Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor ...Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.展开更多
Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects n...Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning （ISO） and oxygen glucose dep- rivation （OGD） model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride （NH4 C1） to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 （cell counting kit-8） and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA （3-Methyladenine） was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.展开更多
Aim Evidence has shown that stimulation of alA-adrenorecetors receptor （alA-AR） or angiotensin II type 1 receptor （AT1R） acutely down-regulates the rapid component of the delayed rectifier K ＋ current （IKr） via...Aim Evidence has shown that stimulation of alA-adrenorecetors receptor （alA-AR） or angiotensin II type 1 receptor （AT1R） acutely down-regulates the rapid component of the delayed rectifier K ＋ current （IKr） via protein kinase C （PKC）. This study was designed to investigate which PKC isozymes mediate down-regulations of IKr by alA-AR and AT1R. Method The whole-cell patch-clamp technique was used to record IKr in native cardio- myocytes and in human embryonic kidney （HEK） 293 cells co-transfected with human ether-a-go-go related gene （hERG） encoding α-subunit of IKr and human alA-AR or AT1R gene. Result In isolated guinea-pig ventricular cardiomyocytes the inhibitory action of Ang II on IKr was little affected by Go6976 （selectively inhibiting PKCα, β and γ） and Go6983 （selectively inhibiting PKCα, β, γ , δ, and ζ）, but was significantly antagonized by an inter- nal dialysis with PKCe-selective inhibitory peptide εV1 -2. In contrast, the inhibitory action of alA-AR agonist A61603 on IKr was remarkably attenuated by Go6976 or Go6983, but not affected by peptide εV1 -2. Moreover, specific PKC-selective inhibitory peptide antagonized the effect of A61603. The results suggested that PKCe and PKCα isoform respectively mediated the inhibitory effect of AT1R and a1A-AR. In heterologous expression system, both PKCα and e-selective activator peptides down regulated hERG current with different manner. PKCα activator peptide shifted the activation curve of the channel to the right, but PKCe-selective activator peptide did not. Simi- larly, A61603 shifted the activation curve to the right, whereas Ang Ⅱ had no effect. In addition, both A61603 and PKCα activator peptide showed inhibitory action on bERG A PKC current （an bERG mutant in which 17 of the 18 ROSITE-predicted PKC acceptor serines/threonines were changed to alanine） with a similar potency to wild type bERG current. But, both Ang Ⅱ and PKCe-selective activator peptide exhibited no effects on bERG △ PKC cur- rent. The results indicated that PKCα and PKCe isoforms down-regulated bERG current through different mecha- nism. Conclusion PKCα and PKCe isoform respectively mediates the inhibition on IKr by stimulation of AT1R and alA-AR via different molecular mechanism.展开更多
OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 depende...OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.展开更多
A protein kinase C inhibitor is found in bovine sper-matozoa. This inhibitor was purified by Sephadex G-200and isoelectrofocusion electrophoresis. The molecularweight of this protein kinase C inhibitor was about 63 00...A protein kinase C inhibitor is found in bovine sper-matozoa. This inhibitor was purified by Sephadex G-200and isoelectrofocusion electrophoresis. The molecularweight of this protein kinase C inhibitor was about 63 000,the isoelectric point of the inhibitor was pH 4.5.展开更多
OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) us...OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.展开更多
Introduction Vascular endothelial (VE)-cadherin is localized to the endothelial borders and the adherens junctions,which are regulated by changes in mitogen activated protein kinases (MAPK),GTPases,and intracellular c...Introduction Vascular endothelial (VE)-cadherin is localized to the endothelial borders and the adherens junctions,which are regulated by changes in mitogen activated protein kinases (MAPK),GTPases,and intracellular calcium. We previously展开更多
