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miRNA375-3p/rapamycin mediates the mTOR pathway by decreasing PS1, enhances microglial cell activity to regulate autophagy in Alzheimer's disease

Wang, Yuxiang, Xiao, Zixuan, Yin, Hanlan, Ren, Zhichao, Ma, Xueting, Wang, Yibo, Zhang, Yan, Fu, Xueqi, Zhang, Fuqiang, Zeng, Linlin·2024·Heliyon
DOI 10.1016/j.heliyon.2024.e37589PMC PMC11461998
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Fig. 1A The mRNA-miRNA interactions were predicted using 63 gene sets related to Alzheimer's disease (AD), autophagy, and microglial cell activation.
Fig. 1A The mRNA-miRNA interactions were predicted using 63 gene sets related to Alzheimer's disease (AD), autophagy, and microglial cell activation.CC BY · PubMed Central
Fig. 1B Venn diagram illustrating the independent and overlapping miRNAs between the predicted proteins in (A) and data sets published by Denk J and others.
Fig. 1B Venn diagram illustrating the independent and overlapping miRNAs between the predicted proteins in (A) and data sets published by Denk J and others.CC BY · PubMed Central
Fig. 1C and D Bubble plots depicting the enrichment of miR-375-3p target proteins using KEGG (C) and GO biological processes. (D) The predicted results were sorted by the number of proteins in respective pathways.
Fig. 1C and D Bubble plots depicting the enrichment of miR-375-3p target proteins using KEGG (C) and GO biological processes. (D) The predicted results were sorted by the number of proteins in respective pathways.CC BY · PubMed Central
Fig. 1E Prediction of miR-375-3p interaction sites with its target proteins.
Fig. 1E Prediction of miR-375-3p interaction sites with its target proteins.CC BY · PubMed Central
Fig. 1F_I Western blot analysis of miR375-3p interacting proteins (results of three replicates). (F) Overexpression of NC or miR375-3p in SH-SY5Y cells and assessment of PS1, Syn, ATG14, and pro-BDNF protein expression levels. (G) Quantification of cell grayscale values using GAPDH as an internal reference via ImageJ. (H) The protein expression levels of PS1, Syn, ATG14, and pro-BDNF in the frontal and temporal lobes of wild-type mice injected with single-stranded NC-agomir or miR375-3p-mimic-agomir were assessed using ImageJ with GAPDH as an internal reference. (I) GAPDH was used as a normalized internal parameter (Fold vec) to analyze the gray values of each group. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 1F_I Western blot analysis of miR375-3p interacting proteins (results of three replicates). (F) Overexpression of NC or miR375-3p in SH-SY5Y cells and assessment of PS1, Syn, ATG14, and pro-BDNF protein expression levels. (G) Quantification of cell grayscale values using GAPDH as an internal reference via ImageJ. (H) The protein expression levels of PS1, Syn, ATG14, and pro-BDNF in the frontal and temporal lobes of wild-type mice injected with single-stranded NC-agomir or miR375-3p-mimic-agomir were assessed using ImageJ with GAPDH as an internal reference. (I) GAPDH was used as a normalized internal parameter (Fold vec) to analyze the gray values of each group. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Fig. 2A and B (A) Western blot analysis of PS1, ATG14, Beclin1, LC3B, MFN2, and Grp75 protein expression levels in SH-SY5Y cells exposed to various Aβ25-35 conditions at different time points. (B) The grayscale values for each group were quantified using ImageJ analysis. GAPDH was set as the internal reference.
Fig. 2A and B (A) Western blot analysis of PS1, ATG14, Beclin1, LC3B, MFN2, and Grp75 protein expression levels in SH-SY5Y cells exposed to various Aβ25-35 conditions at different time points. (B) The grayscale values for each group were quantified using ImageJ analysis. GAPDH was set as the internal reference.CC BY · PubMed Central
Fig. 2C and D Western blot analysis of protein expression in Aβ25-35-induced SH-SY5Y cells after miR375-3p overexpression. (C) Western blot analysis was performed to detect the expression of AD and autophagy marker proteins in each group after SH-SY5Y cells were transfected with single-stranded NC or miR375-3p-mimic and treated with 20 μM Aβ25-35 for 24 h. (D) β-actin was used as an internal reference. For grayscale value quantification and analysis, ImageJ was employed.
