Journal: Drug Design, Development and Therapy

Article Title: Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway

doi: 10.2147/DDDT.S482965

Figure Lengend Snippet: Effect of MZGW on serum SCFA level and the relative expression of tibia CTSK, NFATc1, GPR41, and p-p38 proteins in OVX rats. ( A ) Western blot image of NFATc1 and CTSK protein expression. ( B ) Relative protein expression of NFATc1. ( C ) Relative protein expression of CTSK. ( D ) Changes in the total serum SCFA levels per group. ( E ) Western blot image of GPR41 protein expression. ( F ) Relative protein expression of GPR41. ( G ) Western blot image of p-p38 and p38 protein expression. ( H ) Relative protein expression of p-p38. ( I ) Relative mRNA expression of p38 examined using quantitative reverse transcription PCR (qRT-PCR). Data were expressed as mean ± SD, n=6. # p <0.05 and ## p <0.01 vs the SHAM group; * p <0.05 and ** p <0.01 vs the OVX group.

Article Snippet: After electrophoresis with 8% SDS-PAGE (APPLYGEN), proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk/BSA (APPLYGEN) for 1 h. Subsequently, proteins were incubated with primary antibodies against β-actin (1:10,000, Proteintech, China), GPR41 (1:500, Proteintech), p38 (1:1000, Proteintech), p-p38 (1:1000, Proteintech), CTSK (1:1000, Proteintech), and NFATc1 (1:1000, Proteintech) at 4°C overnight, followed by washing three times rapidly with TBST.

Techniques: Expressing, Western Blot, Reverse Transcription, Quantitative RT-PCR

Journal: Drug Design, Development and Therapy

Article Title: Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway

doi: 10.2147/DDDT.S482965

Figure Lengend Snippet: SCFAs inhibited osteoclast differentiation and decrease the expression of mRNA and proteins associated with the SCFA-GPR41-p38MAPK signaling pathway. ( A ) Representative images of TRAP-stained osteoclasts depicting osteoblast differentiation in each group (scale bar=100 μm). ( B ) Quantitative statistics of osteoclast number (n=6). ( C ) Representative images of toluidine blue-stained osteoclasts in each group depicting bone resorption function. Differentiation of osteoblasts in each group (scale bar=100 μm). ( D ) Measurement of the area of bone resorption lacunae using Image-pro-plus 6.0 software. Data are expressed as mean ± SD, n=6. ( E ) Western blot image of GPR41, p-p38, CTSK, and NFATc1 protein expression. ( F ) Relative protein expression of GPR41. ( G ) Relative protein expression of p-p38. ( H ) Relative mRNA expression of p38. ( I ) Relative protein expression of CTSK. ( J ) Relative protein expression of NFATc1. Data are expressed as mean ± SD, n=6. # p <0.05 and ## p <0.01 vs the NC-S group; ** p <0.01 vs the OVX-S group; ◇ p <0.05 and ◇◇ p <0.01 vs the MZGW-DS group; ☆☆ p <0.01 vs the NC- SCFAs group; Δ p <0.05 and ΔΔ p <0.01 vs the OVX-SCFAs group.

Article Snippet: After electrophoresis with 8% SDS-PAGE (APPLYGEN), proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk/BSA (APPLYGEN) for 1 h. Subsequently, proteins were incubated with primary antibodies against β-actin (1:10,000, Proteintech, China), GPR41 (1:500, Proteintech), p38 (1:1000, Proteintech), p-p38 (1:1000, Proteintech), CTSK (1:1000, Proteintech), and NFATc1 (1:1000, Proteintech) at 4°C overnight, followed by washing three times rapidly with TBST.

Techniques: Expressing, Staining, Software, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway

doi: 10.2147/DDDT.S482965

Figure Lengend Snippet: Knockdown of the GPR41 gene, a key receptor for SCFAs binding in the SCFA-GPR41-p38MAPK pathway, did not reverse osteoclast-induced bone destruction by MZGW drug-containing serum (MZGW-DS). ( A ) Transfection efficiency of knocked down GPR41 protein detected using Western blotting. ( B ) Relative protein expression of GPR41. Data are expressed as mean ± SD, n=6. * p <0.05 and ** p <0.01 vs the OC+siNC group. ( C ) Western blot image depicting GPR41 protein expression upon knockdown of the GPR41 gene. ( D ) Relative protein expression of GPR41. ( E ) Relative expression of p38mRNA in osteoclasts in each group. ( F ) Western blot image depicting p-p38 and p38 protein expression upon knockdown of the GPR41 gene. ( G ) Relative protein expression of p-p38 and p38. ( H ) Quantitative statistics of osteoclast number (n=6). ( I ) Representative images of TRAP-stained osteoclasts in each group (scale bar=100 μm) upon knockdown of the GPR41 gene. ( J ) Western blot image of CTSK protein. ( K ) Relative protein expression of CTSK. ( L ) Western blot image of NFATc1 protein. ( M ) Relative protein expression of NFATc1. Data are expressed as mean ± SD, n=6. # p <0.05 and ## p <0.01 vs the OC+siNC group; * p <0.05 and ** p <0.01 vs the OC+siGPR41 group; ΔΔ p <0.01 vs the OC+siNC+MZGW-DS group.

