3β Activity Research Articles

SARS-CoV-2 propagation to the TPH2-positive neurons in the ventral tegmental area induces cell death via GSK3β-dependent accumulation of phosphorylated tau.

COVID-19, an infectious disease caused by SARS-CoV-2, was declared a pandemic by the WHO in 2020. Psychiatric symptoms including sleep disturbance, memory impairment, and depression are associated with SARS-CoV-2 infection. These symptoms are causes long-term mental and physical distress in recovering patients; however, the underlying mechanism is unclear. In this study, we determined the effects of SARS-CoV-2 infection on brain tissue using k18hACE2 mice. Using brain tissue from 18hACE2 mice infected with SARS-CoV-2 through intranasal administration, SARS-CoV-2 spike protein and RNA were analyzed by immunohistochemical staining and in-situ hybridization. Immunohistochemical analysis revealed that Tryptophan hydroxylase 2 (TPH2)-positive cells and SARS-CoV-2 spike protein were co-localized in the ventral tegmental area of SARS-CoV-2-infected mice. We observed decreased TPH2 expression and increased accumulation of phosphorylated tau protein and Phospho-Histone H2A.X (γH2AX) expression in the ventral tegmental region. In addition, activation of glycogen synthase kinase 3β (GSK3β) was induced by SARS-CoV-2 infection. Overall, our results suggest that SARS-CoV-2 infection of TPH2-positive cells in the ventral tegmental area induces neuronal cell death through increased accumulation of phosphorylated tau. Attenuation of the GSK3β pathway and decreased serotonin synthesis through suppression of TPH2 expression may contribute to the development of neurological symptoms.

The Possible Neuroprotective Effect of Caffeic Acid on Cognitive Changes and Anxiety-Like Behavior Occurring in Young Rats Fed on High-Fat Diet and Exposed to Chronic Stress: Role of β-Catenin/GSK-3B Pathway.

Lifestyle influences physical and cognitive development during the period of adolescence greatly. The most important of these lifestyle factors are diet and stress. Therefore, the aim of this study was to investigate the impact of high fat diet (HFD) and chronic mild stress on cognitive function and anxiety-like behaviors in young rats and to study the role of caffeic acid as a potential treatment for anxiety and cognitive dysfunction. Forty rats were assigned into 4 groups: control, HFD, HFD + stress, and caffeic acid-treated group. Rats were sacrificed after neurobehavioral testing. We detected memory impairment and anxiety-like behavior in rats which were more exaggerated in stressed rats. Alongside the behavioral changes, there were biochemical and histological changes. HFD and/or stress decreased hippocampal brain-derived neurotrophic factor (BDNF) levels and induced oxidative and inflammatory changes in the hippocampus. In addition, they suppressed Wnt/β-catenin pathway which was associated with activation of glycogen synthase kinase 3β (GSK3β). HFD and stress increased arginase 1 and inducible nitric oxide synthase (iNOS) levels as well. These disturbances were found to be aggravated in stressed rats than HFD group. However, caffeic acid was able to reverse these deteriorations leading to memory improvement and ameliorating anxiety-like behavior. So, the current study highlights an important neuroprotective role for caffeic acid that may guard against induction of cognitive dysfunction and anxiety disorders in adolescents who are exposed to HFD and/or stress.

GPAT3 deficiency attenuates corticosterone-caused hepatic steatosis and oxidative stress through GSK3β/Nrf2 signals

The development of nonalcoholic fatty liver disease (NAFLD) may worsen due to chronic stress or prolonged use of glucocorticoids. Glycerol-3-phosphate acyltransferase 3 (GPAT3), has a function in obesity and serves as a key rate-limiting enzyme that regulates triglyceride synthesis. However, the precise impact of GPAT3 on corticosterone (CORT)-induced NAFLD and its underlying molecular mechanism remain unclear. For our in vivo experiments, we utilized male and female mice that were GPAT3−/− and wild type (WT) and treated them with CORT for a duration of 4 weeks. In our in vitro experiments, we transfected AML12 cells with GPAT3 siRNA and subsequently treated them with CORT. Under CORT-treated conditions, the absence of GPAT3 greatly improved obesity and hepatic steatosis while enhancing the expression of genes involved in fatty acid oxidation, as evidenced by our findings. In addition, the deletion of GPAT3 significantly inhibited the production of reactive oxygen species (ROS), increased the expression of antioxidant genes, and recovered the mitochondrial membrane potential in AML12 cells treated with CORT. In terms of mechanism, the absence of GPAT3 encouraged the activation of the glycogen synthase kinase 3β (GSK3β)/nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway, which served as a defense mechanism against liver fat accumulation and oxidative stress. Furthermore, GPAT3 expression was directly controlled at the transcriptional level by the glucocorticoid receptor (GR). Collectively, our findings suggest that GPAT3 deletion significantly alleviated hepatic steatosis and oxidative stress through promoting GSK3β/Nrf2 signaling pathways.

