In Vitro Imaging and Molecular Characterization of Ca2+ Flux Modulation by Nanosecond Pulsed Electric Fields.

In recent years, the application of pulsed electric fields with very short durations (nanoseconds) and extremely high amplitudes (MV/m) has been investigated for novel medical purposes. Various electric protocols have been explored for different objectives, including the utilization of fractionated pulse doses to enhance cell electrosensitization to the uptake of different markers or an increase in apoptosis. This study focused on the use of fluorescence imaging to examine molecular calcium fluxes induced by different fractionated protocols of short electric pulses in neuroblastoma (SH-SY5Y) and mesenchymal stem cells (HaMSCs) that were electroporated using nanosecond pulsed electric fields. In our experimental setup, we did not observe cell electrosensitization in terms of an increase in calcium flux following the administration of fractionated doses of nanosecond pulsed electric fields with respect to the non-fractionated dose. However, we observed the targeted activation of calcium-dependent genes (c-FOS, c-JUN, EGR1, NURR-1, ß3-TUBULIN) based on the duration of calcium flux, independent of the instantaneous levels achieved but solely dependent on the final plateau reached. This level of control may have potential applications in various medical and biological treatments that rely on calcium and the delivery of nanosecond pulsed electric fields.

Incorporation of electroendocytosis and nanosecond pulsed electric field in electrochemotherapy of breast cancer cells.

The study aimed to evaluate the possibility to perform electrochemotherapy using nanosecond pulsed electric field (nsPEF) and low electric field (LEF) monopolar electrical impulses to alleviate the problems of conventional electroporation. Two types of pulses have been used to treat MCF-7 human breast carcinoma cell line: very low voltage (electric field strength) long trains of short unipolar electric pulses, and low frequencies of extremely intense (40kV/cm), ultra-short (10ns) electric pulses. The electropermeabilization efficiency of the formed endocytotic vesicles was measured using the cloning efficacy test. The cell viability was decreased significantly at a repetition frequency begins from 0.01 Hz by ~35% and reached complete cell loss at 1 Hz of nanosecond pulses for cells treated before with monopolar pulses at 20 V/cm in the presence of BLM with 4 µM concentration. The uptake of non-permeant drugs has been done without plasma membrane permeabilization (classical electroporation), but by endocytosis. Nanosecond electric pulses can disrupt the membrane of endocytotic vesicles and release the cytotoxic drug bleomycin.

Caveolin-1 is Involved in Regulating the Biological Response of Cells to Nanosecond Pulsed Electric Fields.

Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC1) and inositol trisphosphate receptor (IP3R)) after exposure to nsPEFs. Release of TRPC1 and IP3R from Cav1 would activate downstream signaling cascades, including store-operated calcium entry, which could explain the influx in calcium after nsPEF exposure. Results of the current study establish a significant relationship between Cav1 and the activation of cell signaling pathways in response to nsPEFs.

A dose-response study of a novel method of selective tissue modification of cellular structures in the skin with nanosecond pulsed electric fields.

BACKGROUND AND OBJECTIVES: This study describes the effects of nanosecond pulsed electric fields (nsPEF) on the epidermis and dermis of normal skin scheduled for excision in a subsequent abdominoplasty. NsPEF therapy applies nanosecond pulses of electrical energy to induce regulated cell death (RCD) in cellular structures, with negligible thermal effects. Prior pre-clinical studies using nsPEF technology have demonstrated the ability to stimulate a lasting immune response in animal tumor models, including melanoma. This first-in-human-use of nsPEF treatment in a controlled study to evaluate the dose-response effects on normal skin and subcutaneous structures is intended to establish a safe dose range of energies prior to use in clinical applications using nsPEF for non-thermal tissue modification. STUDY DESIGN/MATERIALS AND METHODS: Seven subjects with healthy tissue planned for abdominoplasty excision were enrolled. Five subjects were evaluated in a longitudinal, 60-day study of effects with doses of six nsPEF energy levels. A total of 30 squares of spot sizes 25mm2 or less within the planned excision area were treated and then evaluated at 1 day, 5 days, 15 days, 30 days, and 60 days prior to surgery. Photographs were taken over time of each treated area and assessed by three independent and blinded dermatologists for erythema, flaking and crusting using a 5-point scale (0 = low, 4 = high). Punch biopsies of surgically removed tissue were processed and evaluated for tissue changes using hematoxylin and eosin, trichome, caspase-3, microphthalmia transcription factor, and elastin stains and evaluated by a dermatopathologist. The skin of two subjects received additional treatments at 2 and 4 hours post-nsPEF and was evaluated in a similar manner. RESULTS: Most energy settings exhibited delayed epidermal loss followed by re-epithelization by day 15 and a normal course of healing. Histologic analysis identified the appearance of activated caspase-3 at two and four hours after nsPEF treatment, but not at later time points. At the 1-day time point, a nucleolysis effect was observed in epidermal cells, as evidenced by the lack of nuclear staining while the epidermal plasma membranes were still intact. Cellular structures within the treatment zone such as melanocytes, sebaceous glands, and hair follicles were damaged while acellular structures such as elastic fibers and collagen were largely unaffected except for TL6 which showed signs of dermal damage. Melanocytes reappeared at levels comparable with untreated controls within 1 month of nsPEF treatment. CONCLUSIONS: The selective effect of nsPEF treatment on cellular structures in the epidermal and dermal layers suggests that this non-thermal mechanism for targeting cellular structures does not affect the integrity of dermal tissue within a range of energy levels. The specificity of effects and a favorable healing response makes nsPEF ideal for treating cellular targets in the epidermal or dermal layers of the skin, including treatment of benign and malignant lesions. NsPEF skin treatments provide a promising, non-thermal method for treating skin conditions and removing epidermal lesions. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

