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ObjectiveTo observe the effects of the kidney-tonifying and blood-activating prescription on the Wnt/β-catenin signaling pathway and uterine spiral artery remodeling in a mouse model of recurrent miscarriage and to explore its underlying mechanism. MethodA mouse model of normal pregnancy was established by mating CBA/J mice with BALB/c mice. A mouse model of recurrent miscarriage was established by mating CBA/J mice with DBA/2 mice. The modeled mice of recurrent miscarriage were randomized into model， dydrogesterone， and low- and high-dose Chinese medicine groups. The mice in normal pregnancy were used as the control group. Each group consisted of 10 mice， and the drug administration lasted for 14 days. After the treatment， the embryo absorption rate of each group was recorded. Hematoxylin-eosin （HE） staining was employed to observe the pathological morphology of the uterine decidua， and the physiological transformation rate of spiral arteries （SPA） was evaluated. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to determine the mRNA and protein levels， respectively， of matrix metalloproteinases （MMP）-2， MMP-9， vascular endothelial growth factor （VEGF）， and Wnt/β-catenin signaling pathway. ResultCompared with the control group， the model group presented increased embryo absorption rate （P<0.05）， decreased physiological transformation rate of uterine SPA （P<0.05）， cellular swelling， degeneration， and disordered arrangement in the uterine decidua tissue， and down-regulated mRNA and protein levels of key factors involved in SPA remodeling （MMP-2， MMP-9， VEGF） and the Wnt/β-catenin signaling pathway （Wnt2， β-catenin， Cyclin D1， c-Myc） （P<0.05）. Compared with the model group， both the low- and high-dose Chinese medicine reduced embryo absorption rate （P<0.05）， increased SPA physiological transformation rate （P<0.05）， improved uterine decidua tissue morphology， and increased decidua vessel count. Furthermore， they up-regulated the mRNA and protein levels of MMP-2， MMP-9， VEGF， and proteins in the Wnt/β-catenin signaling pathway （P<0.05）. ConclusionRecurrent miscarriage is associated with impaired uterine spiral artery remodeling. The kidney-tonifying and blood-activating prescription can promote uterine spiral artery remodeling by activating the Wnt/β-catenin signaling pathway and promoting the expression of VEGF， MMP-2， and MMP-9， thus treating recurrent miscarriage.
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Aims: To investigate the relationship between E-cadherin, beta-catenin, N-cadherin, ZEB1, and ?SMA as epithelial-mesenchymal transformation markers with tumor stage, lymph node metastasis (LNM), and overall survival (OS) in laryngeal squamous cell carcinomas (LSCC). Materials and Methods: A total of 100 cases diagnosed with LSCC were included in the study. Data about the lymphovascular invasion (LVI), perineural invasion (PNI), necrosis, and LNM were recorded by evaluating hematoxylin-eosin–stained slides. Markers of E-cadherin, beta-catenin, N-cadherin, ZEB1, and ?SMA were applied to the sections prepared from paraffin blocks of tumor samples. Results: Ninety-five male and five female patients were included in the study, and 38 of them exited. A significant relationship was observed between OS with advanced tumor stage, presence of LNM and PNI. A significant relationship was found between increased tumor Zeb1 expression and advanced tumor stage. In univariate and multivariate analyses, a significant negative relationship with OS, and increased Zeb1 expression in tumor and tumor stroma was seen. Any relationship was not observed between E-cadherin, beta-catenin, N-cadherin, and ?SMA and OS. Conclusion: Among the EMT markers, we evaluated in our study, it was seen that Zeb1, which is an EMT transcription factor, is associated with tumor stage, LNM, and OS. Remarkably, Zeb1 expression observed in tumor stroma was also significant for OS. Any similar data reported for LSCCs have not been encountered in the literature, and it was thought that it would be appropriate to support our findings with further studies to be performed on this subject.
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Resumen El cáncer colorrectal (CCR) es una enfermedad compleja determinada por factores de riesgo genéticos, ambientales y de estilo de vida. Los factores genéticos (hereditarios) tienen gran influencia en su desarrollo, sin embargo, la mayoría de los casos de CCR son esporádicos y se desarrollan gradualmente a lo largo de varios años. Los principales factores ambientales de riesgo están asociados a la vía de señalización de β-catenina, entre ellos obesidad, falta de actividad física, consumo de carnes rojas y procesadas, alcoholismo y tabaquismo. La vía está relacionada con la regulación de la homeostasis celular, autorrenovación celular durante la embriogénesis y edad adulta. La principal recomendación para evitar el desarrollo del CCR es reducir los factores de riesgo, aumentar el consumo de frutas, verduras y granos, hacer ejercicio de manera rutinaria y limitar el consumo tanto de alcohol como de tabaco. Dado que los antecedentes familiares y la presencia de un síndrome hereditario aumentan el riesgo, se sugiere hacer exámenes periódicos para detectar CCR y emplear predictores del desarrollo como los marcadores bioquímicos y moleculares, los cuales se presentan en este trabajo.