Structural modification of resveratrol analogue exhibits anticancer activity against lung cancer stem cells via suppression of Akt signaling pathway

BackgroundCompound with cancer stem cell (CSC)-suppressing activity is promising for the improvement of lung cancer clinical outcomes. Toward this goal, we discovered the CSC-targeting activity of resveratrol (RES) analog moscatilin (MOS). With slight structural modification from RES, MOS shows dominant cytotoxicity and CSC-suppressive effect.MethodsThree human lung cancer cell lines, namely H23, H292, and A549, were used to compare the effects of RES and MOS. Cell viability and apoptosis were determined by the MTT assay and Hoechst33342/PI double staining. Anti-proliferative activity was determined by colony formation assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were measured by fluorescence microscopy using DCFH2-DA staining. CSC-rich populations of A549 cells were generated, and CSC markers, and Akt signaling were determined by Western blot analysis and immunofluorescence. Molecular docking and molecular dynamics (MD) simulations were used to predict the possible binding of the compound to Akt protein.ResultsIn this study, we evaluated the effects of RES and MOS on lung cancer and its anti-CSC potential. Compared with RES, its analog MOS more effectively inhibited cell viability, colony formation, and induced apoptosis in all lung cancer cell lines (H23, H292, and A549). We further investigated the anti-CSC effects on A549 CSC-rich populations and cancer adherent cells (A549 and H23). MOS possesses the ability to suppress CSC-like phenotype of lung cancer cells more potent than RES. Both MOS and RES repressed lung CSCs by inhibiting the viability, proliferation, and lung CSC-related marker CD133. However, only MOS inhibits the CSC marker CD133 in both CSC-rich population and adherent cells. Mechanistically, MOS exerted its anti-CSC effects by inhibiting Akt and consequently restored the activation of glycogen synthase kinase 3β (GSK-3β) and decreased the pluripotent transcription factors (Sox2 and c-Myc). Thus, MOS inhibits CSC-like properties through the repression of the Akt/GSK-3β/c-Myc pathway. Moreover, the superior inhibitory effects of MOS compared to RES were associated with the improved activation of various mechanism, such as cell cycle arrest at G2/M phase, production of ROS-mediated apoptosis, and inhibition of Akt activation. Notably, the computational analysis confirmed the strong interaction between MOS and Akt protein. MD simulations revealed that the binding between MOS and Akt1 was more stable than RES, with MM/GBSA binding free energy of − 32.8245 kcal/mol at its allosteric site. In addition, MOS interacts with Trp80 and Tyr272, which was a key residue in allosteric inhibitor binding and can potentially alter Akt activity.ConclusionsKnowledge about the effect of MOS as a CSC-targeting compound and its interaction with Akt is important for the development of drugs for the treatment of CSC-driven cancer including lung cancer.

Necrosulfonamide exerts neuroprotective effect by inhibiting necroptosis, neuroinflammation, and α-synuclein oligomerization in a subacute MPTP mouse model of Parkinson’s disease

Parkinson’s disease (PD) is an incurable movement disorder characterized by dopaminergic cell loss, neuroinflammation, and α-synuclein pathology. Herein, we investigated the therapeutic effects of necrosulfonamide (NSA), a specific inhibitor of mixed lineage kinase domain-like protein (MLKL), in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MLKL is an executor of necroptosis, a programmed cell death pathway that causes inflammation. Repeated administration of NSA resulted in the recovery of impaired motor performance and dopaminergic degeneration. Furthermore, NSA inhibited the phosphorylation, ubiquitylation, and oligomerization of MLKL, all of which are associated with MLKL cell death-inducing activity in dopaminergic cells in the substantia nigra (SN). NSA also inhibited microglial activation and reactive astrogliosis as well as the MPTP-induced expression of proinflammatory molecules such as tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, and cystatin F. Furthermore, NSA inhibited α-synuclein oligomerization and phosphorylation in the SN of MPTP-treated mice by inhibiting the activity of glycogen synthase kinase 3β and matrix metalloproteinase-3. In conclusion, NSA has anti-necroptotic, anti-inflammatory, and anti-synucleinopathic effects on PD pathology. Therefore, NSA is a potential therapeutic candidate for PD.