Fig. 2C and D Western blot analysis of protein expression in Aβ25-35-induced SH-SY5Y cells after miR375-3p overexpression. (C) Western blot analysis was performed to detect the expression of AD and autophagy marker proteins in each group after SH-SY5Y cells were transfected with single-stranded NC or miR375-3p-mimic and treated with 20 μM Aβ25-35 for 24 h. (D) β-actin was used as an internal reference. For grayscale value quantification and analysis, ImageJ was employed.CC BY · PubMed Central
Fig. 2E and F Laser confocal microscopy images of TUNEL staining in cells (400 × magnification) with a scale bar of 50 μm (E). Cell fluorescence intensity was quantified using Image-Pro Plus 6.0 to calculate the apoptosis rate (F).
Fig. 2E and F Laser confocal microscopy images of TUNEL staining in cells (400 × magnification) with a scale bar of 50 μm (E). Cell fluorescence intensity was quantified using Image-Pro Plus 6.0 to calculate the apoptosis rate (F).CC BY · PubMed Central
Fig. 2G and H Cellular ROS immunofluorescence staining results (400 × magnification); Scale bar = 20 μm (G). Fluorescence intensity was quantified to determine ROS levels (H). ROS: reactive oxygen species.
Fig. 2G and H Cellular ROS immunofluorescence staining results (400 × magnification); Scale bar = 20 μm (G). Fluorescence intensity was quantified to determine ROS levels (H). ROS: reactive oxygen species.CC BY · PubMed Central
Fig. 2I and J JC-1 probe staining of cellular monomers (Alexa Fluor 488 nm) and polymers (Alexa Fluor 633) (200 × magnification): scale bar of 100 μm (I). Fluorescence intensity was quantified to assess the JC-1 polymer/monomer ratio and the results are presented relative to the SH + NC group (J).
Fig. 2I and J JC-1 probe staining of cellular monomers (Alexa Fluor 488 nm) and polymers (Alexa Fluor 633) (200 × magnification): scale bar of 100 μm (I). Fluorescence intensity was quantified to assess the JC-1 polymer/monomer ratio and the results are presented relative to the SH + NC group (J).CC BY · PubMed Central
Fig. 2K–N Fluorescence staining results for Aβ, Beclin1, and LC3B. (K) The fluorescence intensity of cells stained with anti-Aβ and anti-Beclin1 was detected at 633 nm, and that of anti-LC3B stained cells was detected at 488 nm; Scale bar = 50 μm. The fluorescence intensities of Aβ (L), Beclin1 (M), and LC3B (N) were quantified and compared across groups. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 2K–N Fluorescence staining results for Aβ, Beclin1, and LC3B. (K) The fluorescence intensity of cells stained with anti-Aβ and anti-Beclin1 was detected at 633 nm, and that of anti-LC3B stained cells was detected at 488 nm; Scale bar = 50 μm. The fluorescence intensities of Aβ (L), Beclin1 (M), and LC3B (N) were quantified and compared across groups. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Fig. 3 A–C Western blot analysis of cellular Beclin1 and LC3B protein expression. (A) Western blot analysis of Beclin1 and LC3B levels was performed using adhered SH-SY5Y cells treated with Rapamycin and Z-VAD for 1 h, followed by Aβ25-35 induction for 24 h. (B and C) Grayscale values for Beclin1 (B) and LC3B (C) in each group were quantified using ImageJ GAPDH was used as the internal reference.
Fig. 3 A–C Western blot analysis of cellular Beclin1 and LC3B protein expression. (A) Western blot analysis of Beclin1 and LC3B levels was performed using adhered SH-SY5Y cells treated with Rapamycin and Z-VAD for 1 h, followed by Aβ25-35 induction for 24 h. (B and C) Grayscale values for Beclin1 (B) and LC3B (C) in each group were quantified using ImageJ GAPDH was used as the internal reference.CC BY · PubMed Central
Fig. 3D–F Western blot analysis of Beclin1 and LC3B protein expression in miR-375-3p overexpressing SH-SY5Y cells. (D) Beclin1 and LC3B expression in SH-SY5Y cells was assessed after cells were transfected with either single-strand NC or miR-375-3p in the presence of Rapamycin and Z-VAD for 1 h, followed by Aβ25-35 induction for 24 h. (E and F) Grayscale values for Beclin1 (E) and LC3B (F) in each group were quantified using ImageJ. GAPDH was used as the internal reference.
