Β-catenin Protein Expression Research Articles

MiR-19b-3p Inhibits Hypoxia-Ischemia Encephalopathy by Inhibiting SOX6 Expression via Activating Wnt/β-catenin Pathway.

Hypoxic-ischemic encephalopathy (HIE) is a detrimental factor in infant death and chronic disease. The specific pathogenesis is not entirely clear. Therefore, exploring the pathogenesis of HIE is critical. The expression of miR-19b-3p and SOX6 in umbilical blood of HIE patients was detected by qRT-PCR assay. HT22 cells were triggered with oxygen-glucose deprivation/reoxygenation (OGD/R) to construct the HIE cell model. Cell Counting Kit-8 (CCK-8) assay was used to estimate viability. SOD and MDA levels were detected by enzyme linked immunosorbent assay. Flow cytometry was implemented to ascertain neurocyte apoptosis. Cellular β-catenin immunofluorescence staining was used to detect the expression and distribution of β-catenin protein. Wnt signaling pathway activation was detected by TOPFlash/FOPFlash luciferase reporter assay. The targeting correlation of SOX6 and miR-19b-3p was corroborated by dual-luciferase reporter gene assay and RNA pull-down assay. MiR-19b-3p expression was once down-regulated, whilst SOX6 expression was up-regulated in HIE patients. MiR-19b-3p overexpression promoted cell proliferation, repressed cell apoptosis, oxidative stress response, and Wnt/β-catenin pathway activation in OGD/R-triggered HT22 cells. MiR-19b-3p negatively regulated SOX6 expression. SOX6 knockdown improved OGD/R-triggered HT22 cells injury via Wnt/β-catenin pathway activation. MiR-19b-3p overexpression suppressed OGD/R-triggered HT22 cell injury via inhibiting SOX6 expression via activating Wnt/β-catenin pathway.

In vitro and Pharmacoinformatics-based phytochemical screening for anticancer impacts of pistachio hull essential oil on AGS, PLC/PRF/5, and CACO2 cell lines.

The essential oil of pistacia vera (cv. Ohadi) hull (PHEO) was checked using gas chromatography mass spectrometry (GC/MS) analysis. It was studied the genes of the wnt pathway with a certain concentration of PHEO on Human gastric cancer (AGS), human hepatocellular carcinoma (PLC/PRF/5), and human colon cancer (CACO2) cell lines. After evaluating the survival rate of cancer cells by MTT test and determining IC50, pistachio hull essential oil (PHEO) was used for 24-hours to treat the cells. After RNA extraction, the expression of wnt pathway genes was evaluated by Real-Time PCR. Considering the crucial role of β-catenin accumulation and its effect on the progression of gastrointestinal cancers, Western blot analysis was also used to determine the effect of PHEO in protein expression of β-catenin inhibition. Also, an in silico analysis was carried out to investigate the effect of PHEO extracted compounds on protein expression of β-catenin and FZD7 inhibition. According to the results, wnt pathway genes were changed in samples treated using PHEO. The results showed the up-regulation of GSK-3β and down-regulation of Wnt-1, LEF-1, TCF1, and CTNNB1 genes compared to the control. We showed inhibition of β-catenin protein in cancer cell lines. Four compounds of PHEO were suggested to have an inhibition effect on β-catenin and FZD7. These compounds can be useful in the treatment of gastrointestinal cancers. Altogether, the inhibitory role of β-catenin protein can be very effective and can be considered one of the therapeutic goals in the treatment of gastrointestinal cancers.

Intervention Mechanism of Niao Du Kang Mixture on the EMT Process of Peritoneal Fibrosis Based on the Wnt/β-Catenin Signaling Pathway.

