Targeted Inhibition of O-Linked ß-N-Acetylglucosamine Transferase as a Promising Therapeutic Strategy to Restore Chemosensitivity and Attenuate Aggressive Tumor Traits in Chemoresistant Urothelial Carcinoma of the Bladder.

Acquisition of acquired chemoresistance during treatment cycles in urothelial carcinoma of the bladder (UCB) is the major cause of death through enhancing the risk of cancer progression and metastasis. Elevated glucose flux through the abnormal upregulation of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) controls key signaling and metabolic pathways regulating diverse cancer cell phenotypes. This study showed that OGT expression levels in two human UCB cell models with acquired resistance to gemcitabine and paclitaxel were significantly upregulated compared with those in parental cells. Reducing hyper-O-GlcNAcylation by OGT knockdown (KD) markedly facilitated chemosensitivity to the corresponding chemotherapeutics in both cells, and combination treatment with OGT-KD showed more severe growth defects in chemoresistant sublines. We subsequently verified the suppressive effects of OGT-KD monotherapy on cell migration/invasion in vitro and xenograft tumor growth in vivo in chemoresistant UCB cells. Transcriptome analysis of these cells revealed 97 upregulated genes, which were enriched in multiple oncogenic pathways. Our final choice of suspected OGT glycosylation substrate was VCAN, S1PR3, PDGFRB, and PRKCG, the knockdown of which induced cell growth defects. These findings demonstrate the vital role of dysregulated OGT activity and hyper-O-GlcNAcylation in modulating treatment failure and tumor aggression in chemoresistant UCB.

Effects of Gallotannin-Enriched Extract of Galla Rhois on the Activation of Apoptosis, Cell Cycle Arrest, and Inhibition of Migration Ability in LLC1 Cells and LLC1 Tumors.

Gallotannin (GT) and GT-enriched extracts derived from various sources are reported to have anti-tumor activity in esophageal, colon and prostate tumors, although their anti-tumor effects have not been determined in lung carcinomas. To investigate the anti-tumor activity of GT-enriched extract of galla rhois (GEGR) against lung carcinomas, alterations in the cytotoxicity, apoptosis activation, cell cycle progression, migration ability, tumor growth, histopathological structure, and the regulation of signaling pathways were analyzed in Lewis lung carcinoma (LLC1) cells and LLC1 tumor bearing C57BL/6NKorl mice, after exposure to GEGR. A high concentration of GT (69%) and DPPH scavenging activity (IC50=7.922 µg/ml) was obtained in GEGR. GEGR treatment exerted strong cytotoxicity, cell cycle arrest at the G2/M phase and subsequent activation of apoptosis, as well as inhibitory effects on the MAPK pathway and PI3K/AKT mediated cell migration in LLC1 cells. In the in vivo syngeneic model, exposure to GEGR resulted in suppressed growth of the LLC1 tumors, as well as inhibition of NF-κB signaling and their inflammatory cytokines. Taken together, our results provide novel evidence that exposure to GEGR induces activation of apoptosis, cell cycle arrest, and inhibition of cell migration via suppression of the MAPK, NF-κB and PI3K/AKT signaling pathways in LLC1 cells and the LLC1 syngeneic model.

Comparison of response to LPS-induced sepsis in three DBA/2 stocks derived from different sources.

Sepsis, one of the most fatal diseases in the world, is known to culminate in multiple organ failure due to an uncontrolled inflammatory response. Hence, the use of animal models in sepsis research is very important to study complex immune responses. The current study was undertaken to compare commercial stocks with KFDA stocks of DBA/2 mice as an animal model for sepsis study. To compare responses of DBA/2 mice to lipopolysaccharides (LPS)-induced sepsis, we measured altered characteristics of various factors associated with sepsis, including survival curves, organ failure and inflammatory response, in DBA/2Korl stock and two commercial stocks (DBA/2A and DBA/2B). Survival rates after LPS exposure were similar for DBA/2Korl and DBA/2B; however, for times over 20 h, survival rates were reduced and concentration dependent in DBA/2A. In order to evaluate multiple organ failure caused by sepsis, H&E stains were evaluated for liver and spleen tissues obtained in the early (2 h) and later (20 h) stages after exposure to LPS; no significant differences were observed between the three stocks. mRNA and protein levels of proinflammatory cytokines were assessed for evaluating inflammatory reactions, and were found to increase in a dose-dependent manner in most DBA/2 mice after LPS treatment. However, no changes were observed in the mRNA levels of proinflammatory cytokines at 20 h after LPS exposure in the DBA/2A stock. The induction of inflammation-mediated factors by LPS exposure did not induce alterations in the mRNA levels of COX-2 and iNOS in all three DBA/2 stocks. Our results indicate that response of DBA/2Korl to LPS-induced sepsis is similar to the two commercial DBA/2 stocks, thus representing its potential as a useful biological resource established in Korea.

