Investigation the mechanism of iron overload-induced colonic inflammation following ferric citrate exposure.

Iron overload occurs due to excessive iron intake compared to the body's demand, leading to iron deposition and impairment of multiple organ functions. Our previous study demonstrated that chronic oral administration of ferric citrate (FC) caused colonic inflammatory injury. However, the precise mechanism underlying this inflammatory response remains unclear. The current study aims to investigate the mechanism by which iron overload induced by FC exposure leads to colonic inflammation. To accomplish this, mice were orally exposed to three different concentrations of FC (71 mg/kg/bw (L), 143 mg/kg/bw (M) and 286 mg/kg/bw (H)) for continuous 16 weeks, with the control group receiving ultrapure water (C). Exposure to FC caused disturbances in the excretory system, altered colonic flora alpha diversity, and enriched pathogenic bacteria, such as Mucispirillum, Helicobacter, Desulfovibrio, and Shigella. These changes led to structural disorders of the colonic flora and an inflammatory response phenotype characterized by inflammatory cells infiltration, atrophy of intestinal glands, and irregular thickening of the intestinal wall. Mechanistic studies revealed that FC-exposure activated the NF-κB signaling pathway by up-regulating TLR4, MyD88, and NF-κB mRNA levels and protein expression. This activation resulted in increased production of pro-inflammatory cytokines, further contributing to the colonic inflammation. Additionally, in vitro experiments in SW480 cells confirmed the activation of NF-κB signaling pathway by FC exposure, consistent with the in vivo findings. The significance of this study lies in its elucidation of the mechanism by which iron overload caused by FC exposure leads to colonic inflammation. By identifying the role of pathogenic bacteria and the NF-κB signaling pathway, this study could potentially offer a crucial theoretical foundation for the research on iron overload, as well as provide valuable insights for clinical iron supplementation.

Potassium dehydroandrographolide succinate regulates the MyD88/CDH13 signaling pathway to enhance vascular injury-induced pathological vascular remodeling.

Pathological vascular remodeling is a hallmark of various vascular diseases. Previous research has established the significance of andrographolide in maintaining gastric vascular homeostasis and its pivotal role in modulating endothelial barrier dysfunction, which leads to pathological vascular remodeling. Potassium dehydroandrographolide succinate (PDA), a derivative of andrographolide, has been clinically utilized in the treatment of inflammatory diseases precipitated by viral infections. This study investigates the potential of PDA in regulating pathological vascular remodeling. The effect of PDA on vascular remodeling was assessed through the complete ligation of the carotid artery in C57BL/6 mice. Experimental approaches, including rat aortic primary smooth muscle cell culture, flow cytometry, bromodeoxyuridine (BrdU) incorporation assay, Boyden chamber cell migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay, were employed to evaluate the influence of PDA on the proliferation and motility of smooth muscle cells (SMCs). Molecular docking simulations and co-immunoprecipitation assays were conducted to examine protein interactions. The results revealed that PDA exacerbates vascular injury-induced pathological remodeling, as evidenced by enhanced neointima formation. PDA treatment significantly increased the proliferation and migration of SMCs. Further mechanistic studies disclosed that PDA upregulated myeloid differentiation factor 88 (MyD88) expression in SMCs and interacted with T-cadherin (CDH13). This interaction augmented proliferation, migration, and extracellular matrix deposition, culminating in pathological vascular remodeling. Our findings underscore the critical role of PDA in the regulation of pathological vascular remodeling, mediated through the MyD88/CDH13 signaling pathway.

Liuwei Dihuang pills attenuate ovariectomy-induced bone loss by alleviating bone marrow mesenchymal stem cell (BMSC) senescence via the Yes-associated protein (YAP)-autophagy axis.

