Proteomic and molecular evidences of Il1rl2, Ric8a, Krt18 and Hsp90b1 modulation during experimental hepatic fibrosis and pomegranate supplementation.

The inspection of variations in the proteomic aspects conspire the biomarker discovery in diagnostics of peculiar diseases. Recent developments in high-throughput proteomic techniques have provided leverage in the discovery of biomarkers during the etiology of various diseases. We identified potential biomarkers by utilizing proteomics, bioinformatics and gene expression studies. Meticulous assessment of collagen and hydroxyproline levels along with the glycogen and protein carbonyl levels exhibited deterioration in the N' - Nitrosodiethylamine (NDEA) administered rat livers and subsequent salubrious effect of pomegranate juice. The immunohistochemical inspection of iNOS and nitrite estimation indicated the peccant fibrotic alterations. 2D proteome profiling and MALDI-TOF MS/MS furthered the significant biomarkers to be analyzed for the gene ontology by PANTHER, cluster analysis by DAVID and network simulation by STRING 10.0. Several genes found relevant after MALDI analysis were evaluated by real-time PCR (RTPCR). Our data revealed CYP2b15, HSP70, TRFE, HPT, Il1rl2, Ric8a, Krt18, Hsp90b1 and iNOS as novel biomarkers for the mechanism of pomegranate against liver fibrosis. It can be inferred that NDEA-induced liver fibrosis actuates various biological pathways by the identified biomarkers and pomegranate juice modifies them.

Effect of Synbiotic and Probiotic Supplementation on Serum Levels of Endothelial Cell Adhesion Molecules in Hemodialysis Patients: a Randomized Control Study.

The aim of this study was to investigate the effect of synbiotic and probiotic supplementation on serum vascular dysfunction and necrosis markers in hemodialysis (HD) patients. In this randomized, double-blind, placebo-controlled trial, 75 HD patients were randomly assigned to either the synbiotic or probiotic or placebo group. The patients in the synbiotic group received 15 g of prebiotics and 5 g probiotic powder containing Lactobacillus acidophilus strain T16 (IBRC-M10785), Bifidobacterium bifidum strain BIA-6, Bifidobacterium lactis strain BIA-6, Bifidobacterium longum strain LAF-5 (2.7 × 107 CFU/g each) in sachets (n = 25), whereas the probiotic group received 5 g probiotics same to the first group with 15 g of maltodextrin powder in sachets (n = 25) and the placebo group received 20 g of maltodextrin powder in sachets (n = 25) for 12 weeks. At baseline and the end of the study, serum concentrations of soluble intercellular adhesion molecule type 1 (sICAM-1), soluble vascular cell adhesion molecule type 1 (sVCAM-1), cytokeratin 18 (CK-18) as the necrosis marker, uric acid, and phosphate levels were measured. Feces also were collected for microbiota colony counting. Serum ICAM-1 level reduced significantly in the synbiotic group after the intervention period (P = 0.02), and this reduction was significantly different in the synbiotic group in comparison to the placebo group (P = 0.03). Serum levels of VCAM-1 and CK-18 were not significantly different between the groups. However, the reduction in serum levels of VCAM-1 in the synbiotic group was significantly higher in comparison to the placebo group (P = 0.01). Multivariate linear regression analysis revealed that ∆ phosphate was the sole independent determinant of ∆ICAM-1 (P = 0 < 001). The study indicated that synbiotic supplementation reduced serum ICAM-1 level, which is a risk factor for cardiovascular diseases in HD patients, but has no effect on the necrosis marker. Trial registration: www.irct.ir (IRCT2017041233393N1).

Yi Guan Jian decoction may enhance hepatic differentiation of bone marrow­derived mesenchymal stem cells via SDF­1 in vitro.

