Journal: Frontiers in Pharmacology

Article Title: A Palmitoylethanolamide Producing Lactobacillus paracasei Improves Clostridium difficile Toxin A-Induced Colitis

doi: 10.3389/fphar.2021.639728

Figure Lengend Snippet: PEA is released in vitro by engineered NAPE-PLD Lactobacillus paracasei under palmitate dose and time dependent boost. (A) PEA release was evaluated in bacterial supernatants at 1, 3, 6, and 12 h, respectively, by HPLC–MS, and the results are expressed as mean ± SEM of n = 4 experiments performed in triplicate. Compared with pLP in absence of palmitate supply, exogenous palmitate (0.000003–0.0003 μg/l) dose- and time-dependently increased PEA release from pNAPE-LP probiotics, *** p < 0.001, ** p < 0.01 vs. both pLP and pLP in presence of palmitate 0.0003 μg/l. PEA levels were undetectable in pLP supernatants, even in the presence of the highest tested doses of exogenous palmitate (0.0003 μg/l). In the same conditions, (B) Western blot analysis of NAPE-PLD expression and relative densitometric analysis of immunoreactive bands show that NAPE-PLD protein expression is time (1, 3, 6, and 12 h) and palmitate concentration (0.000003–0.0003 μg/l) dependent in pNAPE-LP engineered bacteria, whereas no expression was noticeable in pLP alone at the different time points, even in the presence of the highest palmitate doses (0.0003 μg/l). *** p < 0.001 vs. both pLP and pLP + palmitate 0.0003 μg/l n. d., non-detectable.

Article Snippet: After the transfer the membranes were incubated with 10% nonfat dry milk in PBS overnight at 4°C and then exposed, depending on the experiments, with rabbit polyclonal anti-NAPE-PLD (Abcam, Cambridge, United Kingdom) (1:200 v/v), rabbit polyclonal anti–toll-like receptor-4 (TLR4) (Bioss Antibodies, Boston, United States) (1:1,000 v/v), mouse monoclonal anti-RhoA-GTPase (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:100 v/v), rabbit polyclonal anti-p38 mitogen-activated protein kinase (p38 MAPK) (Bioss Antibodies, Boston, United States (1:1,000 v/v), rabbit monoclonal anti-phospho-p-38 (p-p38) MAPK (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:1,000 v/v), rabbit polyclonal anti-NF-κB p65 (Sigma-Aldrich, Milan, Italy) (1:1,000 v/v), mouse monoclonal anti-NF-κB p50 (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:1,000 v/v), mouse monoclonal anti–hypoxia-inducible factor-1-alpha (HIF-1α) (Novus biological, Abingdon, United Kingdom) (1:500 v/v), and rabbit polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Danvers, MA, United States) (1:1,000 v/v) according to standard experimental protocols.

Techniques: In Vitro, Western Blot, Expressing, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: A Palmitoylethanolamide Producing Lactobacillus paracasei Improves Clostridium difficile Toxin A-Induced Colitis

doi: 10.3389/fphar.2021.639728

Figure Lengend Snippet: PEA is released in vivo by engineered NAPE-PLD probiotic under palmitate dose- and time-dependent boost. PEA levels were measured in both wild type (A) and PPARα KO mice (B) colon by HPLC–MS, and results are expressed as mean ± SEM of n = 6 experiments performed in triplicate. TcdA challenge caused PEA increase in both mice types (* p < 0.05 vs. respective controls). Figures (A) and (B) show that pNAPE-LP + palmitate (0.0003 μg/kg), resulted in a significantly increased PEA release as compared to vehicle (both *** p < 0.001 vs. vehicle) and TcdA-treated groups in both wild type and PPARα KO mice ( ooo p < 0.001 and oo p < 0.01 and ° p < 0.05 vs. respective TcdA groups). Figure also shows Western blot analysis of NAPE-PLD expression and relative densitometric analysis of immunoreactive bands in both wild type (C) and PPARα KO mice (D) colon and their relative densitometric quantification (E, F) . Results are expressed as mean ± SEM of n = 6 experiments performed in triplicate. The TcdA challenge caused an increased expression of NAPE-PLD in both mice types (** p < 0.01 vs. respective controls). pNAPE-LP and palmitate (0.0003 μg/kg) supply resulted in a significantly higher NAPE-PLD protein expression in the colon of both untreated mice types (both *** p < 0.001 vs. vehicle) (C–F) , and in both wild type and PPARα KO mice treated with TcdA (both ooo p < 0.001 vs. respective TcdA groups).

Article Snippet: After the transfer the membranes were incubated with 10% nonfat dry milk in PBS overnight at 4°C and then exposed, depending on the experiments, with rabbit polyclonal anti-NAPE-PLD (Abcam, Cambridge, United Kingdom) (1:200 v/v), rabbit polyclonal anti–toll-like receptor-4 (TLR4) (Bioss Antibodies, Boston, United States) (1:1,000 v/v), mouse monoclonal anti-RhoA-GTPase (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:100 v/v), rabbit polyclonal anti-p38 mitogen-activated protein kinase (p38 MAPK) (Bioss Antibodies, Boston, United States (1:1,000 v/v), rabbit monoclonal anti-phospho-p-38 (p-p38) MAPK (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:1,000 v/v), rabbit polyclonal anti-NF-κB p65 (Sigma-Aldrich, Milan, Italy) (1:1,000 v/v), mouse monoclonal anti-NF-κB p50 (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:1,000 v/v), mouse monoclonal anti–hypoxia-inducible factor-1-alpha (HIF-1α) (Novus biological, Abingdon, United Kingdom) (1:500 v/v), and rabbit polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Danvers, MA, United States) (1:1,000 v/v) according to standard experimental protocols.

Techniques: In Vivo, Western Blot, Expressing