Deficiency of complement component 3 may be linked to the development of constipation in FVB/N-C3em1Hlee /Korl mice.

Alterations in complement component 3 (C3) expression has been reported to be linked to several bowel diseases including Crohn's disease, inflammatory bowel disease, and ulcerative colitis; however, the association with constipation has never been investigated. In this study, we aimed to investigate the correlation between C3 regulation and constipation development using a C3 deficiency model. To achieve these, alterations in stool excretion, transverse colon histological structure, and mucin secretion were analyzed in FVB/N-C3em1Hlee /Korl (C3 knockout, C3 KO) mice with the deletion of 11 nucleotides in exon 2 of the C3 gene. The stool excretion parameters, gastrointestinal transit, and intestine length were remarkably decreased in C3 KO mice compared with wild-type (WT) mice, although there was no specific change in feeding behavior. Furthermore, C3 KO mice showed a decrease in mucosal and muscle layer thickness, alterations in crypt structure, irregular distribution of goblet cells, and an increase of mucin droplets in the transverse colon. Mucin secretion was suppressed, and they accumulated in the crypts of C3 KO mice. In addition, the constipation phenotypes detected during C3 deficiency were confirmed in FVB/N mice treated with C3 convertase inhibitor (rosmarinic acid (RA)). Similar phenotypes were observed with respect to stool excretion parameters, gastrointestinal transit, intestine length, alterations in crypt structure, and mucin secretion in RA-treated FVB/N mice. Therefore, the results of the present study provide the first scientific evidence that C3 deficiency may play an important role in the development of constipation phenotypes in C3 KO mice.

Deletion of NKX3.1 via CRISPR/Cas9 Induces Prostatic Intraepithelial Neoplasia in C57BL/6 Mice.

Several techniques have been employed for deletion of the NKX3.1 gene, resulting in developmental defects of the prostate, including alterations in ductal branching morphogenesis and prostatic secretions as well as epithelial hyperplasia and dysplasia. To investigate whether the CRISPR/Cas9-mediated technique can be applied to study prostate carcinogenesis through exon I deletion of NKX3.1 gene, alterations in the prostatic intraepithelial neoplasia (PIN) and their regulatory mechanism were observed in the prostate of NKX3.1 knockout (KO) mice produced by the CRISPR/Cas9-mediated NKX3.1 mutant gene, at the ages of 16 and 24 weeks. The weight of dorsal-lateral prostate (DLP) and anterior prostate (AP) were observed to be increased in only the 24 weeks KO mice, although morphogenesis was constant in all groups. Obvious PIN 1 and 2 lesions were frequently detected in prostate of the 24 weeks KO mice, as compared with the same age wild type (WT) mice. Ki67, a key indicator for PIN, was densely stained in the epithelium of prostate in the 24 weeks KO mice, while the expression of p53 protein was suppressed in the same group. Also, both the 16 and 24 weeks KO mice reveal inhibition of the PI3K/AKT/mTOR pathway in the prostate. However, prostate specific antigen (PSA) levels and Bax/Bcl-2 expressions were decreased in the prostate of 16 weeks KO mice, and were increased in only the 24 weeks KO mice. Taken together, the results of the present study provide additional evidence that CRISPR/Cas9-mediated exon 1 deletion of the NKX3.1 gene successfully induces PIN lesions, along with significant alterations of Ki67 expression, EGFR signaling pathway, and cancer-regulated proteins.

Correction to: Influence of three BALB/c substrain backgrounds on the skin tumor induction efficacy to DMBA and TPA cotreatment.

[This corrects the article DOI: 10.1186/s42826-020-00063-z.].

