Tetrahydroberberrubine retards heart aging in mice by promoting PHB2-mediated mitophagy.

Heart aging is characterized by left ventricular hypertrophy and diastolic dysfunction, which in turn induces a variety of cardiovascular diseases. There is still no therapeutic drug to ameliorate cardiac abnormities in heart aging. In this study we investigated the protective effects of berberine (BBR) and its derivative tetrahydroberberrubine (THBru) against heart aging process. Heart aging was induced in mice by injection of D-galactose (D-gal, 120 mg · kg-1 · d-1, sc.) for 12 weeks. Meanwhile the mice were orally treated with berberine (50 mg · kg-1 · d-1) or THBru (25, 50 mg · kg-1 · d-1) for 12 weeks. We showed that BBR and THBru treatment significantly mitigated diastolic dysfunction and cardiac remodeling in D-gal-induced aging mice. Furthermore, treatment with BBR (40 µM) and THBru (20, 40 µM) inhibited D-gal-induced senescence in primary neonatal mouse cardiomyocytes in vitro. Overall, THBru exhibited higher efficacy than BBR at the same dose. We found that the levels of mitophagy were significantly decreased during the aging process in vivo and in vitro, THBru and BBR promoted mitophagy with different potencies. We demonstrated that the mitophagy-inducing effects of THBru resulted from increased mRNA stability of prohibitin 2 (PHB2), a pivotal factor during mitophagy, thereby upregulating PHB2 protein expression. Knockdown of PHB2 effectively reversed the antisenescence effects of THBru in D-gal-treated cardiomyocytes. On the contrary, overexpression of PHB2 promoted mitophagy and retarded cardiomyocyte senescence, as THBru did. In conclusion, this study identifies THBru as a potent antiaging medicine that induces PHB2-mediated mitophagy and suggests its clinical application prospects.

Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 gene (PVT1) modulates the proliferation and apoptosis of acute lymphoblastic leukemia cells by sponging miR-486-5p.

Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 gene (PVT1) is related to the progress of various cancers. Here, we illuminated the role of PVT1 in acute lymphoblastic leukemia (ALL) cell proliferation and apoptosis. PVT1 was upregulated in plasma samples from patients with ALL and ALL cell lines. PVT1 silencing repressed cell viability and enhanced cell apoptosis in Jurkat and SUP-B15 cells. PVT1 targeted microRNA-486-5p (miR-486-5p) and negatively modulated miR-486-5p expression. Upregulation of miR-486-5p decreased cell viability and increased ALL cell apoptosis. Mastermind Like Transcriptional Coactivator 3 (MAML3) was a downstream molecule of miR-486-5p and miR-486-5p mimic transfection weakened its expression in ALL cells. Rescue experiments proved that reintroduction of PVT1 counteracted the impacts of miR-486-5p in ALL cell proliferation and apoptosis. In vivo, PVT1 silencing repressed the tumor growth of SUP-B15 cells and reduced the expression of MAML3. Altogether, silencing of PVT1 inhibited ALL cell growth and induced cell apoptosis through sponging miR-486-5p.

Kanglexin delays heart aging by promoting mitophagy.

Heart aging is characterized by structural and diastolic dysfunction of the heart. However, there is still no effective drug to prevent and treat the abnormal changes in cardiac function caused by aging. Here, we present the preventive effects of emodin and its derivative Kanglexin (KLX) against heart aging. We found that the diastolic dysfunction and cardiac remodeling in mice with D-galactose (D-gal)-induced aging were markedly mitigated by KLX and emodin. In addition, the senescence of neonatal mouse cardiomyocytes induced by D-gal was also reversed by KLX and emodin treatment. However, KLX exhibited better anti-heart aging effects than emodin at the same dose. Dysregulated mitophagy was observed in aging hearts and in senescent neonatal mouse cardiomyocytes, and KLX produced a greater increase in mitophagy than emodin. The mitophagy-promoting effects of KLX and emodin were ascribed to their abilities to enhance the protein stability of Parkin, a key modulator in mitophagy, with different potencies. Molecular docking and SPR analysis demonstrated that KLX has a higher affinity for the ubiquitin-like (UBL) domain of Parkin than emodin. The UBL domain might contribute to the stabilizing effects of KLX on Parkin. In conclusion, this study identifies KLX and emodin as effective anti-heart aging drugs that activate Parkin-mediated mitophagy and outlines their putative therapeutic importance.

