Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

doi: 10.4103/NRR.NRR-D-23-01414

Figure Lengend Snippet: Isolation and characterization of sEVs from hiPSCs and hiPSC-NPCs. (A) Schematic illustration of direct NPC differentiation of hiPSCs. (B, C) Representative morphology and immunostaining of hiPSCs and hiPSC-NPCs. hiPSCs showed a typical clone morphology with SOX2 and TRA-1-60 expression. hiPSC-NPCs showed a typical epithelium morphology with PAX6 and NESTIN expression. Scale bars: 100 μm. (D) Schematic of sEV isolation protocol. (E) Representative TEM images of NPC-sEVs showed a typical cup-like structure. Scale bar: 200 nm. (F) Size distribution of NPC-sEVs. The inset is a snapshot image of video tracking. (G) Western blot results for positive (SDCBP, TSG101, and ALIX) and negative (calnexin) biomarkers of NPC-sEVs and parental cells. ALIX: Apoptosis-linked gene 2-interacting protein X; hiPSC: human induced pluripotent stem cell; NEM: neural expansion medium; NIM: neural induction medium; NPC: neuronal progenitor cell; PAX6: paired box 6; SDCBP: syndecan binding protein; sEV: small extracellular vesicle; SOX2: SRY-box transcription factor 2; TRA-1-60: tumor-related antigen-1-60; TSG101: tumor susceptibility 101.

Article Snippet: Then immediately after the crush, 200 µg (roughly 2 × 10 11 particles) hiPSC- or NPC-sEVs in PBS were intravitreally injected using a 33 g Hamilton syringe (Hamilton Company, Reno, NV, America).

Techniques: Isolation, Immunostaining, Expressing, Western Blot, Binding Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

doi: 10.4103/NRR.NRR-D-23-01414

Figure Lengend Snippet: Neuroprotective effects of sEVs from hiPSCs and hiPSC-NPCs by OCT and HE staining. (A) Schematic of animal experiments. (B) Representative OCT images of mice at 7 and 14 days post-ONC. Thickness of the retina decreased with time. Scale bar: 200 μm. (C) OCT statistical results of mean thickness of the GCC layer ( n = 16). * P < 0.05, ** P < 0.01, hiPSC-sEVs group vs . PBS group; # P < 0.05, ## P < 0.01, NPC-sEVs group vs . PBS group. (D) Representative image of the GCC layer from mice at 7 and 14 days post-ONC. Similar to the results obtained by OCT, thickness of the retina decreased with time. Scale bars: 20 μm. (E) Statistical results of mean thickness of the GCC layer ( n = 12). (F) Quantification of mean number of cells in the GCL ( n = 12). * P < 0.05, *** P < 0.001. Data are expressed as mean ± SEM (C) or mean ± SD (E and F), and were analyzed by Student’s t -test (C) or one-way analysis of variance followed by Tukey’s post hoc test (E and F). GCC: Ganglion cell complex; GCL: ganglion cell layer; HE: hematoxylin-eosin; hiPSC: human induced pluripotent stem cell; IF: immunofluorescence; iPSC: induced pluripotent stem cell; NPC: neural progenitor cell; ns: not significant; OCT: optical coherence tomography; ONC: optic nerve crush; ONH: optic nerve head; PBS: phosphate buffer saline; sEV: small extracellular vesicle; VEP: visual evoked potential.

Article Snippet: Then immediately after the crush, 200 µg (roughly 2 × 10 11 particles) hiPSC- or NPC-sEVs in PBS were intravitreally injected using a 33 g Hamilton syringe (Hamilton Company, Reno, NV, America).