A previous study reported that Yi Guan Jian (YGJ) may increase the proliferation and differentiation of hepatic oval cells in a rat liver cirrhosis model. The aim of the present study was to investigate the effect and mechanism of action of YGJ on inducing hepatic differentiation in bone marrow­derived mesenchymal stem cells (BM­MSCs) via stromal­cell derived factor­1 (SDF­1). Murine BM­MSCs were isolated with whole bone marrow adherence, then identified by immunocytochemical staining and flow cytometry. Passage 2 cells were divided into 8 groups and their differentiation was induced by cell factors added to the medium, including hepatocyte growth factor (HGF), SDF­1 and YGJ. Each of the cell factors was used alone and any two or three of them were combined to establish different cell microenvironments in the different treatment groups. Albumin (ALB) was selected as a hepatocellular marker and cytokeratin­18 (CK­18) as a cholangiocellular marker. The protein and mRNA expression levels of ALB and CK­18 were used to determine the differentiation of BM­MSCs using immunocytochemical staining, western blotting and reverse transcription­quantitative polymerase chain reaction on days 7, 14, 21 and 28 during induction. The relative expression levels of ALB and CK­18 resulted in time­dependent increases in the groups supplemented only with HGF, SDF­1 or YGJ. Combination treatment of any two HGF, SDF­1 and YGJ led to a higher expression of ALB and CK­18 compared with only one cell factor treatment. Additionally, when all three were used in a combined treatment the expression levels of ALB and CK­18 occurred at an earlier time and was higher overall. Therefore, the present study suggested that YGJ had an effect on inducing hepatic differentiation in BM­MSCs via SDF­1 and may act in a synergistic manner with HGF and SDF­1.

[Keratin 18 phosphorylation increases autophagy of colorectal cancer HCT116 cells and enhanced its sensitivity to oxaliplatin].

OBJECTIVE: To study the correlation between the phosphorylation of keratin 18 (K18) and the autophagy and apoptosis of HCT116 cells under the effect of oxaliplatin (OXA) and investigate its possible mechanism. METHODS: HCT116 cells were transfected with empty plasmid, wild-type K18 expression plasmid and 33, 52 phosphorylation site mutated K18 (Ser33/52A) expression plasmid separately, and all cells were then treated with 60 µmol/L OXA, followed by supplementation of autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin. FITC-conjugated annexin V and propidium iodide (PI) double staining combined with flow cytometry, calcein-AM/PI staining were used to analyze the effects of K18 and its mutants on cell apoptosis; Western blotting was performed to detect the expressions of K18 phosphorylation, autophagy related proteins microtubule associated protein 1 light chain 3 (LC3) and beclin-1. RESULTS: Transfection of Ser33/52A plasmid significantly reduced the level of K18 phosphorylation. After treated with OXA, the apoptosis rate of K18 plasmid transfected group was significantly higher than that of empty plasmid transfected group, while the apoptosis rate of Ser33/52A plasmid transfected HCT116 cells was significantly lower than that of empty plasmid or K18 plasmid transfected group. Compared with empty plasmid group, the autophagy of K18 plasmid transfected group was significantly promoted, while the autophagy in Ser33/52A plasmid transfected group was significantly inhibited. CONCLUSION: K18 overexpression enhanced the autophagy in HCT116 cells and increased its sensitivity to OXA. The decrease of K18 ser33 and ser52 phosphorylation inhibited autophagy and decreased apoptosis of HCT116 cells.

Oblongifolin C inhibits metastasis by up-regulating keratin 18 and tubulins.

Tumor metastasis is the main cause of cancer-related patient death. In this study, we performed a wound healing migration screen to search for a metastatic inhibitor within our library of natural compounds. We found that oblongifolin C (OC), a natural compound extracted from Garcinia yunnanensis Hu, is an effective inhibitor of metastasis in human esophageal squamous carcinoma Eca109 cells. The transwell migration and matrigel invasion assay results also showed that OC inhibits the migration of Eca109 cells and HepG2 cells. OC can increase the expression of tubulin, indicating that OC inhibits metastasis via tubulin aggregation. In addition, the Western blotting, real-time PCR, and immunostaining results indicated that OC increases the expression of keratin18. Furthermore, the knockdown of keratin 18 by small interfering RNAs inhibited the expression of tubulin and increased the metastasis of cancer cells, suggesting that keratin 18 is the upstream signal of tubulin and plays a vital role in metastasis. A subsequent study in a tail vein injection metastasis model showed that OC can significantly inhibit pulmonary metastasis, as revealed by immunohistochemistry staining. Taken together, our results suggest that OC inhibits metastasis through the induction of the expression of keratin 18 and may be useful in cancer therapy.

