Notoginsenoside Ft1 inhibits colorectal cancer growth by increasing CD8+ T cell proportion in tumor-bearing mice through the USP9X signaling pathway.

The management of colorectal cancer (CRC) poses a significant challenge, necessitating the development of innovative and effective therapeutics. Our research has shown that notoginsenoside Ft1 (Ng-Ft1), a small molecule, markedly inhibits subcutaneous tumor formation in CRC and enhances the proportion of CD8+ T cells in tumor-bearing mice, thus restraining tumor growth. Investigation into the mechanism revealed that Ng-Ft1 selectively targets the deubiquitination enzyme USP9X, undermining its role in shielding ß-catenin. This leads to a reduction in the expression of downstream effectors in the Wnt signaling pathway. These findings indicate that Ng-Ft1 could be a promising small-molecule treatment for CRC, working by blocking tumor progression via the Wnt signaling pathway and augmenting CD8+ T cell prevalence within the tumor environment.

Pectolinarigenin ameliorates osteoporosis via enhancing Wnt signaling cascade in PPARß-dependent manner.

BACKGROUND: Osteoporosis is a prevalent metabolic bone disease in older adults. Peroxisome proliferator-activated receptor ß (PPARß), the most abundant PPAR isotype expressed in bone tissues, plays a critical role in regulating the energy metabolism of osteoblasts. However, the botanical compounds targeting PPARß for the treatment of osteoporosis remain largely unexplored. PURPOSE: To discover a potent PPARß agonist from botanical compounds, as well as to investigate the anti-osteoporosis effects and to elucidate the underlying mechanisms of the newly identified PPARß agonist. METHODS: The PPARß agonist effects of botanical compounds were screened by an in vitro luciferase reporter gene assay. The PPARß agonist effects of pectolinarigenin (PEC) in bone marrow mesenchymal stromal cells (BMSCs) were validated by Western blotting. RNA-seq transcriptome analyses were conducted to reveal the underlying osteoporosis mechanisms of PEC in BMSCs. The PPARß antagonist (GSK0660) and Wnt signaling inhibitor (XAV969) were used to explore the role of the PPARß and Wnt signaling cascade in the anti-osteoporosis effects of PEC. PEC or the PEG-PLGA nanoparticles of PEC (PEC-NP) were intraperitoneally administrated in both wild-type mice and ovariectomy-induced osteoporosis mice to examine its anti-osteoporotic effects in vivo. RESULTS: PEC, a newly identified naturally occurring PPARß agonist, significantly promotes osteogenic differentiation and up-regulates the osteogenic differentiation-related genes (Runx2, Osterix, and Bmp2) in BMSCs. RNA sequencing and functional gene enrichment analysis suggested that PEC could activate osteogenic-related signaling pathways, including Wnt and PPAR signaling pathways. Further investigations suggested that PEC could enhance Wnt/ß-catenin signaling in a PPARß-dependent manner in BMSCs. Animal tests showed that PEC-NP promoted bone mass and density, increased the bone cell matrix protein, and accelerated bone formation in wild-type mice, while PEC-NP also played a preventive role in ovariectomy-induced osteoporosis mice via maintaining the expression level of bone cell matrix protein, balancing the rate of bone formation, and slowing down bone loss. Additionally, PEC-NP did not cause any organ injury and body weight loss after long-term use (11 weeks). CONCLUSION: PEC significantly promotes bone formation and reduces bone loss in both BMSCs and ovariectomy-induced osteoporosis mice via enhancing the Wnt signaling cascade in a PPARß-dependent manner, providing a new alternative therapy for preventing estrogen deficiency-induced osteoporotic diseases.

Matrine reduced intestinal stem cell damage in eimeria necatrix-infected chicks via blocking hyperactivation of Wnt signaling.