A dose-response study of nanosecond electric energy pulses on facial skin.

Nanosecond pulsed electric fields, also known as Nano-Pulse Stimulation or NPS, can trigger regulated cell death to clear skin lesions that are cellular in nature. Before treating facial lesions, it is important to demonstrate the effects of these pulses on normal facial skin. Here we have applied a range of NPS energies to the epidermis and dermis of normal facial skin scheduled for excision to establish a safe dose range of energies prior to use in clinical applications. This was an open-label, non-randomized study under the direction of a single Principal Investigator. The time course of the treated tissue changes was determined by histological analysis. All energy settings generated a delayed epidermal loss followed by re-epithelialization by day 7 and a normal course of healing. One day after NPS treatment, the cellular membranes of the treated epidermis were intact, but their nuclei no longer stained with H&E, resulting in a hollow appearance that has been referred to as "ghost cells." Cellular structures in the dermis, such as sebaceous glands and melanocytes, exhibited regulated cell death observed by 1 day post treatment. Melanocytes recovered to their normal density within 7 days. The 60-day samples indicated that epidermis, hair follicles, and eccrine glands appeared normal. The selective effect of NPS treatment on cellular structures in the epidermal and dermal layers suggests that this non-thermal modality of energy delivery is ideal for treating cellular targets including benign and malignant skin lesions. NPS skin treatments provide a promising method for clearing skin lesions with a cellular basis.

Dual-function of Baicalin in nsPEFs-treated Hepatocytes and Hepatocellular Carcinoma cells for Different Death Pathway and Mitochondrial Response.

Nanosecond pulsed electric fields (nsPEFs) is emerged as a potential curative modality to ablate hepatocellular carcinoma (HCC). The application of local ablation is usually limited by insufficiency of liver function. While baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been proven to possess both anti-tumor and protective effects. Our study aimed to estimate different responses of hepatic cancer cells and hepatocytes to the combination of nsPEFs and baicalin. Cell viability, apoptosis and necrosis, mitochondrial transmembrane potential (MTP) and reactive oxygen species (ROS) were examined by CCK-8, FCM, JC-1 and fluorescent probe, respectively. After treatment by nsPEFs, most hepatocytes died by apoptosis, nevertheless, nearly all cancer cells were killed through necrosis. Low concentration of baicalin synergically enhanced nsPEFs-induced suppression and necrosis of HCC cells, nevertheless, the application of baicalin protected normal hepatocytes from the injury caused by nsPEFs, owing to elevating mitochondrial transmembrane potential and reducing ROS generation. Our work provided an advantageous therapy for HCC through the enhanced combination treatment of nsPEFs and baicalin, with which could improve the tumor-ablation effect and alleviate the injury of hepatic tissues simultaneously.

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects.

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.

The biological response of cells to nanosecond pulsed electric fields is dependent on plasma membrane cholesterol.