Effect of allogeneic umbilical cord mesenchymal stem cell transplantation in a rat model of hepatic cirrhosis.

OBJECTIVE: To determine the effects of human umbilical cord mesenchymal stem cell (UCMSC) transplantation, alone or in combination with tanshinone IIA (Tan IIA) on hepatic cirrhosis in rats. METHODS: A rat model of cirrhosis was established. Rats were divided into control, UCMSC, and UCSMC plus Tan IIA groups. Rats in the UCMSC group were injected via the tail vein with 0.2 mL Dil-labeled UCMSC suspension. Intraperitoneal Tan IIA injections (20 mg/kg) were started on the day of UCMSC transplantation in the UCMSC plus Tan IIA group, and continued for 7 consecutive days thereafter. Rats were sacrificed 1 day, 3 days, 1 month, and 3 months after transplantation and the numbers of Dil-labeled UCMSCs colonizing the liver were determined. Albumin (ALB) and alanine aminotransferase (ALT) levels were measured in venous blood, and mRNA and protein expression levels of human ALB and cytokeratin (CK)-18 in liver tissues were determined by reverse transcription-polymerase chain reaction and western blotting, respectively. RESULTS: Serum ALT levels were significantly lower and serum ALB levels significantly higher in rats in the UCMSC group compared with the control group (P < 0.05). Hepatic CK-18 and ALB mRNA and protein expression levels increased after transplantation, and were significantly higher in the UCMSC plus Tan IIA group compared with the UCMSC group (P < 0.05). CONCLUSION: Human UCMSCs transplanted into rats with liver cirrhosis can grow and differentiate into hepatocyte-like cells resulting in improved liver function in vivo. Tan IIA further influenced transplantation outcomes.

Dietary squid ink polysaccharide induces goblet cells to protect small intestine from chemotherapy induced injury.

Gastrointestinal mucositis induced by chemotherapy is associated with alterations of intestinal barrier function due to the potential damage induced by anti-cancer drugs on the epithelial cells. Goblet cells, an important epithelial lining in the intestine, contribute to innate immunity by secreting mucin glycoproteins. Employing a mouse model of chemotherapy induced intestinal mucosal immunity injury by cyclophosphamide, we demonstrated for the first time that polysaccharide from the ink of Ommastrephes bartramii (OBP) enhanced Cyto18, which is a mucin expression in goblet cells. The up-regulation of mucins by OBP relied on the augmented quantity of goblet cells, but not on the changes in the ultrastructure of endoplasmic reticulum (ER). Our results may have important implications for enhanced immunopotentiation function of functional OBP on intestinal mucosal immunity against intestinal disorders involving inflammation and infection.

Proteomic and bioinformatic analyses of possible target-related proteins of gambogic acid in human breast carcinoma MDA-MB-231 cells.

Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.

Effects of ligustrazine on ureteral obstruction-induced renal tubulointerstitial fibrosis.