BACKGROUND: Coccidiosis is a rapidly spreading and acute parasitic disease that seriously threatening the intestinal health of poultry. Matrine from leguminous plants has anthelmintic and anti-inflammatory properties. PURPOSE: This assay was conducted to explore the protective effects of Matrine and the AntiC (a Matrine compound) on Eimeria necatrix (EN)-infected chick small intestines and to provide a nutritional intervention strategy for EN injury. STUDY DESIGN: The in vivo (chick) experiment: A total of 392 one-day-old yellow-feathered broilers were randomly assigned to six groups in a 21-day study: control group, 350 mg/kg Matrine group, 500 mg/kg AntiC group, EN group, and EN + 350 mg/kg Matrine group, EN + 500 mg/kg AntiC group. The in vitro (chick intestinal organoids, IOs): The IOs were treated with PBS, Matrine, AntiC, 3 µM CHIR99021, EN (15,000 EN sporozoites), EN + Matrine, EN + AntiC, EN + Matrine + CHIR99021, EN + AntiC + CHIR99021. METHODS: The structural integrity of chicks jejunal crypt-villus axis was evaluated by hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM). And the activity of intestinal stem cells (ISCs) located in crypts was assessed by in vitro expansion advantages of a primary in IOs model. Then, the changes of Wnt/ß-catenin signaling in jejunal tissues and IOs were detected by Real-Time qPCR,Western blotting and immunohistochemistry. RESULTS: The results showed that dietary supplementation with Matrine or AntiC rescued the jejunal injury caused by EN, as indicated by increased villus height, reduced crypt hyperplasia, and enhanced expression of tight junction proteins. Moreover, there was less budding efficiency of the IOs expanded from jejunal crypts of chicks in the EN group than that in the Matrine and AntiC group, respectively. Further investigation showed that AntiC and Matrine inhibited EN-stimulated Wnt/ß-catenin signaling. The fact that Wnt/ß-catenin activation via CHIR99021 led to the failure of Matrine and AntiC to rescue damaged ISCs confirmed the dominance of this signaling. CONCLUSION: Our results suggest that Matrine and AntiC inhibit ISC proliferation and promote ISC differentiation into absorptive cells by preventing the hyperactivation of Wnt/ß-catenin signaling, thereby standardizing the function of ISC proliferation and differentiation, which provides new insights into mitigating EN injury by Matrine and AntiC.

Dahuang-Wumei decoction protects against hepatic encephalopathy in mice: Behavioural, biochemical, and molecular evidence.

BACKGROUND: Disturbance of the blood‒brain barrier (BBB) and associated inflammatory responses are observed in patients with hepatic encephalopathy (HE) and can cause long-term complications. Dahuang-Wumei decoction (DWD) is a renowned traditional Chinese herbal medicine with a long history of clinical use and has been widely employed as an effective treatment for hepatic encephalopathy (HE). Despite its established efficacy, the precise mechanisms underlying the therapeutic effects of DWD have not been fully elucidated. PURPOSE: The present study aimed to comprehensively explore the potential effects and underlying molecular mechanisms of DWD on HE through an integrated investigation that included both in vivo and in vitro experiments. METHODS: In the present study, carbon tetrachloride (CCl4) and thioacetamide (TAA) were used to establish an HE model in mice. The therapeutic effects of DWD on liver injury, fibrosis, brain injury, behaviour, and consciousness disorders were evaluated in vivo. C8-D1A and bEnd.3 cells were used to construct a BBB model in vitro. The effects of DWD on proinflammatory factor expression, BBB damage and the Wnt/ß-catenin pathway were detected in vivo and in vitro. RESULTS: Our results showed that DWD can improve liver injury and fibrosis and brain damage and inhibit neurofunctional and behavioural disorders in mice with HE. Afterwards, we found that DWD decreased the levels of proinflammatory factors and suppressed BBB disruption by increasing the levels of junction proteins in vivo and vitro. Further studies verified that the Wnt/ß-catenin pathway may play a pivotal role in mediating the inhibitory effect of DWD on HE. CONCLUSION: These results demonstrated that DWD can treat HE by preventing BBB disruption, and the underlying mechanisms involved were associated with the activation of the Wnt/ß-catenin pathway and the inhibition of inflammatory responses.

Film-forming polymer solutions containing cholesterol myristate and berberine mediate pressure ulcer repair via the Wnt/ß-catenin pathway.

Pressure ulcer (PU) is a worldwide problem that is difficult to address because of the related inflammatory response, local hypoxia, and repeated ischaemia/reperfusion, causing great suffering and financial burden to patients. Traditional Chinese medicine turtle plate powder can treat skin trauma, but its composition is complex and inconvenient to use. Here, we combined cholesterol myristate (S8) with berberine (BBR), with anti-inflammatory and antibacterial effects, as a drug and used hydroxypropyl methylcellulose and polyvinylpyrrolidone K30 as carriers to construct a novel film-forming polymeric solution (S8 + BBR FFPS), comprehensively study its reparative effect on PU and explore the potential mechanism in rat PU models. The results showed that S8 + BBR FFPS inhibits excessive inflammatory response, promotes re-epithelialization, and promotes hair follicle growth during the healing process of PU, which may be related to the activation of the Wnt/ß-catenin signalling pathway by S8 + BBR FFPS to mediate hair follicle stem cell proliferation and maintain skin homeostasis. Therefore, S8 + BBR FFPS may be a potential candidate for the treatment of chronic skin injury, and its association with the Wnt/ß-catenin signalling pathway may provide new ideas to guide the design of biomaterial-based wound dressings for chronic wound repair.

Network pharmacology identifies fisetin as a treatment for osteoporosis that activates the Wnt/ß-catenin signaling pathway in BMSCs.

