Deficiency of primary cilia in kidney epithelial cells induces epithelial to mesenchymal transition.

Primary cilium is a microtubule-based non-motile organelle that plays critical roles in kidney pathophysiology. Our previous studies revealed that the lengths of primary cilia decreased upon renal ischemia/reperfusion injury and oxidative stress, and restored with recovery. Here, we tested the hypothesis that lack of primary cilium causes epithelial to mesenchymal transition (EMT) of kidney tubule cells. We investigated the alteration of length of primary cilia in TGF-ß-induced EMT via visualization of primary cilia by fluorescence staining against acetylated α-tubulin. EMT was determined by measuring mesenchymal protein expression using quantitative PCR and indirect fluorescence staining. As a result, TGF-ß treatment decreased ciliary length along with EMT. To test whether defect of primary cilia trigger onset of EMT, cilia formation was disturbed by knock down of ciliary protein using siRNA along with/without TGF-ß treatment. Knock down of Arl13b and Ift20 reduced cilia elongation and increased expression of EMT markers such as fibronectin, α-SMA, and collagen III. TGF-ß-induced EMT was greater as well in Arl13b and Ift20-knock down cells compared to control cells. Taken together, deficiency of primary cilia trigger EMT and exacerbates it under pro-fibrotic signals.

Pathologic HDAC1/c-Myc signaling axis is responsible for angiotensinogen transcription and hypertension induced by high-fat diet.

High-fat diet (HFD)-induced obesity is a cause of resistant hypertension. We have shown a possible link between histone deacetylases (HDACs) and renal angiotensinogen (Agt) upregulation in the HFD-induced hypertension, whereas the underlying mechanisms remain to be elucidated. Here, using a HDAC1/2 inhibitor romidepsin (FK228) and siRNAs, we determined roles of HDAC1 and HDAC2 in HFD-induced hypertension and found the pathologic signaling axis between HDAC1 and Agt transcription. Treatment with FK228 canceled the increased blood pressure of male C57BL/6 mice induced by HFD. FK228 also blocked upregulation of renal Agt mRNA, protein, angiotensin II (Ang II) or serum Ang II. Activation and nuclear accumulation of both HDAC1 and HDAC2 occurred in the HFD group. The HFD-induced HDAC activation was associated with an increase in deacetylated c-Myc transcription factor. Silencing of HDAC1, HDAC2 or c-Myc in HRPTEpi cells decreased Agt expression. However, only HDAC1 knockdown, but not HDAC2, increased c-Myc acetylation, suggesting selective roles in two enzymes. Chromatin immunoprecipitation assay revealed that HFD induced the binding of HDAC1 and deacetylated c-Myc at the Agt gene promoter. A putative c-Myc binding sequence in the promotor region was necessary for Agt transcription. Inhibition of c-Myc downregulated Agt and Ang II levels in kidney and serum, ameliorating HFD-induced hypertension. Thus, the abnormal HDAC1/2 in the kidney may be responsible for the upregulation of the Agt gene expression and hypertension. The results expose the pathologic HDAC1/c-myc signaling axis in kidney as a promising therapeutic target for obesity-associated resistant hypertension.

Antiallergic effect of Ostericum koreanum root extract on ovalbumin-induced allergic rhinitis mouse model and mast cells.

OBJECTIVE: In the present study, the anti-allergic effect of OR extract was evaluated on an ovalbumin (OVA)-induced allergic rhinitis in mice and rat peritoneal mast cells (RPMC). METHODS: Balb/c mice were systemically sensitized to OVA followed by intraperitoneal and nasal allergen challenges. We investigated the effect of OR extract on allergic symptoms, serological marker production and histological changes of the nasal mucosa in a mouse model of allergic rhinitis. We observed mast cell degranulation and detected the production of histamine and inflammatory cytokines by ELISA. RESULTS: Compared to the OVA-control group, oral administration of OR extract at doses of 50 and 100 mg/kg significantly (p < 0.001) decreased the serum levels of histamine, OVA-specific IgE and Th2 cytokine,IIL-4 as well as increasing Th1 cytokine, IFN-gamma. Oral administration of OR extract also attenuated disease progression as determined by nasal symptoms and histological changes of the nasal mucosa in OVA-sensitized mice. Furthermore, treatment with OR extract at doses of 0.2, 0.5 ad 1 mg/mL in RPMC significantly (p <0.01, p <0.001 and p <0.001, respectively) decreased compound 48/80-induced histamine release and suppressed mast cell degranulation. Treatment with OR extract in RPMC also inhibited PMA/A23187-induced production of inflammatory cytokines such as TNF-alpha and IL-6. The mechanism of action underlying OR extract in allergic inflammation appears to be inhibition of the phosphorylation of ERK1/2 and p38 MAPK, in addition to blocking of the NFKB pathway. CONCLUSIONS: These results indicate that OR extract has the potential to be a source of antiallergic agents for use in allergen and/or mast cell-mediated diseases including allergic rhinitis.

