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 d...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.展开更多
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 a...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.展开更多
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 potenc...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.展开更多
Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/th...Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/threonine kinase 11(STK11)mutant non-small cell lung cancer(NSCLC)through an axis inhibition protein 1(AXIN1)-dependent manner.However,the alterations of tumor metabolism and metabolites upon metformin administration remain unclear.Methods:We performed untargeted metabolomics using liquid chromatography(LC)-mass spectrometry(MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis.Results:According to the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database,most metabolites were annotated into metabolism,including nucleotide metabolism.Next,the differentially expressed metabolites in H460(refers to H460 cells),H460_met(refers to metformin-treated H460 cells),and H460_KO_met(refers to metformin-treated Axin1-/-H460 cells)were distributed into six clusters based on expression patterns.The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis.We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated.Furthermore,these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes(STING)pathway independently of AXIN1.Conclusion:Relying on AXIN1,metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC,indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.展开更多
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 produc...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.展开更多
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 c...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,展开更多
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. H...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.展开更多
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 seque...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.展开更多
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 d...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.展开更多
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...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.展开更多
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 ...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.展开更多
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...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.展开更多
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 wi...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.展开更多
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 in...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.展开更多
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 fro...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.展开更多
In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-α (PKC-α) in chronic exposure smoke rats, 54 male Wistar rats were randomly divided into 7 groups: control gro...In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-α (PKC-α) in chronic exposure smoke rats, 54 male Wistar rats were randomly divided into 7 groups: control group (C group), smoke exposure groups (S4w group, S8w group), puerarin groups (P4w group, P8w group), propylene glycol control groups (PC4w group, PC8w group). Rats were exposed to cigarette smoke or air for 4 to 8 weeks. Rats in puerarin groups also received puerarin. To evaluate vascular remodeling, alpha-smooth muscle actin (α-SM-actin) staining was used to count the percentage of completely muscularised vessels to intraacinar pulmonary arteries (CMA/IAPA) which was determined by morphometric analysis of histological sections. Pulmonary artery smooth muscle cell (PASMC) apoptosis was detected by in situ end labeling technique (TUNEL), and proliferation by proliferating cell nuclear antigen (PCNA) staining. Reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and Western blot analysis were done to detect the PKC-α mRNA and protein expression in pulmonary arteries. The results showed that in cigarette smoke-exposed rats the percentage of CMA/IAPA and α-SM-actin expression were increased greatly, PASMC apoptosis was increased and proliferation was markedly increased; Apoptosis indices (AI) and proliferation indices (PI) were higher than in C group; AI and PI were correlated with vascular remodeling indices; The expression of PKC-α mRNA and protein in pulmonary arteries was significantly higher than in C group. In rats treated with puerarin, the percentage of CMA/IAPA and cell proliferation was reduced, whereas PASMC apoptosis was increased; The expression levels of PKC-α mRNA and protein were lower than in smoke exposure rats. There was no difference among all these data between S groups and PC groups. These findings suggested that cigarette smoke-induced pulmonary vascular remodeling was most likely an effect of the imbalance of PASMC proliferation and apoptosis. Puerarin appears to be able to reduce cell proliferation and vascular remodeling possibly through PKC signaling transduction pathway.展开更多
基金Supported by National Natural Science Foundation of China,No.82260211Key Research and Development Project in Jiangxi Province,No.20203BBG73058Chinese Medicine Science and Technology Project in Jiangxi Province,No.2020A0166.
文摘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.
基金supported by the Natural Nature Science Foundation of China,Nos.82030071,81874004the Science and Technology Major Project of Changsha,No.kh2103008(all to JZH).
文摘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.
文摘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.
基金People’s Hospital of Xuyong County-Southwest Medical University Science and Technology Strategic Cooperation Project(2023XYXNYD05)Guangdong Association of Clinical Trials(GACT)/Chinese Thoracic Oncology Group(CTONG)and Guangdong Provincial Key Lab of Translational Medicine in Lung Cancer(2017B030314120)Natural Science Foundation of Chongqing Municipality(CSTB2023NSCQ-MSX0554).
