The Wnt/Ca2+ signaling pathway is essential for the regeneration of GABAergic neurons in planarian Dugesia japonica.

The growth and differentiation of neurons are critical events in the establishment of proper neuron connectivity and function. Planarians have a remarkable ability to completely regenerate a functional nervous system from a pluripotent stem cell population. Thus, planarians provide a powerful model to identify genes required for neuronal differentiation in vivo. The Wnt/Ca2+ signaling pathway is crucial for cancer development, arousing inflammatory responses, and neurodegeneration. We analyzed the expression patterns and RNAi phenotypes for members of the Wnt/Ca2+ signaling pathway in the planarian, Dugesia japonica. The expression of DjWnt5a, DjPLC-ß, DjCamKII, and DjCaln during regeneration was surprisingly similar and revealing in the regenerated brain. RNAi knockdown of DjWnt5a, DjPLC-ß, DjCamKII, and DjCaln led to defects in regenerated brains including brain partial deletions, incompact phenotypes at the posterior of the new brain, and lateral branches, which could not regenerate. Furthermore, the expressions of GAD and the number of GABAergic neurons decreased. Together, these results suggest that the Wnt/Ca2+ signaling pathway is required for GABAergic neuron regeneration.

A novel RhoA-related gene, DjRhoA, contributes to the regeneration of brain and intestine in planarian Dugesia japonica.

A small GTPase, RhoA, plays a variety of functions in the regulation of cellular and developmental events via its downstream effectors, including cytokinesis, cell migration, and phagocytosis. In this study, a novel RhoA-related gene from the planarian Dugesia japonica, DjRhoA, was cloned and characterized. The full-length cDNA of DjRhoA is 869 bp, and the open reading frame encodes a poly-peptide of 194 amino acids. Phylogenetic analysis revealed that DjRhoA clustered with another RhoA-related protein, DjRho2, and located on the base of phylogenetic tree. Whole-mount in situ hybridization results indicated that DjRhoA was expressed in the brain primordia and intestine during regeneration. Knockdown of DjRhoA induces defects in the brain and intestine. These results suggested that DjRhoA was responsible for the regeneration of brain and intestine in Dugesia japonica.

Neuroprotective effects of blueberry anthocyanins against perfluorooctanoic sulfonate on planarian Dugesia japonica.

In this study, the planarian Dugesia japonica was exposed to perfluorooctane sulfonate (PFOS) and blueberry anthocyanins (ANT) for 1-10 days to investigate the protective effects of ANT on neurotoxicity and DNA damage induced by PFOS. The expression of neural related genes (Djnlg, DjFoxD, DjFoxG, DjotxA, and DjotxB) in D. japonica following exposure was determined using quantitative real-time PCR (qPCR). Immunofluorescence was performed to determine the alterations in neural morphology. In addition, ELISA kits were used to measure level of the neurotransmitters Dopamine (DA), serotonin (5-HT) and γ-aminobutyric acid (GABA). Furthermore, single cell gel electrophoresis was measured to analyze DNA damage. In this study, PFOS treatment induced neural morphology defects, alterations in neural-related gene expression, alterations in neurotransmitter levels, and DNA damage. However, co-exposure to ANT and PFOS mitigated the damage to D. japonica induced by PFOS. Restoration of neurotransmitter contents and neural related genes expression were observed in planarians following co-application of ANT and PFOS, immunofluorescence showed that nerve morphology almost recovered, and DNA damage was decreased. The results of this study showed that ANT may have a protective effect against PFOS induced neurotoxicity and DNA damage.

Changes on lipid peroxidation,enzymatic activities and gene expression in planarian (Dugesia japonica) following exposure to perfluorooctanoic acid.