Anticancer effects of the HDAC inhibitor, 3β,6β‑dihydroxyurs‑12‑en‑27‑oic acid, in MCF‑7 breast cancer cells via the inhibition of Akt/mTOR pathways.

Astilbechinensis (A.chinensis) is a perennial herb that is used to treat chronic bronchitis and pain. The anticancer activity of 3β,6β‑dihydroxyurs‑12‑en‑27‑oic acid (ACT‑3), a major component isolated from A.chinensis, has not yet been investigated in detail. The purpose of the present study was to investigate the histone deacetylase (HDAC) inhibitory and anticancer activities of ACT‑3 compared with suberoylanilide hydroxamic acid (SAHA) in MCF‑7 human breast cancer cells. The purity of ACT‑3 was determined using high‑performance liquid chromatography. In the present study, the effects of ACT‑3 on anticancer effects of MCF‑7 cells were determined by measuring the level of apoptotic cell death and cell cycle regulator using flow cytometry analysis and western blot analysis, respectively. The effects of ACT‑3 on HDAC enzyme activity were measured using assay kits. ACT‑3 and SAHA increased the levels of acetylated histone H3 and reduced the levels of HDAC1 and HDAC3 in MCF‑7 cells. ACT‑3 significantly decreased the cell viability in a concentration‑dependent manner and induced different morphological changes at high concentrations. ACT‑3 and SAHA significantly inhibited the colony formation in MCF‑7 cells. ACT‑3 inhibited total HDAC activity in a dose‑dependent manner. ACT‑3 significantly reduced the expression levels of cyclin D1 and cyclin‑dependent kinase 4, and upregulated the expression levels of p21WAF1 and p53. A significant increase in the G1 phase cell population was observed in MCF‑7 cells and ACT‑3 induced apoptosis by reducing the ratio of B‑cell lymphoma‑2 (Bcl‑2)/Bcl‑2‑associated X (Bax) and releasing cleaved caspase 9. Additionally, ACT‑3 significantly increased autophagic cell death by inhibiting the serine‑threonine kinase/mammalian target of the rapamycin pathway. Autophagy induction was confirmed via acridine orange staining. ACT‑3 significantly increased the pERK1/2 and p21 in MCF‑7 cells. Thus, the activated ERK pathway played an important role in cell cycle arrest and apoptosis via ERK‑dependent induction of p21 in MCF‑7 cells. These data indicated that ACT‑3 can be used as a promising anticancer agent to overcome the limitations and reduce the side effects of conventional anticancer drugs.

GSK3β-dependent lysosome biogenesis: An effective pathway to mitigate renal fibrosis with LM49.

Renal fibrosis is an incurable disorder characterised by an imbalance of the extracellular matrix (ECM) favouring excess production over degradation. The identification of actionable pathways and agents that promote ECM degradation to restore ECM homeostasis may help mitigate renal fibrosis. In this study, we identified 5,2′-dibromo-2,4′,5′-trihydroxydiphenylmethanone (LM49), a compound we previously synthesised, as a small-molecule inducer of ECM degradation. LM49 administration efficiently reduced ECM deposition in renal tissue of diabetic nephropathy rats and in transforming growth factor β-treated renal fibroblast cells. LM49 promoted the cytosol-to-nucleus translocation of transcription factor EB (TFEB) to increase lysosome biogenesis, leading to lysosome-based degradation of the ECM. TFEB-mediated lysosome biogenesis was induced by LM49 directly inhibiting the activity of glycogen synthase kinase 3β (GSK3β) rather than mammalian target of rapamycin complex 1. LM49 inhibited GSK3β kinase activity concentration-dependently via competing with ATP. Direct binding between LM49 and GSK3β was confirmed by the bio-layer interferometry assay, cellular thermal shift assay, and drug affinity responsive target stability. A molecular docking and molecular dynamic simulation revealed that LM49 occupied the ATP pocket of GSK3β, which was consistent with the kinase activity assay. In summary, LM49 enhances TFEB-mediated lysosome biogenesis by directly inhibiting GSK3β, leading to the degradation of the ECM by lysosomes. The enhancement of GSK3β-dependent lysosome biogenesis to rebalance the ECM may be a novel strategy to counteract renal fibrosis, and LM49 may be a viable clinical candidate for treating this disorder.