文摘Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/threonine kinase 11(STK11)mutant non-small cell lung cancer(NSCLC)through an axis inhibition protein 1(AXIN1)-dependent manner.However,the alterations of tumor metabolism and metabolites upon metformin administration remain unclear.Methods:We performed untargeted metabolomics using liquid chromatography(LC)-mass spectrometry(MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis.Results:According to the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database,most metabolites were annotated into metabolism,including nucleotide metabolism.Next,the differentially expressed metabolites in H460(refers to H460 cells),H460_met(refers to metformin-treated H460 cells),and H460_KO_met(refers to metformin-treated Axin1-/-H460 cells)were distributed into six clusters based on expression patterns.The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis.We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated.Furthermore,these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes(STING)pathway independently of AXIN1.Conclusion:Relying on AXIN1,metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC,indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.
基金supported by the a grant from China Postdoctoral Science Project,No.801161020425the Natural Science Foundation of China,No.8160010172
文摘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.
文摘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,
基金Supported by the Key Program of Beijing Academy of Health, No. 1998-11-2001-09
文摘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.
文摘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.
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30672161)
文摘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.
基金Major State BasicResearch (973) Program of China, (G1999053905).
文摘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.
基金This study was supported by a grant from the National Nature Science Foun-dation of China (No. 30070747)
文摘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.
基金Supported by National Natural Science Foundation of China (No. 81470609 No. 81170825)+2 种基金the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20123706110003)the Key Project of Natural Science Foundation of Shandong Province (No. ZR2012FZ001) the Youth Project of Natural Science Foundation of Shandong Province (No. ZR2013HQ007)
文摘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.
文摘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.
基金supported by grants from Natural Science Foundation of Hubei Province,China (No. 2010CDB096)the National Key Technology R&D Program of the 12th National Five-year Development Plan of China (No. 2012BAI05B01)
文摘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.
基金the functional analysis of PKC signaling pathway involved in response to salt stress of Dunaliella salinathe National Natural Science Foundation of China (No. 31472260)
文摘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.
基金a grant from the National Natural Sciences Foundation of China (No. 30470759)
文摘In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-α (PKC-α) in chronic exposure smoke rats, 54 male Wistar rats were randomly divided into 7 groups: control group (C group), smoke exposure groups (S4w group, S8w group), puerarin groups (P4w group, P8w group), propylene glycol control groups (PC4w group, PC8w group). Rats were exposed to cigarette smoke or air for 4 to 8 weeks. Rats in puerarin groups also received puerarin. To evaluate vascular remodeling, alpha-smooth muscle actin (α-SM-actin) staining was used to count the percentage of completely muscularised vessels to intraacinar pulmonary arteries (CMA/IAPA) which was determined by morphometric analysis of histological sections. Pulmonary artery smooth muscle cell (PASMC) apoptosis was detected by in situ end labeling technique (TUNEL), and proliferation by proliferating cell nuclear antigen (PCNA) staining. Reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and Western blot analysis were done to detect the PKC-α mRNA and protein expression in pulmonary arteries. The results showed that in cigarette smoke-exposed rats the percentage of CMA/IAPA and α-SM-actin expression were increased greatly, PASMC apoptosis was increased and proliferation was markedly increased; Apoptosis indices (AI) and proliferation indices (PI) were higher than in C group; AI and PI were correlated with vascular remodeling indices; The expression of PKC-α mRNA and protein in pulmonary arteries was significantly higher than in C group. In rats treated with puerarin, the percentage of CMA/IAPA and cell proliferation was reduced, whereas PASMC apoptosis was increased; The expression levels of PKC-α mRNA and protein were lower than in smoke exposure rats. There was no difference among all these data between S groups and PC groups. These findings suggested that cigarette smoke-induced pulmonary vascular remodeling was most likely an effect of the imbalance of PASMC proliferation and apoptosis. Puerarin appears to be able to reduce cell proliferation and vascular remodeling possibly through PKC signaling transduction pathway.