CONTEXT: Liuwei Dihuang pill (LWDH) has been used to treat postmenopausal osteoporosis (PMOP). OBJECTIVE: To explore the effects and mechanisms of action of LWDH in PMOP. MATERIALS AND METHODS: Forty-eight female Sprague-Dawley rats were divided into four groups: sham-operated (SHAM), ovariectomized (OVX), LWDH high dose (LWDH-H, 1.6 g/kg/d) and LWDH low dose (LWDH-L, 0.8 g/kg/d); the doses were administered after ovariectomy via gavage for eight weeks. After eight weeks, the bone microarchitecture was evaluated. The effect of LWDH on the differentiation of bone marrow mesenchymal stem cells (BMSCs) was assessed via osteogenesis- and lipogenesis-induced BMSC differentiation. The senescence-related biological indices were also detected using senescence staining, cell cycle analysis, quantitative real-time polymerase chain reaction and western blotting. Finally, the expression levels of autophagy-related proteins and Yes-associated protein (YAP) were evaluated. RESULTS: LWDH-L and LWDH-H significantly modified OVX-induced bone loss. LWDH promoted osteogenesis and inhibited adipogenesis in OVX-BMSCs. Additionally, LWDH decreased the positive ratio of senescence OVX-BMSCs and improved cell viability, cell cycle, and the mRNA and protein levels of p53 and p21. LWDH upregulated the expression of autophagy-related proteins, LC3, Beclin1 and YAP, in OVX-BMSCs and downregulated the expression of p62. DISCUSSION AND CONCLUSIONS: LWDH improves osteoporosis by delaying the BMSC senescence through the YAP-autophagy axis.

Paeonol Promotes Reendothelialization After Vascular Injury Through Activation of c-Myc/VEGFR2 Signaling Pathway.

Purpose: Dysfunction of endothelium is associated with multiple pathological vascular diseases. However, how to regulate reendothelialization after vascular injury is not well defined. This study aims to determine whether and how Paeonol controls reendothelialization following artery injury. Methods: The endothelium of murine carotid artery was denuded by catheter guide wires injury. H&E staining and IF staining were performed to determine whether Paeonol is critical for reendothelialization. BRDU Incorporation Assay, Boyden Chamber Migration Assay, Tube Formation Assay, and Spheroid Sprouting Assay were used to investigate whether Paeonol is involved in regulating proliferation and migration of endothelial cells. The underlying mechanism of how Paeonol regulates reendothelialization was determined by Molecular docking simulation and CO-IP Assay. Results: Paeonol treatment significantly inhibits neointima formation in carotid artery ligation model by promoting proliferation and migration of endothelial cells. Mechanistically, Paeonol enhances c-Myc expression, consequently interacts with VEGFR2 results in activating VEGF signaling pathway, and eventually promotes reendothelialization after vascular injury. Conclusion: Our data demonstrated that Paeonol plays a critical role in regulating vascular reendothelialization, which may be therapeutically used for treatment of pathological vascular diseases.

Andrographolide suppresses hypoxia-induced embryonic hyaloid vascular system development through HIF-1a/VEGFR2 signaling pathway.

Introduction: Ocular abnormalities and the development of retinal vasculature may cause postnatal retinopathy. In the past decade, tremendous progress has been made in identifying the mechanisms that regulate retina vasculature. However, the means of regulating embryonic hyaloid vasculature development is largely unknown. This study aims to determine whether and how andrographolide regulates embryonic hyaloid vasculature development. Methods: Murine embryonic retinas were used in this study. Whole mount isolectin B4 (IB4) staining, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence staining (IF) were performed to determine whether andrographolide is critical for embryonic hyaloid vasculature development. BrdU incorporation assay, Boyden chamber migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay were performed to evaluate whether andrographolide regulates the proliferation and migration of vascular endothelial cells. Molecular docking simulation and Co-immunoprecipitation assay were used to observe protein interaction. Results: Hypoxia conditions exist in murine embryonic retinas. Hypoxia induces HIF-1a expression; high-expressed HIF-1a interacts with VEGFR2, resulting in the activation of the VEGF signaling pathway. Andrographolide suppresses hypoxia-induced HIF-1a expression and, at least in part, interrupts the interaction between HIF-1a and VEGFR2, causing inhibiting endothelial proliferation and migration, eventually inhibiting embryonic hyaloid vasculature development. Conclusion: Our data demonstrated that andrographolide plays a critical role in regulating embryonic hyaloid vasculature development.