Vitamin C deficiency induces hypoglycemia and cognitive disorder through S-nitrosylation-mediated activation of glycogen synthase kinase 3β

Vitamin C (VC, l-ascorbic acid) is an essential nutrient that plays a key role in metabolism and functions as a potent antioxidant in regulating the S-nitrosylation and denitrosylation of target proteins. The precise function of VC deprivation in glucose homeostasis is still unknown. In the absence of L-gulono-1,4-lactone oxidoreductase, an essential enzyme for the last step of VC synthesis, VC deprivation resulted in persistent hypoglycemia and subsequent impairment of cognitive functions in female but not male mouse pups. The cognitive disorders caused by VC deprivation were largely reversed when these female pups were given glucose. VC deprivation-induced S-nitrosylation of glycogen synthase kinase 3β (GSK3β) at Cys14, which activated GSK3β and inactivated glycogen synthase to decrease glycogen synthesis and storage under the feeding condition, while VC deprivation inactivated glycogen phosphorylase to decrease glycogenolysis under the fasting condition, ultimately leading to hypoglycemia and cognitive disorders. Treatment with Nω-Nitro-l-arginine methyl ester (l-NAME), a specific inhibitor of nitric oxide synthase, on the other hand, effectively prevented S-nitrosylation and activation of GSK3β in female pups in response to the VC deprivation and reversed hypoglycemia and cognitive disorders. Overall, this research identifies S-nitrosylation of GSK3β and subsequent GSK3β activation as a previously unknown mechanism controlling glucose homeostasis in female pups in response to VC deprivation, implying that VC supplementation in the prevention of hypoglycemia and cognitive disorders should be considered in the certain groups of people, particularly young females.

Triclosan Influences on Reproductive Physiology of Climbing Perch in the Spawning Phase

Abstract The endocrine-disrupting effects of triclosan, an antimicrobial agent, and its involvement in reproductive responses mediated through different modes of activity in various fish species have been documented. The present study aims to explore whether triclosan at environmentally relevant and sublethal concentrations could influence the reproductive physiology of the fish Anabas testudineus in the spawning phase. Fish were exposed to triclosan at environmentally relevant (0.009 and 9 µg L−1) and sublethal (176.7 µg L−1) concentrations for 4, 7, 30 and 60 d during the spawning phase for evaluating the reproductive potential of the fish. Triclosan exposure caused a significant (P<0.05) reduction in the level of total protein in gonads and liver tissues while increasing blood plasma to meet the energy demand and overcome metabolic stress. Impairment in gonadal steroidogenesis was evidenced by the reduction in the activities of 3β- and 17β-hydroxysteroid dehydrogenases associated with alteration in the levels of serum gonadotropins, sex steroid hormones and vitellogenin. Triclosan exposure caused a reduction in the rate of fecundity in females and declined sperm counts, motility and viability in males. Histological lesions in gonadal tissues further confirmed the reproductive toxicity of triclosan, which in turn could contribute to the reproductive failure of the fish.

Knockdown of Obg-like ATPase 1 enhances sorafenib sensitivity by inhibition of GSK-3β/β-catenin signaling in hepatocellular carcinoma cells.

To clarify the molecular mechanism of hepatocellular carcinoma (HCC), conducive to developing an effective HCC therapy. Owing to the severe drug resistance, the clinical use of sorafenib, which is approved for HCC treatment, is limited. The precise molecular mechanisms of sorafenib drug resistance remain unclear. In the current work, we evaluated the role of Obg-like ATPase 1 (OLA1) in sorafenib resistance in HCC. The survival of HCC patients between OLA1 expression and sorafenib treatment was analyzed by Kaplan-Meier plotter. Cell viability was measured by cell counting kit-8 (CCK-8) and colony formation assays. Cell death was detected by propidium iodide (PI) and trypan blue staining. The mRNA and protein levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB), respectively. We found that OLA1 was highly correlated with sorafenib resistance of HCC through a public database. Further study showed that knockdown of OLA1 enhanced cell proliferation inhibition and cell death induced by sorafenib, along with a reduction of proliferation-associated proteins (c-Myc and cyclin D1) and increase of apoptosis-related proteins (cleaved caspase-3 and cleaved PARP) in HCC cells. In addition, knockdown of OLA1 reduced the activation of glycogen synthase kinase 3β (GSK-3β)/β-catenin. Our results proved that OLA1 can be a potential target to enhance sorafenib sensitivity in HCC.

