Guanxinning tablet alleviates carotid atherosclerotic plaque injury in rats / 基础医学与临床

Objective To investigate the effect and its mechanism of Guanxinning tablet on carotid atherosclerotic plaque in a rat model.Methods The rats were randomly divided into control group,model group(to construct carot-id atherosclerosis plaque model),Guanxinning groups with low,medium and high dose of Guanxinning tablet(150,300 and 600 mg/kg),atorvastatin group(2 mg/kg),lithiumchloridemonohydrate(LiCl)(Wnt/β-catenin pathway activator)group(15 mg/kg),and Guanxinning plus LiCl group(600 mg/kg Guanxinning tablets+15 mg/kg LiCl),with 12 rats in each group.The intervention began at the third week and was administered once a day for 8 weeks.Olympus Au2700 automatic biochemical analyzer was used to detect the level of total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL)and high-density lipoprotein(HDL)in rat serum;ELISA was applied to detect the level of monocyte chemotactic protein(MCP-1)and hyper-sensitive C-reactive protein(hs-CRP)in rat se-rum;the level of oxidized low density lipoprotein(ox-LDL)and malondialdehyde(MDA)in rat serum were detected by spectrophotometry;HE staining was applied to find pathological changes in the common carotid artery of rats;Western blot was applied to detect the expression of Wnt3a,matrix metalloproteinase-9(MMP-9)and β-catenin in rat common carotid artery.Results Compared with the control group,the level of TG,TC,LDL,MCP-1,hs-CRP,ox-LDL,protein expression of MDA,MMP-9,Wnt3a,β-catenin and total plaque area/total arterial area ratio in-creased,the HDL level decreased in model group(P＜0.05);compared with the model group,the level of TG,TC,LDL,MCP-1,hs-CRP,ox-LDL,MDA,expression of MMP-9,Wnt3a,β-catenin protein and total plaque area/total arterial area ratio in the low,medium,and high dose groups of Guanxinning tablets decreased,the HDL level in-creased.The effect of Guanxinning tablets was dose-dependent,and the change trend of corresponding indicators in the LiCl group was opposite to the above(P＜0.05);compared with the high dose group of Guanxinning tablets,the TG,TC,LDL,MCP-1,hs-CRP,ox-LDL,MDA levels,MMP-9,Wnt3a,β-catenin protein expression,and total plaque area/total arterial area ratio in the high dose+LiCl group of Guanxinning tablets increased,the HDL level de-creased(P＜0.05).Conclusions Guanxinning tablet can inhibit the formation of atherosclerotic plaque in rats and the mechanismis potentially related to the regulation of Wnt/β-catenin pathway.

Expression pattern and signification of Cx43,beta-catenin and Smo in the second heart field / 中国组织工程研究

BACKGROUND:The second heart field is crucial for the development of the embryonic heart.Abnormal development of the second heart field can result in multiple cardiac malformations.After Cx43 gene knockout,reduced formation and proliferation of cells of the second heart field can be observed,but the specific reason remains unclear. OBJECTIVE:(1)To determine whether β-catenin,Smo and Cx43 were co-expressed in the second heart field and the endoderm,we observed the expression patterns of these proteins.(2)To explore whether Cx43 interacts with the Wnt/β-catenin pathway or the Shh pathway in the development of the second heart field. METHODS:Serial paraffin sections of the mouse embryos at embryonic days 10-12 were selected for immunohistochemical staining,hematoxylin-eosin staining and immunofluorescence staining.The primitive gut of mouse embryos at embryonic day 11 was separated for western blot assay and co-immunoprecipitation. RESULTS AND CONCLUSION:(1)Cx43 and Isl1 were co-expressed in some mesenchymal cells on the ventral side of the foregut and dorsal wall of the pericardial cavity of mouse embryos at embryonic days 10-12;Isl1 positive cells increased while Cx43 positive cells increased.(2)Cx43 and β-catenin were co-expressed in the ventral part of the endoderm at embryonic days 10-12.(3)Cx43 and Smo were co-expressed in the endoderm at embryonic days 10-12.(4)The co-immunoprecipitation results confirmed that there was an interaction between Cx43 and β-catenin,which suggested that Cx43 interacted with β-catenin to participate in the development of the second heart field.

Inhibitory effect of curcumin on malignant biological behavior and Wnt/β-catenin pathway of uveal melanoma cells / 中华实验眼科杂志

