Journal: Biology Open

Article Title: Ectopic otoconial formation in the lagena of the pigeon inner ear

doi: 10.1242/bio.034462

Figure Lengend Snippet: Histological sections reveal that the otoconia are enclosed by an acellular membrane and are not innervated. (A) Light micrograph of the pigeon lagena shown in Fig. 1I, with the plane of sectioning indicated by the dashed line. (B) Paraffin section (10 µm) showing the fine crystal structure and membranous margins of the ectopic otoconia. (C) Immunohistological staining with the postmitotic neuronal marker (TuJ1) ( n =3 birds). Staining at the base of lagenar hair cells (HC, black dashed box) was observed, but no signal was observed in the vicinity of the ectopic otoconial mass (OM, white dashed box). (D) Cryo-section (12 µm) showing the ectopic otoconia, magnified in inset. (E) Immunohistological staining with the hair cell marker otoferlin ( n =3 birds). The lagenar hair cells (lower right) were stained. (F) Overlay of the unstained and stained section (D,E) illustrating an absence of positive staining in the vicinity of the ectopic otoconia. Scale bars: 100 µm.

Article Snippet: After a heat-mediated antigen retrieval (H-3301, Vector Laboratories) the sections were washed three times in PBS and primary antibodies against otoferlin (Sc-50159, C-15, Lot #A2930; Santa Cruz, 1:1000) were incubated in 2% milk, 0.1% Triton in PBS overnight at room temperature ( ).

Techniques: Membrane, Paraffin Section, Staining, Marker

Journal: Journal of Inflammation Research

Article Title: P2X7R-NEK7-NLRP3 Inflammasome Activation: A Novel Therapeutic Pathway of Qishen Granule in the Treatment of Acute Myocardial Ischemia

doi: 10.2147/jir.s373962

Figure Lengend Snippet: Figure 1 QSG improved cardiac function in mice after AMI. (A) Experimental protocol for QSG studies in AMI mice. Experimental protocol for QSG studies in AMI mice, LAD ligation was used to made AMI model in mice after 3 d of adaptive feeding, QSG were administered by oral gavage, and NLRP3 inhibitor (MCC950) by intraperitoneal injection from 4 d - 7 d, subsequent cardiac echocardiography and acquisition of relevant parameters. (B) Representative echocardiographic images of mice in each group and analysis of LVEF, LVFS, LVAW; d, LVAW; s, LVPW; d, LVPW; s, LVID; d, LVID; s levels after QSG treatment. (C) ELISA of plasma CKMB and cTnI levels. N = 6 per group. ###P < 0.001 vs sham group, *P < 0.05, **P < 0.01, ***P < 0.001 vs model group.

Article Snippet: After blocking with 0.3% hydrogen peroxide for 15 min, the sections were incubated with primary antibodies anti-IL-1β antibody (AF5103, Affinity, USA), anti-IL-18 antibody (10663-1-AP, Proteintech, China) overnight at 4°C.

Techniques: Ligation, Injection, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Journal of Inflammation Research

Article Title: P2X7R-NEK7-NLRP3 Inflammasome Activation: A Novel Therapeutic Pathway of Qishen Granule in the Treatment of Acute Myocardial Ischemia

doi: 10.2147/jir.s373962

Figure Lengend Snippet: Figure 2 QSG attenuated myocardial tissue inflammatory injury in AMI mice. (A) Representative heart images for each group of HE staining, Scale bar=20 µm. N = 3 per group. (B and C) IHC of IL-18 and IL-1β images of AMI mice in each group and analysis, Scale bar=20 µm. N = 3 per group. (D) ELISA results of IL-1β and IL-18 in cardiac tissue homogenates. N = 6 per group. ###P < 0.001 vs sham group, ***P < 0.001 vs model group.

Article Snippet: After blocking with 0.3% hydrogen peroxide for 15 min, the sections were incubated with primary antibodies anti-IL-1β antibody (AF5103, Affinity, USA), anti-IL-18 antibody (10663-1-AP, Proteintech, China) overnight at 4°C.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: P2X7R-NEK7-NLRP3 Inflammasome Activation: A Novel Therapeutic Pathway of Qishen Granule in the Treatment of Acute Myocardial Ischemia

doi: 10.2147/jir.s373962

Figure Lengend Snippet: Figure 3 QSG inhibited NLRP3 inflamat some activation in cardiac tissue macrophages. (A) Representative NLRP3/ASC/Caspase-1 immunofluorescent staining images of AMI mice in each group. Scale bar=20 µm. (B) Representative CD86/Caspase-1 immunofluorescent staining images of AMI mice in each group. Scale bar=20 µm. (C–I) Western blot analysis showed that QSG treatment reduced the expression of P2X7R, NEK7, NLRP3, ASC, Caspase-1, IL-18, IL-1β in cardiac tissue macrophages. Proteins had been normalized to GAPDH, N = 6 per group. ###P < 0.001 vs sham group, ***P < 0.001 vs model group.

Article Snippet: After blocking with 0.3% hydrogen peroxide for 15 min, the sections were incubated with primary antibodies anti-IL-1β antibody (AF5103, Affinity, USA), anti-IL-18 antibody (10663-1-AP, Proteintech, China) overnight at 4°C.