Influence of three BALB/c substrain backgrounds on the skin tumor induction efficacy to DMBA and TPA cotreatment.

Differences in responsiveness of BALB/c substrains have been investigated in various fields, including diabetes induction, corpus callosum deficiency, virus-induced demyelinating disease, aggressive behavior and osteonecrosis. However, induction efficacy of skin tumor remains untried. We therefore investigated the influence of BALB/c substrain backgrounds on the skin tumor induction efficacy in response to DMBA (7,12-Dimethylbenz[a]anthracene) and TPA (12-O-tetradecanoylphorbol-13-acetate) cotreatment. Alterations in the levels of tumor growth related factors, histopathological structure, and the expression to tumor related proteins were measured in three BALB/c substrains (BALB/cKorl, BALB/cA and BALB/cB) after exposure to DMBA (25 µg/kg) and three different doses of TPA (2, 4 and 8 µg/kg). The average number and induction efficacy of tumors in response to DMBA+TPA treatment were significantly greater in the BALB/cKorl substrain than in BALB/cA and BALB/cB. However, cotreatment with DMBA+TPA induced similar responses for body and organ weights of all three substrains. Few differences were detected in the serum analyzing factors, while similar responsiveness was observed for blood analyzing factors after DMBA+TPA treatment. Furthermore, the three BALB/c substrains exhibited similar patterns in their histopathological structure in DMBA+TPA-induced tumors. The expression levels of apoptotic proteins and tumor related proteins were constantly maintained in all three BALB/c substrains treated with DMBA+TPA. In addition, the responsiveness to cisplatin treatment was overall very similar in the three BALB/c substrains with DMBA+TPA-induced tumors. Taken together, these results indicate that genetic background of the three BALB/c substrains does not have a major effect on the DMBA+TPA-induced skin carcinogenesis and therapeutic responsiveness of cisplatin, except induction efficacy.

Fermented mulberry (Morus alba) leaves suppress high fat diet-induced hepatic steatosis through amelioration of the inflammatory response and autophagy pathway.

BACKGROUND: A novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) is reported to exert anti-obesity activity, although their molecular mechanism during hepatic steatosis has not verified. METHODS: To investigate the role of inflammation and autophagy during the anti-hepatic steatosis effects of EMfC, we measured alterations in the key parameters for inflammatory response and autophagy pathway in liver tissues of the high fat diet (HFD) treated C57BL/6N mice after exposure to EMfC for 12 weeks. RESULTS: Significant anti-hepatic steatosis effects, including decreased number of lipid droplets and expression of Klf2 mRNA, were detected in the liver of the HFD + EMfC treated group. The levels of mast cell infiltration, expression of two inflammatory mediators (iNOS and COX-2), and the MAPK signaling pathway were remarkably decreased in the liver of HFD + EMfC treated group as compared to the HFD + Vehicle treated group. Furthermore, a similar inhibitory effect was measured for the expression levels of pro-inflammatory cytokines, including IL-1ß, IL-6, TNF-α and NF-κB. The expression level of members in the AKT/mTOR signaling pathway (a central regulator in autophagy) was recovered after treatment with EMfC, and autophagy-related proteins (Beclin and LC3-II) were remarkably decreased in the HFD + EMfC treated group compared to the HFD + Vehicle treated group. Moreover, the HFD + EMfC treated group showed decreased transcript levels of autophagy-regulated genes including Atg4b, Atg5, Atg7 and Atg12. CONCLUSIONS: Taken together, findings of the present study provide novel evidences that the anti-hepatic steatosis of EMfC is tightly linked to the regulation of the inflammatory response and autophagy pathway in the liver tissue of HFD-induced obesity mice.

Compensatory role of C3 convertase on the strain difference for C3 protein expression in FVB/N, C3H/HeN and C57BL/6N mice.