Quantification of 24-hour urine protein (24 h-Upro) and serum creatinine (Scr) levels was performed. The protein and mRNA expression levels of E-cadherin, α-SMA, collagen I, β-catenin, Wnt-1, and LEF-1 in peritoneal tissue were measured. In addition, the pathological morphology and ultrastructure of peritoneum were observed. After 5/6 nephrectomy + high glucose peritoneal dialysate + lipopolysaccharide (LPS) treatment, the Scr and 24 h-Upro of rats increased compared with normal rats, and the peritoneal tissue was damaged and thickened, showing fibrotic changes. Compared with the model group, the Scr and 24 h-Upro levels and the levels of E-cadherin, α-SMA, Wnt-1, collagen I, and LEF-1 protein expression in each Niao Du Kang mixture dosage group decreased. The protein expression of β-catenin and the mRNA expression of E-cadherin, α-SMA, Wnt-1, collagen I, β-catenin, and LEF-1 decreased in the high and medium Niao Du Kang mixture dose groups. Hematoxylin and eosin staining showed that the peritoneum of the rats was not only thicker in the model group than in the sham operation group (P < 0.01) but also accompanied by apparent inflammatory cell infiltration, tissue edema, and fibrosis. Compared with the model group, all the Niao Du Kang mixture groups demonstrated various degrees of mitigation in peritoneal thickness and fibrosis (P < 0.01). The strongest effect was observed in the medium-dose group. Transmission electron microscopy showed that the degree of injury of the peritoneal mesothelial cells was ranked as follows: model group > positive drug group > Niao Du Kang mixture high-dose group. The Niao Du Kang mixture may effectively decrease the peritoneal thickness and fibrosis degree through its effect on the Wnt/β-catenin signaling pathway involved in EMT. The present study provides data that assist in elucidating the potential function of the Niao Du Kang mixture in treating or preventing PF.

MiR-141-3p affects β-catenin signalingand apoptosis by targeting Ubtd2 in rats with anorectal malformations.

Anorectal malformations (ARMs) are the most common gastrointestinal malformations. miR-141-3p was obtained from whole-transcriptome sequencing, and Ub domain-containing protein 2 (Ubtd2) was predicted as the target gene. An ARM rat model was induced using ethylenethiourea. Fluorescence in situ hybridization and immunofluorescence were used to detect the spatiotemporal expression of miR-141-3p and Ubtd2, respectively. A dual-luciferase reporter assay confirmed their targeting relationship, and cell proliferation and apoptosis were investigated after transfection in the intestinal epithelium (IEC-6). Additionally, western blotting and co-immunoprecipitation were used to examine the protein levels and the endogenous binding relationship.miR-141-3p was downregulated in the ARM group, whereas Ubtd2 increased and colocalized with TUNEL-positive cells.After miR-141-3p inhibition, protein expression of USP5 and β-catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and β-catenin. Flow cytometry analysisandcaspase 3/7 staining demonstrated that downregulatedmiR-141-3p promoted cell apoptosis through Ubtd2. In summary, targeting Ubtd2 decreased in miR-141-3p and promoted apoptosis of intestinal epithelium and regulated β-catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of β-catenin signaling in the cloaca, further affecting the occurrence of ARMs.

Ketamine modulates neural stem cell differentiation by regulating TRPC3 expression through the GSK3β/β-catenin pathway

Ketamine, a popular anesthetic, is often abused by people for its hallucinogenic effect. Thus, the safety of ketamine in pediatric populations has been called into question for potential neurotoxic effects. However, ketamine also has neuroprotective effects in many brain injury models. The differentiation of neural stem cells (NSCs) was influenced significantly by ketamine, but the molecular mechanism is still unclear. NSCs were extracted from the hippocampi of postnatal day 1 rats and treated with ketamine to induce NSCs differentiation. Our results found that ketamine promoted neuronal differentiation of NSCs dose-dependently in a small dose range (P < 0.001). The main types of neurons from NSCs were cholinergic (51 ± 4 %; 95 % CI: 41–61 %) and glutamatergic neurons (34 ± 3 %; 95 % CI: 27–42 %). Furthermore, we performed RNA sequencing to promise a more comprehensive understanding of the molecules regulated by ketamine. Finally, we combined bioimaging and multiple molecular biology techniques to clarify that ketamine influences NSC differentiation by regulating transient receptor potential canonical 3 (TRPC3) expressions. Ketamine dramatically repressed TRPC3 expression (MD [95 % CI]=0.67 [0.40–0.95], P < 0.001) with a significant increase of phosphorylated glycogen synthase kinase 3β (p-GSK3β; MD [95 % CI]=1.00 [0.74–1.27], P < 0.001) and a decrease of β-catenin protein expression (MD [95 % CI]=0.60 [0.32–0.89], P = 0.001), thereby promoting the differentiation of NSCs into neurons and inhibiting their differentiation into astrocytes. These results suggest that TRPC3 is necessary for ketamine to modulate NSC differentiation, which occurs partly via regulation of the GSK3β/β-catenin pathway.

