Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina

BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.

Critical Role of Protein Kinase C (PKC) in the Onset of Airway Hypersensitivity in Ova-Sensitized Guinea Pig Model of Asthma

Background and Objectives: Protein kinase C (PKC) activation plays an important role in activation of T-lymphocytes in asthma. Airway hypersensitivity is one of the main characteristic features of asthma, the mechanism of onset of which is not clearly understood. Therefore, the objective was to elucidate the role of PKC in etiopathogenesis of airway hypersensitivity in asthma. Methods: Male guinea pigs (n = 30) were sensitized with ovalbumin and day of initial allergen-specific immune response determined by intradermal test, airway hypersensitivity, BALF cytology and lung histopathology. Total PKC activity, PKC isoenzymes and phosphoinositides were assessed in airway smooth muscles (ASM) and peripheral blood lymphocytes. Results: Intradermal test revealed that day 9 was the earliest time of allergen-specific response and onset of airway hypersensitivity to ovalbumin. It was associated with significant increase in total and differential (lymphocytes and eosinophils) BALF counts and grade I peribronchiolar chronic lymphocytic inflammation in lung. On day 14, grade II infiltration of lymphocytes and eosinophils with onset ofstructural remodelingofproximal and distal airways was seen. Total PKC activity, expression of PKCα, PKCε and phosphoinositides increased significantly in ASM and lymphocytes on day 9 and were maximum on day 14. There was no change in PKC-τ expression. Conclusions: Activation of PKC, particularly PKCα and PKCε, mediated signal transduction pathway plays a critical role in lymphocyte infiltration and onset of airway hypersensitivity, airway remodeling and asthma pathophysiology. The present study is the first one on the mechanism of the etiopathogenesis of the disease, which shows a direct evidence of the role of PKC mediated pathway in the initiation and onset of airway hypersensitivity in ovalbumin sensitized guinea pig model.

Latest progress in the research of protein kinase C(PKC)isoform-specific signaling and PKC-modulated autophagy in ischemic stroke

Ischemic stroke is a major cause of morbidity and mortality,and currently there is no effective treatment.The family of protein kinase C(PKCs)could phosphorylate serine or threonine residues of its substrate proteins and play a key role in the ischemia/reperfusion injury.Autophagy is essential for maintaining cell homeostasis under physiological condition and acts as a double-edged sword in the process of ischemic neuronal death.In this article,we reviewed the PKCs isoform-specific signaling pathways and PKC-modulated autophagy in ischemic stroke.

The Effects of Protein Kinase C (PKC) on the Tension of Normal and Passively Sensitized Human Airway Smooth Muscle and the Activity of Voltage-dependent Delayed Rectifier Potassium Channel (Kv)

The effects of protein kinase C （PKC） on the tension and the activity of voltage-dependent delayed rectifier potassium channel （K,,） were examined in normal and passively sensitized human airway smooth muscle （HASM）, by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential （Em） were also detected. The results showed that phorbol 12-myristate 13-acetate （PMA）, a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group （P〈0.05）, and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the Em became more positive, as compared with the DMSO （a PMA menstruum）-treated group （P〈0.01）. This effect could be blocked by Ro31-8220 （P〈0.01 ）. It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.

