Effects of Wumen Gumi Bao Decoction on Ameliorating Bone Loss in Experimentally Induced Osteoporosis in Rats by Regulating Wnt3a/β-Catenin Pathway

[Objectives]To investigate the preventive effects of Wumen Gumi Bao Decoction(WMGBD)on estrogen deficiency-induced bone loss.[Methods]Three-month-old Sprague-Dawley rats were ovariectomized(OVX)and then treated with WMGBD,and their admixtures for six weeks.The bone trabecular microstructure,bone histopathological examination were determined in the rat femur tissue,and serum biomarkers of bone formation and resorption were analyzed by ELISA,and the protein expressions of Wnt3a,β-catenin,and phosphorylatedβ-catenin(p-β-catenin)were analyzed by Western blot.Statistical analysis was conducted by using one-way analysis of variance(ANOVA)followed by LSD post hoc analysis or independent samples t test using the scientific statistic software SPSS version 20.0.[Results]WMGBD could promote osteosis and ameliorate bone loss to improve the repair of cracked bone trabeculae of OVX rats.Furthermore,WMGBD also could prevent OVX-induced decrease in collagen fibers in the femoral tissue of ovariectomized rats and promote the regeneration of new bone or cartilage tissue,while WMGBD could activate the Wnt3a/β-catenin pathway.[Conclusions]WMGBD could ameliorate estrogen deficiency-induced bone loss via the regulation of Wnt3a/β-catenin pathway.

MicroRNA-329-3p inhibits the Wnt/β-catenin pathway and proliferation of osteosarcoma cells by targeting transcription factor 7-like 1

An important factor in the emergence and progre sion of osteosarcoma(OS)is the dysregulated expression of microRNAs(miRNAs).Transcription factor 7-like 1(TCF7LI),a member of the T cell factor/lymphoid enhancer factor(TCF/LEF)transcription factor family,interacts with the Wnt signaling pathway regulator β-catenin and acts as a DNA-specific binding protein.This study sought to elucidate the impact of the interaction between miR 3293p and TCF7L1 on.the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches.MiR329-3p was significantly downregulated,while TCF7L1 was considerably up-regulated in all examined OS cell lines.Additionally,a clinical comparison study was performed using the TCGA database.Subsequently,the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments.When miR 329-3p was transfected into the OS cell line,the expression of TCF7L1 decreased,the proliferation of OS cells was inhibited,the cytoskeleton disintegrated,and the nucleus condensed to fom apoptotic bodies.The expression of proteins that indicate apoptosis increased simultaneously.The cell cycle was arrested in the G0/G1 phase,and the G1/S transition was blocked.The introduction of miR 3293p also inhibited downstream Cyclin D1 of the Wnt pathway.Xenograf experiments indicated that the overexpression of miR-329-3p signi ficanly inhibited the growth of OS xenografts in nude mice,and the expression of TCF7L1 and C-Myc in tumor tssues decreased.MiR 329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo.Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7LI by miR 3293p.Summarizing these results,it can be inferred that miR.3293p exerts anticancer efects in osteosarcoma by inhibiting TCF7L1.

N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway

BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.

萸蓉壮骨膏调控EZH2/Wnt3a/β-catenin通路促进绝经后骨质疏松大鼠成骨分化

目的基于EZH2/Wnt3a/β-catenin通路调控成骨分化探讨萸蓉壮骨膏对绝经后骨质疏松大鼠的治疗作用及机制。方法取SD大鼠复制绝经后骨质疏松模型,模型构建成功后随机分为模型组、阿仑膦酸钠片组[6.3 mg/(kg·周)]及萸蓉壮骨膏高[10.8 g/(kg·d)]、中[5.4 g/(kg·d)]、低剂量[2.7 g/(kg·d)]组,另选同龄大鼠作为假手术组。Micro CT检测骨微结构,HE染色观察股骨组织病理形态;IF法检测大鼠股骨组织中成骨相关蛋白(Runx2、OSX、OPG)表达水平。RT-qPCR及Western blot法检测大鼠胫骨组织中EZH2、Wnt3a、β-catenin mRNA和蛋白表达水平。结果与假手术组比较,模型组大鼠骨微结构(BMD、BV/TV、Tb.Th、Tb.N和Tb.Sp)显著破坏(P<0.05),骨小梁排列稀疏松散,结构出现不连续间断,脂滴累积较多,骨组织中成骨蛋白表达显著降低(P<0.05),EZH2 mRNA和蛋白表达水平显著升高,Wnt3a、β-catenin mRNA和蛋白表达水平显著降低(P<0.05);与模型组比较,各给药组大鼠骨微结构显著改善(P<0.05),各给药组不同程度改善模型大鼠骨组织病理结构,各给药组大鼠骨组织中成骨蛋白表达不同程度增加(P<0.05),各给药组大鼠骨组织中EZH2 mRNA和蛋白表达水平显著降低,Wnt3a、β-catenin mRNA和蛋白表达水平显著增加(P<0.05)。结论萸蓉壮骨膏能够显著改善绝经后骨质疏松大鼠骨流失,其作用机制与萸蓉壮骨膏调控EZH2/Wnt3a/β-catenin通路促进成骨分化有关。

