Constructing Linear-Oriented Pre-Vascularized Human Spinal Cord Tissues for Spinal Cord Injury Repair.

Repairing spinal cord injury (SCI) is a global medical challenge lacking effective clinical treatment. Developing human-engineered spinal cord tissues that can replenish lost cells and restore a regenerative microenvironment offers promising potential for SCI therapy. However, creating vascularized human spinal cord-like tissues (VSCT) that mimic the diverse cell types and longitudinal parallel structural features of spinal cord tissues remains a significant hurdle. In the present study, VSCTs are engineered using embryonic human spinal cord-derived neural and endothelial cells on linear-ordered collagen scaffolds (LOCS). Studies have shown that astrocytes and endothelial cells align along the scaffolds in VSCT, supporting axon extension from various human neurons myelinated by oligodendrocytes. After transplantation into SCI rats, VSCT survives at the injury sites and promotes endogenous neural regeneration and vascularization, ultimately reducing scarring and enhancing behavioral functional recovery. It suggests that pre-vascularization of engineered spinal cord tissues is beneficial for SCI treatment and highlights the important role of exogenous endothelial cells in tissue engineering.

Hedgehog pathway negatively regulated depleted uranium-induced nephrotoxicity.

Depleted uranium (DU) retains the radiological toxicities, which accumulates preferentially in the kidneys. Hedgehog (Hh) pathway plays a critical role in tissue injury. However, the role of Hh in DU-induced nephrotoxicity was still unclear. This study was carried out to investigate the effect of Gli2, which was an important transcription effector of Hh signaling, on DU induced nephrotoxicity. To clarify it, CK19 positive tubular epithelial cells specific Gli2 conditional knockout (KO) mice model was exposed to DU, and then histopathological damage and Hh signaling pathway activation was analyzed. Moreover, HEK-293 T cells were exposed to DU with Gant61 or Gli2 overexpression, and cytotoxicity of DU as analyzed. Results showed that DU caused nephrotoxicity accompanied by activation of Hh signaling pathway. Meanwhile, genetic KO of Gli2 reduced DU-induced nephrotoxicity by normalizing biochemical indicators and reducing Hh pathway activation. Pharmacologic inhibition of Gli1/2 by Gant61 reduced DU induced cytotoxicity by inhibiting apoptosis, ROS formation and Hh pathway activation. However, overexpression of Gli2 aggravated DU-induced cytotoxicity by increasing the levels of apoptosis and ROS formation. Taken together, these results revealed that Hh signaling negatively regulated DU-inducted nephrotoxicity, and that inhibition of Gli2 might serve as a promising nephroprotective target for DU-induced kidney injury.

Soft tissue infection and osteomyelitis caused by Mycobacterium farcinogenes after heart surgery: Case report and literature review of human cases.

Mycobacterium farcinogenes (M. farcinogenes) is rapidly growing mycobacterium, belonging to non-tuberculous mycobacterial (NTM). M. farcinogenes is an exceedingly rare causative agent of human infection. Only seven cases with M. farcinogenes infections in humans were reported. This is a case of soft tissue infection and osteomyelitis caused by M. farcinogenes after heart surgery. Microbial identification was achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The clinical outcome was favorable after surgical debridement and 4-month antibiotics treatment. We also provide a comprehensive literature review on this disease.

Advances in biomaterial-based cardiac organoids.