Abstract Colorectal cancer (CRC) is a complex disease, determined by genetic, environmental and lifestyle-associated risk factors. Genetic (inherited) factors have great influence on its development; however, most cases of CRC are sporadic and gradually develop over several years. The main environmental risk factors are associated with β-catenin signaling pathway, including obesity, lack of physical activity, consumption of red and processed meats, alcoholism, and smoking. The pathway is related to cell homeostasis regulation and cell self-renewal during embryogenesis and adulthood. The main recommendation for preventing the development of CRC is to reduce the risk factors, increase the consumption of fruits, vegetables and grains, exercise regularly and limit the consumption of both alcohol and tobacco. However, family history and the presence of a hereditary syndrome increase the risk, which is why carrying out periodic examinations to detect CRC is suggested, using development predictors such as biochemical and molecular markers, which are discussed in this work.
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Background: Epithelial-mesenchymal transition (EMT) is the heart of invasion. EMT associated with cancer progression and metastasis is known as type III EMT. Beta-catenin, E-cadherin, and MMP9 markers of EMT are routinely employed for diagnostic purposes. Aims: We employed these markers to study EMT by immunohistochemistry (IHC) in gall bladder cancer (GBC) with respect to depth of tumor invasion, clinical outcome, and disease-free survival. Settings and Design: This was a prospective case-control study. Material and Methods: Seventy gall bladders were included (50 GBC and 20 CC). After detailed histology, immunoexpression was studied in terms of percentage and strength of expression. Statistics Analysis Used: Expression was compared between CC and GBC by Student t test and analysis of variance. Kaplan–Meier was used for survival analysis, and the extent of agreement (“Kappa”) was calculated. Results and Conclusions: The age of incidence of GBC was 49.40 (+11.6) years with female predominance (F:M = 4:1). In 88% (44/50) of GBC, the fundus was involved. Moderately differentiated adenocarcinoma was most frequent [54%; 27/50]. Significant downregulation of E-cadherin (P = 0.022) and beta-catenin (P < 0.001) and upregulation in MMP9 (P < 0.001) were seen in GBC with respect to CC with significant association among them. MMP9 expression was significantly associated with higher tumor stage but with chemotherapeutic response. Our results display that epithelial-mesenchymal transition type III plays a role in GBC invasion. MMP9 overexpression and loss of membranous beta-catenin may be considered a marker for poor clinical outcomes and advanced disease.
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Taste is a critical sensory function for human as it supports sustenance and alerts the body to toxins. Taste dysfunction is a common side effect of radiotherapy for the head and neck cancers, which is often accompanied by oral mucositis in the early stage. It is associated with anorexia, anxiety and depression, leading to declined quality of life and treatment tolerance. The incidence of radiation-induced taste dysfunction is high, and its clinical manifestations include increased taste threshold, tastelessness, and persistent bitter, sour or metallic taste, which exert significant effect upon the quality of life. At present, effective therapeutic measures for radiation-induced taste dysfunction are still lacking. In this article, research progresses on clinical characteristics and the potential mechanisms of radiation-induced taste dysfunction were reviewed, aiming to provide reference for the mechanism, prevention and treatment for taste dysfunction.