Article Snippet: After electrophoresis with 8% SDS-PAGE (APPLYGEN), proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk/BSA (APPLYGEN) for 1 h. Subsequently, proteins were incubated with primary antibodies against β-actin (1:10,000, Proteintech, China), GPR41 (1:500, Proteintech), p38 (1:1000, Proteintech), p-p38 (1:1000, Proteintech), CTSK (1:1000, Proteintech), and NFATc1 (1:1000, Proteintech) at 4°C overnight, followed by washing three times rapidly with TBST.

Techniques: Knockdown, Binding Assay, Transfection, Western Blot, Expressing, Staining

Journal: Chinese Medicine

Article Title: Shen-Bai-Jie-Du decoction suppresses the progression of colorectal adenoma to carcinoma through regulating gut microbiota and short-chain fatty acids

doi: 10.1186/s13020-024-01019-4

Figure Lengend Snippet: Comparison of expressions of HDAC1/3 and GPR41/43/109a in colorectal tissue of mice among ND, HFD and SBJDD groups. A GPR41, GPR43 and GPR109a relative expression of mRNA in colon tissues were measured by RT-qPCR analysis. B Western blot analysis of GPR41, GRP43 and GPR109a in colorectal tissues. C Representative microscopic pictures of colorectal tissues for IHC staining of HDAC1 and HDAC3. D HDAC1 and HDAC3 relative expression of mRNA in colon tissues were measured by RT-qPCR. Data are shown as the mean ± SEM of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Anti-GPR41 (FFAR3, no. 66811–1-Ig), anti-GPR43 (FFAR2, no. 19952–1-AP) were purchased from Proteintech Group (Chicago, IL, USA).

Techniques: Comparison, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Chinese Medicine

Article Title: Shen-Bai-Jie-Du decoction suppresses the progression of colorectal adenoma to carcinoma through regulating gut microbiota and short-chain fatty acids

doi: 10.1186/s13020-024-01019-4

Figure Lengend Snippet: The schematic diagram of the mechanism of SBJDD. SBJDD may exert its inhibitory effects on colorectal adenoma carcinogenesis by regulating gut microbiota, promoting the production of SCFAs, activating G protein-coupled receptors GPR43, GPR41 and GPR109a, inhibiting histone deacetylases HDAC1 and HDAC3, inducing M2-like macrophage polarization, reducing intestinal inflammation, inhibiting cancer cell proliferation and restoring intestinal barrier function

Article Snippet: Anti-GPR41 (FFAR3, no. 66811–1-Ig), anti-GPR43 (FFAR2, no. 19952–1-AP) were purchased from Proteintech Group (Chicago, IL, USA).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Sini san regulates intestinal flora and short-chain fatty acids to ameliorate hepatocyte apoptosis and relieve CCl 4 -induced liver fibrosis in mice

doi: 10.3389/fphar.2024.1408459

Figure Lengend Snippet: Effects of SNS on SCFA receptor expression (n = 3). (A) The protein expression of FFAR2 and FFAR3 in liver tissue was detected using Western blotting. (B) The mRNA expression of FFAR2 and FFAR3 in liver tissue was analyzed by RT-qPCR. (C, D) Immunofluorescence detection of the expression levels of FFAR2 and FFAR3 in liver tissue. Data were expressed as the mean ± SEM. * P < 0.05 vs. NC group; # P < 0.05 vs. CCl 4 group.

Article Snippet: Anti-caspase-3 (Cat. 19677-1-AP), anti-FFAR2 (Cat. 19952-1-AP), and anti-FFAR3 (Cat. 66811-1-Ig) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Sini san regulates intestinal flora and short-chain fatty acids to ameliorate hepatocyte apoptosis and relieve CCl 4 -induced liver fibrosis in mice

doi: 10.3389/fphar.2024.1408459

Figure Lengend Snippet: Effect of SCFAs on TNF-a-induced apoptosis in hepatocytes. (A) CCK8 assay of the effects of different concentrations of propionic, butyric, and isovaleric acids on HepG2 viability. (B) The effects of propionic, butyric, and isovaleric acids on FFAR, FFAR3, and caspase-3 protein expression in TNF-a-stimulated HepG2 cells were detected by Western blot. PA: propionic acid; BA: butyric acid; IsA: isovaleric acid. Data are expressed as the mean ± SEM. * P < 0.05 vs. NC group; # P < 0.05 vs. TNF-α group.

Article Snippet: Anti-caspase-3 (Cat. 19677-1-AP), anti-FFAR2 (Cat. 19952-1-AP), and anti-FFAR3 (Cat. 66811-1-Ig) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: CCK-8 Assay, Expressing, Western Blot

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: Primer sequences.