β-catenin ISGylation promotes lipid deposition and apoptosis in ethanol-stimulated liver injury models

ABSTRACT Background The restoration of the Wnt/β-catenin pathway to alleviate alcoholic fatty liver disease (AFLD) progression is under study as a new strategy for alcoholic liver disease (ALD) treatment. Recent studies have indicated that interferon-stimulated gene 15 (ISG15) can covalently bind to β-catenin by HECT E3 ubiquitin ligase 5 (HERC5), leading to ISG degradation and downregulation of β-catenin levels. However, the relationship between β-catenin and the ISG15 system in AFLD remains unclear. Methods Here, we explored the roles of the ISG15 system in β-catenin activation and in the pathogenesis of alcohol-induced liver injury and steatosis. Results In this study, HERC5 silencing upregulated β-catenin protein expression and inhibited lipid metabolism disorders and cell apoptosis. Reduced β-catenin protein expression, increased lipid metabolism disorders, and cell apoptosis were detected in cells induced with HERC5 overexpression, which was reversible with the reactive oxygen species (ROS) inhibitor. All the above results were statistically analyzed. Thus, these observations demonstrate that β-catenin ISGylation is a prominent regulator of ALD pathology, which works by regulating ROS to induce lipid metabolism disorders and cell apoptosis. Conclusion Our findings provided the mechanism involved in the β-catenin ISGylation, allowing for future studies on the prevention or amelioration of liver injury in ALD.

Identification and functional analysis of three novel osteogenic peptides isolated from tilapia scale collagen hydrolysate

On the basis of our previous study that 133 peptides were identified from the tilapia scale peptide-calcium chelate, the potential osteogenic peptide monomers through the calcium-binding properties of peptides and molecular docking were screened, and the osteogenic activity and active mechanism of the peptides were further researched in this study. Three highly osteogenic peptides GPAGPHGPVG (844.4191 Da), APDPFRMY (995.4534 Da), and TPERYY (827.3813 Da) were screened. Molecular docking showed that the three osteogenic peptides had the same interaction sites in the epidermal growth factor receptor (EGFR), namely ARG 285, GLN 8, GLY 317, THR 406, and HIS 409. Compared to the blank control group, within 50 μg/mL of GPAGPHGPVG, APDPFRMY, and TPERYY increased the proliferation of MC3T3-E1 cells by changing the cell proportion in the S and G2/M phases, and the alkaline phosphatase (ALP) activity of MC3T3-E1 cells treated with 50 μg/mL of the three active peptides increased by 25%, 37%, and 56%, respectively. The three active peptides at 10 μg/mL concentration significantly promoted the mineralization of osteoblasts, and the mineralized calcium nodules increased by 166%, 161%, and 111%, respectively. TPERYY significantly increased the expression of osteogenic genes (osteocalcin (OCN), type I collagen (Col I α) and transcription factor (OSX)). Moreover, TPERYY significantly increased the mRNA and protein expression of β-catenin to 1.39 and 2.6 times of the blank control group, respectively, while decreased the mRNA and protein expression of glycogen synthesis kinase (GSK3β) to 1.6 and 2.3 times of the blank control group, respectively. This study provides a theoretical basis for using GPAGPHGPVG, APDPFRMY, and TPERYY peptides as functional foods to prevent osteoporosis.

Comparison of a new thermosensitive rhAm carrier versus traditional PGA carrier for in vitro antibacterial activity and biocompatibility

To compare a new thermosensitive recombinant human amelogenin (rhAm) carrier and traditional propylene glycol alginate (PGA) carrier for their characteristics, antibacterial activity, and biocompatibility with human periodontal membrane fibroblasts. PGA-rhAm was prepared by mixing 3.3% PGA and rhAm, and CS-βGP-rhAm was prepared by mixing 2% chitosan (CS) with rhAm and then with 60% β-sodium glycerophosphate solution (βGP) as the crosslinking agent. The biophysical properties of the prepared carriers were characterized, and their antibacterial activity was assessed by observing Staphylococcus aureus growth. The biocompatibility of the carriers was evaluated in human periodontal membrane fibroblasts (hPDLFs) using CCK8 assay and scratch test, and mRNA and protein expressions of osteogenic genes of the cells incubated with the carriers were detected using RT-qPCR and Western blotting; osteogenic differentiation of the cells was detected using alkaline phosphatase staining. PGA-rhAm had a viscosity value of 3.262±0.055 Pa.s. CS-βGP-rhAm had a solidification capacity of 6 min at 37 ℃ with a pH value close to that of the oral cavity and a swelling rate of about 90%. CS-β GP-rhAm maintained sustained release of rhAm for over 2 weeks with a self-degradation time over 3 weeks. CS-βGPrhAm more effectively inhibited the growth of S. aureus than rhAm-loaded PGA. While PGA did not obviously affect the proliferation of hPDLFs, both CS-βGP and CS-βGP-rhAm significantly promoted the cell proliferation(P < 0.001). Scratch test showed that after rhAm loading, both CS-βGP and PGA promoted cell migration (P < 0.01). CS-βGP-rhAm significantly enhanced the mRNA expressions of RUNX2 and OCN mRNA level and the protein expressions of Ki67, RUNX2, collagen I, and β-catenin (P < 0.05); PGA-rhAm only enhanced RUNX2 (P < 0.05) and OCN (P < 0.01) mRNA expressions without significant effects on the protein expressions. Alkaline phosphatase staining results showed that CS-βGP, but not PGA, promoted osteogenic differentiation of hPDLFs. CS-βGP carrier is capable of sustained release of rhAm, inhibiting the growth of S. aureus, and improving the biological activity of hPDLFs without affecting the bioactivity of rhAm after drug loading.

MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8

BackgroundOur previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear.MethodsWe examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague–Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury.ResultsMiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, β-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats.ConclusionsOur data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

Lanthanum nitrate inhibits adipogenesis in 3T3-L1 preadipocytes with a disorder of mitotic clonal expansion process.

Lanthanum (La) as a rare earth element is widely used in agriculture, industry, and medicine. It has been suggested in several studies that La might influence glycolipid metabolism in vivo. In this study, we used 3T3-L1 preadipocytes as in vitro cell model to elucidate the effects of La(NO3 )3 on adipogenesis and the underlying mechanisms. The results showed that La(NO3 )3 could inhibit the adipogenic differentiation of 3T3-L1 preadipocytes, which showed a decrease in lipid accumulation and the downregulation of specific adipogenic transcription factors. La(NO3 )3 exerted its inhibitory effect mainly at the early differentiation stage. Furthermore, La(NO3 )3 influenced the S-phase entry and cell cycle process during the mitotic clonal expansion and regulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and expressions of the proteins in phosphatidylinositol 3-kinase (PI3K)/Akt pathway at the early stage of differentiation. Besides, La(NO3 )3 upregulated the expressions of wnt10b mRNA and β-catenin protein and promoted the nucleus translocation of β-catenin. Additionally, we found that La(NO3 )3 could promote the growth of 3T3-L1 preadipocytes both with and without MDI (3-isobutyl-1-methylxanthine [IBMX], dexamethasone [Dex], and insulin) stimulation. Collectively, these results indicated that La(NO3 )3 could inhibit adipogenesis in 3T3-L1 preadipocytes and influence cell proliferation.

Effects of plant-based medicinal food on postoperative recurrence and lung metastasis of gastric cancer regulated by Wnt/β-catenin-EMT signaling pathway and VEGF-C/D-VEGFR-3 cascade in a mouse model

BackgroundThe plant-based medicinal food (PBMF) is a functional compound extracted from 6 medicinal and edible plants: Coix seed, L. edodes, A. officinalis L., H. cordata, Dandelion, and G. frondosa. Our previous studies have confirmed that the PBMF possesses anti-tumor properties in a subcutaneous xenograft model of nude mice. This study aims to further investigate the effects and potential molecular mechanisms of the PBMF on the recurrence and metastasis of gastric cancer (GC).MethodsPostoperative recurrence and metastasis model of GC was successfully established in inbred 615 mice inoculated with mouse forestomach carcinoma (MFC) cells. After tumorectomy, 63 GC mice were randomly divided into five groups and respectively subject to different treatments for 15 days as below: model control group, 5-Fu group, and three doses of PBMF (43.22, 86.44, 172.88 g/kg PBMF in diet respectively). The inhibition rate (IR) of recurrence tumor weights and organ coefficients were calculated. Meanwhile, histopathological changes were examined and the metastasis IR in lungs and lymph node tissues was computed. The mRNA expressions related to the canonical Wnt/β-catenin signaling pathway, epithelial-mesenchymal transition (EMT) and lymphangiogenesis were detected by RT-qPCR in recurrence tumors and/or lung tissues. Protein expressions of β-catenin, p-β-catenin (Ser33/37/Thr41), GSK-3β, p-GSK-3β (Ser9), E-cadherin, and Vimentin in recurrence tumors were determined by Western Blot. LYVE-1, VEGF-C/D, and VEGFR-3 levels in recurrence tumors and/or lung tissues were determined by immunohistochemistry staining.ResultsThe mRNA, as well as protein expression of GSK-3β were up-regulated and the mRNA expression of β-catenin was down-regulated after PBMF treatment. Meanwhile, the ratio of p-β-catenin (Ser33/37/Thr41) to β-catenin protein was increased significantly and the p-GSK-3β (Ser9) protein level was decreased. And PMBF could effectively decrease the mRNA and protein levels of Vimentin while increasing those of E-cadherin. Furthermore, PBMF markedly reduced lymphatic vessel density (LVD) (labeled by LYVE-1) in recurrence tumor tissues, and mRNA levels of VEGF-C/D, VEGFR-2/3 of recurrence tumors were all significantly lower in the high-dose group.ConclusionsPBMF had a significant inhibitory effect on recurrence and lung metastasis of GC. The potential mechanism may involve reversing EMT by inhabiting the Wnt/β-catenin signaling pathway. Lymphatic metastasis was also inhibited by PBMF via down-regulating the activation of the VEGF-C/D-VEGFR-2/3 signaling cascade.

