External stimuli, such as radiation, induce inflammatory cytokine and chemokine production in skin, but the mechanisms involved are not completely understood. We previously showed that the P2Y11 nucleotide receptor, p38 MAPK and NF-κB all participate in IL-6 production induced by γ-irradiation. Here, we focused on the transient receptor potential V4 (TRPV4) channel, which is expressed in skin keratinocytes and has been reported to play a role in inflammation. We found that irradiation of human epidermal keratinocytes HaCaT cells with 5 Gy of γ-rays (137Cs: 0.75 Gy/min) induced IL-6 and IL-8 production. HaCaT cells treated with TRPV4 channel agonist GSK1016790A also showed increased IL-6 and IL-8 production. In both cases, IL-6/IL-8 production was not increased at 24 h after stimulation, but was increased at 48 h. ATP was released from cells exposed to γ-irradiation or TRPV4 channel agonist, and the release was suppressed by TRPV4 channel inhibitors. The γ-irradiation-induced increase in IL-6 and IL-8 production was suppressed by apyrase (ecto-nucleotidase), NF157 (selective P2Y11 receptor antagonist) and SB203580 (p38 MAPK inhibitor). GSK1016790A-induced IκB decomposition, which causes NF-κB activation was suppressed by NF157 and SB203580, and γ-irradiation-induced IκB decomposition was suppressed by TRPV4 channel inhibitors. Our results suggest that γ-irradiation of keratinocytes induces ATP release via activation of the TRPV4 channel, and then ATP activates P2Y11 receptor and p38 MAPK-NF-κB signaling, resulting in IL-6/IL-8 production.
We evaluated the suitability of Nagoya Shibata Yasuda (NSY) mice as an animal model for examining the influence of a glucose metabolism disorder on bone integrity, using Institute of Cancer Research (ICR) mice as controls. We selected six NSY and ICR mice each that were matched for weight, and measured serum glucose levels, serum insulin levels, and conducted an oral glucose tolerance test. Histological sections of the femurs of both mouse lines were prepared, and the bone strength, mass, and microstructure of the femur were compared, along with bone metabolism. Serum glucose levels were significantly higher in the NSY mice than in the control mice, but body weight and serum insulin levels did not differ between the groups. Bone mass, microstructure, and strength of the femur, and bone metabolism were lower in the NSY mice than in the control mice. In the cortical bone of the femur in the NSY mice, several parts were not stained with eosin, demonstrating a strong negative correlation between serum glucose levels and bone mineral density; however, there was a negative correlation between serum glucose levels and bone metabolic markers. The bone turnover rate in the NSY mice was decreased by hyperglycemia, resulting in a thinner and shorter femur, reduced cortical and trabecular areas, and lower bone mass compared to those of the control mice. Collectively, these results suggest deteriorated bone strength of the femur in NSY mice, serving as a useful model for studying the link between glucose metabolism and bone integrity.
Upon differentiation of cells, remarkable changes in the structures of glycans linked to lipids on cell surface have been observed. Lactosylceramide (Lac-Cer) serves as a common precursor for a series of glycosphingolipids with diverse structures. In the present study, we examined the underlying mechanism for the biosynthesis of Lac-Cer upon differentiation of 3T3-L1 mouse preadipocytes to adipocytes. TLC analysis showed that the amounts of Lac-Cer decrease in 3T3-L1 adipocytes compared to 3T3-L1 preadipocytes. In accordance with this change, the gene expression level of β4-galactosyltransferase (β4GalT) 5, which was identified as Lac-Cer synthase, decreased drastically upon differentiation of 3T3-L1 preadipocytes. The analysis of the transcriptional mechanism of the β4GalT5 gene demonstrated that the core promoter region is identified between nucleotides -299 and -1 relative to the translational start site. During adipocyte differentiation, the expression levels and promoter activities of the β4GalT5 gene decreased dramatically. Since the Specificity protein 1 (Sp1)-binding sites in the promoter region were critical for the promoter activity, it is suggested that Sp1 plays an important role for the expression of the β4GalT5 gene in 3T3-L1 cells. The gene and protein expression of Sp1 decreased significantly upon differentiation of 3T3-L1 preadipocytes. Taken together, the present study suggest that the expression of the β4GalT5 gene decreases through reduced expression of the Sp1 gene and protein upon differentiation of 3T3-L1 peradipocytes to adipocytes, which may lead to the decreased amounts of Lac-Cer in 3T3-L1 adipocytes.
