Aspirin inhibits the growth of hypertrophic scar in rabbit ears via regulating Wnt/β-catenin signal pathway / 中南大学学报(医学版)

OBJECTIVES@#Steroidal anti-inflammatory drugs have certain side effects in the treatment of hypertrophic scar, and the scar recurrence is easy after withdrawal of steroid anti-inflammatory drugs. Finding reliable alternative drugs is an effective means to improve this defect. Aspirin, a traditional non-steroidal anti-inflammatory drug, is safe for topical use and has anti-inflammatory effects similar to those of steroidal anti-inflammatory drugs, which may have similar effects on the treatment of hypertrophic scar. This study aims to investigate the inhibitory effect of aspirin on the proliferation of hypertrophic scar in rabbit ears and the underlying mechanism.@*METHODS@#The rabbit ear hypertrophic scar models were prepared. The rabbits were randomly divided into a normal skin group (group A), a blank control group (group B), a 0.9% NaCl group (group C), a 0.2% aspirin group (group D), a 0.5% aspirin group (group E), a 2% aspirin group (group F), and a triamcinolone acetonide group (group G). Macroscopic observation of hyperplasia was performed 8 weeks after local injection of the scar, followed by collecting the scar tissue samples for HE staining, Masson staining, and immunohistochemistry, respectively to assess the proliferation of fibroblasts and collagen fibers, and calculate the hypertrophic index, microvessel density, and immunohistochemical score.@*RESULTS@#All rabbit ear hypertrophic scar models were successfully constructed. In groups B and C, the hypertrophic scar edge was irregular, with reddish protruding epidermis, significant contracture and hard touch. In group D, E, and F, with the increase of aspirin administration concentration, the scar became thinner and gradually flat, the proliferation of fibrocytes and collagen fibers was weakened, and the hypertrophic index was gradually decreased (P<0.05). Immunohistochemistry showed that the expression of β-catenin was decreased in the group D, E and F in turn, and the immunohistochemical score was gradually decreased (P<0.05). There was no significant difference in hypertrophic index, microvessel density, and immunohistochemical score (all P>0.05).@*CONCLUSIONS@#Local injection of aspirin can reduce the generation of hypertrophic scar in a dose-dependent manner within a certain concentration range; aspirin inhibits the growth of hypertrophic scar in rabbit ears by inhibiting Wnt/β-catenin signal pathway; 2% aspirin and 40 mg/mL triamcinolone acetonide have similar curative efficacy on hypertrophic scar.

Study on Improvement Effect of Salvianolate on Renal Interstitial Fibrosis Model Rats and Its Mechanism / 中国药房

OBJECTIVE：To study the improve ment effect of salvianolate on renal interstitial fibrosis （RIF）model rats and its possible mechanism. METHODS ：Totally 50 male SD rats were randomly divided into normal group ，model group ，losartan group （positive control group ，9 mg/kg）and salvianolate low-dose and high-dose groups （18，36 mg/kg）according to body weight ，with 10 rats in each group. Except for normal group ，other groups were given adenine 250 mg/kg intragastrically to establish RIF model. After modeling ，administration groups were given relevant medicine intragastrically ，and normal group and model group were given constant volume of normal saline intragastrically ，the volume was 10 mL/kg，once a day ，for consecutive 30 days. After last medication，the serum levels of creatinine （Scr），urea nitrogen （BUN）and 24 h proteinuria （24 h UPro ）were detected by ELISA. HE staining and Masson staining were used to observe the histopathological characteristics and fibrosis of the kidney. The degree of renal tubular injury and glomerulosclerosis were scored ，and the percentage of positive staining area of renal tissue was calculated ； immunohistochemistry and Western blot assay were used to determine the protein expression of Wnt 5a，Wnt5b，and β-catenin. RESULTS：Compared with normal group ，Scr，BUN and 24 h UPro levels ，renal tubular injury score ， glomerulosclerosis score ， the percentage of positive staining area in renal tissue ，the protein expression of Wnt 5a and β-catenin were increased significantly in model group （P＜0.05），while the expression of Wnt 5b protein was decreased significantly （P＜0.05）. Pathological changes such as mesangial hyperplasia ，fibrous tissue increase and inflammatory cell infiltration were observed under microscope. Compared with model group ，above indexes of rats were improved significantly in losartan group ，salvianolate low-dose and high-dose groups （P＜ 0.05），and the effect of salvianolate had dose-dependent trend. CONCLUSIONS ：Salvianolate has the improvement effect on RIF model rats induced by adenine ，and its mechanism may be related to inhibition of Wnt/ β-catenin signal pathway.

