mRNA Expression Of Β-catenin Research Articles

Britannin inhibits hepatocellular carcinoma development and metastasis through the GSK-3β/β-catenin signaling pathway

BackgroundHepatocellular carcinoma (HCC) stands out as a significant contributor of cancer-related death. Traditional Chinese Medicine (TCM) presents numerous advantages in the treatment of HCC. Britannin, a pivotal compound found in Inulae Flos, demonstrates pharmacological effects against various cancers, yet research on its specific impact on anti-hepatocellular carcinoma remains limited. PurposeThis study aims to explore both the anti-hepatocellular carcinoma effects of britannin and its underlying mechanism of action. MethodsMTT assay, clone formation assay and flow cytometry were utilized to detect the cell activity, proliferation ability and apoptosis of britannin against HCC cell lines. Cell migration and invasion abilities of britannin on HCC cell lines were evaluated by wound-healing assay and transwell migration and invasion assay. H22 xenografted tumor mouse model was constructed and britannin treatment was performed to observe the effect of britannin on HCC tumor. The expression levels of liver cancer biomarkers AFP, AFP-L3, APT and TGF-β were detected by Elisa, and the histopathology was observed by HE staining. Network pharmacology and molecular docking were used to predict the possible signaling pathway of anti- hepatocellular carcinoma effect of britannin. The surface plasmon resonance (SPR) experiments was used to verify the interaction between britannin and proteins. The cell kinase activity function experiment was employed to detect the effect of britannin on enzyme activity. RT-qPCR and Western-Blot were used to verify the effect of britannin on the mRNA expressions of key genes and protein levels related to GSK-3β/β-catenin pathway in hepatocellular carcinoma cells and tumor tissues in mice. ResultsIn vitro experiments showed that britannin could inhibit the activity, proliferation, migration and invasion abilities of HCC cells, while promoting their apoptosis. In vivo experiments revealed that britannin exerted inhibitory effects on the growth of transplanted liver cancer tumors, reducing the inflammatory infiltration and the expression levels of AFP, AFP-L3, APT and TGF-β of liver cancer markers in transplanted mice. Network pharmacology and molecular docking predicted that cell adhesion factors and GSK-3β/β-catenin pathway might be the related signaling pathway and had potential docking activity with key proteins. The SPR experiments elucidated the molecular interaction between britannin and GSK-3β. Enzyme activity assays indicated that britannin could modulate the functional activity of GSK-3β kinase. RT-qPCR suggested britannin could regulate the mRNA expressions of β-catenin, GSK-3β, E-cadherin and N-Cadherin. Western-Blot further verified that britannin could significantly up-regulate the expression of GSK-3β and down-regulate the expression of p-GSK-3β and β-catenin. At the same time, the expression of E-cadherin increased and N-Cadherin decreased, thereby reducing the occurrence of EMT and inhibiting the metastasis of hepatocellular carcinoma. ConclusionIn conclusion, britannin could inhibit the growth, development and metastasis of HCC, and its mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway to inhibit epithelial-mesenchymal transition of HCC.

Effects of soluble Klotho and Wnt/β-catenin signaling pathway in vascular calcification in chronic kidney disease model rats and the intervention of Shenyuan granules

Objective This study aimed to investigate the effect of the soluble Klotho (sKlotho)/Wnt/β-catenin signaling pathway on vascular calcification in rat models of chronic kidney disease (CKD) and the intervention effect of Shenyuan granules. Methods Rats with 5/6 nephrectomy and high phosphorus feeding were used to establish the vascular calcification model. The rats were given gradient doses of Shenyuan granules aqueous solution and calcitriol solution by gavage for 8 weeks, which were divided into experimental group and positive control group. Results The 5/6 nephrectomy combined with high phosphorus feeding induced thoracic aortic calcification in rats. Shenyuan granules intervention increased the serum sKlotho level, inhibited the mRNA and protein expression of Wnt1, β-catenin, and Runx2 in the thoracic aorta, and alleviated thoracic aortic media calcification in rats. Conclusion Shenyuan granules may partially regulate the Wnt/β-catenin signaling pathway via serum sKl to interfere with the expression of Runx2, thereby improving vascular calcification in CKD.