GENES EXPRESSION AND SERUM BIOMARKERS FOR DIAGNOSIS OF HEPATOCELLULAR CARCINOMA, CIRRHOSIS AND HEPATITIS C.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Risk factors for HCC include hepatitis C (HCV) and B (HBV) virus infection, alcoholic cirrhosis and genetic alterations that can affect several cellular pathways. This study purposed to analyze the gene and serum protein expression of vascular endothelial growth factor (VEGF), angiogenesis, alpha fetoprotein, cystatin B (CSTB), β-catenin and glypican-3 (GPC3) in groups with HCC, cirrhosis or HCV and controls, and their relation with clinical staging in the HCC and cirrhosis groups, as well its sensitivity and specificity values. A total of 230 individuals were distributed in Group 1 (G1) - 80 patients with HCC; Group 2 (G2) - 76 patients with cirrhosis due to any etiology; Group 3 (G3) - 33 patients with HCV; Group 4 (G4 - controls) - 41 individuals without clinical or biochemical signs of any liver disease. Gene expression was analyzed by qRT-PCR and serum proteins were performed using the ELISA method. Increased VEGF and angiogenesis, alpha fetoprotein expression could be observed in BCLC stage-D patients compared to stage-B patients, and stage-C patients showed higher expression of β-catenin, compared to stage-B patients (P<0.05). For VEGF and GPC3, discriminatory power was observed between HCC patients and controls (AUC =0.71; 0.82, respectively). CSTB showed discriminatory power in the comparison between patients with HCV and controls (AUC =0.74). The present study confirms the sensitivity of serum CSTB in the diagnosis of hepatitis C, and gene expression of VEGF and serum GPC3, confer both sensitivity and specificity for the diagnosis of HCC.

Crocin ameliorates peritoneal fibrosis in rat induced by peritoneal dialysis via Wnt5a/β-Catenin pathway

Peritoneal dialysis is used in the treatment of patients with kidney diseases. Long-term peritoneal dialysis could result in peritoneal fibrosis and recurrent peritonitis, thus leading to failure of ultrafiltration. Crocin is a bioactive carotenoid and isolated from stigma of Crocus sativus, and ameliorates pulmonary and myocardial fibrosis. The role of crocin in peritoneal fibrosis was assessed. Firstly, rats model with peritoneal dialysis was treated with 4.25% peritoneal dialysate. Results showed that injection with peritoneal dialysate induced obvious hyperplasia and increased thickness in peritoneum structure. Rats with peritoneal dialysis were injected with increasing concentrations of crocin at 10, 20, or 40 mg/kg. Crocin ameliorated the pathological changes in the peritoneum of peritoneal dialysate-induced rats. Secondly, crocin attenuated peritoneal dialysate-induced decrease of E-cadherin, increase of fibronectin, α-smooth muscle actin (α-SMA), and collagen I. Moreover, crocin enhanced ultrafiltration volume and reduced glucose transport in rats model with peritoneal dialysis. Thirdly, crocin also reduced levels of Interleukin (IL)-1β, Tumor Necrosis Factor-α (TNF –α), and IL-6 in peritoneal tissues of rats model with peritoneal dialysis. Lastly, protein expression of Wnt5a and β-Catenin in rats model with peritoneal dialysis were also downregulated by crocin. In conclusion, crocin exerted anti-inflammatory and anti-fibrotic effects on rats model with peritoneal dialysis through inactivation of Wnt5a/β-Catenin pathway.