Techniques: Staining, Immunofluorescence, Tomography, Saline

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

doi: 10.4103/NRR.NRR-D-23-01414

Figure Lengend Snippet: RGC survival of ONC mice with or without sEV treatment. (A) Flat-mounted retinas of ONC mice with or without treatment with sEVs on day 7 and day 14 post-ONC. RGC degeneration was progressive after ONC. (B) Schematic diagram of different regions of flat-mounted retinas (central, paracentral, and peripheral). Created with Microsoft PowerPoint Professional 2019. (C) Representative image of central, paracentral, and peripheral retinal regions in ONC model mice with or without treatment with sEVs. Following treatment with hiPSC-sEVs, more RGCs survived in retinas on day 7 post-ONC. However, following treatment with NPC-sEVs, more RGCs survived in retinas on both days 7 and 14. Scale bars: 100 μm. (D) Quantification of mean number of RGCs in different retinal regions at 7 and 14 days ( n = 8/group). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). hiPSC: Human induced pluripotent stem cell; iPSC: induced pluripotent stem cell; NPC: neural progenitor cell; ns: not significant; ONC: optic nerve crush; PBS: phosphate buffer saline; RGC: retinal ganglion cell; sEV: small extracellular vesicle.

Article Snippet: Then immediately after the crush, 200 µg (roughly 2 × 10 11 particles) hiPSC- or NPC-sEVs in PBS were intravitreally injected using a 33 g Hamilton syringe (Hamilton Company, Reno, NV, America).

Techniques: Saline

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

doi: 10.4103/NRR.NRR-D-23-01414

Figure Lengend Snippet: Anti-inflammatory effects of sEVs from hiPSCs and hiPSC-NPCs. (A) Frozen sections of retinas on days 7 and 14. Migrated microglia were reduced in sEV-treated retinas on day 7 but not on day 14 after ONC. Scale bar: 50 μm. (B) Quantification of mean number of IBA1-positive cells per field ( n = 5). (C) Flat-mounted retinas of ONC CX3CR1-GFP mice on day 7. The number of microglia increased in the retina after ONC, but decreased in sEV-treated retinas. (D) Representative images of central, paracentral, and peripheral retinal regions in the ONC model of CX3CR1-GFP mice with or without treatment with sEVs on day 7. The number of microglia and grid-crossed points per microglia were reduced in sEV-treated retinas on day 7. Scale bar: 100 μm. (E) Statistical results of mean number of microglia (CX3CR1-GFP positive cells) per field of different retinal regions ( n = 5). (F) Quantification of mean grid-crossed points per microglia ( n = 5). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). CX3CR1: CX3C chemokine receptor 1; DAPI: 4’,6-diamidino-2-phenylindole; GFP: green fluorescent protein; Iba1: ionized calcium-binding adapter molecule 1; hiPSC: human induced pluripotent stem cell; iPSC: induced pluripotent stem cell; NPC: neural progenitor cell; ns: not significant; ONC: optic nerve crush; PBS: phosphate buffer saline; sEVs: small extracellular vesicles.

Article Snippet: Then immediately after the crush, 200 µg (roughly 2 × 10 11 particles) hiPSC- or NPC-sEVs in PBS were intravitreally injected using a 33 g Hamilton syringe (Hamilton Company, Reno, NV, America).

Techniques: Binding Assay, Saline

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

doi: 10.4103/NRR.NRR-D-23-01414

Figure Lengend Snippet: Protective effect of NPC-sEVs on visual function in ONC mice. (A) Schematic diagram of FVEP. Created with Microsoft PowerPoint Professional 2019. (B) Mean amplitude of N1–P1 waves detected by FVEP on day 7 ( n = 8). Data are expressed as mean ± SD. *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). (C) Overlapping FVEP waves in ONC mice with or without treatment with NPC-sEVs on day 7. FVEP: Flash visual evoked potentials; NPC: neural progenitor cell; ONC: optic nerve crush; PBS: phosphate buffer saline; sEV: small extracellular vesicle.

Article Snippet: Then immediately after the crush, 200 µg (roughly 2 × 10 11 particles) hiPSC- or NPC-sEVs in PBS were intravitreally injected using a 33 g Hamilton syringe (Hamilton Company, Reno, NV, America).

