fig5

Figure 5. CW-DNVs alleviate inflammation through sRNA cargo and terpenoid components. (A) Confocal images and (B) quantification of Raw264.7 cells after incubation with PKH26-labeled CW-DNVs; (C) Expression of Il6, Il1b, and Tnfα mRNA in Raw264.7 cells following pretreatment with CW-DNVs and subsequent LPS stimulation; (D) Confocal images and (E) quantification of primary KCs incubated with PKH26-labeled CW-DNVs; (F) Expression of Il6, Il1b, and Tnfα mRNA in KCs following CW-DNVs pretreatment and LPS stimulation; (G) RNA sequencing read counts for sRNA classes in CW-DNVs; (H) Il6 and Il1b mRNA quantification in KCs transfected with total sRNA isolated from CW-DNVs, followed by LPS stimulation; (I) LC-MS peak-area quantification of terpenoids in CW-DNVs; (J) TLC identification of curdione, curcumenol, atractylenolide II, and germacrone; (K) Effects of individual terpenoids or an equimolar mixture on the expression of LPS-induced inflammatory cytokine genes (Il6, Il1b, and Tnfα) in macrophages. Data are presented as mean ± SEM (n = 3). One-way ANOVA with Tukey’s multiple-comparison test. ^####P < 0.0001 vs. NC group. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001 vs. LPS-only group. CW-DNVs: Curcuma wenyujin-derived nanovesicles; sRNA: small RNA; KCs: Kupffer cells; LPS: lipopolysaccharide; LC-MS: liquid chromatography-mass spectrometry; TLC: thin-layer chromatography; SEM: standard error of the mean; ANOVA: analysis of variance; NC: negative control; ns: not significant.