基于PKC-P2X3信号通路观察电针对神经病理性疼痛小鼠神经修复的作用机制

目的:基于蛋白激酶C(PKC)-配体门控型非选择性离子通道3(P2X3)信号通路观察电针对神经病理性疼痛小鼠神经修复的作用机制。方法:48只小鼠随机分成假手术组、模型组、针刺组与电针组,每组12只。除假手术组外,其余小鼠构建坐骨神经慢性限制性损伤(CCI)模型。术后第8天针刺组与电针组接受针刺与电针干预,连续7 d。于术前、术后3 d、5 d、7 d、10 d、12 d、14 d对各组进行机械缩足反射阈值(MWT)与热刺激缩足反射潜伏期(TWL)测试。术后15 d处死所有小鼠,HE染色观察坐骨神经组织形态,ELISA检测脊髓组织IL-1β、TNF-α水平,Western blot检测脊髓组织PKC、P2X3水平,免疫荧光检测脊髓神经元PKC、P2X3表达情况。结果:假手术组神经元细胞大小不一,细胞膜完整,细胞质呈细小颗粒状,细胞核大而圆,核仁居中清晰可见;模型组可见萎缩的神经元,有髓神经纤维紊乱,轴突肿胀,神经元间形成间隙;与模型组相比,针刺组、电针组萎缩神经元数量减少(P<0.05),有髓神经纤维排布情况改善,电针组较针刺组改善更为明显。与相同时间点的假手术组相比,术后3 d、5 d、7 d、10 d、12 d、14 d模型组、针刺组、电针组患侧后肢的MWT、TWL值明显下降(P<0.05),IL-1β、TNF-α、PKC、P2X3表达量、PKC、P2X3阳性细胞数、平均光密度值升高(P<0.05);与模型组相比,术后10 d、12 d、14 d针刺组、电针组MWT、TWL值明显上升(P<0.05),IL-1β、TNF-α、PKC、P2X3表达量、PKC、P2X3阳性细胞数、平均光密度值降低(P<0.05);与针刺组相比,术后10 d、12 d、14 d电针组MWT、TWL值升高(P<0.05),IL-1β、TNF-α、PKC、P2X3表达量、PKC、P2X3阳性细胞数、平均光密度值降低(P<0.05)。结论:电针可能减轻CCI模型小鼠神经炎症反应,降低PKC、P2X3表达水平,改善坐骨神经细胞形态与有髓神经纤维排布情况,缩小神经元间间隙,提升MWT、TWL值,缓解神经病理性疼痛程度。

脑血栓患者的发病危险因素分析及脑血栓与血清PKC、SOST、血液流变学指标的相关性分析

目的分析脑血栓患者的发病危险因素及脑血栓与血清蛋白激酶C(PKC)、骨硬化蛋白(SOST)、血液流变学指标的相关性。方法回顾性选择2021年1月至2023年7月唐山市人民医院收治的96例脑血栓患者纳入观察组,另选同期收治的96例非脑血栓者纳入对照组。比较两组一般资料(性别、年龄、体重指数、吸烟、饮酒、高血压、糖尿病、高脂血症、冠心病等)、血清PKC、SOST表达水平及血液流变学指标[血浆黏度(PV)、红细胞压积(HCT)、红细胞聚集指数(RAI)、纤维蛋白原(FIB)];采用多因素Logistic回归分析对脑血栓患者的发病危险因素进行分析;根据美国国立卫生研究院卒中量表(NIHSS)评分将脑血栓患者分为轻度组、中度组和重度组,分析血清PKC、SOST表达水平及血液流变学指标在不同严重程度的脑血栓患者间的差异性;采用Pearson相关性分析对血清PKC、SOST表达水平及血液流变学指标与NIHSS评分、改良RANKIN量表(mRS)评分的关系进行分析。结果两组性别构成比、年龄、吸烟患者比例、糖尿病患者比例、冠心病患者比例比较,差异均无统计学意义(P>0.05);观察组的体重指数、饮酒患者比例、高血压患者比例、高脂血症患者比例、血清PKC、SOST水平及PV、HCT、RAI、FIB水平分别为(25.04±1.47)kg/m^(2)、20.83%、54.17%、34.38%、(196.52±45.37)μg/L、(4.65±1.07)ng/mL、(6.75±0.72)mPa·s、(44.82±4.56)%、(5.86±0.71)%、(4.62±0.85)g/L,均高于对照组,差异均有统计学意义(P<0.05)。经多因素Logistic回归分析,高血压、高脂血症、血清PKC、SOST、RAI、FIB均是脑血栓患者发病的独立危险因素(P<0.05)。轻度组、中度组和重度组的血清PKC、SOST表达水平及PV、HCT、RAI、FIB水平比较,差异均有统计学意义(P<0.05)。经Pearson相关性分析,脑血栓患者血清PKC、SOST表达水平及PV、HCT、RAI、FIB水平均与NIHSS评分、mRS评分呈正相关(P<0.05)。结论脑血栓患者的发病受高血压、高脂血症、血清PKC、SOST、RAI、FIB水平影响,其中血清PKC、SOST及血液流变学指标与其病情严重程度及预后有关。

Structure-activity Relationship of Phenothiazines for Inhibition of Protein Kinase C and Reversal of Multidrug Resistance

Studies on structure-activity relationship of phenothiazines (PTZs) forinhibition of protein kinase C (PKC) and reversal of multidrug resistance (MDR) has been made invitro. The results showed that the order of potency of reversal effect of PTZs on MDR is as follows:2-COC_3 H_7 > 2-CF_3 > 2-COCH_3 > H. The type of piperazinyl substitution also significantlyaffected potency against MDR. The results show the order: CH_3 > COOC_2 H_5 > C_2 H_4 OH. Inaddition, PKC plays a marked role in diverse cellular process including MDR. Some derivatives of PTZwas tested for inhibition of PKC, of which PTZ11 showed the highest inhibitory effect of MDR andPKC, implying a potential reversal agent of MDR for tumor therapy in the future. We also tried toexplore the possible binding model of PTZs to PKC. Our molecular-modeling study preliminarilysuggests how these PTZs bind to PKC and provides a structural basis for the design of high affinityPKC-modulator. The infor-mation may be used in the rational design of more effective drugs.

