Exploring the mechanism of anti-hereditary Parkinson's disease of baicalein based on PINK1 RNAi Drosophila model / 药学学报

The aim of this study was to investigate the effect of baicalein on a Drosophila model of hereditary Parkinson's disease caused by gene mutations and to preliminarily elucidate the mechanism of baicalein in delaying hereditary Parkinson's disease. In this paper, PTEN-induced putative kinase 1 (PINK1)-RNAi Parkinson's Drosophila were used as the model group and wild-type Drosophila w1118 were used as the control group. Different doses of baicalein and Madopa were administered to the model group to observe their effects on the life span, motor ability, the abnormal rate of wings, dopamine content and dopaminergic neurons of PINK1-RNAi Parkinson's Drosophila and their effects on mitochondrial dysfunction including adenosine triphosphate (ATP), mitochondrial DNA (mtDNA) and reactive oxygen species (ROS) content. The results showed that the effective administration doses of baicalein were 0.8 mg·mL-1 for low concentration, 1.6 mg·mL-1 for medium concentration and 3.2 mg·mL-1 for high concentration, and the optimal administration dose of the positive drug Madopa was 0.1 μg·mL-1. Baicalein and Madopa could significantly improve the life span, exercise ability and reduce the abnormal rate of wings of PINK1-RNAi male Drosophila (P < 0.05), and low dose baicalein showed the best effect; baicalein could improve the loss of dopaminergic neurons, and the effects of low dose and high dose were the best, but Madopa showed no significant effect; baicalein and Madopa had no significant effect on dopamine content (P > 0.05). Baicalein and Madopa could increase the ATP content of PINK1-RNAi male Drosophila (P < 0.05), and low dose baicalein showed the best effect; middle dose baicalein could significantly increase the mtDNA content of PINK1-RNAi male Drosophila (P < 0.05), but Madopa had no significant effect; baicalein and Madopa had no significant effect on ROS content (P > 0.05).

Mechanism of Cinnamaldehyde in Promoting Wound Healing in Diabetes Rats via PINK1/Parkin-mediated Mitochondrial Autophagy / 中国实验方剂学杂志

ObjectiveTo establish a rat model of diabetic wound by feeding on a high-fat and high-sugar diet combined with intraperitoneal injection of streptozotocin （STZ） and surgical preparation of full-thickness skin defects， observe the effect of cinnamaldehyde on the wound healing of diabetes rats， and explore the therapeutic mechanism of cinnamaldehyde in improving wound healing of diabetes rats based on the PTEN-induced putative kinase （PINK1）/Parkin pathway-mediated mitochondrial autophagy. MethodForty-eight male SD rats were randomly divided into blank group （n=12） and diabetes group （n=36）. The diabetes group was further randomly divided into model group， cinnamaldehyde group， and Beifuxin group， with 12 rats in each group. The blank group and the model group received routine disinfection with physiological saline after creating the wounds， while the cinnamaldehyde group received topical application of polyethylene glycol 400 （PEG 400） gel containing 4 μmol·L-1 cinnamaldehyde， and the Beifuxin group received topical application of Beifuxin gel. Dressings were changed once daily. The wound healing rate of each group was observed. On the 7th and 14th days after intervention， the wound tissues of the rats were collected. Hematoxylin and eosin （HE） staining was performed to observe the pathological changes in the local tissues. Immunohistochemistry （IHC） was used to detect the expression of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， vascular endothelial growth factor （VEGF）， and collagen fibers. Immunofluorescence （IF） and Real-time polymerase chain reaction （Real-time PCR） were used to detect the protein， and mRNA expression of PINK1， Parkin， microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ）. ResultAfter intraperitoneal injection of STZ， compared with the blank group， the random blood glucose values of rats in the diabetic group increased significantly （P<0.01）， all higher than 16.7 mmol·L-1， and persistently hyperglycemic for some time after modeling. Compared with the blank group， the model group showed poor growth and healing of granulation tissue in the wounds， and the wound healing rate decreased （P<0.01）. On the 7th day after intervention， the blank group had squamous epithelial coverage on the wounds. Compared with the blank group， the model group only had a small amount of scab at the wound edges， with a large number of infiltrating inflammatory cells in the wounds. The protein expression levels of IL-6 and TNF-α in the tissues increased （P<0.01）， and the protein and mRNA levels of PINK1， Parkin， and LC3Ⅱ decreased （P<0.01）. On the 14th day after the intervention， the granulation tissue in the wounds of the blank group was mature and well-healed. Compared with the blank group， the model group still had infiltrating inflammatory cells and red blood cell exudation. The protein expression levels of VEGF and collagen fibers in the tissues decreased （P<0.01）， and the protein and mRNA expression levels of PINK1， Parkin， and LC3Ⅱ increased （P<0.01）. Compared with the model group， the cinnamaldehyde group and the Beifuxin group showed better wound healing， with increased wound healing rates （P<0.01）. On the 7th day after intervention， the protein expression levels of IL-6 and TNF-α in the tissues decreased （P<0.01）， and the protein and mRNA expression levels of PINK1， Parkin， and LC3Ⅱ increased （P<0.01）. On the 14th day after intervention， the protein expression levels of VEGF and collagen fibers in the tissues increased （P<0.01）， and the protein and mRNA expression levels of PINK1， Parkin， and LC3Ⅱ decreased （P<0.01）. ConclusionCinnamaldehyde can promote the wound healing of diabetes rats by increasing the wound healing rate， reducing the levels of inflammatory factors IL-6 and TNF-α， and increasing the levels of VEGF and collagen fibers. Its mechanism may be related to the regulation of the PINK1/Parkin signaling pathway， activation of mitochondrial autophagy， inhibition of inflammatory responses， and promotion of angiogenesis and collagen synthesis， thereby promoting the wound healing of diabetes rats.

Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy / 中国实验方剂学杂志

ObjectiveTo observe the effects of Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and their combination on slow transit constipation via PTEN-induced putative kinase 1 （PINK1）/Parkin pathway-mediated mitophagy. MethodFifty-six male SD rats were randomly assigned into normal group， model group， natural recovery group， Aurantii Fructus Immaturus group， Atractylodis Macrocephalae Rhizoma group， Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group， and mosapride group， with 8 rats in each group. Slow transit constipation model was established by gavage with loperamide （3 mg·kg-1·d-1） for 14 days in other groups except the normal group. After successful modeling， except that the model group was continuously induced by loperamide， the normal group and the natural recovery group were administrated with 0.9% normal saline by gavage， and the rats in the Aurantii Fructus Immaturus （1.35 g·kg-1·d-1） group， the Atractylodis Macrocephalae Rhizoma （2.7 g·kg-1·d-1） group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma （4.05 g·kg-1·d-1） group， and the mosapride （1.56 mg·kg-1·d-1） group were administrated with corresponding drugs by gavage for 7 days. The amount of feces， fecal water content， and intestinal propulsion rate of rats were determined. The pathological changes of the colon were evaluated by hematoxylin-eosin （HE） staining and Alcian blue-periodic acid-Schiff （AB-PAS） staining. The activity of respiratory chain complex and the ultrastructure of the colon tissue were determined by ultraviolet spectrophotometry and observed by transmission electron microscopy， respectively. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of PINK1， Parkin， and p62， and Western blot to determine the protein levels of microtubule-associated protein 1 light chain 3 （LC3）， PINK1， and Parkin. ResultCompared with the normal group， the model group and the natural recovery group showed decreases in the amount of feces， fecal water content， intestinal propulsion rate （P<0.05，P<0.01）， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ in the colon tissue （P<0.05，P<0.01）. Further， the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 were up-regulated （P<0.01） and the mRNA level of p62 was down-regulated in the model group （P<0.05） and the natural recovery group. Compared with the model group and the natural recovery group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group showed increased amount of feces， fecal water content， intestinal propulsion rate， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ （P<0.05，P<0.01）. Moreover， the combination meliorated the degree of mitochondrial swelling in the colon tissue， down-regulated the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 （P<0.05，P<0.01）， and up-regulated the mRNA level of p62 （P<0.05）. ConclusionAurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma， and their combination may remedy the colonic motility disorders in rats with slow transit constipation by blocking PINK1/Parkin signaling pathway to inhibit the excessive mitophagy in interstitial cells of Cajal in the colon tissue.