Inhibition of HDACs (Histone Deacetylases) Ameliorates High-Fat Diet-Induced Hypertension Through Restoration of the MsrA (Methionine Sulfoxide Reductase A)/Hydrogen Sulfide Axis.

[Figure: see text].

Histone deacetylase inhibitor CG200745 ameliorates high-fat diet-induced hypertension via inhibition of angiotensin II production.

Obesity is growing rapidly worldwide due to consumption of westernized diet and lack of exercise. Obesity is one of the major risk factors of hypertension. The novel histone deacetylase (HDAC) inhibitor CG200745 was originally developed to treat various cancers. Previous studies showed that CG200745 attenuated hypertension through inhibition of cardiac hypertrophy and fibrosis in deoxycorticosterone acetate-induced hypertensive rat. The purpose of this study is to investigate the role and underlying mechanism of CG200745 in high-fat diet (HFD)-induced hypertension. Nine-week old C57BL/6 mice were fed a normal diet (ND) or HFD for 17 weeks. Each group of mice was treated with vehicle or CG200745 by intraperitoneal injection for 9 days. HFD group showed higher body weight, blood pressure (BP), HDAC activities, angiotensinogen and renin expressions in kidney, angiotensin-converting enzyme (ACE) expression in the lung, serum angiotensin II (Ang II) concentration, and myosin light chain20 (MLC20) phosphorylation in mesenteric artery compared with ND group. CG200745 lowered BP, HDAC activity, renin and angiotensinogen in the kidney, ACE in the lung, serum Ang II level, and phosphorylation of MLC20 in HFD group. In conclusion, CG200745 ameliorated HFD-induced hypertension through inhibition of HDAC/Ang II/vascular contraction axis. Our results offer CG200745 as a novel therapeutic option for HFD-induced hypertension.

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

A novel role for the deubiquitinase USP1 in the control of centrosome duplication.

Defects in the regulation of centrosome duplication lead to tumorigenesis through abnormal cell division and resulting chromosome missegregation. Therefore, maintenance of accurate centrosome number is critical for cell fate. The deubiquitinating enzyme USP1 plays important roles in DNA repair and cell differentiation. Importantly, increased levels of USP1 are detected in certain types of human cancer, but little is known about the significance of USP1 overexpression in cancer development. Here we show that Usp1 plays a novel role in regulating centrosome duplication. The ectopic expression of wild-type Usp1, but not C90S Usp1 (catalytically inactive mutant form), induced centrosome amplification. Conversely, ablation of Usp1 in mouse embryonic fibroblasts (MEFs) showed a significant delay in centrosome duplication. Moreover, Usp1-induced centrosome amplification caused abnormal mitotic spindles, chromosome missegregation and aneuploidy. Interestingly, loss of inhibitor of DNA binding protein 1 (ID1) suppressed Usp1-induced centrosome amplification. Taken together, our results strongly suggest that Usp1 is involved in the regulation of centrosome duplication, at least in part via ID1, and Usp1 may exert its oncogenic activity, partially through inducing centrosome abnormality.

Comparison of the efficacy of KOB03, ketotifen, and montelukast in an experimental mouse model of allergic rhinitis.

KOB03 is a polyherbal medicine derived from an oriental prescription traditionally used to treat allergic diseases. In the present study, we compared the efficacy of KOB03 with modern drugs such as ketotifen and montelukast in an experimental mouse model of allergic rhinitis (AR). Ketotifen is a H1 receptor antagonist and montelukast is a leukotriene receptor antagonist. Mice were treated with KOB03, ketotifen or montelukast in an established AR mouse model using ovalbumin (OVA)-sensitized/challenged BALB/c mice. The treatment of KOB03 had inhibitory effects on symptom scores, serum levels of OVA-specific IgE, histamine, leukotriene C4, IL-4, TNF-α, and IL-1ß in AR mice, and the histolopathological changes of nasal mucosa with mucin release and inflammation. AR mice treated with KOB03 had significantly lower serum levels of OVA-specific IgE, LTC4, IL-4, and IL-1ß than mice treated with ketotifen, whereas they only had significantly lower serum levels of OVA-specific IgE and IL-4 than those treated with montelukast. In addition, the histolopathological changes of nasal mucosa with eosinophil infiltration were significantly lower in the KOB03-treated mice than those in the ketotifen and montelukast-treated group. These results suggest that KOB03 has therapeutic potential for treating AR like other modern medicines.

