The crosstalk between cholangiocytes and hepatic stellate cells promotes the progression of epithelial-mesenchymal transition and periductal fibrosis during Clonorchis sinensis infection.

ABSTRACT: BACKGROUND: Clonorchis sinensis infection is one of the risk factors that provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis and even cholangiocarcinoma (CCA). Disrupted or aberrant intercellular communication among liver-constituting cells leads to pathological states that cause various hepatic diseases. This study was designed to investigate the pathological changes caused by C. sinensis excretory-secretory products (ESPs) in non-cancerous human cell lines (cholangiocytes [H69 cell line] and human hepatic stellate cells [LX2 cell line]) and their intercellular crosstalk, as well the pathological changes in infected mouse liver tissues. METHODS: The cells were treated with ESPs, following which transforming growth factor beta 1 (TGF-ß1) and interleukin-6 (IL-6) secretion levels and epithelial-mesenchymal transition (EMT)- and fibrosis-related protein expression were measured. The ESP-mediated cellular motility (migration/invasion) between two cells was assessed using the Transwell and three-dimensional microfluidic assay models. The livers of C. sinensis-infected mice were stained using EMT and fibrotic marker proteins. RESULTS: Treatment of cells with ESPs increased TGF-ß1 and IL-6 secretion and the expression of EMT- and fibrosis-related proteins. The ESP-mediated mutual cell interaction further affected the cytokine secretion and protein expression levels and promoted cellular motility. N-cadherin overexpression and collagen fiber deposition were observed in the livers of C. sinensis-infected mice. CONCLUSIONS: These findings suggest that EMT and biliary fibrosis occur through intercellular communication between cholangiocytes and hepatic stellate cells during C. sinensis infection, promoting malignant transformation and advanced hepatobiliary abnormalities.

Zinc deficiency triggers hearing loss by reducing ribbon synapses of inner hair cells in CBA/N mice.

Zinc plays a vital role in our metabolism, encompassing antioxidant regulation, immune response, and auditory function. Several studies have reported that zinc levels correlate with hearing loss. We have previously demonstrated that the auditory brainstem response (ABR) threshold increased in mice fed a zinc-deficient diet. However, the effects of zinc deficiency on hearing were not fully elucidated. The present study investigated whether zinc deficiency affects hearing in association with neuronal components or cochlear structures. CBA/N mice were fed a normal or zinc-deficient diet for 8 weeks and assessed for ABR and distortion product otoacoustic emissions (DPOAE). The cochlear sections were stained with hematoxylin and eosin solution. Also, we observed the expression of synaptic ribbons, neurofilaments, and alpha-synuclein (α-Syn). The 8-week zinc-deficient diet mice had an elevated ABR threshold but no changed DPOAE threshold or cochlear structures. A reduced number of synaptic ribbons of inner hair cells (IHCs) and impaired efferent nerve fibers were observed in the zinc-deficient diet mice. The number of outer hair cells (OHCs) and expression of α-Syn remained unchanged. Our results suggest that zinc-mediated hearing loss is associated with the loss of neuronal components of IHCs.

Dexamethasone treatment of murine auditory hair cells and cochlear explants attenuates tumor necrosis factor-α-initiated apoptotic damage.

The most common cause of sensorineural hearing loss is damage of auditory hair cells. Tumor necrosis factor-alpha (TNF-α) is closely associated with sensorineural hearing loss. The present study examined the preconditioning effect of dexamethasone (DEX) on TNF-α-induced ototoxicity in mouse auditory hair cells (HEI-OC1) and cochlear explants. Treatment of HEI-OC1 with 10 ng/ml TNF-α for 24 h decreased cell viability, increased the accumulation of reactive oxygen species (ROS), and induced caspase-mediated apoptotic signaling pathways. Pretreatment with 10 nM DEX for 6 h before TNF-α exposure restored cell viability, decreased ROS accumulation, and attenuated apoptotic signaling activation induced by TNF-α. Incubation of cochlear explants with 20 ng/ml TNF-α for 24 h resulted in significant loss of both inner hair cells (IHCs) and outer hair cells (OHCs) and an increase in apoptotic activation accessed by annexin V staining. The cochlear explants pre-incubated with 10 nM DEX attenuated TNF-α ototoxicity in both IHCs and OHCs and apoptotic cell death. These results indicated that DEX plays a protective role in ototoxicity induced by TNF-α through attenuation of caspase-dependent apoptosis signaling pathway and ROS accumulation.

Zinc is an essential element for the maintenance of redox homeostasis and cell cycle in murine auditory hair cells.