Effect on Danggui Shaoyao Powder on mitophagy in rat model of Alzheimer's disease based on PINK1-Parkin pathway / 中国中药杂志

This study investigated the mechanism of Danggui Shaoyao Powder(DSP) against mitophagy in rat model of Alzheimer's disease(AD) induced by streptozotocin(STZ) based on PTEN induced putative kinase 1(PINK1)-Parkin signaling pathway. The AD rat model was established by injecting STZ into the lateral ventricle, and the rats were divided into normal group, model group, DSP low-dose group(12 g·kg~(-1)·d~(-1)), DSP medium-dose group(24 g·kg~(-1)·d~(-1)), and DSP high-dose group(36 g·kg~(-1)·d~(-1)). Morris water maze test was used to detect the learning and memory function of the rats, and transmission electron microscopy and immunofluorescence were employed to detect mitophagy. The protein expression levels of PINK1, Parkin, LC3BⅠ/LC3BⅡ, and p62 were assayed by Western blot. Compared with the normal group, the model group showed a significant decrease in the learning and memory function(P<0.01), reduced protein expression of PINK1 and Parkin(P<0.05), increased protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05), and decreased occurrence of mitophagy(P<0.01). Compared with the model group, the DSP medium-and high-dose groups notably improved the learning and memory ability of AD rats, which mainly manifested as shortened escape latency, leng-thened time in target quadrants and elevated number of crossing the platform(P<0.05 or P<0.01), remarkably activated mitophagy(P<0.05), up-regulated the protein expression of PINK1 and Parkin, and down-regulated the protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05 or P<0.01). These results demonstrated that DSP might promote mitophagy mediated by PINK1-Parkin pathway to remove damaged mitochondria and improve mitochondrial function, thereby exerting a neuroprotective effect.

Effects of PINK1 mediated mitophagy on senescence and function of rat bone marrow endothelial progenitor cells / 中国药理学通报

Aim To investigate the effects of PTEN-induced putative kinase1(PINK1)mediated mitophagy on senescence and function of rat bone marrow endothelial progenitor cells(EPCs)by using small interfering RNA(siRNA)technology to knock down the PINK1 gene in rat bone marrow EPCs.Methods EPCs from bone marrow in rats were isolated, cultured and identified.After counting, EPCs were randomlydivided into control group, negative control group(NC siRNA), and Pink1 transfection group(PINK1 siRNA).The expression of PINK1 mRNA and protein in cells in various groups were detected by qRT-PCR and Western blot.At the same time, different time points were chosen to simulate the aging process based on the best knock down time.The senescence of cells was detected by SA-β-galactosidase staining and p16 protein expression.The function of cell proliferation, migration and tubule formation was detected by CCK-8, Transwell chamber and in vitro angiogenesis kit.ROS level was detected by flow cytometry.The expressions of PINK1, Parkin, LC3, and p62 were detected by Western blot.Mitochondria and autophagosomes were observed by transmission electron microscope.Results 48 h after PINK1 siRNA transfected, PINK1 was effectively knocked down.Compared with control group, the positive rate of blue staining and the expression of p16 protein in PINK1 siRNA group increased significantly 48 h and 96 h after transfection.The function of cell proliferation, migration and tubule formation decreased significantly.The level of ROS increased significantly, while the expression of PINK1, Parkin and LC3 protein decreased significantly, and p62 protein expression increased significantly.Under the transmission electron microscope, the mitochondria swelled and denatured, and the number of autophagosomes decreased in the PINK1 siRNA group.Conclusions The down-regulation of PINK1 gene can aggravate the senescence of EPCs, and PINK1 mediated mitophagy may participate in the regulation of senescence and function of EPCs.

Nicotinamide mononucleotide attenuates ischemia-reperfusion injury induced by donor liver from cardiac death through Sirt3 / 器官移植