Dietary vitamin B6 supplementation alleviates heat stress-induced intestinal barrier impairment by regulating the gut microbiota and metabolites in broilers

Heat stress (HS) brings great challenges to the poultry industry. Vitamin B6 (VB6) is an essential micro-nutrient for animals to maintain normal physiological functions and possesses antioxidant and anti-inflammatory properties. This study aimed to explore the effect of VB6 on alleviating HS-induced intestinal barrier impairment in broilers. A total of 250 broilers (609.76 ± 0.34 g) were randomly allocated to 5 groups with 5 replicate cages of 10 birds each. The broilers in thermoneutral (TN) group were raised in thermoneutral conditions (23 ± 1°C) and fed with a basal diet. The birds in other four groups were housed under cycle high temperature (34 ± 1°C for 8 h/d) from d 21 to 35 and fed with the basal diet (HS group) or basal diet supplemented with 6, 12, or 24 mg/kg VB6 (HB-6, HB-12, HB-24 groups). The results showed that HS reduced the growth performance, increased ileum inflammatory cytokines levels, and impaired the gut barrier function (P < 0.05). Compared to the HS group, final body weight, average daily gain, and average daily feed intake, and the feed conversion ratio were improved by VB6 supplementation. The diamine oxidase, interleukin (IL)-1β, tumor necrosis factor-α, IL-18, IL-10, and interferon-γ levels were reduced by VB6 supplementation (P < 0.05). Moreover, VB6 supplementation linearly or quadratically enhanced villus height and villus height-to-crypt depth ratio of duodenum and jejunum, and decreased crypt depth of duodenum and ileum. The mRNA expression of Occlaudin, ZO1, Mucin2, Mucin4, E-cadhein, and β-catenin were increased by VB6 treatment (P < 0.05). Furthermore, dietary VB6 altered the diversity and community of gut microbiota (P < 0.05). A total of 83 differential metabolites associated with the amelioration of VB6 were identified, which were primarily enriched in glycerophospholipid metabolism, caffeine metabolism, and glutathione metabolism pathway. Collectively, VB6 may improve the growth performance and intestinal barrier function of heat-stressed broilers by regulating the ileal microbiota and metabolic homeostasis.

Mechanism of Linggui Zhugan Decoction in inhibiting myocardial fibrosis by regulating Wnt/β-catenin pathway

This study aimed to investigate the regulatory mechanism of Linggui Zhugan Decoction(LGZGD)-medicated serum on the fibrosis of cardiac fibroblasts(CFs) and the protein expression of the Wnt/β-catenin signaling pathway. Blank serum and LGZGD-medicated serum were prepared, and primary CFs were isolated and cultured using trypsin-collagenase digestion and differential adhesion method. Immunofluorescence labeling was used to identify primary CFs. Cells were divided into normal control group, model group, 20% blank serum group, and 5%, 10%, and 20% LGZGD-medicated serum groups. Except for the normal control group, all other groups were stimulated with hydrogen peroxide(H_2O_2) after pretreatment with 20% blank serum or 5%, 10%, 20% LGZGD-medicated serum for 12 hours to establish a model of fibrosis in primary CFs. Scratch healing assay was used to observe cell migration ability. ELISA was used to detect the content of collagen type Ⅰ(Col Ⅰ) and type Ⅲ(Col Ⅲ). Western blot was used to detect the protein expression of α-smooth muscle actin(α-SMA), Wnt1, glycogen synthase kinase 3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, and nuclear β-catenin. RT-qPCR was used to detect the gene expression of β-catenin and matrix metalloproteinase 9(MMP9), and immunofluorescence technique was used to detect the expression and localization of key proteins α-SMA and β-catenin. CFs with Wnt1 overexpression were prepared and treated with H_2O_2. The following groups were set up: normal control group, model group, 20% LGZGD-medicated serum group, empty plasmid+20% LGZGD-medicated serum group, and Wnt1 overexpression+20% LGZGD-medicated serum group. ELISA was used to detect the content and ratio of Col Ⅰ and Col Ⅲ. Western blot was used to detect the protein expression of α-SMA, Wnt1, GSK-3β, p-GSK-3β, β-catenin, and nuclear β-catenin. RT-qPCR was used to detect the gene expression of β-catenin and MMP9. Immunofluorescence staining showed that CFs expressed Vimentin positively, appearing green, with blue nuclei and purity greater than 90%, which were identified as primary CFs. RESULTS:: showed that compared with the normal control group, CFs in the model group had enhanced healing rate, increased content of Col Ⅰ and Col Ⅲ, increased ratio of Col Ⅰ/Col Ⅲ, upregulated protein expression of α-SMA, Wnt1, p-GSK-3β, β-catenin, nuclear β-catenin, decreased GSK-3β expression, elevated mRNA expression of β-catenin and MMP9, and enhanced fluorescence intensity and expression of β-catenin and α-SMA. Compared with the model group, 5%, 10%, 20% LGZGD-medicated serum significantly inhibited cell migration ability, reduced the content of Col Ⅰ and Col Ⅲ, decreased ratio of Col Ⅰ/Col Ⅲ, downregulated protein expression of α-SMA, Wnt1, p-GSK-3β, β-catenin, nuclear β-catenin, increased GSK-3β expression, decreased mRNA expression of β-catenin and MMP9, and reduced fluorescence intensity and expression of β-catenin and α-SMA. Compared with the empty plasmid+20% LGZGD-medicated serum group, the effect of LGZGD-medicated serum was significantly reversed after overexpression of Wnt1. LGZGD can reduce excessive deposition of collagen fibers, inhibit excessive proliferation of fibroblasts, and improve the process of myocardial fibrosis. The improvement of myocardial fibrosis by LGZGD is related to the regulation of the Wnt/β-catenin pathway, reduction of collagen deposition, and protection of myocardial cells.

