Particulate matter facilitates amphiregulin-dependent lung cancer proliferation through glutamine metabolism.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) causes lung cancer, the molecular mechanisms underlying the PM-induced increases in lung cancer progression remain unclear. We applied the lung cancer cell line A549 (Parental; A549.Par) to PM for an extended period to establish a mimic PM-exposed lung cancer cell line, A549.PM. Our results indicate that A549.PM exhibits higher cell growth and proliferation abilities compared to A549.Par cells in vitro and in vivo. The RNA sequencing analysis found amphiregulin (AREG) plays a critical role in PM-induced cell proliferation. We observed that PM increases AREG-dependent lung cancer proliferation through glutamine metabolism. In addition, the EGFR/PI3K/AKT/mTOR signaling pathway is involved in PM-induced solute carrier family A1 member 5 (SLC1A5) expression and glutamine metabolism. Our findings offer important insights into how lung cancer proliferation develops upon exposure to PM.

High glucose enhances fibrosis in human annulus fibrosus cells by activating mTOR, PKCδ, and NF-κB signaling pathways.

Low back pain stands as a significant factor in disability, largely resulting from intervertebral disc degeneration (IVDD). High glucose (HG) levels have been implicated in the pathogenesis of IVDD. However, the detailed mechanism of HG in IVDD is largely unknown. Our clinical results revealed that fibrosis markers such as CTGF, Col1a1, ATF4, and EIF2 are highly expressed in advanced-stage IVDD patients. Stimulation of human annulus fibrosus cells (HAFCs) with HG, but not mannitol, promotes fibrosis protein production. Ingenuity Pathway Analysis in the GSE database found that the mTOR, PKCδ, and NF-κB pathways were significantly changed during IVDD. The mTOR, PKCδ, and NF-κB inhibitors or siRNAs all abolished HG-induced fibrosis protein production. In addition, treatment of HAFCs with HG enhances the activation of mTOR, PKCδ, and NF-κB pathways. Thus, HG facilitates fibrosis in IVDD through mTOR, PKCδ, and NF-κB pathways. These results underscore the critical role of HG as a fibrotic factor in the progression of IVDD.

MCP-1 controls IL-17-promoted monocyte migration and M1 polarization in osteoarthritis.

Osteoarthritis (OA) is a low-grade inflammatory joint illness in which monocytes migrate and infiltrate synovial tissue, differentiating into the pro-inflammatory M1 macrophage phenotype. IL-17 is a proinflammatory mediator principally generated by Th17 cells, which is elevated in OA patients; nevertheless, investigators have yet to elucidate the function of IL-17 in M1 polarization during OA development. Our analysis of clinical tissues and results from the open online dataset discovered that the level of M1 macrophage markers is elevated in human OA tissue samples than in normal tissue. High-throughput screening demonstrated that MCP-1 is a potential candidate factor after IL-17 treatment in OA synovial fibroblasts (OASFs). Immunohistochemistry data revealed that the level of MCP-1 is higher in humans and mice with OA than in normal tissues. IL-17 stimulation facilitates MCP-1-dependent macrophage polarization to the M1 phenotype. It also appears that IL-17 enhances MCP-1 synthesis in human OASFs, enhancing monocyte migration via the JAK and STAT3 signaling cascades. Our findings indicate the IL-17/MCP-1 axis as a novel strategy for the remedy of OA.

IL-17 promotes IL-18 production via the MEK/ERK/miR-4492 axis in osteoarthritis synovial fibroblasts.

The concept of osteoarthritis (OA) as a low-grade inflammatory joint disorder has been widely accepted. Many inflammatory mediators are implicated in the pathogenesis of OA. Interleukin (IL)-18 is a pleiotropic cytokine with versatile cellular functions that are pathogenetically important in immune responses, as well as autoimmune, inflammatory, and infectious diseases. IL-17, a proinflammatory cytokine mainly secreted by Th17 cells, is upregulated in OA patients. However, the role of IL-17 in OA progression is unclear. The synovial tissues collected from healthy donors and OA patients were used to detect the expression level of IL-18 by IHC stain. The OA synovial fibroblasts (OASFs) were incubated with recombinant IL-17 and subjected to Western blot, qPCR, and ELISA to examine IL-18 expression level. The chemical inhibitors and siRNAs which targeted signal pathways were used to investigate signal pathways involved in IL-17-induced IL-18 expression. The microRNAs which participated IL-18 expression were surveyed with online databases miRWalk and miRDB, followed by validation with qPCR. This study revealed significantly higher levels of IL-18 expression in synovial tissue from OA patients compared with healthy controls, as well as increased IL-18 expression in OASFs from rats with severe OA. In vitro findings indicated that IL-17 dose-dependently promoted IL-18 production in OASFs. Molecular investigations revealed that the MEK/ERK/miR-4492 axis stimulated IL-18 production when OASFs were treated with IL-17. This study provides novel insights into the role of IL-17 in the pathogenesis of OA, which may help to inform OA treatment in the future.