Activation of the wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions

Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier.However,the potential links between them following ischemic stroke remain largely unknown.The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway.Meanwhile,Wnt/β-catenin pathway activation by the pharmacological inhibito r,TWS119,relieved oxidative stress,increased the levels of cytochrome P4501B1(CYP1B1)and tight junction-associated proteins(zonula occludens-1[ZO-1],occludin and claudin-5),as well as brain microvascular density in cerebral ischemia rats.Moreove r,rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress,suppression of the Wnt/β-catenin pathway,aggravated cell apoptosis,downregulated CYP1B1and tight junction protein levels,and inhibited cell prolife ration and migration.Overexpression ofβ-catenin or knockdown ofβ-catenin and CYP1B1 genes in rat brain mic rovascular endothelial cells at least partly ameliorated or exacerbated these effects,respectively.In addition,small interfering RNA-mediatedβ-catenin silencing decreased CYP1B1 expression,whereas CYP1B1 knoc kdown did not change the levels of glycogen synthase kinase 3β,Wnt-3a,andβ-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivatio n/reoxygenation.Thus,the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling,and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress,increased tight junction levels,and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Silencing of peroxiredoxin 2 suppresses proliferation and Wnt/β-catenin pathway,and induces senescence in hepatocellular carcinoma

Hepatocellular carcinoma(HCC),a common malignancy worldwide,still lacks effective clinical treatment.The study aimed to investigate the oncogenes that affect the progression of HCC and their possible mechanisms.In our study,we initially confirmed a higher level of PRDX2 in the bile of HCC patients compared to those with choledocholithiasis by 2-DE,LC-MS,and ELISA.Subsequently,we demonstrated the high expression of peroxiredoxin 2(PRDX2)in HCC based on the TCGA database and clinical sample analysis.Furthermore,PRDX2 overexpression enhanced the viability of HCC cells.And PRDX2 silencing induced senescence of HCC cells.In vivo,knockdown of PRDX2 significantly reduced the weight of xenograft tumors.PRDX2 also was found to activate the Wnt/β-catenin pathway by inducingβ-catenin nuclear translocation.Consequently,we proved that silencing PRDX2 could inhibit proliferation and Wnt/β-catenin pathway while promoting senescence in HCC cells.

Pachymic acid exerts antitumor activities by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B

Objective:To determine the inhibitory effects of pachymic acid on lung adenocarcinoma(LUAD)cells and elucidate its underlying mechanism.Methods:CCK-8,wound healing,Transwell,Western blot,tube formation,and immunofluorescence assays were carried out to measure the effects of various concentrations of pachymic acid on LUAD cell proliferation,metastasis,angiogenesis as well as autophagy.Subsequently,molecular docking technology was used to detect the potential targeted binding association between pachymic acid and protein tyrosine phosphatase 1B(PTP1B).Moreover,PTP1B was overexpressed in A549 cells to detect the specific mechanisms of pachymic acid.Results:Pachymic acid suppressed LUAD cell viability,metastasis as well as angiogenesis while inducing cell autophagy.It also targeted PTP1B and lowered PTP1B expression.However,PTP1B overexpression reversed the effects of pachymic acid on metastasis,angiogenesis,and autophagy as well as the expression of Wnt3a andβ-catenin in LUAD cells.Conclusions:Pachymic acid inhibits metastasis and angiogenesis,and promotes autophagy in LUAD cells by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B.

