Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Experimental scheme for this study. After gene identification at 1 month of age, 3-month-old male WT and 3×Tg-AD mice were randomly assigned to four groups with 10 animals each and then intragastrically administered either ICA or vehicle for 5 months (WT + vehicle, WT + ICA, 3×Tg-AD + vehicle, 3×Tg-AD + ICA groups). After performing behavior tests, the mice were euthanized. The cerebral cortexes were evaluated using HE and Nissl staining, immunofluorescent staining, and western blot assays to determine the above disease indicators. 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; Aβ: beta-amyloid protein; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; HE: hematoxylin and eosin; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; NeuN: neuronal nuclear antigen; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PSD95: postsynaptic density protein 95; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Staining, Western Blot, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Effects of ICA on impaired insulin signaling in the cerebral cortex of 3×Tg-AD mice. (A) Insulin signaling: IR tyrosine autophosphorylation is stimulated by insulin and triggers IRS1 phosphorylation at tyrosine residues, which represents a positive regulatory mechanism that activates the PI3K/AKT pathway and results in the inhibition of GSK3β. However, serine phosphorylation of IRS1 at specific sites is a negative regulatory mechanism. (B) Representative expression patterns of molecules related to the insulin signaling pathway. (C) Quantification of proteins related to the insulin signaling pathway shown in (B). Protein levels were normalized to those in the WT + vehicle group. The data are presented as the means ± SEM ( n = 4–6). * P < 0.05, vs . WT + vehicle group; # P < 0.05, vs . 3×Tg-AD + vehicle group (one-way analysis of variance followed by the least significant difference test). 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; AKT: protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase 3 beta; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Inhibition, Expressing, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Schematic diagram of the mechanism by which ICA regulates GLUTs and brain insulin signaling to ameliorate memory impairment in AD. Aβ: Amyloid-beta protein; AD: Alzheimer’s disease; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; G-tau: the attachment of O-linked N-acetylglucosamine (O-GlcNAc) on tau; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Stimulation of hair growth by Tianma Gouteng decoction: Identifying mechanisms based on chemical analysis, systems biology approach, and experimental evaluation

doi: 10.3389/fphar.2022.1073392

Figure Lengend Snippet: Molecular docking of core components and Wnt/β-catenin-related targets. Quercetin, luteolin, fisetin, wogonin, oroxylin A, boldine, tetrahydroalstonine, and galangin with (A) β-catenin, and (B) GSK3β. (C) Cluster heatmap of docking results. Numerical value indicated the binding energy ((kcal/mol).

Article Snippet: Antibodies against β-catenin (8480S) and glycogen synthase kinase-3 beta (GSK3β; 12,456) were sourced from Cell Signaling Technology (Boston, United States).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Stimulation of hair growth by Tianma Gouteng decoction: Identifying mechanisms based on chemical analysis, systems biology approach, and experimental evaluation

doi: 10.3389/fphar.2022.1073392

Figure Lengend Snippet: TGD promoted hair shaft elongation in rat vibrissa follicles. (A) Photographs of rat vibrissa follicles cultured with TGD for 0, 3, 6, 9, 12 days; The double arrow area represents the measured hair shaft length. (B) Changes in length of hair shafts in the vibrissa follicles treated with TGD for 12 days. Data are presented as mean ± SEM ( n = 6). ** p < 0.01 compared with the control group. (C–E) The representative images of immunofluorescence staining in rat vibrissa follicles for β-catenin, GSK3β, LEF1 (×40), picture of magnification ×200 in white box.

Article Snippet: Antibodies against β-catenin (8480S) and glycogen synthase kinase-3 beta (GSK3β; 12,456) were sourced from Cell Signaling Technology (Boston, United States).

Techniques: Cell Culture, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Stimulation of hair growth by Tianma Gouteng decoction: Identifying mechanisms based on chemical analysis, systems biology approach, and experimental evaluation

doi: 10.3389/fphar.2022.1073392

Figure Lengend Snippet: (A–E) The representative images and statistical graph of β-catenin, GSK3β, p-GSK3β and LEF1 in DPCs (×200), n = 3 per group, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the control group. (F–H) Western blot analysis of Wnt/β-catenin signaling pathways related protein expression in DPCs, n = 4 per group, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the control group.

Article Snippet: Antibodies against β-catenin (8480S) and glycogen synthase kinase-3 beta (GSK3β; 12,456) were sourced from Cell Signaling Technology (Boston, United States).

