Effect of ganoderma spore on mitochondrial autophagy and apoptosis in testicular tissue of diabetic rats / 中国组织工程研究

BACKGROUND:As a common complication of diabetes mellitus,male reproductive disorders have received increasing attention in recent years.Ganoderma spore have hypoglycemic,antioxidant and anti-inflammatory effects,but the regulatory mechanism for diabetic testicular tissue has not been fully elucidated. OBJECTIVE:To investigate the effect of ganoderma spore on the PTEN-induced kinase 1/E3 ubiquitin ligase pathway and cell apoptosis in testicular tissue of diabetic rats. METHODS:Forty male Sprague-Dawley rats were randomly divided into normal group,high fat and high sugar group,diabetic group and ganoderma spore group,with 10 rats in each group.The latter three groups were given high fat/high sugar diet until the end of the experiment.After 1 month of high fat/high sugar diet,the diabetic and ganoderma spore groups were given intraperitoneal injection of streptozotocin(30 mg/kg per day)to establish type 2 diabetic rat models.After successful modeling,the ganoderma spore group was intragastrically given ganoderma spore(300 mg/kg per day),and the other groups were given the same amount of normal saline for continuous 12 weeks.The sperm number and morphology were detected.The histopathological changes of the testicle were observed.Serum testosterone and oxidative stress levels in testicular tissue were measured.The levels of PTEN-induced kinase 1,E3 ubiquitin ligase,and anti-nucleoporin p62 were analyzed by immunohistochemistry and the expression of PTEN-induced kinase 1,E3 ubiquitin ligase,anti-nucleoporin p62,programmed cell death-1,microtubule-associated protein light chain 3 Ⅱ/Ⅰ,caspase 3,cleaved-caspase 3 were detected by western blot assay. RESULTS AND CONCLUSION:Compared with the normal group and the high fat and high sugar group,the diabetic group had decreased sperm number(P＜0.01),increased sperm malformation rate(P＜0.01),and decreased serum testosterone level(P＜0.01).Compared with the diabetic group,ganoderma spore intervention could increase the sperm number(P＜0.05),decrease the malformation rate(P＜0.01),and increase the serum testosterone level(P＜0.01).Compared with the normal group and the high fat and high sugar group,the malondialdehyde level in testis tissue was increased in the diabetic group(P＜0.01),while the levels of glutathione deoxidase and superoxide dismutase decreased(P＜0.01).Compared with the diabetic group,the malondialdehyde level in testis tissue was decreased in the ganoderma spore group(P＜0.01),and the levels of glutathione deoxidase and superoxide dismutase increased(P＜0.01).Immunohistochemical staining showed that compared with the normal group and the high fat and high sugar group,the positive expressions of PTEN-induced kinase 1 and E3 ubiquitin ligase in testicular tissue were decreased in the diabetic group,while the positive expressions of anti-nucleoporin p62 were increased.Compared with the diabetic group,the positive expressions of PTEN-induced kinase 1 and E3 ubiquitin ligase in testicular tissue e were increased in the ganoderma spore group,while the positive expression of anti-nucleoporin p62 was decreased.Western blot assay results indicated that compared to the normal group and the high fat and high sugar group,the expression of PTEN-induced kinase 1 and E3 ubiquitin ligase,programmed cell death-1 and the ratio of microtubule-associated protein light chain 3 Ⅱ/Ⅰ protein were decreased in the diabetic group(P＜0.05 or P＜0.01),while the expressions of anti-nucleoporin p62,caspase3 and cleaved-caspase3 were increased(P＜0.01).Compared with the diabetic group,ganoderma spore intervention could elevate the expression of PTEN-induced kinase 1 and E3 ubiquitin ligase,programmed cell death-1 and the ratio of microtubule-associated protein light chain 3 Ⅱ/Ⅰ protein(P＜0.05 or P＜0.01)as well as reduce the expressions of anti-nucleoporin p62,caspase3 and cleaved-caspase3(P＜0.05 or P＜0.01).Overall,ganoderma spores may activate the PTEN-induced kinase 1/E3 ubiquitin ligase pathway to enhance autophagy in testicular tissue and reduce apoptosis in tissue cells,so as to protect testicular tissue.

Role of Mitophagy in Myocardial Ischemia/Reperfusion Injury and Chinese Medicine Treatment / 中国结合医学杂志

Mitophagy is one of the important targets for the prevention and treatment of myocardial ischemia/reperfusion injury (MIRI). Moderate mitophagy can remove damaged mitochondria, inhibit excessive reactive oxygen species accumulation, and protect mitochondria from damage. However, excessive enhancement of mitophagy greatly reduces adenosine triphosphate production and energy supply for cell survival, and aggravates cell death. How dysfunctional mitochondria are selectively recognized and engulfed is related to the interaction of adaptors on the mitochondrial membrane, which mainly include phosphatase and tensin homolog deleted on chromosome ten (PTEN)-induced kinase 1/Parkin, hypoxia-inducible factor-1 α/Bcl-2 and adenovirus e1b19k Da interacting protein 3, FUN-14 domain containing protein 1 receptor-mediated mitophagy pathway and so on. In this review, the authors briefly summarize the main pathways currently studied on mitophagy and the relationship between mitophagy and MIRI, and incorporate and analyze research data on prevention and treatment of MIRI with Chinese medicine, thereby provide relevant theoretical basis and treatment ideas for clinical prevention of MIRI.