Investigation on Mechanism of Jianzhong Bushen Xiaozheng Decoction Against Renal Interstitial Fibrosis in Regulating Wnt/β-catenin Signaling Pathway by MiRNA-139 / 中国实验方剂学杂志

Objective:To investigate the mechanism of Jianzhong Bushen Xiaozheng decoction in regulating the effect of miRNA139 on Wnt/<italic>β</italic>-catenin signaling pathway for renal interstitial fibrosis. Method:The 120 mice were randomly divided into sham operation group， unilateral ureteral obstruction （UUO） group， Jianzhong Bushen Xiaozheng decoction low， middle， high dose group， and Niaoduqing group. The UUO animal model was established to observe the morphological changes in mice. Intragastic administration was started from day 3 after modeling. The sham operation group and UUO group received the same amount of distilled water every day. The low， medium and high-dose groups received Jianzhong Bushen Xiaozheng decoction solution at 6，12，24 g·kg^-1·d^-1 respectively. The Niaoduqing group received 6.2 g·kg^-1·d^-1 Niaoduqing granule solution. After 14 d and 21 d， 28 d ， the morphological changes， general signs and renal interstitial fibrosis index of the obstructed side were observed， hematoxylin-eosin （HE） staining and Masson staining were used to observe the pathological changes of renal tissue. Real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） method was used to detect the miRNA-139 expression in renal tissue volume， Western blot was used to detect expression of beta serial proteins （<italic>β</italic>-catenin） and fibrinolytic enzyme activators inhibitor-1 （PAI-1） in renal tissues， and immunohistochemical assay was used for detection of matrix metalloproteinases-7 （MMP-7） protein expression at the obstruction side. Result:After 14， 21 and 28 days， the expression levels of <italic>β</italic>-catenin and PAI-1 in UUO group were higher than those in sham operation group（<italic>P</italic><0.05），while the expression levels of miRNA139 and MMP-7 protein were lower than those of sham operation group （<italic>P</italic><0.05）. The expression levels of <italic>β</italic>-catenin and PAI-1 proteins in mice after treatment in Niaoduqing group and the traditional Chinese medicine groups were lower than those in the UUO group（<italic>P</italic><0.05）， the expression of miRNA139 and MMP-7 proteins increased（<italic>P</italic><0.05）， and the efficacy of high-dose Jianzhong Bushen Xiaozheng decoction group was better than that of other dosage groups or Niaoduqing group（<italic>P</italic><0.05）. Conclusion:Jianzhong Bushen Xiaozheng decoction may regulate miRNA139 to mediate the process of renal interstitial fibrosis through the Wnt/ <italic>β</italic>-catenin pathway and delay the development of renal interstitial fibrosis to improve renal function.

Effect of Huangjingwan on Expressions of Wnt/β-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid with Learning and Memory Disorders / 中国实验方剂学杂志

Objective:To explore the effect of Huangjingwan （HW） on the expressions of Wnt/β-catenin signal pathway-associated proteins in the hippocampus of mice with Alzheimer's disease （AD） induced by D-galactose and okadaic acid with learning and memory disorders， as well as its mechanism. Method:After subcutaneous injection with 1.0% D-galactose （0.14 g·kg-1·d-1） into the back and neck of mice for 4 weeks， the right ventricle of mice was injected with 2 μL（75 ng） of okadaic acid for one time to make AD model， and the successfully modeled AD mice were selected by Morris water maze. Then， the selected AD mice were randomly divided into AD model group， memantine group （1.3×10-3 g·kg-1·d-1） and HW group （2.5 g·kg-1·d-1）. In addition， the sham model control group and the normal control group were set up. At the same time， 2 μL normal saline was injected into the right ventricle of mouse in the sham model control group as the modeling control. Two weeks after molding， the mice in the two experimental drug groups were given the corresponding dose of the experimental drug by gavage for 4 weeks. In addition， after 2 weeks of AD modeling， mice in sham model control group and AD model group were intragastrically administrated with the same amount of normal saline daily for 4 weeks. There was no special treatment in the normal control group. At the end of gavage， the shuttle experiment was performed to detect the differences in learning and memory levels of mice in each group. The changes of β-catenin and GSK-3β positive neurons in CA1 area of hippocampus in each group were tested by immunohistochemistry. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expressions of GSK-3β， β-catenin and CyclinD1 in hippocampus of mice in each group. The Western blot was used to detect the expressions of total GSK-3β （t-GSK-3β）， phosphorylation of GSK-3β at Ser9 （p-Ser9-GSK-3β）， phosphorylation of GSK-3β at Tyr216 （p-Tyr216-GSK-3β）， total β-catenin （t-β-catenin）， phosphorylation of β-catenin （p-β-catenin） and CyclinD1 proteins in hippocampus of mice in each group. Result:Compared with the normal control group， mice in AD model group showed an obvious dementia state， which was characterized by significant declines in learning and memory ability， the number of β-catenin immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-β-catenin and CyclinD1， the protein expressions of p-Ser9-GSK-3β， and the ratio of p-Ser9-GSK-3β/t-GSK-3β and p-Tyr216-GSK-3β/t-GSK-3β in hippocampal region （P<0.01）， and significant increases in the number of GSK-3β immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-GSK-3β， the protein expressions of p-Tyr216-GSK-3β and p-β-catenin， the ratio of p-β-catenin/t-β-catenin in hippocampal region （P<0.01 respectively）. Compared with the AD model group， the dementia symptoms of mice in HW group were significantly alleviated， and the number of β-catenin immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-β-catenin and CyclinD1， the protein level of p-Ser9-GSK-3β， the ratio of p-Ser9-GSK-3β/t-GSK-3β in hippocampal region were all significantly increased （P<0.01 respectively）， whereas the number of GSK-3β immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-GSK-3β， the proteins expressions of p-Tyr216-GSK-3β and p-β-catenin， the ratio of p-β-catenin/t-β-catenin in hippocampal region were all significantly decreased （P<0.01 respectively）， but the ratio of p-Tyr216-GSK-3β/t-GSK-3β has no significant statistical difference. Conclusion:HW shows the role of AD treatment， which can down-regulate the expression of GSK-3β in the hippocampus of AD mice and reduce its protein activity， and up-regulate the expression of β-catenin as well as increase its protein activity， so as to enhance the expression of downstream CyclinD1 and promote the transcription of the target genes. Its mechanism may be related to the activation of Wnt/β-catenin signal pathway.