BACKGROUND: Although fisetin may exist widely in many natural herbs, its anti-OP mechanism is still unclear. The aim of this study is to explore the molecular anti-osteoporosis (OP) mechanism of fisetin based on network pharmacology and cell experiments. METHODS: The target of fisetin was extracted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The targets of OP were obtained by DisGeNET, GeneCards and the Comparative Toxicogenomics Database, and the targets of fisetin in OP were screened by cross-analysis. The protein-protein interaction (PPI) network was constructed by STRING, and the core targets were obtained. We performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on common targets via the Database for Annotation, Visualization and Integrated Discovery. Finally, an in vitro cell experiment was used to verify the anti-OP effect and mechanism of fisetin. RESULTS: There are 44 targets of fisetin related to the treatment of OP. The PPI results suggest that CTNNB1, CCND1, TP53, JUN, and AKT1 are the core targets. A total of 259 biological process, 57 molecular function and 26 cell component terms were obtained from GO enrichment analysis. The results of KEGG pathway enrichment analysis suggested that fisetin treatment of OP may be related to the Wnt signaling pathway, estrogen signaling pathway, PI3K-Akt signaling pathway and other signaling pathways. In vitro cell experiments showed that fisetin significantly increased the expression levels of ALP, collagen I, osteopontin and RUNX2 in bone marrow mesenchymal stem cells (BMSCs) (p < 0.05). Fisetin also increased the gene expression levels of Wnt3 and ß-catenin (CTNNB1) in BMSCs, which indicates that fisetin can regulate the Wnt/ß-catenin signaling pathway and promote the osteogenic differentiation of BMSCs. CONCLUSIONS: Fisetin acts on multiple targets and pathways in the treatment of OP; mechanistically, it regulates the Wnt/ß-catenin signaling pathway, which promotes the osteogenic differentiation of BMSCs and maintains bone homeostasis. The results of this study provide a theoretical basis for further study on the complex anti-OP mechanism of fisetin.

Anticonvulsive and Neuroprotective Effects of Eupafolin in Rats Are Associated with the Inhibition of Glutamate Overexcitation and Upregulation of the Wnt/ß-Catenin Signaling Pathway.

Several plant compounds have been found to possess neuroactive properties. The aim of this study was to investigate the anticonvulsant effect of eupafolin, a major active component extracted from Salvia plebeia, a herb used in traditional medicine for its anti-inflammatory properties. To this end, we assessed the anticonvulsant effects of eupafolin in rats intraperitoneally (i.p.) injected with kainic acid (KA) to elucidate this mechanism. Treatment with eupafolin (i.p.) for 30 min before KA administration significantly reduced behavioral and electrographic seizures induced by KA, similar to carbamazepine (i.p.), a widely used antiepileptic drug. Eupafolin treatment also significantly decreased KA seizure-induced neuronal cell death and glutamate elevation in the hippocampus. In addition, eupafolin notably reversed KA seizure-induced alterations in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR2, glutamate decarboxylase 67 (GAD67, GABAergic enzyme), and Wnt signaling-related proteins, including porcupine, Wnt1, phosphorylated-glycogen synthase kinase-3ß, ß-catenin, and Bcl-2 in the hippocampus. Furthermore, the increased level of Dickkopf-related protein 1 (Dkk-1, a Wnt signaling antagonist) and the decreased level of Disheveled1 (Dvl-1, a Wnt signaling activator) in the hippocampus of KA-treated rats were reversed by eupafolin. This study provides evidence of the anticonvulsant and neuroprotective properties of eupafolin and of the involvement of regulation of glutamate overexcitation and Wnt signaling in the mechanisms of these properties. These findings support the benefits of eupafolin in treating epilepsy.

Icariside II suppressed tumorigenesis by epigenetically regulating the circß-catenin-Wnt/ß-catenin axis in colorectal cancer.

Icariside II, a flavonol glycoside, one of the major components of Traditional Chinese Medicine Herba epimedii. In the present study, we found that Icariside II suppressed the proliferation of CRC by inducing cell cycle arrest and apoptosis in vitro and inhibited tumor growth in vivo. The further mechanism investigation showed that Icariside II suppressed the expression of ß-catenin and led to the functional inactivation of Wnt/ß-catenin signaling. Circß-catenin was considered as a promising candidate for mediating the tumorigenesis and the activation of Wnt/ß-catenin signaling in CRC cells. Furthermore, Icariside II has been proven to suppress the biogenesis of circß-catenin via epigenetically targeting DNA methyltransferases (DNMTs) to decrease global DNA methylation levels in CRC cells. Taken together, our results indicated that Icariside II suppressed tumorigenesis by epigenetically silencing the activation of circß-catenin-Wnt/ß-catenin axis in colorectal cancer. More importantly, the information gained from this study suggest that Icariside II may have great potential to be developed as a therapeutic drug for CRC patients.