Objective To evaluate the effect and mechanism of nicotinamide mononucleotide (NMN) on ischemia-reperfusion injury (IRI) induced by donor liver after cardiac death in rat models. Methods Rat models of orthotopic liver transplantation were established by "magnetic ring + double cuff" method. SD rats were randomly divided into the sham operation group (Sham group), orthotopic liver transplantation group (OLT group), NMN treatment + orthotopic liver transplantation group (NMN group), NMN+sirtuin-3 (Sirt3) inhibitor (3-TYP) + orthotopic liver transplantation group (NMN+3-TYP group), respectively. Pathological changes and hepatocyte apoptosis of the rats were observed in each group. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined. Superoxide dismutase (SOD) and malondialdehyde (MDA) contents in liver tissues were detected. The expression levels of Sirt3, microtubule-associated protein 1 light chain 3 (LC3)Ⅱ, PTEN-induced putative kinase 1 (PINK1), Parkin and translocase of the outer mitochondrial membrane 20 (TOMM20) in liver tissues were measured. Postoperative survival of the rats in each group was analyzed. Results Compared with the Sham group, serum ALT and AST levels were higher in the OLT group. Compared with the OLT group, the levels of ALT and AST were decreased in the NMN group. Compared with the NMN group, the levels of ALT and AST were increased in the NMN +3-TYP group (all P < 0.05). The liver tissue structure of rats in the Sham group was basically normal. In the OLT group, pathological changes, such as evident congestion, vacuolar degeneration and hepatocyte necrosis, were observed in the liver tissues. Compared with the Sham group, Suzuki score and apoptosis rate were higher in the OLT group. Suzuki score and apoptosis rate in the NMN group were lower than those in the OLT group. Suzuki score and apoptosis rate in the NMN+3-TYP group were higher compared with those in the NMN group (all P < 0.05). Compared with the Sham group, the SOD content was decreased, whereas the MDA content was increased in the OLT group. Compared with the OLT group, the SOD content was increased, whereas the MDA content was decreased in the NMN group. Compared with the NMN group, the SOD content was decreased, whereas the MDA content was increased in the NMN+3-TYP group (all P < 0.05). Compared with the Sham group, the relative expression levels of Sirt3 and TOMM20 proteins were down-regulated, whereas those of PINK1, Parkin and LC3Ⅱproteins were up-regulated in the OLT group. Compared with the OLT group, the relative expression levels of Sirt3, PINK1, Parkin and LC3Ⅱproteins were up-regulated, whereas that of TOMM20 protein was down-regulated in the NMN group. Compared with the NMN group, the relative expression levels of PINK1, Parkin and LC3Ⅱproteins were down-regulated, whereas that of TOMM20 protein was up-regulated in the NMN+3-TYP group (all P < 0.05). In the Sham group, the 7 d survival rate of rats was 100%, 50% in the OLT group, 75% in the NMN group and 58% in the NMN+3-TYP group, respectively. Conclusions NMN may enhance the antioxidative capacity of the liver, induce PINK1/Parkin-mediated mitochondrial autophagy, and alleviate IRI of the liver by up-regulating Sirt3, thereby playing a protective role in the donor liver after cardiac death.

Effect of acupuncture on mitophagy of skeletal muscle in rats with exer-cise-induced skeletal muscle damage / 中国病理生理杂志

AIM:The effect of acupuncture on mitophagy-related protein expression in skeletal muscle of rats after heavy-load exercise was investigated to explore the role of acupuncture in the repairment of exercise-induced skeletal muscle damage. METHODS:Male adult Sprague-Dawley rats (n=128) were randomly divided into 4 groups:control (C,n=8) group, exercise (E, n=40) group, acupuncture (A, n=40) group, and exercise and acupuncture (EA, n=40) group. The rats in E group and EA group performed an eccentric exercise,and the rats in A group and EA group immediately after exercise received acupuncture treatment. The rats in the latter 3 groups were further divided into 0 h,12 h,24 h,48 h and 72 h sub-groups(n=8),and soleus muscle was collected at each time point. The transmission electron microscopy was used to observe the ultrastructural changes of the mitochondria in skeletal muscle. The content of citrate synthase (CS) was measured by ELISA. The protein expression of skeletal muscle PTEN-induced putative kinase 1 (PINK1),parkin and microtubule-associated protein 1 light chain 3 (LC3) was determined by Western blot. RESULTS:After the heavy-load exercise,the mitochondria swelled and accumulated under cell membrane. The number of mitophago-somes was increased,and the content of CS was significantly decreased(P<0.05). The expression of PINK1,parkin and LC3 was significantly elevated (P<0.05). However,the acupuncture intervention after exercise promoted the recovery of mitochondrial ultrastructure, attenuated mitophagolysosome formation, maintained CS content and down-regulated the ex-pression of PINK1,parkin and LC3 (P<0.05). CONCLUSION:Heavy-load exercise causes the damages of mitochon-drial structure and function in the skeletal muscle and activates PINK1/parkin pathway to induce excessive occurrence of mitophagy. Acupuncture intervention after exercise is able to alleviate the damage of mitochondria in the skeletal muscle through decreasing the expression of mitochondrial outer membrane protein PINK1,reducing the recruitment of downstream cytoplasmic protein parkin,thereby affecting the combination of LC3 and mitochondria to inhibit the overactivation of mito-phagy.