Loss of Krüppel-like factor-10 facilitates the development of chemical-induced liver cancer in mice.

BACKGROUND: Krüppel-like factor 10 (KLF10) is involved in a positive feedback loop that regulates transforming growth factor ß (TGFß) signaling, and TGFß plays an important role in the pathogenesis of liver disease. Here, we investigated whether KLF10 deletion affects the development of liver fibrosis and hepatocellular carcinoma (HCC). METHODS: We induced KLF10 deletion in C57BL/6 mice. Liver fibrosis was induced by feeding a diet high in fat and sucrose (high-fat diet [HFD]), whereas HCC was produced by intraperitoneal administration of N-diethylnitrosamine (DEN). An in vitro experiment was performed to evaluate the role of KLF10 in the cancer microenvironment using Hep3B and LX2 cells. An immunohistochemical study of KLF10 expression was performed using human HCC samples from 60 patients who had undergone liver resection. RESULTS: KLF10 deletion resulted in an increased DEN-induced HCC burden with significant upregulation of SMAD2, although loss of KLF10 did not alter HFD-induced liver fibrosis. DEN-treated mice with KLF10 deletion exhibited increased levels of mesenchymal markers (N-cadherin and SNAI2) and tumor metastasis markers (matrix metalloproteinases 2 and 9). KLF10 depletion in Hep3B and LX2 cells using siRNA was associated with increased invasiveness. Compared with co-culture of KLF10-preserved Hep3B cells and KLF10-intact LX2 cells, co-culture of KLF10-preserved Hep3B cells and KLF10-depleted LX2 cells resulted in significantly enhanced invasion. Low KLF10 expression in resected human HCC specimens was associated with poor survival. CONCLUSION: The results of this study suggest that loss of KLF10 facilitates liver cancer development with alteration in TGFß signaling.

Opposing roles of HDAC6 in liver regeneration and hepatocarcinogenesis.

Histone deacetylase 6 (HDAC6), a deacetylase of p53, has emerged as a privileged inhibitory target for cancer therapy because of its deacetylating activity for p53 at K120 and K373/382. However, intricate roles of HDAC6 in hepatocellular carcinogenesis have been suggested by recent evidence, namely that HDAC6 ablation suppresses innate immunity, which plays critical roles in tumor immunosurveillance and antitumor immune responses. Therefore, it is valuable to determine whether HDAC6 ablation inhibits hepatocellular carcinogenesis using in vivo animal models. Here, we firstly showed that HDAC6 ablation increased K320 acetylation of p53, known as pro-survival acetylation, in all tested animal models but did not always increase K120 and K373/382 acetylation of p53, known as pro-apoptotic acetylation. HDAC6 ablation induced cellular senescence in primary MEFs and inhibited cell proliferation in HepG2 cells and liver regeneration after two-thirds partial hepatectomy. However, the genetic ablation of HDAC6 did not inhibit hepatocarcinogenesis, but instead slightly enhanced it in two independent mouse models (DEN + HFD and DEN + TAA). Notably, HDAC6 ablation significantly promoted hepatocarcinogenesis in a multiple DEN treatment hepatocellular carcinoma (HCC) mouse model, mimicking chronic DNA damage in the liver, which correlated with hyperacetylation at K320 of p53 and a decrease in inflammatory cytokines and chemokines. Our data from three independent in vivo animal HCC models emphasize the importance of the complex roles of HDAC6 ablation in hepatocellular carcinogenesis, highlighting its immunosuppressive effects.

Anti-Inflammatory Effect of Phytoncide in an Animal Model of Gastrointestinal Inflammation.

Background: Phytoncide is known to have antimicrobial and anti-inflammatory properties. Purpose: This study was carried out to confirm the anti-inflammatory activity of two types of phytoncide extracts from pinecone waste. Methods: We made two types of animal models to evaluate the efficacy, an indomethacin-induced gastroenteritis rat model and a dextran sulfate sodium-induced colitis mouse model. Result: In the gastroenteritis experiment, the expression of induced-nitric oxide synthase (iNOS), a marker for inflammation, decreased in the phytoncide-supplemented groups, and gastric ulcer development was significantly inhibited (p < 0.05). In the colitis experiment, the shortening of the colon length and the iNOS expression were significantly suppressed in the phytoncide-supplemented group (p < 0.05). Conclusions: Through this study, we confirmed that phytoncide can directly inhibit inflammation in digestive organs. Although further research is needed, we conclude that phytoncide has potential anti-inflammatory properties in the digestive tract and can be developed as a functional agent.

HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I.

RIG-I is a key cytosolic sensor that detects RNA viruses through its C-terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While posttranslational modification has been demonstrated to regulate RIG-I signaling activity, its significance for the sensing of viral RNAs remains unclear. Here, we first show that the RIG-I C-terminal region undergoes deacetylation to regulate its viral RNA-sensing activity and that the HDAC6-mediated deacetylation of RIG-I is critical for viral RNA detection. HDAC6 transiently bound to RIG-I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG-I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild-type mice. These findings underscore the critical role of HDAC6 in the modulation of the RIG-I-mediated antiviral sensing pathway.

A Modified Protocol of Diethylnitrosamine Administration in Mice to Model Hepatocellular Carcinoma.

We aimed to create an animal model for hepatocellular carcinoma (HCC) with a short time, a high survival rate, as well as a high incidence of HCC in both males and females than previously reported. The Diethylnitrosamine (DEN) model has an age-related effect. A single dose of DEN treatment is not enough in young mice up to 50 weeks. The same pattern is shown in an adult with multiple-dose trials whether or not there is some promotion agent. In this study, two-week old C57BL6 mice were given a total of eight doses of DEN, initially 20mg/kg body weight, and then 30mg/kg in the third week, followed by 50mg/kg for the last six weeks. The first group is DEN treatment only and the other two groups received thioacetamide (TAA) treatment for four or eight weeks after one week of rest from the last DEN treatment. An autopsy was performed after 24 weeks of the initial dose of DEN in each group. The cellular arrangement of HCC in the entire group was well-differentiated carcinoma and tumor presence with no significant impact on the survival of mice. Increased levels of the biochemical markers in serum, loss of tissue architecture, hepatocyte death, and proliferation were highly activated in all tumor-induced groups. This finding demonstrates an improved strategy to generate an animal model with a high occurrence of tumors combined with cirrhosis in a short time regardless of sex for researchers who want to investigate liver cancer-related.

Hepatocyte-specific clusterin overexpression attenuates diet-induced nonalcoholic steatohepatitis.

Clusterin is a multifunctional glycoprotein that plays important roles and is up-regulated in liver diseases such as hepatitis and hepatocellular carcinoma. However, little is known about the significance of clusterin in the pathogenesis of non-alcoholic steatohepatitis (NASH). The aim of this study is to examine the role of clusterin in progression of steatohepatitis in mice fed a methionine and choline deficient (MCD) diet. We generated hepatocyte-specific clusterin overexpression (hCLU-tg) mice, and hCLU-tg mice showed lower levels of hepatic triglycerides, less infiltration of macrophages and reduction of TNF-α, activation of Nrf-2 than wild-type littermates fed the MCD diet. Also, sustained clusterin expression in liver ameliorated hepatic fibrogenesis by reducing the activation of hepatic stellate cells by MCD diet. Sustained expression of clusterin in liver functioned as a preconditioning stimulus and prevented MCD diet-induced severe steatohepatitis injury via Nrf2 activation. These results demonstrate a novel function of clusterin as an immune preconditioning regulator in various inflammatory diseases including steatohepatitis.

Selective deletion of hepatocyte platelet-derived growth factor receptor α and development of liver fibrosis in mice.

