Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Suppression of intestinal Ticam1 ameliorated MASH via Akkermansia muciniphila QAA37749.1 mediated betaine transformation.

doi: 10.1016/j.bbadis.2024.167571

Figure Lengend Snippet: Fig. 1. The intestinal dysfunction was improved in HFD-fed Ticam1ΔIEC mice compared with Ticam1fl/fl. (A) The immunochemistry staining results of the intestinal epithelial tissues from patients with or without MASH (n = 4). (B) The intestinal mRNA expression level of Ticam1 in wild-type (WT) mice fed with NCD or HFD (n = 6). (C) the H&E staining of the ileum and colon in mice (n = 5). (D) The FD4 intestinal permeability testing results in mice (n = 4 or 5). (E) The immunofluorescence staining of ZO-1 (n = 3 at least). (F) The protein expression level of occludin in the ileum and colon in mice (n = 3) and the quantity analysis of the expression level of occludin in the colon. (G) The mRNA expression level of inflammation factors in mice intestines (n = 5 at least). (H) The level of inflammation factors in mice serum (n = 8 at least). All data are presented as the mean ± SEM. Unpaired Student's two-tailed t-test was used to compare differences between two groups (normal distribution assumed), otherwise, the Mann-Whitney nonparametric test was used. Two-way ANOVA followed by Tukey's post-hoc test was performed for multiple group comparisons (equal variance assumed). Otherwise, the nonparametric Kruskal-Wallis test was used for further analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For in vivo antibiotic treatment, HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice were treated with 50 μg/mL clindamycin (C) (#HY-B0408A, MCE), 50 μg/mL metronidazole (M) (#HY-B0318, MCE), 50 μg/mL penicillin (P) (#HY-B1463, MCE), 25 μg/mL vancomycin (V) (#HY-17362, MCE) and 50 μg/mL neomycin (N) (#HY-B0470, MCE) in sterile drink water at the start of HFD for 12 weeks.

Techniques: Staining, Expressing, Permeability, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Suppression of intestinal Ticam1 ameliorated MASH via Akkermansia muciniphila QAA37749.1 mediated betaine transformation.

doi: 10.1016/j.bbadis.2024.167571

Figure Lengend Snippet: Fig. 2. The MASH was alleviated in HFD-fed Ticam1ΔIEC mice compared with Ticam1fl/fl. (A) The H&E staining of livers in different groups and the MASH activity scores of mice fed with HFD (n = 6 or 7). The arrow represents the spotty necrosis and the asteroid represents the ballooning degeneration. (B) The Sirius red staining of livers in different groups (n = 5). (C) The F4/80 staining of livers in different groups (n = 5). (D) The weight of the liver in different groups (n = 5 at least). (E) The level of ALT, AST, and ALP in mice of different groups (n = 5 at least). (F) The mRNA expression level of inflammation factors and genes related to lipid synthesis in mice liver (n = 5 at least). All data are presented as the mean ± SEM. Unpaired Student's two-tailed t-test was used to compare differences between two groups (normal distribution assumed), otherwise, the Mann-Whitney nonparametric test was used. Two-way ANOVA followed by Tukey's post-hoc test was performed for multiple group comparisons (equal variance assumed). Otherwise, the nonparametric Kruskal-Wallis test was used for further analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For in vivo antibiotic treatment, HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice were treated with 50 μg/mL clindamycin (C) (#HY-B0408A, MCE), 50 μg/mL metronidazole (M) (#HY-B0318, MCE), 50 μg/mL penicillin (P) (#HY-B1463, MCE), 25 μg/mL vancomycin (V) (#HY-17362, MCE) and 50 μg/mL neomycin (N) (#HY-B0470, MCE) in sterile drink water at the start of HFD for 12 weeks.

Techniques: Staining, Activity Assay, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Suppression of intestinal Ticam1 ameliorated MASH via Akkermansia muciniphila QAA37749.1 mediated betaine transformation.