AIM:Regulatory T cells(Tregs)are a specialized subset of CD4^(+)T cells primarily involved in im⁃munosuppressive functions.AMP-activated protein kinase(AMPK)serves as a metabolic sensor that governs the differen⁃tiati...AIM:Regulatory T cells(Tregs)are a specialized subset of CD4^(+)T cells primarily involved in im⁃munosuppressive functions.AMP-activated protein kinase(AMPK)serves as a metabolic sensor that governs the differen⁃tiation,maturation,and immune functions of Tregs through metabolic reprogramming.However,the impact of AMPKα1(the catalytic subunit of AMPK)knockout specifically in Tregs on the host's immune microenvironment remains largely un⁃explored.METHODS:Histological changes in immune organs were assessed using HE staining.The types of immune cells and their relative population percentages in immune organs and blood were quantified through flow cytometry in both AMPKα1flox/flox(AMPKα1^(fl/fl))mice and Treg-specific AMPKα1 knockout mice(AMPKα1^(fl/fl)Foxp3^(cre)mice).RESULTS:Compared to AMPKα1^(fl/fl)mice,the percentage of eosinophils in the bone marrow of AMPKα1^(fl/fl)Foxp3^(cre)mice was significant⁃ly reduced.Additionally,while the thymus of AMPKα1^(fl/fl)Foxp3^(cre)mice exhibited normal structure,both its size and the ratio of thymus weight to body weight were significantly decreased.The knockout of AMPKα1 in Tregs led to a notable reduction in the total percentage of immature double-negative(DN)cells.Consequently,the percentage of CD4^(+)T cells derived from these DN cells also decreased,even though the percentages of DN1 and DN4 cells were higher in the thymus of AMPKα1^(fl/fl)Foxp3^(cre)mice compared to AMPKα1^(fl/fl)mice.Importantly,the proportion of Siglec-F+CD11b^(+)eosinophils in the thymus was significantly lower in AMPKα1^(fl/fl)Foxp3^(cre)mice.Knockout of AMPKα1 in Tregs resulted in a marked increase in the percentage of CD4^(+)T cells in peripheral blood,alongside a decrease in the proportion of mature CD8^(+)T cells.Similarly,the proportion of CD4^(+)T cells in the spleen of AMPKα1^(fl/fl)Foxp3^(cre)mice was elevated compared to AMPKα1^(fl/fl)mice.In contrast,the proportion of neutrophils significantly decreased,while mononuclear cell proportions increased in the spleen of AMPKα1^(fl/fl)Foxp3^(cre)mice.In lymph nodes,the medullary boundaries in AMPKα1^(fl/fl)Foxp3^(cre)mice were blurred,and the lymphoid follicles were missing,a feature not observed in AMPKα1^(fl/fl)mice.Furthermore,the knockout of AMPKα1 in Tregs reduced the CD3^(+)T cell population,particularly the CD8^(+)T cell population,in lymph nodes.Although the mature Treg cell population was significantly lower in AMPKα1^(fl/fl)Foxp3^(cre)mice,the percentage of CD4^(+)T cells was markedly in⁃creased.In contrast,there was no statistically significant difference in granulocyte populations between AMPKα1^(fl/fl)Foxp3^(cre)and AMPKα1^(fl/fl)mice.CONCLUSION:The populations of mature Tregs,CD8^(+)T cells and eosinophils in various im⁃mune organs were significantly altered in mice with Treg-specific AMPKα1 knockout,suggesting a potential remodeling of the host immune microenvironment in response to inflammatory stimuli.展开更多
In order to clarify the mechanism of action of licorice flavonoids in alleviating bone loss caused by osteoporosis,this study compared the effects of four glycyrrhiza flavonoids,naringenin,liquiritigenin,isoliquiritig...In order to clarify the mechanism of action of licorice flavonoids in alleviating bone loss caused by osteoporosis,this study compared the effects of four glycyrrhiza flavonoids,naringenin,liquiritigenin,isoliquiritigenin,and licochalcone A,on osteogenic differentiation and mineralization by molecular docking simulation,alkaline phosphatase(ALP)activity and osteocalcin(OCN)content assays,and Runt-related transcription factor 2(Runx2)expression,and explored their potential molecular mechanisms.The results of molecular docking showed that the docking score of liquiritigenin with the estrogen receptor(ER)was the highest.All four flavonoids up-regulated ALP activity and OCN concentration in MC3T3-E1 cells,thereby elevating the mineralization level,among which liquiritigenin was the most effective.Moreover,treatment with a phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002)inhibited liquiritigenin from inducing increased phosphorylation levels in the PI3K/protein kinase B(AKT)signaling pathway and up-regulation of Runx2 expression,suggesting that PI3K and AKT were involved in osteogenic action.Liquiritigenin reversed bone mineral density loss in a zebrafish osteoporosis model.These findings suggest that liquiritigenin has the most significant osteogenic effect among the four estrogen-like flavonoids,stimulating osteoblast differentiation and bone mineralization through the activation of Runx2 via the PI3K/AKT signaling pathways.In conclusion,this study highlights the great potential of liquiritigenin for preventing and treating osteoporosis.展开更多
OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO)...OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO).Administration of ligustroflavone(10,30,60 mg·kg-1,ig)15 min before ischemia,after which neurological deficit score and infarct volume were detected by longa score and TTC stain.The cell viability and necrosis rate of hypoxia-cultured PC12 cells(O2/N2/CO2,1:94:5,8 h)were evaluated by MTS and LDH release rate.Flow cytometry further verified the mortality rate of PC12 cells.Necroptosis-associated proteins(RIPK1,RIPK3 and MLKL/p-MLKL)were detected by Western blotting.The interaction between RIPK3 and RIPK1 or MLKL were confirmed by immunoprecipitation.Operating Environ⁃ment(MOE)program demonstrated the possible combination of ligustroflavone with RIPK1,RIPK3 and MLKL.RESULTS Ischemic injury(increase in neurological deficit score and infarct volume)and upregulation of necroptosis-associated proteins were showed in rat MCAO model.Administration of ligustroflavone(30 mg·kg^-1,ig)evidently improved neurological func⁃tion,reduced infarct volume,and decreased the levels of necroptosis-associated proteins except the RIPK1.Consistently,hypoxia-cultured PC12 cells caused cellular injury(LDH release and necroposis)concomitant with up-regulation of necroptosis-associated proteins,and these phenomena were blocked in the presence of ligustroflavone(25μmol·L^-1)except the elevated RIPK1 levels.Using the Molecular Operating Environment(MOE)program,we identified RIPK1,RIPK3,and MLKL as potential targets of ligustroflavone.Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced,which was blocked in the presence of ligustroflavone.CONCLUSION Ligus⁃troflavone protects rat brain from ischemic injury,and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1,RIPK3,and/or MLKL.展开更多
The incidence and mortality rate of lung cancer rank among the highest worldwide,severely endangering human health and life.Metformin,an anti-diabetes drug,has been shown to elicit anticancer activities in various tum...The incidence and mortality rate of lung cancer rank among the highest worldwide,severely endangering human health and life.Metformin,an anti-diabetes drug,has been shown to elicit anticancer activities in various tumors.However,its underlying mechanisms remain elusive.In this work,we explore the role of receptor-interacting protein 1(RIP1)which plays a crucial role in the process of cell death,in metformin-induced anticancer activities in lung cancer.Metformin inhibits lung cancer cell proliferation in a dose-dependent manner and promotes apoptotic cell death,as evidenced by metformin-induced PARP and caspase cleavage.Furthermore,the pan-caspase inhibitor z-VAD-fmk reverses metformin-induced cell death.Western blot and qPCR results suggest that metformin markedly downregulates RIP1 expression without affecting its mRNA and ubiquitination levels(0 vs 80 mmol/L,100%vs 20%,100%vs 15%).Additionally,co-immunoprecipitation and immunofluorescence results reveal that metformin may suppress RIP1 expression in an Hsp70-dependent manner,as metformin promotes Hsp70 degradation,and Hsp70 endogenously interacts with RIP1.Subsequent CCK-8,flow cytometry,and Western blot analyses suggest that metformin decreases Hsp70/RIP1 expression through AMPK/PKA/GSK-3βaxis.Consistently,results from a subcutaneous transplant tumor model indicate that metformin retards tumor growth without affecting mouse body weight.Collectively,these data highlight the part of RIP1 in metformin-induced anticancer activities in lung cancer in vitro and in vivo,providing novel strategy for lung cancer administration.展开更多
文摘Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.
