Psoralen induces liver injury and affects hepatic bile acids metabolism in female and male C57BL/6J mice.

Psoralen is a major component of Fructus Psoraleae that could induce liver injury. In this study, C57BL/6J mice were administered with psoralen at doses of 80 mg/kg for 3, 7 and 14 days. Blood and liver samples were collected for serum biochemistry and histopathology examinations, respectively. Psoralen led to liver injury with significantly increased liver weight and liver coefficient and up regulated serum ALT, AST and TG but down regulated serum TC and TP. The expression of bile acid-associated transporters and enzymes was detected by western blot, and the results showed that psoralen significantly down-regulates the expressions of CYP7A1, CYP27A1, BSEP and OSTα protein while up-regulates the expressions of HMGCR and FASN, resulting in the obstacles of bile acid efflux in the liver. The contents of 24 kinds of bile acids in the liver were measured by LC-MS/MS, and the results showed that psoralen led to the accumulation of unconjugated bile acids in the liver, such as ALCA and CA, which were more severe in male mice than female mice. It was indicated that psoralen may disrupt the balance of bile acid metabolism by inhibiting the expression of the efflux transporter, which then leads to liver damage.

Isopsoralen induces different subchronic toxicities and metabolomic outcomes between male and female Wistar rats.

Isopsoralen is a major active and quality-control component of Fructus Psoraleae, but lacks a full safety evaluation. We evaluated the oral toxicity of isopsoralen in Wistar rats treated for 3 months at doses of 0, 3.5, 7.0, and 14 mg/kg. Additionally, the plasma metabolomics of isopsoralen in male and female rats treated for 3 months at doses of 0 and 14 mg/kg were investigated by gas chromatography-mass spectrometry. Many abnormalities were observed in the isopsoralen-treated rats, including suppression of body weight gain, and changes in serum biochemical parameters and visceral coefficients. Histopathological changes in liver, pancreatic, and reproductive system tissues were also observed in the isopsoralen-treated rats. The metabolomic analyses showed alterations in many metabolites (19 in female rats; 28 in male rats) after isopsoralen administration. The significant changes in these metabolites revealed metabolomic alterations in the isopsoralen-treated rats, especially in amino acid metabolism regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Furthermore, fatty acid metabolism comprised the main affected pathways in female rats, while lipid metabolism and energy metabolism were the main affected pathways in male rats.

Optimization of the Time Window of Interest in Ovariectomized Imprinting Control Region Mice for Antiosteoporosis Research.

This study was performed to determine the optimal window of time during which the properties of osteoporosis are obvious and to explore the best region of interest for microstructural evaluation in antiosteoporosis research in an ovariectomized mouse model by examining changes in micro-computed tomography parameters and serum indices. Ovariectomized mice and sham-operated mice were randomly divided into five groups. At the end of the 4th, 8th, 12th, 16th, and 20th weeks after ovariectomy, the microstructure of the proximal tibia and distal femur was scanned by micro-computed tomography and blood samples were collected to detect serum biochemical indicators including alkaline phosphatase, osteocalcin, N-terminal propeptide of type I procollagen (P1NP), and C-terminal telopeptide fragment of type I collagen (CTX1). The trabecular number and connectivity density decreased while the trabecular thickness and trabecular separation increased, indicating substantial changes in the trabecular microstructure of both the tibia and femur and significant changes in bone turnover after ovariectomy, as indicated by lower levels of serum alkaline phosphatase, osteocalcin, and P1NP and higher level of CTX1 in the ovariectomy than sham group. The proximal tibia from weeks 8 to 16 after ovariectomy was optimal for osteoporosis research in this model.

Reassessment of subacute MPTP-treated mice as animal model of Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model remains the most commonly used animal model of Parkinson's disease (PD). There are three MPTP-treatment schemes: acute, subacute and chronic. Considering the advantages of the period and similarity to PD, the subacute model was often chosen to assess the validity of new candidates, but the changes caused by the subacute MPTP treatment and the appropriate positive control for this model remain to be further confirmed. The aim of this study was: to estimate the value of the subacute MPTP mouse model in aspects of behavioral performance, biochemical changes and pathological abnormalities, and to find effective positive drugs. Male C57BL/6 mice were injected with MPTP (30 mg·kg-1·d-1, ip) for 5 consecutive days. Three days before MPTP injection, the mice were orally administered selegiline (3 mg·kg-1·d-1), pramipexole (3 mg·kg-1·d-1), or medopar (100 mg·kg-1·d-1) for 18 days. Behavioral performance was assessed in the open field test, pole test and rotarod test. Neurotransmitters in the striatum were detected using HPLC. Protein levels were measured by Western blot. Pathological characteristics were examined by immunohistochemistry. Ultrastructure changes were observed by electron microscopy. The subacute MPTP treatment did not induce evident motor defects despite severe injuries in the dopaminergic system. Additionally, MPTP significantly increased the α-synuclein levels and the number of astrocytes in the striatum, and destroyed the blood-brain barrier (BBB) in the substantia nigra pars compacta. Both selegiline and pramipexole were able to protect the mice against MPTP injuries. We conclude that the subacute MPTP mouse model does not show visible motor defects; it is not enough to evaluate the validity of a candidate just based on behavioral examination, much attention should also be paid to the alterations in neurotransmitters, astrocytes, α-synuclein and the BBB. In addition, selegiline or pramipexole is a better choice than medopar as an effective positive control for the subacute MPTP model.