A nutrition deficiency is one of the various causes of hearing loss. Zinc is an essential element for cell proliferation, antioxidant reactions, and the maintenance of hearing ability. Our previous studies have reported that the auditory brainstem response (ABR) threshold is increased in mice fed with zinc-deficient diets. However, the molecular mechanism of zinc involved in auditory system remains to be elucidated. In the present study, we examined the detrimental effects of zinc deficiency on cell cycle progression in murine auditory cells (HEI-OC1). The treatment of HEI-OC1 cells with 0.5 µM TPEN (N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine) for 24 h inhibited cell proliferation, accumulation of reactive oxygen species (ROS), and induction of apoptosis. The cell proliferation block was caused by a G1/S phase arrest. Supplementation of the cell growth medium with 5 µM ZnCl2 after exposure to TPEN attenuated ROS accumulation and the arrest caused by the zinc deficiency. The ABR threshold was elevated in mice fed with a zinc-deficient diet. Additionally, we observed an increased expression of p21 and decreased expression of cyclin E and pRb in the spiral ganglion (SG), the organ of Corti (OC), Limbus (L), and stria vascularis (SV) in the zinc-deficient mouse cochlea. These results indicated that zinc is an essential nutrient for proliferation via the cell cycle and that a dysregulation of the cell cycle may cause hearing loss.

The Overactivation of NADPH Oxidase during Clonorchis sinensis Infection and the Exposure to N-Nitroso Compounds Promote Periductal Fibrosis.

Clonorchis sinensis, a high-risk pathogenic human liver fluke, provokes various hepatobiliary complications, including epithelial hyperplasia, inflammation, periductal fibrosis, and even cholangiocarcinogenesis via direct contact with worms and their excretory-secretory products (ESPs). These pathological changes are strongly associated with persistent increases in free radical accumulation, leading to oxidative stress-mediated lesions. The present study investigated C. sinensis infection- and/or carcinogen N-nitrosodimethylamine (NDMA)-associated fibrosis in cell culture and animal models. The treatment of human cholangiocytes (H69 cells) with ESPs or/and NDMA increased reactive oxidative species (ROS) generation via the activation of NADPH oxidase (NOX), resulting in augmented expression of fibrosis-related proteins. These increased expressions were markedly attenuated by preincubation with a NOX inhibitor (diphenyleneiodonium chloride) or an antioxidant (N-acetylcysteine), indicating the involvement of excessive NOX-dependent ROS formation in periductal fibrosis. The immunoreactive NOX subunits, p47phox and p67phox, were observed in the livers of mice infected with C. sinensis and both infection plus NDMA, concomitant with collagen deposition and immunoreactive fibronectin elevation. Staining intensities are proportional to lesion severity and infection duration or/and NDMA administration. Thus, excessive ROS formation via NOX overactivation is a detrimental factor for fibrogenesis during liver fluke infection and exposure to N-nitroso compounds.

Transcriptomic profiling of three-dimensional cholangiocyte spheroids long term exposed to repetitive Clonorchis sinensis excretory-secretory products.

BACKGROUND: Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. Complications are proportional to the intensity and duration of the infection. In addition to mechanical irritation of the biliary epithelia from worms, their excretory-secretory products (ESPs) cause chemical irritation, which leads to inflammation, proliferation, and free radical generation. METHODS: A three-dimensional in vitro cholangiocyte spheroid culture model was established, followed by ESP treatment. This allowed us to examine the intrinsic pathological mechanisms of clonorchiasis via the imitation of prolonged and repetitive in vivo infection. RESULTS: Microarray and RNA-Seq analysis revealed that ESP-treated cholangiocyte H69 spheroids displayed global changes in gene expression compared to untreated spheroids. In ESP-treated H69 spheroids, 185 and 63 probes were found to be significantly upregulated and downregulated, respectively, corresponding to 209 genes (p < 0.01, fold change > 2). RNA-Seq was performed for the validation of the microarray results, and the gene expression patterns in both transcriptome platforms were well matched for 209 significant genes. Gene ontology analysis demonstrated that differentially expressed genes were mainly classified into immune system processes, the extracellular region, and the extracellular matrix. Among the upregulated genes, four genes (XAF1, TRIM22, CXCL10, and BST2) were selected for confirmation using quantitative RT-PCR, resulting in 100% similar expression patterns in microarray and RNA-Seq. CONCLUSIONS: These findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer.

Antioxidant Therapy against Oxidative Damage of the Inner Ear: Protection and Preconditioning.

Oxidative stress is an important mechanism underlying cellular damage of the inner ear, resulting in hearing loss. In order to prevent hearing loss, several types of antioxidants have been investigated; several experiments have shown their ability to effectively prevent noise-induced hearing loss, age-related hearing loss, and ototoxicity in animal models. Exogenous antioxidants has been used as single therapeutic agents or in combination. Antioxidant therapy is generally administered before the production of reactive oxygen species. However, post-exposure treatment could also be effective. Preconditioning refers to the phenomenon of pre-inducing a preventative pathway by subtle stimuli that do not cause permanent damage in the inner ear. This renders the inner ear more resistant to actual stimuli that cause permanent hearing damage. The preconditioning mechanism is also related to the induction of antioxidant enzymes. In this review, we discuss the mechanisms underlying antioxidant-associated therapeutic effects and preconditioning in the inner ear.

Protective Effects of Glucose-Related Protein 78 and 94 on Cisplatin-Mediated Ototoxicity.

