Objective:Buyinqianzheng Formula(BYQZF)is clinically employed in traditional Chinese medicine to treat Parkinson's disease(PD)by improving mitochondrial dysfunction.However,the underlying mechanisms by which BYQZF...Objective:Buyinqianzheng Formula(BYQZF)is clinically employed in traditional Chinese medicine to treat Parkinson's disease(PD)by improving mitochondrial dysfunction.However,the underlying mechanisms by which BYQZF affects mitochondrial morphology remain unknown.Therefore,we observed the effects of BYQZF on mitochondria from the perspective of the PINK1/Parkin pathway.Methods:Cell survival rates were assessed by Cell Counting Kit-8 assay.Expression levels of PINK1 and Parkin mRNA were examined by qRT-PCR.Protein expression levels of PINK1,PINK1-Ser228,Parkin,Parkin-Ser65,Drp1,and Drp1-Ser637 were examined by western blotting.PINK1,Parkin,and MitoTracker?Red CMXRos(MTR)were stained by triple-labeled immunofluorescence,and observed under laser confocal microscopy.Results:Cell survival rate,mitochondrial form factor,mean length and number of mitochondrial network branches,mitochondrial activity,m RNA expression levels of PINK1 and Parkin,and protein expression levels of PINK1,Parkin,and Drp1-Ser637 were reduced after 1-methyl-4-phenylpyridinium(MPP^(+))intervention.In contrast,Pearson's correlation coefficients between PINK1 and Parkin,and between Parkin and MTR,as well as protein expression levels of PINK1-Ser228,Parkin-Ser65,and Drp1 increased significantly after MPP^(+) intervention.Treatment with BYQZF increased cell survival rate,mitochondrial form factor,mean length and number of mitochondrial network branches,mitochondrial activity,m RNA expression levels of PINK1 and Parkin,and expression of PINK1,Parkin,and Drp1-Ser637 proteins.Pearson's correlation coefficients between PINK1 and Parkin,and between Parkin and MTR,as well as protein expression levels of PINK1-Ser228,Parkin-Ser65,and Drp1 decreased after BYQZF treatment.Conclusion:These results demonstrate that BYQZF has a protective effect on mitochondrial molecular mechanisms in the PD cell model,and the mechanism is related to the PINK1/Parkin pathway.展开更多
The vast majority of in vitro studies have demonstrated that PINK1 phosphorylates Parkin to work together in mitophagy to protect against neuronal degeneration.However,it remains largely unclear how PINK1 and Parkin a...The vast majority of in vitro studies have demonstrated that PINK1 phosphorylates Parkin to work together in mitophagy to protect against neuronal degeneration.However,it remains largely unclear how PINK1 and Parkin are expressed in mammalian brains.This has been difficult to address because of the intrinsically low levels of PINK1 and undetectable levels of phosphorylated Parkin in small animals.Understanding this issue is critical for elucidating the in vivo roles of PINK1 and Parkin.Recently,we showed that the PINK1 kinase is selectively expressed as a truncated form(PINK1–55)in the primate brain.In the present study,we used multiple antibodies,including our recently developed monoclonal anti-PINK1,to validate the selective expression of PINK1 in the primate brain.We found that PINK1 was stably expressed in the monkey brain at postnatal and adulthood stages,which is consistent with the findings that depleting PINK1 can cause neuronal loss in developing and adult monkey brains.PINK1 was enriched in the membrane-bound fractionations,whereas Parkin was soluble with a distinguishable distribution.Immunofluorescent double staining experiments showed that PINK1 and Parkin did not colocalize under physiological conditions in cultured monkey astrocytes,though they did colocalize on mitochondria when the cells were exposed to mitochondrial stress.These findings suggest that PINK1 and Parkin may have distinct roles beyond their well-known function in mitophagy during mitochondrial damage.展开更多