Zeaxanthin Attenuates the Vicious Circle Between Endoplasmic Reticulum Stress and Tau Phosphorylation: Involvement of GSK-3β Activation.

Alzheimer's disease (AD) characterized by neurofibrillary tangles caused by hyperphosphorylated tau is the most common cause of dementia. Zeaxanthin (Zea), derived from fruits and vegetables, may reduce the risk of AD. Endoplasmic reticulum stress (ERS) might cause memory impairment in AD. Here, we studied protective role of Zea on the relationship among ERS, activity of glycogen synthase kinase 3β (GSK-3β, tau phosphorylated kinase), and p-Tau (Ser 396 and Thr 231). The results were obtained in non-RA and RA group by using different treatment, such as 9-cis-retinoic acid (RA), TM (ERS inducer), Zea, 4-PBA (ERS inhibitor), and SB216763 (GSK-3β inhibitor). The methods included flow cytometry and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] for the detections of cell cycle and cell viability and western blot as a third measure of proteins in relation to ERS and tau phosphorylation. We have collected and analyzed all the data that suggested application of drugs for the treatment in non-RA and RA group. Zea displays its protection on TM-induced cell injury, upregulation of GRP78 expression, and change of GSK-3β activity and tau phosphorylation when 4-PBA and SB216763 interfere with the process. These studies indicated that Zea is in vicious circle in ERS, GSK-3β, and tau phosphorylation, and further reflect its potential value in AD.

Propolin G-Suppressed Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer Cells via Glycogen Synthase Kinase 3β-Mediated Snail and HDAC6-Regulated Vimentin Degradation.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer with a poor prognosis. The incidence and mortality rate of TNBC are frequently found in younger women. Due to the absence of a good therapeutic strategy, effective remedies for inhibiting TNBC have been developed for improving the cure rate. Epithelial-to-mesenchymal transition (EMT) is a critical mechanism to regulate cancer cell motility and invasion. Furthermore, ectopic expression of EMT molecules correlates with the metastasis and poor prognosis of TNBC. Targeting EMT might be a strategy for the therapy and prevention of TNBC. Propolin G, an active c-prenylflavanone in Taiwanese propolis, has been shown to possess anti-cancer activity in many cancers. However, the anti-metastasis activity of propolin G on TNBC is still unclear. The present study showed that the migration and invasion activities of TNBC cells was suppressed by propolin G. Down-regulated expression of Snail and vimentin and up-regulated expression of E-cadherin were dose- and time-dependently observed in propolin G-treated MDA-MB-231 cells. Propolin G inhibited Snail and vimentin expressions via the signaling pathways associated with post-translational modification. The activation of glycogen synthase kinase 3β (GSK-3β) by propolin G resulted in increasing GSK-3β interaction with Snail. Consequently, the nuclear localization and stability of Snail was disrupted resulting in promoting the degradation. Propolin G-inhibited Snail expression and the activities of migration and invasion were reversed by GSK-3β inhibitor pretreatment. Meanwhile, the outcomes also revealed that histone deacetylase 6 (HDAC6) activity was dose-dependently suppressed by propolin G. Correspondently, the amounts of acetyl-α-tubulin, a down-stream substrate of HDAC6, were increased. Dissociation of HDAC6/Hsp90 with vimentin leading to increased vimentin acetylation and degradation was perceived in the cells with the addition of propolin G. Moreover, up-regulated expression of acetyl-α-tubulin by propolin G was attenuated by HDAC6 overexpression. On the contrary, down-regulated expression of vimentin, cell migration and invasion by propolin G were overturned by HDAC6 overexpression. Conclusively, restraint cell migration and invasion of TNBC by propolin G were activated by the expression of GSK-3β-suppressed Snail and the interruption of HDAC6-mediated vimentin protein stability. Aiming at EMT, propolin G might be a potential candidate for TNBC therapy.

The Reduction of Tau Hyperphosphorylation by Cornel Iridoid Glycosides Is Mediated by Their Influence on Calpain Activity.