To investigate the role of complement C3 (C3) convertase on the strain difference for C3 protein expression in three inbred mice strains, we compared the levels of C2, C3 and C4 mRNA, as well as C3 protein and C3 convertase activity in the serum and liver tissue of FVB/N, C3H/HeN and C57BL/6N mice. The level of mRNA, inactive form (InACF) and active form (ACF) for C3 showed a regular pattern, which they were higher in the FVB/N and C57BL/6N mice than C3H/HeN mice. However, the level of C3b fragments (C3bα and ß) derived from C3 protein were constantly maintained in the liver of FVB/N, C3H/HeN and C57BL/6N mice in spite of the strain difference on the transcriptional and translation level of C3. Especially, a reverse pattern of the level of mRNA, InACF and ACF for C3 was observed on the activity level of C3 convertase activity. The highest level of C3 convertase activity was measured in C3H/HeN mice, followed by C57BL/6N and FVB/N mice. In case of C3 convertase components, the level of C2 mRNA was higher in C3H/HeN mice than FVB/N and C57BL/6 N mice, while levels of C4 mRNA were higher in FVB/N and C57BL/6N mice than C3H/HeN mice. The current study results provide the first scientific evidence that C3 convertase may play complementary role to overcome the strain difference on the C3 protein expression in FVB/N, C3H/HeN and C57BL/6N mice.

Anti-Oxidant Activity of Gallotannin-Enriched Extract of Galla Rhois Can Associate with the Protection of the Cognitive Impairment through the Regulation of BDNF Signaling Pathway and Neuronal Cell Function in the Scopolamine-Treated ICR Mice.

The antibacterial, anti-inflammatory, anti-metastatic/anti-invasion activities and laxative activity of Galla Rhois (GR) are well-known, although the neuropreservation effects of their extracts are still to be elucidated. To investigate the novel therapeutic effects and molecular mechanism of GR on alleviation of cognitive impairment, two different dosages of gallotannin-enriched GR (GEGR) were administered to Korl:ICR mice for three weeks, and to induce memory impairment, scopolamine (SP) was administered during the last seven days of the GEGR treatment period. GEGR showed the high level of the free radical scavenging activity to DPPH and suppressive activity to reactive oxygen species (ROS) in B35 cells as well as enhanced SOD and CAT activity in brains of the SP-induced model. Latency time for memory impairment assessed by the passive avoidance test significantly protected in the SP+GEGR treated group as compared to the SP+Vehicle treated group. Moreover, similar protective effects were observed on the secretion of BDNF in SP+GEGR treated mice. The expression of TrkB receptor, and phosphorylation of PI3K on the TrkB receptor signaling pathway were dramatically protected in the SP-induced model after GEGR treatment, whereas the expression of p75NTR receptor, the phosphorylation of JNK, and expression of Bax/Bcl-2 on the p75NTR receptor signaling pathway was significantly protected in the same group. Furthermore, the GEGR treated SP-induced model showed decreased number of dead neural cells and suppressed acetylcholine esterase (AChE) activity and inhibited inflammatory responses. Taken together, these results indicate that the anti-oxidant activity of GEGR contributes to improving the neuronal cell function and survival during cognitive impairment in the SP-induced model through regulation of BDNF secretion and their receptor signaling pathway.

Constipation in Tg2576 mice model for Alzheimer's disease associated with dysregulation of mechanism involving the mAChR signaling pathway and ER stress response.

BACKGROUND: Although constipation has been researched in various neurological disorders, including Parkinson's disease (PD) and spinal cord injury (SCI), the pathological mechanism of this symptom has not been investigated in Alzheimer's disease (AD) associated with loss of nerve cells in the brain. This study was undertaken to gain scientific evidences for a molecular correlation between constipation and AD. METHODS: To understand the etiology, we measured alterations in various constipation parameters, muscarinic acetylcholine receptors (mAChRs) and endoplasmic reticulum (ER) stress response, in 11-month-old Tg2576 transgenic (Tg) mice showing AD-like phenotypes. RESULTS: A high accumulation of amyloid beta (Aß) peptides, a key marker of AD pathology, were detected in the cortex and hippocampus of Tg mice. Furthermore, significant alterations were observed in various constipation parameters including stool weight, histological structure, cytological structure and mucin secretion in Tg2576 mice. Moreover, M2 and M3 expression and the downstream signaling pathways of mAChRs were decreased in the Tg group, as compared with non-Tg (NT) group. Furthermore, activation of ER stress proteins and alteration of ER structure were also detected in the same group. CONCLUSIONS: The results of the present study provide strong novel evidence that the neuropathological constipation detected in Tg2576 mice is linked to dysregulation of the mAChR signaling pathways and ER stress response.