Effects of Helicobacter pylori and Moluodan on the Wnt/β-catenin signaling pathway in mice with precancerous gastric cancer lesions

BACKGROUND Helicobacter pylori(H.pylori)is the primary risk factor for gastric cancer(GC),the Wnt/β-Catenin signaling pathway is closely linked to tumourigenesis.GC has a high mortality rate and treatment cost,and there are no drugs to prevent the progression of gastric precancerous lesions to GC.Therefore,it is necessary to find a novel drug that is inexpensive and preventive to against GC.AIM To explore the effects of H.pylori and Moluodan on the Wnt/β-Catenin signaling pathway and precancerous lesions of GC(PLGC).METHODS Mice were divided into the control,N-methyl-N-nitrosourea(MNU),H.pylori+MNU,and Moluodan groups.We first created an H.pylori infection model in the H.pylori+MNU and Moluodan groups.A PLGC model was created in the remaining three groups except for the control group.Moluodan was fed to mice in the Moloudan group ad libitum.The general condition of mice were observed during the whole experiment period.Gastric tissues of mice were grossly and microscopically examined.Through quantitative real-time PCR(qRT-PCR)and Western blotting analysis,the expression of relevant genes were detected.RESULTS Mice in the H.pylori+MNU group showed the worst performance in general condition,gastric tissue visual and microscopic observation,followed by the MNU group,Moluodan group and the control group.QRT-PCR and Western blotting analysis were used to detect the expression of relevant genes,the results showed that the H.pylori+MNU group had the highest expression,followed by the MNU group,Moluodan group and the control group.CONCLUSION H.pylori can activate the Wnt/β-catenin signaling pathway,thereby facilitating the development and progression of PLGC.Moluodan suppressed the activation of the Wnt/β-catenin signaling pathway,thereby decreasing the progression of PLGC.

Complement factor Ⅰ knockdown inhibits colon cancer development by affecting Wnt/β-catenin/c-Myc signaling pathway and glycolysis

BACKGROUND Colon cancer(CC)occurrence and progression are considerably influenced by the tumor microenvironment.However,the exact underlying regulatory mechanisms remain unclear.AIM To investigate immune infiltration-related differentially expressed genes(DEGs)in CC and specifically explored the role and potential molecular mechanisms of complement factor I(CFI).METHODS Immune infiltration-associated DEGs were screened for CC using bioinformatics.Quantitative reverse transcription polymerase chain reaction was used to examine hub DEGs expression in the CC cell lines.Stable CFI-knockdown HT29 and HCT116 cell lines were constructed,and the diverse roles of CFI in vitro were assessed using CCK-8,5-ethynyl-2’-deoxyuridine,wound healing,and transwell assays.Hematoxylin and eosin staining and immunohistochemistry staining were employed to evaluate the influence of CFI on the tumorigenesis of CC xenograft models constructed using BALB/c male nude mice.Key proteins associated with glycolysis and the Wnt pathway were measured using western blotting.RESULTS Six key immune infiltration-related DEGs were screened,among which the expression of CFI,complement factor B,lymphoid enhancer binding factor 1,and SRY-related high-mobility-group box 4 was upregulated,whereas that of fatty acid-binding protein 1,and bone morphogenic protein-2 was downregulated.Furthermore,CFI could be used as a diagnostic biomarker for CC.Functionally,CFI silencing inhibited CC cell proliferation,migration,invasion,and tumor growth.Mechanistically,CFI knockdown downregulated the expression of key glycolysis-related proteins(glucose transporter type 1,hexokinase 2,lactate dehydrogenase A,and pyruvate kinase M2)and the Wnt pathway-related proteins(β-catenin and c-Myc).Further investigation indicated that CFI knockdown inhibited glycolysis in CC by blocking the Wnt/β-catenin/c-Myc pathway.CONCLUSION The findings of the present study demonstrate that CFI plays a crucial role in CC development by influencing glycolysis and the Wnt/β-catenin/c-Myc pathway,indicating that it could serve as a promising target for therapeutic intervention in CC.