Cardiovascular disease (CVD) is one of the important causes of death worldwide. The incidence and mortality rates are increasing annually with the intensification of social aging. The efficacy of drug therapy is limited in individuals suffering from severe heart failure due to the inability of myocardial cells to undergo regeneration and the challenging nature of cardiac tissue repair following injury. Consequently, surgical transplantation stands as the most efficient approach for treatment. Nevertheless, the shortage of donors and the considerable number of heart failure patients worldwide, estimated at 26 million, results in an alarming treatment deficit, with only around 5000 heart transplants feasible annually. The existing major alternatives, such as mechanical or xenogeneic hearts, have significant flaws, such as high cost and rejection, and are challenging to implement for large-scale, long-term use. An organoid is a three-dimensional (3D) cell tissue that mimics the characteristics of an organ. The critical application has been rated in annual biotechnology by authoritative journals, such as Science and Cell. Related industries have achieved rapid growth in recent years. Based on this technology, cardiac organoids are expected to pave the way for viable heart repair and treatment and play an essential role in pathological research, drug screening, and other areas. This review centers on the examination of biomaterials employed in cardiac repair, strategies employed for the reconstruction of cardiac structure and function, clinical investigations pertaining to cardiac repair, and the prospective applications of cardiac organoids. From basic research to clinical practice, the current status, latest progress, challenges, and prospects of biomaterial-based cardiac repair are summarized and discussed, providing a reference for future exploration and development of cardiac regeneration strategies.

Sodium butyrate facilitates CRHR2 expression to alleviate HPA axis hyperactivity in autism-like rats induced by prenatal lipopolysaccharides through histone deacetylase inhibition.

Short-chain fatty acids (SCFAs, especially butyric acid) have been demonstrated to play a promising role in the development of autism spectrum disorders (ASD). Recently, the hypothalamic-pituitary-adrenal (HPA) axis is also suggested to increase the risk of ASD. However, the mechanism underlying SCFAs and HPA axis in ASD development remains unknown. Here, we show that children with ASD exhibited lower SCFA concentrations and higher cortisol levels, which were recaptured in prenatal lipopolysaccharide (LPS)-exposed rat model of ASD. These offspring also showed decreased SCFA-producing bacteria and histone acetylation activity as well as impaired corticotropin-releasing hormone receptor 2 (CRHR2) expression. Sodium butyrate (NaB), which can act as histone deacetylases inhibitors, significantly increased histone acetylation at the CRHR2 promoter in vitro and normalized the corticosterone as well as CRHR2 expression level in vivo. Behavioral assays indicated ameliorative effects of NaB on anxiety and social deficit in LPS-exposed offspring. Our results imply that NaB treatment can improve ASD-like symptoms via epigenetic regulation of the HPA axis in offspring; thus, it may provide new insight into the SCFA treatment of neurodevelopmental disorders like ASD. IMPORTANCE Growing evidence suggests that microbiota can affect brain function and behavior through the "microbiome-gut-brain'' axis, but its mechanism remains poorly understood. Here, we show that both children with autism and LPS-exposed rat model of autism exhibited lower SCFA concentrations and overactivation of HPA axis. SCFA-producing bacteria, Lactobacillus, might be the key differential microbiota between the control and LPS-exposed offspring. Interestingly, NaB treatment contributed to the regulation of HPA axis (such as corticosterone as well as CRHR2) and improvement of anxiety and social deficit behaviors in LPS-exposed offspring. The potential underlying mechanism of the ameliorative effect of NaB may be mediated via increasing histone acetylation to the CRHR2 promoter. These results enhance our understanding of the relationship between the SCFAs and the HPA axis in the development of ASD. And gut microbiota-derived SCFAs may serve as a potential therapeutic agent to neurodevelopmental disorders like ASD.

Role of NF-κB signaling pathway in hexavalent chromium-induced hepatotoxicity.