Guizhi Fuling Capsule inhibits uterine fibroids growth by modulating Med12-mediated Wnt/ß-Catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Fuling Capsule (GFC) is a famous traditional Chinese medicine (TCM) formula recorded in Synopsis of the Golden Chamber, which has achieved obvious effects in the treatment of uterine fibroids (UFs). AIM OF STUDY: Mediator complex subunit 12 (Med12) mutations were closely related to UFs in 85% of fibroid cases. The Wnt/ß-Catenin signaling pathway plays an important role in the occurrence and development of UFs. This study aims to explore the pharmacological mechanism of GFC against UFs in which the Med12-mediated Wnt/ß-Catenin pathway is involved. MATERIALS AND METHODS: Med12 was silenced in uterine fibroid cells (UFCs) using a lentivirus-based Med12 gene-specific RNA interference (RNAi) strategy. Cell proliferation was performed by CCK-8 assay, cell apoptosis and cell cycle were measured by flow cytometry. The rat model of UFs was established by injecting estradiol benzoate and progesterone. Forty-eight rats were divided into six groups, the low-dose GFC (L-GFC) group, the medium-dose GFC (M-GFC) group and the high-dose GFC (H-GFC) group were intragastrically treated with GFC solution at 0.25 g/kg, 0.50 g/kg and 1.00 g/kg per day for 8 weeks, the positive control (PC) group was administrated with mifepristone (2.70 mg/kg/day), the normal control (NC) group and the model control (MC) group were given equal volume of normal saline once a day for 8 weeks. The histopathological changes of uterine tissues were evaluated by H&E staining. The expression of Med12 in uterine tissues were detected by immunohistochemistry. The protein and mRNA levels of associated genes were evaluated by western bolt and real time-PCR, respectively. Related indicators involved in Wnt/ß-Catenin pathway, such as Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7 and C-myc, were compared among different groups. RESULTS: The Wnt/ß-Catenin signaling pathway was inhibited after Med12 gene was knocked out in UFCs. GFC-containing serum could induce cell apoptosis, make the cell cycle stagnated in G0/G1 phase to inhibiting the proliferation and reduce the expression of Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7, and C-myc in control-shRNA cells, while had no significant effect on Med12-shRNA cells. Compared with the MC group, the weight, endometrial thickness, and pathological structure of the uterus in the GFC treated groups were significantly improved. The expression of Med12, Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7, and C-myc that related to Wnt/ß-Catenin pathway in the GFC treated groups were decreased with the increase of dosage administration. CONCLUSIONS: GFC inhibited UFs growth, which was directly associated with Med12 modulated Wnt/ß-Catenin signaling pathway. This study provided new perspective to understand the therapeutic mechanism of UFs.

Quercetin promotes cutaneous wound healing in mice through Wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxytropis falcata Bunge is a legume distributed in Northwest China, which is mainly used to treat knife wounds and inflammation. Quercetin is a bioactive flavonoid in O. falcata and becomes a promising healing compound for its angiogenic and anti-inflammatory activities. However, the healing mechanism of quercetin in cutaneous wound remains elusive. AIM OF THE STUDY: The purpose of this study was to evaluate the healing effect of quercetin on cutaneous wound models in vivo and in vitro, and to reveal the Wnt/ß-catenin pathway and Telomerase reverse transcriptase (TERT) involved mechanisms. MATERIALS AND METHODS: The effects of quercetin on the proliferation and migration of 4 kinds of skin cells were determined by CCK-8 and scratch assay. The wound-healing capacity of quercetin was evaluated in cutaneous wound model of C57BL/6 mice and the wound healing degree was observed by histological staining. The expressions of inflammatory factors, growth factors and the related proteins were detected via Western blot and RT-qPCR analyses. The molecular docking was adopted to evaluate the binding ability of quercetin and TERT. RESULTS: Quercetin could promote both proliferation and migration of fibroblasts, and enhance cutaneous wound healing capacity in mice. Compared to the control group, the wound healing rates in low (1.5 mg/mL), medium (3.0 mg/mL) and high dose (6.0 mg/mL) quercetin groups reached 94.67%, 97.31% and 98.42%, respectively. Moreover, the dermal structure in quercetin treated mice restored normal and the content of collagen fiber became abundant after administration. The levels of inflammatory factors, including tumor necrosis factor-α, interleukin-1ß and interleukin-6 were significantly reduced after quercetin administration. Among which, the level of IL-1ß in cutaneous wound was 0.007 times higher than that of the control group when treated with quercetin of high dose (6.0 mg/mL). The improved level of GSH in quercetin treated cutaneous wounds also indicated its higher antioxidant ability. In addition, dose-dependent positive associations were found in the expression levels of vascular endothelial growth factor, fibroblast growth factor and alpha smooth muscle actin in quercetin treated cutaneous wounds. The significantly upregulated protein levels of Wnt and ß-catenin further indicated the important role of quercetin in promoting wound healing in mice. According to molecular docking analysis, the formed hydrogen bonds between quercetin and Ala195, Gln308, Asn369 and Lys372 residues of TERT also indicated the indispensable role of TERT in improving wound healing capacity. CONCLUSION: Quercetin effectively promoted cutaneous wound healing by enhancing the proliferation and migration of fibroblasts, as well as inhibiting inflammation and increasing the expression of growth factors in mice via Wnt/ß-catenin signaling pathway and TERT. It provides a basis for a more thorough understanding of mechanism of action of O. falcata Bunge in the treatment of knife wounds and burns.