Effect of the semen extract of Thuja orientalis on inflammatory responses in transient focal cerebral ischemia rat model and LPS-stimulated BV-2 microglia.

In the central nervous system inflammation is dependent upon the synthesis of various inflammatory mediators by local neurons, astrocytes and especially microglia. In this study, we investigated the anti-inflammatory activities of the semen extract of Thuja orientalis (Thujae Orientalis Semen; TOS) on transient middle cerebral artery occlusion (tMCAO)-induced ischemia in rats and the production of inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE(2)) and proinflammatory cytokine, interleukin (IL)-1ß in lipopolysaccharide (LPS)-stimulated BV-2 mouse microglia. TOS extract significantly decreased the infarction volumes of ischemic brains and also inhibited microglia activation and neuronal death. In addition, TOS extract significantly decreased the production of NO, PGE(2) and IL-1ß in LPS-stimulated BV-2 microglia. TOS extract also attenuated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and IL-1ß mRNAs and proteins in activated microglia. Furthermore, TOS extract significantly suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK, and the nuclear translocation of NF-κB p65 in activated microglia. Our results indicate that TOS extract is capable of inhibiting microglia activation in an ischemic brain through the down-regulation of inflammatory responses, suggesting that the TOS extract may have therapeutic potential as an anti-inflammatory drug for ischemic stroke.

Anti-diabetic effect of Wen-pi-tang-Hab-Wu-ling-san extract in streptozotocin-induced diabetic rats.

OBJECTIVES: Wen-pi-tang-Hab-Wu-ling-san (WHW) is an oriental herbal prescription formulated using 14 herbs and has been used to cure chronic renal failure in Korean oriental medicine. In this study, we investigated the anti-diabetic effect of WHW in the streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Diabetes was induced by streptozotocin (STZ, 60 mg/kg, i.p.) in rats. WHW extract (100 mg/kg) was orally dosed once a day for four weeks. The results were compared with standard antidiabetic drug, glibenclamide (3 mg/kg, p.o). RESULTS: Significant decrease in body weight and insulin levels and increase in blood glucose, triglycerides, urea nitrogen (BUN), and creatinine were detected in STZ-induced diabetic rats with disruption and disappearance of pancreatic and kidney cells and decrease in insulin producing beta cells. However, these diabetic changes were significantly inhibited by treatment with WHW extract. In the oral glucose tolerance test, the extract produced a significant decrease in glycemia 60 minutes after the glucose pulse. CONCLUSIONS: Based on these results, we suggest that WHW extract has favorable effects in protecting the STZ-induced hyperglycemia, renal damage, and beta-cell damage in rats.

Effect of KOB03, a polyherbal medicine, on ovalbumin-induced allergic rhinitis in guinea pigs.

BACKGROUND: KOB03 is a polyherbal medicine that originated from the oriental prescription for the treatment of chronic allergic diseases such as rhinitis and asthma. This study aims to evaluate the effect of KOB03 on ovalbumin (OVA)-induced allergic rhinitis (AR) in guinea pigs. METHODS: Hartley guinea pigs were sensitized to OVA by intraperitoneal injection on days 0, 7, and 14 and challenged with intranasal exposure to OVA three times for 7 days after the last sensitization. KOB03 at doses of 200 and 500 mg/kg were orally administrated to guinea pigs once daily during challenge. The serum levels of histamine, OVA-specific immunoglobulin (Ig) E, eosinophil cationic protein (ECP) and cytokines (TNF-α, IL-4 and IFN-γ) in OVA sensitization/challenge-induced AR guinea pigs were measured. We also observed histological changes in nasal tissues of AR guinea pigs by staining with H&E, Periodic acid-Schiff, and toluidine blue. RESULTS: The administration of KOB03 at a dose of 500 mg/kg significantly decreased the serum levels of histamine (P = 0.001), OVA-specific IgE (P = 0.0017), ECP (P = 0.008), and TNF-α (P = 0.0003) in OVA-sensitized/challenged guinea pigs compared with controls. KOB03 significantly decreased the serum levels of a Th2 cytokine, IL-4 (P = 0.017), while significantly increasing the levels of a Th1 cytokine, IFN-γ (P = 0.0006) in OVA-sensitized/challenged guinea pigs compared with controls. In addition, KOB03 suppressed the epithelial destruction, goblet cell hyperplasia and eosinophilic infiltration into nasal mucosa associated with AR. CONCLUSION: KOB03 may regulate allergic inflammation in AR by inhibiting nasal damage, the release of allergic mediators and modulating the balance of Th1/Th2 cytokines.