Alzheimer's disease (AD) is the most common type of dementia, and the abnormal hyperphosphorylation of the tau protein is the main component of its pathogenesis. Calpain was found to be abnormally activated in neurofibrillary tangles (NFTs) in a previous report. Cornel iridoid glycosides (CIG) have been reported to reduce the hyperphosphorylation of tau protein. Nevertheless, the role of calpain in the reduction tau hyperphosphorylation by CIG remains unclear. In the present study, we investigated the effect of CIG on calpain activity through in vitro and in vivo experiments. Western blotting results suggested that CIG decreased the phosphorylation of tau at Ser 404 and Ser 262 sites in P301S mice. Moreover, CIG inhibited the activity of calpain and glycogen synthase kinase 3β (GSK-3β) and enhanced the activity of protein phosphatase 2A (PP2A) both in vivo and in vitro. CIG also inhibited the activation of PP2A and reduced the GSK-3β activity caused by the calpain activator dibucaine. In addition, the main components of CIG, morroniside and loganin, play an equivalent role in reducing calpain activity, as the effect of their combined use is equivalent to that of CIG. The abovementioned findings revealed that CIG improved PP2A activity and reduced GSK-3β activity by adjusting the activity of calpain 1, leading to a reduction in the phosphorylation of tau. This study highlights the remarkable therapeutic potential of CIG for managing AD.

Ginsenoside Rd Attenuates Tau Phosphorylation in Olfactory Bulb, Spinal Cord, and Telencephalon by Regulating Glycogen Synthase Kinase 3β and Cyclin-Dependent Kinase 5.

Objective Ginseng is a plant of the family Acanthopanaceae. It has been used for thousands of years in China. It is known as the king of hundred herbs. It was recorded first in Shennong Baicao Jing. It has been found that ginsenoside Rd is a neuroprotective agent. This article aims to explore the protective roles of ginsenoside Rd in Alzheimer's disease. Rd, a Chinese herb, may be a promising treatment drug for Alzheimer′s disease (AD) and is also reported to be related to several pathological changes, including the deposition of Aβ and tau hyperphosphorylation in AD as it decreases the deposition of tau hyperphosphorylation in APP transgenic mice. Methods In this study, APP transgenic mice were pretreated with 10 mg/kg Rd for six months, and the effect of Rd on neuropathological deficits in the olfactory bulb, spinal cord, and telencephalon of APP transgenic mice was investigated. The phosphorylation levels of tau (S199/202, S396, S404, and Tau5) and the activities of the proteins glycogen synthase kinase 3β (Tyr216) and cyclin-dependent kinase 5 (P25/P35) were measured. Results The pretreatment of Rd effectively decreased the production and deposition of hyperphosphorylated tau (S199/202, S396, and S404) protein by depressing the expression of glycogen synthase kinase 3β (GSK-3β/Tyr216) and cyclin-dependent kinase 5 (CDK5/P25). Conclusion These findings suggest that ginsenoside Rd could improve the pathological changes of AD in the olfactory bulb, spinal cord, and telencephalon, which further demonstrated the potential therapeutic effect of Rd in early AD.

Transgenerational effects on the fecundity and sperm proteome in rats exposed to a mixture of pyrethroids at doses similar to human consumption

Pyrethroid based pesticide usage for crop protection resulted in percolation of these compounds into the food chain. Toxicological studies that reflect exposure to pyrethroids through food in the human settings are rare. We conducted animal experimentations using a mixture of pyrethroids that is equivalent to the amount consumed by average individual through rice and vegetables in the Indian context. Male rats treated with a mixture of pyrethroids for 1–12 months displayed decreased transgenerational fecundity, sperm count, activities of 3β- and 17β-HSD and perturbed hormonal profile. At the transcriptome level, the expression of genes involved in spermatogenesis, steroidogenesis, germ cell epigenetic modulators and germ cell apoptosis were altered in the testis. In the sperm lysates of control rats, 506 proteins identified by mass spectrometry. The differential expression of these proteins (treated/control ratio) in the pyrethroid exposed rats was analyzed. Among the 506 proteins, 153 had a ratio of 0; 41 had a ratio ranging from >0 to <0.5; and 10 had a ratio >2.0. Interestingly, the differential expression was transgenerational. 26 proteins that were differentially expressed in the sperm of F0 treated rats continued to remain the same in the F1, F2 and F3 generations, while the differential expression was maintained up to F2 and F1 generations for 46 and 2 proteins respectively. Some of the proteins that continued to be differentially expressed in the later generations are reported to have critical roles in male reproduction. These results indicate that the reduced fecundity observed in the later generations could be due to the continued differential expression that was initiated by pyrethroid treatment in the F0 rats. Results of our study, for the first time, provide evidence that long-term exposure to pyrethroids affects transgenerational fecundity manifested by changes in sperm proteome.