Effect of β-catenin RNAi Interference on Signal Mechanism of Huangjingwan in Treating Learning and Memory Impairment Mice / 中国实验方剂学杂志

Objective:To treat mice with Alzheimer's disease （AD） with β-catenin RNA interference （RNAi） Huangjingwan （HW）， so as to explore the neuroprotective signal mechanism of its prevention and treatment of AD. Method:A total of 81 male Kunming mice were randomly divided into normal control group， sham model control group， AD model group， Donepezil group， HW+scrambled group， HW+RNAi group， HW group， with 8 mice in each of donepezil group and HW group， and 13 mice in each of other groups. The AD models were established through injection with D-galactose and scopolamine in the last 5 groups for 5 consecutive weeks. On the 1st day of the 4th week after modeling， 0.75 μL PEI-LMW/β-catenin siRNAs nano-complex was injected into the right lateral ventricle of each mouse in for one time to treat with β-catenin RNAi in mice brains of the HW+RNAi group. The 0.75 μL complex was injected into the right lateral ventricle of each mouse for one time as for β-catenin interference control of the HW+scrambled group. The 0.75 μL normal saline was injected into the right lateral ventricle of each mouse in one time of the sham control group. Two weeks after intracerebroventricular injection， β-catenin RNAi was confirmed to be successful， and Donepezil （6.5×10-4 g·kg-1） was intragastrically administered to each mouse of donepezil group. HW （2.5 g·kg-1） was intragastrically administered to each mouse of HW group， HW+RNAi group and HW+scrambled group. Normal saline （0.5 mL·d-1） was intragastrically administered to each mouse of the sham control group. All gastric perfusion lasted for 4 weeks. At the end of gavage， the difference in learning and memory ability of mice was evaluated by platform jumping test. Nissl staining was used to count the number of neurons in s1Tr area of cerebral cortex and CA1 and CA3 areas of hippocampus of each mouse in each group. The mRNA expressions of Wnt1， DVL2， GSK-3β， β-catenin and CyclinD1 in mice brain of each group were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the expressions of Wnt1， DVL2， GSK-3β， β-catenin and CyclinD1 in mice brain of each group. Result:The expression of β-catenin could be significantly inhibited through the injection with PEI-LMW/β-catenin siRNAs nano-complex into the lateral ventricle of AD mice， and nearly no β-catenin expression could be detected， which successfully achieved gene silencing. Compared with the normal control group， mice in AD model group showed that the learning and memory performance decreased significantly， the number of jumping errors increased （P<0.01）， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus decreased significantly （P<0.01）， the mRNA and protein expressions of Wnt1， DVL2， β-catenin， CyclinD1 in brain decreased significantly （P<0.01）， while the mRNA and protein expressions of GSK-3β increased significantly （P<0.01）. Compared with the AD model group， mice in HW group showed that the learning and memory performance increased significantly， the number of jumping errors decreased， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus increased significantly， the mRNA and protein expressions of Wnt1， DVL2， β-catenin， CyclinD1 in brain increased significantly， while the mRNA and protein expression of GSK-3β decreased significantly （P<0.01）. Compared with the HW group， mice in HW+RNAi group showed that the learning and memory performance decreased significantly， the number of jumping errors increased significantly （P<0.01）， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus decreased significantly （P<0.01）， there was no significant change in mRNA and protein expressions of Wnt1， DVL2， GSK-3β in the brain， and the mRNA and protein expressions of β-catenin， CyclinD1 decreased significantly （P<0.01）. Conclusion:HW can treat and prevent AD by activating Wnt/β-catenin signal pathway.

Advances in the experimental mechanism of acupuncture in the treatment of Alzheimer's disease based on Wnt/β-catenin pathway / 针刺研究

Alzheimer's disease is a neurodegenerative disease closely related to age, which is characterized by cognitive and memory impairment. Extensive studies have confirmed that Wnt/β-catenin signal pathway is involved in the occurrence and development of Alzheimer's disease. With the characteristics of holistic concept and syndrome differentiation, acupuncture is widely used in clinic. Acupuncture plays a role in the treatment of Alzheimer's disease through the regulation of each target and the whole of the pathway. The author reviewed and combed the research on acupuncture treatment of Alzheimer's disease in recent years, and reviewed the regulatory effects of acupuncture on the important components of Wnt/β-catenin signal pathway (Wnt protein, β-catenin, glycogen synthase kinase-3β) and whole, ATP-binding cassette subfamily B member 1 (ABCB1), low density lipoprotein receptor associated protein-1 (LRP-1)．.