文摘Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning （ISO） and oxygen glucose dep- rivation （OGD） model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride （NH4 C1） to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 （cell counting kit-8） and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA （3-Methyladenine） was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.
文摘Aim Evidence has shown that stimulation of alA-adrenorecetors receptor （alA-AR） or angiotensin II type 1 receptor （AT1R） acutely down-regulates the rapid component of the delayed rectifier K ＋ current （IKr） via protein kinase C （PKC）. This study was designed to investigate which PKC isozymes mediate down-regulations of IKr by alA-AR and AT1R. Method The whole-cell patch-clamp technique was used to record IKr in native cardio- myocytes and in human embryonic kidney （HEK） 293 cells co-transfected with human ether-a-go-go related gene （hERG） encoding α-subunit of IKr and human alA-AR or AT1R gene. Result In isolated guinea-pig ventricular cardiomyocytes the inhibitory action of Ang II on IKr was little affected by Go6976 （selectively inhibiting PKCα, β and γ） and Go6983 （selectively inhibiting PKCα, β, γ , δ, and ζ）, but was significantly antagonized by an inter- nal dialysis with PKCe-selective inhibitory peptide εV1 -2. In contrast, the inhibitory action of alA-AR agonist A61603 on IKr was remarkably attenuated by Go6976 or Go6983, but not affected by peptide εV1 -2. Moreover, specific PKC-selective inhibitory peptide antagonized the effect of A61603. The results suggested that PKCe and PKCα isoform respectively mediated the inhibitory effect of AT1R and a1A-AR. In heterologous expression system, both PKCα and e-selective activator peptides down regulated hERG current with different manner. PKCα activator peptide shifted the activation curve of the channel to the right, but PKCe-selective activator peptide did not. Simi- larly, A61603 shifted the activation curve to the right, whereas Ang Ⅱ had no effect. In addition, both A61603 and PKCα activator peptide showed inhibitory action on bERG A PKC current （an bERG mutant in which 17 of the 18 ROSITE-predicted PKC acceptor serines/threonines were changed to alanine） with a similar potency to wild type bERG current. But, both Ang Ⅱ and PKCe-selective activator peptide exhibited no effects on bERG △ PKC cur- rent. The results indicated that PKCα and PKCe isoforms down-regulated bERG current through different mecha- nism. Conclusion PKCα and PKCe isoform respectively mediates the inhibition on IKr by stimulation of AT1R and alA-AR via different molecular mechanism.
文摘OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.
文摘A protein kinase C inhibitor is found in bovine sper-matozoa. This inhibitor was purified by Sephadex G-200and isoelectrofocusion electrophoresis. The molecularweight of this protein kinase C inhibitor was about 63 000,the isoelectric point of the inhibitor was pH 4.5.
文摘OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.