lncRNA MALAT1通过海绵吸附miRNA-141-3p调控Wnt/β-catenin促进卵巢癌的生长及转移

目的 研究长链非编码RNA MALAT1(lncRNA MALAT1)在卵巢癌中的作用及调控机制。方法 实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction, RT-qPCR)检测MALAT1在人正常卵巢上皮细胞系IOSE80及人卵巢癌细胞系SKOV3、OVCA429和HO-8910PM中的表达,选取SKOV3及HO-8910PM细胞进行后续研究。利用siRNA干扰SKOV3和HO-8910PM细胞中MALAT1表达,CCK-8法检测细胞增殖,划痕及Transwell小室检测细胞迁移及侵袭能力,并通过Western blot法检测上皮-间充质转化(epithelial-mesenchymal transformation, EMT)相关指标E-cadherin、N-cadherin的表达。利用生物信息学分析MALAT1与miRNA-141-3p的靶向关系,并利用双荧光素报告基因验证。MALAT1敲低及miRNA-141-3p抑制剂共同作用细胞,检测其对SKOV3及HO-8910PM细胞增殖、侵袭及迁移的影响,并通过Western blot法分析其对Wnt/β-catenin信号通路相关蛋白的表达调控。结果 与人正常卵巢上皮细胞系IOSE80比较,卵巢癌细胞系内MALAT1表达明显上调(P<0.05);而在SKOV3及HO-8910PM中下调MALAT1表达,细胞增殖、侵袭迁移及EMT均受到抑制,同时miRNA-141-3p表达上调(P<0.05)。双荧光素酶报告基因结果证明MALAT1和miRNA-141-3p具有靶向关系。而在下调MALAT1表达的同时使用miRNA-141-3p抑制剂,则可逆转下调MALAT1的所产生的抑制效应(P<0.05)。进一步的研究发现,MALAT1/miRNA-141-3p轴可能通过调控Wnt/β-catenin信号通路影响卵巢癌进展。结论 MALAT1可能通过海绵吸附miRNA-141-3从而调控Wnt/β-catenin影响卵巢癌的发生和发展。

Exosomal miR-320e through wnt2targeted inhibition of the Wnt/β-catenin pathway allevisate cerebral small vessel disease and cognitive impairment

BACKGROUND Exosomal miRNAs play crucial roles in many central nervous system diseases.Cerebral small vessel disease(CVSD)is a small vessel disease that is affected by various factors.This study aimed to investigate the role of exosomal miR-320e in the Wnt/β-catenin pathway stimulated by oxidative stress and assess its clinical correlation with psychiatric symptoms in patients with CVSD.AIM To explore whether exosomal miR-320e could suppress the Wnt/β-catenin pathway and play a protective role in CVSD progression,as well as examine its potential correlation with cognitive impairment and depression in patients with CVSD.METHODS Differentially expressed exosomal miRNAs were filtered by sequencing plasma exosomes from patients with CVSD and healthy controls.Bioinformatics and dual luciferase analyses were used to confirm the binding of miR-320e to Wnt2,and the mRNA and protein levels of downstream components in the Wnt/β-catenin pathway were evaluated when overexpressed or with knockdown of miR-320e under H2O2-induced oxidative stress.In addition,Wnt2-targeting siRNA was used to confirm the role of miR-320e in the Wnt2-mediated inhibition of the Wnt/β-catenin pathway.A retrospective analysis was conducted among patients with CVSD to confirm the correlation between miR-320e expression and the severity of cognitive impairment and depression,which were quantified using the Montreal Cognitive Assessment(MoCA)/Executive Function Assessment(EFA),and the Hamilton Depression Scale(HAMD)/Beck Depression Inventory(BDI),respectively.RESULTS High-throughput sequencing revealed that exosomal miR-320e was downregulated in patients with CVSD.Bioinformatics analysis and dual-luciferase reporter gene experiments showed that exosomal miR-320e inhibited the Wnt/β-catenin pathway in response to oxidative stress by targeting the 3'noncoding region of Wnt2.Uptake of exosomes carrying miR-320e into endothelial cells could also target Wnt2 and inhibit the Wnt2/β-catenin pathway.Elevated miR-320e expression may protect patients with CVSD from relatively severe cognitive impairment and depression,as it was found to have a positive correlation with the MoCA/EFA and HAMD/BDI scores.CONCLUSION Our results suggest that exosomal miR-320e suppresses the Wnt/β-catenin pathway and may play a protective role in CVSD progression.