BACKGROUND: Platelet-derived growth factor receptor α (PDGFRα) expression is increased in activated hepatic stellate cells (HSCs) in cirrhotic liver, while normal hepatocytes express PDGFRα at a negligible level. However, cancerous hepatocytes may show upregulation of PDGFRα, and hepatocellular carcinoma is preceded by chronic liver injury. The role of PDGFRα in non-cancerous hepatocytes and liver fibrosis is unclear. We hypothesized that upon liver injury, PDGFRα in insulted hepatocytes contributes to liver fibrosis by facilitating intercellular crosstalk between hepatocytes and HSCs. METHODS: Hepatocytes were isolated from normal and thioacetamide (TAA)-induced cirrhotic livers for assessment of PDGFRα expression. Conditional knock-out (KO) C57BL/6 mice, in which PDGFRα was selectively deleted in hepatocytes, were generated. Liver fibrosis was induced by injecting TAA for 8 weeks. Hep3B cells were transfected with a small interfering RNA (siRNA) (PDGFRα or control) and co-cultured with LX2 cells. RESULTS: PDGFRα expression was increased in hepatocytes from fibrotic livers compared to normal livers. Conditional PDGFRα KO mice had attenuated TAA-induced liver fibrosis with decreased HSC activation and proliferation. Immunoblot analyses revealed decreased expression of phospho-p44/42 MAPK in TAA-treated KO mice; these mice also showed almost complete suppression of the upregulation of mouse double minute 2. Although KO mice exhibited increased expression of transforming growth factor (TGF)-ß and Smad2/3, this was compensated for by increased expression of inhibitory Smad7. LX2 cells co-cultured with PDGFRα siRNA-infected Hep3B cells showed decreased PDGFRα, α smooth muscle actin, collagen α1(I), TGFß, and Smad2/3 expression. LX2/PDGFRα-deleted hepatocyte co-culture medium showed decreased PDGF-BB and PDGF-CC levels. CONCLUSIONS: Deletion of PDGFRα in hepatocytes attenuated the upregulation of PDGFRα in HSCs after TAA treatment, resulting in decreased liver fibrosis and HSC activation. This suggests that in the event of chronic liver injury, PDGFRα in hepatocytes plays an important role in liver fibrosis by affecting PDGFRα expression in HSCs.

Characterization of the porcine Nanog 5'-flanking region.

OBJECTIVE: Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine. METHODS: To characterize the porcine Nanog promoter, the 5'-flanking region of Nanog was isolated from cells of mini-pig ears. BLAST database search showed that there are two porcine Nanog genomic loci, chromosome 1 and 5, both of which contain an exon with a start codon. Deletion mutants from the 5'-flanking region of both loci were measured using the Dual-Luciferase Reporter Assay System, and a fluorescence marker, green fluorescence protein. RESULTS: Promoter activity was detected in the sequences of chromosome 5, but not in those of chromosome 1. We identified the sequences from -99 to +194 that possessed promoter activity and contained transcription factor binding sites from deletion fragment analysis. Among the transcription factor binding sites, a Sp1 was found to play a crucial role in basal promoter activity, and point mutation of this site abolished its activity, confirming its role in promoter activity. Furthermore, gel shift analysis and chromatin immunoprecipitation analysis confirmed that Sp1 transcription factor binds to the Sp1 binding site in the porcine Nanog promoter. Taken together, these results show that Sp1 transcription factor is an essential element for porcine Nanog basal activity the same as in human and mouse. CONCLUSION: We showed that the porcine Nanog gene is located on porcine chromosome 5 and its basal transcriptional activity is controlled by Sp1 transcription factor.

Characterization of the Nanog 5'-flanking Region in Bovine.

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

Characterization of Bovine NANOG5'-flanking Region during Differentiation of Mouse Embryonic Stem Cells.

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (-420/+181) bovine NANOG 5'-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (-420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

Molecular Characterization and Expression Analysis of the Peroxisome Proliferator Activated Receptor Delta (PPARδ) Gene before and after Exercise in Horse.

While athletic abilities such as speed, endurance and recovery are important in the horse, genes related to these abilities have not been extensively investigated. Here, we characterized the horse peroxisome proliferator-activated receptor delta (PPARδ) gene and analyzed the expression of PPARδ during exercise. PPARδ is a known regulator of ß-oxidation, muscle fiber transformation, and running endurance. Through evolutionary analysis using the synonymous and non-synonymous mutation ratio, it was revealed that positive selection occurred in the horse PPARδ gene. Two important domains related to nuclear hormone receptors, C4 zinc finger and ligand binding domain, were also found to be conserved well in horse PPARδ. Horse PPARδ was expressed ubiquitously in many tissues, but the expression level was various depending on the tissues. In the skeletal muscle, PPARδ increased about 2.5 folds after 30 min of exercise. Unlike in muscle, the increase of PPARδ expression was observed at 60 min but not 30 min of exercise in leukocytes. This finding might be useful for testing the endurance of horse using blood samples. Conclusively, the horse PPARδ gene is evolutionarily conserved well and can be used as a biomarker of endurance in horse.