Exogenous HMGB1 Promotes the Proliferation and Metastasis of Pancreatic Cancer Cells.

Background: Exogenous HMGB1 plays a vital role in tumor recurrence, and HMGB1 is ubiquitous in the tumor microenvironment. However, the mechanism of action is still unclear. We investigated the role of exogenous HMGB1 in tumor proliferation and metastasis using human SW1990 and PANC-1 cells after radiotherapy and explored the possible molecular mechanism.Materials and Methods: Residual PANC-1 cells and SW1990 cells were isolated after radiotherapy. The supernatant after radiotherapy was collected. The relative expression of HMGB1 was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Electron microscope (EMS) was used to collect the images of pancreatic cancer cells pre and post radiotherapy treatment. The proliferation of pancreatic cancer cells which were treated with different radiation doses was measured by Carboxy Fluorescein Succinimidyl Ester (CFSE). The migration rates of pancreatic cancer cells were measured by wound healing assays. Subsequently, the expression of related proteins was detected by Western Blot. In vivo, the subcutaneous pancreatic tumor models of nude mice were established, and therapeutic capabilities were tested.Results: HMGB1 was detected in the supernatant of pancreatic cancer cells after radiotherapy. The results of CFSE showed that exogenous HMGB1 promotes the proliferation and metastasis of pancreatic cancer cells. The western blot results showed activation of p-GSK 3β and up-regulation of N-CA, Bcl-2, and Ki67 in response to HMGB1 stimulation, while E-CA expression was down-regulated in pancreatic cancer cells in response to HMGB1 stimulation. In vivo, ethyl pyruvate (EP, HMGB1 inhibitor) inhibits the growth of tumors and HMGB1 promotes the proliferation of tumors after radiation.Conclusion: Radiotherapy induces HMGB1 release into the extracellular space. Exogenous HMGB1 promotes the proliferation and metastasis of PANC-1 cells and SW1990 cells by activation of p-GSK 3β which is mediated by Wnt pathway.

Parkinson's Disease Dementia: Synergistic Effects of Alpha-Synuclein, Tau, Beta-Amyloid, and Iron.

Parkinson’s disease dementia (PDD) is a common complication of Parkinson’s disease that seriously affects patients’ health and quality of life. At present, the process and pathological mechanisms of PDD remain controversial, which hinders the development of treatments. An increasing number of clinical studies have shown that alpha-synuclein (α-syn), tau, beta-amyloid (Aβ), and iron are closely associated with PDD severity. Thus, we inferred the vicious cycle that causes oxidative stress (OS), due to the synergistic effects of α-syn, tau, Aβ, and, iron, and which plays a pivotal role in the mechanism underlying PDD. First, iron-mediated reactive oxygen species (ROS) production can lead to neuronal protein accumulation (e.g., α-syn andAβ) and cytotoxicity. In addition, regulation of post-translational modification of α-syn by iron affects the aggregation or oligomer formation of α-syn. Iron promotes tau aggregation and neurofibrillary tangles (NFTs) formation. High levels of iron, α-syn, Aβ, tau, and NFTs can cause severe OS and neuroinflammation, which lead to cell death. Then, the increasing formation of α-syn, Aβ, and NFTs further increase iron levels, which promotes the spread of α-syn and Aβ in the central and peripheral nervous systems. Finally, iron-induced neurotoxicity promotes the activation of glycogen synthase kinase 3β (GSK3β) related pathways in the synaptic terminals, which in turn play an important role in the pathological synergistic effects of α-syn, tau and Aβ. Thus, as the central factor regulating this vicious cycle, GSK3β is a potential target for the prevention and treatment of PDD; this is worthy of future study.

Aqueous extract of Paeoniae Radix Rubra prevents deep vein thrombosis by ameliorating inflammation through inhibiting GSK3β activity