Inhibitory effects of the root extract of Dipsacus asperoides C.Y. Cheng et al T.M.Ai on collagen-induced arthritis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipsaci radix, the dried root of Dipsacus asperoides C.Y. Cheng et al T.M.Ai is used as a medicinal plant in oriental clinics for the treatment of bone diseases and functions by strengthening bone and healing bone fractures. AIM OF THE STUDY: This study investigated the therapeutic efficacy of Dipsaci radix in treating rheumatoid arthritis using a type II collagen (CII)-induced arthritis (CIA) mouse model. MATERIALS AND METHODS: Arthritis was induced in male DBA/1 mice by immunization with CII. Dipsaci radix water (DR-W) extract at 50mg/kg and 100mg/kg was orally administered from days to after the induction of arthritis. Arthritic score, serum levels of anti-CII IgG2a, the inflammatory mediator prostaglandin E(2) (PGE(2)), and inflammatory cytokines (TNF-α, IL-1ß and IL-6), and histological changes in the ankle joint were analyzed in CIA mice. RESULTS: Arthritic induction increased the arthritic score, as well as serum levels of anti-CII IgG2a antibody, PGE(2), TNF-α, IL-1ß and IL-6 in mice. However, administration of DR-W extract in CIA mice significantly reduced arthritic scores and serum levels of anti-CII IgG2a antibody, PGE(2), TNF-α, IL-1ß and IL-6 compared with those in vehicle-treated CIA mice. Furthermore, histopathological improvement in joint architecture was also observed in DR-W extract-treated CIA mice. CONCLUSIONS: DR-W extract has anti-inflammatory and anti-arthritic effects in arthritic mice. This suggests that Dipsaci radix might be used as a therapeutic agent for the treatment of human arthritis.

Antiallergic effect of KOB03, a polyherbal medicine, on mast cell-mediated allergic responses in ovalbumin-induced allergic rhinitis mouse and human mast cells.

ETHNOPHARMACOLOGICAL RELEVANCE: KOB03 is a polyherbal medicine consisting of five different herbs and has commonly been used for the treatment of various allergic diseases. However, its precise anti-allergic effect and mechanism remain unknown. AIM OF THE STUDY: The aim of this study was to investigate the effect of KOB03 on allergic responses through the regulation of mast-cell mediated allergic inflammation. MATERIALS AND METHODS: To determine the effect of KOB03 on mast cell-mediated allergic reactions, we investigated the parameter changes of in vivo models such as compound 48/80-induced systemic anaphylaxis and ovalbumin (OVA)-induced allergic rhinitis, and the release of allergic inflammatory mediators such as histamine, immunoglobulin (Ig) E, and inflammatory cytokines via the MAPKs and NF-kappaB pathways. RESULTS: The oral administration of KOB03 at doses of 100 and 200mg/kg inhibited histamine release and mortality in compound 48/80-induced anaphylactic rats. KOB03 also improved rhinitis symptoms, inhibited the histopathological changes of nasal mucosa, and decreased the serum levels of histamine, OVA-specific IgE and TNF-α in OVA-induced allergic rhinitis in mice. In vitro, KOB03 suppressed compound 48/80-induced histamine release by blocking mast cell degranulation. In addition, KOB03 inhibited the production of inflammatory cytokines such as TNF-α, IL-1ß, IL-6 and IL-8 in PMA/A23187-stimulated HMC-1 mast cells by suppressing their gene expression and blocking the ERK1/2 and p38 MAPK and NF-κB pathways. CONCLUSIONS: These results suggest that KOB03 has an anti-allergic effect by modulating mast cell-mediated allergic responses in allergic rhinitis.

Inhibitory effects of the methylene chloride fraction of JP05 on the production of inflammatory mediators in LPS-activated BV2 microglia.

Excessive production of inflammatory mediators such as nitric oxide (NO) and proinflammatory cytokines from activated microglia in the central nervous system contributes to uncontrolled inflammation in neurodegenerative disorders. In this study, we investigated the anti-inflammatory activities of the methylene chloride fraction of JP05 (JP05-MC) on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 mouse microglial cells, and its mechanism of action. JP05-MC significantly inhibited LPS-induced production of NO and the proinflammatory cytokines, TNF-α and IL-6, in BV2 cells. JP05-MC also attenuated the mRNA expression and protein levels of inducible nitric oxide synthase in LPS-induced BV2 cells. JP05-MC significantly attenuated LPS-elicited phosphorylation of the mitogen-activated protein kinase (MAPK), extracellular-signal-regulated kinase 1/2, and nuclear factor-κB (NF-κB) nuclear translocation in BV2 cells. Our results indicate that JP05-MC exerts anti-inflammatory properties via downregulation of inflammatory mediator gene transcription by suppressing the MAPK and NF-κB pathways, suggesting that JP05-MC may have therapeutic potential as an anti-inflammatory agent in neurodegenerative diseases.