Effect of Fushengong Prescription on Wnt/β-catenin Signaling Pathway in Kidney of Rats with Chronic Renal Failure / 中国实验方剂学杂志

Objective:To observe the effects of Fushengong prescription on secretory glycoprotein （Wnt）/β-serial protein （β-catenin） signaling pathway in kidney of rats with chronic renal failure （CRF），and to further explore its mechanism of releasing the aggregation of extracellular matrix（ECM），inhibiting renal tubule interstitial fibrosis （TIF） and prolonging the progression of CRF. Method:A total of 55 SD male rats were randomly divided into the normal group，the model group，and the low， medium and high dose groups of Fushengong prescription，with 11 rats in each group.The normal group was routinely reared and the other 4 groups of rats were used to establish CRF model with 0.5% adenine fodder， fed them continuously for 21 d. After successful modeling，all model rats were switched to conventional feed. Normal saline （NS） was given the normal group and the model group by 20 mL·kg-1·d-1， the low， middle and high dose groups rats of Fushengong prescription were given intragastric administration Fushengong prescription according to the body weight of 4， 8， 16 g·kg-1，once a day，continuous gavage for 30 d. After the experiment，the pathomorphism change of renal tissues of rats was measured by Masson staining， the expression of Wnt4 and β-catenin mRNA in the kidney tissues were observed by the method of Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， the expression of Wnt4，β-catenin and matrix metalloproteinase-7（MMP-7） protein of renal tissues were detected by the methods of Western blot. The expression of Wnt4， β-catenin protein of renal tissues were detected by the methods of immunohistochemistry （IHC）. Result:Compared with normal group，renal tubule interstitial fibrosis of renal tissues increased distinctly and the expression of Wnt4，β-catenin and MMP-7 protein increased significantly in the model group. Wnt4 and β-catenin mRNA also increased significantly in model group（P<0.01）. Compared with model group， the expression of Wnt4， β-catenin and MMP-7 protein in the Fushengong prescription groups decreased obviously （P<0.05）. The expression of Wnt4 and β-catenin mRNA in Fushengong prescription groups also decreased obviously. Conclusion:The mechanism of Fushengong prescription can release the aggregation of ECM，inhibit TIF and delay the progression of CRF，which may be related with the activation of Wnt/β-catenin signal pathway.

Research progress on molecular mechanism of Chinese materia medica affecting bone metabolism / 中草药

Dynamic balance of bone metabolism is one of the important factors to maintain normal osseous tissue function. When the balance is broken, it causes bone damage and even bone metabolic disease. However, the mechanism of bone metabolism is still unclear, the signal pathway is complex, and the treatment of diseases is still under study. The research on the mechanism of bone metabolism by Chinese materia medica has become a new direction in the treatment of bone metabolism diseases. At present, common mechanisms in bone metabolism include OPG/RANKL/RANK signal pathway, Wnt/β-catenin signal pathway, TGF-β/BMP/Smad signal pathway, and NF-κB signal pathway. In this paper, the research on the above pathways was reviewed, so as to provide a reference for further exploring the treatment of bone metabolism diseases with Chinese materia medica.

SNHG16 facilitates proliferation and migration of hepatocellular carcinoma cells by Wnt/β-catenin signaling pathway / 肿瘤

Objective: To investigate the expression of long non-coding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) in hepatocellular carcinoma (HCC) tissues and cells, and to explore the effects of SNHG16 expression regulation on the proliferation and migration of HCC cells as well as the underlying molecular mechanisms. Methods: The cancer tissues and adjacent tissues were collected from 38 patients with HCC. The real-time fluorescent quantitative PCR was used to detected the expression of SNHG16 in 38 cases of clinical HCC tissue samples and their adjacent tissues, 4 kinds of HCC cell lines and normal hepatocellular cell line. The relationship between SNHG16 expression and the clinicopathological features of HCC patients was analyzed. The SNHG16 overexpression or SNHG16-shRNA recombinant lentivirus was infected into Hep-3B or SK-Hep-1 cells, respectively. The up- or down-regulation of SNHG16 expression in Hep-3B or SK-Hep-1 cells was verified by real-time fluorescent quantitative PCR. The effects of SNHG16 expression regulation on the proliferation and migration of HCC cells were determined by CCK-8 and Transwell chamber experiments, respectively. The expressions of key proteins in Wnt/β-catenin signaling pathway were detected by Western blotting. Xenograft tumor experiment was used to determine the effect of SNHG16 on the tumorigenic ability of HCC cells in nude mice. Results: The expression level of SNHG16 in HCC tissues and HCC cells was significantly higher than that in the adjacent tissues (P < 0.001) and normal hepatocellular cells (P < 0.05), respectively. The expression of SNHG16 in HCC tissues was associated with tumor size (P < 0.01), TNM stage (P < 0.01) and alanine aminotransaminase (ALT) expression level (P < 0.05). After the infection with SNHG16 overexpression recombinant lentivirus, the expression of SNHG16 was significantly up-regulated in Hep-3B cells (P < 0.001), the proliferation and migration of Hep-3B cells were significantly promoted (both P < 0.01), and the expressions of β-catenin and c-myc proteins were up-regulated (both P < 0.01). After the infection with SNHG16-shRNA recombinant lentivirus, the expression of SNHG16 was dramatically downregulated in SK-Hep-1 cells (P < 0.001), the proliferation and migration of SK-Hep-1 cells were significantly inhibited (both P < 0.001), and the expressions of β-catenin (P < 0.05) and c-myc (P < 0.01) proteins were down-regulated. In addition, the tumorigenic ability of Hep- 3B cells with SNHG16 over-expression was significantly enhanced in nude mice (P < 0.01), while the tumorigenic ability of SK-Hep-1 cells with SNHG16 knock-down was significantly weakened in nude mice (P < 0.05). Conclusion: SNHG16 is highly expressed in HCC tissues and cell lines, and may promote the proliferation and migration of HCC cells through Wnt/β-catenin signaling pathway.