Hexavalent chromium Cr (VI) is a primary human carcinogen with damaging toxic effects on multiple organs. Cr (VI) exposure can induce hepatotoxicity through oxidative stress, but its exact mechanism of action was still unclear. In our study, a model of acute Cr (VI) induced liver injury was established by exposing mice to different concentrations (0, 40, 80, and 160 mg/kg) of Cr (VI); RNA-seq was used to characterize changes in liver tissue transcriptome of C57BL/6 mice after exposing to 160 mg/kg Bw of Cr (VI). Changes in liver tissue structures, proteins, and genes were observed by hematoxylin and eosin (H&E), western blot, immunohistochemistry and RT-PCR. After Cr (VI) exposure, abnormal liver tissue structure, hepatocyte injury, and hepatic inflammatory response were observed in mice in a dose-dependent manner. RNA-seq transcriptome results indicated that oxidative stress, apoptosis, and inflammatory response pathways were increased after Cr (VI) exposure; KEGG pathway analysis found that activation of NF-κB signaling pathway was significantly upregulated. Consistent with the RNA-seq results, immunohistochemistry showed that Cr (VI) exposure resulted in infiltrating of Kupffer cells and neutrophils, increasing expression of inflammatory factors (TNF-α, IL-6, IL-1ß), and activating of NF-κB signaling pathways (p-IKKα/ß and p-p65). However, ROS inhibitor, N-acetyl-L-cysteine (NAC), could reduce infiltration of Kupffer cells and neutrophils and expression of inflammatory factors. Besides, NAC could inhibit NF-κB signaling pathway activation, and alleviate Cr (VI)-induced liver tissue damage. Our findings strongly suggested that inhibition of ROS by NAC might help in the development of new strategies for Cr (VI)-associated liver fibrosis. Our findings revealed for the first time that Cr (VI) induced liver tissue damage through the inflammatory response mediated by the NF-κB signaling pathway, and inhibition of ROS by NAC might help in the development of new strategies for Cr (VI)-associated hepatotoxicity.

N-acetyl-L-cysteine attenuated the toxicity of ZIF-8 on EA.hy926 endothelial cells by wnt/ß-catenin pathway.

As kinds of porous crystalline compounds, zeolitic imidazolate frameworks (ZIFs) have been developed quickly and attracted considerable attention for use in nano drug delivery systems, which raised concerns about cardiovascular disorders. At the present, the cytotoxic mechanism of ZIFs in cardiovascular disorders was still unclear. Our experiment explored the toxicity of ZIF-8, a typical kind of ZIFs, on human EA.hy926 vascular endothelial cells. The cell viability, ROS formation, apoptosis level, inflammatory response level, wound healing ability and atherosclerosis-related indicators of EA.hy926 endothelial cells were analyzed after ZIF-8 treatment. Meanwhile, we evaluated the ability of antioxidant N-Acetyl-L-cysteine (NAC) to attenuate the toxicity of ZIF-8 on EA.hy926 endothelial cells. As results, NAC attenuated ROS formation, cell apoptosis, LDH formation and endothelial dysfunction caused by ZIF-8. As the Wnt/ß-catenin pathway was involved in endothelial cell dysfunction, we also studied the expression level of ß-catenin and LEF1 in ZIF-8 and/or NAC treated EA.hy926 cells. As expected, ZIF-8 increased the protein expressions of ß-catenin and LEF1in the IC50 group, which was significantly inhibited by co-treatment with NAC. Taken together, this study could help improve our understanding about the mechanism of ZIF-8-induced endothelial cells injury and NAC had therapeutic potential in preventing ZIF-8-associated endothelial dysfunction by wnt/ß-catenin pathway.

A Continuum Robotic Cannula With Tip Following Capability and Distal Dexterity for Intracerebral Hemorrhage Evacuation.

OBJECTIVE: This paper aims to investigate a new continuum robot design and its motion implementation methods appropriate for a minimally invasive intracerebral hemorrhage (ICH) evacuation. METHODS: We propose a continuum robotic cannula, consisting of a precurved body and a 2-degree-of-freedom (DoF) flexible tip, monolithically fabricated. Kinematic model with cable elongation model, and a dedicated design optimization and motion planning algorithm were developed to enable the follow-the-leader (FTL) motion of the cannula. A task-dependent Jacobian-based closed loop control was also designed to track the cannula motion during the insertion and its independent tip motion. RESULTS: Comprehensive experiments were conducted to verify the kinematic model and submillimeter motion coupling between the cannula precurved body and its flexible tip. The cannula was also capable of achieving FTL motion within around 2.5 mm shape deviation and control performance within submillimeter errors. It was finally demonstrated to be capable of the nonlinear insertion and tip manipulation in the brain phantom. CONCLUSION: The new cannula design, together with the proposed algorithms, provides the unique ability to access ICH in a nonlinear trajectory and dexterous tip motion. SIGNIFICANCE: These motion capabilities of the robot in such a slender form factor will lead to more complete ICH evacuation and reduced trauma to the healthy brain tissues.