Progression of diet induced nonalcoholic steatohepatitis is accompanied by increased expression of Kruppel-like-factor 10 in mice.

BACKGROUND: Kruppel-like-factor (KLF) 10 is identified as transforming growth factor (TGF) ß inducible early gene and is reported to suppress lipogenic genes. Although previous studies report that TGFß plays an important role in progression of nonalcoholic steatohepatitis (NASH) by regulating liver fibrosis, the association of KLF10 and NASH has never been explored. Thus we evaluated expressions and changes of KLF10 in diet induced NASH and in NASH which was alleviated by ursodeoxycholic acid (UDCA). We also assessed KLF10 in quiescent and activated hepatic stellate cells (HSCs). METHODS: C57BL/6 mice were given high fat, sucrose diet (HFSD) at least for 12 weeks up to 48 weeks and sacrificed at 12, 24 and 48 weeks thereafter. In other groups, either standard diet (SD) or HFSD was given for 24 weeks at which point mice fed with HFSD were divided into two groups, and were given either UDCA in combination with HFSD or vehicle with HFSD. Mice under SD were given vehicle. HSCs were isolated from C57BL/6 mice in order to evaluated KLF10 expression in activated HSCs. RESULTS: The mice were found to acquire liver steatosis and inflammation starting from week 12 of HFSD feeding, although significant liver fibrosis was noticed by week 24. Increased TGFß and collagen α1(I) (Col1α(I)) expression was also apparent from week 24. However, expression of KLF10 mRNA started to increase from week 12, earlier than TGFß gene. Up-regulation of KLF10 was accompanied by suppressed carbohydrate response element-binding protein (ChREBP) that is known to be protective against insulin resistance. The mice fed with HFSD and UDCA had decreased Colα(I) mRNA that was coincided with reduced TGFß and KLF10 expression. Expression of ChREBP was also recovered by UDCA administration. Enhanced KLF10 was noticed in activated HSCs when quiescent cell showed minimal expression. CONCLUSIONS: Our study demonstrated that KLF10 expression was significantly increased in diet induced NASH and collagen producing activated HSCs. We also noticed that this up-regulation of KLF10 was accompanied by increased TGFß signaling genes and suppressed ChREBP expression. These observations suggest possible association of KLF10 and NASH progression.

Digestive recovery of sulfur-methyl-L-methionine and its bioaccessibility in Kimchi cabbages using a simulated in vitro digestion model system.

BACKGROUND: Sulfur-methyl-L-methionine (SMM) has been known to provide various biological functions such as radical scavenging effect, inhibition of adipocyte differentiation, and prevention of gastric mucosal damage. Kimchi cabbages are known to be a major food source providing SMM but its bioaccessibility has not been studied. The objective of current study was to determine both the digestive stability of SMM and the amount released from Kimchi cabbages under a simulated in vitro digestion model system. RESULTS: The in vitro digestion model system simulating a human gastrointestinal tract was carried out for measuring digestive recovery and bioaccessibility of SMM. SMM was quantified by using high-performance liquid chromatography with a fluorescence detector. Recovery of an SMM standard after digestion was 0.68 and 0.65% for fasted and fed conditions, respectively, indicating that the digestive stability of the SMM standard was not affected by dietary energy or co-ingested food matrix. The SMM standard was also significantly stable in acidic pH (P < 0.05). The bioaccessibility of SMM from Kimchi cabbages was measured under a fasted condition, resulted in 8.83, 14.71 and 10.88%, for salivary, gastric and small intestinal phases, respectively. CONCLUSION: Results from our study suggest that SMM from Kimchi cabbages, a component of food sources, is more bioavailable than SMM by itself.

Antibacterial Activity of Recombinant Pig Intestinal Parasite Cecropin P4 Peptide Secreted from Pichia pastoris.