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Objective:To observe the repair effect and possible mechanism of Dipsacus saponins Ⅵ on tibial fracture model rats.Methods:Thirty Sprague Dawley (SD) rats were randomly divided into model group, intervention group, and combination group, with 10 rats in each group, to establish a tibial fracture rat model using the sawing method. On the second day after surgery, the intervention group was intraperitoneally injected with 10 mg/kg of Chuanduduan saponin Ⅵ; The combination group received intraperitoneal injection of Dipsacus saponins Ⅵ 10 mg/kg and XAV939 1 mg/animal; The model group was intraperitoneally injected with 0.2 ml of physiological saline solution and 0.2 ml of dimethylsulfoxide (DMSO) solution; Once a day, continuous intervention for 14 days. After 2 to 4 weeks of intervention, Micro CT scan and X-ray scan were used to observe the fracture healing status; After 4 weeks of intervention, the wet weight of the tibia was detected; Hematoxylin eosin (HE) staining was used to observe the pathological changes of callus tissue; The Western blot method was used to detect the expression level of callus tissue β- catenin (β-catenin), p-β-catenin, glycogen synthase kinase 3β (GSK-3 β) and Runt related transcription factor 2 (Runx2) protein.Results:After 2 and 4 weeks of intervention, the bone volume fraction (BV/TV), number of trabeculae (Tb.N), Lane Sandhu score, and callus volume in the intervention group were higher than those in the model group (all P<0.05); After 2 and 4 weeks of intervention, the BV/TV, Tb.N, Lane Sandhu score, and callus volume in the combined group were lower than those in the intervention group (all P<0.05). The wet weight of the tibia in the intervention group was higher than that in the model group at 4 weeks after intervention ( P<0.05); The wet weight of the tibia in the combined group was lower than that in the intervention group ( P<0.05). The HE staining results showed that the model group had fibrous tissue hyperplasia and more bone trabeculae, but the maturity was not high and the thickening was not significant; The intervention group formed more bony callus, with orderly arrangement of bone trabeculae, partially mature, and obvious mineralization, consistent with the direction of stress; The combined group formed more cartilaginous and fibrous callus, with more mineralization at the edge of the cartilaginous callus and the formation of bone trabeculae. Abundant capillaries can be observed in the gaps. The expression level of Runx2 and p-β-catenin/β-catenin protein in callus tissue of the intervention group was higher than that of the model group, the protein expression GSK-3 β level was lower than that of the model group (all P<0.05); The expression level of Runx2 and p-β-catenin/β-catenin protein in the callus tissue of the combined group was lower than that of the intervention group; the protein expression level of GSK-3β was higher than that of the intervention group (all P<0.05). Conclusions:Dipsacus saponins Ⅵ can effectively promote fracture repair in tibial fracture model rats; It is possible to plays a role by activating the Wnt/β-catenin signaling pathway.
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OBJECTIVE To investigate whether icari-in(ICA)plays a neuroprotective role by improving glyco-lytic function through activating Wnt/β-catenin signaling pathway.METHODS HT22 cells were treated with Aβ25-35 for 24 h to establish AD cell model,ICA was added in 2 h before Aβ25-35 and the DKK1(a specific inhibitor of the Wnt signaling pathway)was added in 0.5 h before ICA.Pharmacodynamic study:HT22 cells were divided into control group,ICA group(ICA 10 μmol·L-1),model group(Aβ25-3520 μmol·L-1),model + ICA group(Aβ25-3520 μmol·L-1 +ICA 2.5,5,10 μmol·L-1);Mechanism study:HT22 cells were divided into control group,model group,Aβ25-35+ICA 10 μmol·L-1 group,Aβ25-35+DKK1 group,Aβ25-35+DKK1+ ICA group.The cell viability was detected by MTT assay and the cell morphology was obtained by microscope,the lactate content was detected by lactate assay,the ATP content was measured with the chemiluminescence method,the expression levels of HK1,PKM1 and the pro-tein expression of molecules related to the Wnt/β-catenin signaling pathway(Wnt3a,GSK3β,pGSK3β Try216,pGSK3β Ser9,β-catenin,pβ-catenin Ser33/37 Thr41,Active β-catenin and nuclear β-catenin)was assayed by Western blotting.The nuclear translocation of β-catenin was observed by immunofluorescent staining.RESULTS Compared with the control group,the viability of cells in the model group was reduced,the morphology of cells was significantly damaged,the ATP content and lactate content were significantly decreased,and the glycolytic key enzymes:the protein levels of HK1,PKM1 and the protein levels of Wnt3a,pGSK3β Ser9,active β-catenin and nuclear β-catenin were significantly reduced,and the phosphorylation levels of β-catenin Ser33/37 Thr41 were significantly increased.Compared with the model group,the cell morphology was significantly improved and the viability was significantly increased,the ATP and lactate content were significantly increased,the expressions of HK1,PKM1 and Wnt3a,pGSK3β Ser9,active β-catenin and nuclear β-catenin protein were significantly upregulat-ed,and the phosphorylation levels of β-catenin Ser33/37 Thr41 were significantly reduced after ICA treatment.However,when the canonical Wnt signaling was inhibited by DKK1,the above effects of ICA on glycolysis were abolished.CONCLUSION ICA exerts neuroprotective effects on Aβ25-35-induced HT22 cell injury by enhancing the glycolysis function through the activation of the Wnt/β-catenin signaling pathway.