Aerosol inhalation of Mycobacterium vaccae ameliorates airway structural remodeling in chronic asthma mouse model

Background: Airway remodeling is accepted to be a determining component within the natural history of asthma. Nebulized inhalation of Mycobacterium vaccae (M. vaccae) has a protective effect on asthmatic mice. However, little is known regarding the effect of M. vaccae on airway structural remodeling in asthmatic mice. The purpose of this study was to explore the effect and the underlying mechanism of M. vaccae aerosol inhalation on airway structural remodeling in an asthma mouse model. Methods: Chronic asthma mouse models were established by ovalbumin induction. The number of inflammatory cells in bronchoalveolar lavage fluid (BALF), pathological alterations in lung tissue, and levels of associated cytokines (IL-5, IL-13, TNF-α, and ovalbumin-specific immunoglobulin E [OVA-sIgE]) were all assessed after M. vaccae therapy. The relative expression of interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), and Wnt1‐induced signaling protein 1 (WISP1) mRNA were detected. Western blotting and immunohistochemistry detected the expression of Wnt/β-catenin pathway-related proteins in lung tissue. Results: M. vaccae aerosol inhalation relieved airway inflammation, airway hyper-responsiveness, and airway remodeling. M. vaccae reduced the levels of IL-5, IL-13, TNF-α, and OVA-sIgE in and downregulated the expression of IL-1β, TNF-α, NF-κB, and WISP1 mRNA in the pulmonary. In addition, M. vaccae inhibited the expression of β-catenin, WISP1, and Wnt1 protein and upregulated the expression of glycogen synthase kinase-3beta (GSK-3β). Conclusion: Nebulized inhalation of M. vaccae can reduce airway remodeling during asthma.

Enhanced healing of oral chemical burn by inhibiting inflammatory factors with an oral administration of shengFu oil.

ShengFu oil is a compounded Chinese medicinal prescription, and provides antibacterial, anti-inflammatory, and analgesic effects, favoring burn wound repair. In this study, we aimed at investigating the effects of topical applications of ShengFu oil and its active ingredients in oral chemical burns and elucidating its regulatory effects on β-catenin, COX-2, and MMP-9 expression caused by exposure to acid or alkaline agents. ShengFu oil contains 16 components, such as Frankincense, Radix Scutellariae and Radix Rehmanniae, and the main active ingredients from Frankincense are α-pinene, linalool, and n-octanol. Mouse models of oral chemical burns were induced by using glacial acetic acid or sodium hydroxide. Hematoxylin and eosin staining and immunohistochemical staining were used to detect the protein expressions of β-catenin, COX-2, and MMP-9 in wound tissues. They were further quantified by multispectral imaging analysis to clarify the effective mechanism of ShengFu oil for intervening inflammatory factors and active components. Our results indicated that the application of ShengFu oil on oral chemical burns effectively stopped the oral burn bleeding and reduced the inflammatory reaction in the damaged tissues, demonstrating that ShengFu oil can promote wound tissue repair in burns caused by heat, acids, and alkalis. The immunohistochemical staining results illustrated that ShengFu oil and its active ingredients significantly reversed the abnormal changes in inflammation-related proteins in mouse tongue tissues that were caused by chemical burns. Regarding long-term toxic effects of ShengFu oil on the gastrointestinal tract, liver, and kidney system, the results of hematoxylin and eosin staining experiments depicted that ShengFu oil was safe and effective for liver, kidney, intestine, esophagus, and tongue. All of these demonstrated that ShengFu oil and its active ingredients are effective and safe in preventing and treating oral chemical burns by interfering with the inflammatory microenvironment.

The Effect and Mechanism of Curcumin Combined with Carboplatin Chemotherapy Promoting on Apoptosis of Lung Cancer HCC827 Cells.