Antagonistic effects of N-acetylcysteine on lead-induced apoptosis and oxidative stress in chicken embryo fibroblast cells.

Lead (Pb) is a heavy metal that can have harmful effects on the environment, which has severe cytotoxicity in many animal tissues. N-acetylcysteine (NAC) has antioxidant activity, reducing lead-induced oxidative stress and apoptosis, but its role in chicken cells is unknown. The current study explored the antagonistic effect of NAC on lead-induced apoptosis and oxidative stress in chicken embryo fibroblast (CEF). In this study, CEF was used as a model to measure the cytotoxic effects of lead nitrate at different concentrations, demonstrating a dose-dependent effect on CEF activity. Employing inverted microscopy, the investigation of morphological alterations in CEF cells was conducted. Fluorescence staining methodology enabled the assessment of reactive oxygen species (ROS) levels within CEF cells. Moreover, an enzyme-linked immunosorbent assay was utilized to detect the presence of oxidative damage indicators encompassing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activity, malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC) within CEF cells. Furthermore, the determination of the apoptosis rate of CEF cells was accomplished through the utilization of the Hoechst 33258 staining method in combination with the Annexin V-FITC dual staining method. By using RT-qPCR for detection, lead treatment increased expression of pro-apoptotic genes, caspase-3, and caspase-9, and reduced expression of anti-apoptotic genes, Bcl-2, and BI-1. Reduced antioxidant capacity was shown by increased ROS and MDA levels in CEF cells after lead treatment. The results showed that NAC inhibited the expression of caspase-3 and caspase-9 in lead-treated CEF cells, while NAC had a certain inhibitory effect on the relative expression of Bcl-2 and BI-1 mRNA in lead-induced CEF cells. NAC significantly reduced lead-induced oxidative damage and apoptosis. Overall, our results demonstrate a novel protective effect of NAC against lead-induced injury in chicken cells, providing a theoretical basis for future investigations of drugs that are effective in preventing lead poisoning in animals.

Esculetin improves murine mastitis induced by streptococcus isolated from bovine mammary glands by inhibiting NF-κB and MAPK signaling pathways.

Cow mastitis, caused by Streptococcus infection of the mammary glands, is a common clinical disease that can lead to decreased milk quality and threaten animal welfare and performance. Esculetin (ESC) is a coumarin with anti-inflammatory and anti-asthmatic effects. However, whether ESC has therapeutic effects on mastitis remains unexplored. This study was conducted to investigate the protective effect of ESC against murine mastitis caused by Streptococcus isolated from bovine mammary glands and elucidate the underlying mechanisms. Streptococcus uberis was used to construct a mouse model of mastitis. The results showed that the mice exhibited edema and thickening of the acinar wall with inflammatory infiltration after S. uberis treatment. Intraperitoneal injection of ESC significantly reduced inflammatory cell infiltration, restored normal physiological function, and inhibited the production of the inflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that ESC reduced P38 phosphorylation, further inhibited the influence of mammary Streptococcus on cytoplasmic translocation of nuclear factor-κB (P65), and inhibited the transcriptional activation of P65, thus inhibiting the generation of inflammatory cells. Collectively, ESC may inhibit mitogen-activated protein kinase and nuclear factor-κB, thereby highlighting its potential for the treatment and prevention of mastitis.

JNK molecule is a toxic target for IPEC-J2 cell barrier damage induced by T-2 toxin.