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@#Both Wnt/β-catenin signaling pathway and NF-κB signaling pathway are highly conservative pathways that regulate a variety of biological processes, and their cross-regulation have attracted attention in many biological and medical research fields. In this review, we summarize the cross-regulation between Wnt/β-catenin signaling pathway and NF-κB signaling pathway and discuss their involvement in the multi-directional differentiation of mesenchymal stem cells.
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OBJECTIVE To explore the intervention effect and related mechanism of Tongxinluo capsule on renal fibrosis in rats with diabetic nephropathy （DN）. METHODS Eight rats were selected as control group （ordinary feed）， the remaining rats were given high-glucose and high-fat diet combined with ip injection of streptozotocin （35 mg/kg） to induce DN model. Model rats were randomly divided into model group （purified water）， irbesartan group （positive control， 14.12 mg/kg） and Tongxinluo capsule group （0.3 g/kg）， including 12 rats in the model group and 11 rats for each of the other two groups. All groups were given relevant medicine or water intragastrically， once a day， for 16 consecutive weeks. After the last medication， fasting blood glucose and 24 h urinary total protein （24 h UTP） were detected. Pathological changes in renal cortex of rats in each group were observed. Serum levels of tissue-type plasminogen activator （PA） and plasminogen activator inhibitor 1 （PAI-1） were measured. mRNA expressions of transforming growth factor-β（1 TGF-β1）， type Ⅳ collagen（COL-Ⅳ）， Wnt4 and β-catenin in renal cortex of rats were detected. The protein depositions or expressions of TGF-β1， COL-Ⅳ， focal adhesion kinase （FAK）， integrin-linked kinase （ILK）， E-cadherin， PA， PAI-1， Wnt4 and β-catenin in renal cortex of rats were observed or determined. RESULTS Compared with model group， 24 h UTP of rats in Tongxinluo capsule group were all significantly reduced （P＜0.05）； pathological damage and fibrosis of renal cortex were relieved； the expression of PA in serum and renal cortex was significantly increased， while PAI-1 level was significantly reduced （P＜0.05）； the depositions of COL-Ⅳ and TGF-β1 in renal cortex were all reduced， and corresponding mRNA expression was decreased significantly （P＜0.05）； the depositions of ILK and FAK were decreased， while the deposition of E-cadherin was increased； protein and mRNA expressions of Wnt4 and β-catenin were significantly reduced （P＜0.05）. CONCLUSIONS Tongxinluo capsule can relieve pathological damage to renal tissue and renal fibrosis of DN model rats， and reduce extracellular matrix deposition. The mechanism may be related to regulation of fibrinolytic system activity， the decrease of ILK and FAK expression， and inhibition of Wnt/β-catenin signaling pathway.
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Diabetic kidney disease （DKD） is a common complication of diabetes and a leading cause of end-stage kidney disease. Its pathogenesis is complex， and it presents a significant challenge in treatment， gradually becoming a major global public health issue. One of the main pathological changes in DKD is tubulointerstitial fibrosis， clinically characterized by proteinuria and declining kidney function， which severely impacts the daily life of patients. Currently， western medicine commonly uses methods such as controlling blood sugar and blood pressure， and reducing proteinuria to treat DKD， but the efficacy is unsatisfactory， and there are many side effects. As reported， traditional Chinese medicine （TCM） treatment for DKD has many advantages， such as low cost， significant efficacy， and minimal adverse reactions. More researchers focusing on DKD are turning their attention to TCM， and progress has been made in related studies both in China and abroad. The Wnt/β-catenin signaling pathway is relatively evolutionarily conserved and plays a crucial role in normal biological development and the entire life process. Studies have demonstrated that abnormal activation of the Wnt/β-catenin signaling pathway is related to renal fibrosis， which coincides with TCM theory of "collateral diseases". By reviewing relevant literature， this article reviewed the Wnt/β-catenin signaling pathway and its role in DKD and summarized the research status of TCM monomers， single drug extracts， and TCM formulas in improving renal fibrosis and treating DKD through the improvement of glomerular mesangial cells， renal tubular epithelial cells， and podocyte injury， aiming to provide new ideas and directions for TCM treatment of DKD.