We investigated perfluorooctanoic acid (PFOA)-induced stress response in planarians. We administered different concentrations of PFOA to planarians for up to 10 d. PFOA exposure resulted in significant concentration-dependent elevations in lipid peroxidation, glutathione S-transferase and caspase-3 protease activities, and a significant decline in glutathione peroxidase activities compared with control groups. Exposure to PFOA significantly up-regulated the heat shock proteins hsp70 and hsp90, and p53, and down-regulated hsp40 compared with controls. PFOA exposure also increased HSP70 protein levels, as demonstrated by western blot analysis. These alterations indicated that PFOA exposure induced a stress response and affected the regulation of oxidative stress, enzymatic activities and gene expression. These results suggest that these sensitive parameters, together with other biomarkers, could be used for evaluating toxicity, for ecological risk assessment of PFOA in freshwaters.

The protective effect of blueberry anthocyanins against perfluorooctanoic acid-induced disturbance in planarian (Dugesia japonica).

The influence of blueberry anthocyanins on perfluorooctanoic acid (PFOA)-induced stress response in planarian mitochondria was investigated. PFOA at 15mg/L and anthocyanins at 10 or 20mg/L were individually and simultaneously administered to planarians for up to 10d. The results showed PFOA treatment induced an increase in mitochondrial permeability transition pore opening and a decrease antioxidant capacity and enzyme activities. In anthocyanin treated animals, the activity of succinate dehydrogenase, cytochrome oxidase and monoamine oxidase increased, but mitochondrial permeability transition pore opening decreased and total antioxidant capacity increased. An improvement in above-mentioned physiological and biochemical parameters was found in the combined PFOA and anthocyanin treated animals, in a dose-dependent manner. Anthocyanins attenuated the PFOA induced toxicity; antioxidant capacity and enzyme activities are involved in the protective mechanism of anthocyanins.

Planarian myosin essential light chain is involved in the formation of brain lateral branches during regeneration.

The myosin essential light chain (ELC) is a structure component of the actomyosin cross-bridge, however, the functions in the central nervous system (CNS) development and regeneration remain poorly understood. Planarian Dugesia japonica has revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. In this study, the cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. It encodes a deduced protein with highly conserved functionally domains EF-Hand and Ca(2+) binding sites that shares significant similarity with other members of ELC. Whole mount in situ hybridization studies show that DjElc expressed in CNS during embryonic development and regeneration of adult planarians. Loss of function of DjElc by RNA interference during planarian regeneration inhibits brain lateral branches regeneration completely. In conclusion, these results demonstrated that DjElc is required for maintenance of neurons and neurite outgrowth, particularly for involving the brain later branch regeneration.

Preparation of Nanoparticles Loaded with Quercetin and Effects on Bacterial Biofilm and LPS-Induced Oxidative Stress in Dugesia japonica.

Quercetin is a kind of flavonol compound, which has been widely concerned because of its good pharmacological effects. However, its poor water solubility and poor oral absorption limit its application. To address the above problems, the optimal technological conditions for preparing quercetin-loaded chitosan sodium alginate nanoparticles (Q-CSNPs) were obtained through single-factor experiment method. Q-CSNPs were characterized by particle size analyzer, scanning electron microscope (SEM), transmission electron microscope (TEM), and Fourier transform infrared spectroscopy (FTIR). Biofilm experiment evaluated the antibacterial activity of five different concentrations of Q-CSNPs against Escherichia coli and Staphylococcus aureus. DPPH and hydroxyl radical scavenging experiments determined their antioxidant activity. The effect of Q-CSNPs labeled with FITC on the oxidative stress of planarian was determined. The results showed that quercetin was successfully encapsulated and had good antibacterial and antioxidant capacity in vitro. In vivo experiments of planarians also showed that Q-CSNPs could inhibit the oxidative stress induced by lipopolysaccharide (LPS) and especially alleviate the decrease of CAT activity and the increase of MDA content in planarians induced by LPS. After being supported by future in vivo studies, this preparation will provide research possibilities for the development of quercetin nano-drugs, quercetin dietary supplement, and so on.

Exposure to polystyrene microplastics and perfluorooctane sulfonate disrupt the homeostasis of intact planarians and the growth of regenerating planarians.