大蒜素对2型糖尿病大鼠肾脏AGEs/PKC/p66shc通路的影响

目的以腹腔注射链脲佐菌素(streptozotocin,STZ)建立2型糖尿病肾病大鼠模型,探究大蒜素对2型糖尿病大鼠肾脏的保护作用及其机制。方法将30只SD雄性大鼠正常对照组(Control)、糖尿病肾病模型组(Model)、低剂量大蒜素保护组(DT10)、中剂量大蒜素保护组(DT20)、高剂量大蒜素保护组(DT40)5组,利用试剂盒检测肌酐、微量白蛋白、丙二醛的水平以及超氧化物歧化酶的活性。HE、PAS染色观察大鼠肾脏形态学变化。Western Blot检测相关蛋白的表达。结果与模型组相比,大蒜素保护组的肌酐、微量白蛋白、丙二醛的水平明显下降(P<0.05),超氧化物歧化酶的活性明显升高(P<0.05)。HE、PAS染色结果显示,肾组织显微结构改善、肾间质糖原沉积物减少。Western Blot结果显示,与模型组相比,大蒜素保护组的AGEs、p-PKC、p66shc的水平明显降低(P<0.05)。结论大蒜素对糖尿病大鼠肾脏具有一定的保护作用,这种保护作用的机制可能与其调控AGEs/PKC/p66shc信号通路有关。

Calycosin improves cognitive function in a transgenic mouse model of Alzheimer's disease by activating the protein kinase C pathway

The major pathological changes in Alzheimer＇s disease are beta amyloid deposits and cognitive impairment. Calycosin is a typical phy- toestrogen derived from radix astragali that binds to estrogen receptors to produce estrogen-like effects. Radix astragali Calycosin has been shown to relieve cognitive impairment induced by diabetes mellitus, suggesting calycosin may improve the cognitive function of Alzhei- mer＇s disease patients. The protein kinase C pathway is upstream of the mitogen-activated protein kinase pathway and exerts a neuropro- tective effect by regulating Alzheimer＇s disease-related beta amyloid degradation. We hypothesized that calycosin improves the cognitive function of a transgenic mouse model of Alzheimer＇s disease by activating the protein kinase C pathway. Various doses of calycosin （10, 20 and 40 mg/kg） were intraperitoneally injected into APP/PS1 transgenic mice that model Alzheimer＇s disease. Calycosin diminished hippocampal beta amyloid, Tau protein, interleukin-lbeta, tumor necrosis factor-alpha, acetylcholinesterase and malondialdehyde levels in a dose-dependent manner, and increased acetylcholine and glutathione activities. The administration of a protein kinase C inhibitor, cal- phostin C, abolished the neuroprotective effects of calycosin including improving cognitive ability, and anti-oxidative and anti-inflammato- ry effects. Our data demonstrated that calycosin mitigated oxidative stress and inflammatory responses in the hippocampus of Alzheimer＇s disease model mice by activating the protein kinase C pathway, and thereby improving cognitive function.

Effect of gastrin on protein kinase C and its subtype in human colon cancer cell line SW480

INTRODUCTIONGastrin is atrophic gastrointestinal hormone whichis secreted by G cell.Gastrin has long beenconsidered a growth stimulatory hormone formucosa of the gastrointestinal tract.The growthresponses of certain colorectal cancer cells,andxenografts,can be stimulated by endogenousgastrin.Protein kinase C (PKC) is a family ofisozymes that plays a crucial role in transducingsignals of many hormones,growth peptides,

Hyperlipidemia intensifies cerulein-induced acute pancreatitis associated with activation of protein kinase C in rats