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Traditional Chinese medicine （TCM） has certain advantages in the treatment of chronic kidney disease-mineral and bone disorder （CKD-MBD）. In recent years， there have been many studies on the treatment of CKD-MBD by Chinese medicinal compounds and monomers. As revealed by literature retrieval， the research on the mechanism of Chinese medicine in intervening in signaling pathways related to CKD-MBD was mainly based on self-made Chinese medicinal compounds， and the action pathways involved fibroblast growth factor 23/Klotho （FGF23/Klotho） signaling pathway， Wnt/β-catenin signaling pathway， receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin （RANK/RANKL/OPG） system， and other signaling pathways. TCM can improve calcium and phosphorus metabolism and bone metabolism disorder， and regulate inflammatory reaction， oxidative stress， apoptosis， and autophagy by regulating this series of signaling pathways for the treatment of CKD-MBD. This paper introduced the research results of these signaling pathways and the mechanism of TCM in the treatment of CKD-MBD in order to provide ideas and references for the related research of Chinese medicine in the treatment of CKD-MBD.
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Background Research on non-target organ damage of biological pesticides has attracted much attention. Rotenone exposure may be far beyond the occupational environment, and the exposureduring pregnancy may be increased through bioaccumulation, fruit or vegetable residues, and other forms of oral intake. At present, the effects of rotenone on placental development and its mechanism are still unknown. Objective To investigate the developmental damage of rat placenta and evaluate the expression levels of glycogen synthase kinase (GSK-3β) and beta catenin (β-catenin) followed by rotenone exposure through the placental barrier during pregnancy, as well as to propose possible associated mechanisms. Methods Eighteen sexually mature SD female infertile rats without specific pathogens were selected and divided into three groups: blank control group (0.9% saline), corn oil group, and rotenone group (corn oil + 2 mg·kg−1 rotenone) by random number method, six female animals in each group. Another six male rats were selected and mated to the female rats at night with a female to male ratio of 3:1 per cage. Pregnant rats were given 0.9% saline, corn oil, and 2 mg·kg−1 rotenone preparation by isovolumetric gavage once daily for the entire gestation period (19 d), and their conditions were observed after the last dose. The pregnant rats were anesthetized, and the size of the placenta and blood perfusion were detected by ultrasound the next day of the last dose of rotenone. Then, 3 pregnant rats in each group were sacrificed immediately and the placenta and umbilical cord tissues were dissected. The remaining 9 pregnant rats gave birth naturally, and the fetuses were observed for developmental evaluation and weighed. The histopathological changes of umbilical cord and placenta were observed by hematoxylin-eosin staining. The reactive oxygen species levels of placenta tissues were detected by flow cytometry. The Ca2+-ATPase activity of placenta tissues was detected by colorimetric method. The localization and levels of GSK-3β and β-catenin expression of placenta were detected by immunohistochemistry. The p-GSK-3β/GSK-3β and p-β-catenin/β-catenin protein expression in placental tissues were measured by Western blotting. Results No injury or death was recorded during the pregnant rats receiving rotennon administration. Adverse pregnancy outcomes such as fetal absorption and postpartum stillbirth were found in the rotenone group, and the weight of the fetal mice decreased (P<0.05). The B-ultrasound showed disc-shaped placenta with a thick middle and thin edge, smooth fetal surface, rough maternal surface, visible placental lobules, granular echotexture of the placenta with comma-like echogenic densities, and chorionic plate showing deep indentations, no calcification, degeneration, or necrosis in each group. Compared with the corn oil group, the fetal surface diameter of the placenta was reduced in the rotenone group (P<0.05). The Doppler color ultrasound showed that interplacental blood flow was reduced in the rotenone group, while interplacental blood flow was abundant in the blank control and the corn oil groups. The hematoxylin-eosin staining results showed that smooth muscle cells in the umbilical cord tissues of rats were loosely arranged, with fuzzy nuclei and inflammatory infiltration in the rotenone group. The placental trophoblast cells were small in size, disorderly arranged with nuclear fragmentation and cytoplasm turbidity. The tissue reactive oxygen species level in the rotenone group was higher than those in the other two groups (P<0.05). The Ca2+-ATPase activity of placental tissues was reduced in the rotenone group (P<0.05). The immunofluorescence low-magnification observation showed that GSK-3β and β-catenin were expressed in placental tissue, weak fluorescence expression in the decidua basalis, strong fluorescence expression in the labyrinthine layer structure. The labyrinthine layer under high magnification showed that compared with the blank control group and the corn oil group, the brightness of β-catenin fluorescence expression in the rotenone group decreased (P<0.05), and the brightness of GSK-3β expression increased (P<0.05). The Western blotting results showed that the expression of β-catenin and p-GSK-3β proteins decreased (P<0.01), and the expression of GSK-3β protein increased (P<0.01) in the rotenone group. No significant expression of p-β-catenin protein was detected in the placenta tissue of each group. Conclusion Rotenone exposure during pregnancy induces placental hypoperfusion, growth retardation, and oxidative stress in rats, as well as down-regulation of β-catenin and p-GSK-3β protein expression, and up-regulation of GSK-3β protein expression, which may further lead to abnormal pregnancy and fetal restricted growth.