Elevated phosphorylated ubiquitin level elicits neuro-degeneration by aggregating the decline of ubiquitin-dependent proteasomal degradation / 中国药理学与毒理学杂志

OBJECTIVE To determine the roles of phosphorylated ubiquitin(pUb)on ubiquitin-dependent proteasomal(UPS)degradation activity,and the roles of pUb on neurodegeneration.METHODS We use PTEN induced kinase 1(PINK1)to phosphorylate ubiquitin.The Ub/S65A cannot be phosphorylated by PINK1,and was used to antagonize the roles of pUb.The Ub/S65E was used to mimic the roles of pUb.The roles of pUb on UPS degradation activity were determined by immunoflu-orescence,Western blot and TIRF microscope at cellular and protein level.The roles of pUb on neurodegeneration were determined by behavior tests,immunofluorescence,Golgi staining,TEM,Western blot and proteomics sacle in mouse.RESULTS The level of soluble PINK1(sPINK1)and pUb increased in the neurons of aged mouse brain,and in the cells upon the administration of MG132,a proteasome inhibitor.The elevation of sPINK1 and pUb was accompanied by protein aggregation upon aging or the proteasomal inhibition.The pink1 knockout alleviated proteasomal inhibition induced protein aggregation and association of ubiquitinated proteins with proteasome.The over-expression of sPINK1 increased pUb level in hippocampal neuron,which chronically induced protein aggregation,mitochondrial damage and damage the structure of neuronal spines.Such neuronal injury lead to cognitive impairment of mice.The roles of sPINK1 was reversed by co-expression with Ub/S65A,and was mimic by over-expression with Ub/S65E.CONCLUSION The phosphorylation of ubiquitin aggravates UPS degrada-tion,and accelerates neuronal degeneration upon the decline of proteasomal degradation in aging and age-related neuronal diseases.

Effect of Gandou Decoction on Mitophagy in Toxic Milk （TX） Mouse Model of Wilson Disease Based on Pink1/Parkin Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Gandou decoction （GDD） on the mitophagy of hippocampal neurons in toxic milk （TX） mouse model of Wilson disease and explore the protective mechanism of GDD against neuron injury through the PTEN induced kinase 1 （Pink1） /E3 ubiquitin ligase （Parkin） pathway. MethodSixty mice were randomly divided into a blank group， a model group， a penicillamine group （0.09 g·kg-1）， and low- （5.5 g·kg-1）， medium- （11 g·kg-1）， and high-dose （22 g·kg-1） GDD groups， and treated correspondingly by gavage for 8 weeks. Morris water maze， traction test， and pole test were used for the evaluation of animal behaviors. Hematoxylin-eosin （HE） staining and transmission electron microscopy were used to observe cell apoptosis， ultrastructure， autophagy， and mitochondrial structure. The levels of superoxide dismutase （SOD）， reactive oxygen species （ROS）， and malondialdehyde （MDA） were detected by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of Pink1， Parkin， autophagy-associated protein Beclin-1， microtubule-associated protein 1 light chain 3Ⅱ （LC3Ⅱ）， and p62. Western blot was conducted to detect the protein expression of Pink1， Parkin， Beclin-1， LC3Ⅱ/Ⅰ， and p62. ResultCompared with the blank group， the model group showed prolonged escape latency， decreased times of platform crossing， lower score in the traction test， and longer pole climbing time （P<0.01）. Compared with the model group， the medium- and high-dose GDD groups and the penicillamine group showed shortened escape latencies， increased times of platform crossing， higher scores in the traction test， and shortened pole climbing time （P<0.01）. Compared with the blank group， the model group displayed severely damaged neurons and increased autophagosomes. Compared with the model group， the medium- and high-dose GDD groups and the penicillamine group showed improved neuron damage and reduced autophagosomes. The levels of ROS and MDA were higher and SOD was lower in the model group than those in the blank group （P<0.01）， while the levels of the above indicators were reversed by GDD intervention as compared with the model group （P<0.01）. Compared with the blank group， the model group exhibited up-regulated mRNA and protein expression of Pink1， Parkin， LC3Ⅱ， and Beclin-1 and down-regulated p62 （P<0.05）. Compared with the model group， the medium- and high-dose GDD groups showed reduced mRNA and protein expression of Pink1， Parkin， LC3Ⅱ， and Beclin-1 and increased p62 （P<0.05， P<0.01）. ConclusionGDD can significantly inhibit the excessive mitophagy in neurons of TX mice and protect neurons from damage. The mechanism may be related to the regulation of the Pink1/Parkin pathway.

The analysis of polymorphism on PINK1 gene of exon 5 in 50 patients with Parkinson disease / 重庆医学

Objective To study polymorphism of phosphatase and tensinhomologue (PTEN)-induced kinase 1(PINK1) gene of exon 5 in Parkinson patients who located in Guangxi ,and its relationship with Parkinson disease (PD) .Methods PCR ,single strand conformation polymorphism (SSCP) and sequencing analyze were conducted to analyze the PINK 1 gene′s exon 5 polymorphism in 28 cases of early-onset PD patients ,22 cases of late-onset PD patients(early-onset PD patients + late-onset PD patients = PD group) and 55 of control group .Results The intronic intervening sequence 5-5G-A (IVS5-5G-A ) polymorphism and G12164A polymer-phism which were located on PINK1 gene of exon 5 were chain relation .The G/A ,A /A genotype frequency was significantly higher in PD group (42 .0% ) than that in control group (23 .6% ) (χ2 = 4 .034 ,P= 0 .045) .The frequency was also significantly higher in late-onset PD patients (45 .5% )than that of 38 cases who were older than 50 years old in control group(21 .1% )(χ2 = 3 .951 ,P=0 .047) .There were no significant differences in alleles .Conclusion This research suggests that chain relation polymerphism at IVS5-5G-A and G12164A in PINK1 gene may be a susceptible factor for PD patients in Guangxi .