Effect of Jiedu Huayu decoction on oral mucosal Axin and β-catenin expression in oral submucosal fibrosis model rats.

To investigate the protective effect of the Chinese herbal formula of Jiedu Huayu decoction (, JHD) on oral mucosa of rats with oral submucosal fibrosis (OSF) and its potential mechanism of action. Sprague-Dawley male OSF model rats were constructed by injection of betaine and topical rubbing and were randomly grouped and administered by gavage for 4 weeks. Mouth opening and buccal mucosa scores interleukin levels and the expression of Axin and β-catenin proteins or genes were measured before and after drug administration. After treatment with JHD the buccal mucosal lesions of rats were significantly reduced Axin protein and mRNA expression were significantly increased β-catenin protein and mRNA expression were significantly decreased interleukin-1β and interleukin-6 levels were decreased and interleukin-10 levels were increased. The mechanism of action of JHD can effectively alleviate the pathological damage of buccal mucosa in OSF rats which may be related to the promotion of Axin expression and inhibition of β-catenin expression.

Dihydroquercetin improves the proliferation of porcine intestinal epithelial cells via the Wnt/β-catenin pathway

Dihydroquercetin (DHQ), also known as Taxifolin (TA), is a flavanonol with various biological activities, such as anticancer, anti-inflammatory, and antioxidative properties. It has been found to effectively increase the viability of porcine intestinal epithelial cells (IPEC-J2). However, the precise mechanism by which DHQ increases the proliferation of IPEC-J2 cells is not entirely understood. This study aimed to explore the potential pathways through which DHQ encourages the proliferation of IPEC-J2 cells. The findings indicated that DHQ significantly improved the protein expression of tight junction proteins (ZO-1, Occludin, and Claudin1) and a molecular biomarker of proliferation (PCNA) in IPEC-J2 cells. Furthermore, DHQ was found to increase the Wnt/β-catenin pathway-associated β-catenin, c-Myc, and cyclin D1 mRNA expression, and promote the protein expression of β-catenin and TCF4. To confirm the involvement of the Wnt/β-catenin signaling pathway in the DHQ-promoted proliferation of IPEC-J2 cells, the inhibitor LF3, which targets β-catenin/TCF4 interaction, was used. It was found that LF3 inhibited the protein expressions upregulated by DHQ and blocked the promotion of cell proliferation. These results indicate that DHQ positively regulates IPEC-J2 cell proliferation through the Wnt/β-catenin pathway, providing constructive insights into the role of DHQ in regulating intestine development.

Dickopff 1 inhibits cancer stem cell properties and promotes neuronal differentiation of human neuroblastoma cell line SH-SY5Y

Neuroblastomas are pediatric tumors arising from undifferentiated cells of neural crest origin with stem cell-like characteristics. Dysregulation of Wnt/β-catenin signaling has been shown to be linked to the development of various tumors. Activated Wnt signaling results in β-catenin accumulation in the nucleus to support pro-neoplastic traits. DKK1, a secreted glycoprotein, is an inhibitor of Wnt signaling, and the addition of DKKI to the culture medium has been used to suppress the Wnt pathway. This study aimed to analyze the role of Dickopff-1 as a potential differentiating agent for the neuroblastoma cell line SH-SY5Y and neurospheres derived from it. The treatment of SH-5Y5Y derived neurospheres by DKK1 resulted in their disintegration and reduced proliferation markers like Ki67, PCNA. DKK1 treatment to the neurospheres also resulted in the loss of cancer stem cell markers like CD133, KIT and pluripotency markers like SOX2, OCT4, NANOG. DKK1 treatment caused reduction in mRNA expression of β-catenin and TCF genes like TCF4, TCF12. When the SH-SY5Y cancer cells were grown under differentiating conditions, DKKI caused neuronal differentiation by itself, and in synergy with retinoic acid. This was verified by the expression of markers like MAPT, DCX, GAP43, ENO2 and also with changes in neurite length. We concluded that Wnt inhibition, as exemplified by DKK1 treatment, is therefore a possible differentiating condition and also suppresses the proliferative and cancer stemness related properties of SH-SY5Y neuroblastoma cells.