Techniques: Saline

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

doi: 10.4103/NRR.NRR-D-23-01414

Figure Lengend Snippet: miRNA sequencing analysis of sEVs derived from hiPSCs and NPCs. (A) Volcano plot of differentially expressed miRNAs from hiPSC-sEVs and NPC-sEVs. (B, C) KEGG and GO pathway enrichment analyses of differentially expressed miRNAs. (D) Heatmap of differentially expressed miRNAs related to neural pathways. (E) Expression rank of miRNAs in NPC-sEVs. (F) Pie chart showing the percentage of 17 neuroprotective miRNAs in the top 50 miRNAs in term of expression. DEG: Differentially expressed gene; GO: Gene Ontology; hiPSC: human induced pluripotent stem cell; KEGG: Kyoto Encyclopedia of Genes and Genomes; miRNA: microRNA; NPC: neural progenitor cell; sEV: small extracellular vesicle; TPM: tag per million.

Article Snippet: Then immediately after the crush, 200 µg (roughly 2 × 10 11 particles) hiPSC- or NPC-sEVs in PBS were intravitreally injected using a 33 g Hamilton syringe (Hamilton Company, Reno, NV, America).

Techniques: Sequencing, Derivative Assay, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid inhibited NPCs proliferation. (A) Measurement of lactic acid content in NPCs using a lactic acid detection kit (n = 3). (B) The effect of different concentrations or treatment times of lactic acid on the proliferation of NPCs as determined by CCK-8 assay (n = 3). (C-D) The effect of different concentrations of lactic acid on the proliferation of NPCs as determined by EdU staining. Proliferating NPCs appeare red, and nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (E-F) The effect of different concentrations of lactic acid on the cell cycle of NPCs; the box positions represent negative controls (n = 3). (G-H) The effect of different concentrations of lactic acid on DNA double stranded as determined by γ-H2AX; DNA double-stranded-damaged cells appear red, and nuclei were counterstained with DAPI (blue) (scale bar = 50 μm. n = 3). (I-J) The effect of different concentrations of lactic acid on the NPCs senescence as determined by SA-β-gal assay; senescent cells appear blue (scale bar = 200 μm. n = 3). (K-L) Western blotting analysis showing the effect of different lactic acid concentrations on collagen II, MMP9 and MMP13 expression and quantification of the results. La represents lactic acid; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns represents no statistical significance

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques: CCK-8 Assay, Staining, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid inhibited the proliferation of NPCs via the Akt/p21/p27/cyclin D1 pathway. (A-D) Western blotting analysis showing the effect of different lactic acid concentrations on p21, p27, and cyclin D1 expression and quantification of the results (n = 3). (E-H) Western blotting analysis showing the effect of lactic acid and/or SC79 on p21, p27, and cyclin D1 expression and quantification of the results (n = 3). (I-J) The effect of lactic acid and/or SC79 on the proliferation of NPCs as measured by EdU staining. Proliferating NPCs appear red, and nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (K-L) The effect of lactic acid and/or SC79 on the cell cycle of NPCs; the box positions represent negative controls (n = 3). (M-N) The effect of lactic acid and/or SC79 on the senescence of NPCs as determined by SA-β-gal assay. The senescent cells appeared blue (scale bar = 200 μm. n = 3). **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques: Western Blot, Expressing, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: Lactic acid regulates the function of NPCs by binding to specific sites of Akt. (A) Detection of Akt kinase activity in NPCs after treatment with different concentrations of lactic acid (n = 3). (B) Domain structure of Akt. (C-D) Molecular docking diagram of lactic acid and Akt. (E) Western blotting analysis showed Akt expression after knocking down Akt with shRNA and transfecting AktWT, AktK39A, AktL52A and AktK39A,L52A into NPCs respectively. (F) Detection of Akt kinase activity in NPCs of different groups (n = 3). (G-H) The effect of site mutation of Akt on the proliferation of NPCs (red) as determined by EdU staining. Nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (I-J) The effect of lactic acid (10 mM) on the senescence of Akt site-mutated NPCs (blue) as determined by SA-β-gal assay (scale bar = 200 μm. n = 3). (K-L) The effect of lactic acid (10 mM) on ROS production of Akt site-mutated NPCs as determined by a DCFH-DA probe. The FL1 subset was set according to the ROS intensity of the negative control (n = 3). (M-P) Western blotting analysis showing the effect of lactic acid (10 mM) on collagen II, MMP9 and MMP13 expression in Akt site-mutated NPCs and quantification of the results (n = 3). (Q-T) MST analysis of lactic acid in the interaction of NT-647-NHS-labeled Akt. Kd represents the dissociation constant. La represents lactic acid; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns represents no statistical significance