Objective:To explore the inhibitory effect of curcumin on the malignant biological behavior of uveal melanoma (UM) and its possible mechanism.Methods:M23 cells were cultured in curcumin medium with different concentrations (0, 20, 40 and 80 μmol/L) for 48 hours, respectively.The morphological changes of cells were observed under an inverted microscope.The cell survival rate was detected by the cell counting kit-8 (CCK-8) method.The apoptosis, colony formation, migration and invasion of cells were detected by flow cytometry, plate clone formation experiment, cell scratch experiment and Transwell assay, respectively.The relative expressions of genes related to Wnt/β-catenin pathway, c-Myc, Cyclin D1, Survivin and matrix metallo proteinase 9 ( MMP-9) mRNA in cells were detected by real-time fluorescence quantitative PCR.The relative expressions of proteins related to Wnt/β-catenin pathway, c-Myc, Cyclin D1, Survivin, MMP-9 and β-catenin, glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β (p-GSK-3β) and axis inhibition protein 2 (Axin2) proteins were detected by Western blot.Another 20 female BALB/c mice were selected and injected with M23 cell suspension under the subcutaneous fat pad in the left posterior abdomen to establish the in vivo M23 transplanted tumor model.The mice successfully modeled were randomly divided into model group, low-dose curcumin group, medium-dose curcumin group and high-dose curcumin group according to the random number table method, which was intraperitoneally injected with 0, 10, 20 and 40 mg/kg curcumin physiological saline solution respectively.After a continuous injection for 30 days, the subcutaneous tumor was stripped and weighed.The animal experiment process followed the 3Rs principle of animal research and was approved by the Laboratory Animal Ethics Committee of Inner Mongolia Baotou Steel Hospital (No.2021MER-023). Results:The cell survival rate, the number of colony formation, the apoptosis rate, the cell invasion rate and the cell migration rate were (100.00±0.00)%, 128.67±9.18, (1.33±0.29)%, (89.76±4.57)% and 148.33±8.18 in 0 μmol/L curcumin group, (83.78±4.59)%, 100.33±8.73, (14.53±2.04)%, (65.43±3.70)% and 125.33±7.41 in 20 μmol/L curcumin group, (66.09±3.92)%, 58.67±6.55, (27.23±3.56)%, (34.83±2.19)% and 73.67±6.34 in 40 μmol/L curcumin group, and (47.16±3.63)%, 31.67±4.92, (44.73±4.36)%, (18.82±1.99)% and 45.67±5.31 in 80 μmol/L curcumin group.There were statistically significant differences in the survival rate, colony formation number, cell apoptosis rate, migration rate and invasion rate of M23 cells among the four groups ( F=125.321, 97.941, 72.516, 277.097, 139.006; all at P<0.001). With the increase of curcumin concentration, the cell survival rate, colony formation number, cell migration rate and cell invasion number decreased obviously, and the cell apoptosis rate increased obviously, and the pairwise comparisons showed significant differences (all at P<0.05). With the increase of curcumin concentration, the relative expression levels of c-Myc, Cyclin D1, Survivin, MMP-9 mRNA and proteins, β-catenin and p-GSK-3β proteins decreased significantly, while the relative expression level of Axin2 protein increased significantly, showing significant differences in pairwise comparisons (all at P<0.05). The tumor tissue weight of mice decreased with the increase of curcumin dosage, and the pairwise comparisons were statistically significant (all at P<0.05). Conclusions:Curcumin can inhibit the proliferation, migration, invasion and other malignant biological behaviors of UM M23 cells, inhibit tumor growth and promote cell apoptosis.Its mechanism may be related to blocking the activation of Wnt/β-catenin pathway.

Proliferation Inhibitory Activity of Quinones from Blaps rynchopetera Defense Secretion on Colorectal Tumor Cells / 中国结合医学杂志

OBJECTIVE@#To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines.@*METHODS@#Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively.@*RESULTS@#MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC50) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC50 values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC50 values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G1 phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells.@*CONCLUSION@#Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.

Effects of lead exposure on development of heart and spine in zebrafish embryos / 环境与职业医学

Background Lead is widely distributed. Lead exposure interferes with early life development in zebrafish, but the mechanisms by which lead exposure affects skeletal development and cardiac development are not clear as yet. Objective To investigate the molecular mechanisms of bone development and cardiac development toxicity induced by lead acetate exposure. Methods Zebrafish embryos were exposed to different concentrations of lead acetate (0, 6, 12, 24, and 48 μmol·L−1) for 3 h post-fertilization (3 hpf) until 5 d post-fertilization (5 dpf). The malformation phenotypes of 5 dpf were counted, and the mRNA expressions of spinal development-related genes (bmp2b, bmp4, bmp9, runx2a, runx2b) and heart development-related genes (nkx2.5, myh6, myh7) were detected by quantitative PCR (qPCR). Expressions of genes of development-related regulatory pathways including Wnt/β-catenin pathway (wnt5a, wnt8a, wnt10a, β-catenin) and TGF-β pathway (tgf-β1, tgf-β2) as well as key molecule eph of Eph-Ephrin signaling were analyzed. Results At 5 dpf, the zebrafish in the lead acetate treated groups showed deformed phenotypes including spinal curvature and pericardial sac edema compared to the control group. In the lead acetate groups at 24 and 48 μmol·L−1, the spinal curvature deformity rates reached 26.47% and 71.52% (P<0.01) respectively. The qPCR results revealed that the expression levels of spinal development-related genes bmp2b, bmp4, bmp9, runx2a, and runx2b were downregulated in the 48 μmol·L−1 exposure group compared to the control group by 82.8%, 58.0%, 88.7%, 85.5%, and 69.2%, respectively (P<0.05 or P<0.01); the expression levels of heart development-related genes myh6, myh7, and nkx2.5 were down-regulated by 63.7%, 58.9%, and 55.2%, respectively (P<0.01); the expression levels of wnt8a and β-catenin in the Wnt/β-catenin pathway were down-regulated by 71.5% and 47.3% (P < 0.05 or P < 0.01), respectively; the expression level of tgf- β1 in the TGF-β pathway was down-regulated by 67.5% (P<0.01); the expression level of eph was down-regulated by 86.9% (P<0.01). Conclusion Lead acetate exerts developmental toxic effects on zebrafish heart and bone by down-regulating the expressions of genes related to spinal development and heart development, as well as inhibiting development-related Wnt/β-catenin and TGF-β pathways and Eph-Ephrin signaling, causing malformed phenotypes such as spinal curvature and pericardial sac edema.