基金supported by grants from NIH CA-125707 NSF CBET-0729091 (C D ),as well as NIH AI-065566 (A A )
文摘Introduction Vascular endothelial (VE)-cadherin is localized to the endothelial borders and the adherens junctions,which are regulated by changes in mitogen activated protein kinases (MAPK),GTPases,and intracellular calcium. We previously
基金Supported by the National Natural Science Foundation of China(No.81800423)the Guangdong Medical Science and Technology Research project(No.B2022102)。
文摘AIM:Regulatory T cells(Tregs)are a specialized subset of CD4^(+)T cells primarily involved in im⁃munosuppressive functions.AMP-activated protein kinase(AMPK)serves as a metabolic sensor that governs the differen⁃tiation,maturation,and immune functions of Tregs through metabolic reprogramming.However,the impact of AMPKα1(the catalytic subunit of AMPK)knockout specifically in Tregs on the host's immune microenvironment remains largely un⁃explored.METHODS:Histological changes in immune organs were assessed using HE staining.The types of immune cells and their relative population percentages in immune organs and blood were quantified through flow cytometry in both AMPKα1flox/flox(AMPKα1^(fl/fl))mice and Treg-specific AMPKα1 knockout mice(AMPKα1^(fl/fl)Foxp3^(cre)mice).RESULTS:Compared to AMPKα1^(fl/fl)mice,the percentage of eosinophils in the bone marrow of AMPKα1^(fl/fl)Foxp3^(cre)mice was significant⁃ly reduced.Additionally,while the thymus of AMPKα1^(fl/fl)Foxp3^(cre)mice exhibited normal structure,both its size and the ratio of thymus weight to body weight were significantly decreased.The knockout of AMPKα1 in Tregs led to a notable reduction in the total percentage of immature double-negative(DN)cells.Consequently,the percentage of CD4^(+)T cells derived from these DN cells also decreased,even though the percentages of DN1 and DN4 cells were higher in the thymus of AMPKα1^(fl/fl)Foxp3^(cre)mice compared to AMPKα1^(fl/fl)mice.Importantly,the proportion of Siglec-F+CD11b^(+)eosinophils in the thymus was significantly lower in AMPKα1^(fl/fl)Foxp3^(cre)mice.Knockout of AMPKα1 in Tregs resulted in a marked increase in the percentage of CD4^(+)T cells in peripheral blood,alongside a decrease in the proportion of mature CD8^(+)T cells.Similarly,the proportion of CD4^(+)T cells in the spleen of AMPKα1^(fl/fl)Foxp3^(cre)mice was elevated compared to AMPKα1^(fl/fl)mice.In contrast,the proportion of neutrophils significantly decreased,while mononuclear cell proportions increased in the spleen of AMPKα1^(fl/fl)Foxp3^(cre)mice.In lymph nodes,the medullary boundaries in AMPKα1^(fl/fl)Foxp3^(cre)mice were blurred,and the lymphoid follicles were missing,a feature not observed in AMPKα1^(fl/fl)mice.Furthermore,the knockout of AMPKα1 in Tregs reduced the CD3^(+)T cell population,particularly the CD8^(+)T cell population,in lymph nodes.Although the mature Treg cell population was significantly lower in AMPKα1^(fl/fl)Foxp3^(cre)mice,the percentage of CD4^(+)T cells was markedly in⁃creased.In contrast,there was no statistically significant difference in granulocyte populations between AMPKα1^(fl/fl)Foxp3^(cre)and AMPKα1^(fl/fl)mice.CONCLUSION:The populations of mature Tregs,CD8^(+)T cells and eosinophils in various im⁃mune organs were significantly altered in mice with Treg-specific AMPKα1 knockout,suggesting a potential remodeling of the host immune microenvironment in response to inflammatory stimuli.
文摘In order to clarify the mechanism of action of licorice flavonoids in alleviating bone loss caused by osteoporosis,this study compared the effects of four glycyrrhiza flavonoids,naringenin,liquiritigenin,isoliquiritigenin,and licochalcone A,on osteogenic differentiation and mineralization by molecular docking simulation,alkaline phosphatase(ALP)activity and osteocalcin(OCN)content assays,and Runt-related transcription factor 2(Runx2)expression,and explored their potential molecular mechanisms.The results of molecular docking showed that the docking score of liquiritigenin with the estrogen receptor(ER)was the highest.All four flavonoids up-regulated ALP activity and OCN concentration in MC3T3-E1 cells,thereby elevating the mineralization level,among which liquiritigenin was the most effective.Moreover,treatment with a phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002)inhibited liquiritigenin from inducing increased phosphorylation levels in the PI3K/protein kinase B(AKT)signaling pathway and up-regulation of Runx2 expression,suggesting that PI3K and AKT were involved in osteogenic action.Liquiritigenin reversed bone mineral density loss in a zebrafish osteoporosis model.These findings suggest that liquiritigenin has the most significant osteogenic effect among the four estrogen-like flavonoids,stimulating osteoblast differentiation and bone mineralization through the activation of Runx2 via the PI3K/AKT signaling pathways.In conclusion,this study highlights the great potential of liquiritigenin for preventing and treating osteoporosis.
