Fig. 3: Nizatidine reverses the cPLS by inhibiting the HRH2/CREB signaling pathways. | Nature Communications

Fig. 3: Nizatidine reverses the cPLS by inhibiting the HRH2/CREB signaling pathways.

From: A human liver cell-based system modeling a clinical prognostic liver signature for therapeutic discovery

Fig. 3

a Detection of HRH2 in Huh7.5.1dif cells by immunofluorescence. HRH2 is shown in magenta (Alexa FluorTM 647) and nuclei in blue (DAPI). CTRL = cells incubated with AF647-labeled secondary antibody. Scale bar: 10 µm. b PLS assessment upon perturbation of the HRH2/cAMP/PKA axis. PLS induction was determined by GSEA analysis using “Mock” non-treated cells as reference. Results from two experiments performed in triplicate are shown. Simplified heatmaps show PLS global status and PLS poor- and good-prognosis gene expression. FDR false discovery rate. c Intracellular levels of cAMP were assessed by ELISA. Results from two experiments are expressed in % ±SD compared to Mock. (n = 5; *p < 0.05, unpaired t test). d Expression of histidine decarboxylase (HDC) in Huh7.5.1dif cells analyzed by qRT-PCR. Results from three independent experiments are expressed in % ±SD compared to Mock cells and normalized to GAPDH mRNA. (n = 7: **p < 0.01, two-tailed Mann–Whitney U test). e Nizatidine inhibits HCV-mediated CREB1 activation and decreases CREB5 expression. Western blot analysis of phospho-CREB1 (pCREB1) (Ser133), total CREB1, and total CREB5. NT non-treated. Results are representative of one out of two experiments (see Supplementary Fig. 7). f CREB5 is a driver of the HCV-induced cPLS. CREB5 KO was performed as described in “Method”. PLS induction was determined by GSEA analysis using “Mock” non-infected cells as reference. Reversal was assessed by analyzing sgCREB5 VS sgCTRL. g Activation of the HRH2 signaling pathways during chronic liver injury. AC: adenylate cyclase, CRE CREB response element. h Nizatidine reverts the FFA-induced poor-prognosis PLS in culture of patient-derived multicellular spheroids (three patients without history of chronic liver disease). PLS induction was determined by GSEA analysis using “Mock” non-treated spheroids as reference. Simplified heatmaps show PLS global status and PLS poor- and good-prognosis gene expression. i HRH2 expression measured by qRT-PCR in healthy multicellular spheroids. Each experiment shows mean ± SD in percentage compared to Mock spheroids (n = 4), *p < 0.05, unpaired t test. j Nizatidine reverts the histamine-induced poor-prognosis PLS in culture of patient-derived multicellular spheroids. Source data are provided as a Source data file.

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