Juglans regia L. extract promotes osteogenesis of human bone marrow mesenchymal stem cells through BMP2/Smad/Runx2 and Wnt/ß-catenin pathways.

BACKGROUND: The present study investigates the effects of Juglans regia L. (walnut, JRL) leaves extract on osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: hBMSCs were incubated with different concentrations of JRL extract (10, 20, 40, or 80 µM). Cell proliferation was evaluated by Cell Counting Kit-8 assay (CCK-8) assay. ALP activity and Alizarin Red staining were used to assess the osteogenesis of BMSCs. Western blot was performed to measure the levels of proteins. RESULTS: Our results showed all concentrations of JRL extract had no significant effect on cell proliferation. JRL extract concentration-dependently promoted osteoblastic differentiation and cell autophagy of hBMSCs, characterized by the increased expression of pro-osteogenic markers alkaline phosphatase (ALP), osteocalcin (BGLAP), osterin, and osteoprotegerin (OPG) and autophagy marker proteins (LC3II, Beclin-1, and p62). Furthermore, JRL extract stimulated the activation BMP2/Smad/Runx2 and Wnt/ß-catenin signaling pathways in hBMSCs, which play key roles in osteogenesis differentiation. Meanwhile, BMP inhibitor (Noggin) and Wnt antagonist Dickkopf-1 (DKK1) both reversed the increases of BGLAP, osterin, and OPG expression induced by JRL extract. CONCLUSIONS: Our findings indicate that JRL extract regulated osteogenic differentiation and cell autophagy of hBMSCs through the BMP2/Smad/Runx2 and Wnt/ß-catenin pathways.

The effects and mechanism of paeoniflorin in promoting osteogenic differentiation of MC3T3-E1.

BACKGROUND: The incidence of osteoporosis and osteoporotic fractures is increasing every year. Traditional Chinese Medicine (TCM) can shed new light on the treatment of osteoporosis. This study aimed to explore the role and mechanism of paeoniflorin in promoting osteogenic differentiation of an osteoblast precursor cell line (MC3T3-E1). METHODS: MC3T3-E1 cells were cultured in osteogenic induction medium (OIM) and OIM combined with different concentrations of paeoniflorin. The optimal dose of paeoniflorin was assessed by a cell counting kit-8 (CCK-8) assay. Then, alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed to assess the osteogenic capacity of paeoniflorin. The transcription of osteogenic genes and the expression of osteogenic proteins were assessed by RT-PCR and Western blotting, respectively. The transcription of Wnt/ß-catenin signaling pathway genes and proteins was assessed by RT-PCR and Western blotting, respectively. Finally, Dickkopf-1 (DKK-1), a Wnt/ß-catenin signaling pathway inhibitor, was used to identify whether the Wnt/ß-catenin signaling pathway was involved in the osteogenic differentiation of paeoniflorin. Osteoclastogenesis in RAW264.7 cells was identified by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: At concentrations ranging from 0.1 to 100 µM, paeoniflorin was not cytotoxic to MC3T3-E1 cells. Paeoniflorin significantly increased the osteogenic differentiation of MC3T3-E1 cells in a dose-dependent manner. Moreover, paeoniflorin significantly increased osteogenic differentiation gene and protein expression. Through bioinformatic analysis, paeoniflorin-affected genes were found to be involved in different signaling pathways, such as the Wnt/ß-catenin signaling pathway. Paeoniflorin enhanced ß-catenin and CyclinD1 expression compared with that of the control groups. DKK-1 partially reversed the promoting effects of paeoniflorin in promoting osteogenic differentiation of MC3T3-E1 cells. Moreover, paeoniflorin inhibited the osteoclastogenesis of RAW264.7 cells. CONCLUSION: Paeoniflorin promotes osteogenic differentiation in MC3T3-E1 cells by regulating the Wnt/ß-catenin pathway. Paeoniflorin is a potential therapeutic agent for the treatment of osteoporosis.