AIM： To investigate the effects of hyperlipidemia on acute pancreatitis （AP） and the possible mechanisms. METHODS： Rat models of hyperlipidemia and AP were established by Triton WR1339 and cerulein respectively. Human albumin was used to treat AP complicated by hyperlipidemia. In each group, we compared the histological score, volume of ascites, ratio of pancreatic wet/dry weight, serum amylase （AMY） and pancreatic acinar cell apoptosis. The level of protein kinase C （PKC） membrane translocation in pancreatic tissue was detected by Western blot.RESULTS： In the hyperlipidemia model established by Triton WR1339, triglyceride （TG） increased remarkably and reached its peak 6 h after injection, and most rats developed mild acute pancreatitis. Histological score, volume of ascites, ratio of wet/dry weight and serum AMY in AP animals with hyperlipidemia were obviously higher than those in AP animals （P 〈 0.05） and decreased after albumin therapy but not significantly （P 〉 0.05）. Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） increased in AP animals with hyperlipidemia and did not change distinctly after albumin therapy. PKC membrane translocation level increased in AP animals with hyperlipidemia and decreased remarkably after albumin therapy （P 〈 0.05）.CONCLUSION： Hyperlipidemia may induce AP or intensify pancreatic injury. Albumin therapy can not alleviate pancreatic lesion effectively. PKC activation may be one mechanism by which AP is intensified by hyperlipidemia.

Relationship between Invasiveness of Pituitary Somatotrophinomas and Structural Abnormalities of Protein Kinase C Gene in Human

The potential role of the protein kinase C (PKC) transduction systemin controlling proliferation of human pituitary somatotrophinomas was investigat-ed. Twenty somatotrophinomas were studied using PCR and diract sequencing methods. No point mutation within the QPKC gene, previously thought to be as-sociated with invasive pituitary tumors, was found in any of the 20 somatotrophi-nomas. It is concluded that PKC trareduction system may play an important rolein controlling pituitary somatotrophinoma proliferation, but there is no correlation between invasiveness and the previously reported QPKC gene mutation.

GHRP-6 Induces CREB Phosphorylation and Growth Hormone Secretion via a Protein Kinase Cσ-dependent Pathway in GH3 Cells

This study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein （CREB） and the tmderly mechanism. GH3 cells were cultured and subjected to different treatments as follows： GHRP-6, GHRP-6 plus GHRH, phorbol ester （PMA）, an activator of PKC, alone or in combination with GHRP-6, G66983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCσ-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay （ELISA）. The expression of phosphor-CREB, PKCσ, PKC0 and phosphor-PKCo was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors （G66983, rottlerin） and knockdown of PKCσ. PKCσ could be activated by GHRP-6. It is concluded that PKC, especialiy PKCσ, mediates CREB phosphorylation and GHRP-6-induced GH secretion.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Effects of cell membrane phospholipid level and protein kinase C isoenzyme expression on hepatic metastasis of colorectal carcinoma

BACKGROUND: The molecular mechanism of hepaticmetastasis of colorectal cancer is not well understood. Theaim of this study was to assess the relations between phos-pholipid contents of cellular membrane and isoenzyme ex-pression of protein kinase C (PKC) and their effects on he-patic metastasis of colorectal cancer.METHODS: High performance liquid chromatography wasused to detect contents of cell membrane phospholipids:phosphatidylinosital (PI), phosphatidylserine (PS), phos-phatidylethanolamine (PE) and phosphatidylcholine (PC)in primary foci, paratumor mucosa and hepatic metastaticfoci in patients with colorectal carcinoma. The mRNA ex-pression levels of PKC-α, -δ, -ε, -λ, -ξ isoenzymeswere detected with the QRT-PCR technique.RESULTS: The levels of PI, PC and PE in primary foci andhepatic metastatic foci were higher than those in paratumormucosa. The level of PE in hepatic metastatic foci wasmuch higher than that in primary foci (t =98.88, P <0.01);but the levels of PI and PC were not significantly differentbetween primary foci and hepatic metastatic foci (t =1.73 ,1.36, P>0.05). The expression levels of -δ, -ε,-λ, -ξ were enhanced in primary foci and hepatic metasta-tic foci, but the level of PKC-α in primary foci was de-creased as compared with that in paratumor mucosa. Thelevels of PKC-δ, -ε, -λ, -ξ in hepatic metastatic foci werehigher than those in primary foci. A positive correlationwas observed between the expression levels of PI, PC andand also between those of PE and PKC-δ, -ε, -λ,-ξ. However, there was a close negative correlation be-tween PE and PKC-α.CONCLUSION: Increased levels of PI and PC and de-creased ratio of PKC-α to are related to colorectalcancer genesis. Increased levels of PE, increased expressionof PKC-δ, -ε, -λ, -ξ isoenzymes and decreased level ofPKC-α are related to hepatic metastasis in colorectal carci-noma.