The “toxic window” of amoxicillin exposure during pregnancy on long bone development in fetal mice

AimsAmoxicillin is a broad-spectrum beta-lactam antibiotic used to treat infectious diseases in pregnant women. Studies have shown that prenatal amoxicillin exposure (PAmE) has developmental toxicity on fetal development. However, the effect of PAmE on long bone development has not been reported. This study aimed to investigate the “toxic window” of PAmE on long bone development and explore its possible mechanism in fetal mice. Materials and methodsPregnant mice were administered amoxicillin by gavage at different stages (gestational day (GD)10–12 and GD16–18), different doses (150 and 300 mg/kg·d) and different courses (single and multiple courses). Fetal femurs were collected at GD18 and bone development related indicators were detected. Key findingsThe results showed that PAmE significantly reduced the length of the femur and primary ossification center of fetal mice, and inhibited the development of fetal growth plate. Meanwhile, PAmE inhibited the development of bone marrow mesenchymal stem cells, osteoclasts and endothelial cells in fetal long bone. Further, we found the fetal long bone developmental toxicity induced by PAmE was most significant at late-pregnancy (GD16–18), high dose (300 mg/kg·d) and multiple-course group. Besides, PAmE inhibited the expression of Wnt/β-catenin signaling pathway in fetal long bone. The β-catenin mRNA expression was significantly positively correlated with the development indexes of fetal long bone. SignificancePAmE has toxic effects on long bone development, and there was an obvious “toxic window” of PAmE on the long bone development in fetal mice. The Wnt/β-catenin signaling pathway may mediate PAmE-induced fetal long bone development inhibition.

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

The correlation between fecal microbiota profiles and intracellular junction genes expression in young Iranian patients with celiac disease

ABSTRACT Celiac disease (CD) is characterized by the disruption of the intestinal barrier integrity and alterations in the microbiota composition. This study aimed to evaluate the changes in the fecal microbiota profile and mRNA expressions of intracellular junction-related genes in pediatric patients with CD compared to healthy controls (HCs). Thirty treated CD patients, 10 active CD, and 40 HCs were recruited. Peripheral blood (PB) and fecal samples were collected. Microbiota analysis was performed using quantitative real-time PCR (qPCR) test. The mRNA expressions of ZO-1, occludin, β-catenin, E-cadherin, and COX-2 were also evaluated. In active and treated CD patients, the PB expression levels of ZO-1 (p = 0.04 and 0.002, respectively) and β-catenin (p = 0.006 and 0.02, respectively) were lower than in HCs. PB Occludin’s level was upregulated in both active and treated CD patients compared to HCs (p = 0.04 and 0.02, respectively). However, PB E-cadherin and COX-2 expression levels and fecal mRNA expressions of ZO-1, occludin, and COX-2 did not differ significantly between cases and HCs (P˃0.05). Active CD patients had a higher relative abundance of the Firmicutes (p = 0.04) and Actinobacteria (p = 0.03) phyla compared to treated subjects. The relative abundance of Veillonella (p = 0.04) and Staphylococcus (p = 0.01) genera was lower in active patients in comparison to HCs. Researchers should explore the precise impact of the gut microbiome on the molecules and mechanisms involved in intestinal damage of CD. Special attention should be given to Bifidobacteria and Enterobacteriaceae, as they have shown a significant correlation with the expression of tight junction-related genes.

5-Methylcoumarin-4β-glucoside mitigated colon tumor progression in mice with AOM/DSS-induced colon carcinogenesis

ContextThe in vitro cytotoxicity profile of 5-Methylcoumarin-4β-glucoside (5-MC4βG) has been demonstrated to inhibit the proliferation of HT-29 adenocarcinoma cells. Aim: The need for newer and affordable chemotherapeutic agents is critical. Main methodsWe investigated the effect of 5-MC4βG on an azoxymethane /dextran sodium sulfate-induced colon carcinogenesis model in six groups of laboratory BALB/c mice for six weeks. While the first and second groups of mice served as vehicle and disease controls respectively, the third, fourth, and fifth groups were administered oral graded doses (25, 50, and 100 mg/kg) of 5-MC4βG. The sixth group was treated with 5-Flourouracil (15 mg/kg). Key findingsThe 100 mg/kg dose of 5-MC4βG upregulated APC mRNA expression by two-fold and downregulated β-catenin mRNA expression by two-fold compared to their respective disease controls. Furthermore, tumorigenic gene transcripts (MDM2, BCL2, CDK1, and cyclin D1) were downregulated by one-fold (except cyclin D1 which was downregulated by two-fold), whereas pro-apoptotic genes (p53, Bax, and Casp3) were upregulated by two-fold following treatment with 100 mg/kg dose of 5-MC4βG. At the metabolic level, the bioactive compound lowered the expression of classical tumor markers; tissue polypeptide antigen, tumor-associated glycoprotein 72, and carcinoembryonic antigen by at least half. Histologically, 5-MC4βG intervention revealed a reduction in neoplastic cells associated with cellular necrosis. Significance5-MC4βG reduced colon carcinogenesis in mice. Thus, this compound may be a promising candidate for colorectal cancer chemotherapy. Further development of 5-MC4βG will hopefully lead to the development of a potential anti-colon cancer drug candidate.