The most prevalent contaminated mycotoxin in feed and grain is T-2 toxin. The T-2 toxin's primary action target is the gut because it is the main organ of absorption. T-2 toxin can cause intestinal damage, but, few molecular mechanisms have been elucidated. It is important to discover the key pathways by which T-2 toxin causes enterotoxicity. In this research, IPEC-J2 cells are used as a cell model to investigate the function of the MAPK signaling pathway in T-2 toxin-induced intestinal epithelial cell damage. Throughout this research, T-2 toxin results in functional impairment in IPEC-J2 cells by reducing the TJ proteins Claudin, Occludin-1, ZO-1, N-cadherin, and CX-43 expression. T-2 toxin significantly reduced the survival of IPEC-J2 cells and increased LDH release in a dose-dependent way. T-2 toxin induced IPEC-J2 cell oxidative stress by raising ROS and MDA content, and mitochondrial damage was indicated by a decline in MMP and an increase in the opening degree of MPTP. T-2 toxin upregulated the expression of ERK, P38 and JNK, which triggered the MAPK signaling pathway. In addition, T-2 toxin caused IPEC-J2 cell inflammation responses reflected by increased the levels of inflammation-related factors IL-8, p65, P-p65 and IL-6, and down-regulated IL-10 expression level. Inhibition JNK molecule can ease IPEC-J2 cell functional impairment and inflammatory response. In conclusion, as a consequence of the T-2 toxin activating the JNK molecule, oxidative stress and mitochondrial damage are induced, which impair cellular inflammation.

Nesfatin-1 stimulates BMP5 expression and osteoclastogenesis in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common autoimmune disease marked by immune cell activation and chronic inflammation in the synovium accompanied by osteoclast activation and local joint destruction. Increased levels of the adipokine nesfatin-1 in RA synovium are associated with proinflammatory cytokines. Our analysis of datasets from the Gene Expression Omnibus (GEO) database and synovial tissue samples from RA patients revealed that these had higher levels of nesfatin-1 and osteoclast markers compared with normal synovium. These findings were the same in tissue samples from mice with collagen-induced arthritis (CIA) and normal healthy controls. RNA sequencing analysis revealed that nesfatin-1 increased levels of bone morphogenetic protein-5 (BMP5) expression via JAK/STAT signaling in RA synovial fibroblasts. Finally, we found that nesfatin-1 short hairpin RNA reduced BMP5 and osteoclast formation in CIA mice. These findings provide new insights into the pathogenesis of RA.

Increased ß1-adrenergic receptor antibody confers a vulnerable substrate for atrial fibrillation via mediating Ca2+ mishandling and atrial fibrosis in active immunization rabbit models.

BACKGROUND: Autoimmune disorder is the emerging mechanism of atrial fibrillation (AF). The ß1-adrenergic receptor antibody (ß1-AAb) is associated with AF progress. Our study aims to investigate whether ß1-AAbs involves in atrial vulnerable substrate by mediating Ca2+ mishandling and atrial fibrosis in autoimmune associated AF. METHODS: Active immunization models were established via subcutaneous injection of the second extracellular loop (ECL2) peptide for ß1 adrenergic receptor (ß1AR). Invasive electrophysiologic study and ex vivo optical mapping were used to evaluate the changed electrophysiology parameters and calcium handling properties. Phospho-proteomics combined with molecular biology assay were performed to identify the potential mechanisms of remodeled atrial substrate elicited by ß1-AAbs. Exogenous ß1-AAbs were used to induce the cellular phenotypes of HL-1 cells and atrial fibroblasts to AF propensity. RESULTS: ß1-AAbs aggravated the atrial electrical instability and atrial fibrosis. Bisoprolol alleviated the alterations of action potential duration (APD), Ca2+ transient duration (CaD), and conduction heterogeneity challenged by ß1-AAbs. ß1-AAbs prolonged calcium transient refractoriness and promoted arrhythmogenic atrial alternans and spatially discordant alternans, which were partly counteracted through blocking ß1AR. Its underlying mechanisms are related to ß1AR-drived CaMKII/RyR2 activation of atrial cardiomyocytes and the myofibroblasts phenotype formation of fibroblasts. CONCLUSION: Suppressing ß1-AAbs effectively protects the atrial vulnerable substrate by ameliorating intracellular Ca2+ mishandling and atrial fibrosis, preventing the process of the autoimmune associated AF.