Laxative Effect of Spicatoside A by Cholinergic Regulation of Enteric Nerve in Loperamide-Induced Constipation: ICR Mice Model.

Researches on spicatoside A (SpiA)-containing natural products suggest the possibility of SpiA as a potential laxative to alleviate chronic constipation. However, no studies have been conducted with single compound administration of SpiA. To verify the laxative effects and mechanism of action of SpiA on chronic constipation, we investigated alterations in the excretion parameters, histological structure, and cholinergic regulation of the enteric nerve in the colons of Institute of Cancer Research (ICR) mice with loperamide (Lop)-induced constipation after exposure to 20 mg/kg of SpiA. Decrease in the number, weight and water contents of stools in the Lop+Vehicle treated group significantly recovered after SpiA treatment, and alterations in the histological structure and transmission electron microscopy (TEM) images were improved in the Lop+SpiA treated group. Similar recovery effects were observed in the ability for mucin secretion and expression of the membrane water channel gene (aquaporin 8, AQP8). Furthermore, significant improvements were observed in the acetylcholinesterase (AChE) activity and acetylcholine receptors' (AChRs) downstream signaling pathway after treatment of SpiA. The levels of gastrointestinal (GI) hormones including cholecystokinin (CCK) and gastrin were also remarkably enhanced in the Lop+SpiA treated group as compared to the Lop+Vehicle treated group. The expression of receptor tyrosine kinase (C-kit) and protein gene product 9.5 (PGP9.5) in Cajal and neural cells, as well as the phosphorylation of myosin light chain (MLC) in smooth muscle cells, were recovered after SpiA exposure. Taken together, the results of the present study provide the first strong evidence that SpiA improves chronic constipation through muscarinic cholinergic regulation of the enteric nerve in a Lop-induced constipation ICR mice model.

Anti-Inflammatory Response and Muscarinic Cholinergic Regulation during the Laxative Effect of Asparagus cochinchinensis in Loperamide-Induced Constipation of SD Rats.

Several types of saponins and herbal plants containing saponins have been reported to have anti-inflammatory or laxative activities. To verify the therapeutic effects of saponin-enriched extracts of Asparagus cochinchinensis (SPA) on the anti-inflammatory response and on the cholinergic regulation in the gastrointestinal system, an alteration on the constipation phenotypes, on the inflammatory responses, and on the muscarinic cholinergic regulation were investigated in the transverse colons of Sprague Dawley (SD) rats with loperamide (Lop)-induced constipation after the treatment of SPA. Significant increases were observed on the total number of stools, the gastrointestinal transit, the thickness of the mucosal layer, the flat luminal surface, the number of paneth cells, and the lipid droplets in the Lop + SPA-treated group as compared to the Lop + Vehicle-treated group. SPA treatment induced the recovery of inflammatory cytokines (TNF-α, IL-1ß) and IL-6), inflammatory mediators (NF-κB and iNOS), the total number of infiltered mast cells, and mucin secretion. Also, some similar improvements were observed on the levels of acetylcholine esterase (AChE) activity and on the phosphorylation of myosin light chains (MLC) as well as the expression of muscarinic acetylcholine receptors M2/M3 (mAChR M2/M3) and their mediators. The results presented herein provide the first strong evidence that SPA stimulates anti-inflammatory responses and the muscarinic cholinergic regulation when exerting its laxative effects in the chronic constipation of Lop-induced models.

Metabolomics approach to serum biomarker for laxative effects of red Liriope platyphylla in loperamide-induced constipation of SD rats.

Red Liriope platyphylla (RLP) is a known herbal medicine used in the treatment of some chronic diseases including constipation, neurodegenerative disorders, diabetes and obesity. To determine and characterize putative biomarkers that predict the laxative effects induced by RLP treatment, alteration of endogenous metabolites was measured in the serum of loperamide (Lop)-induced constipation rats after administration of RLP extract (EtRLP) using 1H nuclear magnetic resonance (1H NMR) spectral data. The urine volume and amounts, and weights and water contents of stools were significantly recovered in the Lop + EtRLP treated group as compared to the No group, whereas body weight and food intake maintained constant levels. Also, significant recoveries in the thickness of mucosa and muscle were detected in the colon of the Lop + EtRLP treated group. Furthermore, pattern recognition showed absolutely different clustering of the serum analysis parameters when comparing the Lop treated group and Lop + EtRLP treated group. Of the 33 endogenous metabolites, 7 amino acids (alanine, arginine, glutamate, glutamine, glycine, threonine and valine) and 8 endogenous metabolites (betaine, creatine, glucose, taurine, ethanol, lactate, glycerol and succinate) were dramatically increased in the Lop + EtRLP treated SD rats. These results provide the first evidence pertaining to metabolic changes in the constipation rats treated with Lop + EtRLP. Additionally, these findings correlate with changes observed in 15 metabolites during the laxative effects of EtRLP.