Potassium Dehydroandrograpolide Succinate Targets NRP1 Mediated VEGFR2/VE-Cadherin Signaling Pathway to Promote Endothelial Barrier Repair.

Impairment of vascular endothelial integrity is associated with various vascular diseases. Our previous studies demonstrated that andrographolide is critical to maintaining gastric vascular homeostasis, as well as to regulating pathological vascular remodeling. Potassium dehydroandrograpolide succinate (PDA), a derivative of andrographolide, has been clinically used for the therapeutic treatment of inflammatory diseases. This study aimed to determine whether PDA promotes endothelial barrier repair in pathological vascular remodeling. Partial ligation of the carotid artery in ApoE-/- mice was used to evaluate whether PDA can regulate pathological vascular remodeling. A flow cytometry assay, BRDU incorporation assay, Boyden chamber cell migration assay, spheroid sprouting assay and Matrigel-based tube formation assay were performed to determine whether PDA can regulate the proliferation and motility of HUVEC. A molecular docking simulation and CO-immunoprecipitation assay were performed to observe protein interactions. We observed that PDA induced pathological vascular remodeling characterized by enhanced neointima formation. PDA treatment significantly enhanced the proliferation and migration of vascular endothelial cells. Investigating the potential mechanisms and signaling pathways, we observed that PDA induced endothelial NRP1 expression and activated the VEGF signaling pathway. Knockdown of NRP1 using siRNA transfection attenuated PDA-induced VEGFR2 expression. The interaction between NRP1 and VEGFR2 caused VE-Cad-dependent endothelial barrier impairment, which was characterized by enhanced vascular inflammation. Our study demonstrated that PDA plays a critical role in promoting endothelial barrier repair in pathological vascular remodeling.

Ferric citrate-induced colonic mucosal damage associated with oxidative stress, inflammation responses, apoptosis, and the changes of gut microbial composition.

Ferric citrate (FC) has been used as an iron fortifier and nutritional supplement, which is reported to induce colitis in rats, however the underlying mechanism remains to be elucidated. We performed a 16-week study of FC in male healthy C57BL/6 mice (nine-month-old) with oral administration of Ctr (0.9 % NaCl), 1.25 % FC (71 mg/kg/bw), 2.5 % FC (143 mg/kg/bw) and 5 % FC (286 mg/kg/bw). FC-exposure resulted in colon iron accumulation, histological alteration and reduce antioxidant enzyme activities, such as glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC), together with enhanced lipid peroxidation level, including malondialdehyde (MDA) level and 4-Hydroxynonenal (4-HNE) protein expression. Exposure to FC was associated with upregulated levels of the interleukin (IL)- 6, IL-1ß, IL-18, IL-8 and tumor necrosis factor α (TNF-α), while down-regulated levels of IL-4 and IL-10. Exposure to FC was positively associated with the mRNA and protein expressions of cysteine-aspartic proteases (Caspase)- 9, Caspase-3, Bcl-2-associated X protein (Bax), while negatively associated with B-cell lymphoma 2 (Bcl2) in mitochondrial apoptosis signaling pathway. FC-exposure changed the diversity and composition of gut microbes. Additionally, the serum lipopolysaccharide (LPS) contents increased in FC-exposed groups when compared with the control group, while the expression of colonic tight junction proteins (TJPs), such as Claudin-1 and Occludin were decreased. These findings indicate that the colonic mucosal injury induced by FC-exposure are associated with oxidative stress generation, inflammation response and cell apoptosis, as well as the changes in gut microbes diversity and composition.

Andrographolide inhibits murine embryonic neuronal development through PFKFB3-mediated glycolytic pathway.