Objective To investigate the effect and mechanism of curcumin (CUR) killing lung cancer HCC827 cell spheres. Method HCC827 cell spheres were cultured in serum-free medium, and the protein expression of CD133, SOX2, EpCAM, and ABCG2 was detected by western blot. MTT was used to evaluate the cell viability of HCC827 cell spheres and HCC827 cell after they were treated by 1, 2, 5, 10, and 20 mg/mL carboplatin (CBP) for 48 h. The inhibitory effects of 10 μM, 50 μM, 100 μM, and 200 μM CUR on GST (glutathione S-transferase) activity in HCC827 cell spheres were determined by colorimetry. The flow cytometry (FCM), western blot, qPCR, luciferase assay, and microscopy were used to detect the ROS levels, cell pelletization ability, β-catenin, SOX2, and ABCG2 mRNA and the promoter activity of β-catenin upon of HCC827 cell spheres treated with 200 μM CUR for 48 h. The HCC827 cell spheres were infected with β-catenin adenovirus, and then cells were treated with 200 μM CUR (and/or no 5 mg/mL CBP) for 24 h. The mRNA and protein expression of β-catenin, SOX2, and ABCG2 was detected by qPCR and western blot, and cell growth inhibition of HCC827 cell spheres was evaluated by MTT. Result The expression of stem cells marker CD133, SOX2, EpCAM, and drug resistance-related gene ABCG2 mRNA is higher in HCC827 cell spheres, and HCC827 cell spheres resisted the killing effect of difference doses of CBP. The activity of GST of HCC827 cell spheres was inhibited by 10 μM, 50 μM, 100 μM, and 200 μM CUR. It was a dose-dependent manner. After 200 μM CUR had treated HCC827 cell spheres for 48 h, the level of ROS was significantly increased (P < 0.05), and the mRNA and protein expression of β-catenin, SOX2, and ABCG2 and promoter activity of β-catenin were notably decreased (P < 0.05), compared to the control group. Furthermore, the formed-sphere ability of HCC827 sphere was inhibited after cells were treated with 200 μM CUR. 200 μM CUR could suppress the proliferation of HCC827 cell spheres and induced cell apoptosis. The proliferation of HCC827 cell spheres was significantly inhibited, and cell apoptosis rate was increased by 200 μM CUR combined with 5 mg/mL CBP than by 200 μM CUR alone. Upregulation of β-catenin by adenovirus partly reversed the effect of CUR inhibition of the expression of β-catenin, SOX2, and ABCG2, compared to empty vector adenovirus group. Additionally, overexpression of β-catenin significantly remitted the inhibitory effect of 200 μM CUR combined with 5 mg/mL CBP on the proliferation of HCC827 cell spheres. Conclusion CUR inhibited the cell proliferation and stem cell trait and induced apoptosis in HCC827 cell spheres by the inhibition of GST activity and β-catenin expression. CUR is expected to become a treatment for lung cancer and lung cancer stem cells.

Inhibitory effect of protonic bis(5-amino-1,10-phenanthroline) on proliferation of hepatocellular carcinoma and its molecular mechanism

The incidence of HCC continues to increase rapidly in recent years. the principle of developing anti-HCC drugs is to specifically kill tumor cells, without affecting the function of normal cells. Here we synthesized a novel protonic compound bis(5-amino-1,10-phenanthroline) (P-BAP), and the antitumor activity was explored in vitro and in vivo . The results showed that P-BAP could selectively inhibit the proliferation of tumor cells, and induce HCC apoptosis. In HCC-bearing mice, P-BAP could effectively retard the tumor growth, even completely eliminate the tumors at the dose of 5 mg/kg, and meanwhile P-BAP had no significant effect on mouse body weight. Mechanism analysis revealed that P-BAP could down-regulate the protein expressions of pleomorphic adenoma gene like-2 (PLAGL2), HIF-1α and β-catenin, and up-regulate the levels of pro-apoptotic protein BAX and BNIP3. In addition, P-BAP could reduce mitochondrial respiratory chain complex activities, leading to insufficient ATP production. The study provides a new approach for designing selective antitumor drugs, and suggests that P-BAP would be a potential candidate for HCC therapy.

Stromal Transcription Factor 21 Regulates Development of the Renal Stroma via Interaction with Wnt/β-Catenin Signaling.