Hexavalent chromium induces hepatocyte apoptosis via regulation of apoptosis signal-regulating kinase 1/c-Jun amino-terminal kinase signaling.

With the spread of hexavalent chromium (Cr(VI)) contamination, Cr(VI)-induced hepatotoxicity has attracted increasing attention in recent years. To date, however, the exact mechanism of Cr(VI) toxicity remains unclear. In this study, we investigated the role of apoptosis signal-regulating kinase 1 (ASK1)/c-Jun amino-terminal kinase (JNK) in Cr(VI)-induced hepatic toxicity and the possible related mechanisms. AML-12 hepatocyte cell-lines were treated with 0, 1, 4, and 16 µmol/Lof Cr(VI) with or without GS-444271 (an ASK1 inhibitor). Adult male mice were administered with 0, 2, 8, and 32 mg/kg body mass (BM)/day of Cr(VI) for 5 days. The level of hepatocyte apoptosis/proliferation, generation of reactive oxygen species (ROS), and expression levels of mRNAs and proteins related to ASK1/JNK and nuclear factor-E2-related factor 2 (Nrf2) signaling were assessed. Results showed that high Cr(VI) exposure induced hepatocyte apoptosis and liver injury by generation of ROS and down-regulation of Nrf2 signaling. In addition, ASK1/JNK signaling activity was upregulated in the Cr(VI)-treated group. Furthermore, GS-444217 treatment significantly rescued Cr(VI)-induced hepatocyte apoptosis and liver dysfunction in vitro and in vivo by down-regulation of ASK1/JNK signaling. Thus, ASK1/JNK signaling appears to play an important role in Cr(VI)-induced hepatocyte apoptosis and liver injury. This study should help improve our understanding of the mechanism of Cr(VI)-induced liver injury and provide support for future investigations on liver disease therapy.

Yap signalling regulates ductular reactions in mice with CRISPR/Cas9-induced glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in the glucose-6-phosphatase (G6Pase, G6pc) enzyme, which catalyses the final step of gluconeogenesis and glycogenolysis. Accumulation of G6pc can lead to an increase in glycogen and development of fatty liver. Ductular reactions refer to the proliferation of cholangiocytes and hepatic progenitors, which worsen fatty liver progress. To date, however, ductular reactions in GSD-Ia remain poorly understood. Here, we studied the development and potential underlying mechanism of ductular reactions in GSD-Ia in mice. We first generated GSD-Ia mice using CRISPR/Cas9 to target the exon 3 region of the G6pc gene. The typical GSD-Ia phenotype in G6pc -/- mice was then analysed using biochemical and histological assays. Ductular reactions in G6pc -/- mice were tested based on the expression of cholangiocytic markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM). Yes-associated protein 1 (Yap) signalling activity was measured using western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Verteporfin was administered to the G6pc -/- mice to inhibit Yap signalling. The CRISPR/Cas9 system efficiently generated G6pc -/- mice, which exhibited typical GSD-Ia characteristics, including retarded growth, hypoglycaemia, and fatty liver disease. In addition, CK19- and EpCAM-positive cells as well as Yap signalling activity were increased in the livers of G6pc -/- mice. However, verteporfin treatment ameliorated ductular reactions and decreased Yap signalling activity. This study not only improves our understanding of GSD-Ia pathophysiology, but also highlights the potential of novel therapeutic approaches for GSD-Ia such as drug targeting of ductular reactions.