Bibenzyl compound 20c protects against endoplasmic reticulum stress in tunicamycin-treated PC12 cells in vitro.

AIM: Accumulation of α-synuclein (α-syn) in the brain is a characteristic of Parkinson's disease (PD). In this study, we investigated whether treatment with tunicamycin, an endoplasmic reticulum (ER) stress inducer, led to the accumulation of α-syn in PC12 cells, and where α-syn protein was accumulated, and finally, whether bibenzyl compound 20c, a novel compound isolated from Gastrodia elata (Tian ma), could alleviate the accumulation of α-syn and ER stress activation in tunicamycin-treated PC12 cells. METHODS: PC12 cells were treated with tunicamycin for different time (6 h, 12 h, 24 h, 48 h). Cell viability was determined by a MTT assay. Subcellular fractions of ER and mitochondria were extracted with the Tissue Endoplasmic reticulum Isolation Kit. The levels of α-syn protein and ER-stress-associated downstream chaperones were detected using Western blots and immunofluorescence. RESULTS: Treatment of PC12 cells with tunicamycin (0.5-10 µg/mL) dose-dependently increased the accumulation of α-syn monomer (19 kDa) and oligomer (55 kDa), and decreased the cell viability. Accumulation of the two forms of α-syn was observed in both the ER and mitochondria with increasing treatment time. Co-treatment with 20c (10-5 mol/L) significantly increased the viability of tunicamycin-treated cells, reduced the level of α-syn protein and suppressed ER stress activation in the cells, evidenced by the reductions in phosphorylation of eIF2α and expression of spliced ATF6 and XBP1. CONCLUSION: Tunicamycin treatment caused accumulation of α-syn monomer and oligomer in PC12 cells. Bibenzyl compound 20c reduces the accumulation of α-syn and inhibits the activation of ER stress, which protected PC12 cells against the toxicity induced by tunicamycin.

Claulansine F promoted the neuronal differentiation of neural stem and progenitor cells through Akt/GSK-3ß/ß-catenin pathway.

The persistence of neurogenesis raises the idea that neurons produced by the resident or transplanted neural stem cells could replace the neurons lost from brain injury or neurodegenerative disease. Therefore, compounds or methods for promoting neuronal differentiation become the focus of neurodegenerative disease therapy research. Claulansine F (Clau F), a newly discovered carbazole alkaloid, has been showed to induce neuritogenesis in PC12 cells. Herein, we studied the effect of Clau F on neuronal differentiation of neural stem/progenitor cells (NS/PCs). The current study demonstrated that Clau F initiated neuronal differentiation with a significant increase of TuJ1-positive cells and TuJ1 protein levels. We also found that Clau F promoted the maturity and sustainability of neurons by increasing MAP2-positive cells and MAP2 protein levels. At the same time, Clau F significantly inhibited the proliferation of NS/PCs. The underlying mechanism of Clau F was preliminary explored. Clau F treatment resulted in a profound increase of phosphorylation of Akt and GSK-3ß, which led to GSK-3ß inhibition and subsequently the nuclear accumulation of ß-catenin. Further, the interaction between ß-catenin and p300 in the nucleus was enhanced and the transcription of p300/ß-catenin responsive genes were increased significantly (c-jun, fra-1) by Clau F. Importantly, the positive effect of Clau F on neuronal differentiation was abolished by Akti-1/2, a specific inhibitor of Akt-1/2 kinase, which indicated the involvement of Akt/GSK-3ß in Clau F-mediated neuronal differentiation. In conclusion, these data suggested that Clau F promoted neuronal differentiation through Akt/GSK-3ß/ß-catenin signaling pathway in NS/PCs.

20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway.