Expression and prognostic analysis of AEG-1, NKD-1and β-catenin in gastric cancer tissues

Objective: To investigate the expressions of AEG-1, NKD-1 and β-catenin in gastric cancer (GC), and analyze their relationship with clinicopathological features and prognosis and the correlation among the three proteins.Methods: Fifty GC tissues and 50 adjacent normal tissues of patients who underwent radical gastrectomy in the Third Affiliated Hospital of Inner Mongolia Medical University during December 2015 to June 2017 were collected. The expressions of AEG-1, NKD-1 and β-catenin were detected by immunohistochemical SP method and qRT-PCR. The relationship between positive expression and clinical features and survival time of patients was analyzed, and the correlation between the three was analyzed.Results: The positive expression rates of AEG-1 and β-catenin in gastric cancer were 74% (37/50), 60% (30/50), respectively, which was significantly higher than that of paracancer tissue (18% [9/50] and 26% [13/50]). The positive expression rate of NKD1 in gastric cancer tissues was 20% (10/50), which was significantly lower than that in adjacent tissues [88% (44/50)]; and the differences were statistically significant (p &lt; . 05). AEG-1 mRNA expression level (1.0148±0.0019) and β-catenin mRNA expression level (1.0000±0.0005) in gastric cancer tissues were significantly higher than that of AEG-1 mRNA expression level (1.0002±0.0036) and β-catenin expression level in adjacent tissues mRMA expression level (0.9984±0.0012), and the differences were statistically significant (p &lt; .05). The expression level of NKD1 mRNA in gastric cancer tissues (1.0008±0.0073) was significantly lower than that in adjacent tissues (1.0291±0.0041), and the differences were statistically significant (p &lt; .05). The positive expression of AEG-1, NKD1 and β-catenin in gastric cancer tissues was not related to age, sex or tumor size (p &gt; .05), but was related to differentiation degree, TNM stage and lymph node metastasis (p &lt; .05). In gastric cancer, there was a positive correlation between AEG-1 and β-catenin protein expression (r = .678, p &lt; .001), and a negative correlation between AEG-1 and NKD1 protein expression (r = -.323, p = .012). β-catenin expression was negatively correlated with NKD1 expression (r = -.347, p = .007). The survival time of patients with positive expression of AEG-1 and β-catenin was significantly shortened compared with those with negative expression (p &lt; .05), while the survival time of patients with positive expression of NKD1 was longer than that of patients with negative expression of NKD1 (p &lt; .05).Conclusion: The abnormal expression of AEG-1, NKD-1 and β-catenin is closely related with the occurrence, development, invasion, metastasis and prognosis of gastric cancer, which provide a therapeutic target and may become markers for prognosis evaluation.

Chonggu Granules Improve Cartilage Matrix Metabolism in Knee Osteoarthritis via the miR-148a-3p/Wnt/β-Catenin Pathway.

This study aims to explore the effect and underlying mechanism of Chonggu Granules (CGG) in knee osteoarthritis (KOA) in rats. A papain-induced KOA model was established in rats. The pathological alterations of extracellular matrix in rat cartilage tissues were observed through hematoxylin and eosin (H&E) staining, followed by Mankin score for quantitative scoring. The ultrastructure of cartilage extracellular matrix was examined under a transmission electron microscopy (TEM). ELISA was used to measure the levels of IL-6, TNF-α, and IL-1β in rat serum. Immunofluorescence was performed for assessing the levels of MMP-3, MMP-13, and Col2al in rat cartilage. Western blot was used to identify the protein expressions of wnt1, GSK-3β, β-catenin, and Aggrecan in rat cartilage. The mRNA relative expressions of miR-148a-3p, wnt1, β-catenin, and GSK-3β in rat cartilage were detected by RT-PCR. Luciferase reporter gene was used to detect the target genes of miR-148a-3p. CGG significantly improved articular cartilage tissue and extracellular matrix metabolism compared to the model group as indicated by H&E, Mankin score, and TEM data. Moreover, low, medium, and high doses of CGG reduced the levels of IL-6, TNF-α, IL-1β, MMP-3, and MMP-13 in serum to varying degrees but increased the levels of Col2al and Aggrecan. Mechanistically, CGG targeted wnt1 by increasing the expression of miR-148a-3p in a dose-dependent manner, thereby downregulating the mRNA and protein expressions of β-catenin in cartilage tissue and upregulating the mRNA and protein expressions of GSK-3β. CGG may control the miR-148a-3p/wnt/β-catenin signaling pathway to decrease the levels of its downstream target genes MMP-13 and MMP-3, increase the expressions of Col2al and Aggrecan, and downregulate the contents of inflammatory cytokines IL-6, TNF-α, and IL-1β, thereby improving the metabolism of cartilage extracellular matrix and alleviating the degeneration of articular cartilage in KOA.