Bioinformatics analysis of Wnt/β-catenin pathway related regulatory elements in human mesothelin gene and identification of promoter in ovarian cancer cells / 临床与实验病理学杂志

Purpose To analyze the Wnt/β-catenin signaling pathway related TCF/LEF binding sites in human mesothelin gene and to identify the core promoter region of the gene in the ovarian cancer cells. Methods The possible TCF/LEF transcription factor binding sites in the promoter region of human meosothelin gene were analyzed by bioinformatics method. The 1764 bp promoter sequence near the 5'end of the human mesothelin gene were cloned. The fragments was truncated differently at the 5' end and cloned into p GL3-basic report gene vector and transfected into human ovarian cancer cell lines SKOV-3 and 3-AO. The activity of diffetent promoter fragmentis was detected by double luciferase reporter gene system. Results There were multiple potential TCF/LEF binding sites in the promoter region of the human mesothelin gene. Three fragments of mesothelin gene promoter region-1456-+ 308、-164-+ 308、+ 47-+ 308 were cloned and amplified successfully, the p GL3 vector was constructed by sequencing. After transfection of SKOV-3 and 3-AO cells, the double luciferase reporter gene system showed that the-1456-+ 308 fragments and-164-+ 308 framents had high promoting activity in both cell lines, and the activity of + 47-+308 fragments was significantly lower than that of the former two cell lines (P<0.01). Conclusion The-164-+ 47 sequence containing TCF/LEF transcription factor binding site is the core promoter region of mesothelin gene in ovarian cancer, which lays a foundation for further study on the regulation mechanism of mesothelin gene expression in ovarian cancer.

Effects of total flavonoids of Clerodendrum bungei on epithelial-mesenchymal transition (EMT) of A549 cells by regulating Wnt/β-catenin signal pathway / 中草药

Objective To observe the effect of the total flavonoids of Clerodendrum Bungei (TFCB) on proliferation, invasion and migration of A549 cell lines by Wnt/β-catenin signal pathway. Methods Transfecting A549 cell lines with empty vector pcDNA3.1 and β-catenin plasmid which has the mutation at S45 (S45A-β-catenin), which were intervened with TFCB. MTT assay detected cell proliferation, scratch test observed cell migration, Transwell experiment detected the cell invasion. The expression of β-catenin, E-Cadherin, vimentin, Slug, GSK-3β, and p-GSK-3β protein in each group was detected by Western blotting. Results The proliferation, invasion and migration of A549 cells were enhanced significantly after transfected with S45A-β-catenin plasmid (P < 0.05 or 0.01), along with the increasing expression of β-catenin, vimentin, and the reducing expression of E-cadherin, GSK-3β, P-GSK-3β (P < 0.01). TFCB can inhibit the proliferation, migration and invasion of A549 cells (P < 0.05), expecially in S45A-β-catenin group (P < 0.001). A549 cells transfected with empty vector had the ability of up-regulating the expression of E-cadherin, GSK-3β and P-GSK-3β, down-regulating the expression of β-catenin and vimentin with TFCB. A549 cells transfected with S45A-β-catenin plasmid had the ability of down-regulating the expression of β-catenin and vimentin with TFCB. Conclusion The mechanism of inhibiting lung cancer of TFCB maybe associate with the inhibitory expression of β-catenin and regulate the downstream factors, with view to inducing EMT by activating Wnt/β-catenin pathway.

Overexpression of FOXO4 promotes apoptosis of laryngeal carcinoma cells by regulates Wnt/β-catenin signaling pathway / 中国耳鼻咽喉头颈外科

OBJECTIVE To investigate the effect of FOXO4 on proliferation and apoptosis of laryngeal carcinoma cells.METHODS The expression of FOXO4 in laryngeal carcinoma tissues and adjacent tissues was detected by Western blot.Laryngeal carcinoma cells were transfected with FOXO4 overexpression vector (p-EGFP-C1/FOXO4 group) and empty vector (p-EGFP-C1 group),while the non transfection group was established,and transfection reagent was only added in the non transfection group.The level of FOXO4 protein in transfected cells was detected by Western blot.Cell proliferation was detected by MTT,apoptosis was detected by flow cytometry.The levels of Cleaved Caspase-3,Caspase-3,Cleaved Caspase-9,β-catenin,Wntl were detected by Western blot.The transfected laryngeal carcinoma cells with p-EGFP-C1/FOXO4 was activated by Wnt/β-catenin signal pathway activator (activator group),and the proliferation and apoptosis of the cells were detected.RESULTS The expression level of FOXO4 in laryngeal carcinoma tissues was significantly lower than that in adjacent tissues (P=0.000).The expression level of FOXO4 in p-EGFP-C1/FOXO4 group was significantly higher than that in non transfection group (P=0.000).The survival rate and the expression levels of β-catenin and Wnt1 in p-EGFP-C1/FOXO4 group were significantly lower than those in the non transfection group (P=0.002,P=0.004,P=0.006).Cell apoptosis rate and expression levels of Cleaved Caspase-3,Caspase-3,Cleaved Caspase-9,Caspase-9 in p-EGFP-C1/FOXO4 group were significantly higher than those in non transfection group (P=0.002,P=0.001,hP＜0.05,P=0.004,jP＜0.05).Wnt/β-catenin signaling pathway activator can partly reverse the proliferation inhibition and apoptosis promoting effect of FOXO4.CONCLUSION FOXO4 could promote the apoptosis of human laryngeal carcinoma cells and inhibit the proliferation of laryngeal cancer cells.The mechanism may be related to the signal pathway of Wnt/β-catenin.