Dysregulation of neuronal development may cause neurodevelopmental disorders. However, how to regulate embryonic neuronal development and whether this regulation can be medical interrupted are largely unknown. This study aimed to investigate whether and how andrographolide (ANP) regulates embryonic neuronal development. The pregnant mice at embryonic day 10.5 (E10.5) were administrated with ANP, and the embryonic brains were harvested at E17.5 or E18.5. Immunofluorescence (IF), Immunohistochemistry (IHC) performed to determine whether ANP is critical in regulating neuronal development. Real-time quantitative PCR, western blotting, cell counting kit-8 assay, Flow Cytometry assay, Boyden Chamber Migration assay carried out to evaluate whether ANP regulates neuronal proliferation and migration. Protein-protein interaction, CO-immunoprecipitation and IF staining carried out to evaluate whether ANP regulates the interaction between PFKFB3, NeuN and TBR1. Knockdown or overexpression of PFKFB3 by adenovirus infection were used to determine whether ANP inhibits neuronal development through PFKFB3 mediated glycolytic pathway. Our data indicated that ANP inhibited the maturation of embryonic neurons characterized by suppressing neuronal proliferation and migration. ANP regulated the interaction between PFKFB3, NeuN, and TBR1. Knockdown of PFKFB3 aggravated ANP mediated inhibition of neuronal proliferation and migration, while overexpression of PFKFB3 attenuated ANP mediated neuronal developmental suppression. In summary, ANP suppressed the expression of PFKFB3, and interrupted the interaction between TRB1 and NeuN, resulting in suppressing neuronal proliferation, migration and maturation and eventually inhibiting murine embryonic neuronal development.

Cinobufagin induces FOXO1-regulated apoptosis, proliferation, migration, and invasion by inhibiting G9a in non-small-cell lung cancer A549 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufonis (VB), an animal drug called Chansu in China, is the product of the secretion of Bufo gargarizans Cantor or B. melanostictus Schneider. As a traditional Chinese medicine (TCM) for a long time, it has been widely used in the treatment of heart failure, ulcer, pain, and various cancers. Cinobufaginn (CNB), the cardiotonic steroid or bufalene lactone extracted from VB, has the effects of detoxification, detumescence, and analgesia. AIM OF THE STUDY: The present study aimed to define the effects of CNB on non-small-cell lung cancer (NSCLC) and identify the potential molecular mechanisms. MATERIALS AND METHODS: A549 cells were treated with cinobufagin and cell viability, apoptosis, migration, and invasion were then evaluated using Cell Counting Kit-8 (CCK8) assays, flow cytometry, and Transwell assays, respectively. Moreover, the levels of proliferating cell nuclear antigen (PCNA), cytokeratin8 (CK8), poly ADP-ribose polymerase (PARP), Caspase3, Caspase8, B-cell lymphoma/lewkmia-2(Bcl-2), Bcl2-Associated X(Bax), forkhead box O1 (FOXO1), and euchromatic histone-lysine N-methyltransferase2 (G9a, EHMT2) in A549 cells were evaluated using qRT-PCR and/or Western blot analysis (WB), Co-IP, immunofluorescence, and immunohistochemistry. An in vivo imaging system, TUNEL, Immunofluorescence, and immunohistochemistry were also used to detect proliferating cell nuclear antigen(PCNA), Ki67, E-Cadherin(E-Cad), FOXO1, and G9a in mouse xenograft model experiments. RESULTS: CNB suppressed cell proliferation, migration, and invasion but promoted apoptosis in A549 cells in a dose- and time-dependent manner, while cinobufagin had no cytotoxic effect on BEAS-2B cells. In vivo, cinobufagin inhibited the proliferation, migration, and invasion of A549 cells and promoted their apoptosis. The occurrence of the above phenomena was accompanied by an increase in FOXO1 expression and a decrease in G9a expression. In A549 cells, CNB did not reverse the changes in the proliferation, migration, invasion, and apoptosis of A549 cells after FOXO1 was successfully silenced. CONCLUSION: Our study provides the first evidence that cinobufagin suppresses the malignant biological behaviours of NSCLC cells in vivo and in vitro and suggests that mechanistically, this effect may be achieved by inhibiting the expression of the histone methyltransferase G9a and activating the tumour suppressor gene FOXO1. Taken together, our findings provide important insights into the molecular mechanism underlying cinobufagin's anticancer activity, and suggest that cinobufagin could be a candidate for targeted cancer therapy.

Gou Qi Zi inhibits proliferation and induces apoptosis through the PI3K/AKT1 signaling pathway in non-small cell lung cancer.