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing, shRNA, Mutagenesis, Staining, Negative Control, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: Schematic diagram of the signaling pathways involved in the promotion of senescence and oxidative stress in NPCs by and corresponding IVDD by lactic acid

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid damage mitochondria and induce oxidative stress in NPCs. (A-B) The effect of different concentrations of lactic acid on ROS production in NPCs. The FL1 subset was set according to the ROS intensity of the negative control (n = 3). (C-D) The effect of different concentrations of lactic acid on the MMP of NPCs as determined by the CMXRos probe (red); nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 50 μm. n = 3). (E) The effect of different concentrations of lactic acid on the MDA content of NPCs (n = 3). (F) The effect of different concentrations of lactic acid on the mitochondrial morphology of NPCs detected by TEM. **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: An MDA assay kit (S0121, Beyotime) was used to measure the MDA content in NPCs.

Techniques: Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid promote oxidative stress in NPCs via the Akt/Nrf2/HO-1 pathway. (A-D) Western blotting analysis showing the effect of different lactic acid concentrations on Nrf2 in the nucleus (n-Nrf2), Nrf2 in the cytoplasm (c-Nrf2), and HO-1 expression and quantification of the results (n = 3). (E-H) Western blotting analysis showing the effect of lactic acid and/or SC79 on n-Nrf2, c-Nrf2, and HO-1 expression and quantification of the results (n = 3). (I-J) The effect of lactic acid and/or SC79 on the Nrf2 nuclear translocation of NPCs as determined by immunofluorescence. Nrf2 in NPCs appeared red, and nuclei were counterstained with DAPI (blue) (n = 3). (K-L) The effect of lactic acid and/or SC79 on the ROS content of NPCs as determined by flow cytometry. The FL1 subset was set according to the ROS intensity of the negative control (n = 3). (M) The effect of lactic acid and/or SC79 on the MDA content of NPCs as determined by flow cytometry (n = 3). (N-O) The effect of lactic acid and/or SC79 on the MMP of NPCs as determined by the CMXRos probe (red); nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 50 μm. n = 3). (P) The effect of lactic acid and/or SC79 on the mitochondrial morphology of NPCs detected by TEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: An MDA assay kit (S0121, Beyotime) was used to measure the MDA content in NPCs.