Preparative separation of four isomers of synthetic anisodamine by HPLC and diastereomer crystallization.

Anisodamine (654-1), a well-known cholinergic antagonist, is marketed as synthetic anisodamine (mixture of four isomers, 654-2) in China. To preparative resolution and comparison of the bioactivities of the four isomers of synthetic anisodamine, current work explores an economic and effective separation method by using preparative high performance liquid chromatography (HPLC) and diastereomer crystallization. Their absolute configurations were established by single-crystal X-ray diffraction and circular dichroism method. The purities of each isomer were more than 95%. Among them, 654-2-A2 (6R, 2'S configuration) exhibited better effect on cabachol preconditioned small intestine tension more than 654-2 and other isomers. The direct separation method without using HPLC was tried as well, which was still on progress. This is the first report of the method for preparative separation of four isomers of synthetic anisodamine which could be used for large-scale production in industry.

[Reversal Effect of Dihydromyricetin on Drug Resistance of K562/A02 Cell Line to Adriamycin].

OBJECTIVE: To investigate the reversal effect of dihydromyricetin(DMY) on drug resistance of K562/A02 cells to adriamycin and explore its possible mechanism. METHODS: K562 and K562/A02 cells were treated with DMY (5, 10, 20, 40, 60, 80 and 100 mg/L) and ADM (100-0.05 mg/L) for 48 h. The viability of K562 cells and K562/A02 cells was tested and the reversal effect of DMY on drug resistance of K562/A02 cells to adriamycin was analyzed by MTT. The relative concentration of ADM in cells was measured by flow cytometry. Protein expressions of drug resistance related genes including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), glutathione transferase π (GSTπ) and BCL-2 were measured by Western Blot. RESULTS: The proliferation of K562 and K562/A02 cells was significantly decreased by DMY in dose-dependent manner as compared with control group (r1=0.37, r2=0.38). The IC50 of ADM on K562 and K562/A02 cells were 71.23±6.51 and 72.88±5.49 mg/L respectively. DMY (5, 10 and 20 mg/L) was low cytotoxicity. DMY (5, 10 and 20 mg/L) enhanced the sensitivity of K562/A02 cells to ADM in dose-dependent manner (r1=-0.62, r2=-0.71) and the reversal multiples was from 1.38 to 28.591. The relative concentrations of ADM in K562/A02 of DMY (5, 10 and 20 mg/L) group cells were significantly increased in dose-dependent manner compared with the control group (r=0.34). Compared with the control group, the expressions of drug resistance related protein P-gp, MRP1, GSTπ and BCL-2 were significantly decreased in dose-dependent manner in DMY (5, 10 and 20 mg/L) group (r1=-0.41, r2=-0.37, r3=-0.58, r=-0.46). Compared with the ADM group, the protein expressions of drug resistance related genes P-gp, MRP1, GSTπ and BCL-2 in DMY (5, 10 and 20 mg/L)+ADM group were significantly decreased in dose-dependent manner (r1=-0.55, r2=-0.41, r3 =-0.38, r4=-0.44). CONCLUSION: DMY enhances the sensitivity of K562/A02 cells to ADM, its mechanism may be related with decrease of P-gp, MRP1, GSTπ and BCL-2 expressions.

[Gly14]-Humanin Protects Against Amyloid ß Peptide-Induced Impairment of Spatial Learning and Memory in Rats.

Alzheimer disease (AD), a progressive neurodegenerative disorder, is characterized by cognitive decline and the accumulation of senile plaques in the brain. Amyloid ß protein (Aß) in the plaques is thought to be responsible for the memory loss in AD patients. [Gly14]-humanin (HNG), a derivative of humanin (HN), has much stronger neuroprotective effects than natural HN in vitro. However, clarification of the Aß active center and the neuroprotective mechanism of HN still need in vivo evidence. The present study first compared the in vivo biological effects of three Aß fragments (1-42, 31-35, and 35-31) on spatial memory in rats, and investigated the neuroprotective effects and molecular mechanisms of HNG. The results showed that intrahippocampal injection of Aß1-42 and Aß31-35 almost equally impaired spatial learning and memory, but the reversed sequence Aß35-31 did not have any effect; a high dose of Aß31-35 (20 nmol) produced a more detrimental response than a low dose (2 nmol); Aß31-35 injection also disrupted gene and protein expression in the hippocampus, with up-regulation of caspase3 and down-regulation of STAT3; pretreatment with HNG not only protected spatial memory but also rescued STAT3 from Aß-induced disruption; and the neuroprotective effects of HNG were effectively counteracted by genistein, a specific tyrosine kinase inhibitor. These results clearly show that sequence 31-35 in Aß is the shortest active center responsible for the neurotoxicity of Aß from molecule to behavior; and HNG protects spatial learning and memory in rats against Aß-induced insults; and probably involves the activation of tyrosine kinases and subsequent beneficial modulation of STAT3 and caspase3.