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OBJECTIVE@#Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness.@*METHODS@#A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected.@*RESULTS@#Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin+/CD31+ ratio, rather than the number of CD31+ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group.@*CONCLUSION@#Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.
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Humans , Animals , Mice , Carcinoma, Hepatocellular/drug therapy , Proto-Oncogene Proteins c-akt/metabolism , Proliferating Cell Nuclear Antigen/therapeutic use , Mice, Nude , Glycogen Synthase Kinase 3 beta/genetics , beta Catenin/therapeutic use , Liver Neoplasms/drug therapy , Desmin/therapeutic use , Ki-67 Antigen , Cell Line, Tumor , Hypoxia , RNA, Messenger/therapeutic use , Cell ProliferationABSTRACT
OBJECTIVE@#To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD).@*METHODS@#Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins.@*RESULTS@#In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa.@*CONCLUSION@#Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Subject(s)
Humans , Mice , Animals , Caco-2 Cells , beta Catenin/metabolism , Culture Media, Conditioned/pharmacology , Tight Junctions/metabolism , Intestinal Mucosa , Inflammatory Bowel Diseases , Tight Junction Proteins/metabolism , Inflammation/metabolism , Fibroblasts/metabolism , Mice, Inbred C57BL , Glycoproteins/metabolism , Wnt Proteins/pharmacology , Frizzled Receptors/metabolismABSTRACT
Objective:To investigate the regulatory effects of ring finger protein 43 (RNF43) on CD8 + T cell-mediated anti-tumor immune reaction in melanoma. Methods:RNF43 gene was over-expressed and knockdown in mouse melanoma cells line B16-OVA by lentivirus infection; In vivo proliferation of mouse melanoma cells line B16-OVA in the Lv-Ctrl-OE, Lv-RNF43-OE, Lv-Ctrl-KD and Lv-RNF43-KD groups was detected by subcutaneous tumorigenesis assay in mice, and the expression levels of CD8 + T cells perforin and interferon γ (IFN-γ) in tumor immune microenvironment of melanoma were detected by flow cytometry; The expression levels of β-catenin and programmed death-ligand 1 (PD-L1) mRNA in cells were detected by quantitative real-time PCR assay; The effect of RNF43 on the transcriptional regulation of PD-L1 was detected by dual-luciferase reporter gene assay. Results:Stable RNF43 over-expressing and RNF43 knockdown mouse melanoma cells lines Lv-RNF43-OE and Lv-RNF43-KD were successfully constructed. The results of subcutaneous tumorigenesis experiment in mice showed that the tumor mass of the Lv-RNF43-OE group was (0.08±0.06) g, which was significantly smaller than that of the Lv-Ctrl-OE group [ (1.04±0.52) g], with a statistically significant difference ( t=3.71, P=0.032) ; The tumor mass of Lv-RNF43-KD group was (1.94±0.29) g, with no statistically significant difference ( t=-1.70, P=0.164) compared with that of the Lv-Ctrl-KD group (1.15±0.74) g. The flow cytometry results showed that the fluorescence intensity of CD8 + T cell perforin in the Lv-RNF43-OE group was 9 034 ± 2 628, which was significantly higher than that in the Lv-Ctrl-OE group (3 847 ±1 637), with a statistically significant difference ( t=-3.35, P=0.015) ; The fluorescence intensity of CD8 + T cell perforin in the Lv-RNF43-KD group was 966±247, which was significantly lower than that in the Lv-Ctrl-KD group (2 226±646), with a statistically significant difference ( t=3.16, P=0.034) ; The fluorescence intensity of IFN-γ of CD8 + T cell in the Lv-RNF43-OE group was 2 422±429, which was significantly higher than that of 1 688±324 in the Lv-Ctrl-OE group, with a statistically significant difference ( t=-2.73, P=0.034) ; The fluorescence intensity of IFN-γ of CD8 + T cell in the Lv-RNF43-KD group was 614 (454, 863), with a statistically significant difference ( Z=-1.96, P=0.050) compared with 1 159 (1 152, 2 068) in the Lv-Ctrl-KD group. The results of quantitative real-time PCR showed that the relative expression level of β-catenin mRNA in the Lv-RNF43-OE group was 0.67±0.16, which was significantly lower than that of 1.00±0.11 in the Lv-Ctrl-OE group, with a statistically significant difference ( t=2.98, P=0.041) ; The relative expression level of PD-L1 mRNA in the Lv-RNF43-OE group was 0.32±0.09, which