doi: 10.1016/j.bbadis.2024.167571

Figure Lengend Snippet: Fig. 3. The glucose homeostasis and metabolic rate were improved in HFD-fed Ticam1ΔIEC mice compared with Ticam1fl/fl. (A) The pictures of different groups of mice. The recording of mice body weight of different groups. The recording of mice food intake of different groups (n = 5 at least). (B) The H&E staining of iWAT in different groups and the measurement of adipocyte diameters (n = 5). (C) The glucose tolerance test (GTT) of mice in different groups (n = 7 at least) and the quantitative analysis (AUC) of GTT. (D) The insulin tolerance test (ITT) of mice in different groups (n = 7 at least). (E) The level of serum insulin in fasted mice in different groups (n = 5 at least). (F) HOMA-IR of mice in different groups. (G) The whole-body oxygen consumption rate (VO2) recording (the upper image) and the quantity analysis (the lower image) of VO2 (n = 6). (H) The carbon dioxide production rate (VCO2) recording (the upper image) and the quantity analysis (the lower image) of VCO2 (n = 6). (I) The heat production (the upper image) and the quantity analysis (the lower image) of the heat production (n = 6). All data are presented as the mean ± SEM. Two-way ANOVA followed by Tukey's post-hoc test was performed for multiple group comparisons (equal variance assumed). Otherwise, the nonparametric Kruskal-Wallis test was used for further analysis. * p < 0.05, ** p < 0.01.

Article Snippet: For in vivo antibiotic treatment, HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice were treated with 50 μg/mL clindamycin (C) (#HY-B0408A, MCE), 50 μg/mL metronidazole (M) (#HY-B0318, MCE), 50 μg/mL penicillin (P) (#HY-B1463, MCE), 25 μg/mL vancomycin (V) (#HY-17362, MCE) and 50 μg/mL neomycin (N) (#HY-B0470, MCE) in sterile drink water at the start of HFD for 12 weeks.

Techniques: Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Suppression of intestinal Ticam1 ameliorated MASH via Akkermansia muciniphila QAA37749.1 mediated betaine transformation.

doi: 10.1016/j.bbadis.2024.167571

Figure Lengend Snippet: Fig. 4. The composition of gut microbiota was altered in HFD-fed Ticam1ΔIEC mice compared with Ticam1fl/fl. (A) The PCA analysis of gut microbiota in feces from mice of different groups (n = 6). (B) The relative abundance of the significantly differential microbes in HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice (n = 6). (C) The pictures of HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice treated with antibiotics (n = 5). (D) The recording of mice body weight and food intake of HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice treated with antibiotics (n = 5). (E) The H&E staining of livers and MASH scores of HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice treated with antibiotics (n = 5). (F) The level of ALT and AST in serum of HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice treated with antibiotics (n = 5). (G) The liver mRNA expression level of genes related to inflammation response (left) and lipid synthesis (right) (n = 6 at least). (H) The GTT result of HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice treated with antibiotics (n = 6 at least). All data are presented as the mean ± SEM. Unpaired Student's two-tailed t-test was used to compare differences between two groups (normal distribution assumed), otherwise, the Mann-Whitney nonparametric test was used. n.s.: none significance, * p < 0.05, ** p < 0.01.

Article Snippet: For in vivo antibiotic treatment, HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice were treated with 50 μg/mL clindamycin (C) (#HY-B0408A, MCE), 50 μg/mL metronidazole (M) (#HY-B0318, MCE), 50 μg/mL penicillin (P) (#HY-B1463, MCE), 25 μg/mL vancomycin (V) (#HY-17362, MCE) and 50 μg/mL neomycin (N) (#HY-B0470, MCE) in sterile drink water at the start of HFD for 12 weeks.

Techniques: Staining, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Suppression of intestinal Ticam1 ameliorated MASH via Akkermansia muciniphila QAA37749.1 mediated betaine transformation.

doi: 10.1016/j.bbadis.2024.167571

Figure Lengend Snippet: Fig. 5. The deletion of IEC Ticam1 directly supported the growth of Akkermansia muciniphila. (A) Schematic of FMT experiment. (B) The α-diversity analysis of gut microbes from mice after the treatment with antibiotics (A_Ticam1fl/fl, A_ Ticam1ΔIEC) and after the termination of FMT (F_Ticam1fl/fl, F_ Ticam1ΔIEC). Use sobs method, p = 0.00562. (C) The composition of gut microbes in F_Ticam1fl/fl and F_ Ticam1ΔIEC mice (n = 5). (D,E) The recording of body weight and food intake of FMT- Ticam1fl/fl and FMT- Ticam1ΔIEC groups (n = 5). (F) The representative H&E staining for livers of mice in FMT-Ticam1fl/fl and FMT- Ticam1ΔIEC groups. The asteroid represents hepatocyte ballooning degeneration (n = 5). (G) The mRNA expression level of genes related to liver inflammation and lipid synthesis (n = 5). All data are presented as the mean ± SEM. Unpaired Student's two-tailed t-test was used to compare differences between two groups (normal distribution assumed), otherwise, the Mann-Whitney nonparametric test was used. Two-way ANOVA followed by Tukey's post-hoc test was performed for multiple group comparisons (equal variance assumed). Otherwise, the nonparametric Kruskal-Wallis test was used for further analysis. n.s.: none significance, * p < 0.05.