Cecropins (Cec) are antibacterial peptides and their expression is induced in a pig intestinal parasite Ascaris suum by bacterial infection. To explore the usefulness of its activity as an antibiotic, CecP4 cDNA was prepared and cloned into the pPICZ B expression vector and followed by the integration into AOX1 locus in Pichia pastoris. The supernatants from cell culture were collected after methanol induction and concentrated for the test of antimicrobial activity. The recombinant P. patoris having CecP4 showed antimicrobial activity when tested against Staphyllococcus aureus in disc diffusion assay. We selected one of the CecP4 clones (CecP4-2) and performed further studies with it. The growth of recombinant P. pastoris was optimized using various concentration of methanol, and it was found that 2% methanol in the culture induced more antibacterial activity, compared to 1% methanol. We extended the test of antimicrobial activity by applying the concentrated supernatant of CecP4 culture to Pseudomonas aeruginosa and E. coli respectively. Recombinant CecP4 also showed antimicrobial activity against both Pseudomona and E. coli, suggesting the broad spectrum of its antimicrobial activity. After improvements for the scale-up, it will be feasible to use recombinant CecP4 for supplementation to the feed to control microbial infections in young animals, such as piglets.

KLF10, transforming growth factor-ß-inducible early gene 1, acts as a tumor suppressor.

Krüppel-like factor 10 (KLF10) has been suggested to be a putative tumor suppressor. In the present study, we generated KLF10 deficient mice to explore this hypothesis in vivo. KLF10 deficient mice exhibited increased predisposition to skin tumorigenesis and markedly accelerated papilloma development after DMBA/TPA treatment. On the other hand, KLF10 deficient keratinocytes showed increased proliferation and apoptosis. In colony formation assays after oncogenic H-Ras transfection, KLF10 deficient mouse embryonic fibroblasts (MEFs) yielded more colonies than wild-type MEFs. Furthermore, KLF10 dose-dependently activated p21(WAF1/CIP1) transcription, which was independent of p53 and Sp1 binding sites in p21(WAF1/CIP1) promoter. This study demonstrates that KLF10 is a tumor suppressor and that it targets p21(WAF1/CIP1) transcription.

The basal transcriptional activity of the murine Klf10 gene is regulated by the transcriptional factor JunB.

BACKGROUND: Krüppel-like factor 10 (KLF10) belongs to the Sp1-like transcription factor family, which plays an important role in many directions, e.g., cell proliferation, apoptosis, and differentiation. Its 5' upstream regions are conserved across mammalian species. However, the regulatory mechanism has not been elucidated yet. OBJECTIVE: Nonetheless the basal transcriptional regulation mechanisms of these regions are unknown. Here, we characterized it which is indispensable for the basal transcription of the Klf10 gene. METHODS: Seven deletions of 5' upstream DNA fragments from the 10 kb mKlf10 genomic DNA were produced by PCR and cloned into the upstream of the luciferase (Luc) reporter gene in the pGL3 basic plasmid. RESULT: The luciferase reporter assay showed that the DNA sequence at positions from -101 to +68 was required for a principle activity in the promoter of mKlf10 gene, in which transcriptional factor binding motifs, one JunB and two Sp1 sites, are included. Mutations at the sequence of JunB motif, but not at the two Sp1, abrogated the promoter activity completely, suggesting the indispensable role of JunB site for basal transcription of mKlf10 gene. Moreover, electrophoretic mobility and supershift assays (EMSA) uncovered that JunB protein bound to this region specifically. CONCLUSION: Taken together, our study revealed that the JunB but not Sp1 at mKlf10 promoter functions as a positive basic factor for the transcriptional activity of the gene.

Resistin enhances the expansion of regulatory T cells through modulation of dendritic cells.

