OBJECTIVE To investigate the autophagic effect of the compounds from the Chinese medicinal herbs,Radix polygalae as a potential neuroprotective agent that enhances the clearance of mutant Huntingtin andα-synuclein in...OBJECTIVE To investigate the autophagic effect of the compounds from the Chinese medicinal herbs,Radix polygalae as a potential neuroprotective agent that enhances the clearance of mutant Huntingtin andα-synuclein in PC-12 cells.METHODS Radix polygalae was extracted with 75%ethanol using refluxing method,and its quality was assayed by UHPLC-TOF-MS.The autophagic effect of Radix polygalae extract,and its major components including polygalacic acid,senegenin and onjisaponin B were investigated using the green fluorescent protein-light chain 3(GFP-LC3)autophagy detection and Western blot platforms for detecting the autophagic markers,GFP-LC3 puncta formation and LC3Ⅱ expression.The degradation of A53Tα-synuclein and the inhibition of α-synuclein oligo merization related to the Parkinson disease were assayed using Western blot and flow cytometer analysis,respectively.While the cytotoxicity and the degradation of the mutant Huntingtin associated with the Huntingtin disease were investigated using MTT method and flow cytometer analysis.RESULTS Radix polygalae ethanol extract and onjisaponin B improved the GFP-LC3 puncta formation and expression of LC3Ⅱ with time and dose manner,and this induction was activated via AMPK-m TOR and Atg 7 gene pathway.Furthermore,the clearance ofα-synuclein and mutant Huntingtin was enhanced via autophagy induction with the treatment of Radix polygalae ethanol extract and onjisaponin B.CONCLUSION Findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer,onjisaponin B,which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.展开更多
Cyclooxygenase-2(COX-2),an important enzyme,plays a pathological role in diseases,which can be inhibited by proanthocyanidins(PCs) effectively.In this paper,we investigated the inhibitive mechanism of COX-2 performed ...Cyclooxygenase-2(COX-2),an important enzyme,plays a pathological role in diseases,which can be inhibited by proanthocyanidins(PCs) effectively.In this paper,we investigated the inhibitive mechanism of COX-2 performed by PCs.Reverse transcription-polymerase chain reaction(RT-PCR) was performed to identify the mRNA expression level of COX-2 in A549 cell,which was induced by interleukin-1 beta(IL-1β).The pGL3 luciferase reporter vector containing the COX-2 gene promoter fragment(pGL3/COX-2p) was transfected into A549 cell induced by IL-1β,the interference on the COX-2 promoter activity from PCs was analyzed using a dualluciferase reporter assay,and the expressions of the nuclear factor κB composed of subunit p65(NF-κB/p65) and the inhibitor-κB(I-κB) were measured by the Western blotting and immunocytochemistry.The results exhibited that PCs not only inhibited the transcript of COX-2 mRNA and the COX-2 promoter activity,but also suppressed the nuclear translocation of NF-κB/p65 protein and the degradation of I-κB protein.展开更多
基金supported by Macao Foundation(Project code:0212)FDCT grant from the Science and Technology Development Fund of Macao(Project code:076/2011/A3)
文摘OBJECTIVE To investigate the autophagic effect of the compounds from the Chinese medicinal herbs,Radix polygalae as a potential neuroprotective agent that enhances the clearance of mutant Huntingtin andα-synuclein in PC-12 cells.METHODS Radix polygalae was extracted with 75%ethanol using refluxing method,and its quality was assayed by UHPLC-TOF-MS.The autophagic effect of Radix polygalae extract,and its major components including polygalacic acid,senegenin and onjisaponin B were investigated using the green fluorescent protein-light chain 3(GFP-LC3)autophagy detection and Western blot platforms for detecting the autophagic markers,GFP-LC3 puncta formation and LC3Ⅱ expression.The degradation of A53Tα-synuclein and the inhibition of α-synuclein oligo merization related to the Parkinson disease were assayed using Western blot and flow cytometer analysis,respectively.While the cytotoxicity and the degradation of the mutant Huntingtin associated with the Huntingtin disease were investigated using MTT method and flow cytometer analysis.RESULTS Radix polygalae ethanol extract and onjisaponin B improved the GFP-LC3 puncta formation and expression of LC3Ⅱ with time and dose manner,and this induction was activated via AMPK-m TOR and Atg 7 gene pathway.Furthermore,the clearance ofα-synuclein and mutant Huntingtin was enhanced via autophagy induction with the treatment of Radix polygalae ethanol extract and onjisaponin B.CONCLUSION Findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer,onjisaponin B,which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.
基金the Special Supported by Guangdong Key Program (No.GX0307)the Guangdong Science and Technology Planning Project in the Field of Social Development (No.35025)
文摘Cyclooxygenase-2(COX-2),an important enzyme,plays a pathological role in diseases,which can be inhibited by proanthocyanidins(PCs) effectively.In this paper,we investigated the inhibitive mechanism of COX-2 performed by PCs.Reverse transcription-polymerase chain reaction(RT-PCR) was performed to identify the mRNA expression level of COX-2 in A549 cell,which was induced by interleukin-1 beta(IL-1β).The pGL3 luciferase reporter vector containing the COX-2 gene promoter fragment(pGL3/COX-2p) was transfected into A549 cell induced by IL-1β,the interference on the COX-2 promoter activity from PCs was analyzed using a dualluciferase reporter assay,and the expressions of the nuclear factor κB composed of subunit p65(NF-κB/p65) and the inhibitor-κB(I-κB) were measured by the Western blotting and immunocytochemistry.The results exhibited that PCs not only inhibited the transcript of COX-2 mRNA and the COX-2 promoter activity,but also suppressed the nuclear translocation of NF-κB/p65 protein and the degradation of I-κB protein.