Techniques: Western Blot, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effects of Combined Treatment with Acupuncture and Chunggan Formula in a Mouse Model of Parkinson's Disease

doi: 10.1155/2019/3612587

Figure Lengend Snippet: Protective effects of the combined treatment in Parkinson's disease mouse model. The SNpc was isolated and immunoblotted with the indicated antibodies. The intensities of the protein bands were quantitated by densitometry, and the phosphorylated forms were normalized to the total form or β -actin. Changes in (a) pI κ B α , (b) pAKT, (c) pGSK3 β , (d) pERK, (e) pCREB, and (f) BDNF. Data are mean ± standard error. ∗∗∗ p < 0.001 by one-way ANOVA followed by the Newman–Keuls post hoc test ( n = 4 for each group).

Article Snippet: The membranes were washed in tris-buffered saline with 0.1% Tween 20 (TBS-T) three times for 10 min and then blocked in 5% skim milk for 1 h. After blocking, the membranes were activated with antibodies to brain-derived neurotrophic factor (BDNF, 1 : 200, Santa Cruz Biotechnology; sc-546), TH (1 : 3000, Santa Cruz Biotechnology, sc-14007), β -actin (1 : 40,000, Sigma-Aldrich, St. Louis, MO, USA; A1978), phospho-inhibitory kappa B alpha (pI κ B α Ser32, 1 : 500, Cell Signaling Technology, Beverly, MA, USA; #2859), I κ B α (1 : 500, Cell Signaling Technology, #4814), phospho-extracellular signal-regulated kinase (pERK Thr202/Tyr204, 1 : 500, Cell Signaling Technology; #4370), ERK (1 : 500, Cell Signaling Technology, #9102), phospho-protein kinase B (pAKT Ser473, 1 : 1000, Cell Signaling Technology, #4058), AKT (1 : 1000, Cell Signaling Technology, #4691), phospho-cAMP response element-binding protein (pCREB Ser133, 1 : 200, Cell Signaling Technology, #9198), CREB (1 : 500, Cell Signaling Technology, #9197), phospho-glycogen synthase kinase 3 beta (pGSK3 β Ser9, 1 : 250, Cell Signaling Technology, #9336), and GSK3 β (1 : 200, Cell Signaling Technology, #9315) overnight at 4°C.

Techniques: Isolation

Journal: PLoS ONE

Article Title: Microtubule Destabilization Is Shared by Genetic and Idiopathic Parkinson’s Disease Patient Fibroblasts

doi: 10.1371/journal.pone.0037467

Figure Lengend Snippet: (A) Immunoblot and densitometric analyses of (B) total and (C) phosphorylated glycogen synthase kinase 3 beta (GSK3β), p38 MAP Kinase (p38) and p44/42 MAPK (Erk) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 3), mutated parkin (PARK, dark grey bars, N = 3), mutated LRRK2 (LRRK2, light grey bars, N = 3) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of total protein were normalized on the level of GAPDH of the relative sample, whereas the levels of phosphorylated form were normalized on the values of total protein. All values are expressed as mean ± SEM. * p <0.05 and ** p <0.02 vs control, # p <0.05 vs PD according to ANOVA, Tukey HSD post hoc test.

Article Snippet: Membranes were probed with the following antibodies: α-tubulin mouse IgG (clone B-5-1-2, Sigma-Aldrich, St Louis, MO); β-tubulin mouse IgG (clone Tub 2.1, Sigma-Aldrich); deTyr tubulin rabbit IgG (Chemicon, Temecula, CA); Tyr tubulin mouse IgG (clone TUB-1A2, Sigma-Aldrich); Ac tubulin mouse IgG (clone 6-11B-1, Sigma-Aldrich); microtubule-associated protein 1 light chain 3 rabbit IgG (Sigma-Aldrich); vimentin mouse IgG (clone V6, Sigma-Aldrich); actin mouse IgM (N350, Amersham, Little Chalfont, UK); Caspase 3 rabbit IgG (Enzo Life Sciences Ag., Lausen, Switzerland), GADPH mouse IgG (Biogenesis, Poole, UK); Heat Shock Protein 70 mouse IgG (clone 3A3, Chemicon); Glycogen synthase kinase 3 beta rabbit IgG (Abcam, Cambride, UK); Phospho-Glycogen synthase kinase 3 beta (Ser9) rabbit IgG (Cell Signaling Technology, Beverly, MA); p38 alpha MAP Kinase mouse IgG (clone L53F8, Cell Signaling Technology); Phospho-p38 MAP Kinase (Thr180/Tyr182) rabbit IgG (clone 3D7, Cell Signaling Technology); p44/42 MAPK (Erk1/2) rabbit IgG (clone 137F5, Cell Signaling Technology); Phospho-p44/42 MAPK (Thr202/Tyr204) rabbit IgG (clone D13.14.4E, Cell Signaling Technology); parkin mouse IgG (clone prk8, Sigma-Aldrich).

Techniques: Western Blot, Quantitation Assay