BackgroundDeep vein thrombosis (DVT) is a kind of blood stasis syndrome. Paeoniae Radix Rubra (PRR) has long been widely used for eliminating blood stasis in China, but its effect on DVT has not yet been reported. PurposeThe present study aimed to assess the potential inhibitory effect of the aqueous extract of PRR (i.e.,PRR dispensing granule, PRRDG) on DVT and explore the underlying mechanism. Study design/MethodsThe chemical profile of PRRDG was analyzed by high-performance liquid chromatography. Sprague-Dawley rats were intragastrically treated with PRRDG (0.625, 1.25 and 1.875 g crude drug/kg/d) once daily for 7 consecutive days. On the sixth day, a model of inferior vena cava (IVC) stenosis-induced DVT was established. All rats were sacrificed on the seventh day. Serum was collected for enzyme-linked immunosorbent assay. Thrombus-containing IVC was weighed and further processed for histopathologic examination, immunohistochemical analysis and western blotting. LiCl and LY294002 were adopted to block and increase the activity of glycogen synthase kinase 3β (GSK3β), respectively. ResultsThe chemical profile analysis showed that paeoniflorin, benzoylpaeoniflorin, albiflorin, gallic acid and catechin were the main constituents of PRRDG. LiCl decreased thrombus weight, reduced the number of inflammatory cells in thrombus and vein wall, down-regulated phosphorylated NF-κB p65 (p-p65) protein expression. Similarly, PRRDG decreased thrombus weight and tissue factor (TF) protein expression. PRRDG reduced the protein expression levels of P-selectin, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in venous endothelium, serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and the number of inflammatory cells in thrombus and vein wall. Moreover, PRRDG down-regulated p-p65 protein expression and up-regulated phosphorylated GSK3β (p-GSK3β) protein expression. LY294002 abrogated the inhibitory effects of PRRDG on thrombus weight, TF protein expression, TNF-α and IL-1β serum levels, inflammatory cells influxes, and p-p65 protein expression. ConclusionPRRDG prevents DVT by ameliorating inflammation through inhibiting GSK3β activity.

Adiponectin improves amyloid-β 31-35-induced circadian rhythm disorder in mice.

Adiponectin is an adipocyte‐derived hormone, which is closely associated with the development of Alzheimer's disease (AD) and has potential preventive and therapeutic significance. In the present study, we explored the relationship between adiponectin and circadian rhythm disorder in AD, the effect of adiponectin on the abnormal expression of Bmal1 mRNA/protein induced by amyloid‐β protein 31‐35 (Aβ31‐35), and the underlying mechanism of action. We found that adiponectin‐knockout mice exhibited amyloid‐β deposition, circadian rhythm disorders and abnormal expression of Bmal1. Adiponectin ameliorated the abnormal expression of the Bmal1 mRNA/protein caused by Aβ31‐35 by inhibiting the activity of glycogen synthase kinase 3β (GSK3β). These results suggest that adiponectin deficiency could induce circadian rhythm disorders and abnormal expression of the Bmal1 mRNA/protein, whilst exogenous administration of adiponectin may improve Aβ31‐35‐induced abnormal expression of Bmal1 by inhibiting the activity of GSK3β, thus providing a novel idea for the treatment of AD.

Expression of PRIP, a phosphatidylinositol 4,5-bisphosphate binding protein, attenuates PI3K/AKT signaling and suppresses tumor growth in a xenograft mouse model

Cancer is characterized by uncontrolled proliferation resulting from aberrant cell cycle progression. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling, a regulatory pathway for the cell cycle, stabilizes cyclin D1 in the G1 phase by inhibiting the activity of glycogen synthase kinase 3β (GSK3β) via phosphorylation. We previously reported that phospholipase C-related catalytically inactive protein (PRIP), a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding protein, regulates PI3K/AKT signaling by competitively inhibiting substrate recognition by PI3K. Therefore, in this study, we investigated whether PRIP is involved in cell cycle progression.PRIP silencing in MCF-7 cells, a human breast cancer cell line, demonstrated PI(3,4,5)P3 signals accumulated at the cell periphery compared to that of the control. This suggests that PRIP reduction enhances PI(3,4,5)P3-mediated signaling. Consistently, PRIP silencing in MCF-7 cells exhibited increased phosphorylation of AKT and GSK3β which resulted in cyclin D1 accumulation. In contrast, the exogenous expression of PRIP in MCF-7 cells evidenced stronger downregulation of AKT and GSK3β phosphorylation, reduced accumulation of cyclin D1, and diminished cell proliferation in comparison to control cells. Flow cytometry analysis indicated that MCF-7 cells stably expressing PRIP attenuate cell cycle progression. Importantly, tumor growth of MCF-7 cells stably expressing PRIP was considerably prevented in an in vivo xenograft mouse model. In conclusion, PRIP expression downregulates PI3K/AKT/GSK3β-mediated cell cycle progression and suppresses tumor growth. Therefore, we propose that PRIP is a new therapeutic target for anticancer therapy.