Tumor-associated macrophages affect breast cancer endocrine resistance through FOXM1/Wnt/ β-catenin pathway / 中华内分泌外科杂志

Objective:To explore the influence of tumor-associated macrophages (TAMs) on endocrine resistance in breast cancer through the forkhead box M1 (FOXM1) /Wnt/ β-catenin pathway. Methods:Tamoxifen-resistant breast cancer cells were cultured, THP-1 cells were induced into macrophages (MΦ), and further induced into TAMs. After being cultured in the conditioned medium (CM) of MCF-7 cells for 24 hours, MΦ were defined as MS cells. After being cultured in the CM of MCF-7R cells for 24 hours, MΦ were defined as MR cells. MCF-7 cells, after being cultured in the CM of macrophages for 24 hours, were defined as MCF-7 (MΦ) cells. MCF-7 cells, after being cultured in the CM of MS cells for 24 hours, were defined as MCF-7 (MS) cells. MCF-7 cells, after being cultured in the CM of MR cells for 24 hours, were defined as MCF-7 (MR) cells. Cell viability and invasion ability were evaluated using CCK-8 and Transwell assays. The protein levels of CD163, Wnt1, β-catenin, and FOXM1 in different groups were examined by qRT-PCR and Western blot. Results:Compared to the MS group (mRNA: 1.49±0.12, protein: 1.15±0.12), CD163 expression was higher in the MR group (mRNA: 2.33±0.16, protein 1.52±0.11) ( t=7.28, P=0.002) ( t=3.94, P=0.017), indicating that tamoxifen-resistant breast cancer cells can induce polarization of more MΦ into TAMs. TAMs increased the expression of FOXM1 in breast cancer cells, which further activated the Wnt/ β-catenin pathway. Compared to the MCF-7 (MΦ) group, the MCF-7 (MS) and MCF-7 (MR) groups showed enhanced cell viability and invasion, with the most significant increase observed in the MCF-7 (MR) group. Compared with MCF-7 (MΦ) cells, the levels of Wnt1, β-catenin, and FOXM1 in MCF-7 (MS) and MCF-7 (MR) cells were significantly increased, with the highest levels observed in the MCF-7 (MR) group with the most TAM polarization. Compared to the MCF-7 group, both the MCF-7 (MR) and MCF-7+pcDNA-FOXM1 groups showed increased levels of Wnt1 and β-catenin, enhanced cell viability and invasion. Compared to the MCF-7 (MR) group, the MCF-7 (MR) + si-FOXM1 group showed reduced levels of Wnt1 and β-catenin, weakened cell viability and invasion. Conclusion:TAMs promote endocrine resistance in breast cancer by upregulating FOXM1 and activating the Wnt/ β-catenin pathway.

Effect and mechanism of sodium cantharidate on proliferation,migration and invasion of esophageal cancer EC9706 Cells / 新乡医学院学报