Background: Gou Qi Zi (Lycium barbarum) is a traditional herbal medicine with antioxidative effects. Although Gou Qi Zi has been used to prevent premature aging and in the treatment of non-small cell lung cancer (NSCLC), its mechanism of action in NSCLC remains unclear. The present study utilized network pharmacology to assess the potential mechanism of action of Gou Qi Zi in the treatment of NSCLC. Methods: The TCMSP, TCMID, SwissTargetPrediction, DrugBank, DisGeNET, GeneCards, OMIM and TTD databases were searched for the active components of Gou Qi Zi and their potential therapeutic targets in NSCLC. Protein-protein interaction networks were identified and the interactions of target proteins were analyzed. Involved pathways were determined by GO enrichment and KEGG pathway analyses using the Metascape database, and molecular docking technology was used to study the interactions between active compounds and potential targets. These results were verified by cell counting kit-8 assays, BrdU labeling, flow cytometry, immunohistochemistry, western blotting, and qRT-PCR. Results: Database searches identified 33 active components in Gou Qi Zi, 199 predicted biological targets and 113 NSCLC-related targets. A network of targets of traditional Chinese medicine compounds and potential targets of Gou Qi Zi in NSCLC was constructed. GO enrichment analysis showed that Gou Qi Zi targeting of NSCLC was mainly due to the effect of its associated lipopolysaccharide. KEGG pathway analysis showed that Gou Qi Zi acted mainly through the PI3K/AKT1 signaling pathway in the treatment of NSCLC. Molecular docking experiments showed that the bioactive compounds of Gou Qi Zi could bind to AKT1, C-MYC and TP53. These results were verified by experimental assays. Conclusion: Gou Qi Zi induces apoptosis and inhibits proliferation of NSCLC in vitro and in vivo by inhibiting the PI3K/AKT1 signaling pathway.

Critical role of mitochondrial aldehyde dehydrogenase 2 in acrolein sequestering in rat spinal cord injury.

Lipid peroxidation-derived aldehydes, such as acrolein, the most reactive aldehyde, have emerged as key culprits in sustaining post-spinal cord injury (SCI) secondary pathologies leading to functional loss. Strong evidence suggests that mitochondrial aldehyde dehydrogenase-2 (ALDH2), a key oxidoreductase and powerful endogenous anti-aldehyde machinery, is likely important for protecting neurons from aldehydes-mediated degeneration. Using a rat model of spinal cord contusion injury and recently discovered ALDH2 activator (Alda-1), we planned to validate the aldehyde-clearing and neuroprotective role of ALDH2. Over an acute 2 day period post injury, we found that ALDH2 expression was significantly lowered post-SCI, but not so in rats given Alda-1. This lower enzymatic expression may be linked to heightened acrolein-ALDH2 adduction, which was revealed in co-immunoprecipitation experiments. We have also found that administration of Alda-1 to SCI rats significantly lowered acrolein in the spinal cord, and reduced cyst pathology. In addition, Alda-1 treatment also resulted in significant improvement of motor function and attenuated post-SCI mechanical hypersensitivity up to 28 days post-SCI. Finally, ALDH2 was found to play a critical role in in vitro protection of PC12 cells from acrolein exposure. It is expected that the outcome of this study will broaden and enhance anti-aldehyde strategies in combating post-SCI neurodegeneration and potentially bring treatment to millions of SCI victims. All animal work was approved by Purdue Animal Care and Use Committee (approval No. 1111000095) on January 1, 2021.

Acrolein scavenger dimercaprol offers neuroprotection in an animal model of Parkinson's disease: implication of acrolein and TRPA1.