Techniques: Activation Assay, Staining, Western Blot, Expressing

Journal: Journal of Inflammation Research

Article Title: P2X7R-NEK7-NLRP3 Inflammasome Activation: A Novel Therapeutic Pathway of Qishen Granule in the Treatment of Acute Myocardial Ischemia

doi: 10.2147/jir.s373962

Figure Lengend Snippet: Figure 4 QSG inhibited LPS combined with ATP-induced NLRP3 inflammasome activation in RAW264.7 macrophages. (A) Establishment of LPS combined with ATP to induce NLRP3 inflammasome activation model. (B) Immunofluorescence determined that LPS in combination with ATP activates NLRP3 inflammasome. Scale bar=75 µm. (C) Safe concentration of QSG in RAW264.7 macrophages. (D) Experimental protocol for QSG in LPS combined with ATP to induce NLRP3 inflammasome activation in RAW264.7 macrophages. (E) Effective concentration of QSG in RAW264.7 macrophages. (F) MCC950 inhibitor concentration range in RAW264.7 macrophages. (G–M) Western blot analysis showed that QSG treatment reduced the expression of P2X7R, NEK7, NLRP3, ASC, Caspase-1, IL-18, IL-1β in RAW264.7 macrophages. N = 3 per group. ##P < 0.01, ###P < 0.001 vs sham group, *P < 0.05, **P < 0.01, ***P < 0.001 vs model group.

Article Snippet: After blocking with 0.3% hydrogen peroxide for 15 min, the sections were incubated with primary antibodies anti-IL-1β antibody (AF5103, Affinity, USA), anti-IL-18 antibody (10663-1-AP, Proteintech, China) overnight at 4°C.

Techniques: Activation Assay, Immunofluorescence, Concentration Assay, Western Blot, Expressing

Journal: Journal of Inflammation Research

Article Title: P2X7R-NEK7-NLRP3 Inflammasome Activation: A Novel Therapeutic Pathway of Qishen Granule in the Treatment of Acute Myocardial Ischemia

doi: 10.2147/jir.s373962

Figure Lengend Snippet: Figure 5 QSG inhibited the P2X7R-NEK7-NLRP3 inflammasome pathway. (A) Determination of potassium ion concentration in RAW264.7 macrophages supernatant. (B) The effective concentration range of CCK8 to detect AZ. (C) Representative graph of optimal concentration of AZ for WB validation. (D) Representative immuno fluorescence detection images of RAW264.7 macrophages P2X7R expression. Scale bar=25 µm. (E–H) Western blot analysis showed that both QSG and AZ treatment reduced the expression of P2X7R, Caspase-1, IL-18, IL-1β in RAW264.7 macrophages, N = 3 per group. ###P < 0.001 vs sham group, *P < 0.05, **P < 0.01, ***P < 0.001 vs model group.

Article Snippet: After blocking with 0.3% hydrogen peroxide for 15 min, the sections were incubated with primary antibodies anti-IL-1β antibody (AF5103, Affinity, USA), anti-IL-18 antibody (10663-1-AP, Proteintech, China) overnight at 4°C.

Techniques: Concentration Assay, Biomarker Discovery, Fluorescence, Expressing, Western Blot

Journal: Journal of Inflammation Research

Article Title: P2X7R-NEK7-NLRP3 Inflammasome Activation: A Novel Therapeutic Pathway of Qishen Granule in the Treatment of Acute Myocardial Ischemia

doi: 10.2147/jir.s373962

Figure Lengend Snippet: Figure 6 QSG regulates the P2X7R-NEK7-NLRP3 pathway. (A) mP2X7R pcDNA3.1–3xFlag-T2A-EGFP map. (B) WB images of images of RAW264.7 macrophages and analysis of P2X7R. (C) Representative immunofluorescence detection images of Caspase-1. Scale bar=75 µm. (D and E) Western blot images of RAW264.7 macrophages and analysis of IL-18 and IL-1β. (F–H) RAW264.7 macrophages were transfected with P2X7R pcDNA3.1 followed by immunoprecipitation and immunoblot using NEK7 antibody to analyze the protein expression levels of NEK7, NLRP3. The Input represents total protein extracts used for immunoprecipitation. GAPDH was used as a loading control. IB, immunoblot. IP, immunoprecipitation. IgG, negative control. ###P < 0.001 vs pcDNA3.1 group, **P < 0.01, ***P < 0.001 vs P2X7R pcDNA3.1 group.

Article Snippet: After blocking with 0.3% hydrogen peroxide for 15 min, the sections were incubated with primary antibodies anti-IL-1β antibody (AF5103, Affinity, USA), anti-IL-18 antibody (10663-1-AP, Proteintech, China) overnight at 4°C.

Techniques: Immunofluorescence, Western Blot, Transfection, Immunoprecipitation, Expressing, Control, Negative Control

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 3 Immunohistochemical analysis of the distinct presence of Cx43 and BMP-15 in the oophorons of each group (× 200) A-F: Cx43; G-L: BMP-15. A, G: control group; B, H: model group; C, I: positive group; D, J: low dose of BSJPP group; E, K: moderate dose of BSJPP group; F, L: high dose of BSJPP group. Positive group treated with premarin (0.03 mg/kg) once daily for 90 d. Con- trol and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Immunohistochemi- cal analysis clearly revealed the presence of Cx43 and BMP-15 in the oophorons of control mice and those treated with BSJPP (D, E) and premarin compared with the model group. BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Immunohistochemical staining, Control, In Vivo

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 4 Expression Cx43 and BMP-15 mRNA in the ovaries of mice in each group. 1: control group; 2: model group; 3: positive group; 4: low dose of BSJPP group; 5: moderate dose of BSJPP group; 6: high dose of BSJPP group. Positive group treated with pre- marin (0.03 mg/kg) once daily for 90 d. Control and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Compared with the model group, significantly upregulated expression of both Cx43 and BMP-15 was detected in the control group and the groups treated with BSJPP (M and L groups). BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Expressing, Control, In Vivo