Ligustrazine (LIG) is a purified and chemically identified component of the Chinese herb Ligusticum wallichii Franchat. It is a potent blocker of vasoconstriction and has strong scavenger of oxygen free radicals. We investigated the effect of LIG on renal tubulointerstitial fibrosis using a rat model of unilateral ureteral obstruction. Ligustrazine treatment significantly reduced the scores of interstitial collagen deposition, amounts of hydroxyproline, the density of myofibroblasts and macrophages, and amounts of transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF) compared with their level in a saline-treated control group. Using quantitative polymerase chain reaction we found that LIG treatment significantly reduced the mRNA expression of TGF-ß1, CTGF, monocyte chemoattractant protein-1 and osteopontin. Moreover, the mRNA expression of hepatocyte growth factor and bone morphogenetic protein-7 were significantly increased by LIG. In vitro, LIG inhibited the TGF-ß1-induced loss of cytokeratin-18 expression and de novo increase of the expression of α-smooth muscle actin of HK-2 cells in a dose-dependent manner, which suggested that LIG could restrain the process of epithelial-myofibroblast transition of tubular epithelial cells. This study indicates that LIG can attenuate renal tubulointerstitial fibrosis. It might be useful as a potential candidate in the treatment of chronic renal diseases.

Cytokeratin 18 expression inhibits cytokine-induced death of cervical cancer cells.

OBJECTIVES: In cervical cancer, increased cytokeratin 18 (CK18) filament expression is associated with disease progression. However, it may also provide resistance to cytokine-induced apoptosis. The present study tested whether CK18 expression influences susceptibility to cytokine-induced apoptosis. METHODS: The cervical cancer cell lines C-4II (high CK18 expression), ME-180 (low CK18 expression), and 2 subtypes of HeLa cells containing or lacking CK18 expression (CK18+ and CK18- cells, respectively) were exposed to vehicle (control), Fas ligand (FasL) (50 ng/mL), or tumor necrosis factor α (TNF-α; 10 ng/mL) without/with cycloheximide (CHX; 2.5 µg/mL) to test the hypothesis that diminished CK18 expression increases susceptibility to cytokine-induced apoptosis. RESULTS: Flow cytometric analysis of cell death via TUNEL staining revealed that cytokine-induced apoptosis was 2-fold greater in ME-180 cells than C-4II cells in response to FasL+CHX or TNF-α+CHX (P < 0.05). Similarly, there was a higher incidence of FasL-induced apoptosis in CK18- HeLa cells (23% and 91% apoptotic for FasL and FasL+CHX, respectively) than CK18+ HeLa cells (1% and 11%, respectively; P < 0.05). Surprisingly, TNF-α had no effect on either CK18+ or CK18- HeLa cells (P > 0.05). Caspase 3 activity was greater in CK18- HeLa cells than in CK18+ HeLa cells at 8 and 18 hours after FasL treatment (P < 0.05), an effect abrogated by the caspase 8 inhibitor IETD-fmk (P < 0.05). CONCLUSIONS: Cervical cancer cells with diminished CK18 expression are more susceptible to cytokine-induced apoptosis, particularly in response to FasL treatment. These observations suggest that relative CK18 expression is an important factor when considering therapeutic strategies to enhance immune cell-mediated death of cervical cancer cells.

Transgenic zebrafish line with over-expression of Hedgehog on the skin: a useful tool to screen Hedgehog-inhibiting compounds.

We generated a transgenic line Tg(k18:shh:RFP) with overexpression of Sonic hedgehog in the skin epidermis. By 5 day-post-fertilization (dpf), many epidermal lesions were clearly observed, including a swollen yolk sac, epidermis growth malformation around the eyes and at the basement of the pectoral fins. Skin histology revealed embryos derived from Tg(k18:shh:RFP) displayed an elevated Nuclear/Cytoplasmic ratio and pleomorphic nuclei compared to their wild type littermates, suggesting the abnormal growth pattern on the epidermis of Tg(k18:shh:RFP) embryos were dysplasia. Later (by 7 dpf), Tg(k18:shh:RFP) embryos displayed broader pectoral fins which are similar to the polydactyly phenotypes of Nevoid basal cell carcinoma syndrome (NBCCS)/Gorlin patients and polydactylous mice. In addition, treatment with cyclopamine is able to enhance and prolong the survival rates and survival durations of Tg(k18:shh:RFP) embryos. In conclusion, this unique Tg(k18:shh:RFP) fish line, should be an excellent experimental animal for screening for a lower toxicity level of the new Hh-inhibitor and can even be used as a new anti-cancer drug-screening platform.