BACKGROUND: The mechanisms underlying lesions of dopaminergic (DA) neurons, an essential pathology of Parkinson's disease (PD), are largely unknown, although oxidative stress is recognized as a key factor. We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in PD pathology, and that acrolein scavenger hydralazine can reduce the elevated acrolein, mitigate DA neuron death, and alleviate motor deficits in a 6-hydroxydopamine (6-OHDA) rat model. As such, we hypothesize that a structurally distinct acrolein scavenger, dimercaprol (DP), can also offer neuroprotection and behavioral benefits. METHODS: DP was used to lower the elevated levels of acrolein in the basal ganglia of 6-OHDA rats. The acrolein levels and related pathologies were measured by immunohistochemistry. Locomotor and behavioral effects of 6-OHDA injections and DP treatment were examined using the open field test and rotarod test. Pain was assessed using mechanical allodynia, cold hypersensitivity, and plantar tests. Finally, the effects of DP were assessed in vitro on SK-N-SH dopaminergic cells exposed to acrolein. RESULTS: DP reduced acrolein and reversed the upregulation of pain-sensing transient receptor potential ankyrin 1 (TRPA1) channels in the substantia nigra, striatum, and cortex. DP also mitigated both motor and sensory deficits typical of PD. In addition, DP lowered acrolein and protected DA-like cells in vitro. Acrolein's ability to upregulate TRPA1 was also verified in vitro using cell lines. CONCLUSIONS: These results further elucidated the acrolein-mediated pathogenesis and reinforced the critical role of acrolein in PD while providing strong arguments for anti-acrolein treatments as a novel and feasible strategy to combat neurodegeneration in PD. Considering the extensive involvement of acrolein in various nervous system illnesses and beyond, anti-acrolein strategies may have wide applications and broad impacts on human health.

Clioquinol improves motor and non-motor deficits in MPTP-induced monkey model of Parkinson's disease through AKT/mTOR pathway.

Despite decades of research into the pathology mechanisms of Parkinson's disease (PD), disease-modifying therapy of PD is scarce. Thus, searching for new drugs or more effective neurosurgical treatments has elicited much interest. Clioquinol (CQ) has been shown to have therapeutic benefits in rodent models of neurodegenerative disorders. However, it's neuroprotective role and mechanisms in PD primate models and PD patients, especially in the advanced stages, are not fully understood. Furthermore, issues such as spontaneous recovery of motor function and high symptom variability in different monkeys after the same toxic protocol, has not been resolved before the present study. In this study, we designed a chronic and long-term progressive protocol to generate a stabilized PD monkey model showed with classic motor and non-motor deficits, followed by treatment analysis of CQ. We found that CQ could remarkably improve the motor and non-motor deficits, which were based on the reduction of iron content and ROS level in the SN and further improvement in pathology. Meanwhile, we also showed that ferroptosis was probably involved in the pathogenesis of PD. In addition, the study shows a positive effect of CQ on AKT/mTOR survival pathway and a blocking effect on p53 medicated cell death in vivo and in vitro.

Iron overload resulting from the chronic oral administration of ferric citrate induces parkinsonism phenotypes in middle-aged mice.

Iron homeostasis is critical for maintaining normal brain physiological functions, and its mis-regulation can cause neurotoxicity and play a part in the development of many neurodegenerative disorders. The high incidence of iron deficiency makes iron supplementation a trend, and ferric citrate is a commonly used iron supplement. In this study, we found that the chronic oral administration of ferric citrate (2.5 mg/day and 10 mg/day) for 16 weeks selectively induced iron accumulation in the corpus striatum (CPu), substantia nigra (SN) and hippocampus, which typically caused parkinsonism phenotypes in middle-aged mice. Histopathological analysis showed that apoptosis- and oxidative stress-mediated neurodegeneration and dopaminergic neuronal loss occurred in the brain, and behavioral tests showed that defects in the locomotor and cognitive functions of these mice developed. Our research provides a new perspective for ferric citrate as a food additive or in clinical applications and suggests a new potential approach to develop animal models for Parkinson's disease (PD).

The Association of Iron and the Pathologies of Parkinson's Diseases in MPTP/MPP+-Induced Neuronal Degeneration in Non-human Primates and in Cell Culture.