WNT3A结合并稳定FZD2激活Wnt通路促进成骨细胞增殖和分化的分子机制研究

目的:探讨配体WNT3A通过稳定和激活FZD2(Frizzled2)增加成骨细胞骨形成的活性及其分子机制。方法:24只雌性6~8周龄C57BL/6J小鼠随机分为4组,对照(Control)组,假手术(Sham)组,双侧卵巢摘除术(ovariectomy,OVX)诱导骨质疏松症(Osteoporosis,OP)小鼠模型组(OVX组),OVX+雌二醇治疗组(OVX+E2 Treatment组),每组6只小鼠。建立OVX小鼠模型。Western blot法测定小鼠后肢胫骨组织中WNT3A、FZD2、Active-β-Catenin、β-Catenin、ALP和Runx2以及磷酸化(p-)STAT3、STAT3、p-JAK2和JAK2的表达水平。CCK-8测定小鼠胚胎成骨细胞MC3T3-E1的增殖能力。腺病毒-shRNA-FZD2介导敲低MC3T3-E1细胞中的FZD2。免疫共沉淀(co-Immunoprecipitation,co-IP)法测定WNT3A处理MC3T3-E1细胞前后FZD2与泛素(ubiquitin,Ub)的直接结合情况。结果:与OVX组相比,OVX+E2 Treatment组小鼠胫骨组织中WNT3A、FZD2、Active-β-Catenin、β-Catenin、ALP和Runx2的表达水平均被上调(P<0.05)。与Control组相比,WNT3A Treatment组MC3T3-E1细胞中FZD2、Active-β-Catenin、β-Catenin、ALP和Runx2的表达水平均升高(P<0.05);细胞增殖能力增强(P<0.05)。与WNT3A Treatment组相比,WNT3A Treatment+Adv-shRNA-FZD2组FZD2、Active-β-Catenin、β-Catenin、ALP和Runx2的表达水平均降低(P<0.05);p-STAT3和p-JAK2的磷酸化水平均降低(P<0.05);增殖能力降低(P<0.05);而2组细胞中STAT3和JAK2的表达水平无统计学差异(P>0.05)。在IP:Ub组中,与WNT3A处理(-)的细胞相比,WNT3A处理(+)的细胞中FZD2的表达水平升高(P<0.05)。结论:WNT3A的促骨合成代谢活性是通过结合并稳定FZD2激活Wnt/β-Catenin信号通路实现的。

基于Wnt/β-连环蛋白通路探究金天格对肿瘤坏死因子-α诱导的小鼠MC3T3E1细胞生物学功能的影响实验研究

目的:探讨金天格通过调节Wnt/β-连环蛋白(Wnt/β-catenin)通路对肿瘤坏死因子-α(TNF-α)诱导的小鼠成骨细胞(MC3T3E1)细胞生物学功能的影响。方法:体外培养MC3T3E1细胞,分为对照组、TNF-α组(50 ng/ml TNF-α)、L-金天格组(50 ng/ml TNF-α+10^(-6) g/L金天格)、M-金天格组(50 ng/ml TNF-α+10-5 g/L金天格)、H-金天格组(50 ng/ml TNF-α+10^(-4) g/L金天格)、Dickkopf-1(DKK-1)组(50 ng/ml TNF-α+10 ng/ml Wnt/β-catenin通路抑制剂DKK-1)、H-金天格+LiCl组(50 ng/ml TNF-α+10^(-4) g/L金天格+20μmol/L Wnt/β-catenin通路激活剂LiCl)。用CCK-8试剂盒对细胞活性进行检测,用流式细胞仪对细胞凋亡情况进行检测,用酶联免疫吸附试验对细胞白细胞介素-1β(IL-1β)和IL-6水平进行检测,用Western blot对细胞凋亡相关蛋白及Wnt/β-catenin信号通路蛋白表达情况进行检测。结果:与对照组比较,TNF-α组细胞活性、B淋巴细胞瘤-2(Bcl-2)、细胞程序性死亡配体-1(PD-L1)蛋白表达降低,细胞凋亡率、IL-1β、IL-6水平以及B细胞淋巴瘤(Bax)、β-catenin、转录因子7样2(TCF7L2)、细胞周期蛋白D1(Cyclin D1)蛋白表达升高(均P<0.05)。与TNF-α组比较,L-金天格组、M-金天格组、H-金天格组、DKK-1组细胞活性及Bcl-2、PD-L1蛋白表达升高,细胞凋亡率、IL-1β、IL-6水平以及Bax、β-catenin、TCF7L2、Cyclin D1蛋白表达降低(均P<0.05)。与H-金天格组比较,H-金天格+LiCl组细胞活性及Bcl-2、PD-L1蛋白表达降低,细胞凋亡率、IL-1β、IL-6水平以及Bax、β-catenin、TCF7L2、Cyclin D1蛋白表达升高(均P<0.05)。结论:金天格可能通过抑制Wnt/β-catenin通路减轻TNF-α诱导的MC3T3E1细胞损伤。