The present study aims to investigate the roles of StAR in Yangjing Capsule (YC) induced anti-apoptotic effects on Leydig cells and the related mechanism. Leydig tumor cells (MLTC-1) were cultured and treated with YC, and immunofluorescence assay was performed to examine the expression of StAR; furthermore, luciferase reporter assay was conducted to evaluate the impact of YC on StAR promoter; next, MLTC-1 cells were treated with StAR siRNA, and flow cytometry was carried out to examine the effect of StAR siRNA on the apoptosis of the cells; furthermore, qRT-PCR and western blot methods was used to determine the expression of StAR and apoptosis related molecules Bcl-2, Bax and Caspase-3 on both mRNA and protein levels in different groups; finally the secretion of testosterone in different groups was examined by radioimmunoassay. We observed that the YC can increase the expression of StAR in a dose-dependent manner, and YC can activate the promoter of StAR;moreover, transfection of StAR siRNA can block YC induced anti-apoptotic effects and increased production of testosterone. In conclusion, our results suggested that YC might suppress the apoptosis of MLTC-1 cells and enhance the production of testosterone through regulating the expression of StAR.
Tribulus terrestris L(Zygophyllaceae) (TT) is usually used as a cardiotonic, diuretic, and aphrodisiac, as well as for herbal post-stroke rehabilitation in traditional Chinese medicine. However, little is known about the renoprotective effects of TT on obesity-related glomerulopathy (ORG). In this study, 340 monomeric compounds were identified from TT extracts obtain with ethyl acetate combined with 50% methanol. In vitro, IC50 of TT was 912.01 mg/L, and the appropriate concentration of TT against ox-LDL induced human renal glomerular endothelial cells (HRGECs) was 4 mg/L. TT significantly increased the viability (63.2%) and migration (2.33-fold increase) of HRGECs. ORG model rats were induced by a chronic high-fat diet (45%) for 20 weeks and were then treated with TT extract (2.8 g/kg/d) for 8 weeks. Subsequently, the kidneys were removed and their differentially expressed protein profile was identified using two-dimensional electrophoresis coupled with MALDI-TOF-TOF mass spectrometry. Molecular categorization and functional analysis of bioinformatic annotation suggested that excessive energy metabolism, decreased response to stress and low immunity were the potential etiologies of ORG. After TT administration for 8 weeks, body weight, blood pressure, serum cystatin C and cholesterol were decreased. Additionally, TT significantly enhanced the resistance of rats to ORG, decreased energy consumption and the hemorrhagic tendency, and improved the response to acute phase reactants and immunity. In conclusion, TT may play a protective role against ORG in rats.
Calcium alginate (Ca-Alg) is known to suppress the postprandial increase of blood glucose, and therefore may be helpful for preventing lifestyle-related diseases such as diabetes. In this work, we examined the mechanism of this effect. As α-amylase activity and α-glucosidase activity are involved in the digestion of starch, we examined the in vitro inhibitory effect of Ca-Alg on these enzymes. Ca-Alg showed little inhibition of α-amylase, but markedly inhibited α-glucosidase activity. The direct binding affinity of glucose for Ca-Alg was low. Also, Ca-Alg had essentially no effect on the membrane permeability of glucose. Therefore, we considered that the suppression of blood glucose by Ca-Alg is predominantly due to a decrease in the efficiency of starch digestion as a result of inhibition of α-glucosidase, possibly due to increased viscosity of the gastrointestinal contents. Next, we investigated the optimum amount in the diet and the optimum particle size of Ca-Alg for suppressing postprandial blood glucose level in rats orally administered a diet containing starch with various amounts and particle sizes of Ca-Alg. We found that 5% by weight of 270-mesh-pass Ca-Alg was most effective.