Previous work from our laboratory demonstrated nanopore formation in cell membranes following exposure to nanosecond pulsed electric fields (nsPEF). We observed differences in sensitivity to nsPEF in both acute membrane injury and 24h lethality across multiple cells lines. Based on these data, we hypothesize that the biological response of cells to nsPEF is dependent on the physical properties of the plasma membrane (PM), including regional cholesterol content. Results presented in this paper show that depletion of membrane cholesterol disrupts the PM and increases the permeability of cells to small molecules, including propidium iodide and calcium occurring after fewer nsPEF. Additionally, cholesterol depletion concurrently decreases the "dose" of nsPEF required to induce lethality. In summary, the results of the current study suggest that the PM cholesterol composition is an important determinant in the cellular response to nsPEF.

Quantifying pulsed electric field-induced membrane nanoporation in single cells.

Plasma membrane disruption can trigger a host of cellular activities. One commonly observed type of disruption is pore formation. Molecular dynamic (MD) simulations of simplified lipid membrane structures predict that controllably disrupting the membrane via nano-scale poration may be possible with nanosecond pulsed electric fields (nsPEF). Until recently, researchers hoping to verify this hypothesis experimentally have been limited to measuring the relatively slow process of fluorescent markers diffusing across the membrane, which is indirect evidence of nanoporation that could be channel-mediated. Leveraging recent advances in nonlinear optical microscopy, we elucidate the role of pulse parameters in nsPEF-induced membrane permeabilization in live cells. Unlike previous techniques, it is able to directly observe loss of membrane order at the onset of the pulse. We also develop a complementary theoretical model that relates increasing membrane permeabilization to membrane pore density. Due to the significantly improved spatial and temporal resolution possible with our imaging method, we are able to directly compare our experimental and theoretical results. Their agreement provides substantial evidence that nanoporation does occur and that its development is dictated by the electric field distribution.

Nanosecond pulsed electric fields trigger cell differentiation in Chlamydomonas reinhardtii.

Nanosecond pulsed electric fields (nsPEFs) have great potential for biotechnological and medical applications. However, the biological mechanisms causing the cellular responses are still far from understood. We used the unicellular green algae Chlamydomonas reinhardtii as experimental model to dissect the immediate consequences of electroporation from the developmental cellular responses evoked by nsPEFs. We observe that nsPEFs induce a short-term permeabilization of the membrane, accompanied by swelling and oxidative burst. These response are transient, but are followed, several days later, by a second wave of oxidative burst, arrested cell division, stimulated cell expansion, and the formation of an immobile palmella stage. This persistent oxidative burst can be suppressed by specific inhibitor diphenyl iodonium (DPI), but not by the unspecific antioxidant ascorbic acid (Asc). Treated with natural and artificial auxins allow to modulating the cell cycle and cell expansion, and natural auxin can suppress the spontaneous formation of palmella stages. However, when administered prior to the nsPEFs treatment, auxin cannot mitigate the elevated formation of palmella stages induced by nsPEFs. We interpret our findings in terms of a model, where nsPEFs generate a developmental signal that persists, although the other immediate responses remain transient. This signal will initiate, several days later, a developmental programme comprising halted cell cycle, stimulation of cell expansion, a persistent activation of NADPH oxidase activity causing a second wave of oxidative burst, and the irreversible initiation of palmella stages. Thus, a short transient nsPEFs treatment can initiate a stable response of cellular differentiation in Chlamydomonas reinhardtii.

Exploring the Applicability of Nano-Poration for Remote Control in Smart Drug Delivery Systems.

Smart drug delivery systems represent an interesting tool to significantly improve the efficiency and the precision in the treatment of a broad category of diseases. In this context, a drug delivery mediated by nanosecond pulsed electric fields seems a promising technique, allowing for a controlled release and uptake of drugs by the synergy between the electropulsation and nanocarriers with encapsulated drugs. The main concern about the use of electroporation for drug delivery applications is the difference in dimension between the liposome (nanometer range) and the cell (micrometer range). The choice of liposome dimension is not trivial. Liposomes larger than 500 nm of diameter could be recognized as pathogen agents by the immune system, while liposomes of smaller size would require external electric field of high amplitudes for the membrane electroporation that could compromise the cell viability. The aim of this work is to theoretically study the possibility of a simultaneous cell and liposomes electroporation. The numerical simulations reported the possibility to electroporate the cell and a significant percentage of liposomes with comparable values of external electric field, when a 12 nsPEF is used.

Enhanced breast cancer therapy with nsPEFs and low concentrations of gemcitabine.