Fig. 3D–F Western blot analysis of Beclin1 and LC3B protein expression in miR-375-3p overexpressing SH-SY5Y cells. (D) Beclin1 and LC3B expression in SH-SY5Y cells was assessed after cells were transfected with either single-strand NC or miR-375-3p in the presence of Rapamycin and Z-VAD for 1 h, followed by Aβ25-35 induction for 24 h. (E and F) Grayscale values for Beclin1 (E) and LC3B (F) in each group were quantified using ImageJ. GAPDH was used as the internal reference.CC BY · PubMed Central
Fig. 3G and H Tunel staining analysis of SH-SY5Y cell's apoptosis levels. Cells were first treated with Rapamycin for 1 h, followed by overexpression of strand NC or miR-375-3p for 24 h, and then the addition of Aβ25-35 for 24 h. (G) A laser confocal microscope with an Alexa Fluor 488 channel was used to assess the apoptosis rate of each group's cells, which were fixed and permeabilized before Tunel probe treatment for 1 h and then stained with DAPI for 5 min. Scale bar = 50 μm. (H) The fluorescence intensity in each group was quantified using Image-Pro Plus 6.0 to calculate the apoptosis rate.
Fig. 3G and H Tunel staining analysis of SH-SY5Y cell's apoptosis levels. Cells were first treated with Rapamycin for 1 h, followed by overexpression of strand NC or miR-375-3p for 24 h, and then the addition of Aβ25-35 for 24 h. (G) A laser confocal microscope with an Alexa Fluor 488 channel was used to assess the apoptosis rate of each group's cells, which were fixed and permeabilized before Tunel probe treatment for 1 h and then stained with DAPI for 5 min. Scale bar = 50 μm. (H) The fluorescence intensity in each group was quantified using Image-Pro Plus 6.0 to calculate the apoptosis rate.CC BY · PubMed Central
Fig. 3I and J (I) ROS probe to detect intracellular ROS levels. Briefly, cells from each group were treated with the ROS probe for 30 min, washed, and then stained with DAPI for 5 min. The excitation light intensity was measured at 488 nm; Scale bar = 50 μm. (J) The fluorescence intensity in each group was quantified to determine the levels of reactive oxygen species. ROS: Reactive oxygen species.
Fig. 3I and J (I) ROS probe to detect intracellular ROS levels. Briefly, cells from each group were treated with the ROS probe for 30 min, washed, and then stained with DAPI for 5 min. The excitation light intensity was measured at 488 nm; Scale bar = 50 μm. (J) The fluorescence intensity in each group was quantified to determine the levels of reactive oxygen species. ROS: Reactive oxygen species.CC BY · PubMed Central
Fig. 3K and L Western blot analysis of protein expression in Aβ25-35-induced SH-SY5Y cell after miR375-3p overexpression and rapamycin treatment. (K) Protein expression levels of LC3B, PS1, pro-BDNF, MFN2, Grp75, and Beclin1 in each group of cells. (L) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was used as an internal reference.All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 3K and L Western blot analysis of protein expression in Aβ25-35-induced SH-SY5Y cell after miR375-3p overexpression and rapamycin treatment. (K) Protein expression levels of LC3B, PS1, pro-BDNF, MFN2, Grp75, and Beclin1 in each group of cells. (L) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was used as an internal reference.All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Fig. 4A Design of the injection schedule and behavioral testing procedure. The APP/PS1 transgenic mice (28 weeks old) and wild-type (26 weeks old male) mice were used for this assay.
Fig. 4A Design of the injection schedule and behavioral testing procedure. The APP/PS1 transgenic mice (28 weeks old) and wild-type (26 weeks old male) mice were used for this assay.CC BY · PubMed Central
Fig. 4B Rapamycin and miR-375-3p-mimic-agomir were administered through intraperitoneal and tail vein injections in APP/PS1 Tg mice. After treatment completion, organ samples were collected, and tissue sections were prepared for H&E staining analysis; Scale bar = 20 μm.
Fig. 4B Rapamycin and miR-375-3p-mimic-agomir were administered through intraperitoneal and tail vein injections in APP/PS1 Tg mice. After treatment completion, organ samples were collected, and tissue sections were prepared for H&E staining analysis; Scale bar = 20 μm.CC BY · PubMed Central
Fig. 4C APP/PS1 transgenic mice tail veins were injected with 0.8 % physiological saline and miR-375-3p-mimic-agomir-cy3. After 24 h, the mice were anesthetized, and live imaging was conducted using a 633 nm channel.