Microplastics (MPs) and perfluorinated compounds (PFAS) are widespread in the global ecosystem. MPs have the ability to adsorb organic contaminants such as perfluorooctane sulfonate (PFOS), leading to combined effects. The current work aims to explore the individual and combined toxicological effects of polystyrene (PS) and PFOS on the growth and nerves of the freshwater planarian (Dugesia japonica). The results showed that PS particles could adsorb PFOS. PS and PFOS impeded the regeneration of decapitated planarians eyespots, whereas the combined treatment increased the locomotor speed of intact planarians. PS and PFOS caused significant DNA damage, while co-treatment with different PS concentrations aggravated and attenuated DNA damage, respectively. Further studies at the molecular level have shown that PS and PFOS affect the proliferation and differentiation of neoblasts in both intact and regenerating planarians, alter the expression levels of neuronal genes, and impede the development of the nervous system. PS and PFOS not only disrupted the homeostasis of intact planarians, but also inhibited the regeneration of decapitated planarians. This study is the first to assess the multiple toxicity of PS and PFOS to planarians after combined exposure. It provides a basis for the environmental and human health risks of MPs and PFAS.

Chitosan nanoparticles loaded with Eucommia ulmoides seed essential oil: Preparation, characterization, antioxidant and antibacterial properties.

Eucommia ulmoides seed essential oil (EUSO) is a natural plant oil rich in various nutrients, which has been widely used due to its unique medicinal effects. However, it is prone to oxidation and rancidity under many adverse environmental influences. Nanoencapsulation technology can protect and slow down the loss of its biological activity. In this study, chitosan nanoparticles (CSNPs) loaded with EUSO were prepared by emulsification and ionic gel technology. EUSO-CSNPs were characterized by Fourier transform infrared (FTIR) spectroscopy, Thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The results confirmed the success of EUSO encapsulation and the encapsulation rate ranged from 36.95 % to 67.80 %. Nanoparticle size analyzer, Scanning electron microscope (SEM) and Transmission electron microscopy (TEM) showed that CSNPs were spherical particles with a range of 200.6-276.0 nm. The results of in vitro release study indicated that the release of EUSO was phased, and EUSO-CSNPS had certain sustained-release properties. Furthermore, EUSO-CSNPs had higher antioxidant and antibacterial abilities than pure EUSO and chitosan, which was verified through free radical scavenging experiments and bacteria biofilm experiments, respectively. This technology can enhance the medicinal value of EUSO in biomedical and other fields, and will provide support for in vivo research of EUSO-CSNPs in the future.

Effects of N,N-dimethylformamide on behaviour and regeneration of planarian Dugesia japonica.

In this study, the toxicity, behavioural and regeneration effects of dimethylformamide (DMF) on planarian Dugesia japonica were investigated. One control and six different concentrations of DMF (10 ppm, 100 ppm, 500 ppm, 1000 ppm, 5000 ppm and 10,000 ppm) were used in triplicate. The results showed that the mortality was directly proportional to the DMF concentration and planarian locomotor velocity (pLMV) was significantly reduced by increasing the exposure time and DMF concentration. pLMV of D. japonica was significantly reduced at a lower concentration of 10 ppm after 7 days of continuous exposure to DMF. The recovery of the motility of planarians pretreated with DMF was found to be time- and dose dependent, all planarians had complete recovery in their motility after 48 h. The appearance of auricles in regenerating animals was easily affected by DMF exposure in comparison with the appearance of eyespot. The present results suggest that the intact adult mobility in the aquatic planarian D. japonica is a more sensitive biomarker than mortality, and the appearance of auricles in regenerating animals is a more sensitive biomarker than eyespot.

Multiple toxicity evaluations of perfluorooctane sulfonate on intact planarian Dugesia japonica.

Perfluorooctane sulfonate (PFOS) is gaining widespread attention as a persistent organic pollutant with multiple mechanisms of toxicity. In this study, PFOS at different concentrations and different exposure times was used to evaluate the multiple toxicities on intact planarian Dugesia japonica. The proliferation of neoblasts, apoptosis, DNA damage and the expression levels of neuronal genes and the major genes of the Wnt pathway were effectively studied. The results demonstrated that the balance between proliferation and apoptosis of intact planarian cells was disrupted after PFOS exposure, which in turn affected tissue homeostasis and differentiation. PFOS exposure led to increased DNA damage and altered neuronal gene expression. In addition, PFOS exposure could down-regulate the expression of Wnt pathway genes, but the inhibition of the Wnt pathway by PFOS was time- and concentration-dependent. These findings suggest that PFOS has multiple toxic effects on planarians and may interfere with cell proliferation and neurodevelopment by affecting the key gene expression in the Wnt pathway, providing estimable information on the neurodevelopmental toxicity and ecotoxicity of PFOS toxicity in aquatic animals and environments.