Techniques: Western Blot, Expressing, Translocation Assay, Immunofluorescence, Flow Cytometry, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: Lactic acid accumulation in degenerative discs. (A) Magnetic resonance imaging of patients with different Pfirrmann grades and the morphology of the nucleus pulposus tissue removed during surgery. (B) HE staining of nucleus pulposus tissues from people with different degrees of degeneration. Red arrows indicate human NPCs (scale bar = 500 μm). (C) Measurement of lactic acid content in human NP tissues with different degrees of degeneration using a lactic acid detection kit (n = 12). (D) Flow chart and timeline of in vivo experiments. (E-F) MRI detection and Pfirrmann grades of rats after different treatments (n = 9). (G-H) HE staining, SO&FG staining, and histological scores of the intervertebral disc in different groups (scale bar = 500 μm; n = 6). (I-J) Western blotting analysis showing collagen II, MMP9 and MMP13 expression in the nucleus pulposus of the normal and PIDD groups and quantification of the results. (K) Measurement of lactic acid content in rat NP tissues of normal rats and rats with PIDD using a lactic acid detection kit (n = 6). (L) Heatmap of differentially abundant metabolites in the NP tissues of normal rats and rats with PIDD. (M) Metabolomics detection of lactic acid content in the NP tissue of normal rats and rats with PIDD (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: Western blotting Rat nucleus pulposus tissues or NPCs were lysed using RIPA lysis buffer (R0010, Solarbio) supplemented with 1% PMSF (P0100, Solarbio) and then centrifuged at 12,000 × g for 5 min at 4 °C.

Techniques: Magnetic Resonance Imaging, Staining, In Vivo, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid inhibited NPCs proliferation. (A) Measurement of lactic acid content in NPCs using a lactic acid detection kit (n = 3). (B) The effect of different concentrations or treatment times of lactic acid on the proliferation of NPCs as determined by CCK-8 assay (n = 3). (C-D) The effect of different concentrations of lactic acid on the proliferation of NPCs as determined by EdU staining. Proliferating NPCs appeare red, and nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (E-F) The effect of different concentrations of lactic acid on the cell cycle of NPCs; the box positions represent negative controls (n = 3). (G-H) The effect of different concentrations of lactic acid on DNA double stranded as determined by γ-H2AX; DNA double-stranded-damaged cells appear red, and nuclei were counterstained with DAPI (blue) (scale bar = 50 μm. n = 3). (I-J) The effect of different concentrations of lactic acid on the NPCs senescence as determined by SA-β-gal assay; senescent cells appear blue (scale bar = 200 μm. n = 3). (K-L) Western blotting analysis showing the effect of different lactic acid concentrations on collagen II, MMP9 and MMP13 expression and quantification of the results. La represents lactic acid; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns represents no statistical significance

Article Snippet: Western blotting Rat nucleus pulposus tissues or NPCs were lysed using RIPA lysis buffer (R0010, Solarbio) supplemented with 1% PMSF (P0100, Solarbio) and then centrifuged at 12,000 × g for 5 min at 4 °C.

Techniques: CCK-8 Assay, Staining, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid inhibited the proliferation of NPCs via the Akt/p21/p27/cyclin D1 pathway. (A-D) Western blotting analysis showing the effect of different lactic acid concentrations on p21, p27, and cyclin D1 expression and quantification of the results (n = 3). (E-H) Western blotting analysis showing the effect of lactic acid and/or SC79 on p21, p27, and cyclin D1 expression and quantification of the results (n = 3). (I-J) The effect of lactic acid and/or SC79 on the proliferation of NPCs as measured by EdU staining. Proliferating NPCs appear red, and nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (K-L) The effect of lactic acid and/or SC79 on the cell cycle of NPCs; the box positions represent negative controls (n = 3). (M-N) The effect of lactic acid and/or SC79 on the senescence of NPCs as determined by SA-β-gal assay. The senescent cells appeared blue (scale bar = 200 μm. n = 3). **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: Western blotting Rat nucleus pulposus tissues or NPCs were lysed using RIPA lysis buffer (R0010, Solarbio) supplemented with 1% PMSF (P0100, Solarbio) and then centrifuged at 12,000 × g for 5 min at 4 °C.