Objective Keshan disease(KD)is a myocardial mitochondrial disease closely related to insufficient selenium(Se)and protein intake.PTEN induced putative kinase 1(PINK1)/Parkin mediated mitochondrial autophagy regulates ...Objective Keshan disease(KD)is a myocardial mitochondrial disease closely related to insufficient selenium(Se)and protein intake.PTEN induced putative kinase 1(PINK1)/Parkin mediated mitochondrial autophagy regulates various physiological and pathological processes in the body.This study aimed to elucidate the relationship between PINK1/Parkin-regulated mitochondrial autophagy and KD-related myocardial injury.Methods A low Se and low protein animal model was established.One hundred Wistar rats were randomly divided into 5 groups(control group,low Se group,low protein group,low Se+low protein group,and corn from KD area group).The JC-1 method was used to detect the mitochondrial membrane potential(MMP).ELISA was used to detect serum creatine kinase MB(CK-MB),cardiac troponin I(cTnI),and mitochondrial-glutamicoxalacetic transaminase(M-GOT)levels.RT-PCR and Western blot analysis were used to detect the expression of PINK1,Parkin,sequestome 1(P62),and microtubule-associated proteins1A/1B light chain 3B(MAP1LC3B).Results The MMP was significantly decreased and the activity of CK-MB,cTnI,and M-GOT significantly increased in each experimental group(low Se group,low protein group,low Se+low protein group and corn from KD area group)compared with the control group(P<0.05 for all).The mRNA and protein expression levels of PINK1,Parkin and MAP1LC3B were profoundly increased,and those of P62 markedly decreased in the experimental groups compared with the control group(P<0.05 for all).Conclusion Low Se and low protein levels exacerbate myocardial damage in KD by affecting the PINK1/Parkin-mediated mitochondrial autophagy pathway.展开更多
目的探讨金雀根和黄芪配伍对糖尿病肾病(DKD)大鼠的影响。方法将SD大鼠随机分为正常组、模型组、恩格列净组、金雀根组、黄芪组、配伍组,每组10只。采用单侧切除肾脏联合腹腔注射链脲佐菌素(STZ)复制DKD大鼠模型,造模成功后灌胃相应剂...目的探讨金雀根和黄芪配伍对糖尿病肾病(DKD)大鼠的影响。方法将SD大鼠随机分为正常组、模型组、恩格列净组、金雀根组、黄芪组、配伍组,每组10只。采用单侧切除肾脏联合腹腔注射链脲佐菌素(STZ)复制DKD大鼠模型,造模成功后灌胃相应剂量药物8周。第0、4、8周检测24 h尿微量白蛋白(24 h U-mALB);ELISA法检测Scr、BUN、CysC、MDA水平及SOD活性;荧光探针法检测肾组织ROS表达;HE、PAS、Masson、PASM-Masson染色观察肾组织病理结构改变;免疫组织化学法检测肾组织NOX4、Drp1、MFN2、P62表达;Western blot法检测肾组织PINK1、MFN2、Parkin、LC3-Ⅱ/Ⅰ、P62、p-Drp1蛋白表达。结果与模型组比较,各给药组大鼠24 h U-mALB、BUN、Scr、CysC水平均降低(P<0.01),肾组织病理性结构损伤改善;血清和组织MDA水平降低(P<0.01),SOD活性升高(P<0.01);肾组织PINK1、MFN2、Parkin、LC3-Ⅱ/Ⅰ蛋白表达升高(P<0.05,P<0.01),p-Drp1、P62蛋白表达降低(P<0.01),其中配伍组作用优于单用药组(P<0.05,P<0.01)。结论金雀根和黄芪配伍可能是通过调控PINK1/MFN2/Parkin通路改善线粒体动力学,激活线粒体自噬,抑制氧化应激,减轻肾脏病理损伤,改善DKD大鼠肾功能。展开更多
Objective: To explore the protective effect of bloodletting acupuncture at twelve Jing-well points on hand(BAJP) on acute hypobaric hypoxia(AHH)-induced brain injury in rats and its possible mechanisms.Methods: Sevent...Objective: To explore the protective effect of bloodletting acupuncture at twelve Jing-well points on hand(BAJP) on acute hypobaric hypoxia(AHH)-induced brain injury in rats and its possible mechanisms.Methods: Seventy-five Sprague Dawley rats were divided into 5 groups by a random number table(n=15),including control, model, BAJP, BAJP+3-methyladenine(3-MA), and bloodletting acupuncture at non-acupoint(BANA, tail tip blooding) groups. After 7-day pre-treatment, AHH models were established using hypobaric oxygen chambers. The levels of S100B, glial fibrillary acidic protein(GFAP), superoxide dismutase(SOD), and malondialdehyde(MDA) in serum were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method were used to assess hippocampal histopathology and apoptosis. Transmission electron microscopy assay was used to observe mitochondrial damage and autophagosomes in hippocampal tissues. Flow cytometry was used to detect mitochondrial membrane potential(MMP). The mitochondrial respiratory chain complexes Ⅰ, Ⅲ and Ⅳ activities and ATPase in hippocampal tissue were evaluated, respectively. Western blot