Codonopsis pilosula polysaccharides promote osteogenic differentiation and inhibit lipogenic differentiation of rat bone marrow stem cells by activating β-catenin

Aberrant bone marrow mesenchymal stem cell (BMSC) lineage differentiation leads to osteoporosis. Codonopsis pilosula polysaccharides (CPPs) have been widely used in traditional Chinese medicines, due to their multiple pharmacological actions. However, little is known regarding their effects on BMSC differentiation. This study aimed to identify the effects and mechanisms of CPPs on osteogenic and adipogenic differentiation in rat BMSCs. An osteoporosis model was established in Sprague-Dawley (SD) rats through bilateral ovariectomy (OVX), and be applied to observe the effect of CPPs on osteoporosis in vivo. The ability of CPPs to affect rBMSC proliferation was determined using the CCK-8 assay, and the osteogenic differentiation of rBMSCs measured by ALP and Alizarin Red S staining. The adipogenic differentiation of rBMSCs was measured by Oil Red O staining. The mRNA and protein levels related to osteogenesis and adipogenic differentiation of rBMSCs were measured using qRT-PCR and western blotting, respectively. Cellular immunofluorescence was used to detect cytokine expression and localisation in rBMSCs. We observed that CPPs ameliorated bone loss in OVX rats. CPPs considerably enhanced osteogenic differentiation by increasing ALP activity and the prevalence of mineralised nodules and promoting the mRNA and protein expression of osteogenic differentiation markers (RUNX2, COL I, ALP, and OPN). Furthermore, it inhibited the accumulation of lipid vesicles in the cytoplasm and the mRNA and protein expression levels of adipogenic differentiation markers (PPARγ and C/EBPα) in a concentration-dependent manner. Meanwhile, CPPs notably increased the mRNA and protein expression of β-catenin, the core protein of the Wnt/β-catenin signaling pathway, in a concentration-dependent manner. Adding DKK1, a mature inhibitor of the Wnt/β-catenin signaling pathway, partially suppressed CPP-stimulated β-catenin activation, and reversed the acceleration of osteogenic differentiation and the inhibition of lipogenic differentiation. Our observations demonstrated CPPs ameliorate bone loss in OVX rats in vivo, and favour osteogenic differentiation while inhibit adipogenic differentiation of rBMSCs in vitro. The findings suggested that CPPs could serve as functional foods for bone health, and have great potential for the prevention and treatment of osteoporosis.

Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P&lt;0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P&lt;0.05, P&lt;0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P&lt;0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P&lt;0.05, P&lt;0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P&lt;0.01), and the expression of E-cadherin mRNA increased(P&lt;0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.

Novel treatment from a botanical formulation Si-Miao-Yong-an decoction inhibits vasa vasorum angiogenesis and stabilizes atherosclerosis plaques via the Wnt1/β-catenin signalling pathway

Context Si-Miao-Yong-An (SMYA) has been widely used for the clinical treatment of atherosclerosis (AS). Yet, its complete mechanism of action is not fully understood. Objective To investigate the mechanism by which SMYA stabilizes AS plaques from the perspective of inhibiting vasa vasorum (VV) angiogenesis. Materials and methods We used male ApoE-/- mice to establish an AS model. The mice were divided into model, SMYA (11.7 mg/kg/d), and simvastatin (SVTT) (2.6 mg/kg/d) groups. Mice were given SMYA or SVTT by daily gavage for 8 weeks. HE staining, immunofluorescence double-labelling staining, and immunohistochemical staining were used to observe the pathological changes in the plaques. Finally, the protein and mRNA expression levels of the Wnt1/β-catenin signalling pathway were detected by Western blot and qRT-PCR, respectively. Results SMYA significantly attenuated cholesterol crystallization, and lipid accumulation in AS plaques, resulting in smaller plaque size (0.25 mm2 vs. 0.46 mm2), and lowering ratio of plaque to lumen area (20.04% vs. 38.33%) and VV density (50.64/mm2 vs. 98.02/mm2). Meanwhile, SMYA suppressed both the positive area percentage of Wnt1 (2.53 vs. 3.56), β-catenin (3.33 vs. 5.65) and Cyclin D1 (2.10 vs. 3.27) proteins in the aortic root plaques, and mRNA expression of Wnt1 (1.38 vs. 2.09), β-catenin (2.05 vs. 3.25) and Cyclin D1 (1.39 vs. 2.57). Discussion and conclusions SMYA has a protective effect against AS, which may be related to its anti-VV angiogenesis in plaques, suggesting that SMYA has the potential as a novel botanical formulation in the treatment of AS.