Despite much efforts in the last few decades, the mechanism of degeneration of dopamine (DA) neurons in the substantia nigra (SN) in Parkinson's disease (PD) remains unclear. This represents a major knowledge gap in idiopathic and genetic forms of PD. Among various possible key factors postulated, iron metabolism has been widely suggested to be involved with fueling oxidative stress, a known factor in the pathogenesis of PD. However, the correlation between iron and DA neuron loss, specifically in the SN, has not been described in experimental animal models with great detail, with most studies utilizing rodents and, rarely, non-human primates. In the present study, aiming to gain further evidence of a pathological role of iron in PD, we have examined the correlation of iron with DA neuron loss in a non-human primate model of PD induced by MPTP. We report a significant iron accumulation accompanied by both DA degeneration in the SN and motor deficits in the monkey that displayed the most severe PD pathology and behavioral deficits. The other two monkeys subjected to MPTP displayed less severe PD pathologies and motor deficits, however, their SN iron levels were significantly lower than controls. These findings suggest that high iron may indicate and contribute to heightened MPP+-induced PD pathology in late or severe stages of PD, while depressed levels of iron may signal an early stage of disease. Similarly, using a cell culture preparation, we have found that high doses of ferric ammonium citrate (FAC), a factor known to enhance iron accumulation, increased MPP+-induced cell death in U251 and SH-SY5Y cells, and even in control cells. However, at low dose FAC restored or increased the viability of U251 and SH-SY5Y cells in the absence or presence of MPP+. These observations imply that high levels of iron likely contribute to or heighten MPP+ toxicity in the later stages of PD. While we report reduced iron levels in the earlier stages of MPTP induced PD, the significance of these changes remains to be determined.

Endocannabinoid hydrolase and cannabinoid receptor 1 are involved in the regulation of hypothalamus-pituitary-adrenal axis in type 2 diabetes.

Hypothalamus-pituitary-adrenal (HPA) axis, as the key moderator in energy metabolism, plays an important role in diabetes. The endogenous cannabinoid system (eCBs) involves in neuronal functions, and simultaneously cannabinoid receptors are almost expressed in all regions of the hypothalamus according to a spate of reports. However, few data investigate the changes of eCBs and HPA axis in type 2 diabetes. In this study, five diabetes mellitus rhesus monkeys, five prediabetes rhesus monkeys and five healthy rhesus monkeys were observed. In the present study, we detected cell swelling and necrosis extensively in the paraventricular nucleus (PVN) and neurohypophysis in prediabetes and overt diabetes monkeys. The adrenocorticotropic hormone in the pituitary gland, adrenocorticotropic hormone receptor, and 11ß-hydroxysteroid dehydrogenase in the adrenal gland were all hyper-secreted and expressed from healthy to overt diabetes. Meanwhile, the cortisol concentration in the adrenal gland was increased along with the progress of diabetes. It could be concluded that hyperfunction of the HPA axis exists in the type 2 Diabetes pathogenesis. However, we also found a weakened expression and secretion of corticotrophin releasing hormone and glucocorticoids receptor in PVN. The expression of corticotropin releasing hormone receptor 1 in pituitary gland decreased in prediabetes monkeys, but increased in overt diabetes monkeys. The downregulation of cannabinoid receptor 1 and upregulation of monoglycerol lipase and fatty acid amide hydrolase in PVN was involved in the pathogenesis of type 2 diabetes. Collectively, we can conclude that changes in endocannabinoid hydrolase and cannabinoid receptor might indicate the effect of downregulation of eCBs. It can be assumed that hyper-function of the HPA axis from healthy to overt diabetes is due to the undermining inhibition of eCBs. However, the regulatory mechanism of eCBs targets on the HPA axis need to be further explored.

Role of the Death Receptor and Endoplasmic Reticulum Stress Signaling Pathways in Polyphyllin I-Regulated Apoptosis of Human Hepatocellular Carcinoma HepG2 Cells.