Fecal microbiota from children with vitamin A deficiency impair colonic barrier function in germ-free mice: The possible role of alterative bile acid metabolites.

OBJECTIVE: This study explores the effects of fecal microbiota from children with vitamin A (VA) deficiency on colonic mucosal barrier function. METHODS: The composition of gut microbes was identified in children with different VA levels, then feces from children with normal VA or VA deficiency was collected separately and transplanted into germ-free (GF) mice, respectively. Three weeks after transplantation, the colon morphology, colonic tight junction proteins, gut microbes, and metabolites were evaluated. RESULTS: In children, Bifidobacterium and Bacteroides were positively correlated with VA levels. Colonization of VA deficiency fecal microbiota markedly impaired colonic development in GF mice, down-regulated colonic tight junction-related proteins occludin and claudin-1, and reduced immunoglobulin A secretion. Furthermore, fecal microbiota transplantation with different VA levels altered composition of gut microbes and bile acid metabolism pathways in GF mice. CONCLUSION: These data suggest that fecal microbiota from children with VA deficiency attenuates colonic barrier function in GF mice, which may be achieved by changing the bile acid metabolic pathways.

ROCK inhibitor attenuates carbon blacks-induced pulmonary fibrosis in mice via Rho/ROCK/NF-kappa B pathway.

Exposure to carbon blacks (CBs) has been associated with the progression of pulmonary fibrosis, whereas the mechanism is still not clear. We therefore aimed to investigate the effect of RhoA/ROCK pathway on pulmonary fibrosis caused by CBs exposure. Western blot analysis indicated that CBs could promote the activation of RhoA/ROCK pathway and phosphorylation of p65 and IκBα in mice lung. However, ROCK inhibitor Y-27632 could attenuate phosphorylation levels of p65 and IκBα and restore histopathological changes of the lung tissue. Then, we evaluated the effect of RhoA/ROCK pathway on pulmonary fibrosis by detecting the expression levels of α-SMA, vimentin, and Collagen type-I (Col-I), which could be partly inhibited by Y-27632. It was assumed that inhibition of ROCK could be a promising therapeutic candidate for CBs-induced pulmonary fibrosis, which possibly through the blockage of RhoA/ROCK/NF-κB pathway.

Fecal Microbiome Transplantation from Children with Autism Spectrum Disorder Modulates Tryptophan and Serotonergic Synapse Metabolism and Induces Altered Behaviors in Germ-Free Mice.

To determine the relationship of the gut microbiota and its metabolites with autism spectrum disorder (ASD)-like behaviors and preliminarily explore the potential molecular mechanisms, the fecal microbiota from donors with ASD and typically developing (TD) donors were transferred into germ-free (GF) mice to obtain ASD-FMT mice and TD-FMT mice, respectively. Behavioral tests were conducted on these mice after 3 weeks. 16S rRNA gene sequencing of the cecal contents and untargeted metabolomic analysis of the cecum, serum, and prefrontal cortex were performed. Untargeted metabolomics was also used to analyze fecal samples of TD and ASD children. Western blotting detected the protein expression levels of tryptophan hydroxylase 1 (TPH1), serotonin transporter (SERT), and serotonin 1A receptor (5-HT1AR) in the colon and TPH2, SERT, and 5-HT1AR in the prefrontal cortex of mice. ASD-FMT mice showed ASD-like behavior and a microbial community structure different from that of TD-FMT mice. Tryptophan and serotonin metabolisms were altered in both ASD and TD children and ASD-FMT and TD-FMT mice. Some microbiota may be related to tryptophan and serotonin metabolism. Compared with TD-FMT mice, ASD-FMT mice showed low SERT and 5-HT1AR and high TPH1 expression levels in the colon. In the prefrontal cortex, the expression levels of TPH2 and SERT were increased in the ASD-FMT group relative to the TD-FMT group. Therefore, the fecal microbiome of ASD children can lead to ASD-like behaviors, different microbial community structures, and altered tryptophan and serotonin metabolism in GF mice. These changes may be related to changes in some key proteins involved in the synthesis and transport of serotonin.IMPORTANCE The relationship between the gut microbiota and ASD is not yet fully understood. Numerous studies have focused on the differences in intestinal microbial and metabolism profiles between TD and ASD children. However, it is still not clear if these microbes and metabolites cause the development of ASD symptoms. Here, we collected fecal samples from TD and ASD children, transplanted them into GF mice, and found that the fecal microbiome of ASD children can lead to ASD-like behaviors, different microbial community structures, and altered tryptophan and serotonin metabolism in GF mice. We also demonstrated that tryptophan and serotonin metabolism was also altered in ASD and TD children. Together, these findings confirm that the microbiome from children with ASD may lead to ASD-like behavior of GF mice through metabolites, especially tryptophan and serotonin metabolism.