DjFARP Contributes to the Regeneration and Maintenance of the Brain through Activation of DjRac1 in Dugesia japonica.

FERM, RhoGEF, and Pleckstrin domain protein (FARP) mediated RhoGTPase pathways are involved in diverse biological processes, such as neuronal development and tumorigenesis. However, little is known about their role in neural regeneration. We uncovered for the first time that FARP-Rac1 signaling plays an important role in neural regeneration in Dugesia japonica, a planarian that possesses unparalleled regenerative capacities. The planarian FARP homolog DjFARP was primarily expressed in both intact and regenerating brain and pharynx tissue. Functional studies suggested that downregulation of DjFARP with dsRNA in Dugesia japonica led to smaller brain sizes, defects in brain lateral branches, and loss of cholinergic, GABAergic, and dopaminergic neurons in both intact and regenerating animals. Moreover, the Rho GTPase DjRac1 was shown to play a similar role in neural regeneration and maintenance. Rac1 activation assay showed that DjFARP acts as a guanine nucleotide exchange factor (GEF) for DjRac1. Together, these findings indicate that the brain defects seen in DjFARP knockdown animals may be attributable to DjRac1 inactivation. In conclusion, our study demonstrated that DjFARP-DjRac1 signaling was required for the maintenance and proper regeneration of the brain in Dugesia japonica.

Effects of dimethylsulfoxide on behavior and antioxidant enzymes response of planarian Dugesia japonica.

In this study, the toxicity, behavioral and antioxidant activity effects of dimethylsulfoxide (DMSO) on planarian Dugesia japonica were investigated. The results showed that the mortality was directly proportional to the DMSO concentration, and planarian locomotor velocity decreased as the concentration of DMSO increased. The recovery of the motility for planarians pre-exposed to DMSO was found to be time- and dose-dependent, and only those pre-exposed to 0.1-3% DMSO resulted in full recovery. The antioxidant enzymes of planarians in response to long-term DMSO stress was also altered in a time- and dose-dependent manner. Planarians revealed more tolerance to DMSO toxicity at low DMSO (0.1%) level in short- and long-term DMSO stress, in which an efficient antioxidant system was involved and the motility was not affected.

The feedback loop between calcineurin, calmodulin-dependent protein kinase II, and nuclear factor of activated T-cells regulates the number of GABAergic neurons during planarian head regeneration.

Disturbances in the excitatory/inhibitory balance of brain neural circuits are the main source of encephalopathy during neurodevelopment. Changes in the function of neural circuits can lead to depolarization or repeat rhythmic firing of neurons in a manner similar to epilepsy. GABAergic neurons are inhibitory neurons found in all the main domains of the CNS. Previous studies suggested that DjCamkII and DjCaln play a crucial role in the regulation of GABAergic neurons during planarian regeneration. However, the mechanisms behind the regeneration of GABAergic neurons have not been fully explained. Herein, we demonstrated that DjCamkII and DjCaln were mutual negative regulation during planarian head regeneration. DjNFAT exerted feedback positive regulation on both DjCaln and DjCamkII. Whole-mount in situ hybridization (WISH) and fluorescence in situ hybridization (FISH) revealed that DjNFAT was predominantly expressed in the pharynx and parenchymal cells in intact planarian. Interestingly, during planarian head regeneration, DjNFAT was predominantly located in the newborn brain. Down-regulation of DjNFAT led to regeneration defects in the brain including regenerative brain became small and the lateral nerves cannot be regenerated completely, and a decreasein the number of GABAergic neurons during planarian head regeneration. These findings suggest that the feedback loop between DjCaln, DjCamkII, and DjNFAT is crucial for the formation of GABAergic neurons during planarian head regeneration.