analysis was used to detect the protein expressions of Beclin1, autophagy protein 5(ATG5), microtubule-associated protein 1 light chain 3 beta(LC3B), phosphatase and tensin homolog induced kinase 1(PINK1), and Parkin in hippocampal tissues. The mRNA expressions of Beclin1, ATG5 and LC3-Ⅱ were analyzed by quantitative real-time polymerase chain reaction. Results: BAJP treatment reduced hippocampal tissue injury and inhibited hippocampal cell apoptosis in AHH rats. BAJP reduced oxidative stress by decreasing S100B, GFAP and MDA levels and increasing SOD level in the serum of AHH rats(P<0.05 or P<0.01). Then, BAJP increased MMP, the mitochondrial respiratory chain complexes Ⅰ, Ⅲ and Ⅳ activities, and the mitochondrial ATPase activity in AHH rats(all P<0.01). BAJP improved mitochondrial swelling and increased the autophagosome number in hippocampal tissue of AHH rats. Moreover,BAJP treatment increased the protein and mRNA expressions of Beclin1 and ATG5 and LC3-Ⅱ/LC3-Ⅰratio in AHH rats(all P<0.01) and activated the PINK1/Parkin pathway(P<0.01). Finally, 3-MA attenuated the therapeutic effect of BAJP on AHH rats(P<0.05 or P<0.01). Conclusion: BAJP was an effective treatment for AHH-induced brain injury, and the mechanism might be through reducing hippocampal tissue injury via increasing the PINK1/Parkin pathway and enhancement of mitochondrial autophagy.展开更多
基金the National Natural Science Foundation of China(Grant Nos.81573773 and 81774110)。
文摘Objective:Buyinqianzheng Formula(BYQZF)is clinically employed in traditional Chinese medicine to treat Parkinson's disease(PD)by improving mitochondrial dysfunction.However,the underlying mechanisms by which BYQZF affects mitochondrial morphology remain unknown.Therefore,we observed the effects of BYQZF on mitochondria from the perspective of the PINK1/Parkin pathway.Methods:Cell survival rates were assessed by Cell Counting Kit-8 assay.Expression levels of PINK1 and Parkin mRNA were examined by qRT-PCR.Protein expression levels of PINK1,PINK1-Ser228,Parkin,Parkin-Ser65,Drp1,and Drp1-Ser637 were examined by western blotting.PINK1,Parkin,and MitoTracker?Red CMXRos(MTR)were stained by triple-labeled immunofluorescence,and observed under laser confocal microscopy.Results:Cell survival rate,mitochondrial form factor,mean length and number of mitochondrial network branches,mitochondrial activity,m RNA expression levels of PINK1 and Parkin,and protein expression levels of PINK1,Parkin,and Drp1-Ser637 were reduced after 1-methyl-4-phenylpyridinium(MPP^(+))intervention.In contrast,Pearson's correlation coefficients between PINK1 and Parkin,and between Parkin and MTR,as well as protein expression levels of PINK1-Ser228,Parkin-Ser65,and Drp1 increased significantly after MPP^(+) intervention.Treatment with BYQZF increased cell survival rate,mitochondrial form factor,mean length and number of mitochondrial network branches,mitochondrial activity,m RNA expression levels of PINK1 and Parkin,and expression of PINK1,Parkin,and Drp1-Ser637 proteins.Pearson's correlation coefficients between PINK1 and Parkin,and between Parkin and MTR,as well as protein expression levels of PINK1-Ser228,Parkin-Ser65,and Drp1 decreased after BYQZF treatment.Conclusion:These results demonstrate that BYQZF has a protective effect on mitochondrial molecular mechanisms in the PD cell model,and the mechanism is related to the PINK1/Parkin pathway.
基金supported by the National Natural Science Foundation of China,Nos.32070534(to WY),32370567(to WY),82371874(to XL),81830032(to XL),82071421(to SL)Key Field Research and Development Program of Guangdong Province,No.2018B030337001(to XL)+2 种基金Guangzhou Key Research Program on Brain Science,No.202007030008(to XL)Department of Science and Technology of Guangdong Province,Nos.2021ZT09Y007,2020B121201006(to XL)Guangdong Basic and Applied Basic Research Foundation,Nos.2022A1515012301(to WY),2023B1515020031(to WY).