Critical Role of TLR4 on the Microglia Activation Induced by Maternal LPS Exposure Leading to ASD-Like Behavior of Offspring.

Objective: To investigate the role of TLR4 on the microglia activation in the pre-frontal cortex, which leads to autism-like behavior of the offspring induced by maternal lipopolysaccharide (LPS) exposure. Methods: Pregnant TLR4-/- (knockout, KO) and WT (wild type, WT) dams were intraperitoneally injected with LPS or PBS, respectively. The levels of TNFα, IL-1ß, and IL-6 in the maternal serum and fetal brain were assessed with ELISA following LPS exposure. The gestation period, litter size and weight of the offspring were evaluated. Three-chamber sociability test, open field test and olfactory habituation/dishabituation test were used to assess the offspring's autism-like behavior at 7 weeks of age. Western blotting was performed to examine the levels of TLR4, Phospho-NFκB p65, IKKα, IBA-1, iNOS, Arg-1, C3, CR3A, NMDAR2A, and Syn-1 expression in the pre-frontal cortex. The morphological changes in the microglia, the distribution and expression of TLR4 were observed by immunofluorescence staining. Golgi-Cox staining was conducted to evaluate the dendritic length and spine density of the neurons in 2-week-old offspring. Results: Maternal LPS stimulation increased serum TNFα and IL-6, as well as fetal brain TNFα in the WT mice. The litter size and the weight of the WT offspring were significantly reduced following maternal LPS treatment. LPS-treated WT offspring had lower social and self-exploration behavior, and greater anxiety and repetitive behaviors. The protein expression levels of TLR4 signaling pathways, including TLR4, Phospho-NFκB p65, IKKα, and IBA-1, iNOS expression were increased in the LPS-treated WT offspring, whereas Arg-1 was decreased. Maternal LPS treatment resulted in the significant reduction in the levels of the synaptic pruning-related proteins, C3 and CR3A. Moreover, the neuronal dendritic length and spine density, as well as the expression levels of the synaptic plasticity-related proteins, NMDAR2A and Syn-1 were reduced in the WT offspring; however, gestational LPS exposure had no effect on the TLR4-/- offspring. Conclusion: Activation of TLR4 signaling pathway following maternal LPS exposure induced the abnormal activation of microglia, which in turn was involved in excessive synaptic pruning to decrease synaptic plasticity in the offspring. This may be one of the reasons for the autism-like behavior in the offspring mice.

Gli2-regulated activation of hepatic stellate cells and liver fibrosis by TGF-ß signaling.