Colivelin ameliorates amyloid ß peptide-induced impairments in spatial memory, synaptic plasticity, and calcium homeostasis in rats.

Amyloid ß peptide (Aß) has been thought to be neurotoxic and responsible for the impairment of learning and memory in Alzheimer's disease (AD). Humanin (HN), a 24 amino acid polypeptide first identified from the unaffected occipital lobe of an AD patient, is believed to be neuroprotective against the AD-related neurotoxicity. In this study, we investigated the neuroprotective effects of Colivelin (CLN), a novel HN derivative, against Aß by using behavioral test, in vivo electrophysiological recording, and intracellular calcium imaging. Our results showed that intrahippocampal injection of CLN (0.2 nmol) effectively prevented Aß25-35 (4 nmol)-induced deficits in spatial learning and memory of rats in Morris water maze test; the suppression of in vivo hippocampal long term potentiation (LTP) by Aß25-35 was nearly completely prevented by CLN; in addition, CLN pretreatment also effectively inhibited Aß25-35-induced calcium overload in primary cultured hippocampal neurons. These results indicate that CLN has significant neuroprotective properties against Aß, and CLN may holds great promise for the treatment and prevention of AD.

The neuroprotection of Rattin against amyloid ß peptide in spatial memory and synaptic plasticity of rats.

Rattin, a specific derivative of humanin in rats, shares the ability with HN to protect neurons against amyloid ß (Aß) peptide-induced cellular toxicity. However, it is still unclear whether Rattin can protect against Aß-induced deficits in cognition and synaptic plasticity in rats. In the present study, we observed the effects of Rattin and Aß31-35 on the spatial reference memory and in vivo hippocampal Long-term potentiation of rats by using Morris water maze test and hippocampal field potential recording. Furthermore, the probable molecular mechanism underlying the neuroprotective roles of Rattin was investigated. We showed that intra-hippocampal injection of Rattin effectively prevented the Aß31-35-induced spatial memory deficits and hippocampal LTP suppression in rats; the Aß31-35-induced activation of Caspase-3 and inhibition of STAT3 in the hippocampus were also prevented by Rattin treatment. These findings indicate that Rattin treatment can protect spatial memory and synaptic plasticity of rats against Aß31-35-induced impairments, and the underlying protective mechanism of Rattin may be involved in STAT3 and Caspases-3 pathways. Therefore, application of Rattin or activation of its signaling pathways in the brain might be beneficial to the prevention of Aß-related cognitive deficits.

Melatonin protects against amyloid-ß-induced impairments of hippocampal LTP and spatial learning in rats.

Alzheimer's disease (AD), the most prevalent neurodegenerative disease in the elderly, leads to progressive loss of memory and cognitive deficits. Amyloid-ß protein (Aß) in the brain is thought to be the main cause of memory loss in AD. Melatonin, an indole hormone secreted by the pineal gland, has been reported to produce neuroprotective effects. We examined whether melatonin could protect Aß-induced impairments of hippocampal synaptic plasticity, neuronal cooperative activity, and learning and memory. Rats received bilateral intrahippocampal injection of Aß1-42 or Aß31-35 followed by intraperitoneal application of melatonin for 10 days, and the effects of chronic melatonin treatment on in vivo hippocampal long-term potentiation (LTP) and theta rhythm and Morris water maze performance were examined. We showed that intrahippocampal injection of Aß1-42 or Aß31-35 impaired hippocampal LTP in vivo, while chronic melatonin treatment reversed Aß1-42- or Aß31-35-induced impairments in LTP induction. Intrahippocampal injection of Aß31-35 impaired spatial learning and decreased the power of theta rhythm in the CA1 region induced by tail pinch, and these synaptic, circuit, and learning deficits were rescued by chronic melatonin treatment. These results provide evidence for the neuroprotective action of melatonin against Aß insults and suggest a strategy for alleviating cognition deficits of AD.