Objective To investigate the effect and mechanism of sodium cantharidate on the proliferation,migration and invasion of esophageal carcinoma EC9706 cells.Methods Esophageal cancer EC9706 cells were randomly divided into blank control group,low-dose sodium cantharidate group,medium-dose sodium cantharidate group,high-dose sodium canthari-date group and cisplatin group.The EC9706 cells in the low-,medium-and high-dose sodium cantharidate groups were given fi-nal mass concentration of 1.0,2.5 and 5.0 mg·L-1 sodium cantharidate intervention,respectively.The EC9706 cells in the cisplatin group were treated with the final mass concentration of 140 mg·L-1 cisplatin,and the cells in the control group was cultured in Dulbecco's modified Eagle's medium.The cell proliferation rate in each group was detected by cell counting reagent-8 method,the mobility of EC9706 cells in each group was detected by scratch test,the invasion rate of EC9706 cells in each group was detected by Transwell method,and the levels of Wnt3a and β-catenin mRNA in EC9706 cells in each group were detected by real-time quantitative polymerase chain reaction method,and the levels of Wnt3a and β-catenin protein in EC9706 cells in each group were detected by Western blot.Results At 24,48 and 72 h of cultivation,the proliferation rate of EC9706 cells in the low-dose sodium cantharidate group,medium-dose sodium cantharidate group,high-dose sodium canthari-date group and cisplatin group was significantly lower than that in the blank control group(P＜0.05);the proliferation rate of EC9706 cells in the low-dose sodium cantharidate group and medium-dose of sodium cantharidate group was significantly higher than that in the high-dose sodium cantharidate group and cisplatin group(P＜0.05);the proliferation rate of EC9706 cells in the low-dose sodium cantharidate group was significantly higher than that in the medium-dose sodium cantharidate group(P＜0.05);there was no significant difference in the proliferation rate of EC9706 cells between the high-dose sodium cantharidate group and cisplatin group(P＞0.05);the proliferation rate of EC9706 cells in the low-dose sodium cantharidate group,medium-dose sodium cantharidate group,high-dose sodium cantharidate group and cisplatin group was significantly decreased with the extension of culture time(P＜0.05).The mobility rate and invasion rate of EC9706 cells in the low-dose sodium cantharidate group,medium-dose sodium cantharidate group,high-dose sodium cantharidate group and cisplatin group were significantly lower than those in the blank control group(P＜0.05);the mobility rate and invasion rate of EC9706 cells in the low-dose sodium cantharidate group and medium-dose sodium cantharidate group were significantly higher than those in the high-dose sodium cantharidate group and cisplatin group(P＜0.05);the migration rate and invasion rate of EC9706 cells in the low-dose sodium cantharidate group were significantly higher than those in the medium-dose sodium cantharidate group(P＜0.05);there was no significant difference in the mobility rate and invasion rate of EC9706 cells between the high-dose sodium cantharidate group and cisplatin group(P＞0.05).The relative expression levels of Wnt3a and β-catenin mRNA and protein in EC9706 cells in the low-dose sodium cantharidate group,medium-dose sodium cantharidate group,high-dose sodium cantharidate group and cisplatin group were significantly lower than those in the blank control group(P＜0.05);the relative expression levels of Wnt3a and β-catenin mRNA and protein in EC9706 cells in the low-dose sodium cantharidate group and medium-dose sodium cantharidate group were significantly higher than those in the high-dose sodium cantharidate group and cisplatin group(P＜0.05);the relative expressions levels of Wnt3a and β-catenin mRNA and protein in EC9706 cells in the low-dose sodium cantharidate group were significantly higher than those in the medium-dose sodium cantharidate group(P＜0.05);there was no significant difference in the relative expression levels of Wnt3a and β-catenin mRNA and protein in EC9706 cells between the high-dose sodium cantharidate group and cisplatin group(P＞0.05).Conclusion Sodium cantharidate can significantly inhibit the proliferation,migration and invasion of esophageal carcinoma EC9706 cells,and its mechanism may be related to the inhibition of the Wnt3a/β-catenin pathway.

Improvement effect of crocin on cognitive impairment of Alzheimer's disease rats through DKK3 regulation of GSK-3 β/β-Catenin pathway / 中国临床药理学与治疗学

AIM: To explore the improvement effect and mechanism of crocin on cognitive impairmrnt of Alzheimer's disease (AD) rats. METHODS: The hippocampus of SD rats were injected with Aβ 25-35 to establish AD model, then rats were randomly divided into AD group, AD + low, medium, high dose of crocin groups (10, 20, 40 mg/kg) and AD + donepezil group (1 mg/kg), intraperitoneal injection treatment for 4 weeks, set sham group. Dark avoidance test and water maze test were used to evaluate the learning and memory abilities of rats, ELISA was used to detect serum Aβ content, HE staining and Tunel staining were used to determine pathological changes and neuronal apoptosis of hippocampus of rats, immunohistochemistry was used to detect the expression of Brdu, Dcx and NeuN in hippocampus of rats, and Western blot was used to detect the protein expression of Aβ, DKK3, β-catenin, p-GSK-3β/GSK-3β, Caspase-3, Bax, Bcl-2 in hippocampus of rats. RESULTS: Compared to sham group, the learning and memory abilities of AD group rats were decreased, serum Aβ content increased, the pathological change in hippocampus was serious, neuronal apoptosis was increased, the expression of Brdu, Dcx, NeuN were decreased, the protein expression of Aβ, DKK3, p-GSK-3β/GSK-3β, Caspase-3, Bax were increased, protein expression of β-catenin, Bcl-2 were decreased (P<0.01). Compared to AD group, after the treatment of doses of crocin and donepezil, the learning and memory abilities of AD rats were improved, serum Aβ content were increased, and the pathological change in hippocampus were alleviated, neuronal apoptosis were reduced, the expression of Brdu, Dcx, NeuN were decreased, the protein expression of Aβ, DKK3, p-GSK-3β/ GSK-3β, Caspase-3, Bax were decreased, the protein expression of β-catenin, Bcl-2 were increased, notely, dose-dependent effect of crocin was significant. CONCLUSION: Crocin reduced neuronal apoptosis and mediated DKK3 to regulate GSK-3β/ β-catenin pathway to improve the cognitive impairment of AD rats.

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia / 中西医结合学报

OBJECTIVE@#Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment.@*METHODS@#The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway.@*RESULTS@#According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway.@*CONCLUSION@#JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.