Techniques: Western Blot, Expressing, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid affected the phosphorylation of Akt in NPCs. (A) Volcano map of differentially expressed genes in NPCs after treatment with lactic acid. (B) KEGG pathway analysis of differentially expressed genes in NPCs after treatment with lactic acid. (C-E) Western blotting analysis showing the effect of different lactic acid concentrations on p-Akt (Thr308), and p-Akt (Ser473) expression and quantification of the results (n = 3). (F-G) Immunofluorescence staining of p-Akt (Thr308) and p-Akt (Ser473) in NP tissues in different groups (n = 3). **p < 0.01; ****p < 0.0001

Article Snippet: Western blotting Rat nucleus pulposus tissues or NPCs were lysed using RIPA lysis buffer (R0010, Solarbio) supplemented with 1% PMSF (P0100, Solarbio) and then centrifuged at 12,000 × g for 5 min at 4 °C.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: Lactic acid regulates the function of NPCs by binding to specific sites of Akt. (A) Detection of Akt kinase activity in NPCs after treatment with different concentrations of lactic acid (n = 3). (B) Domain structure of Akt. (C-D) Molecular docking diagram of lactic acid and Akt. (E) Western blotting analysis showed Akt expression after knocking down Akt with shRNA and transfecting AktWT, AktK39A, AktL52A and AktK39A,L52A into NPCs respectively. (F) Detection of Akt kinase activity in NPCs of different groups (n = 3). (G-H) The effect of site mutation of Akt on the proliferation of NPCs (red) as determined by EdU staining. Nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (I-J) The effect of lactic acid (10 mM) on the senescence of Akt site-mutated NPCs (blue) as determined by SA-β-gal assay (scale bar = 200 μm. n = 3). (K-L) The effect of lactic acid (10 mM) on ROS production of Akt site-mutated NPCs as determined by a DCFH-DA probe. The FL1 subset was set according to the ROS intensity of the negative control (n = 3). (M-P) Western blotting analysis showing the effect of lactic acid (10 mM) on collagen II, MMP9 and MMP13 expression in Akt site-mutated NPCs and quantification of the results (n = 3). (Q-T) MST analysis of lactic acid in the interaction of NT-647-NHS-labeled Akt. Kd represents the dissociation constant. La represents lactic acid; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns represents no statistical significance

Article Snippet: Western blotting Rat nucleus pulposus tissues or NPCs were lysed using RIPA lysis buffer (R0010, Solarbio) supplemented with 1% PMSF (P0100, Solarbio) and then centrifuged at 12,000 × g for 5 min at 4 °C.

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing, shRNA, Mutagenesis, Staining, Negative Control, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid promote oxidative stress in NPCs via the Akt/Nrf2/HO-1 pathway. (A-D) Western blotting analysis showing the effect of different lactic acid concentrations on Nrf2 in the nucleus (n-Nrf2), Nrf2 in the cytoplasm (c-Nrf2), and HO-1 expression and quantification of the results (n = 3). (E-H) Western blotting analysis showing the effect of lactic acid and/or SC79 on n-Nrf2, c-Nrf2, and HO-1 expression and quantification of the results (n = 3). (I-J) The effect of lactic acid and/or SC79 on the Nrf2 nuclear translocation of NPCs as determined by immunofluorescence. Nrf2 in NPCs appeared red, and nuclei were counterstained with DAPI (blue) (n = 3). (K-L) The effect of lactic acid and/or SC79 on the ROS content of NPCs as determined by flow cytometry. The FL1 subset was set according to the ROS intensity of the negative control (n = 3). (M) The effect of lactic acid and/or SC79 on the MDA content of NPCs as determined by flow cytometry (n = 3). (N-O) The effect of lactic acid and/or SC79 on the MMP of NPCs as determined by the CMXRos probe (red); nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 50 μm. n = 3). (P) The effect of lactic acid and/or SC79 on the mitochondrial morphology of NPCs detected by TEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: Western blotting Rat nucleus pulposus tissues or NPCs were lysed using RIPA lysis buffer (R0010, Solarbio) supplemented with 1% PMSF (P0100, Solarbio) and then centrifuged at 12,000 × g for 5 min at 4 °C.

Techniques: Western Blot, Expressing, Translocation Assay, Immunofluorescence, Flow Cytometry, Negative Control