We conducted a prospective, randomized, double-blind, 3-group, 3-phase crossover study to evaluate the effect of calcium alginate (Ca-Alg) on the postprandial increase of blood glucose in 15 healthy adult subjects who were given udon noodles containing or not containing Ca-Alg (5% or 8%). The value of ΔCmax (difference between the maximum (Cmax) and pre-feeding (C0) blood glucose levels) was significantly reduced in both Ca-Alg groups, and the area under the blood glucose level-time curve over 120 min (ΔAUC, with C0 as the baseline) was also significantly reduced. Thus, supplementation of noodles with Ca-Alg significantly suppressed both the peak postprandial blood glucose level and the total amount of glucose absorption. Blood calcium (Ca) concentration was significantly increased at 120 min after ingestion, but there was no marked change of other parameter values. A questionnaire indicated that addition of Ca-Alg did not affect the acceptability of the noodles. These results indicate that Ca-Alg might a useful food additive for helping to prevent lifestyle-related diseases without adversely affecting individual eating habits.
The intestinal barrier dysfunction is a critical pathological change in irritable bowel syndrome (IBS). The objective of this study was to evaluate the effect of Prim-O-glucosylcimifugin (POG) on intestinal barrier dysfunction and reveal possible molecular mechanisms. Caco-2 cell monolayers induced by tryptase(TRYP) were used to establish an intestinal barrier dysfunction model. Caco-2 cell monolayers from both functional and dysfunctional samples were treated with POG (30, 60 and 120 µg/mL) for 2, 8, 24, 36, 48 and 72h. The Caco-2 cell monolayers were assessed by measurement of trans-epithelial electrical resistance (TEER) and percentage of fluorescein permeation (PFP). The expression of PAR-2 and MLCK mRNA was analyzed by RT-PCR and the level of ZO-1 protein expression was determined by western blot. In addition, the impact of POG on the distribution of the tight juction protein of Occludin was performed by immunofluorescence. Our results showed that POG elevated the TEER and decreased the PFP of the functional Caco-2 cell monolayers, as well as the dysfunctional Caco-2 cell monolayers. Furthermore, POG inhibited the expression of PAR-2 mRNA and MLCK mRNA and increased the level of ZO-1 protein expression in dysfunctional Caco-2 cells. The distribution of the Occludin proteins was ameliorated simultaneously. This study demonstrates that POG can enhances the intestinal barrier function of Caco-2 cell monolayers by inhibiting the expression of PAR-2 and MLCK and up-regulating the expression of ZO-1 protein, and ameliorated the distribution of Occludin protein.
The aim of the present study was to investigate the protective effects of Shenfu injection (SFI) against myocardial ischemia/reperfusion injury (MIRI) in model rats and to explore its mechanism of action. Sprague-Dawley (SD) rats were pretreated with SFI and L-NAME via tail vein injection and then rats were subjected to ischemia by occlusion of the left anterior descending coronary artery for 30min followed by reperfusion for 120min. Left ventricular function was evaluated by echocardiography. Hemodynamic was measured by the Millar pressure - volume system; serum creatine kinase (CK), lactate dehydrogenase (LDH) and serum troponin (TNNI3) levels were determined. Myocardial infarct size was observed by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining; p-Akt/Akt, and p-eNOS/eNOS levels were assessed by western blotting; Nitric oxide (NO) content in serum was determined by the Griess reaction. SFI significantly decreased serum CK, LDH and TNNI3 levels in MIRI rats, while it significantly increased the level of left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), maximal rate of the increase of left ventricular pressure (+dp/dtmax), maximal rate of the decrease of left ventricular pressure (-dp/dtmax), left ventricle ejection fraction percentage (EF), and stroke volume (SV). In addition, SFI significantly reduced myocardial infarction area and activated the phosphorylation of eNOS via Akt. The phosphorylation of eNOS and the concurrent increase of NO production contributed significantly to the protective effects of SFI. These results demonstrate that SFI protects the rat heart against MIRI and that this effect is mediated in part by Akt/eNOS signaling.