Fig. 4C APP/PS1 transgenic mice tail veins were injected with 0.8 % physiological saline and miR-375-3p-mimic-agomir-cy3. After 24 h, the mice were anesthetized, and live imaging was conducted using a 633 nm channel.CC BY · PubMed Central
Fig. 4D–G Morris Water Maze testing of memory in APP/PS1 transgenic mice. (D) The time curve indicates the time each group of mice took to reach the hidden platform during the experimental stages. (E) Cumulative escape latency for each group during the experimental stages. (F) Escape latency for each group on the final day of the experimental stage. (G) Representative images of probe test movement trajectories. (H–J) Y-Maze analysis to test the cognitive abilities in APP/PS1 transgenic mice.
Fig. 4D–G Morris Water Maze testing of memory in APP/PS1 transgenic mice. (D) The time curve indicates the time each group of mice took to reach the hidden platform during the experimental stages. (E) Cumulative escape latency for each group during the experimental stages. (F) Escape latency for each group on the final day of the experimental stage. (G) Representative images of probe test movement trajectories. (H–J) Y-Maze analysis to test the cognitive abilities in APP/PS1 transgenic mice.CC BY · PubMed Central
Fig. 4H Percentage of correct alternations during the 10-min spontaneous alternation test.
Fig. 4H Percentage of correct alternations during the 10-min spontaneous alternation test.CC BY · PubMed Central
Fig. 4I Time spent in the new arm exploration for each group.
Fig. 4I Time spent in the new arm exploration for each group.CC BY · PubMed Central
Fig. 4J Distance traveled in the new arm exploration for each group (n = 5/group). All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 4J Distance traveled in the new arm exploration for each group (n = 5/group). All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Fig. 5A and B Immunohistochemical staining images indicating Aβ aggregation in the hippocampal region of APP/PS1 Tg mice brains. (A) Immunohistochemistry analysis of Anti-Aβ stained sagittal brain tissue sections acquired from whole-brain samples from each group of mice. (B) Image-Pro Plus 6.0 was used to quantitatively process and analyze Aβ-positive expression plaques in each group.
Fig. 5A and B Immunohistochemical staining images indicating Aβ aggregation in the hippocampal region of APP/PS1 Tg mice brains. (A) Immunohistochemistry analysis of Anti-Aβ stained sagittal brain tissue sections acquired from whole-brain samples from each group of mice. (B) Image-Pro Plus 6.0 was used to quantitatively process and analyze Aβ-positive expression plaques in each group.CC BY · PubMed Central
Fig. 5C and D Nissl staining to assess the number of Nissl bodies in the brains of APP/PS1 transgenic mice. (C) The total number of Nissl bodies in the hippocampus and cerebral cortex after brain sections were stained with Nissl solution. (D) Cell counting was performed to determine the number of Nissl bodies in the cerebral cortex of each sample.
Fig. 5C and D Nissl staining to assess the number of Nissl bodies in the brains of APP/PS1 transgenic mice. (C) The total number of Nissl bodies in the hippocampus and cerebral cortex after brain sections were stained with Nissl solution. (D) Cell counting was performed to determine the number of Nissl bodies in the cerebral cortex of each sample.CC BY · PubMed Central
Fig. 5E and F Tunel staining was performed to detect the rate of cell apoptosis in the hippocampal region of APP/PS1 Tg mice brains. (E) Brain sections were treated with a Tunel probe for 60 min, and results were analyzed using a laser confocal microscope with an Alexa Fluor 488 channel. (F) Image-Pro Plus 6.0 was used to quantify the fluorescence intensity in brain sections from each group to calculate the apoptosis rate. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 5E and F Tunel staining was performed to detect the rate of cell apoptosis in the hippocampal region of APP/PS1 Tg mice brains. (E) Brain sections were treated with a Tunel probe for 60 min, and results were analyzed using a laser confocal microscope with an Alexa Fluor 488 channel. (F) Image-Pro Plus 6.0 was used to quantify the fluorescence intensity in brain sections from each group to calculate the apoptosis rate. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Fig. 6A and B Western blot detection of protein expression in the parietal and temporal lobes of the brains of various groups of APP/PS1 Tg mice. (A) Protein expression of AD pathological markers, such as PS1, APP, CTFα, CTFβ, BDNF, and Syn, as well as autophagy markers including Mfn2, Beclin1, Grp75, and LC3B. (B) The grayscale values of each group were quantified using ImageJ. GAPDH was employed as an internal reference.