BACKGROUND: Chemotherapy either before or after surgery is a common breast cancer treatment. Long-term, high dose treatments with chemotherapeutic drugs often result in undesirable side effects, frequent recurrences and resistances to therapy. METHODS: The anti-cancer drug, gemcitabine (GEM) was used in combination with pulse power technology with nanosecond pulsed electric fields (nsPEFs) for treatment of human breast cancer cells in vitro. Two strategies include sensitizing mammary tumor cells with GEM before nsPEF treatment or sensitizing cells with nsPEFs before GEM treatment. Breast cancer cell lines MCF-7 and MDA-MB-231 were treated with 250 65 ns-duration pulses and electric fields of 15, 20 or 25 kV/cm before or after treatment with 0.38 µM GEM. RESULTS: Both cell lines exhibited robust synergism for loss of cell viability 24 h and 48 h after treatment; treatment with GEM before nsPEFs was the preferred order. In clonogenic assays, only MDA-MB-231 cells showed synergism; again GEM before nsPEFs was the preferred order. In apoptosis/necrosis assays with Annexin-V-FITC/propidium iodide 2 h after treatment, both cell lines exhibited apoptosis as a major cell death mechanism, but only MDA-MB-231 cells exhibited modest synergism. However, unlike viability assays, nsPEF treatment before GEM was preferred. MDA-MB-231 cells exhibited much greater levels of necrosis then in MCF-7 cells, which were very low. Synergy was robust and greater when nsPEF treatment was before GEM. CONCLUSIONS: Combination treatments with low GEM concentrations and modest nsPEFs provide enhanced cytotoxicity in two breast cancer cell lines. The treatment order is flexible, although long-term survival and short-term cell death analyses indicated different treatment order preferences. Based on synergism, apoptosis mechanisms for both agents were more similar in MCF-7 than in MDA-MB-231 cells. In contrast, necrosis mechanisms for the two agents were distinctly different in MDA-MB-231, but too low to reliably evaluate in MCF-7 cells. While disease mechanisms in the two cell lines are different based on the differential synergistic response to treatments, combination treatment with GEM and nsPEFs should provide an advantageous therapy for breast cancer ablation in vivo.

Simulation study on electroporation of cancer cells in multicellular system.

Electroporation (EP) of the normal cell and cancer cell both in single-cell and multicellular models was investigated by the meshed transport network method (MTNM) in this paper. The simulation results suggest that the cancer cell undergoes faster and more significant local EP than that of the corresponding normal cell induced by nanosecond pulsed electric fields (nsPEFs) both in single-cell and multicellular models. Furthermore, the results of the multicellular model indicate that there is a unidirectional neighboring effect in the multicellular model, meaning that cells at the center are affected and their pore formation is significantly reduced, but this effect is very weak for cells at the edges of the system. This means that the electric field selectively kills cells in different distribution locations. This work can provide guidance for the selection of parameters for the cancer cell EP process.

Functionally improved mesenchymal stem cells via nanosecond pulsed electric fields for better treatment of osteoarthritis.

Background: Numerous approaches have been utilized to optimize mesenchymal stem cells (MSCs) performance in treating osteoarthritis (OA), however, the constrained diminished activity and chondrogenic differentiation capacity impede their therapeutic efficacy. Previous investigations have successfully shown that pretreatment with nanosecond pulsed electric fields (nsPEFs) significantly enhances the chondrogenic differentiation of MSCs. Therefore, this study aims to explore nsPEFs as a strategy to improve OA therapy by enhancing MSCs' activity and chondrogenic differentiation and also investigate its potential mechanism. Methods: In this study, a million MSCs were carefully suspended within a 0.4-cm gap cuvette and subjected to five pulses of nsPEFs (100 ns at 10 kV/cm, 1 Hz), with a 1-s interval between each pulse. A control group of MSCs was maintained without nsPEFs treatment for comparative analysis. nsPEFs were applied to regulate the MSCs performance and hinder OA progresses. In order to further explore the corresponding mechanism, we examined the changes of MSCs transcriptome after nsPEF pretreatment. Finally, we studied the properties of extracellular vesicles (EVs) secreted by MSCs affected by nsPEF and the therapeutic effect on OA. Results: We found that nsPEFs pretreatment promoted MSCs migration and viability, particularly enhancing their viability temporarily in vivo, which is also confirmed by mRNA sequencing analysis. It also significantly inhibited the development of OA-like chondrocytes in vitro and prevented OA progression in rat models. Additionally, we discovered that nsPEFs pretreatment reprogrammed MSC performance by enhancing EVs production (5.77 ± 0.92 folds), and consequently optimizing their therapeutic potential. Conclusions: In conclusion, nsPEFs pretreatment provides a simple and effective strategy for improving the MSCs performance and the therapeutic effects of MSCs for OA. EVs-nsPEFs may serve as a potent therapeutic material for OA and hold promise for future clinical applications. The translational potential of this article: This study indicates that MSCs pretreated by nsPEFs greatly inhibited the development of OA. nsPEFs pretreatment will be a promising and effective method to optimize the therapeutic effect of MSCs in the future.