Article Snippet: For in vivo antibiotic treatment, HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice were treated with 50 μg/mL clindamycin (C) (#HY-B0408A, MCE), 50 μg/mL metronidazole (M) (#HY-B0318, MCE), 50 μg/mL penicillin (P) (#HY-B1463, MCE), 25 μg/mL vancomycin (V) (#HY-17362, MCE) and 50 μg/mL neomycin (N) (#HY-B0470, MCE) in sterile drink water at the start of HFD for 12 weeks.

Techniques: Staining, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Suppression of intestinal Ticam1 ameliorated MASH via Akkermansia muciniphila QAA37749.1 mediated betaine transformation.

doi: 10.1016/j.bbadis.2024.167571

Figure Lengend Snippet: Fig. 6. The secretion of sIgA was increased in HFD-fed Ticam1ΔIEC mice compared with Ticam1fl/fl. (A) The amount of sIgA in the lumen of intestine and feces in mice (n = 4 at least). (B) The protein expression level of pIgR in mice intestines (n = 4). The quantity analysis of the expression level of pIgR in HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice. (C and D) The detection of IgA-producing B cells and plasma cells within the intestinal lamina propria monocytes (LPMC), Peyer's patches (PPs), and mesenteric lymph nodes (MLNs) and the quantity analysis of these results (n = 6 at least). All data are presented as the mean ± SEM. Two-way ANOVA followed by Tukey's post-hoc test was performed for multiple group comparisons (equal variance assumed). Otherwise, the nonparametric Kruskal-Wallis test was used for further analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For in vivo antibiotic treatment, HFD-fed Ticam1fl/fl and Ticam1ΔIEC mice were treated with 50 μg/mL clindamycin (C) (#HY-B0408A, MCE), 50 μg/mL metronidazole (M) (#HY-B0318, MCE), 50 μg/mL penicillin (P) (#HY-B1463, MCE), 25 μg/mL vancomycin (V) (#HY-17362, MCE) and 50 μg/mL neomycin (N) (#HY-B0470, MCE) in sterile drink water at the start of HFD for 12 weeks.

Techniques: Expressing, Clinical Proteomics

Journal: Molecules

Article Title: Hepatoprotective Effects of Kaempferol-3-O-α-l-Arabinopyranosyl-7-O-α-l-Rhamnopyranoside on d-Galactosamine and Lipopolysaccharide Caused Hepatic Failure in Mice

doi: 10.3390/molecules22101755

Figure Lengend Snippet: Figure 7. The expression of NF-κB and TLR4 signaling pathway-related proteins, such as NF-Kb (A); IκBα (B); p65 (C); TLR4 (D); TRAF6 (E); MyD88 (F); and TRIF (G); were detected using a Western blotting assay in the liver tissues of each group 6 h after D-GalN/LPS administration. The data represented show the mean ± SD. Data of IκBα, p65, and NF-κB are normalized to that of Lamin B, and that of TLR4, TRAF6, MyD88, and TRIF are normalized to β-actin. * p < 0.05, ** p < 0.01 compared to the FHF model group, and # p < 0.05, ## p < 0.01 compared to the control group.

Article Snippet: After blocking in 5% (w/v) non-fat dry milk in PBST for 1 h, the membranes were incubated with primary antibodies for detecting cytochrome C, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, TLR4, TRIF, TAF6, MyD88, p65, NF-κB, and Iκ-Bα (Cell Signaling Technology, Danvers, MA,USA) overnight at 4 °C.

Techniques: Expressing, Western Blot, Control

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Gegen Qinlian Decoction Attenuates Colitis-Associated Colorectal Cancer via Suppressing TLR4 Signaling Pathway Based on Network Pharmacology and In Vivo/In Vitro Experimental Validation.

doi: 10.3390/ph18010012

Figure Lengend Snippet: Figure 9. GQD inhibited TLR4-ralated signaling pathway in LPS-induced RAW264.7 cells. The protein expression of TLR4, Myd88, TRIF, p-IκBα, IκBα, p-P65, P65, p-IRF3, IFR3 in each group. n = 3. * p < 0.05 vs. Control group; # p < 0.05 vs. LPS group. GQD: Gegen Qinlian Decoction; LPS: Lipopolysaccharide.