基金National Natural Science Foundation of China(8157343081872873)Hunan Provincial Natural Science Foundation of China(2015JJ2156)
文摘OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO).Administration of ligustroflavone(10,30,60 mg·kg-1,ig)15 min before ischemia,after which neurological deficit score and infarct volume were detected by longa score and TTC stain.The cell viability and necrosis rate of hypoxia-cultured PC12 cells(O2/N2/CO2,1:94:5,8 h)were evaluated by MTS and LDH release rate.Flow cytometry further verified the mortality rate of PC12 cells.Necroptosis-associated proteins(RIPK1,RIPK3 and MLKL/p-MLKL)were detected by Western blotting.The interaction between RIPK3 and RIPK1 or MLKL were confirmed by immunoprecipitation.Operating Environ⁃ment(MOE)program demonstrated the possible combination of ligustroflavone with RIPK1,RIPK3 and MLKL.RESULTS Ischemic injury(increase in neurological deficit score and infarct volume)and upregulation of necroptosis-associated proteins were showed in rat MCAO model.Administration of ligustroflavone(30 mg·kg^-1,ig)evidently improved neurological func⁃tion,reduced infarct volume,and decreased the levels of necroptosis-associated proteins except the RIPK1.Consistently,hypoxia-cultured PC12 cells caused cellular injury(LDH release and necroposis)concomitant with up-regulation of necroptosis-associated proteins,and these phenomena were blocked in the presence of ligustroflavone(25μmol·L^-1)except the elevated RIPK1 levels.Using the Molecular Operating Environment(MOE)program,we identified RIPK1,RIPK3,and MLKL as potential targets of ligustroflavone.Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced,which was blocked in the presence of ligustroflavone.CONCLUSION Ligus⁃troflavone protects rat brain from ischemic injury,and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1,RIPK3,and/or MLKL.
文摘The incidence and mortality rate of lung cancer rank among the highest worldwide,severely endangering human health and life.Metformin,an anti-diabetes drug,has been shown to elicit anticancer activities in various tumors.However,its underlying mechanisms remain elusive.In this work,we explore the role of receptor-interacting protein 1(RIP1)which plays a crucial role in the process of cell death,in metformin-induced anticancer activities in lung cancer.Metformin inhibits lung cancer cell proliferation in a dose-dependent manner and promotes apoptotic cell death,as evidenced by metformin-induced PARP and caspase cleavage.Furthermore,the pan-caspase inhibitor z-VAD-fmk reverses metformin-induced cell death.Western blot and qPCR results suggest that metformin markedly downregulates RIP1 expression without affecting its mRNA and ubiquitination levels(0 vs 80 mmol/L,100%vs 20%,100%vs 15%).Additionally,co-immunoprecipitation and immunofluorescence results reveal that metformin may suppress RIP1 expression in an Hsp70-dependent manner,as metformin promotes Hsp70 degradation,and Hsp70 endogenously interacts with RIP1.Subsequent CCK-8,flow cytometry,and Western blot analyses suggest that metformin decreases Hsp70/RIP1 expression through AMPK/PKA/GSK-3βaxis.Consistently,results from a subcutaneous transplant tumor model indicate that metformin retards tumor growth without affecting mouse body weight.Collectively,these data highlight the part of RIP1 in metformin-induced anticancer activities in lung cancer in vitro and in vivo,providing novel strategy for lung cancer administration.