Regulatory role of the transcriptional coactivator Mediator 1 in skin hair regeneration and its mechanisms / 中华皮肤科杂志

Objective:To investigate the effect of the transcriptional coactivator Mediator 1 (Med1) on mouse hair regeneration, and to explore potential mechanisms.Methods:Med1 flox/flox C57BL/6J mice were mated with K14-Cre mice, and the mice with epidermis-specific knockout of Med1 gene, namely K14-Cre-expressing Med1 flox/flox mice (knockout group) , were obtained by using the Cre-Loxp system, while Med1 flox/flox mice without K14-Cre expression served as control group. Mice in the two groups (3 mice in each group) were raised together for 8 weeks followed by dorsal hair removal. Hair regeneration was observed for 12 consecutive days after hair removal. After 12 days, all mice in the two groups were sacrificed, their depilated and non-depilated dorsal skin tissues were resected, and total RNA was extracted from the tissues. Real-time quantitative PCR was performed to determine the mRNA expression of hair keratin genes, vitamin D receptor/β-catenin pathway-related genes, and genes associated with maintenance of hair follicle stem cell proliferation and quiescence. Paraffin-embedded sections of depilated and non-depilated mouse skin tissues were prepared, and immunofluorescence staining was conducted to determine the number of stem cells in the hair follicle bulge. Two-independent-sample t test was used for comparisons between two groups. Results:From days 0 to 12 after depilation, hair regeneration was delayed in the depilated skin area in the knockout group compared with the control group. Real-time quantitative PCR showed significantly decreased mRNA relative expression levels of hair keratin genes Ha1 and Krt2-16, vitamin D receptor/β-catenin pathway-related genes S100a3, Dlx3 and Tubb3, and genes associated with maintenance of hair follicle stem cell proliferation and quiescence including Lhx2, Sox9 and Nfatc1 in the depilated skin tissues in the knockout group (22.09 ± 12.32, 2.07 ± 0.20, 0.02 ± 0.01, 12.36 ± 2.12, 1.75 ± 0.46, 0.39 ± 0.02, 4.42 ± 0.76, 0.44 ± 0.07, respectively) compared with the control group (70.53 ± 9.46, 7.76 ± 0.49, 0.05 ± 0.01, 26.16 ± 2.96, 2.60 ± 0.14, 0.71 ± 0.09, 11.93 ± 0.42, 0.75 ± 0.04, respectively; t = 5.40, 18.64, 3.89, 6.57, 3.04, 6.10, 15.03, 6.18, respectively, all P < 0.05) . Immunofluorescence staining showed that the number of CD34 +K15 + hair follicle stem cells in the hair follicle bulge in both depilated and non-depilated skin tissues was significantly lower in the knockout group than in the control group. Conclusion:Med1 gene knockout may down-regulate the expression of downstream genes of the vitamin D receptor/β-catenin pathway and genes associated with maintenance of hair follicle stem cell proliferation and quiescence (Sox9, Nfatc1 and Lhx2) , and reduce the number of hair follicle stem cells, leading to hair follicle differentiation disorder and hair regeneration delay.

Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway / 中国实验方剂学杂志

Objective:To study the effect of Biejiajian Wan on the epithelial-mesenchymal transition （EMT） of rat hepatic oval cells induced by transforming growth factor- β1（TGF-β1）， in order to explore its mechanism in reversing EMT. Method:WB-F344 cells were divided into five groups： blank group， TGF-β1 model group （10 μg·L-1TGF-β1）， low-dose group （10 μg·L-1TGF-β1+0.55 g·kg-1Biejiajian Wan）， medium-dose group （10 μg·L-1TGF-β1+1.1 g·kg-1Biejiajian Wan）， high-dose group （10 μg·L-1TGF-β1+2.2 g·kg-1Biejiajian Wan）. Except blank group， TGF-β1 was used to induce WB-F344 cells in all of the remaining groups to construct an EMT model. After the cells were treated with low， medium and high doses of Biejiajian Wan serum， the changes of migration ability of WB-F344 cells were detected by cell scratching test. The expressions of E-cadherin， N-cadherin and Vimentin were detected by Western blot. Real-time PCR was used to detect the changes in the expression of β-catenin mRNA. The expression of β-catenin was detected by cell immunofluorescence assay. Result:Compared with normal WB-F344 cells， the intercellular space of WB-F344 cells became loose from tight， and the morphology changed from cobblestone to fibroblast after TGF-β1 induced WB-F344 cells for 4 days， and the expression of E-cadherin protein decreased， while the expression of N-cadherin protein increased （P<0.01）， indicating that the EMT model of WB-F344 cells was successfully built. Compared with the blank group， the migration ability of WB-F344 cells in TGF-β1 model group was enhanced （P<0.01）， compared with TGF-β1 model group， Biejiajian Wan could significantly inhibit the migration ability of WB-F344 cells； specifically， the low-dose group had no statistical significance， and the medium and high-dose groups had statistical significance （P<0.05）. Western blot results showed that compared with the blank group， the expression of E-cadherin decreased， whereas those of N-cadherin and Vimentin increased in the TGF-β1 model group （P<0.01）， compared with TGF-β1 model group， E-cadherin protein expression was increased in the low， medium and high-dose groups， while the expressions of N-cadherin and Vimentin was decreased； specifically， the low-dose groups had no statistical significance， and the medium and high dose groups had statistical significance （P<0.05，P<0.01）. Real-time PCR results showed that compared with the blank group， the mRNA expression of β-catenin in the TGF-β1 model group was increased （P<0.05）， whereas compared with TGF-β1 model group， the mRNA expression of β-catenin in the low， medium and high-dose groups of Biejiajian Wan was reduced （P<0.01）. The results of cellular immunofluorescence showed that compared with the blank group， the fluorescence expression of β-catenin in the cell nucleus was enhanced in the TGF-β1 model group； and compared with the TGF-β1 model group， the expression of β -catenin in the cell nucleus of the low， medium and high-dose groups of Biejiajian Wan decreased， and the inhibitory effect of Biejiajian Wan on β-catenin in the cell nucleus was positively correlated with its concentration. Conclusion:Biejiajian Wan may reverse the EMT process that TGF-β1 induced WB-F344 cells， and inhibit the migration of WB-F344 cells by inhibiting Wnt/β-catenin signaling pathway.