Nano-Pulse Stimulation Therapy in Oncology.

Background: Nano-Pulse Stimulation (NPS) therapy applies electric pulses in the nanosecond domain to initiate regulated cell death in the treated tissues. This nonthermal therapy has been used to treat a wide range of murine tumors and has been shown to activate the immune system to inhibit the growth of rechallenge tumors, as well as untreated, abscopal tumors when accompanied by the injection of immune system stimulants into the treated tumors. Clinical trials have begun using NPS to treat basal cell carcinoma and hepatocellular carcinoma. Methods: Murine tumors can be easily imaged when the tumor cells are injected intradermally so that they grow within the mouse skin. Pulling the skin over a translucent light post shines light through the skin and makes it easy to treat the tumor and identify the treatment zone. Results: Original research using murine tumor models is described, including melanoma, squamous cell carcinoma, lung carcinoma, breast carcinoma, and pancreatic carcinoma. The energy required to ablate these tumors has been determined with pancreatic carcinoma and lung carcinoma exhibiting 90% ablation with 240 mJ/mm3, lung carcinoma and squamous cell carcinoma requiring 360 mJ/mm3, and melanoma requiring 480 mJ/mm3. NPS therapy initiated a variable immune response indicated by the rejection of injected rechallenge tumor cells with melanoma and hepatocellular carcinoma exhibiting the strongest response and lung carcinoma, the weakest response. Following the original research data, a review of human clinical trials using NPS therapy is presented. Conclusions: NPS therapy offers a nonthermal, drug-free approach for oncology, which is limited only by applying energy to the tumor. This new immunogenic modality is just beginning to be applied in the clinic. The 87% efficacy of the first large clinical trial conducted by several medical personnel is impressive and indicates that NPS is an effective new modality for cancer treatment.

Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing.

BACKGROUND: Mesenchymal stem cells (MSCs) modulated by various exogenous signals have been applied extensively in regenerative medicine research. Notably, nanosecond pulsed electric fields (nsPEFs), characterized by short duration and high strength, significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways. Consequently, we used transcriptomics to study changes in messenger RNA (mRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA expression during nsPEFs application. AIM: To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs. METHODS: The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing. MSCs were pretreated with 5-pulse nsPEFs (100 ns at 10 kV/cm, 1 Hz), followed by total RNA isolation. Each transcript was normalized by fragments per kilobase per million. Fold change and difference significance were applied to screen the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions, complemented by quantitative polymerase chain reaction verification. RESULTS: In total, 263 DEGs were discovered, with 92 upregulated and 171 downregulated. DEGs were predominantly enriched in epithelial cell proliferation, osteoblast differentiation, mesenchymal cell differentiation, nuclear division, and wound healing. Regarding cellular components, DEGs are primarily involved in condensed chromosome, chromosomal region, actin cytoskeleton, and kinetochore. From aspect of molecular functions, DEGs are mainly involved in glycosaminoglycan binding, integrin binding, nuclear steroid receptor activity, cytoskeletal motor activity, and steroid binding. Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation. CONCLUSION: Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs, 2 miRNAs, and 65 lncRNAs. Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways, which are involved in vesicular transport, calcium ion transport, cytoskeleton, and cell differentiation.

LncRNA Regulation Mechanism in Hepatic Alveolar Echinococcosis with Nanosecond Pulse.