The Hedgehog (Hh) signaling pathway is correlated with hepatic stellate cells (HSCs) activation and liver fibrosis. Gli2 is a key transcription effector of Hh signaling. However, the role of Gli2 in HSC-mediated liver fibrosis progression is largely unknown. In the present study, we investigated the effect of Gli2 on liver fibrogenesis and its possible mechanism using conditional knockout (cKO) Gli2 mice and HSC models. Wild-type (WT) and GFAP-CreERT;Gli2flox/flox male mice were exposed to CCl4 for 1 mo to induce liver fibrosis. Primary HSCs were isolated from mice and the transition of HSCs into a myofibroblastic phenotype was evaluated. Livers from mice underwent histological, immunohistochemical, and immunofluorescence analyses. The expression levels of proteins and genes were evaluated by Western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RNA-seq was used to screen differentially expressed genes. Results showed that CCl4 treatment induced liver fibrosis, promoted HSCs activation and proliferation, and upregulated Hh signaling activity. The cKO of Gli2 in GFAP-CreERT;Gli2flox/flox mice decreased liver fibrosis as well as HSC activation and proliferation. In vitro studies showed that KO of Gli2 in HSCs blocked cell proliferation and activation by decrease of cyclin D1/D2 expression. The RNA-seq results revealed that the expression levels TGF-ß1 ligands were downregulated in Gli2 KO HSCs. Furthermore, overexpression of Gli2 rescued proliferation and activation of HSCs by upregulation of TGF-ß signaling activity. Our data demonstrated that Gli2 regulated HSC activation and liver fibrosis by TGF-ß signaling, thus providing support for future Gli2-based investigations of liver fibrosis therapy.NEW & NOTEWORTHYGli2 is a key transcription effector of Hh signaling. We found that Hh/Gli2 signaling activity was upregulated in CCl4-induced liver fibrosis. Conditional deletion of the Gli2 gene in HSCs ameliorated CCl4-induced liver fibrosis and HSCs activation. Moreover, Gli2 promoted activation of HSCs through upregulation of cyclin expression and TGF-ß signaling activity. Thus, our data provide strong support for future investigations on Gli2 inhibition to slow liver fibrosis progression in humans.

Shh-Yap signaling controls hepatic ductular reactions in CCl4 -induced liver injury.

Carbon tetrachloride (CCl4 ) exposure can induce hepatic ductular reactions. To date, however, the related mechanism remains largely unknown. Sonic hedgehog (Shh) and Yes-associated protein (Yap) signaling are correlated with liver injury and regeneration. Herein, we investigated the role of Shh and Yap signaling in the fate of ductular reaction cells in CCl4 -treated livers and the possible mechanisms. Wild-type and Shh-EGFP-Cre male mice were exposed to CCl4 (2 mL/kg), and then treated with or without the Shh signaling inhibitor Gant61. The level of liver injury, proliferation of ductular reaction cells, and expression levels of mRNA and protein related to the Shh and Yap signaling components were assessed. Results showed that CCl4 treatment induced liver injury and promoted activation and proliferation of ductular reaction cells. In addition, CCl4 induced the expression of Shh ligands in hepatocytes, accompanied by activation of Shh and Yap1 signaling in the liver. Furthermore, administration of Gant61 ameliorated liver regeneration, inhibited hepatic ductular reactions, and decreased Shh and Yap1 signaling activity. Thus, Shh-Yap1 signaling appears to play an integral role in the proliferation of ductular reaction cells in CCl4 -induced liver injury. This study should improve our understanding of the mechanism of CCl4 -induced liver injury and ductular reactions and provide support for future investigations on liver disease therapy.

Establishment of drug screening in human embryonic stem cells based on a high-content screening system.