Dracunculin Inhibits Adipogenesis in Human Bone Marrow-Derived Mesenchymal Stromal Cells by Activating AMPK and Wnt/ß-Catenin Signaling.

Increased bone marrow adiposity is widely observed in patients with obesity and osteoporosis and reported to have deleterious effects on bone formation. Dracunculin (DCC) is a coumarin isolated from Artemisia spp. but, until now, has not been studied for its bioactive potential except antitrypanosomal activity. In this context, current study has reported the anti-adipogenic effect of DCC in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). DCC dose-dependently inhibited the lipid accumulation and expression of adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) in hBM-MSCs induced to undergo adipogenesis. To elucidate its action mechanism, the effect of DCC on Wnt/ß-catenin and AMPK pathways was examined. Results showed that DCC treatment activated Wnt/ß-catenin signaling pathway via AMPK evidenced by increased levels of AMPK phosphorylation and Wnt10b expression after DCC treatment. In addition, DCC treated adipo-induced hBM-MSCs exhibited significantly increased nuclear levels of ß-catenin compared with diminished nuclear PPARγ levels. In conclusion, DCC was shown to be able to hinder adipogenesis by activating the ß-catenin via AMPK, providing potential utilization of DCC as a nutraceutical against bone marrow adiposity.

Erianin suppresses proliferation and migration of cancer cells in a pyruvate carboxylase-dependent manner.

Erianin is a natural small molecule dibenzyl compound extracted from Dendrobium officinale or Dendrobium chrysotoxum. Studies show erianin has many pharmacological functions such as antioxidant, antibacterial, antiviral, improving diabetic nephropathy, relaxing bronchial smooth muscle and anti-tumor. However, the erianin-mediated molecular mechanism is elusive, and the target protein of erianin is not clear yet. Here, we screened and identified that the target protein of erianin in human hepatoma HepG2 cells is human pyruvate carboxylase, and explored the anti-tumor signal pathway regulated by erianin in several cell lines. Firstly, the interaction between human pyruvate carboxylase and erianin was studied by bioinformatics and biochemical methods. Secondly, in vitro, erianin can specifically inhibit the activity of human pyruvate carboxylase, and the purified human pyruvate carboxylase can specifically bind to the activity probe of erianin. Thirdly, human pyruvate carboxylase is highly expressed in a variety of malignant tumors, and the inhibitory effect of erianin on tumor cells is positively correlated with the expression of human pyruvate carboxylase, and erianin can selectively inhibit the activity of pyruvate carboxylase. Finally, erianin can regulate the pyruvate carboxylase-mediated Wnt/ ß- Catenin pathway. All of which provide important data for the further study of the anticancer mechanism of erianin, and lay a solid foundation for the further development and utilization of erianin.

Chemical composition and protective effect of cerebrospinal fluid of Dan-Deng-Tong-Nao capsules on brain microvascular endothelial cells injured by OGD/R.

ETHNOPHARMACOLOGICAL RELEVANCE: Dan-Deng-Tong-Nao Capsules (DDTNC) is a Chinese patent medicine and has been used in treating cerebral ischemic stroke (IS) for a long time in China, protection of brain microvascular endothelial cells (BMECs) is the main treatment strategy. But the holistic chemical information and potential bioactive components of DDTNC on protecting BMECs and its underlying mechanism is still unclear. AIM OF THE STUDY: To identify the active ingredients of DDTNC and to explore the protective effects of DDTNC on BMECs associated with Wnt/ß-catenin pathway. MATERIALS AND METHODS: The components of DDTNC and cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF) were detected by High performance liquid chromatography combined with Diode array detector (HPLC-DAD) and Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), respectively. The experiment rat model was established with middle cerebral artery occlusion (MCAO), the therapeutic effects of DDTNC were assessed by Longa assay and TTC staining. The cerebral micro vessel density was determined by immunofluorescence staining. The injured BMECs caused by oxygen-glucose deprivation and reperfusion (OGD/R) was used to evaluate the protective effect of cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF). The cell survival rate was detected by the method of CCK-8, the intracellular Ca2+ and reactive oxygen species (ROS) was estimated by Fluo-3. Moreover, the proteins of Bax, Bcl-2, Wnt, ß-catenin, GSK-3ß was determined by Western blotting. RESULTS: The RSD values of all methodological studies were less than 3.0%. A total of 20 compounds were detected under the optimized HPLC-DAD chromatographic condition. In the UPLC-Q-TOF-MS negative mode, peak 1 and peak 2 were detecteted in DDTNC-CSF and was identified as Danshensu and Puerarin, respectively. In the UPLC-Q-TOF-MS positive mode, peak 1 and peak 3 were detecteted in DDTNC-CSF and was identified as Danshensu and Scutellarin, respectively. DDTNC significantly decreased the Longa values and infarct volume and significantly increased the cerebral microvessel density of the MCAO rats. The accumulation of intracellular Ca2+ and ROS in BMECs injured by OGD/R decreased significantly in DDTNC-CSF group. The expression of Bcl-2, ß-catenin, wnt-1 was upregulated by DDTNC-CSF and the level of Bax and GSK3ß could be downregulated by DDTNC-CSF. CONCLUSION: The present study provided a scientific basis for revealing the mechanism of DDTNC in the treatment of IS and DDTNC is expected to be an effective drug for the treatment of IS.