BACKGROUND: The mortality of patients infected with hepatic alveolar echinococcosis (HAE) was higher. The aim of this study was to investigate the therapeutic effect of nanosecond pulsed electric fields (nsPEFs) on HAE in rats and explore the related molecular mechanisms. METHODS: Establishment of HAE rat model and the lesions were treated with nsPEFs. The RNA of lesions in the high voltage nsPEFs treatment group and model group were extracted, and lncRNA and mRNA sequence analyses was performed. After obtaining the differentially expressed lncRNAs and mRNAs between the two groups, enrichment analysis was performed for mRNAs. The target genes of lncRNAs were predicted through co-location and co-expression. The expression of important lncRNAs and target genes in lesions was detected by qPCR. RESULTS: The HAE rat model was successfully established. After nsPEFs treatment, the size of lesions was improved significantly. Then, we identified 270 differentially expressed lncRNAs and 1659 differentially expressed mRNAs between the high voltage nsPEFs treatment group and model group. Enrichment analysis showed that the differentially expressed mRNAs were mainly enriched in metabolism and inflammation. Five important lncRNAs regulatory networks were identified, then Cpa1, Cpb1, Cel, Cela2a, and Cela3b were identified as key target genes. Importantly, the expression of 5 lncRNAs and 5 target genes was verified in the lesions. CONCLUSIONS: Preliminary results had shown that HAE treatment with nsPEFs can inhibit the growth of lesions. NsPEFs treatment altered gene expression in the lesions, and some genes were regulated by lncRNAs. The therapeutic mechanism may involve metabolism and inflammation.

Nanosecond pulse effectively ablated hepatocellular carcinoma with alterations in the gut microbiome and serum metabolites.

Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world. Nanosecond pulsed electric fields (nsPEFs) have emerged as a new treatment for cancer. This study aims to identify the effectiveness of nsPEFs in the treatment of HCC and analyze the alterations in the gut microbiome and serum metabonomics after ablation. Methods: C57BL/6 mice were randomly divided into three groups: healthy control mice (n = 10), HCC mice (n = 10), and nsPEF-treated HCC mice (n = 23). Hep1-6 cell lines were used to establish the HCC model in situ. Histopathological staining was performed on tumor tissues. The gut microbiome was analyzed by 16S rRNA sequencing. Serum metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis. Spearman's correlation analysis was carried out to analyze the correlation between the gut microbiome and serum metabonomics. Results: The fluorescence image showed that nsPEFs were significantly effective. Histopathological staining identified nuclear pyknosis and cell necrosis in the nsPEF group. The expression of CD34, PCNA, and VEGF decreased significantly in the nsPEF group. Compared with normal mice, the gut microbiome diversity of HCC mice was increased. Eight genera including Alistipes and Muribaculaceae were enriched in the HCC group. Inversely, these genera decreased in the nsPEF group. LC-MS analysis confirmed that there were significant differences in serum metabolism among the three groups. Correlation analysis showed crucial relationships between the gut microbiome and serum metabolites that are involved in nsPEF ablation of HCC. Conclusion: As a new minimally invasive treatment for tumor ablation, nsPEFs have an excellent ablation effect. The alterations in the gut microbiome and serum metabolites may participate in the prognosis of HCC ablation.

Nanosecond pulsed electric fields prime mesenchymal stem cells to peptide ghrelin and enhance chondrogenesis and osteochondral defect repair in vivo.

Mesenchymal stem cells (MSCs) are important cell sources in cartilage tissue development and homeostasis, and multiple strategies have been developed to improve MSCs chondrogenic differentiation with an aim of promoting cartilage regeneration. Here we report the effects of combining nanosecond pulsed electric fields (nsPEFs) followed by treatment with ghrelin (a hormone that stimulates release of growth hormone) to regulate chondrogenesis of MSCs. nsPEFs and ghrelin were observed to separately enhance the chondrogenesis of MSCs, and the effects were significantly enhanced when the bioelectric stimulation and hormone were combined, which in turn improved osteochondral tissue repair of these cells within Sprague Dawley rats. We further found that nsPEFs can prime MSCs to be more receptive to subsequent stimuli of differentiation by upregulated Oct4/Nanog and activated JNK signaling pathway. Ghrelin initiated chondrogenic differentiation by activation of ERK1/2 signaling pathway, and RNA-seq results indicated 243 genes were regulated, and JAK-STAT signaling pathway was involved. Interestingly, the sequential order of applying these two stimuli is critical, with nsPEFs pretreatment followed by ghrelin enhanced chondrogenesis of MSCs in vitro and subsequent cartilage regeneration in vivo, but not vice versa. This synergistic prochondrogenic effects provide us new insights and strategies for future cell-based therapies.

Corrigendum: Antitumor effect and immune response of nanosecond pulsed electric fields in pancreatic cancer.

[This corrects the article DOI: 10.3389/fonc.2020.621092.].