Nesfatin-1 Stimulates CCL2-dependent Monocyte Migration And M1 Macrophage Polarization: Implications For Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA) is a prototypic inflammatory disease, characterized by the infiltration of proinflammatory cytokines into the joint synovium and the migration of mononuclear cells into inflammatory sites. The adipokine nesfatin-1 is linked to inflammatory events in various diseases, although its role in RA pathology is uncertain. Analysis of the Gene Expression Omnibus GSE55235 dataset revealed high levels of expression of the adipokine nesfatin-1 in human RA synovial tissue. Similarly, our human synovial tissue samples exhibited increasing levels of nesfatin-1 expression and Ccl2 mRNA expression. Nesfatin-1-induced stimulation of CCL2 expression and monocyte migration involved the MEK/ERK, p38, and NF-κB signaling pathways. Notably, nesfatin-1-induced increases in CCL2 expression favored M1 macrophage polarization, which increased the expression of proinflammatory cytokines IL-1ß, IL-6, and TNF-α. Finally, nesfatin-1 shRNA ameliorated the severity of inflammatory disease and reduced levels of M1 macrophage expression in CIA mice. Our studies confirm that nesfatin-1 appears to be worth targeting in RA treatment.

IL-17 Facilitates VCAM-1 Production and Monocyte Adhesion in Osteoarthritis Synovial Fibroblasts by Suppressing miR-5701 Synthesis.

Osteoarthritis (OA) is characterized by the infiltration and adhesion of monocytes into the inflamed joint synovium. Interleukin (IL)-17 is a critical inflammatory mediator that participates in the progression of OA, although the mechanisms linking IL-17 and monocyte infiltration are not well understood. Our analysis of synovial tissue samples retrieved from the Gene Expression Omnibus (GEO) dataset exhibited higher monocyte marker (CD11b) and vascular cell adhesion molecule 1 (VCAM-1) levels in OA samples than in normal, healthy samples. The stimulation of human OA synovial fibroblasts (OASFs) with IL-17 increased VCAM-1 production and subsequently enhanced monocyte adhesion. IL-17 affected VCAM-1-dependent monocyte adhesion by reducing miR-5701 expression through the protein kinase C (PKC)-α and c-Jun N-terminal kinase (JNK) signaling cascades. Our findings improve our understanding about the effect of IL-17 on OA progression and, in particular, VCAM-1 production and monocyte adhesion, which may help with the design of more effective OA treatments.

Double sperm cloning (DSC) is a promising strategy in mammalian genetic engineering and stem cell research.

Embryonic stem cells (ESCs) derived from somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) are promising tools for meeting the personalized requirements of regenerative medicine. However, some obstacles need to be overcome before clinical trials can be undertaken. First, donor cells vary, and the reprogramming procedures are diverse, so standardization is a great obstacle regarding SCNT and iPSCs. Second, somatic cells derived from a patient may carry mitochondrial DNA mutations and exhibit telomere instability with aging or disease, and SCNT-ESCs and iPSCs retain the epigenetic memory or epigenetic modification errors. Third, reprogramming efficiency has remained low. Therefore, in addition to improving their success rate, other alternatives for producing ESCs should be explored. Producing androgenetic diploid embryos could be an outstanding strategy; androgenic diploid embryos are produced through double sperm cloning (DSC), in which two capacitated sperms (XY or XX, sorted by flow cytometer) are injected into a denucleated oocyte by intracytoplasmic sperm injection (ICSI) to reconstruct embryo and derive DSC-ESCs. This process could avoid some potential issues, such as mitochondrial interference, telomere shortening, and somatic epigenetic memory, all of which accompany somatic donor cells. Oocytes are naturally activated by sperm, which is unlike the artificial activation that occurs in SCNT. The procedure is simple and practical and can be easily standardized. In addition, DSC-ESCs can overcome ethical concerns and resolve immunological response matching with sperm providers. Certainly, some challenges must be faced regarding imprinted genes, epigenetics, X chromosome inactivation, and dosage compensation. In mice, DSC-ESCs have been produced and have shown excellent differentiation ability. Therefore, the many advantages of DSC make the study of this process worthwhile for regenerative medicine and animal breeding.

Lead induces apoptosis in mouse TM3 Leydig cells through the Fas/FasL death receptor pathway.