Potential therapeutic efficacy of pachymic acid in chronic kidney disease induced in rats: role of Wnt/ß-catenin/renin-angiotensin axis.

OBJECTIVES: Chronic kidney disease (CKD) is a major public health problem associated with high mortality. The therapeutic effects of pachymic in CKD management and its underlying mechanisms have not been studied. Therefore, we aimed to investigate the possible inhibitory effect of PA on renal Wnt/ß-catenin signalling in CKD. METHODS: CKD was induced in rats by doxorubicin (DOX; 3.5 mg/kg i.p., twice weekly for 3 weeks). Rats were treated orally with PA (10 mg/kg/day), LOS (10 mg/kg/day) or their combination (PA + LOS) for 4 weeks starting after the last dose of DOX. KEY FINDINGS: DOX-induced renal injury was characterized by high serum cystatin-C, and urine albumin/creatinine ratio, renal content of podocin and klotho were decreased. Tumour necrosis factor-α, interleukin-6, interleukin-1ß, Wnt1, active ß-catenin/total ß-catenin ratio and fibronectin along with mRNA expression of RENIN, ACE and AT1 were increased in renal tissues. Treatment with either PA or LOS ameliorated all DOX-induced changes. The combined treatment was more effective in improving all changes than monotherapy. CONCLUSIONS: These results suggest a new therapeutic benefit of PA in ameliorating CKD in rats through its up-regulatory effect on renal klotho thereby preventing Wnt/ß-catenin reactivation and RAS gene expression. PA/LOS combination provided an additional inhibition of Wnt/ß-catenin signalling and its downstream targets.

Silybum marianum total extract, silymarin and silibinin abate hepatocarcinogenesis and hepatocellular carcinoma growth via modulation of the HGF/c-Met, Wnt/ß-catenin, and PI3K/Akt/mTOR signaling pathways.

Hepatocellular carcinoma (HCC) has been identified as one of the most deadly malignancies with limited therapeutic efficacy worldwide. However, understanding the molecular mechanisms of crosstalk between signaling pathways in HCC and predicting cancer cell responses to targeted therapeutic interventions remain to be challenge. Thus, in this study, we aimed to evaluate the anticancerous efficacy of Silybum marianum total extract (STE), silymarin (Sm), and silibinin (Sb) against experimentally-induced HCC in rats. In vitro investigations were also performed and the anticancer effects against HCC cell lines (HepG2 and Huh7) were confirmed. Wistar rats were given diethylnitrosamine (DEN)/2-acetylaminofluorene (AAF)/carbon tetrachloride (CCl4) and were orally treated with STE (200 mg/kg body weight (bw)), Sm (150 mg/kg bw), and Sb (5 mg/kg bw) every other day from the 1st or 16th week to the 25th week of DEN/AAF/CCl4 injection. Treatment with STE, Sm, and Sb inhibited the growth of cancerous lesions in DEN/AAF/CCl4-treated rats. This inhibition was associated with inhibition of Ki-67 expression and repression of HGF/cMet, Wnt/ß-catenin, and PI3K/Akt/mTOR signaling pathways. STE, Sm, and Sb improved liver function biomarkers and tumor markers (AFP, CEA, and CA19.9) and increased total protein and albumin levels in serum. STE, Sm, and Sb treatment was also noted to reduce the hepatic production of lipid peroxides, increase hepatic glutathione content, and induce the activities of hepatic antioxidant enzymes in DEN/AAF/CCl4-treated rats. These results indicate that STE, Sm, and Sb exert anti-HCC effects through multiple pathways, including suppression of Ki-67 expression and HGF/cMet, Wnt/ß-catenin, and PI3K/Akt/mTOR pathways and enhancement of antioxidant defense mechanisms.

Kangxianruangan granule­containing serum mediated inhibition of hepatic oval cell differentiation into hepatocellular carcinoma cells via the Wnt­1/ß­catenin signaling pathway.