文摘The vast majority of in vitro studies have demonstrated that PINK1 phosphorylates Parkin to work together in mitophagy to protect against neuronal degeneration.However,it remains largely unclear how PINK1 and Parkin are expressed in mammalian brains.This has been difficult to address because of the intrinsically low levels of PINK1 and undetectable levels of phosphorylated Parkin in small animals.Understanding this issue is critical for elucidating the in vivo roles of PINK1 and Parkin.Recently,we showed that the PINK1 kinase is selectively expressed as a truncated form(PINK1–55)in the primate brain.In the present study,we used multiple antibodies,including our recently developed monoclonal anti-PINK1,to validate the selective expression of PINK1 in the primate brain.We found that PINK1 was stably expressed in the monkey brain at postnatal and adulthood stages,which is consistent with the findings that depleting PINK1 can cause neuronal loss in developing and adult monkey brains.PINK1 was enriched in the membrane-bound fractionations,whereas Parkin was soluble with a distinguishable distribution.Immunofluorescent double staining experiments showed that PINK1 and Parkin did not colocalize under physiological conditions in cultured monkey astrocytes,though they did colocalize on mitochondria when the cells were exposed to mitochondrial stress.These findings suggest that PINK1 and Parkin may have distinct roles beyond their well-known function in mitophagy during mitochondrial damage.
基金supported by the Natural Science Foundation of Heilongjiang Province(No.LH2021H009).
文摘Objective Keshan disease(KD)is a myocardial mitochondrial disease closely related to insufficient selenium(Se)and protein intake.PTEN induced putative kinase 1(PINK1)/Parkin mediated mitochondrial autophagy regulates various physiological and pathological processes in the body.This study aimed to elucidate the relationship between PINK1/Parkin-regulated mitochondrial autophagy and KD-related myocardial injury.Methods A low Se and low protein animal model was established.One hundred Wistar rats were randomly divided into 5 groups(control group,low Se group,low protein group,low Se+low protein group,and corn from KD area group).The JC-1 method was used to detect the mitochondrial membrane potential(MMP).ELISA was used to detect serum creatine kinase MB(CK-MB),cardiac troponin I(cTnI),and mitochondrial-glutamicoxalacetic transaminase(M-GOT)levels.RT-PCR and Western blot analysis were used to detect the expression of PINK1,Parkin,sequestome 1(P62),and microtubule-associated proteins1A/1B light chain 3B(MAP1LC3B).Results The MMP was significantly decreased and the activity of CK-MB,cTnI,and M-GOT significantly increased in each experimental group(low Se group,low protein group,low Se+low protein group and corn from KD area group)compared with the control group(P<0.05 for all).The mRNA and protein expression levels of PINK1,Parkin and MAP1LC3B were profoundly increased,and those of P62 markedly decreased in the experimental groups compared with the control group(P<0.05 for all).Conclusion Low Se and low protein levels exacerbate myocardial damage in KD by affecting the PINK1/Parkin-mediated mitochondrial autophagy pathway.