Intervention Mechanism of Yishen Huayu Prescription on Glomerular Podocyte Injury in Diabetic Nephropathy Rats Based on EMT Regulated by Wnt/β-catenin Pathway / 中国实验方剂学杂志

Objective:To investigate the intervention mechanism of Yishen Huayu prescription on glomerular podocyte injury in diabetic nephropathy （DN） rats based on epithelialmesenchymal transition （EMT） regulated by Wnt/<italic>β</italic>-catenin pathway. Method:The 60 SD rats were divided into control group， model group， Wnt-C59 group （0.03 g·kg^-1 Wnt/<italic>β</italic>-catenin pathway inhibitor）， low-dose group （8 g·kg^-1）， medium-dose group （16 g·kg^-1） and high-dose group （32 g·kg^-1）. After 12 weeks， various indexes ， including general signs， serum creatinine （SCr）， blood urea nitrogen （BUN）， renal index， urinary protein， blood glucose， renal pathological changes， podocyte and expressions of glomerular basement membrane injury and podocyte injury related proteins ［nephrin， synaptopodin］， Wnt/<italic>β</italic>-catenin pathway related proteins （Wnt1， <italic>β</italic>-catenin）， podocyte EMT related protein ［<italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA）， E-cadherin］， were compared between groups. Result:Compared with the control group， the renal tissue in the model group showed significant pathological changes， including diffuse thickening of glomerular mesangial matrix and severe foot process fusion， and a significant increase in SCr， BUN， renal indexes， urinary protein， blood glucose， Wnt1， <italic>β</italic>-catenin， and <italic>α</italic>-SMA expression levels （<italic>P</italic><0.05） as well as a significant decrease in nephrin， synaptopodin and E-cadherin expression levels（<italic>P</italic><0.05）. Compared with model group， SCr， BUN， renal index， urinary protein， blood glucose， Wnt1， <italic>β</italic>-catenin， and <italic>α</italic>-SMA expression levels in each intervention group significantly decreased （<italic>P</italic><0.05）， while the expression levels of nephrin， synaptopodin and E-cadherin significantly increased （<italic>P</italic><0.05）. Among intervention groups， the improvement of above indexes in high-dose Yishen Huayu prescription group was the most obvious （<italic>P</italic><0.05）， which was similar to the effect in Wnt-C59 group. Conclusion:Yishen Huayu prescription prevents podocyte EMT by inhibiting Wnt/<italic>β</italic>-catenin pathway， thereby repairing glomerular podocyte injury in rats with diabetic nephropathy.

Correlation between miR-206 expression and Wnt/β-catenin pathway in peripheral blood mononuclear cells of patients with hyperthyroidism / 中华地方病学杂志

Objective:To explore the correlation between the expression level of microRNA (miR)-206 and the expression levels of Wnt-1 and β-catenin in Wnt/β-catenin pathway in peripheral blood mononuclear cells of patients with hyperthyroidism.Methods:Using a prospective design, 79 patients with hyperthyroidism admitted to Wuwei City People's Hospital from January 2018 to June 2020 were selected as observation group, and 70 cases of healthy physical examination in Wuwei City People's Hospital from January 2019 to September 2020 were selected as control group. The observation group was treated with anti-thyroid drugs, and tambazole was orally taken 10-20 mg/d during the treatment period; after the serum thyroid hormone was normal, the dosage was reduced and maintained at 5-10 mg/d for 12 months. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of miR-206, Wnt-1 and β-catenin mRNA in peripheral blood mononuclear cells in observation group before and after treatment and control group.Results:The expression level of miR-206 in peripheral blood mononuclear cells of observation group before treatment was lower than that of control group (0.28 ± 0.07 vs 0.79 ± 0.15), and the expression levels of Wnt-1 and β-catenin mRNA were higher than those of control group (6.75 ± 1.39 vs 2.23 ± 0.65, 5.83 ± 1.24 vs 3.21 ± 0.86, P < 0.05). The expression level of miR-206 (0.54 ± 0.13) in peripheral blood mononuclear cells of observation group after treatment was higher than that before treatment, and the expression levels of Wnt-1 and β-catenin mRNA (3.62 ± 1.08, 4.34 ± 1.16) were lower than those before treatment ( P < 0.05). The expression level of miR-206 in peripheral blood mononuclear cells of patients with hyperthyroidism was negatively correlated with the expression levels of Wnt-1 and β-catenin mRNA ( r = - 0.763, - 0.798, P < 0.05). Conclusions:The expression level of miR-206 in peripheral blood mononuclear cells of patients with hyperthyroidism is low, while the expression levels of Wnt-1 and β-catenin mRNA are high. The expression level of miR-206 is negatively correlated with the expression levels of Wnt-1 and β-catenin mRNA.