was significantly lower than that of 1.00±0.09 in the Lv-Ctrl-OE group, with a statistically significant difference ( t=9.13, P=0.001). The results of the dual-luciferase reporter gene assay showed that the PD-L1 promoter luciferase activity in the pCMV6-NC, RNF43, RNF43+β-catenin and β-catenin groups were 1.00±0.00, 0.84±0.00, 1.49±0.00 and 1.57±0.03 ( F=2 218.33, P<0.001). Further pairwise comparison showed that compared with the pCMV6-NC group, PD-L1 promoter luciferase activity was significantly lower in the RNF43 group ( P<0.001) and significantly higher in the RNF43+β-catenin and β-catenin groups ( P<0.001; P=0.003) ; compared with the RNF43 group, PD-L1 promoter luciferase activity was significantly higher in the RNF43+β-catenin group ( P<0.001) . Conclusion:RNF43 may reduce the expression of PD-L1 mRNA in melanoma by inhibiting the expression of β-catenin and promote CD8 + T cell-mediated anti-tumor immune reaction.
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Objective:To investigate the effects of cytoplasmic fragile X mental retardation protein 1 binding protein 2 (CYFIP2) overexpression on the biological functions and Wnt/β-catenin signaling pathways of bladder cancer T24 cells.Methods:The control group was T24 cells transfected with the empty pcDNA3 vector, and the overexpression group was T24 cells transfected with the CYFIP2 overexpression vector. The expression of CYFIP2 mRNA and protein was detected by reverse transcriptase, quantitative polymerase chain reaction, and Western Blot. The effect of CYFIP2 overexpression on T24 cell proliferation was detected by CCK-8. The effect of CYFIP2 overexpression on T24 cell migration and invasion was detected by Transwell. The effects of CYFIP2 overexpression on Wnt/β-catenin signaling pathway in T24 cells were detected by Western Blot.Results:Compared with the control group, the expression levels of CYFIP2 mRNA and protein were increased in the overexpression group (all P < 0.001), and the cell proliferation, migration, and invasion abilities were reduced (all P < 0.01). β-catenin, c-Myc, and Cyclin D1 protein expression were down-regulated in CYFIP2 overexpressed T24 cells (all P < 0.05), while the protein levels of p-β-catenin were increased ( P < 0.05). Conclusions:CYFIP2 overexpression can inhibit T24 cell proliferation, migration, and invasion, and its possible molecular mechanism is related to the inhibition of Wnt/β-catenin signaling pathway.
ABSTRACT
Steroid-induced necrosis of the femoral head （SNOFH） is a common orthopedic disease，which is difficult to cure and has poor clinical prognosis. The number of SNOFH patients in China is still increasing year by year，which seriously threatens human health. Long-term non-standard or short-term extensive use of hormone （GC） is an important reason for the occurrence of this disease. At present，SNOFH is mostly treated by surgical methods such as hip replacement，which has limitations of great harm to patients and high cost. In recent years，with the continuous deepening and innovation of traditional Chinese medicine（TCM） research，the use of TCM to treat SNOFH has been widely used in clinical practice. The main TCM pathogenesis of SNOFH is kidney deficiency and blood stasis. Therefore，TCM monomer and compound compound of tonifying kidney and promoting blood circulation are used to treat SNOFH. And TCM has obvious therapeutic effect，small side effects，less cost and other advantages. Glycoprotein/beta chain protein secretion （Wnt/beta- catenin） signaling pathway as a classic signaling pathway is closely related to the bone，between its by promoting bone marrow mesenchymal stem cell update，enhance the activity of osteoblast and suppress the apoptosis，which adjust the metabolic balance of bone tissue，increase bone density，will play an important role in the process of bone formation. In recent years，the use of TCM monomers and compounds to regulate Wnt/β-catenin signaling pathway to accelerate bone marrow mesenchymal stem cells，promote their transformation into osteoblasts，and maintain bone metabolic balance mechanism to treat SNOFH has become a new research hotspot. This article reviews the research progress of TCM in the prevention and treatment of SNOFH by regulating Wnt/β-catenin signaling pathway，in order to provide reference for the application of TCM in the treatment of SNOFH.