Comparison of responsiveness to cancer development and anti-cancer drug in three different C57BL/6N stocks.

In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1ß, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.

Four amino acids as serum biomarkers for anti-asthma effects in the ovalbumin-induced asthma mouse model treated with extract of Asparagus cochinchinensis.

The butanol extract of Asparagus cochinchinensis roots fermented with Weissella cibaria (BAW) effectively prevents inflammation and remodeling of airway in the ovalbumin (OVA)-induced asthma model. To characterize biomarkers that can predict the anti-asthmatic effects induced by BAW treatment, we measured the alteration of endogenous metabolites in the serum of OVA-induced asthma mice after administration of low concentration BAW (BAWLo, 250 mg/kg) and high concentration BAW (BAWHi, 500 mg/kg) using 1H nuclear magnetic resonance (1H-NMR) spectral data. The number of immune cells and serum concentration of IgE as well as thickness of the respiratory epithelium and infiltration of inflammatory cells in the airway significantly recovered in the OVA+BAW treated group as compared to the OVA+Vehicle treated group. In the metabolic profile analysis, the pattern recognition showed completely separate clustering of serum analysis parameters between the OVA+Vehicle and OVA+BAW treated groups. Of the total endogenous metabolites, 19 metabolites were upregulated or downregulated in the OVA+Vehicle treated group as compared to the Control treated group. However, only 4 amino acids (alanine, glycine, methionine and tryptophan) were significantly recovered after BAWLo and BAWHi treatment. This study provides the first results pertaining to metabolic changes in the asthma model mice treated with OVA+BAW. Additionally, these findings show that 4 metabolites can be used as one of biomarkers to predict the anti-asthmatic effects.

Dose dependence and durability of the therapeutic effects of Asparagus cochinchinensis fermented extract in an ovalbumin-challenged asthma model.

The butanol extract of Asparagus cochinchinensis roots fermented with Weissella cibaria (BAfW) significantly suppressed the inflammatory response induced by lipopolysaccharide (LPS) treatment in RAW264.7 cells. To investigate the dose dependence and durability of BAfW on the anti-asthma effects, alterations in key parameters were measured in ovalbumin (OVA)-challenged Balb/c mice treated with the different doses of BAfW at three different time points. The number of immune cells, OVA-specific IgE level, thickness of respiratory epithelium and mucus score decreased significantly in a dose-dependent manner in response to treatment with 125 to 500 mg/kg BAfW (P<0.05), although the highest level was detected in the 500 mg/kg treated group. Moreover, the decrease in these parameters was maintained from 24 to 48 h in the 500 mg/kg of BAfW treated group. At 72 h, the effects of BAfW on the number of immune cells, OVA-specific IgE level and thickness of respiratory epithelium partially disappeared. Overall, this study provides the first evidence that the anti-asthma effect of BAfW may reach the maximum level in OVA-challenged Balb/c mice treated with 500 mg/kg and that these effects can last for 48 h.

Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA.