Fig. 6A and B Western blot detection of protein expression in the parietal and temporal lobes of the brains of various groups of APP/PS1 Tg mice. (A) Protein expression of AD pathological markers, such as PS1, APP, CTFα, CTFβ, BDNF, and Syn, as well as autophagy markers including Mfn2, Beclin1, Grp75, and LC3B. (B) The grayscale values of each group were quantified using ImageJ. GAPDH was employed as an internal reference.CC BY · PubMed Central
Fig. 6C–L Immunofluorescence staining was performed to detect the protein expression levels of BDNF and Syn in the hippocampal region of APP/PS1 Tg mice. (C, E, G, I, and K) Brain sections from each group were stained with Anti-BDNF (C), Anti-Syn (E), Anti-Beclin1 (G), Anti-LC3B (I), and Anti-PS1 antibodies (K) as well as FITC-conjugated secondary antibodies. (D, F, H, J, and L) The fluorescence intensity of each group was quantified and analyzed using image-pro-plus 6.0. The results were normalized relative to the expression levels of BDNF, Syn, Beclin1, LC3B, and PS1 in wild-type mice.
Fig. 6C–L Immunofluorescence staining was performed to detect the protein expression levels of BDNF and Syn in the hippocampal region of APP/PS1 Tg mice. (C, E, G, I, and K) Brain sections from each group were stained with Anti-BDNF (C), Anti-Syn (E), Anti-Beclin1 (G), Anti-LC3B (I), and Anti-PS1 antibodies (K) as well as FITC-conjugated secondary antibodies. (D, F, H, J, and L) The fluorescence intensity of each group was quantified and analyzed using image-pro-plus 6.0. The results were normalized relative to the expression levels of BDNF, Syn, Beclin1, LC3B, and PS1 in wild-type mice.CC BY · PubMed Central
Fig. 6M and N Immunohistochemistry was performed to detect the protein expression of Beclin1 in the hippocampal region of APP/PS1 Tg mice. (M) Brain sections were stained using Anti-Beclin1 for immunohistochemistry. (N) Image-Pro Plus 6.0 was used to quantify and analyze the positivity rate of Beclin1 in each group.
Fig. 6M and N Immunohistochemistry was performed to detect the protein expression of Beclin1 in the hippocampal region of APP/PS1 Tg mice. (M) Brain sections were stained using Anti-Beclin1 for immunohistochemistry. (N) Image-Pro Plus 6.0 was used to quantify and analyze the positivity rate of Beclin1 in each group.CC BY · PubMed Central
Fig. 6O and P qRT-PCR analysis of the mRNA expression levels in the mice. (O) qRT-PCR results of mRNA levels of AD markers: APP, PS1, BDNF, and Syn. Briefly, total RNA was extracted from the blood of each mouse group, reverse-transcribed, and then used as a template for qRT-PCR. (P) qRT-PCR results of mRNA levels of autophagy markers: LC3B, Beclin1, Pink1, and MFN2. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 6O and P qRT-PCR analysis of the mRNA expression levels in the mice. (O) qRT-PCR results of mRNA levels of AD markers: APP, PS1, BDNF, and Syn. Briefly, total RNA was extracted from the blood of each mouse group, reverse-transcribed, and then used as a template for qRT-PCR. (P) qRT-PCR results of mRNA levels of autophagy markers: LC3B, Beclin1, Pink1, and MFN2. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Fig. 7A and B Western blot analysis was conducted to assess the activation of mTOR pathway-related proteins under different Aβ25-35 induction time conditions. (A) Phosphorylation activation status of mTOR, p70S6, and AKT in SH-SY5Y cells. (B) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was used as an internal reference.
Fig. 7A and B Western blot analysis was conducted to assess the activation of mTOR pathway-related proteins under different Aβ25-35 induction time conditions. (A) Phosphorylation activation status of mTOR, p70S6, and AKT in SH-SY5Y cells. (B) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was used as an internal reference.CC BY · PubMed Central
Fig. 7C and D Western blot evaluation of mTOR pathway-related protein expression in various groups of SH-SY5Y cells. (C) Protein expression of phosphorylated mTOR, ULK1, p70S6, and AKT in SH-SY5Y cells. (D) Grayscale values of each group were quantified using ImageJ with GAPDH as an internal reference.