Polysaccharide from Paris polyphylla improves learning and memory ability in D-galactose-induced aging model mice based on antioxidation, p19/p53/p21, and Wnt/β-catenin signaling pathways

The current study aimed to investigate the effects and mechanisms of Paris polyphylla polysaccharide component 1 (PPPm-1) to improve learning and memory in D-galactose-induced aging model mice. We determined the effects of PPPm-1 on the brain, organ index, and behavior in the aging model mice induced by D-galactose to study learning and memory improvement. UV–Vis spectrophotometry helped determine the PPPm-1 effect on antioxidant parameters associated with learning and memory in the brain and related organs of aging mice. Moreover, in the hippocampi of aging model mice, PPPm-1 effect on the mRNA and protein expressions of p19, p53, p21, P16, Rb, Wnt/1, β-catenin, CyclinD1, TCF-4, and GSK-3β were detected using the quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The results indicated that PPPm-1 could increase the brain and organ indexes, the avoidance latency, the total distance and average speed in the water maze, and the SOD and GSH-PX activities in the brain, liver tissues, and plasma. Moreover, the mRNA and protein expressions of Wnt/1, β-catenin, CyclinD1, and TCF-4 were also elevated in the hippocampi of aging model mice. However, the error times in step-through tests, the MDA content in the brain and liver tissues, the AChE activity in the brain tissue, the protein expressions of P16, Rb in the hippocampi, and the mRNA and protein expressions of p19, p53, p21, and GSK-3β in the hippocampi of aging model mice were significantly decreased. Thus, PPPm-1 significantly enhanced the learning and memory impairment induced by D-galactose in mice. The action mechanisms were associated with anti-oxidative stress, cholinergic nervous system function regulation, LTP enhancement in long-term memory, down-regulated expression of p19/p53/p21 signaling pathway factors, and Wnt/β-catenin signaling pathway activation.

Sevoflurane inhibits cholangiocarcinoma via Wnt/β-catenin signaling pathway

BackgroundCholangiocarcinoma (CCA) is a refractory malignancy derived from bile duct epithelial cells. This study aimed to explore the role and molecular mechanisms of action of sevoflurane in CCA.MethodsCCK-8 assay was used to assess the proliferation of cholangiocarcinoma cells, and flow cytometry was used to detect cholangiocarcinoma cell apoptosis. The effects of sevoflurane on TFK1 and QBC939 cell migration and invasion were investigated using a Transwell assay. Western blotting and RT-qPCR were used to assess the expression of apoptosis-related proteins and genes, and gene expression of the Wnt/β-catenin signaling pathway.ResultsOur study found that sevoflurane inhibited cholangiocarcinoma cell proliferation in a dose-dependent manner. In addition, sevoflurane induced cholangiocarcinoma cell apoptosis, inhibited cholangiocarcinoma cell migration and invasion, as well as the Wnt/β-catenin signaling pathway evidenced by decreased Wnt3a, β-catenin, c-Myc, and Cyclin D1 protein and mRNA expression, reduced p-GSK3β protein expression and p-GSK3β/GSK3β ratio. Further mechanistic studies revealed that Wnt/β-catenin pathway inducer SKL2001 reversed the inhibitory effect of sevoflurane on cholangiocarcinoma cells.ConclusionsSevoflurane induces apoptosis and inhibits the growth, migration, and invasion of cholangiocarcinoma cells by inhibiting the Wnt/β-catenin signaling pathway. This study not only revealed the role of sevoflurane in the development of CCA but also elucidated new therapeutic agents for CCA.

Expression and significance of Shh, Gli1 and β-catenin in triple-negative breast cancer tissues