Article Snippet: Ohwi (C442220601), Scutellaria baicalensis Georgi (C130220702), Coptis chinensis Franch (C129220702), and Glycyrrhiza uralensis Fisch (C118220701) were purchased from Hebei Anjia Pharmaceutical Co., Ltd. (Anguo, China); Ki67 antibody (AB16667) was purchased from Abcam (Cambridge, UK); IRF3 (11904) and p-IRF3 (29047S) antibodies were purchased from the Cell Signaling Technology (Beverly, MA, USA); TLR4 (66350-1- Ig), MyD88 (23230-1-AP), TRIF (23288-1-AP), β-actin (66009-1-Ig), GAPDH (60004-1-Ig), Vinculin (66305-1-Ig), HRP-conjugated Goat Anti-Mouse IgG (H+L) (SA00001-1), HRPconjugated Goat Anti-Rabbit IgG (H+L) (SA00001-2), F4/80 (28463-1-AP) antibodies were all purchased from Proteintech (Wuhan, China). p-IκBα (ABP0038), IκBα (ABP57568), p-P65 (ABP0043), P65 (ABM40111) antibodies were purchased from Abbkine Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Control

Journal: Journal of Virology

Article Title: Microtubule acetylation is not required for HIV-1 infection or TRIM69-mediated restriction of HIV-1 infection

doi: 10.1128/jvi.01026-24

Figure Lengend Snippet: TRIM69 restriction occurs independently from microtubule acetylation. (A) THP-1 cells were stably transduced with a doxycycline-inducible TRIM69 construct and treated with varying concentrations of doxycycline (DOX) for 48 h. Both wild-type (WT) and doxycycline-treated cells were collected and analyzed for protein levels of TRIM69 and GAPDH. (B) Wild-type THP-1 cells were either mock-treated or doxycycline-treated at 5 µg/mL for 48 h. Following treatment, cells were collected and analyzed for protein levels of acetylated tubulin (Ac-Tubulin). As a loading control, protein levels of GAPDH were examined. (C) THP-1 cells were stably transduced with the TRIM69 pcw57 construct and either mock-treated or treated with doxycycline at 5 µg/mL for 48 h. Following treatment, cells were stained and imaged for TRIM69, acetylated tubulin, alpha tubulin, and nuclear staining (DAPI). (D) Transduced THP-1 cells were differentiated with PMA and treated with varying concentrations of doxycycline for 48 h. Subsequently, cells were infected with equal amounts of a firefly luciferase-containing HIV-1 reporter virus. At 48 h post-infection, cells were analyzed via luciferase assay to determine infectivity levels. (E) THP-1 cells were stably transduced with either the aTAT1 V2 CRISPR/Cas9, TRIM69 pcw57, or both constructs. After selection, cells were treated with 5 µg/mL of doxycycline for 48 h. Subsequently, cells were lysed and assayed for protein levels of acetylated tubulin, TRIM69, and GAPDH. (F) THP-1 cells stably expressing an empty V2 CRISPR/Cas9, αTAT1 V2 CRISPR/Cas9, TRIM69 pcw57, or both were differentiated with PMA and treated with 5 µg/mL of doxycycline for 48 h. Following treatment, cells were infected with equal amounts of VSV-g pseudotyped NL4.3 Luc-mCherry HIV-1. At 48 h post-infection, infectivity was determined by flow cytometry analysis to quantify the percentage of mCherry-positive cells. A representative flow experiment is presented on the left, while normalized infectivity across three experiments is provided on the right. Error bars represent ± standard error of the mean (SEM). Statistical significance was determined by a one-tailed t -test, with P < 0.05 considered significant.

Article Snippet: For TRIM69 detection in both immunofluorescence and western blotting, a rabbit polyclonal antibody (Proteintech 12951–1-AP) was utilized.