Fig. 7C and D Western blot evaluation of mTOR pathway-related protein expression in various groups of SH-SY5Y cells. (C) Protein expression of phosphorylated mTOR, ULK1, p70S6, and AKT in SH-SY5Y cells. (D) Grayscale values of each group were quantified using ImageJ with GAPDH as an internal reference.CC BY · PubMed Central
Fig. 7E and F Western blot analysis was conducted to examine the activation of mTOR pathway-related proteins in the parietal and temporal lobes of the brains of APP/PS1 Tg mice. (E) Protein expression levels of phosphorylated mTOR, ULK1, AKT, and ATG14. (F) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was used as an internal reference.
Fig. 7E and F Western blot analysis was conducted to examine the activation of mTOR pathway-related proteins in the parietal and temporal lobes of the brains of APP/PS1 Tg mice. (E) Protein expression levels of phosphorylated mTOR, ULK1, AKT, and ATG14. (F) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was used as an internal reference.CC BY · PubMed Central
Fig. 7G and H Immunofluorescence staining images indicate the protein expression levels of ATG14 in the hippocampal region of APP/PS1 Tg mice. (G) Tissue sections were stained using Anti-ATG14 and FITC-conjugated secondary antibodies. (H) The fluorescence intensity of each group was quantified and analyzed using image-pro-plus 6.0. The results were normalized relative to the expression levels of ATG14 in wild-type mice. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 7G and H Immunofluorescence staining images indicate the protein expression levels of ATG14 in the hippocampal region of APP/PS1 Tg mice. (G) Tissue sections were stained using Anti-ATG14 and FITC-conjugated secondary antibodies. (H) The fluorescence intensity of each group was quantified and analyzed using image-pro-plus 6.0. The results were normalized relative to the expression levels of ATG14 in wild-type mice. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Fig. 8A Prediction of the interaction site between miR-375-3p and CD68 protein.
Fig. 8A Prediction of the interaction site between miR-375-3p and CD68 protein.CC BY · PubMed Central
Fig. 8B–E Western blot analysis was performed to detect CD68 protein expression in SH-SY5Y cells (B) as well as in the parietal and temporal lobes of the brains of wild-type mice after (D) miR375-3p overexpression. Grayscale values of CD68 in each group were quantified using ImageJ. GAPDH levels in SH-SY5Y cells (C) and wild-type mice (E) were assessed and set as an internal reference.
Fig. 8B–E Western blot analysis was performed to detect CD68 protein expression in SH-SY5Y cells (B) as well as in the parietal and temporal lobes of the brains of wild-type mice after (D) miR375-3p overexpression. Grayscale values of CD68 in each group were quantified using ImageJ. GAPDH levels in SH-SY5Y cells (C) and wild-type mice (E) were assessed and set as an internal reference.CC BY · PubMed Central
Fig. 8F and G Immunofluorescence staining was conducted to detect the protein expression levels of CD68 in various groups of SH-SY5Y cells. (F) Staining was performed using Anti-CD68 and PE-conjugated secondary antibodies (400 × ); Scale bar = 50 μm. (G) The fluorescence intensity of each group was quantified and analyzed using image-pro-plus 6.0. The results were normalized relative to the expression levels of CD68 in SH + NC as the folder.
Fig. 8F and G Immunofluorescence staining was conducted to detect the protein expression levels of CD68 in various groups of SH-SY5Y cells. (F) Staining was performed using Anti-CD68 and PE-conjugated secondary antibodies (400 × ); Scale bar = 50 μm. (G) The fluorescence intensity of each group was quantified and analyzed using image-pro-plus 6.0. The results were normalized relative to the expression levels of CD68 in SH + NC as the folder.CC BY · PubMed Central
Fig. 8H and I Western blot analysis was conducted to evaluate the expression of glial cell markers in various groups of SH-SY5Y cells. (H) Protein expression levels of CD80, Iba1, and GFAP. (I) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was employed as an internal reference.
Fig. 8H and I Western blot analysis was conducted to evaluate the expression of glial cell markers in various groups of SH-SY5Y cells. (H) Protein expression levels of CD80, Iba1, and GFAP. (I) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was employed as an internal reference.CC BY · PubMed Central
Fig. 8J and K Western blot analysis was performed to assess the expression of glial cell marker proteins in the parietal and temporal lobes of the brains of APP/PS1 Tg mice. (J) Protein expression levels of GFAP, CD80, CD68, and Iba1. (K) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was employed as an internal reference.