益肾通癃颗粒调控Wnt/β-catenin信号通路对人前列腺癌PC3细胞荷瘤裸鼠的干预作用

目的观察益肾通癃颗粒对人前列腺癌PC3细胞荷瘤裸鼠Wnt/β-catenin信号通路的干预作用。方法利用人前列腺癌骨转移细胞PC3建立前列腺癌荷瘤裸鼠模型,将其随机分为模型对照组、阳性药物组、益肾通癃颗粒低剂量组、益肾通癃颗粒中剂量组、益肾通癃颗粒高剂量组和联合用药组,给予相应的药物干预,连续4周。给药结束后处死动物获取皮下移植瘤瘤体组织样本,分别进行HE染色观察病理变化,免疫组化检测细胞增殖情况,TUNEL检测细胞凋亡情况,Western blot及免疫组织化学法检测Wnt信号通路相关基因蛋白的表达水平。结果益肾通癃颗粒可以有效改善荷瘤裸鼠皮下移植瘤瘤体组织的病理改变,可以显著抑制肿瘤细胞的增殖,促进细胞凋亡的发生,药效呈浓度依赖性。益肾通癃颗粒还可以下调Wnt/β-catenin信号通路相关基因Wnt1、Wnt3a、β-catenin、APC蛋白的表达(P<0.01),上调Wnt/β-catenin信号通路相关基因GSK-3β蛋白的表达(P<0.01),降低p-GSK-3β蛋白的活性(P<0.01),药效与剂量呈正相关性,与Wnt信号通路阻断剂ICG联合时效果最佳(P<0.01)。结论益肾通癃颗粒能够抑制前列腺癌细胞的增殖,促进其凋亡,其干预作用可能与其阻断Wnt/β-catenin信号通路激活密切相关。

TRIB3激活Wnt/β-catenin信号通路对喉癌TU686细胞体外生长增殖及移植瘤小鼠外周免疫抑制分子表达的影响

目的:探讨TRIB3激活Wnt/β-catenin信号通路对喉癌TU686细胞体外生长增殖及移植瘤小鼠外周免疫抑制分子表达的影响。方法:分别在体外细胞(人永生化表皮细胞系HaCat及喉癌细胞TU686)及组织(喉癌及癌旁组织)中检测TRIB3蛋白及RNA表达,随后将TU686细胞分为阴性对照组(NC组)及敲低TRIB3组(sh-TRIB3组),提取细胞总蛋白及RNA,验证两组细胞中TRIB3表达水平。验证成功后,CCK-8、集落克隆实验及流式细胞术检测TU686细胞的增殖能力;Western blot检测两组细胞中Wnt、Cyclin-D1、C-myc、β-catenin、p-β-catenin蛋白表达水平,相关性分析验证TRIB3与Wnt、Cyclin-D1、C-myc、β-catenin、p-β-catenin蛋白表达的相关性。利用敲低TRIB3的核心质粒构建低表达TRIB3的裸鼠移植瘤模型(TRIB3 sgRNA组),并设置平行对照组(Control sgRNA组),观察瘤体的生长体积及重量,ELISA测定移植瘤小鼠血清免疫抑制分子表达。结果:与HaCat细胞及正常癌旁组织相比,TU686细胞及喉癌组织高表达TRIB3。与阴性对照组相比,敲低TRIB3后TU686细胞增殖能力被显著抑制,细胞生长被阻滞于G1/S期;Wnt/β-catenin信号通路相关蛋白Wnt、Cyclin-D1、C-myc、β-catenin表达明显下降,p-β-catenin表达明显上升,TRIB3与Wnt、Cyclin-D1、β-catenin、p-β-catenin蛋白表达存在明显相关性。体内实验结果表明,与Control sgRNA组相比,TRIB3 sgRNA组小鼠肿瘤生长体积及重量均明显降低,血清免疫抑制分子IL-4、IL-6、IL-10、TGF-β及PGE2表达均明显下降。结论:TU686细胞中TRIB3高表达,TRIB3可通过激活Wnt/β-catenin相关信号通路抑制喉癌TU686细胞及移植瘤的生长增殖,并可逆转肿瘤免疫抑制微环境,提示TRIB3可能是治疗喉癌的有效靶标。

人参多糖调控Wnt3/β-catenin/Runx2信号通路改善去卵巢大鼠骨质疏松的作用

目的探究人参多糖改善去卵巢大鼠骨质疏松的作用及对Wnt3/β-catenin/Runx2信号通路的影响。方法将大鼠分为假手术组、模型组、人参多糖低、高剂量组(100、200 mg/kg)及阿仑膦酸钠维生素D3组(6.25 mg/kg),每组10只,采用去卵巢法建立骨质疏松症模型。连续干预12周,记录体质量,观察骨组织病理形态,检测骨密度、骨生物力学、血清骨代谢及生化指标,同时测定骨组织Wnt3、β-catenin及Runx2表达。结果与模型组比较,人参多糖低、高剂量组大鼠体质量明显降低(P<0.01),骨组织病理形态减轻,骨密度、刚度、最大应力及最大载荷均明显增加(P<0.05,P<0.01);血清TRAP-5b含量明显降低(P<0.05,P<0.01),而BALP、OC、OPG及P^(3+)、Ca^(2+)含量明显升高(P<0.05,P<0.01);骨组织Wnt3、β-catenin及Runx2表达均明显增加(P<0.05,P<0.01)。结论人参多糖具有改善去卵巢大鼠骨质疏松的作用,其机制可能与激活Wnt3/β-catenin/Runx2信号通路有关。