Objective: To detect the expression of Shh, Gli1 and β-catenin in triple negative breast cancer tissues and paracancer breast tissues by qRT-PCR and immunohistochemistry and to analyze their correlation with clinicopathological features.Methods: (1) qRT-PCR was used to detect the mRNA expression of Shh, Gli1 and β-catenin in 30 cases of triple negative breast cancer and their paracancer breast tissues, and the correlation among them was analyzed. (2) The expression of Shh, Gli1 and β-catenin proteins in 30 triple negative breast cancer and their paracancer breast tissues was detected by immunohistochemistry, and their correlation with clinicopathological features was analyzed.Results: (1) Shh mRNA expression (1.2334 ± 0.27867), Gli1 mRNA expression (1.2135 ± 0.20636) and β-catenin mRNA expression (1.1421 ± 0.32330) in triple negative breast cancer tissues were higher than that in paracancer breast tissues, i.e., Shh mRNA expression (1.0022 ± 0.06721), Gli1 mRNA expression (1.0003 ± 0.02420) and -catenin mRNA expression (1.0033 ± 0.07920) were significantly different (p &lt; .05). There was a significantly positive correlation between the mRNA expression of Shh and Gli1 (r = .989, p &lt; .001), and between the mRNA expression of Shh and β-catenin (r = .868, p &lt; .001). There was a significantly positive correlation between the mRNA expression of Gli1 and β-catenin expression (r = .869, p &lt; .001). (2) The positive expression rates of Shh, Gli1 and β-catenin in triple negative breast cancer tissues were 93.3% (28/30), 96.7% (29/30) and 93.3% (28/30), respectively, which were higher than those in paracancer tissues 60% (18/30), 73.3% (22/30) and 73.3% (22/30), the differences were statistically significant (p &lt; .05). There was a significantly positive correlation between the mRNA expression of Shh and Gli1 (r = .958, p &lt; .001), and between the mRNA expression of Shh and β-catenin (r = .952, p &lt; .001). There was a significantly positive correlation between the mRNA expression of Gli1 and β-catenin expression (r = .927, p &lt; .001). The expression of Shh, Gli1 and β-catenin protein in triple negative breast cancer was not correlated with age and tumor size (p &gt; .05), but Shh was positively correlated with histological grade (G) (r = .774, p &lt; .001). Furthermore, Gli1 was positively correlated with histological grade (r = .757, p &lt; .001). β-catenin was positively correlated with histological grade (r = .739, p &lt; .001). Shh was positively correlated with TNM staging (r = .460, p = .010). Gli1 was positively correlated with TNM staging (r = .414, p = .023). -catenin was positively correlated with TNM staging (r = .404, p = .027). Shh was positively correlated with lymph node metastasis (r = .540, p = .002). Gli1 was positively correlated with lymph node metastasis (r = .515, p = .004). β-catenin was positively correlated with lymph node metastasis (r = .559, p = .001).Conclusions: (1) The up-regulated expression of Shh, Gli1 and β-catenin proteins in triple negative breast cancer suggests that Shh, Gli1 and β-catenin proteins are involved in tumor genesis. The combined detection of the three proteins may provide a theoretical basis for the diagnosis and prognosis evaluation of triple negative breast cancer. (2) Shh was positively correlated with Gli1 protein expression and β-catenin protein expression. Gli1 was positively correlated with β-catenin protein expression, suggesting that the three types of proteins play a synergistic role in the occurrence and development of TNBC. There may be crosstalk in the Wnt/β-catenin and Hedgehog signaling pathways in TNBC, which may provide a new approach for the treatment of TNBC. (3) The expression of Shh, Gli1 and β-catenin proteins was correlated with the degree of differentiation, TNM staging and lymph node metastasis of triple negative breast cancer, but not correlated with age and tumor size. Therefore, it was predicted that the three types of proteins were related to the invasion, metastasis and prognosis of TNBC.

IL-8 Links NF-κB and Wnt/β-Catenin Pathways in Persistent Inflammatory Response Induced by Chronic &lt;i&gt;Helicobacter pylori&lt;/i&gt; Infection

Helicobacter pylori (H. pylori) infection can cause persistent inflammatory response in human gastric mucosal epithelial cells, which may result in the occurrence of cancer. However, the underlying mechanism of carcinogenesis has not been elucidated yet. Herein, we established the models of chronic H. pylori infection in GES-1 cells and C57BL/6J mice. Interleukin 8 (IL-8) level was detected by ELISA. The expression of NF-κB p65, IL-8, Wnt2 and β-catenin mRNA and proteins was evaluated by real-time PCR, Western blotting, immunofluorescence staining, and immunohistochemistry. The infection of H. pylori in mice was evaluated by rapid urease test, HE staining and Warthin‒Starry silver staining. The morphological changes of gastric mucosa were observed by electron microscopy. Our results showed that in H. pylori infected gastric mucosal cells along with activation of NF-κB signaling pathway and increase of IL-8 level, the expression of Wnt2 was also increased significantly, which preliminarily indicates that IL-8 can positively regulate the expression of Wnt2. Studies in chronic H. pylori infected C57BL/6J mice models showed that there was an increased incidence of premalignant lesions in the gastric mucosa tissue. Through comparing changes of gastric mucosal cell ultrastructure and analyzing the relationship between NF-κB signaling pathway and Wnt2 expression, we found that H. pylori infection activated NF-κB signal pathways, and the massive release of IL-8 was positively correlated with the high expression of Wnt2 protein. Subsequently, the activated Wnt/β-catenin signal pathways may be involved in the malignant transformation of gastric mucosal cells. Collectively, H. pylori chronic infection may continuously lead to persistent inflammatory response: activate NF-κB pathway, promote IL-8 release and thereby activate Wnt/β-catenin pathway. IL-8 probably plays an important role of a linker in coupling these two signal pathways.