AIM: Our preliminary study shows that a bibenzyl compound isolated from Gastrodia elata, 2-[4-hydroxy-3-(4-hydroxybenzyl)benzyl]-4-(4-hydroxybenzyl)phenol (designated 20C), protects PC12 cells against H2O2-induced injury. In this study we investigated whether 20C exerted neuroprotective action in a cell model of Parkinson's disease. METHODS: A cell model of Parkinson's disease was established in PC12 cells by exposure to rotenone (4 µmol/L) for 48 h. Cell viability and apoptosis were assessed, and intracellular ROS level and the mitochondrial membrane potential (MMP) were detected. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome c, cleaved caspase-3, and oxidative stress-related proteins Nrf2, HO-1 and NQO1 were examined using Western blotting. The mRNA levels of HO-1 and NQO1 were determined with RT-PCR. The nuclear translocation of Nrf2 was observed with immunofluorescence staining. RESULTS: Treatment with rotenone significantly increased the number of apoptotic cells, accompanied by marked increases in the Bax/Bcl-2 ratio, cytochrome c release and caspase-3 activation. Rotenone also increased ROS accumulation, reduced MMP, and increased the nuclear translocation of Nrf2 as well as the mRNA and protein levels of the Nrf2 downstream target genes HO-1 and NQO1 in PC12 cells. Co-treatment with 20C (0.01-1 µmol/L) dose-dependently attenuated rotenone-induced apoptosis and oxidative stress in PC12 cells. Nrf2 knockdown by siRNA partially reversed the protective effects of 20C in rotenone-treated PC12 cells. CONCLUSION: The bibenzyl compound 20C protects PC12 cells from rotenone-induced apoptosis, at least in part, via activation of the Nrf2/ARE/HO-1 signaling pathway.

The molecular mechanism of rotenone-induced α-synuclein aggregation: emphasizing the role of the calcium/GSK3ß pathway.

Environmental toxin exposure is associated with the development of Parkinson's disease (PD), and environmental factors can influence the onset of the majority of sporadic PD cases via genetically mediated pathways. Rotenone, a widespread pesticide, induces Parkinsonism and the formation of Lewy bodies in animals; however, the molecular mechanism that underlies α-synuclein aggregation remains unclear. Here, we assessed the aggregation of α-synuclein in PC12 cells with or without cross-linking following rotenone exposure via a variety of methods, including western blotting, immunofluorescence and electron microscopy. We demonstrated that rotenone increased the intracellular calcium levels and induced the aggregation and phosphorylation of α-synuclein in a calcium-dependent manner. Aggregated α-synuclein is typically degraded by autophagy, and rotenone impaired this process. The attenuation of autophagy and α-synuclein alterations were reversed by scavenging calcium. Calcium regulates the activity of AKT-glycogen synthase kinase 3 (GSK3)ß. We demonstrated that rotenone attenuated the phosphorylation of AKT and GSK3ß, and the elimination of calcium reversed these phenomena. As a GSK3ß inhibitor, lithium promoted autophagy and decreased the aggregation and phosphorylation of α-synuclein. GSK3ß activation through overexpression depressed autophagy and increased the total protein level and phosphorylation of α-synuclein. These results suggest that rotenone-induced α-synuclein aggregation is mediated by the calcium/GSK3ß signaling pathway.

Overexpression of human E46K mutant α-synuclein impairs macroautophagy via inactivation of JNK1-Bcl-2 pathway.

Parkinson's disease (PD) is pathologically characterized by selective loss of dopaminergic neurons in the midbrain and the existence of intracellular protein inclusions termed Lewy bodies, largely composed of α-synuclein. Genetic studies have revealed that rare point mutations in the gene encoding α-synuclein including A30P, A53T, and E46K are associated with familial forms of PD, indicating a pathological role for mutant α-synuclein in PD etiology. However, the mechanisms underlying the neuronal toxicity of mutant α-synuclein are still to be elucidated. Growing evidence has suggested a deleterious effect of mutant α-synuclein on the autophagy-lysosome pathway. In this study, we discovered that overexpression of human E46K mutant α-synuclein impaired macroautophagy in mammalian cells. Our data showed that overexpression of E46K mutant α-synuclein impaired autophagy at an early stage of autophagosome formation via the c-Jun N-terminal kinase 1 (JNK1)-Bcl-2 but not the mammalian target of rapamycin (mTOR) pathway. Overexpressed E46K mutant α-synuclein inhibited JNK1 activation, leading to a reduced Bcl-2 phosphorylation and increased association between Bcl-2 and Beclin1, further disrupting the formation of Beclin1/hVps34 complex, which is essential for autophagy initiation. Furthermore, overexpression of E46K mutant α-synuclein increased the vulnerability of differentiated PC12 cells to rotenone treatment, which would be partly due to its inhibitory effects on autophagy. Our findings may shed light on the potential roles of mutant α-synuclein in the pathogenesis of PD.