Cisplatin is a widely used chemotherapeutic drug for treating various solid tumors. Ototoxicity is a major dose-limiting side effect of cisplatin, which causes progressive and irreversible sensorineural hearing loss. Here, we examined the protective effects of glucose-related protein (GRP) 78 and 94, also identified as endoplasmic reticulum (ER) chaperone proteins, on cisplatin-induced ototoxicity. Treating murine auditory cells (HEI-OC1) with 25 µM cisplatin for 24 h increased cell death resulting from excessive intracellular reactive oxygen species (ROS) accumulation and caspase-involved apoptotic signaling pathway activation with subsequent DNA fragmentation. GRP78 and GRP94 expression was increased in cells treated with 3 nM thapsigargin or 0.1 µg/mL tunicamycin for 24 h, referred to as mild ER stress condition. This condition, prior to cisplatin exposure, attenuated cisplatin-induced ototoxicity. The involvement of GRP78 and GRP94 induction was demonstrated by the knockdown of GRP78 or GRP94 expression using small interfering RNAs, which abolished the protective effect of mild ER stress condition on cisplatin-induced cytotoxicity. These results indicated that GRP78 and GRP94 induction plays a protective role in remediating cisplatin-ototoxicity.

Induction of Redox-Active Gene Expression by CoCl2 Ameliorates Oxidative Stress-Mediated Injury of Murine Auditory Cells.

Free radicals formed in the inner ear in response to high-intensity noise, are regarded as detrimental factors for noise-induced hearing loss (NIHL). We reported previously that intraperitoneal injection of cobalt chloride attenuated the loss of sensory hair cells and NIHL in mice. The present study was designed to understand the preconditioning effect of CoCl2 on oxidative stress-mediated cytotoxicity. Treatment of auditory cells with CoCl2 promoted cell proliferation, with increases in the expressions of two redox-active transcription factors (hypoxia-inducible factor 1α, HIF-1α, nuclear factor erythroid 2-related factor 2; Nrf-2) and an antioxidant enzyme (peroxiredoxin 6, Prdx6). Hydrogen peroxide treatment resulted in the induction of cell death and reduction of these protein expressions, reversed by pretreatment with CoCl2. Knockdown of HIF-1α or Nrf-2 attenuated the preconditioning effect of CoCl2. Luciferase reporter analysis with a Prdx6 promoter revealed transactivation of Prdx6 expression by HIF-1α and Nrf-2. The intense immunoreactivities of HIF-1α, Nrf-2, and Prdx6 in the organ of Corti (OC), spiral ganglion cells (SGC), and stria vascularis (SV) of the cochlea in CoCl2-injected mice suggested CoCl2-induced activation of HIF-1α, Nrf-2, and Prdx6 in vivo. Therefore, we revealed that the protective effect of CoCl2 is achieved through distinctive signaling mechanisms involving HIF-1α, Nrf-2, and Prdx6.

HtrA2 suppresses autoimmune arthritis and regulates activation of STAT3.

Rheumatoid arthritis (RA) is an autoimmune disease that is related to the induction of T helper (Th)17 cells, which secrete interleukin-17, and activation of the signal transducer and activator of transcription (STAT) 3. The expression of high-temperature requirement protein A (HtrA) 2, a serine protease involved in apoptosis, was decreased in RA patients nonresponsive to drug treatment of RA. The aim of this study was to determine whether overexpression of HtrA2 has a therapeutic effect on RA. Th17 differentiation, osteoclastogenesis, and lymphocyte activation are increased in motor neuron degeneration (mnd)2 mice, which lack HtrA2 activity because of a missense mutation (Ser276Cys) in the protease domain of HtrA2. The inhibitor of HtrA2 also increased Th17 differentiation. On the other hand, HtrA2 induced cleavage of STAT3 and overexpression of HtrA2 attenuated CIA in a mouse model. HtrA2 overexpression inhibited plaque development as well as the differentiation of Th17 in ApoE-/- mice after immunization with proteoglycans to induce a hyperlipidemia-based RA animal model. The therapeutic function of HtrA2 in inflammatory diseases is linked with Th17 development and the STAT3 pathway in splenocytes. These results suggest that HtrA2 participates in immunomodulatory activity where the upregulation of HtrA2 may shed light on therapeutic approaches to RA and hyperlipidemia.

Effects of H2 and electrochemical reducing power on metabolite production by Clostridium acetobutylicum KCTC1037.

A conventional fermenter (CF), a single-cathode fermenter (SCF), and a double-cathode fermenter (DCF) were employed to evaluate and compare the effects of H2 and electrochemical reducing power on metabolite production by Clostridium acetobutylicum KCTC1037. The source of the external reducing power for CF was H2, for the SCF was electrochemically reduced neutral red-modified graphite felt electrode (NR-GF), and for the DCF was electrochemically reduced combination of NR-GF and platinum plate electrodes (NR-GF/PtP). The metabolites produced from glucose or CO2 by strain KCTC1037 cultivated in the DCF were butyrate, ethanol, and butanol, but ethanol and butanol were not produced from glucose or CO2 by strain KCTC1037 cultivated in the CF and SCF. It is possible that electrochemically reduced NR-GF/PtP is a more effective source of internal and external reducing power than H2 or NR-GF for strain KCTC1037 to produce metabolites from glucose and CO2. This research might prove useful in developing fermentation technology to actualize direct bioalcohol production of fermentation bacteria from CO2.