Kidney formation requires coordinated interactions between multiple cell types. Input from the interstitial progenitor cells is implicated in multiple aspects of kidney development. We previously reported that transcription factor 21 (Tcf21) is required for ureteric bud branching. Here, we show that Tcf21 in Foxd1+ interstitial progenitors regulates stromal formation and differentiation via interaction with β-catenin. We utilized the Foxd1Cre;Tcf21f/f murine kidney for morphologic analysis. We used the murine clonal mesenchymal cell lines MK3/M15 to study Tcf21 interaction with Wnt/β-catenin. Absence of Tcf21 from Foxd1+ stromal progenitors caused a decrease in stromal cell proliferation, leading to marked reduction of the medullary stromal space. Lack of Tcf21 in the Foxd1+ stromal cells also led to defective differentiation of interstitial cells to smooth-muscle cells, perivascular pericytes, and mesangial cells. Foxd1Cre;Tcf21f/f kidney showed an abnormal pattern of the renal vascular tree. The stroma of Foxd1Cre;Tcf21f/f kidney demonstrated marked reduction in β-catenin protein expression compared with wild type. Tcf21 was bound to β-catenin both upon β-catenin stabilization and at basal state as demonstrated by immunoprecipitation in vitro. In MK3/M15 metanephric mesenchymal cells, Tcf21 enhanced TCF/LEF promoter activity upon β-catenin stabilization, whereas DNA-binding deficient mutated Tcf21 did not enhance TCF/LEF promoter activity. Kidney explants of Foxd1Cre;Tcf21f/f showed low mRNA expression of stromal Wnt target genes. Treatment of the explants with CHIR, a Wnt ligand mimetic, restored Wnt target gene expression. Here, we also corroborated previous evidence that normal development of the kidney stroma is required for normal development of the Six2+ nephron progenitor cells, loop of Henle, and the collecting ducts. These findings suggest that stromal Tcf21 facilitates medullary stroma development by enhancing Wnt/β-catenin signaling and promotes stromal cell proliferation and differentiation. Stromal Tcf21 is also required for the development of the adjacent nephron epithelia.

Combined Therapy of Yishen Zhuanggu Decoction and Caltrate D600 Alleviates Postmenopausal Osteoporosis by Targeting FoxO3a and Activating the Wnt/β-Catenin Pathway

Background Postmenopausal osteoporosis (PMO) is the most prevalent metabolic bone disease in women. Yishen Zhuanggu (YSZG) decoction and Caltrate D600 reportedly affects bone formation. This study aimed to investigate the efficacy and mechanism of YSZG decoction combined with Caltrate D600 in PMO treatment. Methods Ovariectomy-induced PMO rat model was treated with YSZG or/and Caltrate D600 for 12 weeks. Femur bone mineral density (BMD), osteoporosis-related protein expression, and serum parameters were measured. Pathological features of femur bone tissues were observed using hematoxylin and eosin staining. Serum levels of oxidative stress parameters were measured using corresponding commercial kits. The mRNA and protein expression of FoxO3a, Wnt, and β-catenin was detected using qRT-PCR and western blotting. Results The BMD and ultimate load of PMO rats were increased after treatment with YSZG. YSZG treatment promoted the bone trabeculae formation of PMO rats. YSZG treatment also induced bone differentiation and suppress oxidative stress in PMO rats, evidenced by the increased BALP, Runx2, OPG, SOD, and CAT levels, as well as the decreased TRACP 5b, RANKL, ROS, and MDA levels. Additionally, YSZG treatment downregulated the FoxO3a expression and upregulated the levels of Wnt and β-catenin in PMO rats. Caltrate D600 addition showed an auxiliary effect for YSZG. Conclusion YSZG decoction exerts the antiosteoporotic effect on PMO by restraining the FoxO3a expression and activating the Wnt/β-catenin pathway, which has an impressive synergistic effect with Caltrate D600.

Ligustrazine Alleviates Kidney Injury in a Rat Model of Uremia via Attenuation of Wnt/β-Catenin Signaling Pathway

Uremia is associated with kidney injury and contributes to chronic renal failure. Ligustrazine, a bioactive alkaloid from traditional Chinese herb Ligusticum wallichii Franchat, exerts renal-protective effect against acute kidney injury. The role of ligustrazine in uremia-associated kidney injury was investigated. To induce uremia, rats were subjected to 5/6 nephrectomy and intravenously administered with saline. Survival rate of rats was decreased by 5/6 nephrectomy, and levels of blood urea nitrogen, serum creatinine, and 24 h urine protein were elevated. Daily administration of ligustrazine to these rats increased the survival rate and reduced levels of blood urea nitrogen, serum creatinine, and 24 h urine protein output. Moreover, analysis of kidney histology showed that ligustrazine also ameliorated pathological changes in kidney. Finally, ligustrazine reduced levels of proinflammatory cytokines and suppressed renal apoptosis in the kidney tissues of uremic rats. In addition, ligustrazine downregulated protein expression of Wnt1 and β-catenin and inhibited nuclear distribution of β-catenin in uremic rats. In conclusion, ligustrazine protects rats against uremia-associated kidney injury and renal inflammation through inactivation of Wnt1/β-catenin pathway.