基于NLRP3/Caspase-1和Wnt/β-catenin信号通路探讨桃核承气汤延缓慢性肾衰竭大鼠肾纤维化的机制

目的基于NLRP3/Caspase-1和Wnt/β-catenin信号通路探讨桃核承气汤延缓慢性肾衰竭(CRF)大鼠肾纤维化的机制。方法将80只SPF级Wistar大鼠随机分为正常组、假手术组、模型组和治疗组,每组20只。模型组和治疗组行5/6肾切除术,假手术组仅做双肾被膜剥离术,正常组不做处理。术后4周开始进行干预,治疗组按10 mL/(kg·d)给予42 g/100 mL桃核承气汤药液灌胃,正常组、假手术组和模型组分别给予等体积生理盐水灌胃,连续干预8周。采用Western blot和qPCR检测肾组织中NOD样受体热蛋白结构域相关蛋白3(NLRP3)、半胱氨酸蛋白酶-1(Caspase-1)、白细胞介素(IL)-18、IL-1β、Wnt4、β-连环蛋白(β-catenin)、基质金属蛋白酶-7(MMP-7)蛋白表达量和mRNA相对表达水平,检测血清肌酐(Scr)、尿素氮(BUN)含量,光镜、电镜下观察肾组织形态。结果光镜观察,模型组肾小球结构固缩,肾间质纤维增生严重,治疗组明显减轻。电镜观察,模型组肾小球内皮细胞、肾小管上皮细胞均水肿,大量炎症细胞浸润,治疗组明显减轻。与正常组和假手术组比较,模型组肾组织NLRP3、Caspase-1、IL-1β、IL-18、Wnt4、β-catenin和MMP-7蛋白表达量和mRNA相对表达水平均明显升高(P<0.05),血清Scr、BUN均明显升高(P<0.05);与模型组比较,治疗组NLRP3、Caspase-1、IL-1β、IL-18、Wnt4、β-catenin和MMP-7蛋白表达量和mRNA相对表达水平均明显降低(P<0.05),血清Scr、BUN均明显降低(P<0.05)。结论桃核承气汤能有效减轻CRF炎症损伤,改善肾纤维化,延缓CRF的进程,其机制可能与调控NLRP3/Caspase-1细胞焦亡通路和Wnt/β-catenin信号通路有关。

左归丸通过FNDC5/Wnt3a/β-catenin通路治疗绝经后骨质疏松症的机制研究

目的对左归丸治疗绝经后骨质疏松症(postmenopausal osteoporosis,PMOP)的机制进行研究。方法通过体外实验制备左归丸低、中、高剂量对应的含药血清,对培养的MC3T3-E1进行药物干预,分为空白对照组、成骨诱导组(OGI组)、OGI+左归丸低、中、高浓度组;对培养的MC3T3-E1细胞进行FNDC5转染后成骨诱导,分为空白对照组、OGI+转染组、OGI+转染+左归丸低、中、高浓度组。对上述细胞培养液进行ALP活力检测及染色。在体内实验中将野生型小鼠随机分为野生型对照组、野生型卵巢切除(OVX)组、野生型治疗组,将FNDC5基因敲除(KO)小鼠随机分为KO对照组、KO-OVX组、KO治疗组,采用OVX方式造模,给予治疗组小鼠灌胃左归丸12周。12周灌胃结束后检测各组股骨骨密度(bone mineral density,BMD),HE染色股骨切片,ELISA检测血清E2、PINP、Irisin含量,实时荧光定量PCR检测FNDC5、Runx2、OPN、OCN、OSX mRNA表达,Western Blot检测FNDC5、Wnt3a、β-catenin蛋白表达。结果左归丸含药血清增加了MC3T3-E1的ALP表达,FNDC5转染后ALP表达下降;左归丸可以显著改善野生型小鼠OVX术后导致的BMD降低及股骨骨小梁损害,缓解E2、PINP、Irisin表达降低,而对于KO小鼠无显著差异;左归丸抑制了OPN的表达,提高了Runx2、OSX、OCN的mRNA表达。对比KO-OVX组,KO治疗组的OPN、Runx2、OSX、OCN等均没有明显差异。左归丸可以上调Wnt3a和β-catenin的表达,当FNDC5被敲除后这种上调效果被显著抑制。结论左归丸可能通过FNDC5/Wnt3a/β-catenin通路治疗PMOP。