Fungus induces the release of IL- 8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways

AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.

Hepatic injury in rats with obstructive jaundice: roles of the protein kinase C signal pathway and cytoprotection of fructose

BACKGROUND: Fructose is cytoprotective during bile salt-induced apoptosis of hepatocytes by regulating protein kinase C (PKC). This study was undertaken to explore the regulating mechanism of hepatic injury in rats with obstructive jaundice, and to detect the PKC signal pathway. METHODS: Rat hepatocytes were isolated by in situ colla-genase perfusion and primary culture, and pretreated with various concentrations of PKC agonist phorbol myristate acetale (PMA) and inhibitor chelerythrine for 20 minutes. After pretreatment, 50 μmol/L glycochenodeoxycholate (GCDC) was added for additional 24 hours. Subsequently, the cells were detected by FCM and TUNEL. After adding with different concentrations of fructose and 100 μmol GCDC , the hepatocytes were evaluated by FCM and TUNEL. Experimental obstructive jaundice was induced with fructose and without fructose via double ligation of the bile duct for 3, 7, 14, and 21 days. Apoptotic status in the liver of all rats was detected with TUNEL, and PKC protein in the liver of obstructive jaundice ( OJ) with the immunohisto-chemistry method. RESULTS: PMA increased GCDC-induced apoptosis and chelerythrine decreased GCDC-induced apoptosis in a concentration-dependent manner. Adding with different concentration of fructose and 100 μmol GCDC, the decreased apoptotic rate was related to the concentration of fructose. The apoptotic rate of the liver was related to times of OJ. PKC and apoptosis index (AI) were the highest after a 14-day ligation of the bile duct without use of fructose. AI and PKC were decreasing from a 14-day ligation of the bile duct with fructose. CONCLUSIONS: PKC takes part in the regulation, occurrence , and progression of hepatic injury in OJ. Fructose is cytoprotective during bile salt-induced apoptosis of hepato-cytes by regulating PKC.

Effects of Mitochondrial ATP-sensitive K^+ Channel on Protein Kinase C Pathway and Airway Smooth Muscle Cell Proliferation in Asthma

The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were investigated.Thirty-six Sprague-Dawley(SD) rats were immunized with saline(controls) or ovalbumin(OVA) with alum(asthma models).ASMCs were cultured from the lung of control and asthma rats.ASMCs were treated with diazoxide(the potent activator of mitoK ATP) or 5-hydroxydencanote(5-HD,the inhibitor of mitoK ATP).Rhodamine-123(R-123) was used to detect Δψm.The expression of PKCα protein was examined by using Western blotting,while PKCα mRNA expression was detected by using real-time PCR.The proliferation of ASMCs was measured by MTT assay and cell cycle analysis.In diazoxide-treated normal ASMCs,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls.The ratio of G 0 /G 1 cells was decreased(P<0.05) in diazoxide-treated ASMCs from normal rats.However,there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group.In untreated and diazoxide-treated ASMCs of asthmatic rats,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group.Furthermore,in comparison to ASMCs from asthmatic rats,these values were considerably increased in asthmatic group treated with diazoxide(P<0.05).After exposure to 5-HD for 24 h,these values were decreased as compared with asthma control group(P<0.05).In ASMCs of asthma,the signal transduction pathway of PKCα may be involved in cell proliferation,which is induced by the opening of mitoK ATP and the depolarization of Δψm.

Characterization and Expression Analysis of Protein Kinase C Gene from Dunaliella salina

Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina (DsPKC) using rapid amplification of cDNA ends (RACE) and RT-PCR methods. And we submitted the mRNA sequence of DsPKC gene to NCBI (Genbank No. JN625213). In the present paper, the DsPKC gene open reading frame obtained by PCR was cloned into pGS-21a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The DsPKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.2 mmol L 1 at 37℃. Under salt stress, the fu- sion protein Green Fluorescent Protein (GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pat- tern of DsPKC gene was analyzed using real-time quantitative PCR, and indicated that DsPKC gene was up-regulated by 3.0 mol L 1 NaCl at 12 h, which was significantly higher than in control values (P < 0.05). These results suggest that the DsPKC gene plays an important role in response to salt stress in D. salina.