Evaluation of joint effects of perfluorooctane sulfonate and wood vinegar on planarians, Dugesia japonica.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant and can cause oxidative stress in animals. Wood vinegar (WV) is the water condensate of smoke produced during wood carbonization. It was used for antibacterial application, pest control, and antioxidant. In the study, PFOS and WV were used to treat the planarian, and then the oxidative stress induced by PFOS on the planarian (Dugesia japonica) and the protective effects of WV on lipid peroxidation, related antioxidant enzyme activity, and mRNA expression in the planarian were studied. PFOS caused an increase in malondialdehyde (MDA) contents, a decrease in superoxide dismutase (SOD) and catalase (CAT) activities, and a change in glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) activities. The mRNA levels of glutathione peroxidase gene (gpx), glutathione S-transferase enzyme gene (gst), and glutathione reductase gene (gr) are upregulated or downregulated to varying degrees. The WV and co-treatment planarians reduced MDA levels, increased the activities of oxidative stress biomarker enzymes, and restored gene expression levels. Our results show that low concentration of WV has protective effects on the oxidative damage caused by PFOS in the planarian.

Neurotoxicity of perfluorooctanoic acid and post-exposure recovery due to blueberry anthocyanins in the planarians Dugesia japonica.

Perfluorooctanoic acid (PFOA) is a widely used synthetic industrial chemical which accumulates in ecosystems and organisms. Our study have investigated the neurobehavioral effects of PFOA and the alleviation effects of PFOA-induced neurotoxicity by blueberry anthocyanins (ANT) in Dugesia japonica. The planarians were exposed to PFOA and ANT for ten days. Researchs showed that exposure to PFOA affected locomotor behavior and ANT significantly alleviated the reduction in locomotion induced by PFOA. The regeneration of eyespots and auricles was suppressed by PFOA and was promoted by ANT. Following exposure to PFOA, acetylcholinesterase activity continually decreased and was unaffected in the ANT group, but was elevated after combined administration of PFOA and ANT. Oxidative DNA damage was found in planarians exposed to PFOA and was attenuated after administration of ANT by the alkaline comet assay. Concentrations of three neurotransmitters increased following exposure to PFOA and decreased after administration of ANT. Furthermore, ANT promoted and PFOA inhibited neuronal regeneration. DjotxA, DjotxB, DjFoxG, DjFoxD and Djnlg associated with neural processes were up-regulated following exposure to PFOA. Our findings indicate that PFOA is a neurotoxicant while ANT can attenuate these detrimental effects.

Blueberry anthocyanin alleviate perfluorooctanoic acid-induced toxicity in planarian (Dugesia japonica) by regulating oxidative stress biomarkers, ATP contents, DNA methylation and mRNA expression.

Blueberry anthocyanin (BA) have strong health benefits as an active natural antioxidant and perfluorooctanoic acid (PFOA) can result in oxidative stress in animals. In our study, the protective effects of BA against stress induced by PFOA was investigated in the planarian Dugesia japonica using oxidative stress biomarkers, ATP contents, ATPase activity, DNA methylation and mRNA expression. PFOA exposure could resulted in malondialdehyde production. At the same time, treatment with BA decreased the production of malondialdehyde in BA-exposed and co-treatment planarians. PFOA caused activities increase in glutathione peroxidase (GPx), glutathione S-transferase (GST) and activities decrease in glutathione reductase (GR). PFOA exposure decreased the GSH and ATP contents. Additionally, it increased the GSSG contents and ATPase activity. BA administration increased the activities of GPx, GST and GR in BA and co-treatment planarians. Meanwhile BA maintained the contents of ATP, ATPase activity, GSH and GSSG by alleviating PFOA toxicity. Moreover, PFOA and BA increased the contents of 5-methylcytosine and decreased 5-hydroxymethylcytosine in all group. In addition, PFOA and BA treated planarians significantly altered the expression of genes associated with above biochemical parameters. The results showed that the mRNA expression of gpx, Djgst, gr, Djnak and dnmt1 were significantly elevated in all groups. Alterations in the mRNA expression levels indicated a stress response to PFOA exposure and anthocyanin protection. These alterations regulated biomarkers of oxidative stress, energy metabolism and DNA methylation levels in planarians. These results indicate that BA attenuated PFOA-induced oxidative stress, energy metabolism, DNA methylation and gene expression disorders.