Nystose attenuates bone loss and promotes BMSCs differentiation to osteoblasts through BMP and Wnt/β-catenin pathway in ovariectomized mice

Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Naturally derived oligosaccharides have shown significant anti-osteoporotic effects.Nystose(NST),an oligosaccharide,was isolated from the roots of Morinda officinalis How.(MO).The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice,and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts.Administration of NST(40,80 and 160 mg/kg)and the positive control of estradiol valerate(0.2 mg/kg)for 8 weeks significantly prevented bone loss induced by ovariectomy(OVX),increased the bone mass density(BMD),improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline(DPD)in ovariectomized mice,while inhibited the increase of body weight without significantly affecting the uterus weight.Furthermore,we found that NST increased osteogenic differentiation,inhibited adipogenic differentiation of BMSCs in vitro,and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways.In addition,Noggin and Dickkopf-related protein-1(DKK-1)reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway.The luciferase activities and the molecular docking analysis further supported the mechanism of NST.In conclusion,these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

结直肠癌患者血清miR-21-5p、miR-377-3p表达与Wnt/β-catenin信号通路和预后的关系分析

目的研究结直肠癌(CRC)患者血清微小RNA(miR)-21-5p、miR-377-3p表达及与Wnt/β-连环蛋白(catenin)通路及预后的关系。方法选取2017年1月—2018年1月中国人民解放军联勤保障部队第九八九医院肿瘤科收治的CRC患者92例作为CRC组,医院同期健康体检者60例作为健康对照组。检测血清miR-21-5p、miR-377-3p表达量及Wnt/β-catenin通路指标Wnt3a、β-catenin、c-myc、细胞周期素D1(Cyclin D1)mRNA表达量。Pearson相关分析miR-21-5p、miR-377-3p与Wnt3a、β-catenin、c-myc、Cyclin D1 mRNA表达的相关性。比较不同临床病理特征CRC患者血清miR-21-5p、miR-377-3p表达差异。Kaplan-Meier曲线及Logrank检验分析不同血清miR-21-5p、miR-377-3p表达CRC患者预后的差异。多因素Cox回归分析CRC患者预后的影响因素。结果与健康对照组比较,CRC组患者血清miR-21-5p、Wnt3a、β-catenin、c-myc、Cyclin D1 mRNA表达量显著升高,miR-377-3p表达量显著降低(t/P=29.202/<0.001、52.006/<0.001、45.973/<0.001、39.196/<0.001、23.119/<0.001、38.120/<0.001)。血清miR-21-5p与Wnt3a、β-catenin、c-myc、Cyclin D1 mRNA表达呈正相关(r/P=0.404/<0.001、0.410/0.002、0.529/<0.001、0.378/<0.001),miR-377-3p与Wnt3a、β-catenin、c-myc、Cyclin D1 mRNA表达呈负相关(r/P=-0.347/0.007、-0.408/<0.001、-0.450/<0.001、-0.419/<0.001)。TNM分期Ⅲ~Ⅳ期和伴淋巴结转移CRC患者血清miR-21-5p表达明显高于TNM分期Ⅰ~Ⅱ期和无淋巴结转移患者,而血清miR-377-3p表达明显低于TNM分期Ⅰ~Ⅱ期和无淋巴结转移患者,差异具有统计学意义(t/P=19.741/<0.001、18.534/<0.001、11.799/<0.001、17.639/<0.001)。血清miR-21-5p高表达患者3年生存率低于低表达患者(χ^(2)/P=17.700/<0.001),miR-377-3p低表达患者3年生存率低于高表达患者(χ^(2)/P=21.380/<0.001)。多因素Cox回归分析显示,肿瘤TNM分期Ⅲ~Ⅳ期、伴淋巴结转移、miR-21-5p升高是CRC患者预后的独立危险因素[OR(95%CI)=1.875(1.322~2.662)、1.662(1.163~2.374)、1.847(1.366~2.500)],miR-377-3p升高是CRC患者预后的独立保护因素[OR(95%CI)=0.518(0.363~0.739)]。结论CRC患者血清miR-21-5p表达升高,miR-377-3p表达降低,两者可通过调控Wnt/β-catenin通路促进CRC的肿瘤进展,可作为辅助评估CRC患者预后的肿瘤标志物。