The study was aimed to investigate the effect of Pb toxicity on mouse Leydig cells and its molecular mechanism. The TM3 cells were cultured in vitro and exposed to Pb at different concentrations for 24h. The effects of Pb on cell proliferation and apoptosis were analyzed with MTT and Annexin V-FITC/PI via flow cytometry, respectively. Expression levels of Fas, Fas-L and caspase-8 in TM3 cells were determined by western blot. As well as the inhibitory effect of the caspase-8 inhibitor Z-IETD-FMK on cell apoptosis. We found that Pb treatment significantly decreased the cellar viability (P<0.05), increased the apoptosis (P<0.01) and the Fas, FasL, and caspase-8 expression levels in Pb-treated cells as compared to the control cells (P<0.05 or P<0.01). Furthermore, the caspase-8 inhibitor effectively block the Pb-induced cell apoptosis. Taken together, our data suggest that Pb-induced TM3 cell toxic effect may involve in the Fas/FasL death receptor signaling pathway.

Effect of LA on the Growth and Development of the Main Organs in Female Mice.

Effects of lead acetate (LA) on the growth and development of major organs in female mice were studied. Female mice were divided randomly into four treatment groups and one control group. In treatment groups, mice were injected with different concentrations of LA solution every 2 days; whereas control-group mice received equal volumes of sterile normal saline. Body weight (BW) and symptoms were recorded every 2 days. After LA exposure, mice were executed by cervical dislocation and main organs (heart, liver, spleen, lung, kidney) collected for evaluation of morphologic and histologic changes. LA could greatly affect increases in BW, and BW decreased with increasing dose and time of exposure to LA. Compared with the control group, organ coefficients in treatment groups were of the order kidney and spleen > liver and lung > heart and demonstrated obvious dose-time effects. LA exposure could damage the heart, liver, spleen, lung, and kidney. Damage to the kidney and spleen was the most severe, followed by that to the liver, heart, and lung. Damage was aggravated with increasing doses and exposure time to LA in an obvious dose-time relationship; when LA dose was ≥20 mg/kg, the growth and development of mice were obviously inhibited. These results suggest that long-term exposure to low-dose LA can result in universal pathologic damage to mouse organs and that severity is dependent on the dose and duration of LA exposure.

Isolation and molecular and phylogenetic analyses of encephalomyocarditis virus from wild boar in central China.

Encephalomyocarditis virus (EMCV) can infect many host species and cause acute myocarditis and respiratory failure in piglets, reproductive failure in pregnant sows. In this study, an EMCV strain, designated JZ1202, was isolated from semi-captive wild boars that presented with acute myocarditis and sudden death in central China. It was identified by hemagglutination inhibition (HI) assay, reverse transcription polymerase chain reaction (RT-PCR) and genome sequencing. The subsequent results showed that the virus could produce a specific cytopathic effect on BHK cells and could cause clinical symptoms and pathological changes in mice. Complete genome sequencing and multiple sequence alignment indicated that JZ1202 strain was 81.3%-99.9% identical with other isolates worldwide. Phylogenetic analysis of the whole genome, ORF, VP3/VP1 and 3D genes using neighbor-joining method revealed that JZ1202 isolate was grouped into lineage 1. The results of this study confirmed that an EMCV strain JZ 1202 isolated from wild boar in central China was fatal to mice and provided new epidemiologic data on EMCV in China.

[Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks].

Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark.

Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides.

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4⁺ T cells and IFN-γ-producing CD8⁺ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3⁺, CD3⁺CD4⁺CD8⁻, and CD3⁺CD4⁻CD8⁺ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

Hypovitaminosis A coupled to secondary bacterial infection in beef cattle.

BACKGROUND: Vitamin A is essential for normal growth, development, reproduction, cell proliferation, cell differentiation, immune function and vision. Hypovitaminosis A can lead to a series of pathological damage in animals. This report describes the case of hypovitaminosis A associated with secondary complications in calves. CASE PRESENTATION: From February to March in 2011, 2-and 3-month old beef calves presented with decreased eyesight, apparent blindness and persistent diarrhea occurred in a cattle farm of Hubei province, China. Based on history inspection and clinical observation, we made a tentative diagnosis of hypovitaminosis A. The disease was confirmed as a congenital vitamin A deficiency by determination of the concentrations of vitamin A in serum and feed samples. Furthermore, pathological and microbiological examination showed that the disease was associated with pathogenic Escherichia coli (E. coli) infection and mucosal barriers damage in intestines. The corresponding treatments were taken immediately, and the disease was finally under control for a month. CONCLUSIONS: To our knowledge, this is the first report of hypovitaminosis A coupled to secondary infection of E. coli in beef cattle, advancing our knowledge of how vitamin A affects infection and immunity in animals. This study could also be contributed to scientific diagnosis and treatments of complex hypovitaminosis A in cattle.