maltolate aluminum exposure, the amplitudes of long-term potentiation(LTP) in hippocampal CA1 region of rats were measured, and the relative expression levels of mRNA and protein of mGluR1, N-methyl-D-aspartate receptor(NMDAR1) and protein kinase C(PKC) in hippocampus tissue of rats were detected by real-time fluorescence quantitative polymerase chain reaction and Western blotting. RESULTS: The amplitude of LTP in hippocampal CA1 region in aluminum group and aluminum agonist group was lower than that in the control group and the aluminum antagonist group(P<0.05). Compared with the control group, the relative expression of mGluR1 mRNA and protein in the aluminum group increased, the relative expression of PKC and NMDAR1 mRNA and protein in the aluminum group decreased(P<0.05). Compared with the aluminum group, the relative expression of mGluR1 mRNA and protein in the aluminum agonist group increased, while the NMDAR1 mRNA decreased(P<0.05); the relative expression of mGluR1 mRNA and protein in the aluminum antagonist group decreased, while the NMDAR1 mRNA and protein increased(P<0.05). Compared with the aluminum agonist group, the relative expression of mGluR1 mRNA and protein decreased, while the NMDAR1 mRNA and protein increased in the aluminum antagonist group(P<0.05). The relative expression level of PKC mRNA and protein in aluminum agonist group and aluminum antagonist group was not statistically significant(P>0.05), and there was no statistical significance in these two groups compared with control group and aluminum group(P>0.05). CONCLUSION: Maltolate aluminum exposure can inhibit synaptic plasticity by inhibiting LTP in hippocampus of rats, and the mechanism may be related to the regulation of NMDAR1 expression by mGluR1.

Effect of ginkgo biloba L. extract on oxidative stress and activity of protein kinase C in spleen tissue of mice with irradiation damage / 吉林大学学报(医学版)

Objective: To investigate the influence of Ginkgo biloba L. extract (GBE) in the expressions of oxidative damage-related proteins in the spleen tissue of the mice with radiation damage, and to clarify its mechanism. Methods: Sixty ICR mice were randomly divided into normal control (N O group, irradiation control (IC) group (4. 0 Gy y-ray radiation), low dose of GBE (IC+GBEL) group (4. 0 Gy y-ray+5. 0 mg • kg-1 GBE), medium dose of GBE (IC + GBEM) group (4.0 Gy y-ray+10. 0 mg • kg-1 GBE) and high dose of GBE (IC + GBEH) group (4. 0 Gy y-ray+20. 0 mg • kg_ 1GBE) (n = 1 2) . After continuous administration of GBE for 14 d, all mice received the whole body radiation of y-ray with the total dose of 4. 0 Gy on the 15th day except for the mice in NC group. The levels of malonaldehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px) in the spleen tissue of the mice in various groups were measured by biochemical method 24 h after radiation. The expression levels of 8-hydroxy-2 deoxyguanosine 8-OHdG) in the spleen tissue of the mice in various groups were examined by immunohistochemistry. The activities of protein kinase C (PKC) in the spleen tissue of the mice in various groups were detected by ELISA. Results: Compared with NC group, the levels of MDA and 8-OHdG in the spleen tissue of the mice in IC and different doses of GBE groups were significantly increased (P < 0 . 05), and the activities of SOD, CAT, GSH-px and PKC were significantly decreased (P < 0 . 05). Compared with IC group, the levels of MDA and 8-OHdG in the spleen tissue of the mice in different doses of GBE groups were significantly decreased (P < 0 . 05), and the activities of SOD, CAT, GSH-px and PKC were significantly increased (P < 0 . 05). Conclusion: GBE can inhibit the oxidative stress and regulate the PKC activity in the spleen tissue of the mice with irradiation damage to play a protective role against the radiation damage.

Research progress of protein kinase C in liver diseases / 中国医师杂志

Protein kinase C (PKC) is a special kinase widely distributed in various tissues and cells of human body and involved in signal transduction of hormones and cytokines.It plays an important role in the pathogenesis of many diseases.Therefore,altering the activity of protein kinase C may be an effective treatment for many diseases.This article reviews the progress of protein kinase C in liver diseases.