Application of blueberry anthocyanins reduces perfluorooctane sulfonate toxicity on planarians (Dugesia japonica) in locomotion, regeneration, and gene expression and contents.

Perfluorooctane sulfonate (PFOS) which has been distributed worldwide is a persistent organic contaminant. Blueberry anthocyanins (ANT) are phytonutrients with antioxidant activities. The influence of different PFOS and ANT concentrations on the behavioral activities, regeneration of planarians (Dugesia japonica), and the expression levels and contents of Djnad6 and Djcox1 genes has been investigated. PFOS treatments affected the gene expression levels, induced a decrease in the planarians' locomotor velocity, and increased the time required for the regeneration of the transverse amputated fragments in a time- and dose-dependent manner. Additionally, ANT treatments, to a certain extent, alleviated the damage caused by PFOS to planarians. ANT increased the planarians' locomotor velocity and the percentage of regenerating planarians with eyespots and auricles. Furthermore, ANT alleviated the expression disorders of Djnad6 and Djcox1 induced by PFOS.

Perfluorooctane sulfonate induced neurotoxicity responses associated with neural genes expression, neurotransmitter levels and acetylcholinesterase activity in planarians Dugesia japonica.

As a persistent and widespread toxic organic pollutant in the environment, perfluorooctane sulfonate (PFOS) has the potential to cause great harm to wildlife. In our study, the effects of PFOS on neurodevelopment gene expression, neurotransmitter content, neuronal morphology, acetylcholinesterase (AChE) activity were examined, and the potential neurotoxicity mechanisms of PFOS were also investigated in planarians, Dugesia japonica. Using quantitative real-time PCR analysis, five neurodevelopmental related genes were measured, among which, DjotxA, DjotxB, DjFoxD, and DjFoxG were found to be down-regulated, while Djnlg was found to be up-regulated, following exposure to PFOS for 10 days compared with control groups. In addition, the neurotransmitters including dopamine, serotonin, and γ-aminobutyricacid as well as the acitivity of AChE were altered by PFOS exposure. Furthermore, PFOS exposure altered brain morphology as well as smaller cephalic ganglia which displayed reduced nerve fiber density decreased brain branches compared to controls. Our results demonstrate that neurotransmission was disturbed after exposure to PFOS and that exposure to this pollutant can cause neurotoxic defects. Results from this study provide valuable information regarding the neuro- and ecological toxicity of PFOS in aquatic animals and aquatic environments.

Effects of perfluorooctanoic acid and perfluorooctane sulfonate on acute toxicity, superoxide dismutase, and cellulase activity in the earthworm Eisenia fetida.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are the two best-known perfluorinated chemicals and have received much attention due to their ubiquity in the environment. In the present study, we evaluated the effects of PFOS and PFOA on acute toxicity, superoxide dismutase (SOD), and cellulase activities in Eisenia fetida. The results of acute toxicity testing using a filter paper contact test and a natural field soil test showed that PFOA and PFOS exhibited acute toxicity in earthworms, and the toxic effect of PFOS was greater than that of PFOA. The results also showed that avoidance behavior is a more sensitive and easy operation biomarker than acute toxicity and will give us information for early diagnosis of soil pollution, well before the lethal effect becomes apparent. Subchronic exposure to PFOA or PFOS resulted in changes in SOD and cellulase activities in E. fetida, and SOD activity was more sensitive than cellulase activity during early exposure. Based on these findings, we suggest that avoidance behavior and SOD activity in earthworms are suitable biomarkers for evaluating the toxicity of PFOA- and PFOS-contaminated soils. These results indicate that exposure to PFOA and PFOS has a potential impact on soil animals and their environment.