Improvement Effects of Polygonatum sibiricum Polysaccharides on Myocardial Injury of Acute Myocardial Infarction Model Rats / 中国药房

OBJECTIVE：To study the imp rovement effects of Polygonatum si biricum polysaccharides（PSP）on the myocardial injury of acute myocardial infarction （AMI）model rats. METHODS ：The rats were randomly divided into blank control group ， model group ，aspirin group （positive control ，25 mg/kg），PSP low-dose ，medium-dose and high-dose groups （0.5，1，2 g/kg）， with 10 rats in each group. Except for blank control group ，AMI model was established by ligation of the anterior descending branch of the left coronary artery of rats in other groups. After modeling ，blank control group and model group were given normal saline intragastrically ，and administration groups were given relevant medicine intragastrically ，once a day ，for consecutive 28 days. After last medication ，left ventricular ejection fraction （LVEF）and left ventricular short axis shorting rate （LVFS）of rats were detected. The morphological changes of myocardial tissue were observed. The levels of oxidative stress indexes （SOD，MDA， ROS）in myocardial tissue of rats were detected by ELISA. The expression of apoptosis-related proteins （Bcl-2，Bax，caspase-3， caspase-8，caspase-9）and the Wnt/ β-catenin pathway-related proteins （Wnt1，β-catenin）in left ventricular anterior wall tissue of rats were detected by Western blot assay. RESULTS ：Compared with model group ，the levels of LVEF and LVFS ，the levels of SOD in myocardial tissue and protein expression of Bcl- 2 in left ventricular anterior wall tissue were increased significantly in PSP medium-dose，high-dose groups and aspirin group （P＜0.05）；the levels of MDA and ROS in myocardial tissue ，the protein expression of Bax ，caspase-3，caspase-8，caspase-9，Wnt1 and β-catenin in left ventricular anterior wall tissue were decreased significantly（P＜0.05）；myocardial tissue structure disorder and inflammatory cell infiltration were reduced. CONCLUSIONS ： PSP can relieve myocardial injury in AMI model rats ；its mechanism may be related to increasing SOD level in myocardial tissue ，decreasing MDA and ROS level ，regulating apoptosis-related proteins and Wnt/ β-catenin pathway-related proteins.

Mechanism of electroacupuncture for regulation of lipid production and improvement in obesity by mediating Wnt/ β-catenin pathway through activating SIRT1 / 中国针灸

OBJECTIVE@#To explore the mechanism of electroacupuncture (EA) for the regulation of lipid production and improvement in obesity by mediating Wnt/β-catenin pathway through activating silent information regulator 1 (SIRT1).@*METHODS@#Of 75 Wistar male rats, 10 rats were selected randomly as the normal group and fed with standard diet. The rest rats were fed with high-fat diet for 8 weeks to establish the obesity model. Forty rats of successful modeling were randomized into a model group, an EA group, an EA plus inhibitor group (EA+I group) and an agonist group, 10 rats in each one. In the EA group, EA was applied at "Guanyuan" (CV 4), "Zhongwan" (CV 12), "Zusanli" (ST 36) and "Fenglong" (ST 40), with continuous wave, 2 Hz in frequency and around 1 mA in intensity. The needles were retained for 20 min. In the EA+I group, sirtinol solution was injected from caudal vein and EA was exerted simultaneously. In the agonist group, resveratrol solution was given by intragastric administration. The intervention of the above three groups was given once every two days, 3 times a week, consecutively for 8 weeks. Before and after intervention, body mass and Lee's index were recorded in the rats of each group. After intervention, the levels of serum total cholesterol (TC), triglyceride (TG) and free fatty acid (FFA) were detected in the rats of each group. After intervention, the mass of white adipose tissue (WAT) and the area of adipocytes were compared in the rats among the 5 groups. Using Western blot method, the protein expressions of SIRT1, glycogen synthase kinase-3β (GSK3β), β-catenin, cyclin D1 and peroxisome proliferators-activated receptor γ (PPARγ) were detected in WAT in the rats of each group.@*RESULTS@#After intervention, compared with the model group, the body mass and Lee's index were reduced in the rats of the EA group and the agonist group (@*CONCLUSION@#Electroacupuncture remarkably improves the body mass, Lee's index and blood lipid metabolism and reduces WAT mass and adipocyte size in obesity model rats, which is probably related to up-regulating the protein expression of SIRT1 in WAT, activating Wnt/β-catenin pathway and inhibiting the expression of PPARγ of downstream lipogenic gene so as to affect lipid production.