Protective effect of protein kinase C inhibitor on rat renal vascular endothelial injury induced by lipopolysaccharide / 中华危重病急救医学

Objective To investigate the protective effect of protein kinase C (PKC) inhibitor rottlerin on rat renal vascular endothelial injury induced by lipopolysaccharide (LPS). Methods Rat renal microvascular endothelial cells cultured for 3-6 generations were divided into three groups according to random number table: blank control group in which cells were not challenged, LPS group in which cells were only stimulated by LPS 10 mg/L for 24 hours, and PKC inhibitor group in which cells were treated with PKC inhibitor rottlerin 2 μmol/L 30 minutes before LPS stimulation. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-1β, IL-8) were determined by enzyme-linked immunosorbent assay (ELISA). Monolayer permeability was determined by Transwell assay. The expressions of PKC, RhoA and vascular endothelial-cadherin (VE-cadherin) were detected by Western Blot. The morphological characteristic and distribution of F-actin was measured by laser confocal fluorescence microscope. Results Compared with blank control group, the levels of inflammatory cytokines at 24 hours after 10 mg/L LPS stimulation were significantly increased in LPS group [TNF-α (ng/L): 397.3±25.4 vs. 46.8±8.9, IL-1β(ng/L): 76.7±11.2 vs. 12.6±3.2, IL-8 (ng/L): 574.5±31.4 vs. 73.2±9.6, all P < 0.05], the permeability of endothelial cells was significantly increased (A value: 1.32±0.03 vs. 0.36±0.02, P < 0.05), while the expressions of PKC and RhoA were significantly up-regulated (PKC/β-actin: 0.88±0.02 vs. 0.61±0.03, RhoA/β-actin: 0.96±0.01 vs. 0.49±0.03, both P < 0.05), VE-cadherin expression was significantly down-regulated (VE-cadherin/β-actin: 0.51±0.01 vs. 0.72±0.04, P < 0.05), and the F-actin distribution disorder had obvious stress fiber formation. Compared with LPS group, the levels of inflammatory cytokines were significantly lowered in PKC inhibitor group [TNF-α (ng/L): 127.4±14.6 vs. 397.3±25.4, IL-1β(ng/L): 43.2±7.8 vs. 76.7±11.2, IL-8 (ng/L): 212.7±18.2 vs. 574.5±31.4, all P < 0.05], the permeability of endothelial cells was significantly decreased (A value: 0.81±0.02 vs. 1.32±0.03, P < 0.05), the expressions of PKC and RhoA were significantly down-regulated (PKC/β-actin: 0.44±0.03 vs. 0.88±0.02, RhoA/β-actin: 0.63±0.05 vs. 0.96±0.01, both P < 0.05), the VE-cadherin expression was significantly up-regulated (VE-cadherin/β-actin: 0.69±0.03 vs. 0.51±0.01, P < 0.05), and the F-actin remodeling and stress fiber formation were significantly reduced. Conclusion PKC inhibitor could significantly attenuate the damage of vascular endothelial barrier induced by LPS, and plays an important role in endothelial cell barrier.

Role of PI3K/Akt signaling pathway in propofol-induced inhibition of migration and invasion ability of human non-small cell lung cancer H1975 cells / 中华麻醉学杂志

Objective To evaluate the role of phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt) signaling pathway in propofol-induced inhibition of migration and invasion ability of human nonsmall cell lung cancer H1975 cells.Methods H1975 cells were divided into 4 groups (n=36 each) using a random number table method:control group (group C),20 μg/ml propofol group (group P),0.5 ng/ml PI3K/Akt signaling pathway activator insulin-like growth factor 1 (IGF-1) group (group IGF-1),and 20 μg/ml propofol plus 0.5 ng/ml IGF-I group (group P+IGF-1).The migration and invasion ability of H1975 cells was determined by wound healing assay and Transwell invasion assay,respectively.The expression of phosphorylated Akt (p-Akt) and matrix metalloproteinase-9 (MMP-9) was assessed by Western blot.Results Compared with group C,and the ability of migration and invasion was significantly reduced,and the expression of p-Akt and MMP-9 was down-regulated in group P,and the ability of migration and invasion was significantly enhanced,and the expression of p-Akt and MMP-9 was up-regulated in group IGF-1 (P＜0.05).Compared with group P,the ability of migration and invasion was significantly enhanced,and the expression of p-Akt and MMP-9 was up-regulated in group P+IGF-1 (P＜0.05).Compared with group IGF-1,the ability of migration and invasion was significantly reduced,and the expression of p-Akt and MMP-9 was down-regulated in group P+IGF-1 (P＜0.05).Conclusion The mechanism by which propofol inhibits migration and invasion ability of human non-small cell lung cancer H1975 cells is related to blocking PI3K/Akt signaling pathway.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.