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Sequencing

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: Gut sections (5 μm) of jejunum, ileum and colon were stained with anti-FFAR2 (red) and anti-FFAR3 (green) antibodies (3 replicates). Furthermore, DAPI (blue) was used for nuclei staining. Jejunal enterocytes of the lamina epithelialis mucosae showed a stronger expression of FFAR3 than FFAR2 in the apical membrane of the cells. A positive FFAR2 and FFAR3 staining at both sides of the enterocytes was observed in ileal enterocytes. In addition, colonocytes were highly positive for both receptors, but in the apical membrane they showed a particular expression in form of two separated lines (arrows) [bar = 20μm].

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Staining, Expressing, Membrane

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: Immunofluorescence labelling was performed with 5μm frozen gut section and were stained with an anti-FFAR2 (red) and anti-FFAR3 (green) antibody. Nuclei were marked with DAPI (blue). The tunica muscularis as well as lamina muscularis mucosae of all three gut segments: jejunum [bar = 100μm], ileum [bar = 20 μm] and colon [bar = 100μm] were found to be FFAR3-positive, but no FFAR2 positive smooth muscle cells were detected in the tunica muscularis.

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Immunofluorescence, Staining

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: Different frozen gut sections (5 μm) were labelled with an anti-FFAR2 (red) and anti-FFAR3 (green) antibody and nuclei staining was performed with DAPI (blue). Endothelial cells of veins (§) and lymphatic vessels showed a very high expression of FFAR3, but no FFAR2 [jejunum: bar = 200μm; ileum: bar = 100μm; colon: bar = 100μm].

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Staining, Expressing

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: Gut sections (5 μm) of jejunum, ileum and colon were stained with anti-FFAR2 (red) and anti-FFAR3 (green) antibodies (3 replicates). Furthermore, DAPI (blue) was used for nuclei staining. Plexus myentericus is located between stratum circulare and stratum longitudinale and was double positive for FFAR2 and FFAR3 in all three gut segments [jejunum: bar = 200μm; ileum: bar = 100μm; colon: bar = 100μm].

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Staining

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: Immunofluorescence labelling was performed with 5μm frozen gut section and were stained with an anti-FFAR2 (red) and anti-FFAR3 (green) antibody. Nuclei were marked with DAPI (blue). We observed cells with a strong autofluorescent granula, which were distributed all over the Peyer`s patches. Lymphocytes with a weak expression of FFAR3 were located in the germinal centre and mantle zone of peyer`s patches [bar = 100μm]. FFAR2 was less expressed in the mantle zone than in the germinal centre. Furthermore, double positive enterocytes but also small double-positive cells with a round nucleus between the enterocytes were detected [bar = 20μm]. Such positive cells were also found in the lamina epithelialis mucosae of the villi and were identified as lymphocytes.

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Immunofluorescence, Staining, Expressing

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: IPEC-J2 cells were stained with FFAR2 (red), FFAR3 (green) and with DAPI (blue) for nuclei staining and analysed with confocal microscopy (N = 3]. No differences between the treatment groups were found in the distribution of FFAR2 and FFAR3 in IPEC-J2 cells. Additionally, a control for unspecific binding of the secondary antibody was performed (= control). Here, all treatment groups of high glucose are exemplarily shown. Both receptors were observed in the cytoplasm independent of the treatment group. Furthermore, expression of both receptors varied strongly within the monolayer of the cells. [bar = 5 μm].

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Staining, Confocal Microscopy, Control, Binding Assay, Expressing

Journal: PLOS ONE

Article Title: Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2

doi: 10.1371/journal.pone.0307411

Figure Lengend Snippet: (A) 100 ng and 2 ng of DNA was used for RT-PCR of FFAR2 and FFAR3. Seven controls were performed: blank [b; no DNA], colon 100/2 ng, jejunum 100/2 ng, ileum 100/2 ng. IPEC-J2 samples of one experiment were exemplified: SMC/wo PA/HIGH 100/2 ng, ALI/wo PA/HIGH 100/2 ng, SMC/wo PA/LOW 100/2 ng, ALI/wo PA/LOW 100/2 ng, SMC/PA/HIGH) 100/2 ng, ALI/PS/HIGH 100/2 ng, SMC/PS/LOW 100/2 ng and (21/22) ALI/PS/LOW 100/2 ng. A strong expression of FFAR2 (100–200 bp) and FFAR3 (200–300 bp) was observed in all gut sections. In IPEC-J2 samples, 2 ng of DNA resulted in no detection of a band. (B) Western blot analyses of FFAR2 and (C) FFAR3 were quantitatively analysed. Therefore, raw intensities were normalized to the loading control β-actin. We found no differentially expression of FFAR2 and FFAR3 with the focus on the application of propionic acid, glucose or through the cultivation system.

Article Snippet: For specific protein identifying different antibodies were utilized: rabbit anti-FFAR2 1:1000, mouse anti-FFAR3 1:1000 (both: proteintech, Manchester, UK) and mouse anti-ß-actin 1:40 000 (Sigma-Aldrich, Munich, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control