Recent studies suggested that excessive Th1/17 cells concomitant with regulatory T cell deficiency might play important roles in Crohn’s disease. Anti-CD52 mAb, which aims on CD52 antigen on mature immunocytes, has both T cell depletion and immunosuppressive activities. In this study, we evaluated the therapeutic effects and possible mechanisms of anti-CD52 treatment on interleukin-10 deficient mouse. Anti-mouse CD52 mAb was administered to C3H.IL-10-/- mice intraperitoneally 20μg per week for 2 weeks. The disease activity index, body weight, the histological grading of colitis, and levels of TNF-α, IFN-γ, IL-17 and IL-6 in colon were quantified after treatment. In addition, CD25, Foxp3 and TGF-β gene as well as the percentage of CD25+Foxp3+ T cells in colon were also measured. The severity of colitis in IL-10-/- mice was significantly decreased by the treatment, with improvement of colon histological grade. The treatment also decreased the TNF-α, IFN-γ, IL-17 and IL-6 levels in colon. Furthermore, the treatment up-regulated the mRNA expression of CD25, Foxp3 and TGF-β gene as well as the percentage of CD25+Foxp3+ T cells in colon LPMCs of IL-10-/- mice. Our data might indicate that anti-CD52 treatment could ameliorate the colitis of C3H.IL-10-/- mice and it might be related to the suppression of Th1/17 related inflammation and the promotion of regulatory T cell differentiation. Thus, our data reveals that anti-CD52 treatment may hold potential for clinical applications for Crohn’s disease treatment.
Temozolomide (TMZ) is currently the first-line drug used for clinical postoperative or non-surgical chemotherapy for glioma, but acquired and intrinsic resistance to TMZ limits its application. The anti-proliferative effect of formononetin on human glioma cells had been confirmed. To improve therapeutic effects of TMZ, we studied the effect of formononetin in combination with TMZ on C6 glioma cells. The anti-proliferative effect of C6 cells was tested by MTT assay, The synergy was evaluated by Chou-Talalay method. Morphological changes were observed by hematoxylin-eosin (HE) staining. The effect of formononetin in combination with TMZ on apoptosis of C6 cells was investigated by flow cytometry. The effect of formononetin in combination with TMZ on migration of cells was investigated by wound healing assay and transwell assay. The expression of proteins related to apoptosis and migration were detected by western blot. These results showed that formononetin or TMZ alone could inhibit the growth of C6 cells in dose-dependent manner and formononetin in combination with TMZ had synergy effect on C6 cells. Further changes in cell morphology could be observed in drug combination by HE staining. Drug combination enhanced the expression of Bax , Cleaved Caspase-3, Cleaved Caspase-9, decreased the expression of Bcl-2, and promoted tumor cells apoptosis. In addition, the expression of matrix metalloproteninase-2 (MMP-2) and matrix metalloproteninase-9 (MMP-9) were down-reguated via drug combination which resulted into inhibit migration of C6 cell. In conclusion, formononetin in combination with TMZ can play a synergistic role in anti-C6 cells, the mechanisms of synergy depended on multiple pathways.
Chrysanthemum zawadskii var. latilobum (CZ) has been used as a traditional medicine in Asian countries for the treatment of inflammatory diseases. Recently, CZ extract was shown to inhibit differentiation of osteoclasts and provide protection against rheumatoid arthritis. The aim of this study was to investigate the molecular mechanisms of BST106, the ethanol extract of CZ, for cartilage protection in monosodium iodoacetate (MIA)-induced osteoarthritis (OA), particularly focusing on apoptosis and autophagy. BST106 (50, 100, and 200 mg/kg) was orally administered once daily to MIA-induced OA rats. Swelling, limping, roentgenography, and histomorphological changes were assessed 28 days after MIA injection. Biochemical parameters for matrix metalloproteinase, apoptosis, and autophagy were also assessed. BST106 ameliorated the severity of swelling and limping after MIA injection. Roentgenographic and histomorphological examinations revealed that BST106 reduced MIA-induced cartilage damage. BST106 decreased MIA-induced increases in MMP-2 and MMP-13 mRNA levels. Increased levels of serum cartilage oligomeric matrix protein and glycosaminoglycan release were attenuated by BST106. Furthermore, BST106 suppressed the protein expression of proapoptotic molecules and increased the protein expression of autophagosome- and autolysosome-related molecules. These findings indicate that BST106 protects against OA-induced cartilage damage by inhibition of the apoptotic pathway and restoration of impaired autophagic flux.
This article has been retracted by the Editorial Committee of The Pharmaceutical Society of Japan because it contains scientific misconduct. Although the data published in this article were generated in part by the first author, the authors violated authorship and sponsorship protocol.