Effect of Catgut Implantation on Spatial Learning-memory Ability, Expression of Hippocampal Protein Kinase C Interacting Protein 1 and GluR 2 and Ca2+ Content in Rats with Chronic Ischemic Cognitive Impairment / 针刺研究

OBJECTIVE: To observe the effect of catgut embedment at "Baihui" (GV 20), "Dazhui" (GV 14), etc. on learning-memory ability, expression of hippocampal protein kinase C interacting protein 1 (PICK 1) and glutamate receptor 2 (GluR 2) proteins and level of calcium ions, so as to explore its mechanism underlying improvement of vascular cognitive impairment. METHODS: A total of 56 male SD rats were randomly divided into sham operation, model, catgut embedment and medication groups (n=14 in each). The chronic ischemic cognitive impairment model was established by permanent occlusion of bilate-ral common carotid arteries. The catgut embedment was applied to GV 20, GV 14, "Shenshu" (BL 23) and "Xuanzhong" (GB 39), once a week, for 4 weeks. Rats of the medication group received intraperitoneal injection of monosialate tetrahexose ganglioside sodium (GM-1, 0.33 mg/kg), once daily for 4 weeks. The rats' learning-memory ability was detected by Morris water maze tasks, pathological changes of hippocampal Nissl's bodies were tested by Nissl staining. The expression levels of PICK 1 and GluR 2 proteins in the hippocampus were detected by Western bolt (WB), and the concentration of calcium ions in the hippocampus tissue was measured by Bicinchoninic acid (BCA) assay. RESULTS: After modeling, the mean escape latencies of place navigation test were significantly increased while the crossing times of target platform quadrant of space probing test notably decreased in the model, catgut embedment and medication groups compared with their own individual pre-modeling (P0.05). CONCLUSION: Catgut implantation at GV 20 etc. can effectively improve the learning-memory ability in rats with chronic ischemic cognitive impairment, which may be related to its effects in down-regulating the expression of PICK 1 and calcium ion concentration and up-regulating the expression of AMPA receptor subunit GluR 2 protein in the hippocampus.

The regulation of tight junction protein via PKCα/β for abnormal permeability of brain microvascular endothelial cells exposed to paraquat / 中华劳动卫生职业病杂志

Objective@#To explore if conventional protein kinase C (cPKC: PKCα and PKCβ) contributes to paraquat (PQ) -induced abnormal permeability of mouse brain microvascular endothelial cells (BMECs) via the regulation of tight junction (TJ) proteins.@*Methods@#The immortalized mouse brain endothelial cell line (bEnd.3) was used to establish a monolayer blood-brain barrier (BBB) model. In order to evaluate the function of the in vitro BBB model, the transendothelial electrical resistance (TEER) and permeability were measured by a Millicell-ERS volt-ohmmeter and sodium fluorescent (Na-FLU) , respectively. MTT assay was used to determine the relative survival rate of cells. The dose-response relationship was determined by treating cells with 0, 50, 100, 200, and 300 μmol/L PQ for 24 hours. The time-response relationship was determined by treating cells with 200 μmol/L PQ for 6, 12, 24, 48, and 72 hours. After the treatment of cells with 0, 100, 200, and 300 μmol/L PQ for 24 hours, the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were measured by immunofluorescence (IF) and quantitative real-time RT-PCR (qRT-PCR) , respectively; the expression of PKCα, PKCβ, phosphorylated (p) -PKCα, and p-PKCβ was determined by Western blot. After the treatment of cells with 200? mol/L PQ for 24 hours following the pretreatment with a classical PKC inhibitor (Go 6983, 1 μmol/L) for 1 hour, the protein expression of ZO-1, Occludin, Claudin-5, p-PKCα, and p-PKCβ was measured by Western blot.@*Results@#The TEER of the bEnd. 3 cells increased gradually with the cell culture time, and reached a peak value of 114.3±6.9 Ω·cm2 on day 6. According to the permeability analysis by Na-FLU, cell permeability gradually decreased with the cell culture time, and reached 1.7±0.2 cm/min on day 6, suggesting a well-behaved barrier function of cells. Compared with the control group, the survival rates of the bEnd.3 cells were significantly reduced after exposure to 100, 200, or 300 μmol/L PQ for 24 hours (P<0.05) , or after exposure to 200 μmol/L PQ for 6, 12, 24, 48, or 72 hours (P <0.05) , indicating a dose-and time-dependent relationship. The IF and qRT-PCR results showed that the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were significantly reduced with the increase in the concentration of PQ (P<0.05) . The Western blot analysis showed that compared with the control group, cells exposed to PQ had significantly higher protein expression of p-PKCα and p-PKCβ and significantly lower protein expression of ZO-1, Occludin, and Claudin-5 (P<0.05) . Compared with the PQ treatment group, the Go 6983 intervention group had significantly higher protein expression of ZO-1, Occludin, and Claudin-5 and significantly lower protein expression of p-PKCα and p-PKCβ (P<0.05) .@*Conclusion@#By activation of cPKC (PKCα and PKCβ) , PQ reduces the protein and mRNA expression of TJ proteins and enhances the permeability of murine BMECs.