BACKGROUND: Resistin, a member of adipokine family, is known to be involved in the modulation of immune responses including inflammatory activity. Interestingly, resistin is secreted by adipocytes in mice and rats whereas it is secreted by leukocytes in humans. However, the mechanism behind the effect of resistin on the expansion of regulatory T cells (Tregs) remains poorly understood. Therefore, we examined regulatory effect of resistin on the induction and cellular modification of Tregs. RESULTS: Both protein and mRNA expression of FoxP3, a representative marker of Tregs, increased in a dose-dependent manner when peripheral blood mononuclear cells were treated with resistin. At the same time, resistin had no direct effect on the induction of FoxP3 in CD4+ T cells, suggesting an indirect role through other cells type(s). Since DCs are an important player in the differentiation of T cells, we focused on the role of DCs in the modulation of Tregs by resistin. Resistin suppressed the expression of interferon regulatory factor (IRF)-1 and its target cytokines, IL-6, IL-23p19 and IL-12p40, in DCs. Furthermore, FoxP3 expression is increased in CD4+ T cells when co-cultured with DCs and concomitantly treated with resistin. CONCLUSION: Our results suggest that resistin induces expansion of functional Tregs only when co-cultured with DCs.

Clusterin overexpression protects against western diet-induced obesity and NAFLD.

Obesity is a significant risk factor for various metabolic diseases and is closely related to non-alcoholic fatty liver disease (NAFLD) characterized by inflammation and oxidative stress. Clusterin is a multi-functional protein that is up-regulated in the pathogenesis of various metabolic diseases, including obesity and NAFLD. Our previous studies indicated that hepatocyte-specific overexpression of clusterin alleviates methionine choline-deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH) by activating nuclear factor erythroid 2-related factor 2 (Nrf2). Here we generated transgenic mice with whole-body clusterin overexpression (wCLU-tg) and investigated the role of clusterin in Western diet-induced obesity and NAFLD. We confirmed that obesity parameters and the spectrum of NAFLD of wCLU-tg mice were improved compared to wild type mice. Contrarily, clusterin deficiency deteriorated metabolic disruptions. We also found that clusterin activates target molecules for obesity and NAFLD, namely Nrf2 and AMPK, suggesting that clusterin protects against Western diet-induced obesity and NAFLD by activating Nrf2 and AMPK.

Gastroprotective effect of phytoncide extract from Pinus koraiensis pinecone in Helicobacter pylori infection.

For centuries, herbs have been used by traditional therapists around the world to treat gastrointestinal tract disorders, such as gastritis. We hypothesized that the anti-Helicobacter pylori properties of phytoncide, which is extracted from pinecone waste, would facilitate use as a natural gastroprotective product to treat gastrointestinal tract disorders. Thus, we investigated in vitro antibacterial efficacy against H. pylori by agar diffusion assay. To determine the gastroprotective properties of phytoncide, we conducted hematoxylin and eosin staining, performed assays for the detection of the cytotoxin gene, and evaluated pro-inflammatory cytokine expression in H. pylori-infected C57BL/6 mice. Phytoncide significantly inhibited the survival of H. pylori in the gastrointestinal system of C57BL/6 mice. Reduction of gastric severity in H. pylori-infected mice was associated with reductions in the expression levels of pro-inflammatory cytokines in the gastric mucosa, and of the cytotoxin CagA gene in phytoncide treated groups (P < 0.05 and P < 0.01). In conclusion, phytoncide significantly inhibited the growth of H. pylori in gastro tissue, possibly due to the abundant α-pinene present in the phytoncide as detected by HPLC analysis. Further studies are needed to validate our findings, but we suggest that phytoncide has the potential to be used as a natural ingredient in anti-H. pylori products.

KLF10 is a modulatory factor of chondrocyte hypertrophy in developing skeleton.

To define the functional role of Krüppel-like factor (KLF) 10 as a modulator of chondrocyte hypertrophy in developing skeleton, the developmental features in the long bone of KLF10 knockout (KO) mice were investigated and the mesenchymal stem cells (MSCs) from KLF10 KO mice were characterized regarding chondrogenesis and osteogenesis. Delayed long bone growth and delayed formation of primary ossification center were observed in an early embryonic stage in KLF10 KO mouse along with very low Indian hedgehog expression in epiphyseal plate. While the chondrogenic potential of mouse MSCs from KLF10 KO mice appeared normal or slight decreased, hypertrophy and osteogenesis were extensively suppressed. These findings suggest that KLF10 is a mediator of chondrocyte hypertrophy in developing skeleton, and that suppression of KLF10 may be employed as a new strategy for preventing hypertrophy in cartilage regeneration using MSCs.