Techniques: Stable Transfection, Transduction, Construct, Control, Staining, Infection, Luciferase, Virus, CRISPR, Selection, Expressing, Flow Cytometry, One-tailed Test

Journal: Journal of Virology

Article Title: Microtubule acetylation is not required for HIV-1 infection or TRIM69-mediated restriction of HIV-1 infection

doi: 10.1128/jvi.01026-24

Figure Lengend Snippet: TRIM69 restricts HIV-1 during reverse transcription. THP-1 cells expressing the inducible TRIM69pcw construct were treated with doxycycline for 48 h prior to infection. Following treatment, cells were infected with DNase-treated HIV-1 for 24 h. Following infection, DNA was isolated and subjected to qRT-PCR analysis using primers specific for LateRT products ( A ) or 2LTR aggregates ( B ). Results are normalized to infected wild-type THP-1. Error bars represent ± standard error of the mean (SEM). Statistical significance was determined by a one-tailed t -test, with P < 0.05 considered significant.

Article Snippet: For TRIM69 detection in both immunofluorescence and western blotting, a rabbit polyclonal antibody (Proteintech 12951–1-AP) was utilized.

Techniques: Reverse Transcription, Expressing, Construct, Infection, Isolation, Quantitative RT-PCR, One-tailed Test

Journal: Journal of Virology

Article Title: Microtubule acetylation is not required for HIV-1 infection or TRIM69-mediated restriction of HIV-1 infection

doi: 10.1128/jvi.01026-24

Figure Lengend Snippet: TRIM69 exhibits broad antiviral activity. THP-1 cells expressing the inducible TRIM69pcw construct were treated with 5 µg/mL doxycycline for 48 h prior to infection. Following treatment, cells were infected with an MOI of 1.0 (ZIKV, VACV, and MP12), 0.1 (CHIKV), or 0.01 (LACV and CVB3). Supernatant was collected 48 h post-infection, and viral titer was determined by plaque assay. Error bars represent ± standard error of the mean (SEM). Statistical significance was determined by a one-tailed t -test, with P < 0.05 considered significant.

Article Snippet: For TRIM69 detection in both immunofluorescence and western blotting, a rabbit polyclonal antibody (Proteintech 12951–1-AP) was utilized.

Techniques: Activity Assay, Expressing, Construct, Infection, Plaque Assay, One-tailed Test

Journal: Journal of Virology

Article Title: Microtubule acetylation is not required for HIV-1 infection or TRIM69-mediated restriction of HIV-1 infection

doi: 10.1128/jvi.01026-24

Figure Lengend Snippet: TRIM69 exhibits broad antiviral activity in the absence of microtubule acetylation. THP-1 cells stably expressing a V2 CRISPR/Cas9 construct targeting αTAT1 and cotransduced with an inducible TRIM69pcw construct were treated with 5 µg/mL doxycycline for 48 h prior to infection. Following treatment, cells were infected with an MOI of 1.0 (ZIKV), 0.1 (CHIKV), and 0.01 (CVB3). Supernatant was collected 48 h post-infection, and viral titer was determined by plaque assay. Error bars represent ± standard error of the mean (SEM). Statistical significance was determined by a one-tailed t -test, with P < 0.05 considered significant.

Article Snippet: For TRIM69 detection in both immunofluorescence and western blotting, a rabbit polyclonal antibody (Proteintech 12951–1-AP) was utilized.

Techniques: Activity Assay, Stable Transfection, Expressing, CRISPR, Construct, Infection, Plaque Assay, One-tailed Test

Journal: PLoS ONE

Article Title: Differences in PGE 2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

doi: 10.1371/journal.pone.0098517

Figure Lengend Snippet: U373-CD14 cells were transfected with control siRNA or with siRNA targeting TRIF or IRF3 (A, C), or TRAM (B, D). Transfected cells were left untreated (solid bars), or were incubated with LPS at 1 ng/ml (shaded bars), with rIL-1β at 100 ng/ml for 18 h (striped bars), or were primed for 1 h with LPS and incubated with rIL-1β for additional 18 h (open bars). Cells were collected and COX-2 mRNA (A, B) and mPGES-1 mRNA (C, D) expressions were assayed by qPCR. The data are shown as fold increase over control PCR with primers specific to β-actin , run in the same samples. PCR was performed using triplicate wells and the data are shown as mean fold increase ± STDEV. This is representative of three experiments.

Article Snippet: For the Western Blotting of the cell lysates prepared from transfected cells and resolved in SDS-PAGE, the following antibodies were used: goat anti-human TRIF Ab (Cat AF6216, R&D Systems), rabbit anti-human TRAM Ab (Cat ab17221, Abcam), and rabbit anti-human IRF3 Ab (Cat 4302, Cell Signaling).