Fig. 8J and K Western blot analysis was performed to assess the expression of glial cell marker proteins in the parietal and temporal lobes of the brains of APP/PS1 Tg mice. (J) Protein expression levels of GFAP, CD80, CD68, and Iba1. (K) Grayscale values of each group were quantified and analyzed using ImageJ. GAPDH was employed as an internal reference.CC BY · PubMed Central
Fig. 8L–O Immunofluorescence staining was conducted to detect the protein expression levels of CD80 and Iba1 in the hippocampal region of APP/PS1 Tg mice. (L and N) Staining was performed using Anti-CD80 (L) and Anti-Iba1 (N) antibodies and FITC-conjugated secondary antibodies (100 × , 400 × ), scale bar: 200 μm, 50 μm. (L and N) The fluorescence intensity of each group was quantified using image-pro-plus 6.0 and analyzed, with results displayed relative to the expression levels of CD80 (M) and Iba1 (O) in wild-type mice as the folder.
Fig. 8L–O Immunofluorescence staining was conducted to detect the protein expression levels of CD80 and Iba1 in the hippocampal region of APP/PS1 Tg mice. (L and N) Staining was performed using Anti-CD80 (L) and Anti-Iba1 (N) antibodies and FITC-conjugated secondary antibodies (100 × , 400 × ), scale bar: 200 μm, 50 μm. (L and N) The fluorescence intensity of each group was quantified using image-pro-plus 6.0 and analyzed, with results displayed relative to the expression levels of CD80 (M) and Iba1 (O) in wild-type mice as the folder.CC BY · PubMed Central
Fig. 8P–S Immunohistochemical staining images indicating the distribution and positive expression of CD80 and Iba1 in the hippocampal region of APP/PS1 Tg mice. (P and R) Brain tissue sections were stained using Anti-CD80 (P) and Anti-Iba1 (R) antibodies for immunohistochemistry. (Q and S) The positive expression patches of CD80 (Q) and Iba1 (S) in each group were quantified and analyzed using image-pro-plus 6.0.
Fig. 8P–S Immunohistochemical staining images indicating the distribution and positive expression of CD80 and Iba1 in the hippocampal region of APP/PS1 Tg mice. (P and R) Brain tissue sections were stained using Anti-CD80 (P) and Anti-Iba1 (R) antibodies for immunohistochemistry. (Q and S) The positive expression patches of CD80 (Q) and Iba1 (S) in each group were quantified and analyzed using image-pro-plus 6.0.CC BY · PubMed Central
Fig. 8T qRT-PCR to measure the mRNA expression levels of CD68, CD80, Iba1, and IL-6 in the mice.
Fig. 8T qRT-PCR to measure the mRNA expression levels of CD68, CD80, Iba1, and IL-6 in the mice.CC BY · PubMed Central
Fig. 8U–W Elisa was performed to detect the protein levels of TMEM119 (U), IL-1β (V), and CD206 (W) in the brains of each group of mice. For each group, three replicates were set. Standard curves were drawn using Origin8.0. Data were summarized and analyzed using image-pro-plus 6.0.
Fig. 8U–W Elisa was performed to detect the protein levels of TMEM119 (U), IL-1β (V), and CD206 (W) in the brains of each group of mice. For each group, three replicates were set. Standard curves were drawn using Origin8.0. Data were summarized and analyzed using image-pro-plus 6.0.CC BY · PubMed Central
Fig. 8X Molecular mechanism of overexpression of miR375-3p combined with rapamycin to clear Aβ deposition in AD environment. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig. 8X Molecular mechanism of overexpression of miR375-3p combined with rapamycin to clear Aβ deposition in AD environment. All data are presented as the mean ± SEM of at least three independent experiments and were compared using one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central

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Metadata

Authors
Wang, Y., Xiao, Z., Yin, H., Ren, Z., Ma, X., Wang, Y., Zhang, Y., Fu, X., Zhang, F., & Zeng, L.
ORCID
Zeng, Linlin
Venue
Heliyon
Publisher
Elsevier BV
Published
2024-10
Volume
10
Issue
19
Pages
e37589
ISSN
2405-8440
DOI
10.1016/j.heliyon.2024.e37589
PMC
PMC11461998
Cited by
5
References
13
Language
en
Licence
Funders
  • Department of Science and Technology of Jilin Province (YDZJ202301ZYTS536)
  • National Key Research and Development Program of China (2021YFA1500400)

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