Hepatocellular carcinoma is a malignancy with poor clinical prognosis. Hepatic oval cells (HOCs) tend to differentiate into cancerous hepatocellular carcinoma cells (HCCs) in the tumor microenvironment. The purpose of the present study was to explore the role of kangxianruangan granule (KXRG)­containing serum in inhibiting the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway. N­methyl­N'­nitro­N­nitrosoguanidine (MNNG) was applied to induce the transformation of the rat HOC cell line WB­F344 into HCCs. The overexpression plasmid, Wnt­1­up, was utilized to increase Wnt­1 expression. Subsequently, high, medium and low concentrations of KXRG were applied to MNNG­treated WB­F344 cells to assess the inhibitory effect of KXRG on cell differentiation. Flow cytometry was conducted to detect the cell cycle distribution, apoptotic rate and expression of cytokeratin­19 (CK­19) protein in cells. An immunofluorescence double staining protocol was used to detect the expression of Wnt­1 and ß­catenin. ELISAs were performed to detect α fetoprotein in the cell supernatants. Reverse transcription­quantitative PCR and western blotting were conducted to detect the mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, matrix metalloproteinase­7 (MMP­7), Axin2 and epithelial cell adhesion molecule (EpCAM) in cells. Compared with the normal group, the apoptotic rate, proportion of S phase cells, concentration of AFP in the cell supernatant, level of CK­19 protein, and mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, MMP­7, Axin2 and EpCAM were all significantly increased in the model group. Addition of KXRG significantly reduced the aforementioned indicators compared with the model group. Moreover, Wnt­1 overexpression further increased the aforementioned indicators compared with the model group, whereas KXRG significantly inhibited these effects. The results indicated that KXRG inhibited the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway, which suggested the potential clinical application of KXRG for the prevention of hepatocellular carcinoma.

In vitro and in silico anti-osteoporosis activities and underlying mechanisms of a fructan, ABW90-1, from Achyranthes bidentate.

Achyranthes bidentata is a traditional Chinese medicine used to treat osteoporosis. AB90, a crude saccharide from A. bidentata, showed excellent osteoprotective effects in ovariectomized rats, and ABW90-1, an oligosaccharide purified from AB90, stimulated significant differentiation of osteoblasts. However, the osteogenic effects and underlying mechanisms of ABW90-1 have remained unknown. In the present study, we found that ABW90-1 significantly promoted ALP activity, mineralization, and the expression of osteogenic markers in MC3T3-E1 cells. ABW90-1 showed strong binding with the WNT signaling complex and BMP2 based on number of interactions, hydrogen bond length, and binding energy in silico. ABW90-1 significantly increased the expression of active-ß-catenin, p-GSK-3ß, LEF-1, BMP2, and p-SMAD1. Importantly, the osteogenic effects of ABW90-1 were partially suppressed by DKK-1 and Noggin, which are specific inhibitors of the WNT and BMP signaling pathways, respectively. Collectively, these findings suggest that ABW90-1 has osteogenic effects through crosstalk between WNT/ß-catenin and BMP2/SMAD1 signaling pathways.

Protective effect of Pai-Nong-San against AOM/DSS-induced CAC in mice through inhibiting the Wnt signaling pathway.

Pai-Nong-San (PNS), a prescription of traditional Chinese medicine, has been used for years to treat abscessation-induced diseases including colitis and colorectal cancer. This study was aimed to investigate the preventive effects and possible protective mechanism of PNS on a colitis-associated colorectal cancer (CAC) mouse model induced by azoxymethane (AOM)/dextran sodium sulfate (DSS). The macroscopic and histopathologic examinations of colon injury and DAI score were observed. The inflammatory indicators of intestinal immunity were determined by immunohistochemistry and immunofluorescence. The high throughput 16S rRNA sequence of gut microbiota in the feces of mice was performed. Western blot was used to investigate the protein expression of the Wnt signaling pathway in colon tissues. PNS improved colon injury, as manifested by the alleviation of hematochezia, decreased DAI score, increased colon length, and reversal of pathological changes. PNS treatment protected against AOM/DSS-induced colon inflammation by regulating the expression of CD4+ and CD8+ T cells, inhibiting the production of HIF-α, IL-6, and TNF-α, and promoting the expression of IL-4 and IFN-γ in colon tissues. Meanwhile, PNS improved the components of gut microbiota, as measured by the adjusted levels of Firmicutes, Bacteroidetes, Proteobacteria, and Lactobacillus. PNS down-regulated the protein expression of p-GSK-3ß, ß-catenin, and c-Myc, while up-regulating the GSK-3ß and p-ß-catenin in colon tissues of CAC mice. In conclusion, our results suggested that PNS exhibits protective effect on AOM/DSS-induced colon injury and alleviates the development of CAC through suppressing inflammation, improving gut microbiota, and inhibiting the Wnt signaling pathway.