Liraglutide protects against amyloid-ß protein-induced impairment of spatial learning and memory in rats.

Type 2 diabetes mellitus is a risk factor of Alzheimer's disease (AD), most likely linked to an impairment of insulin signaling in the brain. Liraglutide, a novel long-lasting glucagon-like peptide 1 (GLP-1) analog, facilitates insulin signaling and shows neuroprotective properties. In the present study, we analyzed the effects of liraglutide on the impairment of learning and memory formation induced by amyloid-ß protein (Aß), and the probable underlying electrophysiological and molecular mechanisms. We found that (1) bilateral intrahippocampal injection of Aß(25-35) resulted in a significant decline of spatial learning and memory of rats in water maze tests, together with a serious depression of in vivo hippocampal late-phase long-term potentiation (L-LTP) in CA1 region of rats; (2) pretreatment with liraglutide effectively and dose-dependently protected against the Aß(25-35)-induced impairment of spatial memory and deficit of L-LTP; (3) liraglutide injection also activated cAMP signal pathway in the brain, with a nearly doubled increase in the cAMP contents compared with control. These results strongly suggest that upregulation of GLP-1 signaling in the brain, such as application of liraglutide, may be a novel and promising strategy to ameliorate the learning and memory impairment seen in AD.

[[Gly14]-humanin protects against Aß31₋35-induced impairment of spatial learning and memory in rats].

Amyloid ß protein (Aß) is closely involved in the pathogenesis of Alzheimer's disease (AD), and one of the main strategies for AD treatment is antagonizing the neurotoxicity of Aß or even clearing the Aß deposited in the brain. The present study was aimed to observe the effects of intrahippocampal injection of Aß31₋35 on the spatial learning and memory of rats by using Morris water maze technique, and explore the neuroprotective effects and possible mechanism of [Gly14]-humanin (HNG) against Aß-induced deficits in learning behavior. The results showed that bilateral intrahippocampal injection of 2.0 nmol Aß31₋35 significantly increased the mean traveled distance of rats in searching for the hidden underwater platform and decreased the distance percentage in the target quadrant in probe test after withdrawal of platform, whereas pretreatment with HNG (0.2 nmol and 2.0 nmol) suppressed Aß31₋35-induced increase in the traveled distance and decrease in swimming distance percentage. Application of Genistein (40 nmol), a specific tyrosine kinase inhibitor, almost completely blocked the antagonistic effects of HNG against Aß31₋35. These results indicate that HNG can dose-dependently prevent against Aß31₋35-induced impairment in spatial learning and memory of rats, and the neuroprotective effects of HNG might involve the activation of endogenous tyrosine kinase pathway, suggesting that up-regulation of the tyrosine kinase signaling by using HNG might be of great significance for the improvement of cognitive function in AD.

Effect of 15-HETE on potassium channels of rabbit pulmonary arterial smooth muscles during hypoxia.

This study investigated the role of 15-hydroxyeicosatetraenoic acid (15-HETE) in rabbit pulmonary arterial smooth muscle cells (PASMCs) under hypoxia by using organ bath and whole cell patch-clamp techniques. Neonatal rabbits born into normoxic environment were transferred after first feeding into normal and hypoxic environments with respectively 0.21 and 0.12 fractional inspired oxygen (FiO2). Pulmonary arteries were extracted after 9 d and cut into rings 1.0 approximately 1.5 mm in length for organ bath experiments. Whole cell patch-clamp technique was used to measure the potassium current in the freshly dispersed rabbit PASMCs. The results showed that 15-HETE-induced vasoconstriction was blocked by 4-aminopyridine (5 mmol/L), a Kv channel blocker. The K(ATP) channel blocker glyburide (1 micromol/L) and the BKCa channel blocker tetraethylammonium (10 mmol/L) did not abolish this vasoconstriction. 15-HETE decreased the whole-cell voltage-gated K+ current in the PASMCs. These findings demonstrate that hypoxia blocks Kv channels through a 15-HETE mediated mechanism, leading to PA vasoconstriction.