High-content screening (HCS) systems can be used for high-throughput screening of drugs in human embryonic stem cells (hESCs). However, hESCs require immunofluorescence staining with stemness markers (e.g., Oct-4) prior to HCS, which can be time consuming and labor intensive. In this study, we employed transgenic hESCs with enhanced green fluorescent protein driven by stemness gene Oct-4 promoter (Oct-4-EGFP-H9), in which the colony area and relative green fluorescence area inferred a state of hESC proliferation and stemness, respectively. The Oct-4-EGFP-H9 transgenic hESCs were cultured in mTeSR medium with different concentrations of 5-Fluorouracil (5-FU), vitamin C (VC), or retinoic acid (RA) for 5-7 days, followed by repeated imaging using the HCS system. Finally, the hESC colony area and green fluorescence area were calculated. Results showed that 5-FU treatment markedly reduced colony area in a dose-dependent manner, whereas VC and RA treatments did not. MTT assay and flow cytometry indicated that 5-FU inhibited the proliferation of hESCs significantly, verifying reliability of the data from the HCS system based on colony area analysis. The green fluorescence to total colony area ratio decreased with RA treatment, suggesting that RA significantly promoted differentiation, whereas 5-FU and VC had almost no effect, as verified by quantitative real-time polymerase chain reaction and western blot analysis. In conclusion, our study established a rapid and efficient drug screening system without the requirement of staining based on HCS.

PM2.5 collecting in a tire manufacturing plant affects epithelial differentiation of human umbilical cord derived mesenchymal stem cells by Wnt/ß-catenin pathway.

Mesenchymal stem cells (MSCs) can differentiate into pulmonary epithelial cells by Wnt/ß-catenin pathway and promote lung repair. However, whether fine particulate matter (PM2.5) could affect Wnt pathway and finally reduce the ability of MSCs to differentiate into epithelial cells is still unknown. This study aimed to investigate whether PM2.5 could inhibit the epithelial differentiation of human umbilical cord-derived MSCs cells (hUCMSCs) and the related underlying mechanism. hUCMSCs were incubated with different concentrations of PM2.5. Then, the cell viability, reactive oxygen species level, and single-cell sphere formation were assessed. The underlying mechanism of PM2.5 in epithelial differentiation of hUCMSCs was further evaluated by co-culturing hUCMSCs with A549 cells. Our results demonstrated that PM2.5 exposures could affect the expressions of ß-catenin and lung epithelial markers (zonula occludens-1 (ZO-1); cytokeratins 5 and 19) in the co-cultured hUCMSCs. The Wnt/ß-catenin pathway is involved in regulating the epithelial differentiation of MSCs. As expected, co-treatment with Wnt3a, which is the activator of the Wnt pathway, attenuated the downregulation of lung epithelial markers (ZO-1; cytokeratins 5 and 19) and paracrine factors (keratinocyte growth factor and hepatocyte growth factor) caused by PM2.5. Altogether, these results demonstrated that PM2.5 could affect the epithelial differentiation of hUCMSCs via the Wnt/ß-catenin pathway.

Hedgehog signaling pathway regulates hexavalent chromium-induced liver fibrosis by activation of hepatic stellate cells.

With the spread of hexavalent chromium [Cr(VI)] contamination, risk of exposure in non-occupational populations is increasing. The liver is the main target organ for Cr(VI) accumulation; however, the effect of long-term Cr(VI) exposure on liver toxicity is largely unknown. In this study, we investigated the effect of chronic Cr(VI) exposure on liver fibrosis and its possible mechanism. Mice were injected with Cr(VI) for two months, and our results showed Cr(VI) treatment caused liver toxicity characterized by liver structure disorganization, liver dysfunction, and antioxidant enzyme system inhibition. The development of liver fibrosis was also found via the emergence of collagen fibril deposition, increased expression of extracellular matrix-related genes, activation of hepatic stellate cells (HSCs) and increase the expression levels of Hedgehog (Hh) signaling pathway-related molecules. To demonstrate the role of Hh signaling in the regulation of Cr(VI)-induced liver fibrosis, genetically modified mice with heterozygous deficiency of Shh (Shh+/-) were used. In the Shh+/- mice, Hh signaling, HSCs activation and liver fibrosis development were all ameliorated. In conclusion, we demonstrated that Cr(VI)-induced liver fibrosis development resulted from Hh pathway-mediated HSCs activation. Our findings strongly suggest that inhibition of Hh pathway may help in the development of new strategies for Cr(VI)-associated liver fibrosis.