Role of protein kinase C in maintenance of chronic inflammatory pain in rats: the relationship with expression of Nav1.8 in dorsal root ganglion / 中华麻醉学杂志

Objective To evaluate the role of protein kinase C in the maintenance of chronic inflammatory pain in rats and the relationship with the expression of Nav1.8 in the dorsal root ganglion (DRG).Methods Thirty pathogen-free healthy female Sprague-Dawley rats,weighing 180-220 g,were divided into 3 groups using a random number table:control group (group C),chronic inflammatory pain group (group CIP) and PKC inhibitor group (group P).Normal saline 20 μl was injected into the plantar surface of the right hindpaw every day for 14 consecutive days in group C.Prostaglandin E2 100 ng was injected into the plantar surface of the right hindpaw every day for 13 consecutive days to establish the model of chronic inflammatory pain,and dimethyl sulfoxide 20μl was injected into the plantar surface of the right hindpaw on 14th day in group CIP.Prostaglandin E2 100 ng was injected into the plantar surface of the right hindpaw every day for 13 consecutive days,and PKC inhibitor GF1O9203X 100 nmol/20 μl was injected into the plantar surface of the right hindpaw on 14th day in group P.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before injection (T0) and 1,3,7 and 14 days after the last injection (T1-4).The DRGs of the lumbar segment (L4.5) were removed for determination of Nav1.8 expression using immunofluorescence and Western blot.Results Compared with group C,the MWT was significantly decreased at T1-4 in CIP and P groups,and the expression of Nav1.8 in DRGs was significantly up-regulated in group CIP (P＜0.05).Compared with group CIP,the MWT was significantly increased at T4,and the expression of Nav1.8 in DRGs was down-regulated in group P (P＜0.05).Conclusion Up-regulated expression of Nav1.8 after PKC activation in DRGs is involved in the maintenance of chronic inflammatory pain in rats.

Effects of fingolimod on sphingosine-1-phosphate signal in osteoclasts / 中华创伤骨科杂志

Objective To study the effects of fingolimod (FTY720) on the sphingosine-1-phosphate (S1P) signaling pathway in osteoclasts.Methods Murine RAW 264.7 macrophages were induced into osteoclasts by dexamethasone and 1 α,25-(OH)2VitD3 and identified by tartrate resistant acid phosphatase Trp staining.After the osteoclasts were divided into 2 groups,the experimental group was treated with 400 ng/mL FTY720-P while the control group was not.The signal expression of extracellular signal-regulated kinase 1 (ERK1),caspase9 and protein kinase(PKC) in the downstream of S1 P pathway was detected at the gene and protein levels,and the expression of bone morphogenetic protein-2(BMP-2) and transforming growth factor-β1 (TGF-β1),which are closely related to bone reconstruction,was detected.Results The RAW264.7 cells were successfully induced into osteoclasts identified by trataric acid phosphatase staining kit (TRAP).The expression of S1PR1 in osteoclasts was higher than that in other S1PRs.After culture for 48 hours,the expression of PKC mRNA in the experimental group was significantly lower than that in the control group while the expression of BMP-2 mRNA and TGF-[β1 mRNA was significantly increased in the experimental group than in the control group (P ＜ 0.05).However,there were no significant differences in expression of ERK1 mRNA or caspase9 mRNA between the 2 groups(P ＞ 0.05).Conclusions FTY720-P can affect the expression of PKC signal in the downstream of osteoclasts by affecting S1P signaling pathway,mainly by binding to S1PR1 signal pathway,and can also promote the expression of TGF-[β1 and BMP-2 in osteoclasts,ultimately affecting the function of osteoclasts.