Effect of recombined human epidermal growth factor combined with alprostadil on Wnt/β-catenin signal pathway in rats with diabetic ulcer / 中国糖尿病杂志

Objective To explore the effect of recombined Human epidermal growth factor (rhEG) combined with alprostadilon Wnt/β-Catenin signal pathway in rats with diabetic ulcer. Methods Rats with diabetic ulcer were randomly divided into four groups :control group ,rhEG group ,epidermal group and combined treatment group. Ulcer skin was smeared by normalsaline in control group ,and by rhEG in rhEG group. Epidermal was administered from caudal vein in epidermal group.Combined treatment group was treated by rhEG and epidermal at the same time. The healing status were observed. The proteinand mRNA expression of Wnt-1 ,β-Catenin and GSK-3β were measured 14 days after treatment. Results The healing rates in combined treatment group were (9.76 ± 2.37 )% ,(35.74 ± 3.65 )% ,(51.37 ± 4.16)% and (84.42 ± 5.35 )% respectivelyin 6th , 10th and 14th day after treatment , which were significantly higher than in rhEG group and epidermal group (P<0.05). The mRNA levels of Wnt-1 ,β-Catenin and GSK-3βin combined treatment group were (1.42 ± 0.19) ,(1.56 ± 0.21) and (0.95 ± 0.15) after treatment for 14 days ,which were significantly higher than in rhEG group and epidermal group (P<0.05). Protein levels of Wnt-1 ,β-Catenin and GSK-3βin combined treatment group were (1.17 ± 0.16) , (1.38 ± 0.18 ) and (0.81 ± 0.13 ) after treatment for 14 days ,which were significantly higher than in rhEG group and epidermal group ( P < 0.05 ). Conclusion rhEG combined with epidermal can significantly accelerate the healing of diabetic ulcer ,and can regulatethe Wnt/β-Catenin signal pathway by increasing the expression of Wnt-1 ,β-Catenin and GSK-3β.

IL-22 inhibits liver fibrosis induced by hepatic stellate cells via Wnt/β-catenin signal pathway / 中国免疫学杂志

Objective:To investigate the effects and mechanisms of interleukin-22(IL-22) on inhibiting liver fibrosis induced by HSC,and explore the role of Wnt/β-catenin pathway in the activation of hepatic stellate cells(HSC).Methods:Rat HSC was activated by TGF-β1,and the mRNA and protein levels of β-catenin and α-SMA were detected by q-PCR and Western blot,respectively.HSC was treated with different hours and concentration of recombinant rat protein IL-22.The cell proliferation rates were detected by CCK8,cell apoptosis rates were tested by flow cytometry.HSC were treated with optimal concentration of IL-22 after activated by TGF-β1,the cell proliferation rates,mRNA and protein levels of β-catenin and α-SMA were compared of before and after intervention.Results:The mRNA and the protein levels of β-catenin and α-SMA were significantly increased after activated by TGF-β1(P0.05).IL-22 significantly inhibited the activation of HSC induced by TGF-β1 and remarkably decreased the mRNA and the protein expression level of β-catenin and α-SMA(P<0.05).Conclusion:The Wnt/β-catenin pathway may participates in the process of HSC activation and α-SMA secretion,and IL-22 inhibits biological function of HSC in a dose-and time-dependent manner.This effect probably via inhibited the Wnt/β-catenin signal pathway.

Inhibitory mechanisms of timosaponin AⅢ on proliferation and growth of human glioblastoma cells / 中国中药杂志

To explore the inhibitory effect of timosaponin AⅢ on the proliferation of human glioblastoma cell line U87MG and investigate its related mechanism. As compared with the model group, the tumor weight was significantly reduced in timosaponin AⅢ-treated group. Timosaponin AⅢinhibited the proliferation of U87MG cell line in a dose-dependent manner. It up-regulated the gene and protein expression levels of p21, meanwhile inhibited the protein expression levels of β-Catenin, Cyclin D1 and Bcl-2. It also inhibited the translocation of β-Catenin into nucleus, suppressed the phosphorylation expression of ERK, but increased the phosphorylation expression of p38 and JNK. Combined use of JNK inhibitor SP600125 and p38 inhibitor SB203580 could decrease p21 and increase β-Catenin protein expressions. Timosaponin AⅢ inhibited the proliferation of human glioblastoma cell line U87MG partly by intervening MAPK and Wnt/β-Catenin signal pathways.