To evaluate the carcinogenicity of p27 knockout (KO) mice with RNA-guided endonuclease (RGENs)-mediated p27 mutant exon I gene (IΔ), alterations in the carcinogenic phenotypes including tumor spectrum, tumor suppressor proteins, apoptotic proteins and cell cycle regulators were observed in p27 (IΔ) KO mice after treatment with 7,12-Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)(DT) for 5 months. The target region (544~571 nt) in exon I of the p27 gene was successfully disrupted in p27 (IΔ) KO mice using the RGEN-induced non-homologous end joining (NHEJ) technique. After DT exposure for 5 months, a few solid tumors (identified as squamous cell carcinoma) developed on the surface of back skin of DT-treated p27 (IΔ) KO mice. Also, squamous cell hyperplasia with chronic inflammation was detected in the skin dermis of DT-treated p27 (IΔ) KO mice, while the Vehicle+p27 (IΔ) KO mice and WT mice maintained their normal histological skin structure. A significant increase was observed in the expression levels of tumor suppressor protein (p53), apoptotic proteins (Bax, Bcl-2 and Caspase-3) and cell-cycle regulator proteins (Cyclin D1, CDK2 and CDK4) in the skin of DT-treated p27 (IΔ) KO mice, although their enhancement ratio was varied. Taken together, the results of the present study suggest that squamous cell carcinoma and hyperplasia of skin tissue can be successfully developed in new p27 (IΔ) KO mice produced by RGEN-induced NHEJ technique following DT exposure for 5 months.

The Anti-Inflammatory Effects of Fermented Herbal Roots of Asparagus cochinchinensis in an Ovalbumin-Induced Asthma Model.

Introduction: Roots of Asparagus cochinchinensis, which have pharmacologically active ingredients, have received great attention because they show good therapeutic effects for various inflammatory diseases without specific toxicity. This study investigated the anti-asthmatic effects of a butanol extract of Asparagus cochinchinensis roots that had been fermented with Weissella cibaria (BAW) and its possible underlying cholinergic regulation. Methods: Alterations of the anti-asthmatic markers and the molecular response factors were measured in an ovalbumin (OVA)-induced asthma model after treatment with BAW. Results: Treatment with BAW decreased the intracellular reactive oxygen species (ROS) production in lipopolysaccharides (LPS) activated RAW264.7 cells. The results of the animal experiments revealed lower infiltration of inflammatory cells and bronchial thickness, and a significant reduction in the number of macrophages and eosinophils, concentration of OVA-specific IgE, and expression of Th2 cytokines in the OVA + BAW treated group. In addition, a significant recovery of goblet cell hyperplasia, MMP-9 expression, and the VEGF signaling pathway was observed upon airway remodeling in the OVA + BAW treated group. Furthermore, these responses of BAW were linked to recovery of acetylcholine esterase (AChE) activity and muscarinic acetylcholine receptor (mAChR) M3 downstream signaling pathway in epithelial cells, smooth muscle cells, and afferent sensory nerves of OVA + BAW-treated mice. Conclusion: Overall, these findings are the first to provide evidence that the therapeutic effects of BAW can prevent airway inflammation and remodeling through the recovery of cholinergic regulation in structural cells and inflammatory cells of the chronic asthma model.

Morusin Functions as a Lipogenesis Inhibitor as Well as a Lipolysis Stimulator in Differentiated 3T3-L1 and Primary Adipocytes.

Conflicting results for morusin activity during adipogenic differentiation are reported in 3T3-L1 adipocytes and cancer cells. To elucidate the influence of morusin on fat metabolism, their anti-obesity effects and molecular mechanism were investigated in 3T3-L1 cells and primary adipocytes. Morusin at a dose of less than 20 µM does not induce any significant change in the viability of 3T3-L1 adipocytes. The accumulation of intracellular lipid droplets in 3T3-L1 adipocytes stimulated with 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, 10 µg/mL insulin in DMEM containing 10% FBS (MDI)-significantly reduces in a dose-dependent manner after morusin treatment. The phosphorylation level of members in the MAP kinase signaling pathway under the insulin receptor downstream also decrease significantly in the MDI + morusin-treated group compared to MDI + vehicle-treated group. Also, the expression of adipogenic transcription factors (PPARγ and C/EBPα) and lipogenic proteins (aP2 and FAS) are significantly attenuated by exposure to the compound in MDI-stimulated 3T3-L1 adipocytes. Furthermore, the decrease in the G0/G1 arrest of cell cycle after culturing in MDI medium was dramatically recovered after co-culturing in MDI + 20 µM morusin. Moreover, morusin treatment induces glycerol release in the primary adipocytes of SD rats and enhances lipolytic protein expression (HSL, ATGL, and perilipin) in differentiated 3T3-L1 adipocytes. Overall, the results of the present study provide strong evidence that morusin inhibits adipogenesis by regulating the insulin receptor signaling, cell cycle and adipogenic protein expression as well as stimulating lipolysis by enhancing glycerol release and lipolytic proteins expression.