Techniques: Transfection, Control, Incubation

Journal: Cell reports. Medicine

Article Title: Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia.

doi: 10.1016/j.xcrm.2024.101782

Figure Lengend Snippet: Figure 4. Morphine-Exo dsRNA mediated spinal microglial activation via the TLR3-TRIF signaling pathways in vitro (A) Double dsRNA immunostaining with NeuN in cultured primary spinal neurons. Scale bars: 50 mm. (B) Representative ADAR1 western blot and quantification results in cultured primary spinal neurons. n = 4 biological replicates, *p < 0.05, non-parametric test (Mann-Whitney test). (C) Quantification of dsRNA immunostaining in cultured primary spinal neurons. n = 5 wells/group, **p < 0.01, non-parametric test (Mann-Whitney test). (D) Detection of dsRNA using ELISA in Exos isolated from conditioned media from cultured primary spinal neurons. n = 5 biological replicates, **p < 0.01, non- parametric test (Mann-Whitney test). (E) Representative IBA1, TLR3, TRIF, and IL-6 western blot and quantification results in cultured primary spinal microglia. n = 4 biological replicates, **p < 0.01, non-parametric test (Mann-Whitney test). Data are shown as the mean ± SEM.

Article Snippet: Rat TLR3 siRNA (NM_198791, Cat#: SR504179) and TRIF siRNA (NM_053588, Cat#: SR510485) were purchased from Origene.

Techniques: Activation Assay, Protein-Protein interactions, In Vitro, Immunostaining, Cell Culture, Western Blot, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Cell reports. Medicine

Article Title: Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia.

doi: 10.1016/j.xcrm.2024.101782

Figure Lengend Snippet: Figure 5. Chronic morphine exposure activated microglia via the TLR3-TRIF signaling pathway in rats (A) Representative TLR3 and TRIF western blot and quantification results in rat spinal dorsal horn. n = 6 rats/group, **p < 0.01, non-parametric test (Mann-Whitney test). (B) Representative TLR3 immunostaining and quantification results on day 7 in spinal cord segments from rats treated with twice-daily intrathecal saline or morphine injections. n = 5 slices/5 rats/group, **p < 0.01, non-parametric test (Mann-Whitney test). Scale bar: 100 mm. (C) Double immunostaining of TLR3 with NeuN, GFAP, and IBA1 on day 7 in spinal cord segments from rats treated with twice-daily intrathecal morphine in- jections. Scale bars: 50 mm.

Article Snippet: Rat TLR3 siRNA (NM_198791, Cat#: SR504179) and TRIF siRNA (NM_053588, Cat#: SR510485) were purchased from Origene.

Techniques: Western Blot, MANN-WHITNEY, Immunostaining, Saline, Double Immunostaining

Journal: Cell reports. Medicine

Article Title: Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia.

doi: 10.1016/j.xcrm.2024.101782

Figure Lengend Snippet: Figure 6. Inhibiting spinal TLR3-TRIF signaling prevented the development of morphine-induced tolerance and hyperalgesia in rats (A) Co-administration of intrathecal morphine and TLR3 inhibitor (10 and 100 mg) attenuated morphine tolerance on days 4–7 as evidenced by the measurement of tail-flick latency. n = 6 rats/group. Morphine plus TLR3 inhibitor (10 mg) vs. morphine plus vehicle, **p < 0.01; morphine plus TLR3 inhibitor (100 mg) vs. morphine plus vehicle, #p < 0.05, ##p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test. (B) Right shift in the dose-response curve for morphine was prevented by TLR3 inhibitor (10 and 100 mg) in the tail-flick test. (C and D) Co-administration of TLR3 inhibitor (10 and 100 mg) blocked morphine-induced decreases in (C) left hind paw withdrawal threshold and (D) latency on day 7. n = 6 rats/group. **p < 0.01, non-parametric test (Mann-Whitney test). (E) Co-administration of TLR3 inhibitor (100 mg) beginning on day 7 reversed morphine-induced reductions in latency on days 9 and 11. n = 6 rats/group. **p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test. (F and G) Co-administration of TLR3 inhibitor (100 mg) beginning on day 7 reversed the reductions in (F) left hind paw withdrawal threshold and (G) latency on day 11. **p < 0.01, non-parametric test (Mann-Whitney test). (H) Co-administration of intrathecal morphine and poly (I:C) (10 mg) accelerated the development of morphine tolerance. n = 6 rats/group, *p < 0.05, **p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Rat TLR3 siRNA (NM_198791, Cat#: SR504179) and TRIF siRNA (NM_053588, Cat#: SR510485) were purchased from Origene.

Techniques: MANN-WHITNEY