Regulation of bifidobacterium on Wnt/β-Catenin signal pathway of intestinal tissue in newborn rats with ne-crotizing enterocolitis / 中华实用儿科临床杂志

Objective To study the effect of bifidobacterium on intestinal tissue of necrotizing enterocolitis (NEC)in newborn rats and its regulation of Wnt/β-Catenin signal pathway.Methods Seventy -five newborn SD rats were randomly divided into 5 groups,and each group had 1 5 rats.Group A was artificial feeding control group;group B was NEC model group;group C was bifidobacterium treatment group;group D was artificial feeding +bifidobacterium control group;group E was rat breast feeding control group.The localization expression of Toll -like re-ceptor 4(TLR4)of ileocecal ileum tissue was detected by immunohistochemical detection,and also the equivalen-tileum tissues were detected for the contents of glycogen synthase kinase -3β(GSK3β)and β-Catenin expression by Wes-tern blot.Comparing the differences of these indicators between the groups,in addition,the data of TLR4,GSK3βandβ-Catenin were analyzed by Bivariate correlations.Results The levels of TLR4 in ileum tissue of 5 groups were 0.36 ±0.03,0.48 ±0.05,0.34 ±0.03,0.37 ±0.04,0.35 ±0.02.The levels of GSK3βin ileum tissue of 5 groups were 0.98 ±0.23,1 .48 ±0.42,0.99 ±0.20,0.56 ±0.1 7,0.60 ±0.1 5.The levels of β-Catenin in ileum tissue of 5 groups were 1 .48 ±0.22,0.64 ±0.55,1 .27 ±0.36,1 .72 ±0.51 ,1 .82 ±0.44.The levels of TLR4 and GSK3βin ileum tissue of group B were significantly increased compared with group E (P <0.05).The levels of β-Catenin sig-nificantly decreased compared with group E (P <0.05).The levels of TLR4 and GSK3βin ileum tissue of group C were significantly decreased compared with group B (P <0.05).The levels of β-Catenin significantly increased com-pared with group B (P <0.05).Negative correlation was observed between the levels of GSK3βand β-Catenin(r =-0.592,P <0.05),while positive correlation was observed between the levels of TLR4 and GSK3β(r =0.295,P <0.05),and negative correlation was observed between the levels of TLR4 and β-Catenin(r =-0.426,P <0.05). Conclusions Bifidobacterium has certain protective effect on the NEC newborn rat intestines,which can reduce the in-cidence of experimental NEC and the severity of intestinal injury.Its effect may be achieved by regulating the Wnt/β-Catenin signal pathway,which decreases the expression of the level of GSK3βand increases the level of repair fac-tor β-Catenin.

Experimental study of osteogenic differentiation of precartilaginous stem cells induced by calcitonin gene-re-lated peptide / 中华物理医学与康复杂志

Objective To investigate the effect of calcitonin gene-related peptide ( CGRP ) in inducing os-teogenic differentiation of rat precartilaginous stem cells in vitro and the underlying mechanisms. Methods Rat pre-cartilaginous stem cells ( PSCs) were cultured in complete osteogenesis medium containing DMEM/F-12 medium and different concentrations (0, 10-8,10-9,10-10mol/L) of CGRP, the morphology changes of PSCs were observed. The proliferation of PSCs was examined at different time points by CCK-8. All the PSCs were then randomly assigned to an experimental group and a control group. The PSCs in the experimental group were cultured in complete osteogenesis medium with 10-10 mol/L CGRP , while the control group cultured merely in complete osteogenesis medium was re-ceived no special intervention. Both groups were stained by Alizarin Red and the expression of alkaline phosphatase (ALP) was detected. The osteogenic genes (RUNX2,OPN and BGP) were measured by use of RT-PCR. The activa-tion of Wnt/β-catenin signaling pathway was tested by using Western blotting to evaluate the effect of CGRP . Results Compared to the control group ( the concentration of CGRP was 0 mol/L) , the concentration of ALP was significantly higher in the experimental group, calcium deposition was significantly more obvious, and the expression of the osteogenic genes such as RUNX2,OPN and BGP as well as theβ-catenin protein expression were up-regulated significantly. However, CGRP had no effect on cell proliferation. Conclusion CGRP activated Wnt/β-catenin sig-nal pathway and induced osteogenic differentiation of precartilaginous stem cells.

Effect of sulindac on human pancreatic cancer cell PANC-1 proliferation and apoptosis and its possible mechanism / 中国生化药物杂志