ABSTRACT
AIM: To scrutinize the role of the Wnt/β-catenin signaling pathway in the epithelial-mesenchymal transition(EMT)of lens epithelial cells under hypoxic conditions, and to further analyze the effect of Dickkopf-1(DKK-1)expression on EMT of lens epithelial cells.METHODS: Human lens epithelial cells(HLEB3 cells)were propagated in vitro and then separated into two groups: one exposed to standard oxygen levels, added DMEM culture solution containing 10% FBS(normoxic group)and another subjected to low oxygen levels(hypoxic group). The hypoxic condition was emulated by applying a concentration of 100 μmol/L cobalt chloride(CoCl2)for 6, 12, 24, and 48h. The utilization of immunofluorescence staining enabled the detection of Wnt3a and DKK-1 expressions, along with the expression and localization of β-catenin protein in these groups. The expression of DKK-1 mRNA was discerned by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS: Immunofluorescence assays indicated an escalating trend in the Wnt3a and DKK-1 protein expression, which corresponded with the increasing duration of hypoxia. Likewise, an intensified nuclear accumulation of β-catenin protein was observed to be directly proportional to the length of hypoxia treatment. The qRT-PCR demonstrated that the difference in DKK-1 mRNA expression between the normoxic group and the group exposed to hypoxia for 6h was not statistically significant(P>0.05), whereas the DKK-1 mRNA expression of the 12, 24, and 48h hypoxia groups were significantly increased(P<0.001).CONCLUSION: Hypoxia can activate Wnt/β-catenin pathway in lens epithelial cells and induce the expression of DKK-1, thus regulating the Wnt/β-catenin pathway and affecting the EMT process.
ABSTRACT
This paper aimed to investigate the effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/peroxisome proliferator-activated receptor γ(PPARγ) pathway in arrhythmic rats. SD rats were randomly divided into a control group, a model group, a low-dose(20 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a medium-dose(40 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a high-dose(80 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a propranolol hydrochloride(2 mg·kg~(-1)) group, with 12 rats in each group. Except the control group, rats in other groups were prepared as models of arrhythmia by sublingual injection of 1 mL·kg~(-1) of 0.002% aconitine. After grouping and intervention with drugs, the arrhythmia, myocardial cells apoptosis, myocardial tissue glutathione peroxidase(GSH-Px), catalase(CAT), malondialdehyde(MDA), serum interleukin-6(IL-6), prostaglandin E2(PGE2) levels, myocardial tissue apoptosis, and Wnt/β-catenin/PPARγ pathway-related protein expression of rats in each group were measured. As compared with the control group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA levels in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels increased significantly in the model group, whereas the GSH-Px and CAT levels, and Bcl-2 and PPARγ protein expression levels in myocardial tissues reduced significantly. As compared with the model group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA leve in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels reduced in the drug intervention groups, whereas the GSH-Px and CAT levels and Bcl-2 and PPARγ protein expression levels in myocardial tissues increased. The groups of total flavonoids of buckwheat flower and leaf were in a dose-dependent manner. There was no significant difference in the levels of each index in rats between the propranolol hydrochloride group and the high-dose group of total flavonoids of buckwheat flower and leaf. The total flavonoids of buckwheat flower and leaf inhibit the activation of Wnt/β-catenin pathway, up-regulate the expression of PPARγ, reduce oxidative stress and inflammatory damage in myocardial tissues of arrhythmic rats, reduce myocardial cell apoptosis, and improve the symptoms of arrhythmia in rats.
Subject(s)
Rats , Animals , PPAR gamma/metabolism , Fagopyrum/genetics , Rats, Sprague-Dawley , bcl-2-Associated X Protein , beta Catenin/metabolism , Interleukin-6 , Flavonoids/pharmacology , Propranolol/pharmacology , Ventricular Fibrillation , Dinoprostone , Wnt Signaling Pathway , Plant Leaves/metabolism , Flowers/metabolism , Apoptosis , Cardiac Complexes, PrematureABSTRACT
OBJECTIVE@#To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition.@*METHODS@#Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide (CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count (WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed; the number of nucleated cells in bone marrow was detected; the serum levels of interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were measured by ELISA; the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method.@*RESULTS@#Compared with the blank group, the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased (P<0.01), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was decreased (P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased (P<0.01, P<0.05), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was increased (P<0.05).@*CONCLUSION@#Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/β-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.