Sex determining region Y-box 9 induced microtubule formation and epithelial⁃mesenchymal transition in human oral squamous cell carcinoma CAL27 cells / 华西口腔医学杂志

OBJECTIVES@#This study aimed to explore the effect of sex determining region Y-box 9 (SOX9) on the microtubule formation and epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (OSCC) CAL27 and the underlying mechanism.@*METHODS@#SOX9-shRNA1 and SOX9-shRNA2 were designed and synthesized and then transfected into CAL27 cells. The expression of SOX9 was detected by quantitative real-time polymerase chain reaction. Microtubule formation assay was used to detect the change in the number of microtubule nodules after interfering with SOX9. Immunofluorescence was used to detect the Vimentin content. Western blot was used to detect the protein expression of EMT marker molecules and Wnt/β-catenin pathway-related proteins, such as E-cadherin, N-cadherin, Fibronectin, Wnt, β-catenin, T-cell factor-4 (TCF-4).@*RESULTS@#The expression level of SOX9 significantly decreased after transfection with SOX9-shRNA1 and SOX9-shRNA2 in CAL27 cells (@*CONCLUSIONS@#Interference with SOX9 decreased Vimentin content and inhibited the microtubule formation and protein expression of EMT marker molecules, as well as the expression of proteins related to the Wnt/β-catenin pathway. Thus, SOX9 can induce microtubule formation and EMT in CAL27, which was related to the inhibition of the Wnt/β-catenin pathway activation.

Effects of sinapine thiocyanate on cutaneous squamous cell carcinoma and its related mechanism / 中国药理学通报

Aim To investigate the effects of sinapine thiocyanate (ST) on the malignant biological behavior of cutaneous squamous cell carcinoma A431 and Colo-16 cells, and its mechanism. Methods The fibroblast cells were treated with 20 μmol · L

Effect of ginkgo biloba extract on behaviors in rat with adolescent sedentariness / 解剖学报

Objective To investigate the effect of Ginkgo biloba extract on behaviors in rat with adolescent sedentariness and its possible mechanism. Methods Twenty-four male SD rats (4-week-old) were randomly divided into normal control (NC), adolescent sedentarines (AS), and Ginkgo biloba extract (GBE) groups. After 4 weeks intervention with GBE, open-field test and elevated plus-maze test were performed to detect the behavioral changes. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in brain were determined by colorimetric method. Protein levels of glycogen synthase kinase 3β(GSK-3β) and β-catenin in cerebral white matter were determined by Western blotting. Results In open-field test, it was shown that the autonomic activity of rats in AS group increased, while the central regional travel time was reduced. Duration and number of residence in the open arm of elevated plus-maze test decreased in AS group. These anxiety-like behaviors were ameliorated by GBE intervention. Compared with the NC group, GSK-3β/ β-catenin ratio and the content of MDA was upregulated in AS group while downregulated in GBE group. And activity of SOD were downregulated in AS group while significantly upregulated in GBE group (P<0. 05). Conclusion Ginkgo biloba extract ameliorate anxiety-like behavior in rat with adolescent sedentariness. Wnt/ β-catenin pathway and antioxidant regulation may play a cerebral protective role.

Social Isolation in Male Rats During Adolescence Inhibits the Wnt/β-Catenin Pathway in the Prefrontal Cortex and Enhances Anxiety and Cocaine-Induced Plasticity in Adulthood / 神经科学通报·英文版

In adult animals, it is well established that stress has a proactive effect on psychostimulant responses. However, whether only a short period of stress during adolescence can also affect cocaine responses later in life and what mechanisms are involved are unknown. Here, we showed that 5 days of social isolation during rat adolescence had a long-term impact on anxiety-like behaviors, cocaine-induced conditioned place preference, and the expression of sensitization during adulthood. At the molecular level, social isolation decreased the activity of the Wnt/β-catenin pathway in the prefrontal cortex (PFC). Furthermore, after the expression of cocaine sensitization, isolated rats showed an increase in this pathway in the nucleus accumbens. Together, these findings suggest that, adolescent social isolation by altering the Wnt/β-catenin pathway in the developing PFC might increase the cocaine responses during adulthood, introducing this pathway as a novel neuroadaptation in the cortical-accumbens connection that may mediate a stress-induced increase in vulnerability to drugs.

Epigenetic deregulations of Wnt/β-catenin and transforming growth factor beta-Smad pathways in esophageal cancer: Outcome of DNA methylation

Background: Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein–protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC. Materials and Methods: Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry. Results: OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/β-catenin and TGF-β-Smad pathways. Conclusions: Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/β-catenin and TGF-β-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/β-catenin and TGF-β signaling pathways