Objective To discuss the influence of different concentration sulindac on pancreatic cancer cell line PANC-1 cell proliferation and apoptosis,and investigate the possible mechanism that sulindac can inhibit the Wnt/beta-catenin pathway to kill pancreatic cancer cells. Methods PANC-1 cell were divided into negative control group (added containing no sulindac DMSO)and experimental group (added sulindac with concentrations of 0.25 ,0.5 ,1 ,1.5 ,2 mM medium,respectively,name as 0.25 mM group,0.5 mM group,1.0 mM group,1.5 mM group,2.0 mM group),and treated with different time,cell proliferation inhibition ratio in each group was detected by MTT assay,cell apoptosis ratio was detected by flow cytometry,the expression ofβ-catenin mRNA and protein were detected by RT-PCR and immunocytochemistry.Results MTT results showed that sulindac can inhibit the cell proliferation of PANC-1 by a dose-and time-dependent manner.Cell apoptosis increased after sulindac treatment in different degrees,and there were statistical differences between 1.5,2.0 mMgroup and control groups (P<0.05).RT-PCR results showed that the expression ofβ-catenin mRNA decreased after the treatment of sulindac,there were statistical differences between 1.5,2.0 mMgroup and control group (P<0.05). In the 2.0mM group,the expression ofβ-catenin decreased along with the time extending (P<0.05 ).ICC results showed that sulindac inhibited the expression ofβ-catenin protein and nuclear accumulation,there were no statistical differences in 0.25 ,0.5 mM group and control group,but there were statistical differences in 1.0,1.5,2.0 mMgroup.Conclusion Sulindac could inhibit cell proliferation and facilitate apoptosis of PANC-1,this effect is dose-and time-dependent.The inhibition of Wnt/beta-catenin signal pathway may be a possible mechanism of its cytotoxicity.

COX-2/PGE2 regulates VEGF expression by activating wnt/β-catenin signal pathway in human colorectal cancer cells / 第二军医大学学报

Objective: To investigate whether COX-2/PGE2 can regulate vascular endothelial growth factor (VEGF) expression through activating Wnt/β-catenin signal pathway in human colorectal cancer cells. Methods: PGE2 and (or) COX-2 selective inhibitor NS-398 was used to treat LoVo cells for 24 h, and then COX-2 and β-catenin protein expression was detected by Western blotting analysis. PGE2 and (or) β-catenin/tcf selective inhibitor FH-535 were used to treat LoVo cells for 24 h, and then the levels of VEGF was determined by ELISA. Routinely cultured LoVo cells were taken as control. Results: Compared with the control group, PGE2 not only increased COX-2 protein expression, but also increased the levels of β-catenin in the total cell, cytosolic and nuclear proteins (being 3. 8 folds, 2. 7 folds, and 3. 0 folds of the control, respectively, P< 0. 01). COX-2 selective inhibitor not only decreased COX-2 protein expression, but also decreased the levels of β-catenin in the total cell, cytosolic and nuclear proteins (being 0. 3 folds, 0. 3 folds, and 0. 2 folds of the control, P<0. 01). Compared with the control group, PGE2 increased VEGF expression in LoVo cells (being 1. 6 folds of the control, P<0. 01), and β-catenin/tcf selective inhibitor decreased VEGF expression in LoVo cells (being 0. 68 folds of the control, P<0. 01). Co-treatment of LoVo cells with β-catenin/tcf selective inhibitor and PGE2 showed no great effect on VEGF expression. Conclusion: COX-2/PGE2 can β-catenin protein level, subsequently activate Wnt/p-catenin signal pathway and promote VEGF expression, which increase might be one of the mechanisms for COX-2/PGE2-induced angiogenesis in colon cancer.

The effect of Wnt/β-catenin signal pathway on multidrug resistance in liver cancer / 中国综合临床

Objective To establish a cisplatin (DDP)-resistant HepG-2 cell line,and to explore the role of Wnt/β-catenin signaling pathway on multidrug resistance in human hepatocellular carcinoma.Methods The HepG-2 cells were exposed in a gradually increasing dose of DDP to establish a cisplatin ( DDP)-resistant HepG-2 cell line.MTT assay was used to detect the cytotoxic activity of DDP against HepG-2 and HepG-2/DDP cells.The mRNA expression of β-catenin was determined by Real-time PCR assay.The small interfering RNA was used to specifically knockdown β-catenin expression in HepG-2/DDP cells.The protein expression was detected by western blot analysis.Results The DDP-resistant cell line HepG-2/DDP was established by gradient DDP induction successfully.The IC50 values of DDP against HepG-2 and HepG-2/DDP cells were (2.29 ± 0.14) μmol/L and ( 20.51 ± 0.84 ) μmol/L,respectively ( t=95.68,P＜0.01 ),HepG-2/DDP cells was 8.96 times than HepG-2 cells on the resistance of cisplatin.The result of real time PCR showed that 2-△Ct value of β-catenin in HepG-2 cells and HepG-2/DDP cells were (0.323±0.065) and (0.674 ±0.097) (P＜0.01 ).And the protein expression of cisplatin in HepG-2/DDP cells was also significantly higher than that in the HepG-2 cells.The expresssion of β-catenin was significantly and specifically depleted by siRNA duplexes(P＜0.01 ).The IC50 values of cisplatin against HepG-2/DDP cells were (21.02 ± 1.64) μmol/L in cisplatin control group,(6.23 ± 0.68 ) μmol/L in SiRNA targeting interference group and ( 20.44 ± 1.26 ) μmol/L in SiRNA negative interference group,and there was significant difference between control group and SiRNA targeting interference group ( P＜0.01 ).Conclusion The Wnt/β-catenin signaling pathway was activated on the cisplatin(DDP)-resistant HepG-2 cell line and down regulation of β-catenin increased the chemosensitivity of HepG-2/DDP cells against cisplatin.It provided a theoretical basis for finding the new targets of multidrug resistance in liver cancer.