Effects of angiotensin Ⅱ on protein kinase Cε and Cα expression in hepatic stellate cells / 中国医师杂志

Objective To investigate the effect of angiotensin Ⅱ on protein kinase Cε (PKCε) and protein kinase Cα (PKCα) expression in hepatic stellate cells.Methods Hepatic stellate cell (HSC)-T6 cells were treated with different concentrations of angiotensin Ⅱ and the proliferation of HSC-T6 cells was detected by methyl thiazolyl tetrazolium (MTT) assay.The expression of PKCε and PKCα was detected by immunofluorescence staining.PKCε and PKCα mRNA levels was detected by real time polymerase chain reaction (PCR).Results Angiotensin Ⅱ concentrated the proliferation of HSC-T6 cells and the level of hydroxyproline (F =25.321,13.283,P ＜ 0.001) and showed a dose-dependent effect.With the increase of angiotensin Ⅱ concentration,PKCε significantly increased and translocated in the cell membrane;PKCα increased significantly,especially in transplanted membrane and cytoplasm (F =21.387,19.431,P ＜0.01),and showed obvious dose effect.Meanwhile,Angiotensin Ⅱ increased the expression of PKCε and PKCα,and induced cell proliferation by up-regulating PKCε and PKCα mRNA levels (F =13.279,15.174,P ＜ 0.05).Conclusions Angiotensin Ⅱ can up-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner,increase the expression of protein kinase Cε and Cα,and promote the proliferation of hepatic stellate cells.

Atorvastatin Attenuates Myocardial Hypertrophy Induced by Chronic Intermittent Hypoxia In Vitro Partly through miR-31/PKCε Pathway / 华中科技大学学报（医学）（英德文版）

Atorvastatin is proven to ameliorate cardiac hypertrophy induced by chronic intermittent hypoxia (CIH).However,little is known about the mechanism by which atorvastatin modulates CIH-induced cardiac hypertrophy,and whether specific hypertrophyrelated microRNAs are involved in the modulation.MiR-31 plays key roles in the development of cardiac hypertrophy induced by ischemia/hypoxia.This study examined whether miR-31 was involved in the protective role of atorvastatin against CIH-induced myocardial hypertrophy.H9c2 cells were subjected to 8-h intermittent hypoxia per day in the presence or absence of atorvastatin for 5 days.The size of cardiomyocytes,and the expression of caspase 3 and miR-31 were determined by Western blotting and RT-PCR,respectively.MiR-31 mimic or Ro 31-8220,a specific inhibitor of protein kinase C epsilon (PKCε),was used to determine the role of miR-31 in the anti-hypertrophic effect of atorvastatin on cardiomyocytes.PKCε in the cardiomyocytes with miR-31 upregulation or downregulation was detected using RT-PCR and Western blotting.The results showed that CIH induced obvious enlargement of cardiomyocytes,which was paralleled with increased atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP),and slow/beta cardiac myosin heavy-chain (MYH7) mRNA levels.All these changes were reversed by the treatment with atorvastatin.Meanwhile,miR-31 was increased by CIH in vitro.Of note,the atorvastatin pretreatment significantly increased the mRNA and protein expression of PKCε and decreased that of miR-31.Moreover,overexpression of miR-31 abolished the anti-hypertrophic effect of atorvastatin on cardiomyocytes.Upregulation and downregulation of miR-31 respectively decreased and increased the mRNA and protein expression of PKCε.These results suggest that atorvastatin provides the cardioprotective effects against CIH probably via up-regulating PKCε and down-regulating miR-31.