Polyphyllin has been reported to exhibit anticancer effects against various types of cancer via the proapoptotic signaling pathway. The aim of the present study was to investigate the role of the endoplasmic reticulum stress and death receptor signaling pathways in PPI-induced apoptosis of human hepatocellular carcinoma HepG2 cells. Analysis demonstrated that PPI could significantly inhibit the proliferation and induce apoptosis of HepG2 cells in a dose- and time-dependent manner. Investigation into the molecular mechanism of PPI indicated that PPI notably mediated ER stress activation via IRE-1 overexpression and activation of the caspase-12 to protect HepG2 cells against apoptosis. In addition, PPI markedly induced the expression of death receptors signaling pathways-associated factors, including tumor necrosis factor (TNF) receptor 1/TNF-α and FAS/FASL. Additionally, suppression of the death receptor signaling pathways with a caspase-8 inhibitor, Z-IETD-FMK, revealed an increase in the death rate and apoptotic rate of HepG2 cells. Collectively, the findings of the present study suggested that the ER stress and death receptor signaling pathways were associated with PPI-induced HepG2 cell apoptosis; however, endoplasmic reticulum stress may serve a protective role in this process. The combination of PPI and Z-IETD-FMK may activate necroptosis, which enhances apoptosis. Therefore, the results of the present study may improve understanding regarding the roles of signaling pathways in PPI regulated apoptosis and contribute to the development of novel therapies for the treatment of HCC.

Pilot study: molecular risk factors for diagnosing sporadic Parkinson's disease based on gene expression in blood in MPTP-induced rhesus monkeys.

Clinical diagnosis of Parkinson's disease (PD) is characterized by the classical features of tremor, bradykinesia and rigidity, which are present only when more than 70%-80% degeneration of dopaminergic (DA) neurons in the substantia nigra. The lack of means for early diagnosis of PD has elicited interest in searching for its risk factors, which, by now, are almost obtained at a single time point in PD process, and little developing risk factors, obtained from completely normal situation to the onset or even advanced stage of PD in individual person which could monitor the progress of PD, are present. Here we have detected some potential factors in the blood of MPTP induced PD monkeys along with the progress of the disease. All the PD monkeys showed mild PD symptoms since the 9th week and gradually reached a classic and stable parkinsonism stage at the 18th week. Our results have found that the expression of Parkin, USP30, MUL1, PINK1, and LRRK2 significantly increased at 1st, 3th, 3th, 5th, and 8th week respectively and remained high till the end; The expression of UCHL1 and TRIM24 significantly increased at the 1st and 18th week, respectively, then gradually decreased and significantly lower than normal value; DJ-1 showed significantly decreased since the 12th week, while SNCA showed no significantly changed excepted at the 5th week. And, the terminal results of whole blood were highly consistent with those of in SN. These results support that these genes change may as biomarkers to monitor the progress of PD, and may facilitate the development of biomarkers for early diagnosis.

Changes in blood anti-oxidation enzyme levels in MPTP-treated monkeys.

Oxidative stress has been implicated in a range of neurodegenerative diseases, in particular, Parkinson's disease (PD). However, the pathogenic mechanism of its influence on PD progression remains unclear. In order to clarify this, PD models are generated by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intramuscular injection in eight rhesus monkeys. During the of progress PD pathogenesis, the activities of some major antioxidant enzymes, such as serum superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione-s-transferase (GST), were measured, which show continuously decrease. While, the GSH and MDA content, as well as the expression of gamma-glutamylcysteine synthetase (GCS) and glutathione-s-transferase pi (GST-pi) exhibit robust increase. Taken together, this study shows the dynamic changes of antioxidant enzymes and lipid peroxidation in blood of PD monkeys during its progress, which will be important for the diagnosis and treatment of PD.

Soy Isoflavones Regulate Lipid Metabolism through an AKT/mTORC1 Pathway in Diet-Induced Obesity (DIO) Male Rats.

The pandemic tendency of obesity and its strong association with serious co-morbidities have elicited interest in the underlying mechanisms of these pathologies. Lipid homeostasis, closely involved in obesity, has been reported to be regulated by multiple pathways. mTORC1 is emerging as a critical regulator of lipid metabolism. Here, we describe that the consumption of soy isoflavones, with a structural similarity to that of estradiol, could mitigate obesity through an AKT/mTORC1 pathway. Fed with soy isoflavones, the diet-induced obesity (DIO) male rats exhibited decreased body weight, accompanied with suppressed lipogenesis and adipogenesis, as well as enhanced lipolysis and ß­oxidation. The phosphorylation of AKT and S6 were decreased after soy isoflavone treatment in vivo and in vitro, suggesting an inhibition effect of soy isoflavones on mTORC1 activity. Our study reveals a potential mechanism of soy isoflavones regulating lipid homeostasis, which will be important for obesity treatment.