文摘近年来,我国脑卒中总体发病率呈上升趋势,其以发病突然、起病急骤的神经功能缺损为主要特征[1]。脑卒中主要包括缺血性脑卒中(ischemic stroke,IS)和出血性脑卒中两大类,而最常见的类型是IS。IS导致神经细胞血液供应不足,所需的葡萄糖和氧气减少,神经细胞稳态被扰乱,从而引发包括兴奋性毒性、氧化应激、炎症、细胞凋亡在内的病理生理过程[2]。研究表明,线粒体自噬可以及时清除受损的线粒体,有效控制线粒体质量和功能,维持神经细胞稳态和防止神经细胞凋亡,在IS病理生理过程中起着不可忽视的作用[3]。现就PTEN诱导推定激酶1(PTEN-induced putative kinase protein 1,PINK1)/人帕金森蛋白2(human Parkinson disease protein 2,Parkin)介导线粒体自噬对IS作用机制的研究进展予以综述。
文摘目的探讨金雀根和黄芪配伍对糖尿病肾病(DKD)大鼠的影响。方法将SD大鼠随机分为正常组、模型组、恩格列净组、金雀根组、黄芪组、配伍组,每组10只。采用单侧切除肾脏联合腹腔注射链脲佐菌素(STZ)复制DKD大鼠模型,造模成功后灌胃相应剂量药物8周。第0、4、8周检测24 h尿微量白蛋白(24 h U-mALB);ELISA法检测Scr、BUN、CysC、MDA水平及SOD活性;荧光探针法检测肾组织ROS表达;HE、PAS、Masson、PASM-Masson染色观察肾组织病理结构改变;免疫组织化学法检测肾组织NOX4、Drp1、MFN2、P62表达;Western blot法检测肾组织PINK1、MFN2、Parkin、LC3-Ⅱ/Ⅰ、P62、p-Drp1蛋白表达。结果与模型组比较,各给药组大鼠24 h U-mALB、BUN、Scr、CysC水平均降低(P<0.01),肾组织病理性结构损伤改善;血清和组织MDA水平降低(P<0.01),SOD活性升高(P<0.01);肾组织PINK1、MFN2、Parkin、LC3-Ⅱ/Ⅰ蛋白表达升高(P<0.05,P<0.01),p-Drp1、P62蛋白表达降低(P<0.01),其中配伍组作用优于单用药组(P<0.05,P<0.01)。结论金雀根和黄芪配伍可能是通过调控PINK1/MFN2/Parkin通路改善线粒体动力学,激活线粒体自噬,抑制氧化应激,减轻肾脏病理损伤,改善DKD大鼠肾功能。
基金the Applied Basic Research Project of Science and Technology Department of Qinghai Province(No.2020-ZJ-760)。
文摘Objective: To explore the protective effect of bloodletting acupuncture at twelve Jing-well points on hand(BAJP) on acute hypobaric hypoxia(AHH)-induced brain injury in rats and its possible mechanisms.Methods: Seventy-five Sprague Dawley rats were divided into 5 groups by a random number table(n=15),including control, model, BAJP, BAJP+3-methyladenine(3-MA), and bloodletting acupuncture at non-acupoint(BANA, tail tip blooding) groups. After 7-day pre-treatment, AHH models were established using hypobaric oxygen chambers. The levels of S100B, glial fibrillary acidic protein(GFAP), superoxide dismutase(SOD), and malondialdehyde(MDA) in serum were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method were used to assess hippocampal histopathology and apoptosis. Transmission electron microscopy assay was used to observe mitochondrial damage and autophagosomes in hippocampal tissues. Flow cytometry was used to detect mitochondrial membrane potential(MMP). The mitochondrial respiratory chain complexes Ⅰ, Ⅲ and Ⅳ activities and ATPase in hippocampal tissue were evaluated, respectively. Western blot analysis was used to detect the protein expressions of Beclin1, autophagy protein 5(ATG5), microtubule-associated protein 1 light chain 3 beta(LC3B), phosphatase and tensin homolog induced kinase 1(PINK1), and Parkin in hippocampal tissues. The mRNA expressions of Beclin1, ATG5 and LC3-Ⅱ were analyzed by quantitative real-time polymerase chain reaction. Results: BAJP treatment reduced hippocampal tissue injury and inhibited hippocampal cell apoptosis in AHH rats. BAJP reduced oxidative stress by decreasing S100B, GFAP and MDA levels and increasing SOD level in the serum of AHH rats(P<0.05 or P<0.01). Then, BAJP increased MMP, the mitochondrial respiratory chain complexes Ⅰ, Ⅲ and Ⅳ activities, and the mitochondrial ATPase activity in AHH rats(all P<0.01). BAJP improved mitochondrial swelling and increased the autophagosome number in hippocampal tissue of AHH rats. Moreover,BAJP treatment increased the protein and mRNA expressions of Beclin1 and ATG5 and LC3-Ⅱ/LC3-Ⅰratio in AHH rats(all P<0.01) and activated the PINK1/Parkin pathway(P<0.01). Finally, 3-MA attenuated the therapeutic effect of BAJP on AHH rats(P<0.05 or P<0.01). Conclusion: BAJP was an effective treatment for AHH-induced brain injury, and the mechanism might be through reducing hippocampal tissue injury via increasing the PINK1/Parkin pathway and enhancement of mitochondrial autophagy.
